ESTUDIO COMPARATIVO DEL CONTROL COLINÉRGICO Y ...

UNIVERSITAT DE LES ILLES BALEARS

Facultat de Ciències

Departament de Biologia Fonamental i Ciències de la Salut

Laboratori de Neurofisiologia

TESIS DOCTORAL

ESTUDIO COMPARATIVO DEL CONTROL COLINÉRGICO Y

SEROTONÉRGICO SOBRE LOS PROCESOS

NEUROFISIOLÓGICOS INVOLUCRADOS EN

EL CICLO SUEÑO-VIGILIA

Memoria para optar al Grado de

Doctor

Programa de doctorado de Ciencias Médicas Básicas del Departamento de Biología Fundamental y Ciencias de la Salud

Presentada por

SILVIA TEJADA GAVELA

Palma de Mallorca, julio de 2010

El interesado Silvia Tejada Gavela

Con el beneplácito de los Directores Dra. Susana Esteban Valdés Profesora Titular de Fisiología Dr. Antoni Gamundí Gamundí Profesor Titular de Fisiología

Por muy alta que sea una colina, siempre hay un sendero hacia su cima

A mis padres y a mi Toni

Agradecimientos

La realización de esta tesis doctoral no hubiese sido posible sin la ayuda,

colaboración, asesoramiento y apoyo de una gran cantidad de personas. Gracias a ellas

he podido superar los angustiosos y duros momentos que he pasado a lo largo de estos

años, pero también gracias a ellos he vivido otros momentos de alegría y con sus

ánimos he podido llegar al final del camino. A todas ellas va dirigido mi

agradecimiento:

Gracias a la Dra. Susana Esteban, directora de esta tesis doctoral, con la cual he

compartido muchas horas escribiendo y discutiendo los experimentos, a veces hasta

horas intempestivas. Gracias por el apoyo brindado tanto a nivel profesional como

personal, y por luchar por cada uno de los trabajos realizados, a todos los niveles.

Gracias al Dr. Antoni Gamundí, director de esta tesis doctoral, por haberme

acogido desde el primer momento y enseñarme sus conocimientos sobre el mundo de la

electrofisiología. Por haberme apoyado y confiado siempre en mi, sobretodo en los

peores momentos a lo largo de todos estos años, tanto a nivel profesional como

personal.

También agradecer al “gran jefe”, el Dr. Rubén V. Rial por ofrecerme la

oportunidad de llevar a cabo esta tesis doctoral en su grupo, ayudarme con los

problemas técnicos y con la revisión final de esta tesis. A la Dra. Cristina Nicolau por

su contagiosa alegría y sus risas en todo momento, que siempre ayudaban en los

momentos difíciles. A Mourad Akaârir, gracias, estuviste conmigo en todo momento, de

manera incondicional, cuando los problemas surgían con la “máquina” siempre tenías

tiempo para dedicarme y ayudar a repararlos.

Agradecer también al Dr. Julián González, por su hospitalidad durante los viajes

a Tenerife, y por su paciencia a la hora de intentar hacerme entender los métodos de

análisis de señales.

Gracias también a los compañeros de laboratorio que han ido pasando a lo largo

de los años: Guillem Mateu, Almudena, María Álvaro, Lluis Gené, Gaspar, Pere

Barceló, Mª Antònia, Bea, Marga. Pero no sólo los que han estado compartiendo

conmigo las tareas del laboratorio me han ayudado; quiero agradecer a gente de otros

grupos que me han prestado su apoyo y siempre me han animado, con los cuales he

llegado a establecer una gran amistad, y que han compartido momentos personales

buenos y malos conmigo: mis chicas “gatunas”, Silvia Terés, Vicky Lladó y Mónica

Higuera, gracias por vuestro apoyo, el trabajo con las células y todas nuestras

confesiones; a la Dra. Salud Deudero, por darme la oportunidad de volver al mundo de

la ciencia y confiar en mí en todo momento; a los chicos de Biología Marina, Toni y

Paqui, Andreu, Piluca y el resto de compañeros que participaban en esas grandes

jornadas de buceo y pesca; a Elisa y Mar así como a todas las chicas de la Feria de la

Ciencia; y a toda la gente de bioquímica que también me han ayudado y apoyado.

Especialmente, quiero agradecer la ayuda, el apoyo, las confidencias recibidas

por dos grandes amigas, gracias a Marian Comas y Cati Roca, por ayudarme a dejar el

lado oscuro de la “Fuerza”. Nuestra historia ya se remonta a muchos años, y hemos

compartido grandes momentos de risas en el laboratorio, en nuestras estancias en el

extranjero y en nuestra vida privada. Muchas gracias a las dos.

I would like to express my special thanks to Professor Ton Coenen for his help

during my stay in Nijmegen. I greatly appreciated his close supervision on my work, his

support in the lab from the first day to the last one and also for creating a very friendly

working environment. I also want to thank Anke Sambeth, for being very supportive

and helpful with the everyday life in the lab, especially with the animal work, which

was more difficult for me being a foreign guest. I also want to acknowledge other lab

colleagues that helped me in my experience abroad: Annika Smit, Saskia van Uum,

Hans Kirjnen and Saskia Hermeling. Being far away from home is an enriching

experience, but one can feel lonely sometimes. That is why I would like to thank my flat

mates and Dutch friends, Loes Thijssen, Silvana Griz, Armen Sargsyan, Aurora and

Eoin, among many others because they made my stay very easy and fun by spending

many nights having dinner, going out and having great conversations.

A mis compañeros de trabajo, gracias al Dr. Noguera por darme la oportunidad

de trabajar a su lado; y a mi nueva compañera de trabajo, Pilar, por estar presente en

estos últimos momentos y ayudarme con los últimos retoques. Y toda la gente con la

que comparto el día a día, a David y a Ignasi, a la gente de la Fundació Mateu Orfila y

del Servicio de Radiología.

También agradecer a esa gente ajena al mundo de la ciencia, con los que sin

querer he acabado compartiendo también los buenos y los malos momentos que he

vivido en todos estos años. Gracias a mis mejores amigas Pili y Rosario, habéis

escuchado todos mis problemas y todos los relacionados con esta tesis, aún sin entender

lo que os contaba, muchas gracias a las dos. También a todos esos amigos, que de una o

de otra manera, me han apoyado con este gran proyecto, gracias Toñy, Cati Fiol, Maties

y Xisca y Miquel Àngel.

Mis gratitudes a mi familia por su comprensión, amor incondicional y paciencia.

Gracias, mamá, has sido mi gran apoyo durante toda mi vida y en especial durante todos

estos años de tesis. Gracias, papá, por darme la oportunidad de poder crearme un

camino. Gracias a mi hermana, por demostrar su orgullo en todo momento. Gracias a

mis abuelos, a Olga, Francisco y a mi niña Andrea. Gracias a toda mi familia en la

Península, y gracias a mi familia política, a mis suegros y a mi cuñada Bel.

Y por último, aunque no por ello menos importante, quiero agradecer todo el

apoyo, ayuda y compresión que he recibido por parte de mi gran amigo, mi compañero,

mi marido. Gracias, Toni. No tengo palabras para agradecer el haberte tenido siempre a

mi lado, sin tu apoyo no hubiese conseguido terminar este largo camino, tú has

entendido cada paso que he dado y has sabido aconsejarme en cada momento. Gracias

por apoyarme incondicionalmente y por darme tu amor.

Muchas gracias a todos.

ÍNDICE / INDEX

Abreviaturas / Abbreviations 15

Resumen / Abstract 17

Lista de artículos originales / List of original papers 21

1. Introducción 23

1.1. Bases fisiológicas del sueño 25

1.1.1. Sueño comportamental 26

1.1.2. Sueño poligráfico 26

1.2. Análisis de las señales electroencefalográficas 29

1.3. Sueño y vigilia: neuroanatomía 31

1.3.1. Áreas responsables de la vigilia 32

1.3.2. Áreas responsables de la pérdida de la vigilia 34

1.3.3. El conmutador sueño-vigilia 36

1.3.4. Control del REM 37

1.3.5. La reacción de alerta 38

1.4. Regulación del ciclo sueño-vigilia 39

1.5. Ciclo sueño-vigilia y sistema colinérgico 40

1.5.1. Organización neurofisiológica del ritmo theta 41

1.5.2. Comunicación entre el hipocampo y la corteza 43

1.5.3. Mecanismos de consolidación de la memoria. Vías implicadas 43

1.5.4. Efectos adversos de la estimulación colinérgica.

Estrés oxidativo 45

1.6. -. Ciclo sueño-vigilia y sistema serotonérgico 47

2. Objetivos 53

3. Métodos generales 57

3.1. Animales de experimentación 59

3.1.1. Ratas 59

3.1.2. Tórtolas 59

3.2. Fármacos y administración 60

3.2.1. Fármacos y productos utilizados 60

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3.2.2. Administración de fármacos 61

3.3. Metodología electrofisiológica 62

3.3.1. Implantación crónica de los electrodos 62

3.3.2. Registros de las señales bioeléctricas 62

3.3.3. Análisis de la señal electroencefalográfica y los parámetros

bioeléctricos 62

3.4. Observaciones comportamentales 64

3.5. Evaluación del estrés oxidativo 65

3.6. Cuantificación de la actividad locomotora espontánea 66

3.7. Valoración de la capacidad cognitiva: Radial maze 67

3.8. Análisis estadístico 68

4. Resultados 69

Manuscript I. Effects of pilocarpine on the cortical and hippocampal

theta rhythm in different vigilance states in rats. 71

Manuscript II. Effects of the muscarinic agonist pilocarpine

on locomotor activity and vigilance states in ring doves. 81

Manuscript III. Effects of serotonergic drugs on locomotor activity

and vigilance states in ring doves. 105

Manuscript IV. Electroencephalogram functional connectivity

between rat hippocampus and cortex after

pilocarpine treatment. 137

Manuscript V. Antioxidant response analysis in the brain

after pilocarpine treatments. 151

Manuscript VI. Antioxidant response and oxidative damage in

brain cortex after high dose of pilocarpine. 159

5. Discusión general 165

6. Conclusiones / Conclusions 179

7. Bibliografía / References 189

14

ABREVIATURAS / ABBREVIATIONS

ACh Acetilcolina / Acetylcholine ANOVA Análisis de la varianza / Analysis of variance AP Anterioposterior / Anterioposterior AW Vigilia activa / Active waking CAT Catalasa / Catalase DV Dorsoventral / Dorsoventral ECG Electrocardiograma / Electrokardiogram EEG Electroencefalograma / Electroencephalogram EOG Electrooculograma / Electrooculogram EMG Electromiograma / Electromyogram FFT Transformada rápida de Fourier / Fast Fourier Transform GP Glutatión peroxidasa / Glutathione peroxidase GR Glutatión reductasa / Glutathione reductase GSH Glutatión reducido / Reduced glutathione GSSG Glutatión oxidado / Oxidized glutathione HPLC Cromatografía Líquida de Alta Resolución / High performance

liquid chromatography Hz Hertzios / Herzts LC Locus ceruleus / Locus ceruleus LDT Latero-dorsal del tegmento / Latero-dorsal tegmental MDA Malondialdehído / Malondialdehyde ML Mediolateral / Mediolateral PCPA Paraclorofenilalanina / Para-chlorophenylalanine PLI Índice de retraso de fase / Phase lag index

15

PPT Pedúnculo-pontino del tegmento / Peduncle-pontine tegmental PW Vigilia pasiva / Passive waking RD Rafe dorsal / Dorsal raphe REM Sueño paradójico / Rapid Eye Movement ROS Especies reactivas del oxígeno / Reactive oxygen species SOD Superóxido dismutasa (CuZnSOD, MnSOD, EC SOD) /

Superoxide dismutase (CuZnSOD, MnSOD, EC SOD) SNC Sistema nervioso central / Nervous central system SWS Sueño de onda lenta / Slow Wave Sleep VLPO Núcleo ventrolateral preóptico / Ventrolateral preoptic nucleus VLPOe Núcleo ventrolateral preóptico extendido / Extended ventrolateral

preoptic nucleus 5-HT Serotonina / Serotonin 5-HTP 5- hidroxitriptófano / 5-hydroxytryptophan 8-OH-DPAT 8-hydroxi-2-(di-n-propilamino) tetralin / 8-hydroxy-2-(di-n-

propylamino) tetralin

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RESUMEN

ESTUDIO COMPARATIVO DEL CONTROL COLINÉRGICO Y

SEROTONÉRGICO SOBRE LOS PROCESOS

NEUROFISIOLÓGICOS INVOLUCRADOS EN

EL CICLO SUEÑO-VIGILIA

Tesis doctoral, Silvia Tejada Gavela, Departament de Biologia Fonmental i Ciències de la Salut.

Laboratorio de Neurofisiología, Universitat de les Illes Balears, Palma de Mallorca, España.

Gran parte de las funciones fisiológicas se encuentran bajo control circadiano.

Entre las más importantes destaca el ciclo sueño-vigilia. En la presente tesis doctoral, el

uso de fármacos que de manera selectiva estimulan o inhiben determinadas vías

nerviosas ha permitido estudiar la regulación neurofisiológica del ciclo sueño-vigilia. Se

ha profundizado en la participación de los sistemas colinérgico y serotonérgico sobre la

estructura de los estados de vigilancia de ratas (Rattus norvegicus) y tórtolas collarizas

(Streptopelia risoria), determinando las variaciones producidas en el patrón

electroencefalográfico (duraciones, número de episodios, latencias, potencia espectral,

coherencias y análisis no lineal). Todo ello, prestando especial atención al ritmo theta

presente en los estados de vigilia y REM, generado principalmente en el hipocampo e

implicado en diferentes procesos cerebrales, como la consolidación de la memoria. Con

el fin de completar los resultados obtenidos a partir de los estudios bioeléctricos, se han

realizado otros estudios como la medición de la actividad locomotora o la valoración de

las funciones cognitivas. Adicionalmente, dado que el agonista colinérgico utilizado es

un fármaco usado en modelos de epilepsia que puede inducir daño neuronal, se ha

valorado el estrés oxidativo en muestras de cerebro para poder descartar la existencia de

artefactos en los resultados obtenidos tras la estimulación colinérgica.

Los resultados mostraron que la estimulación de receptores muscarínicos por

pilocarpina no estaba relacionado con la generación y el mantenimiento del sueño REM,

ni en rata ni en tórtola collariza. Sin embargo, el fármaco colinérgico provocó un claro

incremento de la vigilia y del ritmo theta en ambas especies. En ratas, la pilocarpina

incrementó el ritmo theta y la potencia de esta banda en los estados de vigilia pasiva y

activa en el EEG de corteza cerebral, siempre acompañado de la presencia de ritmo

theta hippocampal. De esta manera, la acción de la pilocarpina se relacionó con la

aparición de ritmo theta en la corteza, indicando un incremento en la transferencia del

17

ritmo theta hipocampal a la corteza durante los estados de vigilia en ratas. Teniendo en

cuenta que la transferencia de oscilación theta entre hipocampo y corteza forma parte de

los circuitos que intervienen en los procesos de aprendizaje y memoria, la pilocarpina

podría estar favoreciendo el flujo de información entre dichas regiones cerebrales, lo

que se comprobó al realizar una evaluación cognitiva mediante el paradigma del

laberinto radial. De esta manera, la estimulación de receptores muscarínicos por

pilocarpina mejoró la realización de esta tarea que constituye uno de los métodos más

utilizados en animales para el estudio del aprendizaje y memoria. Finalmente, las dosis

de pilocarpina usadas en el presente trabajo no han inducido ni manifestaciones

epileptiformes ni daño oxidativo a nivel cerebral.

El papel de la serotonina sobre el ciclo sueño-vigilia es complejo y ha sido

estudiado principalmente en mamíferos, por lo que utilizó como animal de

experimentación la tórtola collariza para comparar los resultados con los previamente

descritos en diversos estudios en mamíferos. La activación del receptor serotonérgico 5-

HT1A mediante la administración del agonista 8-OH-DPAT incrementó la actividad

locomotora espontánea, los estados de vigilia activa y el aseo, disminuyendo los estados

de sueño (SWS y REM). En concordancia con estos resultados, la depleción de

serotonina mediante el tratamiento con PCPA provocó una disminución de la actividad

locomotora espontánea y los estados de la vigilia, mientras que incrementó la duración

y el número de episodios de los estados de sueño. Todo ello refleja un efecto

estimulatorio de la serotonina sobre los estados de vigilia activa, y un efecto inhibitorio

sobre los del sueño, a través del receptor serotonérgico 5-HT1A. Estos resultados están

en la línea con recientes estudios en mamíferos que indican que la actividad

serotonérgica es mayor durante los estados de vigilia, disminuye durante el SWS y es

aún menor durante el REM. Igualmente, los niveles de serotonina en cerebro de rata son

superiores durante la vigilia y disminuyen durante el SWS y REM.

Finalmente, los resultados obtenidos indican que tanto el sistema colinérgico

como el serotonérgico, implicados en el control del ciclo sueño-vigilia, activan

principalmente los estados de vigilia en mamíferos y aves, sugiriendo que los

mecanismos colinérgicos y serotonérgicos que regulan el ciclo sueño-vigilia son

compartidos por ambos grupos de vertebrados reflejando un origen filogenético común.

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ABSTRACT

COMPARATIVE STUDY OF CHOLINERGIC AND

SEROTONERGIC CONTROL ON THE NEUROPHYSIOLOGICAL

PROCESSES INVOLVED IN THE SLEEP-WAKE CYCLE

PhD Thesis, Silvia Tejada Gavela, Departament de Biologia Fonmental i Ciències de la Salut. Laboratorio

de Neurofisiología, Universitat de les Illes Balears, Palma de Mallorca, España.

Most of the physiological functions are under circadian control. One of the more

important is the sleep-wake cycle. In this PhD thesis, the use of drugs that selectively

stimulate or inhibit certain nerve pathways has permitted to study the

neurophysiological regulation of the sleep-wake cycle. It has been examined the

involvement of the cholinergic and serotonergic systems on the structure of the

vigilance states of rats (Rattus norvegicus) and ring doves (Streptopelia risoria),

determining the changes on the EEG pattern (duration, number of episodes, latencies,

spectral power, coherence and nonlinear analysis). Particular attention was focused on

theta rhythm which is present in waking and REM states, it is mainly generated in the

hippocampus and involved in different brain processes, such as memory consolidation.

In order to strengthen the results obtained from bioelectric studies, other studies have

been performed such as the measurement of the spontaneous locomotor activity or the

evaluation of cognitive functions. Additionally, since the chosen cholinergic agonist is a

drug used in epilepsy models that can induce neuronal damage, it has been assessed the

oxidative stress in brain samples in order to rule out the existence of artifacts in the

results obtained after the cholinergic stimulation.

The results showed that the stimulation of the muscarinic receptors by

pilocarpine was not related to the generation and maintenance of REM sleep, neither in

rat nor in ring dove. However, the cholinergic drug caused a marked increase in waking

and theta rhythm in both species. In rats, pilocarpine increased the theta rhythm and the

power of this band in the passive and active waking states in the EEG frontal cortex,

always accompanied by the presence of hippocampal theta rhythm. Consequently, the

action of the pilocarpine was associated to the appearance of theta rhythm in the cortex,

indicating an increase in the transfer of the hippocampal theta rhythm to the cortex

during waking states in rats. Taking into account that theta oscillation transfer between

19

hippocampus and cortex is one of the circuits involved in learning and memory

processes, pilocarpine effects could support the information flow between these brain

regions, hypothesis that was assessed by the evaluation of a cognitive paradigm (radial

maze test). Thus, the simulation of the muscarinic receptors by pilocarpine improved

the performance of this task which is one of the most widely used method for studying

learning and memory in animals. Finally, the dose of pilocarpine used in this study did

not induce neither epileptiform events nor oxidative damage in the brain.

The role of serotonin on sleep-wake cycle is complex and it has been mainly

studied in mammals; therefore, ring doves were used as experimental animals in order

to compare the results with those described in several studies in mammals. The

activation of serotonergic 5-HT1A receptor by the administration of the agonist 8-OH-

DPAT increased the spontaneous locomotor activity, active waking and grooming

states, reducing sleep states (SWS and REM). Consistent with these results, depletion of

serotonin by PCPA treatment caused a decrease of spontaneous locomotor activity and

waking states, while it increased the duration and number of episodes of the sleep states.

Altogether reflects a stimulatory effect of the serotonin on active waking states, and an

inhibitory effect on sleep ones, through the serotonergic 5-HT1A receptor. These results

are in line with recent studies in mammals indicating that the serotonergic activity is

higher during waking states, decreases during SWS and is even lower during REM

sleep. Similarly, the levels of serotonin in rat brain are higher during waking and

decrease during SWS and REM.

Finally, the obtained results indicate that both cholinergic and serotonergic

systems, implicated in controlling the sleep-wake cycle, mainly activate the waking

states in mammals and birds, suggesting that the cholinergic and serotonergic

mechanisms that regulate sleep-wake cycle are shared by both groups of vertebrates,

and reflect a common phylogenetic origin.

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LISTA DE ARTÍCULOS ORIGINALES / LIST OF ORIGINAL PAPERS

1. Tejada, S.; Roca, C.; Sureda, A.; Rial, R.V.; Gamundí, A. and Esteban, S.

(2006) Antioxidant response analysis in the brain after pilocarpine

treatments. Brain Research Bulletin. 69:587-592.

2. Tejada, S.; Sureda, A.; Roca, C.; Gamundí, A. and Esteban, S. (2007)

Antioxidant response and oxidative damage in brain cortex after high dose of

pilocarpine. Brain Research Bulletin. 71:372-375.

3. Tejada, S.; Rial, R. V.; Coenen, A. M. L.; Gamundí, A. and Esteban, S.

(2007) Effects of pilocarpine on the cortical and hippocampal theta rhythm

in different vigilance states in rats. European Journal of Neuroscience.

26:199-206.

4. Tejada, S., González, J.J.; Rial, R.V.; Coenen, A.M.L.; Gamundí, A. and

Esteban, S. (2010) Electroencephalogram functional connectivity between

rat hippocampus and cortex after pilocarpine treatment. Neuroscience.

165:621-631.

5. Tejada, S.; Rial, R.V.; Gamundí, A. and Esteban, S. (2010) Effects of

serotonergic drugs on locomotor activity and vigilance states in ring doves.

Submitted to Behavioural Brain Research.

6. Tejada, S.; Rial, R.V.; Gamundí, A. and Esteban, S. (2010) Effects of the

muscarinic agonist pilocarpine on locomotor activity and vigilance states in

ring doves. Submitted to European Journal of Neuroscience.

21

1. INTRODUCCIÓN

23

INTRODUCCIÓN

1. INTRODUCCIÓN

1.1. Bases fisiológicas del sueño

El sueño ha despertado gran curiosidad y ha sido muy estudiado por las grandes

culturas de todos los tiempos, con poco éxito en sus inicios. El sueño no se entiende sin

la existencia de un estado de vigilia, es decir, la vigilia y el sueño forman un ciclo al

que se denomina “ciclo vigilia-sueño” que en el humano adulto está más o menos

acoplado al ciclo día-noche o ciclo de luz-oscuridad. De hecho, el ritmo biológico más

marcado en toda la filogenia es el de actividad-reposo, o vigilia-sueño. Los dos estados

constituyen comportamientos básicos, ya que todos los demás comportamientos

(sociales, alimentación, sexual) se producen en conexión con ellos (Gottesmann, 1992).

Se tratan de estados particulares en los que acontecen cambios fisiológicos que

involucran distintos sistemas del organismo, regulados por el sistema nervioso central

(SNC). El sueño, a su vez, puede ser subdividido en dos estados -el sueño de onda lenta

(SWS: Slow Wave Sleep) y el sueño paradójico o sueño REM (Rapid Eye Movement)-

con trazados electroencefalográficos característicos y con diferencias marcadas en la

fisiología endocrina y vegetativa en general (Pedemonte, 2000; Pedemonte et al.,

1999).

Por lo tanto, el sueño, lejos de ser un estado de inactividad en donde se queda

reposando en silencio, constituye un estado de gran actividad donde se operan

importantes cambios hormonales, metabólicos, térmicos, bioquímicos y en la actividad

cerebral en general.

El sueño es un estado que puede definirse según diferentes puntos de vista:

• Comportamental, que permite diferenciar el sueño de otros estados de

reposo.

• Poligráfico, a partir del cual se pone de manifiesto que el sueño no es un

estado homogéneo, según las variaciones que se observan en la actividad

eléctrica cerebral y en otros parámetros bioeléctricos.

• Fisiológico, ya que asociados al sueño y a cada fase del mismo se producen

variaciones específicas en las funciones cardiovascular, respiratoria y

termorreguladora; siguiendo los principios de la reostasia y los

correspondientes mecanismos de regulación.

25

INTRODUCCIÓN

1.1.1. Sueño comportamental

La existencia de los estados de actividad-inactividad puede servir para definir el

sueño desde un punto de vista comportamental. Así pues se podría considerar que el

sueño y la baja actividad son equivalentes, de la misma forma que lo son la actividad y

la vigilia. El sueño puede ser definido en términos de criterios comportamentales:

1) Reposo motor (Pieron, 1913).

2) Umbrales sensoriales elevados (Pieron, 1913).

3) Fácil reversibilidad (Pieron, 1913).

4) Adopción de una postura estereotipada (Flanigan, 1974; Flanigan et al., 1974).

5) Uso de lugares específicos donde dormir (Bruce Durie, 1981).

6) Organización cíclica circadiana (Bruce Durie, 1981).

7) Estado regulado, al presentar efectos de privación y saciedad (Tobler, 1985).

Sin embargo, no es necesario que un animal presente todos y cada uno de estos

criterios, cualquier animal que mostrara cuatro o más de estas características se ha

afirmado que presenta sueño comportamental. Por el contrario, hay que suponer que

aquellos que presenten un número menor, simplemente poseerán estados cíclicos de

actividad e inactividad (Bruce Durie, 1981).

De acuerdo con esta definición del sueño, existen muchos animales que tienen

sueño comportamental: entre ellos pueden incluirse numerosos invertebrados y, con

muy pocas excepciones, los vertebrados. Lo que determina que exista este sueño es,

evidentemente, un elevado nivel de complejidad en el comportamiento y, por lo tanto,

en el sistema nervioso.

1.1.2. Sueño poligráfico

De manera resumida, mamíferos y aves muestran un sueño comportamental

completo, con los siete criterios citados con anterioridad. Pero además, este sueño está

dividido en una serie de fases y de estados que se suceden alternándose repetidamente a

lo largo de cada episodio de sueño. Así, se estudió la existencia de actividad bioeléctrica

espontánea en el cerebro y, además, demostrándose que esta actividad se modifica en

respuesta a estímulos auditivos y visuales. Por lo tanto, el sueño no es un estado

homogéneo, sino que aparecen fases que presentan manifestaciones bioeléctricas

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propias (Rechtschaffen y Kales, 1968). Esta actividad bioeléctrica se puede obtener por

medio de registros poligráficos que consisten en recoger de manera simultánea diversas

actividades bioeléctricas las cuales se estudian normalmente en las frecuencias

comprendidas entre los 0,5 y los 50 Hz; aunque es necesario el uso de técnicas como el

electrocardiograma (ECG), el electromiograma (EMG), o el electrooculograma (EOG)

que permiten tener una mayor precisión a la hora de identificar los diferentes períodos.

La actividad electroencefalográfica permite la distinción de diferentes estados de

vigilancia, así como obtener información sobre el funcionamiento del SNC. Muchos

autores separan en el ciclo vigilia-sueño tres estados básicos (vigilia, sueño de onda

lenta y sueño paradójico), aunque una observación más detallada muestra que esos tres

estados se pueden dividir en más. Así, en ratas se pueden diferenciar siete estados

(Gottesmann, 1992), en los cuales se presta especial atención al ritmo theta por estar

presente en diferentes estados de la vigilia y sueño REM:

1- Vigilia activa con ritmo theta. Se corresponde a un estado de atención y/o

vigilia motora activa, en el cual aparecen mayoritariamente frecuencias que varían entre

los 4 y los 8 Hz (banda theta). El EMG presenta alta amplitud.

2- Vigilia activa sin ritmo theta. Se corresponde a un estado de vigilia en el cual

no aparece el rimo theta sino frecuencias superiores a los 8 Hz, y el EMG es de

amplitud alta. Dentro de este estado, se incluiría el comportamiento de aseo o

acicalamiento (grooming), definido como aquel estado en el que el animal se limpia o

acicala.

3- Vigilia pasiva con ritmo theta. Es un estado de vigilia sin atención o actividad

motora; es decir, el sujeto experimental se encuentra en pasividad. Aparece

mayoritariamente ritmo theta con frecuencias comprendidas entre los 4 y 8 Hz. El EMG

presenta una menor amplitud.

4- Vigilia pasiva sin ritmo theta. Es igual que el estado anterior con la diferencia

que el ritmo theta no se observa sino que aparecen frecuencias superiores a los 8 Hz.

5- Sueño de onda lenta (SWS). Es el primer estado que aparece del sueño,

caracterizado por ondas lentas que progresivamente incrementan su amplitud. La

frecuencia del EEG varía entre los 0,5 y los 4 Hz. El EMG disminuye en amplitud.

6- Estado intermedio. Es un estado de corta duración previo al sueño paradójico

y a veces justo después del mismo. Se caracteriza por una asociación inusual de espigas

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de alto voltaje y bajas frecuencias en diferentes regiones del cerebro. A veces constituye

un estado difícil de determinar.

7- Sueño paradójico (PS) o sueño REM. Es el segundo estado del sueño, en el

que se producen movimientos oculares rápidos, movimientos de bigotes, contracciones

súbitas musculares (normalmente correlacionadas con los movimientos oculares), con

frecuencias de EEG que se mueven dentro de la banda theta (4-8 Hz) y en el cual el

EMG pasa a tener una amplitud mínima.

De la misma manera, en aves se pueden diferenciar diferentes estados, en el

presente estudio se han clasificado en función de lo descrito por Fuchs et al. (2006) y

Toledo y Ferrari (1991):

1- Vigilia activa. Se corresponde a un estado de atención y/o vigilia motora

activa con los ojos abiertos, en el cual el EEG presenta una baja amplitud y

mayoritariamente frecuencias altas. El EMG presenta alta amplitud. Dentro de este

estado, se incluiría el comportamiento de aseo o acicalamiento (grooming).

2- Vigilia pasiva. Es un estado de vigilia con un nivel de atención bajo y sin

actividad motora, estando el animal de pie o sentado con los ojos abiertos; es decir, el

sujeto experimental se encuentra en pasividad. Aparece mayoritariamente frecuencias

altas de baja amplitud y el EMG presenta una menor amplitud que en el estado anterior.

3- SWS. Es el primer estado que aparece del sueño en el cual el animal se

encuentra de pie o sentado con los ojos cerrados y algunas veces con el cuello retraído o

recostado sobre el pecho. Se caracteriza por ondas lentas (baja frecuencia) en el EEG

que progresivamente incrementan su amplitud y con una actividad del EMG baja

disminuyendo en amplitud.

4- Sueño REM. Es el segundo estado del sueño, en el que el animal se encuentra

de pie o sentado con los ojos cerrados y durante el cual se producen movimientos

oculares rápidos (actividad del EOG rápida y de alta amplitud), contracciones súbitas

musculares (normalmente correlacionadas con los movimientos oculares), con caídas de

la cabeza esporádicas y bruscas seguidas de la corrección postural de una manera lenta.

Presentan un EEG de baja amplitud y frecuencias altas. El EMG pasa a tener una

amplitud mínima con una alta frecuencia.

De esta manera, gracias a la actividad eléctrica cerebral registrada y en conjunto

con las observaciones comportamentales de mamíferos y aves en el laboratorio, es

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posible desarrollar criterios electrofisiológicos que sirvan de indicador del sueño y de la

vigilia (Campbell y Tobler, 1984).

1.2. Análisis de las señales electroencefalográficas

Una vez obtenida la señal procedente del sujeto de estudio, es preciso proceder a

su análisis. El análisis de los registros de EEG se inicia con un examen visual del

mismo. De esta manera, se separan en fragmentos los diferentes estados en función de

los ritmos observados en el EEG y del comportamiento, pudiéndose realizar así un

actograma, donde se pueden observar los diferentes estados del ciclo sueño-vigilia y su

progresión en el tiempo. Sin embargo, el examen visual para analizar de manera

numérica la información contenida en las señales, contiene un componente subjetivo por

parte del experimentador. Por ello, se recurre al análisis lineal y no lineal de las señales

poligráficas.

Para el estudio de cualquier proceso mediante el análisis de una o más de las

variables asociadas a él, es útil introducir el concepto de sistema. Se denomina sistema a

una unidad funcional que, como respuesta a un conjunto de estímulos (entradas) tanto

externos como internos, es capaz de producir una o más respuestas (salidas).

Una aproximación al estudio de los sistemas que resulta útil en fisiología es

tratarlos como “cajas negras”. Se trata de caracterizar la forma en la que el sistema

transforma las entradas en salida(s) sin necesidad de disponer de un modelo matemático

de su comportamiento ni de su disposición interna. Esto tiene la ventaja de que permite

analizar, dentro de un marco común, sistemas que se correspondan tanto con entidades

físicas reales como con entidades abstractas (Pereda y González, 2002).

Normalmente, los sistemas se pueden dividir en dos grandes grupos, lineales y

no lineales:

A/ Sistema lineal

Un sistema es lineal si su respuesta a un conjunto de n entradas es la suma de las

respuestas a cada una de las entradas individuales. Es decir, se observa una

proporcionalidad entre el estímulo y la respuesta, independientemente de lo que pasa en

el interior de la caja.

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Análisis en el dominio de las frecuencias

El método de análisis de frecuencias más utilizado está basado en la teoría

desarrollada por el matemático francés Joseph Fourier (1768-1830), que define que una

señal periódica de forma compleja puede ser descompuesta en una serie de ondas

simples relacionadas entre si de forma armónica; es decir, la señal se puede

descomponer en la suma de infinitas señales sinusoidales de frecuencias armónicas a la

frecuencia fundamental. Para evaluar el grado de complejidad de la función que

interviene en una señal, se procede al análisis de los armónicos. Por lo tanto, lo que

interesará en este caso no serán las variaciones en el dominio temporal, sino sus

frecuencias, obteniéndose un espectro de potencias, en el que la señal compleja ha sido

descompuesta en señales más simples a través de la transformada de Fourier.

Coherencia

La coherencia es un tipo de análisis lineal que mide la correlación entre dos

señales en el dominio frecuencial (Gerloff y Andres, 2002; Varela et al., 2001; Pereda et

al., 2005) estableciendo un patrón de conectividad entre las actividades del EEG y entre

diferentes regiones cerebrales (Horwitz, 2003), demostrándose que se trata de una

herramienta muy útil para el análisis del EEG. Dos señales tienen una coherencia

elevada a cierta frecuencia cuando se mantienen unas relaciones de fase y amplitud

entre las dos a la frecuencia estudiada. Ya que las medidas de coherencias relacionan los

canales de EEG, es natural esperar que también mida las relaciones funcionales entre las

áreas subyacentes a los electrodos de EEG (Walter y Leuchter, 1997).

Las técnicas neurofisiológicas como el EEG tienen una alta resolución temporal

que permite identificar la sincronización entre bandas de frecuencia dentro de redes

funcionales a gran escala. En los registros electroencefalográficos, la sincronización se

suele cuantificar a través de medidas lineales, como la coherencia. El estudio de las

interacciones funcionales con la finalidad de identificar las interdependencias entre las

series fisiológicas registradas en diversas áreas del cerebro se conoce como

"conectividad funcional".

B/ Análisis no lineal

Si la respuesta a una de las entradas se ve afectada por la existencia o no de las

otras, entonces se dice que el sistema es no lineal. Es decir, las variables que se estudian

dependen unas de otras, y unas pueden tener más peso que las otras, con lo que el valor

de salida no tiene por qué tener una relación directa con las variables iniciales. Esto da

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lugar a que se obtengan respuestas más complejas y ricas. En los sistemas no lineales,

un pequeño cambio en las entradas puede desembocar en grandes cambios en la(s)

salida(s). No hay duda de que las respuestas de los sistemas vivos son no lineales y, por

lo tanto, el análisis lineal sólo puede ser una primera aproximación simplificada.

Índice de retraso de fase (Phase lag index)

A pesar del éxito actual en la caracterización de las redes neuronales

relacionadas con la cognición y diversos trastornos neuropsiquiátricos, existen

limitaciones metodológicas. Las señales que se registran pueden captar la actividad del

electrodo de referencia (debido a la actividad bioeléctrica subyacente), contribuyendo a

incluir en las señales a estudiar componentes similares, lo cual podría dar lugar a

correlaciones falsas entre estas series; de la misma manera que puede influir lo que se

denomina conducción volumétrica. Diversos trabajos han descrito cómo el volumen de

conducción y diferentes tipos de electrodos de referencia podían afectar a los cálculos

de la coherencia (Núñez et al., 1997) y las estimaciones de sincronización de fase en el

EEG (Guevara et al., 2005).

Para abordar el problema del volumen de conducción y electrodos de referencia

en la evaluación de la conectividad funcional, se ha desarrollado una medida alternativa

de las interdependencias entre series temporales, calculándose el índice de retraso de

fase (PLI), una medida nueva para cuantificar la sincronización de fase; es decir, la

asimetría de la distribución de las diferencias de fase entre dos señales (Stam et al.,

2007). Se ha demostrado que el PLI se encuentra menos influenciado por las fuentes

comunes y es superior a la hora de detectar alteraciones en la conectividad de las bandas

de frecuencia (Stam et al., 2007, 2009).

1.3. Sueño y vigilia: neuroanatomía

En la primera mitad del siglo pasado se consideraba que el sueño era un estado

pasivo del cerebro que sobrevenía cuando la estimulación sensorial era insuficiente para

mantener la vigilia. La idea surgió cuando se comprobó que si a un animal se le

practicaba una sección de forma que el telencéfalo quedaba separado del tronco

encefálico, los signos de la vigilia desaparecían por completo en las regiones anteriores

al corte. Esta idea tuvo que abandonarse cuando, en los años 40 y 50 se hicieron nuevas

secciones en regiones posteriores del cerebro, experimentos que se confirmaron con los

de destrucción, de estimulación localizada y con los de registro de la actividad unitaria

de algunos núcleos. Con esto se comprobó que en la parte anterior de la formación

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reticular, en el tegmento mesencefálico-pontino y también en el subtálamo y el

hipotálamo existen regiones capaces de producir la vigilia aún en ausencia de

conexiones sensoriales. En la actualidad se conocen con relativa seguridad la mayor

parte de estas regiones.

También a principios del siglo XX se describieron una serie de casos de

encefalitis letárgica, un proceso que ocasionaba insomnio permanente. Los estudios

realizados sobre los cerebros de estos pacientes demostraron la existencia de

importantes lesiones en el hipotálamo, de lo que se dedujo que en esta región se

encuentra alguno de los núcleos responsables de la producción del sueño. Más tarde se

describieron otras regiones cuya activación determinaba sueño. Entre éstas, merece ser

mencionado el núcleo del tracto solitario, un conjunto de neuronas que reciben

aferencias procedentes del vago y del nervio glosofaríngeo. Este núcleo a su vez envía

fibras hacia un conjunto de núcleos que ha recibido en nombre de sistema límbico

telencefálico e incluye al hipotálamo, el septum, la amígdala y la corteza órbito-frontal,

regiones que cuando son estimuladas también desencadenan el sueño. En conjunto las

áreas responsables del inicio del sueño están distribuidas por todo el encéfalo, aunque

actualmente se está dando una importancia particular al diencéfalo. Experimentos

similares permitieron reconocer que, a su vez, el sueño REM depende de un pequeño

grupo de células situado en el puente, ya que esta fase solamente se muestra en la parte

del cerebro que permanece conectada con esta región, y cuando esta región se destruye

el REM deja de observarse.

1.3.1. Áreas responsables de la vigilia

Se ha comprobado que la actividad neuronal de los núcleos colinérgicos

pedúnculo-pontino y latero-dorsal del tegmento (PPT-LDT) varía de acuerdo con el

estado de vigilancia. Durante la vigilia (Figura 1), su frecuencia de potenciales de

acción es alta, pero esta actividad desaparece durante el SWS y vuelve a elevarse

durante el REM.

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DR (5HT)

LDT-PPT (Acol)

LC (NA)

TB (Acol)

TM (HI)

VLPO (Gaba-Gala)

TálamoHL (Ox)

Figura 1: Conjunto de núcleos y áreas que controlan la vigilia. Las vías colinérgicas que parten del LDT-PPT determinan la activación cortical por intermedio de las regiones también colinérgicas del telencéfalo basal (TB). A la vez, estimulan al tálamo cuyas neuronas entran en modo de disparo tónico. Existen líneas serotonérgicas directas que activan la corteza desde el Rafe Dorsal (RD) y otras noradrenérgicas, histaminérgicas y orexinérgicas, todas indirectas, que también activan la corteza a partir del Locus Ceruleus (LC) del núcleo Tuberomamilar histaminérgico (TM) y del Hipotálamo Lateral (HL), respectivamente. Los núcleos LDT-PPT no sólo tienen capacidad para estimular regiones promotoras de la vigilia, sino que además inhiben al VLPO durante la vigilia.

Fuente: Rial et al., 2006

De estos núcleos parten conexiones ascendentes que al llegar al diencéfalo se

dividen en dos ramas. Una de ellas se proyecta sobre varios núcleos talámicos, entre los

que se cuentan los núcleos intralaminares, los núcleos de relevo sensorial y el núcleo

reticular talámico. Se ha comprobado que la actividad de estas vías es necesaria para

que estas regiones permitan el paso de la información sensorial hasta la corteza. La otra

rama se dirige hacia el hipotálamo, donde se reúne con otras fibras procedentes del

locus ceruleus (LC, noradrenérgico) y los núcleos dorsal y medio del rafe, ambos

serotonérgicos. Este conjunto también se encuentra activo durante la vigilia. Las fibras

procedentes de estas regiones terminan proyectando de forma difusa sobre todo el

encéfalo. Sin embargo, aunque la actividad del LC y de los núcleos del rafe también

depende fuertemente del estado de vigilancia no es idéntica a la del LDT-PPT. Ambos

disparan una alta frecuencia de potenciales de acción durante la vigilia, pero reducen su

actividad durante el SWS y permanecen en silencio durante el REM. Además de los

anteriores, en el hipotálamo lateral se encuentra el núcleo tuberomamilar, cuyas

neuronas son histaminérgicas e igualmente ascienden hacia la corteza determinando

vigilia. Por último, extendidas por varias áreas hipotalámicas, se han encontrado

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neuronas que liberan péptidos, las hipocretinas-orexinas. Se creyó que estas substancias

eran promotoras de la alimentación, despertando sensación de hambre, pero hay datos

que sugieren que su función más importante consiste en activar la actividad locomotora.

La distribución de sus terminales es similar a la de los ya nombrados rafe dorsomedial,

LC y núcleo tuberomamilar; es decir, la liberación de orexinas es alta durante la vigilia

activa, menor durante el reposo motor y nula durante el REM. Con todo, se evidencia

que el perfil de actividad del conjunto de los núcleos descritos hasta este punto es

particular para cada estado de vigilancia, lo que puede comprobarse en la tabla 1.

Vigilia SWS REM

LDT/PPT ↑↑↑ 0 ↑↑↑

LC/DR/TM ↑↑↑ ↑ 0

VLPO 0 ↑↑↑ 0

VLPOe 0 ↑↑ ↑↑↑

HL ↑↑↑ 0 0

Tabla 1 Resumen de la actividad característica de cada una de las regiones más importantes

implicadas en el mantenimiento de la vigilia y las fases de sueño. Abreviaturas: LDT, núcleo

latero dorsal tegmental; PPT, núcleo pedúnculo pontino; LC, Locus ceruleus; DR, rafe doral;

TM núcleo tuberomamilar; VLPO, núcleo ventrolateral preóptico; VLPOe, núcleo ventrolateral

preóptico extendido; HL: hipotálamo lateral.

La mayoría de las áreas promotoras de la vigilancia envían importantes

conexiones inhibidoras hacia el núcleo ventrolateral preóptico (VLPO). La serotonina

(5-HT) y la noradrenalina ejercen inhibición de forma directa. Por el contrario, no se

han encontrado relaciones con la orexina. Por su parte, la histamina liberada durante la

vigilia desde el núcleo tuberomamilar es capaz de inhibir, de forma indirecta, la

actividad del VLPO.

1.3.2. Áreas responsables de la pérdida de vigilia

Todas las áreas descritas hasta el momento reciben fibras procedentes del

hipotálamo VLPO, cuyas neuronas utilizan los neurotransmisores inhibidores GABA y

galanina, por lo que su actividad ejerce efectos inhibidores directos sobre el núcleo

tuberomamilar histaminérgico, los núcleos del rafe y el LC (Figura 2). Además, tienen

capacidad inhibidora indirecta sobre el LDT-PPT. También se ha comprobado que el

VLPO tiene una actividad dependiente de los estados de vigilancia, disparando con la

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máxima frecuencia durante el sueño, tanto SWS como REM. Al contrario, su lesión

ocasiona una pérdida de sueño que es proporcional a la magnitud de la lesión. Por otra

parte, hace ya bastante tiempo que se sabe que las neuronas del VLPO muestran un

aumento en la frecuencia de sus potenciales de acción cuando aumenta la temperatura.

Sin embargo, se ha visto que la mayor propensión para dormir se produce no cuando

sube la temperatura del medio interno, sino todo lo contrario. Es posible que el factor

determinante sea la temperatura cutánea, más que la del medio interno, que presentan

oscilaciones opuestas.

DR (5HT)

LDT-PPT (Acol)LC (NA)

TB (Acol)

TM (HI)

VLPO+VLPOe (Gaba-Gala)

Tálamo nTS

Figura 2: Durante el SWS, el núcleo ventrolateral-preóptico (VLPO) está fuertemente activo, liberando GABA y galanina que inhiben todas las regiones que activan la vigilia. Otra región capaz de activar el SWS es el núcleo del Tracto Solitario (nTS) que produce vías (no representadas) que conectan directamente con el conjunto de regiones que constituyen el telencéfalo víscero-límbico, un conjunto que muestra elevada actividad durante el SWS. Abreviaturas: TB, Telencéfalo Basal; TM, núcleo tuberomamilar; DR, rafe dorsal; LDT-PPT, núcleos laterodorsal tegmental-pedúnculo pontino;LC: Locus ceruleus.


Por otra parte, el núcleo del tracto solitario, una región situada en el

rombencéfalo, envía numerosas conexiones al hipotálamo y al telencéfalo víscero-

límbico. El núcleo del tracto solitario forma parte del sistema nervioso parasimpático y,

por tanto, su actividad está relacionada con las respuestas conservadoras del organismo,

en especial con la alimentación. Sin embargo, no hay evidencias de que la actividad

parasimpática esté relacionada de forma estricta con la producción del sueño.

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1.3.3. El conmutador sueño-vigilia

De la descripción anterior se comprueba que hay varias áreas que mantienen la

vigilia y otras la entrada en el sueño, existiendo abundantes interconexiones de

inhibición recíproca entre ambos grupos, muy especialmente entre las principales áreas

generadoras de vigilancia y el VLPO. Esto ha dado lugar a la hipótesis de que el

conjunto es un sistema biestable, un verdadero conmutador (un “flip-flop”, figura 3) que

imposibilita la actividad simultánea de los dos grupos y que, dentro de ciertos límites,

tanto la vigilia como el sueño se automantienen y se bloquean una al otro, hasta que

llega el momento de una transición. Sobre este conmutador aparecen otras regiones

capaces de modular las transiciones entre sueño y vigilia. Entre las más importantes

destacan las influencias circadianas que están coordinadas desde el núcleo

supraquiasmático, que posee conexiones directas con el VLPO y la glándula pineal

secretora de melatonina, que probablemente actúa directamente sobre las neuronas

hipotalámicas y talámicas responsables de la producción de sueño activando la

liberación del GABA, el principal neurotransmisor inhibidor. Otra influencia notable

sobre el conmutador sueño-vigilia es la que ejercen las hipocretinas/orexinas liberadas

en el hipotálamo lateral; estos péptidos inhiben la actividad del VLPO y,

probablemente, activan los estados en los que existe actividad locomotora, como la

vigilia y en menor proporción el SWS.

Por último, es necesario mencionar la importancia de los factores ambientales

modulando la producción tanto de sueño como de vigilia. Evidentemente, la

estimulación sensorial puede modificar la posición del conmutador en una u otra

dirección.

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DR (5HT)

LDT-PPT (Acol)LC (NA)

TB (Acol)

TM (HI)

VLPO+VLPOe (Gaba-Gala)

Tálamo

nRMTRE

Figura 3: Los núcleos laterodorsal tegmental-pedúnculo pontino (LDT-PPT) activan 1) el VLPO y las neuronas situadas a su alrededor (VLPOe), 2) el tálamo y 3) los núcleos colinérgicos del telencéfalo basal. El VLPO, pero sobretodo el VLPOe, inhiben las regiones promotoras de la vigilancia (TM,núcleo tuberomamilar; RD, rafe dorsal; y LC, Locus ceruleus). Entre la activación talámica y la del telencéfalo basal, la corteza muestra todos los signos de la vigilia. Sin embargo, en ausencia de la activación producida por el RD y el LC, las aferencias sensoriales no llegan a la corteza. Por otra parte, las líneas descendentes del LDT-PPT forman el Tracto Retículo Espinal (TRE) y determinan la atonía muscular típica del REM.


RD (5-HT)

1.3.4. Control del REM

Las secciones del cerebro descritas en párrafos anteriores demostraron que el

área ejecutiva, necesaria y suficiente para producir REM se encuentra a nivel

mediopontino; donde un pequeño conjunto de neuronas colinérgicas, el LDT-PPT,

determina activación del sistema tálamo-cortical que hace desparecer las ondas lentas

del sueño. El LDT-PPT también determina activación en un conjunto difuso de

neuronas situado alrededor de las regiones dorsales y mediales del VLPO, el VLPO

extendido (VLPOe). Esta región contribuye a la reacción de alerta cortical por medio de

sus conexiones activadores con los núcleos del telencéfalo basal, y su lesión reduce el

REM en proporción con el número de neuronas destruidas. En conclusión, el VLPO y

su extensión poseen neuronas especializadas con capacidad para distinguir entre REM y

SWS, aunque la mayor parte de las conexiones inhibitorias que reciben los núcleos del

rafe y el LC proceden del VLPOe. Estas dos regiones reducen su actividad durante el

SWS, y quedan en silencio durante el REM.

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Además, del LDT-PPT surgen otras conexiones. Unas van hacia los núcleos que

controlan los músculos extraoculares y son responsables de los movimientos oculares

rápidos. Otras son descendentes y llegan directamente, pero también de forma indirecta,

a través del núcleo reticular magnocelular (nRM) del bulbo, hasta las motoneuronas

espinales por medio del tracto retículo-espinal (TRE). Estas fibras determinan una fuerte

inhibición de las motoneuronas, con lo que se produce la atonía muscular típica del

REM. Cuando se destruyen las neuronas del núcleo reticular magnocelular en animales

de experimentación, o en el hombre, se pierde la inhibición muscular del REM y se

“ejecutan” los sueños.

1.3.5. La reacción de alerta

Una característica esencial del EEG de la vigilia de mamíferos es la ya descrita

reacción de alerta que aparece tras la estimulación sensorial. Depende de dos tipos de

entrada a la corteza. Se ha comprobado que la atropina, un bloqueador colinérgico y la

para-clorofenilalanina (PCPA), bloqueador específico de la 5-HT, se complementan

para impedir la alerta cortical cuando los animales de experimentación ejecutan ciertos

comportamientos. La activación colinérgica de la corteza procede del telencéfalo basal y

la serotonérgica de los núcleos del rafe. La alerta cortical obedece exclusivamente a

estos dos neurotransmisores porque los dos antagonistas aplicados simultáneamente

impiden la producción de todo tipo de activación.

Se ha comprobado que las neuronas de los núcleos talámicos de relevo tienen

dos estados. En el primero, disparan de forma tónica, lo que se corresponde con una

despolarización también tónica de su membrana. En el segundo estado, se producen

potenciales de acción en forma de salvas que se corresponden con una

hiperpolarización, a la que sigue una despolarización breve de la membrana neuronal.

Se ha comprobado que el modo de disparo tónico se corresponde fundamentalmente con

la vigilia, mientras que el disparo en ráfagas se corresponde sobre todo con el SWS y

con la producción de husos y ondas lentas en el EEG cortical. Por estas razones se ha

considerado que las neuronas talámicas se comportan como un interruptor entre los

sistemas sensoriales y la corteza, interruptor que conecta las vías sensitivas durante la

vigilia y se corresponde con el modo de disparo tónico de las neuronas talámicas. Por el

contrario, la comunicación queda interrumpida durante el sueño, cuando las neuronas

talámicas se encuentran en el modo de disparo en ráfagas. Así, la actividad de las

neuronas talámicas sería determinante del aumento en los umbrales sensoriales típicos

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del sueño. Sin embargo, y a pesar de los numerosos puntos positivos a favor de la

hipótesis, se ha encontrado que un número importante de neuronas talámicas

permanecen en el modo de disparo en salvas durante la vigilia y, por el contrario,

algunas se encuentran tónicamente activadas durante el sueño. Es posible que la

explicación venga del hecho que durante la vigilia pueden existir varios niveles de

activación y que la eficiencia de la transferencia de información entre los sistemas

sensoriales y la corteza cambie según el estado de atención o no atención. En resumen,

está bien aceptado que el tálamo filtra la información con una eficacia que es

dependiente del estado de vigilancia y que regula la transferencia de información hasta

la corteza.

1.4. Regulación del ciclo sueño-vigilia

Las variaciones del ciclo sueño-vigilia son sensibles a un amplio rango de

agentes farmacológicos. Estudios realizados con diferentes fármacos en humanos y

animales de experimentación han permitido una mejor interpretación de la

neurofisiología de los estados de alerta. Existen algunos datos experimentales que

apoyan la hipótesis de que el sueño puede ser el resultado de la acumulación durante la

vigilia de algunos mediadores químicos. Para investigar el papel de los

neurotransmisores clásicos y sus receptores se usan fármacos que se unen a receptores

centrales específicos y que alteran el metabolismo y la distribución de los

neurotransmisores. En esta línea, el estudio de estos fármacos, sus vías y transmisión en

neuronas y sistemas ha permitido ampliar el conocimiento del sueño y de la vigilia, ya

que los cambios que se producen en el ciclo sueño-vigilia se relacionan con agentes

químicos que influencian la comunicación entre las neuronas.

Múltiples sistemas del despertar mantienen al sujeto en vigilia a través de la

acción de neurotransmisores químicos que son liberados y distribuidos en los terminales

nerviosos. Así, la vigilia está mantenida por sistemas múltiples neuronales que usan los

neurotransmisores. Estos sistemas son parcialmente redundantes porque un único

sistema no aparece como el absolutamente necesario para la vigilia, aunque cada uno

contribuye en una única vía en su generación y mantenimiento; con lo cual, las

actividades recíprocas y las interacciones de estos grupos de células, que activan la

vigilia o los grupos celulares que activan el sueño, determinan la alternancia entre la

vigilia y el sueño (Jones, 2005).

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Hay grandes y claras evidencias de la participación de los sistemas colinérgicos

y serotonérgicos en el control del sueño y de la vigilia. Estos sistemas están

perfectamente sincronizados para lograr un funcionamiento adecuado del ciclo sueño-

vigilia.

1.5. Ciclo sueño-vigilia y sistema colinérgico

El sistema colinérgico está implicado en la excitabilidad neuronal durante el

ciclo sueño-vigilia y se considera importante por su implicación como modelo

experimental farmacológico del sueño REM. Las neuronas del sistema colinérgico son

más activas durante la vigilia, disminuyen su actividad durante el SWS y de nuevo

incrementan a su máximo durante el REM al mismo tiempo que se produce una

actividad electroencefalográfica cortical rápida. De hecho, se ha demostrado que los

agonistas colinérgicos muscarínicos producen importantes efectos excitadores al actuar

sobre neuronas de la formación reticular pontina medial, efectos que son análogos a los

cambios de membrana descritos para el estado REM; y que la inyección de agonistas

colinérgicos causa activación cortical acompañada por atonía muscular, un estado que

se parece al REM. Estudios en seres humanos han mostrado que inhibidores de la

acetilcolinesterasa estimulan la activación cortical con una vigilia prolongada cuando se

administra durante la vigilia, pero induce el REM cuando se administra durante el

sueño (Jones, 2005).

La acetilcolina (ACh) es un neurotransmisor específico en las sinapsis del

sistema nervioso somático y en las sinapsis ganglionares del sistema nervioso

autónomo simpático colinérgico, así como en los órganos diana de la división

parasimpática. Los receptores colinérgicos se dividen en muscarínicos y nicotínicos.

Hasta la fecha se han identificado y clonado cinco subtipos de receptores muscarínicos

(M1-M5) (Bonner, 1989; Bymaster et al., 2003a) pertenecientes a la familia de

receptores de membrana que presentan siete dominios transmembranales, acoplados a

proteínas G. A los receptores muscarínicos se les ha adjudicado un papel crítico en la

regulación de las actividades de muchas funciones del SNC y el autónomo. Sin

embargo, no se dispone de agonistas y antagonistas selectivos para cada uno de esos

cinco receptores, lo cual ha podido conllevar resultados discrepantes entre diferentes

trabajos que usan fármacos colinérgicos muscarínicos (Zhang et al., 2002).

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Los efectos de una alta actividad colinérgica están relacionados con la vigilia, el

REM y con la activación cerebral (Gautier-Sauvigne et al., 2005; Marks et al., 2005; Xi

et al., 2004); de hecho, la administración de agonistas muscarínicos han modificado la

cantidad de sueño de onda lenta y REM (Krug et al., 1981) en ratas y en humanos

(Perlis et al., 2002). La pilocarpina es un agonista muscarínico que mimetiza los efectos

de la ACh actuando sobre diferentes receptores (M1, M2 y M5) y que es capaz de

atravesar la barrera hematoencefálica. Este hecho hace que este fármaco pueda alcanzar

el cerebro produciendo cambios en el EEG, pero también tendría un carácter tóxico a

dosis altas, al actuar como modelo inductor de estados similares a los epilépticos.

1.5.1. Organización neurofisiológica del ritmo theta

La información sensorial relacionada con el medio ambiente que rodea al

organismo alcanza el hipocampo vía la corteza entorrinal, mientras que la información

sobre el medio interno es comunicada por las entradas subcorticales (Buzsaki, 1996). La

formación hipocampal ha sido ampliamente implicada en el aprendizaje y la memoria

en seres humanos y otros animales. Se ha reconocido que las trazas de memoria de

eventos pasados son transferidos o re-representados en la neocorteza. Desde el

hipocampo la actividad theta se genera, se propaga en todo momento y es inducida por

la ACh a través de receptores muscarínicos. Dado que el hipocampo presenta esta

actividad lenta rítmica o ritmo theta, es lícito suponer que ejerce sus funciones

cognitivas mediante la sincronización con este ritmo. Revelar los mecanismos

neuronales responsables de la modificación de la conectividad neuronal en la neocorteza

por la formación hipocampal es un interesante campo de estudio.

El ritmo theta es una característica conocida del EEG hipocampal en mamíferos,

presente en roedores, carnívoros y primates en muchos de los estados conductuales, si

bien se muestra en su forma más pura y sincronizada durante la vigilia activa y el sueño

REM (Green y Arduini, 1954; Kocsis et al., 1999, Pedemonte et al., 2001). La actividad

theta se ha relacionado con varias teorías de la función del hipocampo, desde el

procesamiento sensorial al control voluntario del movimiento durante la vigilia y el

sueño (incluyendo sacudidas musculares o twitches, y espigas ponto-geniculo-

occipitales como fenómenos del REM). El análisis del espectro de potencia, a través de

la transformada de Fourier de la actividad bioeléctrica del hipocampo, muestra que la

banda theta está presente en todos los momentos de la vigilia (Pedemonte et al., 2001).

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Así, otra posibilidad es que el ritmo theta del hipocampo actúe como un organizador

temporal en las neuronas sensoriales, es decir, el ritmo theta actuaría como un reloj

interno agregando una dimensión temporal a los procesamientos sensoriales en curso; el

concepto organizador temporal debe ser entendido como un modulador ultradiano que a

su vez es modulado por el ritmo circadiano sueño-vigilia y las entradas sensoriales

(Pedemonte et al., 2001). Adicionalmente, el ritmo theta tendría una influencia

moduladora durante el REM del control autónomo de la frecuencia cardiaca, ya que se

ha observado una correlación entre el ritmo theta y el ECG (Pedemonte et al., 2001). Es

decir, hay una aparente universalidad de la acción rítmica del hipocampo modulando la

actividad neuronal en sistemas muy diversos y distantes dentro del cerebro (auditivo,

visual, autónomo).

Las redes neuronales que acompañan a la oscilación en la banda theta y la

sincronización están muy relacionadas con las vías ascendentes del tronco hacia la

región hipocampal (Bland et al., 2005). Tradicionalmente, la organización del

hipocampo se produce a través de una serie de grupos de células principales que siguen

una vía excitatoria unidireccional con retroalimentación desde la corteza entorrinal. La

señal llega a las células del núcleo dentado granulado a las células piramidales de las

capas CA3 y CA1, y a las neuronas del subículum, para volver a las capas profundas de

la corteza entorrinal. Así, la corteza entorrinal está conectada bidireccionalmente con

casi todas las áreas del manto neocortical (Buzsáki, 1996).

En roedores, las oscilaciones theta se originan desde el circuito hipocampal; de

hecho, las oscilaciones theta tienen su mayor amplitud en las regiones dendríticas

distales de las células piramidales hipocámpicas. Pero la actividad theta ha sido

registrada también localmente en regiones extrahipocampales, incluyendo áreas

corticales; el mecanismo primario de generación del ritmo theta dependería en este caso

de descargas rítmicas de una población de neuronas localizadas en el núcleo del septum

medial, ya que la inactivación de esta región supone la abolición de estas oscilaciones

theta. Por lo tanto, se trata de un circuito marcapasos en el cual el ritmo se genera a

través de las entradas sinápticas provenientes de distintos lugares del cerebro.

Finalmente, las propiedades intrínsecas de membrana de las neuronas del septum medial

así como las del hipocampo contribuyen al mantenimiento y la amplificación del ritmo

theta (Grillner et al., 2005; Pedemonte, 2000; Vyazovskiy y Tobler, 2005).

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1.5.2. Comunicación entre el hipocampo y la corteza

Las redes hipocampales y neocorticales organizan los patrones de activación de

sus neuronas con prominentes oscilaciones en el EEG durante el sueño. Aunque el papel

funcional de estos ritmos no se conoce muy bien, en la rata existe una fuerte correlación

de las descargas neuronales entre el hipocampo y la corteza somatosensorial (Sirota et

al.¸2002).

El hipocampo está especializado en la adquisición a corto plazo de la nueva

información que llega desde los circuitos corticales a través de la corteza entorrinal

durante los períodos en los que los niveles colinérgicos son elevados (como ya se ha

comentado en apartados anteriores, serían los períodos de vigilia y REM). La inervación

colinérgica de la corteza cerebral en ratas procede básicamente del tronco encefálico

(Rye et al., 1984). El flujo de información entre el hipocampo y la corteza está regulado

por la liberación cortical de ACh. Por lo tanto, la señal cortical predomina durante la

vigilia y en los períodos de sueño REM, cuando el feedback hipocampal hacia la corteza

está suprimido por la ACh; es decir, la corteza manda información al hipocampo donde

se produce la consolidación de la memoria durante los períodos de vigilia y REM,

mientras que en el estado de sueño de onda lenta disminuyen los niveles de ACh

permitiendo la vuelta de la información ya consolidada a la corteza (Power, 2004).

1.5.3. Mecanismos de la consolidación de la memoria. Vías implicadas

Aproximadamente durante el último decenio, se ha intentado encontrar las

sustancias que se acumulan durante la vigilia y que son metabolizadas durante el sueño.

Entender cómo las sustancias determinarían la somnolencia podría proveer un

importante discernimiento de la función del sueño. Muchas son las hipótesis sobre la

consolidación de la memoria durante el sueño (Gais y Born, 2004; Power, 2004; Sirota

et al., 2002). Estudios realizados en animales y humanos, sugieren que el procesamiento

de material nuevo, adquirido dentro de las redes hipocampales y neocorticales, tiene

lugar durante el sueño y puede ser la base de la consolidación de la memoria a largo

término (Buzsaki, 1996; Lee y Wilson, 2002; Power, 2004; Figura 4).

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Figura 4: Flujo de información entre el hipocampo y la neocorteza según los niveles de ACh.

Fuente: Power, 2004

Buzsaki (1996) postuló que la transferencia de información neocortical-

hipocampal tiene lugar de manera discontinua temporalmente durante unos minutos

hasta horas o días. La adquisición de la información pasaría muy rápido durante el

estado activo del hipocampo asociado con las oscilaciones theta. Ha sugerido que los

disparos de ondas iniciados en el hipocampo durante el SWS y asociados con las

oscilaciones theta y gamma podría proveer el mecanismo por el cual la información se

devuelve a la corteza durante la consolidación de la memoria. Esta hipótesis es

consistente con la del papel de la ACh como inhibidor del flujo de la información desde

el hipocampo hacia la corteza postulada por Hasselmo (1999) y apoyado por Gais y

Born (2004). El hipocampo está especializado en la rápida adquisición de nueva

información transmitida desde los circuitos corticales a través de la corteza entorrinal

durante períodos de elevados niveles de ACh (Buzsáki, 1996; Hasselmo, 1999). De

hecho, todos estos autores han postulado que el flujo de información entre el hipocampo

y la neocorteza está regulado por la liberación de ACh (Buzsáki, 1996; Hasselmo, 1999;

Power, 2004). La paradoja surge cuando se observa el hecho que durante el SWS se

produce la supresión de la actividad colinérgica, comparado con los altos niveles del

tono colinérgico presente durante la vigilia y el sueño REM (Gais y Born, 2004).

La formación hipocampal se ha implicado largamente en el aprendizaje y la

memoria en animales y humanos. También se ha reconocido que las trazas de memoria

de eventos pasados son eventualmente transferidos o re-representados en la neocorteza

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(Squire, 1992). Así, un trabajo importante a realizar es revelar los mecanismos

neuronales responsables de la modificación de la conectividad neuronal dentro del

hipocampo y la neocorteza por la formación hipocampal.

1.5.4. Efectos adversos de la estimulación colinérgica. Estrés oxidativo

Los fármacos colinérgicos se usan en estudios de arquitectura del sueño (algunos

focalizados en el estudio del ritmo y/o frecuencia theta) (Barnes y Roberts, 1991; Borst

et al., 1987; Bouwman et al., 2005; Cantero et al., 2003; Crouzier et al., 2005; Leung et

al., 1994; Leung, 1998; Yamamoto, 1998). Pero algunos fármacos muscarínicos

también son usados en modelos para inducir un estado epiléptico en roedores. El

modelo epiléptico inducido por la pilocarpina es útil para el estudio del desarrollo y

comprensión de la neuropatología de la epilepsia humana del lóbulo temporal, ya que en

ratas se reproducen alteraciones comportamentales y electroencefalográficas similares a

las de los humanos (Freitas et al., 2004; Turski et al., 1989).

La fisiología de los órganos y los tejidos de los seres vivos depende de un fino

ajuste entre múltiples mecanismos bioquímicos y biofísicos, factores y agentes externos

que regulándose entre si mantienen la homeostasis celular. La homeostasis de las

células sanas se mantiene dentro de unos ciertos límites, que si se sobrepasan, llevan a

la aparición de una lesión celular. Estudios recientes muestran que en muchos procesos

inflamatorios e isquémicos, un metabolismo anormal del oxígeno puede tener un

protagonismo importante en los mecanismos de lesión celular.

El estrés oxidativo es una situación que aparece como consecuencia de la

descompensación entre la generación de especies activadas de oxígeno (ROS) y otros

radicales libres, y las defensas antioxidantes del organismo. La generación de ROS se

produce, en condiciones normales, como resultado de procesos bioquímicos básicos

para el mantenimiento del estado vital como son, por ejemplo, la fosforilación oxidativa

en las mitocondrias y el transporte de oxígeno en sangre (Freitas et al., 2004; Jackson,

2000; Turski et al., 1989).

La relación entre el estado epiléptico y ROS es bien conocida, ya que la

actividad epileptiforme causa una producción excesiva de ROS, implicada en los

mecanismos que dirigen a la muerte celular y a la neurodegeneración (Bellissimo et al.,

2001; Frantseva et al., 2000; Freitas et al., 2004). El agonista colinérgico pilocarpina a

dosis altas produce daño cerebral (Turski et al., 1989), sin embargo, no se conoce si

concentraciones menores de este fármaco, como las usadas en la presente tesis doctoral

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para el estudio del sueño, provocan esa misma actividad epileptogénica y como

consecuencia un daño cerebral.

La toxicidad del oxígeno se explica por la producción de los denominados

radicales libres del oxígeno, altamente reactivos y que son responsables de la lesión e

incluso de la muerte celular en circunstancias patológicas. Además de las ROS, hay

muchas otras moléculas que pueden ser origen de especies reactivas, como las

derivadas del nitrógeno (ver Tabla 2).

Radicales No radicales

O2•- Anión superóxido H2O2 Peróxido de hidrógeno

OH• Radical hidroxilo 1O2 Oxígeno singlete

HO2• Radical hidroxiperoxilo HOCl- Hipoclorito

ROO• Radical peroxilo ONOO- Peroxinitrito

RO• Radical alcoxilo

NO• Óxido nítrico

Tabla 2. Principales especies activas del oxígeno y del nitrógeno relacionadas con el estrés oxidativo

Estas ROS son altamente reactivas y pueden afectar a las propias estructuras

celulares alterando su función (Halliwell, 1994). Para contrarrestar estos radicales libres

las células disponen de un complejo sistema antioxidante que les permite mantener un

balance equilibrado y limitar el daño celular (Clarkson y Thompson, 2000). Debido a

que las ROS se generan de forma continuada es esencial una permanente regeneración

de las defensas antioxidantes para mantener la homeostasis celular (Packer, 1997). La

situación de estrés oxidativo aparece como consecuencia de un desequilibrio entre la

producción de especies reactivas y las defensas antioxidantes del organismo (Todorova

et al., 2004). Este sistema antioxidante de defensa está formado por antioxidantes

exógenos y endógenos, pudiendo ser estos últimos enzimáticos y no enzimáticos.

Los enzimas antioxidantes primarios, que se encargan de eliminar un tipo

particular de ROS, incluyen la superóxido dismutasa (SOD), la glutatión peroxidada

(GP) y la catalasa (CAT). Dentro de los enzimas antioxidantes también se incluyen

otros enzimas como la glutatión reductasa que es importante al regenerar el glutatión

(Powers y Sen, 2000; Figura 5).

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H2O2O2•-

O2•- O2

SOD

Fe2 Fe3

H2

O2 OH- H2O

H+e•

CAT

GSH

GSSG

GP GR

H2O

H2O2O2•-

O2•- O2

SOD

Fe2 Fe3

H2

O2 OH- H2O

H+

H2O2O2•-

O2•- O2

SOD

Fe2 Fe3

H2

O2 OH- H2O

H+e•

CAT

GSH

GSSG

GP GR

H2O

Figura 5: Principales especies reactivas de oxígeno y enzimas antioxidantes

Diferentes efectos surgen a partir de la generación de las ROS. Pueden iniciar

procesos de peroxidación lipídica dañando tanto la estructura como la función de las

membranas, pueden ser responsables de la oxidación de proteínas clave para el

metabolismo y funcionamiento celular, y pueden causar la oxidación de ácidos nucleicos

(Dal-Pizzol et al., 2000; Sah et al., 2002). Todas estas acciones pueden provocar

disfunciones celulares, afectar a la integridad de las células e incluso pueden iniciar

procesos apoptóticos desajustando la regulación del ciclo vital celular.

El cerebro es mucho más susceptible de sufrir daño oxidativo que otros tejidos por

diversas razones. Entre ellas se encuentra su alto consumo de oxígeno (un 20% de la

actividad metabólica total, a pesar de que el cerebro sólo ocupa un pequeño porcentaje de

la masa corporal); por otro lado, el cerebro contiene lípidos y metales que son susceptibles

de ser oxidados, y presenta una capacidad antioxidante baja (Bellissimo et al., 2001; Freitas

et al., 2004; Sah et al., 2002). Freitas y colaboradores observaron que el hipocampo sería la

principal área afectada por los brotes epilépticos y el estado epiléptico inducido por la

pilocarpina (Freitas et al., 2004).

1.6. Ciclo sueño-vigilia y sistema serotonérgico

El neurotransmisor 5-HT ha desempeñado un papel crucial en los últimos 50

años de investigación sobre los mecanismos de control del sueño. De hecho, durante un

breve período en la década de 1960 la 5-HT fue considerada por muchos el

neurotransmisor del sueño, hasta que ello dio un giro de 180°, con los registros de las

neuronas del rafe en animales durmiendo de forma natural, para ser considerado

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neurotransmisor del despertar (Koella y Czicman, 1966). Probablemente, ambas

hipótesis puedan ser correctas.

Desde los primeros estudios de la 5-HT y el sueño, los dos tipos de respuestas, y

respuestas a menudo bifásicas (despertar seguido de sincronía y sueño), se han

registrado siguiendo manipulaciones serotonérgicas. Como un neurotransmisor que

actúa sobre muchos receptores diferentes, la 5-HT puede estar involucrada en muchos

procesos relevantes en el control del sueño y la vigilia, en función de la localización en

el cerebro, el tipo de receptor que activa y el estado del individuo en ese momento. Las

últimas décadas de investigación han demostrado que el sueño es un proceso complejo,

y la idea de que es regido por una estructura cerebral única o por un solo mecanismo,

activado neurofisiológica o neurobioquímicamente, ya no es factible.

Se han descrito efectos bifásicos sobre el sueño tras su administración de 5-HT.

En gatos, se observó un despertar inicial seguido de somnolencia y sueño (Koella y

Czicman, 1966; Koella, 1969). De hecho, en el cerveau isolé de gatos, la inyección de

5-HT indica la excitación inmediata, con signos posteriores de sincronía en el EEG

(Koella y Czicman, 1966). En cambio, lesiones parciales de los núcleos del rafe en

gatos provocaron una reducción del sueño acompañada de una reducción de 5-HT en el

cerebro (Jouvet, 1972; 1999). Estos resultados dieron pie a establecer una primera

hipótesis de la 5-HT y el sueño, postulando que el sistema serotonérgico ascendente era

esencial para SWS (Jouvet, 1972). Sin embargo, las lesiones del rafe en ratas inducen

hiperactividad, pero no pérdida de sueño (Bouhuys y van den Hoodfdakker, 1977);

otros autores observaron que las lesiones del rafe inducían sueño cuando se realizaba

durante la vigilia e inducían vigilia cuando se llevaba a cabo durante el sueño

(Cespuglio et al., 1976).

Muchos de estos experimentos iniciales se realizaron a menudo con métodos

primitivos y fármacos poco selectivos, que de forma aislada podrían explicar los

resultados obtenidos. Los registros de la actividad unitaria de las neuronas del RD

(McGinty y Harper, 1976; Cespuglio et al., 1981) fueron la introducción a una nueva

era en la investigación de la hipótesis 5-HT y sueño. Junto con experimentos de lesiones

del núcleo del rafe (Cespuglio et al., 1976), estos datos fueron los primeros en

cuestionar seriamente la hipótesis 5-HT y sueño. Sin embargo, los resultados

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INTRODUCCIÓN

neurofisiológicos fueron también el fruto de un importante desarrollo de técnicas,

obteniendo resultados de animales no anestesiados, animales que duermen de forma

espontánea (ej: registro unitario, datos de microdiálisis). Esta evolución fue paralela al

desarrollo de la investigación farmacológica en la neurotransmisión serotonérgica, con

la introducción de los inhibidores selectivos de la recaptación de la 5-HT y la

descripción de una variedad de subtipos de receptores serotonérgicos y cada vez más

específicos agonistas y antagonistas de estos receptores. Recientemente, los avances en

biología molecular y la genética también han beneficiado la investigación del sueño.

El primer estudio de registros unitarios de los núcleos del RD, en gatos

durmiendo de forma espontánea, se publicó en 1976 (McGinty y Harper, 1976). Las

neuronas tenían su mayor actividad en la vigilia y la actividad se redujo en SWS

cesando de forma casi completa durante el sueño REM (McGinty y Harper, 1976;

Cespuglio et al., 1981; Trulson y Jacobs, 1979). En ratas el patrón encontrado fue

idéntico (Guzmán-Marín et al., 2000). También se informó que en gatos la estimulación

del núcleo del RD inducía vigilia (Cespuglio et al., 1981). Gracias a técnicas de marcaje

por fluorescencia se demostró que las células responsables del patrón de la actividad

neuronal del RD son serotonérgicas (Jacobs y Azmitia, 1992), idea que se vio reforzada

tras la administración sistémica de un agonista selectivo 5-HT1A que disminuía la

actividad de estas neuronas (Jacobs y Azmitia, 1992).

Al estudiar el contenido extracelular de 5-HT en diversas áreas del cerebro

mediante técnicas de microdiálisis, parece haber una tendencia general de que ésta es

más alta durante la vigilia, se reduce durante SWS y está aún más reducida durante el

sueño REM, lo cual refleja una liberación real en la zona estudiada. Es el caso del

núcleo del RD en gatos (Portas y McCarley, 1994) y ratas (Portas et al., 1998), del

núcleo tegmental pedunculopóntico del tronco encefálico en gatos (Strecker et al.,

1999) y en el hipocampo de ratas (Park et al., 1999). Este patrón coincide

mayoritariamente con los datos sobre actividad eléctrica del núcleo del RD en gatos

(McGinty y Harper, 1976; Cespuglio et al., 1981) y ratas (Guzmán-Marín et al., 2000).

La liberación dendrítica de 5-HT en el núcleo del RD durante el sueño se ha sugerido

que podría ser importante para la reducción de la actividad neuronal del rafe en SWS y

sueño REM, a través de la activación de autorreceptores somatodendríticos inhibitorios

(Cespuglio et al., 1990). Los datos sobre la actividad de las neuronas del rafe, así como

49

INTRODUCCIÓN

los datos de microdiálisis, sugieren una actividad reguladora de las neuronas del rafe, en

fase con el estado del animal.

La PCPA bloquea la triptófano hidroxilasa, enzima que cataliza la síntesis de la

5-HT a partir de su precursor, el aminoácido L-triptófano. Varios estudios han

demostrado que el sueño estaba bloqueado o drásticamente reducido tras la

administración del PCPA. En gatos (Ursin, 1972) y en ratas (Borberly et al., 1981), una

dosis moderada redujo la profundidad del SWS. El insomnio inducido por PCPA se

revertía por 5-hidroxitriptófano (5-HTP), precursor inmediato de la 5-HT (Koella et al.,

1968; Pujol et al., 1971), por L-triptófano (Borberly et al., 1981) o por un inhibidor

selectivo de la recaptación de 5-HT (Ursin et al., 1989). Estos datos de PCPA

implicaban que, sin 5-HT en el cerebro, no hay sueño. Sin embargo, con la

administración crónica de PCPA, el sueño finalmente vuelve a aparecer, a pesar de que

la 5-HT del cerebro es aún muy baja (Dement et al., 1972). Después de la

administración de PCPA en gatos, aparece una actividad ponto-geniculo-occipital

(PGO) que normalmente acompaña el sueño REM, por lo que se supuso que esta

actividad induce vigilia (Dement et al., 1972). Sin embargo, se observó que el insomnio

aparecía mucho antes que el aumento en la actividad PGO (Ursin, 1980).

Los inhibidores selectivos de la recaptación de 5-HT (ISRS) parecen ser los

fármacos ideales para investigar los efectos serotonérgicos, ya que sólo aumentan la 5-

HT en los terminales serotonérgicos cuando la 5-HT se libera ligada a la actividad de las

neuronas serotonérgicas. Cuando se registra poligráficamente el sueño en pacientes

depresivos se observan cambios característicos: menor latencia del sueño REM y SWS

reducido (Ursin, 2002). El efecto más consistente de los ISRS, en todas las especies, es

una reducción del sueño REM, apoyando la hipótesis de inhibición serotonérgica. Se ha

sugerido que los antidepresivos están vinculados a esta hipótesis. Sin embargo, existen

fármacos antidepresivos que no suprimen el sueño REM y que sus efectos varían según

el fármaco y la especie, invalidando esta hipótesis (Vogel et al., 1998). En humanos, el

efecto supresor del sueño REM es prominente. Usualmente no hay efecto sedante, pero

el efecto antidepresivo de los ISRS tiende a mejorar el insomnio en sujetos con

depresión (Simon et al., 1998). Sin embargo, el insomnio también puede ser un efecto

secundario de algunos ISRS, ya que en gatos y ratas se han observados efectos bifásicos

tras la administración aguda de ISRS. En gatos, la vigilia es mayor al principio y luego,

50

INTRODUCCIÓN

con un retraso de alrededor de 2 horas, hay un aumento del SWS (Hilakivi et al., 1987;

Sommerfelt y Ursin, 1991). Todos estos hallazgos sugieren que la estimulación

serotonérgica induce efectos complejos sobre el ciclo sueño-vigilia, tanto inductores

como depresores del sueño (Ursin et al., 1989).

Una cuestión importante es conocer si es posible asignar los efectos divergentes

de la 5-HT sobre el sueño a diferentes subtipos de receptores. La mayoría de los trabajos

en este sentido se han realizado sobre el receptor 5-HT1A y 5-HT2.

Antagonistas del receptor 5-HT2 como la ritanserina promueven sueño en

humanos (Idzikowski et al., 1986) y ratas (Dugovic y Wauquier, 1987), sugiriendo que

la activación de este receptor promueve la vigilia. En gatos se ha observado un efecto

opuesto (Sommerfelt y Ursin, 1993). Sin embargo, la ritanserina no bloquea los efectos

de vigilia de los ISRS lo que sugiere que existen mecanismos diferentes. Uno se puede

encontrar en el núcleo reticular talámico (Lee y McCormick, 1996), ya que la 5-HT,

actuando sobre receptores 5-HT2 talámicos, puede facilitar la vigilia aunque no explica

totalmente por qué la activación serotonérgica induce vigilia.

Los receptores 5-HT1A están localizados somatodendríticamente, de forma

presináptica en las neuronas del rafe y postsinápticamente en otras neuronas. La

estimulación de los autorreceptores somatodendríticos lleva a una reducción de la

actividad neuronal y a una disminución de la liberación de 5-HT. Así, la administración

del agonista 8-OHDPAT en el RD del gato apoya el concepto del control de la

liberación por la activación de dichos receptores en las neuronas del rafe (Portas et al.,

1996). La perfusión de 8-OHDPAT dentro del RD incrementa el sueño REM, pero no

tiene efecto sobre otras fases del sueño (Portas et al., 1996; Bjorvatn et al., 1997). Sin

embargo, la estimulación de los receptores 5-HT1A por administración sistémica de

agonista incrementa la vigilia. En línea con estos datos, el agonista serotonérgico 8-OH-

DPAT aumenta la vigilia y reduce el sueño REM (Bjorvatn et al., 1997).

Así, existen algunos indicios de que los receptores 5-HT pueden facilitar el 1A

sueño o la fenomenología del sueño, al menos en algunas áreas del SNC. El aumento de

la vigilia tras la administración sistémica de agonistas 5-HT no contradice esta teoría 1A

necesariamente. Puede ser debido a efectos serotonérgicos de modulación del sueño en

diferentes localizaciones del SNC (Bjorvatn et al., 1997). Los autorreceptores 5-HT 1A

en las neuronas del RD complican la interpretación de algunos de datos, ya que con la

51

INTRODUCCIÓN

administración sistémica de agentes moduladores del receptor 5-HT , a menudo es 1A

poco claro si el efecto se debe a los receptores postsinápticos o si es el resultado de la

liberación de 5-HT alterada en los terminales de manera secundaria a la modulación de

los autorreceptores.

52

2. OBJETIVOS

53

OBJETIVOS

2. OBJETIVOS

Está ampliamente aceptado que gran parte de las funciones fisiológicas se

encuentran bajo control circadiano. Entre las más importantes destaca el ritmo sueño-

vigilia.

El uso de fármacos que de forma selectiva estimulan o inhiben unas

determinadas vías nerviosas, permite estudiar la regulación neurofisiológica de los

diferentes estados del ciclo sueño-vigilia. En los últimos años, han recibido una gran

atención los sistemas monoaminérgicos en el control de las fases del sueño. En este

trabajo se ha querido profundizar en la participación de los sistemas colinérgico (en

ratas y tórtolas) y serotonérgico (tórtolas) en cada uno de los diferentes estados de

vigilancia. Esta segunda parte, se ha llevado a cabo exclusivamente con tórtolas como

animal de experimentación debido a que este estudio en ratas ha sido ampliamente

desarrollado y existe una base bibliográfica extensa sobre la que comparar los estudios

con tórtolas.

Estudios previos en nuestro laboratorio han puesto de manifiesto el interés de la

tórtola en los estudios circadianos en general, y del sueño en particular, debido a que sus

características circadianas (especie diurna y monocíclica) son mucho más parecidas a

las del ser humano que las de los animales que se utilizan habitualmente en

experimentación, como es el caso de los roedores (especies nocturnas y policíclicas en

general). Los estudios sobre las características neurofisiológicas del sueño de aves y

mamíferos forman parte de una línea de trabajo del grupo de investigación más amplia

que los objetivos de esta tesis doctoral, relacionada con la evolución del sueño.

Por todo ello, los objetivos propuestos en la presente tesis doctoral son:

1.- Caracterizar la influencia del sistema colinérgico sobre la estructura de los

estados de vigilancia en la rata (Rattus norvegicus, variedad Wistar) y tórtola collariza

(Streptopelia risoria). Se determinarán las variaciones producidas por el agonista

colinérgico muscarínico pilocarpina sobre las características comportamentales

correspondientes a los distintos estados de vigilancia, los ritmos de actividad a partir de

las señales del EEG y otras señales bioeléctricas, estableciendo las duraciones, número

de episodios y latencias de dichos estados en ambas especies.

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OBJETIVOS

2.- Caracterizar en la tórtola collariza (S. risoria) la influencia del sistema

serotonérgico sobre la estructura de los estados de vigilancia. Se determinarán las

variaciones producidas por el agonista selectivo del receptor serotonérgico 5-HT1A (8-

OH-DPAT), el antagonista selectivo del receptor serotonérgico 5-HT1A (WAY100635)

y el inhibidor de la síntesis de serotonina PCPA sobre las características

comportamentales correspondientes a los distintos estados de vigilancia, los ritmos de

actividad a partir de las señales del EEG y otras señales bioeléctricas estableciendo las

duraciones, número de episodios y latencias de dichos estados. De forma

complementaria se analizará la actividad locomotora tras la administración de dichos

fármacos.

3- Analizar la conectividad funcional existente entre el hipocampo y la corteza

frontal de ratas tras la administración de pilocarpina, atendiendo a las características

frecuenciales del EEG en los diferentes estados comportamentales mediante técnicas de

análisis lineal y no lineal del EEG. Se prestará especial atención a las bandas de

frecuencia δ y sobretodo θ, debido a la implicación de la acetilcolina en la inducción del

ritmo theta hipocampal, y su proyección a la corteza cerebral en diversos procesos

neurofisiológicos como el aprendizaje.

4- Evaluar los efectos de la administración de pilocarpina sobre la capacidad de

aprendizaje/memoria de ratas mediante un test comportamental (radial-maze), con la

finalidad de establecer una correlación funcional con la transmisión de información

entre el hipocampo y la corteza cerebral bajo los efectos del agonista colinérgico

pilocarpina.

5.- Analizar el estado oxidativo de las regiones cerebrales de interés, hipocampo

y corteza de ratas controles y tratadas con pilocarpina, debido a que la pilocarpina es un

fármaco utilizado en estudios experimentales como inductor de brotes epilépticos y

como consecuencia de daño neuronal, a dosis muy superiores a las utilizadas en el

presente trabajo. Para ello, se usarán ratas controles y tratadas con las dosis de

pilocarpina utilizadas en los objetivos descritos anteriormente, como control positivo

ratas tratadas con dosis elevadas de pilocarpina (con efectos epileptiformes) y como

control negativo ratas tratadas con el agente neuroprotector muscimol.

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3. MÉTODOS GENERALES

57

MÉTODOS GENERALES

3. MÉTODOS GENERALES

3.1. Animales de experimentación

3.1.1. Ratas

Se utilizaron ratas albinas (Rattus novergicus) SPF de la cepa Wistar

procedentes de Charles River, que al llegar se mantuvieron bajo período de cuarentena.

Los animales se estabularon en las estancias del animalario de la Radboud University

Nijmegen o de la Universitat de les Illes Balears (Type III, ILAR), lugar apropiado para

trabajar con animales SPF.

En todos los casos, se utilizaron ratas hembras con un peso comprendido entre

300-375 gramos. Los animales se mantuvieron individualmente en cajas de

polipropileno (Panlab®) translúcidas, con ciclos de 12 horas de luz/oscuridad, bajo

condiciones controladas de temperatura (20-22 °C) y humedad relativa (50-65%).

Recibieron agua y una dieta estándar tipo A04 (Panlab®) ad libitum con un contenido

calórico teórico de 3000 kcal/kg y la siguiente composición en bruto: humedad del 12%,

15,4% de proteína, 2,9% de grasa, 60,5% de glúcidos (de los que un 41% es almidón),

3,9% de fibra y 5,3% de cenizas.

Todos los procedimientos con animales siguieron un protocolo humanitario de

acuerdo con los principios para el cuidado de los animales (Principles of laboratory

animal care; publicación de NIH Nº 85-23, revisado 1996) y fueron aprobados por el

comité bioético de la Radboud University Nijmegen o de la Universitat de les Illes

Balears.

3.1.2. Tórtolas

Como especie aviar se utilizó la tórtola collariza (Streptopelia risoria) de 6

meses de edad y pesos comprendidos entre los 150 y 170 gramos. Las tórtolas se

mantuvieron siempre en el interior de un palomar acondicionado en el laboratorio de

Fisiología Animal de la Universitat de les Illes Balears. Tanto durante su estancia en el

palomar de mantenimiento como durante los experimentos, los animales se mantuvieron

bajo ciclos de 12 horas de luz/oscuridad y en condiciones de temperatura y humedad

controladas (22 ± 2 ºC, 70%). Los animales recibieron agua ad libitum y una dieta

comercial estándar de semillas para palomas. La variedad en la mezcla de semillas es

importante debido a que cada semilla presenta unas propiedades concretas y una

composición diferente, y en su conjunto proporcionan los elementos nutritivos

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MÉTODOS GENERALES

necesarios, evitando enfermedades por alguna carencia. La mezcla de semillas fue: 23%

de mijo, 20% de alpiste, 17% de panizo, 7% de avena, 2% de arroz integral, 3% de maíz

partido, 3% de trigo, 3% de cañamones, 2% de negrillo, 7% de nabino, 2% de perilla,

2% de adormidera, 2% de cártamo, 3% de girasol pequeño, 3% de rábano y 1% de

linaza.

Las tórtolas son animales de hábitos diurnos y monocíclicos, con ritmo de

actividad/inactividad igual al de los seres humanos, teniendo su fase de actividad

durante el día y su fase de reposo durante la noche, a diferencia de lo que ocurre con la

mayoría de los roedores de uso más frecuente en investigación.

Todos los procedimientos con aves siguieron un protocolo humanitario de

acuerdo con los principios para el cuidado de los animales (Principles of laboratory

animal care; publicación de NIH Nº 85-23, revisado 1996) y fueron aprobados por el

comité bioético de la Universitat de les Illes Balears.

3.2. Fármacos y administración

3.2.1. Fármacos y productos utilizados

A lo largo de los diferentes experimentos realizados en la presente tesis doctoral,

se utilizaron diferentes fármacos fundamentalmente colinérgicos y serotonérgicos. Se

usaron: el agonista colinérgico pilocarpina (Sigma-Aldrich Chemie®), el antagonista

colinérgico no selectivo escopolamina (Sigma-Aldrich Chemie®). Como fármacos

serotonérgicos, se utilizó el agonista selectivo del receptor serotonérgico 5-HT1A [8-

hydroxy-2-(di-n-propylamino) tetralin] (8-OH-DPAT; Sigma-Aldrich Chemie®), el

antagonista selectivo del receptor serotonérgico 5-HT1A WAY100635 (Sigma-Aldrich

Chemie®) y el inhibidor de la síntesis de serotonina paraclorofenilalanina (PCPA,

Sigma-Aldrich Chemie®). Además, se utilizó el agonista gabaérgico muscimol (Sigma-

Aldrich Chemie®), seleccionado en este trabajo por sus propiedades neuroprotectoras.

Para la disolución y la administración de los fármacos se utilizó suero salino

como vehículo. Por esta misma razón, se utilizó dicho suero salino en los animales

control.

Para el procedimiento quirúrgico, los animales se anestesiaron por inhalación

utilizándose como anestésico el isofluorano (Forane, Abbot®). Adicionalmente, se les

administró intraperitonealmente el antagonista colinérgico no selectivo atropina

(Braun®) por sus propiedades antisecretoras y antiespasmódicas, evitando un aumento

de secreción salival.

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MÉTODOS GENERALES

3.2.2. Administración de fármacos

La administración intracerebroventricular en ratas de pilocarpina se realizó

mediante una cánula guía implantada crónicamente en el ventrículo lateral del animal

(AP –0.8, ML –2.0, DV –3.3, relativo al bregma) mediante esterotaxia (Paxinos et al.,

1980) y fijada en el cráneo de los animales con cemento acrílico dental, con la finalidad

de asegurar la correcta posición de la cánula; lo que fue corroborado tras realizar un

análisis histológico posterior al registro del EEG. A través de la cánula guía se

introducía otra cánula que estaba unida a una bomba de inyección a través de la cual se

inyectaba el vehículo o el fármaco (1 μl en un intervalo de 5 minutos). Se estudiaron

dos concentraciones diferentes del fármaco colinérgico pilocarpina (120 μg o 360 μg en

1 μl de suero salino).

Para el análisis del estado oxidativo en relación a la administración de

pilocarpina en ratas, se realizó la administración de vehículo (1 ml/kg i.p. o 1 μl i.c.v. de

suero salino), pilocarpina (360 μg i.c.v. y 350 mg/kg i.p.), escopolamina (1 mg/kg i.p.)

inyectada 30 minutos antes de la dosis alta de pilocarpina (350 mg/kg i.p.) con la

finalidad de reducir el número de señales epileptiformes, y el agonista gabaérgico

muscimol (1 mg/kg i.p.) que se utilizó como control negativo por sus propiedades

neuroprotectoras.

Para el estudio de la capacidad cognitiva en ratas tratadas con pilocarpina

mediante el test comportamental del radial maze, se administraron pilocarpina (360 μg

en 1 μl de suero salino, i.c.v.), escopolamina (0,001 mg/kg, i.p.), y un pretratamiento

con escopolamina (0,001 mg/kg i.p.) para antagonizar el efecto de la pilocarpina (360

μg).

La administración del suero salino y los diversos fármacos (colinérgico y

serotonérgicos) en las aves se realizó vía intraperitoneal en todos los casos. Se analizó el

efecto de la administración intraperitoneal de suero salino (1 ml/kg) como control, del

fármaco colinérgico pilocarpina (1 y 3 mg/kg) y de los fármacos serotonérgicos 8-OH-

DPAT (0,5 y 1 mg/kg), WAY100635 (0,5 mg/kg), el efecto del 8-OH-DPAT (0,5

mg/kg) en animales pretratados con WAY100635 (0,5 mg/kg), PCPA (300 mg/kg, y

600 mg/kg administrados en dos inyecciones durante dos días consecutivos) y la

administración de 8-OH-DPAT (0,5 mg/kg) tras los efectos del PCPA. Todos los

tratamientos se realizaron durante la fase de luz.

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MÉTODOS GENERALES

3.3. Metodología electrofisiológica

3.3.1. Implantación crónica de los electrodos

Los registros del electroencefalograma (EEG) fueron obtenidos gracias al

implante crónico de electrodos fijados en el cráneo de los animales con cemento acrílico

dental. En el caso de las ratas en la corteza frontal (AP +2.0, ML +2.5, relativo al

bregma) y en el hipocampo (AP -4.0, ML +2.0, DV –3.0, relativo al bregma) (Paxinos

et al., 1980), con el electrodo de referencia en el cerebelo. En el caso de las tórtolas se

implantaron dos electrodos en la corteza (Karten y Hodos, 1967), con el electrodo de

referencia en el cerebelo. Adicionalmente, se implantaron dos electrodos en los

músculos del cuello para el registro del electromiograma (EMG) en ambas especies y

dos electrodos en la órbita ocular de las tórtolas con la finalidad de registrar el

electrooculograma (EOG).

3.3.2. Registros de las señales bioeléctricas

En el caso de las ratas, las tareas de adquisición y almacenamiento de las señales

bioeléctricas se realizazon a través del paquete informático WINDAQ® (v. 2.29 para

Windows®). Las señales de EEG fueron registradas de forma monopolar, muestreadas a

256 Hz con un ancho de banda de 0,1 a 100 Hz; la señal del EMG fue registrada de

forma bipolar con un ancho de banda de 10 a 500 Hz.

Referente a las aves, las señales bioeléctricas se adquirieron y almacenaron a

través del amplificador y convertidor analógico-digital Digidata 1322A (AxoScope®, v.

8.1 para Windows®). Las señales de EEG de tórtolas se registraron monopolarmente

muestreando a 256 Hz con un ancho de banda de 0,5 a 50 Hz; el EMG y EOG se

registraron de forma bipolar con un ancho de banda de 5-100 Hz y 0,1-50 Hz,

respectivamente.

En todos los casos, se ajustaron los notch filter a 50 Hz. Todos los registros se

realizaron durante el periodo lumínico, en animales no anestesiados que se movían

libremente. Dichos registros fueron almacenados para su posterior análisis.

3.3.3. Análisis de la señal electroencefalográfica y los parámetros bioeléctricos

El análisis visual del EEG, EMG y el EOG, éste último en el caso de las tórtolas,

conjuntamente con la observación del comportamiento registrado mediante el teclado de

voltajes en el mismo archivo de señales bioeléctricas, permitió la división de los

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MÉTODOS GENERALES

diferentes estados del ciclo sueño-vigilia, de acuerdo con los criterios de Gottesman et

al. (1992) en el caso de ratas, y con los de Fuchs et al. (2006) y Toledo y Ferrari (1991)

en el caso de aves. De esta manera, se calculó la duración total, el número de episodios

y las latencias (tiempo en el que aparece el primer episodio) de cada uno de estos

estados; diferenciando aquellos que presentaban ritmo lento y theta.

Los estados comportamentales y electropoligráficos en ratas se clasificaron en 1)

vigilia activa (AW, active waking), definida como la vigilia motora activa en la que el

animal está atento y presenta alta frecuencia y baja amplitud en el EEG y una gran

amplitud en la señal del EMG, 2) vigilia pasiva (PW, passive waking), definida como la

vigilia sin actividad motora en la que el animal está quieto o relajado, con un EEG de

alta frecuencia y baja amplitud y un EMG de alta amplitud, 3) sueño de onda lenta

(SWS, slow wave sleep), caracterizado por ondas lentas en que progresivamente van

incrementando su amplitud y con una disminución de la amplitud del EMG, 4) sueño

REM (rapid eye movements), con un EEG de alta frecuencia y baja amplitud y

caracterizado por movimientos rápidos de los ojos bajo los párpados cerrados que

suelen coincidir con sacudidas musculares y la supresión de la actividad del EMG, y 5)

aseo (grooming), definido como aquel estado en el que el animal se limpia o acicala.

Adicionalmente a esta clasificación clásica, los estados de vigilia se subdividieron a su

vez en estados de vigilia que no presentaban ritmo theta, en aquellos que presentaban

ritmo theta sólo en el canal de EEG hipocampal y en aquellos estados de vigilia que

presentaban dicho ritmo en ambos EEGs, cortical e hipocampal. El ritmo theta fue

definido como una onda sinusoidal con una frecuencia comprendida entre 4 y 9 Hz.

En el caso de las tórtolas, los estados comportamentales se clasificaron en 1)

AW, definido como la vigilia psicomotora con los ojos abiertos, con un EEG de alta

frecuencia y baja amplitud y alta actividad del EMG, 2) PW, definido como aquellos

estados sin actividad locomotora con uno o ambos ojos abiertos en los cuales el animal

está de pie o sentado, con frecuencias altas y de amplitud baja en el EEG y una

actividad EMG alta, 3) SWS, definido como aquellos estados que presentan actividad

de onda lenta de alta amplitud y un baja actividad del EMG mientras el ave se encuentra

sentada o de pie pero con los ojos cerrados, 4) sueño REM, definido por un EEG de alta

frecuencia y baja amplitud, una actividad miográfica baja y una actividad del EOG

rápida y alta (que señala importantes movimientos oculares), todo ello mientras el

63

MÉTODOS GENERALES

animal está sentado o de pie con los ojos cerrados y con caídas de la cabeza esporádicas

y bruscas seguidas de la corrección postural de una manera lenta, y 5) grooming,

definido como aquellos movimientos en los que el animal se frota el pico o las

extremidades por encima o entre las plumas.

El procesamiento y análisis de las señales obtenidas se realizó mediante una

serie de programas informáticos desarrollados en el laboratorio de Biofísica de la

Universidad de La Laguna (Tenerife). El análisis espectral se realizó tras la subdivisión

del registro total (120 minutos) en fragmentos de 5 segundos de duración de todos los

estados comportamentales diferenciados. Estos fragmentos de EEG se analizaron con el

mencionado paquete informático que permitieron el cálculo del espectro de potencia, las

coherencias y el Phase Lag Index para cada uno de los estados poligráficos. La potencia

de banda se determinó para las bandas de frecuencia δ (1-4 Hz), la banda θ (4,1-8 Hz),

la banda α (8,1-12 Hz) y la banda β (12,1-30 Hz). La potencia espectral de los

fragmentos de EEG libres de artefactos se calculó utilizando la transformada rápida de

Fourier (FFT, Fast Fourier Transform). La coherencia es el coeficiente de correlación

lineal normalizada que existe entre la amplitud del poder espectral de dos señales. El

Phase Lag Index es una medida de la sincronización de fase entre dos señales según el

método descrito por Stam et al. (2007). El cálculo se basa en la conversión de las dos

señales a comparar en señales de fase mediante la transformada de Hilbert,

obteniéndose como resultado una señal de diferencia de fase a partir de la cual y usando

la distribución asimétrica de las diferencias de fase se obtiene el Phase Lag Index. En

definitiva, este análisis es una medida de sincronización entre señales que es insensible

al volumen de conducción.

3.4. Observaciones comportamentales

Durante las sesiones de registro, los animales fueron continuamente observados,

a una determinada distancia para no alterar su comportamiento, con el fin de

caracterizar sus estados comportamentales, y se registró a través de un teclado cuyas

teclas emitían diferentes voltajes, de tal manera que los diferentes estados

comportamentales quedaban indicados y almacenados en los mismos archivos que las

señales bioeléctricas.

Adicionalmente, debido a que la pilocarpina es un fármaco con propiedades que

inducen brotes epilépticos e incluso la muerte a altas dosis, la observación de los

64

MÉTODOS GENERALES

animales tratados con pilocarpina se dirigió específicamente a la búsqueda de signos

que pudieran indicar la existencia de un estado epileptiforme. En ningún caso se

observó manifestación alguna ni en ratas ni en tórtolas a las dosis utilizadas para los

objetivos del trabajo.

3.5. Evaluación del estrés oxidativo

Se ha descrito que la pilocarpina es un fármaco que provoca daño oxidativo a

nivel cerebral (Dal-Pizzol et al., 2000; Freitas et al., 2004) a dosis muy altas,

destinadas al estudio de la epilepsia. No se había descrito anteriormente si las

concentraciones usadas en el presente trabajo (120 y 360 μg, i.c.v.) podían provocar

daño cerebral. Gracias a la observación directa realizada sobre los animales se

registraron las posibles señales comportamentales de actividad epileptiforme; sin

embargo, la evaluación del estrés oxidativo nos indicaría si existía daño a nivel tisular

(en hipocampo y en corteza para este caso concreto).

Las ratas fueron sometidas a cirugía para la implantación crónica de una cánula a

través de la cual se introducía una segunda cánula que permitía la administración de las

sustancias a través de una bomba de infusión con un flujo de 1 µl en un intervalo de 5

minutos (ver apartado 3.2.2.). El agonista GABA muscimol se utilizó como control

negativo, ya que se ha descrito que posee propiedades neuroprotectoras (Shuaib et al.,

1993). Como control positivo se utilizó el fármaco objeto de estudio, el agonista

colinérgico pilocarpina a dosis elevadas las cuales han sido descritas como inductoras

de daños epileptiformes en roedores (Turski et al., 1989). Y finalmente, se realizó la

inyección de la pilocarpina a la dosis utilizada en los experimentos electrofisiológicos

(120 μg o 360 μg en 1 μl de suero salino) con la finalidad de comprobar si se produce

daño a nivel tisular.

Las ratas fueron sacrificadas por decapitación y tras ello se procedió a la

extracción del cerebro y la disección del hipocampo y la corteza frontal. Los tejidos

fueron homogeneizados y se determinaron los niveles de malondialdehído (MDA) como

marcador de daño oxidativo, la actividad de los enzimas antioxidantes (catalasa,

superóxido dismutasa, glutatión peroxidasa, glutatión reductasa) en ambas regiones

mediante técnicas espectrofotométricas (Shimadzu® UV-2100), y el enzima Caspasa-3,

al ser uno de los pasos más importantes en la cascada apoptótica responsable de los

cambios morfológicos de la muerte celular programada (Fujikawa et al., 2002),

mediante técnicas espectrofotométricas.

65

MÉTODOS GENERALES

Adicionalmente, se realizó un estudio de los niveles de la vitamina E en la

corteza frontal por cromatografía líquida de alta resolución (HPLC, Shimadzu® detector

de diodos y columna Nova Pak®, C18, 3.9 x 150 mm). También se determinó en la

corteza la expresión génica de los enzimas después del tratamiento con el agonista

colinérgico pilocarpina (350 mg/kg i.p.) expresión de genes para la catalasa, glutatión

peroxidasa y la superóxido dismutasa) mediante PCR a tiempo real (Roche®

Diagnostics).

Todos los resultados se corrigieron según los niveles de proteínas presentes en

las muestras (Biorad Protein Assay®) y todos los materiales usados procedieron de la

misma casa comercial (Sigma-Aldrich Chemie®).

3.6. Cuantificación de la actividad locomotora espontánea

Para medir la actividad locomotora espontánea se mantuvieron a los animales en

una cámara aislada bajo unas condiciones óptimas de temperatura (20-22 °C), humedad

(50-65%) y un fotoperíodo de 12/12 horas de luz/oscuridad. Los animales se colocaron

en jaulas individuales que se colocaron dentro de unos marcos provistos de un sistema

de detección de 2 haces de rayos infrarrojos (λ=950 nm), perpendiculares entre si y

situados a una altura de 7 cm del plano de sustentación. Estos emisores infrarrojos se

excitan a una frecuencia de 4866 Hz y los haces inciden sobre células fotoeléctricas que

responden únicamente a esta longitud de onda, estando sintonizados para responder

únicamente a esta frecuencia para evitar la posible interferencia de otras fuentes

infrarrojas. Este sistema permite trabajar tanto en condiciones de oscuridad como de

baja iluminación natural o artificial. La señal de salida presenta dos niveles o estados:

bajo (L) equivalente a 0 V y alto (H) equivalente a 5 V. Esta señal de salida cambia de

estado cada vez que se detecta un movimiento. El sistema permite tres modos distintos

de detección. Se comprobó que el modo que mejor se adaptaba a la actividad del animal

era el que presenta una salida H cuando no hay ningún haz interrumpido y cambia a

nivel L cuando se interrumpe un solo haz. El sistema también lleva incorporado un

fotómetro y un termómetro para comprobar que se mantienen niveles constantes de luz

y temperatura, y para registrar el momento de cambio de luz. De esta forma, se

cuantificó automáticamente los movimientos horizontales que realizaba el animal dentro

de la caja, gracias al sistema de adquisición de datos DAS_16® (v. 1) desarrollado por el

grupo de Cronobiología de la Universidad de Barcelona.

66

MÉTODOS GENERALES

De esta forma, se monitorizó la actividad locomotora horizontal y espontánea de

los animales durante las 24 h del día. Los animales se mantuvieron 3 días en la cámara

para que se aclimataran. A continuación se empezó a registrar la actividad locomotora y

se dejaron 7 días en los que se cuantificó la actividad basal. Seguidamente se analizó el

efecto de la administración intraperitoneal de suero salino (1 ml/kg) como control, del

fármaco colinérgico pilocarpina (1 y 3 mg/kg) y de los fármacos serotonérgicos 8-OH-

DPAT (0,5 y 1 mg/kg), WAY100635 (0,5 mg/kg), PCPA (300 y 600 mg/kg) y la

administración de 8-OH-DPAT (0,5 mg/kg) tras los efectos del PCPA. Todos los

tratamientos se realizaron durante la fase de luz y se calcularon las actividades absolutas

y relativas acumuladas durante períodos de 1, 2 y 6 horas. En el caso del agonista

serotonérgico 8-OH-DPAT, se calculó el porcentaje de cambio en relación a su

respectivo control (suero salino o el tratamiento previo con PCPA).

3.7. Valoración de la capacidad cognitiva: Radial maze

El laberinto radial está especialmente indicado para estudios de memoria

espacial de trabajo. El laberinto (Panlab®) consiste en una plataforma central octogonal

(32 cm de diámetro) con 8 brazos (50 cm de longitud y 12 cm de ancho) distribuidos

radialmente de forma equidistante. El experimento consiste en la ejecución de un

recorrido en el laberinto en el que el sujeto experimental tiene que recordar en qué

brazos ha entrado previamente (memoria a corto plazo).

El animal utiliza claves visuales para escoger la opción más adecuada y ubicarse

espacialmente en el contexto experimental donde puede ser reforzado positivamente.

Presenta la ventaja, frente al laberinto acuático, de que la inmersión en el agua puede

aumentar el nivel de estrés de los animales, lo que supone una variable importante a

considerar a la hora de aplicar este modelo. Existe una correlación elevada entre los

efectos de las manipulaciones farmacológicas llevadas a cabo en el laberinto de agua y

en el radial (Myhrer, 2003).

Los animales fueron alimentados ad libitum hasta las 48h previas a la realización

el test, con el fin de aumentar el grado de motivación en la búsqueda de alimento en los

brazos del laberinto. Se observaron las entradas que se llevaron a cabo por parte del

animal, cuantificando el tiempo que necesita para recorrer todos los brazos y el número

de errores (entradas repetidas) que comete. El test se considera completado cuando el

animal había entrado en los 8 brazos. En cualquier caso, el experimento se prolongó

como máximo 20 minutos por animal, y al final de este tiempo los brazos en los que no

67

MÉTODOS GENERALES

había entrado se consideraron también errores que se sumaron a las entradas en brazos

repetidos.

Las diferentes variables medidas se registran mediante métodos computerizados

(Smart, Panlab®) y analizadores de imagen digital que pueden valorar además conductas

específicas (distancia a la meta, velocidad de carrera). Pueden determinar también el

“error de búsqueda”, basado en la distribución de la ejecución del animal. Estas medidas

reflejan la mejora para localizar el objetivo. Se pueden valorar distintos parámetros;

para el presente estudio, los errores a la hora de localizar los brazos que contienen el

refuerzo.

3.8. Análisis estadístico

Los resultados obtenidos a lo largo de la presente tesis doctoral se expresan

como valores medios ± error estándar de la media. Se analizaron mediante análisis de la

varianza (ANOVA) univariante y/o mediante prueba T de muestras relacionadas con el

programa estadístico SPSS® (v. 12.0 para Windows®); en ambos casos se consideró

como estadísticamente significativo un nivel mínimo de probabilidad igual o inferior a

p<0.05. Cuando fue necesario se realizaron las comparaciones múltiples (post hoc) de

LSD o Bonferroni para identificar los grupos estadísticamente diferentes.

68

4. RESULTADOS

69

Manuscript I

Effects of pilocarpine on the cortical and hippocampal theta rhythm in different

vigilance states in rats

Tejada, S.; Rial, R.V.; Coenen, M.L.; Gamundí, A. and Esteban, S. (2007) European

Journal of Neuroscience. 26:199-206.

71

Effects of pilocarpine on the cortical and hippocampal thetarhythm in different vigilance states in rats

S. Tejada,1 R. V. Rial,1 A. M. L. Coenen,2 A. Gamundi1 and S. Esteban1

1Laboratori de Neurofisiologia, Departament de Biologia Fonamental i Ciencies de la Salut, Universitat de les Illes Balears, IUNICS,Palma de Mallorca, Spain2Department of Biological Psychology, NICI-Radboud University Nijmegen, The Netherlands

Keywords: EEG analysis, frontal cortex, hippocampus, pilocarpine, theta rhythm

Abstract

It has been suggested that theta rhythm gates the flow of information between the hippocampus and cortex during memoryprocesses. The cholinergic system plays an important role in regulating vigilance states and in generating theta rhythm. This studyaims to analyse the effects of the muscarinic agonist pilocarpine (120 and 360 lg, i.c.v.) on hippocampal and frontal cortical thetarhythm during several vigilance states in rats. Pilocarpine injection increased the duration and number of episodes with theta activity,particularly when theta rhythm appeared during waking states in the cortex and hippocampus simultaneously. It seems that theeffects of pilocarpine are related to the appearance of cortical theta activity in waking states, and suggest that pilocarpine couldmodify the transference rate of information from the hippocampus to cortex in rats during wakefulness states, in relation to thepostulated effect of cholinergic system modulating memory consolidation.

Introduction

Cholinergic mechanisms play an important role in regulating a varietyof behavioural functions, including alertness and some cortical andhippocampal electroencephalographic (EEG) patterns as well as rapideye movement (REM) sleep (Crouzier et al., 2006). Spontaneousrelease of acetylcholine in the pontine reticular formation has beenobserved to be greater during REM sleep and waking when comparedwith slow wave sleep (SWS; Datta & Siwek, 1997). Two cholinergicsystems are involved in the control of the vigilance states; one islocated in the rhombencephalon where an important part of the controlof REM sleep is performed (Velazquez-Moctezuma et al., 1989; Xiet al., 2004), the second depends on the cholinergic innervation of theneocortex and arises primarily from cell groups of the basal forebrain,which can also be activated by other neural systems such as theamygdala (see Dringenberg & Vanderwolf, 1998).

Cholinergic mechanisms also play an important role in generatingtheta rhythm in the EEG (Lee et al., 2005). Theta rhythm is defined asa sinusoidal-like waveform, with a peak frequency of 4–9 Hz and asmall bandwidth (Oddie et al., 1997), and is mainly generated in thepyramidal neurons of the hippocampal formation (van Luijtelaar &Coenen, 1984; Kahana et al., 2001). Rhythmic oscillatory activities attheta frequency in the hippocampus have been found to be involved inseveral brain functions, including cognition, memory and learning(Kahana et al., 2001; Pedemonte et al., 2001; McKinney &Jacksonville, 2005). Theta rhythm is prominent during REM sleepand active waking (AW; Coenen, 1975; Pedemonte et al., 2001; Xiet al., 2004; Shin et al., 2005), and its function is currently accepted tobe similar in both states (Lerma & Garcia-Austt, 1985).

A number of important physiological functions, such as behaviouralarousal and motor control, are regulated by cholinergic muscarinicreceptors (Tayebati et al., 2006). To date, five muscarinic receptorsubtypes have been functionally described and cloned with adifferential expression in different brain areas (Bonner, 1989; Velaz-quez-Moctezuma et al., 1989; Caulfield, 1993; Levey et al., 1995;Bymaster et al., 2003). Pilocarpine is a muscarinic agonist that acts onthe central receptors by activating neural pathways (Takakura et al.,2003), and shows a low affinity for M1 and M2 receptors and a higheraffinity for the M5 receptor subtype (Dong et al., 1995; Seifritz et al.,1998). Recent clinical studies have shown that a selective M1 agonistreduced REM latency sleep and SWS duration with no effects onmemory consolidation (Nissen et al., 2006b). Orally administeredpilocarpine has also been found to shorten the latency of REM sleepand to increase total REM time, the percentage of REM sleep and theduration of the first REM sleep period in humans (Berkowitz et al.,1990). An important role for pilocarpine in preventing the impairmentof memory associated with ageing has also been reported (De-Melloet al., 2005).As theta activity may support information transmission and storage

within and between the hippocampus and cortex during performanceof learned tasks (Muir & Bilkey, 1998), the aim of this study was toanalyse the effects of pilocarpine on sleep–wake architecture and EEGcharacteristics, and on behavioural states with particular attention tothe theta rhythm in the hippocampus as the main generator, and thefrontal cortex as the main hippocampus target.

Materials and methods

Animals

Six adult male Wistar rats, 12 months old, weighing 350–375 g wereused. Animals were housed individually and maintained during all

Correspondence: Dr S. Esteban Valdes, as above.E-mail: [email protected]

Received 24 November 2006, revised 15 May 2007, accepted 21 May 2007

European Journal of Neuroscience, Vol. 26, pp. 199–206, 2007 doi:10.1111/j.1460-9568.2007.05647.x

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd 73

experiments on a 12 : 12 light : dark (LD) cycle (lights on from 15.00 hto 03.00 h). Rats were kept under these light conditions for 1 month tofully adapt to the LD scheme. Standard laboratory animal food andwaterwere available ad libitum. All procedures performed during the darkperiod were carried out under dim red light (< 0.5 Lux).Experiments were performed following the ‘Principles of Laborat-

ory Animal Care’ (NIH Publication no. 85-23, revised 1996) andaccording to the guidelines of the Local Ethics Committee of theRadboud University Nijmegen (The Netherlands).

Surgery

Under isoflurane (Abbot�, The Netherlands) inhalation anaesthesia allanimals were submitted to aseptic surgery for implantation ofelectrodes and a cannula. Isoflurane anaesthesia was administeredusing a ventilated chamber coupled to a mask. Anaesthetic depth waschecked by physiological parameters (immobility, absence of stimulusresponse, body temperature, heart and respiratory rate) of the animal.Atropine (Braun�, The Netherlands) was injected to avoid a rise insalivary secretion (0.05 mg i.m.). Stainless steel tripolar EEGelectrodes (Plastics One Inc., The Netherlands) were placed into thefrontal cortex (AP +2.0, ML +2.5), in the CA4 hippocampal region(AP )4.0, ML +2.0, DV )3.0), both relative to the bregma (Paxinoset al., 1980), with a reference in the cerebellum. Furthermore, onestainless steel cannula guide was placed in the lateral ventricle (AP)0.8, ML )2.0, DV )3.3, relative to the bregma) in order to inject thedrugs. Two additional electrodes were placed over the dorsal neck

muscles for bipolar EMG recording. Electrodes and cannula wereattached to the skull with dental acrylic cement.

Experimental procedure

Animals were allowed to recover from surgery for at least 10 daysbefore the beginning of the experiments. The animals were placed intothe EEG recording boxes the day before the experiment to providehabituation to the experimental conditions. Boxes measuring25 · 24 · 40 cm had walls made of clear Plexiglas, and the top wasopen to facilitate drug administration and the recordings in the freelymoving rats. Rats were connected to the experimental setup through arotating connector, which also prevented twisting of EEG wires. Sixhours after lights off, the period in which theta activity is the lowest (vanLuijtelaar & Coenen, 1984), rats were infused intracerebroventricularlyinside the box without being unhooked from the recording cable. Beforeinfusion, the presence of cortical and hippocampal EEG and elec-tromyogram (EMG) patterns was observed and the recording wasbegun immediately. Each animal was submitted to EEG, EMG andbehavioural recordings for 2 h each, in basal conditions (untreatedanimals), immediately after saline serum injection (1 lL i.c.v.) and afterpilocarpine (Sigma-Aldrich Chemie�, Steinheim, Germany) injections(120 and 360 lg in 1 lL of saline serum, i.c.v.). At least 3 days elapsedbetween the different treatments to allow a complete washout of theadministered substances. Basal recordings were performed in operatedbut untreated animals, whereas the saline control was made up of saline-injected, operated animals. All animals were submitted to the four

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Fig. 1. EEG recording examples in cortical and hippocampal regions after saline and pilocarpine treatment (360 lg i.c.v.) recorded during passive wake.Hippocampal theta is always evident in the hippocampus where it is further enhanced after pilocarpine injection. The drug also provoked the appearance of thetarhythm in the cortex. Similar results were found with 120 lg i.c.v. pilocarpine treatment.

200 S. Tejada et al.

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 26, 199–206

74

treatments on separate days according to a 4 · 4 Latin square designexperiment. Intracerebroventricular administrations were made using aHamilton syringe coupled to a syringe pump (Razel ScientificInstruments�, Stanford, USA) through a polyethylene tube insertedinto the cannula guide, with an injection rate of 1 lL ⁄ 5 min. The dosesof pilocarpine used in the present work were selected on the basis that alack of neuronal damage in rat brain has been previously demonstratedafter the same treatments with pilocarpine as described in the presentstudy (Tejada et al., 2006a,b). As the maximal effect of muscarinicagonists occurs within 2 h (Timofeeva &Gordon, 2001), the recordingswere restricted to this lapse after injection.

At the end of the experiments, animals were killed with an overdoseof sodium pentobarbital (Nembutal�, The Netherlands) and succes-sively submitted to intracardiac perfusion with saline and 10%formaldehyde. The brain was quickly removed for histologicalassessment of the cannula and electrode placement.

Electrographic recording and animal behaviour classification

EEG and EMG recordings were carried out using WINDAQ� (v. 2.29for Windows�, USA). EEGs were filtered with a 1–100 Hz bandpassfilter and sampled at a frequency of 512 Hz with a notch filter at 50 Hz(see Fig. 1 for a representative record). EMG was filtered with a 10–500 Hz bandpass filter.

Behavioural state was recorded by direct observation of the animalsthrough a glass window at a distance of < 1 m. A keyboard was usedto include the behavioural data into the polygraphic recordings and, inthis way, animal behaviour and EEG recordings could later becorrelated by visual inspection. Theta EEG rhythm was recognized asa sinusoidal-like waveform with a peak frequency of 4–9 Hz (Oddieet al., 1997; Kahana et al., 2001), and fragments of the recordingswere submitted to a spectral analysis to evidence the appearance of apeak in the theta range. The behavioural states were classifiedaccording to Gottesman’s (1992) criteria as follows. (1) Passivewaking (PW) or waking without motor activity, which was furtherdivided into three different substates: PW without theta rhythm (PWwithout theta); PW with theta rhythm only in the hippocampus (PWwith hippocampal theta); and PW with theta rhythm in both the frontalcortex and hippocampus (PW with cortical and hippocampal theta).(2) AW or psychomotor AW, which was also divided into threedifferent substates: AW without theta rhythm (AW without theta); AWwith theta rhythm only in the hippocampus (AW with hippocampaltheta); and AW with theta rhythm in both the frontal cortex andhippocampus (AW with cortical and hippocampal theta). (3) SWS. (4)REM sleep.

Additionally, behavioural variables such as number of peripheralcholinergic signs, e.g. tremors, sniffing and clonic movements offorelimbs, were annotated during the 2 h after the treatments.

Data analysis and statistics

Accumulated time spent in theta activity and number of episodes foreach state and treatment, total time spent in each vigilance state andlatencies (defined as the time from the beginning of the recording tothe first appearance of the considered state) were obtained on the basisof EEG, EMG and behavioural parameters. Statistical analysis wascarried out using SPSS� (v. 12.0 for Windows�, Madrid, Spain) usingone-way analysis of variance (anova). Post hoc LSD pairedcomparisons were further made to recognize deviant groups. Resultsare expressed as mean ± SEM, and P < 0.05 was considered statis-tically significant.

Results

Behavioural observations

The animals were visually observed during the time period comprisedbetween pilocarpine administration and the end of the recordingsession. The animals that received pilocarpine i.c.v. (120 or 360 lg) orsaline did not show any behavioural signs characteristic of theepilepticus-like status or seizures.

Effects of pilocarpine on theta EEG rhythm

Figure 1 shows examples of cortical and hippocampal recordings afterthe treatments (1 lL i.c.v.) of serum saline or pilocarpine (360 lg).Independently of the treatment (saline or pilocarpine), theta activitywas more evident in the hippocampus than in the frontal cortex.Cortical theta activity was never shown in the absence of hippocampal

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Fig. 2. Variation in the amount of theta rhythm recorded in EEG (withoutdifferentiation between cerebral regions) during 2 h in basal, saline control(1 lL i.c.v.), pilocarpine 120 lg (P120, contained in 1 lL i.c.v.) andpilocarpine 360 lg (P360, contained in 1 lL i.c.v.) groups. (A) Time spentin theta rhythm (s). (B) Number of episodes presenting theta rhythm.(C) Average duration of the episodes presenting theta rhythm. Bars representmean ± SEM (n ¼ 6). *P < 0.05, **P < 0.01 and ***P < 0.001 when com-pared with the saline control group (one-way anova analysis).

Effects of pilocarpine on EEG theta rhythm 201


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theta activity in all experimental conditions (basal, saline or bothpilocarpine treatments) throughout the 2 h of the recorded EEG.There was no significant difference between basal and saline

control in any parameter studied, evidencing the absence of effectsattributable to the i.c.v. injections (Fig. 2). On the other hand, theeffects of the two administrations of pilocarpine were similar whenboth were compared (Fig. 2). After pilocarpine treatments, total thetarhythm time (without differentiating between cerebral regions)showed a clear increase throughout the 2 h of EEG recording(180%, P < 0.01 when compared with saline; F3,19 ¼ 4.283;Fig. 2A). The number of episodes with theta activity also rose afterboth pilocarpine treatments (128–130%, P < 0.05 compared withsaline; F3,17 ¼ 3.765; Fig. 2B), and the duration of each thetaepisode changed from 5.6 to 7.6 and 6.7 s per episode for 120 and360 lg of pilocarpine, respectively (P < 0.001 when compared withsaline; F3,7391 ¼ 4.299; Fig. 2C).

Effects of pilocarpine on vigilance states

There was no significant difference between basal and saline controlgroups in any sleep–wake parameter (Figs 3 and 4). Additionally, thetwo administrations of pilocarpine did not differ from each other(Figs 3 and 4, Tables 1 and 2).Figure 3 shows SWS and REM sleep durations during the interval

studied. After both pilocarpine treatments, no changes were foundeither in SWS or in REM sleep (F35,177 ¼ 9.126). Similarly, thenumber of SWS and REM sleep episodes did not change afterpilocarpine injections (Table 1). However, a tendency to diminish theSWS duration was observed, although statistical differences were notfound. A greater dose of pilocarpine and ⁄ or number of animals couldperhaps reveal an effect on SWS duration.

Although the total duration of PW and AW did not change afterpilocarpine administration, significant changes did appear betweensubstates (for details, see ‘Electrographic recording and animalbehaviour classification’; Fig. 4, F35,177 ¼ 9.126). PW without thetarhythm and PW with hippocampal theta rhythm durations did notchange after pilocarpine treatments, although a rise in PW withcortical and hippocampal theta rhythm was observed after bothpilocarpine injections (Fig. 4A). With respect to AW, the duration ofAW without theta rhythm diminished after pilocarpine treatments; onthe other hand, AW with hippocampal theta rhythm duration did notchange; and, finally, AW with cortical and hippocampal theta rhythmduration increased after both pilocarpine treatments (Fig. 4B). All inall, pilocarpine injections increased theta rhythm in the cortex.Although the duration of total PW did not change (Fig. 4), the numberof episodes (Table 1) rose at the high administration of pilocarpine(F15,176 ¼ 6.54, P < 0.000). In addition, the number of episodes withsimultaneous theta in the cortex and hippocampus was also increased(F3,20 ¼ 6.401, P < 0.01 for PW and F3,20 ¼ 3.244, P ¼ 0.05 forAW).The effects of pilocarpine on the latencies of the different vigilance

states are shown in Table 2. No differences were found in sleeplatencies, either in SWS (F3,20 ¼ 0.779, P ¼ 0.519) or in REM sleep(F3,20 ¼ 0.348, P ¼ 0.791) after pilocarpine treatments. Moreover,pilocarpine treatments did not change the latency of the PW withouttheta rhythm (F3,20 ¼ 1.491, P ¼ 0.247) or the PW with theta rhythmonly in the hippocampus (F3,20 ¼ 0.707, P ¼ 0.559), but it didshorten the latency of PW when theta rhythm was present in bothcortical and hippocampal regions (F3,20 ¼ 3.077, P ¼ 0.051). On theother hand, latency of AW without theta rhythm increased afterpilocarpine administration (360 lg, i.c.v.; F3,20 ¼ 3.089, P < 0.05),while latency was reduced for AW simultaneously showing theta in

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Fig. 3. Duration of slow wave sleep (SWS) and rapid eye movement (REM) sleep during 2 h of EEG recording (s). Bars represent mean ± SEM (n ¼ 6) in basal,saline (1 lL i.c.v.), pilocarpine 120 lg (P120, contained in 1 lL i.c.v.) and pilocarpine 360 lg (P360, contained in 1 lL i.c.v.) groups. No differences were foundwhen one-way anova was used to comparisons.



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the cortex and hippocampus after both pilocarpine treatments(F3,20 ¼ 3.550, P < 0.05).

Discussion

EEG analysis in freely moving animals is considered a useful methodin order to assess drug effects on behaviour. In the present study, thepharmacological action of pilocarpine on hippocampal and corticalEEG, with particular attention on the occurrence of the theta rhythm,was studied. The results show that the concentrations of pilocarpineused in the present work (120 lg and 360 lg i.c.v., 2 h) inducedsimilar effects: increasing the duration and number of episodes in

which the theta rhythm appeared in the EEG. More particularly, theadministration of pilocarpine increased the duration and number ofepisodes, with a shortened latency of the waking states with thetarhythm appearing simultaneously in both the cortex and hippocampus.Hippocampal theta activity is a characteristic of REM sleep and also

of some waking behavioural states (Bland, 1986; Leung, 1998;Pedemonte et al., 1999; Kahana et al., 2001; Gambini et al., 2002;Bouwman et al., 2005; Lee et al., 2005; Vyazovskiy & Tobler, 2005),and increased hippocampal theta activity has been shown after theadministration of muscarinic agonists (Lawson & Bland, 1993;Yamamoto, 1998; Tai et al., 2006). The rise in the production oftheta EEG observed in the present report was due to an increase in theepisodes presenting theta rhythm and also to the increase in the

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Fig. 4. Duration of waking substates during 2 h of EEG recording (s). (A) Passive wake (PW) duration: total duration (Total PW), PW without theta rhythm (PWwithout h), PW with theta only in hippocampus (PW with hippocampal h), PW with theta in both frontal cortex and hippocampus (PW with cortical and hippocampalh). (B) Active wake (AW) duration: total duration (Total AW), AW without theta rhythm (AW without h), AW with theta only in hippocampus (AW withhippocampal h), and AW with theta in both frontal cortex and hippocampus (AW with cortical and hippocampal h). Bars represent mean ± SEM (n ¼ 6) in basalgroup, saline control (1 lL i.c.v.), pilocarpine 120 lg (P120, contained in 1 lL i.c.v.) and pilocarpine 360 lg (P360, contained in 1 lL i.c.v.). *P < 0.05,**P < 0.01 and ***P < 0.001 when compared with the saline control group (one-way anova analysis).



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duration of each episode. This was observed in both PW and AW, butwas most important in the cortex, while the amount of exclusivehippocampal theta remained unchanged. Taking into account that thetheta rhythm is generated in the hippocampus, and that the increase incortical theta was not accompanied by an increase in hippocampaltheta, these results could suggest a pilocarpine-induced increase in thetheta wave transfer from the hippocampus to the cortex. Thispossibility is consistent with the hypothesis of Buzsaki (1996) whoproposes that a sharp-wave burst initiated in the hippocampus duringSWS and associated with theta oscillation provides the mechanism bywhich information may be relayed back to the cortex during memoryconsolidation. This is also consistent with the role of acetylcholineinhibiting the information flow from the hippocampus to the cortexpostulated by Hasselmo (1999) and supported by Gais & Born (2004).The effects of pilocarpine observed in the present work could berelated with the activation of muscarinic autoreceptors mediating theinhibition of acetylcholine release (Kilbinger et al., 1993), asmuscarinic autoreceptors may belong to the five subtypes cloned todate (Vilaro et al., 1994).There is evidence that the cholinergic system and also the theta

rhythm are related to different aspects of learning and memoryconsolidation. Theta activity may ‘gate’ the flow and storage ofinformation within the hippocampus and neighbouring cortical regionsduring various stages of mnemonic processing (Muir & Bilkey, 1998).Cholinergic antagonist treatments and disruption of cholinergic inputs

have been observed to impair memory consolidation (Stemmelinet al., 1999; Power et al., 2004; Nissen et al., 2006b), whereascholinergic agonists were found to improve it (Smith et al., 1996).Furthermore, a single administration of pilocarpine prevented andreversed age-related learning impairment in rats conducting a spatialtask in a water maze (De-Mello et al., 2005). In addition, theta-patterned stimulation improved memory retention and influenced theinduction of long-term potentiation, a putative memory mechanism(see Muir & Bilkey, 1998). In fact, theta and gamma oscillationsinitiated in the hippocampus may provide the mechanism by whichinformation is sent to the neocortex during memory consolidation(Buzsaki, 1996; Power, 2004). A significant deficit in hippocampus-dependent spatial and non-spatial memory tasks has been observed inmice lacking the muscarinic M5 receptor subtype (Araya et al., 2006),and M3 and M5 muscarinic agonists have been suggested to alleviateamnesia and the decrease in theta power related with ageing(Markowska et al., 1995; De-Mello et al., 2005). As pilocarpinedisplays a high affinity for the M5 receptor subtype, which is abundantin the hippocampus and cortex (Bymaster et al., 2003), the pilocar-pine-induced increase of the theta rhythm appearing simultaneously inthe cortex and hippocampus observed in the present work could bemediated by the M5 receptor subtype.Besides these effects, it is well known that the cholinergic system

is involved in the generation and maintenance of REM sleep, but thecontribution of the individual muscarinic receptors has not been

Table 1. Numbers of episodes observed in the 2 h after the different treatments, in the different states

Number of episodes in 2 h

Basal Saline control Pilocarpine 120 lg Pilocarpine 360 lg F-values

SWS 40.8 ± 6.8 41.7 ± 4.7 40.2 ± 5.4 46.3 ± 9.4 F3,20 ¼ 0.167REM 3.2 ± 2.2 4.5 ± 1 2.5 ± 1.1 2.3 ± 1.1 F3,20 ¼ 0.471Total PW 68.9 ± 10.8 75.7 ± 14.5 94.6 ± 9.4 115.2 ± 15.3** F15,176 ¼ 6.54PW without h 111 ± 19.2 146.2 ± 17.2 114.5 ± 17.3 152.3 ± 30 F3,20 ¼ 0.969PW hippocampal h 56 ± 12.2 54.8 ± 15.6 77.3 ± 18.2 95 ± 22.5 F3,20 ¼ 1.192PW with cortical and hippocampal h 39.7 ± 10.7 26 ± 4.8 92 ± 11.36** 98.2 ± 23.7** F3,20 ¼ 6.401Total AW 44 ± 8.4 55.1 ± 9.9 64.6 ± 8.8 62.1 ± 10 F15,176 ¼ 6.54AW without h 75.3 ± 10.8 103.5 ± 8.6 75.8 ± 20.2 74.7 ± 19.7 F3,20 ¼ 0.808AW hippocampal h 30.3 ± 13 41.5 ± 12.5 53.2 ± 13.5 48.7 ± 13.8 F3,20 ¼ 0.567AW with cortical and hippocampal h 26.3 ± 12 20.3 ± 5.1 64.8 ± 12.2* 63 ± 19.1* F3,20 ¼ 3.244

Data represent mean number of episodes in 2 h ± SEM (n ¼ 6) in basal group, saline control (1 lL i.c.v.), pilocarpine 120 lg (contained in 1 lL i.c.v.) andpilocarpine 360 lg (contained in 1 lL i.c.v.). *P < 0.05 and **P < 0.01 when compared with the saline control group (one-way anova analysis). AW, activewake; PW passive wake; REM, rapid eye movement; SWS, slow wave sleep.

Table 2. Latencies (min) after the different treatments for different states

Latency of onset of each state after treatment (min)

Basal Saline control Pilocarpine 120 lg Pilocarpine 360 lg F-values

SWS 43.69 ± 6.28 38.04 ± 3.43 37.69 ± 2.85 35.37 ± 2.17 F3,20 ¼ 0.779REM 88.91 ± 15.27 70.62 ± 11.56 76.70 ± 14.65 70.58 ± 16.64 F3,20 ¼ 0.348PW without h 37.14 ± 2.48 31.87 ± 0.46 33.83 ± 1.29 34.75 ± 2.20 F3,20 ¼ 1.491PW with hippocampal h 52.69 ± 13.86 41.36 ± 8.08 42.70 ± 5.63 35.31 ± 2.28 F3,20 ¼ 0.707PW with cortical and hippocampal h 46.98 ± 9.89 64.23 ± 12.65 33.72 ± 1.29* 34.64 ± 2.03* F3,20 ¼ 3.077AW without h 34.08 ± 1.26 33.18 ± 0.99 38.13 ± 3.46 51.79 ± 9.02* F3,20 ¼ 3.089AW hippocampal h 68.14 ± 10.70 61.53 ± 13.37 52.90 ± 13.83 53.62 ± 13.50 F3,20 ¼ 0.312AW with cortical and hippocampal h 70.00 ± 15.96 60.38 ± 7.82 36.32 ± 1.93* 36.65 ± 2.69* F3,20 ¼ 3.55

Data represent mean latency ± SEM (n ¼ 6) in basal group, saline control (1 lL i.c.v.), pilocarpine 120 lg (contained in 1 lL i.c.v.) and pilocarpine 360 lg(contained in 1 lL i.c.v.). *P < 0.05 when compared with the saline control group (one-way anova analysis). AW, active wake; PW, passive wake; REM, rapid eyemovement; SWS, slow wave sleep.



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determined. Several authors have reported that muscarinic M2-agonists (oxometrine-M, cisdioxo, carbachol) elicited a significantrise in REM sleep with a shortened latency (Velazquez-Moctezumaet al., 1989; Bueno et al., 2000; Crouzier et al., 2006). Moreover,the muscarinic M1 receptor subtype has been found to be involvedin REM processes (Bueno et al., 2000; Nissen et al., 2006a,b).Pilocarpine in the present work did not produce significantdifferences in the SWS and REM sleep parameters, and aspilocarpine has a low affinity for M1 and M2 receptors and ahigher affinity for the M5 receptor subtype (Dong et al., 1995;Seifritz et al., 1998), it could be inferred that the current effects ofpilocarpine were not mediated by the M1 and M2 receptor subtypes.Human studies, on the other hand, have shown a shortened latencyand an increase in the duration of REM sleep after the oraladministration of pilocarpine (Berkowitz et al., 1990), but thesediscrepancies could be due to a number of methodological variables.That is, the effects of a low dose of pilocarpine might be mediatedby M5 receptors, while a higher dose could be required to activatethe M1 and M2 receptor subtypes. There is scarce informationrelating the M5 subtype with the wake–sleep cycle ) partly due to alack of selective ligands to block or activate M5 receptors (Eglen,2006; Nissen et al., 2006a) ) but m5 mRNA levels have beenreported not to change in a line of rats with an increased REM sleeppattern (Greco et al., 1998).

In conclusion, our results show that the increased theta rhythminduced by the cholinergic agonist pilocarpine does not bear adirect relation on the generation and maintenance of REM sleep.Instead, the main effect of pilocarpine is produced on the amount oftheta activity, a result probably produced in the telencephalicregions (such as the hippocampus) regulating cortical arousal.Moreover, the results suggest pilocarpine may increase the transferof the hippocampal theta rhythm to the frontal cortex duringvigilance in rats, and this could be mediated by the muscarinic M5receptor subtype.

Acknowledgements

This work was supported by grant BFI2002-04583-C02-029 and 36AA ⁄ 04 ofConselleria de Salut i Consum (Govern de les Illes Balears, Spain). SilviaTejada was supported by a FPI grant (Govern de les Illes Balears, Spain). Wealso thank Ma Antonia Comas Soberats for her ideas and help.

Abbreviations

AW, active wake; EEG, electroencephalogram; EMG, electromyogram; LD,light : dark; PW, passive wake; REM sleep, rapid eye movement sleep; SWS,slow wave sleep.

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Effects of the muscarinic agonist pilocarpine on locomotor activity and vigilance

states in ring doves

Tejada, S.; Rial, R.V.; Gamundí, A. and Esteban, S. (Submitted 2010) European Journal

of Neuroscience.

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Effects of the muscarinic agonist pilocarpine on locomotor

activity and vigilance states in ring doves

Tejada, S.; Rial, R.V.; Gamundí, A. and Esteban, S.

Laboratory of Neurophysiology, Department of Biologia Fonamental i Ciències de la

Salut, University of the Balearic Islands, Institut Universitari d’Investigació en Ciències

de la Salut (IUNICS), Palma de Mallorca, Spain

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Abstract

The avian sleep-wake cycle is composed by a number of distinctive stages

defined by EEG, EMG and EOG signs that resemble those present in most of mammals.

The cholinergic system plays an important role in regulating vigilance states and in

generating theta rhythm in mammals. In the present study, the pharmacological action

of the muscarinic agonist pilocarpine (1 and 3 mg/kg, i.p.) on locomotor activity and

EEG rhythm of ring doves was analyzed. The results show that pilocarpine increased

passive waking (135%) and the episodes which presented theta waves (97%). In

contrast, pilocarpine induced a reduction on locomotor activity (74%), active waking

(58%), SWS (53%) and REM (73%) duration, and the episodes in which birds had open

eyes (45%). These results are in good accordance with observations in mammals

suggesting that cholinergic sleep-wake regulating mechanisms shared by the two

vertebrate groups reflects their common phylogenetic origin.

Keywords: pilocarpine, theta rhythm, bird, locomotor activity, EEG rhythm analysis

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Introduction

Classically, cholinergic mechanisms play an important role in regulating a

variety of behavioural functions, including alertness, electroencephalographic (EEG)

patterns or rapid eye movement (REM) sleep (Crouzier et al., 2005). Spontaneous

release of acetylcholine in the pontine reticular formation has been observed to be

greater during REM sleep and waking when compared with slow wave sleep (SWS;

(Datta & Siwek, 1997).

Behavioural sleep can be found in many species, but the electrophysiological

criteria of polygraphic sleep are exclusively met by mammals and birds (Campbell &

Tobler, 1984; Rattenborg, N.C. et al., 2002; Rial et al., 2010). Both groups alternate

behavioural and neural activity patterns of SWS and REM sleep as evidenced by

cortical EEG. Avian REM sleep is defined, like in mammals, by low amplitude with

high frequency EEG similar to the EEG of waking. Rapid eye movements are also

observed during avian REM sleep. However, mammals show atonia of the skeletal

muscles while avian commonly reflect signs of hypotonia (head drooping) by

electrophysiological and behavioural components. Another difference between

mammalian and avian REM sleep is the episode duration. In avian, REM episodes are

very short compared to mammals, remaining for few several seconds (Toledo & Ferrari,

1991; Fuchs et al., 2006).

Cholinergic mechanisms also play an important role in generating theta rhythm

in the EEG (Lee et al., 2005). Theta rhythm is defined as a sinusoidal-like waveform,

with a peak frequency of 4–9 Hz and a small bandwidth (Oddie et al., 1997). In fact,

cholinergic neurons located in the medial septal zone of the reticular formation of the

forebrain diagonal band of Brocca serve as pacemaker for the theta rhythm (Steriade et

al., 1990; Timofeeva & Gordon, 2001). Theta rhythm is prominent during waking and

REM sleep (Coenen, 1975; Pedemonte et al., 2001; Xi et al., 2004; Shin et al., 2005),

and its function is currently accepted to be similar in both states (Lerma & Garcia-Austt,

1985). This activity has been involved in several brain functions, including cognition,

learning and memory (Kahana et al., 2001; Pedemonte et al., 2001; McKinney &

Jacksonville, 2005; Tejada et al., 2007; Tejada et al., 2010).

Pilocarpine is a muscarinic agonist that acts on the central receptors by

activating neural pathways (Takakura et al., 2003), showing low affinity for M1 and M2

receptors and a higher one for the M5 receptor subtype (Dong et al., 1995; Seifritz et

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al., 1998). It has been reported several actions of pilocarpine on the sleep-wake cycle in

humans (Berkowitz et al., 1990; Nissen et al., 2006) and rats (Tejada et al., 2007;

Tejada et al., 2010). Moreover, several works described an improvement of memory

processes under pilocarpine treatment in rats (De-Mello et al., 2005; Tejada et al.,

2010).

As our knowledge, there is not reports relating cholinergic mechanisms to theta

activity in birds, the aim of the current work was to analyse the effects of the muscarinic

agonist pilocarpine administered in ring doves on the spontaneous locomotor activity

and the sleep–wake architecture on the basis of the EEG characteristics and behavioural

states, focusing particular attention on the theta rhythm.

Material and methods

Animals

Adult domestic ring doves (Streptopelia risoria, 160.5 ± 4.8 g body weight)

were used throughout the experiments and maintained in individual cages at a

temperature of 24±2ºC, on a 12:12 Light:Dark (LD) cycle (lights on at 8 a.m.) with free

access to food and water. All procedures were performed during the active light period

(1.2 Klux).

Experiments were performed following the “Principles of Laboratory Animal

Care” (NIH Publication no. 85-23, revised 1996) and according to the guidelines of the

Local Ethics Committee of the University of The Balearic Islands (Spain).

Drugs and injections

Ring doves were submitted to different treatments: A/ serum saline

administration (1 ml/kg i.p.), B/ the muscarinic agonist pilocarpine (1 mg/kg i.p.,

Sigma-Aldrich Chemie, Steinheim, Germany) and C/ pilocarpine (3 mg/kg i.p.). All

administrations were made intraperitoneally. Each animal was submitted to EEG, EOG,

EMG recordings and behavioural observation simultaneously for 2 h after the

treatments. At least 3 days elapsed between the different treatments in order to ensure a

complete washout of the administered substances.

The doses of pilocarpine used in the present work were selected on the basis of

previous works in rats (Tejada et al., 2007; Tejada et al., 2010) and avian (Marley &

Seller, 1974) devoid of epileptiform signs.

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At the end of the experiments, animals were sacrificed with an overdose of

sodium pentobarbital (Nembutal®, Spain).

Two different experimental designs were performed using two different groups

of animals which had similar characteristics (body weight, age and house conditions): 1)

study of spontaneous locomotor activity and 2) study of the vigilance states throughout

EEG recordings.

Study of locomotor activity

Ten adult ring-doves were used. Animals were housed in individual 20x20 cm

cages inside an activity chamber with 12:12 Light:Dark (LD) cycle (lights-on at 9:00),

controlled temperature (23.5 ± 2 ºC) and free access to food and water throughout all

experimental time period. Locomotor activity from the animals was registered using

cross beam infrared interruption system located 70 mm above the floor of the cage,

which detected the movements of the pigeons within its cage. Activity was recorded and

stored in 15 minutes bins on a computerized data-logging system for later analysis.

After habituation to the new environment, activity was continuously measured

during 12 days. Animals were submitted to different treatments: 1) saline administration

(1 ml/kg i.p.), 2) pilocarpine (1 mg/kg i.p.) and 3) pilocarpine (3 mg/kg i.p.).

Administrations were conducted seven hours after lights on.

Surgery

Seven adult ring-doves were used. At least 10 days before the experiments, each

animal was anaesthetized with isoflurane (Abbot®, Spain) inhalation anaesthesia using

a ventilated chamber coupled to a mask. Anaesthetic depth was checked by

physiological parameters (immobility, absence of stimulus response, body temperature,

heart and respiratory rate). Atropine (Braun®, Spain) was injected to avoid a rise in

salivary secretion (0.05 mg i.p.).

Stainless steel bipolar EEG electrodes were stereotaxically placed into the cortex

according to coordinates derived from the brain atlas of the pigeon (Karten & Hodos,

1967) with a reference in the cerebellum. Two bipolar electrodes were also threaded just

above the orbits to detect eye movements (EOG). Two additional electrodes were

placed over the dorsal neck muscles for bipolar EMG recording. All electrodes were

anchored to the skull with jeweller’s screws and fixed with dental cement.

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Electroencephalographic recording

Animals were allowed to recover from surgery for at least 10 days before

beginning the experiments. They were placed in the recording cages 5 hours before the

beginning of the different sessions for habituation to the recording environment.

Immediately after the drug or vehicle injection, the animal returned to its cage and the

recording started. EEG and EMG recordings were carried out using AxoScope (v. 8.1

for Windows®, USA). EEGs were filtered with a 0.5-50 Hz bandpass filter and sampled

at a frequency of 256 Hz with a notch filter at 60 Hz. EMG and EOG were filtered with

a 5-100 Hz and 0.1-50 Hz bandpass filter, respectively. EEG rhythm was recognized as

a sinusoidal-like waveform with different peak frequencies (Oddie et al., 1997; Kahana

et al., 2001), and fragments of the recordings were submitted to a spectral analysis to

evidence the appearance of the different EEG ranges. These conditions are commonly

used to analyse the early effects of pharmacological treatments on sleep (Tejada et al.,

2007; Tejada et al., 2010). As the maximal effect of the muscarinic agonist pilocarpine

occurs within 2 h (Timofeeva & Gordon, 2001), the recordings were restricted to this

lapse time after injection.

Behavioural recording classification

Behavioural state was recorded by direct observation of the animals through a

video system connected to a computer. The observer was at a distance of 3 m to avoid

interference in the animal behaviour. A keyboard was used to include the behavioural

data into the polygraphic recordings and, in this way, animal behaviour and EEG, EOG

and EMG recordings could be later correlated by visual inspection. During the first two

hours after vehicle and drug injections, behaviours were directly observed and

continuously recorded. The behavioural states were classified according to Fuchs et al.

(Fuchs et al., 2006) and Toledo and Ferrari (Toledo & Ferrari, 1991) criteria as follows:

(1) Slow wave states (SWS) as the slow wave activity with high amplitude EEG and

low EMG activity while the animal remains sitting or standing with closed eyes and the

neck retracted and sometimes resting on chest, (2) rapid eye movements sleep (REM)

characterized by a high frequency, low EEG amplitude, fast and high amplitude EOG

activity (indicative of large eye movements), and a low EMG activity while the animal

remains sitting or standing with closed eyes and with sporadic and sudden downward

head drops followed by slow return to an upright posture, (3) passive waking (PW) or

waking without locomotor activity (standing or sitting) with open eyes, high frequency

and low amplitude EEG and high EMG activity, (4) active waking (AW) or

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psychomotor waking with open eyes, high frequency and low amplitude EEG and high

EMG activity, and (5) grooming, which was defined as the rubbing the beak or limbs

over or between the feathers.

Data analysis

Locomotor activity and the parameters of the different vigilance states was

carried out using SPSS® (v. 12.0 for Windows®) using one-way analysis of variance

(ANOVA), with treatment as factor. Accumulated total time spent (duration) and

number of episodes for each vigilance state and treatment were obtained on the basis of

EEG, EOG, EMG and behavioural parameters. Results are expressed as mean ± SEM.

All these tests were followed, when appropriate, by a post-hoc LSD paired comparisons

to recognize deviant groups, and a p<0.05 was accepted as being statistically significant

in these procedures.

Results

Locomotor activity

The acute administration of the muscarinic agonist pilocarpine (1 and 3 mg/kg

i.p.) caused a dose dependent decrease on the spontaneous locomotor activity

(F(11,76)=2.761) accumulated during the first (42-48%) and the second hour (74%) in

ring doves when compared with saline treated animals (Figure 1). A gradual recovery of

control values was observed after 2 hours.


The animals were visually observed during the time period comprised between

pilocarpine administration and the end of the recording session. Animals receiving

vehicle or pilocarpine (1 or 3 mg/kg) treatments did not show any abnormal behavioural

signs or any sign characteristic of the epilepticus-like status or seizures.

Effects of pilocarpine on eye closure

Figure 2 shows the effects of pilocarpine treatments on the duration

(F(11,47)=14.259) and number of episodes (F(11,46)=7.834) when animals had closed

or open eyes. A significant increase of the closed eyes episodes duration (35%-150%)

and number of episodes (43%-139%) occurred after pilocarpine treatment when

compared to saline control. A concomitant decrease on open eyes episodes duration

(31%-45%) and number of episodes (31%-35%) was observed. However, the

muscarinic agonist did not modify significantly one eye closure (right or left) in

comparison with saline control treatment.

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Effects of pilocarpine on EEG rhythm and vigilance states

Figure 3 shows the effects of the muscarinic agonist pilocarpine treatments on

the duration (F(2,91)=6.172), number of episodes (F(2,14)=2.132) and latency

(F(2,11)=0.792) of REM sleep state. A significant decrease of the REM duration (73%)

only occurred after the higher dose of pilocarpine when compared to saline control in

ring doves. Pilocarpine tended to decrease the number of episodes of REM sleep in

comparison with saline control treatment but not statistical significance was obtained.

Also, latencies were not significantly modified after the treatments.

Figure 4 shows the effects of pilocarpine treatments on the duration

(F(17,70)=8.862), number of episodes (F(17,66)=11.228) and time lapse

(F(2,5129)=11.301) of the episodes presenting theta rhythm after the administration of

pilocarpine in ring doves. An increase was observed after the pilocarpine administration

on the duration (97%) and number of episodes (65%) presenting theta rhythm. When

attention was focusing on eyes, the mentioned increases occurred markedly when

animals had the eyes closed (300%-324%). Moreover, in all cases, the time lapse per

episode in those episodes presenting theta rhythm was higher after pilocarpine treatment

than in saline control, independently of the eyes closure. Episodes showing theta rhythm

were mainly observed when animals displayed passive waking (data not shown).

Figure 5 shows the effects of pilocarpine treatments on the duration of the

different vigilance states in ring doves. When vigilance states duration was observed

(Figure 5A, F(17,66)=11.228), a dose dependent increase of the passive waking states

occurred after pilocarpine when compared to saline control (135%), while reductions

were observed on slow wave (53%), active waking (58%) and grooming (69%)

duration. Additionally, the vigilance states were also studied focusing attention on eyes,

when animals showed open eyes (Figure 5B) or closed eyes (Figure 5C). After

pilocarpine administration, when animals had open eyes a reduction on slow wave

(71%, F(35,115)=4.755) and active waking (44%, F(39,92)=8.941) states duration was

observed (Figure 5B). In contrast, when animals had closed eyes, only an increase in

passive waking (230%, F(10,46)=6.56) duration was observed after the higher dose of

pilocarpine (Figure 5C).

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Figure 6 shows the effects of pilocarpine treatments on the number of episodes

of the different vigilance states in ring doves. When vigilance states episodes were

observed (Figure 6A), only a significant increase of the passive waking states occurred

after pilocarpine when compared to saline control (92%, F(17,70)=8.862). Additionally,

the number of episodes of the vigilance states was also studied focusing attention on

eyes, when animals showed open eyes (Figure 6B) or closed eyes (Figure 6C). After

pilocarpine administration, when animals had open eyes a reduction on slow wave

(71%, F(35,116)=4.116) and active waking (56%, F(39,92)=9.776) states episodes was

observed (Figure 5B). When animals had closed eyes only an increase (218%) in

passive waking (F(10,47)=7.602) episodes was observed after the higher dose of

pilocarpine (Figure 6C).

In addition, no differences were found in the latencies of the different vigilance

states of the ring doves after pilocarpine treatments in any case (data not shown).

Discussion

EEG analysis in freely moving animals is considered a useful method in order to

assess drug effects on behavior. In the present study, the pharmacological action of the

muscarinic agonist pilocarpine on locomotor activity and EEG rhythm of ring doves

was analyzed. The results showed that pilocarpine increased passive waking and the

episodes which presented theta waves. In contrast, pilocarpine induced a reduction on

locomotor activity, active waking, SWS and REM duration, and the episodes in which

birds had open eyes. Also a tendency to reduce grooming was observed.

Acetylcholine is one of the most widespread excitatory neurotransmitter in the

central nervous system of mammals and birds (Komarova et al., 2008).

Immunohistochemical studies in the ventrolateral preoptic area of birds have observed

cholinergic fiber and muscarinic (Dietl et al., 1988) and nicotinic cholinorreceptors

(Whiting & Lindstrom, 1986), though the role of these receptors in controlling the

sleep/waking state and their spectral components has not been fully determined in birds

(Komarova et al., 2008). Studies using pilocarpine or other muscarinic agonists in birds

are scarce. It has been described that pilocarpine administration to fowls induced

postural changes in these birds, such as the abduction of wings from the trunk with

lowering of the tail (Marley & Seller, 1974). These postures in avian define some

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characteristics of passive waking which are clearly in agreement with the observations

in the current work. Also, a study in pigeons reported that the administration of the

muscarinic agonist arecoline increased the duration of waking and decreased the

duration of total sleep (Komarova et al., 2008). All these results are in good agreement

with the observations in the current work in ring doves treated with the muscarinic

agonist pilocarpine. Accordingly, in the current work, differences were found during

waking, the duration and number of episodes of passive waking increased after

pilocarpine while the duration of the active waking episodes was diminished. However,

passive waking increased in a great degree that the decrease observed in active waking

resulting in an increase in total waking as observed by (Komarova et al., 2008).

Komarova and coworkers also reported that the activation of muscarinic receptors by

arecoline increased the EEG spectral power during waking without changes during

SWS and REM sleep (Komarova et al., 2008). In birds, it has been characterized four

muscarinic receptor subtypes and named M2-M5 according to sequence homology with

mammal ones (Tietje et al., 1990; Tietje & Nathanson, 1991; Gadbut & Galper, 1994;

Fischer et al., 1998). The few differences in the results obtained by both agonists could

respond to a different pharmacological profile (Messer et al., 1989).

It is accepted that theta activity is characteristic of the waking states and REM

sleep (Bland, 1986; Leung, 1998; Pedemonte et al., 1999; Kahana et al., 2001; Gambini

et al., 2002; Bouwman et al., 2005; Lee et al., 2005; Vyazovskiy & Tobler, 2005). In

the current work, pilocarpine treatment induced an important increase in theta waves

(duration as well as number of episodes) in ring doves. This effect was positively

correlated to the observed increase in passive waking but not with changes observed in

REM sleep. These observations are in good accordance with previous results in

mammals, demonstrating an increase in the duration and number of episodes presenting

theta rhythm during waking in rats treated with pilocarpine (Tejada et al., 2007; Tejada

et al., 2010).

Cholinergic system and theta band are related to different aspects of learning and

memory consolidation (Muir & Bilkey, 1998; Klimesch et al., 2001; Johnson, 2006).

The muscarinic agonist pilocarpine increased the synchronization of the theta activity

between hippocampus (the main generator of this rhythm) and the frontal cortex in rats,

that in accordance with the cholinergic hypothesis of memory consolidation (Buzsaki,

1996; Hasselmo, 1999; Gais & Born, 2004), suggested an increased flow of information

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between these brain structures (Tejada et al., 2010). In fact, in mammals cholinergic

agonists (Smith et al., 1996), and particularly pilocarpine (De-Mello et al., 2005; Tejada

et al., 2010), were found to improve different task related to memory consolidation. In

pigeons, the use of the cholinergic antagonist scopolamine produced a dose-related

decrease on different matching performances (Teal & Evans, 1982; Savage et al., 1994;

Kohler et al., 1996), while the use of the cholinergic agonist aniracetam improved the

accuracy in perform the test in similar manner as in monkeys (Pontecorvo & Evans,

1985). Altogether these observations indicate that cholinergic system is critical for

normal memory function in birds as in mammals, and the increase in theta activity

induced by pilocarpine in the current work could be associated with the improvement of

memory processes.

Acknowledgments

This work was supported by grant BFI2002-04583-C02-029, SAF2007-66878-

CO2-02 (MEC, Madrid, Spain) and 36AA/04 of Conselleria de Salut i Consum (Govern

de les Illes Balears, Spain). Silvia Tejada was supported by a FPI grant (Govern de les

Illes Balears, Spain).

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Figures

Figure 1. Effects of pilocarpine treatments on spontaneous locomotor activity of ring

doves. Bars represent mean ± SEM from 10 animals. * p<0.05, ** p<0.01 (one way

ANOVA analysis).

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Figure 2. Effects of pilocarpine treatments on eyes closure duration and number of

episodes from ring doves. A: Duration of eyes closure, B: Number of episodes with

eyes closure. Bars represent mean ± SEM from 7 ring doves. * pilocarpine (1 or 3

mg/kg) respect to the saline control; # differences between both pilocarpine treatments.

* p<0.05, ** p<0.01, *** p<0.001, # p<0.05 (one way ANOVA analysis).

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Figure 3. Effects of pilocarpine treatments on REM sleep duration, number of episodes

and the latency in ring doves. Bars represent mean ± SEM from 7 ring doves. ** p<0.01

(one way ANOVA analysis).

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Figure 4. Effects of pilocarpine treatments on the episodes presenting theta rhythm in

ring doves. A: Duration of the episodes presenting theta rhythm, B: Number of episodes

presenting theta rhythm, C: Time lapse per episode presenting theta rhythm. Bars

represent mean ± SEM from 7 ring doves. * p<0.05, ** p<0.01, *** p<0.001 (one way

ANOVA analysis).

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Figure 5. Effects of pilocarpine treatments on the duration of the episodes of the

different vigilance states in ring doves. A: Duration of the different vigilance states, B:

Duration of the different vigilance states when animals had open eyes, C: Duration of

the different vigilance states when animals had closed eyes. Bars represent mean ± SEM

from 7 ring doves. * p<0.05, ** p<0.01, *** p<0.001 (one way ANOVA analysis).

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Figure 6. Effects of pilocarpine treatments on the number of episodes of the different

vigilance states in ring doves. A: Number of episodes of the different vigilance states,

B: Number of episodes of the different vigilance states when animals had open eyes, C:

Number of episodes of the different vigilance states when animals had closed eyes. Bars

represent mean ± SEM from 7 ring doves. * p<0.05, ** p<0.01, *** p<0.001 (one way

ANOVA analysis).

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Effects of serotonergic drugs on locomotor activity and vigilance states in ring

doves

Tejada, S.; Rial, R.V.; Gamundí, A. and Esteban, S. (Submitted 2010) Behavioural

Brain Research.

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Effects of serotonergic drugs on locomotor activity and vigilance

states in ring doves

Tejada S., Rial R.V., Gamundí, A. and Esteban, S.

Laboratory of Neurophysiology, Department of Biologia Fonamental i Ciències de la

Salut, University of the Balearic Islands, Institut Universitari d’Investigació en Ciències

de la Salut (IUNICS), Palma de Mallorca, Spain

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Abstract

Serotonergic system has been implicated on sleep-waking states in mammals.

Taking into account that avian in general are monophasic and diurnal species,

characteristics that are similar in humans, ring doves were chosen as experimental

subject in the present work. The role of the neurotransmitter serotonin on vigilance

states were studied in ring doves treated with 8-OH-DPAT, WAY-100635 and para-

chlorophenylalanine (PCPA) by means of behavioural, electrophysiological and infrared

actimetry criteria. Two parallel experiments (locomotor activity and EEG rhythm

analysis) were performed after serotonergic drug administrations, and correlated results

were obtained. 8-OH-DPAT treatment increased locomotor activity, active waking and

grooming states and reduced SWS and REM sleep. Pre-treatment with WAY-100635

prevented the effects induced by 8-OH-DPAT. Serotonin depletion induced by PCPA

treatment reduced locomotor activity, waking and grooming activity while increased

both SWS and REM sleep. Moreover, 8-OH-DPAT in PCPA treated ring doves

produced a marked rise in the locomotor activity and in the active waking and grooming

states while decreased sleep.

Altogether, the results obtained from both experimental schedules showed a

clear relationship, reinforcing the idea that serotonin plays a permissive role on

wakefulness probably through the activation of 5-HT1A receptors, which increases wake

activities and reduces sleep in ring doves.

Keywords:

Serotonin, bird, 8-OH-DPAT, PCPA, locomotor activity, EEG rhythm analysis

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Introduction

The relationship between serotonin (5-hydroxytryptamine, 5-HT) and sleep-

waking states has theoretical and practical importance. Many reports demonstrated that

the serotonergic system is implicated on vigilance states in mammals [1]. Also

serotonergic mechanisms may play an important role in the control of sleep-waking

cycle in avian. Studies in pigeons and chickens have demonstrated that the organization

[2, 3] and functionality [4] of the avian serotonergic system is similar to the mammalian

one. At the contrary to mammals, few pharmacological experiments have studied the

role of 5-HT on vigilance states in birds.

Para-chlorophenylanine (PCPA) which selectively inhibits tryptophan

hydroxylase, the rate-limiting enzyme in the serotonin biosynthetic pathway, results in

depletion of neuronal serotonin stores [5], an effect that has been observed in several

mammalian species including rat [6-8], rabbit [9, 10], cat [11-14], monkeys [15] and

humans [16]. Moreover, in mammals, it was reported that the inhibition of serotonin

synthesis by PCPA induces insomnia facilitating waking and reducing the slow wave

sleep (SWS) (for a review see [1]). Experiments investigating the effects of PCPA in

birds are limited. PCPA significantly reduced whole brain serotonin levels in turkeys

[17], chicks [18, 19] and Coturnix quail [20]. Only studies in parakeets showed that the

systemic administration of PCPA induced prolonged insomnia [21, 22]. These results

allowed to assume a permissive role in the production of sleep for serotonin. However,

the activity of the nucleus of the dorsal raphe is maximal during wakefulness [23]. The

apparent inconsistency between these two effects requires a clear explanation.

The activity of serotonin neurotransmitter in the regulation of sleep-wake cycle

is complex. In mammals, the selective and full agonist of 5-HT1A receptor [8-hydroxy-

2-(di-n-propylamino) tetralin] (8-OH-DPAT) at pre- and postsynaptic sites, and the

high-affinity antagonist of 5-HT1A receptor WAY 100635 have been used to

characterize the role of 5-HT1A receptor in the regulation of the vigilance states. In this

way, it has been reported different results related to the vigilance states in different

species. Some authors described that 5-HT1A agonist 8-OH-DPAT induced a dose-

dependent increase in waking, decrease in SWS, and had no effects on the generation of

the paradoxical sleep in cats, although REM directly occurred after waking, as in

narcolepsy [24]. In other works, 8-OH-DPAT treatment reduced SWS and also REM

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sleep in freely moving rats with an increased waking leading to hyperlocomotion [25],

while other studies reported an increase of REM in rats [26] induced by 8-OH-DPAT.

At the contrary, rats treated with the agonist 5-HT1A 8-OH-DPAT did not show

significant changes on SWS and REM sleep when it was administered alone [27].

On the other hand, the 5HT1A antagonist WAY100635 had no effects on overall

behavioural states in cats [24], but Fornal and col. [28] reported an increased neuronal

activity induced by WAY-100635, in an evident manner during waking when

serotonergic neurons typically display a high activity level, but not during sleep when

neurons display little or no spontaneous activity.

Although serotonin could be important for the control of sleep-waking states,

studies in birds species are scarce. Fuchs and cols. [29] investigated the role of

serotonin in the regulation of REM sleep in pigeons reporting a decrease in the amount

of time spent in this state under the effects of a serotonin reuptake inhibitor. On the

other hand, it has been also reported an increase of the sleep-like postures after

serotonin injections in quails [30]. In a similar manner, administration of 5-HT and

melatonin precursor L-tryptophan before the onset of dark period, reduced the nocturnal

locomotor activity of ring doves improving sleep efficiency which was explained as a

melatonin mediated effect [31]. However, other authors, which studied the effects of the

administration of 5-HT or the selective agonist of 5-HT1A receptors 8-OH-DPAT, did

not report effects on sleep-like behaviours in pigeons [32-34], chickens [35] and turkeys

[35]). Although some of these findings suggest the presence of serotonergic

mechanisms in sleep-waking regulation in birds, there is no general consensus on the

effects of serotonin and the agonist of 5-HT1A receptors in avian sleep-wake cycle

regulation.

Despite of the presence of behavioural sleep in most species, the

electrophysiological criteria of polygraphic sleep is the most appropriate tool for

characterizing the sleep-wake states. Therefore, the present study aims at studying the

effects of different serotonergic drugs (8-OH-DAPT, WAY100635 and PCPA) on the

behavioural sleep and wakefulness and the locomotor activity of ring doves

(Streptopelia risoria) in order to analyse the serotonergic mechanisms in the vigilance

states of these birds using behavioural, electrographic and infrared actimetry criteria.

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Material and methods

Animals

Adult domestic ring doves (Streptopelia risoria, 153.5 ± 7.8 g body weight)

were used throughout the experiments and maintained in individual cages at a

temperature of 24±2ºC, on a 12:12 Light:Dark (LD) cycle (lights on at 8 a.m.) with free

access to food and water. All procedures were performed during the active light period

(1.2 Klux).

Experiments were performed following the “Principles of Laboratory Animal Care”

(NIH Publication no. 85-23, revised 1996) and according to the guidelines of the Local

Ethics Committee of the University of The Balearic Islands (Spain).

Drugs and injections

Ring doves were submitted to different treatments: A/ serum saline

administration (1 ml/kg i.p.), B/ the 5-HT1A receptor agonist 8-OH-DPAT (0.5 and 1

mg/kg i.p., Sigma-Aldrich Chemie, Steinheim, Germany), C/ the 5-HT1A receptor

antagonist WAY100635 (Sigma-Aldrich Chemie) (0.5 mg/kg i.p.), D/ 8-OH-DPAT (1

mg/kg i.p.) after pre-treatment with WAY100635 (0.5 mg/kg i.p., 30 minutes a part), E/

PCPA (Sigma-Aldrich Chemie) treatment (birds received an injection (300 mg/kg i.p.)

daily for two consecutives days 6 hours before to initiate the recordings), and F) 8-OH-

DPAT (0.5 mg/kg i.p.) after pre-treatment with PCPA.All administrations were made

intraperitoneally. Each animal was submitted to EEG, EOG, EMG recordings and

behavioural observation simultaneously for 2 h after the treatments. For recordings after

PCPA treatment, a delay of 6 and 30 hours was allowed to achieve 5-HT depletion. At

least 3 days elapsed between the different treatments in order to ensure a complete

washout of the administered substances.

The doses of PCPA used in the present work were selected on the basis of

previous works in rats and avian [7, 19, 36] showing a large reduction in 5-HT levels.

Also, the doses of 8-OH-DPAT used in the present work were selected on the basis of

previous works in birds [37].

Two different experimental designs were performed using two different groups

of animals which had similar characteristics (body weight, age and house conditions): 1)

study of spontaneous locomotor activity and 2) study of the vigilance states throughout

EEG recordings.

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Study of locomotor activity

Nine adult ring-doves were used. Animals were housed in individual 20x20 cm

cages inside an activity chamber with 12:12 Light:Dark (LD) cycle (lights-on at 9:00),

controlled temperature (23.5 ºC) and free access to food and water throughout all

experimental time period. Locomotor activity from the animals was registered using

cross beam infrared interruption system located 70 mm above the floor of the cage, that

detected the movements of the animals within its cage. Activity was recorded and stored

in 15 minutes bins on a computerized data-logging system for later analysis.

After habituation to the new environment, activity was continuously measured

during 21 days. Animals were submitted to different treatments: 1) saline administration

(1 ml/kg i.p.), 2) 8-OH-DPAT (0.5 and 1 mg/kg i.p.), 3) WAY-100635 (0.5 mg/kg i.p.)

and 4) injections over 2 consecutive days with PCPA treatment (300 mg/kg i.p.).

Moreover the effect of 8-OH-DPAT (0.5 mg/kg i.p.) was analysed after 5) pre-treatment

with WAY-100635 (0.5 mg/kg i.p., 30 minutes before) and 6) after PCPA treatment

(birds received injections over two consecutives days (300 mg/kg i.p.) 6 hours before to

initiate the recordings).

Surgery

Nine adult ring-doves were used. At least 10 days before the experiments, each

animal was anaesthetized with isoflurane (Abbot®, Spain) inhalation anaesthesia using

a ventilated chamber coupled to a mask. Anaesthetic depth was checked by

physiological parameters (immobility, absence of stimulus response, body temperature,

heart and respiratory rate). Atropine (Braun®, Spain) was injected to avoid a rise in

salivary secretion (0.05 mg i.p.).

Stainless steel bipolar EEG electrodes were stereotaxically placed into the

frontal cortex according to coordinates derived from the brain atlas of the pigeon [38]

with a reference in the cerebellum. Two bipolar electrodes were also threaded just above

the orbits to detect eye movements (EOG). Two additional electrodes were placed over

the dorsal neck muscles for bipolar EMG recording. Electrodes were anchored to the

skull with jeweller’s screws and fixed with dental cement.

Electroencephalographic recording

Animals were allowed to recover from surgery for at least 10 days before

beginning the experiments. They were placed in the recording cages 5 hours before the

beginning of the different sessions for habituation to the recording environment. EEG,

EOG and EMG recordings were carried out using AxoScope (v. 8.1 for Windows®,

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USA). EEGs were filtered with a 0.5-50 Hz bandpass filter and sampled at a frequency

of 256 Hz with a notch filter at 50 Hz. EMG and EOG were filtered with a 5-100 Hz and

0.1-50 Hz bandpass filter, respectively. EEG rhythm was recognized as a sinusoidal-like

waveform with different peak frequencies [39, 40], and fragments of the recordings

were submitted to a spectral analysis to evidence the appearance of the different EEG

ranges. These conditions are commonly used to analyse the early effects of

pharmacological treatments on sleep [41, 42]. As the maximal effect of serotonergic

agonists and antagonists occurs within 2 h [25], the recordings were restricted to this

lapse after injection.

Behavioural recording classification

Behavioural state was recorded by direct observation of the animals through a

video system connected to a computer. The observer was at a distance of 3 m to avoid

interference in the animal behaviour. A keyboard was used to include the behavioural

data into the polygraphic recordings and, in this way, animal behaviour and EEG, EOG

and EMG recordings could be later correlated by visual inspection. During the first two

hours after drug injections, behaviours were directly observed and continuously

recorded. The behavioural states were classified according to Fuchs et al. [29] and

Toledo and Ferrari [43] criteria as follows: (1) Slow wave states (SWS) as the slow

wave activity with high amplitude EEG and low EMG activity while the animal remains

sitting or standing with closed eyes and the neck retracted and sometimes resting on

chest, (2) passive waking (PW) or waking without locomotor activity (standing or

sitting) with open eyes, high frequency and low amplitude EEG and high EMG activity,

(3) active waking (AW) or psychomotor waking with open eyes, high frequency and

low amplitude EEG and high EMG activity, (4) grooming, which was defined as the

rubbing the beak or limbs over or between the feathers and (5) rapid eye movements

sleep (REM) characterized by a high frequency, low EEG amplitude, fast and high

amplitude EOG activity (indicative of large eye movements), and a low EMG activity

while the animal remains sitting or standing with closed eyes and with sporadic and

sudden downward head drops followed by slow return to an upright posture.

Data analysis

Locomotor activity was analysed using t-student for unpaired data in all

parameters measured in order to determine the significance of changes induced by the

treatments, and p<0.05 was considered statistically significant.

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Accumulated total time spent (duration) and number of episodes for each

vigilance state and treatment were obtained on the basis of EEG, EOG, EMG and

behavioural parameters. Statistical analysis was carried out using SPSS® (v. 12.0 for

Windows®) using one-way analysis of variance (ANOVA), with treatment as factor.

Results are expressed as mean ± SEM. All these tests were followed, when appropriate,

by a post-hoc LSD paired comparisons to recognize deviant groups, and a p<0.05 was

accepted as being statistically significant in these procedures.

Results

Locomotor activity

The acute administration of the selective agonist of 5-HT1A receptors 8-OH-

DPAT (1 mg/kg i.p.) significantly increased the locomotor activity accumulated during

one hour (30%) in ring doves when compared with saline treated animals (Figure. 1).

Table 1 shows that pre-treatment of animals with the selective 5-HT1A receptors

antagonist WAY-100635 blocked the stimulatory effect of the agonist 8-OH-DPAT on

locomotor activity, demonstrating an effect mediated by 5-HT1A receptors. Additionally,

the antagonist WAY-100635, administered alone, decreased the locomotor activity

(43%) in ring doves when compared to saline control. This inhibitory effect of WAY

seems to indicate the existence of a stimulatory tone on the locomotor activity mediated

by 5HT1A.

Moreover, the treatment with PCPA (300 mg/kg i.p., daily injections in two

consecutive days) produced an important decrease in the locomotor activity (30%-50%)

when compared with saline (Figure 1). After PCPA treatment, the selective agonist 5-

HT1A 8-OH-DPAT (1 mg/kg i.p.) produced an important rise in the activity of animals

(Figure 1).


The animals were visually observed during the time period comprised between

drug administration and the end of the recording session (2h). The animals that received

saline or the different drug treatments did not show any abnormal behavioural signs.

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Effects of serotonergic drugs on eye closure

Figure 2A shows the effects of PCPA treatments, and the effect of the selective

5-HT1A receptor agonist 8-OH-DPAT after saline and after PCPA pre-treatment on the

duration of episodes when animals had open or closed eyes (F(27,99)=13.767). A

significant decrease of the open eyes duration occurred after PCPA treatment when

compared to saline control (78%), while an increase of the closed eyes duration

occurred (133%). 8-OH-DPAT did not modify the eye closure in comparison with

saline control treatment, although the administration of 8-OH-DPAT in PCPA treated

animals increased the open eyes duration (197%) and reduced the close eye duration

(50%).

Figure 2B shows the effects of PCPA treatments, and the effect of the selective


number of episodes when animals had open or closed eyes (F(27,99)=6.055). A

significant decrease of the open eyes episodes occurred after PCPA treatment when

compared to saline control (74%), while an increase occurred when animals had closed

eyes (150%).

8-OH-DPAT did not modify the eye closure in comparison with saline control

treatment, although the administration of 8-OH-DPAT in PCPA treated animals

increased the number of episodes when animal had open eyes (155%).

Effects of serotonergic drugs on vigilance states

Figure 3 shows the effects of PCPA treatments, and the effects of the selective 5-

HT1A receptor agonist 8-OH-DPAT after saline and after PCPA pre-treatment on the

duration (F(5,235)=11.613), number of episodes (F(5,32)=2.683) and latency

(F(5,32)=4.065) of REM sleep states. A significant increase of the REM duration

(57.3%) and number of episodes (273.3%) occurred after PCPA treatments when

compared to saline control, while reduced the latency of REM state (87.1%).

8-OH-DPAT injection reduced REM duration (41.7%) but not the number of episodes

respect to saline control treatment. Moreover, the administration of 5-HT1A receptors 8-

OH-DPAT in PCPA treated animals reduced total duration of REM (42.6%) and the

number of episodes (55%) while increased REM latency (640%).

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Figure 4 shows the effects of PCPA treatments, and the effects of the selective 5-

HT1A receptor agonist 8-OH-DPAT after saline and after PCPA pre-treatment on the

duration of the different vigilance states. When vigilance states duration was observed

(Figure 4A), a significant increase of the slow wave state occurred after PCPA treatment

when compared to saline control (99%, F(31.191)=11.29), while reduced active waking

(78%, F(31,191)=11.29) and grooming (72%, F(6,32)=1.577) states.

No significant differences were found in vigilance states after 8-OH-DPAT

injection respect to saline control treatment, although the administration of 8-OH-DPAT

in PCPA treated animals reduced total duration of slow wave sleep (42%) and

significantly rose active waking (272%) and grooming (230%, F(6,32)=1.577) states

(Figure 4A).

Additionally, the vigilance states were also studied focusing attention on eyes,

when animals showed open eyes (Figure 4B) or closed eyes (Figure 4C). After PCPA

treatment, when animals had open eyes a reduction on slow wave (F(39,306)=7.425),

passive waking (F(39,306)=7.425), active waking (F(29,79)=8.705) and grooming

(F(6,32)=1.577) states duration was observed (Figure 4B). At contrary, when animals

had closed eyes an increase on slow wave and passive waking states (F(39,306)=7.425)

duration was observed (Figure 4C). The administration of 8-OH-DPAT in PCPA treated

animals increased the duration of the time spent in slow wave, active waking and

grooming states when animals had open eyes (Figure 4B) and reduced the duration of

the time spent in slow wave when eyes were closed (Figure 4C).

Figure 5 shows the effects of PCPA treatments, and the effect and the selective


number of episodes observed during the different vigilance states. When vigilance states

were observed (Figure 5A, F(31,191)=4.138), a significant decrease in the active

waking state occurred after PCPA treatment when compared to saline control (73%) and

a significant increase in the number of episodes of active waking (175%) and grooming

(312%) states occurred after the administration of 8-OH-DPAT in PCPA treated

animals compared to saline control.

Additionally, the number of episodes of the vigilance states were also studied

focusing attention on eyes, when animals showed open eyes (Figure 5B) or closed eyes

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(Figure 5C). After PCPA treatment, when animals had open eyes a reduction in the

number of episodes of slow wave, passive waking (F(39,308)=5.817) and active waking

(F(29,79)=7.648) states was observed (Figure 5B). At contrary, when animals had

closed eyes an increase in the number of episodes of slow wave (F(39,308)=5.817) state

was observed (Figure 5C). The administration of 8-OH-DPAT in PCPA treated animals

increased the number of episodes of active waking and grooming states (F(6,27)=1.684)

when animals had open eyes (Figure 5B) while no changes were observed when eyes

were closed (Figure 5C).

Also, latencies of the different vigilance states were analysed but not differences

were found except to the active waking state. PCPA administration increased (79%) the

latency to active wake state respect to the corresponding control (Saline: 32.1 ± 1 min

and PCPA (600 mg/kg): 57.4 ± 9.7 min (p<0.001) data not shown).

Differences in the duration and number of episodes presenting theta rhythm after

the administration of the different serotonergic treatments in ring doves were not found

in any case. Also, latencies were maintained unaltered under the different drug

treatments (data not shown).

Supersensitivity of 5-HT1A receptors induction after PCPA treatment

Table 2 shows the comparative effect of 8-OHDPAT (0.5 mg/kg, i.p.)

administrated alone or after the PCPA treatment on locomotor activity accumulated

during 2h along three consecutive periods. A marked increase in locomotor activity

(until 5 folds) was evidenced after PCPA treatment respect to the effect induced by 8-

OHDPAT in non pre-treated animals, suggesting a development of supersensitivity

mechanisms of 5-HT1A receptors.

Table 3 shows the comparative effect of 8-OHDPAT (0.5 mg/kg, i.p.)

administrated alone or after the PCPA treatment on vigilance states and closure eyes. A

marked increase in the stimulatory or inhibitory effect induced by 8-OHDPAT on the

duration of slow wave sleep (inhibitory effect), active waking and grooming

(stimulatory effects) was evident after PCPA treatment respect to the effect induced by

8-OHDPAT in non PCPA pre-treated animals, suggesting the development of

supersensitivity mechanisms of 5-HT1A receptors. Similarly, a marked increase in the

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effect induced by 8-OHDPAT on the number of episodes of active waking and

grooming (stimulatory effects) was evident after PCPA treatment respect to the effect

induced by 8-OHDPAT in non PCPA pre-treated animals. In addition, a marked

increase in the effect induced by 8-OHDPAT on the duration and number of episodes

when animals had open eyes (stimulatory effect) and closed eyes (inhibitory effect) was

evidenced after PCPA treatment respect to the effect induced by 8-OHDPAT in non

PCPA pre-treated animals, suggesting the induction of supersensitivity mechanisms of

5-HT1A receptors after serotonin depletion by PCPA.

Discussion

The present work analysed the role of the neurotransmitter serotonin (5-

hydroxytryptamine, 5-HT) on the vigilance states of ring doves using behavioural,

electrophysiological and infrared actimetry criteria. The results obtained from the

different experiments showed a clear close correlation, reinforcing the idea that

serotonin during light time plays a permissive role on wakefulness, as well as the

activation of 5-HT1A receptors increases wake activities and reduces sleep in birds.

As a neurotransmitter and neuromodulator, 5-HT influences a multitude of

different, sometimes opposite, physiological responses, depending on the receptor type

and localization in the brain, on whether the receptor is pre- or postsynaptic, and

depending on the current state of the individual [1]. In fact, the activity of 5-HT

neurotransmitter in the regulation of sleep-wake is complex.

Experiments related to the effects of the inhibition of 5-HT synthesis by para-

chlorophenylalanine (PCPA) on sleep-waking states in birds are scarce. However,

PCPA significantly reduced brain serotonin levels in different avian species [17-20] in a

similar manner to the reported in several mammalian species [5-16]. The doses of

PCPA used in the present work were similar or higher to those reported to induce a deep

level of 5-HT depletion in different species [7, 19] (among others).

Several studies have demonstrated that sleep is blocked or heavily reduced in a

period following administration of PCPA in mammals (for a review see [1]). In this

way, PCPA induced insomnia facilitating waking and reducing the slow wave sleep and

REM in cat [44] and rat [8]. Also in parakeets, systemic administration of PCPA

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induced prolonged insomnia [22] but REM sleep was not suppressed as much as SWS

[21]. Nevertheless, the results of the current work are in sharp contrast to the above

mentioned results since PCPA administration neither induced insomnia nor reduced

SWS and REM sleep. On the contrary, the present results after the PCPA administration

increased SWS and REM activity, eyes closure (a correlate of behavioural sleep [45])

and the latency to active waking state when compared with saline controls. In

accordance, also in the present work PCPA administration reduced locomotor activity,

active waking state (duration and number of episodes) and grooming activity (a

behaviour related to active wakefulness). In good agreement, it has been described that

doses of PCPA in rats (150 to 1000 mg/kg) similar to those used in the current work,

also reduced spontaneous locomotor activity, and some wakefulness measures such as

speed and distance per movement [7]. Also, in birds, high doses of PCPA induced a

dose-dependent lethargic behaviour [19]. In this manner, the opposite results often

obtained after PCPA administration on behavioural studies could be related to the use of

different doses and the degree of 5-HT depletion. The first mentioned results from some

studies using PCPA had allowed to assume a permissive role for serotonin in the

production of sleep, it implied that without serotonin in the brain, there is no sleep.

Nevertheless, after the administration of PCPA in mammals, sleep eventually reappears

while brain serotonin was still very low ([46], see [1] for a review). Moreover, the 5-

HT–containing neurons of the reticular formation, the medial and dorsal raphe which

innervate the entire forebrain contribute to maintaining a quiet awake state, and the

activity of these neurons show maximal firing during wakefulness but no activity during

REM sleep [23].

There are many 5-HT receptor subtypes as well as numerous behaviours

attributed to 5-HT transmission, factors that likely have contributed to the confusion

regarding the role of this neurotransmitter in sleep/wake states. Indeed, evidence exists

to support a role of 5-HT both in sleep and wakefulness. However, a consensus has

recently emerged that supports a stronger role in wakefulness because promotion of 5-

HT transmission (eg, reuptake inhibition, precursor loading, etc) results in quiet waking

[47, 48]. Hence, firing activity of serotonergic dorsal raphe neurons was related to the

level of behavioural arousal, since they discharged regularly at a high rate during

waking and at progressively slower rates during slow-wave sleep, and ceased firing

during REM [24]. It seems to be a general tendency that serotonin measured in the

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extracellular fluid, which presumably mirrors the actual release in the area where it is

measured, is highest during waking, is reduced during SWS and more reduced during

REM sleep [49]. This is the case in the different neuronal regions in cats [50], rats [51]

and ring doves [4].

The implication of serotonin receptors of the 5-HT1A type in the regulation of

vigilance states has been the matter of numerous investigations [1]. In the present work,

serotonin seems to play a tonic stimulatory control on locomotor activity mediated by

5HT1A receptors in ring doves since the agonist of 5-HT1A receptors 8-OH-DPAT (1

mg/kg i.p.) increased the locomotor activity. 8-OH-DPAT that is classically considered

as a selective 5-HT1A agonist [52], acts at 5-HT7 receptors at relatively low doses [53].

The results reported in the present study showed that the pre-treatment of ring doves

with the selective 5-HT1A antagonist WAY-100635 blocked the stimulatory effect of the

agonist 8-OH-DPAT on locomotor activity, indicating that the observed stimulatory

effect was mediated by 5-HT1A receptor. This finding and the inhibitory effect on

locomotor activity displayed by PCPA treatment in this work, reinforces the suggestion

that serotonin play a tonic stimulatory role on the locomotor activity of the ring doves.

Moreover in this work, in PCPA treated animals and consequently in the absence

of the basal activity of other serotonergic receptors, the intraperitoneal administration of

8-OH-DPAT in PCPA pre-treated ring doves resulted in a marked stimulatory effect of

8-OH-DPAT on active waking state (duration and episodes) and grooming activity, and

likewise in a marked inhibitory effect on SWS, REM sleep and eyes closure. In line

with the current work, Kostal and Savory [37] showed an increased activity of fowls

during the first hour of the study after the injection of 8-OH-DPAT (1 mg/kg),

concretely in the active waking and grooming (preening). Several works in mammals

are also in line with the current work. The stimulation of 5-HT1A receptors by systemic

administration of agonists in rats has been repeatedly shown to increase waking [54],

and some authors have also reported that 8-OH-DPAT induced a dose-dependent

increase in waking and a decrease in SWS and REM sleep ([1, 25, 55, 56] among

others). Moreover, 8-OH-DPAT perfused into paragigantocellularis lateralis resulted in

fragmented sleep with alternating brief periods of SWS and wake, and an almost

complete elimination of REM sleep in piglets [57, 58]. Also, 8-OHDPAT applied into

the raphe nucleus induced a dose-dependent increase in wakefulness and a decrease in

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SWS [59] and eliminated REM sleep in piglets [57, 60], or have no significant effect on

the generation of REM sleep in cats [24].

Understanding 8-OH-DPAT-induced effects is complicated because the 5-HT1A

receptors are localised both pre-synaptically and post-synaptically [61] and biphasic

effects has been reported for 8-OH-DPAT [62]. According to the model for REM

regulation [63], 5-HT exerts an inhibitory influence on mesopontine cholinergic “REM-

on” neurons [64], notably through postsynaptic 5-HT1A receptors [55, 56], while 5-HT

might also have a facilitatory influence on REM [54, 65] or SWS [24] through the

activation of somatodendritic 5-HT1A autoreceptors in anterior raphe nuclei.

Nevertheless, the inhibitory effect of systemic treatment with various 5-HT1A receptor

agonists, notably the prototypical one 8-OH-DPAT on REM sleep supports the idea that

5-HT1A receptors play an important role in the regulation of this vigilance state in

mammals [62, 66-69].

Reduced REM sleep after 8-OH-DPAT injections is also in good consonance

with the observation that mutant mice that do not express this receptor type (5-HT1A_/_)

showed higher amounts of REM sleep during the entire circadian period than the wild-

type mice [70]. Moreover, the pharmacological blockade of 5-HT1A receptors with

WAY 100635 induced in wild-type mice an increase in REM amounts indicating that

these receptors mediate a tonic inhibitory influence on REM in this species [70]. On the

other hand, the 5-HT1A antagonist WAY100635 had no effects on overall behavioural

states at low concentrations in cats [24] as same of the observed results in the present

work when the attention was focused in the vigilance states. Pigeons clearly tolerate

higher doses of 8-OH-DPAT and other 5-HT1A agonists/antagonists. Based on this

observation, the interpretation of the results obtained after the administration of the 5-

HT1A receptor ligand 8-OH-DPAT alone in ring doves could be related to the fact that

higher doses of 8-OH-DPAT are needed in birds respect to the mammals. Thus, 8-OH-

DPAT administrated alone only modified locomotor activity. However, the

administration of 8-OH-DPAT in PCPA pre-treated ring doves resulted in a more

marked stimulatory effect of 8-OH-DPAT on locomotor activity, active waking states

(duration and episodes) and grooming activity, as well as a marked reduced effect on

SWS, REM sleep and eyes closure, indicating an increased sensitivity of the 5-HT1A

receptors modulating vigilance states as a consequence of the serotonin depletion by

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Manuscript III

PCPA. In this way, similar treatment with PCPA to rats, (300 mg/kg i.p.) increased

specific binding of 3H-5-HT within 24 h by more than 100% [71].

In summary, ring doves as avian in general are monophasic and diurnal species,

characteristics that are similar in humans. Based on electrophysiological,

neurochemical, genetic and neuropharmacological approaches, altogether, the data

support the idea that the serotonergic system, which displays maximal activity and high

serotonin levels during wakefulness, promoted waking and inhibited SWS and REM

throughout activation of 5-HT1A receptors. In contrast, when the level of serotonin went

downwards, the wake was reduced and sleep states increased. The observation that this

phenomenon is present in birds and mammals suggest that serotonergic sleep regulating

mechanisms are shared by the two vertebrate groups reflecting their common

phylogenetic origin [22].

Acknowledgments

This work was supported by grant BFI2002-04583-C02-029, SAF2007-66878-

CO2-02 (MEC, Madrid, Spain) and 36AA/04 of Conselleria de Salut i Consum (Govern

de les IllesBalears, Spain). Silvia Tejada was supported by a FPI grant (Govern de les

Illes Balears, Spain).

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Figures

Figure 1. Effects of PCPA treatments (6 hours after injections in two consecutive days,

300 mg/kg i.p. each), and effect of the 5-HT1A receptor agonist 8-OH-DPAT (0.5 and 1

mg/kg i.p.) after saline and after PCPA treatment on locomotor activity. Bars represent

mean ± SEM from 9 ring doves. * 8-OH-DPAT (0.5 or 1 mg/kg) respect to their

controls; # differences between control treatments. * p<0.05, *** p<0.001, # p<0.05, ##

p<0.01 (t-student for unpaired data).

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mg/kg i.p.) after saline and after PCPA treatment on eyes closure (duration and

episodes). A: Duration of eyes closure, B: Number of episodes of eyes closure. Bars

represent mean ± SEM from 5 ring doves. * 8-OH-DPAT (0.5 or 1 mg/kg) respect to

their controls; # differences between control treatments. * p<0.05, *** p<0.001, #

p<0.05, ### p<0.001 (one way ANOVA analysis).

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mg/kg i.p.) after saline and after PCPA treatment on REM sleep (duration, number of

episodes and latency). Bars represent mean ± SEM from 8 ring doves. * 8-OH-DPAT

(0.5 or 1 mg/kg) respect to their controls; # differences between control treatments. *

p<0.05, *** p<0.001, # p<0.05, ## p<0.01, ### p<0.001 (one way ANOVA analysis).

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mg/kg i.p.) after saline and after PCPA treatment on the duration of the different

vigilance states. A: Duration of the different vigilance states, B: Duration of the

different vigilance states when animals had open eyes, C: Duration of the different

vigilance states when animals had closed eyes. Bars represent mean ± SEM from 5 ring

doves. * 8-OH-DPAT (0.5 or 1 mg/kg) respect to their controls; # differences between

control treatments. * p<0.05, ** p<0.01, *** p<0.001, # p<0.05, ## p<0.01, ###

p<0.001 (one way ANOVA analysis).

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mg/kg i.p.) after saline and after PCPA treatment on the number of episodes of the

different vigilance states. A: Number of episodes of the different vigilance states, B:

Number of episodes of the different vigilance states when animals had open eyes, C:

Number of episodes of the different vigilance states when animals had closed eyes. Bars

represent mean ± SEM from 5 ring doves. * 8-OH-DPAT (0.5 or 1 mg/kg) respect to

their controls; # differences between control treatments. * p<0.05, ** p<0.01, # p<0.05,

## p<0.01, ### p<0.001 (one way ANOVA analysis).

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Table 1. Comparative effect of 8-OH-DPAT injected alone or after WAY100635

treatment on locomotor activity.

Treatment Locomotor activity

Saline 133.5 ± 24.3 8-OH-DPAT (0.5 mg/kg) 89 ± 38

8-OH-DPAT (1 mg/kg) 217.7 ± 40.4* WAY100635 76 ± 16.7**

WAY100635 + 8-OH-DPAT 111.2 ± 23.3* ## Number of movements of nine ring doves accumulated during one hour after the

following treatments: saline, the 5-HT1A receptor agonist 8-OH-DPAT (0.5 and 1

mg/kg, i.p.), the 5-HT1A receptor antagonist WAY100635 (0.5 mg/kg, i.p.) and 8-OH-

DPAT (0.5 mg/kg, i.p.) after pre-treatment with WAY100635 15 min apart (0.5 mg/kg,

i.p.). Values represent mean ± SEM and t-student for unpaired data was used. * p<0.05,

** p<0.01 when compared with control saline; ## p<0.01 when compared with 8-OH-

DPAT (1 mg/kg i.p.) administrated alone.

Table 2. Comparative effect of 8-OH-DPAT injected alone or after PCPA treatment on

locomotor activity.

Treatment 2 hours 4 hours 6 hours

Saline 100 ± 21 100 ± 24 8-OH-DPAT 81.1 ± 9.3 84.4 ± 17.8

PCPA 100 ± 31.9 100 ± 57.1 PCPA + 8-OH-DPAT 214.7 ± 54.5***

100 ± 25.8 113.89 ± 17.2

100 ± 43 646.1 ± 234.73*** 411.8 ± 135.33***

Number of movements of nine ring doves after the treatment with the agonist of 5-HT1A

receptors 8-OH-DPAT (0.5 mg/kg i.p., 2h-4h-6h) after the pre-treatment with saline or

PCPA (300 mg/kg, i.p. during two consecutive days). Values represent the movements

accumulated during 3 successive 2 hr periods after injection as percentage of their

respective control (saline or PCPA). * p<0.001 when compared the effect of 8-OH-

DPAT after saline or PCPA pre-treatment by t-student analysis.

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Table 3. Comparative effect of 8-OH-DPAT injected alone or after PCPA treatment on

vigilance states.

Saline + 8-OH-DPAT PCPA + 8-OH-DPAT

Open eyes 298 ± 27** Closed eyes 50,6 ± 6.6*

Right eye closed 596 ± 220*** Duration open

eyes (%) Left eye closed

78 ± 13 162 ± 54.2

95 ± 20 157 ± 87 42 ± 16.3*

Open eyes 254,7 ± 69,7*** Closed eyes 84.4 ± 7.5

Right eye closed 731 ± 319**

Number of episodes with opened eyes

(%) Left eye closed

75,7 ± 18,9 127 ± 40.7 108 ± 27

167 ± 100 70,7 ± 33 States Opened eyes Closed eyes Opened eyes Closed eyes

Slow wave 127 ± 22.2 73 ± 8.5 168 ± 79 58 ± 12* 302 ± 146* 39 ± 7.4* Passive waking 96 ± 24 73 ± 22 134 ± 56.5 91 ± 21 199 ± 78* 60 ± 13.9* Active waking 95 ± 20 88 ± 20 179 ± 70.7 372 ± 85* 383 ± 83** 377 ± 287.2*

Duration (%)

Grooming 61 ± 15 62 ± 15 0 329 ± 122* 329 ± 122* 0 Slow wave 106 ± 29 73.3 ± 20.2 132.7 ± 43 125 ± 44 287 ± 132* 100 ± 20*

Passive waking 93 ± 22 75 ± 17.8 108 ± 33 117 ± 28 234.7 ± 87* 64 ± 12* Active waking 100 ± 35 78 ± 16.2 231 ± 76 256 ± 37* 330 ± 60.7 166.3 ± 74

Number of episodes (%)

Grooming 62 ± 16 61.8 ± 16.3 0 423 ± 181* 422 ± 180* 0 Ring doves were injected with the selective agonist of 5-HT1A receptors 8-OH-DPAT

(0.5 mg/kg, i.p.) after the pre-treatment with saline or PCPA (300 mg/kg, i.p. during two

consecutive days). Values represent the effects of 8-OH-DPAT on duration and number

of episodes as percentage of their respective control (saline or PCPA). * p<0.001 when

compared the effect of 8-OH-DPAT after saline or PCPA pre-treatment by one-way

ANOVA.

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moving cat. J Neurosci, 1996;16: 2820-2828.

[66] Tissier M, Lainey E, Fattaccini C, Hamon M, Adrien J. Effects of ipsapirone, a 5-

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Manuscript IV

Electroencephalogram functional connectivity between rat hippocampus and

cortex after pilocarpine treatment

Tejada, S.; González, J.J.; Rial, R.V.; Coenen, A.M.L.; Gamundí, A. and Esteban, S.

(2010) Neuroscience. 165:621-631.

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Neuroscience 165 (2010) 621–631

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LECTROENCEPHALOGRAM FUNCTIONAL CONNECTIVITYETWEEN RAT HIPPOCAMPUS AND CORTEX AFTER
ILOCARPINE TREATMENT
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. TEJADA,a J. J. GONZÁLEZ,b R. V. RIAL,a

. M. L. COENEN,c A. GAMUNDÍa AND S. ESTEBANa*

Laboratory of Neurophysiology, Department of Biologia Fonamental iiències de la Salut, University of the Balearic Islands, IUNICS, Palmae Mallorca, Spain

Laboratory of Biophysics, Department of Physiology, Faculty of Med-cine, University of La Laguna, Ctra. La Cuesta-Taco s/n, 38320, Laaguna, Tenerife, Spain

Department of Biological Psychology, NICI-Radboud University Ni-megen, The Netherlands

bstract—The muscarinic agonist pilocarpine has beenhown to increase the duration and total number of episodesresenting theta rhythm—simultaneously in hippocampusnd cortex—in rats during the waking states. Theta waves areuggested to be involved in the flow of information betweenippocampus and cortex during memory processes. Thisork investigates this functional interdependence using thepectral and phase synchronization analysis of the electro-ncephalogram (EEG) theta band recorded in these braintructures of rats after pilocarpine treatment. Pilocarpine wassed at doses devoid of epilepticus-like seizures effects inonscious freely moving rats. The results showed that pilo-arpine administration significantly increased the relativeheta power during the waking states in the cortex, but not inhe hippocampus of rats. Additionally, the EEG coherenceetween the hippocampal EEG theta band and that arising athe frontal cortex increased after pilocarpine treatment butnly during the waking states. This result reveals an increasef the linear correlation between the theta waves of these tworain structures after pilocarpine treatment during the wak-

ng states. Moreover, phase synchronization results showedn effective phase locking with non-zero phase differenceetween hippocampus and frontal cortex theta waves thatemained after pilocarpine treatment. Therefore, pilocarpineeems to reinforce the neural transmission waves from theippocampus toward the cortex during waking. In conclu-ion, the present EEG study could suggest an effect of theuscarinic cholinergic agonist pilocarpine on the hippocam-

al-cortical functional connectivity. © 2010 IBRO. Publishedy Elsevier Ltd. All rights reserved.

ey words: coherence, theta band, hippocampus, frontal cor-ex, phase lag index (phase synchronization), power spec-rum.

Corresponding author. Tel: �34 971173145; fax: �34 971173184.-mail address: [email protected] (S. Esteban).bbreviations: A/D, analogical/digital; ANOVA, analysis of variance;P, anterior posterior; AW, active wake; DV, dorsal ventral; EEG,lectroencephalogram; EMG, electromyogram; FFT, fast Fourier

ransform; Hz, hertz; ML, medial lateral; PLI, phase lag index; PW,

sassive wake; REM, rapid eye movement; SEM, standard error ofean; SWS, slow wave sleep; �g, microgram; �l, microlitre.

306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rightoi:10.1016/j.neuroscience.2009.10.031

621

he cholinergic system plays an important role in the pro-uction and regulation of the sleep-wake cycle (Dringen-erg and Vanderwolf, 1998). Two cholinergic pathways are

nvolved in the control of the vigilance states; the one locatedn the rhombencephalon carries out an important part of rapidye movement (REM) sleep control (Velazquez-Moctezumat al., 1989; Xi et al., 2004). The second depends on theholinergic innervation of the neocortex arising primarilyrom cell groups of the basal forebrain (see Dringenbergnd Vanderwolf, 1998). The cholinergic system is also

nvolved in the generation of the theta rhythm, mainlynitiated in the hippocampus and observed during bothEM sleep and waking (Crouzier et al., 2006). Hippocam-al oscillations in the theta frequency band are mediatedy muscarinic and nonmuscarinic receptors. In this man-er, theta rhythm may represent two separate theta gen-rators in the hippocampus: a lower frequency (4–7 Hz)holinergic theta, and a higher frequency (7–12 Hz) atro-ine-insensitive theta (Kramis et al., 1975).

The hippocampus is a major component of the limbicystem that participates in the control of many physiolog-

cal and behavioural processes. Theta rhythm is the mostrominent activity in the hippocampus, although it has alsoeen recorded in other cortical and limbic structures, re-ponsible for different components of the memory (Kocsist al., 2001). Theta activity may support the transmissionnd storage of information within and between the hip-ocampus and cortex during performance of learned tasksMuir and Bilkey, 1998) and memory processes (Johnson,006; Klimesch et al., 2001) mediated by cholinergicechanisms (Gais and Born, 2004). In a previous work,e observed that the i.c.v. administration of the muscarinicgonist pilocarpine to rats increased—during passiveake (PW) and active wake (AW) states—the duration andumber of episodes with theta rhythm appearing in bothippocampus and cortex simultaneously, without changes

n the hippocampus (Tejada et al., 2007a). These resultsould suggest an increased connectivity between the hip-ocampal and cortical electrical activity as a consequencef pilocarpine administration.

Mathematical models can elucidate information con-erning the neural activity and connections between differ-nt regions. Electroencephalogram (EEG) power spectralensity is used to infer information regarding brain activity

n different areas, and to study functional links betweenrain regions. Coherence functions reflect the connectivityattern analysing the synchronized activity and coopera-ion between different brain regions (Horwitz, 2003) in
patiotemporal terms for a specific frequency band (Gerloff
s reserved.

139

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S. Tejada et al. / Neuroscience 165 (2010) 621–631622

nd Andres, 2002; Varela et al., 2001). The coherencealues depend on the power spectrum between two re-ions and give a measure of the linear correlation betweenwo signals in a specific frequency band. If the brain oscil-ations are synchronized, high coherence values could bexpected (Rosenblum et al., 2001). However, high coher-nce values may be due to the effect of common sourceontamination (volume conduction) of both recording brainites. Thus, phase synchronization analysis has been re-ently proposed as an alternative method to study theoupling between two systems. This new method seems toe less sensitive to volume conduction and is thereforeapable of assessing a real connectivity between themBabiloni et al., 2004; Stam et al., 2007, 2009).

Hence, the aim of the present work was to analyse theower spectrum of EEG frequency bands and the coher-nce and phase synchronization of the theta band be-ween hippocampus and frontal cortex to further investi-ate neural activities and connections after pilocarpinedministration, in freely moving rats at different vigilancetates. Special attention is addressed to changes in thecetylcholine dependent low theta frequency (4–8 Hz).

EXPERIMENTAL PROCEDURES

nimals

even adult male naive Wistar rats (12 months old, 350–375 g),red at the Department of Biological Psychology of the Radboudniversity Nijmegen, were used. Animals were individuallyoused and maintained in standard conditions throughout thexperimental time on a 12:12 light-dark cycle (light period from:00 PM to 3:00 AM). Rats were kept under these light conditions formonth to fully adapt to the light/dark scheme. Standard labora-

ory animal food and water were available ad libitum. All proce-ures were performed during the active dark period and werearried out under dim red light (�0.5 Lux).

Experiments were performed in accordance with the Euro-ean Convention for the Protection of Vertebrate Animals Used forxperimental and Other Scientific Purposes (Directive 86/609/EC) and according to the guidelines of the Local Ethic Commit-

ee of the Radboud University Nijmegen (the Netherlands).

urgery

dequate measures were taken to minimize pain or discomfort.nder isoflurane (Abbot®, the Netherlands) inhalation anaesthe-ia, each rat was submitted to aseptic surgery for implantation oflectrodes and a cannula guide. Anaesthetic depth was checkedy physiological parameters (immobility, absence of stimulus re-ponse, body temperature, heart and respiratory rate) of the animal.tropine (Braun®, the Netherlands) was only injected to avoid a rise

n salivary secretion during the surgery (0.05 mg i.m.). Stainless steelripolar EEG electrodes (Plastics One Inc., the Netherlands) werelaced into the frontal cortex (anterior posterior (AP) �2.0, medial

ateral (ML) �2.5) and into the hippocampal region (AP �4.0, ML2.0, dorsal ventral (DV) �3.0), both relative to the bregma (Paxinost al., 1980), with a reference electrode in the cerebellum. Further-ore, a stainless steel cannula guide was placed in the lateral

entricle (AP �0.8, ML �2.0, DV �3.3, relative to the bregma) inrder to inject the saline serum or pilocarpine. Two additional elec-rodes were attached to the dorsal neck muscles for bipolar electro-yogram (EMG) recording. Electrodes and cannula were attached to

he skull with dental acrylic cement. T

140

xperimental procedure

nimals were allowed to recover from surgery for at least 10 daysefore beginning the experiments. The day before the experiment,

n order to provide habituation to the recording conditions, thenimals were placed into boxes with the top open to allow thedministrations and EEG recordings. Rats were connected with-ut anaesthesia to the experimental setup through a rotatingonnector which also prevented twisting of EEG wires. All recordsegan 6 h after lights off, the period in which theta activity is lowestvan Luijtelaar and Coenen, 1984). Each rat was i.c.v. infusedithout being unhooked from the recording cable. I.c.v. adminis-

rations were made using a Hamilton syringe coupled to a syringeump (Razel scientific instruments®, Stanford, USA) with an in-

ection rate of 1 �l/5 min. Before infusion, the presence of hip-ocampal and cortical EEG and the EMG patterns was observednd recording was immediately initiated. Each animal was sub-itted to EEG, EMG and behavioural recordings for 2 h, immedi-tely after saline serum injection (1 �l i.c.v.) and after pilocarpineSigma-Aldrich Chemie®) injections (120 and 360 �g in 1 �l ofaline serum, i.c.v.). At least 3 days elapsed between the differentreatments to allow a complete wash out of the administeredubstances. All animals were submitted to the three treatments oneparate days according to a 3�3 Latin square design experi-ent. Animals were connected to the EEG machine throughout

he process and were free to move during the administrations tovoid too much hand manipulation. Pilocarpine doses used werehosen from previous studies which did not show neuronalamage in rat brain nor induced epilepticus like seizuresTejada et al., 2006, 2007b) (see the representative record ofig. 1).

As the maximal effect of muscarinic agonists occurs within thewo first hours (Timofeeva and Gordon, 2001), the recordingsere restricted to this lapse after injection. At the end of thexperiments, animals were sacrificed with an overdose of sodiumentobarbital (Nembutal®, the Netherlands, 0.8 ml i.p./animal)nd successively submitted to intracardiac perfusion with salinend 10% formaldehyde. Finally, the brain was quickly removed foristological assessment of the cannula guide and electrodeslacement. Only one animal with incorrect placement of cannulaas discarded.

ecordings

EG and EMG recordings were carried out using WINDAQ® (v..29 for Windows®). The EEG was digitized using a computer withn analogical/digital (A/D) converter. EEGs were filtered with a–100 Hz band pass filter and sampled at a frequency of 256 Hzith a notch filter at 50 Hz (see Fig. 1 for a representative record).MG was filtered with a 10–500 Hz band pass filter. Behaviouraltate was recorded by direct observation of the animals through alass window at a distance of less than 1 m. A keyboard was usedo include the behavioural data in the polygraphic recordings and,n this way, animal behaviour and EEG recordings could be laterorrelated by visual inspection. Behaviour was classed accordingo the following states (Gottesmann, 1992): (1) slow wave sleepSWS). (2) rapid eye movement sleep (REM sleep). (3) passiveaking or waking with non-motivated motor activity, and (4) activeaking or attentive and/or psychomotor active waking. EEG, EMGnd behavioural states were recorded for 2 h after saline orilocarpine administration.

pectral power

he EEG epochs were visually examined and power spectra ofrtefact-free epochs were computed using fast Fourier transformsFFT) in a software programme developed by members of theesearch group. The EEG signals were segmented in 5 s samples.
hese samples were collected from 15 min after injections to 1 h

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S. Tejada et al. / Neuroscience 165 (2010) 621–631 623

f recording attending to the behavioural states (Gottesmann,992). Mean power spectra were divided into various frequencyands, analysing delta (1–4 Hz), theta (4.1–8 Hz), alpha (8.1–12z) and beta (12.1–30 Hz) bands. Relative powers were calcu-

ated by dividing the absolute power within a given frequencyange by corresponding measures of total power.

EG coherence

he coherence is the normalized square of the correlation coeffi-ient between power spectral amplitudes of two signals. Cross-pectrum and power spectrum values are obtained via FFT. Then,oherence is computed for each frequency by the ratio betweenhe cross-spectrum of the two signals and the product of theirower spectra. The coherence in a frequency band is calculatedy averaging the EEG coherence coefficients of such a band.oherence in each band can range from 0 to 1 indicating thetrength of a simple or linear relationship between the two chan-els: a high or low coherence value means that their activities areore or less dependent on each other (Pereda et al., 2002, 2005).he coherence between the hippocampal EEG signal and the

rontal cortex EEG signal was studied for the EEG 5-secondspochs over 1 h. EEG coherence was calculated for delta (1–4z), theta (4.1–8 Hz), alpha (8.1–12 Hz) and beta (12.1–30 Hz)

requency bands.

hase synchronization analysis

software programme developed by members of our researchroup was used to measure phase synchronization between twoignals following the method described by Stam et al. (2007).riefly, two signals are converted into phase signals through theilbert transform, and a phase difference signal is obtained. Then,y using the asymmetric distribution of the phase differences, thehase Lag Index (PLI) is obtained. This PLI analysis is a synchro-ization measure insensitive to volume conduction. The PLI valueanges between 0 and 1, a PLI value equal to zero indicates eithern absence of coupling between the two signals (e.g. volumeonduction) or coupling with a phase difference centred aroundero. A PLI value equal to one indicates effective phase synchro-ization with a phase difference different to zero. PLI was calcu-

ated for delta (1–4 Hz), theta (4.1–8 Hz), alpha (8.1–12 Hz) andeta (12.1–30 Hz) frequency bands.

adial maze trials

he effect of pilocarpine on working memory task was tested innother group of adult rats by means of the eight-arm radial maze.he effect of pilocarpine was analyzed 45 min after the i.c.v. admin-

stration (360 �g in 1 �l, n�8). Also, the effect of pilocarpine was

ig. 1. Representative EEG recording traces in cortical and hippocamassive waking. Power spectra are indicated in percentiles. For interprhe Web version of this article.

valuated after pretreatment (15 min) of rats with a single dose of the �

uscarinic receptor antagonist scopolamine (Sigma-Aldrich Che-ie®, Germany) (0.001 mg/kg i.p., n�8). As scopolamine is known

o induce impairment in memory, we previously test a set of differentoses (0.005–0.2 mg/kg) to choose the adequate dose of the an-agonist, in order to ensure a receptor block but not a compensatoryffect. It was chosen a dose of scopolamine devoid of effects onemory tasks. Thus, the effects of scopolamine alone (0.005 mg/kg

.p., n�4) and saline (n�9) were evaluated.The maze (Panlab, S.L., Barcelona, Spain) consisted of an

ctagonal central platform (32 cm diameter) with eight equallypaced radial arms (50 cm long, 12 cm wide). To test radial mazeemory task, rats were allowed to make an arm choice to obtain foodellets until all eight arms had been visited or 20 min had elapsed.nimals were previously submitted to 48 h fasting. Thus, trials were

udged complete when rats had chosen all 8 baited arms or spent 20in in the trial (time to achieve performance). The sum of non-visitedrms and re-entry into arms was scored as a working memory error.

tatistics

tatistical analysis was carried out using one-way analysis ofariance (ANOVA, SPSS® v. 12.0 for Windows®, Madrid, Spain).ost hoc Bonferroni paired comparisons were further made to

ecognize deviant groups. Results are expressed as mean�SEMnd P�0.05 was considered statistically significant. PLI was ex-ressed as mean and 95% confidence interval.

RESULTS

at behaviour

ild behavioural stress signs (chewing, exploratory move-ents, grooming) were observed after connection to the

ystem in the experimental cage. However, the stressigns disappeared after several minutes indicating adap-ation to the experimental conditions by the normal loco-otion, feeding and grooming activities. The animals were

isually observed during the time period comprised be-ween saline or pilocarpine administration and the end ofhe recording session. No signs of epileptic-like move-ents or seizures were observed after the two pilocarpinedministrations (see Fig. 1).

ippocampal and cortical EEG power spectra

ig. 1 shows an example of hippocampal (upper) andortical (down) recordings after pilocarpine treatment (360

ns of one rat 45 min after pilocarpine treatment (360 �g i.c.v.), duringf the references to color in this figure legend, the reader is referred to

pal regioetation o

g in 1 �l of saline serum i.c.v.). As it can be seen, the

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ajority frequencies were at delta (1–4 Hz) and theta4.1–8 Hz) power band.

Fig. 2 shows a grand average of hippocampal androntal cortex power spectra obtained from EEG fragments

ig. 2. Hippocampal and frontal cortex EEG power spectra grand avef saline (bold line) and pilocarpine (360 �g, grey line) during REM sle

ast Fourier transformation for 5 s periods, and the spectra were print

5 s) in rats after saline control and pilocarpine administra- c

142

ion (360 �g i.c.v.), during REM sleep, passive waking andctive waking states. Differences in REM sleep were notbserved after the pilocarpine treatment either in the hip-ocampal spectrum or in the frontal cortex. When pilo-

fragments extracted from the EEG of 3 rats after i.c.v. administrationsve waking and active waking states. Power spectra were analysed byz intervals.

rage of 8ep, passi

arpine was given, the power spectrum of the hippocam-

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ig. 3. Delta power of hippocampal and frontal cortex EEG in rats forifferent injections of pilocarpine (120 and 360 �g in 1 �l). Values aean�SEM from 7 rats. * pilocarpine 120 �g i.c.v. respect to saline; # pP�0.05, *** P�0.001, # P�0.05, ## P�0.01, ### P�0.001, † P�0.05

ration during REM sleep, passive waking and active t
aking states; while the cortical theta wave peak shifted
riods, and full hour, n�4, after i.c.v. administrations of saline and twosed as a percentage of the total power. Each value represents the360 �g i.c.v. respect to saline; † between both pilocarpine treatments.

.01 (One way ANOVA analysis).

15 min pere expresilocarpine, †† P�0

owards higher amplitude during the PW and AW states.

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Fig. 3 shows the changes of relative delta power bandn hippocampus and frontal cortex in rats during sleepSWS and REM) and waking (passive or active) statesfter saline and pilocarpine (120 and 360 �g) for four 15in periods and for the entire 1 h (for hippocampus:(14,1904)�6.991, P�0.000 for 15–30=; F(14,2336)�9.752, P�0.000 for 30 – 45=; F(14,2364)�19.998,�0.000 for 45–60=; F(14,2538)�22.193, P�0.000 for0–75=; F(14,9104)�58.39, P�0.000 for 15–75=) (for fron-al cortex: F(14,1881)�28.183, P�0.000 for 15–30=;(14,2362)�23.271, P�0.000 for 30–45=; F(14,2412)�9.671, P�0.000 for 45– 60=; F(14,2514)�21.653,�0.000 for 60–75=; F(14,9226)�106, P�0.000 for 15–5=). Differences in the delta power band did not occurither in the hippocampus or in the frontal cortex duringoth sleep states. Similarly, there were no significant dif-erences in the hippocampal delta power during passive orctive waking states, while a very significant decrease inelta power occurred in the cortical power EEG during bothassive and active waking (P�0.001) after the highestilocarpine administration (360 �g).

The results for the EEG spectral power values in theelative theta power after the administration of saline andilocarpine (120 and 360 �g) are shown in Fig. 4. Regard-

ng the hippocampus, spectral power in the theta fre-uency band after injection of saline was not different fromhat obtained after injection of pilocarpine in any of the postnjection periods considered either during sleep (SWS,EM) or during wakefulness (PW, AW) (for hippocampus:(14,1914)�3.052, P�0.000 for 15–30=; F(14,2387)�2.36, P�0.000 for 30–45=; F(14,2341)�5.987, P�0.000

or 45–60=; F(14,2526)�5.589, P�0.000 for 60–75=;(14,9181)�17.03, P�0.000 for 15–75=). However, signif-

cant differences were found for the frontal cortex whenaline was compared to both pilocarpine administrationsuring the waking states (PW and AW); these differenceslearly appeared during the second and third 15-min peri-ds studied, and tended to disappeared at the end of thetudied periods (data not shown) (for frontal cortex:(14,1914)�11.680, P�0.000 for 15–30=; F(14,2363)�2.004, P�0.000 for 30–45=; F(14,2400)�14.829, P�.000 for 45–60=; F(14,2511)�11.736, P�0.000 for 60–5=; F(14,9260)�40.91, P�0.000 for 15–75=). As men-ioned above (Fig. 2) the observed differences betweenilocarpine-treated and saline rats were more important athe theta frequency mainly the defined as cholinergic thetaomponent (Depoortere, 1987; Konopacki et al., 1988).

Changes in the relative frequency bands for alpha andeta were not found after the pilocarpine treatments (dataot shown).

EG coherence between hippocampus and frontalortex

he EEG coherence for theta band between hippocampusnd frontal cortex for 1 h after saline and pilocarpine (120nd 360 �g i.c.v.) treatments are shown in Fig. 5. Saline

nfusion and both pilocarpine treatments did not changehe coherence magnitude during SWS and REM sleep
tates. However, during the PW and AW states, the co- w
144

erence under pilocarpine was significantly greater thannder saline infusion during the post treatment periodsonsidered and the entire hour (For theta F(14,1994)�.145, P�0.000 for 15–30=; F(14,2324)�5.805, P�0.000

or 30–45=; F(14,2290)�6.452, P�0.000 for 45–60=;(14,2402)�9.632, P�0.000 for 60–75=; F(14,9059)�9.995, P�0.000 for 15–75=, 1 h).

Both pilocarpine treatments did not change the EEGoherence in delta, alpha and beta frequency bands duringWS and REM sleep or in waking states (data not shown).

hase synchronization between hippocampus androntal cortex

hase Lag Index (PLI) results between hippocampus androntal cortex in the EEG theta band are shown in Figs. 6nd 7. PLI magnitude during the four vigilance states stud-

ed in control conditions (saline group) was far from zero;ndicating an effective phase locking between hippocam-us-frontal cortex theta waves with a non-zero phase dif-erence (Fig. 6). Significant PLI differences between treat-ents were observed during PW state, when data wererouped in 15-min periods (Fig. 6; F(10,1669)�5.611,�0.000 for 15–30=; F(11,1896)�6.775, P�0.000 for 30–5=; F(11,2019)�3.036, P�0.000 for 45–60=; F(11,1967)�.136, P�0.000 for 60–75=), or after grouping all data overh (Fig. 7; F(2,4028)�10.571, P�0.000). In addition, the

igher PLI mean was obtained after the greater pilocarpineose (Fig. 7). What is more, this non-zero phase synchro-ization observed after saline remained after pilocarpinereatments in the four vigilance states studied (Fig. 6).

In delta, alpha and beta frequency bands, no differ-nces for PLI were obtained during any of the studiedigilance state of rats (data not shown).

adial maze

ilocarpine treated rats showed a decrease in the perfor-ance time (F(3,25)�14.15, P�0.000) and also in theumber of total errors (F(3,25)�7.75, P�0.001) in theight-arm radial maze. Pretreatment of rats with a dose ofhe nonsubtype selective muscarinic antagonist scopol-mine (0.005 mg/kg i.p.), which did not induce an impair-ent in memory tasks, significantly antagonized the im-roving effect of pilocarpine (360 �g in 1 �l of serumaline, i.c.v.) on the spatial memory test (8-arm radialaze), indicating that the observed effects of pilocarpineere mediated by muscarinic receptors (Fig. 8).

DISCUSSION

n the current work, the hippocampal and cortical EEGsere selected for the analysis of the pharmacological ac-

ion of pilocarpine at the theta power band, since phar-aco-EEG analysis has been shown to produce an in-

reased number of episodes with theta activity in cortexfter administration of the muscarinic agonist pilocarpine.he most consistent results in this work showed that pilo-arpine administration in rats increased the theta fre-uency band in the frontal cortex during waking states
ithout changes in the hippocampus, and also pilocarpine

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ncreased the EEG coherence analysis of the theta bandetween hippocampus and frontal cortex during waking inats. Moreover, pilocarpine caused a spatial memory im-

ig. 4. Theta power of hippocampal and frontal cortex EEG in rats forifferent injections of pilocarpine (120 and 360 �g in 1 �l). Values aean�SEM from 7 rats. * pilocarpine 120 �g i.c.v. respect to saline; #

## P�0.001 (One way ANOVA analysis).

rovement in radial maze performance. Altogether, these p

esults suggest an enhanced functional coupling betweenhe recorded areas induced by pilocarpine.

Theta oscillations are mainly generated by the hip-

riods, and full hour, n�4, after i.c.v. administrations of saline and twosed as a percentage of the total power. Each value represents the

ine 360 �g i.c.v. respect to saline. * P�0.05, *** P�0.001, # P�0.05,

15 min pere exprespilocarp

ocampal pyramidal neurons (Kahana et al., 2001; van

145

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uijtelaar and Coenen, 1984). Theta power band (4–8 Hz)s a spectral band typical of both REM sleep and wakingtates (Bouwman et al., 2005; Gambini et al., 2002; Vya-ovskiy and Tobler, 2005), and it has also been demon-trated to be related to cognitive processing and memoryperations (Gais and Born, 2004; Johnson, 2006). Theresent results show that the muscarinic agonist pilo-arpine increased the relative theta power band—mainlyhe cholinergic lower frequency compound of theta—in therontal cortex of rats during waking, and also improved theerformance in the eight arms radial maze. As previouslybserved, pilocarpine induced a rise in the duration and

otal number of episodes presenting theta rhythm in theortex of rats during wakefulness (Tejada et al., 2007a).ltogether, this could indicate a transfer signal from theippocampus to the frontal cortex, related to the postulatedffect of the cholinergic system in modulating memoryonsolidation (Gais and Born, 2004). This suggestion isased on the hypothesis that a sharp-wave burst initiated

n the hippocampus and associated with theta oscillation

ig. 5. Spectral coherence between EEG recordings from hippocampreatments (120 and 360 �g, i.c.v.). Bars represent mean�SEM of fraleep, passive waking and active waking states for 1 h. * P�0.05, ** PNOVA analysis).

rovides the mechanism by which information may be c

146

elayed back to the cortex during memory consolidationBuzsaki, 1996). A role for acetylcholine has been reporteds inhibiting the information flow from the hippocampus tohe cortex (Gais and Born, 2004; Hasselmo, 1999). Mus-arinic antagonists in the hippocampus, which produce aigh level of acetylcholine release (Nilsson et al., 1990),ave been reported to reduce hippocampal theta powernd also to induce impairment in working memory (Givensnd Olton, 1995; Missonnier et al., 2006). On the contrary,uscarinic activation has been seen to improve memorynder impairment conditions (Nilsson et al., 1990). In-reased theta power has been induced by the muscarinicgonists oxotremorine (Markowska et al., 1995) and car-achol attenuating the memory impairment produced byntagonists in rats (Givens and Olton, 1995). In fact, it haseen reported that treatments that restore the normal thetarequency of the hippocampus may optimize its ability torocess new information (Markowska et al., 1995).

Coherence analysis is a measure that reveals corre-ated signals in different networks and deduces functional

ontal cortex from 7 rats per group after saline control and pilocarpine5 s) obtained for theta frequency band during slow wave sleep, REMnd *** P�0.001 when compared to the saline control group (one way

us and frgments (

oupling between these networks; that is, the degree to

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hich two signals show amplitude and phase linear inter-ependence at specific states (Pereda et al., 2002, 2005).ighly coherent oscillations between two structures canccur because they are functionally connected or becausehey share a common input (Kocsis et al., 1999; Moore etl., 2005). In the current work, when coherence analysisompared the spectral power delta band of the fragmentsbtained from the hippocampus and frontal cortex, nohanges were observed after pilocarpine administration inny of the studied vigilance states, indicating a similaregree of connectivity between hippocampal and corticalignals at this frequency band. On the contrary, coherence

ig. 6. Phase Lag Index (PLI) between hippocampal and frontal cor-ex EEG theta waves from 7 rats per group after saline control andilocarpine treatments (120 and 360 �g, i.c.v.). Lines represent meanith a 95% confidence interval of fragments (5 s) obtained for thetaand during slow wave sleep, REM sleep, passive waking and active

† ††
aking states for 1 h. P�0.05 and P�0.01 when compared to bothilocarpine groups (one way ANOVA analysis).
ty

nalysis showed that pilocarpine treatment increased theinear correlation between hippocampal and cortical EEG

ig. 7. Phase Lag Index (PLI) between hippocampus and frontal cortexEG theta band from 7 rats per group after saline control and pilocarpine

reatments (120 and 360 �g, i.c.v.). Lines represent mean with a 95%onfidence interval of fragments (5 s) obtained for theta frequency banduring PW state after all data were grouped along 1 h. * P�0.05 whenompared to saline control group, and ††† P�0.001 when comparing bothilocarpine treatments (one way ANOVA analysis).

ig. 8. Effects of pilocarpine (360 �g in 1 �l) in rats, previouslyubmitted to 48 h fasting, on the time and the numbers of errorserformed to complete the eight-arm radial maze. Rats disposed of aaximal time of 20 min to complete the task. Bars represent the time

equired to perform the test (seconds) and number of errors asean�SEM in the different groups: saline (n�9), pilocarpine injectionlone (n�8, 45 min), pilocarpine after pre-treatment (15 min before)ith a single dose of the scopolamine (0.005 mg/kg i.p., n�8), andcopolamine injection alone (0.005 mg/kg i.p., n�4). ** P�0.01 and** P�0.001 when compared to saline control group; ### P�0.001hen compared to scopolamine alone; ††† P�0.001 when compared

o pilocarpine in scopolamine pretreated rats (one way ANOVA anal-sis followed by Bonferroni’s test was used for statistical evaluation).

147

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heta waves during waking states of rats. A rise in the coher-nce values can be interpreted as evidence of coactive neu-onal populations, functional coupling of the regions involvedn the tasks, and mutual information exchange (Andres and

erloff, 1999; Andrew and Pfurtscheller, 1996). In fact,heta oscillation has been reported to be related to interac-ions between the hippocampus and cortex (Vinogradova,995). Thus, in the present study, the linear correlation at theheta band—mainly the cholinergic lower frequency ofheta—between hippocampus and frontal cortex during theaking states after pilocarpine suggests more efficientoupling within these regions, induced by the muscarinicgonist.

Phase synchronization analysis allows us to look forinear and non-linear phase synchronization between hip-ocampus and cortex. Phase synchronization resultshowed that in basal conditions (saline group) an effectiveon-zero phase locking existed between theta waves ofippocampus and cortex with a phase difference differentrom zero. During active waking, phase synchronization isreater than in the other states. The non-zero phase lock-

ng status remained after pilocarpine treatment in the fourtates studied. In addition, during passive waking, syn-hronization appeared to increase after pilocarpine admin-stration. Therefore, these results confirm the existence ofynchronization in the theta band between hippocampusnd cortex. As coherence measures the linear correlationetween two signals whereas PLI is a non-linear measuref phase synchronization between them, the fact thathase synchronization remains non-zero after pilocarpineakes the increasing coherence after pilocarpine an ef-

ective result with nothing to do with volume conduction.Regarding the sleep states, a lack of effects of pilo-

arpine on delta and theta rhythms has been previouslybserved in hippocampus and cerebral cortex (Tejada etl., 2007a). Moreover, the EEG coherence analysis in theheta frequency band between the studied cerebral regionsas not modified during sleep states after pilocarpine

reatment. Neither did the synchronization index PLI—ower during SWS than REM—change after pilocarpinereatment. Therefore in these states a low interdepen-ence between hippocampus and cortex in the theta bandould exist and it seems not to be affected by pilocarpine.

CONCLUSION

n conclusion, the rise in cholinergic theta power and thereater degree of similarity between the hippocampus and

he frontal cortex, after administration of the muscarinicgonist pilocarpine, revealed a higher degree of synchro-ization of the theta rhythm from both cerebral regionsuring the passive waking and active waking states. TheEG signals coherence and synchronizations analysis be-

ween brain regions is a useful tool to relate the cerebralctivity and functional connectivity with physiological andehavioural processes.

cknowledgments—This work was supported by grant BFI2002-4583-C02-029, SAF2007-66878-CO2-02 (MEC, Madrid, Spain)
nd 36AA/04 of Conselleria de Salut i Consum (Govern de les Illes
148

alears, Spain). Silvia Tejada was supported by a FPI grantGovern de les Illes Balears, Spain).

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(Accepted 14 October 2009)(Available online 22 October 2009)

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Manuscript V

Antioxidant response analysis in the brain after pilocarpine treatments

Tejada, S.; Roca, C.; Sureda, A.; Rial, R.V.; Gamundí, A. and Esteban, S. (2006) Brain

Research Bulletin. 69:587-592.

151

Brain Research Bulletin 69 (2006) 587–592

Antioxidant response analysis in the brain after pilocarpine treatments

S. Tejada a, C. Roca a, A. Sureda b, R.V. Rial a, A. Gamundı a, S. Esteban a,∗a Laboratori de Neurofisiologia, Departament de Biologia Fonamental i Ciencies de la Salut, Universitat de les Illes Balears, Palma de Mallorca, Spain

b Laboratori de Ciencies de l’Activitat Fısica, Departament de Biologia Fonamental i Ciencies de la Salut,Universitat de les Illes Balears, Palma de Mallorca, Spain

Received 11 October 2005; received in revised form 25 November 2005; accepted 4 March 2006Available online 29 March 2006

Abstract

Cholinergic and gabaergic systems play an important role generating electroencephalographic activity and regulating vigilance states. Pilocarpineis a cholinergic agonist commonly used to induce seizures and an epilepticus-like state in rodents. A relationship between status epilepticus andreactive oxygen species has been also suggested which could result in seizure-induced neurodegeneration. The aim of this study was to evaluate theexistence of oxidative damage as well as the antioxidant enzyme response in cortex and hippocampus after the administration of an intraperitoneal(350 mg/kg) and an intracerebroventricular (360 �g, 1 �l) pilocarpine injection in rats. The GABA agonist muscimol (1 mg/kg, i.p.), with describednmiIes©

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europrotective properties, was used as a negative control. Only systemic pilocarpine induced oxidative damage. Malondialdehyde levels, as aarker of lipid peroxidation (LP), increased in both regions (55–56%). Catalase (52–80%) and superoxide dismutase (53–60%) activities also rose

n both regions but glutathione peroxidase activity only increased in cortex (45%). Glutathione reductase and caspase-3 activity did not change.n conclusion, systemic pilocarpine produced oxidative brain damage, whereas local pilocarpine brain injection had no effects. Moreover, thenzymatic determinations performed in this study are a good tool to study brain injury in pharmacological manipulations such as the ones used inhort recording EEG studies.

2006 Elsevier Inc. All rights reserved.

eywords: Antioxidant enzymes; Brain damage; Lipid peroxidation; Pilocarpine; Reactive oxygen species; Status epilepticus

. Introduction

Cells continuously produce free radicals and reactive oxygenpecies (ROS) as part of their metabolic processes [25]. Freeadicals are very reactive chemical species and can readily leado uncontrolled reactions, which may result in oxidative damageo DNA, proteins and lipids [9,44]. ROS can affect ion transportroteins and channels, via protein oxidation or via peroxidationf membrane phospholipids, resulting in deleterious effects ononic homeostasis and neuronal transmission [44]. Free radicalsre neutralized by an elaborate antioxidant defence system con-isting of enzymes such as catalase (CAT), superoxide dismutaseSOD), glutathione peroxidase (GP) and glutathione reductaseGR), and numerous non-enzymatic antioxidants such as glu-athione (GSH). O2

•− is a free radical which is metabolised

∗ Corresponding author at: Laboratori de Neurofisiologia, Universitat de leslles Balears, Crta. Valldemossa, km 7,5, E-07122 - Palma de Mallorca, Balears,pain. Tel.: +34 971173145; fax: +34 971173184.

E-mail address: [email protected] (S. Esteban).

to hydrogen peroxide by SOD, then the hydrogen peroxide isdecomposed to water and molecular oxygen by GP and CATpreventing the generation of hydroxy radicals, the most reactivespecies derivated from oxygen [6,24], and thereby protectingthe cellular constituents from oxidative damage [40]. However,when ROS production is excessive, the intrinsic antioxidantscavenging capacity is overwhelmed resulting in the develop-ment of oxidative stress which can induce tissue injury and mayactivate apoptosis processes [48].

The brain is more vulnerable to oxidative stress damagingeffects than other tissues for several reasons. One reason lies inits high consumption of oxygen [44], which is around 20% ofthe total metabolic activity [9], although the brain only repre-sents a small percentage of the body mass. In addition, whilethe brain contains large amounts of oxidizable lipids and pro-oxidative metals, it has a comparatively low antioxidant capacity[3,15,44].

Pilocarpine is a cholinergic agonist with moderate affin-ity for M1 muscarinic receptors and higher affinity for M5ones. High dose pilocarpine treatment (over 350 mg/kg, i.p.)

361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved.oi:10.1016/j.brainresbull.2006.03.002

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dx.doi.org/10.1016/j.brainresbull.2006.03.002

588 S. Tejada et al. / Brain Research Bulletin 69 (2006) 587–592

is used to induce seizures and an epilepticus-like status (SE) inrodents [49], which can last for several hours [2,15,18,29,30].The epileptic model induced by pilocarpine is a useful ani-mal model to study the development and understanding ofthe neuropathology of human temporal lobe epilepsy becauseit reproduces similar behavioural and electroencephalographicalterations [15,49]. The relationship between SE and ROS is wellknown as the epileptiform activity causes excessive free radicalproduction of ROS, a factor believed to be involved in the mech-anism leading to cell death and neurodegeneration. In fact, freeradicals-mediated reactions may not contribute to the develop-ment of seizures during epilepsy, however they are involved inthe biochemical sequelae of events leading to seizure-inducedcell death [3,14,15].

In addition, high cholinergic activity is related to waking,rapid eye movement (REM) sleep and also to brain activation[19,32,51], showing high levels of brain activity and metabolismwhich can raise the level of oxidative stress up to the point ofit having been proposed that an important function of sleep isthe recovery from the oxidative stress produced during waking[8,10,45]. In fact, the muscarinic cholinergic agonist pilocarpinehas been reported to modify the amount of both REM and slowwave sleep [27,38]. As mentioned above, pilocarpine injectionsproduce brain damage, but low doses of this drug (1–3 mg/kg,i.p.) were found to be useful in EEG studies to induce changes insleep–wake cycle and were presumably devoid of epileptogenica

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of Laboratory Animals Care” (NIH Publication no. 85-23, revised 1996) andaccording to the guidelines of the Balearic Islands University for animal studies.

Rats were divided into five groups (n = 6). Three groups received intraperi-toneal treatment (i.p.) and two groups intracerebroventricular (i.c.v.) to assessthe drug effects directly in the brain. Systemic and intracerebroventricular treat-ment also differed in the doses, the systemic one was a higher dose than the onecorresponding to the i.c.v. treatment.

Two different saline control groups received 0.9% NaCl (1 ml/kg, i.p. or 1 �li.c.v.). The third group received an intracerebroventricular pilocarpine injection(360 �g, 1 �l i.c.v.). The fourth group received an intraperitoneal pilocarpineinjection (350 mg/kg, i.p.) in order to induce brain damage; these animals alsoreceived scopolamine hydrobromide (1 mg/kg, i.p.) 30 min before pilocarpine toavoid peripheral toxicity and death. The last group received muscimol (1 mg/kg,i.p.).

2.2. Surgical techniques

Under isoflurane anaesthesia, two groups of rats were submitted to estereo-taxic surgery under aseptic conditions in order to place a chronic cannula inthe lateral ventricle of the brain (AP −0.8, ML −2.0, DV −3.3, relative to thebregma). Intramuscular atropine (0.1 mg) was injected to avoid excessive auto-nomic activation. The cannula was fixed on the skull with acrylic cement. Atleast 1 week was allowed for recovery after surgery.

Rats were sacrificed by decapitation 2 h after drug administration. The brainswere quickly removed and the cortex and the hippocampus were separated andmaintained at −80 ◦C until analysis. At the end of the experiment, the locationof the cannula was controlled; only the results obtained from a correct placementwere used in the analysis.

2.3. Behavioral observations

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ctivity [18].For this reason, it was considered interesting to dispose of an

asy procedure to determinate oxidative tissue injury. As lipideroxidation is indicative of irreversible biological damage inhe phospholipids of the cellular membrane [16], brain oxidativetress can be quantified by measuring malondialdehyde (MDA)evels, an end-product of lipid peroxidation, and by the effec-iveness of the antioxidant enzyme response. Thus, the aim ofhis study was to evaluate the existence of oxidative damage andhe antioxidant enzyme response in cortical and hippocampusegions after administration of different pilocarpine treatmentsintraperitoneal and intracerebroventricular), in order to observeossible injury in the brain. For comparative purposes, musci-ol, a selective GABAA agonist, was included in the study due to

ts neuroprotective properties, which have been observed in sev-ral brain regions – cerebral cortex and hippocampus included31,46]. The brain regions were chosen since neuropathologyssociated with SE has been described in the cortex, which hasich acetilcholine innervation [39,49], and the hippocampus maye the principal area affected by pilocarpine-induced seizuresnd by SE; hence these regions may be important targets affectedy pilocarpine-induced seizures and by SE [15].

. Materials and methods

.1. Animals and treatments

Thirty adult male Wistar rats (250–350 g) were used. Animals were housedndividually and maintained in standard conditions under 12-h light:12-h darkL:D) schedule, with free access to food and water throughout the experimentaleriod. All efforts were made to minimise animal suffering and to reduce theumber of animals used. Experiments were performed following the “Principles

154

Behavioral variables (number of peripheral cholinergic signs, tremors, sniff-ng and clonic movements of forelimbs) were observed during the 2 h after thereatments.

.4. Lipid peroxidation assay

The cortex and the hippocampus were homogenized in Tris–HCl 10 mM (pH.5) and centrifuged at 10,000 rpm for 10 min. Supernatant was used for the anal-sis. Malondialdehyde (MDA), as a marker of lipid peroxidation, was analyzedy a colorimetric assay kit (Calbiochem, San Diego, CA, USA) following theanufacturer’s instructions. Briefly, samples or standards were placed in glass

ubes containing n-methyl-2-phenylindole (10.3 mM) in acetonitrile:methanol3:1). HCl 12N was added and the samples were incubated for 1 h at 45 ◦C.bsorbance was measured at 586 nm.

.5. Enzymatic determinations

CAT, GP, SOD and GR antioxidant enzyme activities were determined withShimadzu UV-2100 spectrophotometer at 37 ◦C. CAT activity was measuredy the spectrophotometric method of Aebi [1] based on the decomposition of

2O2. SOD activity was measured by an adaptation of the method of McCordnd Fridovich [33]. The xanthine/xanthine oxidase system was used to generatehe superoxide anion (O2

•−). This anion produced the reduction of cytochrome. The SOD of the sample removed the O2

•− and produced an inhibition of theytochrome c reduction. This cytochrome c was monitored at 550 nm. GP activityas measured using an adaptation of the spectrophotometric method of Flohe

nd Gunzler [13]. This activity was determined with H2O2 as the substrate andR and NADPH as enzymatic and non-enzymatic indicators, respectively. GR

ctivity was measured by a modification of the Goldberg and Spooner method22], which required oxidized glutathione (GSSG) as the substrate.

Caspase-3 is the central executioner caspase in the apoptotic cascade con-idered responsible for the morphological changes of apoptotic cell death [17].aspase-3 activity was measured by spectrophotometric assay using the syn-

hetic tetrapeptide DEVD-pNa (Asp-Glu-Val-Asp-nitroanilide) as the specificubstrate for this enzyme [23]. Duplicate samples or blanks were placed in a6-well plate. After addition of the substrate the plate was incubated at 37 ◦C

S. Tejada et al. / Brain Research Bulletin 69 (2006) 587–592 589

for 1 h. DEVD-dependent protease activity was assessed by detection of the freepNA cleaved from the substrates by determining the absorbance at 405 nm.

All results were corrected using the level of protein contents in the sam-ples. Proteins were measured using the Biorad Protein Assay. Unless otherwisespecified, all materials used were provided by Sigma (Spain).

2.6. Statistical analysis

Statistical analysis was carried out using SPSS® (v. 12.0 for Windows®).The statistical significance of the data was compared by one-way analysis ofvariance (ANOVA). Post hoc LSD paired comparisons were further made torecognize deviant groups. Results are expressed as mean ± S.E.M. and p < 0.05was considered statistically significant.

3. Results

3.1. Behavioral observations

The animals were visually observed during the timeperiod comprised between pilocarpine administration and sac-rifice. After the dose of pilocarpine injected intraperitoneally(350 mg/kg), continuous sniffing and some clonic movementsof forelimbs were observed, other peripheral cholinergic signswere abolished by scopolamine injection which counteract theperipheral cholinomimetic effects of pilocarpine that can leadto death. The animals that received pilocarpine i.c.v. (360 �g),saline or muscimol (1 mg/kg, i.p.) did not show any behaviorals

3

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Fig. 2. Catalase activity (mKatals/g of protein) in the supernatant of corticaland hippocampal homogenates. Bars represent mean ± S.E.M. (n = 6) in salinecontrol (S i.p.), pilocarpine 350 mg/kg i.p. (P i.p.), muscimol 1 mg/kg i.p. (M),saline control 1 �l i.c.v. (S i.c.v.) and pilocarpine 360 �g in 1 �l i.c.v. (P i.c.v.).*p < 0.05 and **p < 0.001 when compared with the corresponding saline controlgroup (one-way ANOVA analysis).

3.3. Enzymatic determinations

The increase in MDA concentration in the group treated withintraperitoneal pilocarpine (350 mg/kg, i.p.) could be related tothe activation of the antioxidant defenses of brain tissues toprotect them against oxidative damage induced after treatment.To recognize the state of antioxidant defenses, the activities ofantioxidant enzymes involved in ROS scavenging were mea-sured.

3.3.1. Catalase activityFig. 2 shows CAT activity in the cortex and the hippocam-

pus after the different treatments. CAT activity was significantlyincreased after i.p. pilocarpine treatment in the cortex whencompared with the saline control group (80 ± 10%) and it alsoincreased CAT activity in the hippocampus (52 ± 6%). In con-trast, CAT activity did not change after treatment with the i.c.v.pilocarpine injection (360 �g, 1 �l i.c.v.) or with muscimol(1 mg/ml, i.p.).

3.3.2. Superoxide dismutase activitySOD activity was significantly increased after i.p. pilocarpine

treatment in both the cortical and hippocampal regions (Fig. 3).SOD was increased in the cortex region (53 ± 5%) and in thehippocampus (60 ± 5%) when compared with the saline controlgroup. On the other hand, neither i.c.v. pilocarpine nor muscimolt

3

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igns characteristic of the epilepticus-like status or seizures.


In order to verify the presence of oxidative inbalance inducedy the pilocarpine and muscimol treatments, MDA levels inortex and hippocampus were measured (Fig. 1). Oxidativeamage was only evidenced after the i.p. pilocarpine injection350 mg/kg, i.p.) whereas the i.c.v. one (360 �g, 1 �l i.c.v.) oruscimol (1 mg/kg, i.p.) maintained the basal MDA concentra-

ion. The intraperitoneal dose of pilocarpine increased MDAevels when compared with control (55 ± 8% in cortex and6 ± 2% in hippocampus).

ig. 1. Malondialdehyde (MDA) level (nmol/mg of protein) in the supernatantf cortical and hippocampal homogenates. Bars represent mean ± S.E.M. (n = 6)n saline control (S i.p.), pilocarpine 350 mg/kg i.p. (P i.p.), muscimol 1 mg/kg.p. (M), saline control 1 �l i.c.v. (S i.c.v.) and pilocarpine 360 �g in 1 �l i.c.v.P i.c.v.). *p < 0.01 when compared with the corresponding saline control groupone-way ANOVA analysis).

reatments changed SOD activity.

.3.3. Glutathione peroxidase activityFig. 4 shows GP activity in the cortex and hippocampus after

reatments. This activity was only increased in the cortex afterhe i.p. pilocarpine injection (45 ± 5%) when compared with thealine control group, but not after i.c.v. pilocarpine or muscimol.o changes were observed in hippocampal GP activity.

.3.4. Glutathione reductase activityGlutathione reductase activity did not change saline con-

rol activities in any treatment, with no significant differ- 155


Fig. 3. Superoxide dismutase activity (pKatals/g of protein) in the supernatantof cortical and hippocampal homogenates. Bars represent mean ± S.E.M. (n = 6)in saline control (S i.p.), pilocarpine 350 mg/kg i.p. (P i.p.), muscimol 1 mg/kgi.p. (M), saline control 1 �l i.c.v. (S i.c.v.) and pilocarpine 360 �g in 1 �l i.c.v. (Pi.c.v.). *p < 0.001 when compared with the corresponding saline control group(one-way ANOVA analysis).

Fig. 4. GP activity (nKat/g of protein) in the supernatant of cortical and hip-pocampal homogenates. Bars represent mean ± S.E.M. (n = 6) in saline control(S i.p.), pilocarpine 350 mg/kg i.p. (P i.p.), muscimol 1 mg/kg i.p. (M), salinecontrol 1 �l i.c.v. (S i.c.v.) and pilocarpine 360 �g in 1 �l i.c.v. (P i.c.v.). *p < 0.01when compared with the corresponding saline control group (one-way ANOVAanalysis).

ences between groups (data not shown; one-way ANOVAF(5,42) = 0.58; p = 0.715).

3.3.5. Caspase-3 activityCaspase-3 activity, a marker of apoptosis, was measured in

the i.p. pilocarpine group, where significant oxidative damagewas found. Muscimol treatment was also analyzed for compara-tive purposes. No change in caspase-3 activity was found eitherin the cortex or hippocampus after 2 h of pilocarpine or musci-mol administration when compared with the control saline group(Fig. 5).

4. Discussion

The present work showed the pilocarpine injection(350 mg/kg i.p., 2 h) induced lipid peroxidation and increasedthe response to oxidative stress by enhanced antioxidant enzymeactivities. However, no significant changes were found eitherafter the intracerebroventricular administration of pilocarpine(360 �g, 1 �l, 2 h) or after muscimol (1 mg/kg, i.p.) treatment. In

Fig. 5. Caspase-3 activity (U/mg of tissue) in the supernatant of cortical andhippocampal homogenates. Bars represent mean ± S.E.M. (n = 6) in saline con-trol rats (S i.p.), pilocarpine 350 mg/kg i.p. (P i.p.) and muscimol 1 mg/kg i.p.(M). One-way ANOVA was used to compare experimental groups, however, nodifferences were found.

addition, the animals injected with pilocarpine intraperitoneallyshowed indicative signs of epilepticus-like state while the ani-mals that received pilocarpine i.c.v. or muscimol did not showany behavioral signs characteristic of the epilepticus-like statusor seizures.

A high dose of pilocarpine (350 mg/kg, i.p.), which is withinthe range of doses used to induce the epilepticus state, wasadministered to rats as a positive control to induce oxidativedamage. In the current study, a rise in MDA levels was observedindicating the existence of lipid injury in both cortical and hip-pocampal regions, in agreement with previous works [9,14]. Anincrease in the activities of antioxidant enzymes involved in freeradical scavenging—CAT, SOD and GP – was also observedafter pilocarpine treatment. Together, the results show that braincells try to counteract the pilocarpine induced reactive oxy-gen species (ROS) overproduction and the oxidative damage byincreasing antioxidant enzyme activities. The balance betweenantioxidant enzymes is relevant for cell functions in order tocontrol the damage to neurons by basal ROS production [15].However, the increased levels of MDA showed that the rise inantioxidant capacity was not enough to avoid oxidative damage.

GP activity only increased in the cortex, in agreement withsome studies, which used different drugs to induce a patternof repetitive limbic seizures and SE [9,21,29,31,37,47]. Thesestudies detected free radicals, lipid peroxidation or neuronalapoptosis. Experimental epilepsy induced by kainic acid hasbGscTeafct

c156

een used to induce oxidative stress, using GSH as well as theSH/GSSG ration as markers. As a result, the cerebral cortex

howed the greatest increase in protein oxidation although nohanges were found in glutathione levels in the hippocampus.his suggests that GP plays a minor role as a free radical scav-nger in the cortex [21]. GSH is necessary for GP activity as wells to maintain the GSH/GSSG ratio. However, GR is necessaryor GSH recycling from GSSG. The present results did not showhanges in GR activity, suggesting that its activity was enougho maintain glutathione in the reduced state (GSH).

ROS overproduction is involved in apoptosis processes andaspase-3 is the main protein in the cascade of caspases respon-

S. Tejada et al. / Brain Research Bulletin 69 (2006) 587–592 591

sible for the morphological changes of apoptotic cell death [17].Caspases are activated when the cell presents great damage sothe apoptotic process starts causing the degradation of chromo-somal DNA [11,17]. The present results showed no changeseither in the cortical or hippocampal caspase-3 activity whensystemic pilocarpine injection (350 mg/kg, i.p.) was comparedwith the saline control. This could be related to the unchangedGR activity observed. In fact, a reduced GSH/GSSG ratio hasbeen described to be accompanied by caspase-3 activation andapoptosis [48]. Perhaps, the time (2 h) elapsed between drugadministration and animal sacrifice in the present work was notlong enough to allow the induction of GR and/or the caspase-3activities.

In addition of the well known use of pilocarpine to induce SE,some authors used low doses of pilocarpine to induce changesin the EEG activity in several animals without visible epilepti-cus signs [18,35,52] or in salivation studies [4,29,41]. However,studies of brain injury caused by local administrations of pilo-carpine doses have not been performed. For this reason, in thepresent study, the determination of oxidative damage markersin rats injected with pilocarpine intracerebroventricularly wasincluded. Statistical analysis showed no differences betweeni.c.v. pilocarpine treatment (360 �g, 1 �l) and saline controlgroups in any case, contrasting with the results produced aftersystemic pilocarpine treatment (350 mg/kg, i.p.). In addition,the lack of brain damage observed in animals injected intrac-eobpn[ptRpwa

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lular systems. When ROS production increases, the organismresponds by increasing antioxidant enzyme activities. Takinginto account that only the treatment with pilocarpine i.p., atdoses used commonly to induce the epilepticus state, showedan increased response to oxidative stress, our results are in gen-eral agreement with those indicating that epileptiform activitycauses excessive production of ROS which may be one of thefactors leading to cell death [14,34,43].

In conclusion, high doses of pilocarpine such as those usednormally to study status epilepticus are liable to produce braindamage. In contrast, low doses of pilocarpine, which are usefulin the study of electroencephalographic signals, do not producesuch damage. In addition, the enzymatic determinations per-formed in our study are a good tool to study the brain status inpharmacological brain studies that use short recording periods.

Acknowledgements

This work was supported by grant BFI2002-04583-C02-029.Silvia Tejada was supported by a FPI grant (Govern de les IllesBalears, Spain). We also thank Marıa Alvaro Bartolome for hertechnical assistance.

References

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rebroventricularly showed the surgery per se did not origin thexidative stress. This indicates that low pilocarpine doses coulde a useful tool to study the changes in EEG patterns withoutroducing brain damage. Moreover, muscimol, a GABAA ago-ist also used as a tool in EEG studies and sleep generation28,36,50], has a neuroprotective function [31,46] thus in theresent study it was used as a negative control. In relation withhis property, its administration should not induce a rise either inOS production or in antioxidant defense activation. This pro-osal is in agreement with the results obtained in the presentork since no significant changes between muscimol treated

nimals and the control saline groups were found.Pilocarpine is a cholinergic agonist used as a model to induce

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Manuscript VI

Antioxidant response and oxidative damage in brain cortex after high dose of

pilocarpine

Tejada, S.; Sureda, A.; Roca, C.; Gamundí, A. and Esteban, S. (2007) Brain Research

Bulletin. 71:372-375.

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Brain Research Bulletin 71 (2007) 372–375

Antioxidant response and oxidative damage in brain cortex after highdose of pilocarpine

S. Tejada a, A. Sureda b, C. Roca a, A. Gamundı a, S. Esteban a,∗a Laboratori de Neurofisiologia, Departament de Biologia Fonamental i Ciencies de la Salut, Universitat de les Illes Balears, Crta. Valldemossa,

Km 7.5, E-07122 Palma de Mallorca, Balears, Spainb Laboratori de Ciencies de l’Activitat Fısica, Departament de Biologia Fonamental i Ciencies de la Salut, Universitat de les Illes Balears,

Crta. Valldemossa, Km 7.5, E-07122 Palma de Mallorca, Spain

Received 14 March 2006; received in revised form 6 June 2006; accepted 10 October 2006Available online 7 November 2006

bstract

Pilocarpine is a cholinergic agonist capable to induce seizures and an epilepticus-like state in rodents. This status epilepticus (SE) is an usefulnimal model to study the development and understanding of the neuropathology, behavioural and electroencephalographic alterations of humanemporal lobe epilepsy. It has been suggested a relationship between SE and reactive oxygen species (ROS) that can result in seizure-inducedeurodegeneration. The aim of this study was to evaluate the existence of oxidative damage and the changes in the antioxidant system in cortex afterdministration of a high pilocarpine dose. Rats were injected with pilocarpine (350 mg/kg i.p.) or with saline as control and 2 h after the animals wereacrificed. Malondialdehyde (MDA) levels, as marker of lipid peroxidation, significantly increased (64%) after pilocarpine treatment evidencingxidative damage. Antioxidant enzyme activities – catalase (CAT), glutathione peroxidase (GP) and superoxide dismutase (SOD) – significantly
ncreased in response to pilocarpine (28%, 28% and 21%, respectively). GP and Mn-SOD gene expression were induced by pilocarpine treatment.itamin E concentration in brain cortex decreased (15%) as result of pilocarpine administration. In conclusion, the high dose of pilocarpine, used
n the present study, induces oxidative damage and increases antioxidant enzyme activities and expression in brain cortex. Moreover, increasedipid peroxidation produces the consumption of Vitamin E.

2006 Elsevier Inc. All rights reserved.

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eywords: Pilocarpine; Cortex; Antioxidant response; Vitamin E; Oxidative da

. Introduction

Cholinergic system has been implicated in a variety ofehavioural functions, including learning, control of cognitionnd memory, the circadian cycles synchronization, the controlf the body temperature; and it is also involved in some elec-roencephalographic wave generation as well as in regulatinghe vigilance states (sleep-waking control) [6,25]. Muscarinicholinergic agonists, such as pilocarpine, have effects on slowave sleep and rapid eye movement induction [13]. Moreover,igh-dose of pilocarpine is used to induce a pattern of repetitive
imbic seizures and an epilepticus-like state in rodents, whichan last for several hours [28]. The epileptic model inducedy pilocarpine is an useful animal model to study the devel-
∗ Corresponding author. Tel.: +34 971173145; fax: +34 971173184.E-mail address: [email protected] (S. Esteban).

onCtcef

361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved.oi:10.1016/j.brainresbull.2006.10.005

pment and understanding of the neuropathology of temporalobe epilepsy. This status epilepticus (SE) model is interestingecause reproduces behavioural and electroencephalographiclterations similar to those of human temporal lobe epilepsy12,28].

Cells continuously produce free radicals and reactive oxygenpecies (ROS) as part of metabolic processes [14,15]. ROS haveeen shown to induce damage in all cellular macromolecules,uch as lipids, proteins and DNA [7,23]. Cells contain an elab-rate antioxidant defence system consisting of enzymes such asatalase (CAT), superoxide dismutase (SOD), glutathione per-xidase (GP) and glutathione reductase (GR), and numerouson-enzymatic antioxidants such as Vitamin E and glutathione.ellular antioxidant systems have demonstrated a great adapta-

ion to oxidative stress in order to avoid the oxidative damageaused by reactive oxygen species overproduction. Antioxidantnzymes are regulated by ROS and cytokines, along with otheractors [24,31]. On the other hand Vitamin E is the most potent

161


dx.doi.org/10.1016/j.brainresbull.2006.10.005

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ipid-soluble antioxidant present in biological membranes pre-enting lipid peroxidation [4] and it has a special role in protect-ng mitochondria, the major site of ROS generation in cells [8].xidation of mitochondrial Vitamin E is accompanied by gener-

tion of lipid peroxidation products, altered enzyme activity andlectrical conductance [29]. However, when the ROS produc-ion is excessive, the intrinsic antioxidant scavenging capacitys overwhelmed resulting on the development of oxidative stressnd cellular oxidative damage.

Studies of the EEG using high doses of pilocarpine (over50 mg/kg i.p.) showed the existence of oxidative damage under-ying the SE and rises of the antioxidant enzyme activities haveeen found [12,26]. The brain is more vulnerable to the dam-ging effects of oxidative stress than other tissues because ofeveral reasons. One is its high consumption of oxygen [23].ther reasons are that it contains large quantities of oxidizable

ipids and metals, and has comparatively less antioxidant capac-ty than other tissues [2,12,23]. The cortical region was chosenince neuropathology associated with SE has been described inhe cortex, which has rich acetylcholine innervation [21,28].

The aim of this study was to evaluate the existence of oxida-ive damage and the changes in the antioxidant enzyme activitiesnd Vitamin E concentration in brain cortex after administrationf a high dose of pilocarpine. In addition, we also studied theesponse of the antioxidant enzymes expression to the oxidativetress.

. Materials and methods

.1. Animals and treatments

Twelve adult male Wistar rats (250–350 g) were used. Animals were housedndividually and maintained in standard conditions under 12:12 (L:D) sched-le with free access to food and water during all experimental time period. Allfforts were made to minimise animal suffering and to reduce the number ofnimals used. Experiments were performed following the “Principles of Labo-atory Animals Care” (NIH Publication no 85-23, revised 1996) and accordingo the guidelines of the University of the Balearic Islands for animal studies.

Rats were divided in two groups. The first group (n = 6) was pilocarpinenjected (350 mg/kg i.p.) 30 min after an injection of scopolamine hydrobromide1 mg/kg i.p.) to reduce the number of peripheral cholinergic signs. Animalshowing behavioural variables related to status epilepticus (tremors, sniffingnd clonic movements of forelimbs) were used in the further experiments. Theontrol rats (n = 6) were injected with a physiological salt solution instead ofilocarpine.

The rats were sacrificed by decapitation 2 h after the drug administration andhe brains were quickly removed and dissected. Cortical regions were maintainedt −80 ◦C until determinations.

.2. Enzymatic determinations

Samples were homogenized in Tris–HCl 10 mM (pH 7.5) and centrifuged at0,000 rpm for 10 min. Supernatant was used to the analyses. The CAT, GP andOD activities were determined with a Shimadzu UV-2100 spectrophotometert 37 ◦C. CAT activity was measured by the spectrophotometric method of Aebi1] based on the decomposition of H2O2. GP activity was measured using andaptation of the spectrophotometric method of Flohe and Gunzler [9]. This
ctivity was determined with H2O2 as substrate and GR and NADPH as enzymend non-enzymatic indicators, respectively. SOD activity was measured by andaptation of the method of McCord and Fridovich [18]. The xanthine/xanthinexidase system was used to generate the superoxide anion (O2
•−). This anionroduced the reduction of cytochrome C. The SOD of the sample removed the

Acrn

162

ulletin 71 (2007) 372–375 373

uperoxide anion and produced an inhibition of the cytochrome C reduction.his cytochrome C was monitored at 550 nm. All results were corrected using

he level of proteins content in the samples. Proteins were measured using theiorad Protein Assay.


Malondialdehyde (MDA), as a marker of lipid peroxidation, was analyzedy a colorimetric assay kit (Calbiochem, San Diego, CA, USA) following theanufacturer’s instructions.

.4. Vitamin E determination

Vitamin E (�-tocopherol) was extracted from cortex using n-hexane aftereproteinization with ethanol [3]. Vitamin E concentration was determinedy HPLC in the n-hexane extract of plasma and cell lysates after drying inN2 current and dissolving in ethanol containing BHT 0.02%. The mobile

hase consisted of 550:370:80 acetonitrile:tetrahydrofuran:H2O. The HPLCas a Shimadzu with a diode array detector and a Nova Pak column (C18,.9 mm × 150 mm). Vitamin E was determined at 290 nm and quantified com-aring with a standard of known concentration.

.5. RNA extraction and relative quantitative RT-PCR assay

Antioxidant enzymes mRNA expression was determined by real time RT-CR using rat 18S rRNA as reference. For this purpose, total RNA was isolatedrom the brain cortex by phenol–chloroform extraction. RNA (1 �g) from eachample was reverse transcribed using 50 U of Expand Reverse TranscriptaseRoche Diagnostics, Germany) and 20 pmol oligo (dT) for 60 min at 37 ◦C.he resulting cDNA was amplified. Quantitative PCR was performed using

he LightCycler instrument (Roche Diagnostics, Germany) with DNA-masterYBR Green I kit. Primers used are listed in Table 1. The PCR conditionsere as follows: antioxidant enzymes, 95 ◦C for 10 min, followed by 40 cyclesf amplification at 95 ◦C for 5 s, 60 ◦C for 8 s and 72 ◦C for 12 s, and for8S rRNA 40 cycles at 95 ◦C for 8 s, 62 ◦C for 5 s and 72 ◦C for 12 s. Theelative quantification was performed by standard calculations considering(−��Ct). Antioxidant enzyme levels were normalized to the invariant con-rol 18S rRNA. mRNA levels of the saline group were arbitrarily referredo as 1.

.6. Statistical analysis

Statistical analysis was carried out using SPSS® (v.11.0 for Windows®).esults were expressed as means ± S.E.M. t-Student for unpaired data was used

n all parameters measured, in order to determine the significance of changesnduced by the pilocarpine treatment.

. Results

The presence of oxidative damage induced by the pilocarpinereatment was evidenced by measuring the levels of malondi-ldehyde in cortex 2 h after the treatment respect to saline group.ilocarpine increased the MDA levels (64%) corroborating pre-ious data [26].

This increase in MDA concentration after pilocarpine treat-ent could be related to the activation of antioxidant defences

n brain tissues to protect themselves against oxidative damagenduced after the treatment. Therefore, we measured the activi-ies of antioxidant enzymes involved in free radical scavenging.
ll enzymatic activities measured – CAT, GP and SOD – signifi-
antly increased in response to pilocarpine (28%, 28% and 21%,espectively). These results also corroborated previous determi-ations reported in Tejada et al. [26].


Table 1Primers used to perform PCR

Genes Forward primer Reverse primer

Catalase ATG AAG CAG TGG AAG GAG CA TCA AAG TGT GCC ATC TCG TCGP GGT TCG AGC CCA ATT TTA CA CAT TCC GCA GGA AGG TAA AGMn-SOD ACC GAG GAG AAG TAC CAC GA18s-rRNA GAG GTG AAA TTC TTG GAC CG

Table 2mRNA antioxidant enzyme levels

Fold induction t-Student

Catalase 1.05 ± 0.08GP 1.47 ± 0.15 **Mn-SOD 1.33 ± 0.12 *

t-Student for unpaired data. Asterisk (*) indicates significant differences betweengroups at p < 0.01. Double asterisk (**) indicates significant differences betweengroups at p < 0.05. mRNA levels of the saline group were arbitrarily referred to1.

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ig. 1. Vitamin E concentration (pmol/mg protein) in brain cortex in saline andilocarpine-treated groups. t-Student for unpaired data. Asterisk (*) indicatesignificant differences between groups at p < 0.05.

The antioxidant enzyme gene expression of CAT, GP andOD was determined after pilocarpine treatment (Table 2).P expression significantly increased in the pilocarpineroup (1.47 ± 0.15, p < 0.05), as well as Mn-SOD expression1.33 ± 0.12, p < 0.01). CAT expression was unchanged respecto saline group.

Moreover, the changes in the levels of Vitamin E in brainortex after pilocarpine treatment were determined (Fig. 1). Theitamin E concentration 2 h after the treatment significantlyecreased in pilocarpine-treated group (15%) when comparedith saline.

. Discussion

The cholinergic agonist pilocarpine produces a prolonged sta-us epilepticus that is the cause of neuronal death and excitotoxic
amage [11,12]. Several studies have showed that excitotoxictimulation induces a neuronal lesion in response to an excessroduction of ROS [7,16] being H2O2 one of the main ROSeading to oxidative stress [17]. The cytotoxic effect of H2O2 is
esp

TAG GGC TCA GGT TTG TCC AGG CGA ACC TCC GAC TTT CGT TCT

hought to be caused by hydroxyl radicals, causing damage toembrane lipids [12].We have administered a high dose of pilocarpine (350 mg/kg

.p.), which is within the range of doses used in epileptic studiesn rats to induce oxidative damage. In agreement with otherilocarpine works [7,10], we have observed a rise in MDA levelss indicative of the existence of lipid injury in brain cortex asreviously reported [26].

The increased oxidative stress induced by pilocarpine treat-ent could be reflected in a direct activation of some antioxidant

nzymes. The antioxidant enzymes CAT and GP are the mostmportant enzymes detoxifying H2O2, whereas SOD acts detox-fying superoxide anion and generating H2O2 which must beegraded to prevent oxidative damage. We have observed a sig-ificant increase in the CAT, SOD and GP activities 2 h afterhe high dose pilocarpine treatment. Cells try to counteractilocarpine ROS overproduction and the oxidative damage byncreasing the antioxidant enzyme activities. Moreover, the ele-ation in free radical formation could also induce long-termompensatory mechanism, activating the antioxidant enzymeene expression. However, this increase was not enough to avoidhe pilocarpine-induced oxidative damage. In spite of increasedntioxidant enzyme activities after pilocarpine treatment, thexcess in ROS production can induce brain membrane peroxi-ation, and may activate apoptosis processes [27].

CAT activity increased after pilocarpine treatment, but itsRNA gene expression remained unchanged. It has been evi-

enced that CAT activity could be modified during oxidativetress, possibly by activation through phosphorylation [32]. CATodification could be the responsible of the increased activity

bserved 2 h after pilocarpine administration. GP is importantot only detoxifying H2O2 but rather also participates in theetoxification of lipid hydroperoxides using glutathione. Theigh GP activity and expression after pilocarpine treatment couldndicate a possible induction of GP to repair lipid hydroperox-des in cortex membranes.

The Mn-SOD is the mitochondrial isoform of the enzymend it is important in order to maintain mitochondrial home-stasis. Due to a high content of polyunsaturated phospholipids,he mitochondria is especially sensitive to lipid peroxidation andy its own function [5,30]. The increased expression of Mn-SODfter pilocarpine treatment observed in the present work coulde important in order to avoid mitochondrial dysfunction andnergy failure.

Brain is vulnerable to oxidative damage due to its high lev-ls of polyunsaturated fatty acids in the membranes which areusceptible to suffer from lipid peroxidation. Vitamin E acts byrotecting polyunsaturated fatty acids in biological membranes

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gainst lipid peroxidation, in fact it has been demonstrated tocavenge ROS and decrease the formation of peroxides [19]. Theevels of Vitamin E decreased in response to pilocarpine-inducedxidative stress, suggesting that Vitamin E present in the brainortex is consumed to prevent oxidative damage. A decrease ofitamin E brain concentration in rats subjected to hyperoxia or

reated with convulsants as an oxidative stress inductors has beenvidenced [20,22]. Vitamin E is essential for maintaining func-ional integrity of mitochondria. The oxidant-induced oxidativeamage after pilocarpine treatment could be associated with lossf Vitamin E in mitochondria.

In conclusion, high doses of pilocarpine used normally totudy status epilepticus produced lipid damage in the brain. Thentioxidant system responded by increasing the activities andhe expression of antioxidant enzymes which are not enough tovoid the pilocarpine-induced oxidative damage. Vitamin E isonsumed in response to increased lipid peroxidation in brainembranes.

cknowledgements

This work was supported by grant BFI2002-04583-C02-029.ilvia Tejada was supported by a FPI grant (Govern de les Illesalears, Spain).

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M.G. Naffah-Mazzacoratti, Superoxide dismutase, glutathione peroxidaseactivities and the hydroperoxide concentration are modified in the hip-pocampus of epileptic rats, Epilepsy Res. 46 (2001) 121–128.

[3] G. Brubacher, W. Muller-Mulot, D.A.T. Southgate, Methods for the Deter-mination of Vitamins in Foods, Elsevier Applied Publishers Ltd., NewYork, 1986.

[4] G.W. Burton, M.G. Traber, Vitamin E: antioxidant activity, biokinetics, andbioavailability, Annu. Rev. Nutr. 10 (1990) 357–382.

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[8] J.G. de la Asuncion, M.L. Del Olmo, L.G. Gomez-Cambronero, J. Sastre,F.V. Pallardo, J. Vina, AZT induces oxidative damage to cardiac mito-chondria: protective effect of Vitamins C and E, Life Sci. 76 (2004)47–56.

[9] L. Flohe, W.A. Gunzler, Assays of glutathione peroxidase, Methods Enzy-mol. 105 (1984) 114–121.

10] M.V. Frantseva, J.L. Perez Velazquez, G. Tsoraklidis, A.J. Mendonca, Y.Adamchik, L.R. Mills, P.L. Carlen, M.W. Burnham, Oxidative stress is
involved in seizure-induced neurodegeneration in the kindling model ofepilepsy, Neuroscience 97 (2000) 431–435.
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12] R.M. Freitas, V.S. Nascimento, S.M. Vasconcelos, F.C. Sousa, G.S. Viana,M.M. Fonteles, Catalase activity in cerebellum, hippocampus, frontal cor-tex and striatum after status epilepticus induced by pilocarpine in Wistarrats, Neurosci. Lett. 365 (2004) 102–105.

13] A. Gamundı, M.A. Comas, S. Tejada, M.C. Nicolau, S. Esteban, R.V. Rial,A.M.L. Coenen, Paradoxical dose-dependent of pilocarpine on sleep inrats, sleep-wake, Res. Netherlands 14 (2003) 37–40.

14] M.J. Jackson, in: O. Hanninen, L. Packer, C.K. Sen (Eds.), Handbook ofOxidants and Antioxidants in Exercise, Elsevier Scientific Publishing Com-pany, Amsterdam, 2000, pp. 57–68.

15] R.R. Jenkins, Free radical chemistry. Relationship to exercise, Sports Med.5 (1988) 156–170.

16] L.M. Katz, A.S. Young, J.E. Frank, Y. Wang, K. Park, Regulated hypother-mia reduces brain oxidative stress after hypoxic-ischemia, Brain Res. 1017(2004) 85–91.

17] A. Lueken, U. Juhl-Strauss, G. Krieger, I. Witte, Synergistic DNA damageby oxidative stress (induced by H2O2) and nongenotoxic environmentalchemicals in human fibroblasts, Toxicol. Lett. 147 (2004) 35–43.

18] J.M. McCord, I. Fridovich, Superoxide dismutase. An enzymic functionfor erythrocuprein (hemocuprein), J. Biol. Chem. 244 (1969) 6049–6055.

19] A. Mori, I. Yokoi, Y. Noda, L.J. Willmore, Natural antioxidants may preventposttraumatic epilepsy: a proposal based on experimental animal studies,Acta Med. Okayama 58 (2004) 111–118.

20] K. Onodera, N.O. Omoi, K. Fukui, T. Hayasaka, T. Shinkai, S. Suzuki, K.Abe, S. Urano, Oxidative damage of rat cerebral cortex and hippocampus,and changes in antioxidative defense systems caused by hyperoxia, FreeRadic. Res. 37 (2003) 367–372.

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28] L. Turski, C. Ikonomidou, W.A. Turski, Z.A. Bortolotto, E.A. Caval-heiro, Review: cholinergic mechanisms and epileptogenesis. The seizuresinduced by pilocarpine: a novel experimental model of intractable epilepsy,Synapse 3 (1989) 154–171.

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31] C.W. White, P. Ghezzi, S. McMahon, C.A. Dinarello, J.E. Repine,Cytokines increase rat lung antioxidant enzymes during exposure to hyper-oxia, J. Appl. Physiol. 66 (1989) 1003–1007.

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5. DISCUSIÓN GENERAL

165

DISCUSIÓN

5. DISCUSIÓN GENERAL

El sueño es un estado fisiológico ampliamente estudiado debido a que el hombre

pasa una parte importante de su vida durmiendo (aproximadamente, un tercio de la

misma). Además, las alteraciones del sueño son un tema que preocupa al mundo

occidental, ya que más del 25% de su población mayor de 30 años sufre de estos

trastornos, que conllevan a errores humanos durante el periodo de vigilia (accidentes en

el trabajo, en la conducción, etc.) debido al cansancio (Reinoso Suárez, 1998). Pero el

sueño no es un proceso simple, al dormir el cerebro no está inactivo, sino que pasa por

diferentes estados de la consciencia, manteniéndonos alerta para ciertos estímulos. No

se debe entender el sueño como un cese o desaparición de las funciones que se tienen

estando despierto (estado de vigilia), sino como un estado tan activo o más como lo es

la propia vigilia, sólo que con una actividad cerebral, endocrina, metabólica,... distinta.

Algunas de las funciones, tanto cerebrales como corporales, se ven ralentizadas o

disminuidas, pero no todas; por ejemplo, la respiración, la frecuencia cardíaca o la

temperatura suelen ser más bajas, sin embargo muchas hormonas de nuestro organismo

alcanzan durante el sueño su pico de máxima secreción (melatonina, cortisol, hormona

del crecimiento, hormonas sexuales, etc.).

Pero los mecanismos del sueño son de una gran complejidad y, por ello, hay

numerosos estudios que intentan profundizar en el conocimiento del sueño y conocer

cuál es su función (Siegel, 2005). Para ello, se realizan los estudios polisomnográficos,

que son necesarios para lograr una completa compresión del sueño. Adicionalmente, en

la experimentación con animales se usan técnicas de disociación farmacológica con el

fin de modificar los patrones comunes del ciclo sueño-vigilia y poder profundizar en el

entendimiento de los mecanismos nerviosos y endocrinos que lo regulan.

Sistema colinérgico

El sistema colinérgico está implicado en la inducción del sueño REM y del ritmo

theta. Numerosos estudios han confirmado que los agonistas colinérgicos son capaces

de inducir un estado con todas las características y patrones fisiológicos de actividad

típicas de la vigilia y del sueño paradójico o REM como serían la atonía o movimientos

rápidos de los ojos (Baghdoyan et al., 1993; Xi et al., 2004).

167

DISCUSIÓN

En el presente trabajo se ha utilizado la pilocarpina como agonista muscarínico

colinérgico. En ratas, en condiciones basales o controles la actividad theta hipocampal

fue más frecuente que la cortical. Por ello, los resultados obtenidos son congruentes con

que el hipocampo es el principal generador del ritmo theta (Cantero et al., 2003;

Pedemonte, 2002), ya que, además, la actividad theta cortical aparece conjuntamente

con la hipocampal y nunca sola.

La pilocarpina (120 y 360 μg en 1 μl de suero salino i.c.v.) administrada en ratas

incrementó la duración total de los episodios con ritmo theta a lo largo de las dos horas

de registro. El incremento se debió tanto a un mayor número de episodios presentando

ritmo theta así como en la duración promedia de cada uno de esos episodios con ritmo

theta (Tejada et al., 2007a). Se ha descrito que la actividad theta hipocampal aparece en

los estados REM y de vigilia y que en tales estados el ritmo theta se ve incrementado

tras la administración de agonistas colinérgicos del subtipo M2 (Bland et al., 2005). Sin

embargo, en el presente trabajo con ratas no se produjeron cambios durante los

episodios de sueño, ni para el sueño de onda lenta ni para el sueño REM, ni hubo

cambios en la duración de los episodios ni en el número de veces que aparecían. Estas

diferencias podrían estar relacionadas con el perfil farmacológico de la pilocarpina, que

a diferencia de otros agonistas colinérgicos presenta afinidad por los receptores M1, M2

y sobretodo por el M5 (Dong et al., 1995; Seifritz et al., 1998).

Al observar los estados de vigilia pasiva y activa, se pudieron diferenciar

diferentes episodios desde el punto de vista de la actividad electroencefalográfica:

estados de vigilia en los que no aparecía ritmo theta en ninguna de las dos regiones

estudiadas, episodios en los que aparecía ritmo theta en el registro hipocampal pero no

en el cortical, y episodios en los que aparecía ritmo theta en ambas regiones. Tras la

administración de pilocarpina, no se produjeron cambios durante la vigilia pasiva que

no presentaba ritmo theta en ninguna de las regiones estudiadas. Tampoco aparecieron

cambios en los episodios de vigilia pasiva con ritmo theta únicamente en el hipocampo;

sin embargo, sí se produjo un incremento en la duración de los episodios de vigilia

pasiva con ritmo theta en el hipocampo y la corteza. En el caso de la vigilia activa

disminuyó la duración de los episodios sin ritmo theta, los episodios de vigilia activa

con ritmo theta hipocampal no se vieron modificados, mientras que sí se produjo un

incremento en la duración de los episodios que presentaban simultáneamente ritmo theta

168

DISCUSIÓN

en el hipocampo y en la corteza. El cambio en el número de episodios de estos períodos

de vigilia es paralelo a los que se producen en la duración de los episodios.

Con respecto a las latencias, se incrementaron las de vigilia activa sin ritmo

theta, aunque la latencia de la vigilia pasiva sin ritmo theta no sufrió cambios; las

latencias de las vigilia pasiva o activa cuando aparece ritmo theta sólo en el hipocampo

no sufrieron ningún cambio; pero, de manera complementaria a la duración de los

episodios y del número de veces que aparece un estado, las latencias para las vigilias

pasiva y activa que presentaron simultáneamente ritmo theta en el hipocampo y en la

corteza disminuyeron.

En resumen, la pilocarpina en ratas incrementó la duración y el número de

episodios con ritmo theta en ambas regiones estudiadas, disminuyendo el tiempo de

aparición del primer episodio. Así, la pilocarpina a las dosis utilizadas, no estaría

directamente asociada a la inducción ni al mantenimiento del sueño REM, sino a los

estados de vigilancia pasiva y activa en los que aparece el ritmo theta.

Los efectos colinérgicos descritos en diversos trabajos sobre el sueño REM son

el resultado del uso de agonistas colinérgicos con selectividad por uno de los subtipos

de estos receptores, mayoritariamente el M2 (Bueno et al., 2000; Crouzier et al., 2005;

Velazquez-Moctezuma et al., 1989). La pilocarpina es un fármaco con moderada

afinidad por los receptores M1 y M2 y más alta por el M5 (Dong et al., 1995; Seifritz et

al., 1998). Debido al perfil farmacológico de la pilocarpina por los diferentes subtipos

de receptores, al utilizar concentraciones bajas, el fármaco se uniría con mayor afinidad

al receptor M5, y con menor afinidad al M2, con lo cual esto podría explicar la falta de

cambios en los parámetros estudiados del sueño REM. A favor de esta hipótesis está la

ausencia de manifestaciones epilépticas observadas en el presente trabajo, los cuáles son

atribuidos al receptor M1 (Bymaster et al., 2003a; Hamilton et al., 1997), por el que se

ha comentado que la pilocarpina tiene una afinidad similar a la que presenta por el

subtipo M2 e inferior a la que presenta por el M5. Hay poca información en relación

con la implicación del subtipo M5 sobre el ciclo sueño-vigilia, en parte debido a la falta

de ligandos selectivos de este receptor (Eglen, 2006; Nissen et al., 2006). Si bien no

existen hasta la fecha agonistas selectivos para el receptor M5, los niveles de m5 ARN

mensajero para dicho receptor no se ven alterados en una línea de ratas con un

incrementado sueño REM (Greco et al., 1998). De esta manera, los resultados en ratas

observados en el presente trabajo utilizando una dosis baja de pilocarpina podrían estar

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mediados por el receptor M5, mientras que podrían necesitarse dosis más altas para

activar los subtipos M1 y M2.

Los cambios observados en el presente trabajo suponen un incremento de la

duración y los episodios de vigilancia en los que el ritmo theta aparece simultáneamente

en las dos regiones cerebrales de ratas. Al no observarse cambios en el ritmo theta

hipocampal cuando éste aparece sólo, se sugiere que la pilocarpina induciría un

incremento del ritmo theta en la región cortical. En línea con estas observaciones, la

ACh es un neurotransmisor que está implicado en los procesos de aprendizaje y

consolidación de la memoria. Los niveles de ACh en el cerebro son elevados durante los

periodos de vigilia y también durante el sueño REM; por el contrario, los niveles

disminuyen durante el sueño de onda lenta (Gais y Born, 2004; Power, 2004; Sirota et

al., 2002). El hipocampo está especializado en la adquisición rápida de nueva

información transmitida a los circuitos corticales a través de la corteza entorrinal

durante los períodos de niveles colinérgicos elevados; pero a la inversa, durante el sueño

de onda lenta que viene acompañado de niveles bajos de ACh, se cree que podría

mediar la consolidación de la memoria, cuando la transferencia de información

hipocampal a la neocorteza no está suprimida. De esta manera, la ACh podría modular

la transmisión sináptica para controlar los estados de la dinámica cortical, influyendo en

el flujo de información (Yu y Dayan, 2002) y la transferencia de la misma entre el

hipocampo y la corteza. Teniendo en cuenta que la ACh suprime el flujo de información

entre el hipocampo-corteza, el efecto de la pilocarpina, probablemente actuando sobre

los receptores M5, podría inhibir la liberación de ACh, un efecto sugerido para todos los

receptores muscarínicos, aunque sólo ha sido demostrado para los subtipos M2 y M4

(Zhang et al., 2002).

Como se ha dicho, el incremento en la duración y la frecuencia en que aparecía

ritmo theta durante los períodos de vigilia podría estar asociado a la mayor afinidad de

la pilocarpina al receptor M5. Sin embargo, harían falta más estudios con agonistas y

antagonistas selectivos del receptor M5 para confirmar esta hipótesis. No obstante, la

ausencia de los mismos no permite delinear los papeles fisiológicos y farmacológicos de

todos los subtipos de receptores muscarínicos (Bymaster et al., 2003b; Zhang et al.,

2002); aunque sí existen agonistas y antagonistas selectivos para los tres primeros

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receptores, que ya han sido clonados (Bueno et al., 2000; Crouzier et al., 2005;

Velazquez-Moctezuma et al., 1989).

Por otro lado, los ligandos colinérgicos no sólo son estudiados por los efectos

que producen sobre el EEG, sino que algunos autores han demostrado que alguno de

estos fármacos tiene efectos epileptogénicos que conllevan a un daño cerebral. El

incremento de los niveles del neurotransmisor excitatorio ACh es un evento

neuroquímico que inicia la actividad epileptogénica en los circuitos neuronales

susceptibles a la misma. Así, el inicio y el mantenimiento de los ataques están

controlados por eventos gabaérgicos y colinérgicos. De hecho, la ACh participa en los

patrones típicos del estado epiléptico, al ser ésta la que regula la conductancia de

potasio implicada en la epileptogénesis (Martin et al., 2005; McDonough y Shih, 1997).

El exceso de ACh que se produce en los procesos epilépticos activa los

autorreceptores muscarínicos presinápticos provocando la inhibición de la liberación de

ACh; por esta razón, durante los períodos posteriores al inicio de la actividad

epileptiforme, los niveles extracelulares de ACh son más bajos (McDonough y Shih,

1997). Se ha visto que los antagonistas muscarínicos, activos a nivel central, son

capaces de bloquear los signos epilépticos (Martin et al., 2005; McDonough y Shih,

1997). Inicialmente, la epilepsia se ha propuesto como un desorden de sincronización y

se ha propuesto que las oscilaciones theta podrían ser una manifestación de esta

patología (Cantero et al., 2003). Pero, en realidad, el ritmo theta hipocampal se

corresponde con la resistencia a los ataques, ya que se ha observado que in vivo estos

ataques epilépticos ocurren menos o su inicio se ve inhibido durante la vigilia o el sueño

REM, estados del ciclo sueño-vigilia en los que este ritmo es especialmente abundante

(Cantero et al., 2003; Miller et al., 1994).

Se ha indicado que el daño cerebral debido a los ataques epilépticos es debido a

la generación de las especies reactivas de oxígeno (ROS) (Cavalheiro et al., 1991; Dal-

Pizzol et al., 2000). Para evidenciar que las dosis usadas en los presentes estudios no

producen estos daños, y que los efectos observados en el EEG tras la administración de

pilocarpina no son un artefacto derivado de la actividad epileptogénica del fármaco, se

han realizado estudios adicionales de valoración del estrés oxidativo en el cerebro de

rata tanto en hipocampo como en corteza. El estudio de los enzimas antioxidantes

catalasa, superóxido dismutasa y glutatión peroxidasa ha permitido evidenciar que las

concentraciones de pilocarpina utilizadas para los estudios polisomnográficos, no

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producen daño oxidativo ni cambios significativos en los enzimas antioxidantes. El

mismo resultado se observó al tratar los animales con el agente neuroprotector

muscimol.

Por otro lado, el uso de dosis altas del agonista colinérgico muscarínico

pilocarpina (350 mg/kg i.p.) se usó como control positivo, ya que, de acuerdo con

experimentos previos se ha podido evidenciar que existe daño oxidativo a dosis de

pilocarpina usadas en estudios de inducción de epilepsia experimental (Dal-Pizzol et al.,

2000; Frantseva et al., 2000; Freitas et al., 2004; Peterson et al., 2002). De esta manera,

en el presente trabajo se observó un incremento de las actividades de los enzimas

antioxidantes como respuesta al estrés oxidativo inducido por el agonista colinérgico

muscarínico pilocarpina a altas dosis (350 mg/kg i.p.; Tejada et al., 2006). El

incremento de los niveles de malondialdehído, un marcador de daño oxidativo, indicó la

existencia de daño lipídico en ambas regiones estudiadas, el hipocampo y la corteza.

Las actividades de los enzimas antioxidantes –catalasa, superóxido dismutasa y

glutatión peroxidasa- se midieron dos horas después de la administración de la

pilocarpina, observándose un incremento de sus actividades (Tejada et al., 2006).

La incrementada formación de radicales libres indujo, a largo plazo, mecanismos

compensatorios activando la expresión de los genes para los enzimas antioxidantes.

Tras la administración de la pilocarpina (350 mg/kg i.p.), la expresión de la catalasa se

mantuvo en los niveles basales, pero los niveles de mRNA de la glutatión peroxidasa y

la isoforma Mn-SOD se vieron incrementados (Tejada et al., 2006). Los lípidos son

susceptibles al estrés oxidativo, de tal manera que el incremento en la expresión génica

de la glutatión peroxidasa tras el tratamiento con pilocarpina indicaría una posible

inducción del enzima para reparar los hidroperóxidos lipídicos en las membranas. Por

otro lado, el incremento en la expresión de la Mn-SOD se podría interpretar como un

intento de contrarrestar el daño oxidativo sobre los fosfolípidos de la mitocondria para

evitar la disfunción mitocondrial y un fallo en los mecanismos de producción de

energía; ya que esta isoforma es la que predomina en las mitocondrias. Sin embargo,

este incremento no fue suficiente para evitar el daño oxidativo que produjo la

pilocarpina.

La vitamina E actúa protegiendo los ácidos grasos poliinsaturados en la

membrana contra la peroxidación lipídica. En el presente trabajo, los niveles de

vitamina E disminuyen en respuesta al estrés oxidativo inducido por la pilocarpina,

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sugiriendo que la vitamina E se consume para prevenir el daño oxidativo (Tejada et al.,

2007b).

Se ha descrito que el receptor muscarínico M1 media los ataques epilépticos

inducidos por la pilocarpina, al observarse que ratones deficientes en este receptor eran

resistentes a los efectos convulsivos a altas dosis de pilocarpina (Bymaster et al.,

2003a). Sin embargo, experimentos similares con los otros subtipos de receptores

muscarínicos para los que la pilocarpina presenta afinidad, no han sido realizados

(Bymaster et al., 2003a; Hamilton et al., 1997). La pilocarpina a altas concentraciones

se une a todos los receptores por los cuales presenta afinidad, entre ellos, el M1; pero a

menores concentraciones se une con mayor afinidad al M5, con lo cual, ésta podría ser

la explicación por lo que a bajas concentraciones de pilocarpina no se observa daño

oxidativo a nivel cerebral.

Las tórtolas y, en general las aves, son especies monofásicas y diurnas,

características que son similares en humanos. Y en el caso de la tórtola, los cambios

observados tras la administración del agonista muscarínico pilocarpina fueron similares

a los observados en ratas. Se produjo un incremento de la actividad del ritmo theta, sin

que la duración o episodios del sueño REM incrementaran, de hecho, disminuyó la

duración de dichos episodios (Tejada et al., manuscript II). Sin embargo, como se ha

comentado anteriormente, este ritmo no sólo se asocia a los estados de sueño REM sino

también a los estados de vigilia (Coenen, 1975; Pedemonte et al., 2001; Xi et al., 2004;

Shin et al., 2005); de esta manera, el incremento del ritmo theta observado en tórtola en

el presente trabajo, se asociaría al incremento de la duración y número de episodios de

los estados de vigilia pasiva bajo los efectos del agente colinérgico. Además, la menor

cantidad de actividad locomotora espontánea observada en tórtola en la presente tesis

doctoral bajo la influencia de la pilocarpina, se encuentra en clara relación con la

disminución de los estados de vigilia activa y con el incremento del tiempo que estos

animales tuvieron los ojos cerrados (Tejada et al., manuscript II).

Estudios de inmunohistoquímica en el área preóptica ventrolateral de aves han

indicado la presencia de fibras colinérgicas y colinorreceptores muscarínicos (Dietl et

al., 1988) y nicotínicos (Whiting y Lindstrom, 1986). Sin embargo, el papel del sistema

colinérgico en el control del ciclo sueño-vigilia no se ha determinado en aves. De hecho,

estudios con agonistas muscarínicos en las aves son escasos. Se ha descrito que la

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administración de pilocarpina en aves induce cambios en la postura, como la flexión de

las alas sobre el cuerpo acompañada de la caída de la cola (Marley y Seller, 1974). Estas

posturas se presentan durante la vigilia pasiva, y claramente están de acuerdo con los

resultados obtenidos en el presente trabajo con tórtolas.

En palomas se ha descrito que la administración del agonista muscarínico

arecolina incrementó la duración de la vigilia y disminuyó la duración del sueño total

(Komarova et al., 2008), de acuerdo con los presentes resultados en tórtolas después del

tratamiento con pilocarpina. Así, en las tórtolas, las diferencias encontradas durante la

duración y número de episodios de vigilia pasiva aumentó tras la administración de

pilocarpina, aunque la duración de los episodios de vigilia activa disminuyó; pero la

vigilia pasiva incrementó en gran medida, de tal manera que al juntarla con la

disminución de la vigilia activa, resultó en un aumento de la vigilia total (Tejada et al.,

manuscript II).

En aves se han caracterizado hasta el momento cuatro subtipos de receptores

muscarínicos, denominados M2-M5 de acuerdo con la homología de secuencia que

presentan con los mamíferos (Tietje y Nathanson, 1991; Gadbut y Galper, 1994; Tietje

et al., 1990; Creason et al., 1995; Fischer, 1998). En cerebro y ojo de aves, la arecolina

tiene afinidad por todos los receptores muscarínicos, mientras que la pilocarpina en los

mamíferos tiene afinidad por los M1, M2 aunque mayor para M5. Las pequeñas

diferencias observadas con respecto a los estados de vigilancia por dichos agonistas

podrían responder al perfil farmacológico de ambos.

Ya se ha comentado en varias ocasiones que la actividad theta aparece durante

los estados de vigilia y sueño REM. En los resultados obtenidos en ratas en la presente

tesis doctoral, se ha observado que la pilocarpina indujo un incremento de la duración y

el número de episodios que presentaban ritmo theta durante la vigilia (Tejada et al.,

2007a, 2010); de igual manera, el incremento del ritmo theta relacionado con los

estados de vigilia pasiva también se observó en las tórtolas. También se ha indicado

anteriormente que el sistema colinérgico y la actividad theta se relacionan con diferentes

aspectos del aprendizaje y la consolidación de la memoria (Buzsáki, 1996; Muir y

Bilkey, 1998; Hasselmo, 1999; Klimesch et al., 2001; Gais y Born, 2004; Johnson,

2006); los resultados presentados en ratas tratadas con pilocarpina así parecen indicarlo

al inducir mayor sincronización de la actividad theta entre el hipocampo y la corteza

frontal y, por lo tanto, sugiriendo un mayor flujo de información entre estas estructuras

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cerebrales. De la misma manera, el incremento del ritmo theta observado en tórtolas

(Tejada et al., manuscript II) podría también relacionarse con procesos de aprendizaje y

memoria. De hecho, en paloma (Teal y Evans, 1982; Savage et al., 1994; Kohler et al.,

1995) y monos (Pontecorvo y Evans, 1985) se ha descrito que el antagonista colinérgico

escopolamina disminuye de manera dependiente de la dosis la realización de tareas de

memoria, mientras que el agonista colinérgico aniracetam induce una mejora en la

precisión para realizar la prueba. Estas evidencias indican que el sistema colinérgico es

crítico para la función normal de la memoria tanto en aves como en mamíferos.

Sistema serotonérgico

La serotonina (5-HT) es un neurotransmisor y neuromodulador que es capaz de

influir sobre una gran variedad de respuestas fisiológicas, dependiendo de la

localización en el cerebro y del tipo de receptor al que se une, de su localización

(presináptico o postsináptico) y del estado del individuo (periodo de luz/oscuridad,

actividad/inactividad) (Ursin, 2002). De hecho, la actividad de la 5-HT en la regulación

del ciclo sueño-vigilia es compleja.

Diversos son los estudios realizados en aves sobre los efectos de la inhibición de

la síntesis de 5-HT inducida por para-clorofenilalanina (PCPA), indicando que se

reducen los niveles de 5-HT en el cerebro de diversas especies de aves (El Halawani y

Waibel, 1976; Schrold y Squires, 1971; Balander et al., 1984; Braganza y Wilson,

1978), tal y como se ha descrito en mamíferos (Koe y Weissman, 1966; Weitzman et

al., 1968; Wyatt et al.,1969). Se ha descrito una reducción del sueño y la facilitación de

la vigilia tras la administración del PCPA en mamíferos (Mouret et al., 1968; Koella,

1968; Ursin, 2002). En aves también se ha observado reducción del SWS (Vasconcelos-

Duenas y Guerrero, 1983). Sin embargo, en el presente trabajo, los resultados obtenidos

en tórtolas tratadas con PCPA no indujeron este resultado sino todo lo contrario,

incrementó la duración y episodios de estados de sueño, el cierre de ojos

(correlacionado con el sueño comportamental, Lendrem, 1983) y la latencia de los

estados de vigilia activa (es decir, tardaron más tiempo en aparecer estos estados); por el

contrario, redujo la actividad locomotora espontánea, los estados de vigilia activa y el

aseo (Tejada et al., manuscript III).

Pero los resultados presentados en esta tesis doctoral en relación al PCPA no son

los únicos que crean una contradicción; de hecho, estudios en ratas demostraron una

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reducción de la actividad locomotora espontánea y de algunos comportamientos propios

de la vigilia activa (Dringenberg et al., 1995), y en aves el PCPA produjo

comportamientos letárgicos (Balander et al., 1984). Estos resultados contradictorios

podrían estar relacionados con las dosis de PCPA usadas en cada caso, que inducirían a

una depleción de 5-HT diferente. De hecho, en mamíferos el sueño reaparece después

del tratamiento con PCPA a pesar de que los niveles de 5-HT permanecían bajos

(Dement et al., 1972).

Existen diversos trabajos que conceden a la 5-HT un papel en el ciclo sueño-

vigilia, aunque recientemente se ha relacionado fuertemente con la vigilia debido a que

el incremento de la transmisión de la 5-HT (por ejemplo, inhibición de la recaptación,

administración de precursores de 5-HT) favorece los estados de vigilia pasiva (Jones,

2005; Espana y Scammell, 2004) y el despertar (Koella y Czicman, 1966). Además, la

actividad de las neuronas serotonérgicas del rafe dorsal se relacionan con el nivel del

despertar comportamental, ya que aumenta su actividad eléctrica durante la vigilia y de

manera más lenta durante el SWS llegando a cesar durante el sueño REM (Sakai y

Crochet, 2001; Miller y O'Callaghan, 2006). También, diversos estudios que miden la

cantidad de 5-HT liberada al fluido extracelular, muestran que es mayor durante la

vigilia y menor durante el sueño (Portas et al., 2000) en gatos (Portas y McCarley,

1994), ratas (Portas et al., 1998) y en tórtolas (Garau et al., 2006).

El agonista selectivo de los receptores 5HT1A, 8-OH-DPAT (Hoyer et al., 1994),

que también presenta ligera afinidad por los 5HT7 (Wood et al., 2000), incrementó la

actividad locomotora espontánea en tórtolas en el presente trabajo, lo que indica que la

5-HT juega un control tónico estimulatorio sobre la actividad locomotora. Además, el

pretratamiento con el antagonista selectivo de los receptores 5HT1A WAY100635,

bloqueó el efecto estimulatorio producido por el agonista 8-OH-DPAT sobre la

actividad locomotora espontánea, indicando que el efecto estimulatorio sobre la

actividad locomotora fue mediado por el receptor 5HT1A. Reforzando estos resultados,

se indujo la depleción de 5-HT en tórtolas por medio de PCPA para conseguir la

ausencia de actividad de los receptores serotonérgicos y así estudiar el efecto de la

activación de los receptores 5HT1A tras la administración de su agonista 8-OH-DPAT.

El tratamiento reflejó un efecto estimulatorio del agonista sobre los estados de vigilia

activa y aseo, y un efecto inhibitorio sobre el sueño y el cierre de ojos del animal,

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efectos que fueron más marcados que en el caso de las tórtolas no tratadas con PCPA

(Tejada et al., manuscript III). Algunos autores han descrito efectos similares en aves

(Kostal y Savory, 1996) y mamíferos (Bjorvatn et al., 1997; Horner et al., 1997;

Sorensen et al., 2001; Hoffman et al., 2007), describiendo un incremento en la actividad

durante la primera hora tras la administración del agonista 8-OH-DPAT, concretamente

en la vigilia y el aseo. Todos estos hallazgos refuerzan la sugerencia de que la 5-HT

juega un papel estimulatorio tónico sobre la actividad locomotora espontánea y en los

estados de vigilia de la tórtola collariza igual que en la rata.

Sin embargo, entender los efectos del agonista 8-OH-DPAT es complicado, ya

que los receptores por los que tiene afinidad se encuentran situados tanto presináptica

como postsinápticamente (Kia et al., 1996), lo cual puede explicar los efectos bifásicos

descritos en la literatura (Monti y Jantos, 1992). Es el caso de la neuronas colinérgicas

activadoras del sueño REM (REM-on), en las cuales, la 5-HT tendría un efecto

inhibitorio a través de receptores 5-HT1A postsinápticos (Horner et al., 1997), mientras

que presentaría un efecto estimulatorio a través de los autorreceptores 5-HT1A en los

núcleos del rafe (Bjorvatn et al., 1997; Portas et al.¸1996).

El efecto inhibitorio sobre el sueño REM tras el tratamiento sistémico con

diversos agonistas del receptor 5-HT1A, y en particular el 8-OH-DPAT (Tejada et al.,

manuscript III), apoya la idea de que los receptores 5-HT1A desempeñan un papel

importante en la regulación de los estados de vigilancia en mamíferos (Monti y Jantos,

1992; Tissier et al., 1993; Dzoljic et al., 1992; Quattrochi et al., 1993). La reducción del

sueño REM tras la administración de 8-OH-DPAT (Tejada et al., manuscript III) se

correlaciona con la mayor cantidad de sueño observado en ratones mutantes que no

expresan los receptores 5-HT1A comparado con ratones wild-type (Boutrel et al., 2002).

Al contrario, el bloqueo farmacológico de estos receptores con el antagonista

serotonérgico WAY100635 ha inducido un incremento del sueño REM también en

ratones wild-type, indicando que estos receptores median una influencia inhibitoria

tónica sobre el sueño REM en estas especies (Boutrel et al., 2002). Sin embargo, el

antagonista no provocó efectos en los estados comportamentales a bajas

concentraciones en gatos (Sakai y Crochet, 2001), de igual manera que se ha podido

observar en los resultados presentados sobre los estados de vigilancia de tórtolas.

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Hay que tener en cuenta que se ha descrito que las tórtolas toleran dosis más

altas de 8-OH-DPAT y otros agonistas y antagonistas de los receptores 5-HT1A.

Basándose en este aspecto, posiblemente serían necesarios estudios con dosis más altas

de 8-OH-DPAT en aves, comparadas con las usadas en mamíferos. Por esta razón,

posiblemente, la administración del agonista serotonérgico 8-OH-DPAT modificó sólo

la actividad locomotora espontánea, mientras que su administración en tórtolas

pretratadas con PCPA (que depleciona los niveles de 5-HT impidiendo la actividad

basal de sus receptores), provocó un efecto estimulatorio muy prominente sobre la

actividad locomotora espontánea, los estados de vigilia activa y aseo, así como una

marcada reducción sobre el sueño y el cierre de ojos (Tejada et al., manuscript III).

Estos resultados también podrían indicar que la ausencia de 5-HT por el tratamiento con

PCPA induce una mayor sensibilidad de los receptores 5-HT1A que modulan los estados

de vigilia. En este sentido, tratamientos con PCPA en ratas a dosis parecidas a las

usadas en este trabajo, mostraron un incremento en la densidad de receptores

serotonérgicos del orden del 100% mediante estudios de unión de radioligandos con 3H-

5-HT (Steigrad et al., 1978).

Como conclusión final, basándose en estudios electrofisiológicos,

neuroquímicos, genéticos y neurofarmacológicos, los datos apoyan la idea de que los

sistemas colinérgico y serotonérgico están implicados en el control del ciclo sueño-

vigilia en mamíferos y aves, principalmente en los estados de vigilia. El sistema

colinérgico estaría implicado en el incremento de la actividad theta cuyo principal

generador es el hipocampo sugiriendo un papel importante en la transferencia de la

información desde el hipocampo a la corteza y una relación con los procesos de

aprendizaje y memoria. El sistema serotonérgico promovería la vigilia e inhibiría el

sueño a través de la activación de receptores 5-HT1A.

Finalmente, los resultados sobre la función del hipocampo apoyan la hipótesis de

que la formación del hipocampo en mamíferos y aves son homólogos y que las

características fisiológicas de la formación del hipocampo se han conservado durante la

evolución. De hecho, que estos efectos estén presentes en mamíferos y aves sugiere que

los mecanismos colinérgicos y serotonérgicos que regulan el ciclo sueño-vigilia son

compartidos por ambos grupos de vertebrados reflejando un origen filogenético común

(Mexicano et al., 1992; Rial et al., 2010).

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6. CONCLUSIONES / CONCLUSIONS

179

CONCLUSIONES

6. CONCLUSIONES

1.- En ratas, el agonista colinérgico muscarínico pilocarpina provocó un incremento del

ritmo y potencia de la banda theta durante los estados de vigilia pero no durante los de

sueño, en contradicción con los efectos descritos para otros fármacos agonistas

colinérgicos M2 sobre el sueño REM. Esta diferencia podría estar relacionada con el

perfil farmacológico de la pilocarpina que presenta mayor afinidad por el receptor

muscarínico M5 que por el M2.

2.- Tanto en la vigilia pasiva como en la vigilia activa de ratas, la administración de

pilocarpina indujo un incremento del ritmo theta cuando aparecía simultáneamente en la

región hipocámpica y en la corteza frontal, sin que se viese afectado el ritmo theta

cuando aparecía solo en el hipocampo; sugiriendo un incremento del flujo de

información entre el hipocampo y la corteza.

3.- La pilocarpina incrementó la potencia espectral relativa de la banda de frecuencia

theta durante los estados de vigilancia en la corteza frontal, pero no en el hipocampo de

ratas. Además, la coherencia entre la banda theta del EEG hipocampal y la que emerge

de la corteza frontal incrementó tras la administración de pilocarpina, aunque sólo

durante los estados de vigilia. Estos resultados sugieren que la pilocarpina incrementó la

correlación lineal de esta actividad theta entre hipocampo y corteza durante los estados

de vigilia. De nuevo, la pilocarpina parece reforzar la transmisión neural de la actividad

eléctrica desde el hipocampo hacia la corteza durante los estados de vigilia.

4.- El índice de retraso de fases (PLI) fue mayor durante la vigilia activa en ratas, y la

administración de la pilocarpina mantuvo los diferentes estados comportamentales

estudiados con un acoplamiento de fase distinto a cero, incrementando la sincronización

de las señales de la banda theta entre el hipocampo y la corteza frontal durante la vigilia

pasiva. Estos resultados confirman la existencia de sincronización de la banda de

frecuencia theta entre el hipocampo y la corteza durante los estados de vigilia. De esta

manera, existiría un efecto del agonista colinérgico muscarínico pilocarpina sobre la

conectividad funcional hipocampo-corteza.

181

CONCLUSIONES

5.- El aumento en la conectividad electroencefalográfica entre el hipocampo y la corteza

durante la vigilia se correlacionó con una mejoría en la capacidad de aprendizaje y

memoria de las ratas tratadas con pilocarpina en el test del laberinto radial. El

mecanismo implicado podría ser debido a la inhibición de la liberación de ACh tras la

activación del receptor M5, permitiendo la transmisión de información entre dichas

áreas cerebrales que con niveles altos del neurotransmisor se encuentra bloqueada.

Además, se pudo comprobar que se trataba de un efecto colinérgico, ya que el

antagonista escopolamina de estos receptores impidió que la pilocarpina mejorase la

resolución de las tareas a realizar en el laberinto radial.

6.- En la tórtola collariza, el agonista colinérgico muscarínico pilocarpina provocó un

incremento del ritmo theta y del estado de vigilia pasiva, mientras que indujo una

disminución de la vigilia activa y de los estados de sueño. Estos resultados fueron

similares a los descritos en rata, y podrían también estar relacionados con los efectos del

sistema colinérgico con los procesos de aprendizaje y memoria.

7.- Se ha observado la inducción de daño oxidativo a nivel cerebral tras la

administración de pilocarpina a concentraciones altas, normalmente las usadas como

epileptogénicas. En contraste, las concentraciones menores usadas en el presente trabajo

no produjeron daño oxidativo, por lo que los resultados obtenidos tanto

electroencefalográficos como comportamentales no se debieron a artefactos de la

actividad epileptiforme descrita para la pilocarpina. Se infiere que las determinaciones

enzimáticas para el estudio del estado oxidativo del cerebro resultan de utilidad para

validar los resultados de estudios farmacológicos y electroencefalográficos a nivel

cerebral.

8.- El agonista serotonérgico 5-HT1A 8-OH-DPAT incrementó la actividad locomotora,

los estados de vigilia activa y aseo, reduciendo el SWS y REM en la tórtola collariza. El

pre-tratamiento con el antagonista serotonérgico WAY100635 previno los efectos

inducidos por el 8-OH-DPAT; mientras que administrado solo, disminuyó la actividad

locomotora. El efecto inhibitorio del WAY100635 indicaría la existencia de un tono

estimulatorio sobre la actividad locomotora mediado por 5HT1A.

182

CONCLUSIONES

9.- La depleción de 5-HT inducida por el PCPA en tórtolas redujo la actividad

locomotora, los estados de vigilia y el aseo, incrementando el SWS y REM. La

administración del agonista serotonérgico 8-OH-DPAT en tórtolas pre-tratadas con

PCPA incrementó marcadamente la actividad locomotora y los estados de vigilia activa

y el aseo, mientras que disminuyó los estados de sueño, sugiriendo que en ausencia de

activación de otros receptores serotonérgicos debido a la depleción de 5-HT, la

activación de los receptores 5-HT1A por 8-OH-DPAT tiene un efecto estimulatorio

sobre la vigilia activa e inhibitorio sobre el sueño.

10.- En líneas generales, los resultados obtenidos en tórtola collariza y rata del estudio

del ciclo sueño-vigilia mediante la activación farmacológica de los sistemas colinérgico

y serotonérgico, mostraron efectos similares en ambos grupos taxonómicos, lo que pone

de manifiesto que el ciclo sueño-vigilia está regulado por mecanismos que comparten

ambos grupos de vertebrados con un origen filogenético común.

183

CONCLUSIONS

6. CONCLUSIONS

1.- In rats, the muscarinic cholinergic agonist pilocarpine induced an increase in the

theta rhythm and power band during waking states but not during sleep, in contrast with

the effects described for other M2 cholinergic agonists on REM sleep. This difference

could be related to the pharmacological profile of pilocarpine which has higher affinity

for the M5 muscarinic receptor than for the M2 one.

2.- In both passive and active waking of rats, the administration of pilocarpine induced

an increase of theta rhythm when it simultaneously appeared in the hippocampal region

and frontal cortex, but the theta rhythm only appearing in the hippocampus was not

affected, suggesting an increased flow of information between the hippocampus and

cortex.

3.- Pilocarpine increased the relative spectral power of theta frequency band during

waking states in the frontal cortex, but not in the hippocampus of rats. Moreover,

coherence between hippocampal theta band and the one from the frontal cortex

increased after pilocarpine administration, but only during waking states. These results

suggest that pilocarpine increased the linear correlation between theta activity of the

hippocampus and cortex during waking states. Pilocarpine appears to enhance neural

transmission of the electrical activity from the hippocampus to the cortex during waking

states.

4.- Phase lag index (PLI) was greater during active waking than in other states in rats.

The non-zero phase locking status remained after pilocarpine treatment in the studied

states with an effective non-zero phase; increasing the synchronization of the theta band

between hippocampus and frontal cortex during passive waking. These results confirm

the synchronization of the theta frequency band between hippocampus and cortex

during waking states. Thus, there is an effect of the muscarinic cholinergic agonist

pilocarpine on the functional connectivity of hippocampus-cortex.

5.- The increase in the electroencephalographic connectivity between the hippocampus

and cortex during wakefulness was correlated with the improved learning and memory

abilities of rats treated with pilocarpine in the radial maze test. The involved mechanism

185

CONCLUSIONS

was probably due to inhibition of ACh release after activation of the M5 receptor,

allowing the transmission of information between these brain areas that is blocked by

high levels of the neurotransmitter. In addition, it was demonstrated that it was a

cholinergic effect, since the receptor antagonist scopolamine avoided the effects of

pilocarpine improving the resolution in the radial maze tasks.

6.- In ring dove, the muscarinic cholinergic agonist pilocarpine caused an increase in

theta rhythm and passive waking state, whereas it decreased the active waking and sleep

states. These results were similar to those described in rats, indicating a possible

relationship between the effects of the cholinergic system with the processes of learning

and memory.

7.- It has been observed induction of oxidative damage in the brain after administration

of pilocarpine at high concentrations, usually used as epileptogenic. In contrast, the

lower concentrations used in this study did not produce oxidative damage, so the results

obtained from EEG and behavioral studies were not an artifact due to epileptiform

activity described for the pilocarpine. In addition, enzymatic determinations for the

study of oxidative status of the brain are useful to validate the results of

pharmacological and electroencephalographic studies in the brain.

8.- The serotonergic 5-HT1A agonist 8-OH-DPAT increased the locomotor activity, the

active waking and grooming states, reducing SWS and REM sleep in ring dove. Pre-

treatment with the serotonergic antagonist WAY100635 prevented the effects induced

by 8-OH-DPAT, whereas given alone decreased the locomotor activity. The inhibitory

effect of WAY100635 indicated the existence of a stimulatory tone on locomotor

activity mediated by 5HT1A.

9.- 5-HT depletion induced by PCPA in ring doves reduced the locomotor activity, the

waking and grooming states, increasing SWS and REM sleep. The administration of the

serotonergic agonist 8-OH-DPAT in pigeons pre-treated with PCPA markedly increased

the locomotor activity and waking and grooming states, whereas it decreased sleep

states; suggesting that, with the lack of activation of other serotonergic receptors due to

the depletion of 5-HT, the activation of 5-HT1A receptors by 8 OH-DPAT has a

stimulatory effect on active waking and an inhibitory one on sleep.

186

CONCLUSIONS

10.- Overall, the obtained results in ring dove and rat by the study of the sleep-wake

cycle through the pharmacological activation of the cholinergic and serotonergic

systems, revealed similar effects in both taxonomic groups, which shows that the sleep-

wake cycle is regulated by mechanisms that both groups of vertebrates, with a common

phylogenetic origin, share.

187

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