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UNIVERSIDAD DE GRANADA
INSTITUTO DE NEUROCIENCIAS
DEPARTAMENTO DE PSICOLOGÍA EXPERIMENTAL
Y FISIOLOGÍA DEL COMPORTAMIENTO
TESIS DOCTORAL
DESARROLLO DE LA FUNCIÓN HIPOCAMPAL Y
MEMORIA GUSTATIVA EN RATAS:
PAPEL DEL CONTEXTO TEMPORAL
TATIANA MANRIQUE ZULUAGA
GRANADA, 2008
Editor: Editorial de la Universidad de Granada
Autor: Tatiana Manrique Zuluaga
D.L.: GR.1789-200
ISBN: 978-84-691-5649-0
Dña. MILAGROS GALLO TORRE, PROFESORA TITULAR DE
PSICOBIOLOGÍA DE LA UNIVERSIDAD DE GRANADA
CERTIFICA:
Que el trabajo de investigación titulado “DESARROLLO DE LA
FUNCIÓN HIPOCAMPAL Y MEMORIA GUSTATIVA EN
RATAS: PAPEL DEL CONTEXTO TEMPORAL” ha sido
realizado por Dña. Tatiana Manrique Zuluaga para optar al grado de
Doctor Europeo en Psicología en el Departamento de Psicología
Experimental y Fisiología del Comportamiento de la Facultad de
Psicología de la Universidad de Granada, bajo su dirección.
Y para que conste donde proceda se firma este certificado en
Granada a 10 de Abril de 2008
Fdo. Milagros Gallo Torre
Fdo. Tatiana Manrique Zuluaga
Esta tesis ha sido subvencionada por los siguientes proyectos de
investigación:
BSO2002-01215 del Ministerio de Ciencia y
Tecnología de España, SEJ2005-01344 del Ministerio de Educación
y Ciencia de España, ambos parcialmente subvencionados por el
Fondo Europeo de Desarrollo Regional y el proyecto HUM 02763
de la Junta de Andalucía.
Esta tesis está dedicada a una mujer, a quien respeto y admiro
profundamente … a Milagros.
AGRADECIMIENTOS
Un sueño hecho realidad, cuando llegué de Colombia hace casi 9 años, tenía
grandes ilusiones y deseos, sólo contaba con el apoyo de mi familia y mis
ideales. Tenía claro que en España encontraría lo que estaba buscando, no fue
fácil pero a pesar de diversos obstáculos encontré el camino con la ayuda de
Milagros, con su profesionalismo y apoyo incondicional logramos no solo este
valioso documento sino una maravillosa amistad.
Pero todo esto no es un trabajo sólo de dos, a mis compañeros de laboratorio
colegas y amigos, un agradecimiento sincero por compartir sus experiencias y
su tiempo conmigo … Nacho, Nines, Fernando, Ana, David y Rosa gracias por
tan espléndida colaboración.
A aquellos docentes que siempre confiaron en mi trabajo y en mis destrezas,
que condujeron cada una de mis inquietudes e hicieron que amara cada día
más esta profesión, Isabel de Brugada, Felisa González, Antonio Cándido,
Antonio Maldonado, Andrés Catena, André Fenton, hoy y siempre gracias por
estar ahí.
A Esteban compañero y amigo, por su colaboración en la diagramación y
expresión gráfica de posters y charlas presentadas en congresos, al igual que
por su apoyo, gracias.
Finalmente a mi madre, a mis hermanas y a mi tía gracias por estar ahí, por
darme siempre una voz de aliento y por no dejarme desfallecer ni dar un paso
atrás.
A la memoria de mi padre hoy le ofrezco más que un logro, una satisfacción
por todas sus enseñanzas y buen ejemplo de la vida.
A todos mil gracias, sin ustedes no hubiera sido posible…
DESARROLLO DE LA FUNCIÓN HIPOCAMPAL Y MEMORIA
GUSTATIVA EN RATAS:
PAPEL DEL CONTEXTO TEMPORAL
La presente tesis doctoral está compuesta de seis capítulos. Cada uno de
ellos contiene un artículo original de la autora de esta tesis ya publicado
(capítulos 1, 2, 3 y 6) o que se encuentra en proceso de revisión en una revista
científica (capítulos 4 y 5). En conjunto los experimentos contenidos en esta
tesis están dirigidos al estudio de la función hipocampal y su relación con el
aprendizaje en ratas desde una aproximación de desarrollo, entendido el
término en su sentido más amplio desde las etapas tempranas de formación del
Sistema Nervioso hasta el envejecimiento.
El capítulo 1 presenta una
introducción general al aprendizaje aversivo gustativo, modelo de aprendizaje
empleado en la mayor parte de la tesis (capítulos 1-5), y su valor para los
estudios de desarrollo. Este material fue publicado (en castellano) en una
reconocida revista de divulgación científica (Mente y cerebro, 2006, 11, 3940). El capítulo 2, que contiene una revisión de los efectos del envejecimiento
normal sobre el aprendizaje aversivo gustativo, consiste en una revisión sobre
el tema ya publicada (Chemical Senses, 2007). El resto de los capítulos
incluyen las aportaciones experimentales que forman el grueso de esta tesis. El
capítulo 3 incluye la demostración de que un cambio de hora puede actuar
como contexto en aprendizaje aversivo gustativo, interfiriendo con el
fenómeno de inhibición latente, hallazgo publicado en Neurobiology of
Learning and Memory, (2004, 82, 77-80). En el capítulo 4 se demuestra la
utilidad de aplicar el protocolo empleado en el capítulo anterior en ratas
envejecidas intactas y con lesiones del hipocampo, poniendo de manifiesto
tanto la ausencia de modulación por parte del contexto temporal, como la
facilitación de formas alternativas de modulación, respectivamente. El capítulo
5 contiene un estudio dirigido a investigar la ontogenia de la inhibición latente
y su modulación por el contexto temporal. Los resultados ponen de manifiesto
cursos de desarrollo independientes para el fenómeno de inhibición latente, la
especificidad contextual de la aversión gustativa y la especificidad contextual
del fenómeno de inhibición latente en aprendizaje aversivo gustativo. Por
último, el capítulo 6 está formado por un estudio ya publicado en
Developmental Psychobiology (2005, 46, la 340-349) que demuestra la
relevancia no sólo de las experiencias de aprendizaje previas sobre el
desarrollo de las capacidades de aprendizaje adultas sino también de sus
parámetros temporales.
DESARROLLO DE LA FUNCIÓN HIPOCAMPAL Y
MEMORIA GUSTATIVA EN RATAS:
PAPEL DEL CONTEXTO TEMPORAL
Contents
CONTENTS
Resumen .........................................................................................................................1
Introducción.......................................................................................................3
Aprendizaje Aversivo Gustativo (Capítulo 1) ......................................6
Envejecimiento y Aprendizaje Aversivo Gustativo (Capítulo 2) .......10
Justificación y objetivos .....................................................................14
Inhibición Latente y Contexto Temporal (Capítulo 3) .......................17
Ontogenia de la inhibición latente y de los efectos del contexto
temporal en aprendizaje aversivo gustativo (Capítulo 5) ...................22
Envejecimiento, Hipocampo y Contexto Temporal (Capítulo 4) .......28
Fracaso Temprano y Aprendizaje Adulto (Capítulo 6).......................32
Conclusiones....................................................................................................41
Referencias ......................................................................................................44
Chapter 1. Neurobiología del Aprendizaje Aversivo Gustativo...................................63
Chapter 2. Hippocampus, Ageing, and Taste Memories..............................................71
Abstract ...........................................................................................................73
2.1. Introduction .............................................................................................74
2.2. Ageing and taste memories......................................................................79
2.3. Hippocampus and taste memories ...........................................................84
Tatiana Manrique
2.4. Hippocampal decline and ageing impact on taste memories ...................89
2.5. Conclusions .............................................................................................91
References .......................................................................................................93
Chapter 3. Time of day-dependent latent inhibition of conditioned taste aversions in
rats ..............................................................................................................................101
Abstract..........................................................................................................103
3.1. Introduction ...........................................................................................104
3.2. Materials and methods...........................................................................105
3.3. Results ...................................................................................................108
3.4. Discussion..............................................................................................111
References .....................................................................................................114
Chapter 4. Hippocampus, Aging and Segregating memories.....................................117
Abstract..........................................................................................................119
4.1. Introduction ...........................................................................................120
4.2. Materials and Methods ..........................................................................123
4.2.1. Behavioural Protocol .............................................................124
4.2.2. Dorsal Hippocampal Lesion ..................................................126
4.3. Results ...................................................................................................129
4.3.1. Experiment 1..........................................................................129
4.3.1.1. Results....................................................................129
Contents
4.3.2. Experiment 2........................................................................135
4.3.2.1. Results....................................................................136
4.4. Discussion..............................................................................................139
References .....................................................................................................146
Chapter 5. Peculiar modulation of taste aversion learning by the time of day in
developing rats ...........................................................................................................151
Abstract .........................................................................................................153
5.1. Introduction ...........................................................................................154
5.2. Experiment 1 .........................................................................................158
5.2.1. Method...................................................................................160
5.2.1.1. Subjects..................................................................160
5.2.1.2. Apparatus ...............................................................161
5.2.2. Procedure ...............................................................................162
5.2.3. Results and Discussion ..........................................................164
5.3. Experiment 2 .........................................................................................167
5.3.1. Subjects..................................................................................168
5.3.2. Procedure ...............................................................................169
5.3.3. Results and Discussion ..........................................................170
5.4. General Discussion ................................................................................178
References .....................................................................................................189
Tatiana Manrique
Chapter 6. Early Learning Failure Impairs Adult Learning in Rats ..........................203
Abstract..........................................................................................................205
6.1. Introduction ...........................................................................................206
6.2. Experiment 1..........................................................................................208
6.2.1. Methods .................................................................................208
6.2.1.1. Subjects ..................................................................208
6.2.1.2. Apparatus ...............................................................210
6.2.1.3. Procedure ...............................................................211
6.2.2. Results....................................................................................213
6.2.2.1. Morris Water Maze ................................................213
6.2.2.2. Avoidance Task......................................................215
6.2.3. Discussion..............................................................................217
6.3. Experiment 2..........................................................................................218
6.3.1. Method ...................................................................................220
6.3.1.1. Subjects ..................................................................220
6.3.1.2. Procedure ...............................................................221
6.3.2. Results.....................................................................................221
6.3.2.1. Morris Water Maze ................................................221
6.3.2.2. Avoidance Task......................................................225
6.3.3. Discussion..............................................................................228
6.4. General Discussion ................................................................................230
References .....................................................................................................238
RESUMEN
INTRODUCCIÓN
El estudio de la organización anatómica y funcional de los circuitos
neuronales responsables del aprendizaje y la memoria se beneficia no sólo de la
investigación sobre el cerebro adulto (para una revisión ver Gallo y Manrique,
2007) sino también de una aproximación de desarrollo. Del mismo modo que
las técnicas de lesión permanente o reversible (Gallo, 2007) permiten disociar
procesos de aprendizaje dependientes de regiones cerebrales diferentes,
el
estudio de la emergencia y decaimiento de distintas capacidades de aprendizaje,
así como de las modificaciones que dichas capacidades sufren a lo largo de la
vida, representa una herramienta excepcional para establecer disociaciones de
forma natural entre los múltiples procesos involucrados.
Las diferentes capacidades de aprendizaje surgen a lo largo del
desarrollo temprano a medida que maduran los circuitos cerebrales, y se
modifican a lo largo de la vida incorporando los efectos de la experiencia previa
y siendo, algunas de ellas, especialmente sensibles al envejecimiento. Aquellos
tipos de aprendizaje que requieren la participación de memoria declarativa o
explícita, como es el caso del aprendizaje espacial en ratas, son abolidos
selectivamente por lesiones del hipocampo, muestran una emergencia tardía
durante el desarrollo, de acuerdo con el prolongado curso de maduración
hipocampal, y suelen decaer a edades avanzadas, como consecuencia de la
Resumen
vulnerabilidad hipocampal a los efectos del envejecimiento. Sin embargo, otros
tipos de aprendizaje que requieren memoria no-declarativa o implícita, tales
como distintas formas de condicionamiento clásico, no requieren un hipocampo
intacto, aparecen a edades más tempranas dependiendo de la maduración de los
sistemas sensoriales implicados y son resistentes a los efectos del
envejecimiento. Ello es congruente con la existencia de múltiples sistemas de
memoria con bases cerebrales disociables, en el sentido propuesto por Squire
(1994) y otros autores.
De este modo es posible distinguir entre tipos de
aprendizaje basados en memoria declarativa, que dependen de la maduración del
sistema hipocampal y áreas corticales asociadas (Alvarado y Bachevalier, 2000;
Nelson, 1998), y una diversidad de tipos de aprendizaje con circuitos neurales
independientes, que son maduros a edades más tempranas y que no decaen con
la edad (Gerhardstein, Adle y Rovee-Collier, 2000).
Adicionalmente, la organización peculiar de los sistemas de memoria
durante el desarrollo temprano, debido tanto a la falta de maduración de ciertas
regiones cerebrales como al exceso de sinapsis previo a los procesos de poda
axónica, facilita capacidades de aprendizaje peculiares no observadas en el
adulto (Barr, Marrott, y Rovee-Collier, 2003; Bordner y Spear, 2006; Brasser y
Spear, 2004; Campbell y Spear, 1972; Hoffmann y Spear, 1988; Molina,
Hoffmann, Serwatka y Spear, 1991). Del mismo modo, el cerebro envejecido
muestra una organización peculiar que va más allá del decaimiento funcional de
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Memoria, Contexto e Hipocampo
ciertas regiones cerebrales y que se corresponde con una reorganización de las
capacidades de aprendizaje y memoria (Erickson y Barnes, 2003; Kelly et al.,
2006).
Sin embargo, el empleo de diversos tipos de aprendizaje en la
investigación sobre la evolución de los sistemas de memoria a lo largo de la vida
plantea dificultades de interpretación dadas las diferentes exigencias sensoriales,
motoras, motivacionales, emocionales, etc., que pueden resultar, asimismo,
afectadas de forma diferencial por la edad. Los problemas de interpretación se
reducen cuando se emplea una única tarea de aprendizaje a la que se van
añadiendo mayores exigencias de aprendizaje y/o de memoria. Aproximaciones
de este tipo han sido seguidas con éxito por diversos grupos empleando
paradigmas de aprendizaje tales como condicionamiento palpebral o miedo
condicionado (para una revisión Hunt et al., 2007). Los resultados han mostrado
una emergencia diferencial de nuevas posibilidades durante el desarrollo
siguiendo una secuencia en la que a la capacidad de establecer asociaciones
básicas entre estímulo condicionado e incondicionado, le sigue la posibilidad de
introducir mayores dilaciones entre estímulos y aparición de otros fenómenos de
aprendizaje, como inhibición latente, y modulación por el contexto. Del mismo
modo, se ha reportado deterioro inducido por el envejecimiento en aquellas
capacidades de aprendizaje de aparición más tardía.
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Resumen
Una aproximación de este tipo es especialmente eficaz si cumple una
serie de requisitos. En primer lugar, debe emplear modalidades estimulares que
exhiban un desarrollo tan temprano como sea posible. Ello es especialmente
importante es especies altriciales como la rata que no son capaces de oír y ver
hasta la segunda semana postnatal. En segundo lugar, las modalidades
sensoriales empleadas no deberían resultar afectadas por el envejecimiento. Es
bien conocido que el envejecimiento está frecuentemente asociado a una pérdida
de visión, audición y olfato. En tercer lugar, es especialmente útil el empleo de
un tipo de aprendizaje básico de aparición muy temprana y que no sufra
deterioro con la edad. Por último, debe tratarse de un tipo de aprendizaje que
permita el estudio de fenómenos de diversa complejidad mediante pequeñas
modificaciones del procedimiento que no alteren los procesos sensoriales,
motores, motivacionales y emocionales implicados.
Aprendizaje Aversivo Gustativo (Capítulo 1)
La adquisición de aversiones gustativas aprendidas representa un tipo de
aprendizaje de especial interés para una aproximación de desarrollo. García,
Kimeldorf y Hunt (1956) fueron los primeros en la observación de este tipo de
aprendizaje en ratas. Estos autores descubrieron que el consumo de agua y
comida disminuía durante la exposición a dosis relativamente bajas de radiación
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Memoria, Contexto e Hipocampo
gamma de baja intensidad.
Sugirieron que el cambio progresivo en el
comportamiento de consumo durante exposiciones repetidas podría ser, en parte,
una respuesta condicionada en la que la evitación de agua y comida es reforzada
debido a la asociación con las consecuencias de la exposición radioactiva. El
uso de una solución de sacarina demostró la eficacia de la radiación iónica como
estímulo incondicionado en el comportamiento animal. Las ratas tendían a evitar
el estímulo gustativo asociado a la radiación.
En la tarea básica se establece una asociación entre un sabor desconocido
y el malestar gastrointestinal posterior, generalmente inducido mediante una
inyección i.p. de cloruro de litio (LiCl). Como consecuencia, el sabor se
convierte en desagradable, induce patrones de respuesta característicamente
aversivos y es rechazado en posteriores ocasiones, protegiendo al organismo de
la ingestión repetida de sustancias nocivas. Las modalidades sensoriales
químicas involucradas (gustativo-olfativa y visceral) son de las primeras en ser
funcionales durante la vida prenatal y, en el caso de la modalidad gustativa, no
sufre cambios críticos a edades avanzadas. El aprendizaje aversivo gustativo se
ha descrito ya en la etapa fetal en ratas (Smotherman y Robinson, 1985;
Smotherman, 2002a, 2002b) y persiste, e incluso resulta potenciado, a edades
avanzadas (Cooper, McNamara y Thompson, 1980; Peterson, Valliere, Misanin
y Hinderliter, 1985; Misanin y Hinderliter, 1994, 1995; Misanin, Hoefel, Riedy
y Hinderliter, 1997; Misanin, Hoefel, Riedy, Wilson y Hinderliter, 2000;
7
Resumen
Misanin et al, 2002; Morón, Ballesteros, Cándido y Gallo, 2002). Por tanto, se
trata de un tipo de condicionamiento clásico presente a lo largo del ciclo vital,
que ofrece un procedimiento de aprendizaje aplicable desde la etapa prenatal
hasta edades avanzadas.
El aprendizaje aversivo gustativo exhibe una serie de características
peculiares que, además de representar en su momento un reto para la teoría del
aprendizaje obligando a su remodelación, son especialmente adecuadas para una
aproximación de desarrollo. En primer lugar, es posible inducir intensas
aversiones selectivas a un sabor en un único ensayo, lo que evita la necesidad de
entrenamientos largos que impidan determinar con precisión el momento de
desarrollo en que surgen las capacidades de aprendizaje. Ello es de especial
importancia en la investigación con ratas infantes dado el rápido curso de
desarrollo que permite el inicio de la adolescencia al final del primer mes (día
postnatal 28, según Spear, 2000). En segundo lugar, no requiere respuestas
motoras, ya que es posible inducirlo incluso en animales inconscientes. Ello
facilita la investigación a lo largo de toda la vida, incluyendo periodos en los
que, debido a inmadurez o deterioro, puedan existir deficiencias motoras. Por
último, en el adulto permite introducir largas dilaciones entre los estímulos a
asociar. La duración de dicho intervalo varía en función de la edad,
incrementándose gradualmente durante el desarrollo temprano y ampliándose
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Memoria, Contexto e Hipocampo
durante el envejecimiento (Misanin et al, 2002). Así, es posible investigar la
adquisición gradual de nuevas posibilidades de aprendizaje.
A pesar de exhibir características peculiares, que no están presentes
juntas en ningún otro tipo de condicionamiento clásico, el aprendizaje aversivo
gustativo exhibe los fenómenos de aprendizaje descritos en otras tareas de
condicionamiento clásico. Se ha descrito inhibición latente, bloqueo
condicionado y efectos de modulación por el contexto, entre una variedad de
fenómenos (para una revisión véase Gallo, Ballesteros, Molero y Morón, 1999).
Por tanto, permite investigar la emergencia y deterioro de las funciones
relevantes para la aparición de dichos fenómenos.
El circuito neural básico responsable de este tipo de aprendizaje incluye
áreas cerebrales en distintos niveles del sistema nervioso (Yamamoto, 1993,
1994; Yamamoto, Shimura, Sakai y Ozaki, 1994; Spector, 1995; Bures,
Bermudez-Rattoni y Yamamoto, 1998; Bernstein, 1999; Gallo et al., 1999;
Bermudez-Rattoni,
2004), siendo al parecer la interacción entre el núcleo
parabraquial y la corteza gustativa crítica para el establecimiento de la
asociación en ratas adultas (Gallo et al., 1992). A su vez, ciertos fenómenos de
aprendizaje complejos implican la función hipocampal en el adulto, como es el
caso del fenómeno de bloqueo (Gallo y Cándido, 1995a, 1995b; Gallo,
Valouskova y Cándido, 1997; Morón, Ballesteros, Valouskova y Gallo, 2001) o
los efectos del contexto. El hecho de que el circuito neural del que depende la
9
Resumen
adquisición de aversiones a los sabores integre áreas cerebrales situadas en
diferentes niveles de organización e interaccione con el sistema hipocampal
(Manrique y Gallo, 2005)
permite investigar la formación y evolución
dependiente de la edad de los sistemas de memoria, gracias a la imposición de
nuevas exigencias mediante modificaciones del procedimiento comportamental.
Cuando se trata de estudiar el efecto del envejecimiento sobre la función
hipocampal, el aprendizaje aversivo gustativo es especialmente adecuado. A
diferencia de lo que ocurre en otras tareas de aprendizaje, el aprendizaje
aversivo gustativo básico no sólo no muestra deterioro sino que resulta
facilitado. Ello facilita la interpretación de los efectos del envejecimiento sobre
fenómenos complejos.
Envejecimiento y Aprendizaje Aversivo Gustativo (Capítulo 2)
El deterioro cognitivo generalmente relacionado con el envejecimiento
no involucra un decaimiento general en el funcionamiento de los sistemas de
memoria.
En términos generales, algunos tipos de aprendizaje están
preservados, mientras que aquellos que requieren memoria declarativa
usualmente decaen durante el envejecimiento (Gallagher y Rapp, 1997).
Con respecto al aprendizaje aversivo gustativo el envejecimiento no
parece afectar ni a la percepción ni a la memoria de los sabores habitualmente
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Memoria, Contexto e Hipocampo
empleados. No se han observado deficiencias en la respuesta neofóbica, en su
habituación, ni en el fenómeno de inhibición latente en ratas envejecidas
(Gallagher and Burwell, 1989; Morón and Gallo, 2007; Morón, Ballesteros, et
al., 2002). De hecho, la capacidad de asociar sabores con consecuencias
gastrointestinales negativas resulta potenciada en ratas de edades avanzadas
(Misanin, Collins, et al., 2002; Misanin, Goodhart, et al., 2002; Morón,
Ballesteros, et al., 2002). Sin embargo, el envejecimiento induce un deterioro
selectivo sobre el bloqueo de una aversión aprendida a un sabor que se presenta
en compuesto con otro sabor previamente condicionado (Gallo et al., 1997;
Morón et al., 2001; Morón, Ballesteros, et al., 2002).
Se trata, por tanto, de un panorama complejo que incluye capacidades
preservadas, potenciadas y deterioradas.
Ello es congruente con una
interpretación del envejecimiento que va más allá de un mero decaimiento de los
sistemas de aprendizaje y memoria. Por el contrario, la senescencia se presenta
como un periodo de la vida con necesidades de adaptación propias y que se
beneficia de una larga historia aprendida previamente (Morón and Gallo, 2007).
Dado el efecto pernicioso del envejecimiento sobre el bloqueo del
aprendizaje aversivo gustativo, fenómeno que requiere la integridad del
hipocampo en la rata adulta, el aprendizaje aversivo gustativo se presenta como
adecuado para el estudio de la función hipocampal a edades avanzadas. Sin
embargo, esta aproximación no ha sido objeto de estudio con anterioridad y los
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resultados sobre el efecto de la lesión del hipocampo empleando animales
adultos en este tipo de aprendizaje son también escasos. Ello se ha debido
probablemente al hecho de que lesiones permanentes o reversibles del
hipocampo no impiden (Gallo and Cándido, 1995a) e incluso pueden potenciar
(Stone, Grimes y Katz, 2005) la adquisición de aversiones gustativas aprendidas
en adultos. Resultados similares se han obtenido con respecto a la inhibición
latente del aprendizaje aversivo gustativo. Existen reportes tanto de ausencia de
efecto (Gallo and Cándido, 1995a) como facilitación (Reilly, Harley y Revusky,
1993; Purves, Bonardi y Hall, 1995; Stone et al., 2005) inducida por lesiones
hipocampales en adultos.
Ello ha podido determinar la pérdida de interés en la exploración del
efecto del daño hipocampal en la modulación del aprendizaje aversivo gustativo
por el contexto, función hipocampal ya demostrada en otros tipos de aprendizaje
(Honey and Good, 1993; Holland and Bouton, 1999; Maren and Holt, 2000).
Efectivamente, el contexto en el que la exposición al sabor tiene lugar modula la
adquisición de aversiones aprendidas (Puente, Cannon, Best y Carrell, 1988;
Bonardi, Honey y Hall, 1990; Loy, Alvarez, Rey y López, 1993; Boakes,
Westbrook, Elliot y Swinbourne, 1997) y modula también el fenómeno de
inhibición latente del aprendizaje aversivo gustativo (Hall and Channell, 1986;
Rosas and Bouton, 1997) en ratas adultas.
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Memoria, Contexto e Hipocampo
Sin embargo, la investigación de los efectos del envejecimiento sobre
esta función hipocampal se enfrenta con una dificultad relacionada con la
definición habitual de “contexto” en términos de estimulación externa, haciendo
referencia a las condiciones del lugar de la situación de aprendizaje, lo cual
plantea los problemas de interpretación previamente mencionados. En este
sentido, puede resultar especialmente interesante el empleo de contextos
independientes de la estimulación exteroceptiva. De hecho, se ha demostrado
que la hora del día puede emplearse bien como parte del contexto (Bonardi et
al., 1990; Hall and Channell, 1986) o como el único contexto (Morón et al.,
2002) en aprendizaje aversivo gustativo, abriendo nuevas posibilidades para una
aproximación de desarrollo en el estudio de la función hipocampal.
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Justificación y objetivos
El estudio de la emergencia y decaimiento de distintas capacidades de
aprendizaje, así como de las modificaciones que dichas capacidades sufren a lo
largo de la vida, representa una herramienta excepcional para investigar la
organización anatómica y funcional de los circuitos neuronales responsables del
aprendizaje y la memoria.
La función hipocampal es necesaria para aquellos tipos de aprendizaje
que requieren la participación de memoria declarativa o explícita. Se trata de un
tipo de memoria que muestra una emergencia tardía durante el desarrollo,
revelando un prolongado curso de maduración hipocampal, y suele decaer a
edades avanzadas, como consecuencia de la vulnerabilidad hipocampal a los
efectos del envejecimiento. Adicionalmente, el sistema hipocampal ejerce
efectos moduladores sobre los circuitos neurales responsables de aprendizajes de
aparición temprana y resistencia a los efectos de la edad, como evidencia su
papel crucial en ciertos fenómenos de aprendizaje complejos, tales como los
efectos del contexto.
El aprendizaje aversivo gustativo muestra una temprana emergencia
durante la vida prenatal y persiste, e incluso resulta potenciado, a edades
avanzadas, y, sus características peculiares, tales como la adquisición en un
ensayo e independencia de la maduración motora, son especialmente adecuadas
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Memoria, Contexto e Hipocampo
para una aproximación de desarrollo. El hecho de que presente fenómenos de
aprendizaje que requieren la función hipocampal, tales como bloqueo
condicionado y modulación por el contexto, permite que sea empleado para
investigar la emergencia y deterioro de las funciones relevantes para la aparición
de dichos fenómenos.
La definición habitual de contexto en términos de estimulación externa,
suele hacer referencia al lugar en que se produce la situación de aprendizaje, lo
cual plantea los problemas de interpretación cuando se aplica a estudios de
desarrollo en ratas, ya que involucra funciones sensoriales de maduración tardía
y/o especialmente vulnerables a los efectos del envejecimiento, así como
procesamiento espacial. Por ello, en el estudio de la función hipocampal desde
una aproximación de desarrollo resulta especialmente interesante el empleo de
contextos independientes de la estimulación exteroceptiva. En este sentido,
estudios previos indican que la hora del día puede actuar como un contexto en sí
misma (Arvanitogiannis, Sullivan y Amir, 2000). De hecho, se ha demostrado
que la hora del día puede emplearse bien como parte del contexto (Bonardi, et
al, 1990; Hall and Channell, 1986) o como el único contexto (Morón et al.,
2002) en aprendizaje aversivo gustativo, abriendo nuevas posibilidades para una
aproximación de desarrollo en el estudio de la función hipocampal.
La presente tesis doctoral pretende emplear la modulación del
aprendizaje aversivo gustativo por parte de la hora del día como herramienta
15
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fundamental para investigar el desarrollo de la función hipocampal a lo largo de
la vida, desde la infancia y adolescencia hasta el envejecimiento.
Adicionalmente, se pretenden emplear otras modalidades de aprendizaje con el
fin de explorar interacciones entre sistemas de memoria. Para ello se plantean
los siguientes objetivos:
1.
Investigar la posibilidad de emplear la hora del día, en ausencia de
otro tipo de estímulos externos, para explorar la especificidad
contextual del fenómeno de inhibición latente en aprendizaje aversivo
gustativo, empleando ratas adultas.
2.
Explorar la emergencia ontogenética en crías de rata del fenómeno de
inhibición latente y su especificidad temporal, empleando tareas de
aprendizaje aversivo gustativo similares a las aplicadas en ratas
adultas.
3.
Investigar el efecto del envejecimiento sobre la especificidad temporal
del fenómeno de inhibición latente en aprendizaje aversivo gustativo
en ratas de 25 meses de edad.
4.
Investigar el efecto de la lesión hipocampal sobre la modulación
temporal del fenómeno de inhibición latente en ratas senescentes.
5.
Evaluar las consecuencias del fracaso temprano, inducido mediante la
exposición de ratas de 18 días a una tarea hipocampal irresoluble a esa
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Memoria, Contexto e Hipocampo
edad, sobre la capacidad de aprendizaje adulta en una tarea de
evitación independiente, que no requiere la integridad del hipocampo.
6.
Evaluar las consecuencias del entrenamiento de ratas de 25 días
durante un periodo crítico de desarrollo hipocampal a una tarea
hipocampal que comienza a ser resoluble a dicha edad, sobre la
capacidad de aprendizaje adulta en una tarea de evitación
independiente, que no requiere la integridad del hipocampo.
Inhibición Latente y Contexto Temporal (Capítulo 3)
Como acaba de mencionarse, una de las funciones atribuidas al sistema
hipocampal es mediar los diversos efectos del contexto en las tareas de memoria
(Holland y Bouton, 1999).
En las tareas de memoria de reconocimiento
gustativo, los efectos del contexto se han puesto de manifiesto empleando
procedimientos que incluyen preexposición al estímulo gustativo. Por un lado,
se ha investigado la dependencia del contexto en el fenómeno de inhibición
latente (IL). Este fenómeno pone de manifiesto cómo la experiencia previa a un
sabor sin consecuencias aversivas retrasa la adquisición posterior de una
aversión condicionada a dicho sabor (Lubow, 1989). Los estudios de Hall y
Channell (1986), y posteriormente de otros autores (Maren y Holt, 2000; De la
Casa y Lubow, 2001; Wesbrook, Jones, Bailey y Harris, 2000), han demostrado
17
Resumen
que el cambio de contexto entre la preexposición y el condicionamiento
interrumpe el fenómeno. Por otro lado, en tareas de discriminación condicionada
se ha planteado que la recuperación de una aversión gustativa aprendida puede
ser dependiente del contexto (Bonardi, et al., 1990; Loy, et al., 1993),
especialmente cuando el sabor ha sido previamente preexpuesto sin
consecuencias (Puente et al., 1988; Boakes et al., 1997).
En conjunto, la
evidencia indica que las señales contextuales pueden facilitar la recuperación
tanto de recuerdos gustativos apetitivos como aversivos.
Es importante destacar que al hablar de contexto pueden incluirse claves
externas e internas, así como un sentido del tiempo (Bouton, 1993). Sin
embargo, la relevancia de las señales temporales como contexto ha sido muy
poco explorada. Existe un estudio en el que se reporta a la hora del día como un
contexto en sí misma, mostrando la dependencia temporal de la expresión de
una sensibilización comportamental a la anfetamina (Arvanitogiannis, et al.,
2000). Experimentos previos en nuestro laboratorio (Morón, Manrique, Molero,
Ballesteros, Gallo y Fenton, 2002b) fueron diseñados con el fin de evaluar la
dependencia contextual espacial y temporal de la inhibición latente. Los
resultados indicaron que el cambio de contexto espacial y temporal tiene efectos
similares sobre el fenómeno de condicionamiento aversivo gustativo (Morón,
2002).
Se demostró que la recuperación de la aversión fue específica de
contexto, demostrando por primera vez que las ratas adultas usan la hora del día
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Memoria, Contexto e Hipocampo
para modular la expresión de una aversión gustativa adquirida en un solo ensayo
de forma similar a lo que ocurrió cuando se empleó un contexto espacial sin
cambios temporales.
El procedimiento usado por Morón et al (2002) incluía 2 días de
habituación a beber agua durante 15 minutos en la mañana y en la tarde, dos
sesiones de preexposición a una solución salina isotónica, restricción a 6 ml de
consumo el día de condicionamiento y 5 sesiones de prueba con botella única.
El experimento del capítulo 3 de esta tesis (publicado en Neurobiology of
Learning and Memory, 2004) se diseño con el objetivo de evaluar si la
inhibición latente del condicionamiento aversivo gustativo depende del cambio
de hora del día. Para ello se hicieron sutiles modificaciones al procedimiento.
Se usaron 4 grupos de ratas Wistar en un diseño 2 x 2 (Preexposición x
Grupo). Los grupos Preexpuestos (Pre), pero no los Controles (Ctrl) recibieron
2 preexposiciones a la solución salina isotónica antes del condicionamiento. En
cada condición (Pre o Ctrl) los animales fueron asignados a dos grupos: un
grupo Same en el que las preexposiciones, el condicionamiento y los tests
tuvieron lugar a la misma hora del día (la sesión de tarde), y un grupo Diff que
fue condicionado a una hora diferente de las preexposiciones y los tests.
Las
modificaciones del procedimiento usado por Morón et al (2002) fueron dos: se
aplicaron 5 días de habituación en lugar de 2 y se les permitió beber libremente
durante el condicionamiento.
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Resumen
Los resultados de este experimento indicaron que el cambio de hora del
día entre la preexposición y el condicionamiento interrumpió el fenómeno de
inhibición latente. El grupo que fue preexpuesto a la solución salina a una hora
diferente del condicionamiento adquirió una aversión de similar intensidad a la
de los grupos control no preexpuestos.
Sin embargo, la reducción en la
magnitud de la aversión fue evidente en los grupos que fueron preexpuestos,
condicionados y probados a la misma hora del día. Las sutiles modificaciones al
procedimiento de Morón et al (2002) aplicadas en el capítulo 3 permitieron
observar la dependencia contextual de la inhibición latente, a diferencia de la
dependencia contextual de la aversión observada en Morón et al (2002). Por lo
que sabemos, esta es la primera demostración de que las ratas usan la hora del
día como un contexto para modular el fenómeno de inhibición latente. Es de
destacar que usando dos procedimientos similares, que básicamente difieren en
la duración de la habituación al contexto y en la existencia o no de restricción
del consumo durante el condicionamiento, se obtienen patrones de ingestión
opuestos en los grupos preexpuestos, sin que haya diferencias entre los grupos
controles no-preexpuestos.
Hall y Channell (1986) propusieron que la duración y el tipo de
habituación al contexto pueden determinar su habilidad para modular el
aprendizaje.
Los datos obtenidos en este capítulo 3 (Manrique, Molero,
Ballesteros, Morón, Gallo y Fenton, 2004) pueden interpretarse en el sentido de
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Memoria, Contexto e Hipocampo
que una habituación más larga, incrementando la saliencia y discriminación
entre sesiones de mañana y tarde, ha facilitado la aparición de la dependencia
contextual de la inhibición latente.
Además, el consumo libre durante el
condicionamiento puede ser una característica adicional para el incremento en la
saliencia temporal del contexto.
En conjunto, los resultados obtenidos por Morón et al (2002) y Manrique
et al (2004) ponen de manifiesto que la hora del día, en ausencia de cambios
externos, actúa como contexto en el condicionamiento aversivo gustativo,
permitiendo el estudio de la dependencia contextual de la aversión y de la
inhibición latente mediante el mismo procedimiento con sutiles modificaciones.
Ello apoya la validez del aprendizaje aversivo gustativo como modelo adecuado
para estudiar efectos contextuales en aprendizaje. A su vez, esto permite
emplear tareas de aprendizaje aversivo gustativo para investigar tanto circuitos
de memoria hipocampales como no hipocampales en ratas adultas, tal y como ha
sido previamente propuesto (Gallo et al, 1999). Aunque la integridad del sistema
hipocampal no sea necesaria para el condicionamiento aversivo gustativo (Gallo
y Cándido, 1995a), el hipocampo puede tener un papel crítico en la mediación
de otros efectos en aprendizaje gustativo, tales como los efectos del contexto.
Aún más, el empleo de la hora del día como contexto permite investigar la
función hipocampal a lo largo de la vida, incluyendo tanto etapas tempranas
21
Resumen
como envejecimiento y eliminando las grandes dificultades de interpretación
asociadas a los cambios evolutivos en las capacidades sensoriales y motoras.
Ontogenia de la inhibición latente y de los efectos del contexto temporal en
aprendizaje aversivo gustativo (Capítulo 5)
Ontogenéticamente, el condicionamiento aversivo gustativo es un tipo de
aprendizaje asociativo primitivo y tempranamente desarrollado. La habilidad
para asociar sabores con el malestar visceral inducido por cloruro de litio y
mostrar aversiones condicionadas en presentaciones posteriores ha sido
reportada tan tempranamente como en fetos de rata (Abate, Pepino, Domínguez,
Spear y Molina 2000; Smotherman, 2002a, 2002b; Smotherman y Robinson,
1985). Ratas recién nacidas y predestetadas son capaces de aprender aversiones
a olores y sabores (Arias y Chotro, 2006; Nizhnikov, Petrov y Spear, 2002;
Rudy y Cheatle, 1977).
Efectivamente, se han observado aversiones a
soluciones saladas o dulces inducidas mediante inyecciones de cloruro de litio
en crías de 5 días evaluadas 5 o 16 días después (Kehoe y Blass, 1986).
Sin embargo, el fenómeno de inhibición latente del condicionamiento
aversivo gustativo muestra una emergencia tardía durante el desarrollo.
Algunos estudios muestran inhibición latente en ratas de 20 a 25 días después de
un gran número de preexposiciones (Franchina, Donato, Patsiokas y Griesemer,
22
Memoria, Contexto e Hipocampo
1980), mientras que otros estudios reportan déficits en la inhibición latente del
condicionamiento aversivo gustativo antes de los 20-25 días de edad (Klein,
Mikulka, Domato, y Hallstead, 1977; Misanin, Blatt, y Hinderliter, 1985;
Misanin, Guanowsky, y Riccio, 1983; Wilson y Riccio, 1973). De hecho,
Nicolle, Barry, Veronesi, y Stanton (1989) en un estudio que incluía todos los
controles adecuados no encontraron evidencia de inhibición latente a soluciones
de café y sacarina en ratas menores de 32 días aplicando 4 preexposiciones
intraorales. Adicionalmente, si el hipocampo es el responsable del
procesamiento del contexto y la hora del día puede considerarse como contexto,
es de esperar que el desarrollo tardío del sistema hipocampal en ratas induzca un
retraso en la ontogenia de los efectos moduladores del aprendizaje aversivo
gustativo. Dada la aparición tardía del fenómeno de inhibición latente es de
esperar que la dependencia temporal de la aversión condicionada aparezca antes
que la dependencia temporal del fenómeno de inhibición latente durante el
desarrollo.
En el capítulo 5 de esta tesis, se presenta un estudio usando el
condicionamiento aversivo gustativo con el fin de explorar la ontogenia de la
inhibición latente (Experimento 1) y su especificidad contextual (Experimento
2) en ratas destetadas aplicando un protocolo de aprendizaje que ha sido
apropiado para inducir la inhibición latente específica de la hora del día en el
23
Resumen
condicionamiento aversivo gustativo en ratas adultas (Capítulo 3, Manrique, et
al, 2004).
Con relación a la ontogenia del fenómeno de inhibición latente, los
resultados del experimento 1 mostraron que la inhibición latente del
condicionamiento aversivo gustativo es evidente a los 32 pero no a los 24 días
de edad aplicando un procedimiento de dos días de habituación a beber agua dos
veces al día durante 15 minutos, dos preexposiciones a la solución salina (0.1%)
y una tercera exposición al sabor seguida de una inyección intraperitoneal de
cloruro de litio (LiCl, 0.15M, 2% peso corporal), idéntico al usado en ratas
adultas. En el experimento 2 se usaron 5 días de habituación en lugar de 2, y se
confirmó la presencia de inhibición latente a los 32 días de edad.
Los resultados son congruentes con los de Nicolle et al (1989) quienes
no encontraron evidencia de inhibición latente en ratas menores de 32 días en el
aprendizaje aversivo gustativo usando cuatro preexposiciones al sabor aplicadas
durante dos días. Estos autores atribuyeron el retraso en la aparición de la
inhibición latente a la inmadurez del sistema hipocampal, dado que la sección
del fórnix llevada a cabo en ratas predestetadas interrumpió la emergencia de la
inhibición latente a los 32 días de edad.
Sin embargo, hasta ahora no ha habido evidencia que ponga de
manifiesto un papel crítico del hipocampo en la inhibición latente del
condicionamiento aversivo gustativo (para una revisión ver Buhusi, Gray y
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Memoria, Contexto e Hipocampo
Schmajuk, 1998; Gallo, Ballesteros, Molero y Morón, 1999). Estudios de lesión
en ratas adultas no muestran efecto (Gallo y Cándido, 1995a) o muestran un
aumento de la inhibición latente (Purves, Bonarde y Hall, 1995; Reilly, Harley y
Revusky, 1993). Aún más, se ha reportado que la sección del fórnix, similar a la
empleada por Nicolle et al. (1989), no interrumpe la inhibición latente en ratas
adultas (Weiner, Feldon, Tarrasch, Harrison y Joel, 1998). Es posible plantear
que la sección del fórnix durante el desarrollo temprano tenga efectos más
generales sobre la organización de los circuitos neuronales obstaculizando una
explicación simple de sus efectos en crías de rata.
El rango de edad elegido en este capítulo para estudiar la ontogenia de la
especificidad contextual de la inhibición latente cubrió el período completo de
adolescencia, desde los 32 hasta los 64 días, lo cual está de acuerdo con
propuestas previas que aplican un criterio laxo en términos de cambios
comportamentales (Spear, 2000). Los resultados mostraron una emergencia
tardía de la modulación del condicionamiento aversivo gustativo por el contexto
temporal, la cual no es evidente en ratas de 32 días. Sin embargo, la modulación
del condicionamiento aversivo gustativo por el contexto temporal en ratas de 48
y 64 días de edad difirió dramáticamente de la observada en el grupo de ratas
adultas.
En las ratas adultas el cambio de contexto temporal interrumpió la
inhibición latente del condicionamiento aversivo gustativo, mostrando
25
Resumen
aversiones similares a las exhibidas por los grupos no preexpuestos. Por lo
tanto, el grupo condicionado a una hora diferente (Diff) de la preexposicion
mostró una aversión más intensa que la del grupo preexpuesto y condicionado a
la misma hora del día (Same). Esta especificidad contextual de la inhibición
latente en ratas adultas confirma los resultados presentados en el capítulo 3,
usando el mismo procedimiento.
Sin embargo, la inhibición latente del condicionamiento aversivo
gustativo no se interrumpió al aplicar el condicionamiento a una hora diferente
de las preexposiciones en los grupos de 48 y 64 días de edad. Los grupos
preexpuestos Same y Diff mostraron aversiones más débiles que sus grupos
control no preexpuestos, indicando la presencia del fenómeno. No obstante, la
aversión fue más intensa en los grupos Same que en los grupos Diff, en los que
el cambio de hora del día indujo un patrón de diferencias opuesto al observado
en los grupos de ratas adultas. Ese patrón de diferencias entre los grupos
preexpuestos Same y Diff se ha reportado previamente en ratas adultas (Morón
et al, 2002) aplicando un protocolo comportamental de dos días de habituación
en lugar de 5 y consumo restringido durante el condicionamiento en lugar del
consumo libre usado en el presente estudio. Una aversión más débil en aquellos
grupos condicionados y evaluados a una hora diferente del día que aquellos
condicionados y evaluados a la misma hora del día puede interpretarse como
especificidad contextual de la aversión en los grupos preexpuestos. Por tanto, la
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Memoria, Contexto e Hipocampo
especificidad contextual de la inhibición latente y la especificidad contextual de
la aversión son fenómenos evolutivamente disociables, mostrando el primero
una emergencia más tardía que el segundo. Una organización peculiar de los
sistemas de aprendizaje y memoria durante la adolescencia, que facilite la
formación de la representación del compuesto sal y hora del día, podría explicar
el hecho de que el mismo procedimiento que permite detectar la especificidad
contextual de la inhibición latente en ratas adultas pueda permitir la
especificidad contextual de la aversión en ratas adolescentes. De esta manera, es
posible observar la especificidad contextual de aversiones gustativas aprendidas
que en ratas adultas sólo es evidente con un protocolo de habituación corta.
En conjunto, los resultados confirman hallazgos previos que indican una
emergencia tardía del fenómeno de inhibición latente en aprendizaje aversivo
gustativo, durante la adolescencia temprana a los 32 días de edad en ratas.
Además, muestran una disociación ontogenética entre el efecto del cambio de
contexto temporal sobre la aversión condicionada, que es evidente a los 48 días
de edad, y sobre el fenómeno de inhibición latente, efecto que únicamente
aparece en el grupo adulto. Ello parece implicar un largo periodo durante la
adolescencia desde 48 hasta 64 días de edad en la rata, durante el cual la
peculiar organización de los sistemas de aprendizaje y memoria facilita
determinados efectos contextuales sobre el aprendizaje e impide la especificad
contextual de la inhibición latente. De acuerdo con la prolongada duración de
27
Resumen
los procesos de maduración hipocampal podría proponerse que la especificidad
temporal de la inhibición latente muestra una emergencia tardía debido a su
dependencia de una determinada función que aún no puede soportar el
hipocampo en desarrollo.
Envejecimiento, Hipocampo y Contexto Temporal (Capítulo 4)
Recientemente ha sido demostrado que la capacidad para segregar
elementos de una experiencia en representaciones internas separadas es
dependiente del hipocampo.
Kubik y Fenton (2005) han estudiado dicha
función segregadora usando tareas espaciales en ratas adultas. Estos autores
proponen que una función fundamental del hipocampo es facilitar la segregación
de representaciones de estímulos en un compuesto, incluso cuando los estímulos
a segregar no sean espaciales (Kesner, Lee y Gilbert, 2004; Wesierska, Dockery
y Fenton, 2005).
En este sentido, el empleo de tareas de condicionamiento aversivo
gustativo con cambio de hora del día puede resultar especialmente adecuado
para poner a prueba esta hipótesis, ya que en ellas es difícil identificar
componente espacial alguno, siendo la hora del día la única variable que permite
distinguir la experiencia de condicionamiento entre los diferentes grupos
preexpuestos. Puede proponerse que la especificad temporal de la inhibición
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Memoria, Contexto e Hipocampo
latente, observada en la rata adulta, requiere dicha segregación entre el sabor y
la hora del día, mientras que para que se produzca la especificidad temporal de
la aversión, que ya es evidente en la rata adolescente, el sabor y la hora del día
son probablemente representados como un único estímulo compuesto. Esta
podría ser la explicación de que en el primer caso durante los tests, llevados a
cabo a la misma hora de la preexposición (contexto seguro), se recupere una
intensa aversión, mientras que en el segundo caso, sea necesario que el sabor se
presente a la hora del condicionamiento para que se incremente la aversión.
Como se mencionó antes, el deterioro cognitivo relacionado con el
envejecimiento no involucra un decaimiento generalizado en el funcionamiento
de los sistemas de memoria. Algunos tipos de memoria están preservados,
mientras otros usualmente decaen durante el envejecimiento normal (Gallagher
y Rapp, 1977; Manrique, Morón, Ballesteros, Guerrero y Gallo, 2007). De
hecho, son aquellas tareas que requieren un hipocampo intacto las que sufren
mayor deterioro a edades avanzadas, probablemente debido a modificaciones
bien conocidas en la función hipocampal (Rosenzweig y Barnes, 2003; Wilson
et al, 2003). Puede proponerse, en consecuencia, que las ratas envejecidas
deben mostrar deficiencias en la modulación temporal de la inhibición latente
empleando aprendizaje aversivo gustativo. El capítulo 5 de esta tesis es un
artículo actualmente en revisión, diseñado para evaluar el efecto del
29
Resumen
envejecimiento y de la lesión del hipocampo dorsal en la modulación temporal
del aprendizaje aversivo gustativo.
Aplicando el procedimiento usado en el Capítulo 3 (Manrique et al.,
2004) se estudió la dependencia contextual de la inhibición latente en ratas
envejecidas (25 meses), cuya ejecución fue comparada con la de ratas adultas
(4-5 meses). En cada edad se usaron únicamente dos grupos preexpuestos, uno
condicionado a la misma hora de las preexposiciones y los test y otro
condicionado a una hora diferente. Los resultados indicaron que a diferencia de
las ratas adultas, las envejecidas no fueron sensibles a la hora del día en la que
fueron condicionadas (Manrique, Gámiz, Morón, Ballesteros y Gallo, 2008, en
revisión).
La magnitud de la aversión no difirió entre estos grupos y su
intensidad fue intermedia a la aversión expresada por las ratas adultas que
fueron condicionadas a una hora familiar y diferente.
Por tanto, mientras las ratas envejecidas adquirieron aversiones
condicionadas al sabor de una magnitud similar a las ratas adultas, no mostraron
modulación de la aversión en función de la hora del día. Este resultado podría
explicarse por la posibilidad de que ratas envejecidas tengan alterada la
detección de señales temporales, pero esta explicación fue descartada en el
Experimento 2. En el segundo experimento se comparó la ejecución de ratas
envejecidas intactas con aquellas que recibieron lesiones excitotóxicas del
hipocampo dorsal, mediante la inyección intracerebral de NMDA (0.077M).
30
Memoria, Contexto e Hipocampo
Sorprendentemente, las ratas lesionadas, a diferencia de las intactas, usaron la
hora del día para modular la expresión de la aversión condicionada al sabor,
aunque mostraron un patrón de ingestión opuesto al de las ratas adultas intactas
del primer experimento. El grupo preexpuesto, condicionado y probado a la
misma hora del día mostró una mayor aversión a la solución salina en
comparación con el grupo condicionado a una hora diferente. Se trata de un
patrón similar al observado en ratas adolescentes, como se ha descrito en el
capítulo anterior, que puede interpretarse como especificidad temporal de la
aversión condicionada.
El hecho de que la lesión del hipocampo dorsal en ratas envejecidas
facilite la modulación de la aversión gustativa aprendida por parte de la hora del
día demuestra que las ratas envejecidas pueden usar la hora del día en
representaciones de la experiencia. Sin embargo, la pérdida de la especificidad
temporal del fenómeno de inhibición latente en animales lesionados parece
indicar que la habilidad para codificar, mantener o usar representaciones
segregadas del sabor y las señales temporales requiere un hipocampo intacto.
Por otro lado, los resultados ponen de manifiesto que los efectos del
envejecimiento no son comparables a los producidos por la lesión hipocampal,
ya que no se observó modulación temporal alguna en los animales envejecidos,
mientras que el efecto se invirtió con respecto a los adultos en ratas envejecidas
con lesión hipocampal. La ausencia de efecto del cambio de hora en ratas
31
Resumen
senescentes intactas refleja un deterioro cognitivo que puede ser el resultado de
alteraciones en la función hipocampal provocadas por la edad. Además, la
facilitación de la especificidad temporal de la aversión aprendida por parte de la
lesión hipocampal es consistente con otras evidencias experimentales que
sugieren interacciones entre múltiples sistemas de memoria. En este caso, los
resultados apoyan la existencia de interacciones competititivas, mostrando un
efecto inhibidor por parte del sistema hipocampal sobre los circuitos cerebrales
responsables de la modulación temporal de la aversión aprendida, inhibición que
es eliminada por la lesión, pero no por la disfunción hipocampal asociada al
envejecimiento. La naturaleza de la interacción entre sistemas de aprendizaje y
memoria diferentes puede verse modificada a lo largo de la vida en función de
las experiencias vividas y de las modificaciones inducidas por la edad.
Fracaso Temprano y Aprendizaje Adulto (Capítulo 6)
Como se ha mencionado previamente, ciertas tareas de aprendizaje y
memoria que en el adulto dependen del sistema hipocampal, tales como
desigualación a la muestra demorada y aprendizaje espacial, muestran
emergencia tardía durante el desarrollo temprano y decaimiento asociado al
envejecimiento. En las tareas de aprendizaje espacial se han reportado déficits
en ratas predestetadas menores de 20 días en la adquisición de la tarea de
32
Memoria, Contexto e Hipocampo
búsqueda de plataforma oculta en el laberinto acuático (Morris, 1981), debido a
la incompleta maduración del sistema hipocampal, el cual permite integrar
señales distales para la realización de esta tarea. En general, la ejecución en esta
tarea decae a edades avanzadas (Jones, Barnes, Kirkby y Higgins, 1995; Quirón
et al, 1995; Greferath, Bennie, Kourakis y Barret 2000; Smith, Al-Khamees,
Costall, Naylor y Smythe, 2002; Yau et al., 2002) y ello se ha relacionado con
disfunciones hipocampales asociadas al envejecimiento normal (Gallagher,
Nagahara y Burwell, 1995; Zhong et al., 2000).
Teniendo en cuenta la posibilidad de interacción entre distintos sistemas
de memoria planteada previamente y el hecho de que el hipocampo pueda jugar
un papel crítico en dicha interacción, como pone de manifiesto su participación
en diversos tipos de aprendizaje cuando se emplean fenómenos complejos,
resulta de especial interés explorar la posibilidad de modificar capacidades de
aprendizaje generales actuando sobre el sistema hipocampal a edades tempranas.
Ello podría realizarse sometiendo a los animales a tareas hipocampales durante
el periodo crítico de aparición de la capacidad de aprendizaje. El procedimiento
a seguir podría consistir en entrenar a ratas de alrededor de 20 días en una tarea
espacial de búsqueda de plataforma oculta en laberinto acuático. Si la
maduración hipocampal a esta edad permitiera el aprendizaje de la tarea, el
procedimiento representaría la estimulación temprana del sistema hipocampal, y
podría ejercer un efecto beneficioso sobre el desarrollo posterior de las
33
Resumen
capacidades de aprendizaje, en general. Por el contrario, si la inmadurez
hipocampal a esa edad impide el aprendizaje, el animal se encontraría expuesto
a una situación irresoluble, con las consecuencias emocionales que conlleva la
frustración. En efecto, la exposición a eventos incontrolables puede dificultar la
capacidad para aprender en tareas posteriores.
En el paradigma convencional de indefensión aprendida las ratas son
expuestas a choques incontrolables y luego evaluadas en tareas de escape o
evitación (Drugan, Basile, Ha, Healy y Ferland, 1997; Vollmayr y Henn, 2001).
Aunque el fenómeno de indefensión aprendida ha sido reportado usando
diferentes tareas (para una revisión ver Maier y Seligman, 1976; Overmier,
2002), parece ser que un factor importante es la incontrolabilidad, ya sea a la
hora de eventos aversivos inescapables o problemas de discriminación
irresolubles (Hiroto y Seligman, 1975). Por tanto, la incontrolabilidad puede ser
inducida aplicando una tarea irresoluble o con demandas de aprendizaje
excesivas en una etapa temprana del desarrollo maduracional y sabiendo que las
experiencias tempranas con eventos estresantes incontrolables pueden tener
efectos a largo plazo (Hannum, Rosellini y Seligman, 1976; Seligman y
Visintainer, 1985).
El Capítulo 6 de esta tesis presenta un estudio diseñado con el propósito
de evaluar el efecto de someter a ratas en desarrollo a una tarea hipocampal
resoluble, bien a una edad en que comienzan a ser capaces de resolverla o bien a
34
Memoria, Contexto e Hipocampo
una edad más temprana en la que es irresoluble debido a la inmadurez cerebral,
sobre la capacidad para aprender una tarea de evitación independiente, que no
requiere la integridad del hipocampo, una vez que son adultas. Esta
investigación longitudinal ha sido publicada en Developmental Psychobiology
en 2005 por Manrique, Molero, Cándido y Gallo.
En un primer experimento piloto se comparó la ejecución de ratas
adultas, que habían sido entrenadas durante la infancia en una tarea espacial de
búsqueda de plataforma oculta en laberinto acuático, en la adquisición de una
respuesta de evitación ante un choque pareado a un tono. De acuerdo con la
experiencia temprana de aprendizaje los sujetos fueron asignados a tres grupos
contrabalanceados por sexo: PN17 y PN25, teniendo lugar la primera sesión de
la tarea espacial los días postnatales 17 y 25 respectivamente. El grupo control
(Ctrl) no fue entrenado durante el desarrollo. Al terminar esta fase, las ratas
permanecieron agrupadas por sexo en grupos de tres o cuatro hasta los 3 meses,
edad en la que fueron individualizados para iniciar el entrenamiento de evitación
de un choque en presencia de un tono. Los resultados del aprendizaje temprano
en la tarea de laberinto acuático de Morris (Morris, 1981) revelaron que el grupo
con 25 días aprendió a localizar la plataforma oculta en 10 bloques de ensayos
(2 bloques diarios de 4 ensayos cada uno) mientras que el de 17 días no lo hizo.
Estos resultados confirman informes previos sobre un desarrollo tardío del
aprendizaje espacial en ratas (Carman y Mactutus, 2001; Kraemer y Randall,
35
Resumen
1995; Rudy y Paylor, 1988; Rudy, Staedler-Morris y Albert, 1987). Rudy et al
(1987, 1988) reportaron déficits maduracionales en ratas con menos de 20 días
en habilidades espaciales requeridas para aprender la relación entre la
plataforma oculta y claves distales, aunque fueron capaces de aprender la
localización de la plataforma usando claves proximales. Adicionalmente, los
resultados confirman otros reportes (Carman y Mactutus, 2001; Kraemer y
Randall, 1995) mostrando una ejecución inferior en crías menores de 20 días
comparada con ratas mayores, incluso cuando se usan procedimientos de
aprendizaje con ensayos espaciados, lo cual facilita el aprendizaje al reducir la
fatiga. Sin embargo, en nuestros experimentos el déficit en aprendizaje espacial
fue facilitado por el gran tamaño de la piscina.
A los tres meses de edad los grupos (PN17, PN25, Ctrl) fueron
individualizados y entrenados en la tarea de evitación con salto en el aire
siguiendo el procedimiento descrito en Cándido, Maldonado y Vila (1988). En
resumen, los sujetos debían saltar ante la presencia de una señal auditiva de
aviso para evitar una descarga en las patas. El entrenamiento terminó cuando
alcanzaron 10 respuestas consecutivas de evitación (Consecutive Avoidance
Responses, CARs, sus siglas en inglés) o hasta un máximo de 240 ensayos en 4
sesiones (cada una de 60 ensayos). Los resultados de este entrenamiento adulto
mostraron que los sujetos que fracasaron en la resolución de la tarea espacial a
la edad de 17 días debido a su desarrollo inmaduro, necesitaron un mayor
36
Memoria, Contexto e Hipocampo
número de ensayos para adquirir la respuesta de evitación que el grupo
entrenado a los 25 días en la tarea de laberinto acuático, pero también que el
grupo control sin experiencia previa. No se observó mejora significativa sobre el
aprendizaje adulto inducida por el éxito en la resolución de la tarea en el
laberinto acuático de Morris, en el grupo que fue entrenado a los 25 días de
edad.
Se diseñó entonces un segundo experimento con el fin de replicar los
resultados del experimento piloto añadiendo dos grupos control para excluir
explicaciones alternativas en términos de incapacidad para aprender una tarea
resoluble o haber sido sometidos a ejercicio. El entrenamiento fue similar al del
experimento piloto con tres diferencias. Primero, en lugar de 10 bloques de
ensayos en el laberinto acuático se aplicaron 8, dado que se inició la tarea a los
18 días de edad permitiendo un mayor desarrollo sensorial.
Segundo, se
añadieron dos grupos controles, uno en el que la plataforma cambiaba de
cuadrante al azar en cada ensayo, convirtiendo la tarea en irresoluble para ambos
grupos de edad, y otro sin plataforma dentro del agua, para explorar los efectos
del ejercicio. Cada sujeto de estos grupos fue igualado en sexo y punto de
partida en el laberinto a un sujeto del grupo experimental permaneciendo el
mismo tiempo en el agua. Tercero, se añadió un test de retención sin plataforma
al terminar el octavo bloque de ensayos.
37
Resumen
De forma congruente con el experimento 1, las ratas adultas que fueron
entrenadas a los 18 días para localizar la plataforma oculta en el laberinto
acuático, necesitaron un mayor número de ensayos para alcanzar los criterios de
aprendizaje (CARs 3, 5 y 10) en la tarea de evitación del choque con salto en el
aire que aquellas ratas que recibieron un entrenamiento previo similar a la edad
de 25 días. En ambos experimentos los grupos más jóvenes mostraron déficits
en aprendizaje espacial, evidenciando latencias más largas a la hora de localizar
la plataforma, y en el experimento 2, menos tiempo de búsqueda en el cuadrante
objetivo durante la prueba, comparado con los grupos de mayor edad.
Adicionalmente, el grupo entrenado a los 25 días de edad en la tarea espacial sin
plataforma mostró un retraso en la adquisición del aprendizaje de evitación,
evidente sólo cuando se incremento la dificultad. Dado el patrón de inmovilidad
observado en esta condición en el grupo mayor, pero no en el menor, los
resultados pueden interpretarse en términos de un efecto general de la inducción
de indefensión aprendida en periodos tempranos del desarrollo sobre el
aprendizaje adulto.
De hecho, la magnitud del déficit adulto observado en la tarea de
evitación parece estar relacionada con la percepción de fracaso en el aprendizaje
más que con el resultado de la tarea espacial temprana, ya que el efecto no
aparece en la tarea irresoluble con posiciones variables de la plataforma.
Resultados previos
38
muestran
que
la
experiencia
con
problemas
de
Memoria, Contexto e Hipocampo
discriminación irresolubles puede llevar a la indefensión aprendida y a déficits
de aprendizaje posteriores (Hiroto y Seligman, 1975) dando apoyo a esta
propuesta.
Esta interpretación podría implicar que las ratas más jóvenes
discriminan entre la tarea espacial con plataforma fija como resoluble, pero
identifican como irresolubles la plataforma al azar y la tarea sin plataforma.
Tanto la plataforma fija como al azar implican colocar al animal en la
plataforma después de cada ensayo si no la encuentra durante el ensayo de 60
segundos. Es de esperar que las ratas más jóvenes sean capaces de identificar la
localización de la plataforma basandose en claves proximales. Podrían entonces
percibir la condición de plataforma fija como una tarea resoluble a pesar de ser
incapaces de resolverla, puesto que la tarea requiere un procesamiento complejo
de claves distales. Sin embargo, los animales pueden percibir la condición al
azar como una tarea irresoluble debido a que la localización de la plataforma
cambia. Así, aunque el resultado es similar en ambas condiciones, es decir, el
fracaso en la localización de la plataforma, la evaluación cognitiva de la
situación puede ser diferente. Se ha propuesto que la experiencia en situaciones
donde la percepción es que las demandas superan los recursos puede ser una
importante fuente de estrés (Kemeny, 2003). Se puede plantear entonces que el
efecto pernicioso del entrenamiento temprano en la tarea con plataforma fija
sobre el aprendizaje adulto puede deberse a un nivel de demanda excesivo,
llevando a la indefensión aprendida.
39
Resumen
Los resultados de estos experimentos abren nuevos conocimientos sobre
el efecto de la intervención temprana durante periodos sensibles del desarrollo
en la modificación de la organización de los circuitos neuronales, que
conduzcan a cambios permanentes que influencian el aprendizaje y la
plasticidad neuronal adulto (Kolb, Gibb y Robinson, 2003; Mlynarik, Johansson
y Jezova, 2004). Los resultados del grupo experimental más joven en la tarea
espacial muestran que el entrenamiento espacial temprano tuvo lugar durante un
periodo de desarrollo crítico para la función hipocampal, en el que las
habilidades de aprendizaje estaban emergiendo. Por lo tanto, la formación de
circuitos cerebrales específicos relevantes para esta tarea podría haber sido
influenciada por el fracaso en el aprendizaje temprano. Sin embargo, el hecho
de que los déficits adultos fueran encontrados en una tarea de aprendizaje
independiente dos meses y medio después del entrenamiento temprano apunta a
un efecto pernicioso general sobre los mecanismos plásticos cerebrales, que
pudiera ser mediado por circuitos emocionales. Esto podría implicar que el
entrenamiento temprano en una tarea, antes de que los circuitos cerebrales
específicos involucrados alcancen el estado maduracional necesario para
resolverla, puede tener efectos de larga duración sobre el aprendizaje adulto. En
conjunto, los resultados de esta investigación apuntan al papel de las
experiencias de aprendizaje y la historia temprana de éxito o fracaso en la
formación de habilidades de aprendizaje adulto en general.
40
Memoria, Contexto e Hipocampo
Conclusiones
1. La hora del día actúa como un contexto en la modulación del aprendizaje
aversivo gustativo en ratas adultas.
1.1 Un cambio de contexto temporal entre la preexposición y el
condicionamiento
interrumpe
la
inhibición
latente
del
condicionamiento aversivo gustativo en ratas adultas, si se
emplea un protocolo que incluye 5 días de habituación a beber
dos veces al día y no se restringe la cantidad ingerida durante la
sesión de condicionamiento.
1.2 Cambios en la duración del procedimiento de habituación a los
contextos temporales usados modifican el efecto del cambio de
hora entre exposición, condicionamiento y tests, poniendo de
manifiesto la especificidad temporal ora de la aversión aprendida,
ora del fenómeno de inhibición latente.
2
El fenómeno de inhibición latente, la especificidad temporal de la aversión
aprendida y la especificidad temporal de la inhibición latente del aprendizaje
aversivo gustativo muestran cursos de desarrollo ontogenético disociables
que se suceden durante la adolescencia en ratas.
41
Resumen
2.1 El fenómeno de inhibición latente del aprendizaje aversivo
gustativo es evidente en ratas de 32 pero no de 24 días de edad,
cuando se emplea un protocolo idéntico al aplicado en adultos.
2.2 La especificidad temporal del fenómeno de inhibición latente del
aprendizaje aversivo gustativo muestra una aparición tardía, no
siendo evidente en ratas de 48 ni 64 días de edad.
2.3 La modulación de la memoria de reconocimiento gustativo por la
hora del día durante la adolescencia muestra características
peculiares. La especificidad temporal de la aversión gustativa
aprendida resulta facilitada en ratas de 48 y 64 días de edad,
poniéndose de manifiesto con el procedimiento comportamental
que en ratas adultas evidencia la especificidad temporal de la
inhibición latente.
3
El envejecimiento deteriora la especificidad temporal del fenómeno de
inhibición en aprendizaje aversivo gustativo. Las ratas senescentes
sometidas a la sesión de condicionamiento a una hora diferente de la de las
preexposiciones y tests adquirieron aversiones gustativas de la misma
magnitud que aquellas en las que todas las sesiones tuvieron lugar a la
misma hora.
4
La lesión neurotóxica mediante inyección intracerebral de NMDA en ratas
senescentes facilita la especificidad temporal de la aversión gustativa,
42
Memoria, Contexto e Hipocampo
haciéndose evidente con el procedimiento comportamental que en ratas
adultas induce especificidad temporal de la inhibición latente.
5
El entrenamiento en tareas irresolubles durante el periodo crítico de
desarrollo hipocampal, en el que dichas capacidades de aprendizaje están
emergiendo, puede ejercer un amplio y duradero efecto sobre los sistemas de
memoria alterando la capacidad de aprendizaje adulto.
5.1 Ratas de 18 días de edad que fracasaron en una tarea hipocampal
irresoluble, como es la búsqueda de plataforma oculta en el
laberinto acuático, debido a la inmadurez del sistema
hipocampal, mostraron un retraso en la adquisición de una
respuesta de evitación cuando fueron adultas.
5.2 No se observaron efectos facilitadores del éxito temprano en la
tarea de búsqueda de plataforma oculta en el laberinto acuático
sobre el aprendizaje de evitación adulto.
5.3 El entrenamiento en la misma tarea a los 25 días, edad en que
comienza a ser resoluble, no alteró la capacidad de aprendizaje de
evitación adulta. Sin embargo, el grupo que a la misma edad fue
sometido a ejercicio en ausencia de la plataforma mostró retraso
en el aprendizaje adulto con mayor nivel de dificultad.
43
Resumen
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61
CHAPTER 1
Manrique, T. y Gallo, M. (2005).
Neurobiología del aprendizaje aversivo gustativo.
Mente y Cerebro, 11: 39-40.
Neurobiología del aprendizaje aversivo gustativo
¿Por qué ciertos sabores nos resultan desagradables mientras que otros nos
producen sensaciones placenteras? ¿Qué mecanismos cerebrales permiten
aprender nuevas aversiones a los sabores?
Nuestro bienestar depende de la selección de una dieta adecuada que
excluya sustancias nocivas para el organismo. Para distinguir las sustancias
beneficiosas de las perjudiciales nos valemos del sentido del gusto. Pero el olor
adquiere también especial relevancia cuando se presenta en combinación con
información gustativa dando lugar a lo que denominamos “sabor”.
A lo largo de la evolución se han venido desarrollando preferencias por
los sabores dulce y salado, propios de los hidratos de carbono y sales minerales
necesarios para el organismo. Los sabores amargo y ácido, que suelen ir
asociados a venenos y alimentos en mal estado, tienden a resultar
desagradables. Sin embargo, cada individuo desarrolla su propio repertorio de
preferencias y aversiones en el curso de la vida. Comenzó a forjarse en la etapa
fetal y continúa enriqueciéndose hasta edades avanzadas, gracias a diversos
procesos de aprendizaje gustativo.
Entre tales procesos, la adquisición de
aversiones gustativas representa un tipo de aprendizaje de especial interés, pues
Chapter 1
permite establecer en un solo ensayo asociaciones selectivas entre el sabor de
lo que hemos comido o bebido y efectos nocivos que no se manifiestan de
forma inmediata. Se produce siempre que la ingestión de un sabor desconocido
vaya seguida de malestar gastrointestinal, que, a su vez, suele ir acompañado
de náuseas y vómitos. Ante esa experiencia, el sabor se convierte en
desagradable y es rechazado en posteriores ocasiones, protegiendo de
sustancias nocivas al organismo.
Pionero en la investigación de tales asociaciones fue John García, de la
Universidad de California. Experimentando con ratas, en los años cincuenta y
sesenta del siglo pasado, las conclusiones a que llegó pusieron en tela de juicio
los principios generales del aprendizaje tal y como eran postulados en la época:
contigüidad temporal, ensayos repetidos y equipotencialidad de los eventos a
asociar. La controversia desencadenada a raíz de los hallazgos de García
estimuló la investigación experimental de los mecanismos cerebrales
implicados en ese tipo de aprendizaje. En España, a finales de los setenta,
Amadeo Puerto, hoy en la Universidad de Granada, inició una línea de trabajo
que ha sido continuada por varias generaciones de psicobiólogos.
El cerebro muestra una sorprendente capacidad para asociar sensaciones
de malestar gastrointestinal con sabores, una facultad menos evidente cuando
se trata de otras modalidades sensoriales. Basta que el malestar visceral se
produzca después de haber probado un sabor desconocido en una sola ocasión
66
Neurobiología del aprendizaje aversivo gustativo
para que se desarrolle una intensa aversión gustativa. Tras ese fenómeno podría
hallarse la estrecha relación anatómica entre los sistemas sensoriales
implicados. En efecto, las vías cerebrales que procesan la información
gustativa y visceral se superponen desde el primer nivel de relevo sensorial.
Este tipo de aprendizaje lo observamos en toda la escala filogenética, así como
en etapas precoces del desarrollo ontogenético del individuo. Se trata, pues, de
un carácter primitivo; tesis que no se debilita porque los cerebros menos
desarrollados no soporten largas dilaciones entre los estímulos. Las pruebas
obtenidas en roedores sobre sus bases cerebrales por diversos laboratorios,
incluido el nuestro, ha permitido situar el locus asociativo básico en el área
parabraquial, segundo relevo de información gustativa y visceral.
Esa
estructura se halla en el tronco cerebral, una de las subdivisiones más antiguas
del encéfalo. En experimentos con ratas, la inactivación reversible del área
parabraquial después de la sesión de condicionamiento impide la formación de
aversiones gustativas, tanto si el malestar ha sido inducido mediante
inyecciones de cloruro de litio como si ha sido provocado por rotación
corporal, sin que se haya interrumpido el procesamiento gustativo, visceral o
ambos.
67
Chapter 1
Sin embargo, la capacidad de establecer asociaciones gustativoviscerales no puede explicarse exclusivamente por convergencia anatómica en
la zona asociativa cuando median minutos, e incluso horas en condiciones
óptimas, entre el sabor y el malestar visceral. Se requieren mecanismos de
memoria gustativa que permitan la existencia de convergencia temporal,
además de anatómica.
Al parecer, las conexiones recíprocas directas de la
corteza insular gustativa y la amígdala con el área parabraquial añaden nuevas
posibilidades de memoria y posibilitan un procesamiento más complejo de los
estímulos a asociar. A su vez, este circuito neural especializado se beneficia de
68
Neurobiología del aprendizaje aversivo gustativo
la interacción con sistemas generales de aprendizaje y memoria; en particular,
si tales sistemas intervienen en la formación de aversiones moderadas en la
vida cotidiana, cuando se trata de sabores conocidos y no aparecen aislados,
sino en complejas combinaciones. En estos casos la experiencia adquirida
modula la adquisición de nuevas aversiones.
Aunque el hipocampo dorsal no forma parte del circuito básico
necesario para adquirir aversiones gustativas, podría participar en la
modulación del aprendizaje aversivo gustativo, relacionado con la experiencia
previa y las condiciones ambientales. A diferencia del aprendizaje aversivo
gustativo básico, algunas de estas funciones decaen con la edad. Nuestra
investigación ha demostrado que pueden ser restablecidas en ratas de edad
avanzada o que han sufrido daño hipocampal mediante trasplantes de tejido
precursor hipocampal embrionario.
En conjunto, los resultados obtenidos en nuestro laboratorio, junto a M.
a
Angeles Ballesteros, Ignacio Morón y Andrés Molero, sugieren que el
aprendizaje aversivo gustativo podría constituir un modelo privilegiado para
estudiar la organización de los sistemas de aprendizaje y memoria. El complejo
circuito neural del que depende la adquisición de aversiones a los sabores
integra áreas cerebrales situadas en diferentes niveles de organización e
interacciona con otros sistemas de memoria independientes.
69
CHAPTER 2
Manrique, T., Morón, I., Ballesteros, MA., Guerrero, RM.
and Gallo, M. (2007).
Hippocampus, ageing and taste memories.
Chemical Senses, 32(1): 111-117.
Hippocampus, Ageing, and Taste Memories
a,b
Manrique, T., a,b Morón, I, a Ballesteros, M.A., a,b Guerrero, R. and a,b Gallo,
M.
a
Department of Experimental Psychology and Physiology of Behavior, University of
Granada, Campus Cartuja, Granada-18071, Spain.
b
Institute of Neurosciences F.
Oloriz, University of Granada, Spain.
ABSTRACT
Previous studies have shown that ageing may induce deficits in hippocampaldependent learning and memory tasks, the spatial task being most extensively applied
in rats. It is proposed that taste learning and memory tasks may assist in
understanding the ageing of memory systems, giving access to a more complete
picture. Taste learning tasks allow us to explore a variety of learning phenomena in
safe and aversive memories using similar behavioral procedures. In demanding the
same sensory, response, and motivational requirements, this approach provides
reliable comparisons between the performance of hippocampal lesioned and aged rats
in different types of memory. Present knowledge on the effect of both ageing and
hippocampal damage in complex taste learning phenomena is reviewed. Besides
inducing deficits in hippocampal-dependent phenomena, such as blocking of
conditioned taste aversion, while at the same time leaving intact non-hippocampaldependent effects, such as latent inhibition, ageing is also associated with an
increased neophobia by previous aversive taste memories and enhanced taste aversion
conditioning which cannot be explained by age-related changes in taste or visceral
distress sensitivity. In all, the results indicate a peculiar organization of the memory
systems during aging that cannot be explained by a general cognitive decline or
exclusively by the decay of the hippocampal function.
Chapter 2
This article was supported by the CICYT grants BSO2002-01215 (Ministerio de Ciencia y
Tecnología, Spain) and SEJ2005-01344 (Ministerio de Educación y Ciencia, Spain) which are
both partially supported by Fondo Europeo de Desarrollo Regional.
Key words: ageing, conditioned blocking, context, hippocampal, latent inhibition,
taste aversion, taste recognition memory, time of day, rat
2.1. Introduction
Ageing is a developmental process that offers a privileged opportunity
to study the plasticity of the memory systems induced by both a long-life
learning experience, together with changes in some body functions and new
adaptive requirements. The cognitive decline related to ageing does not involve
a general decay in the functioning of the memory systems. Some types of
memories are spared, whereas others usually decay during normal ageing
(Gallagher and Rapp, 1997). This complex picture has arisen from research
using a variety of different learning procedures that may involve different
sensory modalities and different response requirements. Well-known changes
in the hippocampal function (Rosenzweig and Barnes, 2003; Wilson et al.,
2005) have been related to a lower performance of aged rats in various memory
tasks, there being the spatial tasks most extensively studied. However, the
conventional behavioral tasks used to study the hippocampal functions have
several pitfalls (Eichenbaum and Fortin, 2003) that are magnified when applied
74
Hippocampus, Ageing and Taste Memories
to aged animals. The main criticism concerns the fact that some of these tasks
may involve sensory, motor, and motivational requirements that may decline
during ageing, Thus, a worse performance in a learning and memory task by
old-age rats compared with young adult rats could have several interpretations,
not necessarily related to the hippocampal involvement in learning and
memory. For example, considerable research has focused on the use of the
hidden platform water maze task on which performance is impaired in both
aged and hippocampal-damaged young adult rats. Due to the visual component
of the task, a worse performance of old rats in this task could be due to the well
described effects of visual system degeneration by ageing. Even if the aged rats
behave as young adults in cued or visible platform control versions of the task,
lower visual in the control tasks compared with those requiring processing of
several environmental distal cues in the experimental task cannot be excluded.
Motor deficits in aged rats can be another confounding variable if the
escape latency is measured because swimming speed may decrease in aged
rats. Even if path length is used to test learning and memory of the platform
location, escape latency is a useful measure to assess the potential motivational
changes induced by aging. Motivational differences between old and young
adult rats have been stressed as a major point of concern in ageing studies
using the water maze task as poorer thermoregulation of aged rats may affect
either the motivation to escape from the water or the emotional reactivity to the
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test situation (Van der Staay, 2002). In all, cued or visible platform versions of
the task proposed as the best control for motivational variables are only useful
if the escape latencies of young adult and old rats are similar, which should not
be expected in most of the cases.
We have previously proposed taste memory tasks as a suitable model
for the study of hippocampal and non-hippocampal brain memory circuits in
adult rats (Gallo et al., 1999). We also propose taste memory tasks as a choice
paradigm for studying ageing-induced changes in different neural systems and
cognitive domains. Taste recognition memory tasks allow us to study aversive
and safe memories with dissociable neural, cellular, and molecular mechanisms
(Bermúdez-Rattoni, 2004). Aversive and safe taste memories have been widely
studied in the laboratory using novel tastes which produce an innate response
called neophobia. This consists of a tendency to reject a novel taste when it is
first presented to the animal (Lubow, 1989; Morón and Gallo, 2007).
Safe taste memories are learned when a novel taste is presented without
visceral malaise. As a consequence, there will be an increase in taste
consumption in successive presentations of the taste. This learning process is
called habituation of neophobia (Lubow, 1989).
Conditioned taste aversions (CTAs) are learned when a taste is followed
by aversive visceral consequences. The basic procedure for inducing aversive
taste memories in the laboratory involves a single pairing of a novel taste (CS)
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Hippocampus, Ageing and Taste Memories
and a malaise-inducing treatment (US). An intraperitoneal injection of lithium
chloride (LiCl) is typically used as the US. As a consequence, the taste
becomes aversive inducing orofacial aversive reactions and being avoided in
later presentations.
Besides basic aversive and safe memories that depend on the
consequences of ingesting novel tastes, the development of taste memories in
daily life is profoundly modulated by previous and other ongoing experiences
with the same or different tastes. Thus, understanding the effect of previous
taste experience plays a major role in investigating taste memory and its role in
diet selection at an advanced age. This can be investigated in the laboratory by
using modified behavioral procedures that have been thoroughly studied in a
variety of learning tasks. Contrary to early indications, CTA can access a
variety of so-called complex learning phenomena, relying on the effect of
previous experience, such as latent inhibition (LI), the US preexposure effect,
and blocking, and also exhibits sensitivity to the context. LI refers to reduced
conditioning if a familiar taste solution previously exposed without aversive
consequences is used as the CS. The effect of the US exposure refers to a
similar reduced conditioning if the US, LiCl injection for instance, was
previously applied without being associated with the conditioned taste.
Blocking consists of a reduced conditioning of a taste if it is presented
during the conditioning trial in compound with a second taste that had
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previously been paired with the US. Moreover, both safe and aversive taste
memories are sensitive to the context where they are established. For instance,
a context change either between preexposure and conditioning or between
conditioning and testing can disrupt learning. In the former case, the context
change disrupts LI, and conditioning proceeds as if a novel taste was used. In
the latter case, the context change impairs retrieval of the taste aversion. For a
summary of the behavioral procedures used see Table 1. Demonstrating each of
the above-mentioned learning phenomena requires at least 2 groups of animals
as the experimental group should show a different strength aversion than a
control group without previous experience or not subjected to the context
change. In order to detect differences in consumption between groups and to
avoid ceiling effects, one-bottle tests are required. Lower US dosage and
several trials are also applied for some of the effects to appear.
Although basic CTA does not involve the hippocampus, more complex
learning phenomena may either be hippocampal or non-hippocampal
dependent. Thus, taste memory may be a valuable tool for exploring the aging
impact on hippocampal-dependent cognition.
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Hippocampus, Ageing and Taste Memories
2.2. Ageing and taste memories
Although ageing does not affect some taste memory abilities, it may
induce either an enhancement or impairment of other taste memory tasks.
First, ageing does not impair unconditioned reactions to tastes. No effect of
ageing has been reported in neophobia to a grape juice solution (Gallagher and
Burwell, 1989) or to sodium saccharin (0.1%) (Morón and Gallo, 2007),
sodium chloride (0.5%), and cider vinegar (3%) solutions (Morón et al.,
2002a). The acquisition of safe taste memories seems also to be largely spared
in aged rats. Habituation of the neophobic response, implying an increased
intake of the now familiar taste solution, is also evident in aged rats. Although
a diminished habituation of grape juice neophobia in aged rats has been
reported (Gallagher and Burwell, 1989), we have found similar habituation of
neophobia in adult and aged rats using a low concentration (0.5%) sodium
chloride solution (Morón et al., 2002a). Moreover, the LI phenomenon is not
impaired by ageing. Both in young adult and ageing Wistar rats, the acquired
safe taste memory after 6 preexposures to a saline solution interferes with the
acquisition of a LiCl-induced aversion to this familiar taste. The experimental
preexposed group showed weaker saline aversions than the control nonpreexposed group, as demonstrated by a higher saline intake in a one-bottle test
79
Chapter 2
(Morón, et al., 2002b).
Second, far from being impaired, the acquisition of aversive taste
memories is even potentiated in aged rats (Misanin et al., 2002a; Misanin et al.,
2002b; Morón et al., 2002a). Saccharin (0.1%) taste aversions assessed in a 24h saccharin–water choice test can be induced in 24- to 30-month-old Wistar
rats using longer delays between the taste and the LiCl injections than in
younger rats. Wistar rats of 2 and 2.5 years of age, but not those of 0.25, 1, and
1.5 years of age, developed taste aversions using a 360¬min delay between
saccharin and lithium (Misanin et al., 2002a). It has been consistently shown
that when using a conventional 15-min delay between the taste solution and the
lithium injection, a stronger taste aversion is found in ageing Wistar rats
compared with young adult rats provided that ceiling effects are avoided by
presenting a palatable 0.5% saline solution previously exposed (Morón et al.,
2002a), a 3% cider vinegar solution in compound with a 0.1% sodium
saccharin solution (Morón et al., 2002a), or by using a low LiCl dosage (1%
body weight [b.w.], 0.15 M) after a saccharin solution intake (Morón and
Gallo, 2007). Although the latter aversions were assessed in one¬bottle tests,
an interpretation based on an age-related unspecific reduction of fluid intake
seems to be excluded by the absence of differences between aged and young
adult rats in taste consumption during the conditioning session.
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Hippocampus, Ageing and Taste Memories
Several explanations that could account for this enhancement of taste
aversion learning in aged rats can be excluded. An enhanced sensitivity to the
CS, leading to greater taste intensity, is not supported by the fact that the
preference for the 0.1% saccharin solution over water does not change in 30
month-old rats compared with 3- and 12-month-old rats (Misanin et al., 2002a).
Moreover, the amount of the taste solution ingested during conditioning
follows a pattern of differences that does not correspond to the age differences
during testing (Misanin et al., 2002a). In fact, it seems unlikely that ageing
would increase taste sensitivity because the opposite, that is, decreased taste
sensitivity, would be expected. A second explanation based on an increased
effect of the US in aged rats is not supported by the results. For instance, an
account based on there being a more intense US in aged rats because the higher
amount of LiCl injected in heavier animals can be discarded because a fixed
LiCl amount (2.3 ml) induced greater aversions in aged than in young adult rats
(Misanin and Hinderliter, 1994). Also, an increased sensitivity to LiCl in aged
rats due to physical deterioration, such as renal dysfunction, this leading to a
more intense US, cannot explain why the interval over which long-trace
conditioning is evident can be extended by increasing the unconditioned
stimulus intensity in old-age rats but not in young adult rats. (Misanin et al.,
2002b). Finally, an effect of an increased familiarity with the cage context due
to extended life experiences which could have reduced interference by the
81
Chapter 2
context in aged rats may not account for the aversions acquired with CS–US
delays longer than in young adult rats. Aged rats but not young adult rats show
aversions irrespective of the context in which they were kept during the
interstimulus interval (Misanin and Hinderlitter, 1995). Slowing down of a
pacemaker that shortens the time between events has also been proposed as the
explanation for the longer CS–US interval at advanced age (Misanin et al.,
2002b). However, this would not explain the reported enhanced taste aversion
at conventional CS–US delays.
In all, the age-related potentiation of CTA can be considered as a
learning superiority, which may represent an advantage for survival because
aged rats may be less able to deal with poisoning. Moreover, it cannot be
discarded that the effect of previous learning experiences during an extended
life may play a role in the development of this adaptive age difference, but
more research is needed to unveil the underlying mechanisms. This is
consistent with the effect of previously learned taste aversions on later
neophobia in 27-to 28-month-old Wistar rats. An enhanced effect of a previous
saccharin aversion induced by a 1% b.w. injection of LiCl (0.15 M) on the later
neophobic response to a 1% saline solution has been recently reported in aged
rats (Morón and Gallo, 2007). The increased neophobic response does not
seem to be related to the enhanced saccharin aversion in aged rats because a
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Hippocampus, Ageing and Taste Memories
similar strength aversion induced by a 2% b.w. LiCl injection in young adult
rats did not induce a similar saline neophobia. Thus, this seems to be another
example of age-related potentiation of taste memory functions.
Third, although ageing does not affect some complex taste learning
phenomena such as LI, old rats do show impairments in other tasks such as
blocking (Gallo et al., 1997; Morón et al., 2001; Morón et al., 2002a) and the
effect of unconditioned stimulus preexposure on later learning (Misanin et al.,
1997).
Previous research in our laboratory has shown that blocking may be a
sensitive assay for detecting age-induced cognitive deficits. We have found
that blocking is absent in ageing rats (Morón et al., 2001; Morón et al., 2002a).
In 15-to 17-month-old rats, a previously learned saccharin (0.1%) aversion did
not reduce the magnitude of a cider vinegar (3%) aversion presented in
compound with saccharin during the conditioning trial, the vinegar aversion
being as strong as if no previous experience had taken place. This deficit may
appear as early as 8 months in Wistar rats (Gallo et al., 1997). Similarly, the
US preexposure effect seems to be also disrupted by ageing. Misanin et al.
(1997) have reported that 6 daily 1% b.w. injections of 0.15 M LiCl interfered
with the acquisition of a saccharin (0.1%) aversion induced by a similar LiCl
83
Chapter 2
injection in weanling (20–25 days) and young adult (90–105 days) but not in
aged (635–725 days) rats.
Thus, taste learning and memory tasks reveal a complex but complete
picture of ageing as inducing a different organization of the learning abilities
instead of mere decay. Both enhanced and preserved functions besides those
deteriorated represent useful tools to study the ageing brain.
2.3. Hippocampus and taste memories
The basic brain circuit required for CTA involves several brain areas,
such as the nucleus of the solitary tract, the parabrachial nucleus, the insular
gustatory cortex, and the amygdala (for reviews see Bernstein, 1999; Gallo et
al., 1999; Bermúdez-Rattoni, 2004; Reilly and Bornovalova, 2005). The
hippocampal integrity is not required for basic CTA. In fact, permanent lesions
of the dorsal hippocampus do not interfere with taste aversion learning (Gallo
and Cándido, 1995a). Moreover, enhanced taste aversion learning after
temporary dorsal hippocampal inactivation by muscimol infusions has been
reported (Stone, Grimes and Kats, 2005). However, the hippocampus may have
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Hippocampus, Ageing and Taste Memories
a critical role in mediating some of the effects of previous experience on
subsequent taste learning.
On the one hand, the role played by the hippocampus in LI has been
controversial, especially using taste learning tasks, and this remains to be
clarified. Disruption, no effect (Gallo and Cándido, 1995a), or enhanced
(Reilly, Harley and Revusky, 1993; Purves, Bonardi and Hall, 1995; Stone et
al., 2005) LI of taste aversion learning by hippocampal lesions or by
inactivation have all been reported (for a critical review of early studies
reporting disruption, see Gallo et al., 1999). These discrepancies have been
attributed to differences in the behavioral procedures used and total time of CS
exposure which may interact with the hippocampal lesion, thus affecting CS
novelty (Buhusi, Gray and Schmajuk, 1998). However, a hippocampal role in
LI of CTA different to that observed in LI when using other learning tasks
cannot be discarded because the facilitatory effect of hippocampal lesions on
LI has been reported only using a taste aversion procedure (Buhusi et al.,
1998). In general, the present evidence does not support a critical hippocampal
role on LI of taste aversion learning, but some modulatory function cannot be
excluded.
On the other hand, previous results obtained in our laboratory have
shown that the hippocampus becomes critically involved in other complex taste
85
Chapter 2
learning phenomena such as blocking and the contextual modulation of
learning. Consistent with findings from other learning procedures, permanent
electrolytic lesions (Gallo and Cándido, 1995a) or reversible inactivation by
TTX injections (Gallo and Cándido 1995b) of the dorsal hippocampus, while
not affecting LI of either saccharin (0.1%) or saline (0.5%) aversions, impair
blocking of cider vinegar (3%) aversions when presented in compound with a
previously conditioned saccharin solution in adult rats. This impairment can be
reversed by hippocampal fetal transplants in lesioned rats (Gallo et al., 1997).
In addition to the above, some effects of the contextual in-formation on
various memory tasks have also been demonstrated to be hippocampal
dependent in adult rats (Honey and Good, 1993; Holland and Bouton, 1999;
Maren and Holt, 2000), but this remains to be investigated using taste learning
tasks. In fact, both aversive (Puente, Cannon, Best and Carrell, 1988; Bonardi,
Honey and Hall, 1990; Loy, Alvarez, Rey and López, 1993; Boakes,
Westbrook, Elliot and Swinbourne, 1997) and safe (Hall and Channell, 1986;
Rosas and Bouton, 1997) taste memories are bound to the external environment
in which learning occurred.
The context dependency of taste aversive memories was probably not
revealed in the early studies due to ceiling effects induced by the conventional
one-trial taste aversion learning protocol. Consistently, Bonardi et al., (1990)
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Hippocampus, Ageing and Taste Memories
used NaCl (1%) and HCl (1%) for testing the contextual specificity of taste
aversions induced by a low (1% b.w.) dose of LiCl (0.3 M) after a previous
habituation session with each of 2 contexts which included visual, tactile,
auditory, spatial, and temporal differences. They reported no differences
between the groups tested in the same or different context throughout 6 onebottle extinction tests in a single trial protocol. However, after 5 conditioning
pairings, a weaker aversion was evident in the group tested in a different
context by the third extinction test as the aversion began to diminish. Similarly,
other studies showing a context specificity of learned taste aversions have used
several conditioning trials (Puente et al. 1988; Loy et al. 1993; Boakes et al.,
1997). However, a study reporting negative results applied a single saccharin–
LiCl pairing (Rosas and Bouton, 1997). Although it has been proposed that a
single pairing might not be sufficient for establishing the context as a
conditional cue controlling the CS–US association (Bonardi et al., 1990), we
have demonstrated the context dependency of a saline (1%) aversion after a
single conditioning trial by using a behavioral procedure that included 2
habituation days to the contexts and 2 saline preexposures. The reduced saline
aversion when tested in a different context was clear if a place context was
used and reached significance in the second extinction test when the time of
day was used as a context (Morón et al., 2002b). The hippocampus does not
seem to play a role in the contextual specificity of taste aversions because N87
Chapter 2
methyl-D-aspartate (NMDA) induced lesions of the dorsal hippocampus do not
disrupt the effect when changes of the temporal context are used (Gallo 2005).
On the contrary, the hippocampus may be involved in the contextual
specificity of safe taste memories. Both the development of a safe taste
memory during extinction and the LI effect depending on the safe taste
memory developed during preexposure have been demonstrated to be context
specific. Returning to the conditioning context after extinction in a different
context may lead to a renewal of the previously learned taste aversion (Rosas
and Bouton, 1997; Morón et al., 2002b). Also, a context change between preexposure and conditioning disrupts LI, leading to increased aversions in the
group subjected to the context change com-pared with that preexposed and
conditioned in the same context. The context dependency of LI is evident using
either 6 (Hall and Channell, 1986) or 5 (Manrique et al., 2004) habituation days
to the contexts used. Moreover, the effect appears not only using a mixture of
spatial, visual, texture, and time cues to conform the context (Hall and
Channell, 1986) but also using the time of day itself (Manrique et al., 2004).
This latter effect has been reported to be hippocampal dependent because
NMDA lesions of the dorsal hippocampus disrupt it in young adult rats.
88
Hippocampus, Ageing and Taste Memories
2.4. Hippocampal decline and ageing impact on taste memories
The hippocampus of the rat shows changes in its functional
organization during ageing without significant neuron loss (Erickson and
Barnes, 2003; Kelly et al., 2006). Anatomical studies have shown no cell loss
that could be related to memory impairment in any of the aged hippocampus
fields (Rapp and Gallagher, 1996; Rasmussen, Schliemann, Sorensen, Zimmer
and West, 1996). However, ageing is associated with changes in connectivity
and functional responsiveness of the hippocampal neurons (Erickson and
Barnes, 2003; Rosenzweig and Barnes, 2003; Kelly et al., 2006; Wilson et al.,
2005). Alterations in connectivity have been reported, including a decline in
functional cholinergic transmission, fewer but compensatory increases in the
strengths of remaining synapses in the dentate granule cells, loss of functional
synapses in CA1 pyramidal cells, and increased gap junctional connectivity.
Moreover, the aged hippocampus shows alterations in different forms of
plasticity. In addition to a reduced persistence of long-term potentiation (LTP)
and LTP induction deficits using perithreshold parameters, long-term
depression is more easily induced in aged rats.
The variety of neurophysiological and biochemical alterations in the
hippocampal functions during ageing may account for the failure to support
89
Chapter 2
some complex learning tasks. Thus, impaired performance of aged rats has
been reported in a variety of learning and memory tasks requiring an intact
hippocampus (Gallagher and Rapp, 1997; Erickson and Barnes, 2003).
We have compared the performance of adult hippocampal and old rats
in a variety of taste memory tasks (Morón et al., 2002a). In accordance with an
explanation of the age-related cognitive impairment based on the decline of the
hippocampal function, LI, but not blocking, was preserved both in aged and
hippocampal rats. Moreover, blocking was reestablished by fetal hippocampal
transplants both in hippocampal lesioned and in intact aged rats (Morón et al.,
2001). However, aged, but not adult hippocampal lesioned, rats showed an
enhancement of taste aversion learning (Morón et al., 2002a). Moreover,
hippocampal grafts, which reinstated blocking, did not reverse this age-induced
enhancement (Morón et al., 2001). This suggests that, in addition to
hippocampal-related impairments, ageing induces independent changes in the
brain circuit required for basic taste aversion learning, which may be
responsible for enhanced taste memory functions.
90
Hippocampus, Ageing and Taste Memories
2.5. Conclusions
Taste recognition memory may be proposed as a choice model for the
study of the ageing impact on memory. Taste learning tasks represent useful
behavioral tools for studying ageing-related changes in cognition because they
allow us to investigate the participation of different types of memory by
introducing variations in the same basic procedure. Thus, sensory, motor,
motivational, and emotional requirements are shared, and this facilitates
comparisons. The results show impaired, preserved, and enhanced functions in
aged rats, indicating alterations in the organization of the memory systems
during ageing. Some of the effects of dorsal hippocampal lesions in adult rats
are also seen in aged rats. Both aged and hippocampal adult rats show an intact
LI effect. Similarly, conditioned blocking is absent in both aged and
hippocampal adult rats. Thus, it is conceivable that the aged hippocampus is
unable to support certain types of taste memory modulation. However, with the
behavioral procedure used, aged rats exhibited an enhancement of basic taste
aversion that is not induced by hippocampal lesions in young adult rats.
In all, the results confirm that the impact of age on memory is complex
and cannot be explained by a general cognitive decline or exclusively by
91
Chapter 2
hippocampal function decay. Rather, the present results suggest that there is
reorganization within the brain memory systems during the ageing process.
Acknowledgements
The authors wish to thank to Ms. Ana Molina for her technical help with the
animal care and are grateful to Dr Michelle Symonds for reviewing the
manuscript and for helpful suggestions with the English.
92
Hippocampus, Ageing and Taste Memories
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Reilly, S., Harley, C., and Revusky S. (1993). Ibotenate lesions of the
hippocampus enhance latent inhibition in conditioned taste aversion and
increase resistance to extinction in conditioned taste preference.
Behavioral Neuroscience, 107, 996–1004.
Rosas, J. M., and Bouton, M. E. (1997). Renewal of a conditioned taste
aversion upon return to the conditioning context after extinction in
another cue. Learning and Motivation, 28, 216–229.
Rosenzweig, E. S., and Barnes, C. A. (2003). Impact of aging on hippocampal
function: plasticity, network dynamics, and cognition. Progress in
Neurobiology, 69, 143–179.
Stone, M. E., Grimes, B. S., and Katz, D. B. (2005). Hippocampal inactivation
enhances taste learning. Learning and Memory, 12, 547–548.
Van der Staay, F. J. (2002). Assesment of age-associated cognitive deficits in
rats: a tricky business. Neuroscience and Biobehavioral Reviews, 26,
753–759.
Wilson, I.A., Ikonen, S., Gallagher, M., Eichenbaum, H., and Tanila, H.
(2005). Age¬associated alterations of hippocampal place cells are
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Manrique, T., Molero, A., Morón, I., Ballesteros, M.A.,
Gallo, M. and Fenton, A. (2004).
Time of day-dependent latent inhibition of conditioned taste
aversions in rats.
Neurobiology of Learning and Memory, 82 (2): 77-80.
Time of day-dependent latent inhibition of conditioned taste
aversions in rats
a,b
Manrique, T., a Molero, A., a,b Morón, I., a Ballesteros, M. A., a,b Gallo, M.
and c,d Fenton, A.
a
Department of Experimental Psychology and Physiology of Behavior, University of
Granada, Campus Cartuja, Granada-18071, Spain.
Oloriz, University of Granada, Spain.
Sciences, Prague, Czech Republic.
d
c
b
Institute of Neurosciences F.
Institute of Physiology, Czech Academy of
Department of Physiology and Pharmacology,
State University of New York, Downstate Medical Center, Brooklyn, NY, USA
ABSTRACT
We have determined that the temporal context of drinking can modulate latent
inhibition of learned saline aversions in Wistar rats by changing the time of
day of drinking of the preexposure and conditioning phases. Latent inhibition
was absent in the group preexposed and conditioned to saline at different times
of the day, but not in the group that was preexposed and conditioned at the
same time of day. The results confirm a previous report that the time of day can
modulate taste aversion learning independently of other environmental cues. It
is proposed that the features and duration of the habituation training to the
temporal contexts used may be critical for time-dependent latent inhibition to
appear.
This research was supported by the CICYT Grant BSO200201215 (MICYT, Spain).
Keywords: context; latent inhibition; learning; rat; taste aversion; time of day.
Chapter 3
3.1. Introduction
The role of the time of day in animal learning and memory is being
thoroughly studied in time-place learning tasks. Although controversial
(McDonald, Hong, Ray, and Ralph, 2002) and not always easy to demonstrate
(Lukoyanov, Pereira, Mesquita, and Andrade, 2002; Means, Arolfo, Ginn,
Pence, and Watson, 2000b; Thorpe, Bates, and Wilkie, 2003), some results
show that, after extensive training, rats are able to use timing cues to locate a
reinforcer (Carr and Wilkie, 1997, 1999; Means, Ginn, Arolfo, and Pence,
2000a, 2000b; Mistlberger, de Groot, Bossert, and Marchant, 1996; Thorpe et
al., 2003). It has been proposed that the time-of-day acts as contextual stimulus
in these tasks (Lukoyanov et al., 2002; Means et al., 2000a, 2000b). The fact
that the time of day may itself form a context is also supported by a report
showing a time of daydependent expression of the behavioral sensitization to
amphetamine (Arvanitogiannis, Sullivan, and Amir, 2000).
Latent inhibition (LI) of conditioned taste aversion (Bures, BermúdezRattoni, and Yamamoto, 1998), i.e., a reduced learned aversion to a taste that
was previously experienced without aversive consequences, has been reported
to show a contextual dependence in rats (Lubow, 1989). Although there are
some negative results (Best and Meachum, 1986; Kurz and Levitsky, 1982), it
104
Time of day, Latent Inhibition and CTA
has been reported that a change of external context between preexposure and
conditioning disrupts LI (Hall and Channell, 1986; Rudy, Rosenberg, and
Sandell, 1977). The ability of a context change between preexposure and
conditioning to interfere with LI seems to depend on the familiarity of the
context (Hall and Channell, 1986; Rudy et al., 1977). In a previous report, we
reported that the time of day itself may act as a context to modulate the
extinction of conditioned taste aversions (CTA). However, we found no effect
of a time of day change between preexposure and conditioning on LI (Morón et
al, 2002).
In the present experiment, we speifically examine if LI of CTA may
depend on the time of day if a longer habituation period that allows differences
in the amount of ingested fluid is applied. We used a 5 day habituation period,
which was longer than the 2 days applied in our previous report.
3.2. Materials and methods
Seventy-one naïve male Wistar rats (280–320 gr) from the breeding
colony of the University of Granada were used. To eliminate external timing
cues the animals were individually housed in an isolated room with constant
temperature and a 12:12 h light-dark cycle (lights on at 9:00 and off at 21:00).
105
Chapter 3
Food was available ad libitum, but water availability depended on the
behavioral procedure.
Animals had two daily 15 min drinking sessions.
Morning (10:00) and evening (20:00) drinking sessions were used to
maximally differentiate the temporal contexts. The procedures were approved
by the University of Granada Ethics Committee for Animal Research, and were
in accordance with both the NIH of the United States guidelines for the ethical
treatment of animals, and the European Communities Council Directive of 24
November 1986 (86/609/EEC).
Four groups of rats (n = 18 per group except Ctrl-Same n = 17) were
used in a 2 x 2 design (Table 1). Preexposed groups (Pre), but not control
groups (Ctrl) received two non-reinforced saline preexposures before
conditioning. Animals in each group were then assigned to one of two groups:
The ‘‘Same’’ group was preexposed and aversively conditioned to saline and
tested in their home cages at the same time of day. The ‘‘Different’’ group was
aversively conditioned to saline at a different time from the preexposure and
testing.
The behavioral procedure had five phases: Temporal Context
Habituation, Taste Preexposure, Conditioning and Extinction. The amount of
fluid ingested was recorded to the nearest 0.1 ml in all phases. The Temporal
Context Habituation phase lasted 5 days.
During the first 3 days all the
animals drank water ad libitum during the morning and evening sessions. On
the last 2 days, morning and evening intake was equated by limiting morning
106
Time of day, Latent Inhibition and CTA
water ingestion to the average amount drunk in the previous six sessions.
Table 1. Behavioral procedures for the different groups A/B subgroups in
each behavioral group as a consequence of balancing; AM/PM, Temporal
contexts; HC, Home cage; LiCl, Lithium Chloride; Pre-1 and Pre-2,
Preexposure 1 and 2; Sal, Isotonic saline; W, Water.
Pre-1 and 2
Conditioning
Recovery
Extinction
1–5
Sal A (n=9) AM
B (n=9) PM
Sal-LiCl A PM B
AM
W (AM/PM)
Sal A AM B
PM
W AM/PM
Sal-LiCl A (n= 9)
PM B (n =9) AM
W (AM/PM)
Sal A AM B
PM
Pre-Same
Sal A (n=9) AM
B (n= 9) PM
Sal-LiCl A AM B
PM
W (AM/PM)
Sal A AM B
PM
Ctrl-Same
W (AM/PM)
Sal-LiCl A (n=9)
AM B (n=9) PM
W (AM/PM)
Sal A AM B
PM
PreDifferent
CtrlDifferent
The Preexposure phase lasted 2 days. The time of day was
counterbalanced, i.e., half of the animals in each preexposed group (Pre-Same
and Pre-Different) were allowed to drink saline (1%) for 15 min during the
morning drinking session while the rest of the animals drank saline during the
evening session. The non-preexposed control groups (Ctrl-Same and CtrlDifferent) were allowed to drink water during the preexposure phase.
During the conditioning session, the animals were allowed to drink the
107
Chapter 3
saline solution either at the same time of day as during the preexposure
sessions (Pre-Same) or at a different time (Pre-Diffrent). The non-preexposed
(Ctrl) groups drank at the same time as their corresponding preexposed group
i.e., half of each group drank saline during the morning and half during the
evening drinking session. Immediately, after the 15 min of saline drinking, all
the animals received an i.p. injection of lithium chloride (LiCl, 0.15 M; 2%
body weight) and returned to the home cage. The next day they were allowed
to recover from the LiCl-induced visceral distress with access to water during
both drinking periods.
The extinction phase began the day after recovery. The preexposed rats
and their controls were allowed to drink the saline solution at the same time of
day as their saline preexposure sessions. The extinction phase lasted 5 days.
The volume of consumed saline was compared across groups and across days.
The average values ±SEM are reported.
3.3. Results
No differences were found among the groups in the water intake during
habituation. Prior to conditioning, the only difference between the groups, was
the expected preference for saline in the rats of the preexposed groups
108
Time of day, Latent Inhibition and CTA
compared with the water intake by the control non-preexposed groups during
the second preexposure session (F(1,67)= 25.88; p < 0.01). Fig. 1 summarizes
the results of the conditioning and testing phases. Although there were no
differences in the amount of saline solution drank by the different groups
during conditioning, only the group preexposed and conditioned at the same
time of day showed latent inhibition, i.e., reduced saline aversions compared
with non-preexposed groups.
Fig. 1. Mean (+SEM) saline intake of the different groups during the
conditioning and extinction retention phases (Cond, conditioning; D, Different
Groups; S, Same Groups; Pre, Saline Preexposed Groups; Ctrl, Control nonpreexposed groups; E1–E5, Extinction tests 1–5).
109
Chapter 3
A 2 x 2 (Preexposure x Time) ANOVA of the different groups’ saline
intake in the conditioning session showed no significant effect of Preexposure
(F(1,67 )= 2.68; p > 0.1), Time (F(1,67)= 1.27; p > 0.2) or the interaction
Preexposure x Time (F(1,67)= 1.22; p > 0.2). Extinction was studied by
measuring saline intake, which was during the drinking session at the same
time of saline preexposures. The saline intake over five extinction tests was
analysed using a 2 x 2 x 5 (Preexposure x Time x Days) ANOVA. There were
significant main effects of Preexposure (F(1,67)= 18.76; p < 0.01), Time (F(1,67)
= 6.05; p < 0.01), Days (F(4,268) = 66.57; p < 0.01) and the interaction
Preexposure x Time (F(1,67)= 4.52; p < 0.05). No other interaction approached
significance. Newman–Keuls post hoc analyses of the Days effect showed a
significant increase of saline intake across the first four extinction tests p’s <
0.01. The extinction of the aversion stabilised after the Day 4 saline intake
because Test 4 and Test 5 did not differ (p > 0.7). Analysis of the interaction
Preexposure x Time showed the absence of LI in those groups that were
preexposed and conditioned at a different time of day, as there was no
significant effect of Preexposure (F(1,34)= 2.68; p > 0.1). However, a clear
latent inhibition effect appeared in the groups Same, as the Preexposed group
had a reduced saline aversion compared to the non-preexposed control group
(F(1,33)= 18.98; p < 0.01). There were no differences between the Ctrl-Same and
Ctrl-Different groups across the five extinction sessions (F(1,33)= 0.05; p > 0.8),
110
Time of day, Latent Inhibition and CTA
but the group Pre-Same had a higher saline intake compared to the group PreDifferent (F(1,34)= 11.24; p < 0.01).
3.4. Discussion
The results indicate that, a change in the time of day between
preexposure and conditioning disrupted latent inhibition of CTA. The group
that was preexposed to saline at a different time of conditioning acquired saline
aversions that were similar to those expressed by the non-preexposed groups.
Reduced saline aversions were evident, however, in the rats that were
preexposed at the same time of day as they were conditioned. Consistent with
this finding, the most cited previous study that reported a contextual
dependence of latent inhibition in CTA included the time of day as part of the
contextual change (Hall and Channell, 1986).
The procedural differences between the Morón et al. (2002) experiment
and the present experiment may be critical for explaining the presence of timedependent latent inhibition that was absent in the previous report. Hall and
Channell (1986) proposed that the duration and type of habituation to the
context may determine its ability to modulate learning. Previous findings using
physical contexts have shown that the prior experience with the context
reduces its ability to interfere with LI of CTA (Rudy et al., 1977). This was
111
Chapter 3
attributed to a reduction of the con-textual associability as the animals learned
it was an irrelevant cue. However, our data show that a longer habituation, 5
days compared to the 2 days in Morón et al (2002) may instead have facilitated
the time of day dependency of LI. It is conceivable that the salience and
distinction of the drinking sessions time of day increased throughout the
habituation period, and thus revealed the contextual dependence of LI.
Moreover, as our context habituation included changes in the amount that the
animal was allowed to drink, it can be proposed to have provided an additional
feature to increase the salience of temporal context.
To our knowledge, this is the first evidence of time-of-day dependent
latent inhibition in the absence of other environmental changes. It shows that
rats use the time of day as a context to modulate the effect of previous
experience with the to be learned stimuli. It cannot be ruled out that the rat
strain used plays a critical role in this effect. Recent evidence has demonstrated
that Wistar but not Long Evans rats are sensitive to time-of-day modulation of
conditioned place preference (Cain, Ko, Chalmers, and Ralph, 2004). This
would explain the failure of McDonald et al. (2002) to find time-stamping in
spite of the extensive exposure to the temporal contexts. The fact that testing
took place at the time of saline preexposure does not support an occasion
setting function of the time of day during testing. Rather the change of time of
day may have favoured the CS–US association during conditioning, either by
112
Time of day, Latent Inhibition and CTA
increasing the CS novelty or removing the interference by a previously formed
CS-no US association. The experiment does not allow the determination of the
specific timing cues that may act as a context. It has been reported that rats are
able to use different strategies based either on circadian phase (Mistlberger et
al., 1996; Thorpe et al., 2003), or interval or ordinal timing (Carr and Wilkie,
1997) in tasks requiring time-of-day discriminations.
In all, the results are consistent with previous findings (Arvanitogiannis
et al., 2000; Carr and Wilkie, 1997, 1999; Means et al., 2000a, 2000b;
Mistlberger et al., 1996; Morón et al., 2002; Thorpe et al., 2003) showing that
time of day can modulate learning and memory in rats. The time of day
dependency of LI described here provides a paradigm for research into the
neural mechanisms that underlie how memories are stored and modulated by a
broad sense of context (Holland and Bouton, 1999).
113
Chapter 3
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conditioned
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to
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behavioral
sensitization
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modulation of conditioned place preference in rats depends on the strain
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Carr, A. R., and Wilkie, D. M. (1997). Rats use an ordinal timer in a daily
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on taste mediated environmental conditioning: Potentiation and
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Hall, G., and Channell, S. (1986). Context Specificity of latent inhibition in
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Holland, P. C., and Bouton, M. E. (1999). Hippocampus and context in
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Kurz, E. M., and Levitsky, D. A. (1982). Novelty of contextual cues in taste
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Lubow, R. E. (1989). Latent inhibition and conditioned theory. Cambridge:
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Means, L. W., Arolfo, M. P., Ginn, S. R., Pence, J. D., and Watson, N. P.
(2000b). Rats more readily acquire a time-of-day go no-go
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Mistlberger, R. E., de Groot, H. M., Bossert, J. M., and Marchant, E. G. (1996).
Discrimination of circadian phase in intact and supraquiasmatic nucleiablated rats. Brain Research, 739, 12–18
Morón, I., Manrique, T., Molero, A., Ballesteros, M. A., Gallo, M., and Fenton,
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116
CHAPTER 4
Manrique, T., Morón, I, Ballesteros, M. A., Guerrero, R.M.,
Fenton, A.A., and Gallo, M. (2008)
Hippocampus, Ageing and Segregating memories
(en revision)
Hippocampus, Aging and Segregating memories
a,b
Manrique, T., a,b Morón, I, a Ballesteros, M. A., a,b Guerrero, R.M., , c Fenton,
A.A., and a,b Gallo, M. *
a
Department of Experimental Psychology and Physiology of Behavior. University of
Granada. Spain, bInstitute of Neurosciences F. Oloriz. University of Granada. Spain,
c
Department of Physiology and Pharmacology, the Robert F. Furchgott Center for
Neural and Behavioral Science, SUNY, Downstate Medical Center, USA
ABSTRACT
Rats use time-of-day cues to modulate learned taste aversion memories. If adult
rats are accustomed to drinking saline in the evening and they receive a lithium
chloride injection after drinking saline in the morning, they form a stronger
aversion to saline than rats that were conditioned after drinking saline at the
familiar time. The difference indicated the rats formed segregated
representations of saline taste and the time of day the saline was consumed. This
was inferred because the modulation of learning by time of day was observed
when the aversions were tested at the familiar evening drinking time. If the rats
had formed a compound representation of saline taste and the time of day it was
consumed, the opposite pattern of differences would be expected. We used this
modulation of learning by time of day to assay whether aged rats have an
impaired ability to form segregated representations of experience. We find that
aged rats had similar saline aversions if they were conditioned at either the
familiar or the unfamiliar time of day. Furthermore, dorsal hippocampal lesions
in the aged rats caused greater saline aversions if the rats were conditioned
after drinking saline at the familiar time of day. This indicated that aged rats
are aware of the time of day but after the lesion they act as if they do not
segregate saline taste from the time of day it was consumed. The results suggest
that the ability to form segregated representations of a complex experience is
impaired in aging and abolished by hippocampal lesions.
Grant sponsor: MICYT, MEC and Junta de Andalucía (Spain), Grant number: BSO200201215, SEJ2005-01344 and HUM 02763
Chapter 4
KEY WORDS, aging, hippocampus, latent inhibition, taste aversion, taste
recognition memory, time-of-day, rat, cognitive segregation.
4.1. Introduction
Episodic memory content includes the place and the time of an
experience. Evidence from rat studies suggests the hippocampal representation
of space provides spatial episodic content (reviewed by Kentros, 2006; Smith
and Mizumori, 2006). Much less is known about temporal episodic content,
which has been difficult to study in rats. Recently, Morón et al. (2002b) and
Manrique et al., (2004) found that the time of day modulates learned taste
aversions in rats. Rats accustomed to drinking water in the mornings ("AM")
and the evenings ("PM"), drank saline in the evenings for two days. The next
day, half the rats (group SAME) received lithium chloride injections after
drinking saline in the evening. The other half (group DIFF) received lithium
chloride injections after drinking saline in the morning, at a different time-ofday. Subtle modifications of the procedure seem to drastically modify the role of
the temporal context. In the short-habituation protocol, there were only two days
of prior habituation training to drink water twice a day. Drinking was also
120
Hippocampus, Ageing and Segregating memories
restricted during the conditioning session. Rats in the SAME group had a greater
aversion than those in the DIFF group when tested in the evening (Morón, et al.,
2002). This suggests that a compound conditioned stimulus representation
("saline-AM" or "saline-PM") had been formed. However, the results were
opposite if the duration of the habituation to the temporal context was increased
and the animals were also allowed to drink freely during the conditioning
session. In this long-habituation protocol, rats in the DIFF group had a greater
aversion than those in the SAME group when tested in the evening (Manrique et
al., 2004).
In the long-habituation protocol it was unlikely that the time of day
combined with the taste to form a representation of a compound conditioned
stimulus ("saline-AM" or "saline-PM") that was associated with malaise. In this
case, saline-PM would be associated with malaise in the SAME group and
saline-AM would be associated with malaise in the DIFF group. If this was the
case, the SAME group should have had a greater saline aversion when tested in
the evening. The opposite difference was observed, indicating the rats
represented the saline taste separately from the time of day it was experienced.
This hippocampus-dependent ability to segregate elements of an
experience into separate internal representations was recently demonstrated to
be distinct from the role of hippocampus in encoding associations. Kubik and
121
Chapter 4
Fenton (2005) demonstrated hippocampus-dependent segregation using tasks
that depend on spatial information processing, which most researchers agree is a
fundamental function of hippocampus, analogous to visual information
processing being a fundamental function of striate cortex. Excitotoxic lesions of
dorsal hippocampus have been used to assess whether the hippocampus also
participates in the segregation of taste and time-of-day memories, independent
of its role in processing spatial features of experience.
In these CTA
experiments it is difficult to identify a spatial component of the information
processing. This is because the time of day of the saline ingestion was the only
variable that distinguished the conditioning experiences (Gallo, 2005). Dorsal
hippocampal lesions did not interfere with the effect of a time change in the
short-habituation procedure (Gallo, 2005). However, the lesion selectively
disrupted the effect of a time-of-day change in the long-habituation procedure.
The lesion abolished the difference between the “Same” and “Different” groups;
the DIFF and SAME groups expressed similarly strong saline aversions on the
first extinction test (Gallo, 2005). Furthermore, additional extinction tests
revealed the opposite difference (unpublished data). The saline aversion was
stronger in the SAME group. This indicated that the hippocampus was not
crucial for processing time-of-day information itself. Furthermore, and
unexpectedly, the results indicated that taste and the time of day are more likely
to be represented as a compound stimulus (saline-AM or saline-PM) and
122
Hippocampus, Ageing and Segregating memories
associated with malaise after hippocampal lesion. This is consistent with the
idea that a fundamental function of hippocampus is to facilitate the segregation
of stimulus representations even when the stimuli to segregate are not spatial
(Kesner, Lee, and Gilbert, 2004). We now report that aging compromises this
fundamental feature of hippocampal function.
4.2. Materials and Methods
The procedures were approved by the University of Granada Ethics
Committee for Animal Research and were in accordance with the European
Communities Council Directive 86/609/EEC. Male Wistar rats were housed on a
12:12 light:dark cycle (lights on 8:00; lights off 20:00). The rats were subjected
to the long-habituation procedure of Manrique et al. (2004), which is
summarized in Table 1 and explained below. The consumed amount of fluid was
recorded as the dependent measure.
123
Chapter 4
4.2.1. Behavioural Protocol
Habituation (days 1-5): The rats were habituated to consume their daily
intake of water in two 15-min water drinking sessions. One session was in the
morning (9:00) and the other in the evening (19:00).
Preexposure (days 6-7): After habituation, all the animals were allowed
to drink a 1% saline solution instead of water during the following two evening
drinking sessions. Groups that were not preexposed were not included because
the present experiments were not designed to study latent inhibition (see Morón
et al., 2002b).
Conditioning (day 8): The rats were conditioned either at the same
(SAME) or at a different time of day (DIFF) than preexposure. The conditioning
session took place during the evening session for the groups in the SAME
condition and during the morning session for those in the DIFF condition. The
lithium chloride injection (LiCl, 0.15 M; 2% b.w.; i.p.) was administered to all
animals after the saline drinking session. Groups that received saline instead of
LiCl were not included because prior work indicated that exposure to saline at a
novel or familiar time of day did not differentially alter subsequent saline intake
(Manrique et al., 2004; see also Fig 1 A, C).
124
Hippocampus, Ageing and Segregating memories
Recovery (days 9-10): The rats were allowed to drink water during the
morning and evening drinking sessions.
Test (days 11-13): Throughout the extinction test days, all the animals
were allowed to drink water during the morning drinking sessions according to
the procedure of Manrique et al (2004). Three extinction tests were given during
the evening sessions. Only the saline solution was available to all the animals.
The volume of saline consumed was recorded and analysed in separate
ANOVAs, because each test session acts to extinguish the aversion. Note, we
used an asymmetric experimental design with saline preexposure occurring only
in the evenings. We previously studied the symmetric design, with different rats
preexposed to saline in the mornings and evenings and found no difference in
the time-of-day modulation of conditioned saline aversion if preexposure was
given in the morning or evening (Manrique et al., 2004).
125
Chapter 4
Table 1. Behavioral procedure used in Experiments 1 and 2. The water
drinking sessions during preexposure, conditioning, recovery (days 9-10)
and testing are not represented. All the drinking sessions lasted 15
minutes. Abbreviations: am = 9 h; pm = 19 h; LiCl = Lithium Chloride.
The SAME and DIFF groups differed in the time-of-day (bold font) they
drank saline on the conditioning day.
Group
HABITUATION
PREEXPOSURE
CONDITIONING
TESTS
5 sessions
2 sessions
1 session
3 sessions
(days 1-5)
(days 6-7)
(day 8)
(days 11-13)
Water
Saline
Saline
am/pm
pm
LiCl pm
Water
Saline
Saline
am/pm
pm
LiCl am
Saline
Same
(SAME)
Different
(DIFF)
pm
Saline
pm
4.2.2. Dorsal Hippocampal Lesion
Bilateral dorsal hippocampal lesions were made by infusing 0.6 µl
NMDA (0.077M) at -2.3 and -3.3 posterior, 1.5 lateral and 3.4 ventral to bregma
(Paxinos and Watson, 1986). The rats were anesthetized with sodium
pentobarbital (50 mg/kg i.p.) and mounted in a stereotaxic frame. A midline
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suture was used to expose the skull and 1 mm holes were drilled through the
bone to provide access to the infusion sites. Sham-operated animals were
subjected to the same surgical procedure but vehicle (0.9% saline) was injected
instead of NMDA. A 30ga cannula was stereotaxically positioned at the infusion
sites. The cannula was attached to a 10 µl Hamilton syringe by Tygon tubing
and the solution was infused during 1 min at each location. Ten days after
surgery the rats were subjected to the same behavioural procedure as in
Experiment 1 (see Table 1).
The rats were deeply anesthetized with sodium pentobarbital (100
mg/kg, i.p.) at the end of the behavioural procedure and transcardially perfused
with saline then formalin solutions. Their brains were removed and processed
for histological verification of the lesions.
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Chapter 4
Figure 2. Diagrams taken from Paxinos and Watson atlas (1986) of coronal
sections depicting the largest and smallest acceptable hippocampal lesion.
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Hippocampus, Ageing and Segregating memories
4.3. Results
4.3.1. Experiment 1
This experiment tested whether adult and aged rats differ in their ability
to form segregated representations of taste and time of day. Taste aversions were
studied in ADULT rats (4-5 months old, weighing 397.5 SEM 13.6 g) and
AGED rats (25 months old, weighing 527.5 SEM 11.3 g). The rats were
conditioned either at the same (ADULT SAME; n=6 and AGED SAME; n=8) or
at a different time of day (ADULT DIFF; n=6 and AGED DIFF; n=10) than
preexposure and testing.
4.3.1.1. Results
Water intake in the adult and aged groups did not differ in the
habituation and preexposure sessions. The (Age x Group x Days) repeated
measures ANOVA of the water intake comparing the last habituation evening
session (Adult mean = 8.49; SEM ± 0.69; Aged mean = 8.36; SEM ± 0.58) and
saline intake during the first (Adult mean = 8.01; SEM ± 1.18; Aged mean =
5.87; SEM ± 0.53) and second (Adult mean = 10.52; SEM ± 0.71; Aged mean =
10.3; SEM ± 0.76) preexposure sessions revealed a significant effect of days
(F(2,52)= 12.62; p < 0.01), but no effects of age, group, or their interactions. Posthoc LSD comparisons indicated that saline intake during the first saline
129
Chapter 4
preexposure was lower (mean = 6.73; SEM ± 1.23) than water intake during the
previous evening session (mean = 8.42; SEM ± 1.54), indicating a neophobic
response to the novel saline taste at both ages. Saline intake during the second
preexposure session was higher (mean = 10.39; SEM ± 1.9) than both water and
saline intake during the previous sessions. This indicated a recovery from
neophobia and a preference for saline at both ages. Importantly, prior to
conditioning, the groups had a similar saline preference.
Figure 1a shows the average (±SEM) saline intake during the
conditioning and test phases. Exposure to the CS was similar across the groups
because saline intake was not different between the groups in the conditioning
session. This was confirmed by a two way Age x Group ANOVA comparing the
effects of age and the time of day on saline intake during conditioning. The
effects of Age (F(1,26)= 0.12; p > 0.7), Group (F(1,26)= 2.79; p > 0.1) and the Age x
Group interaction (F(1,26) = 3.29; p > 0.08) were all not significant. Water intake
was also similar during the recovery phase (data not shown), suggesting that the
aversive effect of the US was also similar across the groups.
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Hippocampus, Ageing and Segregating memories
131
Chapter 4
Figure 1. Time-of-day modulation of learned saline aversions. Mean (+SEM)
saline consumption during conditioning and testing of the different groups.
Experiment 1: (a) Adult rats were sensitive to the time-of-day saline was
ingested before LiCl injection but aged rats were insensitive to the time-of-day.
Adult rats (DIFF) that drank saline at an unfamiliar time-of-day learned a greater
saline aversion than adult rats (SAME) that drank the saline at a familiar time of
day. The inset highlights the key comparisons on the first retention test.
Experiment 2: (b) Cresyl violet-stained coronal sections showing representative
sham and NMDA dorsal hippocampal (HC) lesions at different rostro-caudal
levels in aged rats. The coordinates relative to bregma are indicated. (c) Aged
rats with NMDA lesions of the dorsal hippocampus were sensitive to the timeof-day saline was ingested before lithium injection. However, the pattern of
differences between SAME and DIFF lesion groups is opposite to the pattern
that was observed in the Experiment 1 adult rats. The inset highlights the key
comparisons on the first retention test. Abbreviations: SAME = preexposure,
conditioning and testing at the same time of day; DIFF = conditioning at a
different time of day as preexposure and testing; HC = Hippocampus lesion
group; EXT1,2,3 = extinction tests 1, 2, 3. The asterisk indicates a significant
difference (p < 0.05) from all other groups.
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Hippocampus, Ageing and Segregating memories
On the test for the learned saline aversion, the rats in the ADULT DIFF
group had the strongest aversions; rats in the ADULT SAME group had the
weakest aversions, and the two AGED groups had intermediate aversions. This
pattern was confirmed by the Age x Group ANOVA comparing the effects of
age and the time of day of the conditioning on saline intake during the first test
session. The effect of Group (F(1,26)= 7.41; p < 0.01) and the Age x Group
interaction (F(1,26)= 9.07; p < 0.01) were significant. LSD comparisons indicated
greater saline aversion in the ADULT DIFF group compared to the other groups
(ADULT SAME, and both the AGED groups; p’s < 0.05). Importantly, there
was no difference between the AGED SAME and AGED DIFF groups (p > 0.8).
The ADULT SAME group had a weaker saline aversion than the ADULT DIFF
group as well as both the aged groups (p < 0.05).
This pattern of aversions was robust as it was also observed on the
second and third extinction tests. The Age x Group ANOVA of the saline intake
during extinction test 2 confirmed significant effects of Age (F(1,26)= 7.02; p <
0.01), Group (F(1,26)= 8.18; p < 0.01) and the Age x Group interaction (F(1,26)=
5.07; p < 0.05). LSD comparisons indicated differences between the ADULT
SAME and ADULT DIFF groups (p < 0.01) but not between the AGED SAME
and AGED DIFF groups (p > 0.6). The ADULT SAME group had the weakest
133
Chapter 4
aversion because these rats drank more saline than all of the other groups (p <
0.01).
On the third extinction test the effects of Age (F(1,26)= 12.41; p < 0.01),
Group (F(1,26)= 10.73; p < 0.01) and the interaction Age x Group (F(1,26)= 6.68; p
< 0.05) were significant. Once again, LSD comparisons confirmed the greater
aversion in the ADULT DIFF group than the ADULT SAME group and both
the AGED groups (p < 0.01) but no difference between the AGED SAME and
AGED DIFF groups (p > 0.5).
In summary, in all the extinction tests, adult rats had the greatest
aversions when conditioning occurred at a different time of day than the
preexposure and tests. In contrast, rats in the aged groups acquired saline
aversions that were insensitive to the time of day. The failure to find a difference
between the aged groups can be attributed to a failure of aged animals to
segregate the representations of saline taste and the time of day the taste was
experienced. Alternatively, the difference between the response in the adult and
aged groups might be explained by an aging-related deficit in processing timeof-day cues.
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Hippocampus, Ageing and Segregating memories
4.3.2. Experiment 2
Experiment 2 was performed to determine whether a segregation deficit
or a deficit in sensing time of day could account for the results of Experiment 1.
Adult rats with hippocampal lesions failed to segregate taste and the time of day
it was experienced. However, they expressed learned saline aversions that were
modulated by time of day (unpublished data). If aged rats have an impaired
sense of time of day, then with a hippocampal lesion they should not express
learned saline aversions that are modulated by the time of day the saline was
tasted.
Thirty-eight naïve 27-month-old aged rats were used. Half received a
hippocampal lesion (HC) and the other half received a sham (SHAM) lesion.
The animals were assigned to the following groups based on the behavioural
procedure: SHAM SAME (n=9), SHAM DIFF (n=10), HC SAME (n=9) and
HC DIFF (n=10).
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Chapter 4
4.3.2.1. Results
Representative brains from the HC and SHAM groups are presented in
Figure 1b. Most of the rats in the HC groups had large lesions with cell loss in
the dorsal hippocampus that extended throughout CA1 in all cases. In most
cases, the ventral part of the dentate gyrus was intact. The CA3 subfield was
also damaged in some but not all brains. Figure 2 depicts the maximum and
minimum extent of the lesions. No hippocampal damage was observed in the
SHAM groups but both the HC and SHAM animals sustained damaged to the
corpus callosum and the cortex dorsal to the hippocampus. Additionally, the loss
of hippocampal cells was associated with a collapse and distortion of the cortex
dorsal to the hippocampus. Consequently, the overlying cortex was more altered
in the HC than the SHAM groups.
The groups did not differ in the habituation and preexposure sessions.
The (Lesion x Group x Days) repeated measures ANOVA of the water intake
comparing the last habituation evening session and saline intake during the first
and second preexposure sessions revealed a significant effect of Days (F(2,68)=
17.63; p < 0.01), but no effects of Lesion, Group, or any of their interactions.
Significant LSD comparisons indicated that saline intake during the first saline
preexposure was higher (mean = 9.34 ml; SEM =0.53) than water intake during
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Hippocampus, Ageing and Segregating memories
the previous evening session (mean = 7.43 ml; SEM ± 0.43). This indicated
there was a preference for saline. Saline intake during the second preexposure
session was even higher (mean = 10.39 ml; SEM ± 0.57). This increased
preference for saline was presumably because of reduced neophobia. Prior to
conditioning, animals in all the groups had a similar saline preference.
Figure 1c illustrates saline intake during conditioning and testing. Fluid
intake did not differ between the groups on the conditioning or recovery days
(data not shown). This was confirmed by the two-way Lesion x Group ANOVA
that compared the effects of the lesion and the time of day on saline intake
during conditioning. None of the effects of Lesion (F(1,34)= 2.42; p > 0.1), Group
(F(1,34)= 2.45; p > 0.1) and the Age X Group interaction (F(1,34)= 0.01; p > 0.9)
were significant.
In contrast, the aged rats with a hippocampus lesion exhibited stronger
saline aversions if they were conditioned at the same time of testing than the rats
that were conditioned at a different time. No differences were found between the
SHAM SAME and SHAM DIFF aged groups, reproducing this Experiment 1
result. The Lesion X Group ANOVA of saline intake on extinction test 1
revealed a significant effect of Group (F(1,34)= 4.03; p < 0.05) but no other
effects. The SAME animals that were conditioned after drinking saline at a
familiar time of day had stronger saline aversions (mean = 2.88; SEM ± 0.61)
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Chapter 4
than the DIFF animals that were conditioned after drinking saline at an
unfamiliar time of day (mean = 4.99; SEM ± 0.78). LSD comparisons indicated
that this effect was due to weaker aversions in the HC DIFF group compared to
the HC SAME group (p < 0.05) because the SHAM SAME and SHAM DIFF
groups did not differ (p > 0.4).
Although the aversion began to extinguish, the pattern of differences
persisted on extinction test 2. There was a significant Lesion X Group
interaction (F(1,34)= 4.04; p < 0.05) but no effect of Lesion or Group. LSD
comparisons indicated that the HC DIFF group drank more saline than the HC
SAME and the SHAM DIFF groups (p < 0.05). This indicated there was a
weaker aversion in the aged lesion group, but only if they were conditioned at a
different time of day. No other differences were significant.
The differences in saline aversion were no longer apparent on test 3. The
Lesion x Group ANOVA confirmed there were no significant effects.
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Hippocampus, Ageing and Segregating memories
4.4. Discussion
Experiment 1 indicated that unlike adult rats, aged rats were insensitive
to the time of day in which they were conditioned to avoid the taste of saline.
The aged rats expressed the same magnitude of the learned saline aversion
regardless of whether the saline was paired with visceral distress after drinking
saline at a familiar or different time of day. Importantly, the magnitude of
aversion in the aged rats was intermediate to the aversion expressed by the adult
rats that were conditioned after drinking saline at a familiar or different time.
This indicates that while aged rats acquire conditioned taste aversions of a
magnitude like adult rats, they failed to express the aversion differentially as a
function of the time of day. This result can be explained by the possibility that
aged rats have a compromised sense of the time of day, but this explanation is
unlikely because Experiment 2 demonstrated that aged rats do use time of day to
modulate the expression of conditioned taste aversion. Lesions were made of the
dorsal hippocampus that also affected the overlying neocortex. In aged rats,
these lesions caused a greater saline aversion if drinking saline at the familiar
(same) time of day was paired with lithium chloride injection compared to the
saline aversion conditioned at an unfamiliar (different) time of day. Since aging
did not occlude the effect of the lesion in the aged rats, it is unlikely that the
behavioural effects of aging can be fully explained by a decline of hippocampal
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Chapter 4
function. It will therefore be valuable to explore the contribution of other
memory systems in the time-of-day modulation of memory.
Our findings are consistent with evidence suggesting that there are
interactions amongst multiple memory systems as well as evidence for a
hippocampal role in forming compound stimulus representations. The normal
modulation of hippocampus-independent taste memories by the time of day
depends on adult hippocampal function (Gallo, 2005). Similarly, Experiment 2,
demonstrated that lesion of the aged dorsal hippocampus facilitated the time-ofday modulation of learning. The demonstration in aged rats that hippocampal
damage may result in an abnormal tendency to form compound-stimulus
representations has also been reported in adult rats (Eichembaum, Mathews and
Cohen, 1989) and monkeys (Saksida, Bussey, Buckmaster, and Murray, 2007).
These studies used simultaneous-cue odor discrimination learning and
transverse-patterning tasks, respectively. These data are consistent with reports
that there are competitive interactions between memory systems in adult rats
(Poldrack and Packard, 2003). There is also evidence that reversible inactivation
of the dorsal hippocampus, which attenuates the acquisition of a place task,
enhances the acquisition of a non-hippocampal response task in adult rats
(Schroeder, Wingard and Packard, 2002). These and other similar results
obtained in adult rats (Poldrack and Packard, 2003) provide evidence of a
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Hippocampus, Ageing and Segregating memories
functional interdependence between hippocampal and non-hippocampal
memory systems, which may compete, leading to enhanced learning after
hippocampal lesions. As far as we know, the present work is the first time that
such an enhancing effect of hippocampal lesion has been reported in aged rats,
which suggests that the notion of interference between memory systems may
extend also to the aged brain.
It therefore appears that aged rats can use the time of day in
representations of experience but that their ability to encode, maintain or use
segregated representations of non-spatial experience is different from this ability
in adult rats. Because this ability to segregate representations of experience is a
function of the adult hippocampus (Gallo, 2005), we have determined that
impaired non-spatial sensory segregation occurs in aged rats and this cognitive
impairment may be the result of aged hippocampal function.
We now consider alternative explanations of the results. First, the
behavioural protocol (Table 1) preexposed all the rats to saline and as a
consequence, after conditioning, all groups expressed less saline aversion than if
there was no preexposure to saline. Rats that received the same behavioural
protocol without saline preexposure drank no saline on the first test and only 1
ml ± 0.67 ml on the third test (data not shown; Morón et al., 2002b). Thus the
present data can be considered in terms of latent inhibition, where more saline
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Chapter 4
aversion indicates less latent inhibition. The results may therefore also be
interpreted as a moderate aging-related deficit of latent inhibition with
hippocampal lesion causing an even more severe deficit. This interpretation,
however, fails to account for why the modulation of latent inhibition by time of
day is in opposite directions in normal adult rats (SAME expressed more latent
inhibition than DIFF) and rats with hippocampal lesions (DIFF expressed more
latent inhibition than SAME). Moreover, this interpretation contradicts the
results of taste aversion (Morón et al., 2002a; Manrique et al., 2007) and active
avoidance (Francès et al., 2001) studies that failed to observe evidence of a
latent inhibition deficit in aged rats or in adult rats with lesions of the dorsal
hippocampus (Gallo and Cándido, 1995). The possibility that we observed
impaired latent inhibition would also contradict the enhanced latent inhibition of
taste aversion that has been repeatedly observed in adult rats with permanent
hippocampal lesions (Purves, Bonardi and Hall, 1995; Reilly, Harley and
Revusky, 1993) and temporary (Stone, Grimes and Katz, 2005) hippocampal
inactivation. We thus reject the interpretation that our results reflect aging
and/or hippocampus lesion deficits of latent inhibition. However, latent
inhibition is not a simple, uniform process. It is affected by the context, time of
day and type of information being processed. Thus, the performance of the aged
rats could be interpreted as a disruption of a temporal context dependency of
latent inhibition (Manrique et al., 2004). This would be consistent with other
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Hippocampus, Ageing and Segregating memories
effects of aging on the dependence of taste neophobia on previous learning
experiences (Morón and Gallo, 2007). However, this interpretation has difficulty
to explain the pattern of results in Experiment 2. The fact that the lesion aged
SAME group showed no signs of latent inhibition could be explained by the
neocortical damage together with the hippocampal lesion (Lewis and Gould,
2007). But this interpretation is also unlikely because the lesion did not disrupt
the latent inhibition that presumably also occurred in the DIFF groups.
A second alternative explanation of the present data is based on the fact
that aging is accompanied by alterations in the diurnal secretion of hormones,
which could be one of the salient components of the time of day that were
available to the rats. It is therefore possible that because aged rats may have a
compromised sense of time of day, they may fail to modulate taste memories by
time of day. This is consistent with the reported disruption of diurnal circadian
rythmicity, which has been related to age differences in hippocampal-dependent
memory processes (Winocur and Hasher, 2004). Results from Experiment 2
suggest that if this interpretation were correct, then hippocampal lesions have
the ability to restore the salience of time-of-day cues. Although this would be a
surprising possibility, it is nonetheless possible.
In contrast to explanations based on latent inhibition and a compromised
sense of time of day in aged rats, the results are all consistent with the
143
Chapter 4
interpretation that the hippocampus is important for segregating components of
experience into distinct representations that can be individually associated with
consequences to efficiently direct subsequent behaviours (Kesner et al., 2004;
Kubik and Fenton, 2005; Wesierska, Dockery and Fenton, 2005). This
viewpoint leads us to conclude that this segregation function is compromised in
aged rats. Furthermore, this interpretation is consistent with accumulating data
that the electrical activity of the aged hippocampus does not readily distinguish
between environments (Wilson et al., 2003) spatial reference frames
(Rosenzweig, Redish, McNaughton and Barnes, 2003), or behavioural episodes
(Shen, Barnes, McNaughton, Skaggs and Weaver, 1997). Different rats, like
people, develop different cognitive abilities and impairments at the same
chronological age. It is therefore possible that the deficit we observed in the
aged group reflects a deficit in only a subset of the aged subjects.
Importantly, the present data demonstrate there are reliable cognitive
deficits in aged rats before and after hippocampal lesion. The deficits are
difficult to explain by the widely acknowledged views that the hippocampus is
important for the normal processing of spatial information (O’Keefe and Nadel,
1978) or relational memory (Squire, 1992; Cohen and Eichenbaum, 1993). The
present results suggest that comprehensive accounts of hippocampal function
might consider highlighting its role in segregating experience into separately
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Hippocampus, Ageing and Segregating memories
stored representations. Such appropriately segregated representations can later
be recombined to form associations with other relevant representations.
Segregated representations of this sort, can also be separated from potentially
irrelevant representations. Incorporating this segregation perspective may help
to account for why aging and hippocampal dysfunction compromise episodic
memory ability.
145
Chapter 4
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149
CHAPTER 5
Manrique, T., Gámiz, F, Morón, I., Ballesteros, M. A., and
Gallo, M. (2008)
Peculiar modulation of taste aversion learning by the time of
day in developing rats
(en revision)
Peculiar modulation of taste aversion learning by the time of
day in developing rats
a,b
Manrique, T., , a,bGámiz, F, a,bMorón, I., aBallesteros, M. A., and a,bGallo, M.
a
Department of Experimental Psychology and Physiology of Behavior. University of
Granada. Spain, bInstitute of Neurosciences F. Oloriz. University of Granada. Spain
ABSTRACT
The ontogeny of the temporal context modulation of conditioned taste aversion
was studied in male Wistar rats using a palatable 1% NaCl solution. A
procedure that included two saline preexposures, a single pairing salinelithium chloride (0,15M; 1% b.w.) either at the same or a different time of day
of preexposures and a one-bottle test at the same time than preexposure was
applied. Five age groups (PN24, PN32, PN48, PN64 and PN100) covering the
complete range from preadolescence to the adult period were tested in two
experiments. The results showed no effect of a temporal context shift both in
PN24 and PN32. A peculiar enhancement of temporal context-specific saline
aversions was exhibited by PN48 and PN64 rats, while the adult typical
temporal context dependency of latent inhibition was only evident in PN100
rats. The results are discussed in terms of the peculiar brain functional
organization during a protracted adolescence period.
This article was supported by the Ministerio de Educación y Ciencia. Spain (SEJ2005-01344)
and Junta de Andalucía. Spain (HUM 02763)
Chapter 5
KEY WORDS: taste aversion, temporal context, adolescence, rats, latent
inhibition, post-weaning, neophobia.
5.1. Introduction
Conditioned taste aversions (CTA) are readily acquired in one-trial by
adult rats if intake of a taste solution is followed by visceral distress induced by
lithium chloride. The learned response consists in a shift of the taste hedonic
value, thus becoming unpalatable and being avoided in later encounters (Bures,
Bermúdez-Rattoni, and Yamamoto, 1988). Among other factors, such as
palatability, taste novelty is a potent modulator of CTA. Previous exposure to
the taste without consequences retards CTA acquisition, a well known
phenomenon called latent inhibition (Lubow, 1989). Context may also
modulate CTA. In addition to context aversions that may be induced by lithium
chloride if several conditioning trials are applied (Boakes, Westbrook, and
Barnes, 1992; Rodríguez, López, Symonds and Hall, 2000; Symonds, and Hall,
1997), both latent inhibition (LI) of CTA and CTA itself may exhibit contextdependency under certain circumstances. First, a context change between
preexposure and conditioning may attenuate the latent inhibition effect (Hall
and Channell, 1986; Rudy, Rosenberg, and Sandell, 1977). Second, a context
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Time of day, Temporal context and Ontogeny
change between conditioning and testing may interfere with learned taste
aversions retrieval (Boakes, Westbrook, Elliot and Swinbourne, 1997; Bonardi,
Honey and Hall, 1990; Loy, Álvarez, Rey, and López, 1993; Puente, Cannon,
Best, and Carell, 1988).
We have previously reported that a time of day shift may also act as a
context, thus modulating CTA in absence of other environmental changes
(Morón et al., 2002; Manrique et al., 2004). Using a behavioural procedure
that included changing the temporal context between preexposure and testing
and also between conditioning and testing, we have demonstrated the temporal
context dependency of both latent inhibition and conditioned taste aversion.
Evidencing the former or the latter phenomenon depends on subtle
modifications of the same basic behavioural procedure. Thus, if the procedure
includes a long habituation period to drink twice a day and non restricted
intake during conditioning those groups preexposed and conditioned at a
different time of day exhibit stronger aversions than those preexposed and
conditioned at the same time of day, showing that a time of day shift between
preexposure and conditioning interferes with LI (Manrique et al., 2004).
However, the opposite pattern is seen by applying only two days of previous
habituation to drink water twice a day and restricted drinking during the
conditioning session. The group conditioned and tested at the same time of day
155
Chapter 5
showed stronger aversions than that conditioned and tested at a different time
of day (Morón et al., 2002). This showed that the same time of day of
conditioning facilitates retrieval of the aversive taste memory during testing
(Manrique, et al., 2004).
Ontogenetically, CTA is a primitive and early developing type of
associative learning. The ability to associate flavour cues with subsequent
lithium-induced visceral distress and to exhibit conditioned flavour aversions
in later encounters has been reported in rat foetuses (Abate, Pepino,
Domínguez, Spear, and Molina, 2000; Smotherman, 2002a, 2002b;
Smotherman and Robinson, 1985). Neonatal rats are able to learn odour and
taste aversions. Lemon-quinine pairings 3-5 hours after birth result in odour
aversions that reduce both attachment to a surrogate nipple and milk intake in
the odour presence (Nizhnikov, Petrov, and Spear, 2002). Rudy and Cheatle
(1977) reported aversions in 8-day-old rat pups exposed to an odour-lithium
chloride pairing at the age of two days. Although the nursing situation may
interfere with the acquisition of learned taste aversions (Alberts and Gubernick,
1984; Martin and Alberts, 1979), aversions to sweet and salty solutions
followed by lithium chloride injections are evident in five-day-old rat pups
when tested 5 or 16 days later (Kehoe and Blass, 1986). Hoffmann, Molina,
156
Time of day, Temporal context and Ontogeny
Kucharski, and Spear (1987) also demonstrated saccharin and sucrose
aversions induced by lithium chloride pairings in 5- and 9-day old pups.
As it has been described in other types of learning, new CTA
capabilities emerge at older ages, supporting both longer retention intervals
(Gregg, Kittrell, Domjan, and Amsel, 1978; Guanoswsky, Misanin, and Riccio,
1983; Schweitzer and Green, 1982) and the appearance of more complex
associative phenomena, such as latent inhibition or second-order conditioning
(Ader and Peck, 1977; Cheatle and Rudy, 1979). In fact, latent inhibition of
CTA shows a late emergence during development. In spite of previous results
showing LI of sucrose aversions after a high number of preexposures in 20-25
day old rats (Franchina, Donato, Patsiokas, and Griesemer, 1980), a number of
early studies reported deficits in latent inhibition of CTA before 20-25 days of
aged (Klein, Mikulka, Domato, and Hallstead, 1977; Misanin, Blatt, and
Hinderliter, 1985; Misanin, Guanowsky, and Riccio, 1983; Wilson and Riccio,
1973). Nicolle, Barry, Veronesi, and Stanton (1989) performed a well
controlled study which included 18- 25- and 32-day-old rats in order to assess
LI of CTA to coffee and saccharin solutions. By applying four preexposures to
the solutions throughout intraoral cannulae along 2 days and lithium injection
after a fifth solution infusion the next day they did not found evidence of LI of
157
Chapter 5
CTA in rats younger than 32 days of age, being the aversion tested in a onebottle test performed four days later.
To the best of our knowledge no data are available on the ontogeny of
the LI contextual specificity using CTA. Moreover, studies on conditioned
emotional response (CER) provide conflicting results. Rudy (1994) reported
context-specific LI in 23-day-old rats, while Zuckerman, Rimmerman, and
Weiner (2003) showed that 35-day-old rats exhibited LI “resistant” to a context
shift between preexposure and conditioning.
In the present experiments we have applied CTA in order to explore the
ontogeny of latent inhibition (Exp. 1) and its contextual specificity (Exp. 2) in
postweaning rats by using a taste aversion learning protocol that has been
proven appropriate to induce time-of-day specific LI of CTA in adult rats.
5.2. Experiment 1
Latent inhibition emerges at different developmental points depending
on the learning procedure. Latent inhibition has been reported in 18-day-old
rats applying fear conditioning to an auditory stimulus (Kraemer and Randall,
158
Time of day, Temporal context and Ontogeny
1992; Rudy, 1994), an odour (Richardson, Fan, and Parnas, 2003; Yap and
Richardson, 2005) and in fear potentiated startle (Richardson et al., 2003). The
emergence of latent inhibition of CTA seems to be dependent on the number of
preexposures, among other parameters of the behavioural procedure. Nicolle et
al (1989) who applied throughout oral cannulae four taste preexposures
demonstrated latent inhibition of CTA in rats at the age of 32 but not 25 or 18
days. However, by increasing the number of preexposure LI of CTA has been
reported in 20-25-day-old rats (Franchina et al, 1980).
This experiment examined whether 24 and 32-day-old (PN24 and
PN32) rats demonstrate latent inhibition of CTA in a conventional protocol
including voluntary intake identical to that previously applied to adult rats
(Morón et al., 2002). The behavioural procedure included two non-reinforced
saline exposures and a single saline-lithium chloride pairing. Given that
Nicolle, Barry, Veronesi, and Stanton (1989) showed that 25-day-old fail to
exhibit latent inhibition after four taste preexposures, we predicted that 24-dayold rats would not exhibit the preexposure effect, i.e., that preexposed and
control non-preexposed groups would exhibit similar strength aversions.
However, we hypothesized that 32-day-old rats would exhibit latent inhibition,
thus showing the preexposed group weaker aversions than the control nonpreexposed group.
159
Chapter 5
5.2.1. Method
5.2.1.1. Subjects
Thirty-six naïve male Wistar rats obtained from ten dams were used.
All the rats were bred at the colony of the University of Granada weighed a
mean of 62.6 gr (range = 46.2 – 81.6 gr) for 24 day-old group and 85.8 gr
(range = 66 – 108 gr) for 32 day-old group. The female pregnant rats were
checked daily for new births being the first postnatal day (PN0) the morning in
which the new litters were first observed. Three days after birth each litter was
culled to ten pups (being the males always preserved) and housed with their
dams in standard clear polyethylene hanging cages. Weaning took place on
postnatal day 19 (PN19). The litters were individually housed in an isolated
room with constant temperature (22-24ºC) and a 12:12 h. light-dark cycle
(lights on at 8:00 am and off at 8:00 pm). Food was available ad libitum, but
water availability depended on the behavioural procedure.
Half of the animals were conditioned at the age of 24 ±1 days (PN24)
while the other half at 32 ±1 days (PN32). In addition, half of the animals in
each group received previous preexposures (Pre) while the other half were nonpreexposed (Ctrl). Thus, the rats were randomly assigned to one of 4 groups:
160
Time of day, Temporal context and Ontogeny
PrePN24 (n=10), CtrlPN24 (n=8), PrePN32 (n=9) and CtrlPN32 (n=8). The
behavioural procedures were approved by the University of Granada Ethics
Committee for Animal Research, and were in accordance with both the NIH of
the United States guidelines for the ethical treatment of animals, and the
European
Communities
Council
Directive
of
24
November
1986
(86/609/EEC).
5.2.1.2. Apparatus
During the behavioural training the rats remained on individual home
cages, which consisted of one chamber (30 x 15 x 30 cm) made of four walls:
two opposing walls were made of opaque polyethylene; the front and back
walls were made of clear polyethylene. The front wall had two holes of 1.6 cm
of diameter, placed to equal distance from the centre and 10 cm from the
bottom of the cage, thus allowing us to introduce the graduated burettes
containing the taste solution. The luminance provided by the lights located on
the ceiling of the room provided 40 nit. A ventilation fan that was located on
the side wall of the room produced a low-level background noise of 66 dB.
161
Chapter 5
5.2.2. Procedure
The animals were subjected to the short-habituation procedure
described by Morón et al. (2002). Throughout the behavioural procedure they
had two daily 15 min drinking sessions. Morning (9:00 am) and evening (7:00
pm) drinking sessions were used to maximally differentiate the temporal
contexts. The procedure consisted in five phases: Habituation, Taste
Preexposure, Conditioning, Recovery and Testing (see table 1).
After being habituated for 2 days to the water deprivation procedure,
the rats belonging to the preexposed groups (Pre) were allowed to drink saline
solution (NaCl, 1% diluted in distilled water) for 15 min during the evening
session for the following 2 days while those belonging to the non-preexposed
groups (Ctrl) were allowed to drink water.
The next day conditioning took place during the evening session. All
the rats were allowed to drink 4 ml of a sodium chloride solution (1%) for 15
min. Fifteen minutes later they received an intraperitoneal (i.p.) injection of
lithium chloride (LiCl 0.15M; 2% body weight) and were returned to the home
cage.
After two recovery days with water available during the drinking
sessions, a one-bottle test was applied during the evening drinking session. The
amount of fluid ingested was recorded to the nearest 0.1 ml. In order to
162
Time of day, Temporal context and Ontogeny
facilitate comparisons between different aged groups and different experiments
a test intake rate was calculated (test/CTA * 100), representing 100 no
aversion, since the animals would have drunk the same amount during testing
than during conditioning and 0 maximum aversion.
Table 1.
Behavioural procedure. am/pm = temporal contexts
morning/evening; Sal = Isotonic saline, 1%; LiCl = Lithium Chloride. Pre
= Preexposed group; Ctrl = Control group; Same = groups with
preexposure, conditioning and testing during evening session; Diff =
groups with conditioning during morning session.
HABITUATION PREEXPOSURE CONDITIONING RECOVERY
TESTING
(Exp 1: 2 days;
(2 days)
(1 session)
(2 sessions)
(1 session)
Exp 2: 5 days)
am and pm
am: Water
am: Water
am and pm
am: Water
Water
pm: Sal
pm: Sal ± LiCl
Water
pm: Sal
am and pm
am: Water
am: Sal ± LiCl
am and pm
am: Water
Water
pm: Sal
pm: Water
Water
pm: Sal
am and pm
am and pm
am: Water
am and pm
am: Water
Water
Water
pm: Sal ± LiCl
Water
pm: Sal
am and pm
am and pm
am: Sal ± LiCl
am and pm
am: Water
Water
Water
Water
pm: Sal
Pre-Same
Pre-Diff
(only Exp 2)
Ctrl-Same
Ctrl-Diff
(only Exp 2)
pm: Water
163
Chapter 5
5.2.3. Results and Discussion
A 2 x 2 (Age x Preexposure) between groups ANOVA analysis of the
rats weight at the beginning of the experiment showed only a significant main
effect of Age (F(1,31)= 32.55; p < 0.01), indicating that PN24 rats weighted less
(62.73 gr ± 2.58) than the PN32 rats (85.79 gr ± 3.18). There was not a
significant effect of Preexposure (F(1,31)= 2.01; p > 0.17) nor interaction Age x
Preexposure (F(1,31)= 0.05; p > 0.82).
Consistently, there was a significant
effect of Age (F(1,33)= 18.7; p < 0.01) in the water intake during the second
habituation evening session, that was taken as the baseline, PN24 rats drunk
3.65 ml (SEM ± 0.2) while PN32 drunk 4.79 ml (SEM ± 0.2).
A mixed 2 x 3 (Age x Days) ANOVA analyses of the amount ingested
by the preexposed groups including the water intake during the baseline and
saline intake during the preexposure sessions was applied in order to explore
the presence of neophobia. The analysis showed a main effect of Days (F(2,32)=
19.9; p < 0.01) but no significant effect of Age (F(1,16)= 2.31; p > 0.15) nor
interaction Age x Days (F(2,32)= 0.49; p > 0.62). Post hoc LSD comparisons
indicated higher consumption of saline during the first preexposure than water
intake during the baseline water drinking session ( p < 0.01) and no differences
164
Time of day, Temporal context and Ontogeny
between the first and second preexposure (p > 0.55) . The increased saline
consumption compared with water shows a high acceptance of the low NaCl
concentration used. The steady intake in both preexposures, i.e., the absence of
increase in the amount ingested during the second preexposure session can be
interpreted as absence of neophobia to the low concentration of saline solution,
although a ceiling effect can not be discarded.
Since the saline intake during conditioning was restricted to 4 ml, there
were no differences between the groups. Fig. 1 summarizes the results of the
conditioning and testing phases using a test intake rate (%). A 2 x 2 (Age x
Preexposure) ANOVA analysis of the test intake rate (%) showed only a
significant main effect of Preexposure (F(1,31)= 5.38; p < 0.05), but not
significant effects of Age (F(1,31)= 1.32; p > 0.26) nor interaction Age x
Preexposure (F(1,31)= 0.62; p > 0.44). In spite of the fact that the interaction
was not significant, LSD planned comparisons showed no differences between
Pre and Ctrl groups in the younger PN24 condition (p > 0.27) indicating
absence of latent inhibition. In contrast, at the older age (PN32) the nonpreexposed Ctrl group showed a significantly lower test intake rate, i.e.,
stronger aversion than the non-preexposed Ctrl group (p < 0.05), thus showing
the latent inhibition effect.
165
Chapter 5
Figure 1. Saline solution test intake rates (Test/CTA *100) of preexposed
and control groups receiving a taste-lithium pairing at postnatal day 24
(PN24) and postnatal day 32 (PN32) in the Experiment 1. The average
values ± SEM are reported.
In all, the results of experiment 1 showed three main findings. First,
there was no evidence of neophobic response to a low concentration saline
solution at any of the ages tested. On the contrary, the saline solution intake
was higher than that of water baseline and no subsequent evidence of
habituation of neophobia was found, since there was no further increase in the
amount of saline solution consumption during the second preexposure.
166
Time of day, Temporal context and Ontogeny
Second, both age groups exhibited reliable taste aversion to a highly
accepted low concentration saline aversion. The absence of aversion would
have yielded test intake rates higher than 100, due to the restricted intake
during conditioning. Third, 32- but not 24-day-old rats showed latent inhibition
of CTA using a behavioural procedure that included two saline preexposures
twenty four hours apart between each of them and conditioning. This finding
confirms and extends previous results (Nicolle et al., 1989) showing that rats
younger than 32 days of age do not exhibit LI of CTA. Nicolle, et al. (1989)
applied three preexposures to a intraorally infused coffee solution separated
between them by two hours and a fourth exposure sixteen hours later,
conditioning taking place ten hours after the last preexposure. In the present
experiment the absence of LI in 25-day-old was demonstrated using a
procedure that included two saline preexposures and we had previously
demonstrated to induce LI in adult rats.
5.3. Experiment 2
The results of experiment 1 demonstrated LI of learned saline aversions
in 32- but not 24- day-old rats. By using the time of day as context and a
167
Chapter 5
modified procedure that included five days of habituation to drink twice a day
and non-restricted intake during conditioning, we have previously reported
context-specific LI of conditioned saline aversions in adult rats (Manrique et
al., 2004). Experiment 2 examined the emergence of the temporal context
dependency of LI during the adolescence period, which has been widely
defined as covering a wide ages range from PN28 to around PN60 (Spear,
2000). Thus, the performance of three adolescent groups (PN32, PN48, PN64)
and an adult three-month-old group (PN100) was compared using an identical
procedure to that used by Manrique et al. (2004).
5.3.1. Subjects
One hundred and forty one male Wistar rats obtained from 31 litters
were used. The litters were culled to 10 pups and weaning took place on PN19
following the procedure described in Experiment 1. After weaning, the rats
were housed in groups of three to four subjects until the training procedure
required individual housing. According to the 4 x 2 x 2 (Age x Preexposure x
Group) design the pups were randomly assigned to one of the sixteen groups
(see table 1). The behavioural procedures were approved by the University of
Granada Ethics Committee for Animal Research, and were in accordance with
168
Time of day, Temporal context and Ontogeny
both the NIH of the United States guidelines for the ethical treatment of
animals, and the European Communities Council Directive of 24 November
1986 (86/609/EEC).
5.3.2. Procedure
The general behavioural procedure was similar to that described in
Experiment 1 except for two differences: the habituation phase to drink water
twice a day lasted five days instead of two, and the rats were allowed to drink
the saline solution during the 15 minutes conditioning session without
restriction (see table 1).
The behavioural procedure has been previously described in detail
(Manrique et al., 2004). Briefly, in order to test LI and its contextual
specificity, each age group was further divided in four groups according to the
presence or absence both of a time of day shift during conditioning and of
saline preexposures.
Same groups were conditioned during the evening
drinking session while Diff groups were conditioned during the morning
drinking session, i.e., at a different time of preexposure and testing. Control
groups had no previous saline preexposures while Pre groups received two
saline preexposures. In order to facilitate comparisons between different age
169
Chapter 5
groups a test intake rate was calculated (test/CTA * 100), representing 100 no
aversion, since the animals would have drunk the same amount during testing
than during conditioning and 0 maximum aversion.
5.3.3. Results and Discussion
Table 2 shows the mean (± SEM) body weights of the different groups
at the behavioural procedure onset. A 4 x 2 x 2 (Age x Preexposure x Group)
ANOVA analysis evidenced a significant main effect of Age (F(3,125)= 847.86;
p < 0.01), showing an expected weight increase related with increasing age.
Thus, post hoc LSD comparisons showed differences between all the age
groups (p < 0.01). No other effects were significant.
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Time of day, Temporal context and Ontogeny
Table 2.
Experimental design including number of subjects in each group and
mean ± SEM of weight by age.
PN32
PN48
PN64
ADULT
Same
Preexposed
Control
n=8
n=9
n=9
n = 10
83.47 gr ± 5.35 41.22 gr ± 6.28 71.13 gr ± 6.69 255.84 gr ± 4.6
Diff
n=8
n = 10
n = 10
n = 10
85.03 gr ± 3.35 54.16 gr ± 6.15 55.93 gr ± 5.06 57.82 gr ± 4.31
Same
n=8
n=9
n=8
n=9
86.7 gr ± 4.48 9.98 gr ± 4.03 61.02 gr ± 4.71 57.71 gr ± 3.04
n=8
n=8
n=9
Diff
n=8
84.32 gr ± 3.66 45.15 gr ± 3.85 56.16 gr ± 5.53 53.89 gr ± 4.18
A 4 x 2 x 2 (Age x Preexposure x Group) ANOVA analysis of the water
consumption during the baseline showed a significant main effect of Age
(F(3,127)= 12.96; p < 0.01, but no other main effects nor interactions were
significant. Post hoc LSD comparisons indicated that PN32 group consumed
less water than the rest of the groups (p < 0.01). In spite of the age-related
weight increase, no differences were seen among the rest of the groups in the
water intake.
In order to explore the neophobic response and its potential habituation
a 4 x 4 (Age x Days) mixed ANOVA analysis of the amount of water and
saline solution drunk during the baseline, preexposures and conditioning
sessions in those groups conditioned at the same time of preexposures (Pre171
Chapter 5
Same) was performed. There was a significant effect of Age (F(3,32)= 11.29; p <
0.01), Days (F(3,96)= 24.90; p < 0.01) and the interaction Age x Days (F(9,96)=
2.11; p < 0.05)
Repeated measures ANOVA analyses of the amount drunk by each age
group including the baseline, preexposures and conditioning sessions revealed
a significant main effect of Days in each group, PN32 (F(3,21)= 7.84; p < 0.01) ,
PN48 (F(3,24)= 3.47; p < 0.05), PN64 (F(3,24)= 11.50; p < 0.01) and Adult
(F(3,27)= 15.96; p < 0.01). Post-hoc LSD comparisons showed increase of
saline intake during the first preexposure compared with the previous baseline
water drinking session in the each of the age groups (p < 0.05), confirming the
results obtained in Experiment 1. An increase of saline consumption during
the second preexposure compared with the first preexposure was evident only
in PN32 (p < 0.01) and Adult groups (p < 0.01). However, PN32 rats reduced
again the saline intake during the third preexposure with no significant
differences in comparison with the first preexposure, while adult rats
maintained a high saline intake with no differences between the second and
third preexposures, thus confirming attenuation of neophobia only in adult rats.
Unexpectedly, PN64 rats that did not showed attenuation of neophobia in the
second preexposure, exhibited a later increase in saline solution intake during
the third preexposure, i.e., the conditioning session compared with the previous
172
Time of day, Temporal context and Ontogeny
preexposure session (p < 0.01) which did not appear in the younger groups.
This increase could not be detected in Experiment 1 since saline intake was
restricted during the conditioning session, but it allows us a different
conclusion concerning the presence of the neophobic response.
Regarding the ontogeny of taste neophobia, the data support not only
the presence of a neophobic reaction to a highly accepted low saline
concentration, response that requires two exposures to habituate in Adult
groups, but also a saline neophobic response at 64 days of age, that required
three exposure to habituate. No evidence of neophobic responses were found in
the younger groups.
Figure 2 shows the mean (± SEM) test intake rates of the different
groups. A 4 x 2 x 2 (Age x Preexposure x Group) ANOVA analysis showed a
significant effect of the interaction Age x Preexposure x Group (F(3,125)= 6.12;
p < 0.01). Therefore, 2 x 2 (Preexposure x Group) two way ANOVA analyses
were performed in each age group.
173
Chapter 5
Figure 2. Test Saline solution test intake rates (Test/CTA *100) of the different
groups receiving a taste-lithium pairing at postnatal day 24 (PN24), postnatal day 32
(PN32), postnatal day 48 (PN48), postnatal day 64 (PN64) and postnatal day 100
(Adult) in the Experiment 2. (Pre = preexposed groups; Ctrl = non-preexposed
groups; Same = groups receiving preexposures, conditioning and testing at the same
time of day; Diff = groups conditioned at a different time of day of preexposure and
testing.)
In PN32 there was a significant effect of Preexposure (F(1,28)= 16.14; p
< 0.01), evidencing the non-preexposed groups stronger aversions (mean:
60.26 ± 7.17) than the preexposed groups (mean: 24.30 ± 4.9), but no effect
of time of day shift (F(1,28)= .21; p > 0.65), nor interaction (F(1,28)= 0.04; p >
0.84). Thus, the results revealed the presence of latent inhibition and no effect
of the temporal context shift on LI of CTA at this age.
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Time of day, Temporal context and Ontogeny
In PN48 there was a significant main effect of Preexposure (F(1,32)=
33.77; p < 0.01), Group (F(1,32)= 5.25; p < 0.05) and the interaction Preexposure
x Group (F(1,32)= 4.76; p < 0.05). Post-hoc LSD comparisons revealed that the
effect could be attributed to the differences between the preexposed groups,
since no differences were seen between the Ctrl non-preexposed groups (p >
0.94) which exhibited a low test intake rate, i.e., strong saline aversions. LI was
evident in both preexposed groups, since both Pre-Same (p < 0.01) and PreDiff (p < 0.05) showed higher intake rates than their respective control nonpreexposed groups. The Pre-diff group showed also a weaker aversion than
Pre-Same group (p < 0.01). This pattern of results supported the presence of
context specific saline aversions in the preexposed animals and absence of
context specific LI.
Unexpectedly, similar results were obtained in PN64 rats. Again, there
was a significant main effect of Preexposure (F(1,31)= 39.01; p < 0.01), Group
(F(1,31)= 6.71 p < 0.01) and the interaction Preexposure x Group (F(1,31)= 10.74;
p < 0.01). Post-hoc LSD comparisons confirmed that the effect could be
attributed to the differences between the preexposed groups, since no
differences were seen between the Ctrl non-preexposed groups (p > 0.64)
which exhibited a low test intake rate, i.e., strong saline aversions. LI was
evident in both preexposed groups, since both Pre-Same (p < 0.05) and Pre175
Chapter 5
Diff (p < 0.01) showed higher intake rates than their respective control nonpreexposed groups. Again, the Pre-diff group showed also a weaker aversion
than Pre-Same group (p < 0.01).
The results obtained with the adult group confirmed previously reported
results showing that a time of day shift during conditioning disrupt LI
(Manrique et al., 2004). There was a significant main effect of Preexposure
(F(1,34)= 15.39; p < 0.01), Group (F(1,34)= 4.06; p < 0.05) and the interaction
Preexposure x Group (F(1,34)= 6.19; p < 0.05). Post-hoc LSD comparisons
revealed that the effect could be attributed to the differences between the
preexposed groups, since no differences were seen between the Ctrl nonpreexposed groups (p > 0.75) which exhibited a low test intake rate, i.e., strong
saline aversions. Interestingly, the pattern of results shown by the preexposed
groups was opposite to that found in the younger groups. LI was evident only
in the Pre-Same group (p < 0.01), but not in the Pre-Diff group (p > 0.32). In
fact, the Pre-diff group showed also a stronger aversion than Pre-Same group
(p < 0.01).
Taken together the results indicated a late emergence of the adult timeof-day specific LI (Manrique et al., 2004), which is only observed in the adult
group. Consistent with a context-independent LI, the deleterious effect of
saline preexposures on later CTA was resistant to a time of day shift in PN32.
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Time of day, Temporal context and Ontogeny
However, the time of day shift induced a peculiar strong effect both in PN48
and PN64. Those preexposed groups conditioned at a different time of day of
preexposure and testing exhibited weaker saline aversions than those
conditioned at the same time of day of preexposure and testing. This is an
effect previously reported by using a short-habituation procedure in adult rats
(Morón et al., 2002), but never obtained with the present long-habituation
protocol in adult rats (Manrique et al., 2004).
Regarding the ability to learn a saline aversion, a one-way ANOVA
analysis of the test intake rates showed by the Ctrl non-preexposed groups at
the different ages showed a significant main effect of Age (F(3,59)= 16.38; p <
0.01), evidencing weaker aversions in the younger PN32 group than in the rest
of the groups (p < 0.01). No differences were seen among the rest of the
groups. There was not significant effects of Group (F(1,59)= 0.24; p > 0.63) nor
interaction Age x Group (F(3,59)= 0.39; p > 0.76). This result evidenced that the
learning ability improved with age, having reached the adult level by 48 days
of age.
177
Chapter 5
5.4. General Discussion
Several findings regarding the ontogeny of taste neophobic responses,
the ability to learn taste aversions, the latent inhibition phenomenon and the
effects of context modulation on CTA are reported using a low concentration
NaCl solution as the taste stimulus in rats.
First, regarding the ontogeny of the neophobic response to a 1% sodium
chloride (NaCl) solution, the results of both Experiment 1 and Experiment 2
showed increased intake of the novel salty solution during the first preexposure
session in all the age groups. We have previously reported similar results in
adult and aged Wistar rats (Morón and Gallo, 2007). These data confirm the
high palatability of this low saline concentration solution for rats. Accordingly,
previous results have shown that this solution is preferable to water (Bare,
1949; Kare, Fregly, and Bernard, 1980; Kiefer and Grijalva, 1980; Pfaffmann,
1960; Weiner and Stellar, 1951).
Although taste neophobia is defined as the reluctance to consume
novel-taste solutions (Bures, et al. 1998), the demonstration of such reluctance
requires an increase in taste consumption induced by the successive
presentations
178
without
negative
consequences
(neophobia
attenuation),
Time of day, Temporal context and Ontogeny
regardless of previous water consumption. This is especially true if a palatable
solution is used, as in our case. Therefore, we determined the absence of the
neophobic response to a highly palatable NaCl solution not only by
comparisons
between
saline
consumption
and
previous-day
water
consumption, but also by further comparisons with saline intake on a second
presentation (Exp. 1 and 2) as proposed by Reilly and Bornovalova (2005), and
even on a third presentation (Exp. 2).
The results of Experiment 1 confirmed the absence of saline neophobia
both in PN24 and PN32, since no further saline increase was seen in the second
preexposure session. Similar results were obtained in Experiment 2 at 48 and
64 days of age. The transitory increase of saline consumption observed in
PN32 group during the second exposition does not seem to reflect attenuation
of neophobia, since it disappeared during the following presentation in which
the volume ingested decreased to the first preexposure level. However, in adult
rats an increase in saline ingestion during the second presentation, which was
maintained on the following presentation, evidenced the presence of
neophobia.
Thus, the data of both experiments concerning previous water intake
and two saline presentations could be initially interpreted in terms of absence
of saline neophobia until the age of 64 days. However, the analysis of the
179
Chapter 5
saline intake in Experiment 2 which included a third presentation, i.e., the
saline intake during the conditioning session in those groups preexposed and
conditioned at the same time of day, revealed the presence of neophobia not
only in the adult group but also in the 64-day-old group, since an increase of
saline intake was seen during the conditioning session. No such increase
occurred in the younger groups confirming the absence of neophobia. The
possibility that determining the presence of neophobic responses to highly
palatable solutions may require a minimum of three taste presentations instead
of two as previously proposed (Reilly and Bornovalova, 2005) should be
considered.
There are three different explanations for the absence of neofobia to a
NaCl solution in the younger groups aged from 24 to 48 days. On the first
place, the absence of neophobia could be attributed to immaturity of the
gustatory system. Important changes in the sensitivity of the gustatory system
to NaCl take place during development as shown by neurophysiological data. It
has been reported that functional responses of the nucleus of the solitary tract,
which is the first relay level of the gustatory system in rats, are not mature until
after 35 days of age (Hill, Bradley, and Mistretta, 1983). Thus responsiveness
to salts changes dramatically during a prolonged developmental period.
Compared with adult rats, younger 25-day-old rats drink higher amounts of
180
Time of day, Temporal context and Ontogeny
high concentration NaCl solutions that are aversive to mature rats (Midkiff and
Bernstein, 1983). This could be interpreted as the younger rats failing to
perceive qualitative or quantitative features of the stimulus. However, in the
present experiment all the age groups demonstrated the ability to detect the
saline solution, because learned aversions were evident. While a reduced
sensitivity to the low saline concentration used in the present experiment
cannot be fully discarded in 24- and 32 day-old rats, groups that showed
weaker saline aversions than adult rats, this explanation is not supported in the
48-day-old group exhibiting robust saline aversions similar to that of older age
groups.
On the second place, the absence of saline neophobia in the younger
groups could be due to an unspecific effect of early handling and training after
weaning. It has been reported a decrease in neophobia induced by early
handling (Weinberg, Smotherman, and Levine, 1978). However, all the groups
were similarly treated concerning weaning and early handling.
Finally, the most feasible explanation for the absence of neophobia, at
least in PN32 and PN48 groups, is that based in the increased novelty seeking
associated to adolescence (Spear, 2000). Reduced neophobic responses have
been reported in adolescent rats (Darmani, Shaddy and Gerdes, 1996; Spear,
2000; Spear, Shalaby and Brick, 1980). Although a reduced salt sensitivity
181
Chapter 5
cannot be discarded in the younger 24- and 32-day-old groups, the absence of
neophobia in 48-day-old rats can be more likely due to the peculiar features of
the adolescent behaviour leading to novelty seeking. If this were so, the results
of Experiment 2 support that 64-day-old rats may reflect the transition to the
mature behavioral pattern regarding responses to taste novelty.
With respect to the ontogeny of CTA, the ability to learn aversions to a
low saline concentration was present in 24-day-old rats but it seemed to
increase at older ages, reaching the mature level at 48 days of age. Experiment
1 showed stronger aversions in the older PN32 group than the younger PN24
group. This finding was extended in the second experiment. The youngest
PN32 group exhibited weaker aversion than the rest of the groups that did not
differ between them. The results confirm previous reports showing lithiuminduced aversions to salty solutions at early ages (Kehoe and Blass, 1986), but
they indicate a developmental course of CTA capability throughout the
preadolescent and early adolescent period. As mentioned above, the protracted
developmental course of the brain mechanisms involved in NaCl perception
which may have led to a potential reduced sensitivity to the taste solution, as
well as the delayed maturation of the associative and memory processes
required (Vogt and Rudy, 1984), can provide a feasible explanation for the
improvement in long-delay CTA ability from 24 to 48 days.
182
Time of day, Temporal context and Ontogeny
Third, regarding the ontogeny of the latent inhibition phenomenon, the
results of Experiment 1 showed that latent inhibition of CTA is evident at 32
but not 24 days of age. Experiment 2 confirmed the presence of LI from 32
days of age. The results are consistent with those of Nicolle et al. (1989) who
did not found evidence of latent inhibition of CTA using four taste
preexposures applied along two days in rats younger than 32 days of age. The
authors attributed the delayed onset of latent inhibition to immaturity of the
hippocampal system, since fornix transections performed in preweanling rats
disrupted the emergence of latent inhibition at 32 days of age. However, at
present, there is no evidence for a critical hippocampal involvement in latent
inhibition of CTA (for reviews see Buhusi, Gray, and Schmajuk, 1998; Gallo,
Ballesteros, Molero, and Morón., 1999). Lesion studies in adult rats showed no
effect (Gallo and Cándido, 1995) or enhancement of latent inhibition (Purves,
Bonardi, and Hall, 1995; Reilly, Harley, and Revusky, 1993). Moreover, no
disruption of LI by fornix transections has been reported in adult rats (Weiner,
Feldon, Tarrasch, Hairston, and Joel, 1998). It can be envisaged that fornix
transection during the early development can have widespread effects on the
neural networks organization hindering a simple explanation.
Finally, the most outstanding finding in the present study is the
modification of the temporal context effect on CTA throughout the adolescence
183
Chapter 5
period. The range of developmental ages chosen, from PN24 to PN64, covered
the complete adolescence period in rats, according to the most wide
behavioural criteria (Spear, 2000). The results show a late emergence of the
temporal context modulation of CTA, which is not evident in 32-day-old rats.
Moreover, the temporal context modulation of CTA in PN48 and PN64
dramatically differs of that seen in the adult group. In fact, a time of day shift
during the conditioning session induced a similar pattern of results at 48 and 64
days that was opposite to that seen at 100 days of age. In adult rats the
temporal context change disrupted LI of CTA, thus showing a similar aversion
to that exhibited by the non-preexposed groups. Therefore, the group
conditioned at a different time of preexposure (Diff) showed a stronger
aversion than the group preexposed and conditioned at the same time of day
(Same). This temporal context specificity of LI has been previously reported in
adult rats using an identical procedure (Manrique et al, 2004).
However, the latent inhibition of CTA was not disrupted by
conditioning the saline aversion at a different time of day than the preexposure
sessions both in PN48 and PN64 groups. The Pre-Same and Pre-Diff groups
both had weaker aversions than their respective non-pre-exposed control
groups. Context non-specific LI has also been previously reported (Hall and
Channell, 1986; Kurz and Levitsky, 1983; Rudy, Rosenberg, and Sandell,
184
Time of day, Temporal context and Ontogeny
1977). Moreover, in these age groups the time of day shift induced an opposite
pattern of differences, with stronger aversions in the Pre-Same groups than in
the Pre-Diff groups. We have previously reported a similar pattern of
differences between Same and Diff preexposed groups in adult rats (Morón et
al., 2002) applying the behavioural protocol described in Experiment 1, which
included only two habituation days and restricted drinking during conditioning.
A weaker aversion in those groups conditioned and tested at a different time of
day than in those conditioned and tested at the same time of day may be the
result of a temporal context specific aversion in the preexposed groups. The
absence of differences between the Ctrl Pre and Ctrl Diff non-preexposed
groups confirms previous results using one-trial CTA and one-bottle tests
(Bonardi, Honey, and Hall, 1990; Rosas and Bouton, 1997) and it is consistent
with previous findings showing that taste familiarity increases the contextual
control of the aversion (Boakes et al., 1997; Puente et al., 1988).
It can be proposed that the contextual specificity of LI and the
contextual specificity of CTA are developmentally dissociable phenomena,
showing the former a later emergence than the later. This is consistent with
data showing that NMDA lesions of the dorsal hippocampus in adult rats
disrupted the context specificity of LI but they did not impair the context
specificity of CTA (Gallo, 2005). A bulk of results has pointed to a delayed
185
Chapter 5
emergence during development of learning and memory functions requiring a
mature hippocampus (Bachevalier and Vardha-Khadem, 2005; Stanton, 2000).
In fact, context dependent learning effects show a late emergence during
development in other aversive learning tasks, such as fear conditioning (Rudy,
1994; Rudy and Morledge, 1993; Yap and Richardson, 2007). Furthermore, it
has also been suggested that different functions of context cues in learning and
memory (Holland and Bouton, 1999) may show different developmental
courses (Carew and Rudy, 1991; Rudy, 1993).
Nonetheless, the above reasoning is not enough to explain how the
behavioural procedure that evidenced the temporal context specificity of LI in
adult rats may have disclosed the temporal context specificity of CTA in
developing rats, leading to the opposite pattern of differences seen in
adolescent rats. The explanation has to be based in a peculiar organization of
the learning and memory systems during this developmental period that
facilitated the formation of a compound representation of saline and time-ofday. Thus, the temporal context specificity of learned taste aversions which in
adult rats is only seen using a short habituation learning protocol was
displayed. Brain development involves not only progressive but also critical
regressive processes, taking place at different developmental stages in different
brain areas. Thus, the developing brain systems involved in learning and
186
Time of day, Temporal context and Ontogeny
memory exhibit peculiar patterns of organization that are not seen in adults and
that may promote unique types of learning at early developmental stages.
Among the peculiar features of learning demonstrated in developing rats is the
enhancement of the ability to establish associations between stimuli not found
in adults (Campbell and Spear, 1972; Hoffmann and Spear, 1988; Molina,
Hoffmann, Serwatka and Spear, 1991; Bordner and Spear, 2006), the
potentiation of the contextual conditioning by CS conditioning (Brasser and
Spear, 2004) and facilitation of sensory preconditioning (Barr, Marrott, and
Rovee-Collier, 2003).
It can be proposed that the contextual modulation of CTA seen in the
adolescent groups in our study involves associations between the time of day
and the taste CS. These would have been facilitated due to the diffuse brain
activation produced by an excessive number of synapses before the protracted
axonal pruning taking place throughout the adolescence. However, adult rats
would have represented the saline taste separately from the time of day it was
experienced, since the opposite pattern of results was evident. Thus, the time of
day specificity of LI could depend of the ability for segregating elements of an
experience into separate internal representations, a function which has been
attributed to the late developing hippocampus (Kubik and Fenton, 2005).
Whatever the explanation, the results of Experiment 2 support a peculiar
187
Chapter 5
organization of the learning and memory systems which is still evident at 64
days of age. This shows a protracted time frame of adolescence which is
consistent with the extended course of axonal pruning and myelination taking
place in the developing brain both in rats (Bockhorst et al., 2008) and humans
(Durston and Casey, 2006).
In summary, the results reported in the present study show a dissociated
developmental emergence of the phenomena studied. Conditioned taste
aversion (CTA) was evident in the younger PN24 group, while latent inhibition
did not appear until 32 days of age. Neophobic responses to the palatable saline
solution showed a later emergence being evident the attenuation of neophobia
at the third exposure in PN64 while at the second exposure in adult rats. No
effect of a temporal context shift was seen either in PN24 or PN32. Moreover,
a peculiar enhancement of a temporal context specific CTA was exhibited only
by PN48 and PN64 rats, while the adult typical temporal context-dependency
of LI was evident in PN100 rats.
188
Time of day, Temporal context and Ontogeny
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neurodevelopmental models of schizophrenia. Psychopharmacology,
169(3-4), 298-307.
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Manrique, T., Molero, A., Cándido, A. and Gallo, M. (2005).
Early learning faillure impairs adult learning in rats.
Developmental Psychobiology, 46: 340-349.
Early Learning Failure Impairs Adult Learning in Rats
a,b
Manrique, T., a Molero, A, a Cándido, A. and a,b Gallo, M.
a
Department of Experimental Psychology and Physiology of Behavior, University of
Granada, Campus Cartuja, Granada-18071, Spain.
b
Institute of Neurosciences F.
Oloriz, University of Granada, Spain.
ABSTRACT
Early life experiences may affect adult learning ability. In two experiments we tested
the effect of early learning failure on adult performance in Wistar rats. In the first
experiment 17-day-old rats (PN17), but not 25-day-old rats (PN25), trained in a
hidden platform water maze task showed deficits in tone-shock avoidance learning
when they were 3-months-old. The second experiment, which included randomplatform and non-platform control groups, confirmed the effect of early (PN18)
spatial learning failure on adult avoidance learning. However, postweaning training
(PN25) without platform also tended to induce adult learning deficits as long as the
adult task difficulty was increased. The older non-platform group did not differ from
the impaired group which received early training in a fixed hidden platform task. The
results are discussed in terms of the relevance of early learning outcome and
developmental stage on adult general learning deficits which may be related to the
learned helplessness phenomenon and developmental neural plasticity. 2005 Wiley
Periodicals, Inc. Dev Psychobiol 46: 340–349, 2005.
This research was supported by the CICYT grant BSO2002-01215 (MICYT, Spain)
Key words: weaning, learning, morris water maze, avoidance, learned
helplessness.
Chapter 6
6.1. Introduction
Exposure to uncontrollable aversive events may impair later learning
ability in rats. It has been suggested that the animal learns to be helpless,
suffering motivational, cognitive, and emotional changes that lead to passivity
and impaired learning (Overmier and Seligman, 1967; Maier and Seligman,
1976). The conventional procedure to induce learned helplessness involves
exposure to inescapable shocks and later testing in an avoidance task (see for
example Drugan, Basile, Ha, Healy, and Ferland, 1997; Vollmayr and Henn,
2001). However, the learned helplessness phenomenon has been reported using
different tasks (see Maier and Seligman, 1976; Overmier, 2002 for review). A
critical factor seems to be uncontrollability, either experiencing inescapable
aversive events or unsolvable discrimination problems (Hiroto and Seligman,
1975).
It can be proposed that uncontrollability may be induced either by
applying an unsolvable task or by imposing excessive learning demands at an
early stage of developmental maturation. It is well known that early
experiences with uncontrollable stressful events may have long lasting effects
on adult performance. On one hand, it has been reported that inescapable
shocks received as a weanling profoundly impair adult escape behavior
(Hannum, Rosellini, and Seligman, 1976) and undermine the ability of adults
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rats confronted with shocks to reject tumors (Seligman and Visintainer, 1985).
On the other hand, positive effects of early handling which can be considered a
mildly stressful stimulation, have been described in a variety of avoidance
tasks (Chapillon, Patin, Roy, Vincent, and Caston, 2002; Gschanes,
Eggenreich, Windisch, and Crailsheim, 1995; Nuñez et al., 1995; Tejedor-Real,
Costela, and Gubert-Rahola, 1998). Thus, it seems interesting to test the
potential effect on adult learning ability of training infant rats in a task that they
are not able to solve due to developmental immaturity. Learning to locate a
hidden platform in the Morris water maze has several advantages for this
purpose (Morris, 1981). First, this learning ability appears late during
development. Significant acquisition deficits have been reported in preweaning
rats younger than 20 days of age. These deficits range from a complete failure
to express spatial learning (Rudy and Paylor, 1988; Rudy, Staedler-Morris, and
Albert, 1987; Schenk, 1985) to inferior performance relative to adults (Carman
and Mactutus, 2001; Kraemer and Randall, 1995) depending on the procedure
requirements. Thus, applying the conventional spatial learning task to 17–19
day old rats results in a learning failure due to incomplete brain maturation.
Second, the task involves learning to avoid a mild stressor. Finally, it does not
require food deprivation in contrast to spatial tasks in other mazes, avoiding
constraints to control motivation in lactating rats.
The present study was designed to test whether early failure in spatial
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Chapter 6
learning may induce a long-lasting effect on an independent aversive learning
task. Adult performance on a tone-shock avoidance task by rats that were
trained in a spatial task at an early age was tested. Thus, if there is a general
effect of early training outcome on adult learning it should appear in a task
which involves not only different aversive stimuli (water vs. shock avoidance)
but also different sensory (visual vs. auditory) and motor (swimming vs.
jumping) requirements.
6.2. Experiment 1
A pilot experiment was designed to compare adult performance in the
acquisition of a tone-shock avoidance response in three groups of rats. Two
groups were previously trained in a hidden platform water maze task, one of
these groups after weaning (postnatal day 25), and the other before weaning
(postnatal day 17), i.e., at an age reported as too early to solve this task. A third
group received no early training.
6.2.1. Methods
6.2.1.1. Subjects
Subjects were 11 male and 12 female Wistar rats bred at the University
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of Granada. All the rats used belonged to three litters. The female pregnant
rats were checked daily for new births being the first postnatal day (PN0) the
morning in which the new litters were first observed. Three days after birth
each litter was culled out to a maximum of eight pups (sex was balanced) and
housed with their dams in standard clear polyethylene hanging cages. Weaning
took place on postnatal day 22 (PN22) and the litters remained together. Once
they were 30-days-old (PN30) the pups were housed in groups of three to four
subjects of the same sex.
At 3 months of age the animals were housed
individually before the adult training experience onset. Food and water were
available ad libitum in the home cage. The animals were maintained on a
12:12 hr light-dark cycle, training being performed during the light phase of
the cycle.
According to the early training experience the subjects were assigned to
three sex-counterbalanced groups (n = 8): groups PN17 (four males and four
females) and PN25 (three males and five females) were trained on the spatial
tasks taking place the first session on postnatal days 17 and 25, respectively. A
control group (Ctrl, four males and four females) had no early training
experience. At the age of 3 months all the animals were trained to avoid a
shock by jumping in the presence of a tone.
All the experimental procedures were approved by the University of
Granada Ethics Committee, and were in accordance with the European
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Chapter 6
Communities Council Directive of 24 November 1986 (86/609/EEC).
6.2.1.2. Apparatus
For the early training, the water maze was located in a 4 x 5 m room
containing a great amount of extra-maze cues (electrophysiological
instruments, posters, lights, video-camera, etc.) visible to the swimming
animal. The pool consisted of a 200 cm diameter and 30 cm deep circular
plastic tank with a removable 11 cm diameter circular platform. The
temperature of the water was maintained at 24–26oC. The water level was 22
cm and the platform was placed 1 cm below the water surface. In order to
ensure that the platform was invisible the water surface was covered by small
pieces of white polyethylene. The pool was divided conceptually into four
quadrants, and the platform was placed approximately 35 cm from the pool
border in the center of each quadrant depending on the behavioral conditions.
The experimental apparatus used for the adult training consisted of a
modified rat operant conditioning chamber (LETICA LI-200) made of four
walls: two opposing walls (31 x 28 cm) were made of clear polyethylene; the
other walls (31 x 23.5 cm) were opaque polyethylene and modular aluminum
plates, respectively. The floor was formed by a grid of 19 stainless steel rods 4
mm in diameter and were positioned 2 cm center-to-center; these were
connected in series to a LETICA LI 2700 shock-source module designed to
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Early Learning Failure
produce continuous scrambled current of 1 mA. Five photo-electric cells were
mounted 25 cm above the grid at 5 cm intervals, beginning 5 cm from the
aluminum wall. Corresponding lights (5 mm in diameter) were mounted in the
opposite wall. Lights and cells formed an electrical circuit connected to a
response recorder. A vertical jump interrupted the circuit and it was recorded
as a response (Cándido, Catena, and Maldonado, 1984; Cándido, Maldonado,
and Vila, 1991; Cándido, González, and de Brugada, 2004). A buzzer,
producing 80 dB SPL at 24 V, was used as the warning signal. It was installed
in the center of the aluminum wall at a height of 2.5 cm. The chamber was
placed in a sound-attenuating box 70 x 46 x 53.5 cm; a fan produced a
background noise of 70 dB SPL, measured inside the chamber.
Avoidance and escape latencies were measured by a LETICA LE
130/100 digital chronometer, accurate to 0.1 s. The temporal sequence of the
events was controlled by the LI 2700 module connected to a computer.
6.2.1.3. Procedure
Morris water maze task. A spaced learning procedure was applied for
minimizing fatigue, as proposed by Kraemer and Randall (1995). However, as
it has been shown that reducing the task requirements may allow spatial
learning in preweanlings (Carman and Mactutus, 2001; Carman, Booze, and
Mactutus, 2002), a large circular pool was used in order to increase the
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Chapter 6
difficulty of the task. Each subject received 10 blocks of training, applied in
two daily sessions, morning and afternoon, during 5 consecutive days. The two
daily blocks were separated by a 5 hr interval. Each block consisted of four
trials. There was an inter-trial interval of 5 min. Each trial began by placing the
subject into the water facing the pool wall at one of the four compass
conditions (east, west, north, or south). Each subject was released once from
each of the four compass points during a block of four trials. The order varied
randomly. The animal was allowed to swim freely for 60 s or until it climbed
onto the hidden platform. If it did not find the platform, after 60 s it was placed
on the platform by the experimenter and remained there for 15 s. During the
inter-trial intervals the subjects were group-housed in a cage lined with an
electric heating pad behind a large column which hided the swimming pool.
During the inter-block intervals, the preweaning rats were returned to their
home cage with the dam in the vivarium. The postweaning group was housed
with the rest of the litter. Latencies to reach the platform were recorded.
Avoidance task. Before the onset of the adult training experience, at the
age of 3 months, the animals were housed in individual cages. The subjects
were randomly assigned to morning or afternoon session and counter-balanced
by sex and group. The learning procedure followed was that described in detail
by Cándido, Maldonado, and Vila (1988). The daily session consisted of 60
trials. Each rat was placed into the chamber and was allowed 5 min to explore
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Early Learning Failure
it before the trial began. Each trial consisted of a warning signal followed 5 s
later by a 1 mA electric shock. Both continued for 30 s or until the rat showed
an escape response breaking the photocell beam by jumping. An avoidance
response was one that occurred within 5 s of the warning signal onset. Training
lasted until the rat reached the acquisition criterion of 10 consecutive
avoidance responses (CARs) or until a maximum of 240 trials (four sessions).
Those animals that failed to escape the shock in five consecutive trials were
eliminated and training did not continue. The number of trials required to reach
3, 5, and 10 CARs were used as the dependent variables.
6.2.2. Results
6.2.2.1. Morris Water Maze
Figure 1 shows the mean latencies to reach the platform of the groups
trained in the Morris water maze. A 2 x 2 x 10 (Age x Sex x Blocks of Trials)
analysis of variance (ANOVA), the within subject factor being the latency to
reach the platform in the last trial of each block, showed significant main
effects of Age (F(1, 12)= 22.96; p < 0.01), Blocks of Trials (F(9, 108)= 8.22; p <
0.01), and the interaction Age x Blocks of Trials (F(9,
108)=
3.27; p < 0.01).
Analyses of the interaction revealed shorter latencies to reach the platform of
the older group (PN25) compared to the younger group (PN17) in trial 8 (F(1,
213
Chapter 6
14)=
5.05; p < 0.05) and trial 10 (F(1, 14)= ¼8.18; p < 0.01). Repeated measures
ANOVA analyses indicated spatial learning both in the younger (F(9, 63) ¼ 3.30;
p < 0.01) and in the older group (F(9, 63) ¼ 5.81; p < 0.01). However, Newman–
Keuls post-hoc analyses showed faster learning in the older group, the latencies
to reach the platform for blocks 8 (p < 0.05), 9 and 10 (p < 0.01) being
significantly shorter than those of the first blocks of trials. However, the
younger group showed reduced latencies only in block 9 (p < 0.01), and only in
comparison with the first block in the case of block 10 (p < 0.05).
FIGURE 1. Mean (+SEM) search time of the two groups trained in the hidden
platform water maze task in Experiment 1.
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Early Learning Failure
6.2.2.2. Avoidance Task
Figure 2 shows the number of trials required by each group for reaching
3, 5, and 10 consecutive conditioned avoidance responses (CARs). None of the
subjects had to be excluded due to absence of avoidance responses. A 2 x 3
(Sex x Group) analysis of variance (ANOVA) showed only a significant effect
of group using CARs 5 as the learning criterion (F(2, 18)= ¼ 4.02; p < 0.05).
Newman– Keuls post-hoc comparisons showed that the group trained in the
spatial task at PN17 required a significantly higher number of trials to reach the
learning criterion than both the non-trained control group and the group trained
at PN25 (p < 0.05). Similar tendencies appeared using CARs 3 and 10 as
learning criteria, but the differences did not reach a significant level. There
were no other significant effects.
215
Chapter 6
FIGURE 2. Mean (±SEM) number of trials needed by each group to reach the 3
(a), 5 (b), and 10 (c) consecutive conditioned avoidance responses criteria
(CARs) in Experiment 1.
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Early Learning Failure
6.2.3. Discussion
The results of the early training in the water maze task revealed that the
younger group was impaired in finding the hidden platform location compared
to the older group. These results are congruent with previous data showing a
late development of spatial learning in rats (Carman and Mactutus, 2001;
Kraemer and Randall, 1995; Rudy and Paylor, 1988; Rudy et al., 1987). Our
procedure was designed in order to minimize fatigue, including a spaced
distribution of trials as proposed by Kraemer and Randall (1995). This fact
may have favored learning in the younger group, as can be seen in the last two
trials. Spatial learning at this age has been reported employing a procedure
adapted to the pups requirements, such as a reduced size of the pool (Carman
and Mactutus, 2001; Carman et al., 2002).
However, using the present
behavioral procedure the differences in latencies to locate the platform between
both age groups were evident. Seventeen-day-old pups are less proficient than
25-day¬old in learning to find the platform.
The results of the adult avoidance training showed that the group
trained in the spatial task at the age of 17 days needed a higher number of trials
for acquiring an avoidance response than both the control non-trained group
and the group trained at the age of 25 days. Although the tendency appeared
217
Chapter 6
using different learning criteria, the fact that the differences were significant
only using a CARs 5 criterion may reflect floor and ceiling effects when the
task is too easy (CARs 3) or too difficult (CARs 10).
Thus, the results point to a deleterious effect on adult learning ability of
having been trained in a different task before reaching the developmental stage
required for solving it. No effect of being efficiently trained at a later
developmental stage was evident compared to the control group that did not
receive early training.
6.3. Experiment 2
The results of Experiment 1 showed that those rats which failed to solve
a spatial task due to developmental immaturity exhibited as adults acquisition
deficits in a different avoidance task. Experiment 2 was designed to replicate
this finding. In addition, control groups were added in order to exclude
alternative interpretations. On one hand, in spite of the fact that we used the
procedure of Kraemer and Randall (1995) in order to minimize the contribution
of fatigue, due to the large size of the pool, the possibility of increased fatigue
in the younger group in relation to the older cannot be excluded. Having been
subjected to fatigue producing exercise during infancy may then be responsible
for the adult learning impairment. On the other hand, irrespective of
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Early Learning Failure
developmental stage, the learning impairment found in adults may be due to
those rats having been trained in an unsolvable task. In order to examine
whether the adult learning impairment was due to inability to learn a solvable
task or whether similar results could be found after training the animals in an
unsolvable task at a later developmental stage, or after being subjected to
exercise, random platform and non-platform yoked control groups were added.
One animal of each control condition (random and the non-plat) was yoked to
each animal of the experimental group. These were matched for the starting
point and swimming time. Moreover, in order to test retention in the early
spatial task, a probe trial without platform was added. Additionally, the
immobility time in this probe trial was recorded as a measure of ‘‘behavioral
despair,’’ a construct related to learned helplessness. If learning failure was due
to premature developmental stage, the preweaning group trained in the spatial
task would show learning deficits in the adult task. However, if training age is
not a factor, those groups performing either an unsolvable task or exercise
should be impaired.
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Chapter 6
6.3.1. Method
6.3.1.1. Subjects
Eighty-three Wistar rats (44 males and 39 females) obtained from nine
litters were housed as described in Experiment 1. The subjects were randomly
assigned to four sex counterbalanced behavioral groups labeled as follows:
Experimental (Exp; PN18: six males and four females; PN25: seven males and
seven females), Random (PN18: five males and five females; PN25: four males
and five females), Non-platform (non-plat; PN18: five males and five females;
PN25: six males and four females), and Control (PN18: six males and four
females; PN25: five males and five females). The Exp group was trained to
find a platform in a fixed location. The location of the platform varied
randomly in the random group and there was no platform in the non-plat group.
Each animal of both yoked random and non-plat groups were treated exactly in
the same way concerning starting point and swimming time than the
experimental group. The control group had no early training. Each group was
divided in two age conditions (PN18 vs. PN25) depending on the first day of
early training.
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Early Learning Failure
6.3.1.2. Procedure
All subjects received an early training procedure similar to that used in
Experiment 1, except for the three following differences. First, training
consisted of only eight blocks of trials, the number being reduced in order to
delay preweaning training onset, in this way allowing a greater sensory
maturation. Second, random and non-platform control groups were added.
Animals from the random group remained in the platform 15 s after each trial,
whether or not they had found the platform by themselves. Third, a retention
test without platform was added. This probe trial took place the morning
following the last block of trials (block 8). On the probe trial the subject was
allowed to swim freely for 60 s after being released from a fixed starting
location. The video recordings of the probe trials were used to measure search
time in the target quadrant and immobility time. Three seconds was set as the
minimum criterion for each immobility period. The adult avoidance task was
performed as described in Experiment 1.
6.3.2. Results
6.3.2.1. Morris Water Maze
Acquisition. Figure 3a shows the main latencies to reach the platform of
the two experimental groups. The rest of the groups are not represented
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Chapter 6
because they were yoked. A 2 x 2 x 3 x 8 (Sex x Age x Group x Block of
Trials) analysis of variance (ANOVA) showed significant main effects of Age
(F(1, 52)= 79.32; p < 0.01), Block of Trials (F(7,364)= 56.61; p < 0.01), and the
interaction Age x Block of Trials (F(7, 364)= 13.8; p < 0.01). The analysis of
interaction Age x Block of Trials revealed a significant decrease of the
latencies to reach the platform along the blocks both in PN18 group (F(7, 203)=
28.55; p < 0.01) and in PN25 (F(7, 231)= 47.44; p < 0.01). Post-hoc Newman–
Keuls comparisons of the younger group showed no significant differences
between blocks 1 and 5 but they differed on blocks 6, 7, and 8 (p < 0.01). The
latencies to reach the platform decreased progressively between blocks 6 and 7
(p < 0.05) and blocks 7 and 8 (p < 0.01). In contrast with the PN18 group, the
older group (PN25) showed progressive and faster decline of the latencies to
reach the platform between blocks 1 and 2 (p < 0.05), 2 and 3 (p < 0.01), 3 and
4 (p < 0.05), 4 and 5 (p < 0.01), 6 and 7 (p < 0.05) but no significant
differences between 7 and 8 showing a potential floor effect. The rest of
differences between blocks of trials were significant (p < 0.01). The younger
group showed longer latencies than the older group in blocks 2– 8 (p < 0.01).
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Early Learning Failure
FIGURE 3. (a) Mean (+SEM) search time of the two experimental groups during
training in the fixed location hidden platform water maze task in Experiment 2.
The data of the yoked groups trained with random platform location and nonplatform are not presented. (b) Mean (+SEM) search time in target quadrant of
the different groups during the probe trial
Probe trial. Figure 3b shows the main search time in target quadrant for
all the groups. A 2 x 2 x 3 (Sex x Age x Group) ANOVA of the time spent in
the quadrant that had previously contained the platform during the probe trial
showed a significant main effect of group (F(2,
52)=
22.43; p < 0.01) and the
223
Chapter 6
interaction between Age x Group (F(2, 52)= 7.14; p < 0.01). There were no other
significant effects. The analysis of interaction Age x Group revealed that PN25
experimental group spent significantly more time searching the platform than
the younger experimental group (F(1,
22)=
10.26; p < 0.01). PN18 non-plat
group also spent significantly more time in the platform quadrant than PN25
non-plat group (F(1, 18)= 5,19; p < 0.04). There were no significant differences
between random groups (F(1, 18)= 0,61; p > 0.44). For those conditioned at 25
days of age there was a significant effect of group (F(2,31)= 38.44; p < 0.01).
Post-hoc Newman–Keuls comparisons showed that experimental, random and
non-plat groups differed significantly (p < 0.01). No effect of group was seen
in the younger groups. Besides, post-hoc Newman–Keuls comparisons showed
that PN25 experimental group remained searching for the platform in the target
quadrant longer than the rest of the groups (p < 0.01).
Table 1 presents the mean immobility time of each group during the
probe trial. The 2 x 2 x 3 (Sex x Age x Group) ANOVA revealed a significant
main effects of age (F(1, 52)= 5.66; p < 0.05), group (F(1, 52)= 7.10; p < 0.01), and
the interaction of Age x Group (F(2,52)= 8,56; p < 0.01). The analysis of
interaction Age x Group showed that the younger experimental group was
significantly different from the PN25 experimental group (F(1,22)= 5.40; p <
0.05), which remained swimming during the whole test. There were no
significant differences between random groups (F(1,
224
18)=¼1;
p > 0.33). The
Early Learning Failure
older non-platform group remained immobile longer than the younger nonplatform group (F(1,18)= 5.20; p < 0.03). An ANOVA of the younger groups
revealed no significant group effect (F(1, 27)= 1,48; p > 0.25). An ANOVA of
PN25 groups yield a significant effect (F(1,31)= 6,68; p < 0.01). Post-hoc
comparisons showed that the non-platform group spent almost 30% of the
probe trial immobile, behaving significantly different from the rest of the
groups (p < 0.01). Besides, post-hoc Newman–Keuls comparisons showed that
this group differed significantly from the rest of the groups regardless the age.
Table 1. Immobility Time (s) of the Different Groups during the Probe Trial of
the Morris Water Maze in Experiment 2
PN18 (Mean ±SEM)
PN25 (Mean ±SEM)
Experimental
3.5 ± 1.71
0±0
Random
0.97 ± 0.92
0±0
Non-platform
0.75 ± 0.71
29.9 ± 12.11
6.3.2.2. Avoidance Task
Figure 4 presents the main number of trials required by each group to
reach the 3, 5, and 10 consecutive CARs criteria. None of the subjects had to
be excluded due to absence of avoidance responses. There were not significant
225
Chapter 6
sex effects in the global analysis. Thus it was not included in further analyses.
The 2 x 3 x 4 (Age x Group x Acquisition Criteria) analysis of variance
(ANOVA) in CARs 3 revealed significant effects of Age (F(1, 75)= ¼ 4.14; p <
0.05), Group (F(3,
75)=
5.68; p < 0.01), and the interaction Age x Group
(F(3,75)=¼5,17; p < 0.01). There were significant differences between both ages
in the experimental group (F(1, 22)= 15.94; p < 0.01) but not in the rest of the
groups. For those conditioned at 18 days, there was a significant effect of
Group (F(3, 36)= 6.81; p < 0.01). Post-hoc Newman–Keuls comparisons revealed
that the younger experimental group performed significantly worse than the
rest of the groups (p < 0.01). There were no other significant effects.
The analysis of CARs 5 showed only a significant effect of the
interaction Age x Group (F(3, 75)= 5.42; p < 0.01). Again, there were significant
differences between both ages in the experimental group (F(1, 22)= 23.07; p <
0.01) but not in the rest of the groups. For those conditioned at 18 days, there
was a significant effect of Group (F(3, 36)= 4.81; p < 0.01). Post-hoc Newman–
Keuls analyses revealed that the younger experimental group performed
significantly worse than the rest of the groups (p < 0.01). The group effect did
not reach significance in PN25 group (F(3, 39)= 2.73; p < 0.06). Again, post-hoc
Newman–Keuls comparisons revealed that the younger experimental group
performed significantly worse than the rest of the groups (p < 0.01). There
were no other significant differences.
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Early Learning Failure
FIGURE 4. Mean (+SEM) number of trials needed by each group to reach the 3
(a), 5 (b), and 10 (c) consecutive conditioned avoidance responses criteria
(CARs) in Experiment 2.
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Chapter 6
Thus, both in CARs 3 and 5 the younger experimental group needed
significantly more trials to reach the learning criteria than the rest of the
groups. The analysis of variance (ANOVA) of CARs 10 revealed again only a
significant effect of the interaction Age x Group (F(3,75)= 6.53; p < 0.01). There
were significant differences between PN18 and PN25 experimental groups (F(1,
22)=
16,79; p < 0.01), but not between the random (F(1,17)= 0.21; p > 0.65), non-
plat (F(1,18)= 2.52; p > 0.13), and control (F(1,18)= 0.01; p > 0.9) groups. For the
young group there were a significant group effect (F(3,36)= 5.47; p < 0.01). As
in the case of the previous learning criteria, the experimental group performed
significantly worse than the rest of the groups (p < 0.01). The group effect did
not reach significance in PN25 group (F(3,39)= 2.45; p < 0.08). Post-hoc
Newman–Keuls analyses showed significant differences between the Exp
PN18 and the rest of groups (p < 0.03) except the PN25 non-plat group (p >
0.28). This later group showed also marginal differences with the PN25
experimental group (p < 0.08).
6.3.3. Discussion
These results confirm the main findings of Experiment 1 concerning the
deleterious effect of early learning failure on the acquisition of a different
avoidance response during adulthood, but also contribute new data.
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Early Learning Failure
The latencies to reach the platform during acquisition as well as the
search times in the target quadrant during the probe trial showed spatial
learning deficits in the younger (PN18) experimental group compared to the
older experimental group (PN25). Although the younger group latencies to
reach the platform declined during the last acquisition trials, during the probe
trial these animals showed significantly lower search time in the target
quadrant than the older group. No evidence of a search pattern targeting a
specific quadrant was found in either random or non-platform groups. The
typical search pattern shown by the random groups included exploration and
crossings throughout the four quadrants and no age differences were evident.
However, the search pattern of the non-plat groups during the probe trial
seemed to differ between the age groups. The younger PN18 group seemed to
exhibit a pattern similar to the random groups, with active swimming and
crossings of the four quadrants. However, the older PN25 group showed
reduced exploration and long periods of floating without swimming. The
immobility time results during the probe trial in Morris water maze showed
that the PN25 group, trained without platform, remained immobile
significantly longer than the rest of the groups.
Thus, the absence of the escape platform during training seemed to lead
to different outcomes depending on the age of the pups. Passivity in the older
group could be interpreted as learned helplessness because the situation was
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Chapter 6
inescapable. Immobility may be an adaptive response if the animal learned to
wait until the end of the trial to be taken away (Glazer and Weiss, 1976).
Accordingly, the older group trained without platform tended to perform worse
in the adult avoidance task when the difficulty was increased. This did not
seem to be the case in the younger group trained without platform which
showed immobility times similar to the rest of the groups and no deficits in the
adult avoidance task. However, the differences between both groups did not
reach significance.
Taken together, these results show that training the animals in a
solvable task before the maturational requirements are met may have a higher
impact on later adult learning than exposing them to an inescapable mildly
stressful situation.
6.4. General Discussion
The main finding reported in the present experiments is that learning
difficulties at an early age, due to deficits in solving a spatial task, may induce
long-lasting deleterious effects on learning a different avoidance task in adult
rats. Adult rats trained to locate a hidden platform in a water maze at the age of
17 (Experiment 1) or 18 (Experiment 2) days required a higher number of trials
to reach the learning criteria in a shock avoidance task than those rats receiving
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a similar training at the age of 25 days, regardless the difficulty of the task. In
both experiments the younger group of trained rats showed spatial learning
deficits, i.e., longer latencies to locate the platform and reduced search time in
the target quadrant during the probe trial, compared to the older group.
However, in both experiments the latencies to reach the platform of the
younger groups decreased during the last acquisition trials. This fact could be
interpreted as emergent new learning abilities, although they were not yet
evident in the probe trial of Experiment 2.
These results are in agreement with previous studies reporting a late
ontogeny of some types of spatial learning. Rudy et al. (1987, 1988) reported a
maturational deficit in rats younger than 20 days in the spatial abilities required
for learning the relationship between the hidden platform and distal cues,
although they were able to learn the platform location using proximal cues.
Additionally, the results confirm other reports (Carman and Mactutus, 2001;
Kraemer and Randall, 1995) showing an inferior performance in pups younger
than 20 days compared to older rats, even when a spaced trials learning
procedure was used, which facilitate learning by reducing fatigue. Moreover, in
the present experiments the spatial learning deficit was facilitated by the large
size of the pool.
Using different learning criteria adult learning deficits of the young
experimental group in the shock avoidance task were evident. These deficits
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cannot be attributed to unspecific effects arising from the exposure of the pups
to aversive events or handling, nor to exercise or fatigue. It is therefore
conceivable that in younger rats evidencing emergent albeit inefficient learning
abilities, the perceived difficulty in learning a solvable task may be a critical
factor in explaining adult learning deficits. This explanation is supported by the
greater impairment in those groups trained at an early age in Experiment 2
compared to Experiment 1. It can be seen that the PN18 experimental group in
Experiment 2 required a higher number of trials in reaching the avoidance
learning criteria than the PN17 group in Experiment 1. However, in the early
learning task the PN18 group of Experiment 2 performed better than the PN17
group in the previous experiment, showing evidence of emerging learning
abilities. It is thus suggested that in Experiment 1 the animals would not have
had as much chance to perceive the spatial task as solvable as the animals in
Experiment 2. The magnitude of the deficit observed in the adult toneavoidance task can therefore be related to the perceived learning failure rather
than to the objective outcome of the early spatial task. Previous results showing
that experiencing unsolvable discrimination problems may lead to learned
helplessness and later learning deficits (Hiroto and Seligman, 1975) lend
support to this proposal.
Such an interpretation would imply that the younger rats discriminate
between the solvable fixed platform spatial task and the unsolvable random
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platform and non-platform tasks. Both fixed and random platform conditions
involved placing the animal on the platform after each trial if they had not
found it before. It can be expected that the younger animals are able to identify
the platform location based on proximal cues. They may therefore perceive the
fixed platform condition as a solvable task in spite of the fact that they are
unable to solve it because this task requires complex processing of distal cues.
However, the animals may perceive the random condition as an unsolvable task
due to the changing location of the platform. Thus, although the outcome is
similar in both conditions, i.e., failure in reaching the platform, the cognitive
appraisal of the situations may be different. It has been proposed that
experiencing situations in which the demands are perceived to outweigh the
resources may be an important source of stress (Kemeny, 2003). The
deleterious effect of early training in the fixed location platform task on adult
learning may therefore be due to an excessive level of demand, leading to
learned helplessness.
An interpretation in terms of learned helplessness may also account for
the tendency to adult avoidance learning deficits evidenced in the group trained
in the non-platform condition at 25 days of age. This tendency approached
significant levels when task difficulty was increased using CARS 5 and 10
criteria. Moreover, the older random group performance in the adult avoidance
task did not differ from the impaired younger group, which received early
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training in the fixed located platform task. The reduced activity or immobility
seen in most rats belonging to the older random group during the probe trial
suggest that such reduced responding may be related to learned immobility
produced by the contingency between staying immobile and waiting to be
taken away. Similar reinforcement mechanisms of immobility have been reported by Glazer and Weiss (1976) for inescapable shocks. A conservative
explanation central to the learned helplessness phenomenon is the assumption
that prior action-outcome non-contingency experience produces interference
with subsequent learning, subsequent action outcome relationships being
judged as non-contingent or as less strongly related than they are (Reed,
Frasquillo, Colkin, Liemann, and Colbert, 2001). Additionally, according to
Maier and Seligman (1976), prior experience with action-outcome noncontingency will also produce motivational deficits and short-term emotional
disturbances. Such a motivational deficit would prevent the initiation of
responding, which may be the case in the PN25 group trained without platform.
However, both the active swimming pattern and the absence of adult learning
deficits in the non-platform younger group indicates that this condition may
have been perceived differently by rats of different maturational stages.
It could be proposed that the younger pups do not experience swimming
in the absence of platform as a stressful avoidance unsolvable task, but rather
as exercise, which may be reinforcing in itself at this developmental stage. In
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Early Learning Failure
fact, developing mice aged 14–21 days are able to acquire preferences for a
place paired with administration of D-amphetamine at doses that induce lower
increases in locomotor activity than at postweaning ages (Cirulli and Laviola,
2000). Moreover, physical activity has been reported to be naturally reinforcing
for children if multiple, short exercise bouts are used, as it is the case in the
present experiments (Epstein, Kilanowski, Consalvi, and Paluch, 1999).
There is an alternative epigenetic explanation of the results which
would rely on specific changes in the brain learning systems. It is well known
that early stimulation during sensitive developmental periods may modify the
organization of the neuronal circuitry thus leading to permanent changes that
influence adult behavior (Kolb, Gibb, and Robinson, 2003). The results of the
younger experimental group performance in the spatial task showed that
training took place during a critical developmental period in which learning
abilities were emerging. Shaping of the specific brain circuits relevant for this
task could therefore have been influenced by the perceived early learning
failure. However, the fact that the adult deficits were found in an independent
learning task points to a more general deleterious effect on brain plastic
mechanisms. This would imply that early training in a learning task, before the
specific brain circuits involved are able to solve it, may modify the synaptic
organization compromising plastic changes in a variety of learning systems.
Whatever the relevant mechanism may be, the adult learning deficits
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shown by the early trained animals are more pronounced than those exhibited
by the animals trained in the random condition at a later age. The group trained
at an early developmental stage in the fixed location platform showed
significant deficits in the adult tone avoidance task at different levels of
difficulty. However, the deficits of those rats trained in the non-platform
condition at 25 days of age approached significance only when the difficulty of
the task was increased. This is consistent with previous findings reporting a
higher beneficial effect of combined tactile-visual stimulation in the first
postnatal week compared to the fourth on adult passive avoidance (Gschanes,
Eggenreich, Windisch, and Crailsheim, 1998). Although infantile amnesia is a
fairly pervasive phenomenon (Spear and Riccio, 1994) and retention of spatial
navigation tasks does not exceed several days in preweaning rats (Carman and
Mactutus, 2001; Carman et al., 2002; Kraemer and Randall, 1995), in the
present study early learning training effects were evident 73 days later. This
shows that there are long-lasting effects of early stimulation on either emotion
and/or learning related brain systems organization and that these effects are
independent of memory recall.
Although more research will be needed to explain the mechanisms and
processes involved, it may be concluded that early training in a spatial task,
before achieving the maturational stage required to solve it, may have long
lasting effects on adult learning. These effects do not necessarily arise from
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early stressful events. The present findings thus point to the role of early
learning experiences and infant history of success and failure in shaping adult
learning abilities in general.
Acknowledgements
The authors are thankful to Dr. M. Burnett and Dr. F. Tornay for their
helpful suggestions with the English and statistics, respectively. The authors
are also greatly indebted to Prof. A. Maldonado for his insightful comments on
the manuscript.
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