ISSN Paper Edition: 0214-9915
1999. Vol. 11, nº 3
ACTIVE AVOIDANCE CONDITIONING IN RATS: ABSENCE OF SEX DIFFERENCE AND ESTROUS EFFECT
Sandra Rubio, Rubén Miranda, Marcelino Cuesta, Azucena Begega,Luis J. Santín y Jorge L. Arias
Universidad de Oviedo y * Universidad de Málaga
In this paper, possible differences between male and female rats (Rattus
norvegicus) in the acquisition and consolidation of 2-way active avoidance learning
are studied. In addition to monitoring behavioural parameters (escape and avoidance), the
phase of the estrous cycle of the female rats was also recorded in order to establish
whether this physiological phase affects the animals performance. Other authors
reported differential behaviour between the sexes and a modulatory effect of the estrous
cycle. In contrast, in our study we did not find any differences in the escape and
avoidance responses between the sexes and no influence of the estrous cycle. It was only
observed a greater male than female intertrial activity. This contradicts the hypothesis
about an organisational or activational effect of the sex hormones on shuttle-box
Condicionamiento de evitación activa en ratas: ausencia de efecto
del sexo y ciclo estral. En este artículo se estudian las posibles diferencias entre
las ratas macho y hembra (Rattus norvegicus) en la adquisición y consolidación
del aprendizaje de evitación de 2 vías. Además de monitorizar los parámetros
conductuales (escapes y evitaciones), se determinó la fase del ciclo estral en que se
encontraban diariamente las ratas hembras para establecer si el momento del ciclo afecta a
la ejecución de los animales. Otros autores han señalado diferencias conductuales entre
los sexos y un efecto modulador del ciclo estral. En contraste con estos datos, en nuestro
estudio no encontramos ninguna diferencia en las respuestas de evitación y escape entre
los sexos y tampoco ninguna influencia del ciclo estral sobre las mismas. Tan sólo
observamos una mayor actividad entre ensayos de los machos frente a las hembras. Estos
resultados contradicen las hipótesis previas sobre un efecto organizacional o
activacional de las hormonas sexuales en el aprendizaje de evitación activa.
Correspondencia: Jorge L. Arias
Facultad de Psicología
Universidad de Oviedo
33003 Oviedo (Spain)
Sex differences between male and female rats in reproductive and
non-reproductive behaviors have been reported in several studies. This behavioral
dimorphism has been registered in running wheel activity, reactivity to footshock,
intracranial self stimulation (Diaz-Veliz et al., 1989), spatial learning (Warren et al.,
1990), lever-pressing (Van Haaren et al., 1990) and classical conditioning (Wood and Shors
1998). However, other works have discarded the existence of behavioral dimorphism in some
of these tasks, such as the Morris water maze (Berry et al., 1997) or studies of operant
conditioning (Van Haaren et al., 1990). In avoidance conditioning tasks, females avoid
more shocks than males (Beatty and Beatty, 1970), acquire avoidance response more quickly
and have a slower extinction of the response (Van Haaren et al., 1990). Differences in the
active avoidance conditioning according to the estrous cycle phase have also been found
(Diaz-Veliz et al., 1989; Sfikakis et al., 1978). However, not all of the studies
corroborate these results (Denti and Epstein, 1972).
The aim of this work was to determine whether sex differences exist in
the acquisition and consolidation of the active avoidance response and if the estrous
cycle of the rat influences avoidance behavior during learning. We used a two-way
shuttle-box active avoidance procedure that made it possible to acquire the response in
only one day. In this way, we were able to analyze the influence of gender and cycle on
acquisition in spite of the shortness of the cycle phases. Once the avoidance response had
been acquired, the task was prolonged for 4 more days to observe the possible influences
of the fluctuating hormones on the consolidation of the task.
This behavioral analysis provided us with information on the
performance of the females in different phases on the same day of learning. In addition,
we were able to compare the performance of the same animal during its cycle since, in the
final days, the avoidance response was totally consolidated and potential variations in
the avoidance response could only be attributed to fluctuations of the gonadal hormones.
Materials and methods
Ten male and ten female, three month old Wistar rats (weighing 250 ±
50 g) from the University of Oviedo vivarium were used. The animals were kept in
independent cages and were maintained on a 12-hour light-dark cycle (8:00-20:00) at a
constant temperature (21 ± 1 ºC) and with free access to food and water. Care of the
animals was in strict accordance with current guidelines on the care and use of
experimental animals established by the A.P.A. on the 2nd of August, 1985.
Estrous Cycle Determination
Daily vaginal epithelium samples were taken from each female rat before
behavioral testing following the Feder method in order to determine the state at estrous
cycle (Feder, 1981). The rats were classified according to whether they were in one of the
four phases corresponding to the estrous cycle: proestrus, estrus, metestrus and diestrus.
The learning test consisted of a two-way active avoidance task
performed in a shuttle-box (Letica Scientific Instruments, Spain). The box (53 cm x 71 cm)
was divided into two compartments, and a light and a sound source could be used jointly or
separately in each of the compartments. Furthermore, the floor grid could be electrified
independently in each compartment (see illustration I). The learning program carried out
in the box was controlled by an associated module. The shuttle-box was covered by a black
curtain when the animals were introduced into it. By this way the animals are kept in
semi-darkness during the habituation and learning period.
The male and female animals, were handled prior to the experiment. The
learning process took place during the light period between 9:00-12:00 A.M. over 6 days,
the first day of which was for habituation. The habituation consisted of maintaining the
animal inside the shuttle-box for 15 minutes during which period it could freely explore.
The schedule for the remaining days was as follows: the animals were kept in semi-darkness
and in a soundproof area for 30 seconds (intertrial interval) after which they were
subjected to a sound stimulus (80dB, 1400Hz) (Conditioned Stimulus -CS). During this
second period, which lasted for 5 seconds, while the tone is present the animal had to
change of chamber to avoid an electric footshock (0.8mA) (Unconditioned Stimulus -US). If
the animals did not change of chamber (avoidance response) the electric shock was
maintained during 5 secs. or until the rat escape to the other chamber (see table I). The
animal, therefore, learned that the sound predicted the shock. There were 50 trials daily.
The number of responses of each animal recorded in the intertrial period was considered as
an index of exploratory activity. Three variables were recorded daily: intertrial
responses (A), number of changes of chamber per animal during the intertrial interval;
avoidances (B), changes of chamber during the presentation of the tone; and escapes (C),
changes of chamber during the presentation of the shock.
Analysis of variance (two-way ANOVA with repeated measures) was used to
compare the differences between the sexes of each variable. The independent variable was
sex (male or female) and the dependent variable corresponded to the different measurements
(A,B,C) recorded on each learning day. The post hoc test used was the Tukey HSD (honestly
significant difference) test. There were performed three independent analysis for each
The number of intertrial responses differed significantly
between sexes (F(1,4)= 9.67, p<0.0l) and between the days (F(1,4)= 5.75, p<0.01).
There were more intertrial responses on day 3 than on the first and last day (p<0.01),
since the responses of the males increased until this day (p<0.01) and decreased
from then on (see fig. 2).
There were no significant differences in the avoidances or escapes
between the male and female rats. However, differences were found between the days
(F(1,4)= 31.4, p<0.01). The level of avoidance responses increased after the
first day (p<0.01) (see fig. 1).
The estrous cycle analysis involved comparison of the females on
each of the learning days. In this way, possible differences between the four phases of
the estrous cycle were determined on each day. This analysis was, therefore, repeated 5
times, once on each learning day. The Kruskal-Wallis test showed no differences between
the different phases of the estrous cycle in any of the three parameters (see fig. 3).
The present study showed no sex differences in active avoidance
conditioning. Both sexes rapidly acquired avoidance behavior and an optimum level of
avoidance response was observed on day 2, this level was maintained during the learning
period. These data coincide with those of Brush et al. (1985) who did not find any sex
differences either in high-avoidance line and low-avoidance line rats. By the other hand,
Beatty et al. (1970) observed a worse performance in the males, regardless of the shock
intensity, while on the other hand. They find that the sexual hormones can have hight
activational effects on avoidance acquisition in both sexes (Beatty, 1992). However,
discrepancies between the results of the different studies can be explained by the fact
that experimental procedures do not use either the same shock intensity or duration, the
same conditioned stimulus (CS) or even the same number of trials or strain, and the data
are, therefore, not comparable.
In our study, consolidation and acquisition of the avoidance response,
and the intertrial responses were examined in both sexes and during the estrous cycle
and no differences were found. Furthermore, other studies did not detect any relationship
between the estrous cycle and activity level or the avoidance conditioning reached (Denti
and Epstein, 1972; Kristal et al., 1978). Some authors found estrous cycle interference on
learning, although no agreement has been reached on the phases in which facilitation or
deterioration of the response in the two-way avoidance learning is produced. Sfikakis et
al. (1978) report facilitation during proestrus and impairment during the diestrus and
estrus phases. Later studies, found facilitation of acquisition during diestrus and
impairment in the proestrus and estrus phases (Diaz-Veliz et al., 1989). In order to
explain this controversy, we must, once again, look at the variability of the procedures
used for this type of learning and memory studies. Thus, Sfikakis et al. (1978) used an
avoidance task of one single day, using light and 45v electric shock as the CS. In another
work, Diaz-Veliz et al. (1989) performed a learning test in which a discharge of 0.2 mA
was presented after a 5-second sound. If the animal did not avoid the shock, it was
maintained until it escaped. Both procedures differ substantially from that used in this
work, which could explain the differences found.
On the other hand, it has been demonstrated that both the amygdala as
well as the hippocampus show certain plasticity in response to fluctuations of the
circulating gonadal hormones in female rats. The amygdala is related to fear conditioning
and the hippocampus is necessary for complex data processing, such as the details of the
spatial environment (LeDoux, 1992). As avoidance behavior requires the participation of
both structures, it could be affected by these variations (LeDoux, 1992)
Variations in synaptic pattern of dendrite spine synapses (Nishizuka
and Arai 1983 ) and in serotonin receptor density (Biegon and McEwen, 1982) have been
found in the amygdala of female rats. Such changes seem to be related with the regulation
of estrous rhythm in rats (Chateau et al., 1984) and with the control that serotonin
exerts on the release of prolactin, LH and FSH during the estrous cycle (Becu de
Villalobos et al., 1984). Different changes have also been found in the hippocampus, among
them a decrease in the GABA and glutamate levels, facilitation of the long-term
potentiation (LTP) during proestrus (Warren et al., 1995) and a 30% decrease in the
density of the hippocampal dendritic spines in CA1 during estrus (Woolley and McEwen,
1992). Rats with an inborn high (HP) learning capacity to perform in a shuttle-box
avoidance paradigm present a lower threshold for inducing long-term potentiation (LTP)
(Keller et al., 1992). However, it has recently been pointed out that the experience
dependent increase in the synapses is beneficial for the learning capacity in the spatial
hippocampal task, but that caused by the gonadal hormones has no beneficial effect on
these (Warren and Juraska, 1997). We were also unable to verify that these changes are
relevant for active avoidance conditioning. However, morphological alterations in the
hippocampus of the female rats have been detected in Sprague-Dawley rats (Warren et al,
1995), a different strain from that used in this study.
Our data reveal sex differences in the number of intertrial responses.
Female activity was more constant over the learning days whereas the males showed more
variable activity. We could think that this greater variability exists because the males
are more sensitive to the electrical shock. However, Beatty et al. (1970) did not find any
sex differences in the intertrial responses during the avoidance test although they did
record differences in the sensitivity threshold to electrical shock (1mA). Brush et al.
(1985) did not find any relationship between the intertrial responses and sensitivity to
pain either since the low-avoidance line rats had fewer responses than the high-avoidance
line rats. However, neither rat differed in regards to the electrical sensitivity
thresholds. In our case, it is also possible that the variability in intertrial responses
was not influenced by a different sensitivity to shock.
In other anxiety models, aversion provokes different levels of activity
between male and female rats, although these variations seem to depend on the procedure
used. Thus, in the Vogel punished drinking test, it could be concluded that the males are
less anxious than the females (Johnston and File, 1991), but in the elevated plus-maze
test, the opposite occurs (Johnston and File, 1991). In all these models, the behavioral
differences between sexes are generally described in terms of the presence or absence of
behavioral inhibition in response to aversive stimulation (Van Haaren et al, 1990). In our
work, neither of the sexes presented behavioral inhibition since both emitted responses
without interference of the variable activity presented by the males in the intertrial
period. The intensity of the aversive stimulus was 0.8 mA and this did not provoke
paralysation and when it was associated with the sound, avoidance responses were possible.
Since the greater exploratory activity of the males can not be
explained by differences in anxiety or in behavioral inhibition it could possibly reflect
a search for other response strategies. Thus, the increase in male activity is greater
than the increase in female activity during the intertrial period. Once learning is
consolidated, the activity is reduced to the same level as the females. The animals have
learnt that only the responses given fortuitously with the sound (CS) are effective at
avoiding the shock.
In conclusion, our results imply that no differences exist between male
and female rats in the acquisition and maintenance of active avoidance learning, although
the number of intertrial responses varies between sexes, possibly due to a greater
exploratory activity of the males. In addition, it was also impossible to establish that
the estrous cycle exerts an influence on two-way shuttle-box active avoidance
The authors thank Miss Piedad Burgos and Mrs. Begoña Diaz for their
technical assistance, Caroline Coope for translating this article into English and Marcos
Begega for the graphic design. This study was supported by grants from the FICYT
(PB97-SAL10), and MEC (DGES PB96-0318). Spain.
Beatty, W. W., and Beatty, P. A. (1970). Hormonal determinants of sex
differences in avoidance behavior and reactivity to electric shock in the rat. J. Comp.
Physiol. Psychol. 73, 446-455.
Beatty, W. W. (1992). Gonadal hormones and sex differences in
nonreproductive behaviors. In: A.A., Gerall, H.Moltz and I.L. Ward (Editors), Seuxal
Differentiation, vol. 11 of Handbook of Behavioral Neurobiology, Plenum Press,
New York, p.p. 85-128.
Becu de Villalobos, D., Lux, V. A., Lacau de Mengido, I., and Libertun,
C. (1984). Sexual differences in the serotonergic control of prolactin and luteinizing
hormone secretion in the rat. Endocrinology. 115(1), 84-89.
Berry, B., McMahan, R., and Gallagher, M. (1997). Spatial learning and
memory at defined points of the estrous cycle : effects on performance of a
hippocampal-dependent task. Behav. Neurosci. 111(2), 267-274.
Biegon, A. and McEwen, B. S. (1982). Modulation by estradiol of
serotonin receptors in brain. J. Neurosci. 2(2), 199-205.
Brush, F. R., Baron, S., Froechlich, J. C., Ison, J. R., Pellegrino, L.
J., Phillips, D. S., Sakellaris, P. C., and Williams, V. N. (1985). Genetic differences in
avoidance learning by rattus norvergicus: escape/avoidance responding, discrimination
learning and open-field behavior. J. Comp. Psychol. 9, 60-73.
Chateau, D, Kauffmann, M. T., and Aron, C. (1984). Are the amygdaloid
projections to the hypothalamic ventromedial nucleus involved in estrous rhythm regulation
in the female rat?. Exp. Clin. Endocrinol. 83(3), 303-309.
Denti, A., and Epstein, A. (1972). Sex differences in the acquisition
of two kinds of avoidance behavior in rats. Physiol. Behav. 8, 611-615.
Diaz-Veliz, G., Soto, V., Dussaubat, N., and Mora, S. (1989). Influence
of the estrous cycle, ovariectomy and estradiol replacement upon the acquisition of
conditioned avoidance responses in rats. Physiol. Behav. 46(3), 397-401.
Feder, H. H. (1981). Estrous cyclicity in mammals. In: N.J. Adler
(Editor), Neuroendocrinology of reproduction physiology and behavior. Plenum Press,
New York, pp. 279-348.
Johnston, A. L., and File, S. E. (1991). Sex differences in animal
tests of anxiety. Physiol. Behav. 49(2), 245-250.
Keller, E. A., Borghese, C. M., Carrer, H. F., and Ramirez, O. A.
(1992). The learning capacity of high or low performance rats is related to the
hippocampus NMDA receptors. Brain Res. 576(1), 162-164.
Kristal, M. B., Axelrod, S., and Noonan, M. (1978). Learning in
escape/avoidance tasks in female rats does not vary with reproductive condition. Physiol.
Behav. 21(2), 251-256.
LeDoux, J.E. (1992). Brain mechanism of emotion and emotional learning.
Current Opinion in Neurobiology. 2(2), 191-197.
Nishizuka, M., and Arai, Y. (1983). Regional difference in sexually
dimorphic synaptic organization of the medial amygdala. Exp. Brain. Res. 49(3),
Sfikakis, A., Spyraki, C., Sitaras, N., and Varonos, D. (1978).
Implications of the estrous cycle on conditioned avoidance behavior in the rat. Physiol.
Behav. 21, 441-446.
Van Haaren, F., Van Hest, A., and Heinsbroek, R. P. (1990). Behavioral
differences between male and female rats: Effects of gonadal hormones on learning and
memory. Neurosci. Biobehav. Rev. 14(1), 23-33.
Warren, S.G., Wilson, L.A. and Nadel, L. (1990). Sexually dimorphic
spatial abilities in the Morris water task. Society for Neuroscience Abstract, 16, 1.321.
Warren, S.G., Humphreys, A., Juraska, J. M., and Greenough, W. T.
(1995). LTP varies across the estrous cycle : Enhanced synaptic plasticity in
proestrus rats. Brain Res. 703, 26-30.
Warren, S.G., and Juraska, J. M. (1997). Spatial and nonspatial
learning across the rat estrous cycle. Behav. Neurosci. 111(2), 259-266.
Wood, G.E. and Shors, T. J. (1998). Stress facilitates classical
conditioning in males, but impairs classical conditioning in females through activational
effects of ovarian hormones. Proc. Natl. Acad. Sci. 95(7) , 4.066-4.071.
Woolley, C. S., and McEwen, B. S. (1992). Estradiol mediates
fluctuation in hippocampal synapse density during estrous cycle in the adult rat. J.
Neurosci. 12(7), 2.548-2.554.
Aceptado el 18 de marzo de 1999