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Seasonal peculiarities of behavior of ground squirrel Citellus undulatus in holeboard and open field tests
Behavioural Processes 56 (2001) 195– 200 www.elsevier.com/locate/behavproc
Seasonal peculiarities of behavior of ground squirrel Citellus undulatus in holeboard and open field tests T.P. Semenova *, I.A. Anoshkina, B.M. Khomut 1, S.G. Kolaeva Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia Received 26 September 2000; received in revised form 11 September 2001; accepted 13 September 2001
Abstract Seasonal changes in the behavior of ground squirrels Citellus undulatus (n = 35) in holeboard and open field tests were studied. It was found that the number of head dips and rearings in the holeboard and the number of crossed squares, rearings, and runs across the field center in the open field in ground squirrels during arousal between bouts at the end of the hibernation period, in the middle of March, were lower than these indices in active summer animals. A rapid restoration of all behavioral parameters after the arousal of animals from hibernation in mid-April was shown. In some cases, the behavioral parameters of activity in the holeboard and open field tests and the habituation reaction reached the values characteristic of active summer animals in a very short period of time, sometime within 1 day. By contrast, the locomotory and exploratory activities of animals tested in autumn (at the beginning of September and in the middle of October) were significantly lower than in summer animals, i.e. these indices decreased to minimum values 1.5–2 months prior to their entry into hibernation. The results of behavioral experiments indicate that the preparation of the CNS of hibernators for seasonal changes in the functional states is under the control of endogenous regulators. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Annual cycle; Ground squirrels; Hibernation; Holeboard; Open field; Exploratory activity; Locomotory activity
1. Introduction Hibernation is an adaptive response of animals to unfavorable environmental conditions: a lowering of ambient temperature, a shortening of the photoperiod, absence of food, etc. The hibernation process affects all levels of organization of living organisms, from the molecular and cellular * Corresponding author. Fax: + 7-967-790509. E-mail address: [email protected] (T.P. Semenova). 1 Present address: Knowledge Exchange Institute, West Nyack, NY 10994, USA.
to the systemic, including the behavior of animals (Slonim, 1979; Liman et al., 1982; Heller, 1989). The behavior of hibernators provides the maintenance of vital activity under unfavorable conditions and consists in realizing the genetic programs that are triggered by the corresponding exogenous (photoperiod, temperature, availability of food) and endogenous factors (circannual rhythms of neurotransmitters, hormones and peptides). Hibernators exhibit a wide range of neuronal activity of the brain, from profound inactivity
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(torpor) of deep hibernation to all the central activity states during their non-hibernating (euthermic) phase, thereby providing a unique system capable of regulating the full range of mammalian CNS activity states (Beckman and Stanton, 1982). Biochemical studies revealed appreciable seasonal variations in the functional activity of the neurochemical regulatory systems responsible for the synthesis of endogenous regulators of hibernation such as serotonin, catecholamines, neuropeptides, and enzymes involved in amino acid metabolism (Haak et al., 1991; Popova et al., 1993; Nurnberger, 1995; Cai et al., 1996; Semenova and Kolaeva, 1999). However, it remains unclear how the behavior of hibernators changes in different phases of their annual cycle. There are a few papers devoted to learning and memory in hibernators. However, these studies were performed in the summer period, and the researchers were focused on the effect of natural hypothermia on various forms of memory in hibernators (McNamara and Riedesel, 1973; Nuritdinov, 1987; Mateo and Johnston, 2000; Millesi et al., 2001). Observations in field conditions during the transitional stage from activity to hibernation revealed a drastic impairment of the major activity, movement co-ordination and circadian rhythms of activity (Slonim 1979). The goal of the present work was to study seasonal changes in the behavior of Jakutian ground squirrel, Citellus undulatus, in holeboard and open field tests.
2. Materials and methods
2.1. Subjects Adult ground squirrels (C. undulatus) of both sexes weighing 350– 600 g (n =35) were used. The animals were trapped in the valley near Jakutsk and kept for 2–3 weeks under the care of veterinarians at the vivarium of the Institute of Biology (Siberian Division of the Russian Academy of Sciences) for 2–3 weeks. At the end of August they were transported to our Institute. The animals were housed individually, in 35× 40 ×20 cm cages with free access to dry food, fresh vegetables, herb, and drinking water. The
light cycle was 12-h on:12-h off (07:00 h in the morning to 07:00 h in the evening). At the end of October, the ground squirrels were placed to a special dark hibernation room, where they were kept until arousal. The temperature in the room was + 4 °C, which is comparable to natural conditions. Experiments were carried out with five groups of animals in different phases of their annual cycle: Group I: during arousal between bouts at the end of the hibernation period (in the middle of March); Group II: after exit from hibernation (in the middle of April); Group III: in the phase of activity (in the middle of June); Groups IV and V (in early September and in the middle of October, respectively): prior to the entry into hibernation. The behavioral experiments were carried out between 09:00 and 11:00 h in the morning. The experiments were conducted in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ as adopted and promulgated by the Declaration of Helsinki.
2.2. Experimental arrangement The locomotory and exploratory activities of animals were studied using a holeboard and an open field arena. The holeboard consisted of a Plexiglas box (40×40× 40 cm), with 16 equally spaced holes (2.5 cm in diameter) in the floor. The holeboard test was performed in a dimly-lighted room. A test animal was placed from the home cage to the center of the floor, and its behavior was examined during 10 min. At the end of the experimental period, the animal was placed to the center of the open field arena. The open field setup was a 1 m2 area of restricted by barriers 40 cm in height. The field was delineated into 100 squares (10×10 cm). The sixteen squares most remote from the barriers formed the center of the field. The field was illuminated with a 200 W lamp fixed 1 m above the center (Semenova et al., 1987, 2000). At the end of the experiment, the animal was returned to its home cage. Between the test sessions, both apparatus were cleaned thoroughly with a disinfectant to remove feces, urine and other scents.
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The behavior of each animal was examined successively for 10 min in the holeboard and 10 min in the open field. The number of rearings and head-dips in the holeboard, per 5 min, served as an indicator of exploratory activity (Boissier and Simon, 1962; File and Wardill, 1975). The number of squares crossed by the animal in the open field and the number of runs across the field center during the first 3 min served as indicators of locomotory activity and the number of rearings as a measure of exploratory activity (Markel et al., 1988). The dynamics of the extinction of locomotory activity during 6 min reflected the habituation reaction (Dewsbury, 1978; Sokolov, 1965).
2.3. Statistics The data were expressed as the mean9 S.E.M. Each group consisted of a minimum of six ground squirrels. Differences in the numbers of crossed squares, rearings, and head-dips between Group III and the other groups were analyzed with the non-parametric Mann– Whitney U-test.
3. Results Analysis of the results revealed significant seasonal variations in the animal behavior. Table 1 presents the data characterizing the exploratory activity of ground squirrels in the holeboard test. It can be seen that both behavioral indicators of exploratory activity (the number of rearings and head-dips in the holeboard) were maximal in summer (Group III). A significant reduction in the number of head-dips and rearings took place in
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normothermic animals during arousal between bouts in the middle of March (Group I). However, there were no significant differences between the summer animals (Group III) and the animals arousing in mid-April (Group II). The number of head-dips registered at the beginning of September (Group IV) and in mid-October (Group V) were 3–4.5 times less than in summer animals. Table 2 shows the data characterizing the locomotory and exploratory activity of ground squirrels in the open field. It can be seen that the animals in Group II and III have higher values of all parameters compared with animals in Groups I, IV, and V: the number of crossed squares, rearings, and runs across the center in Groups II and III are much greater. Normothermic animals arousing between bouts (Group I) have an activity 1.9 (PB 0.05) times lower than summer animals (Group III). Early and mid-autumn ground squirrels (Group IV and V) have an activity that is, respectively, 2.3 and 2.8 (P B0.01) times lower than in summer animals. It is quite remarkable that the parameters characterizing the behavior of the animals in Groups II–III and IV–V are so much alike. In ground squirrels arousing from hibernation (Group II), the parameters reach the values typical of the behavior of animals during their maximum activity in summer (Group III) within a very short time, sometimes within a few hours. By contrast, the behavioral indicators of animals tested at the beginning of September (Group IV) and in midOctober (Group V) were significantly lower than in the summer animals (Group III) despite the fact that these animals both in nature and under the conditions of a vivarium enter hibernation at the end of November.
Table 1 Seasonal changes in the exploratory activity of animals in the holeboard test Group Group Group Group Group Group
I II III IV V
N
Number of rearings during 3 min
Number of head dips during 3 min
6 6 9 8 6
2.29 0.6* 3.0 9 0.6 3.89 0.5 0.9 9 0.2** 0.89 0.3**
1.5 9 0.6* 2.0 9 0.6 2.7 9 0.9 1.1 9 0.3* 0.7 9 0.2**
N, number of animals in the group. The values represent mean 9S.E.M. of the experiments indicated (N). Significance of difference between data for Group III an other animal groups according to the Mann–Whitney U-test: *, PB0.05, **, PB0.01.
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Table 2 Seasonal changes in the activity of animals in the open field Group Group Group Group Group Group
I II III IV V
N
Number of crossed squares
Number of rearings
Number of crossed centers
6 6 9 8 6
66.19 7.5* 124.2 9 15.0 128.1 9 14.0 56.79 11.2*** 46.4 9 4.5***
3.390.9* 5.5 9 1.1 6.8 91.1 2.1 9 0.9* 1.8 90.5**
2.8 9 0.7 2.5 9 0.6 3.2 9 0.6 1.1 9 0.3*** 0.4 9 0.2**
The values represent mean 9 S.E.M. of the number of experiments indicated (N). Significance of differences between data for group III and other groups according to the Mann–Whitney U-test: *, PB0.05; **, PB0.01; ***, PB0.001.
Fig. 1 presents curves characterizing season-related changes in the dynamics of extinction of locomotory activity of ground squirrels in the open field (observation period 6 min). In the animals of Groups II and III, the habituation is more pronounced than in animals of Groups I, IV and V. The seasonal characteristics of habituation are consistent with the changes in exploratory behavior noted previously.
4. Discussion Thus, taken together, these data suggest that the behavior of hibernators in holeboard and open field assays undergoes apparent seasonal variations. The results of our investigation are in agreement with the data on the behavior of ground squirrels in freely living populations. Long-tail ground squirrels C. undulatus in Jakutia arouse from hibernation at the end of March or at the beginning of April. Animals living on the northern slopes of Alatoo mountains enter hibernation in the beginning of October whereas on the southern slopes of these mountains and in the valley near Jakutsk they stay active until November. Seasonal variations in the circadian rhythm of activity in this species of ground squirrels were observed (Vinokurov and Ahrimenko, 1982). It was shown that the dynamics of their activity had two peaks during all seasons. In July, ground squirrels left their holes from 06:00 to 11:30 h in the morning and from 03:30 to 07:30 h in the evening. The total time of their presence on the ground was 8.5–9.5 h. In September, the activity
of ground squirrels drastically decreased. They left their holes at around 12:00 h in the evening for a short period and again from 03:30 to 07:00 h in the evening. In autumn, the time of their presence on the ground was no more than 4–4.5 h (Vinokurov and Ahrimenko, 1982). Variations in behavioral patterns, including investigation, locomotion and social encounters, in different periods of the non-hibernating period, especially after emergence in the spring and prior to entrance into hibernation, were also observed in another species, the 13-lined ground squirrel (Wistrand, 1974). In our experiments, we examined the modifications of ground squirrel behavior more thoroughly than in previous studies. Although the conceptual distinction between locomotory and exploration activities has been widely accepted, the separation between these forms of activity is not always easy to achieve in test situations using rodents. The holeboard apparatus was first introduced by Boissier and Simon (1962) for studying the exploratory reactions in rats. According to these authors, head-dipping is a measure that reflects both of these factors. Later, it was shown that frequency and duration of head-dips reflect the exploration rather than activity (File and Wardill, 1975). Although several studies have been performed on the behavior of laboratory rodents in the open field, it is difficult to arrive at a general conclusion due to considerable variations in experimental design and behavioral parameters used (Archer, 1973). In the open-field test the number of crossed squares and rearings in a novel and unusual environment has been used as a measure of both
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exploration and locomotory activity. On the basis of behavioral observations of rats in the open field (Archer, 1973; Markel et al., 1988), it seems more justified to regard the number of squares crossed by ground squirrels as a measure of locomotory activity, and the number of rearings as an indicator of exploratory behavior. We believe that the peak in the number of head-dips and rearings in the holeboard test as well as the number of rearings, center crossings and square crossings in the open field in animals from Group II reflect a peak in exploratory and locomotory activities which is generally observed in the summer period.
Fig. 1. Seasonal variations in the dynamics of extinction of the activity in ground squirrels as compared with summer animals (Group III). (A) Group I (early March); (B) Group II (middle of April); (C) Group IV (early September); (D) Group V (middle of October). ( ) Summer animals (Group III); () animals of other groups. On the ordinate, the number of squares in the open field crossed by animals; on the abscissa, time of observation in min.
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In ground squirrels arousing between bouts during the hibernation season, all behavioral indicators of exploratory activity in holeboard and of locomotory and exploratory activities in the open field tests were lower than in active summer animals. However, after exit from hibernation (Group II), a strikingly rapid restoration of all behavioral parameters of activity occurs: in some cases the parameters reach the values characteristic of summer animals even within the first day. The decrease in all behavioral parameters in autumn approached minimum values long before the entry into hibernation (45 days– 2 months). The analysis of the data suggests that the locomotory and exploratory activities of ground squirrels at the beginning of autumn are very similar to those of animals tested at the end of November immediately prior to entry into hibernation (Semenova and Kolaeva, 1999). The distinct decrease in the rate of the locomotory activity in the animals from Groups II and III indicates that habituation capacities of ground squirrels in these seasons are better than in others seasons. It is noteworthy that the functional activity of monoaminergic and peptidergic systems of the brain of hibernators begins to decrease during the normothermic period, long before the animals enter hibernation (Haak et al., 1991; Popova et al., 1993; Wang, 1993; Nurnberger, 1995; Semenova et al., 2000), whereas its enhancement occurs before complete arousal of animals from hibernation. These data, as well as, the results of our behavioral experiments, indicate that the preparation of the CNS of hibernators for seasonal changes in the functional states is under the control of endogenous regulators. It can be assumed that the seasonal variations in functional activity of the neurochemical regulatory systems play a key role in the reorganization of the behavior of hibernators in different phases of the annual cycle.
Acknowledgements We thank Galina Bliskovka for secretarial assistance. This work was supported by the Russian Foundation for Basic Research (Grant N01-04-
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48199) and the program ‘Integration’ (Grant A No. 0055). References Archer, J., 1973. Tests for emotionality in rats and mice: a review. Anim. Behav. 21, 205 –235. Beckman, A.L., Stanton, T.L., 1982. Properties of the CNS during the State of Hibernation. In: Beckman, A.L. (Ed.), Neural Basis of Behavior. Spectrum, New York, pp. 19 – 45. Boissier, J.R., Simon, P., 1962. La reaction d’exploration chez la souris. Therapie 17, 1225 –1232. Cai, Y.-P., Lee, T.-F., Wang, L.C.H., 1996. State-dependent change in thermoregulatory responses to intraseptal 5-hydroxytryptamine injection in ground squirrels. In: Geiser, F., et al. (Eds.), Adaptation to the Cold: Tenth International Hibernation Symposium. University of New England Press, Armidal, pp. 179 –184. File, S.E., Wardill, A.G., 1975. Validity of head-dipping as a measure of exploration in a modified holeboard. Psychpharmacologia 44, 53 –59. Dewsbury, D., 1978. Comparative Animal Behavior. Mc Graw-Hiller Book Company, New York, St. Louis, San Francisco, pp. 363 – 389. Haak, L., Mignot, E., Kilduff, T., Dement, W., Heller, H., 1991. Regional changes in central monoamine and metabolite levels during the hibernation cycle in the golden-mantled ground squirrel. Brain Res. 563, 215 –220. Markel, A.L., Galaktionov, J.u.K., Efimov, V.M., 1988. Factor analysis of rats behavior in open field test. Zh. Vyschej Nervnoj Dejatelnosty 38, 855 –863 In Russian. Mateo, J.M., Johnston, R.E., 2000. Retention of social recognition after hibernation in Belding’s ground squirrels. Anim. Behav. 59, 491 –499. McNamara, M.C., Riedesel, M.L., 1973. Memory and hibernation in Citellus lateralis. Science 179, 92 – 94. Millesi, T., Prossinger, H., Dittami, J.P., Fieder, M., 2001. Hibernation effects on memory in European ground squirrels (Spermophilus citellus). J. Biol. Rhythms 16, 264 – 271.
Nuritdinov, E.N., 1987. The peculiarity of higher nervous activity in ground squirrels Citellus Ful6us in active period. Zh. Evol. Biochim. Fiziol 23, 749 – 754 In Russian. Nurnberger, F., 1995. The neuroendocrine system in hibernating mammals: present knowledge and open questions. Cell Tissue Res. 281, 391 – 412. Popova, N.K., Voronova, I.P., Kulikov, A.V., 1993. Involvement of brain tryptophan hydroxilase in the mechanism of hibernation. Pharmacol. Biochem. Behav. 46, 9 – 13. Semenova, T.P., Kolaeva, S.G., 1999. The role of endogenous and exogenous factors in the regulation of the brain functional state in hibernators. In: Trufakin, V.A., Shochenko, K.A. (Eds.), The Ecological Physiology. Nauka, Novosibirsk, pp. 213 – 219 In Russian. Semenova, T.P., Gromova, E.A., Grishchenko, N.I., Bragin, A.G., Nesterova, I.V., Smirnova, G.A., Tretyk, T.M., Vinogradova, O.S., 1987. Behavioural, biochemical and hishoshemical effects of locus coeruleus transplantation in rats with neurotoxic lesions of the catecholaminergic system. Neuroscience 22, 993 – 1002. Semenova, T.P., Anoshkina, I.A., Dolgacheva, L.P., Abzhalelov, B.B., Kolaeva, S.G., 2000. Seasonal variations in monoaminergic control of behavior in hibernating animals. Russian J. Physiol. 86, 1188 – 1194 In Russian. Slonim, A.D., 1979. Physiological states (hibernation and estivation). In: Slonim, A.D., et al. (Eds.), Ecological Physiology of Animals. P.1. General Ecological Physiology and Physiology of Adaptation. Nauka, Leningrad, pp. 183 – 246 In Russian. Sokolov, E.N., 1965. Habituation as a simplest form of conditioned reflex. Zh. Vyschej Nervnoj Dejatelnosty 15, 249 – 259 In Russian. Vinokurov, V.N., Ahrimenko, A.K., 1982. Ecology of the Long-Tail Ground Squirrels in Jakutia. Nauka, Jakutsk, p. 164 In Russian. Wang, L.C.H., 1993. Neurochemical Regulation of Arousal From Hibernation. In: Carey, C., et al. (Eds.), Living in the Cold. Bulder, Westview, pp. 559 – 561. Wistrand, H., 1974. Individual, social and seasonal behavior of the thirteen-lined ground squirrels (Spermophilus tridecmillineatus). J. Mammalogy 55, 329 – 347.
Seasonal peculiarities of behavior of ground squirrel Citellus undulatus in holeboard and open field tests T.P. Semenova *, I.A. Anoshkina, B.M. Khomut 1, S.G. Kolaeva Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia Received 26 September 2000; received in revised form 11 September 2001; accepted 13 September 2001
Abstract Seasonal changes in the behavior of ground squirrels Citellus undulatus (n = 35) in holeboard and open field tests were studied. It was found that the number of head dips and rearings in the holeboard and the number of crossed squares, rearings, and runs across the field center in the open field in ground squirrels during arousal between bouts at the end of the hibernation period, in the middle of March, were lower than these indices in active summer animals. A rapid restoration of all behavioral parameters after the arousal of animals from hibernation in mid-April was shown. In some cases, the behavioral parameters of activity in the holeboard and open field tests and the habituation reaction reached the values characteristic of active summer animals in a very short period of time, sometime within 1 day. By contrast, the locomotory and exploratory activities of animals tested in autumn (at the beginning of September and in the middle of October) were significantly lower than in summer animals, i.e. these indices decreased to minimum values 1.5–2 months prior to their entry into hibernation. The results of behavioral experiments indicate that the preparation of the CNS of hibernators for seasonal changes in the functional states is under the control of endogenous regulators. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Annual cycle; Ground squirrels; Hibernation; Holeboard; Open field; Exploratory activity; Locomotory activity
1. Introduction Hibernation is an adaptive response of animals to unfavorable environmental conditions: a lowering of ambient temperature, a shortening of the photoperiod, absence of food, etc. The hibernation process affects all levels of organization of living organisms, from the molecular and cellular * Corresponding author. Fax: + 7-967-790509. E-mail address: [email protected] (T.P. Semenova). 1 Present address: Knowledge Exchange Institute, West Nyack, NY 10994, USA.
to the systemic, including the behavior of animals (Slonim, 1979; Liman et al., 1982; Heller, 1989). The behavior of hibernators provides the maintenance of vital activity under unfavorable conditions and consists in realizing the genetic programs that are triggered by the corresponding exogenous (photoperiod, temperature, availability of food) and endogenous factors (circannual rhythms of neurotransmitters, hormones and peptides). Hibernators exhibit a wide range of neuronal activity of the brain, from profound inactivity
0376-6357/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 6 3 5 7 ( 0 1 ) 0 0 1 9 5 - 4
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(torpor) of deep hibernation to all the central activity states during their non-hibernating (euthermic) phase, thereby providing a unique system capable of regulating the full range of mammalian CNS activity states (Beckman and Stanton, 1982). Biochemical studies revealed appreciable seasonal variations in the functional activity of the neurochemical regulatory systems responsible for the synthesis of endogenous regulators of hibernation such as serotonin, catecholamines, neuropeptides, and enzymes involved in amino acid metabolism (Haak et al., 1991; Popova et al., 1993; Nurnberger, 1995; Cai et al., 1996; Semenova and Kolaeva, 1999). However, it remains unclear how the behavior of hibernators changes in different phases of their annual cycle. There are a few papers devoted to learning and memory in hibernators. However, these studies were performed in the summer period, and the researchers were focused on the effect of natural hypothermia on various forms of memory in hibernators (McNamara and Riedesel, 1973; Nuritdinov, 1987; Mateo and Johnston, 2000; Millesi et al., 2001). Observations in field conditions during the transitional stage from activity to hibernation revealed a drastic impairment of the major activity, movement co-ordination and circadian rhythms of activity (Slonim 1979). The goal of the present work was to study seasonal changes in the behavior of Jakutian ground squirrel, Citellus undulatus, in holeboard and open field tests.
2. Materials and methods
2.1. Subjects Adult ground squirrels (C. undulatus) of both sexes weighing 350– 600 g (n =35) were used. The animals were trapped in the valley near Jakutsk and kept for 2–3 weeks under the care of veterinarians at the vivarium of the Institute of Biology (Siberian Division of the Russian Academy of Sciences) for 2–3 weeks. At the end of August they were transported to our Institute. The animals were housed individually, in 35× 40 ×20 cm cages with free access to dry food, fresh vegetables, herb, and drinking water. The
light cycle was 12-h on:12-h off (07:00 h in the morning to 07:00 h in the evening). At the end of October, the ground squirrels were placed to a special dark hibernation room, where they were kept until arousal. The temperature in the room was + 4 °C, which is comparable to natural conditions. Experiments were carried out with five groups of animals in different phases of their annual cycle: Group I: during arousal between bouts at the end of the hibernation period (in the middle of March); Group II: after exit from hibernation (in the middle of April); Group III: in the phase of activity (in the middle of June); Groups IV and V (in early September and in the middle of October, respectively): prior to the entry into hibernation. The behavioral experiments were carried out between 09:00 and 11:00 h in the morning. The experiments were conducted in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ as adopted and promulgated by the Declaration of Helsinki.
2.2. Experimental arrangement The locomotory and exploratory activities of animals were studied using a holeboard and an open field arena. The holeboard consisted of a Plexiglas box (40×40× 40 cm), with 16 equally spaced holes (2.5 cm in diameter) in the floor. The holeboard test was performed in a dimly-lighted room. A test animal was placed from the home cage to the center of the floor, and its behavior was examined during 10 min. At the end of the experimental period, the animal was placed to the center of the open field arena. The open field setup was a 1 m2 area of restricted by barriers 40 cm in height. The field was delineated into 100 squares (10×10 cm). The sixteen squares most remote from the barriers formed the center of the field. The field was illuminated with a 200 W lamp fixed 1 m above the center (Semenova et al., 1987, 2000). At the end of the experiment, the animal was returned to its home cage. Between the test sessions, both apparatus were cleaned thoroughly with a disinfectant to remove feces, urine and other scents.
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The behavior of each animal was examined successively for 10 min in the holeboard and 10 min in the open field. The number of rearings and head-dips in the holeboard, per 5 min, served as an indicator of exploratory activity (Boissier and Simon, 1962; File and Wardill, 1975). The number of squares crossed by the animal in the open field and the number of runs across the field center during the first 3 min served as indicators of locomotory activity and the number of rearings as a measure of exploratory activity (Markel et al., 1988). The dynamics of the extinction of locomotory activity during 6 min reflected the habituation reaction (Dewsbury, 1978; Sokolov, 1965).
2.3. Statistics The data were expressed as the mean9 S.E.M. Each group consisted of a minimum of six ground squirrels. Differences in the numbers of crossed squares, rearings, and head-dips between Group III and the other groups were analyzed with the non-parametric Mann– Whitney U-test.
3. Results Analysis of the results revealed significant seasonal variations in the animal behavior. Table 1 presents the data characterizing the exploratory activity of ground squirrels in the holeboard test. It can be seen that both behavioral indicators of exploratory activity (the number of rearings and head-dips in the holeboard) were maximal in summer (Group III). A significant reduction in the number of head-dips and rearings took place in
197
normothermic animals during arousal between bouts in the middle of March (Group I). However, there were no significant differences between the summer animals (Group III) and the animals arousing in mid-April (Group II). The number of head-dips registered at the beginning of September (Group IV) and in mid-October (Group V) were 3–4.5 times less than in summer animals. Table 2 shows the data characterizing the locomotory and exploratory activity of ground squirrels in the open field. It can be seen that the animals in Group II and III have higher values of all parameters compared with animals in Groups I, IV, and V: the number of crossed squares, rearings, and runs across the center in Groups II and III are much greater. Normothermic animals arousing between bouts (Group I) have an activity 1.9 (PB 0.05) times lower than summer animals (Group III). Early and mid-autumn ground squirrels (Group IV and V) have an activity that is, respectively, 2.3 and 2.8 (P B0.01) times lower than in summer animals. It is quite remarkable that the parameters characterizing the behavior of the animals in Groups II–III and IV–V are so much alike. In ground squirrels arousing from hibernation (Group II), the parameters reach the values typical of the behavior of animals during their maximum activity in summer (Group III) within a very short time, sometimes within a few hours. By contrast, the behavioral indicators of animals tested at the beginning of September (Group IV) and in midOctober (Group V) were significantly lower than in the summer animals (Group III) despite the fact that these animals both in nature and under the conditions of a vivarium enter hibernation at the end of November.
Table 1 Seasonal changes in the exploratory activity of animals in the holeboard test Group Group Group Group Group Group
I II III IV V
N
Number of rearings during 3 min
Number of head dips during 3 min
6 6 9 8 6
2.29 0.6* 3.0 9 0.6 3.89 0.5 0.9 9 0.2** 0.89 0.3**
1.5 9 0.6* 2.0 9 0.6 2.7 9 0.9 1.1 9 0.3* 0.7 9 0.2**
N, number of animals in the group. The values represent mean 9S.E.M. of the experiments indicated (N). Significance of difference between data for Group III an other animal groups according to the Mann–Whitney U-test: *, PB0.05, **, PB0.01.
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Table 2 Seasonal changes in the activity of animals in the open field Group Group Group Group Group Group
I II III IV V
N
Number of crossed squares
Number of rearings
Number of crossed centers
6 6 9 8 6
66.19 7.5* 124.2 9 15.0 128.1 9 14.0 56.79 11.2*** 46.4 9 4.5***
3.390.9* 5.5 9 1.1 6.8 91.1 2.1 9 0.9* 1.8 90.5**
2.8 9 0.7 2.5 9 0.6 3.2 9 0.6 1.1 9 0.3*** 0.4 9 0.2**
The values represent mean 9 S.E.M. of the number of experiments indicated (N). Significance of differences between data for group III and other groups according to the Mann–Whitney U-test: *, PB0.05; **, PB0.01; ***, PB0.001.
Fig. 1 presents curves characterizing season-related changes in the dynamics of extinction of locomotory activity of ground squirrels in the open field (observation period 6 min). In the animals of Groups II and III, the habituation is more pronounced than in animals of Groups I, IV and V. The seasonal characteristics of habituation are consistent with the changes in exploratory behavior noted previously.
4. Discussion Thus, taken together, these data suggest that the behavior of hibernators in holeboard and open field assays undergoes apparent seasonal variations. The results of our investigation are in agreement with the data on the behavior of ground squirrels in freely living populations. Long-tail ground squirrels C. undulatus in Jakutia arouse from hibernation at the end of March or at the beginning of April. Animals living on the northern slopes of Alatoo mountains enter hibernation in the beginning of October whereas on the southern slopes of these mountains and in the valley near Jakutsk they stay active until November. Seasonal variations in the circadian rhythm of activity in this species of ground squirrels were observed (Vinokurov and Ahrimenko, 1982). It was shown that the dynamics of their activity had two peaks during all seasons. In July, ground squirrels left their holes from 06:00 to 11:30 h in the morning and from 03:30 to 07:30 h in the evening. The total time of their presence on the ground was 8.5–9.5 h. In September, the activity
of ground squirrels drastically decreased. They left their holes at around 12:00 h in the evening for a short period and again from 03:30 to 07:00 h in the evening. In autumn, the time of their presence on the ground was no more than 4–4.5 h (Vinokurov and Ahrimenko, 1982). Variations in behavioral patterns, including investigation, locomotion and social encounters, in different periods of the non-hibernating period, especially after emergence in the spring and prior to entrance into hibernation, were also observed in another species, the 13-lined ground squirrel (Wistrand, 1974). In our experiments, we examined the modifications of ground squirrel behavior more thoroughly than in previous studies. Although the conceptual distinction between locomotory and exploration activities has been widely accepted, the separation between these forms of activity is not always easy to achieve in test situations using rodents. The holeboard apparatus was first introduced by Boissier and Simon (1962) for studying the exploratory reactions in rats. According to these authors, head-dipping is a measure that reflects both of these factors. Later, it was shown that frequency and duration of head-dips reflect the exploration rather than activity (File and Wardill, 1975). Although several studies have been performed on the behavior of laboratory rodents in the open field, it is difficult to arrive at a general conclusion due to considerable variations in experimental design and behavioral parameters used (Archer, 1973). In the open-field test the number of crossed squares and rearings in a novel and unusual environment has been used as a measure of both
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exploration and locomotory activity. On the basis of behavioral observations of rats in the open field (Archer, 1973; Markel et al., 1988), it seems more justified to regard the number of squares crossed by ground squirrels as a measure of locomotory activity, and the number of rearings as an indicator of exploratory behavior. We believe that the peak in the number of head-dips and rearings in the holeboard test as well as the number of rearings, center crossings and square crossings in the open field in animals from Group II reflect a peak in exploratory and locomotory activities which is generally observed in the summer period.
Fig. 1. Seasonal variations in the dynamics of extinction of the activity in ground squirrels as compared with summer animals (Group III). (A) Group I (early March); (B) Group II (middle of April); (C) Group IV (early September); (D) Group V (middle of October). ( ) Summer animals (Group III); () animals of other groups. On the ordinate, the number of squares in the open field crossed by animals; on the abscissa, time of observation in min.
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In ground squirrels arousing between bouts during the hibernation season, all behavioral indicators of exploratory activity in holeboard and of locomotory and exploratory activities in the open field tests were lower than in active summer animals. However, after exit from hibernation (Group II), a strikingly rapid restoration of all behavioral parameters of activity occurs: in some cases the parameters reach the values characteristic of summer animals even within the first day. The decrease in all behavioral parameters in autumn approached minimum values long before the entry into hibernation (45 days– 2 months). The analysis of the data suggests that the locomotory and exploratory activities of ground squirrels at the beginning of autumn are very similar to those of animals tested at the end of November immediately prior to entry into hibernation (Semenova and Kolaeva, 1999). The distinct decrease in the rate of the locomotory activity in the animals from Groups II and III indicates that habituation capacities of ground squirrels in these seasons are better than in others seasons. It is noteworthy that the functional activity of monoaminergic and peptidergic systems of the brain of hibernators begins to decrease during the normothermic period, long before the animals enter hibernation (Haak et al., 1991; Popova et al., 1993; Wang, 1993; Nurnberger, 1995; Semenova et al., 2000), whereas its enhancement occurs before complete arousal of animals from hibernation. These data, as well as, the results of our behavioral experiments, indicate that the preparation of the CNS of hibernators for seasonal changes in the functional states is under the control of endogenous regulators. It can be assumed that the seasonal variations in functional activity of the neurochemical regulatory systems play a key role in the reorganization of the behavior of hibernators in different phases of the annual cycle.
Acknowledgements We thank Galina Bliskovka for secretarial assistance. This work was supported by the Russian Foundation for Basic Research (Grant N01-04-
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