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Effects of thyroid hormone deficiency on behavior in rat strains with different predisposition to catalepsy
Physiology & Behavior 75 (2002) 733 – 737
Effects of thyroid hormone deficiency on behavior in rat strains with different predisposition to catalepsy N.N. Barykinaa, V.F. Chuguya, T.A. Alekhinaa, V.G. Kolpakova, A.V. Maksiutovab, A.V. Kulikovb,* a
Laboratory of Evolutionary Genetics, Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 10 Lavrentiev Avenue, Novosibirsk 630090, Russia b Laboratory of Behavioral Phenogenetics, Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 10 Lavrentiev Avenue, Novosibirsk 630090, Russia Received 18 June 2001; received in revised form 30 November 2001; accepted 28 January 2002
Abstract The effects of thyroidectomy on anxiety-related behavior in the elevated plus-maze test, locomotor activity, and defecation in the openfield test and duration of cataleptic freezing were studied in rats of two strains differing in predisposition to catalepsy: cataleptic strain GC and its ancestor strain Wistar. Total thyroxine level was significantly decreased in control GC rats compared to that in control Wistar rats. Control Wistar and GC rats did not differ either in the percentages of open-arm entries or the time spent therein in the elevated plus-maze test or in defecation score in the open-field test. At the same time, control Wistar rats showed more locomotor activity compared to control GC rats in the open-field test. Thyroid hormone deficiency did not affect the percentages of open-arm entries and the time spent therein in the elevated plus-maze test as well as defecation score in both strains. Thyroidectomy did not alter significantly locomotor activity in Wistar rats, but produced a nearly twofold increase in locomotor activity in GC rats. The most important finding is that thyroidectomy significantly increased the expression of catalepsy in Wistar rats, which points to a role of thyroid hormones in the regulation of predisposition to cataleptic reaction. D 2002 Published by Elsevier Science Inc. Keywords: Catalepsy; Thyroxine; Plus-maze; Open-field; Rat
1. Introduction In spite of numerous clinical data that point to a close link between thyroid dysfunction and mental disorders [8,9,17], the mechanisms of influence of thyroid hormones on brain functions and behavior remain obscure. Avoidance behavior anomalies are considered to be a probable biological basis of depressive disorders [4]. There is experimental evidence that early [22], as well as adult [16], hypothyroidism produce an avoidance behavior deficit. Recently, it was shown that hypothyroid rats were more immobile in the forced swim test compared with controls [15]. Immobility or freezing is a common adaptive defensive strategy of coping with inescapable threats in some mammalian species and birds [4]. It is therefore interesting * Corresponding author. Tel.: +7-3832-34-47-53; fax: +7-3832-33-1278. E-mail address: [email protected] (A.V. Kulikov). 0031-9384/02/$ – see front matter D 2002 Published by Elsevier Science Inc. PII: S 0 0 3 1 - 9 3 8 4 ( 0 2 ) 0 0 6 6 2 - 5
whether a decreased thyroid function is a common feature of predisposition to freezing reactions. A rat strain GC (designated so by the initials of words ‘genetic’ and ‘catalepsy’), was produced from an outbred Wistar stock by breeding for an increased predisposition to freezing with elements of catalepsy in response to stressful stimulation. More than 50% of GC rats maintain for more than 10 s an imposed immobile vertical posture [2]. Besides, GC rats display a lower locomotor and exploratory activity in the open-field [11,23] and an increased immobility in the forced swimming test [18] as compared to Wistar rats. The present study aimed to compare the effects of thyroid hormone deficiency on various behavioral responses to stressful stimuli in GC and Wistar rats. It was planned (1) to compare serum thyroid hormone concentration in GC and Wistar rats and (2) to study the effects of thyroidectomy on behavior under mild aversive stimulation in open-field and plus-maze tests as well as on manifestation of cataleptic freezing reaction in rats of these strains.
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2. Materials and methods
2.3. Statistics
2.1. Animals and surgery
The data were expressed as mean ± S.E.M. and were compared by two-way ANOVA followed by the Bonferroni – Dunn test.
The studies were made on 24 Wistar and 22 GC males at the age of 2 months (at the beginning of the experiment) weighing 200 ± 12 g taken from respective stocks maintained at the Institute of Cytology and Genetics of the Russian Academy of Sciences (Novosibirsk, Russia). In 12 Wistar males and 10 GC males, the thyroid and parathyroid glands were surgically removed. The other 12 Wistar males and 12 GC males were sham-operated and served as controls. The surgery was carried out under sodium pentobarbital anesthesia (35 mg/kg ip). Animals were housed four per cage in 60 40 20-cm cages at a regulated temperature of 22 °C with a 12:12-h light/dark cycle. All thyroidectomized rats were given 0.5% CaCl2 in water. Rats were kept and used strictly in accordance with guidelines of the European Community. The tests started 31 days after surgery. Two days before the tests, the animals were isolated in cages 60 40 20 cm. 2.2. The test for catalepsy To induce catalepsy, the rat was lifted by its forepaws with a stick in a corner of the cage and then the stick was removed and the period (in seconds) during which the animal maintained the forced vertical posture was recorded. Catalepsy was estimated by the period during which the animal maintained the imposed vertical posture and the number of animals that maintained this posture for at least 10 s. The plus-maze test was performed on the day after the test for catalepsy in a device made of Perspex, with two opposite open arms (50 10 cm) and two opposite closed arms of the same size with walls measuring 40 cm in height. The whole device was elevated 50 cm above a white floor and exposed to dim illumination. Each animal was placed in the central square facing the enclosed arm. The number of entries into and time spent in each arm were recorded for 5 min. An arm entry was scored when all four paws were in the arm. The percentages of open-arm entries and the time spent therein related to the total number of entries and time spent in both types of arms were calculated and used as indices of anxiety-related behavior [13]. The open-field test was performed on the day after the plus-maze test in a device (140 70 45 cm) made of Perspex and colored white. The floor, brightly lit (300 lx), was divided into 98 squares (10 10 cm). Each rat was placed in the centre of the arena, tested for 6 min, and the number of crossed squares and the number of fecal boli (defecation score) were recorded and used as indices of locomotor activity and autonomic reactivity, respectively. Total thyroxine concentration (nM) was estimated in serum by a immunoenzymatic technique using the ThyroidIEA-Tyroxine Kit (Peterburg, Russia).
3. Results Total serum thyroxine level was significantly lower in sham-operated GC rats compared to that in sham-operated Wistar rats (47.5 ± 2.3 nM in Wistar vs. 34.4 ± 1.9 nM in GC, P < .001). Thyroidectomy produced about a sevenfold decrease in serum thyroxine level in GC rats (down to 5 ± 2.5 nM, P < .001) and a twofold (down to 25.5 ± 2.8 nM, P < .001) decrease in Wistar rats with respect to that in the sham-operated control (Fig. 1). A significant influence of both genotype [ F(1,39) = 43.8, P < .001] and thyroidectomy [ F(1,39) = 107.3, P < .001] on serum thyroxine concentration was found, but the interaction between these two factors was not significant [ F(1,39) = 2.6, P > .1]. In the plus-maze test sham-operated Wistar and GC rats did not differ significantly in percentages of open-arm entries and time spent therein. Tyroidectomy did not alter these traits either in Wistar or GC rats (Table 1). There were no effects of genotype [ F(1,42) = 0.06, P>.8], thyroidectomy [ F(1,42) = 0.98, P >.3], or interaction of these factors [ F(1,42) = 0.54, P >.4] on the percentage of open-arm entries. No effects of genotype [ F(1,42 = 3.3, P > .05], thyroidectomy [ F(1,42 = 0.32, P > .5], or factors interaction [ F(1,42 = 2.2, P >.1] on the percentage of time spent in open arms were found. Sham-operated rats from both strains did not differ in the defecation score (3.9 ± 0.4 in Wistar vs. 4.6 ± 0.9 in GC). Thyroidectomy did not alter this trait either in Wistar (3.9 ± 0.4) or GC rats (3.0 ± 0.7). There were no effects of genotype [ F(1,42) = 1.6, P > .2], thyroidectomy
Fig. 1. Influence of thyroidectomy on serum thyroxine level in Wistar and GC rats. *** P < .001, thyroidectomized rats vs. respective sham-operated control. ###P < .001, sham-operated Wistar vs. sham-operated GC.
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Table 1 Effect of thyroidectomy on percentages of open-arm entries and time spent therein in the plus-maze test in Wistar and GC rats Strain Treatment
Wistar
GC
Percentage of open-arm entries Sham 18.8 ± 8.7 (n = 12) Thyroidectomized 15.3 ± 6.6 (n = 12)
24.5 ± 9.2 (n = 12) 11.5 ± 4.8 (n = 10)
Percentage of time spent in open arms Sham 30.1 ± 9.5 (n = 12) Thyroidectomized 38.2 ± 11.2 (n = 12)
26.6 ± 8.4 (n = 12) 8.4 ± 3.8 (n = 10)
[ F(1,42) = 0.04, P >.8], or interaction of these factors [ F(1,42) = 1.9, P >.2] on defecation score. Sham-operated GC rats showed significantly less locomotion in the open field than sham-operated Wistar rats did (82.4 ± 17.3 vs. 159.8 ± 28.2, respectively, P < .05) (Fig. 2). No main effects of strain [ F(1,42) = 0.01, P >.9] and thyroidectomy [ F(1,42) = 1.01, P >.32] on locomotor activity in the open-field test was shown. However, there was a significant interaction between the strain (genotype) and thyroidectomy factors in the determination of locomotor activity [ F(1,42) = 12.6, P < .001]. Thyroidectomy did not alter significantly locomotor activity in Wistar rats, but produced a twofold increase of locomotion in GC rats (up to 184.5 ± 24.4, P < .01). Thyroidectomy increased the predisposition to catalepsy in Wistar rats: while only 2 of 12 (16.6%) sham-operated rats displayed catalepsy, such reaction was observed in 7 of 12 of thyroidectomized rats [58.3%, t(22) = 2.2, P < .05]. The duration of cataleptic reaction in thyroidectomized Wistar rats was also increased as compared to sham-operated control (11.58 ± 3.6 vs. 2.3 ± 1.5 s, P < .05) up to the level of shamoperated GC rats (9.9 ± 2.8 s, Fig. 3). Thyroidectomy also produced a nonsignificant increase of cataleptic immobility time in GC (up to 17.1 ± 3.3 s, P >.05, Fig. 3). A significant
Fig. 2. Influence of thyroidectomy on open-field locomotion in Wistar and GC rats. ** P < .01, thyroidectomized rats vs. respective sham-operated control. #P < .05, sham-operated Wistar vs. sham-operated GC.
Fig. 3. Influence of thyroidectomy on the duration of catalepsy in Wistar and GC rats. * P < .05, thyroidectomized rats vs. respective sham-operated control. #P < .05, sham-operated Wistar vs. sham-operated GC.
influence of both genotype [ F(1,42) = 5.16, P < .03] and thyroidectomy [ F(1,42) = 8.09, P < .007], but not of interaction of these two factors [ F(1,42) = 0.14, P >.7] on the duration of catalepsy was found.
4. Discussion Significant interstrain differences in total serum thyroxine level were found. While the thyroxine concentration in control Wistar rats (47.5 nmol/l) was very close to that shown by other authors (40 – 60 nmol/l) [5,20], in control GC rats the hormone concentration was significantly lower (34.4 nmol/l). It may be hypothesized that GC rats have a kind of genetically determined borderline primary hypothyroidism. However, more evidence including serum triiodothyronine and thyroid-stimulating hormone assays are needed to verify this hypothesis. The surgical removal of thyroid gland resulted in a dramatic decrease of thyroxine concentration in rats of both strains. In Wistar rats, thyroidectomy produced a twofold decrease in serum thyroxine level. This result is close to that found by Redei et al. [20]. Although in GC rats thyroidectomy produced a sevenfold decrease in serum thyroxine concentration, we may not assert that GC rats are more vulnerable to thyroidectomy compared with Wistar rats, because ANOVA failed to show a statistically significant interaction between strain and thyroidectomy factors. There were no differences between control Wistar and GC rats in the percentages of open-arm entries and time spent therein. Thyroidectomy also failed to produce any statistically significant alteration of these traits in both strains. It is commonly assumed that the percentages of open-arm entries and the time spent therein are closely correlated with anxiety [3]. Therefore, it appears that both breeding for catalepsy and thyroid hormone deficiency did not affect this manifestation of anxiety-related behavior.
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There were no differences between control Wistar and GC rats in defecation score in the open-field. Thyroidectomy did not change the defecation score either in Wistar or in GC rats. Since defecation is usually considered as an index of autonomic reactivity [21], it suggests that neither breeding for catalepsy nor thyroid hormone deficit has influenced this kind of reaction of the autonomic system to the stressful situation. GC rats with inherited thyroxine deficit are less active in the open-field test compared to Wistar rats, and this finding agrees with earlier published data [11,23]. It has been found that adult mice with genetically determined hypothyroidism also showed a decreased locomotion as compared with their euthyroid counterparts [1]. Thyroidectomy did not alter locomotor activity in the open-field test in Wistar rats, but produced its marked increase in GC rats. It is noteworthy that other authors also found that thyroid hormone deficit did not change [7] or even decreased [6] the locomotor activity in adult rats. At present, we cannot give a definite explanation of this paradoxical increase of locomotor activity in thyroidectomized GC rats. However, one may hypothesize that the unexpected increase in motor activity of cataleptic rats induced by thyroidectomy may reflect an increase in the expression of the hyperkinetic pole of the catatonic syndrome, which has been observed in GC rats [12]. The most important finding was that thyroidectomy increased predisposition to catalepsy and freezing duration in Wistar rats. Earlier, it has been shown that thyroidectomized rats usually have a deficit of performance in avoidance and escape learning [22]. Earlier, we found that hypothyroid rats were more immobile in the forced swim test compared to euthyroid control [15]. Hypothyroid rats needed more unconditioned stimuli (footshocks) to acquire conditioned avoidance [24]. GC rats, which we found to have a thyroxine deficit, showed less active swimming in the Porsolt test [18]. Therefore, taking together the data present in literature and our own results, it may be hypothesized that a deficit in thyroid hormones decreases active avoidance behavior. What is the mechanism of the phenomenon of increase of cataleptic-like immobility found in hypothyroid rats? At present, there is no information sufficient to answer this question. However, a promising idea came from the study of Levine et al. [16] who studied the effect of thyroidectomy in rats on the course of learning to escape a footshock by pressing the lever. They found that while control rats improved their performance, thyroidectomized rats showed a marked decrease in successful responses. Using Markov transition analysis, the authors concluded that in contrast to control rats, thyroidectomized rats were able to learn not to escape. It does not seem too improbable to accept a very attractive idea that thyroid hormones serve as a ‘switch’ between active (flight) and passive (crypting) strategies. Catalepsy is known to be an exaggerated form of passive defense reaction, e.g., crypting [4], and has been shown to be regulated by various neurotransmitters and neuropeptides [10]. Now, thyroid hormones may be added to the list of
compounds able to affect catalepsy. Among the wide range of neurochemical substances that affect the expression of catalepsy, there are two that attract most attention: dopamine, which is traditionally associated with cataleptic reactions [10], and serotonin, which is associated with genetic predisposition to catalepsy [19]. Indeed, the predisposition to catalepsy in GC rats and in CBA mice is accompanied by a decrease in brain 5-HT2A receptor density [19], and 5-HT2A receptor density is decreased in the cortex of thyroidectomized rats [14]. Taking these two facts together, one may hypothesize that thyroid hormone deficiency increases the proneness to cataleptic response to aversive stimuli by means of decreasing the brain 5-HT2A receptor density. Thus, analysis of the existing literature as well as of the present experimental results leads to suggestion that thyroid hormone deficiency makes the animal resort to passive (crypting) strategy of coping with stressful environment. Therefore, it may be hypothesized that animals in which genetically determined thyroid hypofunction and passive defense strategy are combined may be candidates for animal models to study the biological mechanisms of the interaction between thyroid dysfunctions and defensive behavior disorders.
Acknowledgments The study was supported by RFFI grant N 99-04-49945.
References [1] Adams A, Stein SA. The effects of congenital hypothyroidism using the hyt/hyt mouse on locomotor activity and learned behavior. Horm Behav 1993;27:418 – 33. [2] Barykina NN, Chepkasov IL, Alekhina TA, Kolpakov VG. Breeding of Wistar rats for predisposition to catalepsy. Genetika 1983;19: 2014 – 21 (in Russian). [3] Dawson RD, Tricklebank DT. Use of elevated plus maze in the search for novel anxiolytic agents. TIPS 1995;16:33 – 6. [4] Dixon AK. Ethological strategies for defence in animals and humans: their role in some psychiatric disorders. Br J Med Psychol 1998;71: 417 – 45. [5] Escobar-Moreale HF, Escobar del Rey F, Moreale de Escobar G. Only the combined treatment with thyroxine and triiodothyronine ensures euthyroidism in all tissues of the thyroidectomized rat. Endocrinology 1996;137:2490 – 502. [6] Fundaro A. Behavioral modifications in relation to hypothyroidism and hyperthyroidism in adult rats. Neuropharmacology 1986;13: 927 – 40. [7] Gordon CJ. Behavioral and autonomic thermoregulation in the rat following propylthiouracil induced hypothyroidism. Pharmacol, Biochem Behav 1997;58:231 – 6. [8] Joffe RT, Levitt AJ. The thyroid and depression. In: Joffe RT, Levitt AJ, editors. The thyroid axis and psychiatric illness. Washington (DC): American Psychiatric Press, 1993. pp. 195 – 253. [9] Kirkegaard C, Faber J. The role of thyroid hormones in depression. Eur J Endocrinol 1998;138:1 – 9. [10] Klemm WR. Drugs effects on active immobility responses: what the
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focus on the thyroid and adrenal system. Br J Psychiatry 1996;168: 123 – 8. Nikulina EM, Popova NK, Kolpakov VG, Alekhina TA. Brain dopaminergic system in rats with a genetic predisposition to catalepsy. Biog Amines 1987;4:399 – 406. Popova N, Kulikov A. On the role of brain serotonin in expression of genetic predisposition to catalepsy in animal models. Am J Med Genet 1995;60:214 – 20. Redei EE, Solberg LC, Kluczynski JM, Pare WP. Paradoxical hormonal and behavioral responses to hypothyroid and hyperthyroid states in the Wistar – Kyoto rats. Neuropsychopharmacology 2000;24:632 – 9. Royce JR. On the construct validity of open-field measures. Psychol Bull 1977;84:1098 – 106. Schalock RL, Brown WJ, Smith RL. Long-term effects of propylthiouracil-induced neonatal hypothyroidism. Psychobiology 1979;12: 187 – 99. Shtilman NI, Alekhina TA, Barykina NN, Kolpakov VG, Shtark MB. Learning ability and protein metabolism in some brain structures of rats genetically predisposed to catalepsy. Izv Sib Otd Akad Nauk SSSR 1988;20:86 – 92 (in Russian). Tamary V, Meisami E, Du JZ, Timiras PS. Exploratory behaviour learning activity and thyroid hormonal responses to stress in female rats rehabilitating from postnatal hypothyroidism. Psychobiology 1986;19:537 – 53.
Effects of thyroid hormone deficiency on behavior in rat strains with different predisposition to catalepsy N.N. Barykinaa, V.F. Chuguya, T.A. Alekhinaa, V.G. Kolpakova, A.V. Maksiutovab, A.V. Kulikovb,* a
Laboratory of Evolutionary Genetics, Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 10 Lavrentiev Avenue, Novosibirsk 630090, Russia b Laboratory of Behavioral Phenogenetics, Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 10 Lavrentiev Avenue, Novosibirsk 630090, Russia Received 18 June 2001; received in revised form 30 November 2001; accepted 28 January 2002
Abstract The effects of thyroidectomy on anxiety-related behavior in the elevated plus-maze test, locomotor activity, and defecation in the openfield test and duration of cataleptic freezing were studied in rats of two strains differing in predisposition to catalepsy: cataleptic strain GC and its ancestor strain Wistar. Total thyroxine level was significantly decreased in control GC rats compared to that in control Wistar rats. Control Wistar and GC rats did not differ either in the percentages of open-arm entries or the time spent therein in the elevated plus-maze test or in defecation score in the open-field test. At the same time, control Wistar rats showed more locomotor activity compared to control GC rats in the open-field test. Thyroid hormone deficiency did not affect the percentages of open-arm entries and the time spent therein in the elevated plus-maze test as well as defecation score in both strains. Thyroidectomy did not alter significantly locomotor activity in Wistar rats, but produced a nearly twofold increase in locomotor activity in GC rats. The most important finding is that thyroidectomy significantly increased the expression of catalepsy in Wistar rats, which points to a role of thyroid hormones in the regulation of predisposition to cataleptic reaction. D 2002 Published by Elsevier Science Inc. Keywords: Catalepsy; Thyroxine; Plus-maze; Open-field; Rat
1. Introduction In spite of numerous clinical data that point to a close link between thyroid dysfunction and mental disorders [8,9,17], the mechanisms of influence of thyroid hormones on brain functions and behavior remain obscure. Avoidance behavior anomalies are considered to be a probable biological basis of depressive disorders [4]. There is experimental evidence that early [22], as well as adult [16], hypothyroidism produce an avoidance behavior deficit. Recently, it was shown that hypothyroid rats were more immobile in the forced swim test compared with controls [15]. Immobility or freezing is a common adaptive defensive strategy of coping with inescapable threats in some mammalian species and birds [4]. It is therefore interesting * Corresponding author. Tel.: +7-3832-34-47-53; fax: +7-3832-33-1278. E-mail address: [email protected] (A.V. Kulikov). 0031-9384/02/$ – see front matter D 2002 Published by Elsevier Science Inc. PII: S 0 0 3 1 - 9 3 8 4 ( 0 2 ) 0 0 6 6 2 - 5
whether a decreased thyroid function is a common feature of predisposition to freezing reactions. A rat strain GC (designated so by the initials of words ‘genetic’ and ‘catalepsy’), was produced from an outbred Wistar stock by breeding for an increased predisposition to freezing with elements of catalepsy in response to stressful stimulation. More than 50% of GC rats maintain for more than 10 s an imposed immobile vertical posture [2]. Besides, GC rats display a lower locomotor and exploratory activity in the open-field [11,23] and an increased immobility in the forced swimming test [18] as compared to Wistar rats. The present study aimed to compare the effects of thyroid hormone deficiency on various behavioral responses to stressful stimuli in GC and Wistar rats. It was planned (1) to compare serum thyroid hormone concentration in GC and Wistar rats and (2) to study the effects of thyroidectomy on behavior under mild aversive stimulation in open-field and plus-maze tests as well as on manifestation of cataleptic freezing reaction in rats of these strains.
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2. Materials and methods
2.3. Statistics
2.1. Animals and surgery
The data were expressed as mean ± S.E.M. and were compared by two-way ANOVA followed by the Bonferroni – Dunn test.
The studies were made on 24 Wistar and 22 GC males at the age of 2 months (at the beginning of the experiment) weighing 200 ± 12 g taken from respective stocks maintained at the Institute of Cytology and Genetics of the Russian Academy of Sciences (Novosibirsk, Russia). In 12 Wistar males and 10 GC males, the thyroid and parathyroid glands were surgically removed. The other 12 Wistar males and 12 GC males were sham-operated and served as controls. The surgery was carried out under sodium pentobarbital anesthesia (35 mg/kg ip). Animals were housed four per cage in 60 40 20-cm cages at a regulated temperature of 22 °C with a 12:12-h light/dark cycle. All thyroidectomized rats were given 0.5% CaCl2 in water. Rats were kept and used strictly in accordance with guidelines of the European Community. The tests started 31 days after surgery. Two days before the tests, the animals were isolated in cages 60 40 20 cm. 2.2. The test for catalepsy To induce catalepsy, the rat was lifted by its forepaws with a stick in a corner of the cage and then the stick was removed and the period (in seconds) during which the animal maintained the forced vertical posture was recorded. Catalepsy was estimated by the period during which the animal maintained the imposed vertical posture and the number of animals that maintained this posture for at least 10 s. The plus-maze test was performed on the day after the test for catalepsy in a device made of Perspex, with two opposite open arms (50 10 cm) and two opposite closed arms of the same size with walls measuring 40 cm in height. The whole device was elevated 50 cm above a white floor and exposed to dim illumination. Each animal was placed in the central square facing the enclosed arm. The number of entries into and time spent in each arm were recorded for 5 min. An arm entry was scored when all four paws were in the arm. The percentages of open-arm entries and the time spent therein related to the total number of entries and time spent in both types of arms were calculated and used as indices of anxiety-related behavior [13]. The open-field test was performed on the day after the plus-maze test in a device (140 70 45 cm) made of Perspex and colored white. The floor, brightly lit (300 lx), was divided into 98 squares (10 10 cm). Each rat was placed in the centre of the arena, tested for 6 min, and the number of crossed squares and the number of fecal boli (defecation score) were recorded and used as indices of locomotor activity and autonomic reactivity, respectively. Total thyroxine concentration (nM) was estimated in serum by a immunoenzymatic technique using the ThyroidIEA-Tyroxine Kit (Peterburg, Russia).
3. Results Total serum thyroxine level was significantly lower in sham-operated GC rats compared to that in sham-operated Wistar rats (47.5 ± 2.3 nM in Wistar vs. 34.4 ± 1.9 nM in GC, P < .001). Thyroidectomy produced about a sevenfold decrease in serum thyroxine level in GC rats (down to 5 ± 2.5 nM, P < .001) and a twofold (down to 25.5 ± 2.8 nM, P < .001) decrease in Wistar rats with respect to that in the sham-operated control (Fig. 1). A significant influence of both genotype [ F(1,39) = 43.8, P < .001] and thyroidectomy [ F(1,39) = 107.3, P < .001] on serum thyroxine concentration was found, but the interaction between these two factors was not significant [ F(1,39) = 2.6, P > .1]. In the plus-maze test sham-operated Wistar and GC rats did not differ significantly in percentages of open-arm entries and time spent therein. Tyroidectomy did not alter these traits either in Wistar or GC rats (Table 1). There were no effects of genotype [ F(1,42) = 0.06, P>.8], thyroidectomy [ F(1,42) = 0.98, P >.3], or interaction of these factors [ F(1,42) = 0.54, P >.4] on the percentage of open-arm entries. No effects of genotype [ F(1,42 = 3.3, P > .05], thyroidectomy [ F(1,42 = 0.32, P > .5], or factors interaction [ F(1,42 = 2.2, P >.1] on the percentage of time spent in open arms were found. Sham-operated rats from both strains did not differ in the defecation score (3.9 ± 0.4 in Wistar vs. 4.6 ± 0.9 in GC). Thyroidectomy did not alter this trait either in Wistar (3.9 ± 0.4) or GC rats (3.0 ± 0.7). There were no effects of genotype [ F(1,42) = 1.6, P > .2], thyroidectomy
Fig. 1. Influence of thyroidectomy on serum thyroxine level in Wistar and GC rats. *** P < .001, thyroidectomized rats vs. respective sham-operated control. ###P < .001, sham-operated Wistar vs. sham-operated GC.
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Table 1 Effect of thyroidectomy on percentages of open-arm entries and time spent therein in the plus-maze test in Wistar and GC rats Strain Treatment
Wistar
GC
Percentage of open-arm entries Sham 18.8 ± 8.7 (n = 12) Thyroidectomized 15.3 ± 6.6 (n = 12)
24.5 ± 9.2 (n = 12) 11.5 ± 4.8 (n = 10)
Percentage of time spent in open arms Sham 30.1 ± 9.5 (n = 12) Thyroidectomized 38.2 ± 11.2 (n = 12)
26.6 ± 8.4 (n = 12) 8.4 ± 3.8 (n = 10)
[ F(1,42) = 0.04, P >.8], or interaction of these factors [ F(1,42) = 1.9, P >.2] on defecation score. Sham-operated GC rats showed significantly less locomotion in the open field than sham-operated Wistar rats did (82.4 ± 17.3 vs. 159.8 ± 28.2, respectively, P < .05) (Fig. 2). No main effects of strain [ F(1,42) = 0.01, P >.9] and thyroidectomy [ F(1,42) = 1.01, P >.32] on locomotor activity in the open-field test was shown. However, there was a significant interaction between the strain (genotype) and thyroidectomy factors in the determination of locomotor activity [ F(1,42) = 12.6, P < .001]. Thyroidectomy did not alter significantly locomotor activity in Wistar rats, but produced a twofold increase of locomotion in GC rats (up to 184.5 ± 24.4, P < .01). Thyroidectomy increased the predisposition to catalepsy in Wistar rats: while only 2 of 12 (16.6%) sham-operated rats displayed catalepsy, such reaction was observed in 7 of 12 of thyroidectomized rats [58.3%, t(22) = 2.2, P < .05]. The duration of cataleptic reaction in thyroidectomized Wistar rats was also increased as compared to sham-operated control (11.58 ± 3.6 vs. 2.3 ± 1.5 s, P < .05) up to the level of shamoperated GC rats (9.9 ± 2.8 s, Fig. 3). Thyroidectomy also produced a nonsignificant increase of cataleptic immobility time in GC (up to 17.1 ± 3.3 s, P >.05, Fig. 3). A significant
Fig. 2. Influence of thyroidectomy on open-field locomotion in Wistar and GC rats. ** P < .01, thyroidectomized rats vs. respective sham-operated control. #P < .05, sham-operated Wistar vs. sham-operated GC.
Fig. 3. Influence of thyroidectomy on the duration of catalepsy in Wistar and GC rats. * P < .05, thyroidectomized rats vs. respective sham-operated control. #P < .05, sham-operated Wistar vs. sham-operated GC.
influence of both genotype [ F(1,42) = 5.16, P < .03] and thyroidectomy [ F(1,42) = 8.09, P < .007], but not of interaction of these two factors [ F(1,42) = 0.14, P >.7] on the duration of catalepsy was found.
4. Discussion Significant interstrain differences in total serum thyroxine level were found. While the thyroxine concentration in control Wistar rats (47.5 nmol/l) was very close to that shown by other authors (40 – 60 nmol/l) [5,20], in control GC rats the hormone concentration was significantly lower (34.4 nmol/l). It may be hypothesized that GC rats have a kind of genetically determined borderline primary hypothyroidism. However, more evidence including serum triiodothyronine and thyroid-stimulating hormone assays are needed to verify this hypothesis. The surgical removal of thyroid gland resulted in a dramatic decrease of thyroxine concentration in rats of both strains. In Wistar rats, thyroidectomy produced a twofold decrease in serum thyroxine level. This result is close to that found by Redei et al. [20]. Although in GC rats thyroidectomy produced a sevenfold decrease in serum thyroxine concentration, we may not assert that GC rats are more vulnerable to thyroidectomy compared with Wistar rats, because ANOVA failed to show a statistically significant interaction between strain and thyroidectomy factors. There were no differences between control Wistar and GC rats in the percentages of open-arm entries and time spent therein. Thyroidectomy also failed to produce any statistically significant alteration of these traits in both strains. It is commonly assumed that the percentages of open-arm entries and the time spent therein are closely correlated with anxiety [3]. Therefore, it appears that both breeding for catalepsy and thyroid hormone deficiency did not affect this manifestation of anxiety-related behavior.
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There were no differences between control Wistar and GC rats in defecation score in the open-field. Thyroidectomy did not change the defecation score either in Wistar or in GC rats. Since defecation is usually considered as an index of autonomic reactivity [21], it suggests that neither breeding for catalepsy nor thyroid hormone deficit has influenced this kind of reaction of the autonomic system to the stressful situation. GC rats with inherited thyroxine deficit are less active in the open-field test compared to Wistar rats, and this finding agrees with earlier published data [11,23]. It has been found that adult mice with genetically determined hypothyroidism also showed a decreased locomotion as compared with their euthyroid counterparts [1]. Thyroidectomy did not alter locomotor activity in the open-field test in Wistar rats, but produced its marked increase in GC rats. It is noteworthy that other authors also found that thyroid hormone deficit did not change [7] or even decreased [6] the locomotor activity in adult rats. At present, we cannot give a definite explanation of this paradoxical increase of locomotor activity in thyroidectomized GC rats. However, one may hypothesize that the unexpected increase in motor activity of cataleptic rats induced by thyroidectomy may reflect an increase in the expression of the hyperkinetic pole of the catatonic syndrome, which has been observed in GC rats [12]. The most important finding was that thyroidectomy increased predisposition to catalepsy and freezing duration in Wistar rats. Earlier, it has been shown that thyroidectomized rats usually have a deficit of performance in avoidance and escape learning [22]. Earlier, we found that hypothyroid rats were more immobile in the forced swim test compared to euthyroid control [15]. Hypothyroid rats needed more unconditioned stimuli (footshocks) to acquire conditioned avoidance [24]. GC rats, which we found to have a thyroxine deficit, showed less active swimming in the Porsolt test [18]. Therefore, taking together the data present in literature and our own results, it may be hypothesized that a deficit in thyroid hormones decreases active avoidance behavior. What is the mechanism of the phenomenon of increase of cataleptic-like immobility found in hypothyroid rats? At present, there is no information sufficient to answer this question. However, a promising idea came from the study of Levine et al. [16] who studied the effect of thyroidectomy in rats on the course of learning to escape a footshock by pressing the lever. They found that while control rats improved their performance, thyroidectomized rats showed a marked decrease in successful responses. Using Markov transition analysis, the authors concluded that in contrast to control rats, thyroidectomized rats were able to learn not to escape. It does not seem too improbable to accept a very attractive idea that thyroid hormones serve as a ‘switch’ between active (flight) and passive (crypting) strategies. Catalepsy is known to be an exaggerated form of passive defense reaction, e.g., crypting [4], and has been shown to be regulated by various neurotransmitters and neuropeptides [10]. Now, thyroid hormones may be added to the list of
compounds able to affect catalepsy. Among the wide range of neurochemical substances that affect the expression of catalepsy, there are two that attract most attention: dopamine, which is traditionally associated with cataleptic reactions [10], and serotonin, which is associated with genetic predisposition to catalepsy [19]. Indeed, the predisposition to catalepsy in GC rats and in CBA mice is accompanied by a decrease in brain 5-HT2A receptor density [19], and 5-HT2A receptor density is decreased in the cortex of thyroidectomized rats [14]. Taking these two facts together, one may hypothesize that thyroid hormone deficiency increases the proneness to cataleptic response to aversive stimuli by means of decreasing the brain 5-HT2A receptor density. Thus, analysis of the existing literature as well as of the present experimental results leads to suggestion that thyroid hormone deficiency makes the animal resort to passive (crypting) strategy of coping with stressful environment. Therefore, it may be hypothesized that animals in which genetically determined thyroid hypofunction and passive defense strategy are combined may be candidates for animal models to study the biological mechanisms of the interaction between thyroid dysfunctions and defensive behavior disorders.
Acknowledgments The study was supported by RFFI grant N 99-04-49945.
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