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Anxiolytic actions of diazepam, but not of buspirone, are influenced by gender and the endocrine stage
Behavioural Brain Research 88 (1997) 213 – 218
Research report
Anxiolytic actions of diazepam, but not of buspirone, are influenced by gender and the endocrine stage A. Ferna´ndez-Guasti a,b,*, O. Picazo c a
Seccio´n de Terape´utica Experimental, Departamento de Farmacologı´a y Toxicologı´a, CINVESTAV, Apartado Postal 22026, Me´xico 14 000 D.F., Me´xico b Instituto Mexicano de Psiquiatrı´a, Me´xico D.F., Me´xico c Escuela Superior de Medicina del Instituto Polite´cnico Nacional, Me´xico D.F., Me´xico Received 6 September 1996; received in revised form 21 February 1997; accepted 24 February 1997
Abstract The effect of diazepam (1.0 mg/kg, ip) and buspirone (5.0 mg/kg, ip) on the burying behaviour latency (denoting actions on the animals’ reactivity) and on the cumulative burying behaviour (directly reflecting the experimental anxiety levels), were analyzed in male-, intact females, at proestrus and metoestrus, and in neonatally-androgenized-rats. Androgenization was performed by injecting 60 mg/rat of testosterone propionate on day 5 after delivery. Two main groups of neonatally-androgenized rats were established: A group of animals showing permanent oestrus from the vaginal opening (acyclic females) and a group presenting the delayed anovulatory syndrome. Diazepam produced a clear reduction in experimental anxiety in males and neonatally-androgenized-females. Particularly important was the anxiolytic effect of diazepam on acyclic females that was accompanied by a significant increase in burying behaviour latency. Conversely, buspirone induced a clear reduction in burying behaviour, without modifying its latency, in all groups regardless of the gender and the neonatal treatment. Data are discussed on the basis of the androgen participation on the anxiolytic drug effects. A possible age-related benzodiazepine actions in females is suggested. © 1997 Elsevier Science B.V. Keywords: Anxiety; Burying behaviour; Diazepam; Buspirone; Gender differences; Neonatal androgenization
1. Introduction It has been demonstrated that the action of various anxiolytic drugs vary depending on the gender. Thus, we have shown that diazepam, at the same dose (1.0 mg/kg) produces anxiolytic-like effect in males but not in females. Conversely, the injection of the serotonergic anxiolytics, buspirone and 8-OH-DPAT reduced burying behaviour (a response interpreted as a reduction in
* Corresponding author. Tel.: + 52 5 130432; fax: + 52 5 130432; e-mail: [email protected] 0166-4328/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 4 3 2 8 ( 9 7 ) 0 0 0 4 7 - 8
anxiety) similarly regardless of the gender [5]. Additionally, Lynda Uphouse and coworkers established that some actions of these same serotonergic agonists are gender dependent and also may vary according to the female oestrous cycle phase [29]. In line with the differential drug effects along the endocrine cycle, variations in the burying behaviour response to diazepam, have been reported [5]. Moreover, we have recently observed blockade of the anxiolytic action of 8-OH-DPAT in lactating rats [8] and others have reported on the absence of the inhibition of 8-OH-DPAT on female sexual response in estrogen pretreated rats [12]. All these data, taken together, suggest that the action of
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benzodiazepines and serotonergic anxiolytics may differ depending both, upon the gender of the neural structures on which they act to produce their behavioural effects, and on the hormonal milieu circumscribing such structures. An additional factor that could modify the anxiolytic action of serotonergic drugs is age. Thus, we have found that in 3 week young male animals no reduction in burying behaviour is found because of presumably a floor-effect, while in relatively old animals ipsapirone and buspirone loose its tranquilizing properties whereas 8OH-DPAT and indorenate still produce their actions [14]. In 1964 Swanson and van der Werff ten Bosch [25] reported that administration of low doses of androgens during the critical period of sexual differentiation, result in a greatly reduced period of fertility. This effect is manifested in two different manners: A large population of females showing persistent vaginal cornification from puberty onwards and a small but very consistent population of females that cease ovulating and exhibited permanent vaginal oestrus in young adulthood. This last phenomenon is referred to as the ‘delayed anovulatory syndrome’ (DAS) [9]. One hypothesis for the permanent effects of exogenous androgen exposure on the neonatal hypothalamus is that select aspects of neural and endocrine age-related changes are accelerated [25]. Thus, androgen-sterilized female rats pass into an endocrine state at the juvenile period (persistent vaginal cornification) that would typically occur during their midlife [30]. Although controversial as an experimental animal model of reproductive senescence [4,13,30], such manipulation has the main advantage of including females with intact components of their physiology for the expression of various behavioural cues independently of alterations in their neuroendocrine functions. Thus, for example, it has been demonstrated that various behaviours, useful for establishing experimental anxiety in laboratory animals such as the defensive burying behaviour gradually declines after a peak present during youth [27,28]. On these bases, the general purpose of the present work was to analyze whether injection of diazepam or buspirone, tested at single effective doses, may cause differential effects in animals in various endocrine states: Normal cycling females (at proestrus or metoestrus), intact males, acyclic females and females with one or two cycles that develop the DAS. These last two groups neonatally received low doses of androgens to produce such a state. The experimental anxiety was determined in the burying behaviour test, a paradigm which robustness has been established under various physiological and pharmacological conditions [6,27,28].
2. Materials and methods.
2.1. Animals and procedure Male and female Wistar rats were used in this study. All animals were kept in a room under inverted and controlled light-dark cycle conditions (lights on at 2200 h). Rats were housed seven per cage with ad libitum access to water and Purina Rat Chow all over the experiments. All male and females were born in our bioterium. The day of birth was designated as day 1 and the sex of the newborn established. On the fifth day females were divided into two main groups: Testosterone-propionate (TP)- and oil-treated-females; the former group received a sc injection of 60 mg/rat of TP dissolved in 0.1 ml of corn oil, the latter received the same volume of the vehicle. Three weeks after injection females were checked for vaginal opening. Once their vagina opened a daily vaginal smear was taken to establish the oestrous cycle phase. All females were registered for their vaginal cytology at least for three consecutive weeks. To control for manipulation effects on the anxiety test, male rats were handled similarly to females for three consecutive weeks and thereafter treated with anxiolytics. The control neonatally-oil-treated females were selected for pharmacological treatment when in proestrus or metoestrus. These phases were recognized according to the vaginal cells present. The neonatally-TP-treated females showed two different vaginal cytology profiles: A major group of females was in permanent oestrus from the vaginal opening onwards (acyclic females), the rest showed one, two or a maximum of three normal 4–5 day cycles followed by persistent oestrus. As a criterium for the present investigation, these females must show at least 9 days of persistent vaginal cornification to be pharmacologically tested. After the anxiety test all females were ovariectomized under pentobarbital anesthesia. The ovaries were carefully dissected and observed under the microscope to establish their general appearance.
2.2. Drugs and steroids Testosterone propionate (TP) was purchased from Sigma Chemical Co., St Louis, MI. Diazepam and buspirone were kind gifts from Hoffman La Roche and Mead Johnson, Me´xico City, Me´xico, respectively. TP was dissolved in corn oil and injected sc in 0.1 ml to 5 day old females. Diazepam (0 and 1.0 mg/kg) was dissolved in propylene glycol 40% and injected ip to adult animals 30 min before the tests. Buspirone (0 and 5 mg/kg) was dissolved in saline and injected ip with a latency of 20 min. Proper control groups were injected with vehicle. These doses were selected on the basis of previous studies [5].
A. Ferna´ndez-Guasti, O. Picazo / Beha6ioural Brain Research 88 (1997) 213–218
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Fig. 1. Effect of diazepam (1.0 mg/kg) and buspirone (5.0 mg/kg) on the cumulative burying behaviour of proestrus- (P), metoestrus- (M), delayed anovulatory- (DAS), acyclic- (AC) female and male (MA) rats. Figure shows mean 9S.E. expressed in seconds. The number of animals per group ranged between 7 and 12. Mann Whitney U test, *P B0.05; **PB0.02; ***PB0.01.
2.3. Anxiety test
2.4. Statistics
Males, neonatally oil-treated females, and androgenized-females, all weighing (250 – 300 g) and in groups ranging from 7–12 were tested for burying behaviour. The burying behaviour test has been previously described in detail and validated as a useful and selective model for establishing animal’s experimental anxiety [5 – 7,14,18,19,27,28]. This paradigm consists of an acrylic cage (27x16x23 cm) with a prod (7 cm long) emerging from one of its walls (2 cm above the bedding material) and the floor covered with fine sawdust. Through the prod the animal receives an electric shock of 0.3 mA. The electric source consisted of a constant current shocker (LaFayette Instruments Co., model 5806). During the test the animal was placed in the cage and its behaviour recorded during a 10 min period. Each time the animal touched the prod it received an electric shock and typically displayed the burying behaviour that consists in a series of rapid and alternating movements of its forepaws, moving and pushing a pile of bedding material over the aversive stimulus. Two main parameters were registered: The burying behaviour latency (time between the first shock and the burying behaviour display), and the cumulative burying behaviour (cumulative time that the animal spends burying the prod during a 10 min test). It has been considered that burying behaviour latency inversely reflects the animals’ reactivity, while the cumulative burying behaviour directly denotes the experimental anxiety levels [6,14,19,27]
The statistical analysis for the burying behaviour latency and the cumulative burying behaviour was performed by the two-way analysis of variance followed by the Mann-Whitney U test. For other comparisons the Student’s t-test was employed [22,23].
3. Results Regarding some parameters of the general physiology of the females included in this study it is interesting to mention that virilized females showed a retarded vaginal opening when compared with control females (mean age (days) 9 S.E.: control, 35.9 9 0.3 versus androgenized, 40.59 0.2, PB 0.001). Additionally, these two main groups showed an important difference between their ovaries; thus, all neonatally oil-treated females showed normal ovaries with several corpora lutea, while all those treated neonatally with TP present very small atrophic polifollicular ovaries. Finally, all non treated females present normal 4–5 day cycles while all virilized females showed persistent vaginal cornification. However, within this group two main populations were found: A small proportion 24.3% (28 out of 115) of females showing the delayed anovulatory syndrome and 75.6% (87 out of 115) of females showing permanent oestrus from the vaginal opening. The effect of the anxiolytic drugs on the cumulative burying behaviour of females in various endocrine stages and males is shown in Fig. 1. Regarding the
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Fig. 2. Effect of diazepam (1.0 mg/kg) and buspirone (5.0 mg/kg) on the burying behaviour latency of proestrus- (P), metoestrus- (M), delayed anovulatory- (DAS), acyclic- (AC) female and male (MA) rats. Figure shows mean 9 S.E. expressed in seconds. The number of animals per group ranged between 7 and 12. Mann Whitney U test, **PB 0.02; ***PB 0.01.
control condition, it is worth mentioning that all females (independently of their endocrine status) showed very similar values of burying behaviour, except of those in proestrus, such females, as previously reported [5,6], exhibited lower levels (statistically different from females in metoestrus and from the DAS group, PB 0.05 for both). The results from the two way ANOVA for the main effects of endocrine stage and anxiolytic drug on cumulative burying behaviour were: F =3.93, P B 0.01 and F = 38.49, P B0.01, respectively and F = 3.50, P B0.01 for endocrine stage X anxiolytic drug interaction d.f.(4,215). Fig. 1 shows that the administration of diazepam (1.0 mg/kg) resulted in differential effects according to the gender and the endocrine status. Thus, such treatment produced no effect in proestrus- or metoestrus-females, a clear statistical significant decrease in burying behaviour in males and DAS-females. Interestingly, in acyclic females a very drastic reduction in this parameter was observed after diazepam injection. Conversely, buspirone (5.0 mg/kg) produces similar anxiolytic responses regardless of the endocrine state or the gender. As forementioned, the burying behaviour latency is proposed to inversely reflect the animals’ reactivity. The effect of anxiolytic drugs on the burying behaviour latency of females in various endocrine stages and males is shown in Fig. 2. The results from the two way ANOVA for the main effects of endocrine stage and anxiolytic drug on burying behaviour latency were as follows: F =4.74, PB 0.01 and F = 22.51, P B 0.01,
respectively and F= 5.60, PB 0.01 for endocrine stage X anxiolytic drug interaction d.f.(4,215). Clearly, diazepam (1.0 mg/kg) produced an increase in this parameter in males and in acyclic females, in this last group this benzodiazepine produced the highest prolongation of the latency. Such effect resulted statistically different (PB 0.01) as compared with all other groups. Buspirone (5.0 mg/kg), by contrast, did not affect the latency in any group.
4. Discussion The main findings of the present investigation can be summarized as follows: 1. The basal burying behaviour levels between metoestrus-, acyclic- and females showing the delayed anovulatory syndrome were very similar. However, males and females in proestrus, showed lower values of this behaviour. 2. Diazepam (1.0 mg/kg) produced clear reduction in burying behaviour in males and females showing the delayed anovulatory syndrome. A much drastic reduction, after this treatment, was observed in acyclic females than in the other groups. 3. An increase in burying behavior latency after diazepam (1.0 mg/kg) was found in males and acyclic females. 4. Buspirone (5.0 mg/kg) induced a clear and analogous reduction in burying behaviour in all groups tested, without modifying the burying behaviour latency.
A. Ferna´ndez-Guasti, O. Picazo / Beha6ioural Brain Research 88 (1997) 213–218
In a previous publication we showed gender differences in the cumulative burying behaviour (considered as a reduction in experimental anxiety) [5]. Thus, as here shown, males presented lower levels of burying behaviour than females in metoestrus and dioestrus. These gender differences have been reported for other behaviours and seem to be either dependent or independent of the presence of testosterone in the neonatal period of sexual differentiation. As examples of the former, it has been shown that males had slower extinction rates than females in a conditioned taste aversion test [21], a paradigm proposed for studying anxiety levels [27]. In this test, females exposed to testosterone during the perinatal period showed prolonged extinction when adults [21]. By contrast with these data, the nature of burying behaviour does not seems to depend upon the presence of androgens in the neonatal period then being gender specific. According with this idea, neonatally-androgenized-females, regardless of the presence of vaginal cycles during adulthood, showed burying behaviour levels similar to those displayed by intact females in metoestrus or ovariectomized rats [6]. Recently, we have reported that in late proestrus females [6] and in 14th-day-pregnant rats [18] a clear reduction in burying behaviour was observed as compared with ovariectomized- or metoestrus-dioestrus-animals. Such reduction most likely is due to the presence of high levels of progesterone or its metabolites with anxiolytic activity during these particular phases [1,2,6,19]. Interestingly, in this line of thought we failed to observe a reduction in this behaviour after allopregnanolone (5-a, 3-adihydroprogesterone) in neonatallyandrogen-treated as compared with normal-females [7]. A similar result was published by Rodrı´guez Sierra, et al. [20] who showed that in neonatally-androgen-treated female rats the anxiolytic effect of chlordiazepoxide, but not of progesterone, was evident. These data, together with present results show a clear action of diazepam and buspirone in neonatally-androgen-treated-females, indicating that virilized females are able to display anxiolytic responses elicited by benzodiazepines or serotonergic agonists, but not by steroid hormones. These results suggest an insensitivity of virilized females to progesterone or its derivatives. Further experiments should be performed to fully confirm this proposition. It has been previously shown that the behavioural action of drugs may differ generically [5,29] and between cycling- and androgenized-females. Regarding these last groups, Molina, et al. [17] recently reported that blockade of opiate receptors with naloxone decreased the sensitivity to a noxious stimuli in males and androgenized females but not in normal females. Results from the present report also show that diazepam, at the dose used, produced a clear reduction in burying behaviour in males and neonatally-androgen-treated-females and lacked such action in neonatally non-treated females.
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It is at present considered that the anxiolytic action of benzodiazepines is mediated after the stimulation of a binding site coupled to the GABAA receptor. Interestingly, Szwarcfarb, et al. [26] reported that the administration of aminooxyacetic acid, that increases hypothalamic GABA concentrations, induced a significant increase in LH levels in female rats, which was completely abolished by neonatal androgenization. Additionally, McCarthy and coworkers [3,16] hypothesized that during development, hormone dependent alterations of the GABAergic system contribute to the establishment of sex specific phenotypes in the adult rat brain. In their study is revealed a significant sex difference in mRNA content for the a1 and 2 subunits of the GABAA receptor that at least partially may explain the gender differences in the effects of benzodiazepine anxiolytics. Furthermore, they found an important decreased density of this mRNA in the amygdala accompanied by a significant increased density in the preoptic area of androgenized females that was not present neither in normal females nor in males. These data suggest that neonatally androgenized females, may differentially express the quantity of the a1 and 2 subunits of the GABAA receptor possibly involved in the anxiolytic actions of benzodiazepines in adulthood. Remarkably, buspirone produced the same reduction in burying behaviour regardless of the gender [5] and the neonatal hormonal exposure. In line with these results, Hockl, et al. [11] demonstrated that a single injection of buspirone increased serum prolactin (PRL) levels in both sexes and that neonatal androgenization of females or orchidectomy of males failed to modify the PRL-releasing action of buspirone. Accordingly, clinical data reveal no gender differences in the anxiolytic action of buspirone [10]. These data would suggest that the anxiolytic action of drugs acting on the 5-HT1A receptor are gender- and neonatally-hormoneindependent. The differential effects of anxiolytics in females under various hormonal conditions might be of interest since these stages may reflect certain physiological or pathological conditions. Thus, it has been proposed that neonatal androgenization could produce either the delayed anovulatory syndrome [25], that in turn could be used as an animal model of reproductive senescence [4,13], or permanent anoestrus, that has been proposed as a paradigm for studying polycystic ovarian diseases [15]. To our knowledge, little is known on the antianxiety drug effects in old subjects. Some clinical data, however, reveal that benzodiazepines are commonly used in old patients, particularly women [24], even its use is specifically counterindicated since they may produce adverse effects, such as cognitive impairment and sedation [31], which might be related with the prolongation of the burying behaviour latency in acyclic rats.
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In summary, this paper shows important differences in the anxiolytic properties of diazepam between males, females and neonatally-androgenized-females. Conversely, buspirone, produced similar actions in all groups. The possible role of hormones during the neonatal period of sexual differentiation in the anxiolytic drug action should be addressed.
Acknowledgements Authors wish to thank Mr Vı´ctor Flores Montoya for animal caring and Mr Facundo Moreno for technical assistance. Present investigation was partially supported by a grant (No. 2338P) to A.F.-G. from the ‘Consejo Nacional de Ciencia y Tecnologı´a’.
References [1] D. Bitran, R.J. Hilvers, C.K. Kellogg, Anxiolytic effects of 3a-hydroxy-5a[ß]-pregnan-20-one: Endogenous metabolites of progesterone that are active at the GABAA receptor, Brain Res. 561 (1991) 157 – 161. [2] D. Bitran, M. Shiekh, M. McLeod, Anxiolytic effect of progesterone is mediated by the neurosteroid allopregnanolone at brain GABA(A) receptors, J. Neuroendocrinol. 7 (1995) 171–177. [3] A.M. Davis, M.M. McCarthy, Sex differences in GABAA receptor subunit mRNA levels in neonatal rat brain, 28th. Ann. Conf. Reprod. Behav., Concordia University, Montre´al, Canada, 1996, pp. 33. [4] A. Farrell, A.A. Gerall, M.J. Alexander, Age-related decline in receptivity in normal, neonatally androgenized female and male hamsters, Exp. Aging Res. 3 (1977) 117–128. [5] A. Ferna´ndez-Guasti, O. Picazo, The actions of diazepam and serotonergic anxiolytics vary according to the gender and the oestrous cycle phase, Pharmacol. Biochem. Behav. 37 (1990) 77 – 81. [6] A. Ferna´ndez-Guasti, O. Picazo, Changes in burying behavior during the estrous cycle: effect of estrogen and progesterone, Psychoneuroendocrinology 17 (1992) 681–689. [7] A. Ferna´ndez-Guasti, O. Picazo, Differential allopregnanolone anxiolytic actions in males, females and neonatally androgenized females, Horm. Behav., Submitted. [8] A. Ferna´ndez-Guasti, O. Picazo, A. Ferreira, Blockade of the anxiolytic action of 8-OH-DPAT in lactating rats, Pharmacol. Biochem. Behav., in press. [9] R.A. Gorski, The neuroendocrinology of reproduction, Biol. Reprod. 20 (1979) 111–127. [10] D.E. Hilleman, S.M. Mohiuddin, M.G. Delcore, Comparison of fixed-dose transdermal nicotine, tapered-dose transdermal nicotine, and buspirone in smoking cessation, J. Clin. Pharmacol. 34 (1994) 222 – 224. [11] P.F. Hockl, G.S. Dı´az, C. Libertun, Prolactin- releasing effect of buspirone in developing and adult male and female rats, Proc. Soc. Exp. Biol. Med. 202 (1993) 447–450. [12] A. Jackson, L. Uphouse, Prior treatment with estrogen attenuates the effect of the 5-HT1A agonist, 8-OH-DPAT, on lordosis behavior, Horm. Behav. 30 (1996) 145–152.
.
[13] J.R. Lehman, D.A. McArthur, S.E. Hendricks, Pharmacological induction of ovulation in old and neonatally androgenized rats, Exp. Gerontol. 13 (1978) 107 – 114. [14] C. Lo´pez-Rubalcava, A. Ferna´ndez-Guasti, R. Urba´-Holmgren, Age-dependent differences in the rat’s conditioned defensive burying behavior: Effect of 5-HT1A compounds, Dev. Psychobiol. 29 (1996) 157 – 169. [15] D.K. Mahajan, Polycystic ovarian disease: Animal models, Endocrinol. Metab. Clin. North. Am. 17 (1988) 705 – 732. [16] M.M. McCarthy, A.M. Davis, H.R. Besmer, J.A. Mong, R.L. Kurzweil, Mechanisms of sexual differentiation of the brain: does transient excitation mediate masculinization? 28th. Ann. Conf. Reprod. Behav., Concordia University, Montre´al, Canada, 1996 p. 16. [17] N. Molina, M.T. Bedran de Castro, J.C. Bedran de Castro, Sex-related differences in the analgesic response to the rat tail immersion test, Braz. J. Med. Biol. Res. 27 (1994) 1669–1672. [18] O. Picazo, A. Ferna´ndez-Guasti, Changes in experimental anxiety during pregnancy and lactation, Physiol. Behav. 54 (1993) 295 – 299. [19] O. Picazo, A. Ferna´ndez-Guasti, Antianxiety effects of progesterone and some of its reduced metabolites: an evaluation using the burying behavior test, Brain Res. 680 (1995) 135 – 141. [20] J.F. Rodrı´guez-Sierra, M.T. Hagley, S.E. Hendricks, Anxiolytic effects of progesterone are sexually dimorphic, Life Sci. 38 (1986) 1841 – 1845. [21] C.B. Sengstake, K.C. Chambers, Sensitivity of male, female, and androgenized female rats to testosterone during extinction of a conditioned taste aversion, Behav. Neurosci. 105 (1991) 120– 125. [22] S. Siegel, Nonparametric Statistics for the Behavioral Sciences, McGraw Hill, New York, 1956. [23] R.G.D. Steel, J.H. Torrie, Principles and Procedures of Statistics. A Biometrical Approach, McGraw Hill, New York, 1985. [24] R.B. Stewart, R.G. Marks, P.D. Padgett, W.E. Hale, Benzodiazepine use in an ambulatory elderly population: A 14-year overview, Clin. Ther. 16 (1994) 118 – 124. [25] H.E. Swanson, J.J. van der Werff ten Bosch, The ‘early-androgen’ syndrome; differences in response to pre-natal and post-natal administration of various doses of testosterone propionate in female and male rats, Acta Endocrinologica 47 (1964) 37–50. [26] B. Szwarcfarb, S. Carbone, M.L. Stein, J. Medina, J.A. Moguilevsky, Sexual differences in the effect of the GABAergic system on LH secretion and in the hypothalamic ontogenesis of GABAA receptors in prepubertal rats, Brain Res. 646 (1994) 351 – 355. [27] D. Treit, J.P.J. Pinel, H.C. Fibiger, Conditioned defensive burying: A new paradigm for the study of anxiolytic agents, Pharmacol. Biochem. Behav. 15 (1981) 619 – 626. [28] D. Treit, Animal models for the study of anti-anxiety agents: A review, Neurosc. Behav. Rev. 9 (1985) 203 – 222. [29] L. Uphouse, S. Salamanca, M. Caldarola-Pastuszka, Gender and estrous cycle differences in the response to the 5-HT1A agonist 8-OH-DPAT, Pharmacol. Biochem. Behav. 40 (1991) 901–906. [30] F.S. vom Saal, C.E. Finch, Reproductive senescence: phenomena and mechanisms in mammals and selected vertebrates, in: E. Knobil, J. Neill et al. (Eds.), The Physiology of Reproduction, Raven Press, New York, 1988. [31] S.M. Willcox, D.U. Himmelstein, S. Woolhandler, Inappropriate drug prescribing for the community-dwelling elderly, JAMA 272 (1994) 292 – 296.
Research report
Anxiolytic actions of diazepam, but not of buspirone, are influenced by gender and the endocrine stage A. Ferna´ndez-Guasti a,b,*, O. Picazo c a
Seccio´n de Terape´utica Experimental, Departamento de Farmacologı´a y Toxicologı´a, CINVESTAV, Apartado Postal 22026, Me´xico 14 000 D.F., Me´xico b Instituto Mexicano de Psiquiatrı´a, Me´xico D.F., Me´xico c Escuela Superior de Medicina del Instituto Polite´cnico Nacional, Me´xico D.F., Me´xico Received 6 September 1996; received in revised form 21 February 1997; accepted 24 February 1997
Abstract The effect of diazepam (1.0 mg/kg, ip) and buspirone (5.0 mg/kg, ip) on the burying behaviour latency (denoting actions on the animals’ reactivity) and on the cumulative burying behaviour (directly reflecting the experimental anxiety levels), were analyzed in male-, intact females, at proestrus and metoestrus, and in neonatally-androgenized-rats. Androgenization was performed by injecting 60 mg/rat of testosterone propionate on day 5 after delivery. Two main groups of neonatally-androgenized rats were established: A group of animals showing permanent oestrus from the vaginal opening (acyclic females) and a group presenting the delayed anovulatory syndrome. Diazepam produced a clear reduction in experimental anxiety in males and neonatally-androgenized-females. Particularly important was the anxiolytic effect of diazepam on acyclic females that was accompanied by a significant increase in burying behaviour latency. Conversely, buspirone induced a clear reduction in burying behaviour, without modifying its latency, in all groups regardless of the gender and the neonatal treatment. Data are discussed on the basis of the androgen participation on the anxiolytic drug effects. A possible age-related benzodiazepine actions in females is suggested. © 1997 Elsevier Science B.V. Keywords: Anxiety; Burying behaviour; Diazepam; Buspirone; Gender differences; Neonatal androgenization
1. Introduction It has been demonstrated that the action of various anxiolytic drugs vary depending on the gender. Thus, we have shown that diazepam, at the same dose (1.0 mg/kg) produces anxiolytic-like effect in males but not in females. Conversely, the injection of the serotonergic anxiolytics, buspirone and 8-OH-DPAT reduced burying behaviour (a response interpreted as a reduction in
* Corresponding author. Tel.: + 52 5 130432; fax: + 52 5 130432; e-mail: [email protected] 0166-4328/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 4 3 2 8 ( 9 7 ) 0 0 0 4 7 - 8
anxiety) similarly regardless of the gender [5]. Additionally, Lynda Uphouse and coworkers established that some actions of these same serotonergic agonists are gender dependent and also may vary according to the female oestrous cycle phase [29]. In line with the differential drug effects along the endocrine cycle, variations in the burying behaviour response to diazepam, have been reported [5]. Moreover, we have recently observed blockade of the anxiolytic action of 8-OH-DPAT in lactating rats [8] and others have reported on the absence of the inhibition of 8-OH-DPAT on female sexual response in estrogen pretreated rats [12]. All these data, taken together, suggest that the action of
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benzodiazepines and serotonergic anxiolytics may differ depending both, upon the gender of the neural structures on which they act to produce their behavioural effects, and on the hormonal milieu circumscribing such structures. An additional factor that could modify the anxiolytic action of serotonergic drugs is age. Thus, we have found that in 3 week young male animals no reduction in burying behaviour is found because of presumably a floor-effect, while in relatively old animals ipsapirone and buspirone loose its tranquilizing properties whereas 8OH-DPAT and indorenate still produce their actions [14]. In 1964 Swanson and van der Werff ten Bosch [25] reported that administration of low doses of androgens during the critical period of sexual differentiation, result in a greatly reduced period of fertility. This effect is manifested in two different manners: A large population of females showing persistent vaginal cornification from puberty onwards and a small but very consistent population of females that cease ovulating and exhibited permanent vaginal oestrus in young adulthood. This last phenomenon is referred to as the ‘delayed anovulatory syndrome’ (DAS) [9]. One hypothesis for the permanent effects of exogenous androgen exposure on the neonatal hypothalamus is that select aspects of neural and endocrine age-related changes are accelerated [25]. Thus, androgen-sterilized female rats pass into an endocrine state at the juvenile period (persistent vaginal cornification) that would typically occur during their midlife [30]. Although controversial as an experimental animal model of reproductive senescence [4,13,30], such manipulation has the main advantage of including females with intact components of their physiology for the expression of various behavioural cues independently of alterations in their neuroendocrine functions. Thus, for example, it has been demonstrated that various behaviours, useful for establishing experimental anxiety in laboratory animals such as the defensive burying behaviour gradually declines after a peak present during youth [27,28]. On these bases, the general purpose of the present work was to analyze whether injection of diazepam or buspirone, tested at single effective doses, may cause differential effects in animals in various endocrine states: Normal cycling females (at proestrus or metoestrus), intact males, acyclic females and females with one or two cycles that develop the DAS. These last two groups neonatally received low doses of androgens to produce such a state. The experimental anxiety was determined in the burying behaviour test, a paradigm which robustness has been established under various physiological and pharmacological conditions [6,27,28].
2. Materials and methods.
2.1. Animals and procedure Male and female Wistar rats were used in this study. All animals were kept in a room under inverted and controlled light-dark cycle conditions (lights on at 2200 h). Rats were housed seven per cage with ad libitum access to water and Purina Rat Chow all over the experiments. All male and females were born in our bioterium. The day of birth was designated as day 1 and the sex of the newborn established. On the fifth day females were divided into two main groups: Testosterone-propionate (TP)- and oil-treated-females; the former group received a sc injection of 60 mg/rat of TP dissolved in 0.1 ml of corn oil, the latter received the same volume of the vehicle. Three weeks after injection females were checked for vaginal opening. Once their vagina opened a daily vaginal smear was taken to establish the oestrous cycle phase. All females were registered for their vaginal cytology at least for three consecutive weeks. To control for manipulation effects on the anxiety test, male rats were handled similarly to females for three consecutive weeks and thereafter treated with anxiolytics. The control neonatally-oil-treated females were selected for pharmacological treatment when in proestrus or metoestrus. These phases were recognized according to the vaginal cells present. The neonatally-TP-treated females showed two different vaginal cytology profiles: A major group of females was in permanent oestrus from the vaginal opening onwards (acyclic females), the rest showed one, two or a maximum of three normal 4–5 day cycles followed by persistent oestrus. As a criterium for the present investigation, these females must show at least 9 days of persistent vaginal cornification to be pharmacologically tested. After the anxiety test all females were ovariectomized under pentobarbital anesthesia. The ovaries were carefully dissected and observed under the microscope to establish their general appearance.
2.2. Drugs and steroids Testosterone propionate (TP) was purchased from Sigma Chemical Co., St Louis, MI. Diazepam and buspirone were kind gifts from Hoffman La Roche and Mead Johnson, Me´xico City, Me´xico, respectively. TP was dissolved in corn oil and injected sc in 0.1 ml to 5 day old females. Diazepam (0 and 1.0 mg/kg) was dissolved in propylene glycol 40% and injected ip to adult animals 30 min before the tests. Buspirone (0 and 5 mg/kg) was dissolved in saline and injected ip with a latency of 20 min. Proper control groups were injected with vehicle. These doses were selected on the basis of previous studies [5].
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Fig. 1. Effect of diazepam (1.0 mg/kg) and buspirone (5.0 mg/kg) on the cumulative burying behaviour of proestrus- (P), metoestrus- (M), delayed anovulatory- (DAS), acyclic- (AC) female and male (MA) rats. Figure shows mean 9S.E. expressed in seconds. The number of animals per group ranged between 7 and 12. Mann Whitney U test, *P B0.05; **PB0.02; ***PB0.01.
2.3. Anxiety test
2.4. Statistics
Males, neonatally oil-treated females, and androgenized-females, all weighing (250 – 300 g) and in groups ranging from 7–12 were tested for burying behaviour. The burying behaviour test has been previously described in detail and validated as a useful and selective model for establishing animal’s experimental anxiety [5 – 7,14,18,19,27,28]. This paradigm consists of an acrylic cage (27x16x23 cm) with a prod (7 cm long) emerging from one of its walls (2 cm above the bedding material) and the floor covered with fine sawdust. Through the prod the animal receives an electric shock of 0.3 mA. The electric source consisted of a constant current shocker (LaFayette Instruments Co., model 5806). During the test the animal was placed in the cage and its behaviour recorded during a 10 min period. Each time the animal touched the prod it received an electric shock and typically displayed the burying behaviour that consists in a series of rapid and alternating movements of its forepaws, moving and pushing a pile of bedding material over the aversive stimulus. Two main parameters were registered: The burying behaviour latency (time between the first shock and the burying behaviour display), and the cumulative burying behaviour (cumulative time that the animal spends burying the prod during a 10 min test). It has been considered that burying behaviour latency inversely reflects the animals’ reactivity, while the cumulative burying behaviour directly denotes the experimental anxiety levels [6,14,19,27]
The statistical analysis for the burying behaviour latency and the cumulative burying behaviour was performed by the two-way analysis of variance followed by the Mann-Whitney U test. For other comparisons the Student’s t-test was employed [22,23].
3. Results Regarding some parameters of the general physiology of the females included in this study it is interesting to mention that virilized females showed a retarded vaginal opening when compared with control females (mean age (days) 9 S.E.: control, 35.9 9 0.3 versus androgenized, 40.59 0.2, PB 0.001). Additionally, these two main groups showed an important difference between their ovaries; thus, all neonatally oil-treated females showed normal ovaries with several corpora lutea, while all those treated neonatally with TP present very small atrophic polifollicular ovaries. Finally, all non treated females present normal 4–5 day cycles while all virilized females showed persistent vaginal cornification. However, within this group two main populations were found: A small proportion 24.3% (28 out of 115) of females showing the delayed anovulatory syndrome and 75.6% (87 out of 115) of females showing permanent oestrus from the vaginal opening. The effect of the anxiolytic drugs on the cumulative burying behaviour of females in various endocrine stages and males is shown in Fig. 1. Regarding the
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Fig. 2. Effect of diazepam (1.0 mg/kg) and buspirone (5.0 mg/kg) on the burying behaviour latency of proestrus- (P), metoestrus- (M), delayed anovulatory- (DAS), acyclic- (AC) female and male (MA) rats. Figure shows mean 9 S.E. expressed in seconds. The number of animals per group ranged between 7 and 12. Mann Whitney U test, **PB 0.02; ***PB 0.01.
control condition, it is worth mentioning that all females (independently of their endocrine status) showed very similar values of burying behaviour, except of those in proestrus, such females, as previously reported [5,6], exhibited lower levels (statistically different from females in metoestrus and from the DAS group, PB 0.05 for both). The results from the two way ANOVA for the main effects of endocrine stage and anxiolytic drug on cumulative burying behaviour were: F =3.93, P B 0.01 and F = 38.49, P B0.01, respectively and F = 3.50, P B0.01 for endocrine stage X anxiolytic drug interaction d.f.(4,215). Fig. 1 shows that the administration of diazepam (1.0 mg/kg) resulted in differential effects according to the gender and the endocrine status. Thus, such treatment produced no effect in proestrus- or metoestrus-females, a clear statistical significant decrease in burying behaviour in males and DAS-females. Interestingly, in acyclic females a very drastic reduction in this parameter was observed after diazepam injection. Conversely, buspirone (5.0 mg/kg) produces similar anxiolytic responses regardless of the endocrine state or the gender. As forementioned, the burying behaviour latency is proposed to inversely reflect the animals’ reactivity. The effect of anxiolytic drugs on the burying behaviour latency of females in various endocrine stages and males is shown in Fig. 2. The results from the two way ANOVA for the main effects of endocrine stage and anxiolytic drug on burying behaviour latency were as follows: F =4.74, PB 0.01 and F = 22.51, P B 0.01,
respectively and F= 5.60, PB 0.01 for endocrine stage X anxiolytic drug interaction d.f.(4,215). Clearly, diazepam (1.0 mg/kg) produced an increase in this parameter in males and in acyclic females, in this last group this benzodiazepine produced the highest prolongation of the latency. Such effect resulted statistically different (PB 0.01) as compared with all other groups. Buspirone (5.0 mg/kg), by contrast, did not affect the latency in any group.
4. Discussion The main findings of the present investigation can be summarized as follows: 1. The basal burying behaviour levels between metoestrus-, acyclic- and females showing the delayed anovulatory syndrome were very similar. However, males and females in proestrus, showed lower values of this behaviour. 2. Diazepam (1.0 mg/kg) produced clear reduction in burying behaviour in males and females showing the delayed anovulatory syndrome. A much drastic reduction, after this treatment, was observed in acyclic females than in the other groups. 3. An increase in burying behavior latency after diazepam (1.0 mg/kg) was found in males and acyclic females. 4. Buspirone (5.0 mg/kg) induced a clear and analogous reduction in burying behaviour in all groups tested, without modifying the burying behaviour latency.
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In a previous publication we showed gender differences in the cumulative burying behaviour (considered as a reduction in experimental anxiety) [5]. Thus, as here shown, males presented lower levels of burying behaviour than females in metoestrus and dioestrus. These gender differences have been reported for other behaviours and seem to be either dependent or independent of the presence of testosterone in the neonatal period of sexual differentiation. As examples of the former, it has been shown that males had slower extinction rates than females in a conditioned taste aversion test [21], a paradigm proposed for studying anxiety levels [27]. In this test, females exposed to testosterone during the perinatal period showed prolonged extinction when adults [21]. By contrast with these data, the nature of burying behaviour does not seems to depend upon the presence of androgens in the neonatal period then being gender specific. According with this idea, neonatally-androgenized-females, regardless of the presence of vaginal cycles during adulthood, showed burying behaviour levels similar to those displayed by intact females in metoestrus or ovariectomized rats [6]. Recently, we have reported that in late proestrus females [6] and in 14th-day-pregnant rats [18] a clear reduction in burying behaviour was observed as compared with ovariectomized- or metoestrus-dioestrus-animals. Such reduction most likely is due to the presence of high levels of progesterone or its metabolites with anxiolytic activity during these particular phases [1,2,6,19]. Interestingly, in this line of thought we failed to observe a reduction in this behaviour after allopregnanolone (5-a, 3-adihydroprogesterone) in neonatallyandrogen-treated as compared with normal-females [7]. A similar result was published by Rodrı´guez Sierra, et al. [20] who showed that in neonatally-androgen-treated female rats the anxiolytic effect of chlordiazepoxide, but not of progesterone, was evident. These data, together with present results show a clear action of diazepam and buspirone in neonatally-androgen-treated-females, indicating that virilized females are able to display anxiolytic responses elicited by benzodiazepines or serotonergic agonists, but not by steroid hormones. These results suggest an insensitivity of virilized females to progesterone or its derivatives. Further experiments should be performed to fully confirm this proposition. It has been previously shown that the behavioural action of drugs may differ generically [5,29] and between cycling- and androgenized-females. Regarding these last groups, Molina, et al. [17] recently reported that blockade of opiate receptors with naloxone decreased the sensitivity to a noxious stimuli in males and androgenized females but not in normal females. Results from the present report also show that diazepam, at the dose used, produced a clear reduction in burying behaviour in males and neonatally-androgen-treated-females and lacked such action in neonatally non-treated females.
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It is at present considered that the anxiolytic action of benzodiazepines is mediated after the stimulation of a binding site coupled to the GABAA receptor. Interestingly, Szwarcfarb, et al. [26] reported that the administration of aminooxyacetic acid, that increases hypothalamic GABA concentrations, induced a significant increase in LH levels in female rats, which was completely abolished by neonatal androgenization. Additionally, McCarthy and coworkers [3,16] hypothesized that during development, hormone dependent alterations of the GABAergic system contribute to the establishment of sex specific phenotypes in the adult rat brain. In their study is revealed a significant sex difference in mRNA content for the a1 and 2 subunits of the GABAA receptor that at least partially may explain the gender differences in the effects of benzodiazepine anxiolytics. Furthermore, they found an important decreased density of this mRNA in the amygdala accompanied by a significant increased density in the preoptic area of androgenized females that was not present neither in normal females nor in males. These data suggest that neonatally androgenized females, may differentially express the quantity of the a1 and 2 subunits of the GABAA receptor possibly involved in the anxiolytic actions of benzodiazepines in adulthood. Remarkably, buspirone produced the same reduction in burying behaviour regardless of the gender [5] and the neonatal hormonal exposure. In line with these results, Hockl, et al. [11] demonstrated that a single injection of buspirone increased serum prolactin (PRL) levels in both sexes and that neonatal androgenization of females or orchidectomy of males failed to modify the PRL-releasing action of buspirone. Accordingly, clinical data reveal no gender differences in the anxiolytic action of buspirone [10]. These data would suggest that the anxiolytic action of drugs acting on the 5-HT1A receptor are gender- and neonatally-hormoneindependent. The differential effects of anxiolytics in females under various hormonal conditions might be of interest since these stages may reflect certain physiological or pathological conditions. Thus, it has been proposed that neonatal androgenization could produce either the delayed anovulatory syndrome [25], that in turn could be used as an animal model of reproductive senescence [4,13], or permanent anoestrus, that has been proposed as a paradigm for studying polycystic ovarian diseases [15]. To our knowledge, little is known on the antianxiety drug effects in old subjects. Some clinical data, however, reveal that benzodiazepines are commonly used in old patients, particularly women [24], even its use is specifically counterindicated since they may produce adverse effects, such as cognitive impairment and sedation [31], which might be related with the prolongation of the burying behaviour latency in acyclic rats.
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In summary, this paper shows important differences in the anxiolytic properties of diazepam between males, females and neonatally-androgenized-females. Conversely, buspirone, produced similar actions in all groups. The possible role of hormones during the neonatal period of sexual differentiation in the anxiolytic drug action should be addressed.
Acknowledgements Authors wish to thank Mr Vı´ctor Flores Montoya for animal caring and Mr Facundo Moreno for technical assistance. Present investigation was partially supported by a grant (No. 2338P) to A.F.-G. from the ‘Consejo Nacional de Ciencia y Tecnologı´a’.
References [1] D. Bitran, R.J. Hilvers, C.K. Kellogg, Anxiolytic effects of 3a-hydroxy-5a[ß]-pregnan-20-one: Endogenous metabolites of progesterone that are active at the GABAA receptor, Brain Res. 561 (1991) 157 – 161. [2] D. Bitran, M. Shiekh, M. McLeod, Anxiolytic effect of progesterone is mediated by the neurosteroid allopregnanolone at brain GABA(A) receptors, J. Neuroendocrinol. 7 (1995) 171–177. [3] A.M. Davis, M.M. McCarthy, Sex differences in GABAA receptor subunit mRNA levels in neonatal rat brain, 28th. Ann. Conf. Reprod. Behav., Concordia University, Montre´al, Canada, 1996, pp. 33. [4] A. Farrell, A.A. Gerall, M.J. Alexander, Age-related decline in receptivity in normal, neonatally androgenized female and male hamsters, Exp. Aging Res. 3 (1977) 117–128. [5] A. Ferna´ndez-Guasti, O. Picazo, The actions of diazepam and serotonergic anxiolytics vary according to the gender and the oestrous cycle phase, Pharmacol. Biochem. Behav. 37 (1990) 77 – 81. [6] A. Ferna´ndez-Guasti, O. Picazo, Changes in burying behavior during the estrous cycle: effect of estrogen and progesterone, Psychoneuroendocrinology 17 (1992) 681–689. [7] A. Ferna´ndez-Guasti, O. Picazo, Differential allopregnanolone anxiolytic actions in males, females and neonatally androgenized females, Horm. Behav., Submitted. [8] A. Ferna´ndez-Guasti, O. Picazo, A. Ferreira, Blockade of the anxiolytic action of 8-OH-DPAT in lactating rats, Pharmacol. Biochem. Behav., in press. [9] R.A. Gorski, The neuroendocrinology of reproduction, Biol. Reprod. 20 (1979) 111–127. [10] D.E. Hilleman, S.M. Mohiuddin, M.G. Delcore, Comparison of fixed-dose transdermal nicotine, tapered-dose transdermal nicotine, and buspirone in smoking cessation, J. Clin. Pharmacol. 34 (1994) 222 – 224. [11] P.F. Hockl, G.S. Dı´az, C. Libertun, Prolactin- releasing effect of buspirone in developing and adult male and female rats, Proc. Soc. Exp. Biol. Med. 202 (1993) 447–450. [12] A. Jackson, L. Uphouse, Prior treatment with estrogen attenuates the effect of the 5-HT1A agonist, 8-OH-DPAT, on lordosis behavior, Horm. Behav. 30 (1996) 145–152.
.
[13] J.R. Lehman, D.A. McArthur, S.E. Hendricks, Pharmacological induction of ovulation in old and neonatally androgenized rats, Exp. Gerontol. 13 (1978) 107 – 114. [14] C. Lo´pez-Rubalcava, A. Ferna´ndez-Guasti, R. Urba´-Holmgren, Age-dependent differences in the rat’s conditioned defensive burying behavior: Effect of 5-HT1A compounds, Dev. Psychobiol. 29 (1996) 157 – 169. [15] D.K. Mahajan, Polycystic ovarian disease: Animal models, Endocrinol. Metab. Clin. North. Am. 17 (1988) 705 – 732. [16] M.M. McCarthy, A.M. Davis, H.R. Besmer, J.A. Mong, R.L. Kurzweil, Mechanisms of sexual differentiation of the brain: does transient excitation mediate masculinization? 28th. Ann. Conf. Reprod. Behav., Concordia University, Montre´al, Canada, 1996 p. 16. [17] N. Molina, M.T. Bedran de Castro, J.C. Bedran de Castro, Sex-related differences in the analgesic response to the rat tail immersion test, Braz. J. Med. Biol. Res. 27 (1994) 1669–1672. [18] O. Picazo, A. Ferna´ndez-Guasti, Changes in experimental anxiety during pregnancy and lactation, Physiol. Behav. 54 (1993) 295 – 299. [19] O. Picazo, A. Ferna´ndez-Guasti, Antianxiety effects of progesterone and some of its reduced metabolites: an evaluation using the burying behavior test, Brain Res. 680 (1995) 135 – 141. [20] J.F. Rodrı´guez-Sierra, M.T. Hagley, S.E. Hendricks, Anxiolytic effects of progesterone are sexually dimorphic, Life Sci. 38 (1986) 1841 – 1845. [21] C.B. Sengstake, K.C. Chambers, Sensitivity of male, female, and androgenized female rats to testosterone during extinction of a conditioned taste aversion, Behav. Neurosci. 105 (1991) 120– 125. [22] S. Siegel, Nonparametric Statistics for the Behavioral Sciences, McGraw Hill, New York, 1956. [23] R.G.D. Steel, J.H. Torrie, Principles and Procedures of Statistics. A Biometrical Approach, McGraw Hill, New York, 1985. [24] R.B. Stewart, R.G. Marks, P.D. Padgett, W.E. Hale, Benzodiazepine use in an ambulatory elderly population: A 14-year overview, Clin. Ther. 16 (1994) 118 – 124. [25] H.E. Swanson, J.J. van der Werff ten Bosch, The ‘early-androgen’ syndrome; differences in response to pre-natal and post-natal administration of various doses of testosterone propionate in female and male rats, Acta Endocrinologica 47 (1964) 37–50. [26] B. Szwarcfarb, S. Carbone, M.L. Stein, J. Medina, J.A. Moguilevsky, Sexual differences in the effect of the GABAergic system on LH secretion and in the hypothalamic ontogenesis of GABAA receptors in prepubertal rats, Brain Res. 646 (1994) 351 – 355. [27] D. Treit, J.P.J. Pinel, H.C. Fibiger, Conditioned defensive burying: A new paradigm for the study of anxiolytic agents, Pharmacol. Biochem. Behav. 15 (1981) 619 – 626. [28] D. Treit, Animal models for the study of anti-anxiety agents: A review, Neurosc. Behav. Rev. 9 (1985) 203 – 222. [29] L. Uphouse, S. Salamanca, M. Caldarola-Pastuszka, Gender and estrous cycle differences in the response to the 5-HT1A agonist 8-OH-DPAT, Pharmacol. Biochem. Behav. 40 (1991) 901–906. [30] F.S. vom Saal, C.E. Finch, Reproductive senescence: phenomena and mechanisms in mammals and selected vertebrates, in: E. Knobil, J. Neill et al. (Eds.), The Physiology of Reproduction, Raven Press, New York, 1988. [31] S.M. Willcox, D.U. Himmelstein, S. Woolhandler, Inappropriate drug prescribing for the community-dwelling elderly, JAMA 272 (1994) 292 – 296.