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Regulation of mating behaviour in the female rat by gonadotropin-releasing hormone in the ventral tegmental area: effects of selective destruction of the A10 dopamine neurones
Brain Research, 374 (1986) 167-173 Elsevier
167
BRE 21537
Regulation of mating behaviour in the female rat by gonadotropin-releasing hormone in the ventral tegmental area: effects of selective destruction of the AIO dopamine neurones D.J.S. SIRINATHSINGHJI, P.E. WHITI'INGTON and A.R. AUDSLEY Section for Neuroendocrinology and Behavioural Neurobiology, AFRC Neurobiology Programme, Agricultural and Food Research Council, Langford, Bristol BS18 7D Y (U. K. ) (Accepted January 14th, 1986) Key words: gonadotropin-releasing hormone - - ventral tegmental area - - A10 dopamine neurone - 6-hydroxydopamine - - lordosis behaviour - - female rat
Microinfusions of gonadotropin-releasing hormone (GnRH) into the ventral tegmental area (VTA) potentiated lordosis behaviour in oestrogen-primed ovariectomised female rats. Facilitation was observed within 5 min after the infusion and lasted for about 90 rain. When GnRH was infused into the VTA of oestrogen-primed animals which were previously subjected to 6-hydroxydopamine treatment (to destroy the A 10 dopamine cells), it produced a marked facilitation of lordosis lasting for about 24 h. These results suggest that the A10 dopamine neurones in the VTA may be critically involved in the mechanisms by which GnRH may modulate midbrain circuits involved in the regulation of lordosis behaviour in the female rat. The lesion studies also imply that the A10 dopamine neurones function as inhibitory neurones regulating lordosis behaviour by suppressing the activity of those cells in the VTA which are sensitive to GnRH. Removal of this inhibitory input leads to an exaggerated response.
G o n a d o t r o p i n releasing h o r m o n e ( G n R H ) is widely distributed throughout the central nervous system (CNS) 20'26'27"38 suggesting, in c o m m o n with
bic structures and to prefrontal, cingulate and entorhinal cortices 1428'29"34. These neurones have been
other regulatory peptides, extrahypophysiotropic roles, i.e., in the regulation of behavioural re-
implicated in adaptive, cognitive and emotive functions ls'23'3° as well as in the pathogenesis of psychopathological states 32'33'35'36.
sponses. In this respect, hypothalamic G n R H neuronal projections to the midbrain 2° have attracted much attention. Indeed, microinfusions of G n R H into the
A wide body of evidence also indicates that dopamine functions as an inhibitory neurotransmitter in the control of female sexual behaviour tl. It is feasible
dorsal region of the central grey (CG) have been shown to exert potent facilitatory effects on lordosis behaviour in the oestrogen-treated ovariectomised female rat25; moreover, i m m u n o n e u t r a l i s a t i o n of endogenous G n R H in the CG with a specific antiserum 25'31 or blockade of putative G n R H receptors on
that G n R H may be crucially involved in this regulation whereby dopamine could inhibit female sexual behaviour by suppressing the action of G n R H . This rationale may be compatible with the consensus that dopamine plays an inhibitory role in the control of G n R H and anterior pituitary luteinising h o r m o n e (LH) secretion 2'1° although the dopamine cell groups
GnRH-sensitive n e u r o n e s in the C G with an antagonist analogue 31 of G n R H abolish lordosis behaviour in the female rat. G n R H fibres have also been detected in the ventral tegmental area ( V T A ) 2°, the site of A10 dopamine n e u r o n e s of the mesolimbic and mesocortical d o p a m i n e systems which project to lim-
involved in this process (A12 in the arcuate and periventricular hypothalamic nuclei 3'5'~ and A l l , A13 and A14 belonging to incertohypothalamic system 4`s) are different from those being investigated in this study.
Correspondence: D.J.S. Sirinathsinghji, Department of Neuroendocrinology, AFRC Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, U.K. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
168 The presence of G n R H fibres in the VTA suggests that in this midbrain area there may be neurones sensitive to G n R H (although there is as yet no evidence for this assumption) and that there could be communication between G n R H and the A10 dopamine neutones in the VTA in the regulation of female sexual behaviour. A suppression or removal of these inhibitory dopamine neurones may thus greatly facilitate the action of G n R H . This study examines this hypothesis by assessing the effects of G n R H in the VTA on lordosis behaviour in oestrogen-treated ovariectomised female rats with (1) intact A10 dopamine neurones and (2) selective A10 dopamine cell loss induced by the catecholamine neurotoxin, 6-hydroxydopamine (6-OHDA). Stainless-steel 24-gauge guide cannulae (1.0 cm long) with 31-gauge inner stylets (1.0 cm long) were chronically implanted bilaterally 1.0 mm dorsal to the VTA in 24 ovariectomised Wistar female rats (220-250 g) under sodium pentobarbital anaesthesia (30 mg/kg b. wt.) using a Kopf stereotaxic instrument fitted with atraumatic ear-bars. The coordinates of K6nig and Klippe117 were used (A: 2.2, L: +0.6, V: 6.3 mm) but the modifications of Wishaw et 81. 37 were employed to maintain in each rat an angle of 5 ° below the horizontal as measured between the interaural line and the rostral edge of the upper incisor bar. During the same surgery 12 of these animals also received bilateral microinjections of 6 - O H D A (8 ~g) (Sigma) into the VTA via 31-gauge inner cannulae which protruded 1.0 mm beyond the tips of the guide shafts. The drug was prepared for injection by dissolving 6 - O H D A hydrobromide (2/~g/~tl) in 0.9% saline containing 0.1% ascorbic acid to retard oxidation of the neurotoxin and microinfused at a rate of 0.5/A/min. Each infusion cannula was connected to a Hamilton microsyringe (10 kd) via a 40-cm length of silastic medical grade tubing (Dow Corning) which was filled with saline containing 0.1% ascorbic acid. The syringes were driven by a Harvard syringe pump (Model 975). This procedure allowed the simultaneous delivery of the neurotoxin into both sides of the brain. Desmethylimipramine (25 mg/kg, i.p.) was given 30 rain before the 6 - O H D A injection to protect noradrenaline (NE)-containing neurones. The other 12 animals received control injections of the vehicle (0.1% ascorbic acid in 0.9% saline) into the VTA.
Behavioural tests on all the animals were conducted two weeks later. Each female received 5 ug oestradiol benzoate in peanut oil (0.1 ml) 96 h belore the test. Lordosis behaviour was observed in circular Plexiglass cages 3-10 h after lights-off under dim redlight illuminations (25 W). Immediately after saline or G n R H infusion, each female was paired with ~ sexually vigorous cage-adapted male amt the lordosis response assessed in terms of the intensity of lordosis (lordosis reflex score, LS) and the lordosis quotient (LQ; number of lordoses × 100/number of mounts). In order to ensure vigorous mounts with the experimental females, males were first exposed to stimulusreceptive females and permitted two or 3 intromissions. The stimulus females were then replaced with the experimental females. If during the test session the quality of the male's mounts declined, it was replaced with another experienced mate. For each mount with thrusting of the male, the female received a lordosis intensity rating of 0 = no lordosis (no dorsiflexion), 1 = lordosis with slight dorsiflexion, 2 = lordosis with moderate dorsiflexion and 3 = strongest possible response (full dorsiflexion). The LS and LQ were assessed immediately after the infusion and then every 30 rain until lordosis returned to preinfusion levels. The LQ was calculated as percent lordoses per 20 mounts. The average of 20 ratings of the reflex score was collected independently by 3 investigators. G n R H (NIH, Bethesda, MD. U.S.A.) was stored in aliquots of 5/~g at -20 °C. It was prepared immediately before use to give a final concentration of t00 ng//A. G n R H was infused in a volume of 0.5 ul over a period of 1.0 rain. At the end of the behavioural observations the animals were decapitated, the brains rapidly removed (within 1 rain of decapitation) and frozen on dry ice. Serial 200 ~m coronal sections were cut in the stereotaxic plane of K6nig and Klippe117 in a cryostat at -15 °C. Samples of tissue from the nucleus accumbens (a major projection area of the mesolimbic dopamine pathway originating from the A10 dopamine neurones in the VTA 8) and the caudate nucleus (terminal area of the nigrostriatal pathway originating from the A9 dopamine neurones in the pars compacta of the substantia nigra 8) were punched out from the sections according to the method of Palkovits 24 using
169 stainless-steel needles (0.85 mm i.d.). The samples were stored in Eppendorf microtubes at -70 °C for a maximum of 4 days until assayed for dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid, H V A ) and NE. This procedure allowed an assessment of the extent of the 6OHDA-induced destruction of the A10 dopamine neurones in the VTA. Coronal sections (20 ktm thickness) of the remaining portion of the brain were cut in the cryostat and stained with cresyl violet to verify cannulae placements in the VTA. The determination of DA, D O P A C , H V A and NE in the brain micropunches was carried out by highperformance liquid chromatography (HPLC) with electrochemical detection based essentially on the method described by Hegstrand and Eichelman 15. Brain punches were homogenised in 380/~1 of 0.1 mM perchloric acid containing 0.1 mM E D T A to which was added 10 ~tl of dihydroxybenzylamine (800 ng/ml), the internal standard. After centrifugation at 10,000 g for 10 min at 4 °C, the supernatants were used for injection into the HPLC (10-25 ktl). Each pellet was dissolved in 100/~1 of 1 M N a O H for determination of protein content by the method of Lowry et al. 19. The chromatographic equipment consisted of a DuPont 870 triple-head pump module, a C18 reverse-phase column (Rainin Microsorb 5/~, 250 × 4.6 mm, Anachem, U.K.), an electrochemical detector (LC-4; Bioanalytical Systems, West Lafayette, IN, U.S.A.), a glassy carbon electrode (Bioanalytical Systems) and a graphic integrator (3380 A; Hewlett Packard, Avondale, PA, U.S.A.). The detector potential was set at +0.72 V vs an Ag/AgCI reference electrode. The flow rate was maintained at 1.25 ml/min and the pressure at 2610 psi. The mobile phase was a solution containing 0.1 M potassium phosphate (pH 3.0), 0.2 mM sodium octyl sulfonate and 0.1 mM E D T A and 5% methanol. The column was used at a temperature of 35 °C. Water was deionised and distilled. The buffer and methanol were fitted through Millipore H A W P 0.45-/~m and 0.50/~m filters, respectively. The buffer was degassed using a DuPont flotation degassing accessory. Under these conditions the retention times were 3.77 min for NE, 6.35 min for D H B A , 10.04 min for DA, 14.03 rain for D O P A C and 33.40 min for HVA.
All the lordosis ratings and quotients were converted by arc-sine transformation. The data from treatment and control groups were compared using the Student's t-test after two-way analysis of variance. The levels of DA, DOPAC, H V A and NE in non-lesioned and lesioned animals were compared by the Student's t-test after one-way analysis of variance. When infused into the VTA of animals with intact A10 dopamine neurones (i.e. animals in which the VTA were not subjected to bilateral infusions of 6O H D A ) , G n R H (100 ng; 50 ng bilaterally) significantly potentiated lordosis behaviour in oestrogenprimed ovariectomised female rats. Facilitation oc100
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Fig. 1. Effect of saline ( i ) and G n R H (O) infusions into the V T A of control, non-lesioned, oestrogen-treated, ovariectomised female rats. Each time point denotes the m e a n + S.E.M. of 8 rats. Asterisks (* P < 0.05, ** P < 0.01, *** P < 0.001, analysis of variance and Student's t-test) denote statistically significant differences between G n R H and saline-infused animals.
170
curred within 10 min and lasted for about 91~ min (Fig. 1). In animals (n = 4) in which one guide cannula was found lying outside the V T A , in the most medial part of the substantia nigra, the lordotic responses to G n R H were p o o r (data not shown). These animals were omitted from both the behavioural and neurochemical analyses. The lordosis responses (96 h after oestrogen treatment) with saline infusion into the V T A of 6 - O H D A treated animals were generally higher than non-lesioned animals but at no time point were they significantly different (Fig. 2). H o w e v e r , in response to G n R H (100 ng; 50 ng bilaterally) infusions into the
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Fig. 2. Effect of saline (11) and GnRH (O) infusions into the V T A of oestrogen-treated ovariectomised female rats previously subjected to bilateral infusions of 6-OHDA into the VTA. Each time point denotes the mean + S.E.M, of 12 rats. Asterisks (* P < 0.05, ** P < 0.01, *** P < 0.001, analysis of variance and Student's t-test) denote statisticallysignificant differences between GnRH and saline-infused animals.
V T A of 6 - O H D A - t r e a t e d animals, :t marked and long-lasting facilitation of lordosis was t4~servcd, bacilitation was evident within 5 min. Maximum taciliration persisted for 21 h after the single infusion of G n R H and then declined gradually although facilitation was still significant 24 h after ( i n R H inlusion (Fig. 2). The mean duration of the facilitation ot the lordosis response induced bv G n R H was 23.5 ~: IL9 h (range: 19-27 h). During the period of maximal facilitation, the animals showed no aggressix e, defensive or resistive behavioural responses, e.g. squealing, kicking or rolling over and fending off die male during mounting attempts. A d d i t i o n a l l y it was observed that the average duration of maintaining a full iordosis was greatly prolonged by G n R H : some animals would hold this posture for about 10 s. However, despite this m a r k e d sexual receptivity none of the G n R H - t r e a t e d 6 - O H D A - l e s i o n e d animals showed any proceptive or active soliciting behavioural re-, sponses, e.g. hopping, darting or ear-wiggling. All the animals (n = 12) treated with ,~-OHDA in the V T A had correctly placed cannulae as confirmed by histological examination. This is substantiated by neurochemical data (Table I) showing that all the animals had severe depletion of d o p a m i n e , D O P A C and H V A (75, 94 and 94~:'; respectively) m the nucleus accumbens when c o m p a r e d to control animals not treated with 6 - O H D A . The NE content in the nucleus accumbens in the tS-OHDA-treated animals were not affected indicating the effectiveness of desmethylimipramine in protecting N k neuronal systems. The levels of D A . D O P A C , H V A and NE in the caudate nucleus in the O - O H D A - t r e a t e d animals were not significantly different from the levels in non-lesioned animals thus indicating that the neurotoxin was restricted to the V T A and did not diffuse to the adjacent d o p a m i n e neurones in the substantia nigra which project to the caudate nucleus ~ The results obtained in this study are important m two respects. Firstly, they give the first d e m o n s t r a tion that neurones in the V T A sensitive h~ G n R H are involved in midbrain circuits regulating lordosis behaviour in the female rat. Although G n R H fibres have been detected in the V T A suggesting a role for G n R H in this brain area in the regulation of reproductive behaviour, no studies, however, have as yet been p e r f o r m e d to establish whether iontophoretically applied G n R H can alter the firing rate of neuro-
171 TABLE I Levels of dopamine, its metabolites and N E in micropunches of the nucleus accurnbens and caudate nucleus of female rats
The lesioned rats were subjected to bilateral infusions of 6-OHDA in the VTA and the non-lesioned rats were saline-infused. Assays were performed using HPLC liquid chromatography with electrochemical detection. Values shown are means _+S.E.M. in ng/mg protein. DA
Nucleus accumbens Control (n = 8) Lesion (n = 12) Nucleus caudatus Control (n = 8) Lesion (n = 12)
26.38 + 3.14 6.56 + 0.55* 50.98 + 2.97 54.67 _+3.43
D 0 PAl C
9.15 + 1.02 0.52 + 0.07* 13.27 _+0.53 13.84 + 0.57
HVA
NE
7.23 _+0.43 0.39 + 0.05*
7.92 + 0.55 8.55 _+0.35
6.87 +_0.54 7.39 _+0.37
8.44 _+0.42 8.54 _+0.35
* P < 0.00l vs control animals (one-way analysis of variance and Student's t-test).
nes in the V T A . Such studies are now in progress in this l a b o r a t o r y and should provide an electrophysiological basis for the behavioural d a t a o b t a i n e d here. Secondly, this study has given the first d e m o n s t r a t i o n that the action of G n R H can be greatly e n h a n c e d after selective destruction of a p o p u l a t i o n of chemically identified neurones, in this case, the A10 d o p a m i n e neurones. This finding may imply that the A10 dopamine neurones function as inhibitory neurones in the regulation of lordosis b e h a v i o u r by suppressing the activity of those neurones in the V T A which are excited by G n R H . R e m o v a l of this inhibitory influence leads to an e x a g g e r a t e d lordosis response. The findings in this study may thus lend support to a n u m b e r of studies which indicate that d o p a m i n e functions as an inhibitory n e u r o t r a n s m i t t e r in the regulation of lordosis b e h a v i o u r in the female rat (in contrast to the male rat where it is excitatory to copulatory performance11). F o r example, d o p a m i n e receptor blockers such as pimozide or haloperidol increase the frequency and duration of lordosis in oest r o g e n - p r i m e d ovariectomised female rats 1"~3 whereas lordosis is suppressed by agonists such as a m p h e t a mine 21'22 and a p o m o r p h i n e 9'12. In addition, intracerebroventricular injections of 6 - O H D A (which produce m o d e r a t e depletion of both d o p a m i n e and N E ) have been shown to p r o d u c e an increase in the intensity, frequency and duration of lordosis in oestrogentreated ovariectomised female rats while severely disrupting the proceptive patterns of behaviour6"l~; such t r e a t m e n t also severely suppresses the sexual behaviour of male rats 7. Such observations have led to the proposal that midbrain d o p a m i n e neurones
may be involved in much b r o a d e r aspects of sensorim o t o r function which may not be specifically sexlinked 6. Thus, suppressing d o p a m i n e r g i c activity disrupts activated m o t o r patterns such as male sexual behaviour or female soliciting patterns of behaviour while enhancing responses requiring immobility such as lordosis behaviour. In this respect, it is interesting to note that as o b s e r v e d in the present study, while G n R H infusions into the 6 - O H D A - t r e a t e d V T A produced long-lasting facilitation of lordosis in oestrogen-primed rats, no soliciting behavioural patterns (hopping, darting) were observed. It is thus recognised that the integration of sensory information and the a p p r o p r i a t e m o t o r responses which comprise the lordosis reflex d e p e n d s upon the integrity of both the mesolimbic and nigrostriatal dopamine neurones 616. H o w e v e r , the participation of a n e u r o h u m o r a l c o m p o n e n t in this process cannot be excluded. It is feasible that the e n h a n c e m e n t of sexual receptivity o b s e r v e d following suppression of dopaminergic activity in o e s t r o g e n - t r e a t e d ovariectomised female rats, could be due, in part, to a release of the inhibitory influence of d o p a m i n e on G n R H secretion. I n d e e d , this study has d e m o n s t r a t e d that removal of the inhibitory influence exerted by a specific d o p a m i n e cell group (A10) greatly facilitates the action of G n R H in the V T A in potentiating lordosis behaviour in o e s t r o g e n - p r i m e d ovariectomised female rats. This localisation of a specific interaction between the A10 d o p a m i n e neurones and G n R H neuronal systems in the V T A may provide some insight into the role of the A10 d o p a m i n e neurones in sensor i m o t o r - n e u r o e n d o c r i n e integration.
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Regulation of mating behaviour in the female rat by gonadotropin-releasing hormone in the ventral tegmental area: effects of selective destruction of the AIO dopamine neurones D.J.S. SIRINATHSINGHJI, P.E. WHITI'INGTON and A.R. AUDSLEY Section for Neuroendocrinology and Behavioural Neurobiology, AFRC Neurobiology Programme, Agricultural and Food Research Council, Langford, Bristol BS18 7D Y (U. K. ) (Accepted January 14th, 1986) Key words: gonadotropin-releasing hormone - - ventral tegmental area - - A10 dopamine neurone - 6-hydroxydopamine - - lordosis behaviour - - female rat
Microinfusions of gonadotropin-releasing hormone (GnRH) into the ventral tegmental area (VTA) potentiated lordosis behaviour in oestrogen-primed ovariectomised female rats. Facilitation was observed within 5 min after the infusion and lasted for about 90 rain. When GnRH was infused into the VTA of oestrogen-primed animals which were previously subjected to 6-hydroxydopamine treatment (to destroy the A 10 dopamine cells), it produced a marked facilitation of lordosis lasting for about 24 h. These results suggest that the A10 dopamine neurones in the VTA may be critically involved in the mechanisms by which GnRH may modulate midbrain circuits involved in the regulation of lordosis behaviour in the female rat. The lesion studies also imply that the A10 dopamine neurones function as inhibitory neurones regulating lordosis behaviour by suppressing the activity of those cells in the VTA which are sensitive to GnRH. Removal of this inhibitory input leads to an exaggerated response.
G o n a d o t r o p i n releasing h o r m o n e ( G n R H ) is widely distributed throughout the central nervous system (CNS) 20'26'27"38 suggesting, in c o m m o n with
bic structures and to prefrontal, cingulate and entorhinal cortices 1428'29"34. These neurones have been
other regulatory peptides, extrahypophysiotropic roles, i.e., in the regulation of behavioural re-
implicated in adaptive, cognitive and emotive functions ls'23'3° as well as in the pathogenesis of psychopathological states 32'33'35'36.
sponses. In this respect, hypothalamic G n R H neuronal projections to the midbrain 2° have attracted much attention. Indeed, microinfusions of G n R H into the
A wide body of evidence also indicates that dopamine functions as an inhibitory neurotransmitter in the control of female sexual behaviour tl. It is feasible
dorsal region of the central grey (CG) have been shown to exert potent facilitatory effects on lordosis behaviour in the oestrogen-treated ovariectomised female rat25; moreover, i m m u n o n e u t r a l i s a t i o n of endogenous G n R H in the CG with a specific antiserum 25'31 or blockade of putative G n R H receptors on
that G n R H may be crucially involved in this regulation whereby dopamine could inhibit female sexual behaviour by suppressing the action of G n R H . This rationale may be compatible with the consensus that dopamine plays an inhibitory role in the control of G n R H and anterior pituitary luteinising h o r m o n e (LH) secretion 2'1° although the dopamine cell groups
GnRH-sensitive n e u r o n e s in the C G with an antagonist analogue 31 of G n R H abolish lordosis behaviour in the female rat. G n R H fibres have also been detected in the ventral tegmental area ( V T A ) 2°, the site of A10 dopamine n e u r o n e s of the mesolimbic and mesocortical d o p a m i n e systems which project to lim-
involved in this process (A12 in the arcuate and periventricular hypothalamic nuclei 3'5'~ and A l l , A13 and A14 belonging to incertohypothalamic system 4`s) are different from those being investigated in this study.
Correspondence: D.J.S. Sirinathsinghji, Department of Neuroendocrinology, AFRC Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, U.K. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
168 The presence of G n R H fibres in the VTA suggests that in this midbrain area there may be neurones sensitive to G n R H (although there is as yet no evidence for this assumption) and that there could be communication between G n R H and the A10 dopamine neutones in the VTA in the regulation of female sexual behaviour. A suppression or removal of these inhibitory dopamine neurones may thus greatly facilitate the action of G n R H . This study examines this hypothesis by assessing the effects of G n R H in the VTA on lordosis behaviour in oestrogen-treated ovariectomised female rats with (1) intact A10 dopamine neurones and (2) selective A10 dopamine cell loss induced by the catecholamine neurotoxin, 6-hydroxydopamine (6-OHDA). Stainless-steel 24-gauge guide cannulae (1.0 cm long) with 31-gauge inner stylets (1.0 cm long) were chronically implanted bilaterally 1.0 mm dorsal to the VTA in 24 ovariectomised Wistar female rats (220-250 g) under sodium pentobarbital anaesthesia (30 mg/kg b. wt.) using a Kopf stereotaxic instrument fitted with atraumatic ear-bars. The coordinates of K6nig and Klippe117 were used (A: 2.2, L: +0.6, V: 6.3 mm) but the modifications of Wishaw et 81. 37 were employed to maintain in each rat an angle of 5 ° below the horizontal as measured between the interaural line and the rostral edge of the upper incisor bar. During the same surgery 12 of these animals also received bilateral microinjections of 6 - O H D A (8 ~g) (Sigma) into the VTA via 31-gauge inner cannulae which protruded 1.0 mm beyond the tips of the guide shafts. The drug was prepared for injection by dissolving 6 - O H D A hydrobromide (2/~g/~tl) in 0.9% saline containing 0.1% ascorbic acid to retard oxidation of the neurotoxin and microinfused at a rate of 0.5/A/min. Each infusion cannula was connected to a Hamilton microsyringe (10 kd) via a 40-cm length of silastic medical grade tubing (Dow Corning) which was filled with saline containing 0.1% ascorbic acid. The syringes were driven by a Harvard syringe pump (Model 975). This procedure allowed the simultaneous delivery of the neurotoxin into both sides of the brain. Desmethylimipramine (25 mg/kg, i.p.) was given 30 rain before the 6 - O H D A injection to protect noradrenaline (NE)-containing neurones. The other 12 animals received control injections of the vehicle (0.1% ascorbic acid in 0.9% saline) into the VTA.
Behavioural tests on all the animals were conducted two weeks later. Each female received 5 ug oestradiol benzoate in peanut oil (0.1 ml) 96 h belore the test. Lordosis behaviour was observed in circular Plexiglass cages 3-10 h after lights-off under dim redlight illuminations (25 W). Immediately after saline or G n R H infusion, each female was paired with ~ sexually vigorous cage-adapted male amt the lordosis response assessed in terms of the intensity of lordosis (lordosis reflex score, LS) and the lordosis quotient (LQ; number of lordoses × 100/number of mounts). In order to ensure vigorous mounts with the experimental females, males were first exposed to stimulusreceptive females and permitted two or 3 intromissions. The stimulus females were then replaced with the experimental females. If during the test session the quality of the male's mounts declined, it was replaced with another experienced mate. For each mount with thrusting of the male, the female received a lordosis intensity rating of 0 = no lordosis (no dorsiflexion), 1 = lordosis with slight dorsiflexion, 2 = lordosis with moderate dorsiflexion and 3 = strongest possible response (full dorsiflexion). The LS and LQ were assessed immediately after the infusion and then every 30 rain until lordosis returned to preinfusion levels. The LQ was calculated as percent lordoses per 20 mounts. The average of 20 ratings of the reflex score was collected independently by 3 investigators. G n R H (NIH, Bethesda, MD. U.S.A.) was stored in aliquots of 5/~g at -20 °C. It was prepared immediately before use to give a final concentration of t00 ng//A. G n R H was infused in a volume of 0.5 ul over a period of 1.0 rain. At the end of the behavioural observations the animals were decapitated, the brains rapidly removed (within 1 rain of decapitation) and frozen on dry ice. Serial 200 ~m coronal sections were cut in the stereotaxic plane of K6nig and Klippe117 in a cryostat at -15 °C. Samples of tissue from the nucleus accumbens (a major projection area of the mesolimbic dopamine pathway originating from the A10 dopamine neurones in the VTA 8) and the caudate nucleus (terminal area of the nigrostriatal pathway originating from the A9 dopamine neurones in the pars compacta of the substantia nigra 8) were punched out from the sections according to the method of Palkovits 24 using
169 stainless-steel needles (0.85 mm i.d.). The samples were stored in Eppendorf microtubes at -70 °C for a maximum of 4 days until assayed for dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid, H V A ) and NE. This procedure allowed an assessment of the extent of the 6OHDA-induced destruction of the A10 dopamine neurones in the VTA. Coronal sections (20 ktm thickness) of the remaining portion of the brain were cut in the cryostat and stained with cresyl violet to verify cannulae placements in the VTA. The determination of DA, D O P A C , H V A and NE in the brain micropunches was carried out by highperformance liquid chromatography (HPLC) with electrochemical detection based essentially on the method described by Hegstrand and Eichelman 15. Brain punches were homogenised in 380/~1 of 0.1 mM perchloric acid containing 0.1 mM E D T A to which was added 10 ~tl of dihydroxybenzylamine (800 ng/ml), the internal standard. After centrifugation at 10,000 g for 10 min at 4 °C, the supernatants were used for injection into the HPLC (10-25 ktl). Each pellet was dissolved in 100/~1 of 1 M N a O H for determination of protein content by the method of Lowry et al. 19. The chromatographic equipment consisted of a DuPont 870 triple-head pump module, a C18 reverse-phase column (Rainin Microsorb 5/~, 250 × 4.6 mm, Anachem, U.K.), an electrochemical detector (LC-4; Bioanalytical Systems, West Lafayette, IN, U.S.A.), a glassy carbon electrode (Bioanalytical Systems) and a graphic integrator (3380 A; Hewlett Packard, Avondale, PA, U.S.A.). The detector potential was set at +0.72 V vs an Ag/AgCI reference electrode. The flow rate was maintained at 1.25 ml/min and the pressure at 2610 psi. The mobile phase was a solution containing 0.1 M potassium phosphate (pH 3.0), 0.2 mM sodium octyl sulfonate and 0.1 mM E D T A and 5% methanol. The column was used at a temperature of 35 °C. Water was deionised and distilled. The buffer and methanol were fitted through Millipore H A W P 0.45-/~m and 0.50/~m filters, respectively. The buffer was degassed using a DuPont flotation degassing accessory. Under these conditions the retention times were 3.77 min for NE, 6.35 min for D H B A , 10.04 min for DA, 14.03 rain for D O P A C and 33.40 min for HVA.
All the lordosis ratings and quotients were converted by arc-sine transformation. The data from treatment and control groups were compared using the Student's t-test after two-way analysis of variance. The levels of DA, DOPAC, H V A and NE in non-lesioned and lesioned animals were compared by the Student's t-test after one-way analysis of variance. When infused into the VTA of animals with intact A10 dopamine neurones (i.e. animals in which the VTA were not subjected to bilateral infusions of 6O H D A ) , G n R H (100 ng; 50 ng bilaterally) significantly potentiated lordosis behaviour in oestrogenprimed ovariectomised female rats. Facilitation oc100
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Fig. 1. Effect of saline ( i ) and G n R H (O) infusions into the V T A of control, non-lesioned, oestrogen-treated, ovariectomised female rats. Each time point denotes the m e a n + S.E.M. of 8 rats. Asterisks (* P < 0.05, ** P < 0.01, *** P < 0.001, analysis of variance and Student's t-test) denote statistically significant differences between G n R H and saline-infused animals.
170
curred within 10 min and lasted for about 91~ min (Fig. 1). In animals (n = 4) in which one guide cannula was found lying outside the V T A , in the most medial part of the substantia nigra, the lordotic responses to G n R H were p o o r (data not shown). These animals were omitted from both the behavioural and neurochemical analyses. The lordosis responses (96 h after oestrogen treatment) with saline infusion into the V T A of 6 - O H D A treated animals were generally higher than non-lesioned animals but at no time point were they significantly different (Fig. 2). H o w e v e r , in response to G n R H (100 ng; 50 ng bilaterally) infusions into the
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Fig. 2. Effect of saline (11) and GnRH (O) infusions into the V T A of oestrogen-treated ovariectomised female rats previously subjected to bilateral infusions of 6-OHDA into the VTA. Each time point denotes the mean + S.E.M, of 12 rats. Asterisks (* P < 0.05, ** P < 0.01, *** P < 0.001, analysis of variance and Student's t-test) denote statisticallysignificant differences between GnRH and saline-infused animals.
V T A of 6 - O H D A - t r e a t e d animals, :t marked and long-lasting facilitation of lordosis was t4~servcd, bacilitation was evident within 5 min. Maximum taciliration persisted for 21 h after the single infusion of G n R H and then declined gradually although facilitation was still significant 24 h after ( i n R H inlusion (Fig. 2). The mean duration of the facilitation ot the lordosis response induced bv G n R H was 23.5 ~: IL9 h (range: 19-27 h). During the period of maximal facilitation, the animals showed no aggressix e, defensive or resistive behavioural responses, e.g. squealing, kicking or rolling over and fending off die male during mounting attempts. A d d i t i o n a l l y it was observed that the average duration of maintaining a full iordosis was greatly prolonged by G n R H : some animals would hold this posture for about 10 s. However, despite this m a r k e d sexual receptivity none of the G n R H - t r e a t e d 6 - O H D A - l e s i o n e d animals showed any proceptive or active soliciting behavioural re-, sponses, e.g. hopping, darting or ear-wiggling. All the animals (n = 12) treated with ,~-OHDA in the V T A had correctly placed cannulae as confirmed by histological examination. This is substantiated by neurochemical data (Table I) showing that all the animals had severe depletion of d o p a m i n e , D O P A C and H V A (75, 94 and 94~:'; respectively) m the nucleus accumbens when c o m p a r e d to control animals not treated with 6 - O H D A . The NE content in the nucleus accumbens in the tS-OHDA-treated animals were not affected indicating the effectiveness of desmethylimipramine in protecting N k neuronal systems. The levels of D A . D O P A C , H V A and NE in the caudate nucleus in the O - O H D A - t r e a t e d animals were not significantly different from the levels in non-lesioned animals thus indicating that the neurotoxin was restricted to the V T A and did not diffuse to the adjacent d o p a m i n e neurones in the substantia nigra which project to the caudate nucleus ~ The results obtained in this study are important m two respects. Firstly, they give the first d e m o n s t r a tion that neurones in the V T A sensitive h~ G n R H are involved in midbrain circuits regulating lordosis behaviour in the female rat. Although G n R H fibres have been detected in the V T A suggesting a role for G n R H in this brain area in the regulation of reproductive behaviour, no studies, however, have as yet been p e r f o r m e d to establish whether iontophoretically applied G n R H can alter the firing rate of neuro-
171 TABLE I Levels of dopamine, its metabolites and N E in micropunches of the nucleus accurnbens and caudate nucleus of female rats
The lesioned rats were subjected to bilateral infusions of 6-OHDA in the VTA and the non-lesioned rats were saline-infused. Assays were performed using HPLC liquid chromatography with electrochemical detection. Values shown are means _+S.E.M. in ng/mg protein. DA
Nucleus accumbens Control (n = 8) Lesion (n = 12) Nucleus caudatus Control (n = 8) Lesion (n = 12)
26.38 + 3.14 6.56 + 0.55* 50.98 + 2.97 54.67 _+3.43
D 0 PAl C
9.15 + 1.02 0.52 + 0.07* 13.27 _+0.53 13.84 + 0.57
HVA
NE
7.23 _+0.43 0.39 + 0.05*
7.92 + 0.55 8.55 _+0.35
6.87 +_0.54 7.39 _+0.37
8.44 _+0.42 8.54 _+0.35
* P < 0.00l vs control animals (one-way analysis of variance and Student's t-test).
nes in the V T A . Such studies are now in progress in this l a b o r a t o r y and should provide an electrophysiological basis for the behavioural d a t a o b t a i n e d here. Secondly, this study has given the first d e m o n s t r a t i o n that the action of G n R H can be greatly e n h a n c e d after selective destruction of a p o p u l a t i o n of chemically identified neurones, in this case, the A10 d o p a m i n e neurones. This finding may imply that the A10 dopamine neurones function as inhibitory neurones in the regulation of lordosis b e h a v i o u r by suppressing the activity of those neurones in the V T A which are excited by G n R H . R e m o v a l of this inhibitory influence leads to an e x a g g e r a t e d lordosis response. The findings in this study may thus lend support to a n u m b e r of studies which indicate that d o p a m i n e functions as an inhibitory n e u r o t r a n s m i t t e r in the regulation of lordosis b e h a v i o u r in the female rat (in contrast to the male rat where it is excitatory to copulatory performance11). F o r example, d o p a m i n e receptor blockers such as pimozide or haloperidol increase the frequency and duration of lordosis in oest r o g e n - p r i m e d ovariectomised female rats 1"~3 whereas lordosis is suppressed by agonists such as a m p h e t a mine 21'22 and a p o m o r p h i n e 9'12. In addition, intracerebroventricular injections of 6 - O H D A (which produce m o d e r a t e depletion of both d o p a m i n e and N E ) have been shown to p r o d u c e an increase in the intensity, frequency and duration of lordosis in oestrogentreated ovariectomised female rats while severely disrupting the proceptive patterns of behaviour6"l~; such t r e a t m e n t also severely suppresses the sexual behaviour of male rats 7. Such observations have led to the proposal that midbrain d o p a m i n e neurones
may be involved in much b r o a d e r aspects of sensorim o t o r function which may not be specifically sexlinked 6. Thus, suppressing d o p a m i n e r g i c activity disrupts activated m o t o r patterns such as male sexual behaviour or female soliciting patterns of behaviour while enhancing responses requiring immobility such as lordosis behaviour. In this respect, it is interesting to note that as o b s e r v e d in the present study, while G n R H infusions into the 6 - O H D A - t r e a t e d V T A produced long-lasting facilitation of lordosis in oestrogen-primed rats, no soliciting behavioural patterns (hopping, darting) were observed. It is thus recognised that the integration of sensory information and the a p p r o p r i a t e m o t o r responses which comprise the lordosis reflex d e p e n d s upon the integrity of both the mesolimbic and nigrostriatal dopamine neurones 616. H o w e v e r , the participation of a n e u r o h u m o r a l c o m p o n e n t in this process cannot be excluded. It is feasible that the e n h a n c e m e n t of sexual receptivity o b s e r v e d following suppression of dopaminergic activity in o e s t r o g e n - t r e a t e d ovariectomised female rats, could be due, in part, to a release of the inhibitory influence of d o p a m i n e on G n R H secretion. I n d e e d , this study has d e m o n s t r a t e d that removal of the inhibitory influence exerted by a specific d o p a m i n e cell group (A10) greatly facilitates the action of G n R H in the V T A in potentiating lordosis behaviour in o e s t r o g e n - p r i m e d ovariectomised female rats. This localisation of a specific interaction between the A10 d o p a m i n e neurones and G n R H neuronal systems in the V T A may provide some insight into the role of the A10 d o p a m i n e neurones in sensor i m o t o r - n e u r o e n d o c r i n e integration.
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