Brain Research, 88 (1975) 281-293
281
© ElsevierScientificPublishing Company,Amsterdam- Printed in The Netherlands
5-HYDROXYTRYPTAMINE IN THE CENTRAL NERVOUS SYSTEM AND SEXUAL RECEPTIVITY OF FEMALE RHESUS MONKEYS*
P. B. GRADWELL, B. J. EVERITT AND J. HERBERT (P.B. G.) Department of Anatomy, University of Rhodesia, Salisbury (Rhodesia) and ( B.J.E. and J.H. ) Department of Anatomy, Downing Street, Cambridge (Great Britain)
(Accepted December 2nd, 1974)
SUMMARY
The role of 5-hydroxytryptamine (5-HT) in the control of sexual receptivity in female rhesus monkeys has been studied in 24 adult females paired with 6 adult males, p-Chlorophenylalanine (PCPA, 75 mg/kg or 100 mg/kg, every fourth day), a selective inhibitor of 5-HT, was found to reverse unreceptivity induced by adrenalectomy in ovariectomised, oestrogen-treated females. PCPA-treated females presented more frequently and initiated more sexual behaviour, or else they refused fewer of the male's attempts to mount. These effects were in turn reversed by 5-hydroxytryptophan (5-HTP, 20 mg/kg every second day), when this was given to PCPAtreated animals. In addition, 5-HTP given alone to ovariectomised oestrogen-treated females reduced their receptivity. Parallel biochemical experiments showed that PCPA in the doses used lowered the levels of 5-HT in the brain as measured by the levels of 5-hydroxyindole-3-acetic acid (5-HIAA) in the CSF, and that these were restored by 5-HTP. Both oestradiol benzoate (15/~g/day for 10 days) and testosterone propionate (250/zg/day or 400/~g/day for 10 days) lowered the turn-over rates of 5-HT in the brain (as measured by the probenecid test) in ovariectomised female monkeys. These effects of oestradiol on turnover were antagonised by progesterone (15 mg/day for 10 days, given with oestradiol). A substance other than an adrenal androgen has thus been found to reverse the effects of adrenalectomy on sexual receptivity in female monkeys. It is therefore possible that androgens regulate receptivity in female monkeys by modifying the activity of 5-HT-containing neural systems.
* These experiments were carried out in the Department of Anatomy,Universityof Birmingham, Birmingham, Great Britain. Address reprint requests to B, J. Everitt.
282 INTRODUCTION
Androgens, secreted mainly from the adrenal cortex, appear to control sexual receptivity in female rhesus monkeys. Ovariectomised female monkeys are made unreceptive if their adrenals are removed 16 or suppressed with dexamethasone 15, but their sexual receptivity is restored by systemic injections of either testosterone propionate or androstenedione. Neither oestradiol nor adrenal secretions such as cortisol or progesterone are effective15,16. Very small amounts of testosterone propionate implanted stereotaxically into the anterior hypothalamus-preoptic area of the brain also induced sexual receptivity in adrenalectomised female monkeys, whereas similar implants in other sites (including the posterior hypothalamus) did not alter behaviour 17. Thus at least one site of action of these androgens in the brain is known. What is not clear is how androgens act on the nervous system. In sub-primate mammals monoamines, particularly 5-hydroxytryptamine (5-HT), have been implicated in the neural control of oestrous behaviour of the rat z7-4°,42,54, the cat 2'~ and the hamster4L Since different hormones appear to regulate receptivity in primates and non-primates, it is important to determine whether the underlying neural mechanisms are correspondingly different. The site of action of the effective hormones seems similar (see above). This paper describes experiments carried out to determine whether 5-HT has a role in controlling sexual receptivity in female primates. MATERIALS AND METHODS
Six adult male and 24 adult female rhesus monkeys (Macaca mulatta) were used. The males were intact, but the females had all been bilaterally ovariectomised several months before the experiments started. In addition, 10 of the females were then bilaterally adrenalectomised, using a technique described in detail elsewhere 16. The animals were carefully watched in the post-operative period, and all made excellent recoveries. Seven females had stainless steel intracranial cannulae fixed to the skull, their tips lying in the hypothalamus, as the result of a previous experiment 17. The cannulae were empty, and the behaviour of these females was undistinguishable from animals without such cannulae.
Behavioural observations Sexual behaviour between male-female pairs of monkeys was measured by methods given in detail elsewhere 1~,z~,23. Females were housed individually in cages (85 cm x 75 cm × 100 cm high) in a communal monkey house. Each female was paired with a male (always the same one) in a large observation cage (150 cm × 75 cm × 90 cm high) for 30 min daily and observed through a one-way-vision mirror. The following indices were derived from each series of 10 observations, as in previous work~1,24,25.
Sexual behaviour For the female. (a) Mean number of sexual presentations (invitations to the
283 male to mount); (b) proportion of mounts initiated by the female; (c) mean female's acceptance ratio (i.e. proportion of the male's attempts to mount allowed by the female); (d) total number of refusals to allow the male to mount during the series of 10 tests. For the male. (a) Mean number of mounts; (b) mean ejaculation time (from the first mount to ejaculation); (c) mean number of thrusts per intromitted mount; (d) mean mounting rate (mounts/min); (e) mean male's acceptance ratio (i.e. proportion of the female's presentations eliciting a mounting attempt by the male). This measure is a good indicator of the attractiveness of the female to the male, providing some presentations are made 24. The other components of the male's sexual activity are not considered in detail in this paper, since this is primarily concerned with alterations in the female's behaviour.
Aggressive behaviour The total number of 30-sec periods in which an aggressive gesture was shown by either animal. Social behaviour The total number of minutes monkeys spent grooming each other, or sitting in proximity within arm's length of one another, was recorded, together with the number of grooming invitations made by each animal. Treatments Females received oestradiol benzoate (in arachis oi1-10~o benzyl alcohol) 15 #g or 25 #g daily s.c. throughout the experiment. p-Chlorophenylalanine (PCPA) as the methyl ester hydrochloride, a potent inhibitor of 5-HT synthesis 30, was dissolved in Sorensen's phosphate buffer (pH 7.3). Monkeys received 75 or 100 mg/kg base every fourth day, beginning 4 days before the first observations, receiving 4 injections per series in all. L-5-Hydroxytryptophan (5-HTP) as the methyl ester hydrochloride was also dissolved in Sorensen's phosphate buffer (pH 7.8). Monkeys received 20 mg/kg base every second day, beginning 2 days before the first observation. Adrenalectomised animals were maintained on cortisol, given as hydrocortisone sodium succinate (20 mg/day) and deoxycorticosterone trimethylacetate (25 mg once monthly by deep intramuscular injection), and remained in excellent health throughout the experiments. Eight females were first tested while receiving either 15 #g or 25 #g oestradiol per day. They were then adrenalectomised, and together with 2 other adrenalectomised females from a previous experiment 17 they were tested again with the same males. All 10 then received PCPA. Finally, 5 received both PCPA and 5-HTP. Four ovariectomised females were tested while receiving oestradiol alone and then treated with 5-HTP. All females received the same dose of oestradiol under all treatment conditions.
284 A Presentations It It
20 ¸
.2
It It 15
10-
It It It m
e-
E
It tt
5It
It
It
i
i
O-
Female-initiated mounts .~ IO0It
$
E O1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8 9 1 0
1 2 3 4 5 6 7 8 9 1 0
125
610
Identity of female Fig. 1. Changes in the females' presentations (above) and the proportion (~) of mounts they initiate by presenting to the males (below). Treatments are A: ovariectomy alone; B: ovariectomy + adrenalectomy (with cortisol replacement); C: as B but receiving PCPA; D: as C but receiving 5-HTP. Doses used given in the text. Data are means per series (10 observations in each block): Identity of females: (1) 1886; (2) 2259; (3) 2263; (4) 2219; (5) 2261; (6) 2260; (7) 1887; (8) 2264; (9) 1992; (10) 1265. * P < 0.05. ** P < 0.01 compared with series B (Wilcoxon test). Females received oestradiol benzoate throughout the experiment. Biochemical measurements
Cerebrospinal fluid (CSF) was withdrawn from the cisterna magna of female monkeys under ketamine hydrochloride anaesthesia (7-8 mg/kg) between 09.00 and 11.00 h BST (British Standard Time) on the d a y of assay. Levels of 5-hydroxyindole-3-acetic acid (5-HIAA), the primary metabolite o f 5-HT in the CSF 6 were assayed spectrophotofluorimetrically, using the method of Ashcroft and SharmanS. In addition, turnover rates of 5-HT were measured using the probenecid test 6, 31,32. Levels of 5-HIAA were measured immediately before, and then 2 h after, an intravenous injection of probenecid sodium (20 mg/kg, p H 7.7-7.9) and turnover rates of 5-HT were then expressed as the percentage increase of 5-HIAA levels in the CSF during this 2-h period. Since sampling the CSF would have interfered with behaviourat observations,
285 TABLE I REFUSALS BY FEMALE TO ALLOW MALE TO MOUNT
Data given are total number of refusals per series (10 observations). Figures in parentheses correspond with those identifying females in Figs. 1 and 2. Treatments: A: ovariectomised; B: ovariectomised and adrenalectomised; C: as B but given PCPA; D: as C but given 5-HTP; E: given 5-HTP alone. Doses used given in text. - - indicates no data. Female no.
1886 2259 2263 2219 2261 2260 1887 2264 1992 1265
315 316 317 318
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
(11) (12) (13) (14)
Treatment
A
B
C
D
0 0 0 0 1 0 0 0 ---
3 1 7 2 4 70 28 0 0 4
0 1 0 2 1 10 4 0 0 0
6 19 --13 92 ---0
A
E
0 0 0 0
0 0 0 1
treatments given to animals during the behavioural tests were repeated where necessary. 5-HIAA levels were measured in 9 ovariectomised female monkeys, 7 of which had been adrenalectomised as well (and had taken part in the behavioural tests) under the following treatments: (1) no gonadal hormones, at least 2 months after operation; (2) receiving oestradiol alone; (3) after 4, 8 and 12 days on PCPA; (4) after I and 2 weeks following withdrawal of PCPA; (5) after 2 and 12 days of PCPA plus 5-HTP; and (6) 1 week after the withdrawal of PCPA plus 5-HTP treatment. F o u r ovariectomised animals were given 5-HTP alone, and measures were made (1) with no hormone treatment; (2) receiving oestradiol alone; (3) after 2 and 12 days o f 5-HTP treatment; and (4) 1 week after withdrawing 5-HTP. In the final experiment, 10 (different) ovariectomised female monkeys were used. Both the levels o f 5-HIAA and the turnover rates of 5-HT were measured under the following conditions: (1) no gonadal hormones, at least 2 months after ovariectomy; (2) after receiving 15/~g/day oestradiol for 10 days; (3) after 15 #g/day oestradiol plus 15 mg progesterone/day for 10 days; (4) after 250/~g or 400 #g/day testosterone propionate for 10 days; and (5) after receiving no hormones for at least 2 months. Two monkeys received treatment (4) before (2) and (3); this change in order had no effect on the results.
286 T A B L E 11 THE MALES~ ACCEPTANCE RATIOSOF THE FEMALES~ PRESENTATIONS See text for definition. - - indicates too few presentations to calculate ratio. See Table I for further explanation.
Female no.
1886 2259 2263 2219 2261 2260 1887 2264 1992 1265
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
Mean % per series A
B
C
D
69.2 66.2 94.7 76.0 94.7 75.0 83.3 87.9
57.1 80.0 --100 -92.3 100 100 75.0
68.2 67.7 90 91.7 93.3 -66.7 75.0 78.6 52.0*
61.4 100
100 --
76.0
* P < 0.01 (Wilcoxon test) compared to series B.
Statistical methods The behavioural data were subjected to a Friedman two-way analysis of variance. In those cases where the X~ statistic was associated with a probability of 0.05 or less the difference between individual treatments was tested by the Wilcoxon matchedpairs signed-ranks test49. A two-way analysis of variance was carried out on the biochemical data. Where the F statistic was associated with a probability of 0.05 or less, paired t-tests were carried out on the relevant comparisons. RESULTS
Behavioural observations Removing the adrenals from ovariectomised oestrogen-treated females caused a marked decrease in their sexual receptivity, confirming our earlier observationslS,16. All presented significantly less to the males and initiated a lower proportion of the sexual interaction (Fig, 1A and B). Eight females began to refuse the male's attempts to mount (Table I) whereas only one had ever done so before operation. PCPA reversed the effects of adrenatectomy. In 7 out of 10 cases, females presented significantly more and 6 initiated a greater proportion of sexual interaction; their behaviour after PCPA therefore resembled that before adrenalectomy, or (in earlier experiments) in adrenalectomised monkeys given testosterone (Fig. 1C)1~. Five females continued to refuse the male occasionally, much less frequently than before treatment with PCPA (Table I). Giving 5-HTP to 5 PCPA-treated females reversed the effects of PCPA by
287
.~
A Presentations
E
lit
E
i
Female-initiated: mounts
•~ 100-
0 ~. 50c E
O.
11121314
11121314
Identity of females
Fig. 2. Changes in the females' presentations (above) and the proportion of mounts they initiate by presenting to the males (below). Treatments are A: ovariectomy + oestradiol benzoate; E: as A but with 5-HTP added. Doses used given in the text. Identity of females: (11) 315; (12) 316; (13) 317; (14) 318. See Fig. 1 for further explanation. Statistical comparisons were made between treatments A and E. causing them to become unreceptive again. Four of the females' presentations were no longer significantly greater than after adrenalectomy (series B) and in 3 the proportion of female-initiated mounts decreased; 4 refused the males more often than they had after PCPA alone (Table I). Females received oestradiol throughout this experiment and so remained sexually attractive to the males, as shown by the consistently high males' acceptance ratios (Table II). Giving 5-HTP alone to 4 ovariectomised oestrogen-treated females made them less receptive (Fig. 2). Three presented significantly less and initiated less sexual interaction than they had before this treatment; there were similar changes in the behaviour of the fourth female, but these were not statistically significant (Fig. 2). However, the females' refusals o f the males' attempts to mount were not increased when they were given 5-HTP (Table I). Aggressive and social behaviour Neither PCPA nor PCPA + 5-HTP nor 5-HTP treatment alone had any clear effect on aggressive or social behaviour. Only one female (on one day) showed increased aggression toward the male when she was given PCPA -+- 5-HTP (female 1886). There were no consistent effects o f PCPA, PCPA + 5-HTP or 5-HTP alone on grooming or proximity behaviour. 5-HT levels and turnover PCPA in these doses considerably reduced levels of 5-HT in the brain, as
288 TABLE I11 LEVELSOF 5-HIAA (ng/ml) tN CSF OF FEMALERHESUSMONKEYSAFTERTREATMENTWITHPCPA AN[) 5-HTP Doses given in text. Number of monkeys
Levels (means 4- S.D.)
Oestradiol benzoate alone
9
68.2 i 9.87
PCPA added 4 days 8 days 12 days
9 9 9
31.9 i 6.49** 28.9 ± 6.99** 27.2 i 4.40**
PCPA + 5-HTP 2 days 12 days
5 5
70.2 ± 13.05 107.7 ± 15.86
PCPA + 5-HTP both withdrawn 7 days 5
35.3 ± 12.79
Oestradiol benzoate alone
4
51.8 i 6.18
5-HTP added 2 days 12 days
4 4
5-HTP alone withdrawn 7 days
4
89.4 i 13.27" 157.6 i 19.74'* 58.9 ± 4.86
* P < 0.05. P < 0.01 (t-test) compared with values during treatment with oestradiol benzoate alone.
**
measured by 5-HIAA levels in the CSF, to between 30 ~o and 58 ~ of the oestrogentreated condition (Table HI). It was noticeable that each successive injection lowered 5-HIAA levels still further. After 4 days (at the time of the second PCPA injection) levels of 5-HIAA were 47 ~ of the oestradiol-treated condition, after 8 days, 42 ~ , and by the fourth injection (after 12 days), 40 ~ of normal. The levels of 5-HIAA in the CSF were still depressed (55 ~ ) after treatment with PCPA had been discontinued for a week and remained so even after 2 weeks without treatment (68 ~ o f normal). 20 mg/kg 5-HTP given every second day to animals receiving PCPA was sufficient to restore 5-HIAA in the CSF to levels not significantly different from those during oestrogen-treatment alone (Table III). After 12 days of combined 5-HTP and PCPA treatment, levels seemed well above, but were not significantly different from, the oestrogen-treated condition (158~o). However, the effect of 5-HTP was relatively transitory, and a week after the termination of 5 - H T P - P C P A treatment the average level recorded reflected only the inhibitory effects of PCPA (52 ~ of normal). In 4 ovariectomised oestrogen-treated females given 5-HTP, 5.HIAA increased significantly in the CSF to 173 ~o (after 2 days) and then to 205 ~ of its original levels
289 TABLE IV LEVELS (ng/ml) AND TURNOVERSOF 5-HIAA IN CSF OF 10 OVARIECTOMISEDRHESUSMONKEYSGIVEN STEROID HORMONES
Turnovers measured 2 h after probenecid as % of initial value. Levels
No treatment Oestradiol benzoate (15 beg or 25 #g/day) Oestradiol q- progesterone (15 #g q- 15 mg/day) Testosterone propionate (250/~g or 400 pg/day)
Turnover
Mean
Range
Mean
Range
76.1 72.2 78.4 67.4*
57.1-113.7 57.2-94.6 60.4-118.4 52.1-97.9
207.1 149.8' 177.4 130.6"
174.4-236.4 127.1-185.4 133.9-226.6 102.1-169.3
* P < 0.01 (Wilcoxon test, two-tailed) compared to values in untreated monkeys.
(after 12 days) (Table III). This findingis of significance in the context of the behavioural results obtained with these animals (see above). The results of administering oestradiol, oestradiol + progesterone or testosterone propionate on the levels of 5-HIAA in the CSF are shown in Table IV. Although oestradiol and testosterone both lowered 5-HIAA, the result was significant only after the latter hormone. However, clear alterations in turnover emerged (Table IV). Both oestradiol and testosterone propionate lowered the turnover rates of 5-HT, according to the method used. The effects of oestradiol were attenuated by the addition of progesterone. DISCUSSION
These results show that depletion of 5-HT by giving PCPA to female rhesus monkeys is able to reverse sexual unreceptivity induced by adrenalectomy and that this effect is itself reversed by giving 5-HTP. The only other substances which have, so far, been shown to counteract the effects of adrenalectomy1~ or adrenal suppression 15 in comparable ways have been testosterone propionate and androstenedione given systemically or very small amounts of testosterone propionate implanted intracerebrally within the anterior hypothalamus-preoptic area~L It seems very likely that PCPA exerts its effects on receptivity as a direct consequence of reducing cerebral 5-HT levels. PCPA is a relatively specific depletor of 5-HT 3° and, in these experiments, its effects on behaviour ran parallel with those on 5-HIAA levels in the CSF. Furthermore, adding 5-HTP (whose formation is prevented by PCPA) reversed the latter's effects on both behaviour and on 5-HIAA in the CSF. Finally, giving receptive females 5-HTP alone in doses which appeared to raise cerebral 5-HT to above normal levels reduced their sexual receptivity. Changes in catecholamines may occur after PCPA treatment3°, and have been implicated in the neural control of sexual behaviour in rats1, ~. However, administering 5-HTP would not replenish these substances. Any role which the latter may have in the sexual behaviour of female primates is still to be investigated.
290 There are few other reports of the effects of PCPA on the behaviour of monkeys. Maas et al. a4 gave 150 mg/kg orally for 2 weeks to 5 normal male and female stumptailed macaques (M. speciosa) caged in groups, and report reduced sexual activity in one male. Boelkins 5 gave 330-735 mg/kg for 7-17 days to 3 immature crab-eating macaques (M. fascieularis) caged together and also found decreased sexual interaction. Zitrin a5 reports experiments which appear to show that a single oral dose of 400 mg/kg given to male rhesus (M. mulatta) had inconsistent results on mounting activity, and that other experiments on adolescent males and females were also without effect. The animals studied in these previous experiments and those reported in this paper differ in 3 ways. Firstly, many of the monkeys used in the former were immature and this decreases the reliability of observations on sexual behaviour. Secondly, the dose of PCPA was very much larger and many of the animals (in the first 2 experiments at least) apparently showed toxic effects (e.g. weight loss, lassitude, ataxia) which make changes in behaviour relatively uninformative. Thirdly, and most important of all, PCPA was given to normal animals whereas, in our experiment, it was administered to monkeys in which a particular pattern of behaviour (receptivity) had been specifically reduced by withdrawing androgens. PCPA may thus be unable to stimulate behaviour to supra-normal levels in intact animals but be capable of restoring normal levels in hormone-deprived ones. A parallel finding has been reported to follow the administration of increasing doses of testosterone to castrated male guinea pigs, in which this hormone activated mounting behaviour to a maximum determined by the animals' somatic constitution 5a. However, PCPA has also been found to increase mounting behaviour in castrated male rats only if they received sub-maximal doses of testosterone 36. Sexual behaviour has been reported to be stimulated by treating ratslS, 19,~° and cats 2° with PCPA. It can substitute for the action of progesterone in female rats ~2 and induce mounting behaviour of both females 4v and other males by male rats 44 and cats 2°. Removal of the adrenals is said to prevent its effects on the behaviour of rats t4, though the operation itself possibly has marked effects on 5-HT levels as the animals were not given replacement corticosteroids, and changes in the latter may themselves alter 5-HT metabolism 4. A number of studies have now suggested that androgens play a considerable role in regulating receptivity in non-human primates as well as, it seems, in human females 45,46,4s. Since depleting 5-HT or giving androgens both restore receptivity to adrenalectomised (i.e. androgen-deprived) female monkeys, is it possible that androgens normally have their effect by acting upon a 5-HT-containing neural system ? Though no certain conclusions are possible at present, there are arguments in favour. Testosterone given to female monkeys lowered 5-HT turnover in the experiments reported here so far as this can be judged by the probenecid test. However, steroids might interact in some way with the effect of probenecid on the amine metabolite transportation mechanism. In so far as the results obtained reflect changes in the 5-HT turnover in those parts of the brain bordering the ventricles, they could equally be related to the effects of the hormone on other neural systems, such as those controlling the function of the anterior pituitary 21. The latter explanation is more likely
291 to apply to the similar (though lesser) effect of oestradiol on 5-HT turnover, since oestrogen is well known to alter gonadotrophin secretion in female monkeys2~,5~ but is yet to be proved to have an effect on their receptivity. In the anterior hypothalamus-preoptic area, where small implants of testosterone propionate activate sexual receptivity, the hormone is likely to act either on postsynaptic mechanisms or on the preterminal release or re-uptake of 5-HT in the anterior hypothalamus, though an additional direct action on the 5-HT-containing neuron cell bodies in the mesencephalon has not yet been investigated. It is interesting that progesterone has opposite effects on 5-HT turnover in female monkeys and rats 19. This could be correlated with the different role this hormone plays in sexual behaviour in the 2 species. In monkeys, progesterone has been reported to cause unreceptivity48 whereas, in rats, its initial effect is to potentiate the action of oestrogen in inducing oestrous behaviourS,9,sL 5-HT has been implicated in the neural control of a number of other behaviours, including eating33, aggression1° and states of arousal 27,2s,z5 and it should not be forgotten that depleting 5-HT may cause changes in sexual (and other) behaviour by a less specific mechanism. Animals may be rendered generally more sensitive to sensory stimuli (e.g. olfactory, visual or tactile) which elicit behavioural responses 51. Electric shocks, which presumably have a non-specific arousing effect on an animal's behaviour, have been found to induce sexual activity in male rats paired with females, but aggressive behaviour if the second animal was a maleL The conditions used in the experiments reported here are particularly conducive to sexual interaction and thus changes in this category of behaviour would be expected to occur. This interpretation does not, however, necessarily exclude the more specific one given above. Oestrogen has a well established and specific role in the control of sexual behaviour in species such as the rat, yet it also has effects on a number of other bodily functions, including eating12 and locomotionla. Furthermore, it may be that 5-HT-containing neural systems are involved in a variety of behaviours, in which case we need to understand how a hormone (such as testosterone) activates a particular category of behaviour (such as receptivity) by acting on only one part of the 5-HT system. Taken in the light of our previous work, female primates seem to differ from nonprimate mammals with regard to the hormones that regulate receptivity (androgen in the primate, oestrogen in the non-primate), but the neural site at which they act (anterior hypothalamus-preoptic area) and the mechanism of action (on a 5-HTcontaining neural system) may be similar. ACKNOWLEDGEMENTS
Supported in part by a grant from the Medical Research Council. P.B.G. was in receipt of a fellowship from the British Council. We thank the Department of Anatomy, University of Birmingham for excellent research facilities, Mr. J. D. Hamer for help with the adrenalectomies, Dr. R. F. Long (Roche Products) for supplying some of the PCPA, Mr. R. Overhill for the illustrations and Dr. E. B. Keverne for commenting on the manuscript.
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281
© ElsevierScientificPublishing Company,Amsterdam- Printed in The Netherlands
5-HYDROXYTRYPTAMINE IN THE CENTRAL NERVOUS SYSTEM AND SEXUAL RECEPTIVITY OF FEMALE RHESUS MONKEYS*
P. B. GRADWELL, B. J. EVERITT AND J. HERBERT (P.B. G.) Department of Anatomy, University of Rhodesia, Salisbury (Rhodesia) and ( B.J.E. and J.H. ) Department of Anatomy, Downing Street, Cambridge (Great Britain)
(Accepted December 2nd, 1974)
SUMMARY
The role of 5-hydroxytryptamine (5-HT) in the control of sexual receptivity in female rhesus monkeys has been studied in 24 adult females paired with 6 adult males, p-Chlorophenylalanine (PCPA, 75 mg/kg or 100 mg/kg, every fourth day), a selective inhibitor of 5-HT, was found to reverse unreceptivity induced by adrenalectomy in ovariectomised, oestrogen-treated females. PCPA-treated females presented more frequently and initiated more sexual behaviour, or else they refused fewer of the male's attempts to mount. These effects were in turn reversed by 5-hydroxytryptophan (5-HTP, 20 mg/kg every second day), when this was given to PCPAtreated animals. In addition, 5-HTP given alone to ovariectomised oestrogen-treated females reduced their receptivity. Parallel biochemical experiments showed that PCPA in the doses used lowered the levels of 5-HT in the brain as measured by the levels of 5-hydroxyindole-3-acetic acid (5-HIAA) in the CSF, and that these were restored by 5-HTP. Both oestradiol benzoate (15/~g/day for 10 days) and testosterone propionate (250/zg/day or 400/~g/day for 10 days) lowered the turn-over rates of 5-HT in the brain (as measured by the probenecid test) in ovariectomised female monkeys. These effects of oestradiol on turnover were antagonised by progesterone (15 mg/day for 10 days, given with oestradiol). A substance other than an adrenal androgen has thus been found to reverse the effects of adrenalectomy on sexual receptivity in female monkeys. It is therefore possible that androgens regulate receptivity in female monkeys by modifying the activity of 5-HT-containing neural systems.
* These experiments were carried out in the Department of Anatomy,Universityof Birmingham, Birmingham, Great Britain. Address reprint requests to B, J. Everitt.
282 INTRODUCTION
Androgens, secreted mainly from the adrenal cortex, appear to control sexual receptivity in female rhesus monkeys. Ovariectomised female monkeys are made unreceptive if their adrenals are removed 16 or suppressed with dexamethasone 15, but their sexual receptivity is restored by systemic injections of either testosterone propionate or androstenedione. Neither oestradiol nor adrenal secretions such as cortisol or progesterone are effective15,16. Very small amounts of testosterone propionate implanted stereotaxically into the anterior hypothalamus-preoptic area of the brain also induced sexual receptivity in adrenalectomised female monkeys, whereas similar implants in other sites (including the posterior hypothalamus) did not alter behaviour 17. Thus at least one site of action of these androgens in the brain is known. What is not clear is how androgens act on the nervous system. In sub-primate mammals monoamines, particularly 5-hydroxytryptamine (5-HT), have been implicated in the neural control of oestrous behaviour of the rat z7-4°,42,54, the cat 2'~ and the hamster4L Since different hormones appear to regulate receptivity in primates and non-primates, it is important to determine whether the underlying neural mechanisms are correspondingly different. The site of action of the effective hormones seems similar (see above). This paper describes experiments carried out to determine whether 5-HT has a role in controlling sexual receptivity in female primates. MATERIALS AND METHODS
Six adult male and 24 adult female rhesus monkeys (Macaca mulatta) were used. The males were intact, but the females had all been bilaterally ovariectomised several months before the experiments started. In addition, 10 of the females were then bilaterally adrenalectomised, using a technique described in detail elsewhere 16. The animals were carefully watched in the post-operative period, and all made excellent recoveries. Seven females had stainless steel intracranial cannulae fixed to the skull, their tips lying in the hypothalamus, as the result of a previous experiment 17. The cannulae were empty, and the behaviour of these females was undistinguishable from animals without such cannulae.
Behavioural observations Sexual behaviour between male-female pairs of monkeys was measured by methods given in detail elsewhere 1~,z~,23. Females were housed individually in cages (85 cm x 75 cm × 100 cm high) in a communal monkey house. Each female was paired with a male (always the same one) in a large observation cage (150 cm × 75 cm × 90 cm high) for 30 min daily and observed through a one-way-vision mirror. The following indices were derived from each series of 10 observations, as in previous work~1,24,25.
Sexual behaviour For the female. (a) Mean number of sexual presentations (invitations to the
283 male to mount); (b) proportion of mounts initiated by the female; (c) mean female's acceptance ratio (i.e. proportion of the male's attempts to mount allowed by the female); (d) total number of refusals to allow the male to mount during the series of 10 tests. For the male. (a) Mean number of mounts; (b) mean ejaculation time (from the first mount to ejaculation); (c) mean number of thrusts per intromitted mount; (d) mean mounting rate (mounts/min); (e) mean male's acceptance ratio (i.e. proportion of the female's presentations eliciting a mounting attempt by the male). This measure is a good indicator of the attractiveness of the female to the male, providing some presentations are made 24. The other components of the male's sexual activity are not considered in detail in this paper, since this is primarily concerned with alterations in the female's behaviour.
Aggressive behaviour The total number of 30-sec periods in which an aggressive gesture was shown by either animal. Social behaviour The total number of minutes monkeys spent grooming each other, or sitting in proximity within arm's length of one another, was recorded, together with the number of grooming invitations made by each animal. Treatments Females received oestradiol benzoate (in arachis oi1-10~o benzyl alcohol) 15 #g or 25 #g daily s.c. throughout the experiment. p-Chlorophenylalanine (PCPA) as the methyl ester hydrochloride, a potent inhibitor of 5-HT synthesis 30, was dissolved in Sorensen's phosphate buffer (pH 7.3). Monkeys received 75 or 100 mg/kg base every fourth day, beginning 4 days before the first observations, receiving 4 injections per series in all. L-5-Hydroxytryptophan (5-HTP) as the methyl ester hydrochloride was also dissolved in Sorensen's phosphate buffer (pH 7.8). Monkeys received 20 mg/kg base every second day, beginning 2 days before the first observation. Adrenalectomised animals were maintained on cortisol, given as hydrocortisone sodium succinate (20 mg/day) and deoxycorticosterone trimethylacetate (25 mg once monthly by deep intramuscular injection), and remained in excellent health throughout the experiments. Eight females were first tested while receiving either 15 #g or 25 #g oestradiol per day. They were then adrenalectomised, and together with 2 other adrenalectomised females from a previous experiment 17 they were tested again with the same males. All 10 then received PCPA. Finally, 5 received both PCPA and 5-HTP. Four ovariectomised females were tested while receiving oestradiol alone and then treated with 5-HTP. All females received the same dose of oestradiol under all treatment conditions.
284 A Presentations It It
20 ¸
.2
It It 15
10-
It It It m
e-
E
It tt
5It
It
It
i
i
O-
Female-initiated mounts .~ IO0It
$
E O1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8 9 1 0
1 2 3 4 5 6 7 8 9 1 0
125
610
Identity of female Fig. 1. Changes in the females' presentations (above) and the proportion (~) of mounts they initiate by presenting to the males (below). Treatments are A: ovariectomy alone; B: ovariectomy + adrenalectomy (with cortisol replacement); C: as B but receiving PCPA; D: as C but receiving 5-HTP. Doses used given in the text. Data are means per series (10 observations in each block): Identity of females: (1) 1886; (2) 2259; (3) 2263; (4) 2219; (5) 2261; (6) 2260; (7) 1887; (8) 2264; (9) 1992; (10) 1265. * P < 0.05. ** P < 0.01 compared with series B (Wilcoxon test). Females received oestradiol benzoate throughout the experiment. Biochemical measurements
Cerebrospinal fluid (CSF) was withdrawn from the cisterna magna of female monkeys under ketamine hydrochloride anaesthesia (7-8 mg/kg) between 09.00 and 11.00 h BST (British Standard Time) on the d a y of assay. Levels of 5-hydroxyindole-3-acetic acid (5-HIAA), the primary metabolite o f 5-HT in the CSF 6 were assayed spectrophotofluorimetrically, using the method of Ashcroft and SharmanS. In addition, turnover rates of 5-HT were measured using the probenecid test 6, 31,32. Levels of 5-HIAA were measured immediately before, and then 2 h after, an intravenous injection of probenecid sodium (20 mg/kg, p H 7.7-7.9) and turnover rates of 5-HT were then expressed as the percentage increase of 5-HIAA levels in the CSF during this 2-h period. Since sampling the CSF would have interfered with behaviourat observations,
285 TABLE I REFUSALS BY FEMALE TO ALLOW MALE TO MOUNT
Data given are total number of refusals per series (10 observations). Figures in parentheses correspond with those identifying females in Figs. 1 and 2. Treatments: A: ovariectomised; B: ovariectomised and adrenalectomised; C: as B but given PCPA; D: as C but given 5-HTP; E: given 5-HTP alone. Doses used given in text. - - indicates no data. Female no.
1886 2259 2263 2219 2261 2260 1887 2264 1992 1265
315 316 317 318
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
(11) (12) (13) (14)
Treatment
A
B
C
D
0 0 0 0 1 0 0 0 ---
3 1 7 2 4 70 28 0 0 4
0 1 0 2 1 10 4 0 0 0
6 19 --13 92 ---0
A
E
0 0 0 0
0 0 0 1
treatments given to animals during the behavioural tests were repeated where necessary. 5-HIAA levels were measured in 9 ovariectomised female monkeys, 7 of which had been adrenalectomised as well (and had taken part in the behavioural tests) under the following treatments: (1) no gonadal hormones, at least 2 months after operation; (2) receiving oestradiol alone; (3) after 4, 8 and 12 days on PCPA; (4) after I and 2 weeks following withdrawal of PCPA; (5) after 2 and 12 days of PCPA plus 5-HTP; and (6) 1 week after the withdrawal of PCPA plus 5-HTP treatment. F o u r ovariectomised animals were given 5-HTP alone, and measures were made (1) with no hormone treatment; (2) receiving oestradiol alone; (3) after 2 and 12 days o f 5-HTP treatment; and (4) 1 week after withdrawing 5-HTP. In the final experiment, 10 (different) ovariectomised female monkeys were used. Both the levels o f 5-HIAA and the turnover rates of 5-HT were measured under the following conditions: (1) no gonadal hormones, at least 2 months after ovariectomy; (2) after receiving 15/~g/day oestradiol for 10 days; (3) after 15 #g/day oestradiol plus 15 mg progesterone/day for 10 days; (4) after 250/~g or 400 #g/day testosterone propionate for 10 days; and (5) after receiving no hormones for at least 2 months. Two monkeys received treatment (4) before (2) and (3); this change in order had no effect on the results.
286 T A B L E 11 THE MALES~ ACCEPTANCE RATIOSOF THE FEMALES~ PRESENTATIONS See text for definition. - - indicates too few presentations to calculate ratio. See Table I for further explanation.
Female no.
1886 2259 2263 2219 2261 2260 1887 2264 1992 1265
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
Mean % per series A
B
C
D
69.2 66.2 94.7 76.0 94.7 75.0 83.3 87.9
57.1 80.0 --100 -92.3 100 100 75.0
68.2 67.7 90 91.7 93.3 -66.7 75.0 78.6 52.0*
61.4 100
100 --
76.0
* P < 0.01 (Wilcoxon test) compared to series B.
Statistical methods The behavioural data were subjected to a Friedman two-way analysis of variance. In those cases where the X~ statistic was associated with a probability of 0.05 or less the difference between individual treatments was tested by the Wilcoxon matchedpairs signed-ranks test49. A two-way analysis of variance was carried out on the biochemical data. Where the F statistic was associated with a probability of 0.05 or less, paired t-tests were carried out on the relevant comparisons. RESULTS
Behavioural observations Removing the adrenals from ovariectomised oestrogen-treated females caused a marked decrease in their sexual receptivity, confirming our earlier observationslS,16. All presented significantly less to the males and initiated a lower proportion of the sexual interaction (Fig, 1A and B). Eight females began to refuse the male's attempts to mount (Table I) whereas only one had ever done so before operation. PCPA reversed the effects of adrenatectomy. In 7 out of 10 cases, females presented significantly more and 6 initiated a greater proportion of sexual interaction; their behaviour after PCPA therefore resembled that before adrenalectomy, or (in earlier experiments) in adrenalectomised monkeys given testosterone (Fig. 1C)1~. Five females continued to refuse the male occasionally, much less frequently than before treatment with PCPA (Table I). Giving 5-HTP to 5 PCPA-treated females reversed the effects of PCPA by
287
.~
A Presentations
E
lit
E
i
Female-initiated: mounts
•~ 100-
0 ~. 50c E
O.
11121314
11121314
Identity of females
Fig. 2. Changes in the females' presentations (above) and the proportion of mounts they initiate by presenting to the males (below). Treatments are A: ovariectomy + oestradiol benzoate; E: as A but with 5-HTP added. Doses used given in the text. Identity of females: (11) 315; (12) 316; (13) 317; (14) 318. See Fig. 1 for further explanation. Statistical comparisons were made between treatments A and E. causing them to become unreceptive again. Four of the females' presentations were no longer significantly greater than after adrenalectomy (series B) and in 3 the proportion of female-initiated mounts decreased; 4 refused the males more often than they had after PCPA alone (Table I). Females received oestradiol throughout this experiment and so remained sexually attractive to the males, as shown by the consistently high males' acceptance ratios (Table II). Giving 5-HTP alone to 4 ovariectomised oestrogen-treated females made them less receptive (Fig. 2). Three presented significantly less and initiated less sexual interaction than they had before this treatment; there were similar changes in the behaviour of the fourth female, but these were not statistically significant (Fig. 2). However, the females' refusals o f the males' attempts to mount were not increased when they were given 5-HTP (Table I). Aggressive and social behaviour Neither PCPA nor PCPA + 5-HTP nor 5-HTP treatment alone had any clear effect on aggressive or social behaviour. Only one female (on one day) showed increased aggression toward the male when she was given PCPA -+- 5-HTP (female 1886). There were no consistent effects o f PCPA, PCPA + 5-HTP or 5-HTP alone on grooming or proximity behaviour. 5-HT levels and turnover PCPA in these doses considerably reduced levels of 5-HT in the brain, as
288 TABLE I11 LEVELSOF 5-HIAA (ng/ml) tN CSF OF FEMALERHESUSMONKEYSAFTERTREATMENTWITHPCPA AN[) 5-HTP Doses given in text. Number of monkeys
Levels (means 4- S.D.)
Oestradiol benzoate alone
9
68.2 i 9.87
PCPA added 4 days 8 days 12 days
9 9 9
31.9 i 6.49** 28.9 ± 6.99** 27.2 i 4.40**
PCPA + 5-HTP 2 days 12 days
5 5
70.2 ± 13.05 107.7 ± 15.86
PCPA + 5-HTP both withdrawn 7 days 5
35.3 ± 12.79
Oestradiol benzoate alone
4
51.8 i 6.18
5-HTP added 2 days 12 days
4 4
5-HTP alone withdrawn 7 days
4
89.4 i 13.27" 157.6 i 19.74'* 58.9 ± 4.86
* P < 0.05. P < 0.01 (t-test) compared with values during treatment with oestradiol benzoate alone.
**
measured by 5-HIAA levels in the CSF, to between 30 ~o and 58 ~ of the oestrogentreated condition (Table HI). It was noticeable that each successive injection lowered 5-HIAA levels still further. After 4 days (at the time of the second PCPA injection) levels of 5-HIAA were 47 ~ of the oestradiol-treated condition, after 8 days, 42 ~ , and by the fourth injection (after 12 days), 40 ~ of normal. The levels of 5-HIAA in the CSF were still depressed (55 ~ ) after treatment with PCPA had been discontinued for a week and remained so even after 2 weeks without treatment (68 ~ o f normal). 20 mg/kg 5-HTP given every second day to animals receiving PCPA was sufficient to restore 5-HIAA in the CSF to levels not significantly different from those during oestrogen-treatment alone (Table III). After 12 days of combined 5-HTP and PCPA treatment, levels seemed well above, but were not significantly different from, the oestrogen-treated condition (158~o). However, the effect of 5-HTP was relatively transitory, and a week after the termination of 5 - H T P - P C P A treatment the average level recorded reflected only the inhibitory effects of PCPA (52 ~ of normal). In 4 ovariectomised oestrogen-treated females given 5-HTP, 5.HIAA increased significantly in the CSF to 173 ~o (after 2 days) and then to 205 ~ of its original levels
289 TABLE IV LEVELS (ng/ml) AND TURNOVERSOF 5-HIAA IN CSF OF 10 OVARIECTOMISEDRHESUSMONKEYSGIVEN STEROID HORMONES
Turnovers measured 2 h after probenecid as % of initial value. Levels
No treatment Oestradiol benzoate (15 beg or 25 #g/day) Oestradiol q- progesterone (15 #g q- 15 mg/day) Testosterone propionate (250/~g or 400 pg/day)
Turnover
Mean
Range
Mean
Range
76.1 72.2 78.4 67.4*
57.1-113.7 57.2-94.6 60.4-118.4 52.1-97.9
207.1 149.8' 177.4 130.6"
174.4-236.4 127.1-185.4 133.9-226.6 102.1-169.3
* P < 0.01 (Wilcoxon test, two-tailed) compared to values in untreated monkeys.
(after 12 days) (Table III). This findingis of significance in the context of the behavioural results obtained with these animals (see above). The results of administering oestradiol, oestradiol + progesterone or testosterone propionate on the levels of 5-HIAA in the CSF are shown in Table IV. Although oestradiol and testosterone both lowered 5-HIAA, the result was significant only after the latter hormone. However, clear alterations in turnover emerged (Table IV). Both oestradiol and testosterone propionate lowered the turnover rates of 5-HT, according to the method used. The effects of oestradiol were attenuated by the addition of progesterone. DISCUSSION
These results show that depletion of 5-HT by giving PCPA to female rhesus monkeys is able to reverse sexual unreceptivity induced by adrenalectomy and that this effect is itself reversed by giving 5-HTP. The only other substances which have, so far, been shown to counteract the effects of adrenalectomy1~ or adrenal suppression 15 in comparable ways have been testosterone propionate and androstenedione given systemically or very small amounts of testosterone propionate implanted intracerebrally within the anterior hypothalamus-preoptic area~L It seems very likely that PCPA exerts its effects on receptivity as a direct consequence of reducing cerebral 5-HT levels. PCPA is a relatively specific depletor of 5-HT 3° and, in these experiments, its effects on behaviour ran parallel with those on 5-HIAA levels in the CSF. Furthermore, adding 5-HTP (whose formation is prevented by PCPA) reversed the latter's effects on both behaviour and on 5-HIAA in the CSF. Finally, giving receptive females 5-HTP alone in doses which appeared to raise cerebral 5-HT to above normal levels reduced their sexual receptivity. Changes in catecholamines may occur after PCPA treatment3°, and have been implicated in the neural control of sexual behaviour in rats1, ~. However, administering 5-HTP would not replenish these substances. Any role which the latter may have in the sexual behaviour of female primates is still to be investigated.
290 There are few other reports of the effects of PCPA on the behaviour of monkeys. Maas et al. a4 gave 150 mg/kg orally for 2 weeks to 5 normal male and female stumptailed macaques (M. speciosa) caged in groups, and report reduced sexual activity in one male. Boelkins 5 gave 330-735 mg/kg for 7-17 days to 3 immature crab-eating macaques (M. fascieularis) caged together and also found decreased sexual interaction. Zitrin a5 reports experiments which appear to show that a single oral dose of 400 mg/kg given to male rhesus (M. mulatta) had inconsistent results on mounting activity, and that other experiments on adolescent males and females were also without effect. The animals studied in these previous experiments and those reported in this paper differ in 3 ways. Firstly, many of the monkeys used in the former were immature and this decreases the reliability of observations on sexual behaviour. Secondly, the dose of PCPA was very much larger and many of the animals (in the first 2 experiments at least) apparently showed toxic effects (e.g. weight loss, lassitude, ataxia) which make changes in behaviour relatively uninformative. Thirdly, and most important of all, PCPA was given to normal animals whereas, in our experiment, it was administered to monkeys in which a particular pattern of behaviour (receptivity) had been specifically reduced by withdrawing androgens. PCPA may thus be unable to stimulate behaviour to supra-normal levels in intact animals but be capable of restoring normal levels in hormone-deprived ones. A parallel finding has been reported to follow the administration of increasing doses of testosterone to castrated male guinea pigs, in which this hormone activated mounting behaviour to a maximum determined by the animals' somatic constitution 5a. However, PCPA has also been found to increase mounting behaviour in castrated male rats only if they received sub-maximal doses of testosterone 36. Sexual behaviour has been reported to be stimulated by treating ratslS, 19,~° and cats 2° with PCPA. It can substitute for the action of progesterone in female rats ~2 and induce mounting behaviour of both females 4v and other males by male rats 44 and cats 2°. Removal of the adrenals is said to prevent its effects on the behaviour of rats t4, though the operation itself possibly has marked effects on 5-HT levels as the animals were not given replacement corticosteroids, and changes in the latter may themselves alter 5-HT metabolism 4. A number of studies have now suggested that androgens play a considerable role in regulating receptivity in non-human primates as well as, it seems, in human females 45,46,4s. Since depleting 5-HT or giving androgens both restore receptivity to adrenalectomised (i.e. androgen-deprived) female monkeys, is it possible that androgens normally have their effect by acting upon a 5-HT-containing neural system ? Though no certain conclusions are possible at present, there are arguments in favour. Testosterone given to female monkeys lowered 5-HT turnover in the experiments reported here so far as this can be judged by the probenecid test. However, steroids might interact in some way with the effect of probenecid on the amine metabolite transportation mechanism. In so far as the results obtained reflect changes in the 5-HT turnover in those parts of the brain bordering the ventricles, they could equally be related to the effects of the hormone on other neural systems, such as those controlling the function of the anterior pituitary 21. The latter explanation is more likely
291 to apply to the similar (though lesser) effect of oestradiol on 5-HT turnover, since oestrogen is well known to alter gonadotrophin secretion in female monkeys2~,5~ but is yet to be proved to have an effect on their receptivity. In the anterior hypothalamus-preoptic area, where small implants of testosterone propionate activate sexual receptivity, the hormone is likely to act either on postsynaptic mechanisms or on the preterminal release or re-uptake of 5-HT in the anterior hypothalamus, though an additional direct action on the 5-HT-containing neuron cell bodies in the mesencephalon has not yet been investigated. It is interesting that progesterone has opposite effects on 5-HT turnover in female monkeys and rats 19. This could be correlated with the different role this hormone plays in sexual behaviour in the 2 species. In monkeys, progesterone has been reported to cause unreceptivity48 whereas, in rats, its initial effect is to potentiate the action of oestrogen in inducing oestrous behaviourS,9,sL 5-HT has been implicated in the neural control of a number of other behaviours, including eating33, aggression1° and states of arousal 27,2s,z5 and it should not be forgotten that depleting 5-HT may cause changes in sexual (and other) behaviour by a less specific mechanism. Animals may be rendered generally more sensitive to sensory stimuli (e.g. olfactory, visual or tactile) which elicit behavioural responses 51. Electric shocks, which presumably have a non-specific arousing effect on an animal's behaviour, have been found to induce sexual activity in male rats paired with females, but aggressive behaviour if the second animal was a maleL The conditions used in the experiments reported here are particularly conducive to sexual interaction and thus changes in this category of behaviour would be expected to occur. This interpretation does not, however, necessarily exclude the more specific one given above. Oestrogen has a well established and specific role in the control of sexual behaviour in species such as the rat, yet it also has effects on a number of other bodily functions, including eating12 and locomotionla. Furthermore, it may be that 5-HT-containing neural systems are involved in a variety of behaviours, in which case we need to understand how a hormone (such as testosterone) activates a particular category of behaviour (such as receptivity) by acting on only one part of the 5-HT system. Taken in the light of our previous work, female primates seem to differ from nonprimate mammals with regard to the hormones that regulate receptivity (androgen in the primate, oestrogen in the non-primate), but the neural site at which they act (anterior hypothalamus-preoptic area) and the mechanism of action (on a 5-HTcontaining neural system) may be similar. ACKNOWLEDGEMENTS
Supported in part by a grant from the Medical Research Council. P.B.G. was in receipt of a fellowship from the British Council. We thank the Department of Anatomy, University of Birmingham for excellent research facilities, Mr. J. D. Hamer for help with the adrenalectomies, Dr. R. F. Long (Roche Products) for supplying some of the PCPA, Mr. R. Overhill for the illustrations and Dr. E. B. Keverne for commenting on the manuscript.
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