Physiology&Behavior,Voi. 23, pp. 701-707. PergamonPress and Brain Research Publ., 1979. Printed in the U.S.A.
Parachlorophenylalanine and Copulatory Behaviour in Neonatally Castrated Male Rats J A N E T E. B O O T H , P I E T E R V A N D E R S C H O O T * A N D P E T E R M O L E M A N t
Department of Physiology, Charing Cross Hospital Medical School, Fulham Palace Road, London W6 8RF, England and *Departments of Endocrinology, Growth and Reproduction and tPsychiatry, Faculty of Medicine, Erasmus University, Rotterdam, The Netherlands R e c e i v e d 5 O c t o b e r 1978 BOOTH, J. E., P. VAN DER SCHOOT AND P. MOLEMAN. Parachlorophenylalanine and copulatory behaviour in neonatally castratedmalerats. PHYSIOL. BEHAV. 23(4) 701-707, 1979.--Masculine copulatory behaviour was studied in rats which had been castrated as adults or at birth to examine the hypothesis that the behavioural defects shown by neonatally castrated rats result from changes in brain systems involving the neurotransmitter 5-hydroxytryptamine (5-HT). One group of neonatally castrated rats was injected with the non-aromatizable androgen dihydrotestosterone propionate (DHTP) for the first 5 or 15 days after birth; a second group received no androgen at this stage. Animals castrated as adults or castrated at birth and treated with DHTP during infancy showed normal genital development in response to injections of testosterone propionate (TP); genital development was reduced in the neonatally castrated rats which did not receive DHTP. Copulatory behaviour was studied in adulthood in all three groups of rats during treatment with TP with or without injections of the drug parachlorophenylalanine (PCPA), a specific depictor of brain concentrations of 5-HT. PCPA increased the intromission frequency and reduced the ejaculation latency amongst the rats castrated as adults. The drug reduced the numbers of intromissions before the first ejaculation and facilitated ejaculation in most, but not all of the rats castrated at birth and treated with DHTP during infancy. However, the copulatory behaviour of rats castrated at birth and not given DHTP was almost unaffected by PCPA. These results suggest that normal penis development is necessary for PCPA to stimulate masculine copulatory behaviour in rats, Attempts to correlate the different effects of PCPA in the three groups of rats with differences in the concentrations of 5-HT in their brain during adulthood, or with differences in their sensitivities to PCPA were unsuccessful. In all three groups, both the initial concentrations of 5-HT and the specific dose-related reduction in response to PCPA were similar. These results do not support therefore the hypothesis that altered development of systems involving 5-HT is responsible for the behavioural defects of male rats castrated at birth. Part of this study has been published as an abstract [5]. Neonatal castration
Ejaculation
5 a-Dihydrotestosterone
M A L E rats which have been castrated at birth are deprived of testicular androgens at a critical stage of development. When, as adults, they are injected with testosterone propionate (TP), they will readily mount oestrous female rats but fail to ejaculate [2]. This failure may arise because the genitalia of neonatally castrated rats remain poorly developed even after prolonged treatment with TP during adulthood [1]. However, insufficient genital development cannot provide the full explanation for the absence of ejaculation. The reduced androgen dihydrotestosterone and the synthetic androgen fluoxymesterone each appear to act as an adequate substitution for the neonatal testicular hormones on the developing genitalia: neonatally castrated rats which have received these steroids during infancy show normal penis development after treatment with TP as adults. Such rats can achieve frequent penile intromissions; however, they rarely ejaculate [4, 10, 11, 23]. These findings suggest that the central nervous mechanisms which control masculine copulatory behaviour, especially ejaculation, are defective in rats which have matured without the influence of testicular an-
5-Hydroxytryptamine
Parachlorophenylalanine
drogens. Several studies have suggested that the neurotransmitter 5-hydroxytryptamine (5-HT) plays a role both in the early post-natal organization of the neural substrate [ 12,15] and in the display of masculine copulatory behaviour during adulthood [18, 19, 22, 24, 26]. Also, masculine copulatory behaviour, including ejaculation, is facilitated in female rats by reducing the brain concentrations of 5-HT [7, 21, 25]. In these studies parachlorophenylalanine (PCPA) was the agent used to cause specific depletion of 5-HT in the brain [13]. We therefore set out to compare the effects of PCPA on copulatory behaviour and on brain concentrations of 5-HT in rats which were castrated at birth with those in rats castrated as adults. GENERAL METHOD
Animals The experiments were performed with locally bred (RxU)F~ hybrid rats derived from two inbred Wistar sub-
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/100701-07501.20/0
702 strains. The rats were maintained in controlled environment (lights on 05:00-19:00 hr; temperature 20-23°C) and were allowed free access to pellet food and tap water.
Neonatal Treatment Two experiments involved the castration of newborn rats. Male rats were castrated under cryoanaesthesia within 2-16 hr after birth (=Day 1). The testes were removed via a single midline incision in the abdominal wall just cranial to the top of the penis. The incision was sutured with atraumatic sewing material. Survival rate of the procedure was above 9(1%. Rats in which the suturing of the abdominal wall was apparently incomplete so that the intestines bulged through the abdominal muscular wall beneath the skin were eliminated from the behavioural studies. In one experiment neonatally castrated rats were left untreated or injected with arachis oil SC (0.05 ml) on Days 1,3, and 5 (day of birth=Day 1). In the other experiment the rats received SC injections of dihydrotestosterone propionate (DHTP) in 0.05 ml arachis oil (0.1 mg DHTP on Days 1, 3, and 5 or 1 mg DHTP on Days I, 3, 5, 10, and 15). The animals were weaned on Days 22-25 and kept together in groups of 4-6.
Treatment During Adulthood Two to three weeks before the start of behavioural tests the animals were transferred to a room with a reversed lighting scheme (lights on 10:00--20:00 hr). From this time on and throughout the period of testing they were injected with TP (0.7 mg per week divided over three weekly injections of 0.2, 0.2, and 0.3 mg respectively). Behavioural tests were conducted during the dark period of the light cycle. For behavioural testing, the animals were placed in individual semicircular glass-fronted observation cages. They were allowed to adapt for 30-60 min before a receptive female rat was introduced (an ovariectomized animal pretreated with oestradiol and progesterone). The following behaviour items were recorded: mount, mount without penile intromission but with pelvic thrusting; intromission, mount with penile intromission; ejaculation, mount with a final deep intromission followed by slow dismounting, genital grooming, and a period of sexual inactivity. The contact latency (the period between the introduction of the female and the first mount or intromission), the ejaculation latency (the period between the first mount or intromission and the first ejaculation), and the post-ejaculatory interval (PEI, the time between the first ejaculation and the first mount or intromission of the next series of copulations) were also measured. Whenever possible, the numbers of mounts and intromissions to the second ejaculation were recorded also as well as the second ejaculation latency (the period between the end of the PEI and the second ejaculation). The observations lasted 30 rain (in one study 60 rain) but were stopped earlier if two ejaculations had occurred within this period.
Statistical Analysis Statistical analysis comprised one-way or two-way analysis of variance of behavioural data followed by Student's t-tests or Tuckey's HSD-test. Data obtained with the analysis of the brain concentrations of 5-HT were analysed by Kruskal-Wallis analysis of variance and Mann-Whitney U-tests or with Student's t-test. Differences were judged
BOOTH, VAN DER SCHOOT AND MOLEMAN significant if they were associated with two-lailed probabilities of 5% or less. EXPERIMENT 1: PCPA AND COPULATORY BEHAVIOUR IN RATS CASTRATED AS ADUt,TS
Method Twelve 4-month-old rats were castrated and injected with TP. After three weeks their copulatory behaviour with receptive female rats was recorded during a single 30 rain period. After that session the animals were injected IP with 0.5 ml saline or with PCPA (Labkemi, G6teborg, Sweden; 30 mg/rat, i.e. 100 + l0 mg per kilogram bodyweight, in accordance to doses used in earlier studies [7, 22, 261) dissolved in saline. Daily observations, followed the first three days by daily injections of saline or the same amount of PCPA, were conducted for the next four days. There were three more tests during the subsequent week.
Results The results are presented in Fig. 1. There was an immediate reduction of the first ejaculation latency 24 hr after the first injection of PCPA. This effect of PCPA persisted during the whole period of treatment but had disappeared at seven days after cessation of treatment. The number of intromissions to the first ejaculation seemed to be lower during treatment with PCPA, but this difference was not statistically significant, Treatment with PCPA also reduced the second ejaculation latency. The number of intromissions to the second ejaculation was significantly reduced during the last test with PCPA and the first test after cessation of treatment. The obvious effect of PCPA on ejaculation latency, but not on numbers of intromissions to ejaculation, was reflected in the increased intromission frequency (number of intromissions/min). During treatment with PCPA, the mean (-+ SEM) intromission frequency before the first ejaculation was 3.3 -+ 0.2, and 3.0 _+ 0.2 before the second ejaculation. The values during treatment with saline were 1.6 -+ 0.2 and 2.0 _+ 0.2 respectively. Treatment with PCPA did not significantly reduce the length of the PEI. EXPERIMENT 2: PCPA AND COPULATORY BEHAVIOUR IN RATS CASTRATED AT BIRTH
Method Nineteen rats were castrated at birth. Ten rats received injections of arachis oil on Days 1, 3, and 5; the remaining 9 rats were left untreated. From about the age of 90 days the rats were injected with TP. After 3--5 preliminary tests with receptive female rats---during which tests the males all showed frequent mounting behaviour, some intromissions, but no ejaculation--the males were injected with the same amount of PCPA as used in Experiment 1, They were tested the next day. Daily observations of copulatory behaviour, followed by daily injections of PCPA, were performed throughout the next four days. Two more behavioural tests took place during the subsequent week. Seven of the rats were then injected with double the dose of TP. After three weeks they were subjected to a series of behaviour tests identical to that described in Experiment 1.
Results The results are presented in Table 1. There were signifi-
COPULATORY BEHAVIOUR IN NEONATALLY CASTRATED RATS
numberof intromlssionsuntil first ejoculatlon
20 -
703
numberof intromissions until secondejaculation
T
/
-10
16.
12-
8-
4-
0-
!
,
.
,
I0 min,
!
,
!
u
v
u
I
e'~iaculatlatency n sec°nd~u a~~niY
i
0
5mln.
t,,r" 0
0
8-
m
i
n
.
post-ejaculatory interval ~
O!
2
3
4
.5
6
7
8 testnumber
FIG. 1. PCPA and copulatory behaviour in rats castrated as adults. Copulatory behaviour was tested prior to (Test 1), during (Tests 2-5) and after (Tests 6-8) daily treatment with 30 mg PCPA/rat (----) or saline only ( ). Statistically significant effects of PCPA appeared as significant groups × tests interactions in two-way analysis of variance. Significant interactions were further analysed with t-ratio tests. PCPA significantly reduced the numbers of intromissions to the second ejaculation, F(7,70)=2.32, p
Parachlorophenylalanine and Copulatory Behaviour in Neonatally Castrated Male Rats J A N E T E. B O O T H , P I E T E R V A N D E R S C H O O T * A N D P E T E R M O L E M A N t
Department of Physiology, Charing Cross Hospital Medical School, Fulham Palace Road, London W6 8RF, England and *Departments of Endocrinology, Growth and Reproduction and tPsychiatry, Faculty of Medicine, Erasmus University, Rotterdam, The Netherlands R e c e i v e d 5 O c t o b e r 1978 BOOTH, J. E., P. VAN DER SCHOOT AND P. MOLEMAN. Parachlorophenylalanine and copulatory behaviour in neonatally castratedmalerats. PHYSIOL. BEHAV. 23(4) 701-707, 1979.--Masculine copulatory behaviour was studied in rats which had been castrated as adults or at birth to examine the hypothesis that the behavioural defects shown by neonatally castrated rats result from changes in brain systems involving the neurotransmitter 5-hydroxytryptamine (5-HT). One group of neonatally castrated rats was injected with the non-aromatizable androgen dihydrotestosterone propionate (DHTP) for the first 5 or 15 days after birth; a second group received no androgen at this stage. Animals castrated as adults or castrated at birth and treated with DHTP during infancy showed normal genital development in response to injections of testosterone propionate (TP); genital development was reduced in the neonatally castrated rats which did not receive DHTP. Copulatory behaviour was studied in adulthood in all three groups of rats during treatment with TP with or without injections of the drug parachlorophenylalanine (PCPA), a specific depictor of brain concentrations of 5-HT. PCPA increased the intromission frequency and reduced the ejaculation latency amongst the rats castrated as adults. The drug reduced the numbers of intromissions before the first ejaculation and facilitated ejaculation in most, but not all of the rats castrated at birth and treated with DHTP during infancy. However, the copulatory behaviour of rats castrated at birth and not given DHTP was almost unaffected by PCPA. These results suggest that normal penis development is necessary for PCPA to stimulate masculine copulatory behaviour in rats, Attempts to correlate the different effects of PCPA in the three groups of rats with differences in the concentrations of 5-HT in their brain during adulthood, or with differences in their sensitivities to PCPA were unsuccessful. In all three groups, both the initial concentrations of 5-HT and the specific dose-related reduction in response to PCPA were similar. These results do not support therefore the hypothesis that altered development of systems involving 5-HT is responsible for the behavioural defects of male rats castrated at birth. Part of this study has been published as an abstract [5]. Neonatal castration
Ejaculation
5 a-Dihydrotestosterone
M A L E rats which have been castrated at birth are deprived of testicular androgens at a critical stage of development. When, as adults, they are injected with testosterone propionate (TP), they will readily mount oestrous female rats but fail to ejaculate [2]. This failure may arise because the genitalia of neonatally castrated rats remain poorly developed even after prolonged treatment with TP during adulthood [1]. However, insufficient genital development cannot provide the full explanation for the absence of ejaculation. The reduced androgen dihydrotestosterone and the synthetic androgen fluoxymesterone each appear to act as an adequate substitution for the neonatal testicular hormones on the developing genitalia: neonatally castrated rats which have received these steroids during infancy show normal penis development after treatment with TP as adults. Such rats can achieve frequent penile intromissions; however, they rarely ejaculate [4, 10, 11, 23]. These findings suggest that the central nervous mechanisms which control masculine copulatory behaviour, especially ejaculation, are defective in rats which have matured without the influence of testicular an-
5-Hydroxytryptamine
Parachlorophenylalanine
drogens. Several studies have suggested that the neurotransmitter 5-hydroxytryptamine (5-HT) plays a role both in the early post-natal organization of the neural substrate [ 12,15] and in the display of masculine copulatory behaviour during adulthood [18, 19, 22, 24, 26]. Also, masculine copulatory behaviour, including ejaculation, is facilitated in female rats by reducing the brain concentrations of 5-HT [7, 21, 25]. In these studies parachlorophenylalanine (PCPA) was the agent used to cause specific depletion of 5-HT in the brain [13]. We therefore set out to compare the effects of PCPA on copulatory behaviour and on brain concentrations of 5-HT in rats which were castrated at birth with those in rats castrated as adults. GENERAL METHOD
Animals The experiments were performed with locally bred (RxU)F~ hybrid rats derived from two inbred Wistar sub-
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/100701-07501.20/0
702 strains. The rats were maintained in controlled environment (lights on 05:00-19:00 hr; temperature 20-23°C) and were allowed free access to pellet food and tap water.
Neonatal Treatment Two experiments involved the castration of newborn rats. Male rats were castrated under cryoanaesthesia within 2-16 hr after birth (=Day 1). The testes were removed via a single midline incision in the abdominal wall just cranial to the top of the penis. The incision was sutured with atraumatic sewing material. Survival rate of the procedure was above 9(1%. Rats in which the suturing of the abdominal wall was apparently incomplete so that the intestines bulged through the abdominal muscular wall beneath the skin were eliminated from the behavioural studies. In one experiment neonatally castrated rats were left untreated or injected with arachis oil SC (0.05 ml) on Days 1,3, and 5 (day of birth=Day 1). In the other experiment the rats received SC injections of dihydrotestosterone propionate (DHTP) in 0.05 ml arachis oil (0.1 mg DHTP on Days 1, 3, and 5 or 1 mg DHTP on Days I, 3, 5, 10, and 15). The animals were weaned on Days 22-25 and kept together in groups of 4-6.
Treatment During Adulthood Two to three weeks before the start of behavioural tests the animals were transferred to a room with a reversed lighting scheme (lights on 10:00--20:00 hr). From this time on and throughout the period of testing they were injected with TP (0.7 mg per week divided over three weekly injections of 0.2, 0.2, and 0.3 mg respectively). Behavioural tests were conducted during the dark period of the light cycle. For behavioural testing, the animals were placed in individual semicircular glass-fronted observation cages. They were allowed to adapt for 30-60 min before a receptive female rat was introduced (an ovariectomized animal pretreated with oestradiol and progesterone). The following behaviour items were recorded: mount, mount without penile intromission but with pelvic thrusting; intromission, mount with penile intromission; ejaculation, mount with a final deep intromission followed by slow dismounting, genital grooming, and a period of sexual inactivity. The contact latency (the period between the introduction of the female and the first mount or intromission), the ejaculation latency (the period between the first mount or intromission and the first ejaculation), and the post-ejaculatory interval (PEI, the time between the first ejaculation and the first mount or intromission of the next series of copulations) were also measured. Whenever possible, the numbers of mounts and intromissions to the second ejaculation were recorded also as well as the second ejaculation latency (the period between the end of the PEI and the second ejaculation). The observations lasted 30 rain (in one study 60 rain) but were stopped earlier if two ejaculations had occurred within this period.
Statistical Analysis Statistical analysis comprised one-way or two-way analysis of variance of behavioural data followed by Student's t-tests or Tuckey's HSD-test. Data obtained with the analysis of the brain concentrations of 5-HT were analysed by Kruskal-Wallis analysis of variance and Mann-Whitney U-tests or with Student's t-test. Differences were judged
BOOTH, VAN DER SCHOOT AND MOLEMAN significant if they were associated with two-lailed probabilities of 5% or less. EXPERIMENT 1: PCPA AND COPULATORY BEHAVIOUR IN RATS CASTRATED AS ADUt,TS
Method Twelve 4-month-old rats were castrated and injected with TP. After three weeks their copulatory behaviour with receptive female rats was recorded during a single 30 rain period. After that session the animals were injected IP with 0.5 ml saline or with PCPA (Labkemi, G6teborg, Sweden; 30 mg/rat, i.e. 100 + l0 mg per kilogram bodyweight, in accordance to doses used in earlier studies [7, 22, 261) dissolved in saline. Daily observations, followed the first three days by daily injections of saline or the same amount of PCPA, were conducted for the next four days. There were three more tests during the subsequent week.
Results The results are presented in Fig. 1. There was an immediate reduction of the first ejaculation latency 24 hr after the first injection of PCPA. This effect of PCPA persisted during the whole period of treatment but had disappeared at seven days after cessation of treatment. The number of intromissions to the first ejaculation seemed to be lower during treatment with PCPA, but this difference was not statistically significant, Treatment with PCPA also reduced the second ejaculation latency. The number of intromissions to the second ejaculation was significantly reduced during the last test with PCPA and the first test after cessation of treatment. The obvious effect of PCPA on ejaculation latency, but not on numbers of intromissions to ejaculation, was reflected in the increased intromission frequency (number of intromissions/min). During treatment with PCPA, the mean (-+ SEM) intromission frequency before the first ejaculation was 3.3 -+ 0.2, and 3.0 _+ 0.2 before the second ejaculation. The values during treatment with saline were 1.6 -+ 0.2 and 2.0 _+ 0.2 respectively. Treatment with PCPA did not significantly reduce the length of the PEI. EXPERIMENT 2: PCPA AND COPULATORY BEHAVIOUR IN RATS CASTRATED AT BIRTH
Method Nineteen rats were castrated at birth. Ten rats received injections of arachis oil on Days 1, 3, and 5; the remaining 9 rats were left untreated. From about the age of 90 days the rats were injected with TP. After 3--5 preliminary tests with receptive female rats---during which tests the males all showed frequent mounting behaviour, some intromissions, but no ejaculation--the males were injected with the same amount of PCPA as used in Experiment 1, They were tested the next day. Daily observations of copulatory behaviour, followed by daily injections of PCPA, were performed throughout the next four days. Two more behavioural tests took place during the subsequent week. Seven of the rats were then injected with double the dose of TP. After three weeks they were subjected to a series of behaviour tests identical to that described in Experiment 1.
Results The results are presented in Table 1. There were signifi-
COPULATORY BEHAVIOUR IN NEONATALLY CASTRATED RATS
numberof intromlssionsuntil first ejoculatlon
20 -
703
numberof intromissions until secondejaculation
T
/
-10
16.
12-
8-
4-
0-
!
,
.
,
I0 min,
!
,
!
u
v
u
I
e'~iaculatlatency n sec°nd~u a~~niY
i
0
5mln.
t,,r" 0
0
8-
m
i
n
.
post-ejaculatory interval ~
O!
2
3
4
.5
6
7
8 testnumber
FIG. 1. PCPA and copulatory behaviour in rats castrated as adults. Copulatory behaviour was tested prior to (Test 1), during (Tests 2-5) and after (Tests 6-8) daily treatment with 30 mg PCPA/rat (----) or saline only ( ). Statistically significant effects of PCPA appeared as significant groups × tests interactions in two-way analysis of variance. Significant interactions were further analysed with t-ratio tests. PCPA significantly reduced the numbers of intromissions to the second ejaculation, F(7,70)=2.32, p
