Female lordosis behavior in estrogen-primed male rats treated with p-chlorophenylalanine or α-methyl-p-tyrosine

HORMONES AND BEHAVIOR

3,181-189

(1972)

Female Lordosis Behavior in Estrogen-Primed Male Rats Treated with p-Chlorophenylalanine or a-Methyl-p-Tyrosine

PER SijDERSTEN

and SVEN AHLENIUS

Departments of Psychology and Pharmacology, University of GBteborg, Sweden

The injection of a-methyl-p-tyrosine methylester hydrochloride, 100 mg/kg ip, facilitated lordosis in castrated male rats, treated daily with 50 pg/kg estradiol benzoate. Injection of p-chlorophenylalanine methylester hydrochloride, 150 rndkg ip, did not affect the lordosis behavior. The effect of a-methyl-p-tyrosine was abolished by the administration of L-3,4dihydroxyphenylalanine methylester hydrochloride, 50 mg/kg ip, in combination with an inhibitor of peripheral dopa decarboxylase, Nf-(DL-seryl)@-(2,3,4_trihydroxybenzyl) hydrazine, 50 mg/kg ip. The results suggest that catecholamine synthesis inhibition is responsible for the facilitation of lordosis behavior seen in estrogen-pretreated male rats after administration of central monoaminedepleting agents. The mechanism whereby drugs that interfere with central monoamine neurotransmission facilitate lordosis behavior in rats is discussed.

Boling and Blandau (1939) and Beach (1942) demonstrated that receptivity in ovariectomized female rats may be induced with combined estrogenprogesterone treatment. More recently Davidson et al. (1968) showed that repeated estrogen injections alone can stimulate female sex behavior. Adult male rats, castrated at 30 days of age, will not display lordosis in response to combined estrogen-progesterone treatment (Grady, Phoenix, and Young, 1965). However, repeated treatment with high doses of estrogen will result in lordosis behavior in male rats that differs little from that ordinarily seen in the female (Davidson, 1969; A&n-Engelbrektsson et al., 1970). Thus, the male rat is capable of showing the female lordosis response, provided estrogen is given in doses appreciably larger than those necessary in the female, whereas progesterone if given in addition to estrogen apparently does not facilitate this behavior (Davidson and Levine, 1969). Meyerson (1964) has shown that the facilitatory action of progesterone on estrogen-activated lordosis behavior in the female rat can be substituted for by drugs known to interfere with central monoaminergic neurotransmission. Thus, drugs which deplete central monoamines (reserpine and tetrabenazine) will facilitate lordosis if administered to estrogen-primed ovariectomized females (Meyerson, 1964), and treatment with a monoamine oxidase inhibitor Copyright 0 1972 by Academic Press, Inc. 181 All rights of reproduction

in any form reserved.

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(pargyline), which increases central levels of monoamines, will impair estrogen-progesterone-activated estrous behavior when combined with a low dose of 5hydroxytryptophan (Meyerson, 1964). We have confirmed that tetrabenazine facilitates lordosis in the female rat (Ahlenius et al., 1972) and also demonstrated that castrated estrogen-treated male rats will show increments in lordosis behavior if injected with tetrabenazine (Larsson and Sodersten, 1971). This facilitatory action of tetrabenazine was blocked if the rats were pretreated with nialamide (Larsson and Sodersten, 1971). Thus, in the male as well as in the female low levels of central monoamines are correlated with increased lordosis behavior, and conversely high levels of central monoamines result in suppression of lordosis behavior. In our initial report on males (Larsson and Sodersten, 1971) nothing was said about the relative involvement of different central monoamines. The experiments herein reported were designed to investigate the respective roles of central catecholamines and 5hydroxytryptamine (5-HT), for the facilitatory action on lordosis behavior seen in male rats treated with a central monoamine-depleting agent. This was accomplished by the use of inhibitors of catecholamine and 5HT synthesis, i.e., a-methyl-p-tyrosine methylester hydrochloride (Corrodi and Hansson, 1966) and p-chlorophenylalanine methylester hydrochloride (Koe and Weissman, 1966).

METHODS Subjects. Sixty-nine male rats from a locally maintained strain of albinos were used in this series of experiments. Procedure. All rats were castrated at 30 days of age. When 75-80 days old and weighing approximately 280 g, the animals were given daily injections of 50 fig/kg estradiol benzoate. This hormone treatment was maintained throughout the experiments. Injected materials. Estradiol benzoate (Dimenformon @ Organon) was dissolved in oleum arachidis and injected in a volume of 1 .O ml/kg SCp-chlorophenylalanine methylester hydrochloride (H 69/ 17, Hassle, PCPA), a-methylp-tyrosine methylester hydrochloride (H 44/68, Hassle, a-MT), L-3,4-dihydroxyphenylalanine methylester hydrochloride (H 19/6 1, Hassle, L -dopa) and N’ -(DL-Seryl)-~2-(2,3,4-trihydroxybenzyl) hydrazine (Ro 4-4602, Hoffman-La Roche) were dissolved in isotonic saline, and the drugs were injected intraperitoneally in a volume of 2.0 ml/kg. Experiments with PCPA. After 10 days of hormone treatment, 34 rats were randomly divided into two groups. One group (N=17) received injections of 150 mg/kg PCPA, and the other (J/=17) served as a saline-injected control group. Behavioral tests were conducted according to the time schedule presented below (behavioral testing procedure).

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Experiments with a-MT. As in the above experiment, after 10 daily hormone injections, 35 rats were randomly assigned to either of two groups, an experimental (N=18) and a control (W17) group. Experimental subjects were thereafter injected with 100 mg/kg a-MT, and controls were injected with saline. Behavioral testing procedure is presented below. After finding that a-MT facilitates lordosis in male rats, we tried to reverse this effect by administrating the catecholamine precursor L-dopa after a-MT treatment. All animals that took part in the experiment with a-MT alone were used. They received estrogen for 3 additional days and were thereafter randomly divided into three groups, group I (N=12), group II (N=12) and group III (N=ll). These groups were then injected with a-MT, a-MT + L-dopa, and with saline, respectively. All rats received an injection of an inhibitor of peripheral dopa decarboxylase Ro 4-4602. a-MT was given at a dose of 100 mg/kg, L-dopa at 50 mg/kg and Ro 4-4602 at 50 mglkg. The drug injection schedule in relation to tests for lordosis is outlined in Table 1. Behavioral tests were given as described in the section below. Finally, to see whether t-dopa alone affects the display of lordosis in male rats, all rats in group III (i.e., saline-injected controls) were given an injection of Ro 4-4602 50 mg/kg followed lh hr later by 50 mglkg L-dopa, and then tested for lordosis 1 hr after L-dopa treatment. This experiment was performed 1 day after the experiment described above (combined a-MT and L-dopa experiment) and the animals served as their own controls. Behavioral testing procedure. Sex behavior tests were started 4 days after the first estrogen injection, and thereafter given daily at Days 5-9. As in female rats (Ahlenius et al., 1972) such prolonged hormonal treatment of castrated male rats results in a steady submaximal response level, and is therefore suitable for investigating drug effects over time. Ten days after the onset of hormone treatment the animals were injected with drugs as described above. PCPA- and a-MT-treated rats were then tested 2, 8, and 26 hr later. Animals injected with a-MT, a-MT + L-dopa, and saline were tested only once, 2 hr after the first injection (see Table I). A circular (50 cm diam) Plexiglas cage with a sawdust-covered floor was used as a test arena. All test animals were presented to two sexually vigorous stimulus males. An experimental rat remained with the stimulus males until mounted 10 times. If the stimulus males failed to mount, the test male was TABLE

1

Drug Injection Schedule in Relation to Test for Lordosis Treatment at

0 hr

30 min

1 hr

2 hr

Group I Group II Group III

a-MT a-MT NaCl

Ro 4-4602 Ro 4-4602 Ro 4-4602

Ldopa NaCl NaCl

Test for lordosis

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removed and placed with two other males until mounted the required 10 times. To be scored as displaying lordosis, an animal had to show concave back flexion, tail deviation, and maximal neck extension, i.e., those behavioral components normally seen in a lordosing receptive female rat. A lordosis quotient (lordosis to mount ratio, i.e., total number of lordosis displayed divided by total times mounted, in this case 10) was thereafter calculated. This measureis a sensitiveindicator of female receptivity (Beach, 1942). Female sexual behavior in estrogen-treated male rats does not include those behaviors, collectively described as soliciting behaviors, which are normally seen in highly receptive females. Thus, neither “ear wiggling,” “hopping,” nor “darting” are seenin these males(A&n-Engelbrektsson et al., 1970). These“higher level female receptive behaviors” can easily be elicited in a female rat in heat by rapid forefinger flank palpation. On the last preexperimental tests (i.e., 9 days after the onset of estrogen administration, and on the day before combined a-MT and L-dopa treatment), and on tests after drug administration, all maleswere subjected to such manual stimulation in an attempt to elicit any of these female estrousbehaviors. RESULTS Effects of PCPA Ninety-seven percent of the malesdisplayed lordosis at least once in the preexperimental tests. The overall mean lordosis quotient during these tests was 0.39. Figure 1 presents the effects of PCPA on lordosis behavior. The last preexperimental tests (i.e., the day before injection of PCPA, -24 HRS in the

PCPA

cl

I-IRS

-24 NS

CONTROL

2

8

26

NS

MS

NS

Fig. 1. Mean lordosis quotient after injection of PCPA (150 mg/kg ip). The animals were tested 2, 8, and 26 hr after drug administration. -24 HRS refers to the test on the preceding day. Vertical bars are standard deviations. Group comparisons were performed with the Mann-Whitney U test (Siegel, 1956).

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figure) is shown together with the tests after drug administration. As can be seen there were no effects at any of the time intervals studied. Within-group comparisons also failed to reveal significant differences at any time interval. Manual stimulation of the lumbar region failed to elicit any female soliciting behaviors. Effects of a-MT

In the preexperimental tests 74% of the animals displayed at least one lordosis. The group mean lordosis quotient was 0.23. Figure 2 shows the effects of a-MT on the number of lordoses displayed. There was a statistically significant group difference both at the 2and at the 8-hr test (p < .OS on both occasions, Mann-Whitney U test). Twenty-six hours after drug treatment the difference had disappeared. Withingroup comparison (lordosis quotient on the day before, i.e., -24 HRS in the figure, was used as control level) revealed a statistically significant increase for a-MT-treated rats at the 2-hr test (p < .02, Wilcoxon T test), and no significant differences for controls at any time. Interestingly, at both the 2and 8-hr tests, manual stimulation elicited ear wiggling in one of the experimental males. Three days later the rats were subjected to a combined a-MT and L-dopa treatment as described in the Methods section. Figure 3 shows the effects in relation to the test on the day before (pretest in the figure). An analysis of variance revealed an overall statistically significant heterogeneity (JJ< .05, Kruskal-Wallis one-way analysis of variance). Between-group comparisons showed a-MT-treated rats to be more responsive than both a-MT- t L-dopa-treated rats (p < .05) and controls (p < .05). There was no difference

HRS P

-24 NS

2

a

26

-05

-=05

NS

Fig. 2. Mean lordosis quotient after injection of c&MT (100 mg/kg ip). The animals were tested 2, 8, and 26 hr after drug administration. -24 HRS refers to the test on the preceding day. Vertical bars are standard deviations. Group comparisons were performed with the Mann-Whitney U test (Siegel, 1956).

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2 .a0

PLMT+L-D~PA

a’ 52 60

“0’ P

9 A0

f Y

t

.20

PRETEST

DRUGTEST

Fig. 3. Mean lordosis quotient after administration of &MT (100 mg/kg ip); GMT (lOO,mg/kg ip) + L-dopa (50 mg/kg ip), and saline, respectively. The animals were tested 2 hr after the first injection. Pretest refers to the test on the preceding day. Vertical bars are standard deviations. Group comparisons were performed with the Mann-Whitney U test (Siegel, 1956).

between u-MT- + L-dopa-treated and control animals. Within-group comparisons revealed no significant changes in the control or the a-MT- + L-dopa treated subjects but a statistically reliable increase in lordosis quotient was noted for u-MT-treated rats (p < .Ol). Again manual stimulation elicited vigorous ear wiggling in one of the u-MT-treated animals (this was not the same animal that responded positively in the above experiment). Finally, all rats used as controls in the last experiment were injected with L-dopa a day later and the test results compared with their own performance a day earlier. This experiment was done to see whether L-dopa alone impairs lordotic performance in estrogen-primed males. A slight nonsignificant decrease from a mean lordosis quotient of 0.24 to 0.17 was observed. All animals remained in excellent health throughout the experiments, and no signs of sedation or debilitation were observed after any of the drug treatments described. In summary: No effects on intensity of lordosis were noted at any time interval after the administration of PCPA. a-MT, however, facilitated lordosis behavior within 2 hr after injection, and 26 hr after administration this effect was gone. The effect of u-MT was effectively blocked by administration of L-dopa 1 hr after the injection of u-MT.

DISCUSSION The data presented here focus attention on central levels of catecholamines for the facilitation of lordosis behavior seen in castrated, estrogen-

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primed male rats treated with central monoamine-depleting agents (i.e., tetrabenazine or reserpine). Thus, PCPA had no effect on estrogen-induced lordosis behavior in male rats. On the other hand, inhibition of catecholamine synthesis by a-MT administration, resulted in facilitation of lordosis in estrogenprimed males. The finding that L-dopa counteracted this effect further suggests the involvement of catecholamines in estrogen-drug-acticated lordosis behavior in male rats. Meyerson, after a series of experiments (see Meyerson and Lewander, 1970) suggest that, in female rats, lowering of central levels of serotonin rather than catecholamines would account for the facilitation of lordosis observed after combined estrogen-reserpine (or estrogen-tetrabenazine) treatment. Malmnas and Meyerson (1971) demonstrated that PCPA facilitates mounting behavior in castrated male rats, treated with a submaximal dose of testosterone. From these results on males and females Malmnh and Meyerson (197 1) discuss the possibility that male and female sexual behavior, regardless of the sex of the test animal, is under the influence of a central serotonergicinhibiting mechanism. In a previous report from this laboratory (Ahlenius et al., 1971) we also reported facilitation of male copulatory behavior in male rats treated with PCPA. However, we suggested a different mechanism of action than that proposed by Malmnas and Meyerson (1971). The data presented here suggest the involvement of catecholamines rather than 5HT for drug-facilitated lordosis behavior in male rats. This closely parallels our results in females (Ahlenius et QL, 1972; AhIenius et al., in preparation). In this series of experiments we have demonstrated a close correlation between central levels of catecholamines and the display of lordosis. Thus, a high dose of tetrabenazine (10 mg/kg) facilitates lordosis in estrogen-pretreated females and depletes central catecholamines and 5-HT, whereas a low dose (2 mg/kg) likewise facilitates lordosis but depletes only dopamine significantly (Ahlenius et al., 1972). a-MT and PCPA also facilitate lordosis behavior and deplete central catecholamines and, in the case of PCPA, also 5-HT. The effect of PCPA, however, is confined to the time interval when this drug lowers brain levels of catecholamines and 26 hr after injection when catecholamine levels are back to normal and 5-HT levels are significantly reduced, there is no facilitation of lordosis (Ahlenius et al., in preparation). These data clearly demonstrate the involvement of brain catecholamines for drug-induced facilitation of lordosis in female rats. The data on male rats presented in this communication are in line with our findings in females. The mechanism whereby drugs act to facilitate lordosis in estrogentreated rats remains to be determined. It has been shown in female mice (Uphouse, Wilson, and Schlesinger, 1970) and female rats (Paris, Resko, and Goy, 1971) that the reserpine-induced facilitation of lordosis after estrogen priming is abolished by adrenalectomy and dexamethasone treatment, respectively, and these workers suggest that reserpine acts by eliciting secretion

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of adrenal progesteronevia ACTH secretion from the anterior pituitary. The demonstration by Feder and Ruf (1969) that ACTH is capable of facilitating lordosis behavior via the mechanism described, supports this hypothesis. Furthermore, it was recently shown that ACTH secretion in the rat might be inhibited by an adrenergic mechanism(van Loon et al, 1971). Our data that catecholamine synthesis inhibition facilitates lordosis in estrogen-primed females, thus, leads to the hypothesis that drugs that interfere with central monoamine neurotransmission facilitate lordosis behavior in estrogen-pretreated female rats by lowering brain contents of catecholamines, thereby eliciting adrenal progesterone secretion via stimulation of ACTH secretion from the adenohypophysis. Whether this same mechanismcan explain the results for male lordotic behavior remains a speculation, since male rats have not been shown to respond to progesterone(seeIntroduction).

ACKNOWLEDGMENTS This work was supported by grants from the Swedish Council for Social Research, grant number 223/71 p, and Riksbankens Jubileumsfond, grant number 68/50:1, to Professor Knut Larsson. We thank Dr. Hans Corrodi, Hassle Biochemical Laboratories, Goteborg, for generous supply of H 69/17, H 44/68 and H 19/61. We are indebted to Dr. Alfred Pletscher, Hoffman-La Roche, Basel, Switzerland, for the supply of Ro 44602. For estrogen we thank Organon, The Netherlands. The skillful technical assistance of Mr. Nils Carlsson is thankfully acknowledged. REFERENCES Ahlenius, S., Eriksson, H., Larsson, K., Modigh, K., and Siidersten, P. (1971). Mating behavior in the male rat treated with p-chlorophenylalanine methylester alone and in combination with pargyline. Psychopharmacologiu 20, 383-388. Ahlenius, S., Engel, J., Eriksson, H., and Sodersten, P. (1972). Effects of tetrabenazine on lordosis behaviour and on brain monoamines in the female rat. J. Neural Transmiss., in press. Aren-Engelbrektsson, B., Larsson, K., Sodersten, P., and Wilhelmsson, M. (1970). The female lordosis pattern induced in male rats by estrogen. Horm. Behav. 1, 181-188. Beach, F. A. (1942). Importance of progesterone to induction of sexual receptivity in spayed female rats. Proc. Sot. Exp. Biol. Med. 51, 367-371. Boling, J. L., and Blandau, R. J. (1939). The estrogen-progesterone induction of mating responses in the spayed female rat. Endocrinology 25, 259-264. Corrodi, H., and Hansson, L. C. F. (1966). Central effects of an inhibitor of tyrosine hydroxylation. Psychopharmacologia 10, 116-125. Davidson, J. M., Smith, E. R. Rodgers, C. H., and Bloch, G. 3. (1968). Relative thresholds of behavioral and somatic responses to estrogen. Physiol. Behav. 3, 227-229.

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Davidson, J. M., and Levine, S. (1969). Progesterone and heterotypical sexual behavior in male rats. J. Endocrinol. 44, 129-130. Feder, H. H., and Ruf, K. B. (1969). Stimulation of progesterone release and estrous behavior by ACTH in ovariectomized rodents. Endocrinology 84, 171-174. Grady, K. L., Phoenix, C. H., and Young, W. C. (1965). Role of the developing rat testis in differentiation of the neural tissues mediating mating behavior. J. Comp. Physiol. PsychoL 59, 176-182. Koe, B. K., and Weissman, A. (1966). pChlorophenylalanine: A specific depletor of brain serotonm. J. Pharmacol. Exp. Ther. 154, 499-516. Larsson, K., and Sodersten, P. (1971). Lordosis behavior in male rats treated with estrogen in combination with tetrabenazine and nialamide. Psychopharmacologia 21, 13-16. Malmnas, C. O., and Meyerson, B. J. (1971). pChlorophenylalanine and copulatory behavior in the male rat. Nature (London] 232, 398400. Meyerson, B. J. (1964). Central nervous monoamines and hormone induced estrus behavior in the spayed rat. Acta Physiol. Stand. 63, Suppl. 241. Meyerson, B. J., and Lewander, T. (1970). Serotonin synthesis inhibition and estrous behavior in female rats. Life Sci 9, 661-671. Paris, C. A., Resko, J. A., and Coy, R. W. (1971). A possible mechanism for the induction of lordosis by reserpine in spayed rats. Biol. Reprod. 4, 23-30. Siegel, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. Uphouse, L. L., Wilson, J. R., and Schlesinger, K. (1970). Induction of estrus in mice: the possible role of adrenal progesterone. Horm. Behav. 1, 255-264. van Loon, G. R., Scapagnini, U., Moberg, G. P., and Ganong, W. F. (1971). Evidence for central adrenergic neural inhibition of ACTH secretion in the rat. Endocrinology 89, 1464-1469.