Brain Research, 166 (1979) 369-380 © Elsevier/North-HollandBiomedicalPress
369
NORADRENERGIC TRANSI~HSSION AND FEMALE SEXUAL BEHAVIOR OF GUINEA PIGS
BRUCE NOCK and HARVEY H. FEDER Institute of Animal Behavior, Rutgers-The State University, Newark, N.J. 07102 (U.S.A.)
(Accepted August 10th, 1978)
SUMMARY Treatment with the dopamine beta-hydroxylase (DBH) inhibitor U-14,624 (50, 100, or 150 mg/kg) blocked the induction of lordosis behavior by estradiol benzoate (EB) and progesterone (P) in ovariectomized guinea pigs. After treatment with U14,624 (100 mg/kg), norepinephrine (NE) content of medial basal hypothalamus, preoptic area and cortex was reduced (by 55 %) and dopamine (DA) content of medial basal hypothalamus was increased (by 155 %) during the period when females treated with EB and P normally display lordosis. Treatment with the NE receptor stimulator clonidine (1.0 mg/kg) restored lordosis behavior in females treated with EB, P, and U14,624 (100 mg/kg), but the putative DA and serotonin (5-HT) receptor blockers pimozide (!.0 mg/kg) and methysergide (20.0 mg/kg) were ineffective in this respect. Thus, inhibition of lordosis after treatment with U-14,624 appeared to be attributable primarily to a reduction in NE neurotransmission, rather than to an increase in DA or 5-HT activity. Because clonidine induced lordosis in females treated with EB, P, and U-14,624, it seemed unlikely that the facilitatory effects of clonidine on lordosis were mediated by activation of presynaptic alpha-adrenergic receptors (i.e. inhibitory NE autoreceptors) rather than by postsynaptic alpha-receptors. In addition, pretreatment with the postsynaptic alpha-adrenergic antagonist phenoxybenzamine (20.0 mg/kg) blocked the facilitation of lordosis by clonidine (1.0 mg/kg) in females primed with EB alone and with EB plus P. Thus, the facilitatory effects of clonidine on lordosis appear to be mediated by activation of postsynaptic alpha-adrenergic (i.e. NE) receptors. The results of this study provide further evidence that NE neurotransmission facilitates the expression of female sexual behavior in guinea pigs.
INTRODUCTION In guinea pigs, the expression of female sexual behavior depends on the
370 sequential action of estrogen and progesterone. Ovariectomized guinea pigs usually do not display sexual behavior unless they are treated with estrogen followed 36-48 h later by progesterone 25. In a recent study, we reported that the noradrenergic agonist clonidine induced lordosis behavior in females treated with estrogen alone 7. In females rendered sexually receptive by estrogen and progesterone, clonidine increased the duration of individual lordosis responses, while the alpha-adrenergic antagonist phenoxybenzamine abolished ongoing lordosis behavior6. These results suggest that in the guinea pig activation of noradrenergic mechanisms may, in part, underlie the induction of sexual receptivity by ovarian hormones. The experiments reported here are a further test of this hypothesis. In the first experiment, females primed with estrogen and progesterone were treated with the dopamine beta-hydroxylase (DBH) inhibitor U-14,62418. Our prediction was that suppression of norepinephrine synthesis by U-14,624 would inhibit lordosis behavior. In the second experiment, the effects of U-14,624 on regional brain norepinephrine (NE) and dopamine (DA) content were assessed with a radioenzymatic assay. In a third experiment, we injected estrogen and progesterone primed females with U-14,624 and and clonidine. Our prediction was that clonidine, by activating NE receptors, would override any inhibitory effects on lordosis that U-14,624 might have. In the fourth experiment, females primed with either estrogen alone or estrogen and progesterone were treated with phenoxybenzamine prior to administration of clonidine. This experiment was designed to determine whether the known facilitatory effects of clonidine on lordosis behavior are mediated by activation of postsynaptic NE receptors. METHODS
Animals Adult Hartley strain, albino female guinea pigs (Camm Research Laboratories, Wayne, N.J.) that weighed 350400 g when they arrived in our laboratory were used in this study. The guinea pigs were group-housed in cages measuring 90 cm x 60 cm x 23 cm with no more than 10 animals per cage. Purina guinea pig chow and water were available ad libitum, and cabbage was supplied once a week. Lights in the colony room were on from 05.00 to 19.00 h and the temperature was maintained at about 20 °C. Surgery Shortly after they arrived, all animals were ovariectomized under a combination of Innovar-vet (Pitman-Moore, Washington Crossing, N.J., 0.05 ml/animal, i.m.) and Chloropent (Fort Dodge Laboratories, Fort Dodge, Ia., 1.7 ml/kg, i.p.). All experiments were conducted approximately three weeks after surgery. Drugs and hormones Estradiol-17fl benzoate (EB, 1.5 #g/animal) and progesterone (P, 0.5 mg/animal) were injected s.c. in 0.1 ml sesame oil. All drugs were injected i.p. at a volume of 2.0 ml/kg. U-14,624 was suspended in 0.25 ~ methylcellulose (15 centipoises). Phenoxybenzamine hydrochloride was dis-
371 solved in 50 ~ propylene glycol. Clonidine hydrochloride and methysergide maleate were dissolved in isotonic saline. Pimozide was dissolved in 75 #1 glacial acetic acid and the pH was adjusted to 4.0 with 2 N NaOH. This solution was then diluted to the final volume with bidistilled water. Drug dosages are reported in the procedural section of each experiment. Dosages for clonidine and methysergide refer to the salt, for phenoxybenzamine the base. EB and P were generously supplied by the Schering Corporation, clonidine by Boehringer Ingelheim Ltd, phenoxybenzamine by Smith Kline and French Labs., pimozide by McNeil Laboratories, Inc. and methysergide by Sandoz. U-14,624 was obtained from Regis Biochemical Company. Behavioral testing Tests for lordosis were conducted in the home cage using the manual manipulation technique described by Goy and Young 1~. Each animal was tested hourly beginning 40 h after EB administration and continuing until the end of heat or until 5 h after the final drug treatment (see the procedural section of each experiment). An animal was considered 'in heat' if it displayed a lordosis response with a duration of 2 sec or more during at least two tests. Latency, duration, and maximum were used to assess the the quality of heat. These quality measures are defined as follows. Latency: the time (h) from the first test to the first lordosis response. Duration: the total time (h) that an animal displayed lordosis. Maximum: the time (sec) that the longest lordosis position was held. Experiment 1 - - inhibition of dopamine beta-hydroxylase Rationale Administration of phenoxybenzamine, an alpha-adrenergic antagonist4,~, abolished lordosis behavior in female guinea pigs rendered sexually receptive by EB plus p6. In order to determine whether the inhibition of lordosis by phenoxybenzamine is peculiar to this drug or is a property of drugs that interfere with noradrenergic synaptic transmission in general, females were treated with EB, P, and the DBH inhibitor U-14,62418. Because DBH catalyzes the hydroxylation of DA to NE, inhibition of this enzyme decreases the rate of NE synthesis and ultimately the availability of NE for synaptic transmission. If the inhibition of lordosis by phenoxybenzamine results from an interference with NE neurotransmission then U-14,624 should also inhibit lordosis. Procedure The design of this experiment is outlined in Table I. Basically, all groups received 1.5 #g EB at 0 h and 0.5 mg P 40 h later. Group 1, the control group, received an injection of the vehicle for U-14,624 1 h after P. Experimental groups 2, 3, and 4 received 50, 100, and 150 mg/kg U-14,624 respectively, 1 h after P. Hourly tests for lordosis continued until every animal failed to respond to manual manipulation with a display of lordosis.
372 TABLE I Effects of U-14,624 ( U) on sexual behavior induced by estradiol benzoate ( EB) and progesterone (P)
Dosage of U is given in mg/kg as subscript. Veh = 0.25 ~ methylcellulose vehicle. Group
1 2 3 4
Treatment
N
0 (h)
40 (h)
41 (h)
EB EB EB EB
P P P P
Veh Us0 U10o Uas0
12 11 11 11
% in heat Latency*
75.0 27.3** 27.3** 9.1 ***
(X ± S.E., h)
Duration* (X ~z S.E., h)
Maximum* (X ± S.E., see)
5.6 ± 6.0 ± 5.7 ± 7.0 ±
6.4 ± 7.0 ± 6.0 ± 8.0 ±
12.4i 2.25 16.7± 3.18 13.3 ± 2.72 7.0 22 0.00
0.50 1.16 0.88 0.00
0.71 2.00 0.58 0.00
* Based on animals that displayed heat. ** P < 0.05 versus group 1 (Fisher Exact Probability Test). *** P < 0.005 versus group 1 ('Fisher Exact Probability Test).
Results
The percentage of animals displaying heat after treatment with EB and P was decreased by all doses of U-14,624 tested (compare groups 2, 3, and 4 with group 1 in Table I). The drug-treated animals showed no signs of a general behavioral disturbance and actively resisted manual manipulation by running away. Because very few drug-treated females displayed heat, the effects of U-14,624 on the quality of heat (latency, duration, and maximum) could not be assessed. Experiment 2 - - catecholamine content o f several brain areas after treatment with U14,624 Rationale
In Experiment 1, treatment with U-14,624 inhibited lordosis behavior in females treated with EB and P. Presumably, this inhibition was associated with a drug-induced decrease in NE synthesis. To confirm this, the catecholamine (CA) content of 4 brain areas was determined after animals were treated with EB, P, and U-14,624, as in Experiment 1. Brain areas were selected that (a) take up and retain estrogen and/or progesterone from the general circulation, (b) contain a high density of NE terminals, (c) and/or have been implicated in the regulation of female sexual behavior by hormone-implant, lesion, and drug studies. The caudate-putamen, preoptic area, medial basal hypothalamus, and cortex were selected and assayed with a sensitive radioenzymatic assay. Procedure
All animals received 1.5/~g EB at 0 h and 0.5 mg P 40 h later. One hour after the administration of P, animals received either U-14,624 (100 mg/kg) or the methylcellulose vehicle. At 2, 6, and 24 h after drug treatment, animals were decapitated and their brains quickly removed and dissected under dry ice. The preoptic area (POA),
373 TABLE II Effects of U-14,624 (100 mg/kg) on norephinephrine content (in ng/mg tissue) of medial basal hypothalamus ( MBH) , cortex ( CO RT) , and pre-optic area ( PO A ) at 2,6, and24 h after administration
Values are the mean of 5 samples plus or minus the standard error. Hours M B H Control
2 6 24
CORT U-14,624
% Control Control
POA U-14,624
% Control Control
U-14,624
% Control
3.56±0.21 1.76±0.31' (49.4) 0.25-4-0.02 0.16-4-0.03 (64.0) 2.754-0.20 1.784-0.30 (64.7) 3.37-4-0.26 1.52.4.0.55" (45.1) 0.284-0.01 0.134-0.03" (46.4) 2.844-0.41 1.20.4.0.49" (42.2) 3.254-0.41 2.10.4.0.54 (64.6) 0.24-4-0.04 0.204-0.04 (83.3) 3.23-I_0.57 1.79.4.0.43' (55.4)
* P < •.•5versusc•ntr••va•uesatthesametime(Ana•ysis•fVariancef••••wedbyDuncan•sMu•tip•eRange Test). caudate-putamen (C-P), medial basal hypothalamus (MBH), and a section of frontal cortex (CORT) were taken and assayed for N E and D A content with the radioenzymatic assay of Cuello, Hiley and Iversen 8 as modified by Zschaeck and Ramirez 26. Results
The effects of U-14,624 on regional brain N E and DA content of EB plus P primed females are presented in Tables II and III, respectively. N E content of C-P and D A content of C O R T were not detectable. U-14,624 caused a significant reduction in N E content of MBH, CORT, and POA within 6 h. U-14,624 had no effect on D A content of POA or C-P. D A content of M B H was increased at 2, 6, and 24 h after administration of U-14,624. Experiment 3
restoration o f lordosis behavior after treatment with U-14,624 by drugs
that affect monoamine receptors Rationale
Treatment with D B H inhibitor U-14,624 inhibited lordosis behavior in females treated with EB and P. However, in addition to causing a significant decrease in brain N E levels, treatment with U-14,624 caused a 155 ~ increase in D A content of M B H (see Experiment 2). Further, inhibition of D B H has previously been shown to increase brain serotonin (5-HT) synthesis 19,z°. Because D A and 5-HT agonists suppress lordosis behavior in female guinea pigs% the inhibition of lordosis induced by U14,624 may have been attributable to an increase in D A and 5-HT activity rather than to a decrease in N E activity. In order to distinguish between these alternatives, the following experiment was designed. Females were treated with EB, P, and U-14,624, as in Experiments 1 and 2. Five hours after treatment with U-14,624 animals received pimozide a D A receptor
374
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0
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- H ~ -q
-H ~H -H
~S
~C ea .,~- u-.~
-Hq-H
::3
d~Sd
-H -H -H 'c
xs
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375 TABLE IV Effects of clonidine ( C), pimozide (Pi), and methysergide ( M) on sexual behavior of animals treated with estradiol benzoate ( EB) , progesterone (P) and the D B H inhibitor U-14,624 (U)
Veh at 41 h - 0.25 % methylcellulose vehicle; Veh at 46 h - isotonic saline vehicle. Group Treatment
N
% in heat Maximum*
(X :k S.E., sec)
0 (h)
40 (h)
41 (h)
46 (h)
1 2 3
EB EB EB
P P P
Veh U U
Veh Veh C
14 13 13
71.4 0.0 69.2**
10.5 ± 1.33 -11.2 ± 1.81
4 5
EB EB
P P
U U
Pi M
14 10
14.3 10.0
10.5 :k 4.5 7.0 ± 0.0
* Based on animals that displayed heat. ** P < 0.005 versus group 2 (Fisher Exact Probability Test). blocker 2, methysergide - - a 5-HT receptor blocker 16,17, or clonidine - - a N E receptor stimulatorL If the inhibition of lordosis by U-14,624 resulted from an increase in D A activity, then treatment with pimozide should induce lordosis behavior in animals treated with EB, P, and U-14,624. If the inhibition resulted from an increase in 5-HT activity, then methysergide should induce lordosis. If, on the other hand, the U14,624-induced decrease in N E synthesis was responsible for inhibition of lordosis, then treatment with clonidine should induce lordosis behavior. Pro cedure
The design of this experiment is outlined in Table IV. Basically, all groups received 1.5/~g EB at 0 h, 0.5 mg P at 40 h, and two additional injections (the first at 41 h and the second at 46 h). Groups 1 and 2 were control groups. Group 1 received an injection of the vehicle for U-14,624 at 41 h and isotonic saline at 46 h. G r o u p 2 received U-14,624 (100 mg/kg) at 41 h and saline at 46 h. Experimental groups 3, 4, and 5 all received U-14,624 (100 mg/kg) at 41 h followed by clonidine (1.0 mg/kg), pimozide (1.0 mg/kg), or methysergide (20.0 mg/kg), respectively, at 46 h. Because U-14,624 did not inhibit lordosis in 100 % of females treated with EB and P (73 % inhibited, see Experiment 1), any animal treated with this drug that displayed a lordosis response prior to the injection at 46 h was eliminated from the experiment. This procedure reduced the possibility of incorrectly attributing a lordosis response induced by EB and P to the action of one of the three experimental drugs. Hourly tests for lordosis continued until 5 h after the final drug treatment. In addition to the hourly tests, animals were tested at 46.5 and 47.5 h. Results
Of the three experimental drugs (i.e. clonidine, pimozide and methysergide), only clonidine induced lordosis behavior in females treated with EB, P, and U-14,624 (compare ' % in heat' for groups 3, 4, and 5 with group 2 in Table IV). The animals
376 treated with EB, P, U-14,624 and clonidine did not differ from those treated with only EB and P in terms of the percentage of animals displaying heat or the longest lordosis duration (compare group 3 with group 1 in Table IV). The clonidine-treated animals began displaying lordosis 1.67 ~ 0.40 h after the injection of clonidine and continued showing the response for 2.72 ~z 0.29 h (values are ~ ~ S.E.). Experiment 4 - - effects of pretreatment with an alpha-adrenergic antagonist on clonidine facilitation of female sexual behavior Rationale In previous studies, clonidine facilitated the expression of sexual receptivity in female guinea pigs treated with EB alone 7 and EB plus p6. In Experiment 3 of this study, clonidine induced lordosis behavior in animals treated with EB, P, and the DBH inhibitor U-14,624. Under all of these conditions, the facilitation of female sexual behavior presumably resulted from the activation of postsynaptic noradrenergic receptors by clonidine. In order to test this, EB and EB plus P primed females were treated with the alpha-adrenergic blocker phenoxybenzamine4, 9 prior to treatment with clonidine. If clonidine facilitates lordosis behavior by stimulation of alphaadrenergic receptors, then blockade of these receptors with phenoxybenzamine should block or reduce the facilitation of lordosis by clonidine. Procedure The design of this experiment is outlined in Table V. Basically, groups 1-3 received 1.5/zg EB at 0 h and 0.5 mg P 40 h later. Groups 4-6 received 1.5 #g EB at 0 h and no P. At 41 h after EB, phenoxybenzamine (20.0 mg/kg, groups 2, 3, and 6) or the 50 % propylene glycol vehicle (groups 1, 4, and 5) was given. Five hours later, at 46 h, each group received clonidine (1.0 mg/kg, groups 3, 5, and 6) or saline (groups 1, 2, and 4). Hourly tests for lordosis continued until 5 h after the final drug treatment (i.e. 51 h). In addition to the hourly tests, the animals were tested at 46.5 and 47.5 h. Results Treatment with clonidine induced lordosis behavior in animals treated with EB alone (compare '% in heat' for group 5 with group 4 in Table V). The animals treated with EB plus clonidine began displaying lordosis 0.89 ~ 0.18 h after the injection of clonidine and continued showing the response for 2.39 ~: 0.22 h (values are ~ ± S.E.). The lordosis responses of females treated with EB plus clonidine were generally less intense than those displayed by females treated with EB and P (compare 'maximum' for group 5 with group 1 in Table V) and usually consisted of a weak flattening of the back, brief display and dilatation of the vaginal region, and occasional rumbling vocalizations. Pretreatment with phenoxybenzamine blocked the induction of lordosis behavior by clonidine in EB primed females (compare' % in heat' for group 6 with group 5 in Table V). Treatment with phenoxybenzamine inhibited lordosis behavior in animals
377 TABLE V Effects of phenoxybenzamine (Pb) on the facilitation of sexual behavior by clonidine ( C) in animals treated with estradiol benzoate (EB) and progesterone (P) or EB alone
Veh at 41 h = 50~ propylene glycol vehicle; Veh at 46 h = isotonic saline. Group Treatment O(h)
40(h)
41(h)
46(h)
1 2 3 4 5
EB EB EB EB EB
P P P ---
6
EB
--
Veh Pb Pb Veh Veh Pb
Veh Veh C Veh C C
N
% in heat Maximum* (X ± S.E., see)
8 7 8 9 11 10
100.0 17.62 ± 3.79 0.0"* - 25.0 7.50 ± 3.50 0.0 -81.8"** 6.67 4- 1.12§ 10.0§§ 8.00 4- 0.00
* Based on animals that displayed heat. ** P < 0.005 versus group 1 (Fisher Exact Probability Test). *** P < 0.005 versus group 4 (Fisher Exact Probability Test). § P < 0.01 versus group 1 (t-test, two-tailed). §§ P < 0.05 versus group 5 (Fisher Exact Probability Test).
treated with EB and P (compare' ~ in heat' for group 2 with group 1 in Table V). The percentage of EB plus P primed animals displaying heat after treatment with phenoxybenzamine and clonidine did not differ from the percentage displaying heat after phenoxybenzamine without clonidine (compare group 3 with group 2 in Table V). DISCUSSION This study demonstrated that U-14,624 (a D B H inhibitor), given 1 h after progesterone, blocks induction of lordosis behavior by estrogen and progesterone in ovariectomized guinea pigs. The effects of U-14,624 on CA content of MBH, POA, CP, and C O R T were assessed at 2, 6, and 24 h after drug administration. Within 6 h, U14,624 caused a 55 ~ depletion of NE in MBH, POA, and C O R T (NE was not detectable in C-P). Females treated with estrogen and progesterone normally begin to display lordosis about 4-6 h after administration of progesterone. Thus, NE levels were significantly reduced after treatment with U-14,624 during the period when females are usually sexually receptive. On the basis of these data alone, we were unable to conclude that the inhibition of lordosis by U-14,624 was a consequence solely (or even primarily) of depletion of NE. U-14,624 also caused a 1 5 5 ~ increase in D A content of MBH. Effects of U-14,624 on 5-HT were not examined in the guinea pig, but in rats inhibition of D B H not only interferes with noradrenergic transmission but also increases brain 5-HT synthesis19, 20. Because D A and 5-HT agonists inhibit lordosis in guinea pigs 6, the inhibition of lordosis induced by U-14,624 may have been attributable to an increase in D A or 5-HT activity rather than to a decrease in N E activity. In order to distinguish between these alternatives, we attempted to restore lordosis behavior in estrogen plus progesterone primed females treated with U-14,624
378 by (a) activating NE receptors with clonidine, (b) blocking DA receptors with pimozide, and (c) blocking 5-HT receptors with methysergide. Of these drugs, only clonidine restored lordosis behavior. Thus, inhibition of lordosis after administration of U-14,624 appears to be attributable solely to a reduction in noradrenergic transmission. However, it may be premature to conclude that changes in DA and/or 5HT activity do not contribute to the U-14,624-induced inhibition of lordosis. Although the dosages of pimozide and methysergide used in this study are relatively high and facilitate lordosis behavior in rats12,21, we lack evidence that these drugs effectively block DA and 5-HT receptors in guinea pigs. In previous studies, clonidine facilitated the expression of sexual receptivity in female guinea pigs primed with estrogen alone 7, and with estrogen and progesterone 6. In this study, clonidine induced lordosis in females primed with estrogen alone and also restored lordosis in females treated with estrogen, progesterone and U-14,624. Under all of these conditions, the facilitation of female sexual behavior by clonidine presumably resulted from the activation of postsynaptic alpha-adrenergic (noradrenergic) receptors. However, under some conditions (e.g. low doses in rats) clonidine may preferentially activate presynaptic alpha-adrenergic receptors10,23,24. These presynaptic receptors or autoreceptors are thought to mediate a local negative feedback through which interneuronal NE can inhibit its own further release2L Because clonidine facilitated lordosis behavior in female guinea pigs with already reduced noradrenergic transmission (as a result of U-14,624), it seems unlikely that facilitation of lordosis by clonidine is mediated by activation of presynaptic alpha-adrenergic receptors. In addition, pretreatment with the postsynaptic alpha-adrenergic antagonist phenoxybenzamine11,23 blocked the facilitation of lordosis by clonidine in both estrogen and estrogen plus progesterone primed females. The results of this study and those of previous experiments6, 7 suggest that NE neurotransmission facilitates the expression of female sexual behavior in guinea pigs. Thus far, few studies have examined the role of NE in the regulation of female sexual behavior in other species. In rats, NE may also facilitate the expression of female sexual behavior but this is far from certain. Everitt and coworkers 12-14 reported that in estrogen-primed female rats the CA releaser amphetamine increases the facilitatory effects of DA receptor blockade on lordosis. Presumably the facilitation of lordosis by amphetamine under these conditions is attributable to a selective enhancement of NE receptor activity. However, activation of NE receptors with clonidine suppresses ongoing lordosis responding induced by estrogen and progesterone in rats 1°. This is in marked contrast to the facilitatory effects of clonidine on lordosis behavior in guinea pigs. Davis and KohP 0 have suggested that the inhibitory effects of clonidine on lordosis in rats might be mediated by activation of inhibitory presynaptic NE receptors rather than by postsynaptic NE receptors. This suggestion seems plausible because pretreatment with the putative presynaptic NE receptor antagonist yohimbine, but not the postsynaptic NE receptor antagonist phenoxybenzamine, blocks the inhibitory effects of clonidine on lordosis in rats 1°. However, inhibition of DBH (with FLA 63) or treatment with phenoxybenzamine has little or no effect on lordosis behavior induced by estrogen and progesterone in rats 1°,12-14. In fact, Ahlenius et al. 1
379 have reported that the DBH inhibitor FLA 63 increases lordosis behavior in rats primed with estrogen alone. Thus, at present it is unclear whether NE facilitates female sexual behavior in rats or other species as it does in guinea pigs. We also do not know what neurochemical systems NE influences to facilitate sexual behavior of female guinea pigs. Antelman and Caggiula 5 have recently suggested that NE may influence the expression of a number of 'activated or stressinduced' behaviors (i.e. stimulant-induced stereotypy or motor activity, shuttle-box avoidance, several types of aggressive behaviors, electrical self-stimulation of the brain, and stress-related eating) by modulating brain (perhaps nigrostriatat) DA transmission. Perhaps a similar N E - D A interaction is involved in the regulation of sexual behavior in female guinea pigs. ACKNOWLEDGEMENTS
We thank Dr. V. D. Ramirez for providing facilities to learn the radioenzymatic assay and C. Reboulleau and P. Albietz for their expert technical assistance. Supported by N I H Research G r a n t HD-04467, N I M H Career D e v e l o p m e n t A w a r d MH-29006 a n d a grant from the Busch F o u n d a t i o n , Rutgers University (all to H.H.F.). C o n t r i b u t i o n N u m b e r 314 of the Institute of A n i m a l Behavior.
REFERENCES 1 Ahlenius, S., Engel, J., Eriksson, H., Modigh, K. and Sodersten, P., Involvement ofmonoamines in the mediation of lordosis behavior. In M. Sandier and G. L. Gessa (Eds.), Sexual Behavior: Pharmacology and Biochemistry, Raven Press, New York, 1975, pp. 137-145. 2 And6n, N.-E., Butcher, S. G., Corrodi, H., Fuxe, K. and Ungerstedt, U., Receptor activity and turnover of dopamine and noradrenaline after neuroleptics, Europ. J. PharmacoL, 11 (1970) 303314. 3 And6n, N.-E., Corrodi, H., Fuxe, K., Hokfelt, B., Hokfelt, T., Rydin, C. and Svenson, T., Evidence for a central noradrenaline stimulation by clonidine, Life Sci., 9 (1970) 513-523. 4 And6n, N.-E. and Strombom, U., Adrenergic receptor blocking agents: Effects on central noradrenalineand dopamine receptors and on motor activity, Psychopharmacologia (Berl.), 38 (1974) 91-103. 5 Antelman, S. M. and Caggiula, A. R., Norepinephrine-dopamine interactions and behavior, Science, 195 (1977) 646-653. 6 Crowley, W. R., Feder, H. H. and Morin, L. P., Role ofmonoaminesinsexual behavior ofthe female guinea pig, Pharmacol. Biochem. Behav., 4 (1976) 67-71. 7 Crowley, W. R., Nock, B. L. and Feder, H. H., Facilitation of lordosis behavior by clonidine in female guinea pigs, PharmacoL Biochem. Behav., 8 (1978) 207-209. 8 Cuello, A. C., Hiley, R. and Iversen, L. L., Use of catechol O-methyl-transferase for the enzyme radiochemical assay of dopamine, J. Neurochem., 21 (1973) 1337-1340. 9 Dairman, W., Gordon, R., Spector, S., Sjoerdsma and Udenfriend, A., Increased synthesis of catecholamines in the intact rat following administration of alpha-adrenergic blocking agents, Molec. Pharmacol., 4 (1968) 457-468. 10 Davis, G. A. and Kohl, R., The influence of alpha-receptors on lordosis in the female rat, Pharmacol. Biochem. Behav., 6 (1977) 47-53. 11 Doxey, J. C., Smith, C. F. C. and Walker, J. M., Selectivity of blocking agents for pre- and postsynaptic alpha-adrenoceptors, Brit. J. Pharmacol., 60 (1977) 91-96. 12 Everitt, B. J., Fuxe, K. and Hokfelt, T., Inhibitory role of dopamine and 5-hydroxytryptamine in the sexual behavior of female rats, Europ. J. PharmacoL, 29 (1974) 187-191.
380 13 Everitt, B. J., Fuxe, K. and Hokfelt, T., Serotonin,catecholamines and sexual receptivity of female rats, J. Pharmacol. (Paris), 6 (1975) 269-276. 14 Everitt, B. J., Fuxe, K., Hokfelt, T. and Jonsson, G., Role of monoamines in the control by hormones of sexual receptivity in the female rat, J. comp. Physiol. PsychoL, 89 (1975) 556-572. 15 Goy, R. W. and Young, W. C., Strain differences in the behavioral responses of female guinea pigs to alpha-estradiol benzoate and progesterone, Behaviour, 10 (1957) 340-353. 16 Gyermek, L., 5-Hydroxytryptamine antagonist, Pharmacol. Rev., 13 (1961) 399-439. 17 Jacobs, B. L., Evidence for the functional interaction of two neurotransmitters, Psychopharmacologia (Berl.), 39 (1974) 81-86. 18 Johnson, G. A.,Boukma, S. J. and Kim,E. G.,Inhibition ofdopamine beta-hydroxylase by aromatic and alkyl thioureas, J. Pharmacol. exp. Ther., 168 (1969) 229-234. 19 Johnson, G. A. and Kim, E. G., Increase of brain levels oftryptophaninduced by inhibition ofdopamine beta-hydroxylase, J. Neuroehem., 20 (1973) 1761-1764. 20 Johnson, G. A., Kim, E. G. and Boukma, S. J., 5-Hydroxyindole levels in rat brain after inhibition of dopamine beta-hydroxylase, J. Pharmacol. exp. Ther., 180 (1972) 539-546. 21 Rodriguez-Sierra, J. F. and Davis, G. A., Progesterone does not inhibit lordosis through interference with estrogen priming, Life Sci., 22 (1978) 373-378. 22 Starke, K., Taube, H. D. and Borowski, E., Presynaptic receptor systems in catecholaminergic transmission, Biochem. PharmacoL, 26 (1977) 259-268. 23 Strombom, U., On the functional role of pre- and postsynaptic catecholamine receptors in brain, Acta Physiol. Scand., 431, Suppl. (1975) 2-43. 24 Svensson, T. H., Bunney, B. S. and Aghajanian, G. K., Inhibition of both noradrenergic and serotonergic neurons in brain by the alpha-adrenergic agonist clonidine, Brain Research, 92 (1975) 291306. 25 Young, W. C., Psychobiology of sexual behavior in the guinea pig. In D. S. Lehrman, R. A. Hinde and E. Shaw (Eds.), Advances in the Study of Behavior, VoL 2, Academic Press, New York, 1969, pp. 1-110. 26 Zschaeck, L. L. and Ramirez, V. D., A rapid and simple micromethod to measure picograms of norepinephrine (NE) and dopamine (DA) in small samples of brain tissue and pituitary gland, J. Neural Transmiss., 39 (1976) 291-200.
369
NORADRENERGIC TRANSI~HSSION AND FEMALE SEXUAL BEHAVIOR OF GUINEA PIGS
BRUCE NOCK and HARVEY H. FEDER Institute of Animal Behavior, Rutgers-The State University, Newark, N.J. 07102 (U.S.A.)
(Accepted August 10th, 1978)
SUMMARY Treatment with the dopamine beta-hydroxylase (DBH) inhibitor U-14,624 (50, 100, or 150 mg/kg) blocked the induction of lordosis behavior by estradiol benzoate (EB) and progesterone (P) in ovariectomized guinea pigs. After treatment with U14,624 (100 mg/kg), norepinephrine (NE) content of medial basal hypothalamus, preoptic area and cortex was reduced (by 55 %) and dopamine (DA) content of medial basal hypothalamus was increased (by 155 %) during the period when females treated with EB and P normally display lordosis. Treatment with the NE receptor stimulator clonidine (1.0 mg/kg) restored lordosis behavior in females treated with EB, P, and U14,624 (100 mg/kg), but the putative DA and serotonin (5-HT) receptor blockers pimozide (!.0 mg/kg) and methysergide (20.0 mg/kg) were ineffective in this respect. Thus, inhibition of lordosis after treatment with U-14,624 appeared to be attributable primarily to a reduction in NE neurotransmission, rather than to an increase in DA or 5-HT activity. Because clonidine induced lordosis in females treated with EB, P, and U-14,624, it seemed unlikely that the facilitatory effects of clonidine on lordosis were mediated by activation of presynaptic alpha-adrenergic receptors (i.e. inhibitory NE autoreceptors) rather than by postsynaptic alpha-receptors. In addition, pretreatment with the postsynaptic alpha-adrenergic antagonist phenoxybenzamine (20.0 mg/kg) blocked the facilitation of lordosis by clonidine (1.0 mg/kg) in females primed with EB alone and with EB plus P. Thus, the facilitatory effects of clonidine on lordosis appear to be mediated by activation of postsynaptic alpha-adrenergic (i.e. NE) receptors. The results of this study provide further evidence that NE neurotransmission facilitates the expression of female sexual behavior in guinea pigs.
INTRODUCTION In guinea pigs, the expression of female sexual behavior depends on the
370 sequential action of estrogen and progesterone. Ovariectomized guinea pigs usually do not display sexual behavior unless they are treated with estrogen followed 36-48 h later by progesterone 25. In a recent study, we reported that the noradrenergic agonist clonidine induced lordosis behavior in females treated with estrogen alone 7. In females rendered sexually receptive by estrogen and progesterone, clonidine increased the duration of individual lordosis responses, while the alpha-adrenergic antagonist phenoxybenzamine abolished ongoing lordosis behavior6. These results suggest that in the guinea pig activation of noradrenergic mechanisms may, in part, underlie the induction of sexual receptivity by ovarian hormones. The experiments reported here are a further test of this hypothesis. In the first experiment, females primed with estrogen and progesterone were treated with the dopamine beta-hydroxylase (DBH) inhibitor U-14,62418. Our prediction was that suppression of norepinephrine synthesis by U-14,624 would inhibit lordosis behavior. In the second experiment, the effects of U-14,624 on regional brain norepinephrine (NE) and dopamine (DA) content were assessed with a radioenzymatic assay. In a third experiment, we injected estrogen and progesterone primed females with U-14,624 and and clonidine. Our prediction was that clonidine, by activating NE receptors, would override any inhibitory effects on lordosis that U-14,624 might have. In the fourth experiment, females primed with either estrogen alone or estrogen and progesterone were treated with phenoxybenzamine prior to administration of clonidine. This experiment was designed to determine whether the known facilitatory effects of clonidine on lordosis behavior are mediated by activation of postsynaptic NE receptors. METHODS
Animals Adult Hartley strain, albino female guinea pigs (Camm Research Laboratories, Wayne, N.J.) that weighed 350400 g when they arrived in our laboratory were used in this study. The guinea pigs were group-housed in cages measuring 90 cm x 60 cm x 23 cm with no more than 10 animals per cage. Purina guinea pig chow and water were available ad libitum, and cabbage was supplied once a week. Lights in the colony room were on from 05.00 to 19.00 h and the temperature was maintained at about 20 °C. Surgery Shortly after they arrived, all animals were ovariectomized under a combination of Innovar-vet (Pitman-Moore, Washington Crossing, N.J., 0.05 ml/animal, i.m.) and Chloropent (Fort Dodge Laboratories, Fort Dodge, Ia., 1.7 ml/kg, i.p.). All experiments were conducted approximately three weeks after surgery. Drugs and hormones Estradiol-17fl benzoate (EB, 1.5 #g/animal) and progesterone (P, 0.5 mg/animal) were injected s.c. in 0.1 ml sesame oil. All drugs were injected i.p. at a volume of 2.0 ml/kg. U-14,624 was suspended in 0.25 ~ methylcellulose (15 centipoises). Phenoxybenzamine hydrochloride was dis-
371 solved in 50 ~ propylene glycol. Clonidine hydrochloride and methysergide maleate were dissolved in isotonic saline. Pimozide was dissolved in 75 #1 glacial acetic acid and the pH was adjusted to 4.0 with 2 N NaOH. This solution was then diluted to the final volume with bidistilled water. Drug dosages are reported in the procedural section of each experiment. Dosages for clonidine and methysergide refer to the salt, for phenoxybenzamine the base. EB and P were generously supplied by the Schering Corporation, clonidine by Boehringer Ingelheim Ltd, phenoxybenzamine by Smith Kline and French Labs., pimozide by McNeil Laboratories, Inc. and methysergide by Sandoz. U-14,624 was obtained from Regis Biochemical Company. Behavioral testing Tests for lordosis were conducted in the home cage using the manual manipulation technique described by Goy and Young 1~. Each animal was tested hourly beginning 40 h after EB administration and continuing until the end of heat or until 5 h after the final drug treatment (see the procedural section of each experiment). An animal was considered 'in heat' if it displayed a lordosis response with a duration of 2 sec or more during at least two tests. Latency, duration, and maximum were used to assess the the quality of heat. These quality measures are defined as follows. Latency: the time (h) from the first test to the first lordosis response. Duration: the total time (h) that an animal displayed lordosis. Maximum: the time (sec) that the longest lordosis position was held. Experiment 1 - - inhibition of dopamine beta-hydroxylase Rationale Administration of phenoxybenzamine, an alpha-adrenergic antagonist4,~, abolished lordosis behavior in female guinea pigs rendered sexually receptive by EB plus p6. In order to determine whether the inhibition of lordosis by phenoxybenzamine is peculiar to this drug or is a property of drugs that interfere with noradrenergic synaptic transmission in general, females were treated with EB, P, and the DBH inhibitor U-14,62418. Because DBH catalyzes the hydroxylation of DA to NE, inhibition of this enzyme decreases the rate of NE synthesis and ultimately the availability of NE for synaptic transmission. If the inhibition of lordosis by phenoxybenzamine results from an interference with NE neurotransmission then U-14,624 should also inhibit lordosis. Procedure The design of this experiment is outlined in Table I. Basically, all groups received 1.5 #g EB at 0 h and 0.5 mg P 40 h later. Group 1, the control group, received an injection of the vehicle for U-14,624 1 h after P. Experimental groups 2, 3, and 4 received 50, 100, and 150 mg/kg U-14,624 respectively, 1 h after P. Hourly tests for lordosis continued until every animal failed to respond to manual manipulation with a display of lordosis.
372 TABLE I Effects of U-14,624 ( U) on sexual behavior induced by estradiol benzoate ( EB) and progesterone (P)
Dosage of U is given in mg/kg as subscript. Veh = 0.25 ~ methylcellulose vehicle. Group
1 2 3 4
Treatment
N
0 (h)
40 (h)
41 (h)
EB EB EB EB
P P P P
Veh Us0 U10o Uas0
12 11 11 11
% in heat Latency*
75.0 27.3** 27.3** 9.1 ***
(X ± S.E., h)
Duration* (X ~z S.E., h)
Maximum* (X ± S.E., see)
5.6 ± 6.0 ± 5.7 ± 7.0 ±
6.4 ± 7.0 ± 6.0 ± 8.0 ±
12.4i 2.25 16.7± 3.18 13.3 ± 2.72 7.0 22 0.00
0.50 1.16 0.88 0.00
0.71 2.00 0.58 0.00
* Based on animals that displayed heat. ** P < 0.05 versus group 1 (Fisher Exact Probability Test). *** P < 0.005 versus group 1 ('Fisher Exact Probability Test).
Results
The percentage of animals displaying heat after treatment with EB and P was decreased by all doses of U-14,624 tested (compare groups 2, 3, and 4 with group 1 in Table I). The drug-treated animals showed no signs of a general behavioral disturbance and actively resisted manual manipulation by running away. Because very few drug-treated females displayed heat, the effects of U-14,624 on the quality of heat (latency, duration, and maximum) could not be assessed. Experiment 2 - - catecholamine content o f several brain areas after treatment with U14,624 Rationale
In Experiment 1, treatment with U-14,624 inhibited lordosis behavior in females treated with EB and P. Presumably, this inhibition was associated with a drug-induced decrease in NE synthesis. To confirm this, the catecholamine (CA) content of 4 brain areas was determined after animals were treated with EB, P, and U-14,624, as in Experiment 1. Brain areas were selected that (a) take up and retain estrogen and/or progesterone from the general circulation, (b) contain a high density of NE terminals, (c) and/or have been implicated in the regulation of female sexual behavior by hormone-implant, lesion, and drug studies. The caudate-putamen, preoptic area, medial basal hypothalamus, and cortex were selected and assayed with a sensitive radioenzymatic assay. Procedure
All animals received 1.5/~g EB at 0 h and 0.5 mg P 40 h later. One hour after the administration of P, animals received either U-14,624 (100 mg/kg) or the methylcellulose vehicle. At 2, 6, and 24 h after drug treatment, animals were decapitated and their brains quickly removed and dissected under dry ice. The preoptic area (POA),
373 TABLE II Effects of U-14,624 (100 mg/kg) on norephinephrine content (in ng/mg tissue) of medial basal hypothalamus ( MBH) , cortex ( CO RT) , and pre-optic area ( PO A ) at 2,6, and24 h after administration
Values are the mean of 5 samples plus or minus the standard error. Hours M B H Control
2 6 24
CORT U-14,624
% Control Control
POA U-14,624
% Control Control
U-14,624
% Control
3.56±0.21 1.76±0.31' (49.4) 0.25-4-0.02 0.16-4-0.03 (64.0) 2.754-0.20 1.784-0.30 (64.7) 3.37-4-0.26 1.52.4.0.55" (45.1) 0.284-0.01 0.134-0.03" (46.4) 2.844-0.41 1.20.4.0.49" (42.2) 3.254-0.41 2.10.4.0.54 (64.6) 0.24-4-0.04 0.204-0.04 (83.3) 3.23-I_0.57 1.79.4.0.43' (55.4)
* P < •.•5versusc•ntr••va•uesatthesametime(Ana•ysis•fVariancef••••wedbyDuncan•sMu•tip•eRange Test). caudate-putamen (C-P), medial basal hypothalamus (MBH), and a section of frontal cortex (CORT) were taken and assayed for N E and D A content with the radioenzymatic assay of Cuello, Hiley and Iversen 8 as modified by Zschaeck and Ramirez 26. Results
The effects of U-14,624 on regional brain N E and DA content of EB plus P primed females are presented in Tables II and III, respectively. N E content of C-P and D A content of C O R T were not detectable. U-14,624 caused a significant reduction in N E content of MBH, CORT, and POA within 6 h. U-14,624 had no effect on D A content of POA or C-P. D A content of M B H was increased at 2, 6, and 24 h after administration of U-14,624. Experiment 3
restoration o f lordosis behavior after treatment with U-14,624 by drugs
that affect monoamine receptors Rationale
Treatment with D B H inhibitor U-14,624 inhibited lordosis behavior in females treated with EB and P. However, in addition to causing a significant decrease in brain N E levels, treatment with U-14,624 caused a 155 ~ increase in D A content of M B H (see Experiment 2). Further, inhibition of D B H has previously been shown to increase brain serotonin (5-HT) synthesis 19,z°. Because D A and 5-HT agonists suppress lordosis behavior in female guinea pigs% the inhibition of lordosis induced by U14,624 may have been attributable to an increase in D A and 5-HT activity rather than to a decrease in N E activity. In order to distinguish between these alternatives, the following experiment was designed. Females were treated with EB, P, and U-14,624, as in Experiments 1 and 2. Five hours after treatment with U-14,624 animals received pimozide a D A receptor
374
xs 0
0
c5 t'-,I
¢-q
- H ~ -q
-H ~H -H
~S
~C ea .,~- u-.~
-Hq-H
::3
d~Sd
-H -H -H 'c
xs
t~
E q4 -H -H 0
c~do
o~5c5 -H~-H
;>
~S 0
d V eq
375 TABLE IV Effects of clonidine ( C), pimozide (Pi), and methysergide ( M) on sexual behavior of animals treated with estradiol benzoate ( EB) , progesterone (P) and the D B H inhibitor U-14,624 (U)
Veh at 41 h - 0.25 % methylcellulose vehicle; Veh at 46 h - isotonic saline vehicle. Group Treatment
N
% in heat Maximum*
(X :k S.E., sec)
0 (h)
40 (h)
41 (h)
46 (h)
1 2 3
EB EB EB
P P P
Veh U U
Veh Veh C
14 13 13
71.4 0.0 69.2**
10.5 ± 1.33 -11.2 ± 1.81
4 5
EB EB
P P
U U
Pi M
14 10
14.3 10.0
10.5 :k 4.5 7.0 ± 0.0
* Based on animals that displayed heat. ** P < 0.005 versus group 2 (Fisher Exact Probability Test). blocker 2, methysergide - - a 5-HT receptor blocker 16,17, or clonidine - - a N E receptor stimulatorL If the inhibition of lordosis by U-14,624 resulted from an increase in D A activity, then treatment with pimozide should induce lordosis behavior in animals treated with EB, P, and U-14,624. If the inhibition resulted from an increase in 5-HT activity, then methysergide should induce lordosis. If, on the other hand, the U14,624-induced decrease in N E synthesis was responsible for inhibition of lordosis, then treatment with clonidine should induce lordosis behavior. Pro cedure
The design of this experiment is outlined in Table IV. Basically, all groups received 1.5/~g EB at 0 h, 0.5 mg P at 40 h, and two additional injections (the first at 41 h and the second at 46 h). Groups 1 and 2 were control groups. Group 1 received an injection of the vehicle for U-14,624 at 41 h and isotonic saline at 46 h. G r o u p 2 received U-14,624 (100 mg/kg) at 41 h and saline at 46 h. Experimental groups 3, 4, and 5 all received U-14,624 (100 mg/kg) at 41 h followed by clonidine (1.0 mg/kg), pimozide (1.0 mg/kg), or methysergide (20.0 mg/kg), respectively, at 46 h. Because U-14,624 did not inhibit lordosis in 100 % of females treated with EB and P (73 % inhibited, see Experiment 1), any animal treated with this drug that displayed a lordosis response prior to the injection at 46 h was eliminated from the experiment. This procedure reduced the possibility of incorrectly attributing a lordosis response induced by EB and P to the action of one of the three experimental drugs. Hourly tests for lordosis continued until 5 h after the final drug treatment. In addition to the hourly tests, animals were tested at 46.5 and 47.5 h. Results
Of the three experimental drugs (i.e. clonidine, pimozide and methysergide), only clonidine induced lordosis behavior in females treated with EB, P, and U-14,624 (compare ' % in heat' for groups 3, 4, and 5 with group 2 in Table IV). The animals
376 treated with EB, P, U-14,624 and clonidine did not differ from those treated with only EB and P in terms of the percentage of animals displaying heat or the longest lordosis duration (compare group 3 with group 1 in Table IV). The clonidine-treated animals began displaying lordosis 1.67 ~ 0.40 h after the injection of clonidine and continued showing the response for 2.72 ~z 0.29 h (values are ~ ~ S.E.). Experiment 4 - - effects of pretreatment with an alpha-adrenergic antagonist on clonidine facilitation of female sexual behavior Rationale In previous studies, clonidine facilitated the expression of sexual receptivity in female guinea pigs treated with EB alone 7 and EB plus p6. In Experiment 3 of this study, clonidine induced lordosis behavior in animals treated with EB, P, and the DBH inhibitor U-14,624. Under all of these conditions, the facilitation of female sexual behavior presumably resulted from the activation of postsynaptic noradrenergic receptors by clonidine. In order to test this, EB and EB plus P primed females were treated with the alpha-adrenergic blocker phenoxybenzamine4, 9 prior to treatment with clonidine. If clonidine facilitates lordosis behavior by stimulation of alphaadrenergic receptors, then blockade of these receptors with phenoxybenzamine should block or reduce the facilitation of lordosis by clonidine. Procedure The design of this experiment is outlined in Table V. Basically, groups 1-3 received 1.5/zg EB at 0 h and 0.5 mg P 40 h later. Groups 4-6 received 1.5 #g EB at 0 h and no P. At 41 h after EB, phenoxybenzamine (20.0 mg/kg, groups 2, 3, and 6) or the 50 % propylene glycol vehicle (groups 1, 4, and 5) was given. Five hours later, at 46 h, each group received clonidine (1.0 mg/kg, groups 3, 5, and 6) or saline (groups 1, 2, and 4). Hourly tests for lordosis continued until 5 h after the final drug treatment (i.e. 51 h). In addition to the hourly tests, the animals were tested at 46.5 and 47.5 h. Results Treatment with clonidine induced lordosis behavior in animals treated with EB alone (compare '% in heat' for group 5 with group 4 in Table V). The animals treated with EB plus clonidine began displaying lordosis 0.89 ~ 0.18 h after the injection of clonidine and continued showing the response for 2.39 ~: 0.22 h (values are ~ ± S.E.). The lordosis responses of females treated with EB plus clonidine were generally less intense than those displayed by females treated with EB and P (compare 'maximum' for group 5 with group 1 in Table V) and usually consisted of a weak flattening of the back, brief display and dilatation of the vaginal region, and occasional rumbling vocalizations. Pretreatment with phenoxybenzamine blocked the induction of lordosis behavior by clonidine in EB primed females (compare' % in heat' for group 6 with group 5 in Table V). Treatment with phenoxybenzamine inhibited lordosis behavior in animals
377 TABLE V Effects of phenoxybenzamine (Pb) on the facilitation of sexual behavior by clonidine ( C) in animals treated with estradiol benzoate (EB) and progesterone (P) or EB alone
Veh at 41 h = 50~ propylene glycol vehicle; Veh at 46 h = isotonic saline. Group Treatment O(h)
40(h)
41(h)
46(h)
1 2 3 4 5
EB EB EB EB EB
P P P ---
6
EB
--
Veh Pb Pb Veh Veh Pb
Veh Veh C Veh C C
N
% in heat Maximum* (X ± S.E., see)
8 7 8 9 11 10
100.0 17.62 ± 3.79 0.0"* - 25.0 7.50 ± 3.50 0.0 -81.8"** 6.67 4- 1.12§ 10.0§§ 8.00 4- 0.00
* Based on animals that displayed heat. ** P < 0.005 versus group 1 (Fisher Exact Probability Test). *** P < 0.005 versus group 4 (Fisher Exact Probability Test). § P < 0.01 versus group 1 (t-test, two-tailed). §§ P < 0.05 versus group 5 (Fisher Exact Probability Test).
treated with EB and P (compare' ~ in heat' for group 2 with group 1 in Table V). The percentage of EB plus P primed animals displaying heat after treatment with phenoxybenzamine and clonidine did not differ from the percentage displaying heat after phenoxybenzamine without clonidine (compare group 3 with group 2 in Table V). DISCUSSION This study demonstrated that U-14,624 (a D B H inhibitor), given 1 h after progesterone, blocks induction of lordosis behavior by estrogen and progesterone in ovariectomized guinea pigs. The effects of U-14,624 on CA content of MBH, POA, CP, and C O R T were assessed at 2, 6, and 24 h after drug administration. Within 6 h, U14,624 caused a 55 ~ depletion of NE in MBH, POA, and C O R T (NE was not detectable in C-P). Females treated with estrogen and progesterone normally begin to display lordosis about 4-6 h after administration of progesterone. Thus, NE levels were significantly reduced after treatment with U-14,624 during the period when females are usually sexually receptive. On the basis of these data alone, we were unable to conclude that the inhibition of lordosis by U-14,624 was a consequence solely (or even primarily) of depletion of NE. U-14,624 also caused a 1 5 5 ~ increase in D A content of MBH. Effects of U-14,624 on 5-HT were not examined in the guinea pig, but in rats inhibition of D B H not only interferes with noradrenergic transmission but also increases brain 5-HT synthesis19, 20. Because D A and 5-HT agonists inhibit lordosis in guinea pigs 6, the inhibition of lordosis induced by U-14,624 may have been attributable to an increase in D A or 5-HT activity rather than to a decrease in N E activity. In order to distinguish between these alternatives, we attempted to restore lordosis behavior in estrogen plus progesterone primed females treated with U-14,624
378 by (a) activating NE receptors with clonidine, (b) blocking DA receptors with pimozide, and (c) blocking 5-HT receptors with methysergide. Of these drugs, only clonidine restored lordosis behavior. Thus, inhibition of lordosis after administration of U-14,624 appears to be attributable solely to a reduction in noradrenergic transmission. However, it may be premature to conclude that changes in DA and/or 5HT activity do not contribute to the U-14,624-induced inhibition of lordosis. Although the dosages of pimozide and methysergide used in this study are relatively high and facilitate lordosis behavior in rats12,21, we lack evidence that these drugs effectively block DA and 5-HT receptors in guinea pigs. In previous studies, clonidine facilitated the expression of sexual receptivity in female guinea pigs primed with estrogen alone 7, and with estrogen and progesterone 6. In this study, clonidine induced lordosis in females primed with estrogen alone and also restored lordosis in females treated with estrogen, progesterone and U-14,624. Under all of these conditions, the facilitation of female sexual behavior by clonidine presumably resulted from the activation of postsynaptic alpha-adrenergic (noradrenergic) receptors. However, under some conditions (e.g. low doses in rats) clonidine may preferentially activate presynaptic alpha-adrenergic receptors10,23,24. These presynaptic receptors or autoreceptors are thought to mediate a local negative feedback through which interneuronal NE can inhibit its own further release2L Because clonidine facilitated lordosis behavior in female guinea pigs with already reduced noradrenergic transmission (as a result of U-14,624), it seems unlikely that facilitation of lordosis by clonidine is mediated by activation of presynaptic alpha-adrenergic receptors. In addition, pretreatment with the postsynaptic alpha-adrenergic antagonist phenoxybenzamine11,23 blocked the facilitation of lordosis by clonidine in both estrogen and estrogen plus progesterone primed females. The results of this study and those of previous experiments6, 7 suggest that NE neurotransmission facilitates the expression of female sexual behavior in guinea pigs. Thus far, few studies have examined the role of NE in the regulation of female sexual behavior in other species. In rats, NE may also facilitate the expression of female sexual behavior but this is far from certain. Everitt and coworkers 12-14 reported that in estrogen-primed female rats the CA releaser amphetamine increases the facilitatory effects of DA receptor blockade on lordosis. Presumably the facilitation of lordosis by amphetamine under these conditions is attributable to a selective enhancement of NE receptor activity. However, activation of NE receptors with clonidine suppresses ongoing lordosis responding induced by estrogen and progesterone in rats 1°. This is in marked contrast to the facilitatory effects of clonidine on lordosis behavior in guinea pigs. Davis and KohP 0 have suggested that the inhibitory effects of clonidine on lordosis in rats might be mediated by activation of inhibitory presynaptic NE receptors rather than by postsynaptic NE receptors. This suggestion seems plausible because pretreatment with the putative presynaptic NE receptor antagonist yohimbine, but not the postsynaptic NE receptor antagonist phenoxybenzamine, blocks the inhibitory effects of clonidine on lordosis in rats 1°. However, inhibition of DBH (with FLA 63) or treatment with phenoxybenzamine has little or no effect on lordosis behavior induced by estrogen and progesterone in rats 1°,12-14. In fact, Ahlenius et al. 1
379 have reported that the DBH inhibitor FLA 63 increases lordosis behavior in rats primed with estrogen alone. Thus, at present it is unclear whether NE facilitates female sexual behavior in rats or other species as it does in guinea pigs. We also do not know what neurochemical systems NE influences to facilitate sexual behavior of female guinea pigs. Antelman and Caggiula 5 have recently suggested that NE may influence the expression of a number of 'activated or stressinduced' behaviors (i.e. stimulant-induced stereotypy or motor activity, shuttle-box avoidance, several types of aggressive behaviors, electrical self-stimulation of the brain, and stress-related eating) by modulating brain (perhaps nigrostriatat) DA transmission. Perhaps a similar N E - D A interaction is involved in the regulation of sexual behavior in female guinea pigs. ACKNOWLEDGEMENTS
We thank Dr. V. D. Ramirez for providing facilities to learn the radioenzymatic assay and C. Reboulleau and P. Albietz for their expert technical assistance. Supported by N I H Research G r a n t HD-04467, N I M H Career D e v e l o p m e n t A w a r d MH-29006 a n d a grant from the Busch F o u n d a t i o n , Rutgers University (all to H.H.F.). C o n t r i b u t i o n N u m b e r 314 of the Institute of A n i m a l Behavior.
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