HORMONES
AND
BEHAVIOR
26,
451-473 (1992)
,x-, a-, and K-Opioid Receptor Agonists Selectively Modulate Sexual Behaviors in the Female Rat: Differential Dependence on Progesterone JAMES G. PFAUS AND DONALD
W. PFAFF
Laboratory of NeurobioIogy and Behavior, The RockefeUer University, 1230 York Avenue, New York, New York 10021 Previous studies suggested that opioid receptor agonists infused into the lateral ventricles can inhibit (through /L receptors) or facilitate (through S receptors) the lordosis behavior of ovariectomized (OVX) rats treated with estrogen and a low dose of progesterone. The present study investigated the behavioral and hormonal specificity of those effects using more selective opioid receptor agonists. Sexually experienced OVX rats were implanted stereotaxically with guide cannulae aimed at the right lateral ventricle. One group of rats was treated with estradiol benzoate (EB, 10 pg) 48 hr and progesterone (P, 250 pg) 4 hr before testing, whereas the other group was treated with EB alone. Rats were infused with different doses of the selective p-receptor agonist DAMGO, the selective a-receptor agonist DPDPE, or the selective K-receptor agonist U50-488. The females were placed with a sexually vigorous male in a bilevel chamber (Mendelson and Gorzalka, 1987) for three tests of sexual behavior, beginning 15, 30, and 60 min after each infusion. DAMGO reduced lordosis quotients and magnitudes significantly in rats treated with EB and P, but not in rats treated with EB alone. In contrast, DPDPE and U50-488H increased lordosis quotients and magnitudes significantly in both steroid-treatment groups. Surprisingly, measures of proceptivity, rejection responses, and level changes were not affected significantly by w or K agonists, although proceptivity and rejection responses were affected by DPDPE treatment. These results suggest that the effects of lateral ventricular infusions of opioid receptor agonists on the sexual behavior of female rats are relatively specific to lordosis behavior. Moreover, the facilitation of Iordosis behavior by F- or Kreceptor agonists is independent of progesterone treatment, whereas the inhibitory effect of p-receptor agonists on lordosis behavior may require the presence of progesterone. 0 1992 Academic Press. Inc.
Opioids exert complex controls over reproductive functions in the female rat. Selective opioid receptor agonists and endogenous opioid peptides inhibit pituitary LH secretion (Cicero, Shainker, and Meyer, 1979; Kalra and Kalra, 1984; Leadem and Kalra, 1985). This effect is mediated by an opioid-induced inhibition of GnRH-containing neurons in the medial preoptic area (MPOA), inhibition of GnRH release in the arcuate nucleus/median eminence (Bicknell, 1985; Kalra, Allen, and Kalra, 1989), 457 0018-506x/92 $4.00 All
Copyright 0 1992 by Academic Press. Inc. rights of reproduction in any form reserved.
458
PFAUS AND PFAFF
or possibly a direct inhibitory action of opioids on pituitary gonadotrophs (Blank, Fabbri, Catt, and Dufau, 1986; Simantov and Snyder, 1977). However, under certain experimental conditions opioid agonists can stimulate GnRH release and LH secretion (Kalra, Crowley, and Kalra, 1986; Leadem and Kalra, 1983; Piva, Limonta, Maggi, and Martini, 1986). Opioids may also inhibit or facilitate the sexual behavior of female rats, depending upon the receptor type or brain area stimulated. For example, p-endorphin can inhibit or facilitate lordosis behavior in female rats following the infusion of comparable doses into the third ventricle (Wiesner and Moss, 1986a,b) or lateral ventricles (Pfaus and Gorzalka, 1987a), respectively. However, low doses of both ,&endorphin and the p-selective agonist morphiceptin inhibit lordosis behavior, whereas only high doses of these agonists facilitate lordosis following infusion into the lateral ventricles (Pfaus and Gorzalka, 1987b). In contrast, the relatively selective a-receptor agonist n-Ser’,Leu5,Thr6-enkephalin (a-receptor peptide; DSLET) or the selective K-receptor agonist leumorphin produce a dosedependent facilitation of lordosis behavior following infusion into the lateral ventricles (Pfaus and Gorzalka, 1987b; Suda, Nakao, Sakamoto, Morii, Sugawara, and Imura, 1986). Infusions of morphine or @-endorphin to the ventromedial hypothalamus (VMH) or mesencephalic reticular formation also inhibit lordosis, whereas infusions to the mesencephalic central grey (MCG), dorsomedial hypothalamus, amygdala, or striatum are without effect (Vathy, van der Plas, Vincent, and Etgen, 1991; Wiesner and Moss, 1989; but see Sirinathsinghji, 1984). /3-Endorphin has also been reported to inhibit lordosis following infusion into the MPOA (Sirinathsinghji, 1986). The inhibitory effect of both /3-endorphin and morphiceptin may occur through an interaction with ,ul sites, as the selective ,+ antagonist naloxazone reversed the inhibitory effect of these peptides (Pfaus, Pendleton, and Gorzalka, 1986; Wiesner and Moss, 1989). Because pendorphin possesses equal affinity for cr. and F receptors (Paterson, Robson, and Kosterlitz, 1983), and high doses of morphiceptin and leumorphin also bind to 6 receptors (Chang et al., 1981), Pfaus and Gorzalka (1987b) suggested that p-receptor activation may inhibit, whereas a-receptor activation may facilitate, lordosis. The precise role of K receptors in lordosis is unclear, and it is not known to what extent the effects of p or 6 agonists depend upon the presence of progesterone. It has also been unclear whether the effects of opioid receptor agonists are specific to lordosis behavior or reflect indirect actions or more general modulations of social or locomotor behaviors. The sexual behavior of female rats typically is examined in small unilevel chambers. Lordosis quotients and magnitudes are commonly employed as measures of sexual receptivity. Measures of sexual proceptivity (e.g., presenting, hopping, and darting) and rejection (e.g., fighting, boxing, or defensive postures) can also be recorded in these chambers, although such measures generally
OPIOIDS AND FEMALE
SEXUAL
BEHAVIOR
459
do not convey the rich pattern of solicitation displayed by female rats mating in open fields (e.g., Beach, 1976; McClintock, 1984; Madlafousek and Hlinak, 1978). It has also been common to measure locomotor behavior as a control separately from sexual behavior. The bilevel chambers designed by Mendelson and Gorzalka (1987) provide an opportunity to assess female sexual behavior in a situation where the females can control or “pace” their copulatory contact with a male (Mendelson and Pfans, 1989; Pfaus, Mendelson, and Phillips, 1990). Typically, female rats force males to chase them from level to level, reminiscent of the pacing behavior displayed by female rats in a large open field. This level-to-level locomotion by the female can be counted prior to each mount or intromission by the male, and may constitute a measure of pacing behavior that can be used in conjunction with measures of sexual receptivity, proceptivity, and rejection to assess the full pattern of female sexual behavior. The present study was undertaken to examine whether the effects of lateral ventricular infusions of highly selective opioid receptor agonists on female sexual behavior are specific to lordosis, and whether those effects depend upon the presence of progesterone. Accordingly, we examined the effects of different doses of the selective p-receptor agonist nAla*,MePhe”,Gly-ol’-enkephalin (DAMGO), the selective S-receptor agonist D-Pen’,&Pet?-enkephalin (DPDPE), or the selective K-receptor agonist U50-488 on lordosis quotients, lordosis reflexes, proceptive behaviors, rejection responses, and level changes displayed in bilevel chambers by OVX rats primed with either estrogen and progesterone or estrogen alone. METHOD Animals and surgery. Ovariectomized female Sprague-Dawley rats (250 g) and intact male Long-Evans rats (300-500 g) were obtained from Charles River, Inc. (Wilmington, MA). Prior to surgery, the females were given at least six 30-min acquisition tests of sexual behavior with sexually vigorous male Long-Evans rats in bilevel chambers to allow them to acquire the complete pattern of sexual behavior in these chambers (see below). Following this acquisition period, each female was anesthetized with sodium pentobarbital (60 mg/kg) and implanted stereotaxically with a 23-gauge stainless-steel guide cannula (Plastics One) aimed at the right lateral ventricle according to the atlas of Pellegrino, Petlegrino, and Cushman (1979). Following surgery, the females were housed individually in Plexiglas cages in a colony room maintained on a reversed 12: 12 hr light/dark cycle (lights off at 1O:OO AM) at approximately 21°C. Sexually vigorous Long-Evans males were housed in groups of three in suspended wire-mesh cages in the same colony room. All rats were given free access to lab chow and water. The placement and flow of each cannula was tested 2 days before and 2 days after each experiment by replacing the
460
PFAUS AND PFAFF
dummy cannula with an injection cannula and infusing 2 pg of angiotensin II into the right lateral ventricle of each rat (Pfaus and Gorzalka, 1987b). Because infusions of angiotensin II into the lateral ventricles reliably induce thirst (Epstein, Fitzsimons, and Rolls, 1970), only those females that displayed vigorous drinking within 5 min of infusion served as subjects in each experiment. Drug and hormone treatments. Estradiol benzoate (EB) and progesterone (P) were purchased from Sigma (St. Louis, MO), dissolved in sesame oil, and injected subcutaneously in 0.1 ml. Angiotensin II (Bachem) was dissolved in physiological saline at a concentration of 1 pg/pl. DAMGO (Bachem), DPDPE (Bachem), and U50-488 (a gift from Drs. Kelly Standifer and Gavril Pasternak of the Memorial Sloan-Kettering Institute) were dissolved in physiological saline to obtain concentrations of 10, 100, or 1000 ng/pl, each of which was infused in a constant volume of 2 ~1. Control rats received infusions of 2 ,ul of physiological saline. All central infusions were made using an electrically driven infusion pump (Sage Instruments Model 3HlA) at a rate of 2 pl/min. The injection cannula was left in place for an additional minute to assure diffusion of the drug. Dummy cannulae were replaced after each infusion. Behavioral testing. Experimental tests of sexual behavior involved placing each female into a bilevel testing chamber with a sexually vigorous male until 10 mounts with pelvic thrusting had occurred. Lordosis quotients (LQ; calculated as the percentage of mounts with pelvic thrusting that resulted in a lordosis posture), lordosis reflex scores (LS; calculated for each lordosis response using a modified method of Hardy and DeBold (1971), in which each lordosis response was ranked in intensity from 1 to 3 according to the extent of dorsiflexion observed), proceptive behaviors/mount (counts of hopping, darting, and presenting prior to each mount) rejection responses/mount (counts of boxing, fighting, and defensive postures prior to each mount), and the number of level changes prior to each mount, were calculated during each test for each female. All testing occurred during the middle third of the rats’ dark cycle in a room with dim illumination. Following surgery, the females were given two saline-infusion pretests of sexual behavior in the bilevel chambers at 4-day intervals prior to the first experiment to assure that surgery did not affect their ability to perform sexual behaviors. Following the pretest period, the females received infusions of either 20, 200, or 2000 ng of each drug once every 4 days in a latinized design, such that all females were administered each dose of a drug over a 16day period. Within each experiment, sexual behaviors were observed 15, 30, and 60 min after infusion. One group of females was primed with EB (10 fig) 48 hr and P (250 pg) 4 hr before each infusion. This steroidpriming regimen has been used in previous experiments to produce a moderate degree of lordosis responding (LQs of 50 to 70) in rats with
OPIOIDS AND FEMALE SEXUAL BEHAVIOR DAMGO
DAMGO (EB alone)
461
(EB+P)
104 Ii-,
10
20
30
40
50
60
10
20
Time (r&n)
30
40
50
63
Time (mm)
FIG. 1. Effects of different doses of DAMGO on lordosis quotients (LQ) and lordosis scores (LS) in OVX rats primed with EB alone (left side; n = 8) or EB and P (right side, )I = 8). Statistical differences among the groups are described fully in the text.
chronic guide cannulae aimed at the lateral ventricles (Pfaus and Gorzalka, 1987b). The other group of females was primed with EB (10 pg) 48 hr before each test. Statistical analysis. Two-way within-subjects analyses of variance (ANQVAs) were conducted for the effects of dose, time, and the dose x time interaction for each of the five behavioral measures. For each significant ANOVA, pairwise comparisons among the means were made using the Tukey method, P < 0.05. RESULTS Effect of p-receptor activation with DAMGB. The effects of differenr doses of DAMGO on measures of female sexual behavior displayed by OVX rats primed with EB and P or EB alone are shown in Fig. 1 and Table 1. DAMGO produced a dose-dependent reduction in the LQs of rats primed with EB and P (fig. 1, top right). The ANOVA detected a significant main effect of dose, F(3, 21) = 5.18, P < 0.008, and time, F(2, 14) = 25.13, P < 0.0001, but not a significant interaction of dose and time. Post-hoc comparisons of dose revealed that the 200- and 2000ng doses of DAMGO significantly reduced the LQs in these females compared with the control. Post-hoc comparisons of the time points re-
462
PFAUS
Effects
of DAMGO
EB alone Dose of DAMGO
on Level
Min
AND
PFAFF
TABLE 1 Changes, Proceptivity,
and Rejection
Responses
Level
changes
Proceptivity
1.5 30 60
2.18 1.70 1.12
+ 0.50 -c 0.20 + 0.22
0.02 0.10 0.13
+ 0.01 + 0.09 f 0.04
0.00 0.13 0.05
15 30 60
1.78 1.20 1.80
+ 0.51 c 0.31 -+ 0.56
0.10 0.28 0.13
+ 0.08 + 0.16 + 0.08
0.07 0.00 0.03
+ 0.07
200 ng
15 30 60
2.08 1.17 1.76
+ 0.39 t 0.30 f 0.47
0.10 0.32 0.22
?z 0.10 2 0.18 !I 0.07
0.03 0.10 0.07
2 0.03 f 0.10 + 0.07
2000 ng
1.5 30 60
0.86 0.90 1.38
+ 0.40 + 0.33 + 0.38
0.13 0.17 0.12
+ 0.07 + 0.09 k 0.05
0.00 0.03 0.03
+ 0.03 2 0.03
Control
20 ng
EB + P Dose of DAMGO
Min
Control
Level
changes’
Proceptivity
Rejection
+ 0.13 r 0.05
f
0.03
Rejection
15 30 60
1.53 + 0.22 1.35 + 0.09 1.41 k 0.21
0.06 0.14 0.09
t 0.04 + 0.06 + 0.04
0.08 0.05 0.02
+ 0.07 + 0.04 + 0.02
15 30 60
1.05 + 0.32 0.99 I!I 0.17 1.03 + 0.16
0.11 0.09 0.04
+ 0.10 k 0.04 + 0.02
0.00 0.01 0.00
+ 0.01
200 ng
15 30 60
0.90 k 0.19 0.78 + 0.28 1.15 + 0.19
0.03 0.04 0.02
+ 0.02 t 0.04 + 0.02
0.01 0.00 0.00
2 0.01
2000 ng
15 30 60
0.35 0.55 1.31
0.03 0.03 0.00
+ 0.02 f 0.02
0.01 0.01 0.02
+ 0.01 f 0.01 + 0.02
20 ng
Note.
Values
are means
+ SEM.
f 0.12* f 0.16* + 0.36
*P < 0.05 from
control.
vealed that the LQs were higher in all dose conditions at 30 and 60 min compared to 15 min. Although none of the doses of DAMGO decreased the LSs in these rats, there was a significant effect of time on the LSs, F(2, 14) = 7.07, P < 0.008 (Fig. 1, bottom right). No significant interaction of dose and time occurred for the LS. DAMGO did not affect level changes, proceptivity, or rejection responses significantly (Table 1). In contrast, DAMGO did not affect the LQs significantly of rats primed with E alone (Fig. 1, top left). The ANOVA failed to detect a significant effect of dose, although a significant effect of time was detected for these rats, F(2, 10) = 49.02, P < 0.0001. No significant interaction of dose
OPIOIDS
AND FEMALE
DPDPE (EB alone)
10 20 30 40 50 60 Time (min)
SEXUAL
BEHAVIOR
463
DPDPE (EB+P)
10 20 30 40 50 60 Time (min)
FIG. 2. Effects of different doses of DPDPE on lordosis quotients (LQ) and iordosis scores (LS) in OVX rats primed with EB alone (left side, n = 10) or EB and P (right srde, n = 11). Statistical differences among the groups are described fully in the text.
and time was detected. Post-hoc comparisons of the effect of time revealed that the LQs were higher in all dose conditions at 30 and 60 min compared to 15 min. Similarly, DAMGO did not affect the LSs, although a significant effect of time was detected, F(2, 10) = 3.99, P < 0.05 (Fig. 1, bottom left). Post-hoc comparisons revealed that the LSs at 60 min were significantly higher than those at 15 min. DAMGO also reduced the number of level changes per mount (Table 1). The ANOVA detected a significant main effect of dose, F(3, 21) = 3.44, P < 0.04, and time, F(2, 14) = 3.51, P < 0.05, and a significant interaction of dose and time, F(h, 42) = 2.37, P < 0.05. Post-hoc comparisons of the effects of dose and time did not reveal any overall differences among the marginal means. However, post-hoc comparisons of the interaction effect revealed that the 2000-ng dose of DAMGO reduced level changes significantly 15 and 30 min after infusion compared to the control values. DAMGO did not affect proceptivity or rejection responses significantly (Table 1). Effect of &receptor activation with DPDPE. The effect of different doses of DPDPE on measures of female sexual behavior displayed by OVX rats primed with EB and P or EB alone are shown in Fig. 2 and Table 2. In contrast to the effect of DAMGO, all doses of DPDPE facilitated the LQs of rats primed with EB and P (Fig. 2, top right). The ANQVA
464
PFAUS
Effects EB alone Dose of DPDPE
of DPDPE
on Level
AND
PFAFF
TABLE 2 Changes, Proceptivity,
and Rejection
Responses
Min
Level
changes
15 30 60
1.51 1.11 1.70
+ 0.22 + 0.20 k 0.25
0.01 0.01 0.09
+ 0.01 f 0.01 ” 0.03
0.32 0.10 0.03
f 0.11 + 0.05 * 0.02
20 ng
1.5 30 60
1.93 1.74 1.82
+ 0.17 r 0.29 + 0.27
0.15 0.13 0.12
r 0.06 + 0.05 + 0.05
0.11 0.05 0.03
f t f
200 ng
15 30 60
1.86 1.59 1.81
2 0.17 + 0.20 ?z 0.19
0.08 0.20 0.12
f 0.08 + 0.05 c 0.04
0.08 0.10 0.05
+ 0.05 + 0.05 + 0.04
2000 ng
15 30 60
1.95 1.26 1.35
? 0.26 f 0.20 2 0.12
0.08 0.11 0.17
+ 0.05 f 0.03 f 0.06
0.04 0.03 0.00
f 0.03* k 0.02
Min
Level
changes
Control
EB + P Dose of DPDPE Control
Proceptivity
Proceptivity
Rejection
0.05 0.04 0.03
Rejection
15 30 60
1.60 k 0.33 1.31 + 0.20 1.10 f 0.22
0.20 0.18 0.20
f 0.05 + 0.04 iz 0.08
0.17 0.10 0.08
+ 0.10 c 0.07 + 0.08
20 ng
15 30 60
1.43 1.43 1.61
k 0.16 k 0.23 + 0.38
0.37 0.38 0.29
-t- 0.15 +- 0.17 2 0.11
0.03 0.05 0.04
+ 0.03 + 0.05 + 0.04
200 ng
15 30 60
2.06 1.26 1.18
+ 0.31 + 0.26 -r- 0.22
0.49 0.54 0.32
f 0.09* 2 0.10* + 0.09*
0.01 0.04 0.04
+ 0.01 + 0.03 + 0.04
2000 ng
15 30 60
2.13 1.26 1.28
+- 0.26 + 0.09 + 0.20
0.32 0.50 0.39
+ 0.08* + 0.10* +- 0.09*
0.00 0.00 0.00
Note.
Values
are means
f
SEM.
*P < 0.05 from
control.
detected a significant main effect of dose, F(3, 27) = 15.24, P < 0.0001, and time, F(2, 18) = 19.31, P < 0.0002, and a significant interaction of the two, F(6,54) = 2.40, P < 0.04. Post-hoc comparisons of dose revealed that all doses of DPDPE increased the LQs compared to the control; however, there were no significant differences between doses of DPDPE. Post-hoc comparisons of time revealed that the LQs were significantly higher at 30 and 60 min compared to 15 min. Post-hoc comparisons of the interaction of dose and time revealed that all doses of DPDPE increased the LQs at 15 and 30 min compared to the control. By 60 min, however, the LQs did not differ significantly among the dose conditions.
OPIOIDS AND FEMALE
SEXUAL
BEHAVIOR
465
DPDPE also facilitated proceptive behaviors (Table 1). The ANOVA detected a significant main effect of dose, F(3, 27) = 4.12, P < 0.02, but no main effect of time or interaction. Post-hoc comparisons revealed that both 200 and 2000 ng of DPDPE increased proceptive behaviors significantly across time compared to the effect of the control. DPDPE did not affect the LSs (Fig. 2, bottom right), level changes, or rejection responses significantly (Table 2). Similarly, DPDPE facilitated the LQs of females primed with EB alone (Fig. 2, top left). The ANOVA detected a significant main effect of dose, F(3, 30) = 21.88, P < 0.0001, and time, F(2, 30) = 73.75, P < 0.0001, and a significant interaction of the two, F(6, 60) = 2.84, P < 0.02. Posthoc comparisons of dose revealed that all doses of DPDPE increased the LQs compared to the control; however, there were no significant differences between doses of DPDPE. Post-hoc comparisons of time revealed that the LQs were significantly higher in all dose conditions at 30 and 60 min compared to 15 min. Post-hoc comparisons of the interaction of dose and time revealed that all doses of DPDPE increased the LQs significantly from the control dose at every time point. DPDPE also facilitated the LSs in these rats (Fig. 2, bottom left). The ANOVA detected a signifjcant main effect of dose, F(3, 30) = 3.95, P < 0.02, and time, F(2, 20) = 12.71, P < 0.0005, and a significant interaction of the two, F(6, 60) = 3.78, P < 0.005. Post-hoc comparisons of dose revealed that all doses of DPDPE increased the LSs significantly compared to control. Post-hoc comparisons of time revealed that the LSs were significantly increased at 60 min compared to 15 min. Post-hoc comparisons of the interaction of dose and time revealed that all doses of DPDPE increased the LSs significantly from control at 15 min, but not at 30 or 60 min. DPDPE also reduced the number of rejection responses displayed by these rats (Table 2). The ANOVA detected a significant main effect of dose, F(3, 30) = 3.57, P < 0.04, and a significant interaction of dose and time, F(6, 60) = 3.18, P < 0.01. Post-hoc comparisons of the interaction effect revealed that the 2000-ng dose reduced rejection responses significantly from the control dose at 15 min, but not at 30 or 60 min. DPDPE did not affect level changes or proceptivity significantly (Table 2).
Eflect of K-receptor activation with U50-488. The effect of different doses of U50-488 on measures of female sexual behavior displayed by OVX rats primed with EB and P or EB alone are shown in Fig. 3 and Table 3. U50-488 facilitated the LQs of rats primed with EB and P (Fig. 3, top right). The ANOVA detected a significant main effect of dose, F(3, 15) = 3.57, P < 0.05, and time, F(2, 10) = 10.65, P < 0.004, but no significant interaction of the two. Post-hoc comparisons of dose revealed that the 200- and 2000-ng doses increased the LQs significantly compared to the 20-ng dose and control. Post-hoc comparisons of time revealed that the
466
PFAUS AND PFAFF
0.01------l 10 20
30
40
50
Time (min)
60
Time (mid
FIG. 3. Effects of different doses of U50-488 on lordosis quotients (LQ) and lordosis scores (LS) in OVX rats primed with EB alone (left side, n = 6) or EB and P (right side, n = 6). Statistical differences among the groups are described fully in the text.
LQs were significantly higher at 60 min compared to 15 min. U50-488 did not affect the LSs (Fig. 3, bottom right), level changes, proceptivity, or rejection responses significantly (Table 3). U50-488 also facilitated the LQs of OVX rats primed with EB alone (Fig. 3, top left). The ANOVA detected a significant main effect of dose, F(3, 15) = 7.43, P < 0.004, and time, F(2, 10) = 80.63, P < 0.0001, but no significant interaction of the two. Post-hoc comparisons of dose revealed that all doses of U50-488 increased the LQs significantly compared to control. Post-hoc comparisons of time revealed that the LQs were increased significantly at 30 and 60 min compared to 15 min. U50488 did not affect the LSs (Fig. 3, bottom left), level changes, proceptivity, or rejection responses significantly (Table 3). DISCUSSION
The results of this study provide strong evidence that opioids inhibit or facilitate the lordosis behavior of female rats depending upon the receptor subtype stimulated and the hormonal state of the animal. Consistent with previous reports (Pfaus and Gorzalka, 1987b; Hammer, Dornan, and Bloch, 1989), p-receptor stimulation inhibited lordosis quotients selectively and dose-dependently in OVX rats primed with EB and P.
OPIOIDS
Effects
of U50-488
El3 alone Dose of U50-488
AND
on Level
Min
Control
FEMALE
SEXUAL
BEHAVIOR
TABLE 3 Changes, Proceptivity,
Level
changes
and Rejection
Proceptivity
467
Responses
Rejection
15 30 60
1.45 + 0.31 1.90 + 0.11 1.70 + 0.27
0.00 0.03 0.02
t 0.03 k 0.02
0.13 0.10 0.03
t 0.07 4 0.06 t 0.03
15 30 60
1.97 f 0.42 1.72 + 0.25 1.43 -c- 0.15
0.00 0.05 0.07
f 0.01 -c 0.07
0.10 0.12 0.10
k 0.08 r 0.09 Ifr 0.11
1.5 30 60
2.13 1.76 1.75
r f f
0.35 0.42 0.26
0.00 0.06 0.06
r 0.05 ir 0.04
0.02 0.03 0.00
r 0.02 + 0.03
15 30 60
2.30 1.68 1.28
+ 0.18 + 0.33 -c 0.17
0.02 0.06 0.03
IiT 0.02 t 0.05 t 0.03
0.06 0.00 0.00
f
Min
Level
changes
Proceptivity
15 30 60
1.87 1.72 2.40
+ 0.32 zt 0.21 f 0.35
0.06 0.05 0.07
zt 0.03 + 0.03 t 0.02
0.02 0.00 0.07
2 0.02
20 ng
15 30 60
2.33 k 0.41 1.50 k 0.29 1.87 k 0.36
0.10 0.05 0.03
f 0.07 f 0.03 k 0.02
0.03 0.05 0.08
f 0.03 i- 0.05 + 0.08
200 ng
15 30 60
1.45 1.58 1.86
+- 0.28 + 0.24 t 0.27
0.12 0.15 0.07
-i- 0.05 k 0.07 r 0.03
0.02 0.00 0.00
+ 0.02
2000 ng
15 30 60
2.00 2.25 2.36
2. 0.38 +- 0.49 2 0.56
0.05 0.13 0.03
k 0.02 + 0.10 + 0.03
0.00 0.00 0.00
20 ng
200 ng
2000 ng
EB + P Dose of U50-488 Control
Note.
Values
are means
+ SEM.
*P < 0.05 from
0.06
Rejection
t 0.05
control
However, this effect was not evident in rats primed with EB alone, suggesting that progesterone treatment may be required for the inhibitory effect of p-receptor stimulation as we have examined it. Such an interaction with progesterone is reminiscent of the inhibitory effect of p-chlorophenylalanine (Gorzalka and Whalen, 1975) or cholecystokinin (Mendelson and Gorzalka, 1984) on lordosis behavior. In contrast, the facilitatory effect of 6- or K-receptor stimulation on lordosis behavior occurred independently of progesterone treatment. With few exceptions, DAMGO, DPDPE, and U50-488 did not affect lordosis reflex intensities or the complex patterns of level changes, pro-
468
PFAUS AND PFAFF
ceptivity, or rejection responses displayed in the bilevel chambers. This suggests that the effects of these drugs were relatively specific to the frequency of lordosis behavior, and not secondary to a more general effect on arousal, social behavior, or locomotor activity during copulation. However, the highest dose of DAMGO reduced level changes significantly in females treated with EB and P. Although those data could be interpreted as evidence of a more general sedative effect of DAMGO, it is common for drugs that reduce pacing or proceptivity, e.g., haloperidol, to increase, rather than decrease, the frequency and magnitude of lordosis behavior (Caggiula, Antelman, Chiodo, and Lineberry, 1979). The lack of effect of DAMGO on proceptivity was surprising, especially given evidence by Wiesner and Moss (1986a,b) that infusions of P-endorphin to the third ventricle reduced measures of receptivity and proceptivity in OVX rats primed with EB and P and mated in small unilevel chambers. It is possible that the behavioral constraints imposed in situations where females cannot easily control the copulatory contact (e.g., in unilevel chambers) reveals effects of opioid agonists that are not manifested in the bilevel chambers. Indeed, female rats with sexual experience in the bilevel chambers primarily use level changing to regulate their copulatory contact with males, rather than the more complex forms of defensive or proceptive behaviors observed in unilevel chambers (Mendelson and Gorzalka, 1987; Mendelson and Pfaus, 1989; Pfaus et al., 1990). The generally low frequency of proceptive or rejection responses may therefore have precluded a significant depressing effect of these drugs. However, it is noteworthy that DPDPE facilitated proceptivity counts in OVX rats primed with EB and P and reduced the number of rejection responses of OVX rats primed with EB alone. These data suggest that, in addition to increasing the frequency of lordosis, S-receptor activation facilitates copulatory contact in females. There are several possible sites for the inhibitory action of CL-opioid receptor agonists on lordosis behavior. The MPOA, bed nucleus of stria terminalis, and MCG contain p-opioid binding sites (Desjardins, Brawer, and Beaudet, 1990; Goodman, Adler, and Pasternak, 1988; Mansour, Khachaturian, Lewis, Akil, and Watson, 1987). Infusions of the selective p-receptor antagonist p-funaltrexamine into the MPOA of OVX rats primed with EB facilitates lordosis (Hammer et al., 1989), although preliminary data indicate that infusions of morphine to the MPOA do not affect lordosis (Vathy et al., 1991). The VMH does not contain detectable populations of DAMGO binding sites (Mansour et al., 1987); however, it binds the selective p-agonist n-Ala2,N-Me-Phe”,Met-(0)5-ol-enkephalin (FK-33824) with low affinity (Desjardins et al., 1990; McLean, Rothman, and Herkenham, 1986). Infusions of p-endorphin or morphine to the VMH consistently inhibit lordosis behavior in OVX rats primed with EB
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and P (Vathy et al., 1991; Wiesner and Moss, 1989), an effect that likely reflects p-receptor suppression of endogenous norepinephrine release (Vathy et al., 1991). Infusions of morphine (Vathy et al., 1991) or met’enkephalin in conjunction with the enkephalinase inhibitor kelotorphan (Bednar, Forsberg, and Sodersten, 1987) to the MCG also inhibit lordosis in OVX rats primed with EB and P, although the mechanism by which p agonists exert this effect is unknown. Little is known about the possible sites for the facilitation of lordosis by selective 6 or K agonists. Areas of high-to-moderate 6 binding in rat brain include the cortex, dorsal and ventral striatum, MPOA, bed nucleus and stria terminalis, diagonal band, medial amygdala, suprachiasmatic nucleus, and the VMH (Desjardins et al., 1990; Goodman et al., 1988; Mansour et al., 1987). Low-affinity binding sites for DPDPE have also been detected in the MCG (Blackburn, Cross, Hille, and Slater, 1988). K-Receptor binding is relatively high in cortex, dorsal and ventral striatum, MPOA, suprachiasmatic nucleus, VMH, and MCG (Desjardins et d, 1990; Mansour et al., 1987). Infusions of the opioid receptor antagonist naloxone to the lateral ventricles inhibits lordosis (Lindblom, Forsberg, and Sodersten, 1986) and infusions of /3-endorphin to the amygdala produce a nonsignificant increase in the lordosis quotients of OVX rats primed with EB and P (Wiesner and Moss, 1989). It is possible that selective 6or K-receptor activation could facilitate lordosis in one or more of these sites. However, the lack of a clear dose-response effect for DPDPE on lordosis suggests that it may exert its effect by binding to a population of high-affinity 6 receptors near the lateral ventricles. There is also extensive evidence that opioid receptor binding varies during the estrous cycle of intact female rats (Hammer, 1990; Limonta, Maggi, Dondi, Martini, and Piva, 1987) or following treatment of OVX rats with EB and P (Weiland and Wise, 1990). Many of the brain areas that express 6 or K receptors also express p receptors in varying concentration (Desjardins et al., 1990) and are terminal regions for opioid-producing neurons (Goodman et al., 1988; Mansour et al., 1988). Hammer (1990) reported that 13H]DAMG0 binding in the MPOA decreased significantly in intact, cycling females during proestrus and estrus, suggesting that ,u-receptor downregulation in this region may contribute to a “disinhibition” of lordosis. However, in that study the specific binding of [3H]bremazocine to K receptors did not change significantly in the MPOA during the estrous cycle. Given that endogenous opioid peptides bind to more than one opioid receptor subtype (Patterson et al., 1983), and given the facilitatory effect of U50-488 in the present experiment, it is tempting to suggest that the relative proportion of p to K sites could shift the direction of endogenous opioid action from inhibition to facilitation within the MPOA of female rats during estrus. The MPOA also contains a small
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population of 6 receptors (Blackburn et al., 1988; Mansour et al., 1987); however, it is not known whether a similar shift in the number of or. receptors might enhance opioid action through 6 receptors in this region. In addition to a downregulation of p receptors, estrogen also increases the synthesis of proenkephalin in the VMH (Lauber, Romano, Mobbs, Howells, and Pfaff, 1990; Romano, Harlan, Shivers, Howells, and Pfaff, 1988; Romano, Mobbs, Howells, and Pfaff, 1989) and increases the number of immunoreactive me?-enkephalin-containing neurons in the interstitial nucleus of the stria terminalis, globus pallidus, medial and lateral preoptic nuclei, and MCG (DuPont et al., 1980; Watson, Hoffmann, and Wiegand, 1986). The VMH sends efferents to many of these regions (Krieger, Conrad, and Pfaff, 1979; Morrell and Pfaff, 1982) some of which are enkephalin-containing fibers (Yamano et al., 1986). Enkephalinimmunoreactive neurons in the VMH also concentrate estrogen (Akesson and Micevych, 1991). During the estrous cycle of rats, the number of immunoreactive met5-enkephalin-containing neurons increases in the anterior hypothalamic-preoptic area, mediobasal hypothalamus, and anterior pituitary in the morning of proestrus but returns to baseline throughout the evening of proestrus and morning of estrus, suggesting that increased enkephalin turnover in these regions precedes the onset of estrus (Kumar, Chen, and Muther, 1979). It is thus conceivable that estrogen could prime opioid systems that facilitate lordosis both by increasing enkephalin tone and modulating the relative proportion of receptors upon which enkephalins act. More work is required to elucidate the enkephalin-containing pathways that may facilitate lordosis. ACKNOWLEDGMENTS This research was supported by USPHS National Research Service Award NS08621 to J.G.P. and NIH HD 05071 to D.W.P. The authors thank Dr. Kelly Standifer and Dr. Gavril Pasternak of the Memorial Sloan-Kettering Institute for their generous gift of U50-488.
REFERENCES Akesson, T. R., and Micevych, P. E. (1991). Endogenous opioid-immunoreactive neurons of the ventromedial hypothalamic nucleus concentrate estrogen in male and female rats. J. Neurosci. Res. 28, 359-366. Beach, F. A. (1976). Sexual attractivity, proceptivity, and receptivity in female mammals. Horm. Behav. 7, 105-138. Bednar, I., Forsberg, G., and Sodersten, P. (1987). Inhibition of sexual behavior in female rats by intracerebral injections of met-enkephalin in combination with an inhibitor of enkephalin degrading enzymes. Neurosci. Len. 79, 341-345. Bicknell, R. J. (1985). Endogenous opioid peptides and hypothalamic neuroendocrine neurons. J. Endocrinol. 107,437-446. Blackbum, T. P., Cross, A. J., Hille, C., and Slater, P. (1988). Autoradiographic localization of delta opiate receptors in rat and human brain. Neuroscience 27, 497-506. Blank, M. S., Fabbri, A., Catt, K., and Dufau, M. (1986). Inhibition of luteinizing hormone
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118,2097-2102. Caggiula, A. R., Antelman, S. M., Chiodo, L. A., and Lineberry, C. 6. (1979). Brain dopamine and sexual behavior: Psychopharmacological and electrophysiological evidence for an antagonism between active and passive components. In E. Usdin, 1. J. Kopin, and J. Barchas (Eds.), Catecholamines: Basic and clinical frontiers, pp. 1?651767. Pergamon, New York. Chang, K.-J., Killian, A., Hazum, E., Cuatrecasas, P., and Chang, J.-K. (1981). Morphiceptin (NH?-Tyr-Pro-Phe-Pro-CONI&): A potent and specific agonist for morphine (y) receptors. Science 212, 75-77. Cicero, T. J., Schainker, B. A., and Meyer, E. R. (1979). Endogenous opioids participate in the regulation of the hypothalamic-pituitary-luteinizing hormone axis and testosterone’s negative feedback control of luteinizing hormone. Endocrinology 104,12861291. Desjardins, G. C., Brawer, J. R., and Beaudet, A. (1990). Distribution of cc, 6, and K opioid receptors in the hypothalamus of the rat. Bruin Res. 536, 114-123. DuPont, A., Barden, N., Cusan, L., Merand, Y., Labrie, F., and Vaudry, H. (1980). pendorphin, and met-enkephalins: Their distribution, modulation by estrogens, and role in neuroendocrine control. Fed. Proc. 39, 2.544-2550. Epstein, A., Fitzimmons, J., and Rolls, B. (1970). Drinking induced by injection of angiotensin in the brain of the rat. J. Physiol. 210,457-474. Goodman, R. R., Adler, B. A., and Pasternak, G. W. (1988). Regional distribution of opioid receptors. In G. W. Pasternak (Ed.), The opiate receptors, pp. 197-228. Humana Press, Clifton, NJ. Gorzalka, B. B., and Whalen, R. (1975). Inhibition, not facilitation, of sexual behavior by PCPA. Pharmacol. Biochem. Behav. 3, 511-513. Hammer, R. P. (1990). F-Opiate receptor binding in the medial preoptic area is cyclical and sexually dimorphic. Brain Res. 515, 187-192. Hammer, R. P., Doman, W. A., and Bloch, G. J. (1989). Sexual dimorphism and function of p-opiate receptors in rat medial preoptic area: Involvement in regulation of lordosis behavior. Abstracts of the International Conference on Hormones, Brain, and Behaviour (European Society for Comparative Physiology and Biochemistry, Liege, Belgium), 7778. Hardy, D. F., and DeBold, J. F. (1971). Effects of mounts without intromission upon the behavior of female rats during the onset of estrogen-induced heat. Physiol. Behav. 7, 643445.
Kalra, S. P., and Kalra, P. S. (1984). Neural regulation of luteinizing hormone secretion in the rat. Endocrinol. Rev. 4, 311-351. Kalra, S. P., Allen, L. G., and Kalra, P. S. (1989). Opioids in the steroid-adrenergic circuit regulating LH secretion: Dynamics and diversities. In R. G. Dyer and R. J. Bicknell (Eds.), Brain opioid systems in reproduction, pp. 95-111. Oxford Univ. Press, Oxford. Kalra, P. S., Crowley, W. R., and Kalra, S. P. (1986). Differential in vitro stimulation by naloxone of LHRH and catecholamine release from the hypothalami of intact and castrated rats. Endocrinology 120, 178-185. Krieger, M. S., Conrad, L. C. A., and Pfaff, D. W. (1979). An autoradiographic study of the efferent connections of the ventromedial hypothalamus. J. Camp. Neural. 183,785-
815. Kumar, M. S. A., Chen, C. L., and Muther, T. F. (1979). Changes in the pituitary and hypothalamic content of methionine-enkephalin during the estrous cycle of rats. Ltfe Sci. 25, 1687-1696. Lauber, A. H., Romano, G. J., Mobbs, C. V., Howells, R. D., and Pfaff, D. W. (1990).
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Estradiol induction of proenkephalin messenger RNA in hypothalamus: Dose-response and relation to reproductive behavior in the female rat. Mol. Bruin Res. 8, 47-54. Leadem, C. A., and Kalra, S. P. (1985). Effects of endogenous opioid peptides and opiates on luteinizing hormone and prolactin secretion in ovariectomized rats. Neuroendocrinology
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Limonta, P., Maggi, R., Dondi, D., Martini, L., and Piva, F. (1987). Gonadal steroid modulation of brain opioid systems. J. Steroid Biochem. 27, 691-698. Lindblom, C., Forsberg, G., and Sodersten, P. (1986). The effect of naloxone on sexual behavior in female rats depends on the site of injection. Neurosci. Lett. 70, 97-100. Madlafousek, J., and Hlinak, Z. (1978). Sexual behaviour of the female laboratory rat: Inventory, patterning, and measurement. Behaviour 63, 129-173. Mansour, A., Khachaturian, H., Lewis, M. E., Akil, H., and Watson, S. J. (1987). Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain. J. Neurosci. 7, 2445-2464. Mansour, A., Khachaturian, H., Lewis, M. E., Akil, H., and Watson, S. J. (1988). Anatomy of CNS opioid receptors. Tr. Neural. Sci. 11, 308-314. McClintock, M. K. (1984). Group mating in the domestic rat as a context for sexual selection: Consequences for the analysis of sexual behavior and neuroendocrine responses. Adv. Stud.
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McLean, S., Rothman, R. B., and Herkenham, M. (1986). Autoradiographic localization of p- and &opiate receptors in the forebrain of the rat. Bruin Rex 378, 49-60. Mendelson, S. D., and Gorzalka, B. B. (1984). Cholecystokinin-octapeptide produces inhibition of lordosis in the female rat. Pharmacol. Biochem. Behav. 21, 755-759. Mendelson, S. D., and Gorzalka, B. B. (1987). An improved chamber for the observation and analysis of the sexual behavior of the female rat. Physiol. Behav. 39, 67-71. Mendelson, S. D., and Pfaus, J. G. (1989). Level searching: A new assay of sexual motivation in the male rat. Physiol. Behav. 45, 337-341. Morrell, J. I., and Pfaff, D. W. (1982). Characterization of estrogen-concentrating hypothalamic neurons by their axonal projections. Science 217, 1273-1276. Paterson, S. J., Robson, L. E., and Kosterlitz, H. W. (1983). Classification of opioid receptors. Br. Med. Bull. 39, 31-36. Pellegrino, L. J., Pellegrino, A. S., and Cushman, A. J. (1979). A stereotaxic atlas of the rat brain. Plenum, New York. Pfaus, J. G., and Gorzalka, B. B. (1987a). Opioids and sexual behavior. Neurosci. Biobehav. Rev. 11, l-34. Pfaus, J. G., and Gorzalka, B. B. (1987b). Selective activation of opioid receptors differentially affects lordosis behavior in female rats. Peptides 8, 309-317. Pfaus, J. G., Mendelson, S. D., and Phillips, A. G. (1990). A correlational and factor analysis of anticipatory and consummatory measures of sexual behavior in the male rat. Psychoneuroendocrinology 15, 329-340. Pfaus, J. G., Pendleton, N., and Gorzalka, B. B. (1986). Dual effect of morphiceptin on lordosis behavior: Possible mediation by different opioid receptor subtypes. Pharmacol. Biochem. Behav. 24, 1461-1464. Piva, F., Limonta, P., Maggi, R., and Martini, L. (1986). Stimulatory and inhibitory effects of the opioids on gonadotropin secretion. Neuroendocrinology 42, 504-512. Romano, G. J., Harlan, R. E., Shivers, B. D., Howells, R. D., and Pfaff, D. W. (1988). Estrogen increases proenkephalin messenger ribonucleic acid levels in the ventromedial hypothalamus of the rat. Mol. Endocrinol. 2, 1320-1328. Romano, G. J., Mobbs, C. V., Howells, R. D., and Pfaff, D. W. (1989). Estrogen regulation of proenkephalin gene expression in the ventromedial hypothalamus of the rat: Temporal qualities and synergism with progesterone. Mol. Bruin Res. 5, 51-58.
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Simantov, R., and Snyder, S. H. (1977). Opiate receptor binding in the pituitary gland. Brain Res. 124, 178-184. Sirinathsinghji, D. J. S. (1984). Modulation of lordosis behavior of female rats by naloxone, P-endorphin and its antiserum in the mesencephalic central grey: Possible mediation via GnRH. Neuroendocriaology 39, 222-230. Sirinathsinghji, D. J. S. (1986). Regulation of lordosis behaviour in the female rat by corticotropin-releasing factor, @endorphin/corticotropin and luteinizing hormone-releasing hormone neuronal systems in the medial preoptic area. Bruin Res. 375, 49-56. Suda, M., Nakao, K., Sakamoto, M., Mot%, N., Sugawara, A., and Imura, H. (1986). Changes in the immunoreactivities of an opioid peptide leumorphin in the hypothalamus and anterior pituitary during the estrous cycle of the rat and their relation to sexual behavior. Brain Res. 374, 236-243. Vathy, I., van der Plas, J., Vincent, P. A., and Etgen, A. M. (1991). Intracranial dialysis and microinfusion studies suggest that morphine may act in the ventromedial hypothalamus to inhibit female sexual behavior. Harm. Behav. 25, 354-366. Yamano, M., Inagaki, S., Kito, S., Matsuzaki, T., Shmohara, Y., and Tohyama, M. (1986). Enkephalinergic projection from the ventromedial hypothalamic nucleus to the midbrain central gray matter in the rat: An immunocytochemical analysis. Bruin Res. 398, 337346. Watson, R. E., Hoffmann, G. E., and Wiegand, S. J. (1986). Sexually dimorphic opioid distribution in the preoptic area: Manipulation by gonadal steroids. Bruin Res. 398, 157-163. Weiland, N. G., and Wise, P. M. (1990). Estrogen and progesterone regulate opiate receptor densities in multiple brain regions. Endocrinology l26, 804-808. Wiesner, J. B., and Moss, R. L. (1986a). Suppression of receptive and proceptive behavior in ovariectomized, estrogen-progesterone-primed rats by intraventricular P-endorphin: Studies of behavioral specificity. Neuroendocrinology 43, 57-62. Wiesner, J. B., and Moss, R. L. (1986b). Behavioral specificity of ,!3-endorphin suppression of sexual behavior: Differential receptor antagonism. Pharmncol. Biochem. Behuv. 24, 1235-1239. Wiesner, J. B., and Moss, R. L. (1989). A psychopharmacological characterization of the opioid suppression of sexual behaviour in the female rat. In R. G. Dyer and R, J. Bicknell (Eds.), Brain opioid systems in reproduction, pp. 187-202. Oxford Univ. Press, Oxford.
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,x-, a-, and K-Opioid Receptor Agonists Selectively Modulate Sexual Behaviors in the Female Rat: Differential Dependence on Progesterone JAMES G. PFAUS AND DONALD
W. PFAFF
Laboratory of NeurobioIogy and Behavior, The RockefeUer University, 1230 York Avenue, New York, New York 10021 Previous studies suggested that opioid receptor agonists infused into the lateral ventricles can inhibit (through /L receptors) or facilitate (through S receptors) the lordosis behavior of ovariectomized (OVX) rats treated with estrogen and a low dose of progesterone. The present study investigated the behavioral and hormonal specificity of those effects using more selective opioid receptor agonists. Sexually experienced OVX rats were implanted stereotaxically with guide cannulae aimed at the right lateral ventricle. One group of rats was treated with estradiol benzoate (EB, 10 pg) 48 hr and progesterone (P, 250 pg) 4 hr before testing, whereas the other group was treated with EB alone. Rats were infused with different doses of the selective p-receptor agonist DAMGO, the selective a-receptor agonist DPDPE, or the selective K-receptor agonist U50-488. The females were placed with a sexually vigorous male in a bilevel chamber (Mendelson and Gorzalka, 1987) for three tests of sexual behavior, beginning 15, 30, and 60 min after each infusion. DAMGO reduced lordosis quotients and magnitudes significantly in rats treated with EB and P, but not in rats treated with EB alone. In contrast, DPDPE and U50-488H increased lordosis quotients and magnitudes significantly in both steroid-treatment groups. Surprisingly, measures of proceptivity, rejection responses, and level changes were not affected significantly by w or K agonists, although proceptivity and rejection responses were affected by DPDPE treatment. These results suggest that the effects of lateral ventricular infusions of opioid receptor agonists on the sexual behavior of female rats are relatively specific to lordosis behavior. Moreover, the facilitation of Iordosis behavior by F- or Kreceptor agonists is independent of progesterone treatment, whereas the inhibitory effect of p-receptor agonists on lordosis behavior may require the presence of progesterone. 0 1992 Academic Press. Inc.
Opioids exert complex controls over reproductive functions in the female rat. Selective opioid receptor agonists and endogenous opioid peptides inhibit pituitary LH secretion (Cicero, Shainker, and Meyer, 1979; Kalra and Kalra, 1984; Leadem and Kalra, 1985). This effect is mediated by an opioid-induced inhibition of GnRH-containing neurons in the medial preoptic area (MPOA), inhibition of GnRH release in the arcuate nucleus/median eminence (Bicknell, 1985; Kalra, Allen, and Kalra, 1989), 457 0018-506x/92 $4.00 All
Copyright 0 1992 by Academic Press. Inc. rights of reproduction in any form reserved.
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or possibly a direct inhibitory action of opioids on pituitary gonadotrophs (Blank, Fabbri, Catt, and Dufau, 1986; Simantov and Snyder, 1977). However, under certain experimental conditions opioid agonists can stimulate GnRH release and LH secretion (Kalra, Crowley, and Kalra, 1986; Leadem and Kalra, 1983; Piva, Limonta, Maggi, and Martini, 1986). Opioids may also inhibit or facilitate the sexual behavior of female rats, depending upon the receptor type or brain area stimulated. For example, p-endorphin can inhibit or facilitate lordosis behavior in female rats following the infusion of comparable doses into the third ventricle (Wiesner and Moss, 1986a,b) or lateral ventricles (Pfaus and Gorzalka, 1987a), respectively. However, low doses of both ,&endorphin and the p-selective agonist morphiceptin inhibit lordosis behavior, whereas only high doses of these agonists facilitate lordosis following infusion into the lateral ventricles (Pfaus and Gorzalka, 1987b). In contrast, the relatively selective a-receptor agonist n-Ser’,Leu5,Thr6-enkephalin (a-receptor peptide; DSLET) or the selective K-receptor agonist leumorphin produce a dosedependent facilitation of lordosis behavior following infusion into the lateral ventricles (Pfaus and Gorzalka, 1987b; Suda, Nakao, Sakamoto, Morii, Sugawara, and Imura, 1986). Infusions of morphine or @-endorphin to the ventromedial hypothalamus (VMH) or mesencephalic reticular formation also inhibit lordosis, whereas infusions to the mesencephalic central grey (MCG), dorsomedial hypothalamus, amygdala, or striatum are without effect (Vathy, van der Plas, Vincent, and Etgen, 1991; Wiesner and Moss, 1989; but see Sirinathsinghji, 1984). /3-Endorphin has also been reported to inhibit lordosis following infusion into the MPOA (Sirinathsinghji, 1986). The inhibitory effect of both /3-endorphin and morphiceptin may occur through an interaction with ,ul sites, as the selective ,+ antagonist naloxazone reversed the inhibitory effect of these peptides (Pfaus, Pendleton, and Gorzalka, 1986; Wiesner and Moss, 1989). Because pendorphin possesses equal affinity for cr. and F receptors (Paterson, Robson, and Kosterlitz, 1983), and high doses of morphiceptin and leumorphin also bind to 6 receptors (Chang et al., 1981), Pfaus and Gorzalka (1987b) suggested that p-receptor activation may inhibit, whereas a-receptor activation may facilitate, lordosis. The precise role of K receptors in lordosis is unclear, and it is not known to what extent the effects of p or 6 agonists depend upon the presence of progesterone. It has also been unclear whether the effects of opioid receptor agonists are specific to lordosis behavior or reflect indirect actions or more general modulations of social or locomotor behaviors. The sexual behavior of female rats typically is examined in small unilevel chambers. Lordosis quotients and magnitudes are commonly employed as measures of sexual receptivity. Measures of sexual proceptivity (e.g., presenting, hopping, and darting) and rejection (e.g., fighting, boxing, or defensive postures) can also be recorded in these chambers, although such measures generally
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do not convey the rich pattern of solicitation displayed by female rats mating in open fields (e.g., Beach, 1976; McClintock, 1984; Madlafousek and Hlinak, 1978). It has also been common to measure locomotor behavior as a control separately from sexual behavior. The bilevel chambers designed by Mendelson and Gorzalka (1987) provide an opportunity to assess female sexual behavior in a situation where the females can control or “pace” their copulatory contact with a male (Mendelson and Pfans, 1989; Pfaus, Mendelson, and Phillips, 1990). Typically, female rats force males to chase them from level to level, reminiscent of the pacing behavior displayed by female rats in a large open field. This level-to-level locomotion by the female can be counted prior to each mount or intromission by the male, and may constitute a measure of pacing behavior that can be used in conjunction with measures of sexual receptivity, proceptivity, and rejection to assess the full pattern of female sexual behavior. The present study was undertaken to examine whether the effects of lateral ventricular infusions of highly selective opioid receptor agonists on female sexual behavior are specific to lordosis, and whether those effects depend upon the presence of progesterone. Accordingly, we examined the effects of different doses of the selective p-receptor agonist nAla*,MePhe”,Gly-ol’-enkephalin (DAMGO), the selective S-receptor agonist D-Pen’,&Pet?-enkephalin (DPDPE), or the selective K-receptor agonist U50-488 on lordosis quotients, lordosis reflexes, proceptive behaviors, rejection responses, and level changes displayed in bilevel chambers by OVX rats primed with either estrogen and progesterone or estrogen alone. METHOD Animals and surgery. Ovariectomized female Sprague-Dawley rats (250 g) and intact male Long-Evans rats (300-500 g) were obtained from Charles River, Inc. (Wilmington, MA). Prior to surgery, the females were given at least six 30-min acquisition tests of sexual behavior with sexually vigorous male Long-Evans rats in bilevel chambers to allow them to acquire the complete pattern of sexual behavior in these chambers (see below). Following this acquisition period, each female was anesthetized with sodium pentobarbital (60 mg/kg) and implanted stereotaxically with a 23-gauge stainless-steel guide cannula (Plastics One) aimed at the right lateral ventricle according to the atlas of Pellegrino, Petlegrino, and Cushman (1979). Following surgery, the females were housed individually in Plexiglas cages in a colony room maintained on a reversed 12: 12 hr light/dark cycle (lights off at 1O:OO AM) at approximately 21°C. Sexually vigorous Long-Evans males were housed in groups of three in suspended wire-mesh cages in the same colony room. All rats were given free access to lab chow and water. The placement and flow of each cannula was tested 2 days before and 2 days after each experiment by replacing the
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dummy cannula with an injection cannula and infusing 2 pg of angiotensin II into the right lateral ventricle of each rat (Pfaus and Gorzalka, 1987b). Because infusions of angiotensin II into the lateral ventricles reliably induce thirst (Epstein, Fitzsimons, and Rolls, 1970), only those females that displayed vigorous drinking within 5 min of infusion served as subjects in each experiment. Drug and hormone treatments. Estradiol benzoate (EB) and progesterone (P) were purchased from Sigma (St. Louis, MO), dissolved in sesame oil, and injected subcutaneously in 0.1 ml. Angiotensin II (Bachem) was dissolved in physiological saline at a concentration of 1 pg/pl. DAMGO (Bachem), DPDPE (Bachem), and U50-488 (a gift from Drs. Kelly Standifer and Gavril Pasternak of the Memorial Sloan-Kettering Institute) were dissolved in physiological saline to obtain concentrations of 10, 100, or 1000 ng/pl, each of which was infused in a constant volume of 2 ~1. Control rats received infusions of 2 ,ul of physiological saline. All central infusions were made using an electrically driven infusion pump (Sage Instruments Model 3HlA) at a rate of 2 pl/min. The injection cannula was left in place for an additional minute to assure diffusion of the drug. Dummy cannulae were replaced after each infusion. Behavioral testing. Experimental tests of sexual behavior involved placing each female into a bilevel testing chamber with a sexually vigorous male until 10 mounts with pelvic thrusting had occurred. Lordosis quotients (LQ; calculated as the percentage of mounts with pelvic thrusting that resulted in a lordosis posture), lordosis reflex scores (LS; calculated for each lordosis response using a modified method of Hardy and DeBold (1971), in which each lordosis response was ranked in intensity from 1 to 3 according to the extent of dorsiflexion observed), proceptive behaviors/mount (counts of hopping, darting, and presenting prior to each mount) rejection responses/mount (counts of boxing, fighting, and defensive postures prior to each mount), and the number of level changes prior to each mount, were calculated during each test for each female. All testing occurred during the middle third of the rats’ dark cycle in a room with dim illumination. Following surgery, the females were given two saline-infusion pretests of sexual behavior in the bilevel chambers at 4-day intervals prior to the first experiment to assure that surgery did not affect their ability to perform sexual behaviors. Following the pretest period, the females received infusions of either 20, 200, or 2000 ng of each drug once every 4 days in a latinized design, such that all females were administered each dose of a drug over a 16day period. Within each experiment, sexual behaviors were observed 15, 30, and 60 min after infusion. One group of females was primed with EB (10 fig) 48 hr and P (250 pg) 4 hr before each infusion. This steroidpriming regimen has been used in previous experiments to produce a moderate degree of lordosis responding (LQs of 50 to 70) in rats with
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63
Time (mm)
FIG. 1. Effects of different doses of DAMGO on lordosis quotients (LQ) and lordosis scores (LS) in OVX rats primed with EB alone (left side; n = 8) or EB and P (right side, )I = 8). Statistical differences among the groups are described fully in the text.
chronic guide cannulae aimed at the lateral ventricles (Pfaus and Gorzalka, 1987b). The other group of females was primed with EB (10 pg) 48 hr before each test. Statistical analysis. Two-way within-subjects analyses of variance (ANQVAs) were conducted for the effects of dose, time, and the dose x time interaction for each of the five behavioral measures. For each significant ANOVA, pairwise comparisons among the means were made using the Tukey method, P < 0.05. RESULTS Effect of p-receptor activation with DAMGB. The effects of differenr doses of DAMGO on measures of female sexual behavior displayed by OVX rats primed with EB and P or EB alone are shown in Fig. 1 and Table 1. DAMGO produced a dose-dependent reduction in the LQs of rats primed with EB and P (fig. 1, top right). The ANOVA detected a significant main effect of dose, F(3, 21) = 5.18, P < 0.008, and time, F(2, 14) = 25.13, P < 0.0001, but not a significant interaction of dose and time. Post-hoc comparisons of dose revealed that the 200- and 2000ng doses of DAMGO significantly reduced the LQs in these females compared with the control. Post-hoc comparisons of the time points re-
462
PFAUS
Effects
of DAMGO
EB alone Dose of DAMGO
on Level
Min
AND
PFAFF
TABLE 1 Changes, Proceptivity,
and Rejection
Responses
Level
changes
Proceptivity
1.5 30 60
2.18 1.70 1.12
+ 0.50 -c 0.20 + 0.22
0.02 0.10 0.13
+ 0.01 + 0.09 f 0.04
0.00 0.13 0.05
15 30 60
1.78 1.20 1.80
+ 0.51 c 0.31 -+ 0.56
0.10 0.28 0.13
+ 0.08 + 0.16 + 0.08
0.07 0.00 0.03
+ 0.07
200 ng
15 30 60
2.08 1.17 1.76
+ 0.39 t 0.30 f 0.47
0.10 0.32 0.22
?z 0.10 2 0.18 !I 0.07
0.03 0.10 0.07
2 0.03 f 0.10 + 0.07
2000 ng
1.5 30 60
0.86 0.90 1.38
+ 0.40 + 0.33 + 0.38
0.13 0.17 0.12
+ 0.07 + 0.09 k 0.05
0.00 0.03 0.03
+ 0.03 2 0.03
Control
20 ng
EB + P Dose of DAMGO
Min
Control
Level
changes’
Proceptivity
Rejection
+ 0.13 r 0.05
f
0.03
Rejection
15 30 60
1.53 + 0.22 1.35 + 0.09 1.41 k 0.21
0.06 0.14 0.09
t 0.04 + 0.06 + 0.04
0.08 0.05 0.02
+ 0.07 + 0.04 + 0.02
15 30 60
1.05 + 0.32 0.99 I!I 0.17 1.03 + 0.16
0.11 0.09 0.04
+ 0.10 k 0.04 + 0.02
0.00 0.01 0.00
+ 0.01
200 ng
15 30 60
0.90 k 0.19 0.78 + 0.28 1.15 + 0.19
0.03 0.04 0.02
+ 0.02 t 0.04 + 0.02
0.01 0.00 0.00
2 0.01
2000 ng
15 30 60
0.35 0.55 1.31
0.03 0.03 0.00
+ 0.02 f 0.02
0.01 0.01 0.02
+ 0.01 f 0.01 + 0.02
20 ng
Note.
Values
are means
+ SEM.
f 0.12* f 0.16* + 0.36
*P < 0.05 from
control.
vealed that the LQs were higher in all dose conditions at 30 and 60 min compared to 15 min. Although none of the doses of DAMGO decreased the LSs in these rats, there was a significant effect of time on the LSs, F(2, 14) = 7.07, P < 0.008 (Fig. 1, bottom right). No significant interaction of dose and time occurred for the LS. DAMGO did not affect level changes, proceptivity, or rejection responses significantly (Table 1). In contrast, DAMGO did not affect the LQs significantly of rats primed with E alone (Fig. 1, top left). The ANOVA failed to detect a significant effect of dose, although a significant effect of time was detected for these rats, F(2, 10) = 49.02, P < 0.0001. No significant interaction of dose
OPIOIDS
AND FEMALE
DPDPE (EB alone)
10 20 30 40 50 60 Time (min)
SEXUAL
BEHAVIOR
463
DPDPE (EB+P)
10 20 30 40 50 60 Time (min)
FIG. 2. Effects of different doses of DPDPE on lordosis quotients (LQ) and iordosis scores (LS) in OVX rats primed with EB alone (left side, n = 10) or EB and P (right srde, n = 11). Statistical differences among the groups are described fully in the text.
and time was detected. Post-hoc comparisons of the effect of time revealed that the LQs were higher in all dose conditions at 30 and 60 min compared to 15 min. Similarly, DAMGO did not affect the LSs, although a significant effect of time was detected, F(2, 10) = 3.99, P < 0.05 (Fig. 1, bottom left). Post-hoc comparisons revealed that the LSs at 60 min were significantly higher than those at 15 min. DAMGO also reduced the number of level changes per mount (Table 1). The ANOVA detected a significant main effect of dose, F(3, 21) = 3.44, P < 0.04, and time, F(2, 14) = 3.51, P < 0.05, and a significant interaction of dose and time, F(h, 42) = 2.37, P < 0.05. Post-hoc comparisons of the effects of dose and time did not reveal any overall differences among the marginal means. However, post-hoc comparisons of the interaction effect revealed that the 2000-ng dose of DAMGO reduced level changes significantly 15 and 30 min after infusion compared to the control values. DAMGO did not affect proceptivity or rejection responses significantly (Table 1). Effect of &receptor activation with DPDPE. The effect of different doses of DPDPE on measures of female sexual behavior displayed by OVX rats primed with EB and P or EB alone are shown in Fig. 2 and Table 2. In contrast to the effect of DAMGO, all doses of DPDPE facilitated the LQs of rats primed with EB and P (Fig. 2, top right). The ANQVA
464
PFAUS
Effects EB alone Dose of DPDPE
of DPDPE
on Level
AND
PFAFF
TABLE 2 Changes, Proceptivity,
and Rejection
Responses
Min
Level
changes
15 30 60
1.51 1.11 1.70
+ 0.22 + 0.20 k 0.25
0.01 0.01 0.09
+ 0.01 f 0.01 ” 0.03
0.32 0.10 0.03
f 0.11 + 0.05 * 0.02
20 ng
1.5 30 60
1.93 1.74 1.82
+ 0.17 r 0.29 + 0.27
0.15 0.13 0.12
r 0.06 + 0.05 + 0.05
0.11 0.05 0.03
f t f
200 ng
15 30 60
1.86 1.59 1.81
2 0.17 + 0.20 ?z 0.19
0.08 0.20 0.12
f 0.08 + 0.05 c 0.04
0.08 0.10 0.05
+ 0.05 + 0.05 + 0.04
2000 ng
15 30 60
1.95 1.26 1.35
? 0.26 f 0.20 2 0.12
0.08 0.11 0.17
+ 0.05 f 0.03 f 0.06
0.04 0.03 0.00
f 0.03* k 0.02
Min
Level
changes
Control
EB + P Dose of DPDPE Control
Proceptivity
Proceptivity
Rejection
0.05 0.04 0.03
Rejection
15 30 60
1.60 k 0.33 1.31 + 0.20 1.10 f 0.22
0.20 0.18 0.20
f 0.05 + 0.04 iz 0.08
0.17 0.10 0.08
+ 0.10 c 0.07 + 0.08
20 ng
15 30 60
1.43 1.43 1.61
k 0.16 k 0.23 + 0.38
0.37 0.38 0.29
-t- 0.15 +- 0.17 2 0.11
0.03 0.05 0.04
+ 0.03 + 0.05 + 0.04
200 ng
15 30 60
2.06 1.26 1.18
+ 0.31 + 0.26 -r- 0.22
0.49 0.54 0.32
f 0.09* 2 0.10* + 0.09*
0.01 0.04 0.04
+ 0.01 + 0.03 + 0.04
2000 ng
15 30 60
2.13 1.26 1.28
+- 0.26 + 0.09 + 0.20
0.32 0.50 0.39
+ 0.08* + 0.10* +- 0.09*
0.00 0.00 0.00
Note.
Values
are means
f
SEM.
*P < 0.05 from
control.
detected a significant main effect of dose, F(3, 27) = 15.24, P < 0.0001, and time, F(2, 18) = 19.31, P < 0.0002, and a significant interaction of the two, F(6,54) = 2.40, P < 0.04. Post-hoc comparisons of dose revealed that all doses of DPDPE increased the LQs compared to the control; however, there were no significant differences between doses of DPDPE. Post-hoc comparisons of time revealed that the LQs were significantly higher at 30 and 60 min compared to 15 min. Post-hoc comparisons of the interaction of dose and time revealed that all doses of DPDPE increased the LQs at 15 and 30 min compared to the control. By 60 min, however, the LQs did not differ significantly among the dose conditions.
OPIOIDS AND FEMALE
SEXUAL
BEHAVIOR
465
DPDPE also facilitated proceptive behaviors (Table 1). The ANOVA detected a significant main effect of dose, F(3, 27) = 4.12, P < 0.02, but no main effect of time or interaction. Post-hoc comparisons revealed that both 200 and 2000 ng of DPDPE increased proceptive behaviors significantly across time compared to the effect of the control. DPDPE did not affect the LSs (Fig. 2, bottom right), level changes, or rejection responses significantly (Table 2). Similarly, DPDPE facilitated the LQs of females primed with EB alone (Fig. 2, top left). The ANOVA detected a significant main effect of dose, F(3, 30) = 21.88, P < 0.0001, and time, F(2, 30) = 73.75, P < 0.0001, and a significant interaction of the two, F(6, 60) = 2.84, P < 0.02. Posthoc comparisons of dose revealed that all doses of DPDPE increased the LQs compared to the control; however, there were no significant differences between doses of DPDPE. Post-hoc comparisons of time revealed that the LQs were significantly higher in all dose conditions at 30 and 60 min compared to 15 min. Post-hoc comparisons of the interaction of dose and time revealed that all doses of DPDPE increased the LQs significantly from the control dose at every time point. DPDPE also facilitated the LSs in these rats (Fig. 2, bottom left). The ANOVA detected a signifjcant main effect of dose, F(3, 30) = 3.95, P < 0.02, and time, F(2, 20) = 12.71, P < 0.0005, and a significant interaction of the two, F(6, 60) = 3.78, P < 0.005. Post-hoc comparisons of dose revealed that all doses of DPDPE increased the LSs significantly compared to control. Post-hoc comparisons of time revealed that the LSs were significantly increased at 60 min compared to 15 min. Post-hoc comparisons of the interaction of dose and time revealed that all doses of DPDPE increased the LSs significantly from control at 15 min, but not at 30 or 60 min. DPDPE also reduced the number of rejection responses displayed by these rats (Table 2). The ANOVA detected a significant main effect of dose, F(3, 30) = 3.57, P < 0.04, and a significant interaction of dose and time, F(6, 60) = 3.18, P < 0.01. Post-hoc comparisons of the interaction effect revealed that the 2000-ng dose reduced rejection responses significantly from the control dose at 15 min, but not at 30 or 60 min. DPDPE did not affect level changes or proceptivity significantly (Table 2).
Eflect of K-receptor activation with U50-488. The effect of different doses of U50-488 on measures of female sexual behavior displayed by OVX rats primed with EB and P or EB alone are shown in Fig. 3 and Table 3. U50-488 facilitated the LQs of rats primed with EB and P (Fig. 3, top right). The ANOVA detected a significant main effect of dose, F(3, 15) = 3.57, P < 0.05, and time, F(2, 10) = 10.65, P < 0.004, but no significant interaction of the two. Post-hoc comparisons of dose revealed that the 200- and 2000-ng doses increased the LQs significantly compared to the 20-ng dose and control. Post-hoc comparisons of time revealed that the
466
PFAUS AND PFAFF
0.01------l 10 20
30
40
50
Time (min)
60
Time (mid
FIG. 3. Effects of different doses of U50-488 on lordosis quotients (LQ) and lordosis scores (LS) in OVX rats primed with EB alone (left side, n = 6) or EB and P (right side, n = 6). Statistical differences among the groups are described fully in the text.
LQs were significantly higher at 60 min compared to 15 min. U50-488 did not affect the LSs (Fig. 3, bottom right), level changes, proceptivity, or rejection responses significantly (Table 3). U50-488 also facilitated the LQs of OVX rats primed with EB alone (Fig. 3, top left). The ANOVA detected a significant main effect of dose, F(3, 15) = 7.43, P < 0.004, and time, F(2, 10) = 80.63, P < 0.0001, but no significant interaction of the two. Post-hoc comparisons of dose revealed that all doses of U50-488 increased the LQs significantly compared to control. Post-hoc comparisons of time revealed that the LQs were increased significantly at 30 and 60 min compared to 15 min. U50488 did not affect the LSs (Fig. 3, bottom left), level changes, proceptivity, or rejection responses significantly (Table 3). DISCUSSION
The results of this study provide strong evidence that opioids inhibit or facilitate the lordosis behavior of female rats depending upon the receptor subtype stimulated and the hormonal state of the animal. Consistent with previous reports (Pfaus and Gorzalka, 1987b; Hammer, Dornan, and Bloch, 1989), p-receptor stimulation inhibited lordosis quotients selectively and dose-dependently in OVX rats primed with EB and P.
OPIOIDS
Effects
of U50-488
El3 alone Dose of U50-488
AND
on Level
Min
Control
FEMALE
SEXUAL
BEHAVIOR
TABLE 3 Changes, Proceptivity,
Level
changes
and Rejection
Proceptivity
467
Responses
Rejection
15 30 60
1.45 + 0.31 1.90 + 0.11 1.70 + 0.27
0.00 0.03 0.02
t 0.03 k 0.02
0.13 0.10 0.03
t 0.07 4 0.06 t 0.03
15 30 60
1.97 f 0.42 1.72 + 0.25 1.43 -c- 0.15
0.00 0.05 0.07
f 0.01 -c 0.07
0.10 0.12 0.10
k 0.08 r 0.09 Ifr 0.11
1.5 30 60
2.13 1.76 1.75
r f f
0.35 0.42 0.26
0.00 0.06 0.06
r 0.05 ir 0.04
0.02 0.03 0.00
r 0.02 + 0.03
15 30 60
2.30 1.68 1.28
+ 0.18 + 0.33 -c 0.17
0.02 0.06 0.03
IiT 0.02 t 0.05 t 0.03
0.06 0.00 0.00
f
Min
Level
changes
Proceptivity
15 30 60
1.87 1.72 2.40
+ 0.32 zt 0.21 f 0.35
0.06 0.05 0.07
zt 0.03 + 0.03 t 0.02
0.02 0.00 0.07
2 0.02
20 ng
15 30 60
2.33 k 0.41 1.50 k 0.29 1.87 k 0.36
0.10 0.05 0.03
f 0.07 f 0.03 k 0.02
0.03 0.05 0.08
f 0.03 i- 0.05 + 0.08
200 ng
15 30 60
1.45 1.58 1.86
+- 0.28 + 0.24 t 0.27
0.12 0.15 0.07
-i- 0.05 k 0.07 r 0.03
0.02 0.00 0.00
+ 0.02
2000 ng
15 30 60
2.00 2.25 2.36
2. 0.38 +- 0.49 2 0.56
0.05 0.13 0.03
k 0.02 + 0.10 + 0.03
0.00 0.00 0.00
20 ng
200 ng
2000 ng
EB + P Dose of U50-488 Control
Note.
Values
are means
+ SEM.
*P < 0.05 from
0.06
Rejection
t 0.05
control
However, this effect was not evident in rats primed with EB alone, suggesting that progesterone treatment may be required for the inhibitory effect of p-receptor stimulation as we have examined it. Such an interaction with progesterone is reminiscent of the inhibitory effect of p-chlorophenylalanine (Gorzalka and Whalen, 1975) or cholecystokinin (Mendelson and Gorzalka, 1984) on lordosis behavior. In contrast, the facilitatory effect of 6- or K-receptor stimulation on lordosis behavior occurred independently of progesterone treatment. With few exceptions, DAMGO, DPDPE, and U50-488 did not affect lordosis reflex intensities or the complex patterns of level changes, pro-
468
PFAUS AND PFAFF
ceptivity, or rejection responses displayed in the bilevel chambers. This suggests that the effects of these drugs were relatively specific to the frequency of lordosis behavior, and not secondary to a more general effect on arousal, social behavior, or locomotor activity during copulation. However, the highest dose of DAMGO reduced level changes significantly in females treated with EB and P. Although those data could be interpreted as evidence of a more general sedative effect of DAMGO, it is common for drugs that reduce pacing or proceptivity, e.g., haloperidol, to increase, rather than decrease, the frequency and magnitude of lordosis behavior (Caggiula, Antelman, Chiodo, and Lineberry, 1979). The lack of effect of DAMGO on proceptivity was surprising, especially given evidence by Wiesner and Moss (1986a,b) that infusions of P-endorphin to the third ventricle reduced measures of receptivity and proceptivity in OVX rats primed with EB and P and mated in small unilevel chambers. It is possible that the behavioral constraints imposed in situations where females cannot easily control the copulatory contact (e.g., in unilevel chambers) reveals effects of opioid agonists that are not manifested in the bilevel chambers. Indeed, female rats with sexual experience in the bilevel chambers primarily use level changing to regulate their copulatory contact with males, rather than the more complex forms of defensive or proceptive behaviors observed in unilevel chambers (Mendelson and Gorzalka, 1987; Mendelson and Pfaus, 1989; Pfaus et al., 1990). The generally low frequency of proceptive or rejection responses may therefore have precluded a significant depressing effect of these drugs. However, it is noteworthy that DPDPE facilitated proceptivity counts in OVX rats primed with EB and P and reduced the number of rejection responses of OVX rats primed with EB alone. These data suggest that, in addition to increasing the frequency of lordosis, S-receptor activation facilitates copulatory contact in females. There are several possible sites for the inhibitory action of CL-opioid receptor agonists on lordosis behavior. The MPOA, bed nucleus of stria terminalis, and MCG contain p-opioid binding sites (Desjardins, Brawer, and Beaudet, 1990; Goodman, Adler, and Pasternak, 1988; Mansour, Khachaturian, Lewis, Akil, and Watson, 1987). Infusions of the selective p-receptor antagonist p-funaltrexamine into the MPOA of OVX rats primed with EB facilitates lordosis (Hammer et al., 1989), although preliminary data indicate that infusions of morphine to the MPOA do not affect lordosis (Vathy et al., 1991). The VMH does not contain detectable populations of DAMGO binding sites (Mansour et al., 1987); however, it binds the selective p-agonist n-Ala2,N-Me-Phe”,Met-(0)5-ol-enkephalin (FK-33824) with low affinity (Desjardins et al., 1990; McLean, Rothman, and Herkenham, 1986). Infusions of p-endorphin or morphine to the VMH consistently inhibit lordosis behavior in OVX rats primed with EB
OPIOIDS
AND
FEMALE
SEXUAL
BEHAVIOR
469
and P (Vathy et al., 1991; Wiesner and Moss, 1989), an effect that likely reflects p-receptor suppression of endogenous norepinephrine release (Vathy et al., 1991). Infusions of morphine (Vathy et al., 1991) or met’enkephalin in conjunction with the enkephalinase inhibitor kelotorphan (Bednar, Forsberg, and Sodersten, 1987) to the MCG also inhibit lordosis in OVX rats primed with EB and P, although the mechanism by which p agonists exert this effect is unknown. Little is known about the possible sites for the facilitation of lordosis by selective 6 or K agonists. Areas of high-to-moderate 6 binding in rat brain include the cortex, dorsal and ventral striatum, MPOA, bed nucleus and stria terminalis, diagonal band, medial amygdala, suprachiasmatic nucleus, and the VMH (Desjardins et al., 1990; Goodman et al., 1988; Mansour et al., 1987). Low-affinity binding sites for DPDPE have also been detected in the MCG (Blackburn, Cross, Hille, and Slater, 1988). K-Receptor binding is relatively high in cortex, dorsal and ventral striatum, MPOA, suprachiasmatic nucleus, VMH, and MCG (Desjardins et d, 1990; Mansour et al., 1987). Infusions of the opioid receptor antagonist naloxone to the lateral ventricles inhibits lordosis (Lindblom, Forsberg, and Sodersten, 1986) and infusions of /3-endorphin to the amygdala produce a nonsignificant increase in the lordosis quotients of OVX rats primed with EB and P (Wiesner and Moss, 1989). It is possible that selective 6or K-receptor activation could facilitate lordosis in one or more of these sites. However, the lack of a clear dose-response effect for DPDPE on lordosis suggests that it may exert its effect by binding to a population of high-affinity 6 receptors near the lateral ventricles. There is also extensive evidence that opioid receptor binding varies during the estrous cycle of intact female rats (Hammer, 1990; Limonta, Maggi, Dondi, Martini, and Piva, 1987) or following treatment of OVX rats with EB and P (Weiland and Wise, 1990). Many of the brain areas that express 6 or K receptors also express p receptors in varying concentration (Desjardins et al., 1990) and are terminal regions for opioid-producing neurons (Goodman et al., 1988; Mansour et al., 1988). Hammer (1990) reported that 13H]DAMG0 binding in the MPOA decreased significantly in intact, cycling females during proestrus and estrus, suggesting that ,u-receptor downregulation in this region may contribute to a “disinhibition” of lordosis. However, in that study the specific binding of [3H]bremazocine to K receptors did not change significantly in the MPOA during the estrous cycle. Given that endogenous opioid peptides bind to more than one opioid receptor subtype (Patterson et al., 1983), and given the facilitatory effect of U50-488 in the present experiment, it is tempting to suggest that the relative proportion of p to K sites could shift the direction of endogenous opioid action from inhibition to facilitation within the MPOA of female rats during estrus. The MPOA also contains a small
470
PFAUS AND PFAFF
population of 6 receptors (Blackburn et al., 1988; Mansour et al., 1987); however, it is not known whether a similar shift in the number of or. receptors might enhance opioid action through 6 receptors in this region. In addition to a downregulation of p receptors, estrogen also increases the synthesis of proenkephalin in the VMH (Lauber, Romano, Mobbs, Howells, and Pfaff, 1990; Romano, Harlan, Shivers, Howells, and Pfaff, 1988; Romano, Mobbs, Howells, and Pfaff, 1989) and increases the number of immunoreactive me?-enkephalin-containing neurons in the interstitial nucleus of the stria terminalis, globus pallidus, medial and lateral preoptic nuclei, and MCG (DuPont et al., 1980; Watson, Hoffmann, and Wiegand, 1986). The VMH sends efferents to many of these regions (Krieger, Conrad, and Pfaff, 1979; Morrell and Pfaff, 1982) some of which are enkephalin-containing fibers (Yamano et al., 1986). Enkephalinimmunoreactive neurons in the VMH also concentrate estrogen (Akesson and Micevych, 1991). During the estrous cycle of rats, the number of immunoreactive met5-enkephalin-containing neurons increases in the anterior hypothalamic-preoptic area, mediobasal hypothalamus, and anterior pituitary in the morning of proestrus but returns to baseline throughout the evening of proestrus and morning of estrus, suggesting that increased enkephalin turnover in these regions precedes the onset of estrus (Kumar, Chen, and Muther, 1979). It is thus conceivable that estrogen could prime opioid systems that facilitate lordosis both by increasing enkephalin tone and modulating the relative proportion of receptors upon which enkephalins act. More work is required to elucidate the enkephalin-containing pathways that may facilitate lordosis. ACKNOWLEDGMENTS This research was supported by USPHS National Research Service Award NS08621 to J.G.P. and NIH HD 05071 to D.W.P. The authors thank Dr. Kelly Standifer and Dr. Gavril Pasternak of the Memorial Sloan-Kettering Institute for their generous gift of U50-488.
REFERENCES Akesson, T. R., and Micevych, P. E. (1991). Endogenous opioid-immunoreactive neurons of the ventromedial hypothalamic nucleus concentrate estrogen in male and female rats. J. Neurosci. Res. 28, 359-366. Beach, F. A. (1976). Sexual attractivity, proceptivity, and receptivity in female mammals. Horm. Behav. 7, 105-138. Bednar, I., Forsberg, G., and Sodersten, P. (1987). Inhibition of sexual behavior in female rats by intracerebral injections of met-enkephalin in combination with an inhibitor of enkephalin degrading enzymes. Neurosci. Len. 79, 341-345. Bicknell, R. J. (1985). Endogenous opioid peptides and hypothalamic neuroendocrine neurons. J. Endocrinol. 107,437-446. Blackbum, T. P., Cross, A. J., Hille, C., and Slater, P. (1988). Autoradiographic localization of delta opiate receptors in rat and human brain. Neuroscience 27, 497-506. Blank, M. S., Fabbri, A., Catt, K., and Dufau, M. (1986). Inhibition of luteinizing hormone
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AND FEMALE
SEXUAL
BEHAVIOR
471
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