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Steroid priming promotes oxytocin-induced norepinephrine release in the ventromedial hypothalamus of female rats
189
Brain Research, 620 (1993) 189-194 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19131
Steroid priming promotes oxytocin-induced norepinephrine release in the ventromedial hypothalamus of female rats P a t r i c i a A. V i n c e n t a n d A n n e M. E t g e n Departments of Psychiatry and Neuroscience, Albert Einstein College of Medicine, Bronx, IVY 10461 (USA) (Accepted 23 March 1993)
Key words: Oxytocin; Norepinephrine release; Estradiol; Progesterone; Ventromedial hypothalamus; Lordosis; Prazosin
In vivo microdialysis was used to detect norepinephrine (NE) release in the ventromedial hypothalamus of estradiol (E2)- or E 2 plus progesterone (P)-treated female rats injected with 1.0 IU of oxytocin (OXY). Dialysates were collected before and after OXY administration on 3 consecutive days and analyzed for NE content by high performance liquid chromatography with electrochemical detection. After the last sample was collected on day 1, animals were injected with 3 ~tg E z benzoate or oil. On day 3, E2-primed animals received 200/zg of P and control females received oil prior to OXY administration. OXY administration did not induce NE release on day 1. When OXY was administered to animals that received E 2 approximately 20 h earlier, increased release of NE was not consistently seen. In contrast, E2-primed animals that received P on day 3 displayed significant increases in the release of NE after OXY administration compared to their own basal levels and to NE levels in control animals. To distinguish whether E 2 priming is sufficient to promote OXY-induced release of NE without the addition of P, NE content of VMH dialysates in a second group of animals was examined following exposure to vehicle or E 2 alone. When OXY was administered 24 or 48 h after estrogen priming, only 1 of 4 E2-primed females had modestly elevated dialysate NE levels. To evaluate the interactions between OXY and NE in the regulation of reproductive behavior, lordosis responses were observed in hormone-primed female rats receiving systemic injections of OXY, the C~l-adrenoceptor antagonist prazosin, or both OXY and prazosin. OXY enhanced lordosis behavior in females primed with subthreshold doses of E 2 and P. Prazosin abolished lordosis behavior in rats primed with behaviorally effective doses of E 2 and P and significantly inhibited lordosis in steroid-primed females given OXY. These data suggest that after priming with both E 2 and P together, but not with E z alone, OXY may facilitate lordosis behavior through activation of NE transmission.
INTRODUCTION Noradrenergic transmission has been shown to exert a facilitatory influence on hormone-dependent sexual behavior in female rodents 11'34'3s. One brain area where norepinephrine (NE) is thought to produce this effect is the ventromedial hypothalamus (VMH) 1°'33 where noradrenergic nerve terminals originating in the A-1 and A-2 noradrenergic cell groups project densely to the dendritic fields surrounding the ventromedial nucleus of the hypothalamus23'24. The VMH is also known to be a site of estrogen-concentrating neurons 27 and a locus where crystalline implants of dilute estradiol (E 2) facilitate sexual receptivity 2'27. More recent work studying the influence of the neuropeptide oxytocin (OXY) on reproductive behavior has found that OXY
can also enhance reproductive behavior (for reviews see refs. 7, 15), possibly through actions in the VMH 4'29'37. Further supporting this notion are findings that OXY receptors are highly concentrated in the VMH and that their number and distribution are modulated by both estrogen and progestin 9'tS't6,29. The neuroanatomical convergence of noradrenergic nerve terminals, estrogen-concentrating ceils and OXY receptors, together with the reported facilitatory influence that these three systems have on reproductive behavior, strongly suggest a complex interactive relationship. Thus, our aim in the experiments presented here was to examine the relationship between OXY and NE in the facilitation of lordosis behavior and to test the hypothesis that OXY modulates NE release from the VMH following ovarian steroid priming.
Correspondence: A.M. Etgen, Albert Einstein College of Medicine, Department of Psychiatry, 1300 Morris Park Ave., Bronx, NY 10461, USA. Fax: (1) (718) 918-9274.
190 MATERIALS AND METHODS Animals Female Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing 200-250 g were maintained on a 14 h light/10 h dark cycle with water and food available ad libitum. Four to 7 days prior to hormone injection for behavioral testing or implantation of microdialysis probes, animals were ovariohysterectomized (OVX) bilaterally under Metofane anesthesia (Pitman-Moore, Inc., Atlanta, GA).
Materials E z benzoate (EB) and progesterone (P) were purchased from Steraloids, Inc. (Wilton, NH), dissolved in peanut oil and injected subcutaneously (s.c.) in a volume of 0.1 ml. O X Y (Sigma, St. Louis, MO) was dissolved in saline, and 1.0 IU was injected intraperitoneally (i.p.) in a volume of 0.1 ml. This dose was chosen based on reports that changes in female sexual behavior 22, feeding and drinking 3 can be observed in rats following systemic administration of comparable O X Y doses. Prazosin-HCI was dissolved in saline containing 25% propylene glycol and administered i.p.
In t,iL,o brain microdialysis Loop-type microdialysis probes were assembled using a modification of the procedure of Ungerstedt 31 as described previously 32 using cellulose dialysis m e m b r a n e ( S p e c t r a / P o t Hollow Fiber Bundle, o.d. 0.25 mm; mol. wt. 6,000 cutoff; Fisher Scientific, Springfield, NJ). Approximately 1.0-1.5 m m of dialysis m e m b r a n e was exposed at the dialysis site. Before implantation, these probes were determined to provide 5 - 1 0 % recovery of NE in vitro at the flow rate (2 ~ l / m i n ) used in the dialysis experiments. To the 40 /xl of dialysate that were collected every 20-25 min, 100 pg of 3,4-dihydroxybenzylamine (DHBA) was added to monitor any degradation of monoamines during sample storage. Just prior to injection onto the chromatographic column, 0.5 U of ascorbate oxidase was added to eliminate the ascorbic acid. Using this procedure, recovery of NE was consistently high, ranging from 80-100%. Monoamines in the dialysates were separated on a reverse-phase ODS column (C18, 5 /zm particle size, Bioanalytical Systems, West Lafayette, IN) and quantified using a Waters Model 460 electrochemical detector (Waters Associates, Milford, MA) with the working electrode set at + 0.70 V. The mobile phase consisted of: 0.3 m M Na2-EDTA, 0.1 m M KH2PO4, 0.25 m M octyl sodium sulfate brought to pH 3.5 using 6 N citric acid, plus 8% methanol. This system provides a low level detection limit of approximately 1 pg of NE.
or 3 /zg of EB. In the first experiment, baseline samples were collected on day 3 followed by administration of 200 /Lg of P to E2-primed females or oil vehicle to control females. O X Y was injected after P or oil, and sampling continued. In the second experiment, the effects of E 2 priming without added P were evaluated. Therefore, neither control nor estrogen-treated females received P or oil prior to O X Y administration on day 3.
Histological t,erification of probe placement Following collection of the last dialysate sample on day 3, animals were anesthetized with sodium pentobarbital and decapitated. Brains were removed, frozen in 1-methylbutane and stored at - 7 0 ° C . A cryostat microtome was used to cut 25 /xm sections throughout the hypothalamus. The slide-mounted and dried brain sections were stained with thionin and cover-slipped for storage. Determination of the probe placement was made by both investigators using a projection microscope and the atlas of Pellegrino et al. 26.
Effects of OXY and prazosin on lordosis behaL,ior In the first experiment, O V X rats (n = 14) were administered 3 ~ g of EB followed approximately 40 h later by 200 /zg of P. This hormone regime was chosen to produce a moderate level of sexual receptivity and was identical to that used in the microdialysis experiments. Animals also received 1.0 IU of O X Y (n = 7) or saline (n = 7) i.p. 1 h prior to behavioral testing. Behavioral testing was conducted 4 h after the P injection. Females were placed in an observation chamber with a sexually active stimulus male rat until the male m o u n t e d the female 10 times. The n u m b e r of times females assumed the lordosis posture when m o u n t e d was recorded and converted to a lordosis quotient (LQ = number lordosis r e s p o n s e s / n u m b e r mounts × 100), and the quality of the lordosis posture was ranked using a scale of 0 - 3 as described by Hardy and DeBold 13. In the second experiment, O V X females (n = 27) received the same hormone treatment described above. Control animals (n = 6) were injected with vehicle 1 h before P and 1 h after P. The remaining animals were split into 3 groups: O X Y animals (n = 7) received 1.0 IU of O X Y 1 h after P; prazosin animals (n = 6 ) received 0.5 m g / k g of the al-adrenoceptor blocker prazosin 1 h prior to P; the final group of rats (n = 8) received both prazosin 1 h before and O X Y 1 h after P. Behavior testing was carried out 4 h after P as described above. The dose and timing of prazosin administration were based on pilot studies indicating that prazosin administered 1 h before but not 1 h after P effectively abolished hormonefacilitated lordosis (data not shown).
Statistical analysis Stereotaxic surgery Animals were anesthetized with sodium pentobarbital (32 m g / k g ) and placed in a Kopf stereotaxic apparatus (Tujunga, CA) with the nosepiece set at +5.0 mm. Microdialysis probes were aimed at the ventrolateral VMH, the site of densest NE innervation. With respect to interaural zero, V M H coordinates were: anterior/posterior, + 0.2 mm; medial/lateral _+ 1.0 mm; dorsal/ventral - 9.3 m m 26. After the microdialysis probe was lowered to the desired location, dental cement and jeweler's screws were used to secure the probe to the skull surface. The incision was closed with wound clips. Microdialysis tubing and sample collecting vials were attached to the microdialysis probe, and constant perfusion was initiated with artificial cerebrospinal fluid containing 147 m M NaCI, 2.8 m M KCI, 2.0 m m CaCI2, 0.62 m M KH2PO4, pH 6.95, plus 113.5/xM ascorbic acid (to prevent oxidation of monoamines) at a flow rate of 2 /zl/min. Animals were allowed to recover from surgery overnight and were able to move freely around their cages throughout the remainder of the experiment.
Effects of steroids and OXY on NE release At the beginning of each experimental day (days 1-3), 2 to 3 baseline samples were collected from each animal. Animals were then injected with 1.0 IU of OXY, and samples were collected every 20 to 25 rain until a total of 10 samples were obtained. After the last sample was collected on day 1, animals were injected with either oil
Behavioral data were analyzed for percent of animals displaying lordosis and showing increased NE release using Fisher's Exact Probability test. Student's t-test or one-way A N O V A was used to analyze LQs and lordosis quality scores. NE content of microdialysis samples in control and hormone-treated females before and after O X Y administration was determined by a mixed-design A N O V A with hormone treatment as the between-subjects factor and sample n u m b e r as the within-subjects factor. The N e w m a n - K e u l ' s test was used for planned post hoc comparisons. For data from an animal in the microdialysis experiment to be included in the analysis, the microdialysis probe had to be placed within 0.5 m m of the ventrolateral border of the ventromedial nucleus. To be considered statistically significant, the probability values had to be less than 0.05.
RESULTS
OXY effects on NE release in animals with confirmed probe placements in the VMH The effects of combined administration of E~ and P were examined first. Table I shows the n u m b e r of animals in which i.p. administration of O X Y increased N E content of V M H dia|ysates, defined as an increase
191 TABLE I Number o f animals in which O X Y increased N E content of 1/MH dialysates
Criterion for increased NE = NE content greater than 4 pg above basal within 6 samples (approximately 2 h) after OXY injection. In both experiments, E 2 treatment was 3 p,g of EB given s.c. after sample collection on day l (see arrows). Vehicle controls received an s.c. injection of peanut oil at the same time. In experiment 1, 200 ~g of P was administered to EB-primed rats and oil was administered to vehicle controls prior to OXY injection on day 3 (see arrows). Day 1
Expt. 1: E 2 + P Vehicle controls E2 + P
E 2 or oil
Expt. 2: E 2 alone Vehicle controls E2
E 2 or oil
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* Significantly different from vehicle control, P = 0.03, Fisher's Exact Probability test in N E c o n t e n t greater than 4 pg above basal levels within 6 samples (2 h) of O X Y injection, during the 3 days of microdialysis sampling. O n day 1, i.e., prior to the first injection o f oil or EB, O X Y failed to increase N E release in any animal. O n day 2, approximately 20 h after E B or vehicle injection, O X Y increased dialysate N E content in 5 / 8 E B - t r e a t e d females and 2 / 5 oil-injected controls. O n day 3, 7 / 8 E B + P-treated females showed increased N E release after O X Y injection, whereas only 1 / 5 animals in the oil-treated g r o u p released N E in response to O X Y (Fisher's Exact Probability test, P = 0.03). T h e elevation in N E c o n t e n t of V M H dialysates f r o m estrogen plus progestin-treated rats was significantly higher than their own basal N E content and than N E c o n t e n t in dialysates f r o m controls ( P < 0 . 0 5 , N e w m a n - K e u l ' s ) during the third t h o u g h fifth p o s t - O X Y sample collection periods on day 3 (Fig. 1). In control females, O X Y did not significantly increase N E c o n t e n t of V M H dialysates at any post-injection time. T h e second study evaluated the effects of E 2 alone in the absence of a d d e d P on day 3 (Table I). As in the first experiment, O X Y administration failed to alter N E content of V M H dialysates in any animal on day 1. O n day 2, N E content was u n c h a n g e d in the 2.5 h following O X Y administration to the 4 vehicle-treated females. Similarly, only 1 of 4 E2-primed females displayed any response to O X Y administration, and this was an increase of 7 pg above basal only in the sixth sample after O X Y (approx. 2 h post-injection). O n day 3, O X Y increased N E c o n t e n t of the V M H dialysate by 6 pg above basal levels approximately 2 h post-injection (i.e. sixth p o s t - O X Y sample) in one control female. In the E 2 group, the female which had r e s p o n d e d 2 h after O X Y administration on day 2 exhibited increased
dialysate N E content (from 4 - 9 pg above basal) in the second t h r o u g h fourth samples after O X Y administration. T h e o t h e r 3 Ea-treated females showed no c h a n g e in dialysate N E levels after O X Y injection on day 3. H o r m o n a l facilitation o f O X Y - i n d u c e d N E release also a p p e a r e d to be neuroanatomically limited. Animals with p r o b e placements anterior, lateral or dorsolateral to the V M H frequently had detectable levels of N E in their dialysates, but N E content was not altered by O X Y administration in either control or h o r m o n e treated conditions. However, it is notable that 2 animals with p r o b e placements dorsal to the anterior V M H , specifically in or near the lateral paraventricular nucleus, exhibited substantial (e.g. greater than 20 pg above basal) increases in dialysate N E content following O X Y administration. This response to O X Y was
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Sample Number Fig. 1. Effects of OXY administration on NE content of VMH dialysates. OVX female rats received daily i.p. injections of 1.0 IU of OXY at the times indicated by arrows. Days 1-3 of dialysis sampling are separated by gaps between samples 9-10 and 18-19. At the end of dialysis sampling on day 1, animals were injected with oil vehicle (upper panel, n = 5) or with 3/xg of EB (lower panel, n = 8). Prior to dialysis sampling on day 3, oil animals received a second vehicle injection while EB-primed animals received 200 /~g of P. Values represent means + S.E.M. * Significantly greater than basal in EB + P animals and than corresponding samples in OIL controls, P < 0.05 (Newman-Keul's).
192 d e m o n s t r a t e d b o t h in the p r e s e n c e a n d a b s e n c e of ovarian s t e r o i d injection. T h r o u g h o u t the study, levels o f d o p a m i n e , 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyind o l e a c e t i c acid ( 5 - H I A A ) , a n d 3-methoxy-4-hydroxyphenylglycol ( M H P G ) w e r e also m e a s u r e d a n d a n a lyzed. T h e m e t a b o l i t e s D O P A C , M H P G a n d 5 - H I A A were p r e s e n t in V M H dialysates from m a n y animals, but no significant c h a n g e s in a b s o l u t e c o n t e n t or in the p a t t e r n of r e l e a s e of t h e s e a m i n e s could b e c o r r e l a t e d to the time o f O X Y a d m i n i s t r a t i o n or steroid p r i m i n g ( d a t a not shown). D o p a m i n e was d e t e c t e d in dialysates o f s o m e animals, a n d it also failed to vary as a function of O X Y or steroid a d m i n i s t r a t i o n ( d a t a not shown).
Behauioral results Fig. 2 shows that i.p. a d m i n i s t r a t i o n of the O X Y dose used in the microdialysis e x p e r i m e n t s significantly increases the p e r c e n t a g e o f E B + P - t r e a t e d a n i m a l s showing lordosis q u o t i e n t s g r e a t e r t h a n 20 from 43 to 100% ( F i s h e r ' s Exact P r o b a b i l i t y test, P < 0.05). M o r e over, O X Y - t r e a t e d animals also h a d h i g h e r lordosis quality scores t h a n saline controls (t-test, P < 0.05). A s e c o n d e x p e r i m e n t investigated i n t e r a c t i o n s b e t w e e n N E and O X Y . In this e x p e r i m e n t , O X Y failed to facilitate lordosis above c o n t r o l levels, b e c a u s e E B + P - t r e a t e d f e m a l e s were a l r e a d y maximally r e c e p t i v e (Fig. 3). T h e c~l-adrenergic r e c e p t o r a n t a g o n i s t prazosin had no effect on the n u m b e r of a n i m a l s showing lordosis or on the lordosis score when it was a d m i n i s t e r e d 1 h after P ( d a t a not shown). H o w e v e r , p r a z o s i n a d m i n i s t e r e d 1 h p r i o r to P a b o l i s h e d lordosis when
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Fig. 2. OXY facilitation of EB + P-facilitated lordosis behavior. OVX female rats were injected with 3/zg of EB followed 40 h later by 200 ~tg of P. Half of the animals (n = 7) received saline vehicle (VEH) and the other half (n = 7) received 1.0 IU of OXY i.p. 1 h before lordosis testing, which occurred approximately 4 h after the P injection. Open bars: percent lordosis, defined as the percent of animals demonstrating LQs > 20. Solid bars: lordosis score, mean + S.E.M. *P < 0.05 vs. VEH (Fisher's Exact Probability test for percent lordosis; t-test for lordosis score).
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Fig. 3. Effects of OXY and prazosin (PRAZ) on lordosis behavior in EB + P-treated female rats. OVX female rats received the same EB and P treatments described in Fig. 2. VEH animals were injected with saline vehicle 1 h before and 1 h after P; OXY animals received 1.0 IU of OXY 1 h after P; PRAZ animals received 0.5 mg/kg prazosin 1 h before P; and OXY + PRAZ animals were injected with PRAZ 1 h before and with OXY 1 h after P (n =6-8/group). Lordosis testing took place approximately 4 h after the P injection, and data represent means_+S.E.M. * Significantly less than VEH, P < 0.05, Newman-Keuls. * * Significantly less than VEH, P < 0.01, Newman-Keuls. #Significantly less than OXY and greater than PRAZ, P < 0.05, Newman-Keuls.
given a l o n e ( P < 0.01 vs. vehicle, N e w m a n - K e u l ' s ) a n d significantly i n h i b i t e d lordosis in O X Y - t r e a t e d f e m a l e s ( P < 0.05 vs. O X Y , N e w m a n - K e u l ' s ) . DISCUSSION T h e p r e s e n t findings s u p p o r t the h y p o t h e s i s that t h e facilitatory actions of O X Y on r e p r o d u c t i v e b e h a v i o r in s t e r o i d - p r i m e d f e m a l e rats may involve e n h a n c e m e n t of n o r a d r e n e r g i c n e u r o t r a n s m i s s i o n in the V M H . Previous o b s e r v a t i o n s on the b e h a v i o r a l actions of O X Y have i n d i c a t e d t h a t w h e n m o d e r a t e doses o f e s t r o g e n a r c used, O X Y facilitation o f b e h a v i o r a l receptivity is o b s e r v e d only if P is also a d m i n i s t e r e d t'12"29. H e n c e , o u r o b s e r v a t i o n that O X Y induces significant N E release from the V M H following a d m i n i s t r a t i o n o f low doses o f b o t h E B a n d P, b u t not after E B alone, m a y a c c o u n t for e a r l i e r r e p o r t s that O X Y facilitation of receptivity in rats r e q u i r e s e x p o s u r e to b o t h o v a r i a n s t e r o i d s (for review see ref. 15). It is n o t a b l e that the V M H is also a m a j o r b r a i n site at which E2, in d o s e s known to p r i m e r e p r o d u c t i v e behavior, i n c r e a s e s O X Y r e c e p t o r c o n t e n t in f e m a l e rats 9'15'16'29. T h e a d d i t i o n of P to e s t r o g e n - p r i m e d rats t h e n a p p e a r s to cause O X Y r e c e p t o r s to r e d i s t r i b u t e into regions l a t e r a l to the v e n t r o m e d i a l nucleus 29, i.e. into t h e r e g i o n w h e r e o u r V M H dialysis p r o b e s are located. It has also b e e n r e p o r t e d that in g o n a d a l l y intact rats: (1) the V M H o f e s t r o u s f e m a l e s show h i g h e r levels o f O X Y b i n d i n g t h a n t h e V M H of n o n - e s t r o u s f e m a l e s 15, a n d (2) O X Y
193 release into the pituitary portal system peaks on the afternoon of proestrus, before the onset of behavioral receptivity 28. Previous microdialysis work from this laboratory showed that the onset of steroid-facilitated copulatory behavior in female rats is accompanied by pronounced release of NE in the ventrolateral VMH 33. In those studies, the enhancement of catecholamine secretion in sexually receptive females rats was observed only in animals with confirmed probe placements in the VMH and was specific for NE (i.e. dopamine release was not correlated with sexual receptivity). Recent studies with hypothalamic slices maintained in vitro demonstrated that priming doses of estrogen attenuate a2-adrenergic receptor-mediated autoinhibition of NE release in the hypothalamus17. Taken together, the evidence supports a model wherein OXY released from the posterior pituitary a n d / o r CNS sites, perhaps in response to vaginal a n d / o r cervical stimulation from the mounting male, acts in the VMH to promote NE release. The augmentation of NE release initiated by OXY may then be prolonged due to the relative absence of negative feedback inhibition by az-autoreceptors. The current experiments also examined the possibility that OXY facilitation of lordosis behavior may be causally related to NE release. We showed previously that implants of the a~-adrenergic antagonist prazosin directly into the VMH inhibited estrogen plus progestin-dependent lordosis in female rats 1°. Therefore, we attempted to block OXY facilitation of lordosis using systemic administration of prazosin. In the present study, prazosin alone completely inhibited lordosis behavior when given 1 h prior to P, and it partially attenuated lordosis in steroid-primed rats given OXY 1 h after P. These data could be interpreted as evidence that OXY facilitation of lordosis is mediated by NE, specifically via NE interactions with a~-adrenoceptors. However, this conclusion must be considered preliminary for two reasons. First, OXY facilitation of lordosis was not demonstrated in this part of the study, because control animals given neither OXY nor prazosin were already exhibiting nearly maximal levels of sexual receptivity (see Fig. 3). Second, it could be argued that the inhibitory effects of prazosin are partially attenuated by the administration of OXY, suggesting that the facilitatory actions of NE on lordosis might be mediated by OXY release. In this regard, it is interesting that NE has been reported to influence OXY secretion in non-VMH brain sites 8'19'36 and in ovarian granulosa cells 21. Indeed, Parker and Crowley 25 have suggested that al-adrenoceptors mediate NE stimulation of OXY secretion from the paraventricular and supraoptic nuclei in lactating female rats. Thus, it is tempting to
speculate that OXY and NE may act synergistically within the VMH, each promoting the secretion of the other in an ovarian steroid-dependent manner, forming a local positive feedback loop. Steroid-dependent interactions between OXY and NE have also been reported in the medial preoptic area (MPOA) and mediobasal hypothalamus of sheep. Kendrick and colleagues 19 have shown that both OXY and NE release increase in the MPOA of ewes during parturition and that in estrogen-primed ewes, OXY infusions augment NE content of MPOA dialysates. These authors propose that OXY and NE may act together in the MPOA to reduce maternal aggression toward newborn lambs in the immediate postpartum period. Another report from this group demonstrated that either vaginocervical stimulation or OXY infusion into the MBH of progestin + estrogen-primed ewes enhances NE release TM. Other investigators have reported markedly increased OXY release from the MPOA-anterior hypothalamus of ovariectomized, estrogen-primed rats approximately 4 h after P administration 6. The same authors reported two peaks of OXY release from the rostral VMH of female rats on the afternoon of proestrus. Such findings support the proposal that OXY may act in the MPOA as well as the VMH to facilitate sexual receptivity 5'6. Since we have not monitored NE content of MPOA dialysates as a function of ovarian hormone treatment, our findings neither support nor refute this possibility. It should also be emphasized that our data cannot be construed as evidence that OXY promotes NE release only in the VMH, because we observed robust, OXY-induced increases in extracellular NE when dialysis probes were located in or near the paraventricular nucleus. The NE response to OXY in the paraventricular nucleus differed from that in the VMH in that it was hormone-independent. The present data clearly do not rule out the possibility that OXY facilitation of female sexual behavior involves direct actions of the neuropeptide on VMH neuronal excitability. Inenaga et al. 14 have shown that nanomolar concentrations of OXY enhance excitation in approximately half of the spontaneously active neurons recorded from VMH slices of guinea pigs. The excitatory effects of OXY were mimicked by an OXY receptor agonist and persisted when synaptic activity was blocked, suggesting a direct action of OXY on VMH neurons. Similar conclusions were reached by Kow et al. 2°, who superfused OXY agonists and antagonists onto VMH slices from female rats. The latter authors showed further that estrogen priming increased the percentage of VMH neurons excited by low concentrations of OXY and increased the number
194 of neurons Therefore,
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dosis behavior.
Acknowledgements. This work was supported by NSF Grant BNS 8607247, DHHS Grants MH41414 and RSDA MH00636 to A.M.E. and by the Department of Psychiatry, Albert Einstein College of Medicine. P.A.V. was supported by NRSA MH15788. The authors wish to thank Robert Goldstein for his technical assistance in the microdialysis experiments, William Paredes for assistance with statistical analyses, and Nikolas Karkanias for assistance with preparation of figures.
REFERENCES 1 Arletti, R. and Bertolini, A., Oxytocin stimulates lordosis behavior in female rats, Neuropeptides, 6 (1986) 247-253. 2 Barfield, R.J., Rubin, B.S., Glaser, J.H. and Davis, P.G., Sites of action of ovarian hormones in the regulation of oestrous responsiveness in rats. In J. Balthazart, E. Prove and R. Gilles (Eds.), Hormones and Behaviour in Higher Vertebrates, Springer-Verlag, Berlin, 1982, pp. 2-18. 3 Benelli, A., Bertolini, A. and Arleni, R., Oxytocin-induced inhibition of feeding and drinking: no sexual dimorphism in rats, Neuropeptides, 20 (1991) 57-62. 4 Caldwell, J.D., Prange Jr., A.J. and Pedersen, C.A., Oxytocin facilitates the sexual receptivity of estrogen-treated female rats, Neuropeptides, 7 (1986) 175-189. 5 Caldwell, J.D., Jirikowski, G.F., Greet, E.R. and Pedersen, C.A., Medial preoptic area oxytocin and female sexual receptivity, Behat:. Neurosci., 103 (1989) 655-662. 6 Caldwell, J.D., Mason, G.A., Walker, C.H. and Pedersen, C.A., Analysis of oxytocin release by microdialysis and interactions with progesterone, Ann. NYAcad. Sci., 652 (1992) 437-439. 7 Carter, C.S., Oxytocin and sexual behavior, Neurosci. Biobehav. Ret,., 16 (1992) 131-144. 8 Carter, D.A. and Lightman, S.L., Modulation of oxytocin secretion by ascending noradrenergic pathways: sexual dimorphism in rats, Brain Res., 406 (1987) 313-316. 9 de Kloet, E.R., Voorhuis, D.A.M., Boschma, Y. and Elands, J., Estradiol modulates the density of putative "oxytocin receptors" in discrete rat brain regions, Neuroendocrinology, 44 (1986) 415421. 10 Etgen, A.M., Intrahypothalamic implants of noradrenergic antagonists disrupt lordosis behavior in female rats, Physiol. Behat,., 48 (1990) 31-36. 11 Etgen, A.M., Ungar, S. and Petitti, N., Estradiol and progesterone modulation of norepinephrine neurotransmission: implications for the regulation of female reproductive behavior, J. NeuroendocrinoL, 4 (1992) 255-271. 12 Gorzalka, B.B. and Lester, G.L.L., Oxytocin-induced facilitation of lordosis behavior in rats is progesterone-dependent, Neuropeptides, 10 (1987) 55-65. 13 Hardy, D.F. and DeBold, J.F., Effects of coital stimulation upon behavior of the female rat, J. Comp. Physiol. Psychol., 78 (1972) 400-408. 14 Inenaga, K., Kannan, H., Yamashita, H., Tribollet, E., Raggenbass, M. and Dreifuss, J.J., Oxytocin excites neurons located in the ventromedial nucleus of the guinea-pig hypothalamus, J. Neuroendocrinol., 3 (1991) 569-573. 15 lnsel, T.R., Oxytocin--a neuropeptide for affiliation: evidence from behavioral, receptor autoradiographic, and comparative studies, Psychoneuroendocrinology, 17 (1992) 3-35. 16 Johnson, A.E., Coirini, H., Ball, G.F. and McEwen, B.S., Anatomical localization of the effects of 17/3-estradiol on oxy-
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tocin receptor binding in the ventromedial hypothalamic nucleus, Endocrinology, 124 (1989) 207-211. Karkanias, G.B. and Etgen, A.M., Estradiol attenuates o~2-adrenoceptor-mediated inhibition of hypothalamic norepinephrine release, J. Neurosci., in press. Kendrick, K.M., Fabre-Nys, C., Blache, D., Goode, J.A. and Broad, K.D., The role of oxytocin release in the mediobasal hypothalamus of the sheep in relation to female sexual receptivity, J. Neuroendocrinol., (1993) 13-21. Kendrick, K.M., Keverne, E.B., Hinton, M.R. and Goode, J.A., Oxytocin, amino acid and monoamine release in the region of the medial preoptic area and bed nucleus of the stria terminalis of the sheep during parturition and suckling, Brain Res., 569 (1992) 199-209. Kow, L.-M., Johnson, A.E., Ogawa, S. and Pfaff, D.W., Electrophysiologic actions of oxytocin on hypothalamic neurons in vitro: neuropharmacologic characterization and effects of ovarian steroids, Neuroendocrinology, 54 (1991) 526-535. Luck, M.R. and Munker, M., Beta adrenoceptors mediate the catecholamine-induced stimulation of oxytocin secretion from cultured bovine granulosa cells, Reprod. FertiL Dev., 3 (1991) 715-723. Moody, K.M., Steinman, J.L., Komisaruk, B.R. and Adler, N.T., Oxytocin-induced facilitation of lordosis is mediated by the pelvic nerve, Soc. Neurosci. Abstr., 17 (1991) 1416. Moore, R.Y. and Bloom, F.E., Central catecholamine neuron systems: anatomy and histology of the norepinephrine and epinephrine systems, Annu. Ret,. Neurosci., 2 (1979) 113-168. Moore, R.Y. and Card, J.P., Noradrenaline-containing neuron systems. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, VoL 2, Elsevier, New York, 1984, pp. 123-156. Parker, S.L. and Crowley, W.R., Central stimulation of oxytocin release in the lactating rat: interaction of neuropeptide Y with oq-adrenergic mechanisms, Endocrinology, 132 (1993) 658-666. Pellegrino, L.J., Pellegrino, A.S. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, Plenum, New York, 1979. Pfaff, D.W., Estrogens and Brain Function, Springer, New York, 1980. Sarkar, D.K. and Gibbs, D.M., Cyclic variation of oxytocin in the blood of pituitary portal vessels of rats, Neuroendocrinology, 39 (1984) 481-483. Schumacher, M. Coirini, H., Frankfurt, M. and McEwen, B.S., Localized actions of progesterone in hypothalamus involve oxytocin, Proc. Natl. Acad. Sci. USA, 86 (1989) 6798-6801. Tribollet, E., Audigier, S., Dubois-Dauphin, M. and Dreifuss, J.J., Gonadal steroids regulate oxytocin receptors but not vasopressin receptors in the brain of male and female rats. An autoradiographical study, Brain Res., 511 (1990) 129-140. Ungerstedt. U., Measurement of neurotransmitter release by intracranial dialysis. In C.A. Marsden (Ed.), Measurement of Neurotransmitter Release In ~vo, Wiley and Sons, New York, 1984, pp. 81-105. Vathy, I. and Etgen, A.M., Ovarian steroids and hypothalamic norepinephrine release: studies using in vivo brain microdialysis, Life Sci., 43 (1988) 1493-1499. Vathy, I. and Etgen, A.M., Hormonal activation of female sexual behavior is accompanied by hypothalamic norepinephrine release, J. NeuroendocrinoL, 1 (1989) 383-388. Vincent, P.A., Thornton, J.E. and Feder, H.H., Possible role of alpha- 2 receptors in the modulation of lordosis behavior in the female guinea pig, Neuroendocrinology, 46 (1987) 10-13. Vincent, P.A. and Feder, H.H., Alpha-~ and alpha- 2 noradrenergic receptors modulate lordosis behavior in female guinea pigs, Neuroendocrinology, 48 (1988) 477-481. Wakerley, J.B., Clarke, G. and Summerlee, A.J.S., Milk ejection and its control. In E. Knobil, J. Neill et al. (Eds.), The Physiology of Reproduction, New York, Raven, 1988, pp. 2283-2321. Witt, D.M. and lnsel, T.R., A selective oxytocin antagonist attenuates progesterone facilitation of female sexual behavior, Endocrinology, 128 (1991) 3269-3276.
Brain Research, 620 (1993) 189-194 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19131
Steroid priming promotes oxytocin-induced norepinephrine release in the ventromedial hypothalamus of female rats P a t r i c i a A. V i n c e n t a n d A n n e M. E t g e n Departments of Psychiatry and Neuroscience, Albert Einstein College of Medicine, Bronx, IVY 10461 (USA) (Accepted 23 March 1993)
Key words: Oxytocin; Norepinephrine release; Estradiol; Progesterone; Ventromedial hypothalamus; Lordosis; Prazosin
In vivo microdialysis was used to detect norepinephrine (NE) release in the ventromedial hypothalamus of estradiol (E2)- or E 2 plus progesterone (P)-treated female rats injected with 1.0 IU of oxytocin (OXY). Dialysates were collected before and after OXY administration on 3 consecutive days and analyzed for NE content by high performance liquid chromatography with electrochemical detection. After the last sample was collected on day 1, animals were injected with 3 ~tg E z benzoate or oil. On day 3, E2-primed animals received 200/zg of P and control females received oil prior to OXY administration. OXY administration did not induce NE release on day 1. When OXY was administered to animals that received E 2 approximately 20 h earlier, increased release of NE was not consistently seen. In contrast, E2-primed animals that received P on day 3 displayed significant increases in the release of NE after OXY administration compared to their own basal levels and to NE levels in control animals. To distinguish whether E 2 priming is sufficient to promote OXY-induced release of NE without the addition of P, NE content of VMH dialysates in a second group of animals was examined following exposure to vehicle or E 2 alone. When OXY was administered 24 or 48 h after estrogen priming, only 1 of 4 E2-primed females had modestly elevated dialysate NE levels. To evaluate the interactions between OXY and NE in the regulation of reproductive behavior, lordosis responses were observed in hormone-primed female rats receiving systemic injections of OXY, the C~l-adrenoceptor antagonist prazosin, or both OXY and prazosin. OXY enhanced lordosis behavior in females primed with subthreshold doses of E 2 and P. Prazosin abolished lordosis behavior in rats primed with behaviorally effective doses of E 2 and P and significantly inhibited lordosis in steroid-primed females given OXY. These data suggest that after priming with both E 2 and P together, but not with E z alone, OXY may facilitate lordosis behavior through activation of NE transmission.
INTRODUCTION Noradrenergic transmission has been shown to exert a facilitatory influence on hormone-dependent sexual behavior in female rodents 11'34'3s. One brain area where norepinephrine (NE) is thought to produce this effect is the ventromedial hypothalamus (VMH) 1°'33 where noradrenergic nerve terminals originating in the A-1 and A-2 noradrenergic cell groups project densely to the dendritic fields surrounding the ventromedial nucleus of the hypothalamus23'24. The VMH is also known to be a site of estrogen-concentrating neurons 27 and a locus where crystalline implants of dilute estradiol (E 2) facilitate sexual receptivity 2'27. More recent work studying the influence of the neuropeptide oxytocin (OXY) on reproductive behavior has found that OXY
can also enhance reproductive behavior (for reviews see refs. 7, 15), possibly through actions in the VMH 4'29'37. Further supporting this notion are findings that OXY receptors are highly concentrated in the VMH and that their number and distribution are modulated by both estrogen and progestin 9'tS't6,29. The neuroanatomical convergence of noradrenergic nerve terminals, estrogen-concentrating ceils and OXY receptors, together with the reported facilitatory influence that these three systems have on reproductive behavior, strongly suggest a complex interactive relationship. Thus, our aim in the experiments presented here was to examine the relationship between OXY and NE in the facilitation of lordosis behavior and to test the hypothesis that OXY modulates NE release from the VMH following ovarian steroid priming.
Correspondence: A.M. Etgen, Albert Einstein College of Medicine, Department of Psychiatry, 1300 Morris Park Ave., Bronx, NY 10461, USA. Fax: (1) (718) 918-9274.
190 MATERIALS AND METHODS Animals Female Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing 200-250 g were maintained on a 14 h light/10 h dark cycle with water and food available ad libitum. Four to 7 days prior to hormone injection for behavioral testing or implantation of microdialysis probes, animals were ovariohysterectomized (OVX) bilaterally under Metofane anesthesia (Pitman-Moore, Inc., Atlanta, GA).
Materials E z benzoate (EB) and progesterone (P) were purchased from Steraloids, Inc. (Wilton, NH), dissolved in peanut oil and injected subcutaneously (s.c.) in a volume of 0.1 ml. O X Y (Sigma, St. Louis, MO) was dissolved in saline, and 1.0 IU was injected intraperitoneally (i.p.) in a volume of 0.1 ml. This dose was chosen based on reports that changes in female sexual behavior 22, feeding and drinking 3 can be observed in rats following systemic administration of comparable O X Y doses. Prazosin-HCI was dissolved in saline containing 25% propylene glycol and administered i.p.
In t,iL,o brain microdialysis Loop-type microdialysis probes were assembled using a modification of the procedure of Ungerstedt 31 as described previously 32 using cellulose dialysis m e m b r a n e ( S p e c t r a / P o t Hollow Fiber Bundle, o.d. 0.25 mm; mol. wt. 6,000 cutoff; Fisher Scientific, Springfield, NJ). Approximately 1.0-1.5 m m of dialysis m e m b r a n e was exposed at the dialysis site. Before implantation, these probes were determined to provide 5 - 1 0 % recovery of NE in vitro at the flow rate (2 ~ l / m i n ) used in the dialysis experiments. To the 40 /xl of dialysate that were collected every 20-25 min, 100 pg of 3,4-dihydroxybenzylamine (DHBA) was added to monitor any degradation of monoamines during sample storage. Just prior to injection onto the chromatographic column, 0.5 U of ascorbate oxidase was added to eliminate the ascorbic acid. Using this procedure, recovery of NE was consistently high, ranging from 80-100%. Monoamines in the dialysates were separated on a reverse-phase ODS column (C18, 5 /zm particle size, Bioanalytical Systems, West Lafayette, IN) and quantified using a Waters Model 460 electrochemical detector (Waters Associates, Milford, MA) with the working electrode set at + 0.70 V. The mobile phase consisted of: 0.3 m M Na2-EDTA, 0.1 m M KH2PO4, 0.25 m M octyl sodium sulfate brought to pH 3.5 using 6 N citric acid, plus 8% methanol. This system provides a low level detection limit of approximately 1 pg of NE.
or 3 /zg of EB. In the first experiment, baseline samples were collected on day 3 followed by administration of 200 /Lg of P to E2-primed females or oil vehicle to control females. O X Y was injected after P or oil, and sampling continued. In the second experiment, the effects of E 2 priming without added P were evaluated. Therefore, neither control nor estrogen-treated females received P or oil prior to O X Y administration on day 3.
Histological t,erification of probe placement Following collection of the last dialysate sample on day 3, animals were anesthetized with sodium pentobarbital and decapitated. Brains were removed, frozen in 1-methylbutane and stored at - 7 0 ° C . A cryostat microtome was used to cut 25 /xm sections throughout the hypothalamus. The slide-mounted and dried brain sections were stained with thionin and cover-slipped for storage. Determination of the probe placement was made by both investigators using a projection microscope and the atlas of Pellegrino et al. 26.
Effects of OXY and prazosin on lordosis behaL,ior In the first experiment, O V X rats (n = 14) were administered 3 ~ g of EB followed approximately 40 h later by 200 /zg of P. This hormone regime was chosen to produce a moderate level of sexual receptivity and was identical to that used in the microdialysis experiments. Animals also received 1.0 IU of O X Y (n = 7) or saline (n = 7) i.p. 1 h prior to behavioral testing. Behavioral testing was conducted 4 h after the P injection. Females were placed in an observation chamber with a sexually active stimulus male rat until the male m o u n t e d the female 10 times. The n u m b e r of times females assumed the lordosis posture when m o u n t e d was recorded and converted to a lordosis quotient (LQ = number lordosis r e s p o n s e s / n u m b e r mounts × 100), and the quality of the lordosis posture was ranked using a scale of 0 - 3 as described by Hardy and DeBold 13. In the second experiment, O V X females (n = 27) received the same hormone treatment described above. Control animals (n = 6) were injected with vehicle 1 h before P and 1 h after P. The remaining animals were split into 3 groups: O X Y animals (n = 7) received 1.0 IU of O X Y 1 h after P; prazosin animals (n = 6 ) received 0.5 m g / k g of the al-adrenoceptor blocker prazosin 1 h prior to P; the final group of rats (n = 8) received both prazosin 1 h before and O X Y 1 h after P. Behavior testing was carried out 4 h after P as described above. The dose and timing of prazosin administration were based on pilot studies indicating that prazosin administered 1 h before but not 1 h after P effectively abolished hormonefacilitated lordosis (data not shown).
Statistical analysis Stereotaxic surgery Animals were anesthetized with sodium pentobarbital (32 m g / k g ) and placed in a Kopf stereotaxic apparatus (Tujunga, CA) with the nosepiece set at +5.0 mm. Microdialysis probes were aimed at the ventrolateral VMH, the site of densest NE innervation. With respect to interaural zero, V M H coordinates were: anterior/posterior, + 0.2 mm; medial/lateral _+ 1.0 mm; dorsal/ventral - 9.3 m m 26. After the microdialysis probe was lowered to the desired location, dental cement and jeweler's screws were used to secure the probe to the skull surface. The incision was closed with wound clips. Microdialysis tubing and sample collecting vials were attached to the microdialysis probe, and constant perfusion was initiated with artificial cerebrospinal fluid containing 147 m M NaCI, 2.8 m M KCI, 2.0 m m CaCI2, 0.62 m M KH2PO4, pH 6.95, plus 113.5/xM ascorbic acid (to prevent oxidation of monoamines) at a flow rate of 2 /zl/min. Animals were allowed to recover from surgery overnight and were able to move freely around their cages throughout the remainder of the experiment.
Effects of steroids and OXY on NE release At the beginning of each experimental day (days 1-3), 2 to 3 baseline samples were collected from each animal. Animals were then injected with 1.0 IU of OXY, and samples were collected every 20 to 25 rain until a total of 10 samples were obtained. After the last sample was collected on day 1, animals were injected with either oil
Behavioral data were analyzed for percent of animals displaying lordosis and showing increased NE release using Fisher's Exact Probability test. Student's t-test or one-way A N O V A was used to analyze LQs and lordosis quality scores. NE content of microdialysis samples in control and hormone-treated females before and after O X Y administration was determined by a mixed-design A N O V A with hormone treatment as the between-subjects factor and sample n u m b e r as the within-subjects factor. The N e w m a n - K e u l ' s test was used for planned post hoc comparisons. For data from an animal in the microdialysis experiment to be included in the analysis, the microdialysis probe had to be placed within 0.5 m m of the ventrolateral border of the ventromedial nucleus. To be considered statistically significant, the probability values had to be less than 0.05.
RESULTS
OXY effects on NE release in animals with confirmed probe placements in the VMH The effects of combined administration of E~ and P were examined first. Table I shows the n u m b e r of animals in which i.p. administration of O X Y increased N E content of V M H dia|ysates, defined as an increase
191 TABLE I Number o f animals in which O X Y increased N E content of 1/MH dialysates
Criterion for increased NE = NE content greater than 4 pg above basal within 6 samples (approximately 2 h) after OXY injection. In both experiments, E 2 treatment was 3 p,g of EB given s.c. after sample collection on day l (see arrows). Vehicle controls received an s.c. injection of peanut oil at the same time. In experiment 1, 200 ~g of P was administered to EB-primed rats and oil was administered to vehicle controls prior to OXY injection on day 3 (see arrows). Day 1
Expt. 1: E 2 + P Vehicle controls E2 + P
E 2 or oil
Expt. 2: E 2 alone Vehicle controls E2
E 2 or oil
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Day 2
Day 3 P or oil
2/5 5/8
~ 1/5 7/8 *
0/4 1/4
1/4 1/4
* Significantly different from vehicle control, P = 0.03, Fisher's Exact Probability test in N E c o n t e n t greater than 4 pg above basal levels within 6 samples (2 h) of O X Y injection, during the 3 days of microdialysis sampling. O n day 1, i.e., prior to the first injection o f oil or EB, O X Y failed to increase N E release in any animal. O n day 2, approximately 20 h after E B or vehicle injection, O X Y increased dialysate N E content in 5 / 8 E B - t r e a t e d females and 2 / 5 oil-injected controls. O n day 3, 7 / 8 E B + P-treated females showed increased N E release after O X Y injection, whereas only 1 / 5 animals in the oil-treated g r o u p released N E in response to O X Y (Fisher's Exact Probability test, P = 0.03). T h e elevation in N E c o n t e n t of V M H dialysates f r o m estrogen plus progestin-treated rats was significantly higher than their own basal N E content and than N E c o n t e n t in dialysates f r o m controls ( P < 0 . 0 5 , N e w m a n - K e u l ' s ) during the third t h o u g h fifth p o s t - O X Y sample collection periods on day 3 (Fig. 1). In control females, O X Y did not significantly increase N E c o n t e n t of V M H dialysates at any post-injection time. T h e second study evaluated the effects of E 2 alone in the absence of a d d e d P on day 3 (Table I). As in the first experiment, O X Y administration failed to alter N E content of V M H dialysates in any animal on day 1. O n day 2, N E content was u n c h a n g e d in the 2.5 h following O X Y administration to the 4 vehicle-treated females. Similarly, only 1 of 4 E2-primed females displayed any response to O X Y administration, and this was an increase of 7 pg above basal only in the sixth sample after O X Y (approx. 2 h post-injection). O n day 3, O X Y increased N E c o n t e n t of the V M H dialysate by 6 pg above basal levels approximately 2 h post-injection (i.e. sixth p o s t - O X Y sample) in one control female. In the E 2 group, the female which had r e s p o n d e d 2 h after O X Y administration on day 2 exhibited increased
dialysate N E content (from 4 - 9 pg above basal) in the second t h r o u g h fourth samples after O X Y administration. T h e o t h e r 3 Ea-treated females showed no c h a n g e in dialysate N E levels after O X Y injection on day 3. H o r m o n a l facilitation o f O X Y - i n d u c e d N E release also a p p e a r e d to be neuroanatomically limited. Animals with p r o b e placements anterior, lateral or dorsolateral to the V M H frequently had detectable levels of N E in their dialysates, but N E content was not altered by O X Y administration in either control or h o r m o n e treated conditions. However, it is notable that 2 animals with p r o b e placements dorsal to the anterior V M H , specifically in or near the lateral paraventricular nucleus, exhibited substantial (e.g. greater than 20 pg above basal) increases in dialysate N E content following O X Y administration. This response to O X Y was
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Sample Number Fig. 1. Effects of OXY administration on NE content of VMH dialysates. OVX female rats received daily i.p. injections of 1.0 IU of OXY at the times indicated by arrows. Days 1-3 of dialysis sampling are separated by gaps between samples 9-10 and 18-19. At the end of dialysis sampling on day 1, animals were injected with oil vehicle (upper panel, n = 5) or with 3/xg of EB (lower panel, n = 8). Prior to dialysis sampling on day 3, oil animals received a second vehicle injection while EB-primed animals received 200 /~g of P. Values represent means + S.E.M. * Significantly greater than basal in EB + P animals and than corresponding samples in OIL controls, P < 0.05 (Newman-Keul's).
192 d e m o n s t r a t e d b o t h in the p r e s e n c e a n d a b s e n c e of ovarian s t e r o i d injection. T h r o u g h o u t the study, levels o f d o p a m i n e , 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyind o l e a c e t i c acid ( 5 - H I A A ) , a n d 3-methoxy-4-hydroxyphenylglycol ( M H P G ) w e r e also m e a s u r e d a n d a n a lyzed. T h e m e t a b o l i t e s D O P A C , M H P G a n d 5 - H I A A were p r e s e n t in V M H dialysates from m a n y animals, but no significant c h a n g e s in a b s o l u t e c o n t e n t or in the p a t t e r n of r e l e a s e of t h e s e a m i n e s could b e c o r r e l a t e d to the time o f O X Y a d m i n i s t r a t i o n or steroid p r i m i n g ( d a t a not shown). D o p a m i n e was d e t e c t e d in dialysates o f s o m e animals, a n d it also failed to vary as a function of O X Y or steroid a d m i n i s t r a t i o n ( d a t a not shown).
Behauioral results Fig. 2 shows that i.p. a d m i n i s t r a t i o n of the O X Y dose used in the microdialysis e x p e r i m e n t s significantly increases the p e r c e n t a g e o f E B + P - t r e a t e d a n i m a l s showing lordosis q u o t i e n t s g r e a t e r t h a n 20 from 43 to 100% ( F i s h e r ' s Exact P r o b a b i l i t y test, P < 0.05). M o r e over, O X Y - t r e a t e d animals also h a d h i g h e r lordosis quality scores t h a n saline controls (t-test, P < 0.05). A s e c o n d e x p e r i m e n t investigated i n t e r a c t i o n s b e t w e e n N E and O X Y . In this e x p e r i m e n t , O X Y failed to facilitate lordosis above c o n t r o l levels, b e c a u s e E B + P - t r e a t e d f e m a l e s were a l r e a d y maximally r e c e p t i v e (Fig. 3). T h e c~l-adrenergic r e c e p t o r a n t a g o n i s t prazosin had no effect on the n u m b e r of a n i m a l s showing lordosis or on the lordosis score when it was a d m i n i s t e r e d 1 h after P ( d a t a not shown). H o w e v e r , p r a z o s i n a d m i n i s t e r e d 1 h p r i o r to P a b o l i s h e d lordosis when
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Fig. 2. OXY facilitation of EB + P-facilitated lordosis behavior. OVX female rats were injected with 3/zg of EB followed 40 h later by 200 ~tg of P. Half of the animals (n = 7) received saline vehicle (VEH) and the other half (n = 7) received 1.0 IU of OXY i.p. 1 h before lordosis testing, which occurred approximately 4 h after the P injection. Open bars: percent lordosis, defined as the percent of animals demonstrating LQs > 20. Solid bars: lordosis score, mean + S.E.M. *P < 0.05 vs. VEH (Fisher's Exact Probability test for percent lordosis; t-test for lordosis score).
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[ OXY+PRAZ
Fig. 3. Effects of OXY and prazosin (PRAZ) on lordosis behavior in EB + P-treated female rats. OVX female rats received the same EB and P treatments described in Fig. 2. VEH animals were injected with saline vehicle 1 h before and 1 h after P; OXY animals received 1.0 IU of OXY 1 h after P; PRAZ animals received 0.5 mg/kg prazosin 1 h before P; and OXY + PRAZ animals were injected with PRAZ 1 h before and with OXY 1 h after P (n =6-8/group). Lordosis testing took place approximately 4 h after the P injection, and data represent means_+S.E.M. * Significantly less than VEH, P < 0.05, Newman-Keuls. * * Significantly less than VEH, P < 0.01, Newman-Keuls. #Significantly less than OXY and greater than PRAZ, P < 0.05, Newman-Keuls.
given a l o n e ( P < 0.01 vs. vehicle, N e w m a n - K e u l ' s ) a n d significantly i n h i b i t e d lordosis in O X Y - t r e a t e d f e m a l e s ( P < 0.05 vs. O X Y , N e w m a n - K e u l ' s ) . DISCUSSION T h e p r e s e n t findings s u p p o r t the h y p o t h e s i s that t h e facilitatory actions of O X Y on r e p r o d u c t i v e b e h a v i o r in s t e r o i d - p r i m e d f e m a l e rats may involve e n h a n c e m e n t of n o r a d r e n e r g i c n e u r o t r a n s m i s s i o n in the V M H . Previous o b s e r v a t i o n s on the b e h a v i o r a l actions of O X Y have i n d i c a t e d t h a t w h e n m o d e r a t e doses o f e s t r o g e n a r c used, O X Y facilitation o f b e h a v i o r a l receptivity is o b s e r v e d only if P is also a d m i n i s t e r e d t'12"29. H e n c e , o u r o b s e r v a t i o n that O X Y induces significant N E release from the V M H following a d m i n i s t r a t i o n o f low doses o f b o t h E B a n d P, b u t not after E B alone, m a y a c c o u n t for e a r l i e r r e p o r t s that O X Y facilitation of receptivity in rats r e q u i r e s e x p o s u r e to b o t h o v a r i a n s t e r o i d s (for review see ref. 15). It is n o t a b l e that the V M H is also a m a j o r b r a i n site at which E2, in d o s e s known to p r i m e r e p r o d u c t i v e behavior, i n c r e a s e s O X Y r e c e p t o r c o n t e n t in f e m a l e rats 9'15'16'29. T h e a d d i t i o n of P to e s t r o g e n - p r i m e d rats t h e n a p p e a r s to cause O X Y r e c e p t o r s to r e d i s t r i b u t e into regions l a t e r a l to the v e n t r o m e d i a l nucleus 29, i.e. into t h e r e g i o n w h e r e o u r V M H dialysis p r o b e s are located. It has also b e e n r e p o r t e d that in g o n a d a l l y intact rats: (1) the V M H o f e s t r o u s f e m a l e s show h i g h e r levels o f O X Y b i n d i n g t h a n t h e V M H of n o n - e s t r o u s f e m a l e s 15, a n d (2) O X Y
193 release into the pituitary portal system peaks on the afternoon of proestrus, before the onset of behavioral receptivity 28. Previous microdialysis work from this laboratory showed that the onset of steroid-facilitated copulatory behavior in female rats is accompanied by pronounced release of NE in the ventrolateral VMH 33. In those studies, the enhancement of catecholamine secretion in sexually receptive females rats was observed only in animals with confirmed probe placements in the VMH and was specific for NE (i.e. dopamine release was not correlated with sexual receptivity). Recent studies with hypothalamic slices maintained in vitro demonstrated that priming doses of estrogen attenuate a2-adrenergic receptor-mediated autoinhibition of NE release in the hypothalamus17. Taken together, the evidence supports a model wherein OXY released from the posterior pituitary a n d / o r CNS sites, perhaps in response to vaginal a n d / o r cervical stimulation from the mounting male, acts in the VMH to promote NE release. The augmentation of NE release initiated by OXY may then be prolonged due to the relative absence of negative feedback inhibition by az-autoreceptors. The current experiments also examined the possibility that OXY facilitation of lordosis behavior may be causally related to NE release. We showed previously that implants of the a~-adrenergic antagonist prazosin directly into the VMH inhibited estrogen plus progestin-dependent lordosis in female rats 1°. Therefore, we attempted to block OXY facilitation of lordosis using systemic administration of prazosin. In the present study, prazosin alone completely inhibited lordosis behavior when given 1 h prior to P, and it partially attenuated lordosis in steroid-primed rats given OXY 1 h after P. These data could be interpreted as evidence that OXY facilitation of lordosis is mediated by NE, specifically via NE interactions with a~-adrenoceptors. However, this conclusion must be considered preliminary for two reasons. First, OXY facilitation of lordosis was not demonstrated in this part of the study, because control animals given neither OXY nor prazosin were already exhibiting nearly maximal levels of sexual receptivity (see Fig. 3). Second, it could be argued that the inhibitory effects of prazosin are partially attenuated by the administration of OXY, suggesting that the facilitatory actions of NE on lordosis might be mediated by OXY release. In this regard, it is interesting that NE has been reported to influence OXY secretion in non-VMH brain sites 8'19'36 and in ovarian granulosa cells 21. Indeed, Parker and Crowley 25 have suggested that al-adrenoceptors mediate NE stimulation of OXY secretion from the paraventricular and supraoptic nuclei in lactating female rats. Thus, it is tempting to
speculate that OXY and NE may act synergistically within the VMH, each promoting the secretion of the other in an ovarian steroid-dependent manner, forming a local positive feedback loop. Steroid-dependent interactions between OXY and NE have also been reported in the medial preoptic area (MPOA) and mediobasal hypothalamus of sheep. Kendrick and colleagues 19 have shown that both OXY and NE release increase in the MPOA of ewes during parturition and that in estrogen-primed ewes, OXY infusions augment NE content of MPOA dialysates. These authors propose that OXY and NE may act together in the MPOA to reduce maternal aggression toward newborn lambs in the immediate postpartum period. Another report from this group demonstrated that either vaginocervical stimulation or OXY infusion into the MBH of progestin + estrogen-primed ewes enhances NE release TM. Other investigators have reported markedly increased OXY release from the MPOA-anterior hypothalamus of ovariectomized, estrogen-primed rats approximately 4 h after P administration 6. The same authors reported two peaks of OXY release from the rostral VMH of female rats on the afternoon of proestrus. Such findings support the proposal that OXY may act in the MPOA as well as the VMH to facilitate sexual receptivity 5'6. Since we have not monitored NE content of MPOA dialysates as a function of ovarian hormone treatment, our findings neither support nor refute this possibility. It should also be emphasized that our data cannot be construed as evidence that OXY promotes NE release only in the VMH, because we observed robust, OXY-induced increases in extracellular NE when dialysis probes were located in or near the paraventricular nucleus. The NE response to OXY in the paraventricular nucleus differed from that in the VMH in that it was hormone-independent. The present data clearly do not rule out the possibility that OXY facilitation of female sexual behavior involves direct actions of the neuropeptide on VMH neuronal excitability. Inenaga et al. 14 have shown that nanomolar concentrations of OXY enhance excitation in approximately half of the spontaneously active neurons recorded from VMH slices of guinea pigs. The excitatory effects of OXY were mimicked by an OXY receptor agonist and persisted when synaptic activity was blocked, suggesting a direct action of OXY on VMH neurons. Similar conclusions were reached by Kow et al. 2°, who superfused OXY agonists and antagonists onto VMH slices from female rats. The latter authors showed further that estrogen priming increased the percentage of VMH neurons excited by low concentrations of OXY and increased the number
194 of neurons Therefore,
that
responded
NE and OXY
the same population rons to enhance
to both
OXY
and
might act independently
of lordosis-relevant, VMH
NE. on
17
neu-
e x c i t a b i l i t y a n d t h e r e b y f a c i l i t a t e lor-
dosis behavior.
Acknowledgements. This work was supported by NSF Grant BNS 8607247, DHHS Grants MH41414 and RSDA MH00636 to A.M.E. and by the Department of Psychiatry, Albert Einstein College of Medicine. P.A.V. was supported by NRSA MH15788. The authors wish to thank Robert Goldstein for his technical assistance in the microdialysis experiments, William Paredes for assistance with statistical analyses, and Nikolas Karkanias for assistance with preparation of figures.
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