The role of norepiinephrine in mediating luteinizing hormone release in response to blockade of κ-opioid receptors in the medial preoptic area

BRAIN RESEARCH Brain Research 698 (1995) 121-129

ELSEVIER

Research report

The role of norepinephrine in mediating luteinizing hormone release in response to blockade of K-opioid receptors in the medial preoptic area Shanjun Zhen, Robert V. Gallo * Department of Physiology and Neurobiology, Universi~ of Connecticut, 3107 Horsebarn Hill Road, Box U-154. Storrs, CT 06269-4154. USA Accepted 28 June 1995

Abstract Our previous study [32] indicated that blockade of K-opioid receptors with nor-binaltorphimine (nor-BNl) in the medial preoptic area (MPOA) produced two different LH responses during midpregnancy in the rat: an increase in basal pulsatile LH secretion, followed in many cases by a larger and/or sustained increase in LH release. In the present study, two experiments were conducted to examine the role of norepinephrine (NE) in mediating these different LH responses. In experiment 1, the effects of NE synthesis inhibition with FLA-63 on nor-BNI induced LH secretion were examined. In 5 of 9 vehicle pretreated rats, nor-BNI perfusion in the MPOA produced only an increase in basal pulsatile LH secretion. In the remaining 4 animals blockade of MPOA K-receptors produced not only an increase in basal LH secretion, but also a large/sustained release of LH. Pretreatment with FLA-63 had no effect on the nor-BNI induced increase in basal pulsatile LH secretion, but completely prevented the occurrence of the large/sustained release of LH. The objective of experiment 2 was to determine whether any change in NE release occurred at the site of nor-BNl perfusion in rats showing this large/sustained increase in plasma LH levels, by measuring in vivo NE release at that site. No significant change in perfusate NE levels was observed during perfusion of the MPOA with nor-BNI alone or in combination with desipramine, a NE reuptake blocker, in rats that showed this type of LH response. These results demonstrate that while NE does not mediate the increase in basal pulsatile LH release produced by nor-BNI perfusion in the MPOA, it is essential for the large/sustained elevation in LH secretion seen in response to blockade of K-opioid receptors at this site. This latter type of LH secretory response is not, however, associated with an increase in NE release directly at the site of K-opioid receptor blockade in the MPOA in pregnant rats. Keywords: Luteinizing hormone; Opioid receptors; Norepinephrine; Medial preoptic area: Pregnancy; Push-pull perfusion: HPLC

I. Introduction Previous studies in this laboratory demonstrated that r-opioid receptors in both the medial basal hypothalamus (MBH) and the medial preoptic area (MPOA) are involved in the suppression of luteinizing hormone (LH) release during midpregnancy in the rat, since intraventricular administration [8] as well as direct perfusion of either site with nor-binaltorphimine (nor-BNI) [32], a specific ropioid receptor antagonist [21,22,26], stimulated increases in LH secretion. Moreover, the LH release patterns were different depending on the site where K-opioid receptors were blocked. While an increase in basal pulsatile LH release was observed in response to nor-BNI perfusion in the MBH or MPOA, only during blockade of r-receptors in the MPOA was this latter effect often followed by a

* Corresponding author. Fax: (1) (203) 486-3303. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V, All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 8 7 4 - 8

large a n d / o r sustained increase in LH secretion, that was of greater magnitude and longer duration than a typical LH pulse [32]. Thus, blockade of M P O A K-receptors results in either an increase in basal pulsatile LH release, a large/sustained increase in LH secretion, or both responses. The stimulatory effect of nor-BNI on LH release could be exerted directly on LH-releasing hormone (LHRH) neurons, since in addition to r-receptors [5,19], in the rat the M P O A and MBH contain cell bodies and axons, respectively, of LHRH neurons [24]. However, a recent study employing GTI-1 cells, an LHRH neuronal cell line, indicated that while LHRH neurons contain 8-opioid receptors, they lack r-receptors [17]. Alternatively, antagonism of M P O A or MBH r-receptors could increase LH release by altering the activity of neuronal systems that are involved in regulating LHRH, and thus LH secretion, since both areas contain neurotransmitter systems involved in regulating LH release [28].

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S. Zhen, R.V. Gallo/Brain Research 698 (1995) 121-129

Other studies have suggested that the increase in LH release produced by naloxone, an opioid receptor antagonist, is exerted by increasing the stimulatory input of noradrenergic neurons on the LHRH neuronal system [1,12,13,15,27]. During early pregnancy the stimulatory effect of naloxone on LH secretion can be prevented by inhibition of NE but not epinephrine synthesis [18]. Moreover, activation of K-opioid receptors has been shown to suppress NE release in the MPOA of male rats [10], in the guinea pig cortex [29,30], and in the rabbit hippocampus [2]. Taken together, these findings raised the possibility that the increased LH release induced by blockade of ~c-opioid receptors in the MBH or MPOA may be due to an increase in the release of NE. However, we recently demonstrated that NE does not mediate the increase in basal pulsatile LH release in response to blockade of K-opioid receptors in the MBH [33]. There is no information available about the role of NE, if any, in mediating the two different LH responses occurring during blockade of K-opioid receptors in the MPOA. A recent study involving microdialysis in the MPOA demonstrated that no change in NE release occurred at this site in response to either local or systemic administration of naloxone in intact and castrated male rats, although these naloxone treatments produced increases in LH secretion [31]. The first objective of the present study was to determine whether NE mediates either the increase in basal pulsatile LH release or the large/sustained increase in LH secretion occurring during blockade of K-opioid receptors in the MPOA. If NE were shown to be involved, the second objective was to determine whether an increase in NE release at the site of nor-BNI perfusion was associated with the increased LH secretion.

was inserted so that its tip was flush with the tip of the guide cannula. Daily vaginal smears were continued. Implantation performed on the day of estrus resulted in minimal disruption of the estrous cycle. After 1-3 weeks of recovery from surgery, rats were housed with an adult male rat at 15:00-16:00 h on the afternoon of proestrus. Mating was confirmed by the presence of sperm in the vaginal smear the next morning. This was designated day 1 of pregnancy. The inner stylette was removed 3 - 7 days before the push-pull perfusion experiment, and a new stylette inserted at that time so that its tip extended 1 mm beyond the tip of the guide cannula. 2.3. Experimental protocols

Between 09:00-10:00 h on the day before the experiment, rats were anesthetized with ether and a polyethylene (PE50) cannula was inserted into or near the right atrium via the external jugular vein. Twenty-four h later blood sampling and push-pull perfusion of the MPOA were done. The technique of push-pull perfusion was similar to that previously employed in our laboratory [32,33]. On the morning of the experiment the flow rates of the push and pull peristaltic pumps (Pharmacia, Piscataway, NJ) were carefully balanced and set to deliver 10/xl/min. The inner stylette was then removed, and a push-pull cannula assembly was inserted into the outer cannula. The tip of the inner cannula extended 0.75 mm beyond the tip of the guide cannula. The push end of the tubing was inserted into a test tube containing the perfusion solution, and the pull end was inserted into a collecting test tube kept on ice and containing 6 /xl 0.25 N perchloric acid to prevent oxidation of NE in the perfusate samples in experiment 2 when perfusate NE concentrations were measured. Two experiments were done.

2. Materials and methods 2.1. Animals

Adult female Sprague-Dawley CD rats (Charles River Laboratories, Wilmington, MA) were maintained on a 14 h light : 10 h dark schedule (lights on at 05:00 h), and fed rat chow and water ad libitum. Estrous cycle stages were determined by daily examination of vaginal smears. The experimental protocols were approved by the local University Animal Care and Use Committee. 2.2. Push-pull cannula implantation

Rats displaying 4-day estrous cycles were anesthetized with pentobarbital and a 21-gauge stainless steel guide cannula was stereotaxically implanted into the MPOA (7.8 mm anterior to the interaural line, 0.8 mm lateral to the midline, and 6.8 mm down from the surface of the brain), according to the atlas of deGroot [4]. The cannula was cemented in place, and a removable stylette (26 gauge)

Experiment 1 To examine the effect of NE synthesis inhibition on LH release induced by nor-BNI perfusion in the MPOA, animals were divided into 3 groups: (1) vehicle (26% ethanol, 1 ml/kg, s.c.) given 3 h prior to the onset of a 4.5 h period of perfusion in the MPOA with artificial CSF (NaCI 140 mM, KCI 4 mM, CaC12 2.3 raM, MgSO 4 1 mM, Na2HPO 4 1.2 mM, NaH2P Q 0.3 mM, glucose 3.4 mM, pH 7.4); (2) vehicle given 2.5-3 h prior to the onset of 4.5 h of perfusion of the MPOA with CSF containing nor-BNI, a specific antagonist of K-opioid receptors [21,22,26], (40 /zg/h, Research Biochemicals, Natick, MA); or (3) bis(4methyl-l-homopiperazinylthiocarbonyl) disulfide (FLA-63, a dopamine-/3-hydroxylase inhibitor [3], ICN Biomedicals, Irvine, CA, 25 m g / k g s.c.) given 2.5-3 h prior to the onset of a 4 h period of perfusion in the MPOA with CSF containing nor-BNI (40 /zg/h). This laboratory has shown that such treatment with FLA-63 produced a 70% depletion in hypothalamic-preoptic area levels of NE and a blockade of naloxone-induced LH release [18].

S. Zhen, R.V. Gallo/Bram Research 698 (1995) 121-129

Experiment 2 To monitor in vivo NE release in the MPOA in response to blockade of K-receptors, push-pull perfusion of the MPOA involved a 1.5 h period of perfusion with CSF, followed immediately by 4.5 h of CSF containing nor-BNI (40 /zg/h). Since NE reuptake is a major means for removal of NE from the synaptic cleft [11], it was possible that nor-BNl could have produced a small increase in NE release, but removal of the released NE from the synaptic cleft by a presynaptic NE transporter might prevent the detection of such a rise. Therefore, in two additional groups, desipramine (DMI), a NE reuptake blocker [14,23,25], (10 /xM, Sigma, St. Louis, MO) was added to the CSF solution, and two consecutive periods of perfusion were done with either (a) CSF containing DMI for 2 h, followed immediately by another 4.5 h period of DMI, or (b) CSF containing DMI for 2 h, followed by 4.5 h of CSF containing DMI + nor-BNI (40-88 /zg/h). Perfusate samples were collected every 10 rain for 5-5.5 h, starting 20-30 rain after the onset of perfusion with CSF alone, and 1 h after the onset of DMI perfusion. Both experiments were performed on days 13-17 of gestation in rats weighing 379_+ 5 g (mean + S.E.M.; experiment 1) and 351 _+ 6 g (experiment 2). Rats were injected with heparin and bled continuously through a peristaltic pump. In experiment 1, blood samples were collected at a rate of 150 p.l/12 min for 2.5-3 h starting 1.5 h after the onset of nor-BNI perfusion. In experiment 2, blood samples were collected at a rate of 150 /xl/15 min for 5-5.5 h, coincident with the onset of perfusate collection. Hematocrits were stable with both these bleeding protocols. At the end of the experiments blood samples were centrifuged, and the plasma was separated and stored at - 7 0 ° C until assayed for LH by RIA. The animals were anesthetized with pentobarbital, and pregnancy was confirmed by laparotomy. In addition, the brains were fixed in 10% formalin + 1% CaC12. Frozen 50 p.m serial sections were subsequently stained with a Nissl stain using basic fuchsin, and the location of the cannula tip was determined.

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pies were kept at 4°C, and 20 #1 were injected into the HPLC system by an ESA model 460 autosampler. Chromatograms were analyzed by a Hewlett Packard model 3390A reporting integrator. NE standard was obtained from Sigma (St. Louis, MO) and was prepared in mobile phase with pH adjusted to 3.0. NE in the standard preparation and perfusate samples kept at 4°C in the autosampler was stable for at least 24 h. Assay sensitivity was 0.5-1 p g / 2 0 /11. The inter- and intraassay variations for 10 pg N E / 2 0 /zl (n = 23) were 2.4 and 1.6%, respectively.

2.5. Radioimmunoassay Plasma samples were analyzed for LH by the ovine : ovine rat LH double antibody RIA of Niswender et al. [20] as previously described [6]. The sensitivity of the assay was 7-10 pg/tube. Inter- and intraassay variations determined at a mean plasma LH level of 1.25 + 0.05 ng/ml (n = 15) were 16.5 and 13.9%, respectively. LH values (ng/ml plasma) were expressed in terms of the NIDDK rat LH-RP-2 standard.

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2.4. Chromatography The HPLC system employed a Coulochem II dual electrode electrochemical detector (ESA, Inc., Bedford, MA) in conjunction with an ESA model 5014 analytical cell and a Hypersil ODS column (150 × 3 mm, 3 /zm; Keystone Scientific, Bellefonte, PA). The degassed and filtered mobile phase (75 mM NaH2PO 4 • H20, 1.7 mM l-octane sulfonic acid, 20 /zM EDTA, 100 p,1/1 triethylamine, 3% acetonitrile, pH adjusted to 3.0 with H3PO 4) was delivered by an ESA model 420 pump at a flow rate of 0.5 ml/min. Detection of NE was achieved by sequenlial oxidation : oxidation of the samples. Guard cell potential was 350 mV, and analytical cell potentials were - 170 mV (detector 1) and 200 mV (detector 2). Peffusate sam-

t Fig. 1. Sagittal (top) and transverse (bottom) sections through the rat brain indicating the location of push-pull cannula tips (shaded areas) in the M P O A where perfusion with nor-BNl increased LH secretion. The arrows indicate the level of the transverse sections. Sections were labeled according to the atlas of deGroot [4]. A C = anterior commissure: A H A = anterior hypothalamic area; DBB = diagonal band of Broca; DMH = dorsomedial hypothalamic nucleus; FX = fornix; OC = optic chiasm; P O A = preoptic area; SC = s u p r a c h i a s m a t i c nucleus; V M H = ventromedial hypothalamic nucleus; V = third ventricle.

S. Zhen, R.V. Gallo/Brain Research 698 (1995) 121-129

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vehicle

2.6. Data analysis In experiment 1, differences in mean plasma LH levels between all 4 groups were first compared by analysis of variance followed by Duncan's multiple range test. Since the variance in LH values within animals showing the large a n d / o r sustained increase in LH secretion in response to nor-BNI perfusion in the MPOA was significantly greater than in the other 3 groups of animals showing only basal pulsatile LH release, differences in LH pulse frequency and mean plasma LH levels were further analyzed in these latter 3 groups alone by analysis of variance and Duncan's multiple range test. Differences between groups in the percent occurrence of the large and/or sustained increase in LH secretion were determined by Fishers exact probability test. To analyze LH pulsatility, the coefficient of variation (CV) method was used to define an LH pulse, as previously described [6]. The CV and mean LH level were determined for all LH values which comprised the ascending and descending phases of each potential pulse (i.e., not just the nadir and peak values), and a pulse was defined when this CV was 1.5 times greater than the intra-assay CV determined at a comparable mean plasma LH level. In this study, the intra-assay CVs for LH levels in the ranges of 0.53-0.93 n g / m l (n = 7), 1.03-1.32 n g / m l (n = 9), and 1.39-1.60 n g / m l (n = 4) were 18.1, 14.0, and 12.2, respectively. Therefore, the minimum CVs accepted for the definition of a pulse for mean plasma LH levels in the above ranges were 27.1, 21.0, and 18.2. respectively. Pulsatile LH re-

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Fig. 3. Representative examples of the plasma LH responses in rats pretreated with vehicle followed by perfusion of the MPOA with either CSF (a and b) or nor-BNI (c and d, e and f), and in rats pretreated with FLA-63 followed by perfusion with nor-BNI (g and h) during midgestation. Arrows indicate defined LH pulses. Filled circles indicate plasma LH concentrations beyond the linear portion of the LH standard curve. Note the change in scale for plasma LH concentrations in c and d.

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No. of Rats Fig. 2. Effects of pretreatment 2.5-3 h earlier with either vehicle or FLA-63 on the LH responses to perfusion in the MPOA with CSF alone or CSF containing nor-BNI during midpregnancy in the rat. Animals pretreated with vehicle and peffused with nor-BNI were divided into two groups (n = 5,4) because of the different LH responses observed (see text). Vehicle + CSF: P < 0.05 vs vehicle + nor-BNI (either group), FLA63 + nor-BNI. Vehicle + nor-BNI (n = 4): P < 0.05 vs. vehicle + nor-BNl (n - 5), FLA-63 + nor-BNI.

lease was characterized by calculating mean plasma LH levels (the mean of all plasma samples collected from each rat during the bleeding period was first determined, and then the average plasma LH level was determined for the experimental group from the individual means), pulse amplitude (the difference between the nadir and ensuing peak in LH levels), and pulse frequency (the number of pulses/3 h). Pulse amplitude could not be determined in rats with no defined LH pulses; frequency in such cases was considered as zero pulses/3 h [16]. All values are given as the means _+ S.E.M. In experiment 2, differences in mean plasma LH and perfusate NE levels between the control and treatment period were first compared by Student's paired t-test. Then mean values during each individual h of the treatment period were compared to the control by analysis of variance followed by Dunnett's t-test. Time 0 in experiment 2 (Fig. 5) represented the time when DMI and/or nor-BNI reached the MPOA at the onset of the second part of the perfusion period. In order to determine the relationship between peffusate NE and plasma LH levels, analysis of LH for each individual hour, and for the entire treatment

S. Zhen, R. V. Gallo / Brain Research 698 (1995) 121-129

period, excluded the plasma sample taken at 15 min after time 0 and began with the following sample, since the response time for intraventricularly administered NE to increase LH secretion is 15-20 min [7,9].

summarized in Fig. 2. In rats pretreated with vehicle and perfused with CSF in the MPOA, plasma LH levels in most samples were below assay sensitivity. Two representative examples are shown in Fig. 3a,b. As in our previous study [32], two different LH responses were observed during nor-BNI perfusion in the MPOA. In 5 of 9 rats. nor-BNI perfusion produced an increase only in basal pulsatile LH secretion. Mean plasma LH levels were elevated ( P < 0.05) due to a small but significant increase in LH pulse frequency compared to the CSF controls (1.0 + 0.4 vs. 0.1 _+ 0.1 pulses/3 h, P < 0.05). Changes in LH pulse amplitude were not compared due to the lack of sufficient LH pulses in CSF control rats. Representative examples are shown in Fig. 3e,f. In the remaining 4 rats, nor-BNI perfusion produced an additional large elevation in plasma LH levels, which was much higher in magnitude and longer in duration than the LH pulses observed in the other 5 animals. Plasma LH levels in these 4 rats were significantly increased compared to those in CSF control animals ( P <0.05), as well as when compared to LH levels obtained in the 5 nor-BNI perfused rats that showed only an increase in basal pulsatile LH release ( P < 0.05).

3. Results

3.1. Histology The location of the push-pull cannula tips in the MPOA where blockade of K-opioid receptors stimulated increases in LH release is shown in Fig. 1. There was no difference in the location of cannula tips between our previous study in the MPOA [32] and the present report, nor between rats that did or did not show the large a n d / o r sustained rise in LH release during nor-BNI perfusion of the MPOA.

3.2. Experiment 1 The effects of nor-BNI perfusion in the MPOA on LH secretion in rats pretreated with vehicle or FLA-63 are

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Fig. 4. Effects of push-pull perfusion of the MPOA with either (1) CSF followed by nor-BNI (left), (2) two consecutive periods of DMI (middle), or (3) DMI followed by DMI + nor-BNl (right) on plasma LH concentrations and M P O A perfusatc NI~ levels during midpregnancy in the rat. The numbcr of animals in each group is given at the base of each bar. * P < 0.05 vs. the control period; c = control; 1 - 4 = whole treatment period of 4 h; 1,2,3, and 4 - individual h of treatment. Note the change in scale for plasma LH concentrations on the left side of both the left and right panels.

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S. Zhen, R. V. Gallo / Brain Research 698 (1995) 121-129

Moreover, during the approximate 2 h period during norBNI perfusion in these 4 rats when plasma LH levels were relatively stable i.e. not undergoing the large elevation, the frequency of LH pulses (0.9 +__0 . 3 / 2 h) was similar to that which occurred in the previous 5 rats, when the 3 h pulse frequency in those 5 animals (1.0 + 0.4) is expressed in terms of pulses/2 h (0.7 + 0.3). Representative examples are shown in Fig. 3c,d. Pretreatment of 9 rats with FLA-63 had no effect on the nor-BNI induced increase in basal pulsatile LH release (Fig. 2). In these animals mean plasma LH levels were elevated above those in vehicle pretreated, CSF controls ( P < 0.05) due to an increase in LH pulse frequency (1.0 + 0.3 vs. 0.1 + 0.1 pulses/3 h, P <0.0-5). Mean plasma LH levels as well as LH pulse frequency were the same as those values observed in the 5 rats that had been pretreated with vehicle, and which showed only an increase in basal pulsatile LH secretion in response to norBNI. However, the large/sustained increase in LH secretion, which was not seen in any of the 10 vehicle pretreated rats perfused with CSF, but which occurred in 45%

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(4 of 9) of the vehicle pretreated, nor-BNI perfused animals ( P < 0.05), was completely prevented in these 9 nor-BNI perfused rats by pretreatment with FLA-63 ( P < 0.05 vs vehicle pretreated rats perfused with nor-BNI). The absence of this large/sustained LH increase in response to nor-BNI in FLA-63 pretreated rats resulted in plasma LH levels that were less than those in the 4 vehicle pretreated, nor-BNI perfused animals which did demonstrate this LH response ( P < 0.05). Representative examples of LH profiles observed during nor-BNI perfusion in the MPOA in FLA-63 pretreated rats are shown in Fig. 3g,h.

3.3. Experiment 2 The objective of experiment 2 was to determine if NE involvement in the large and/or sustained increase in LH release during K-receptor blockade in the MPOA involved an increased release of NE at the site of nor-BNI perfusion. Changes in plasma LH concentrations and perfusate NE levels between the control and treatment period are summarized in Fig. 4. In agreement with experiment 1,

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S. Zhen, R. V. Gallo /Brain Research 098 (1995) 121-129

perfusion with nor-BNI alone produced two types of LH responses (Fig. 4 left, top). An increase only in basal pulsatile LH secretion was seen in 10 rats, while a large elevation in plasma LH levels, occurring primarily during the last 2 h of perfusion, was seen in 2 rats. Due to the small number of animals in this group showing the latter type of LH response, there was no significant difference in mean plasma LH levels between the CSF control period and either the whole treatment period or each individual hour. However, nor-BNI perfusion did not produce any change in perfusate NE levels compared to the CSF control period, regardless of whether rats did or did not show the large a n d / o r sustained increase in LH secretion (Fig. 4 left, bottom). Representative examples are shown in Fig. 5 left, top and middle. Addition of DMI to the perfusion solution increased perfusate NE levels to a new steady state value after 1 h (data not shown), as no further increase in perfusate NE levels was observed between the 2nd hour of DMI perfusion (control period for these experiments) and the last 4.5 h of DMI perfusion (Fig. 4 middle, bottom). However, despite the elevated perfusate NE levels seen during both periods of DM! perfusion as compared to that seen during perfusion with CSF alone, plasma LH levels (Fig. 4 middle, top) were not different from values obtained during perfusion with CSF alone. A representative example of an LH and NE profile during DMI perfusion of the MPOA is shown in Fig. 5 left, bottom. Lastly, perfusion of the MPOA with DMI + nor-BNI also produced two types of LH responses (Fig. 4 right, top). An increase only in basal pulsatile LH release was obtained in 12 animals, while an additional large a n d / o r sustained elevation in LH secretion occurred in 4 rats. Due to the large variation in plasma LH levels during the last 2 h of perfusion in the latter 4 animals, only the increase in basal LH secretion was significant. Nevertheless, although perfusate NE levels now tended to increase in response to perfusion with DMI + nor-BNI, there was no difference in the magnitude of this increase, regardless of whether animals showed only a small increase in basal pulsatile LH secretion, or the additional large a n d / o r sustained elevation in plasma LH levels (Fig. 4 right, bottom). Varying the dose of nor-BNI from 40 to 88 / z g / h made no difference in either plasma LH concentrations or perfusate "qE levels (data not shown). Three representative examples ~tre shown in Fig. 5, right.

4. Discussion

The results of experiment 1 demonstrate a role for NE in mediating only one of the two types of LH responses occurring in response to blockade of K-opioid receptors in the MPOA in the pregnant rat. While the nor-BNI induced increase in basal pulsatile LH release was not affected by NE synthesis inhibition with FLA-63, pretreatment with

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FLA-63 completely prevented the occurrence of the large a n d / o r sustained elevation in LH secretion. Thus the normal functioning of the noradrenergic system is important in mediating only this latter type of LH response during K-receptor blockade in the MPOA, but not the increase in basal pulsatile LH secretion. With respect to the lack of involvement of NE in mediating the increase in basal pulsatile LH release in response to MPOA K-receptor blockade, this conclusion is in agreement with that reached in our previous study involving a different CNS site, the MBH [33]. In that report we demonstrated that neither inhibition of NE synthesis with FLA-63, nor blockade of o~-adrenergic receptors with phentolamine, had any effect on the increase in basal pulsatile LH release induced by blockade of K-opioid receptors in the MBH. Our studies clearly demonstrate that the increase in basal pulsatile LH secretion produced by blockade of K-opioid receptors in either the MBH [33], or MPOA (present report), does not require the participation of the noradrenergic system. Additional experiments, involving measurement of in vivo NE release at the MPOA site of nor-BNI perfusion, were done to determine if the importance of NE to the large, sustained increase in LH release involved an increased release of NE at the site of perfusion. When compared with our earlier study [32], or the results of experiment 1 in the present report, the percentage of rats demonstrating the large a n d / o r sustained increase in LH secretion during blockade of MPOA K-receptors was unexpectedly low in experiment 2 (23% vs. 62 and 45%, respectively). Nevertheless, whether the large/sustained increase in LH release was produced by perfusion of the MPOA with nor-BNI alone, or whether it occurred during treatment with DMI + nor-BNI, the NE responses in these two groups of animals were similar to those seen in rats also perfused with nor-BNI alone or in combination with DMI, but which demonstrated only the increase in basal puisatile LH secretion. NE released in the MPOA may be merely permissive to the induction of the large and/or sustained increase in LH secretion occurring in response to blockade of K-opioid receptors in the MPOA during pregnancy, i.e. only the presence of basal NE secretion is required. Alternatively, MPOA K-receptor blockade may activate neuronal pathways capable of triggering an increase in NE release outside the MPOA, an increase that is capable of generating an elevation in LHRH secretion and a large/sustained increase in LH release. Interestingly, preliminary studies in a recent report involving microdialysis in the MPOA also found no change in NE release at that site in response to either local or systemic administration of naloxone, although this opioid antagonist stimulated an increase in LH secretion in intact and castrated male rats [31]. Reuptake is the major mechanism for terminating noradrenergic synaptic transmission, by removing most of the released NE from the synaptic cleft [11]. As in the MBH

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[33], the present data clearly s h o w the presence of an efficient N E reuptake m e c h a n i s m in the M P O A , since D M I increased perfusate N E levels at this site. H o w e v e r , n o r - B N I applied to the M B H was ineffective in increasing perfusate N E levels whether administered alone or in c o m b i n a t i o n with the N E reuptake blocker D M I [33]. In contrast to the M B H , b l o c k a d e of K-opioid receptors in the M P O A was m o r e likely to result in an increase in perfusate N E levels w h e n administered in c o m b i n a t i o n with the N E reuptake blocker, rather than w h e n administered alone. H o w e v e r , it is important to indicate that the small but significant increase in perfusate N E levels that did occur in response to b l o c k a d e of K-opioid receptors in the M P O A , in rats s h o w i n g an increase in basal pulsatile L H release, could not have been related to this increased L H secretion, since inhibition of N E synthesis had no effect on the ability of n o r - B N I applied to this site to produce the latter type of L H response. This n o r - B N I induced increase in N E secretion must be related to another physiological process regulated by an interaction b e t w e e n K-receptors and the noradrenergic system in the M P O A . In s u m m a r y , the results f r o m our present study d e m o n strate that w h i l e N E does not mediate the increase in basal pulsatile L H release p r o d u c e d by K-receptor b l o c k a d e in the M P O A , it does participate in the induction of the large a n d / o r sustained elevation in L H secretion occurring in response to antagonism of K-opioid receptors at this site during m i d p r e g n a n c y in the rat. H o w e v e r , the importance of N E in this regard is not expressed as an increased release of this neurotransmitter at the site of K-opioid receptor blockade.

Acknowledgements W e w o u l d like to thank Dr. G.D. N i s w e n d e r for antiovine L H # 1 5 , Dr. H. P a p k o f f for o v i n e L H for iodination, Dr. A.F. Parlow and the N I D D K for the rat L H used as a reference preparation, and M a t t h e w J. Smith and Peter C h o u h a n for technical assistance. This w o r k was supported by N I H Grant H D - 1 7 7 2 8 .

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