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The effect of blockade of κ-opioid receptors in the medial preoptic area on the luteinizing hormone surge in the proestrous rat
Brain Research 768 Ž1997. 111–119
Research report
The effect of blockade of k-opioid receptors in the medial preoptic area on the luteinizing hormone surge in the proestrous rat Matthew J. Smith, Robert V. Gallo
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Department of Physiology and Neurobiology, The UniÕersity of Connecticut, 3107 Horsebarn Hill Rd., Box U-154, Storrs, CT 06269-4154, USA Accepted 30 April 1997
Abstract The present study examined whether blockade of k-opioid receptors in the medial preoptic area ŽMPOA. prior to the critical period on the afternoon of proestrus could prematurely evoke an ovulatory luteinizing hormone ŽLH. surge, and if so, whether norepinephrine ŽNE. is involved in mediating this response. In the first experiment, push–pull perfusion of the MPOA with nor-binaltorphimine Žnor-BNI., a specific k-opioid receptor antagonist, was done in rats between 10.30 and 13.50 h on proestrus. To determine whether any resulting ovulation was due to a nor-BNI-induced increase in LH release, rats were injected with pentobarbital at 13.55 h to block the afternoon LH surge. In 7 of 10 rats, nor-BNI in the MPOA produced a large increase in LH release beginning between 12.30 and 13.30 h, and 5 of 7 ovulated. During MPOA perfusion with cerebrospinal fluid in our normal colony between 14.00 and 17.00 h, surges of LH release began in the majority of rats between 15.30 and 16.30 h. Thus blockade of MPOA k-opioid receptors advanced the LH surge by 3 h. The next experiment examined the effect of NE synthesis inhibition with bisŽ4-methyl-1-homopiperazinylthiocarbonyl. disulfide ŽFLA-63., or a-adrenergic receptor blockade with phenoxybenzamine ŽPBZ., on the nor-BNI-induced LH response. In 5 of 6 vehicle-treated rats, blockade of MPOA k-opioid receptors elicited a large increase in LH release and all 5 ovulated. In contrast, only 3 of 8 rats pretreated with FLA-63 had a large increase in LH release and ovulated, and PBZ prevented the nor-BNI-induced LH increase and ovulation in 4 of 4 rats. PBZ also prevented the afternoon LH surge and ovulation in 4 of 4 rats in our normal colony. Finally, HPLC measurement of NE levels in MPOA push–pull perfusate indicated no increase in NE release during the nor-BNI-induced or normal afternoon LH surges. These results indicate that antagonism of k-opioid receptors in the MPOA can prematurely evoke an ovulatory LH surge prior to the critical period on the afternoon of proestrus. Furthermore, the nor-BNI-induced as well as the normal afternoon LH surges are dependent on the proper functioning of central noradrenergic neurons, but do not involve increased NE release within the MPOA. q 1997 Elsevier Science B.V. Keywords: Medial preoptic area; Luteinizing hormone surge; Norepinephrine; Push–pull perfusion; k-opioid receptor; Nor-binaltorphimine
1. Introduction Endogenous opioid peptides exert an inhibitory influence on luteinizing hormone ŽLH. release, and a significant decrease in this inhibitory tone Ždisinhibition. is viewed as a critical event underlying induction of the LH surge on the afternoon of proestrus Žfor review, see w16x.. This ‘disinhibition’ hypothesis is based in part on the ability of systemic administration of the general opioid receptor antagonist naloxone to prematurely evoke a LH surge prior to the critical period on the afternoon of proestrus w1,2x. Evidence in the literature supports a role for b-endorphin originating in the arcuate nucleus and acting via m-opioid receptors in this suppression of proe)
Corresponding author. Fax: q1 Ž860. 4863303.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 6 2 2 - 7
strous LH secretion. Thus, the concentration of b-endorphin in hypophyseal portal blood w30x, as well as arcuate levels of the mRNA for proopiomelanocortin Žthe precursor for b-endorphin which binds to m-opioid receptors. w3,37x, decrease prior to the increase in LH release on the afternoon of proestrus. That this reduction in opioid tone may also be due, in part, to a decrease in opioid receptors, is supported by the finding that naloxone binding to hypothalamic tissue decreases during the afternoon of the steroid-induced LH surge w15x, as does the number of hypothalamic m-opioid receptors on the afternoon of proestrus w21x. k-Opioid receptors w23x, as well as LH-releasing hormone ŽLHRH. cell bodies w38x, are located in the medial preoptic area ŽMPOA., a site known to play an essential role in generating the neural signal that triggers the LH
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surge w9x. Our laboratory recently demonstrated that blockade of k-opioid receptors in the MPOA during midpregnancy with nor-binaltorphimine Žnor-BNI., a specific kopioid receptor antagonist, produced a large increase in LH release w41,43x. As a follow-up to experiments in the pregnant rat, and taking into consideration the ‘disinhibition’ hypothesis applied to the preovulatory LH surge, in the present study we examined whether antagonism of k-opioid receptors in the MPOA prior to the critical period on the afternoon of proestrus could prematurely evoke an ovulatory LH surge. The preovulatory LH surge depends on the functional integrity of central noradrenergic neurons. Inhibition of norepinephrine ŽNE. synthesis or blockade of a-adrenergic receptors prevents the ovulatory surge of LH Žfor review, see w18x.. In addition, k-opioid receptor activation has been shown to inhibit NE release in the MPOA of male rats w14x and in the guinea pig cortex w34,35x. Thus if blockade of k-opioid receptors in the MPOA were found to prematurely elicit an ovulatory LH surge, the next objective was to determine whether NE was involved in mediating this LH response.
2. Materials and methods 2.1. Animals Adult female rats in our colony, derived from Charles River Sprague-Dawley CD rats, and weighing 270–320 g at the time of cannula implantation, were maintained on a 14:10 h lightrdark schedule Žlights on at 05.00 h., and fed rat chow and water. Estrous cyclicity was determined by daily examination of vaginal smears. The experimental procedures 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 ŽPlastics One, Roanoke, VA. was stereotaxically implanted into the MPOA Žcoordinates: 7.8 mm anterior to the interaural line, 0.8 mm lateral to the midline, and 6.8 mm below the surface of the brain., according to the atlas of deGroot w7x. The cannula was cemented in place, and a removable stylette Ž26 gauge. was inserted so that its tip was flush with the tip of the guide cannula. Cannula implantation on the day of estrus resulted in minimal disruption of the estrous cycle. Daily vaginal smears were continued and animals displaying at least two consecutive 4-day estrous cycles were used in the following experiments. The inner stylette was removed 4–7 days before the day of push–pull perfusion, and a new stylette was inserted so that its tip extended 1 mm beyond the tip of the guide cannula.
2.3. Experimental protocols Between 08.00 and 10.00 h on the day before the experiment Ždiestrous day 2., rats were anesthetized briefly with ether, and a polyethylene ŽPE50. cannula was inserted into or near the right atrium via the external jugular vein. The next day rats having nucleated epithelial smears characteristic of proestrus were used in experiments. The technique of push–pull perfusion was similar to that previously used in our laboratory w41–43x. On the morning of the experiment, the flow rates of the push and pull peristaltic pumps ŽPharmacia, Piscataway, NJ. were carefully balanced and set to deliver 10 mlrmin. This was accomplished by setting the push pump to deliver 10 mlrmin, and then adjusting the flow rate of the pull pump until a drop of fluid at the tip of the push–pull cannula system remained unchanged in size for 1 h. 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 tube kept on ice and containing 12 ml 0.25 N perchloric acid to adjust the perfusate pH to 3.0 in Experiment 2, in which perfusate NE levels were measured. Constancy of flow rates during the perfusion was verified gravimetrically by weighing the perfusate. The following experiments were done. 2.3.1. Experiment 1 The objective of this experiment was to determine whether antagonism of k-opioid receptors in the MPOA can prematurely evoke an ovulatory LH surge prior to the critical period on the afternoon of proestrus. The critical period is the time during which a neural stimulus normally triggers the LH surge on proestrus. Push–pull perfusion of the MPOA with either artificial cerebrospinal fluid ŽCSF; NaCl 140 mM, KCl 4 mM, CaCl 2 2.3 mM, MgSO4 1 mM, Na 2 HPO4 1.2 mM, NaH 2 PO4 0.3 mM, glucose 3.4 mM, pH 7.4. or CSF containing nor-binaltorphimine Žnor-BNI., a selective k-opioid receptor antagonist w27,33x, Ž40 mgrh, Research Biochemicals, Natick, MA. was done in rats between 10.30 and 13.50 h on proestrus. This dose has been shown to produce a large increase in LH release when applied to the MPOA during midpregnancy w41,43x. During the perfusion period, blood and perfusate samples were collected every 20 min, the latter for measurement of NE levels. This was done to determine the relationship, if any, between NE release and the nor-BNI-induced LH surge as part of Expt. 2. At 13.55 h, prior to the onset of the critical period, the animals were injected with pentobarbital Ž3.5 mgr100 g. b.wt., i.p.. to block the normal afternoon LH surge w10x. This was done in order to determine whether any resulting ovulation was due to the nor-BNI-induced increase in LH release. Additional blood samples were taken every 30–60 min between 14.00 and
M.J. Smith, R.V. Gallo r Brain Research 768 (1997) 111–119
17.00 h on proestrus. At the end of the experiment, rats were returned to the animal quarters overnight. The following morning Žestrus. the ovaries were removed, the oviducts were separated from the ovaries, and ovulation was verified by counting the ova with the aid of a low power microscope. In another group of rats, push–pull perfusion of the MPOA with CSF was done between 14.00 and 17.00 h. Blood and perfusate samples were taken at 20 min intervals during this time to determine the onset of the preovulatory LH surge in our colony of rats as part of Expt. 1, and to relate any changes in NE release in the MPOA to the time of onset of the normal afternoon LH surge as part of Expt. 2. At 17.00 h rats were returned to the animal quarters overnight. Ovulation was checked the next morning. 2.3.2. Experiment 2 To examine a possible role for NE in the nor-BNI-induced LH increase, the effects of NE synthesis inhibition or a-adrenergic receptor blockade on the LH response to nor-BNI were examined. Push–pull perfusion of the MPOA with CSF containing nor-BNI Ž40 mgrh. was done between 10.30 and 13.50 h on proestrus in rats pretreated with either Ž1. bisŽ4-methyl-1-homopiperazinylthiocarbonyl. disulfide ŽFLA-63, a dopamine-b-hydroxylase inhibitor w6x, ICN Biomedicals, Irvine, CA.; Ž2. phenoxybenzamine ŽPBZ., an a-adrenergic receptor blocker w25x, ŽResearch Biochemicals.; or Ž3. their respective vehicles. FLA-63 was dissolved in 26% EtOH and was given s.c. 2.5–3 h prior to the onset of perfusion at a dose of 25 mgrkg. Our laboratory has previously shown that this dose produces a 72% decrease in hypothalamic-MPOA NE levels, and prevents the naloxone-induced increase in LH secretion w22x as well as the large increase in LH release occurring in response to blockade of MPOA k-opioid receptors during midpregnancy w43x. PBZ was dissolved in acid saline ŽpH 5.0., and was given i.p. 1 h prior to the onset of perfusion at a dose of 20 mgrkg. This dose of PBZ has been shown to suppress pulsatile LH secretion in diestrous rats w12x, as well as prevent the steroid-induced LH surge w8x. During the perfusion period, blood samples were collected at 20-min intervals. Animals were injected with pentobarbital at 13.55 h. Additional blood samples were taken every 30–60 min between 14.00 and 17.00 h on proestrus and the animals were then returned to their quarters. Ovulation was checked the next morning. PBZ was also given to another group of rats at 13.00 h on proestrus to examine the role of NE in the normal afternoon LH surge. Blood samples were taken at 30 min intervals from 14.00 to 17.00 h, and ovulation was checked the following morning. Animals in all experiments were injected with 400 U heparin before blood samples Ž150 ml each. were collected continuously through a peristaltic pump every 20 min, or withdrawn with a syringe at 30–60-min intervals. At the
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end of each experiment blood samples were centrifuged, and the plasma was separated and stored at y708C until assayed for LH by radioimmunoassay. In addition, the animals were anesthetized with pentobarbital and their brains were fixed in 10% formalin q1% CaCl 2 . Frozen 50-mm serial sections were stained with a Nissl stain using basic fuchsin, and the location of the cannula tip was determined. 2.4. Chromatography The HPLC system used a Coulochem II dual electrode electrochemical detector ŽESA, Bedford, MA. in conjunction with an ESA model 5014 analytical cell and a Hypersil column Ž150 = 3 mm, 3 mm; Keystone Scientific, Bellefonte, PA.. The degassed and filtered mobile phase Ž75 mM NaH 2 PO4 P H 2 O, 1.7 mM 1-octane sulfonic acid, 20 mM EDTA, 100 mlrl triethylamine, 1.5–3% acetonitrile, pH adjusted to 3.0 with H 3 PO4 . was delivered by an ESA model 420 pump at a flow rate of 0.5 mlrmin. Detection of NE was achieved by sequential oxidation:oxidation of the sample. Guard cell potential was 350 mV, and analytical cell potentials were y170 mV Ždetector 1. and 200 mV Ždetector 2.. 20–30-ml samples were kept at 48C and injected by an ESA model 460 autosampler. Chromatograms were analyzed by a Hewlett-Packard model 3390A reporting integrator. NE standard was obtained from Sigma ŽSt. Louis, MO. and was prepared in CSF with pH adjusted to 3.0 with perchloric acid. Assay sensitivity was 1 pg NE. The inter- and intraassay variations for 10 pg NEr20–30 ml Ž n s 15. were 6% and 2.5%, respectively. 2.5. Radioimmunoassay Plasma samples were analyzed for LH by the ovine:ovine rat LH double antibody radioimmunoassay of Niswender et al. w26x, as previously described w11x. The sensitivity of the assay was 5–10 pgrtube. Inter- and intraassay variations determined at a mean plasma LH level of 2.21 " 0.09 ngrml Ž n s 11. were 13.8% and 13.2%, respectively. LH values Žngrml plasma. were expressed in terms of the NIDDK rat LH-RP-2 standard. 2.6. Data analysis Mean plasma LH or perfusate NE levels for each experimental group were determined by first calculating the mean of all the blood or perfusate samples collected from each rat during the perfusion period, and then determining the average plasma LH or perfusate NE level for the experimental group from the individual means. Significant differences between groups in mean plasma LH levels were determined by Student’s unpaired t-test in Expt. 1, and analysis of variance followed by Dunnett’s t-test in Expt. 2. Differences between groups in the percent occur-
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rence of the large increase in LH release were determined by Fisher’s exact probability test. Differences between groups in perfusate NE levels were determined by analysis of variance followed by Duncan’s multiple range test. Results were considered significant at P - 0.05. All results are expressed as the mean " S.E.M.
3. Results 3.1. Experiment 1 In 5 rats perfused in the MPOA between 10.30 and 13.50 h with CSF alone, LH release was basal during this time, and plasma LH levels remained low between 14.00 and 17.00 h. All 5 rats failed to ovulate the next morning, demonstrating that the injection of pentobarbital they received at 13.55 h was completely effective in preventing the normal LH surge. In contrast, push–pull perfusion of the MPOA with nor-BNI produced a surge increase in LH release in 7 of 10 rats Ž70%; P - 0.02 vs. 0% in rats perfused with CSF alone.. This LH response began between 12.30 and 13.30 h, or about 2–3 h after the onset of nor-BNI perfusion. Five of these 7 rats ovulated the next morning with an average of 16.0 " 0.9 ova. Mean plasma LH levels during nor-BNI perfusion Ž3.71 " 1.07 ngrml.
Fig. 2. Mean plasma LH concentrations Ž`; ns8. and MPOA perfusate NE levels Ž^; ns 7. in rats from our normal colony perfused in the MPOA with CSF between 14.00 and 17.00 h on the afternoon of proestrus. Standard error bars are not shown when smaller than the corresponding symbols.
were significantly greater than those in CSF-perfused rats Ž0.58 " 0.06 ngrml; P - 0.05.. In the 7 rats with norBNI-induced increases in LH secretion, peak plasma LH levels of at least 20.6 " 6.3 ngrml were reached before the end of the perfusion period. Following pentobarbital administration at 13.55 h, in these 7 rats plasma LH levels gradually declined between 14.00 and 17.00 h, and in the 3 rats with no nor-BNI-induced LH increase plasma LH
Fig. 1. Representative examples of LH secretion in rats during push–pull perfusion of the MPOA with CSF, or CSF containing nor-BNI, on the morning of proestrus. The filled circle indicates a plasma LH value beyond the linear portion of the LH standard curve. Note the change in scale for plasma LH concentrations in the nor-BNI examples.
M.J. Smith, R.V. Gallo r Brain Research 768 (1997) 111–119
levels remained low during this 3-h period Ždata not shown.. Representative examples of LH responses during MPOA perfusion with CSF or nor-BNI are given in Fig. 1. Plasma LH values in 8 rats in our normal colony bled between 14.00 and 17.00 h on the afternoon of proestrus are given in Fig. 2. Surges of LH release generally began between 15.30 and 16.30 h, and these rats ovulated with an average of 14.0 " 0.4 ova. Thus the nor-BNI-induced LH increase represented a 3 h advancement in the time of the LH surge. 3.2. Experiment 2 In animals pretreated with vehicle alone, blockade of MPOA k-opioid receptors with nor-BNI elicited a surge increase in LH release in 83% Ž5 of 6. of the rats. These 5 rats ovulated the next morning with an average of 14.0 "
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1.5 ova. In contrast, only 3 of 8 FLA-63-pretreated rats displayed a large nor-BNI-induced increase in LH release and ovulation, with an average of 9.0 " 2.0 ova. The percentage of FLA-63-pretreated rats demonstrating the large increase in LH release was reduced Ž38% vs. 83%., but this difference was not statistically significant. However, mean plasma LH levels in response to nor-BNI in FLA-63-pretreated animals Ž1.93 " 0.44 ngrml. were significantly less than those in animals pretreated with vehicle alone Ž3.88 " 0.87 ngrml; P - 0.05.. In rats pretreated with PBZ, the nor-BNI-induced increase in plasma LH levels was prevented; none of the 4 rats perfused with nor-BNI showed an increase in LH release Ž0%; P - 0.03 vs. 83% in rats perfused with nor-BNI and pretreated with vehicle., and none ovulated. In addition, mean plasma LH levels in PBZ pretreated animals in response to nor-BNI Ž0.37 " 0.03 ngrml. were also significantly lower than
Fig. 3. Representative examples of LH secretion during push–pull perfusion of the MPOA with nor-BNI on the morning of proestrus in rats pretreated with vehicle Žtop., FLA-63 Žmiddle., or PBZ Žbottom.. The filled circles indicate plasma LH values beyond the linear portion of the LH standard curve. Note the change in scale for plasma LH concentrations in one of the two vehicleq nor-BNI examples.
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those in animals pretreated with vehicle alone Ž3.88 " 0.87 ngrml; P - 0.01.. Following pentobarbital administration at 13.55 h in Expt. 2, plasma LH levels between 14.00 and 17.00 h either gradually declined if LH secretion had increased in response to nor-BNI between 10.30 and 13.50 h, or remained low during this time if no nor-BNI-induced LH surge had occurred Ždata not shown.. Representative examples of LH responses during MPOA perfusion with nor-BNI in rats pretreated with vehicle, FLA-63, or PBZ are given in Fig. 3. PBZ also prevented the normal afternoon LH surge and ovulation from occurring in all 4 rats tested in our normal colony. As previously indicated in Expt. 1, MPOA perfusion with nor-BNI produced a surge increase in LH release in 7 animals. Although NE release in the MPOA was monitored in only 3 of these rats showing large increases in LH release Žmean s 5.67 " 1.89 ngrml., mean perfusate NE levels in these animals Ž20.4 " 4.5 pgr20 min. were not different from those found in 5 rats perfused in the MPOA between 10.30 and 13.50 h with CSF alone Ž19.2 " 4.0 pgr20 min. and showing basal plasma LH levels Ž0.58 " 0.06 ngrml.. Perfusate NE levels did not increase in association with the nor-BNI-induced LH surge in any of the 3 rats. Representative examples of perfusate NE and plasma LH levels during MPOA perfusion with CSF or nor-BNI between 10.30 and 13.50 h are shown in Fig. 4 Žtop.. Moreover, in 7 of the 8 rats perfused in the MPOA with CSF between 14.00 and 17.00 h on proestrus and demonstrating a normal afternoon LH surge, NE release in the MPOA was also monitored. Plasma LH levels and MPOA perfusate NE values between 14.00 and 17.00 h on proestrus for these animals are given in Fig. 2, and a representative example is shown in Fig. 4 Žbottom.. None
Fig. 5. Sagittal Žtop. and tranverse Žbottom. sections through the rat brain indicating the location of push–pull cannula tips Žshaded areas. in the MPOA where perfusion with nor-BNI increased LH secretion. The arrows indicate the level of the tranverse sections. Sections are labelled according to the atlas of deGroot w7x. AC, anterior commissure; AHA, anterior hypothalamic area; DBB, diagonal band of Broca; DMH, dorsomedial hypothalamic nucleus; FX, fornix; OC, optic chiasm; POA, preoptic area; SC, suprachiasmatic nucleus; V, third ventricle; VMH, ventromedial hypothalamic nucleus.
Fig. 4. Representative examples of plasma LH concentrations Ž`. and MPOA perfusate NE levels Ž^. during push–pull perfusion of the MPOA with either CSF Žleft. or CSF containing nor-BNI Žright. prior to the critical period on the afternoon of proestrus Žtop., or CSF during the normal afternoon LH surge Žbottom.. Filled circles indicate plasma LH values beyond the linear portion of the LH standard curve. Note the change in scale for plasma LH concentrations during the nor-BNI-induced or afternoon LH surges.
M.J. Smith, R.V. Gallo r Brain Research 768 (1997) 111–119
of the 7 rats showed an increase in perfusate NE values in association with the rise in plasma LH levels, and mean perfusate NE levels Ž16.2 " 1.3 pgr20 min. were similar to those in rats perfused with CSF or nor-BNI between 10.30 and 13.50 h on proestrus. 3.3. Histology The locations of push–pull cannula tips where perfusion with nor-BNI resulted in large increases in LH release are summarized in Fig. 5. All locations were within the MPOA, in the same region where we have previously shown perfusion with nor-BNI produces a large increase in LH release during midpregnancy in the rat w41,43x. Furthermore, no difference was observed in the location of cannula tips in rats that did or did not exhibit a large increase in LH secretion in response to nor-BNI.
4. Discussion The present results demonstrate that selective blockade of MPOA k-opioid receptors prior to the critical period on the afternoon of proestrus can prematurely evoke an ovulatory LH surge. These findings are consistent with the hypothesis that a significant decrease Ždisinhibition. in existing inhibitory opioid tone on the afternoon of proestrus is a key event underlying generation of the LH surge Žfor review, see w16x.. Systemic administration of the general opioid receptor antagonist naloxone w1,2x, or selective blockade of MPOA k-opioid receptors Žpresent data., are each able to advance the time of onset of the LH surge on proestrus. The evidence reviewed by Kalra w16x supported a role for b-endorphin, originating in arcuate nucleus neurons, and acting via m-opioid receptors in this inhibition of proestrous LH secretion. The present data extend this hypothesis by demonstrating that k-opioid receptors in the MPOA may also be important in regulating the timing of the LH surge. Interestingly, we have previously shown that antagonism of k-opioid receptors in the MPOA can produce a large increase in LH release during mid-pregnancy w41,43x. Thus our work indicates that this k-opioid receptor-mediated restraint on LH secretion is expressed in different reproductive states of the rat, i.e., midpregnancy and the morning of proestrus, and may serve to prevent an inappropriate large increase in LH release from taking place at these times. The present data also demonstrate that NE is involved in mediating the premature LH surge occurring in response to blockade of MPOA k-opioid receptors on proestrus. Pretreatment with the NE synthesis inhibitor FLA-63 partially reduced the occurrence of the nor-BNI-induced LH surge, and blockade of a-adrenergic receptors with PBZ completely prevented its occurrence. Furthermore, our results show that the importance of NE in this k-opioid
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receptor-mediated mechanism is not expressed as an increased release of this neurotransmitter at the site of perfusion. These results are in agreement with those of our previous study in which we found that NE was essential for the large increase in LH secretion occurring in response to blockade of MPOA k-opioid receptors during midpregnancy w43x, but that this LH response was not associated with increased NE release at the site of perfusion. One possibility is that NE released in the MPOA may be simply permissive to the induction of the nor-BNI-induced LH surge on proestrus, and only the presence of basal NE release is necessary. Alternatively, blockade of MPOA k-opioid receptors may activate neuronal pathways capable of triggering NE release outside the site of perfusion, an increase capable of stimulating LHRH release and the nor-BNI-induced LH surge. Interestingly, another study reported an increase in LH secretion, but no change in NE release in the MPOA, in response to the local or systemic administration of naloxone in the castrated or intact male rat w39x. In addition to being essential to the nor-BNI-induced LH surge, our results also indicate that NE is important for generation of the normal afternoon LH surge, since PBZ completely prevented its occurrence. This observation is consistent with data from other laboratories demonstrating a role for NE in the LH surge Žfor review, see w18x.. An increased NE turnover rate in the MPOA has been reported to occur in association with the proestrous LH surge w28x, or the steroid-induced LH surge in ovariectomized rats w36x. This increased turnover rate is an indirect estimate of increased noradrenergic neuronal activity. Using push–pull perfusion to directly assess NE release in the MPOA, Mohankumar et al. w24x demonstrated increased MPOA perfusate NE levels in conjunction with the proestrous LH surge. However, we observed no change in perfusate NE levels in the MPOA occurring prior to or during the LH surge normally seen on the afternoon of proestrus. Although NE reuptake is an effective means of removal of NE from the synaptic cleft w19,29,32x, this cannot explain the lack of increase in perfusate NE levels in our report, since neither their study nor our own used desipramine, an inhibitor of NE reuptake, in the perfusion solution. While the reasonŽs. for the difference between this latter result and ours is not apparent, in our study measurement of NE release in the MPOA was done in the same animals in which plasma LH levels were determined. Nevertheless, the present results are in agreement with literature demonstrating that NE is clearly involved in generation of the normal afternoon LH surge. Although previous reports have implicated a role for b-endorphin in opioid inhibition of the LH surge w3,30,37x, the inference from our nor-BNI data is that dynorphin is the neuropeptide ligand active at the MPOA. Dynorphin rather than b-endorphin is the endogenous opioid with a high binding affinity for brain k-opioid receptors w4,5x. The terminals of dynorphin-containing fibers w13x and high
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concentrations of dynorphin w40x, as well as k-opioid receptors w23x, are found in the MPOA. With respect to the regulation of LH secretion, intraventricular injections of dynorphin suppress LH secretion in gonadectomized rats w17,20x, and antisera to dynorphin injected into the medial basal hypothalamus increased LH release in immature 12-day-old female rats w31x. Further studies are planned to determine if dynorphin is the endogenous ligand active at k-opioid receptors in the MPOA in mediating suppression of the LH surge. In summary, these results demonstrate that blockade of k-opioid receptors at the level of the MPOA can prematurely evoke an ovulatory LH surge. Furthermore, the nor-BNI-induced as well as the normal afternoon LH surges are dependent on the proper functioning of central noradrenergic neurons, but do not involve increased NE release within the MPOA.
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Acknowledgements
w18x
We would like to thank Kevin Shaw, Cassandra Soder and Josh Farber for their technical assistance, Dr. G.D. Niswender for antiovine LHa15, Dr. H. Papkoff for ovine LH for iodination, and Dr. A.F. Parlow and the NIDDK for the rat LH used as a reference preparation. This study was supported by NIH Grant HD-17728.
w19x
References w1x L.G. Allen, S.P. Kalra, Evidence that a decrease in opioid tone may evoke preovulatory luteinizing hormone release in the rat, Endocrinology 118 Ž1986. 2375–2381. w2x L.G. Allen, E. Hahn, D. Caton, S.P. Kalra, Evidence that a decrease in opioid tone on proestrus changes the episodic pattern of luteinizing hormone ŽLH. secretion: implications in the preovulatory LH hypersecretion, Endocrinology 122 Ž1988. 1004–1013. w3x H.C.L. Bohler, H. Tracer, G.R. Merriam, S.L. Petersen, Changes in proopiomelanocortin messenger ribonucleic acid levels in the rostral periarcuate region of the female rat during the estrous cycle, Endocrinology 128 Ž1991. 1265–1269. w4x C. Chavkin, A. Goldstein, Specific receptor for the opioid peptide dynorphin: structure-activity relationships, Proc. Natl. Acad. Sci. USA 78 Ž1981. 6543–6547. w5x C. Chavkin, I.F. James, A. Goldstein, Dynorphin is a specific endogenous ligand of the k opioid receptor, Science 215 Ž1981. 413–415. w6x H. Corrodi, K. Fuxe, L. Hamberger, A. Ljungdahl, Studies on central and peripheral noradrenaline neurons using a new dopamine b-hydroxylase inhibitor, Eur. J. Pharmacol. 12 Ž1970. 145–155. w7x J. deGroot, The Rat Forebrain in Stereotaxic Coordinates, North Holland Publ., Amsterdam, 1959, pp. 1–40. w8x S.V. Drouva, E. Laplante, C. Kordon, a 1 -adrenergic receptor involvement in the LH surge in ovariectomized estrogen-primed rats, Eur. J. Pharmacol. 81 Ž1982. 341–344. w9x J.W. Everett, Preoptic stimulative lesions and ovulation in the rat: ‘Thresholds’ and LH-release time in late diestrus and proestrus, in: E. Bajusz, G. Jasmin ŽEds.., Major Problems in Neuroendocrinology, S. Karger, Basel, 1964, pp. 346–366. w10x J.W. Everett, C.H. Sawyer, A 24-hour periodicity in the ‘LH-release
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droxytryptamine in slices of the midbrain-hypothalamus region of the rat, Acta Pharmacol. Toxicol. 36 Ž1975. 395–408. D.K. Sarkar, S.S.C. Yen, Changes in b-endorphin-like immunoreactivity in pituitary portal blood during the estrous cycle and after ovariectomy in rats, Endocrinology 116 Ž1985. 2075–2079. R. Schulz, A. Wilhelm, K.M. Pirke, C. Gramsch, A. Herz, b-Endorphin and dynorphin control serum luteinizing hormone level in immature female rats, Nature 294 Ž1981. 757–759. D.N. Thomas, R.B. Holman, A microdialysis study of the regulation of endogenous noradrenaline release in the rat hippocampus, J. Neurochem. 56 Ž1991. 1741–1746. F.C. Tortella, E. Echevarria, A.W. Lipkowski, A.E. Takemori, P.S. Portoghese, J.W. Holaday, Selective k antagonist properties of nor-binaltorphimine in the rat MES seizure model, Life Sci. 44 Ž1989. 661–665. L.L. Werling, S.R. Brown, B.M. Cox, Opioid receptor regulation of the release of norepinephrine in brain, Neuropharmacology 26 Ž1987. 987–996. L.L. Werling, P.N. MacMahon, P.S. Portoghese, A.E. Takemori, B.M. Cox, Selective opioid antagonist effects on opioid-induced-inhibition of release of norepinephrine in guinea pig cortex, Neuropharmacology 28 Ž1989. 103–107. P.M. Wise, N. Rance, C.A. Barraclough, Effects of estradiol and progesterone on catecholamine turnover rates in discrete hypothalamic regions in ovariectomized rats, Endocrinology 108 Ž1981. 2186–2193.
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w37x P.M. Wise, K. Scarborough, N.G. Wieland, G.H. Larson, Diurnal pattern of proopiomelanocortin gene expression in the arcuate nucleus of proestrous, ovariectomized and steroid-treated rats: a possible role in cyclic luteinizing hormone secretion, Mol. Endocrinology 4 Ž1990. 886–892. w38x J.W. Witkin, C.M. Paden, A.-J. Silverman, The luteinizing hormone-releasing hormone ŽLHRH. systems in the rat brain, Neuroendocrinology 35 Ž1982. 429–438. w39x A.M. Wolfe, T. Porka-Heiskanen, J.R. Norgle, J.E. Levine, Evidence that norepinephrine release in the preoptic area does not mediate effects of opiate receptor antagonism of LH secretion: an in vivo microdialysis study, Soc. Neurosci. Abstr. 131.4 Ž1993. 311. w40x N. Zamir, M. Palkovits, M.J. Brownstein, Distribution of immunoreactive dynorphin in the central nervous system of the rat, Brain Res. 280 Ž1983. 81–93. w41x S. Zhen, R.V. Gallo, The effect of blockade of k-opioid receptors in the medial basal hypothalamus and medial preoptic area on luteinizing hormone release during midpregnancy in the rat, Endocrinology 131 Ž1992. 1650–1656. w42x S. Zhen, R.V. Gallo, Lack of catecholamine involvement in the increased luteinizing hormone release due to blockade of k-opioid receptors in the medial basal hypothalamus during midpregnancy in the rat, Brain Res. 642 Ž1994. 178–184. w43x S. Zhen, R.V. Gallo, The role of norepinephrine in mediating luteinizing hormone release in response to blockade of k-opioid receptors in the medial preoptic area, Brain Res. 698 Ž1995. 121–129.
Research report
The effect of blockade of k-opioid receptors in the medial preoptic area on the luteinizing hormone surge in the proestrous rat Matthew J. Smith, Robert V. Gallo
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Department of Physiology and Neurobiology, The UniÕersity of Connecticut, 3107 Horsebarn Hill Rd., Box U-154, Storrs, CT 06269-4154, USA Accepted 30 April 1997
Abstract The present study examined whether blockade of k-opioid receptors in the medial preoptic area ŽMPOA. prior to the critical period on the afternoon of proestrus could prematurely evoke an ovulatory luteinizing hormone ŽLH. surge, and if so, whether norepinephrine ŽNE. is involved in mediating this response. In the first experiment, push–pull perfusion of the MPOA with nor-binaltorphimine Žnor-BNI., a specific k-opioid receptor antagonist, was done in rats between 10.30 and 13.50 h on proestrus. To determine whether any resulting ovulation was due to a nor-BNI-induced increase in LH release, rats were injected with pentobarbital at 13.55 h to block the afternoon LH surge. In 7 of 10 rats, nor-BNI in the MPOA produced a large increase in LH release beginning between 12.30 and 13.30 h, and 5 of 7 ovulated. During MPOA perfusion with cerebrospinal fluid in our normal colony between 14.00 and 17.00 h, surges of LH release began in the majority of rats between 15.30 and 16.30 h. Thus blockade of MPOA k-opioid receptors advanced the LH surge by 3 h. The next experiment examined the effect of NE synthesis inhibition with bisŽ4-methyl-1-homopiperazinylthiocarbonyl. disulfide ŽFLA-63., or a-adrenergic receptor blockade with phenoxybenzamine ŽPBZ., on the nor-BNI-induced LH response. In 5 of 6 vehicle-treated rats, blockade of MPOA k-opioid receptors elicited a large increase in LH release and all 5 ovulated. In contrast, only 3 of 8 rats pretreated with FLA-63 had a large increase in LH release and ovulated, and PBZ prevented the nor-BNI-induced LH increase and ovulation in 4 of 4 rats. PBZ also prevented the afternoon LH surge and ovulation in 4 of 4 rats in our normal colony. Finally, HPLC measurement of NE levels in MPOA push–pull perfusate indicated no increase in NE release during the nor-BNI-induced or normal afternoon LH surges. These results indicate that antagonism of k-opioid receptors in the MPOA can prematurely evoke an ovulatory LH surge prior to the critical period on the afternoon of proestrus. Furthermore, the nor-BNI-induced as well as the normal afternoon LH surges are dependent on the proper functioning of central noradrenergic neurons, but do not involve increased NE release within the MPOA. q 1997 Elsevier Science B.V. Keywords: Medial preoptic area; Luteinizing hormone surge; Norepinephrine; Push–pull perfusion; k-opioid receptor; Nor-binaltorphimine
1. Introduction Endogenous opioid peptides exert an inhibitory influence on luteinizing hormone ŽLH. release, and a significant decrease in this inhibitory tone Ždisinhibition. is viewed as a critical event underlying induction of the LH surge on the afternoon of proestrus Žfor review, see w16x.. This ‘disinhibition’ hypothesis is based in part on the ability of systemic administration of the general opioid receptor antagonist naloxone to prematurely evoke a LH surge prior to the critical period on the afternoon of proestrus w1,2x. Evidence in the literature supports a role for b-endorphin originating in the arcuate nucleus and acting via m-opioid receptors in this suppression of proe)
Corresponding author. Fax: q1 Ž860. 4863303.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 6 2 2 - 7
strous LH secretion. Thus, the concentration of b-endorphin in hypophyseal portal blood w30x, as well as arcuate levels of the mRNA for proopiomelanocortin Žthe precursor for b-endorphin which binds to m-opioid receptors. w3,37x, decrease prior to the increase in LH release on the afternoon of proestrus. That this reduction in opioid tone may also be due, in part, to a decrease in opioid receptors, is supported by the finding that naloxone binding to hypothalamic tissue decreases during the afternoon of the steroid-induced LH surge w15x, as does the number of hypothalamic m-opioid receptors on the afternoon of proestrus w21x. k-Opioid receptors w23x, as well as LH-releasing hormone ŽLHRH. cell bodies w38x, are located in the medial preoptic area ŽMPOA., a site known to play an essential role in generating the neural signal that triggers the LH
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surge w9x. Our laboratory recently demonstrated that blockade of k-opioid receptors in the MPOA during midpregnancy with nor-binaltorphimine Žnor-BNI., a specific kopioid receptor antagonist, produced a large increase in LH release w41,43x. As a follow-up to experiments in the pregnant rat, and taking into consideration the ‘disinhibition’ hypothesis applied to the preovulatory LH surge, in the present study we examined whether antagonism of k-opioid receptors in the MPOA prior to the critical period on the afternoon of proestrus could prematurely evoke an ovulatory LH surge. The preovulatory LH surge depends on the functional integrity of central noradrenergic neurons. Inhibition of norepinephrine ŽNE. synthesis or blockade of a-adrenergic receptors prevents the ovulatory surge of LH Žfor review, see w18x.. In addition, k-opioid receptor activation has been shown to inhibit NE release in the MPOA of male rats w14x and in the guinea pig cortex w34,35x. Thus if blockade of k-opioid receptors in the MPOA were found to prematurely elicit an ovulatory LH surge, the next objective was to determine whether NE was involved in mediating this LH response.
2. Materials and methods 2.1. Animals Adult female rats in our colony, derived from Charles River Sprague-Dawley CD rats, and weighing 270–320 g at the time of cannula implantation, were maintained on a 14:10 h lightrdark schedule Žlights on at 05.00 h., and fed rat chow and water. Estrous cyclicity was determined by daily examination of vaginal smears. The experimental procedures 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 ŽPlastics One, Roanoke, VA. was stereotaxically implanted into the MPOA Žcoordinates: 7.8 mm anterior to the interaural line, 0.8 mm lateral to the midline, and 6.8 mm below the surface of the brain., according to the atlas of deGroot w7x. The cannula was cemented in place, and a removable stylette Ž26 gauge. was inserted so that its tip was flush with the tip of the guide cannula. Cannula implantation on the day of estrus resulted in minimal disruption of the estrous cycle. Daily vaginal smears were continued and animals displaying at least two consecutive 4-day estrous cycles were used in the following experiments. The inner stylette was removed 4–7 days before the day of push–pull perfusion, and a new stylette was inserted so that its tip extended 1 mm beyond the tip of the guide cannula.
2.3. Experimental protocols Between 08.00 and 10.00 h on the day before the experiment Ždiestrous day 2., rats were anesthetized briefly with ether, and a polyethylene ŽPE50. cannula was inserted into or near the right atrium via the external jugular vein. The next day rats having nucleated epithelial smears characteristic of proestrus were used in experiments. The technique of push–pull perfusion was similar to that previously used in our laboratory w41–43x. On the morning of the experiment, the flow rates of the push and pull peristaltic pumps ŽPharmacia, Piscataway, NJ. were carefully balanced and set to deliver 10 mlrmin. This was accomplished by setting the push pump to deliver 10 mlrmin, and then adjusting the flow rate of the pull pump until a drop of fluid at the tip of the push–pull cannula system remained unchanged in size for 1 h. 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 tube kept on ice and containing 12 ml 0.25 N perchloric acid to adjust the perfusate pH to 3.0 in Experiment 2, in which perfusate NE levels were measured. Constancy of flow rates during the perfusion was verified gravimetrically by weighing the perfusate. The following experiments were done. 2.3.1. Experiment 1 The objective of this experiment was to determine whether antagonism of k-opioid receptors in the MPOA can prematurely evoke an ovulatory LH surge prior to the critical period on the afternoon of proestrus. The critical period is the time during which a neural stimulus normally triggers the LH surge on proestrus. Push–pull perfusion of the MPOA with either artificial cerebrospinal fluid ŽCSF; NaCl 140 mM, KCl 4 mM, CaCl 2 2.3 mM, MgSO4 1 mM, Na 2 HPO4 1.2 mM, NaH 2 PO4 0.3 mM, glucose 3.4 mM, pH 7.4. or CSF containing nor-binaltorphimine Žnor-BNI., a selective k-opioid receptor antagonist w27,33x, Ž40 mgrh, Research Biochemicals, Natick, MA. was done in rats between 10.30 and 13.50 h on proestrus. This dose has been shown to produce a large increase in LH release when applied to the MPOA during midpregnancy w41,43x. During the perfusion period, blood and perfusate samples were collected every 20 min, the latter for measurement of NE levels. This was done to determine the relationship, if any, between NE release and the nor-BNI-induced LH surge as part of Expt. 2. At 13.55 h, prior to the onset of the critical period, the animals were injected with pentobarbital Ž3.5 mgr100 g. b.wt., i.p.. to block the normal afternoon LH surge w10x. This was done in order to determine whether any resulting ovulation was due to the nor-BNI-induced increase in LH release. Additional blood samples were taken every 30–60 min between 14.00 and
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17.00 h on proestrus. At the end of the experiment, rats were returned to the animal quarters overnight. The following morning Žestrus. the ovaries were removed, the oviducts were separated from the ovaries, and ovulation was verified by counting the ova with the aid of a low power microscope. In another group of rats, push–pull perfusion of the MPOA with CSF was done between 14.00 and 17.00 h. Blood and perfusate samples were taken at 20 min intervals during this time to determine the onset of the preovulatory LH surge in our colony of rats as part of Expt. 1, and to relate any changes in NE release in the MPOA to the time of onset of the normal afternoon LH surge as part of Expt. 2. At 17.00 h rats were returned to the animal quarters overnight. Ovulation was checked the next morning. 2.3.2. Experiment 2 To examine a possible role for NE in the nor-BNI-induced LH increase, the effects of NE synthesis inhibition or a-adrenergic receptor blockade on the LH response to nor-BNI were examined. Push–pull perfusion of the MPOA with CSF containing nor-BNI Ž40 mgrh. was done between 10.30 and 13.50 h on proestrus in rats pretreated with either Ž1. bisŽ4-methyl-1-homopiperazinylthiocarbonyl. disulfide ŽFLA-63, a dopamine-b-hydroxylase inhibitor w6x, ICN Biomedicals, Irvine, CA.; Ž2. phenoxybenzamine ŽPBZ., an a-adrenergic receptor blocker w25x, ŽResearch Biochemicals.; or Ž3. their respective vehicles. FLA-63 was dissolved in 26% EtOH and was given s.c. 2.5–3 h prior to the onset of perfusion at a dose of 25 mgrkg. Our laboratory has previously shown that this dose produces a 72% decrease in hypothalamic-MPOA NE levels, and prevents the naloxone-induced increase in LH secretion w22x as well as the large increase in LH release occurring in response to blockade of MPOA k-opioid receptors during midpregnancy w43x. PBZ was dissolved in acid saline ŽpH 5.0., and was given i.p. 1 h prior to the onset of perfusion at a dose of 20 mgrkg. This dose of PBZ has been shown to suppress pulsatile LH secretion in diestrous rats w12x, as well as prevent the steroid-induced LH surge w8x. During the perfusion period, blood samples were collected at 20-min intervals. Animals were injected with pentobarbital at 13.55 h. Additional blood samples were taken every 30–60 min between 14.00 and 17.00 h on proestrus and the animals were then returned to their quarters. Ovulation was checked the next morning. PBZ was also given to another group of rats at 13.00 h on proestrus to examine the role of NE in the normal afternoon LH surge. Blood samples were taken at 30 min intervals from 14.00 to 17.00 h, and ovulation was checked the following morning. Animals in all experiments were injected with 400 U heparin before blood samples Ž150 ml each. were collected continuously through a peristaltic pump every 20 min, or withdrawn with a syringe at 30–60-min intervals. At the
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end of each experiment blood samples were centrifuged, and the plasma was separated and stored at y708C until assayed for LH by radioimmunoassay. In addition, the animals were anesthetized with pentobarbital and their brains were fixed in 10% formalin q1% CaCl 2 . Frozen 50-mm serial sections were stained with a Nissl stain using basic fuchsin, and the location of the cannula tip was determined. 2.4. Chromatography The HPLC system used a Coulochem II dual electrode electrochemical detector ŽESA, Bedford, MA. in conjunction with an ESA model 5014 analytical cell and a Hypersil column Ž150 = 3 mm, 3 mm; Keystone Scientific, Bellefonte, PA.. The degassed and filtered mobile phase Ž75 mM NaH 2 PO4 P H 2 O, 1.7 mM 1-octane sulfonic acid, 20 mM EDTA, 100 mlrl triethylamine, 1.5–3% acetonitrile, pH adjusted to 3.0 with H 3 PO4 . was delivered by an ESA model 420 pump at a flow rate of 0.5 mlrmin. Detection of NE was achieved by sequential oxidation:oxidation of the sample. Guard cell potential was 350 mV, and analytical cell potentials were y170 mV Ždetector 1. and 200 mV Ždetector 2.. 20–30-ml samples were kept at 48C and injected by an ESA model 460 autosampler. Chromatograms were analyzed by a Hewlett-Packard model 3390A reporting integrator. NE standard was obtained from Sigma ŽSt. Louis, MO. and was prepared in CSF with pH adjusted to 3.0 with perchloric acid. Assay sensitivity was 1 pg NE. The inter- and intraassay variations for 10 pg NEr20–30 ml Ž n s 15. were 6% and 2.5%, respectively. 2.5. Radioimmunoassay Plasma samples were analyzed for LH by the ovine:ovine rat LH double antibody radioimmunoassay of Niswender et al. w26x, as previously described w11x. The sensitivity of the assay was 5–10 pgrtube. Inter- and intraassay variations determined at a mean plasma LH level of 2.21 " 0.09 ngrml Ž n s 11. were 13.8% and 13.2%, respectively. LH values Žngrml plasma. were expressed in terms of the NIDDK rat LH-RP-2 standard. 2.6. Data analysis Mean plasma LH or perfusate NE levels for each experimental group were determined by first calculating the mean of all the blood or perfusate samples collected from each rat during the perfusion period, and then determining the average plasma LH or perfusate NE level for the experimental group from the individual means. Significant differences between groups in mean plasma LH levels were determined by Student’s unpaired t-test in Expt. 1, and analysis of variance followed by Dunnett’s t-test in Expt. 2. Differences between groups in the percent occur-
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rence of the large increase in LH release were determined by Fisher’s exact probability test. Differences between groups in perfusate NE levels were determined by analysis of variance followed by Duncan’s multiple range test. Results were considered significant at P - 0.05. All results are expressed as the mean " S.E.M.
3. Results 3.1. Experiment 1 In 5 rats perfused in the MPOA between 10.30 and 13.50 h with CSF alone, LH release was basal during this time, and plasma LH levels remained low between 14.00 and 17.00 h. All 5 rats failed to ovulate the next morning, demonstrating that the injection of pentobarbital they received at 13.55 h was completely effective in preventing the normal LH surge. In contrast, push–pull perfusion of the MPOA with nor-BNI produced a surge increase in LH release in 7 of 10 rats Ž70%; P - 0.02 vs. 0% in rats perfused with CSF alone.. This LH response began between 12.30 and 13.30 h, or about 2–3 h after the onset of nor-BNI perfusion. Five of these 7 rats ovulated the next morning with an average of 16.0 " 0.9 ova. Mean plasma LH levels during nor-BNI perfusion Ž3.71 " 1.07 ngrml.
Fig. 2. Mean plasma LH concentrations Ž`; ns8. and MPOA perfusate NE levels Ž^; ns 7. in rats from our normal colony perfused in the MPOA with CSF between 14.00 and 17.00 h on the afternoon of proestrus. Standard error bars are not shown when smaller than the corresponding symbols.
were significantly greater than those in CSF-perfused rats Ž0.58 " 0.06 ngrml; P - 0.05.. In the 7 rats with norBNI-induced increases in LH secretion, peak plasma LH levels of at least 20.6 " 6.3 ngrml were reached before the end of the perfusion period. Following pentobarbital administration at 13.55 h, in these 7 rats plasma LH levels gradually declined between 14.00 and 17.00 h, and in the 3 rats with no nor-BNI-induced LH increase plasma LH
Fig. 1. Representative examples of LH secretion in rats during push–pull perfusion of the MPOA with CSF, or CSF containing nor-BNI, on the morning of proestrus. The filled circle indicates a plasma LH value beyond the linear portion of the LH standard curve. Note the change in scale for plasma LH concentrations in the nor-BNI examples.
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levels remained low during this 3-h period Ždata not shown.. Representative examples of LH responses during MPOA perfusion with CSF or nor-BNI are given in Fig. 1. Plasma LH values in 8 rats in our normal colony bled between 14.00 and 17.00 h on the afternoon of proestrus are given in Fig. 2. Surges of LH release generally began between 15.30 and 16.30 h, and these rats ovulated with an average of 14.0 " 0.4 ova. Thus the nor-BNI-induced LH increase represented a 3 h advancement in the time of the LH surge. 3.2. Experiment 2 In animals pretreated with vehicle alone, blockade of MPOA k-opioid receptors with nor-BNI elicited a surge increase in LH release in 83% Ž5 of 6. of the rats. These 5 rats ovulated the next morning with an average of 14.0 "
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1.5 ova. In contrast, only 3 of 8 FLA-63-pretreated rats displayed a large nor-BNI-induced increase in LH release and ovulation, with an average of 9.0 " 2.0 ova. The percentage of FLA-63-pretreated rats demonstrating the large increase in LH release was reduced Ž38% vs. 83%., but this difference was not statistically significant. However, mean plasma LH levels in response to nor-BNI in FLA-63-pretreated animals Ž1.93 " 0.44 ngrml. were significantly less than those in animals pretreated with vehicle alone Ž3.88 " 0.87 ngrml; P - 0.05.. In rats pretreated with PBZ, the nor-BNI-induced increase in plasma LH levels was prevented; none of the 4 rats perfused with nor-BNI showed an increase in LH release Ž0%; P - 0.03 vs. 83% in rats perfused with nor-BNI and pretreated with vehicle., and none ovulated. In addition, mean plasma LH levels in PBZ pretreated animals in response to nor-BNI Ž0.37 " 0.03 ngrml. were also significantly lower than
Fig. 3. Representative examples of LH secretion during push–pull perfusion of the MPOA with nor-BNI on the morning of proestrus in rats pretreated with vehicle Žtop., FLA-63 Žmiddle., or PBZ Žbottom.. The filled circles indicate plasma LH values beyond the linear portion of the LH standard curve. Note the change in scale for plasma LH concentrations in one of the two vehicleq nor-BNI examples.
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those in animals pretreated with vehicle alone Ž3.88 " 0.87 ngrml; P - 0.01.. Following pentobarbital administration at 13.55 h in Expt. 2, plasma LH levels between 14.00 and 17.00 h either gradually declined if LH secretion had increased in response to nor-BNI between 10.30 and 13.50 h, or remained low during this time if no nor-BNI-induced LH surge had occurred Ždata not shown.. Representative examples of LH responses during MPOA perfusion with nor-BNI in rats pretreated with vehicle, FLA-63, or PBZ are given in Fig. 3. PBZ also prevented the normal afternoon LH surge and ovulation from occurring in all 4 rats tested in our normal colony. As previously indicated in Expt. 1, MPOA perfusion with nor-BNI produced a surge increase in LH release in 7 animals. Although NE release in the MPOA was monitored in only 3 of these rats showing large increases in LH release Žmean s 5.67 " 1.89 ngrml., mean perfusate NE levels in these animals Ž20.4 " 4.5 pgr20 min. were not different from those found in 5 rats perfused in the MPOA between 10.30 and 13.50 h with CSF alone Ž19.2 " 4.0 pgr20 min. and showing basal plasma LH levels Ž0.58 " 0.06 ngrml.. Perfusate NE levels did not increase in association with the nor-BNI-induced LH surge in any of the 3 rats. Representative examples of perfusate NE and plasma LH levels during MPOA perfusion with CSF or nor-BNI between 10.30 and 13.50 h are shown in Fig. 4 Žtop.. Moreover, in 7 of the 8 rats perfused in the MPOA with CSF between 14.00 and 17.00 h on proestrus and demonstrating a normal afternoon LH surge, NE release in the MPOA was also monitored. Plasma LH levels and MPOA perfusate NE values between 14.00 and 17.00 h on proestrus for these animals are given in Fig. 2, and a representative example is shown in Fig. 4 Žbottom.. None
Fig. 5. Sagittal Žtop. and tranverse Žbottom. sections through the rat brain indicating the location of push–pull cannula tips Žshaded areas. in the MPOA where perfusion with nor-BNI increased LH secretion. The arrows indicate the level of the tranverse sections. Sections are labelled according to the atlas of deGroot w7x. AC, anterior commissure; AHA, anterior hypothalamic area; DBB, diagonal band of Broca; DMH, dorsomedial hypothalamic nucleus; FX, fornix; OC, optic chiasm; POA, preoptic area; SC, suprachiasmatic nucleus; V, third ventricle; VMH, ventromedial hypothalamic nucleus.
Fig. 4. Representative examples of plasma LH concentrations Ž`. and MPOA perfusate NE levels Ž^. during push–pull perfusion of the MPOA with either CSF Žleft. or CSF containing nor-BNI Žright. prior to the critical period on the afternoon of proestrus Žtop., or CSF during the normal afternoon LH surge Žbottom.. Filled circles indicate plasma LH values beyond the linear portion of the LH standard curve. Note the change in scale for plasma LH concentrations during the nor-BNI-induced or afternoon LH surges.
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of the 7 rats showed an increase in perfusate NE values in association with the rise in plasma LH levels, and mean perfusate NE levels Ž16.2 " 1.3 pgr20 min. were similar to those in rats perfused with CSF or nor-BNI between 10.30 and 13.50 h on proestrus. 3.3. Histology The locations of push–pull cannula tips where perfusion with nor-BNI resulted in large increases in LH release are summarized in Fig. 5. All locations were within the MPOA, in the same region where we have previously shown perfusion with nor-BNI produces a large increase in LH release during midpregnancy in the rat w41,43x. Furthermore, no difference was observed in the location of cannula tips in rats that did or did not exhibit a large increase in LH secretion in response to nor-BNI.
4. Discussion The present results demonstrate that selective blockade of MPOA k-opioid receptors prior to the critical period on the afternoon of proestrus can prematurely evoke an ovulatory LH surge. These findings are consistent with the hypothesis that a significant decrease Ždisinhibition. in existing inhibitory opioid tone on the afternoon of proestrus is a key event underlying generation of the LH surge Žfor review, see w16x.. Systemic administration of the general opioid receptor antagonist naloxone w1,2x, or selective blockade of MPOA k-opioid receptors Žpresent data., are each able to advance the time of onset of the LH surge on proestrus. The evidence reviewed by Kalra w16x supported a role for b-endorphin, originating in arcuate nucleus neurons, and acting via m-opioid receptors in this inhibition of proestrous LH secretion. The present data extend this hypothesis by demonstrating that k-opioid receptors in the MPOA may also be important in regulating the timing of the LH surge. Interestingly, we have previously shown that antagonism of k-opioid receptors in the MPOA can produce a large increase in LH release during mid-pregnancy w41,43x. Thus our work indicates that this k-opioid receptor-mediated restraint on LH secretion is expressed in different reproductive states of the rat, i.e., midpregnancy and the morning of proestrus, and may serve to prevent an inappropriate large increase in LH release from taking place at these times. The present data also demonstrate that NE is involved in mediating the premature LH surge occurring in response to blockade of MPOA k-opioid receptors on proestrus. Pretreatment with the NE synthesis inhibitor FLA-63 partially reduced the occurrence of the nor-BNI-induced LH surge, and blockade of a-adrenergic receptors with PBZ completely prevented its occurrence. Furthermore, our results show that the importance of NE in this k-opioid
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receptor-mediated mechanism is not expressed as an increased release of this neurotransmitter at the site of perfusion. These results are in agreement with those of our previous study in which we found that NE was essential for the large increase in LH secretion occurring in response to blockade of MPOA k-opioid receptors during midpregnancy w43x, but that this LH response was not associated with increased NE release at the site of perfusion. One possibility is that NE released in the MPOA may be simply permissive to the induction of the nor-BNI-induced LH surge on proestrus, and only the presence of basal NE release is necessary. Alternatively, blockade of MPOA k-opioid receptors may activate neuronal pathways capable of triggering NE release outside the site of perfusion, an increase capable of stimulating LHRH release and the nor-BNI-induced LH surge. Interestingly, another study reported an increase in LH secretion, but no change in NE release in the MPOA, in response to the local or systemic administration of naloxone in the castrated or intact male rat w39x. In addition to being essential to the nor-BNI-induced LH surge, our results also indicate that NE is important for generation of the normal afternoon LH surge, since PBZ completely prevented its occurrence. This observation is consistent with data from other laboratories demonstrating a role for NE in the LH surge Žfor review, see w18x.. An increased NE turnover rate in the MPOA has been reported to occur in association with the proestrous LH surge w28x, or the steroid-induced LH surge in ovariectomized rats w36x. This increased turnover rate is an indirect estimate of increased noradrenergic neuronal activity. Using push–pull perfusion to directly assess NE release in the MPOA, Mohankumar et al. w24x demonstrated increased MPOA perfusate NE levels in conjunction with the proestrous LH surge. However, we observed no change in perfusate NE levels in the MPOA occurring prior to or during the LH surge normally seen on the afternoon of proestrus. Although NE reuptake is an effective means of removal of NE from the synaptic cleft w19,29,32x, this cannot explain the lack of increase in perfusate NE levels in our report, since neither their study nor our own used desipramine, an inhibitor of NE reuptake, in the perfusion solution. While the reasonŽs. for the difference between this latter result and ours is not apparent, in our study measurement of NE release in the MPOA was done in the same animals in which plasma LH levels were determined. Nevertheless, the present results are in agreement with literature demonstrating that NE is clearly involved in generation of the normal afternoon LH surge. Although previous reports have implicated a role for b-endorphin in opioid inhibition of the LH surge w3,30,37x, the inference from our nor-BNI data is that dynorphin is the neuropeptide ligand active at the MPOA. Dynorphin rather than b-endorphin is the endogenous opioid with a high binding affinity for brain k-opioid receptors w4,5x. The terminals of dynorphin-containing fibers w13x and high
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concentrations of dynorphin w40x, as well as k-opioid receptors w23x, are found in the MPOA. With respect to the regulation of LH secretion, intraventricular injections of dynorphin suppress LH secretion in gonadectomized rats w17,20x, and antisera to dynorphin injected into the medial basal hypothalamus increased LH release in immature 12-day-old female rats w31x. Further studies are planned to determine if dynorphin is the endogenous ligand active at k-opioid receptors in the MPOA in mediating suppression of the LH surge. In summary, these results demonstrate that blockade of k-opioid receptors at the level of the MPOA can prematurely evoke an ovulatory LH surge. Furthermore, the nor-BNI-induced as well as the normal afternoon LH surges are dependent on the proper functioning of central noradrenergic neurons, but do not involve increased NE release within the MPOA.
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Acknowledgements
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We would like to thank Kevin Shaw, Cassandra Soder and Josh Farber for their technical assistance, Dr. G.D. Niswender for antiovine LHa15, Dr. H. Papkoff for ovine LH for iodination, and Dr. A.F. Parlow and the NIDDK for the rat LH used as a reference preparation. This study was supported by NIH Grant HD-17728.
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