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Departments of Neuroscience (S.P., S.P.K.) and Physiology (P.S.K.) and University of Florida Brain Institute, University of Florida College of Medicine, Gainesville, Florida 32610; and the Department of Obstetrics and Gynecology, Yale University School of Medicine (T.L.H. S.D., F.N.), New Haven, Connecticut 06510
Address all correspondence and requests for reprints to: Dr. Satya P. Kalra, Department of Neuroscience, University of Florida College of Medicine, P.O. Box 100244, Gainesville, Florida 32610-0244. E-mail: SKALRA{at}neocortex.health.ufl.edu
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Introduction |
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Opioid peptides, especially those released from the projections of ß-endorphin (ßEND)-producing neurons located in the arcuate nucleus of the hypothalamus, have been shown to exert a tonic inhibitory control on GnRH secretion in rats (1, 2, 18, 19, 20, 21). Thus, administration of naloxone (NAL), an opiate receptor antagonist, rapidly stimulates the release of hypothalamic GnRH and pituitary LH in intact rats (1, 2, 22, 23, 24, 25). If the assumption that GnRH secretion is regulated by a dual control comprised of excitatory and inhibitory neural systems is correct, and the glutaminergic neurons represent an excitatory component (10, 11, 15), it appeared likely that the opioidergic neurons may constitute a significant component of the network engaged in restraining NO efflux in the hypothalamus. Therefore, we explored the existence of communication between opioids and NO in two ways. In the first series of experiments, we evaluated the effects of opioid receptor blockade with NAL on NO efflux in the medial preoptic area (MPOA) where NO- and GnRH-producing neurons are coextensive (3, 4, 5). NO efflux was assessed by analyzing extracellular cGMP, which parallels the increase in NOS activity in response to stimulations (12, 26) and has been shown by us and a number of other investigators to be a reliable index of NO output in vivo (reviewed in Refs. 12 and 27–29). Basal and NAL-induced cGMP effluxes were measured by microdialysis in freely moving, conscious rats (27). In the second series of experiments, we determined the morphological relationship between ßEND- and NOS-containing neurons by employing correlated light and electron microscopic, double immunostaining.
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Materials and Methods |
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Effect of naloxone on NO efflux: Intact and castrated rats (2 weeks postorchidectomy) were prepared for microdialysis as described previously (27). Briefly, rats were stereotaxically implanted with a guide cannula (CMA/12, CMA Microdialysis, Acton, MA) aimed at the MPOA (0.5 mm posterior to the bregma, 0.5 mm lateral from the midline, and 5.5 mm ventral from the dura) (27) under ketamine plus xylazine anesthesia (100 and 15 mg/kg, respectively, ip). After 48 h, a CMA/12 microdialysis probe with a 2-mm membrane length and a 20-kDa Mr cut-off was inserted into the guide cannula and perfused with artificial cerebrospinal fluid (127.6 mM NaCl, 2.5 mM KCl, 1.4 mM CaCl2, 1.0 mM MgSO4, and 12.0 mM phosphate buffer) at a rate of 5 µl/min. During dialysis the rats were unanesthetized and freely moving in their cages. Dialysates were collected on ice at 20-min intervals, lyophilized, and stored at -20 C until analysis of cGMP by RIA. The in vitro cGMP recovery rate, determined with known concentrations of cGMP and [3H]cGMP, was 10.25% at a flow rate of 5 µl/min. Saline (SAL; as vehicle control) or NAL (2 mg/kg; DuPont Pharmaceuticals, Wilmington, DE) was injected sc 100 min after initiation of microdialysis, and dialysate samples were collected for an additional 100 min. In an additional group of intact animals, morphine sulfate (45 mg/kg; Alkenes-Sinn, Cherry Hill, NJ) was injected sc immediately before NAL injection to verify the specificity of opioid receptor involvement. The doses were selected on the basis of previous reports indicating that this NAL dose produced optimal stimulation of LH in intact rats, which was blockable by morphine (45 mg/kg) (30). At the end of the experiment, rats were anesthetized with pentobarbital-Na (60 mg/kg) and transcardially perfused with 10% formol SAL. Brains were removed and processed for histological examination of the microdialysis probe placement as described previously (27). Only animals that showed correctly placed microdialysis probe in the MPOA were included in the analysis of results. Microdialysis probe placements outside the MPOA were found in two rats in the SAL plus SAL, one rat in the SAL plus NAL, and one rat in the morphine plus SAL treatment groups. cGMP efflux was not altered by these treatments in these rats.
Effect of NAL on LH secretion: Intact and castrated (2 week postorchidectomy) rats received either SAL or morphine sulfate (45 mg/kg), followed immediately by a sc injection of either SAL or NAL (2 mg/kg). Rats were killed 20 min later by decapitation, and trunk blood was collected and processed for the determination of plasma LH levels by RIA.
RIAs. The levels of cGMP in dialysates were measured with RIA kits (DuPont-New England Nuclear, Boston, MA). The acetylation protocol provided by the manufacturer was used to enhance the sensitivity of the RIA to 1 fmol at 89% binding. LH levels in plasma were determined by RIA kit provided by Dr. A. F. Parlow and the National Hormone and Pituitary Program, NIDDK. Iodinated LH was purchased from Corning Hazelton Laboratories (Vienna, VA). Concentrations of LH were expressed in terms of rLH RP-2. The assay sensitivity was 5 pg/tube at 90% binding.
Statistical analyses. One-factor ANOVA with repeated measures was used to compare the changes in cGMP levels over time, followed by Fisher’s protected least significant difference test for multiple group comparison to determine significant differences between posttreatment and pretreatment cGMP levels (average of first five samples before any treatment). Two-way ANOVA (time x treatment) was used to compare the cGMP levels between treatment and control groups. Statistically significant differences between the groups were determined by Fisher’s protected least significant difference at each time point. To facilitate comparison of responses to different treatments, the area under the curve (AUC) for fractions 6–10 was calculated, and the basal AUC (fractions 1–5) was subtracted from this value. Single factor ANOVA was used to determine significant differences in AUC and LH levels between the groups. The level of significance was set at P < 0.05.
Exp 2: morphological studies between NOS- and ßEND-immunoreactive
pathways
These studies were performed at the Department of Obstetrics and
Gynecology, Yale University School of Medicine (New Haven, CT). Normal
cycling, adult female Sprague-Dawley rats (Charles River, Wilmington,
MA; 200–250 g BW) were used in this experiment. Animals were kept
under standard laboratory conditions, with tap water and regular rat
chow ad libitum and a 12-h light/dark cycle.
Fixation. Rats were killed under ether anesthesia by transaortic perfusion with 60 ml heparinized SAL followed by 250 ml fixative. The fixative consisted of either 5% acrolein or 4% paraformaldehyde, 15% picric acid, and 0.2% glutaraldehyde in 0.1% phosphate buffer (PB), pH 7.4. The brains were dissected out, and 3-mm thick coronal blocks containing the hypothalamus were postfixed for an additional 1–2 h in glutaraldehyde-free fixative.
Tissue preparation and immunostaining. Tissue blocks were rinsed in several changes of PB, and 50-mm vibratome sections were prepared and rinsed four times for 15 min each time in PB. Sections for electron microscopy were transferred into vials containing 0.5 ml 10% sucrose (in PB) and rapidly frozen by immersing the vial in liquid nitrogen to enhance antibody penetration. They were then thawed to room temperature and repeatedly washed in PB. Subsequently, sections for both light and electron microscopy were treated with 1% sodium borohydride in PB for 10 min to eliminate unbound aldehydes from the tissue (31).
Light microscopic double immunostaining for ßEND and NOS was carried out according to the following protocol. Incubation in rabbit anti-ßEND (32) (1:5000 in PB containing 1% normal goat serum and 0.3% Triton X-100) for 38 h at 4 C was followed by several washes in PB; sections were incubated in the second antibody (biotinylated goat antirabbit IgG, 1:240 in PB; Vector Laboratories, Burlingame, CA) for 2 h at room temperature, then rinsed in PB three times for 10 min each time and incubated for 2 h at room temperature with avidin-biotin-peroxidase (1:250 in PB; ABC Elite Kit, Vector Laboratories) followed by a modified version of the nickel-diaminobenzidene (Ni-DAB) reaction (15 mg DAB, 0.12 mg glucose oxidase, 12 mg ammonium chloride, 600 ml 0.05 M nickel ammonium sulfate, and 600 ml 10% ß-D-glucose in 30 ml PB for 6–10 min at room temperature; dark blue reaction product) to visualize the tissue-bound peroxidase. After several rinses in PB, sections were further immunostained for NOS (33, 34). In this procedure, after a 48-h incubation at 4 C in rabbit antibrain NOS (brain) antiserum (Transduction Laboratories, Lexington, KY, catalogue no. N31030; 1:500 in PB containing 0.1% sodium azide), the sections were further processed using the peroxidase-antiperoxidase technique: incubation in the secondary antibody (goat antirabbit IgG; 1:50 in PB) for 2 h at room temperature, followed by rabbit peroxidase-antiperoxidase (1:100 in PB). Between each incubation step, sections were rinsed three times for 15 min each time in PB. The tissue-bound peroxidase was visualized by a light brown DAB reaction (25 mg DAB and 165 ml 0.3% H2O2 in 30 ml PB, 5–10 min at room temperature; brown reaction product). After immunostaining of the second tissue antigen, sections were thoroughly rinsed in PB, placed on gelatin-coated slides, dehydrated through increasing ethanol concentrations, and mounted with Permount (Fisher Scientific Co., FairLawn, NJ). In control double immunostaining experiments in which one of the primary antibodies was replaced with normal serum, only single immunostaining could be detected.
For electron microscopic analysis, sections were processed the same way as for light microscopy, except that the labeling of NOS was carried out using 5 nm gold-conjugated goat antirabbit IgG (1:10 in PB for 48 h at 4 C; Polysciences, Warrington, PA) as secondary antiserum. Subsequently, sections were postosmicated (1% OsO4 in PB) for 30 min, dehydrated through increasing ethanol concentrations (using 1% uranyl acetate in the 70% ethanol, 30 min), flat-embedded in Araldite between liquid release (Electron Microscopy Science, Ft. Washington, PA)-coated slides and coverslips, and placed in an oven to polymerize for 48 h at 60 C. The blocks were trimmed, and ribbons of ultrathin sections (Reichert-Jung ultramicrotome, Leica, Inc., Deerfield, IL) were collected on Formvar-coated single slot grids and examined using a Philips CM-10 electron microscope (Philips Electronics, Mahway, NJ).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a representative photomicrograph
illustrating placement of the microdialysis probe in the MPOA. The
results of experiments to examine a potential interaction of opioids
with cGMP/NO efflux in intact and castrated rats are summarized in
Figs. 2 and 3. The basal cGMP efflux from
the MPOA in the interval before treatments varied slightly (Fig. 2);
however, the differences among the three groups were not statistically
significant (P > 0.05). The cumulative basal rate of
cGMP efflux among these groups was also not statistically significant
(P > 0.05). As evident, blockade of opiate receptors
with NAL increased extracellular concentrations of cGMP in the MPOA of
intact rats (by one-factor ANOVA). The cGMP increments began
immediately after NAL injection and persisted over the course of the
experiment (Fig. 2A). Pretreatment with morphine, the µ opiate
receptor agonist, abolished the NAL-induced increments in the MPOA cGMP
response (by two-way ANOVA). This dose of NAL was also effective in
stimulating LH release, and morphine significantly blunted this
response (Fig. 2B). In contrast, NAL was completely ineffective in
raising plasma LH levels in castrated rats (Fig. 3B), and
interestingly, the rate of cGMP release was also not changed in
response to NAL (Fig. 3A).
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In contrast, NOS-immunopositive neuronal cell bodies were found in the
medial septum, diagonal band of Broca, MPOA, anterior periventricular
region, supraoptic and paraventricular nuclei (PVN; in both
parvocellular and magnocellular regions), and dorsomedial hypothalamic
nucleus (Fig. 4).
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). Both the anti-ßEND and anti-NOS antisera were generated in the rabbit, raising the possibility that cross-labeling of the antigens may have occurred during this study. However, labeling the ßEND profiles first with a dark blue-black Ni-DAB reaction assures that in the case of cross-reaction during the second staining for NOS, the light brown DAB product will only further darken the previously labeled ßEND profiles, but will not change the dark blue color to light brown, resulting in two distinct colorizations of ßEND and NOS.
Electron microscopy. Upon electron microscopic examination of
the double stained material, it was clear that the close apposition
represented classic synaptic connections between ßEND axon terminals
and NOS neurons in all the areas where light microscopy indicated the
presence of putative contacts between the two systems (Fig. 4). Both
axo-somatic and axo-dendritic synaptic connections between ßEND- and
NOS-immunopositive networks were observed, and the vast majority of
these synaptic membrane specializations were symmetrical (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This inference is of considerable significance in view of the previous findings that EAA enhance NO efflux through activation of NMDA receptors located on NOS-immunoreactive cells (10, 11, 14, 17). Seemingly, NOS-immunoreactive cells receive direct neuronal inputs from both an excitatory NMDA and an inhibitory ßEND pathway, and the interplay between the opposing actions of these messengers provides a temporal and quantitative precision to impart periodicities in GnRH secretion (1, 2).
This dual control of NO efflux may have important pathophysiological and physiological implications. It is highly likely that ßEND keeps in check the overshoot of the excitatory influence of EAA on NO efflux, thereby protecting target cells in the vicinity from NO toxicity (14, 15, 16). On the other hand, in physiological conditions that require neurohormone discharge for a long period of time, such as the preovulatory GnRH surge, a timely reduction in opioid restraint on NOS neurons may permit robust NO activation by EAA, resulting in extended GnRH secretion. In fact, an antecedent decrease in the inhibitory opioid tone (2, 20, 25) is crucial for increased activity in the excitatory neural pathways composed of EAA and peptidergic systems, such as neuropeptide Y (NPY), which activate GnRH hypersecretion (37, 38). More recently, we have observed that the rate of cGMP/NO efflux in the MPOA is augmented in association with the preovulatory and ovarian steroid-induced hypersecretion on NPY and GnRH (39). As NO can stimulate NPY release (40), we envision the following sequence of neural events associated with the induction of preovulatory GnRH surge. An initial restraint of ßEND’s inhibitory influence, perhaps induced by the timing mechanism (2), concomitant with an increase in NMDA receptor activation results in a robust and protracted cGMP/NO efflux, leading to neural events that promote NPY and GnRH hypersecretion.
NMDA, NO, and NPY elicit GnRH secretion from GT1–7 immortalized cells in vitro (8, 41, 42, 43), thereby implying that there is a potential for these neurotransmitters to act independently on GnRH neurons, possibly to elicit a synergistic response. However, the in vivo evidence showing that selective blockade of synthesis, release, or postsynaptic action of NPY abolished the GnRH surge (2, 44), argues in favor of the view that the NPY system is interposed between the NMDA receptor-containing subpopulation of NOS neurons and GnRH neurons. Thus, as alluded to earlier, initially a decrease in ßEND’s influence facilitates EAA-induced NO efflux, which leads to activation of the NPY to GnRH line of communication, culminating in the preovulatory GnRH and LH surges.
The morphological relationship between ßEND- and NOS-immunoreactive neurons was documented in female rats in the current study. There is no reason to believe that similar anatomical relationships may not exist in male rats, which showed increased MPOA cGMP efflux in response to NAL. The possibility that the NAL-induced cGMP response will be different in female rats is unlikely because we have not observed any difference in the basal pattern of MPOA cGMP efflux in male and cycling female rats (unpublished).
NAL is a general opiate receptor antagonist, with a predominant µ receptor subtype involvement in the central nervous system (1, 2, 36). Because morphine, the µ receptor agonist, completely abolished NAL-induced cGMP/NO efflux, and the effects of ßEND are mediated through µ receptor subtype, these observations imply that the µ receptor subtype probably mediates the opioid restraint on NOS-containing neurons, although not completely ruling out the role of other opiate receptor subtypes. In this study, we noted an interesting dichotomy of morphine antagonism of the NAL-induced cGMP/NO and LH responses. Whereas morphine completely counteracted the NAL stimulation of cGMP, it was partially effective in blocking the LH response. This suggests that NAL’s action in stimulating LH release under the current experimental conditions may involve other opiate receptor subtypes or nonopiate receptors not blockable by morphine (1, 36). This interesting dissociation of the effects of NAL on cGMP and LH responses needs further analysis.
It is important to remark upon the effects of gonadal steroids on basal and NAL-induced cGMP responses. Consistent with earlier studies (27), the basal average cGMP output before SAL injection in intact rats (4.99 ± 0.88 fmol/20 min) was significantly higher (P < 0.02) than that in castrated rats (1.97 ± 0.34 fmol/20 min). The inability of NAL to stimulate cGMP efflux and LH release in castrated rats is also quite obvious in our studies. In our recent study, NMDA was unable to stimulate cGMP efflux in the MPOA or LH release in steroid-deficient milieu (27). There are reports that gonadal steroids augment immunoreactive levels of NOS in hypothalamic sites, including the MPOA, and enhance levels of calcium-dependent NOS messenger RNA in other brain sites (45, 46). Taken together, these findings suggest that increased NOS abundance, at least in the MPOA of intact rats, may play a role in the augmented cGMP/NO efflux in response to NAL or NMDA, which, in turn, stimulates GnRH secretion in amounts sufficient to increase pituitary LH discharge.
These results have given us significant new information regarding the regulatory influence of ßEND and gonadal steroids on cGMP/NO release in the hypothalamus. It is possible now to propose dual regulation of NO release exercised by excitatory NMDA receptors and inhibitory µ opioid receptors directly at the level of NOS neurons. Because NO has been shown to play a key role in the regulation of GnRH secretion, this dual control has broad-based implications; it provides an extrinsic counterbalancing input on NO-producing neurons to possibly generate two modalities in GnRH secretion as well as to protect against neurotoxic effects of excessive NO released by glutamate receptor activation. The important task ahead is to understand the cellular and molecular events that mediate the restraint exercised by ßEND on NO release.
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Footnotes |
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Received October 24, 1996.
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