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Endocrinology Vol. 145, No. 11 5322-5331
Copyright © 2004 by The Endocrine Society

Septopreoptic µ Opioid Receptor Mediation of Hindbrain Glucoprivic Inhibition of Reproductive Neuroendocrine Function in the Female Rat

Sushma R. Singh and Karen P. Briski

Department of Basic Pharmaceutical Sciences, School of Pharmacy, College of Health Sciences, University of Louisiana, Monroe, Louisiana 71209

Address all correspondence and requests for reprints to: Dr. K. P. Briski, 356 Sugar Hall, School of Pharmacy, 580 University Avenue, Monroe, Louisiana 71209. E-mail: briski{at}ulm.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Central glucostasis is a critical monitored variable in neuroendocrine regulation of pituitary LH secretion. Glucoprivic signals originating within the caudal hindbrain suppress LH. Septopreoptic µ opioid receptors (µ-R) function within neural pathways maintaining basal LH levels and mediate the effects of diverse physiological stimuli on hormone release. To identify potential sites in the septopreoptic area where ligand neuromodulatory actions may occur in response to hindbrain glucoprivic signaling, the present studies evaluated the distribution of µ-R-immunoreactive (-ir) neurons in the septopreoptic area that are genomically activated in response to caudal fourth ventricular (CV4) delivery of the glucose antimetabolite, 5-thioglucose (5TG). The effects of lateral ventricular pretreatment with the selective µ-R antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), on LH secretory and GnRH neuronal transcriptional responses to hindbrain glucoprivation were also evaluated. Estradiol benzoate- and progesterone-primed, ovariectomized female rats were treated by CV4 administration of 5TG or the vehicle, saline, at the onset of the afternoon LH surge. The inhibitory effects of hindbrain glucoprivation on mean plasma LH levels as well as colabeling of rostral preoptic GnRH neurons for Fos-ir were attenuated in animals pretreated by lateral ventricular delivery of CTOP. Dual immunocytochemical labeling for septopreoptic µ-R-ir and Fos-ir demonstrated a robust induction of Fos expression by receptor-positive neurons within discrete septopreoptic sites in response to CV4 5TG, a genomic response that was diminished by CTOP pretreatment. The current studies provide novel evidence for the transcriptional activation of neuroanatomically characterized, µ-R-expressing neurons by decreased hindbrain glucose utilization and show that the functional status of µ-R is critical for maximal induction of the Fos stimulus-transcription cascade in these cells by central glucoprivic signaling. The finding that receptor antagonist-mediated suppression of this genomic response is correlated with increased reproductive neuroendocrine output supports a role for these discrete µ-R-expressing neuron populations as substrates for ligand regulatory effects on the GnRH-pituitary LH axis during neuroglucopenia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
METABOLIC DEFICITS INHIBIT mammalian reproduction (1). Reports that pituitary gonadotropin secretion is diminished by insulin-induced hypoglycemia or pharmacological glucoprivation emphasize the physiological impact of glucostasis on reproductive endocrine function (2, 3, 4, 5, 6). Evidence that plasma LH levels are decreased by intracerebroventricular (icv) administration of the glycolytic enzyme inhibitor, 2-deoxy-D-glucose (2DG) (5, 6, 7), are consistent with the idea of central monitoring of glucose availability and suggest that pathways governing LH are activated by decreased production of glycolytic intermediates and/or end products within glucose sensor structures accessible from the ventricular system. Current studies implicate the periventricular caudal brainstem as a source of glucoprivic stimuli regulating LH release (6, 7, 8) and provide novel evidence that lactate uptake and/or catabolism may be involved in local mechanisms of transduction of cellular substrate fuel imbalance into regulatory signaling that impacts reproductive neuroendocrine function (9).

Central opioid receptor function is required for glucoprivic suppression of pituitary LH secretion. Inhibition of pulsatile LH release in gonadectomized male rats by systemic 2DG injection is reversible by icv pretreatment with the nonselective opioid receptor antagonist, naltrexone (6). Central delivery of this antagonist is also reported to attenuate suppression of the LH surge and coincident immediate-early gene expression by GnRH neurons in steroid-primed, ovariectomized (OVX) female rats by systemic or icv antimetabolite administration (8). Although µ-, {kappa}-, and {delta} opioid receptor agonists inhibit LH release (10, 11), the specific receptor subtype(s) involved in glucoprivic suppression of reproductive neuroendocrine function is as yet unknown. The proopiomelanocortin peptide product and high affinity µ receptor (µ-R) ligand, ß-endorphin, tonically inhibits LH (12) via receptor-mediated actions at the level of GnRH neuronal perikarya and axon terminals within the adjoining septal, preoptic, and anterior hypothalamic areas and the medial-basal hypothalamus (MBH), respectively (13, 14, 15, 16, 17). Evidence that GnRH neurons do not express µ-R suggests that endorphinergic modulation of these cells is apparently accomplished via afferent input to these cells (18).

Recent studies have described the distribution of µ-R-immunoreactive neurons (µ-R-ir) within the contiguous septal and preoptic areas, e.g. septopreoptic area, and hypothalamus of the rat brain, reporting localization of antigenicity to the lateral septum (LS), medial septum (MS), bed nucleus of the stria terminalis (BST), medial (MPN) and median (MEPO) preoptic nuclei, anterior hypothalamic periventricular nucleus, and lateral hypothalamic area (19, 20, 21). The studies described below used the selective µ-R antagonist, CTOP, as a pharmacological tool to characterize the involvement of these receptors in central mechanisms underlying hindbrain glucoprivic inhibition of LH release and to investigate the role of individual, neuroanatomically defined µ-R populations in this metabolic regulatory process. Immunocytochemical methods were employed to evaluate the genomic responsiveness of discrete µ-R-ir neuron populations within the septopreoptic area of steroid-primed, OVX female rats to suppression of glucose utilization within the caudal hindbrain and to determine whether µ-R antagonist-mediated inhibition of this transcriptional activation is correlated with restored reproductive neuroendocrine function during central neuroglucopenia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult female Sprague Dawley rats (210–240 g body weight) were maintained under a 14-h light, 10-h dark schedule and allowed free access to standard laboratory rat chow and water. On d 1 of the experiment, the animals were bilaterally OVX under ketamine/xylazine anesthesia (0.1 ml/100 g body weight, ip; 100 mg ketamine and 10 mg xylazine/ml; Henry Schein, Inc., Port Washington, NY) and implanted with hand-held PE-20 cannulas aimed at the left lateral ventricular (LV; coordinates: 2.0 mm lateral to midline, –1.0 mm posterior to bregma, 5.0 mm ventral to skull surface) and caudal fourth ventricles (CV4; coordinates: 0 mm lateral to midline, –12.8 mm posterior to bregma, 6.6 mm ventral to skull surface). The animals were subsequently injected sc with estradiol benzoate (E; 10 µg/0.1 ml safflower oil) at 1000 h on d 14 and 17 and with progesterone (P; 2.0 mg/0.2 ml safflower oil) at 1100 h on d 18.

Experimental design
After P administration on d 18, E- and P-primed OVX rats were divided into three groups (n = 6/group). At 1350 h on the same day, groups of animals received LV injections of either 3.0 µl saline (groups I and II) or 10 µg CTOP (Sigma-Aldrich Corp., St. Louis, MO) in an equivalent volume (groups III and IV). At 1400 h, rats were injected with either 210 µg 5-thioglucose (5TG) in 3.0 µl saline (group II and IV) or saline alone (groups I and III) into the CV4. At 1600 h, the rats were transcardially perfused with 2.0% sodium nitrite in 0.9% saline, followed by 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.6, under deep pentobarbital anesthesia. Trunk blood was obtained by cardiac puncture immediately before perfusion, and the plasma stored for LH and glucose measurements. This time point was verified to coincide with the ascending phase of the steroid-induced LH surge by assessment of the onset and the peak of hormone release in separate groups of E- plus P-primed OVX animals decapitated at 1400, 1500, 1600, and 1700 h. At the conclusion of each perfusion, the brain was dissected out, postfixed overnight in fresh fixative, sunk in 30% sucrose, and cut into 20-µm serial sections on a sliding Reichart-Jung microtome. Sections were stored at –20 C in cryoprotectant containing 30% sucrose until processed as follows.

Dual-label immunocytochemistry
Separate populations of GnRH-ir-positive neurons located within the rostral preoptic area (rPO) at the level of the organum vasculosum of the lamina terminalis, MS, and anteroventral preoptic nucleus (AVP) were processed for colabeling for nuclear Fos-ir, as previously described (8). Sections collected at 160-µm intervals through these brain regions were stringently washed with 0.05 M Tris-buffered saline (TBS), pH 7.6, and incubated with TBS containing 3% H2O2 to quench endogenous peroxidase activity. After preincubation with normal goat serum (Vector Laboratories, Inc., Burlingame, CA) for 30 min at room temperature, the tissues were incubated with a rabbit polyclonal antiserum against human Fos4–17 (Ab-5, 1:100,000; Oncogene Research Products, Cambridge, MA) diluted in TBS with 0.05% Triton X-100, for 48 h at 4 C. After sequential 60-min incubations with biotinylated goat antirabbit secondary antibody and avidin-biotin-peroxidase complex reagent (Vector Elite rabbit ABC kit, Vector Laboratories, Inc.), nuclear antigenic sites were visualized by processing with filtered Sigma Fast cobalt chloride-intensified 3,3'-diaminobenzidine tetrahydrochloride tablet sets (Sigma-Aldrich Corp.). After preincubation in normal goat serum (Vector Laboratories, Inc.) for 30 min, the sections were incubated with a rabbit polyclonal antiserum against GnRH (1:25,000; Dr. R. Benoit, Québec, Canada) for 48 h at 4 C. Immunofluorescence demonstration of cytoplasmic GnRH-ir was accomplished by incubation with AlexaFluor 568 goat antirabbit antiserum (A-11036, Molecular Probes, Inc., Eugene, OR) for 2 h. For each animal, an additional one in eight series of sections collected through the rostrocaudal extent of the septopreoptic area was processed for dual localization of nuclear Fos- and µ-R-ir by avidin-biotin immunoperoxidase staining for Fos, as described above, followed by indirect immunofluorescence labeling of µ-R using a rabbit polyclonal primary antiserum (PC165L, 1:3000; Oncogene Research Products) and AlexaFluor 568 goat antirabbit secondary antibodies. After mounting on gelatin-covered glass slides, the sections were dried and coverslipped with Slowfade Antifade kit reagents (Molecular Probes). Immunostaining controls included substitution of primary antisera with nonimmune sera and elimination of biotinylated or fluorochrome-labeled secondary reagents or ABC reagent from the protocol. Digital images were captured with a Nikon E400 upright microscope (Nikon, Melville, NY) equipped with epifluorescence accessories and a CoolSNAP-cf monochrome digital camera (Photometrics, Tucson, AZ) and were processed with Metamorph imaging software (Downingtown, PA) (Table 1Go).


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  		TABLE 1. Coordinates of neuroanatomical structures evaluated and approximate number of sections examined per structure

 
LH RIA
Plasma LH was measured using kit reagents provided by the National Hormone and Pituitary Agency, as previously described (22). The intra- and interassay coefficients of variation of this assay are 6% and 11%, respectively. Hormone concentrations were expressed as nanograms per milliliter in terms of the reference preparation, rLH-RP-3.

Blood glucose measurements
Plasma glucose levels were measured using Glucose (Trinder) Assay Kit reagents according to the manufacturer’s instructions (Sigma-Aldrich Corp.).

Statistics
Mean values for plasma glucose or LH levels were analyzed by one-way ANOVA, followed by Duncan’s multiple range test. For each brain site evaluated, mean cell counts of GnRH-ir-positive, GnRH- and Fos-ir-positive, µ-R-ir-positive, or µ-R-/Fos-ir-positive neurons were each analyzed by ANOVA, followed by Duncan’s multiple range test. Differences were considered significance at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of CV4 administration of 5TG on blood glucose and LH levels
As shown in Fig. 1Go, E- plus P-primed OVX rats treated with 5TG at 1400 h on the day of the induced LH surge exhibited a significant increase in mean blood glucose levels at 1600 h compared with non-5TG-treated controls. The data also indicate that this hyperglycemic response to CV4 antimetabolite delivery was reversed in animals pretreated at 1350 h by administration of CTOP into the LV. Blood glucose levels in the CTOP- plus 5TG-treated rats were significantly decreased vs. those in the vehicle- plus 5TG-treated group and were not statistically different from levels measured in the saline- plus saline-treated controls. Basal glucose values were not altered by administration of CTOP alone. The data in Fig. 2Go show that mean plasma LH concentrations in samples obtained at 1600 h during the ascending phase of the afternoon surge were significantly lower in animals treated at 1400 h with 5TG than in those injected with vehicle at that time. This inhibitory LH response to 5TG was attenuated by LV delivery of CTOP; circulating LH levels were significantly higher in animals pretreated with CTOP vs. vehicle before antimetabolite administration into the CV4. Plasma LH levels were not different between groups treated with saline plus saline vs. those treated with CTOP plus saline.



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  		FIG. 1. Effects of LV pretreatment with CTOP on blood glucose levels in E- plus P-primed OVX female rats after CV4 administration of the glucose antimetabolite, 5TG. At 1350 h, groups of rats received LV injections of either vehicle (groups 1 and II) or CTOP (groups III and IV), and at 1400 h, the animals were injected with vehicle (groups I and III) or 5TG (groups II and IV) into the CV4. Bars represent the mean glucose value ± SEM for six animals per group. *, P < 0.05 compared with the saline plus saline controls.

 


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  		FIG. 2. Suppression of E- plus P-induced LH release in OVX rats by 5TG; effects of CTOP pretreatment. Bars depict the mean plasma hormone level ± SEM at 1600 h for six animals per group. *, P < 0.05 compared with the saline plus saline controls; #, P < 0.05 compared with the saline plus 5TG group.

 
Effects of CV4 administration of 5TG on colabeling of GnRH-ir neurons for Fos
Immunolabeling of tissue sections obtained from E- plus P-primed OVX rats at 1600 h identified distinct populations of GnRH-ir neurons within the rPO, MS, and AVP; the mean numbers of these neurons in each site were not different among treatment groups. In Fig. 3Go, the matched dark- and brightfield images in E and F depict dual labeling for cytoplasmic GnRH- and nuclear Fos-ir, respectively, within the rPO of a non-5TG-injected rat, whereas the paired photomicrographs in G and H show that this colocalization was reduced by antimetabolite administration. The quantitative data in Fig. 4Go indicate that 5TG administration caused a significant decline in mean numbers of GnRH- plus Fos-ir-positive neurons in the rPO, but did not alter colabeling of GnRH neurons in either the MS or AVP. Animals pretreated with CTOP exhibited greater numbers of GnRH-ir- plus Fos-ir-positive neurons in the rPO vs. rats pretreated with vehicle only before 5-TG. Mean numbers of rPO neurons that were colabeled for GnRH- and Fos-ir were not different between animals treated with saline plus saline vs. CTOP plus saline. In the MS and AVP, the mean numbers of GnRH neurons that were colabeled for Fos-ir did not differ between treatment groups.



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  		FIG. 3. Effect of the glucose antimetabolite, 5TG, on gonadal steroid-positive feedback induction of Fos immunoexpression by septopreoptic GnRH-ir-positive neurons. A–D, Coronal maps of rat brain, adapted from Ref.35 , that depict the distribution of GnRH-ir neurons in the septopreoptic area of the rat brain. A and B, Distribution of GnRH neurons in the rPO, located lateral and dorsal to the organum vasculosum of the lamina terminalis (OVLT). C, Medial septum, dorsal to the anterior commissure (ACO) and rostral to the OVLT. D, AVP: GnRH-ir neurons located dorsolaterally to the optic chiasm. Maps are adapted and modified from the Swanson rat brain atlas (35 ). {blacksquare}, Two or three GnRH-ir neurons. The darkfield photomicrograph in E reveals immunofluorescence labeling for GnRH-ir within the rPO of a vehicle-injected, E- plus P-primed OVX rat, whereas the corresponding brightfield image of the same field of view (F) depicts Fos immunoexpression. Arrowheads indicate neurons that are colabeled for GnRH- and Fos-ir. G and H, Matched dark- and brightfield images of GnRH- and Fos-ir, respectively, in the rPO of an E- plus P-primed OVX rat injected with 5TG. Arrowheads indicate colabeling for GnRH- and Fos-ir; arrows depict non-Fos-ir-positive GnRH neurons. Note that only one GnRH-ir neuron visible in the field is colabeled for Fos. Scale bars, 50 µm.

 


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  		FIG. 4. Effects of CTOP pretreatment on glucose antimetabolite inhibition of Fos immunoexpression by GnRH-ir-positive neurons in the rPO, medial septum (MS), and AVP in E- plus P-primed, OVX female rats. Bars represent mean total cell count ± SEM for six animals per group. *, P < 0.05 compared with the saline plus saline controls; #, P < 0.05 compared with the saline- plus 5TG-treated group.

 
Effects of CV4 5TG on colabeling of septopreoptic µ-R-ir neurons for Fos-ir
Neuronal µ-R-ir was visualized by immunofluorescence labeling within the LS, MS, MEPO, MPN, and BST. The matched dark- and brightfield images in G and H of Fig. 5Go depict patterns of µ-R and Fos immunostaining in the LS of a non-5TG-treated, E- plus P-primed OVX rat. The paired images in I and J show that Fos immunoexpression by µ-R-containing neurons in this site was increased by administration of 5TG into the CV4. The data in Fig. 6Go indicate that mean numbers of neurons labeled for µ-R in each brain site were not modified by antimetabolite treatment. Dual-label immunochemistry showed that few µ-R-ir neurons were colabeled for Fos-ir in response to CV4 delivery of vehicle alone. However, injection of 5TG into that site resulted in robust Fos expression in µ-R-ir neurons in the LS, MS, and MEPO, significantly increasing the mean numbers of µ-R-expressing neurons that were colabeled for Fos-ir in these brain sites compared with those in the non-5TG-treated controls. In contrast, 5TG did not induce Fos immunoexpression by µ-R-ir neurons in BST and MPN. Pretreatment with CTOP significantly diminished colabeling of µ-R-ir neurons in LS, MS, and MEPO for Fos-ir compared with that in the vehicle- plus 5TG-treated group.



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  		FIG. 5. Effects of CTOP pretreatment on glucose antimetabolite suppression of Fos immunoexpression by septopreoptic µ-R-ir neurons. A–F, Coronal brain maps illustrating µ-R-ir neurons in the septopreoptic area. A, Lateral septum (LS), µ-R-ir neurons located adjacent to the LV in an arched distribution; B and C, µ-R-ir neurons located in the MEPO immediately dorsal to the third ventricle; D, medial septum (MS); E, medial preoptic nucleus; F, BST, ventral to the LV and dorsal to the anterior commissure (ACO). {blacksquare}, Two or three µ-R-ir neurons. G, Immunofluorescence-labeled µ-R-ir neurons located in the LS of a steroid-primed OVX rat treated by delivery of vehicle into the CV4; the brightfield image of this field of view in H shows that Fos labeling of these receptor-expressing neurons is minimal. I and J, Corresponding dark- and brightfield views of µ-R-ir and Fos-ir in the LS of a 5TG-treated, steroid-primed OVX rat, which show that colabeling of µ-R-expressing neurons in that structure is decreased by antimetabolite administration. Arrowheads indicate µ-R-/For-ir-positive neurons; arrows depict µ-R-ir neurons that are Fos-ir-negative. Scale bars, 50 µm.

 


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  		FIG. 6. Effects of CTOP pretreatment on 5TG-mediated inhibition of Fos immunoexpression by µ-R-immunopositive cells within discrete septopreoptic structures. The data represent the mean cell count ± SEM in the lateral septum (LS), medial septum (MS), BST, MPN, and MEPO for six animals per treatment group. *, P < 0.05 compared with the saline- plus saline-treated group; #, P < 0.05 compared with the vehicle- plus 5TG-treated animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies confirm and extend previous reports that the LH hormonal surge in female rats is attenuated by glucoprivic signals of hindbrain origin and provide new evidence that µ-R exist within the neural circuitry that regulates the GnRH-pituitary LH neuroendocrine axis during hindbrain glucodeprivation. Our immunocytochemical data show that glucose antimetabolite administration into CV4 elicits site-specific transcriptional activation of µ-R-immunopositive neuron populations within the rat septopreoptic area, a region of the brain characterized by the presence of µ-R that inhibit LH release. The current findings that this genomic response by neuroanatomically discrete cell groups is inhibited by pharmacological µ-R blockade and that this neuronal deactivation coincides with enhanced LH secretion in 5TG-treated animals suggest that these functionally characterized, µ-R-expressing neurons may be substrates for hindbrain glucoprivic regulation of reproductive neuroendocrine function.

There is consistent evidence that disruption of glucostasis within the periventricular hindbrain impairs reproductive hormone secretion. The present observations that CV4 delivery of 5TG diminishes mean LH release during the ascending afternoon phase of the estrogen- plus P-induced agree with our recent findings that fourth ventricular infusion of the oxidizable metabolic fuel, lactate, reverses the inhibitory effects of systemic glucoprivation on the proestrous LH surge in intact female rats (9). In light of evidence that glucose-sensitive neurons also occur within the lateral hypothalamic area and ventromedial hypothalamic nucleus (23), it is possible that the reproductive neuroendocrine axis may receive combined input on glucose availability from these neuroanatomically diverse sites. Indeed, intrahypothalamic administration of glucose is reported to attenuate insulin-induced hypoglycemic suppression of pulsatile LH release in OVX female rats (24). However, the robust reduction in plasma LH levels achieved here by glucose antimetabolite delivery into CV4 emphasizes the sensitivity of the steroid-positive, feedback-activated GnRH-pituitary LH axis to hindbrain glucopenia.

The present data show that estrogen plus P priming induced Fos expression by GnRH neurons in the rPO, MS, and AVP of the OVX rat brain, but this genomic response was attenuated only within the rPO as a result of 5TG treatment. These results extend previous observations of the unique responsiveness of this discrete subpopulation of GnRH to more widespread induction of neuroglucopenia within the brain (8). Taken together, these findings suggest that afferent signaling of central nervous system glucostasis may be restricted to GnRH neurons located in the rPO, or alternatively, that metabolic regulatory input to GnRH neurons in other brain sites is of insufficient magnitude to modulate steroidal transcriptional activation of those cells. The traditional view holds that positive feedback stimulation of the reproductive neuroendocrine axis does not involve direct action on GnRH neurons, but, rather, is mediated by a facilitatory cascade that is activated by estrogen-sensitive neurons located within the anteroventral periventricular nucleus (25, 26). However, recent studies provide molecular and immunocytochemical evidence for GnRH neuronal sensitivity to estrogen (27). An unresolved issue concerns whether hindbrain-activated, glucoprivic regulatory stimuli and estrogen-activated facilitative signals constitute independent input to rPO GnRH neurons, or if these pathways converge upstream of this cell population.

The µ opioid receptor antagonist, CTOP, is a cyclic octapeptide with high affinity (50% inhibitory concentration, 2.8 nM) for a single binding site in the rat brain and is exceptionally selective ({delta} vs. µ affinity ratio, 4829) (28). The present studies show that pretreatment by LV administration of CTOP significantly increased mean plasma LH levels as well as numbers of Fos-immunopositive rPO GnRH neurons in rats injected with 5TG compared with animals pretreated with vehicle alone, but did not fully restore either measure to levels detected in the non-5TG-treated controls. It exhibits minimal interaction with other opioidergic ({delta} and {kappa}) as well as nonopioidergic systems, including somatostatin, substance P, cholinergic, {alpha}- and ß-adrenergic, {gamma}-aminobutyric-ergic, and dopaminergic systems. The results reported here may indicate that µ-R-independent as well as µ-R-containing pathways convey hindbrain glucoprivic signals to the GnRH-LH axis or, alternatively, may reflect incomplete inhibition of all pertinent µ-R populations by the method for CTOP administration used here. Previous evidence from our laboratory that LV delivery of the nonselective opioid receptor antagonist, naltrexone, only partially reversed glucose antimetabolite suppression of gonadal steroid-induced LH release (8) supports the possibility that nonopiatergic mechanisms may be involved in this metabolic regulatory signaling.

As shown in Fig. 3Go, 5TG administration caused an overall increase in Fos immunostaining in the rPO. Because the present studies do not shed light on the neurochemical identity(ies) of GnRH-ir-negative neurons in the rPO that are labeled for Fos after icv glucose antimetabolite administration, we can only speculate on the potential regulatory influence of these cells on neighboring GnRH neurons. Because the rPO participates in the regulation of diverse physiological and behavioral functions, and neuroglucopenia elicits numerous compensatory responses in addition to suppression of the reproductive neuroendocrine axis, it is plausible that glucoprivic activation of non-GnRH neurons in this brain site may reflect in part activation of circuitries that have no direct impact on reproduction. Given the increasing availability of information on local neurochemicals that have demonstrable modulatory effects on reproductive neuroendocrine function, the present results emphasize the need for phenotypic characterization of these additional Fos-positive rPO neurons, information that could shed light on potential local regulatory interactions.

Despite evidence implicating central µ-R in neural mechanisms linking glucose substrate homeostasis to LH release, the precise neuroanatomical location(s) of those receptors that mediate glucoprivic inhibitory effects on gonadotropin secretion is not known. µ-R that function to regulate basal LH levels exist within the septopreoptic-anterior hypothalamic area and MBH, as determined by assessment of in vivo LH responses to agonist/antagonist delivery into specific areas of the rat brain or agonist effects on GnRH release from tissue fragments in vitro (13, 14, 15, 16, 17). Recent immunocytochemical mapping studies show that µ-R-ir is present within discrete loci in the septopreoptic-anterior hypothalamus, including the LS, MS, BST, MEPO, MPN, and lateral preoptic area, but is not demonstrable within the MBH (19, 20, 21). These discrepant outcomes of experimental strategies examining physiological vs. antigenic end points may reflect the presence of noncross-reactive receptor variants within the MBH, artifactual diffusion of in vivo administered compounds beyond the confines of the MBH, and/or involvement of non-µ-R mechanisms in reproductive neuroendocrine responses to in vivo or in vitro pharmacological manipulation of the MBH.

The current examination of the transcriptional status of discrete µ-R-ir neurons in the female rat septopreoptic area shows that hindbrain glucoprivation elicited colabeling of receptor-immunopositive neurons in the LS, MS, and MEPO for Fos, but that receptor-bearing neurons in the BST and MPN were Fos-ir-negative in the antimetabolite-treated animals. The finding that this genomic activation of these neuron populations is suppressed by icv administration of the selective µ-R antagonist, CTOP, suggests that receptor stimulation is a prerequisite for maximal induction of the Fos stimulus-transcription cascade within these cells. The coincident enhancement of LH release by this pharmacological manipulation supports the view that the functional state of these receptor-expressing neurons is critical for hindbrain glucoprivic inhibition of gonadotropin secretion. The present results show that µ-R-containing neurons in the LS, MS, and/or MEPO may be the source of direct (monosynaptic) or indirect (polysynaptic) signals that inhibit GnRH neurons. Our data provide a useful neuroanatomical map for future investigation of the neurochemical phenotype(s) that either functions as cellular substrates for endorphinergic stimulation or, alternatively, serves to link such cells with downstream GnRH neurons. Although the present results emphasize a role for discrete septopreoptic µ-R populations in the neural circuitry linking hindbrain glucose-sensitive neurons to the reproductive neuroendocrine axis, these data do not exclude the involvement of additional µ-R located within the MBH in this regulation. Additional studies aimed at examining the effects of CTOP administration within the individual septopreoptic loci identified here on hormone release in antimetabolite-treated animals will be necessary to evaluate the role of local µ-R in hindbrain glucoprivic regulation of LH.

Evidence for glucoprivic activation of hypothalamic structures of demonstrated significance for both energy homeostasis and reproductive endocrine function, e.g. hypothalamic paraventricular nucleus, lateral hypothalamic area, and arcuate nucleus, suggest that local neurons may govern multiple compensatory responses to this metabolic challenge. Data implicating µ-R in both glucoprivic hyperphagia (29, 30) and inhibition of the GnRH-pituitary LH axis suggest that arcuate nucleus proopiomelanocortin neurons may be substrates for integration of these and possibly other functions. Although systemic insulin or 2DG injections promote Fos expression by several hypothalamic neurochemical phenotypes that regulate reproductive neuroendocrine function, including orexin-, vasopressin-, melanin-concentrating hormone-, and NADPH-synthesizing neurons (31, 32, 33, 34), the present studies did not determine whether CV4 antimetabolite administration elicits this genomic response within those neural sites where these cell populations occur or colabeling of these neurons for Fos-ir. Ongoing studies in our laboratory aim to determine whether these phenotypic cell groups reside within pathways that mediate the effects of central glucoprivation on the GnRH-LH hormonal axis, and if those circuitries include µ-R.

Because neuronal genomic activation does not occur solely in response to induction of c-fos gene expression, our findings that BST and MPN µ-R-containing neurons are not colabeled for Fos after caudal ventricular 5TG administration do not preclude the possibility that hindbrain glucoprivation may elicit genomic responses of these neurons via activating protein-1-independent mechanisms, namely phosphorylated cAMP response element-binding protein-regulated gene activity involving the cAMP response element. Previous studies in our laboratory showed that ip glucose antimetabolite injection results in Fos expression by µ-R neurons in the male rat BST, and that this response is diminished by CTOP pretreatment (35). The present findings that these neurons are Fos-ir-negative in female rats treated by CV4 antimetabolite administration may reflect differential, e.g. more widespread, induction of inhibitory neural pathways by systemic vs. central glucopenia or, alternatively, gender disparities in the involvement of BST µ-R-expressing neurons in central mechanisms mediating the glucoprivic regulatory influence on reproductive neuroendocrine function.

Although the current studies show that pharmacological induction of neuroglucopenia within the periventricular caudal hindbrain suppresses reproductive neuroendocrine function, these data do not reveal the specific neuroanatomical site(s) of origin of this regulatory signaling. However, recent functional mapping of the hindbrain by site-specific glucose antimetabolite infusion indicates that rare structures in this area of the brain, including the periventricular nucleus tractus solitarius, activate neural pathways that compensate for glucoprivation (36). Corroborative evidence for the unique responsiveness of catecholaminergic neurons in the nucleus tractus solitarius and adjacent area postrema to glucopenia is provided by electrophysiological assessments of synaptic activity and immunocytochemical demonstration of immediate-early gene expression by neurons located at these sites (37, 38, 39, 40). Pharmacological evidence that {alpha}2-adrenergic receptor neurotransmission is required for fasting-induced inhibition of basal LH release in OVX, E-treated rats supports this view (41). The finding that insulin-induced hypoglycemic suppression of LH release is prevented by surgical removal of the area postrema suggests that glucose-sensing neurons and/or pathways mediating glucoprivic regulatory effects on gonadotropin secretion reside within this periventricular structure (42). Additional studies are warranted to elucidate the neuroanatomical and neurochemical mechanisms by which hindbrain catecholaminergic signaling of glucostasis is conveyed to µ-R-expressing neurons within the septopreoptic area.

In summary, the present studies show that hindbrain neuroglucopenia suppresses the LH hormonal surge in female rats via mechanisms that involve central µ-R and provide novel immunocytochemical evidence implicating neuroanatomically discrete, septopreoptic µ-R-containing neuron populations in the inhibitory metabolic regulatory circuitry that opposes steroid-positive feedback activation of the GnRH-pituitary LH axis. These findings provide new insight concerning the neurochemical basis and in situ sites of action underlying central glucoprivic regulation of reproductive neuroendocrine function.


   Footnotes
 
Abbreviations: AVP, Anteroventral preoptic nucleus; BST, bed nucleus of the stria terminalis; CTOP, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2; CV4, caudal fourth ventricular; 2DG, 2-deoxy-D-glucose; E, estradiol benzoate; icv, intracerebroventricular; -ir, immunoreactive, immunoreactivity; LS, lateral septum; LV, lateral ventricular; MBH, medial-basal hypothalamus; MEPO, median preoptic nucleus; MPN, preoptic nucleus; MS, medial septum; OVX, ovariectomized; P, progesterone; µ-R, opioid receptor; rPO, rostral preoptic area; TBS, Tris-buffered saline; 5TG, 5-thioglucose.

Received February 2, 2004.

Accepted for publication August 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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