Rat Medial Basal Hypothalamus Research Articles

Cadmium-Induced Disruption in 24-h Expression of Clock and Redox Enzyme Genes in Rat Medial Basal Hypothalamus: Prevention by Melatonin

In a previous study we reported that a low daily p.o. dose of cadmium (Cd) disrupted the circadian expression of clock and redox enzyme genes in rat medial basal hypothalamus (MBH). To assess whether melatonin could counteract Cd activity, male Wistar rats (45 days of age) received CdCl2 (5 ppm) and melatonin (3 μg/mL) or vehicle (0.015% ethanol) in drinking water. Groups of animals receiving melatonin or vehicle alone were also included. After 1 month, MBH mRNA levels were measured by real-time PCR analysis at six time intervals in a 24-h cycle. In control MBH Bmal1 expression peaked at early scotophase, Per1 expression at late afternoon, and Per2 and Cry2 expression at mid-scotophase, whereas neither Clock nor Cry1 expression showed significant 24-h variations. This pattern was significantly disrupted (Clock, Bmal1) or changed in phase (Per1, Per2, Cry2) by CdCl2 while melatonin counteracted the changes brought about by Cd on Per1 expression only. In animals receiving melatonin alone the 24-h pattern of MBH Per2 and Cry2 expression was disrupted. CdCl2 disrupted the 24-h rhythmicity of Cu/Zn- and Mn-superoxide dismutase (SOD), nitric oxide synthase (NOS)-1, NOS-2, heme oxygenase (HO)-1, and HO-2 gene expression, most of the effects being counteracted by melatonin. In particular, the co-administration of melatonin and CdCl2 increased Cu/Zn-SOD gene expression and decreased that of glutathione peroxidase (GPx), glutathione reductase (GSR), and HO-2. In animals receiving melatonin alone, significant increases in mean Cu/Zn and Mn-SOD gene expression, and decreases in that of GPx, GSR, NOS-1, NOS-2, HO-1, and HO-2, were found. The results indicate that the interfering effect of melatonin on the activity of a low dose of CdCl2 on MBH clock and redox enzyme genes is mainly exerted at the level of redox enzyme gene expression.

Effect of cadmium on 24-hour pattern in expression of redox enzyme and clock genes in rat medial basal hypothalamus

The effect of cadmium (Cd) in the brain has been attributed to an increase in reactive oxygen species in cells, particularly when high amounts of the metal are given. In this study we examined the effect of a low dose of Cd (7.5 microg/day) on 24-h changes in expression of redox pathway enzyme and circadian genes in rat medial basal hypothalamus (MBH). Rats receiving CdCl(2) (5 ppm in drinking water) or tap water for 1 month were killed at six different time intervals throughout a 24 h cycle. MBH mRNA levels were measured by real-time PCR analysis. In CdCl(2) treated rats a disruption of 24-h pattern of hypothalamic gene expression of nitric oxide synthase (NOS)-1 and -2, heme oxygenase (HO)-1 and -2, Mn- superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione reductase was detectable. Mean levels of MBH mRNA for HO-2, Mn-SOD and catalase augmented after Cd intake, whereas those of NOS-2 decreased. After CdCl(2) intake rats the 24-h pattern of clock gene expression in MBH seen in controls was significantly suppressed (Bmal1) or changed in phase (Per1, Per2, Cry2) while in the case of Clock significant 24-h variations were induced. The results are compatible with the view that a low amount of Cd given in tap water brought about significant changes in circadian expression of redox enzyme and clock genes in rat MBH.

Effect of cadmium on 24-h pattern expression of redox enzyme genes in rat medial basal hypothalamus

The mechanism of action of nuclear factor-kappa B (NF-κB)’s has been widely studied in nervous system damage and disease since 2000. NF-κB is widely present in eukaryotic cells, and inactive NF-κB is present in the cytoplasm. Activated NF-κB transfers into the nucleus and induces expression of several target genes. Through the action of different downstream effectors, NF-κB plays a bi-directional interactive role in central nervous system damage, as well as learning and memory. While NF-κB can induce pro-inflammation factor release and exacerbate brain injury, it can also exert protective effects on neurons in central nervous system diseases and injuries by upregulating endothelial nitric oxide synthase (eNOS), Mn-superoxide dismutase (Mn-SOD), B-cell lymphoma/leukaemia-2 (Bcl-2), and inhibitors of apoptosis protein (IAPs). Moreover, NF-κB is required for spatial learning and memory and passive avoidance training. However, a decrease in NF-κB activity in the hippocampal dentate gyrus is necessary for active avoidance training. This paper reviews NF-κB’s bi-directional interactive role in these processes.

Effect of cadmium on 24-h pattern in expression of clock genes in rat medial basal hypothalamus

Effect of cadmium on 24-h pattern in expression of clock genes in rat medial basal hypothalamus

Hypoglycemia Affects the Activation State of Glycogen Synthase Kinase‐3beta in the Rat Hypothalamus

Adaptations in brain signaling pathways may underlie the altered detection and response to hypoglycemia seen in hypoglycemic unawareness. Glycogen synthase kinase-3beta (GSK3ß), a key regulator of numerous metabolic processes, responds to changes in insulin and glucose. The objective of this study was to examine the phosphorylation of GSK3ß by Akt (protein kinase B) in the rat medial basal hypothalamus (MBH) following acute and recurrent episodes of insulin-induced hypoglycemia. MBH brain regions were obtained from male Sprague-Dawley rats 30 minutes after a first-time (acute) or fourth-time (recurrent) 2 U/kg IV insulin injection. Expression of Ser-9 phosphorylated and total GSK3ß and Thr-308 phosphorylated and total Akt were measured from homogenates by Western blot analysis. Following acute hypoglycemia, phosphorylation of GSK3ß was decreased ~40% (p=0.05) as compared to saline-treated controls. The phosphorylation of Akt was decreased ~50% (p=0.03) after first-time hypoglycemia. Levels of phosphorylated GSK3ß were similar (p=0.34) to saline controls after recurrent hypoglycemia. There was no change (p=.98) in Akt phosphorylation to a 4th hypoglycemic episode. Total GSK3ß and Akt levels were not changed by acute or recurrent hypoglycemia. The decrease in phosphorylation and inactivation of hypothalamic GSK3ß to insulin-induced hypoglycemia was absent after 3 previous episodes of hypoglycemia. These results are consistent with a possible role of GSK3ß in hypoglycemic unawareness. NIH-DK59755

Antibodies obtained by xenotransplantation of organ-cultured median eminence specifically recognize hypothalamic tanycytes.

Tanycytes are specialized ependymal cells lining the infundibular recess of the third ventricle of the cerebrum. Early and recent investigations involve tanycytes in the mechanism of gonadotropin-releasing hormone (GnRH) release to the portal blood. The present investigation was performed to obtain a specific immunological marker of tanycytes and to identify the compound(s) responsible for this labeling. After 30 days of organ culture, explants of bovine median eminence formed spherical structures mostly constituted by tanycytes. These tanycyte spheres were xenotransplanted to rats, and the antibodies raised by the host animals against the transplanted living tanycytes were used for immunochemical studies of the bovine and rat median eminence. This antiserum immunoreacted with two compounds of 60 kDa and 85 kDa present in extracts of bovine and rat median eminence. The individual immunoblotting analysis of rat medial basal hypothalami showed a decrease in the amount of the 85-kDa compound in castrated rats as compared to control rats processed at oestrus and dioestrus. The antiserum, labeled as anti-P85, when used for immunostaining of sections throughout the rat central nervous system, immunoreacted specifically with the hypothalamic tanycytes. Within tanycytes, P-85 immunoreactivity was exclusively present in the basal processes. It is suggested that the 85-kDa and 60-kDa compounds correspond to two novel proteins selectively expressed by tanycytes. The possibility that they are secretory proteins involved in GnRH release is discussed. Anti-P85 appears to be the first specific marker of hypothalamic tanycytes.

Methylene Blue Inhibits the Increase of Inducible Nitric Oxide Synthase Activity Induced by Stress and Lipopolysaccharide in the Medial Basal Hypothalamus of Rats

In infection bacterial products such as lipopolysaccharides (LPS) induce inducible nitric oxide synthase (iNOS) that produces large quantities of NO toxic to the invading organisms, but also often has toxic effects on host cells. Therefore, inhibition of iNOS activity might be beneficial in combatting these adverse effects. To determine if methylene blue (MB), an oxidizing agent that inactivates iNOS, would reduce the iNOS levels in the medial basal hypothalami (MBH) of conscious male rats, LPS (5 mg/kg) was injected intravenously (i.v.), and after 3 h they were injected i.v. with either MB (3 mg/kg) or saline and the effects on iNOS in the MBH determined. iNOS was measured by conversion of labeled arginine into citrulline by incubating MBH in the absence of calcium (Ca²⁺) since iNOS does not require Ca²⁺ for activation. The results indicate that iNOS was induced by the injection of saline, but the induction by LPS was much greater, an increase of 10-fold above that of control sham-operated animals. Both the induction of iNOS from the stress of saline injections and LPS were completely eliminated by MB indicating that MB might be beneficial in preventing injury to brain tissue following LPS injection. There was no effect of either LPS or MB on the Ca²⁺-dependent constitutive NOS activity.

L-Deprenyl stimulates the release of catecholamines in the rat medial basal hypothalamus in vivo

In vivo release of catecholamines in the medial basal hypothalamus (MBH) by l-deprenyl, a monoamine oxidase-B (MAO-B) inhibitor, was measured in young male Sprague–Dawley rats with stereotaxically implanted push-pull cannulae in the MBH and perfused with 0 (control), 1.5, 2.5 or 10.0 μg deprenyl in 20 μl of saline. Perfusate samples were collected at 20-min intervals and analyzed for norepinephrine (NE) and dopamine (DA) by high performance liquid chromatography (HPLC)-EC. NE release in the MBH was enhanced following perfusion with 2.5 and 10.0 μg deprenyl while DA release was augmented after infusion of 10.0 μg of deprenyl. There were no significant alterations in the release of NE and DA in the control and 1.5 μg deprenyl groups. These results suggest that deprenyl-induced in vivo release of catecholamines in the MBH may be involved in the reversal of some of the reproductive aging processes.

Physiological concentrations of melatonin inhibit the norepinephrine-induced activation of prostaglandin E2 and cyclic AMP production in rat hypothalamus: a mechanism involving inhibiton of nitric oxide synthase.

In this paper, we summarize the results of in vitro studies showing that physiological concentrations of melatonin inhibit the norepinephrine-induced activation of prostaglandin E2 (PGE2) and cyclic AMP production in rat medial basal hypothalamus (MBH). Interestingly, a concentration of melatonin as low as 1 nM, which is roughly equivalent to the nocturnal serum physiological concentration of the hormone in the rat, significantly inhibit PGE2 and cyclic AMP production in the MBH. The suppressive effect of melatonin may be mediated by an inhibition of nitric oxide synthase (NOS) activity, since the stimulatory effect of sodium nitroprusside (SNP), a spontaneous generator of NO, was not prevented by melatonin. Melatonin also inhibited NOS activity in rat MBH in a dose-dependent manner. The results suggest the existence of a new or an ancillary means by which melatonin may regulate the physiology of the hypothalamus-pituitary unit.

Differential proopiomelanocortin gene expression in the medial basal hypothalamus of rats during pregnancy and lactation.

Hypothalamic proopiomelanocortin (POMC) gene expression was determined using in situ hybridization histochemistry (ISHH) during pregnancy and lactation in rats with and without prior reproduction experience. POMC mRNA levels in the arcuate nucleus were compared between primigravid (first pregnancy) and multigravid (second pregnancy) and primiparous and multiparous lactating rats, and between these groups and age-matched, regularly cycling, nulliparous females in diestrus. Hybridizations were performed using a digoxigenin-labeled riboprobe complementary to 837 bp of the POMC gene. The number of cells expressing POMC mRNA in the arcuate nucleus decreased in primiparous rats on day 12 of lactation when compared with the number of POMC cells in the arcuate nucleus of nulliparous rats in diestrus. In addition, the number of cells expressing POMC mRNA in multigravid animals was significantly less than in the primigravid group on days 7 and 21 of pregnancy, and on day 12 of lactation in primiparous animals. Repeated reproductive experience affected the number of POMC mRNA positive cells; there were fewer cells expressing POMC mRNA in the multigravid females on day 7 of pregnancy and an increase in the number of POMC cells in the multiparous group on day 12 of lactation compared to the primiparous animals. Optical density measurements revealed a significant increase in reaction product in the labeled cells on all days of pregnancy compared with virgin females in diestrus and a significant decrease in reaction product on day 12 of lactation in the multiparous group. The results of the present study indicate that POMC gene expression changes across pregnancy and lactation and that repeated reproductive experience has long-term, possibly permanent, effects on the endogenous opioid system.

Expression of the orphan receptor steroidogenic factor-1 mRNA in the rat medial basal hypothalamus.

Steroidogenic factor-1 (SF-1), an orphan receptor of the nuclear hormone receptor family, binds to the AAGGTCA motif in the promoter elements of several diverse target genes, including some that mediate steroidogenesis and sexual differentiation. In addition, SF-1 is expressed in embryonic forebrain, suggesting that it plays a role in neural development. This study was undertaken to study the distribution and regulation of SF-1 mRNA expression in the rat brain. SF-1 mRNA levels were measured in tissue dissections by ribonuclease protection assay. A 452 nt 32P-labeled cRNA probe, complementary to the putative ligand-binding domain of the rat SF-1 mRNA, was synthesized from the rat SF-1 cDNA inserted into pBluescript II KS, using a Sty 1 fragment and T3 polymerase. The probe protected a single 390 nt transcript in the medial basal hypothalamus (MBH) and peripheral steroidogenic tissues of the male rat. The size of this protected band corresponded to that of the protected sense RNA standard (HindIII fragment of the SF-1 cDNA transcribed with T7 polymerase). No SF-1 mRNA was detected in the preoptic area, amygdala or cingulate cortex. The levels of SF-1 mRNA in MBH were not affected by gonadectomy or androgen treatment, nor was there a sex difference in its expression in adults. In situ hybridization histochemistry revealed that SF-1 was localized to the ventromedial nucleus of the adult hypothalamus. The levels of SF-1 mRNA were high on gestational day 18 after which they fell by approximately 30% and remained constant throughout gestation, the first week of neonatal life, and into adulthood. These results demonstrate that the gene encoding SF-1 is expressed in a discrete region of the rat hypothalamus and appears to be developmentally regulated, but not affected by gonadal hormones in adults.

Twenty-four hour rhythms of serum prolactin, growth hormone and luteinizing hormone levels, and of medial basal hypothalamic corticotropin-releasing hormone levels and dopamine and serotonin metabolism in rats neonatally administered melatonin.

To obtain information on long-term circadian consequences of administering melatonin neonatally to rats, we assessed the 24-hour rhythms of 1) serum prolactin (PRL), luteinizing hormone (LH), and growth hormone (GH), and 2) medial basal hypothalamic dopamine (DA) and serotonin (5-HT) metabolism and corticotropin-releasing hormone (CRH) content in 60-day old male rats injected with 100 micrograms of melatonin on the 5th day of life. Controls receiving vehicle alone showed serum PRL concentration (when 60 days of age) attaining its maximum at the end of the light period (i.e., at 2000 hr), while in melatonin-injected rats high PRL values were found between 1200 and 2000 hr. Twenty-four hour changes in serum LH levels exhibited a maximum at noon, and to a similar extent in vehicle- and melatonin-treated rats. Neonatal melatonin injection did not affect serum GH concentration when the rats were adult. In the medial basal hypothalamus (MBH), the dihydroxyphenyl acetic acid (DOPAC)/ DA ratio attained a maximum at midnight, its amplitude being significantly higher in melatonin- than in vehicle-treated rats. Neonatally melatonin-injected rats also exhibited a second maximum in DOPAC/DA ratio at noon, coinciding with a minimum in DA levels of MBH. The 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio in MBH showed significant diurnal variations in vehicle-injected controls with maxima at 1200 and 0400 hr, while in melatonin-treated rats a single maximum occurred at 2400 hr. These maximum correlated with minima in 5-HT content of MBH. Neonatal melatonin injection brought about a significant increase in the CRH content of MBH, as well as distortion of its diurnal rhythmicity, a maximum being found at noon in controls and at 1800 hr in melatonin-treated rats. The results indicate that exposure to melatonin early in life affects subsequent diurnal rhythmicity of PRL release, and of DA and 5-HT turnover and CRH content in the MBH of rats.

Sex-specific changes in preoptic regulatory factor-1 and preoptic regulatory factor-2 mRNA expression in the rat brain during development

Preoptic regulatory factor-1 (Porf-1) and Preoptic regulatory factor-2 (Porf-2) are two novel peptide genes which are expressed in the central nervous system. Expression is modified by age and by hormones of the reproductive system. In this study nuclease protection assays were employed to investigate Porf-1 and Porf-2 mRNA expression in the cerebral cortex (CC), hippocampus (HIPP), preoptic area (POA) and medial basal hypothalamus (MBH) of male and female rats, aged 15, 30 and 60 days. Porf-1 and Porf-2 mRNA expression tended to decrease from 15 to 60 days, with two exceptions. Porf-2 in the hippocampus of female rats, and Porf-1 in the MBH of the male rats, were found instead to increase with age. There were distinctive sex differences inporf-1 andporf-2 gene expression. Consistently higher mRNA levels were measured for both genes in the POA of female rats at all ages examined, and this difference reached statistical significance for Porf-1 at the age of 60 days. In contrast, levels of Porf-2 mRNA were higher in MBH of male than female rat MBH at 15 days, and Porf-1 mRNA was substantially more abundant in male than in female rat MBH at 30 and 60 days of age. These results indicate that there is sexual dimorphism and regional specificity in the developmental expression of these genes.

Parathyroid hormone-induced dopamine turnover in the rat medial basal hypothalamus

The parathyroid hormone (PTH) gene is expressed and translated in the rat hypothalamus, and the possibility that PTH may modulate neural activity was therefore examined in anesthetized rats. Intracerebroventricular (ICV) injections of 1.0 or 10.0 μg rat, human, or bovine PTH(1–34) was followed 60 min later by increased concentrations of DOPAC (dihydroxyacetic acid) and the DOPAC:dopamine (DA) ratio in the medial basal hypothalamus (MBH), but not in other (brainstem, cerebral cortex, cerebellum) regions of the brain. Tissue concentrations of norepinephrine and serotonin were unchanged by ICV. PTH administration, although MBH concentrations of 5-hydroxyindolacetic acid (5-HIAA) were increased following PTH administration. An increase in MBH DA turnover (as indicated by an increased DOPAC: DA ratio) was also induced by the ICV injection of 10 μg PTH-related protein [PTHrP(1–34)]. Pretreatment with the receptor antagonists PTH(7–34) or PTHrP(7–34) completely blocked the subsequent DOPAC response to ICV PTH or PTHrP, respectively. The DOPAC concentrations in hypothalamic extracellular fluid (ECF), sampled by microdialysis, were also increased within 20 min of PTH(1–34) perfusion, in the absence of changes in the ECF concentrations of 5-HIAA. These results demonstrate that PTH and PTH-like peptides specifically increase DA turnover in the rat MBH and suggest novel roles for these hormones in neural regulation.

Dopaminergic actions of parathyroid hormone in the rat medial basal hypothalamus in vitro.

Parathyroid hormone (PTH) modulates dopamine (DA) metabolism in the rat medial basal hypothalamus (MBH) in vivo. Direct effects of PTH on MBH DA metabolism were therefore investigated in vitro. Incubation of rat MBHs for 60 min with 10(-7)-10(-5) M human PTH1-34 consistently reduced the tissue DA content and increased the DOPAC (dihydroxyphenylacetic acid) to DA ratio. This ratio was further increased in tissues incubated in 10(-5) M PTH1-34, as a result of an increase in DOPAC content. The tissue content of DOPAC and DA was unaffected by 10(-9) M PTH. The serotonin (5HT) content of the MBH was reduced by 10(-5) M PTH1-34, but concentrations of 5HT, 5-hydroxyindolacetic acid, and norepinephrine were otherwise unaffected by 10(-9)-10(-5) M PTH1-34. Concentrations of DA in the incubation media were reduced after exposure to 10(-6) or 10(-5) M PTH1-34. The uptake of 3H-labelled DA by incubated tissues was also reduced by 10(-6) M PTH1-34, as was the metabolism of 3H-labelled DA into tissue and media DOPAC. Monoamine oxidase (MAO) activities A and B were significantly increased after the incubation of the MBH with 10(-6) or 10(-5) M PTH1-34. These results further demonstrate neuromodulatory actions of PTH on dopaminergic neurons within the rat MBH in vitro, and suggest neural and/or neuroendocrine roles of PTH of central or peripheral origin.

Rat hypothalamic proopiomelanocortin messenger RNA is unaffected by adrenalectomy

The negative feedback control of hypothalamic cortocotrophin releasing factor (CRF) and anterior pituitary proopiomelanocortin (POMC) by corticosteroids is well understood. However, less is known about the mechanisms that regulate POMC gene expression in the arcuate nuclei in the medial basal hypothalamus (MBH). Using a sensitive and specific S1 endonuclease protection assay, we have examined the effect of adrenalectomy on POMC mRNA in the rat MBH and pituitary. Our results show that adrenalectomy does not change POMC mRNA levels in the MBH at 7 or 14 days post surgery. The neurointermediate lobe of the pituitary was similarly unaffected by adrenalectomy, while in the anterior lobe, POMC mRNA increased 7-10 fold at both time points, effects that were prevented by dexamethasone treatment. We conclude that while POMC mRNA in the anterior lobe of the pituitary is regulated by plasma glucocorticoids, in the MBH and neurointermediate lobe, it is not.

A role for central postsynapticα 2-adrenoceptors in glucoregulation

Central or peripheral administration of theα 2-adrenoceptor agonist clonidine causes marked hyperglycemia in the rat. It is not clear whether this effect is mediated within the brain at either pre- or postsynapticα 2-adrenoceptors or whether it is due to peripheralα 2-agonist actions. We employed computerized mass spectrometry to measure noradrenaline (NA) and its primary neuronal metabolite 3,4-dihydroxyphenylglycol (DHPG) in the medial basal hypothalamus of rats treated acutely with clonidine, theα 2-antagonist yohimbine, the postganglionic noradrenergic blocker guanethidine and the neuroglycopenic agent 2-deoxy- d-glucose (2-DG). That clonidine's hyperglycemic effect was due, in part, to an action at centralα 2-adrenoceptors was indicated by the ability of guanethidine to significantly inhibit the glucose response. Because of clonidine's inhibition of hypothalamic NA release (assessed by the DHPG/NA ratio), presumably by presynaptic agonism, these data indicated that postsynaptic receptor stimulation by clonidine was involved in activating glucose release. Yohimbine markedly increased the hypothalamic DHPG/NA ratio, reflecting presynaptic stimulation of NA release, but at the same time inhibited the hyperglycemic response due to 2-DG administration. This latter effect to block hyperglycemia is consistent with antagonism of postsynapticα 2-adrenoceptors involved in mediating hepatic glucose output. These data indicate a major role for postsynapticα 2-adrenoceptors in glucoregulation.

Endotoxin-Induced Release of Interleukin-6 from Rat Medial Basal Hypothalami*

Interleukin-6 (IL-6) is an inflammatory cytokine that stimulates T-cell activation and B-cell differentiation. We recently reported that picomolar concentrations of IL-6 stimulated PRL, GH, and LH release in vitro. These data suggested that IL-6 may function as a hypothalamic releasing factor for anterior pituitary hormones. Medial basal hypothalami (MBH) were incubated for 60-90 min in Krebs-Ringer bicarbonate buffer supplemented with 0.025% BSA, and the conditioned medium was assayed for IL-6 concentrations by the 7TD1 cell growth factor assay. It was found that MBH released IL-6 in vitro. Although depolarizing concentrations of K+ (56 mM) did not increase IL-6 release, somatostatin release from the MBH was increased significantly. The bacterial endotoxin lipopolysaccharide (LPS; 1-100 ng/ml) induced significant increases in IL-6 release from the MBH. The presence of IL-6 in the hypothalamus suggested a possible role for this cytokine in the regulation of neuropeptide release; however, the release of somatostatin was not affected by 20 ng/ml IL-6. Comparison studies of neural and neuroendocrine tissues revealed that the anterior and posterior pituitaries released larger amounts of bioactive IL-6 than the MBH or parietal cortex during a 4-h incubation; induction of IL-6 release by endotoxin occurred only in the anterior pituitary and hypothalamus. IL-6 mRNA was detectable in the MBH and anterior pituitary tissue after a 4-h incubation; however, no IL-6 mRNA was detectable in freshly isolated tissues. LPS (100 ng/ml) and (Bu)2cAMP (1 mM) increased IL-6 mRNA accumulation in and IL-6 release from the MBH and anterior pituitary. These data suggest that the MBH synthesizes and releases IL-6 via a nonneuronal source in vitro.

Hypothalamic Opioid Mechanisms in Experimental Uremic Hypogonadism*

Previous studies have demonstrated that abolition of the naloxone-stimulated increase in plasma LH levels is characteristic of hypothalamic dysfunction in experimental uremia. This study aimed to further characterize the nature of the defect in hypothalamic opiatergic mechanisms in experimental uremia. Specifically, we have tested the hypothesis that naloxone resistance was due to either opioid receptor dysfunction or diminished opioid peptide levels. Administration of naloxone (2 mg/kg, iv) to cannulated freely mobile rats confirmed previous observations that despite marked increases in plasma LH in control rats, plasma LH levels were unaffected in uremic male rats (P = 0.001 for group x time interaction). In a second experiment, morphine (2 mg/kg) or saline diluent was given quasi-continuously as small aliquots before each blood sample during the pulse studies of castrate mature male rats that had undergone either subtotal nephrectomy or sham operation. After the administration of morphine, uremic rats exhibited a 60% reduction in mean LH levels (14.9 +/- 1.4 vs. 6.0 +/- 0.7 ng/ml) attributable to a 42% reduction in LH pulse frequency (3.6 +/- 0.4 vs. 2.1 +/- 0.5 peaks/3 h) and a 60% reduction in LH pulse amplitude (4.7 +/- 0.5 vs. 1.9 +/- 0.3 ng/ml). The preservation of sensitivity to morphine despite complete naloxone resistance raised the alternate hypothesis of depletion of endogenous opiate peptide levels in the uremic hypothalamus. This hypothesis was tested by measuring the beta-endorphin content of the medial basal hypothalamus (MBH) in a rat beta-endorphin RIA. Rat MBH beta-endorphin content was not significantly altered specifically by either uremia or castration. We conclude, therefore, that naloxone resistance of plasma LH in experimental uremia is not due to either defects in opioid receptor function or reduced hypothalamic beta-endorphin content. Instead, we suggest that uremia may diminish the release of endogenous opioid peptides that interact with GnRH neurons from the MBH.

Ultrastructural Characterization of Prolactin-Like Immunoreactivity in Rat Medial Basal Hypothalamus

Prolactin-like immunoreactivity has been reported in the medial basal hypothalamus at the light microscopic level, in hypophysectomized rats. Here, with preembedding immunocytochemistry at the electron microscopic level, we have observed prolactin-immunoreactive neurons and synapses in the hypothalamus. Reaction product was discovered in medial basal hypothalamic neurons, which had typical large nucleoli and received axosomatic synapses. In the cytoplasm, reaction product was distinctly granular. Immunoreactive neurons were usually surrounded by nonreactive cells. Reaction product was also seen in dendrites, some of which had spines. Some axons in the hypothalamus contained reaction product, usually surrounded by nonreactive axons, and immunopositive synapses were detected both in the hypothalamus and in the midbrain. In a small number of cases immunoreactive axons could be seen synapsing on immunoreactive dendrites.