Glucose responsiveness in a novel adult-derived GnRH cell line, mHypoA-GnRH/GFP: involvement of AMP-activated protein kinase.

Glucose regulates energy homeostasis and reproductive function within the hypothalamus. The underlying mechanisms responsible for glucose regulation of GnRH gene transcription were investigated using a novel murine immortalized, adult-derived hypothalamic cell line, mHypoA-GnRH/GFP. Analysis of GnRH mRNA synthesis and secretion following agonist treatment demonstrated that the mHypoA-GnRH/GFP cell line is a representative model of in vivo GnRH neurons. c-fos mRNA levels, following glucose exposure, indicated that these neurons were responsive to low (0.5mM) and high (5mM) glucose, and high glucose stimulated GnRH mRNA transcription in a metabolism-dependent manner. Glucose inhibited AMPK activity, and was linked to the downstream stimulation of GnRH mRNA levels. The effect was confirmed with an AMPK antagonist, Compound C. Collectively, these findings demonstrate that glucose can directly regulate GnRH transcription, while implicating the AMPK pathway as an essential mediator of nutritional signaling in a novel GnRH neuronal cell model.

Cellular leptin resistance impairs the leptin-mediated suppression of neuropeptide Y secretion in hypothalamic neurons.

Evidence shows that neuropeptide Y (NPY) neurons are involved in mediating the anorexigenic action of leptin via neuronal circuits in the hypothalamus. However, studies have produced limited data on the cellular processes involved and whether hypothalamic NPY neurons are susceptible to cellular leptin resistance. To investigate the direct regulation of NPY secretion by leptin, we used novel NPY-synthesizing, immortalized mHypoA-NPY/green fluorescent protein and mHypoA-59 hypothalamic cell lines derived from adult hypothalamic primary cultures. We report that leptin treatment significantly suppressed NPY secretion in the cells by approximately 20%. We found a decrease in c-fos expression upon leptin exposure, indicating deactivation or hyperpolarization of the neurons. Protein analysis indicated that leptin inhibits AMP-activated protein kinase (AMPK) activity and activates acetyl-coenzyme A carboxylase in NPY neurons, supporting the hypothesis of an AMPK-dependent mechanism. Inhibiting both AMPK with Compound C or phosphatidylinositol 3 kinase (PI3K) with 2-(4-morpholinyl)-8-phenyl-1(4H)-1-benzopyran-4-one hydrochloride prevented the leptin-mediated decrease in NPY secretion, indicating both AMPK- and PI3K-mediated mechanisms. Further, NPY secretion was stimulated by 30% by the AMPK activator, aminoimidazole carboxamide ribonucleotide. Importantly, prolonged leptin exposure in the mHypoA-NPY/green fluorescent protein cells prevented leptin-induced changes in AMPK phosphorylation and suppression of NPY secretion, indicating that NPY neurons are susceptible to leptin resistance. Our studies indicate that AMPK and PI3K pathways are involved in leptin action in NPY neurons and that leptin resistance blocks the feedback response likely required to maintain energy homeostasis.

Ciliary neurotrophic factor recruitment of glucagon-like peptide-1 mediates neurogenesis, allowing immortalization of adult murine hypothalamic neurons.

The distinct lack of cell lines derived from the adult brain is evident. Ciliary neurotrophic factor (CNTF) triggers neurogenesis in primary culture from adult mouse hypothalamus, as detected by bromodeoxyuridine and Ki67 immunostaining. Using SV-40 T-antigen, we immortalized dividing neurons and generated clonal cell lines expressing neuropeptides and receptors involved in neuroendocrine function. We hypothesized that proglucagon-derived peptides may be the mechanistic downstream effectors of CNTF due to documented neuroprotective and proliferative effects. Indeed, proglucagon gene expression was induced by CNTF, and exposure of primary cells to glucagon-like peptide-1 receptor (GLP-1) agonist, exendin-4, induced cell proliferation. Intracerebroventricular injection of CNTF into adult mice caused increased expression of proglucagon peptide in the hypothalamus. Using a specific GLP-1-receptor antagonist, we found that neurogenesis was significantly attenuated and primary culture from GLP-1-receptor-knockout mice lacked CNTF-mediated neuronal proliferation, thus linking the induction of neurogenesis in the hypothalamus to GLP-1-receptor signaling.

Neurobiology of stress-induced reproductive dysfunction in female macaques.

It is now well accepted that stress can precipitate mental and physical illness. However, it is becoming clear that given the same stress, some individuals are very vulnerable and will succumb to illness while others are more resilient and cope effectively, rather than becoming ill. This difference between individuals is called stress sensitivity. Stress sensitivity of an individual appears to be influenced by genetically inherited factors, early life (even prenatal) stress, and by the presence or absence of factors that provide protection from stress. In comparison to other stress-related diseases, the concept of sensitivity versus resilience to stress-induced reproductive dysfunction has received relatively little attention. The studies presented herein were undertaken to begin to identify stable characteristics and the neural underpinnings of individuals with sensitivity to stress-induced reproductive dysfunction. Female cynomolgus macaques with normal menstrual cycles either stop ovulating (stress sensitive) or to continue to ovulate (stress resilient) upon exposure to a combined metabolic and psychosocial stress. However, even in the absence of stress, the stress-sensitive animals have lower secretion of the ovarian steroids, estrogen and progesterone, have higher heart rates, have lower serotonin function, have fewer serotonin neurons and lower expression of pivotal serotonin-related genes, have lower expression of 5HT2A and 2C genes in the hypothalamus, have higher gene expression of GAD67 and CRH in the hypothalamus, and have reduced gonadotropin-releasing hormone transport to the anterior pituitary. Altogether, the results suggest that the neurobiology of reproductive circuits in stress-sensitive individuals is compromised. We speculate that with the application of stress, the dysfunction of these neural systems becomes exacerbated and reproductive function ceases.

Ovarian steroid treatment decreases corticotropin-releasing hormone (CRH) mRNA and protein in the hypothalamic paraventricular nucleus of ovariectomized monkeys.

Corticotropin-releasing hormone (CRH) gene and protein expression were examined in the paraventricular nucleus (PVN) of ovariectomized female macaques treated with placebo or hormone therapy (HT) consisting of either estrogen (E) for 28 days, or progesterone (P) for the last 14 of 28 days, or E for 28 days supplemented with P for the last 14 of 28 days using Silastic capsules implanted s.c. in the periscapular region (n=4/group). Perfusion fixed sections (25 microm) at five levels of the PVN (rostral to caudal at 250 microm intervals) were immunostained (ICC) with an antibody to human CRH or processed in an in situ hybridization (ISH) assay with a monkey specific CRH riboprobe. The immunostained CRH-positive area was quantified with a Marianas Stereology Workstation and Slidebook 4.2. There was a significant decrease in the immunological CRH signal with E, P, and E+P treatment as measured by total or average pixels and microns (analysis of variance (ANOVA), p<0.002; Student-Newman-Keul's post hoc test versus placebo control group, p<0.05). There was also a decrease in the number of detectable CRH neurons (ANOVA, p<0.03) with HT. The sections processed for ISH were exposed to autoradiographic films. The CRH mRNA signal was analyzed with NIH Image. The average optical density and positive pixel area of the CRH mRNA signal was significantly suppressed by ovarian HT (ANOVA p<0.002; Student-Newman-Keul's post hoc test versus placebo control group, p<0.05). In summary, 1 month of stable treatment with a moderate dose of E, P or E+P significantly reduced CRH mRNA and protein in the PVN of ovariectomized monkeys. These results suggest that this hormone treatment regimen may increase stress resilience in surgically menopausal primates.

Corticotropin-releasing hormone and pro-opiomelanocortin gene expression in female monkeys with differences in sensitivity to stress.

BACKGROUND/AIMS: The expressions of corticotropin-releasing hormone (CRH) and pro-opiomelanocortin (POMC) were assessed in brain tissue collected from nonstressed female cynomolgus monkeys previously categorized as highly stress resilient (HSR), medium stress resilient (MSR), or stress sensitive (SS) with respect to stress-induced anovulation. METHODS: In situ hybridization and quantitative image analysis was used to measure mRNAs coding for CRH in the hypothalamic paraventricular nucleus (PVN) and thalamic center median-subfascicular complex (CM-Sf). Then, CRH neurons in the PVN were immunostained and the area of immunostaining was measured. Also, CRH fibers were immunostained in the central nucleus of the amygdala and the area of immunostaining was obtained. Finally, POMC mRNA expression was characterized in the hypothalamic infundibular nucleus. The groups were compared with ANOVA and Student-Newman-Keul's (SNK) post hoc comparison. RESULTS: CRH mRNA was significantly elevated in the caudal PVN in the MSR and SS animals compared to HSR animals (p < 0.05, SNK). There was a significant increase in average and total CRH-positive area in the MSR and SS groups compared to the HSR group (p < 0.05, SNK). There was also a significant increase in CRH volume in the MSR and SS groups compared to the HSR group (p < 0.05, SNK). In the CM-Sf, the average CRH optical density was significantly higher in the MSR and SS groups than in the HSR group (p < 0.05, SNK). In the central nucleus of the amygdala, the area of CRH fiber staining was significantly higher in the SS group than in the MSR or HSR groups (p < 0.05, SNK). There was no difference between the groups in POMC mRNA expression in the mediobasal hypothalamus. CONCLUSION: Macaques that exhibit immediate suppression of reproductive function upon stress are considered stress sensitive. These animals have elevated CRH in the hypothalamus and limbic structures, which may play a role in suppressing the hypothalamic-gonadal axis upon stress initiation.

Hypothalamic expression of serotonin 1A, 2A and 2C receptor and GAD67 mRNA in female cynomolgus monkeys with different sensitivity to stress.

Like women, female cynomolgus monkeys show differential sensitivity to stress-induced reproductive dysfunction. A combined social and metabolic stress (mild diet+moderate exercise+relocation) will rapidly induce anovulation in a third of female cynomolgus monkeys (stress-sensitive; SS); a third will ovulate once and then become anovulatory (medium stress-resilient; MSR) and a third are highly stress-resilient (HSR) and exhibit normal menstrual cycles through two stressed menstrual cycles. In a non-stressed menstrual cycle, SS animals have lower levels of estrogen and progesterone, lower activity of the serotonin system and lower expression of genes related to the serotonin system in the dorsal raphe nucleus. In this study, we examined the expression of 5HT1A, 5HT2A, 5HT2C receptors and GAD67 in the hypothalamus of SS, HSR and MSR monkeys using in situ hybridization. SS monkeys exhibited higher expression of 5HT2A mRNA in the paraventricular nucleus (PVN), higher expression of 5HT2C and GAD67 in the infundibulum, as well as higher expression of GAD67 in the posterior hypothalamus (PH), compared with HSR monkeys. However, the expression of 5HT1A mRNA in the ventromedial nucleus (VMN) was not different between groups. We speculate that the serotonin and GABA systems may be altered in the stress-response and reproductive-related circuits of SS monkeys, and may be participating in altering the sensitivity of the reproductive system to stress in these individuals.

Serotonin in microdialysate from the mediobasal hypothalamus increases after progesterone administration to estrogen primed macaques.

Estrogen and progesterone act on gene and protein expression in serotonin neurons in a manner that suggests serotonin neurotransmission should increase. However, measurement of extracellular serotonin in macaques was lacking. Elevated prolactin secretion can be an indicator of increased serotonergic function and prolactin is increased by combined estrogen and progesterone treatment. We examined extracellular serotonin by microdialysis in a well-characterized macaque model of steroid-induced prolactin secretion. Monkeys were fitted with 2 guide tubes directed to the arcuate nucleus of the hypothalamus. Samples (75 microl/15-minute interval) were obtained via a tether-swivel device through sample lines into an adjoining room. Serotonin was measured with a modified commercial enzyme linked immunoassay (ELISA) kit. Fenfluramine infused through the probe (300 microM for 2 h; n=2 trials) or administered intravenously (2.5 mg/kg; n=2 trials) caused a marked increase in extracellular serotonin and verified the efficacy of the procedure. Three monkeys were maintained with an estrogen implant for 2 weeks. Each monkey was injected with 20 mg of progesterone s.c. in oil at 1500 h; microdialysis was initiated the next morning and samples were obtained for 24 h. There was a significant increase in serotonin between 40 and 43 h after the progesterone injection (P<0.001, ANOVA). Serotonin averaged 59+/-1 pg/sample from 18-30 h post-progesterone injection, and averaged 76+/-2 pg/sample from 30-48 h post-progesterone injection (P<0.0001; t-test). Since the increase in serotonin is delayed by approximately 40 h after progesterone-injection, we speculate that the action of progesterone may involve either nuclear progestin receptors or membrane progestin receptors.

Estradiol increases alpha7 nicotinic receptor in serotonergic dorsal raphe and noradrenergic locus coeruleus neurons of macaques.

Acetylcholine, acting on presynaptic nicotinic receptors (nAChRs), modulates the release of neurotransmitters in the brain. The rat dorsal raphe nucleus (DR) and the locus coeruleus (LC) receive cholinergic input and express the alpha7nAChR. In previous reports, we demonstrated that estradiol (E) administration stimulates DR serotonergic and LC noradrenergic function in the macaque. In addition, it has been reported that E induces the expression of the alpha7nAChR in rats. We questioned whether E increased the expression of the alpha7nAChR in the macaque DR and LC. We utilized double immunostaining to study the effect of a simulated preovulatory surge of E on the expression of the alpha7nAChR in the DR and the LC and to determine whether alpha7nAChR colocalizes with serotonin and tyrosine hydroxylase (TH) in macaques. There was no difference in the number of alpha7nAChR-positive neurons between ovariectomized (OVX) controls and OVX animals treated with a silastic capsule containing E (Ecap). However, supplemental infusion of E for 5-30 hours to Ecap animals (Ecap + inf) significantly increased the number of alpha7nAChR-positive neurons in DR and LC. In addition, supplemental E infusion significantly increased the number of neurons in which alpha7nAChR colocalized with serotonin and TH. These results constitute an important antecedent for study of the effects of nicotine and ovarian steroid hormones in the physiological functions regulated by the DR and the LC in women.

Expression of alpha 4 and alpha 7 nicotinic receptors in the brainstem of female rabbits after coitus.

Coital signaling in the female rabbit involves sequential events in the brainstem and hypothalamus, resulting in a massive release of hypothalamic gonadotropin-releasing hormone (GnRH) that peaks within 1-2 h after mating. The neural connections between coitus and GnRH release involves norepinephrine (NE) and acetylcholine (ACh) since administration of antagonists against NE (dibenamine or phentolamine) or ACh (atropine, alpha-bungarotoxin (alpha-BTX) or scopolamine) blocks or attenuates ovulating events. Moreover, hypothalamic NE release and brainstem tyrosine hydroxylase (TH, the rate-limiting enzyme for NE synthesis) expression in the noradrenergic areas increase prior to, or in concert with, the preovulatory GnRH surge. How ACh is involved in the control of ovulation in the rabbit is lesser known. In the present study, the number of brainstem neurons expressing TH, alpha4 and alpha7 subunits of the nicotinic ACh receptor (nAChR) before and after coitus was determined by immunocytochemistry. Compared to non-mated female rabbits, the number of alpha4, alpha7 and TH single-labeled neurons as well as alpha4/TH and alpha7/TH double-labeled neurons increased in the A1, A2 and A6 brainstem noradrenergic areas at 1 h, but not 2 h, after coitus. The results suggest that the participation of ACh in the control of coitus-induced ovulation may include activation of alpha4beta2 and alpha7 nAChRs in neurons within or adjacent to the brainstem noradrenergic areas in female rabbits.