The neuropeptides kisspeptin, neurokinin B, and dynorphin A (collectively abbreviated as KNDy) are, respectively, encoded by KiSS-1, NKB, and PDYN and are coexpressed by neurons of the hypothalamic arcuate nucleus (ARC). Here, using quantitative real-time PCR, we examined agerelated changes in the expression of genes encoding KNDy and associated receptors G proteincoupled receptor 54 (encoded by GPR54), neurokinin 3 receptor (encoded by NK3), and -opioid receptor (encoded by KOR), in the female rhesus macaque ARC-median eminence (ARC-ME). Expression of KiSS-1 and NKB was highly elevated in old perimenopausal compared with young or middle-aged premenopausal animals. To test whether these age-related changes could be attributed to perimenopausal loss of sex steroids, we then examined KNDy, GPR54, NK3, and KOR expression changes in response to ovariectomy (OVX) and exposure to 17␤-estradiol (E 2 ). Shortterm (7 months) OVX (with or without 1 month of estrogen replacement) failed to modulate the expression of any of the KNDy-related genes. In contrast, long-term (ϳ4 yr) OVX significantly increased KiSS-1 and NKB expression, and this was reversed by E 2 administration. Finally, we examined the expression of KNDy-related genes in young adult females during the early follicular, late follicular, or midluteal phases of their menstrual cycle but found no difference. Together, the results suggest that short-term alterations in circulating E 2 levels, such as those occurring during the menstrual cycle, may have little effect on the ARC-ME expression of KNDy and associated receptors. Nevertheless, they clearly demonstrate that loss of ovarian steroid negative feedback that occurs during perimenopause plays a major role in modulating the activity of KNDy circuits of the aging primate ARC-ME. (Endocrinology 151: 3783-3794, 2010) M enopause marks the end of reproductive function for women and is characterized by a disruption of the hypothalamic-pituitary-gonadal (HPG) axis. In contrast to rodents, the age-associated decline in circulating sex-steroid hormone concentrations in primates is thought to stem primarily from a depletion of ovarian follicles, rather than from sex-steroid-independent changes within the hypothalamus (1). Even so, hypothalamic changes do occur in perimenopausal women and may have a major impact on the activity of the HPG axis as well as other physiological functions. Currently, there is clear evidence that a population of cells located in the arcuate nucleus (ARC) of the hypothalamus coexpress kisspeptin, neurokinin B, and dynorphin A (2); here, we refer to these neuropeptides collectively by the abbreviation KNDy (2). Importantly, these neurons play a major role in modulating the release of GnRH from the hypothalamus and also show marked changes in their activity

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, and the responsiveness of neurons to GABA can be modulated by sex steroids. To better understand how ovarian steroids influence GABAergic system in the primate brain, we evaluated the expression of genes encoding GABA receptor subunits, glutamic acid decarboxylase (GAD) and a GABA transporter in the brains of female rhesus macaques. Ovariectomized adults were subjected to a hormone replacement paradigm involving either 17β-estradiol (E), or E plus progesterone (E+P). Untreated animals served as controls. Using GeneChip® microarray analysis and real-time RT-PCR (qPCR), we examined gene expression differences within and between the amygdala (AMD), hippocampus (HPC) and arcuate nuclei of the medial basal hypothalamus (MBH). The results from PCR corresponded with results from representative GeneChip® probesets, and showed similar effects of sex steroids on GABA receptor subunit gene expression in the AMD and HPC, and a more pronounced expression than in the MBH. Exposure to E+P attenuated GAD1, GAD2 and SLC32A1 gene expression in the AMD and HPC, but not in the MBH. GABA receptor subunit gene expression was generally higher in the AMD and HPC than in the MBH, with the exception of receptor subunits ε and γ2. Taken together, the data demonstrate differential regulation of GABA receptor subunits and GABAergic system components in the MBH compared to the AMD and HPC of rhesus macaques. Elevated ε and reduced δ subunit expression in the MBH supports the hypothesis that the hypothalamic GABAergic system is resistant to the modulatory effects of sex steroids.

Background: The hypothalamus plays a key role in mediating the effects of estrogen on many physiological functions, including reproduction, metabolism, and thermoregulation. We have previously observed marked estrogen-dependent gene expression changes within the hypothalamus of rhesus macaques during aging, especially in the KNDy neurons of the arcuate-median eminence (ARC-ME) that produce kisspeptin, neurokinin B, and dynorphin A. Little is known, however, about the mechanisms involved in mediating the feedback from estrogen onto these neurons. Methods: We used quantitative real-time PCR to profile age- and estrogen-dependent gene expression changes in the rhesus macaque hypothalamus. Our focus was on genes that encode steroid receptors (ESR1, ESR2, PGR, and AR) and on enzymes that contribute to the local synthesis of 17β-estradiol (E₂; STS, HSD3B1/2, HSD17B5, and CYP19A). In addition, we used RT² Profiler™ PCR Arrays to profile a larger set of genes that are integral to hypothalamic function. Results: KISS1, KISS1R, TAC3, and NPY2R mRNA levels increased in surgically menopausal (ovariectomized) old females relative to age-matched ovariectomized animals that received E₂ hormone therapy. In contrast, PGR, HSD17B, GNRH2, SLC6A3, KISS1, TAC3, and NPY2R mRNA levels increased after E₂ supplementation. Conclusion: The rhesus macaque ARC-ME expresses many genes that are responsive to changes in circulating estrogen levels, even during old age, and these may contribute to causing the normal and pathophysiological changes that occur during menopause.

Reduced activity has been linked to age-associated physiological changes but the underlying root cause is unclear. The goal of the present study was to compare the orexin neuronal system of old (23–29 years) female rhesus macaques with either active or sedentary 24-hour locomotor activity patterns. Using immunohistochemistry we counted the number of orexin A and orexin B neurons in the lateral hypothalamic area (LHA) of each animal. Overall, we observed no difference in the distribution pattern or number of either orexin A or orexin B immune-positive neurons between animals in the two groups. Thus, reduced activity in the elderly is unlikely to stem from a loss of orexin neuronal perikarya in the LHA. This, however, does not rule out the possibility that the reduced activity stems from reduced orexin neuronal projections to arousal centers of the brain, such as the locus coeruleus, or from attenuated release of orexin.