STUDIES ON THE NEUROTRANSMITTER REGULATION OF KISSPEPTIN NEURONS

Abstract

Background: Kisspeptin is a neuropeptide which acts through the activation of previously orphan G-protein coupled receptor 54 (GPR54). One of the most important functions of kisspeptin neurons is to stimulate gonadotropin-releasing hormone (GnRH) secretion which further regulates reproduction. Although a lot of studies have been done about the kisspeptin regulation of hypothalamic-(brain)-pituitary-gonadal (HPG) axis, data available about the regulation of kisspeptin itself are very scarce. Therefore, the present research work was designed to study the neurotransmitter (glutamate and GABA) regulation of kisspeptin neurons in adult male rhesus macaque under different physiological conditions such as during breeding and non-breeding season and during normal fed and 48 h fasted states. Additionally, expression of gonadal steroid receptor was examined on a specific hypothalamic kisspeptin neuronal population in adult male and female mice to better understand the steroidal regulation of kisspeptin signaling. Materials and Methods: In monkey studies, hypothalamic blocks containing medio basal hypothalamus (MBH) and preoptic area (POA) were obtained from five intact adult male rhesus monkeys (Macaca mulatta), captured from wild during the breeding (n=3; October; TV: 69.00 ± 1.00 ml) and non-breeding season (n=2; July; TV: 12.88 ± 0.31 ml) and from four adult male monkeys during fed (n=2) and 48 h fasting (n=2) states. Hemi-hypothalamic blocks were placed in a fixative for immunocytochemistry and other hemi-hypothalamus was quickly immersed in liquid nitrogen and then placed at -80 ̊C until RNA isolation. Blood samples were also drawn from animals during breeding and non-breeding seasons to measure plasma testosterone levels and during fed and fasting states to measure plasma testosterone and blood glucose levels. Real time polymerase chain reaction ( PCR) was carried out by using specific primers to quantify the levels of Kiss1, Kiss1r, NR1 and glutamic acid decarboxylase 67 (GAD67) mRNA during different reproductive seasons and metabolic states. Double label immunofluorescence using specific antibodies against kisspeptin and NR1 was also performed to examine the interaction of kisspeptin neurons with NR1 subunit of N-methyl-D, L-aspartate (NMDA) receptors in hypothalamic MBH of adult male rhesus macaque during the breeding and non-breeding seasons and during normal feeding and 48 h fasting states. In mice studies, ten adult male and ten adult female C57BL/6J mice were used. Dual label chromogen immunocytochemistry was performed on brain sections by using specific primary antibodies directed against kisspeptin and estrogen receptor α (ERα)/ androgen receptor (AR) in RP3V region of male and female mice. Kisspeptin immunoreactivity was revealed by treating the brain sections with diaminobenzidine hydrochloride (DAB) (brown cytoplasmic staining) and ERα/AR immunoreactivity was revealed by treating the brain sections with nickel-enhanced diaminobenzidine hydrochloride (NiDAB) (black nucleus staining). Results: Our results demonstrated that the expression of Kiss1, Kiss1r and NR1 mRNA levels were significantly (p<0.05) increased whereas expression of GAD67 mRNA levels were significantly (p<0.05) decreased in the MBH and POA of adult male rhesus monkey during the breeding season and vice versa during the non- breeding season. Plasma testosterone levels (p<0.01) and testicular volumes (p<0.001) were significantly increased during the breeding season as compared to the non-breeding season. The number of kisspeptin neurons, and the interactions of kisspeptin neurons with NR1 expressing elements were significantly (p<0.05) increased during the breeding season as compared to non-breeding season. In the other experiment, plasma testosterone and blood glucose levels were significantly (p<0.05) decreased after short term fasting. Hypothalamic expression of Kiss1, Kiss1r and NR1 mRNA levels were significantly (p<0.05) reduced in adult male rhesus monkeys which were fasted for 48 h as compared to those which were fed ad libitum while there was no clear difference in GAD67 mRNA expression levels between the two groups. The number of kisspeptin neurons was significantly (p<0.05) reduced after 48 h fasting, and the percentage of kisspeptin neurons expressing NR1 was also reduced after 48 h fasting. In the mice study, kisspeptin neurons residing in rostral periventricular area of third ventricle (RP3V) expressed ERα and AR in both male and female mice. Percentage of RP3V kisspeptin neurons expressing AR was significantly higher (p<0.05) in males than females. About 65% and 37% of RP3V kisspeptin neurons expressed ERα in adult male and female mice, respectively. However, this difference in percentage of ERα expressing RP3V kisspeptin neurons between male and female mice was statistically non-significant (p>0.05). Percentage of kisspeptin neurons expressing ERα was significantly (p<0.05) greater than the percentage of kisspeptin neurons expressing AR in the female mouse. However, percentage of kisspeptin neurons expressing ERα and AR was statistically similar in the male mouse. Conclusions: Taken together the results of the current research work suggest that kisspeptin signaling is stimulated by glutamate and inhibited by GABA neurotransmitters in adult male rhesus macaques during different photoperiods and metabolic states. This research work strengthens our knowledge about the excitatory (glutamate) and inhibitory (GABA) neurotransmitters regulation of kisspeptin signaling especially in higher primates. In addition, present study extend our knowledge about the steroid regulation of kisspeptin neurons by demonstrating putative involvement of both androgen and estrogen on a subgroup of hypothalamic kisspeptin population in male and female rodents.