Kisspeptin (Kiss1) and neurokinin B (NKB) (encoded by the Kiss1 and Tac2 genes, respectively) are indispensable for reproduction. In the female of many species, Kiss1 neurons in the arcuate nucleus (ARC) coexpress dynorphin A and NKB. Such cells have been termed Kiss1/NKB/Dynorphin (KNDy) neurons, which are thought to mediate the negative feedback regulation of GnRH/LH secretion by 17β-estradiol. However, we have less knowledge about the molecular physiology and regulation of Kiss1/Kiss1-expressing neurons in the ARC of the male. Our work focused on the adult male mouse, where we sought evidence for coexpression of these neuropeptides in cells in the ARC, assessed the role of Kiss1 neurons in negative feedback regulation of GnRH/LH secretion by testosterone (T), and investigated the action of NKB on KNDy and GnRH neurons. Results showed that 1) the mRNA encoding Kiss1, NKB, and dynorphin are coexpressed in neurons located in the ARC; 2) Kiss1 and dynorphin A mRNA are regulated by T through estrogen and androgen receptor-dependent pathways; 3) senktide, an agonist for the NKB receptor (neurokinin 3 receptor, encoded by Tacr3), stimulates gonadotropin secretion; 4) KNDy neurons express Tacr3, whereas GnRH neurons do not; and 5) senktide activates KNDy neurons but has no discernable effect on GnRH neurons. These observations corroborate the putative role for KNDy neurons in mediating the negative feedback effects of T on GnRH/LH secretion and provide evidence that NKB released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in the male.

The medial septum/diagonal band (MSDB), which gives rise to the septohippocampal pathway, is a critical locus for the mnemonic effects of muscarinic drugs. Infusion of muscarinic cholinergic agonists into the MSDB enhance learning and memory processes both in young and aged rats and produce a continuous theta rhythm in the hippocampus. Intraseptal muscarinic agonists also alleviate the amnesic syndrome produced by systemic administration of muscarinic receptor antagonists. It has been presumed, but not proven, that the cellular mechanisms underlying the effects of muscarinic agonists in the MSDB involve an excitation of septohippocampal cholinergic neurons and a subsequent increase in acetylcholine (ACh) release in the hippocampus. Using a novel fluorescent labeling technique to selectively visualize live septohippocampal cholinergic neurons in rat brain slices, we have found that muscarinic agonists do not excite septohippocampal cholinergic neurons, instead they inhibit a subpopulation of cholinergic neurons. In contrast, unlabeled neurons, confirmed to be noncholinergic, septohippocampal GABA-type neurons using retrograde marking and double-labeling techniques, are profoundly excited by muscarine. Thus, the cognition-enhancing effects of muscarinic drugs in the MSDB cannot be attributed to an increase in hippocampal ACh release. Instead, disinhibitory mechanisms, caused by increased impulse flow in the septohippocampal GABAergic pathway, may underlie the cognition-enhancing effects of muscarinic agonists.

Activation of the G-protein-coupled receptor GPR54 by kisspeptins during normal puberty promotes the central release of gonadotropinreleasing hormone (GnRH) that, in turn, leads to reproductive maturation. In humans and mice, a loss of function mutations of GPR54 prevents the onset of puberty and leads to hypogonadotropic hypogonadism and infertility. Using electrophysiological, morphological, molecular, and retrograde-labeling techniques in brain slices prepared from vGluT2-GFP and GnRH-GFP mice, we demonstrate the existence of two physiologically distinct subpopulations of GnRH neurons. The first subpopulation is comprised of septal GnRH neurons that colocalize vesicular glutamate transporter 2 and green fluorescent protein and is insensitive to metabotropic glutamate receptor agonists, but is exquisitely sensitive to kisspeptin which closes potassium channels to dramatically initiate a long-lasting activation in neurons from prepubertal and postpubertal mice of both sexes. A second subpopulation is insensitive to kisspeptin but is uniquely activated by group I metabotropic glutamate receptor agonists. These two physiologically distinct classes of GnRH cells may subserve different functions in the central control of reproduction and fertility.

The novel hypothalamic peptides avian gonadotropin inhibitory hormone (GnIH) and its mammalian analogue RFRP-3, are emerging as key negative regulators of reproductive functions across species. GnIH/RFRP-3 reduces gonadotropin release and may play an inhibitory role in ovulation and seasonal reproduction, actions opposite to that of the puberty-promoting kisspeptins. GnIH directly inhibits gonadotropin release from the anterior pituitary in birds. GnIH/RFRP-3-immunoreactive fibres also abut the preoptic-septal gonadotropin-releasing hormone (GnRH) neurons, suggesting an additional site of action that has not been studied at the cellular level. Using anatomical labelling and electrophysiological recordings in septal brain slices from GnRH-GFP, vGluT2-GFP and GAD67-GFP mice, we report inhibitory actions of GnIH/RFRP-3 on kisspeptin-activated vGluT2 (vesicular glutamate transporter 2)-GnRH neurons as well as on kisspeptin-insensitive GnRH neurons, but not on cholinergic or GABAergic neurons (n = 531). GnIH and RFRP-3 produced a strikingly similar non-desensitizing hyperpolarization following brief 15 s applications (GnIH: 9.3 ± 1.9 mV; RFRP-3: 9.0 ± 0.9 mV) with IC 50 values of 34 and 37 nm, respectively. The inhibitory effect was mediated via a direct postsynaptic Ba 2+ -sensitive K + current mechanism and could prevent or interrupt kisspeptin-induced activation of vGluT2-GnRH neurons. GnIH-immunoreactive fibres were in apparent contact with vGluT2-GFP neurons. Thus, GnIH/RFRP-3 could reduce GnRH and glutamate release in target brain regions and in the median eminence via a direct inhibition of vGluT2-GnRH neurons. This in turn could suppress gonadotropin release, influence reproductive development and alter sex behaviour.