Sex‐ and sub region‐dependent modulation of arcuate kisspeptin neurones by vasopressin and vasoactive intestinal peptide

Abstract

A population of kisspeptin neurones located in the hypothalamic arcuate nucleus (ARN) very likely represent the gonadotrophin‐releasing hormone pulse generator responsible for driving pulsatile luteinising hormone secretion in mammals. As such, it has become important to understand the neural inputs that modulate the activity of ARN kisspeptin (ARNKISS) neurones. Using a transgenic GCaMP6 mouse model allowing the intracellular calcium levels ([Ca2+]i) of individual ARNKISS neurones to be assessed simultaneously, we examined whether the circadian neuropeptides vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) modulated the activity of ARNKISS neurones directly. To validate this methodology, we initially evaluated the effects of neurokinin B (NKB) on [Ca2+]i in kisspeptin neurones residing within the rostral, middle and caudal ARN subregions of adult male and female mice. All experiments were undertaken in the presence of tetrodotoxin and ionotropic amino acid antagonists. NKB was found to evoke an abrupt increase in [Ca2+]i in 95%‐100% of kisspeptin neurones throughout the ARN of both sexes. By contrast, both VIP and AVP were found to primarily activate kisspeptin neurones located in the caudal ARN of female mice. Although 58% and 59% of caudal ARN kisspeptin neurones responded to AVP and VIP, respectively, in female mice, only 0%‐8% of kisspeptin neurones located in other ARN subregions responded in females and 0%‐12% of cells in any subregion in males (P < 0.05). These observations demonstrate unexpected sex differences and marked heterogeneity in functional neuropeptide receptor expression amongst ARNKISS neurones organised on a rostro‐caudal basis. The functional significance of this unexpected influence of VIP and AVP on ARNKISS neurones remains to be established.

Direct inhibition of arcuate kisspeptin neurones by neuropeptide Y in the male and female mouse

“…Acute brain slices were prepared from adult male Kiss1::GCaMP6 mice in which 90%‐95% of ARN KISS neurones display robust NKB‐evoked increases in GCaMP6 fluorescence in ARN KISS neurones (Figure 4A). 18 Using the two‐stimulation NKB protocol, the second NKB‐evoked elevation in [Ca] i was found to be higher than the first, generating an S2/S1 of 1.09 ± 0.09 (Figure 4B). Inclusion of 100 nmol L ‐1 NPY at the same time as the second NKB stimulus generated a marked decrease in evoked [Ca] i rise, with the resultant S2/S1 being 0.20 ± 0.05 (n = 30 from three mice; P < 0.001, Wilcoxon signed rank test) (Figure 4).…”

“…Experiments were performed in the presence of tetrodotoxin and a cocktail of amino acid receptor antagonists to block any indirect actions of NPY. Although we used 50 nmol L ‐1 NKB in the electrophysiological studies, 100 nmol L ‐1 NKB is found to provide a more consistent stimulation in calcium imaging investigations 18 . Acute brain slices were prepared from adult male Kiss1::GCaMP6 mice in which 90%‐95% of ARN KISS neurones display robust NKB‐evoked increases in GCaMP6 fluorescence in ARN KISS neurones (Figure 4A).…”

“…Kiss1::GCaMP6 mice were generated by crossing Kiss1 ‐Cre +/− and homozygous floxed GCaMP6f (Ai95(RCL‐GCaMP6f)‐D) 16 lines to generate mixed background 129S6Sv/Ev C57BL6 Kiss1‐Cre::GCaMP6f‐lox‐STOP‐lox mice. Both Kiss1::tGFP and Kiss1::GCaMP6 mouse lines have been shown previously to express green fluorescent protein (GFP) or GCaMP6 selectively in ARN KISS neurones 17,18 . Adult mice were group‐housed in individually‐ventilated cages with environmental enrichment under a 12:12 hour light/dark photocycle (lights on 6.00 am ) at 22 ± 2°C and access to food (Teklad Global 18% Protein Rodent Diet 2918; Envigo, Indianapolis, IN, USA) and water available ad lib.…”

“…For assessing the effects of neuropeptides on ARN kisspeptin neurones, 100 nmol L ‐1 NPY (Tocris Bioscience, St Louis, MO, USA) or 500 nmol L ‐1 αMSH was applied into the bathing solution aCSF when recording basal or 50 nmol L ‐1 neurokinin B (NKB)‐evoked firing. For the latter, an S2/S1 experimental protocol was used in which two 1‐minute 50 nmol L ‐1 NKB‐activations were given 5‐10 minutes apart, with the second stimulation (S2) occurring in the presence or absence of the neuropeptide being tested 18 . Previous studies have shown that 100 nmol L ‐1 NPY 19,20 and 500 nmol L ‐1 αMSH 20,21 concentrations are effective in modulating the activity of hypothalamic and other neurones in acute brain slice studies.…”

Time‐of‐day‐dependent sensitivity of the reproductive axis to RFamide‐related peptide‐3 inhibition in female Syrian hamsters

“…To modulate the balance of negative and positive E2 feedback, the SCN coordinates the activity of two opposing ERα-expressing neuropeptidergic systems that lie upstream of the GnRH system: the stimulatory neuropeptide, kisspeptin, and the inhibitory neuropeptide, RFamide-related peptide-3 (RFRP-3; the mammalian orthologue of avian gonadotrophin-inhibitory hormone [GnIH]). 4,11,18,[23][24][25][26][27][28][29][30] The SCN coordinates cellular activity of RFRP-3 neurones to suppress the reproductive axis outside the time window of the LH surge and to allow for the transient suppression of E 2 -negative-feedback around the time of the LH surge. 11,18,23 RFRP-3 neurones are concentrated in the dorsomedial hypothalamus (DMH) and project broadly to hypothalamic loci that contain GnRH neurones and fibres (ie, medial septum, diagonal band of Broca, preoptic area, anterior hypothalamus and arcuate nucleus [ARC]) in addition to the ventromedial nucleus of the hypothalamus and brainstem.…”