Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) is a neuromodulator implicated in anxiety, metabolism and reproductive behavior. PACAP global knockout mice have decreased fertility and PACAP modulates LH release. However, its source and role at the hypothalamic level remain unknown. We demonstrate that PACAP-expressing neurons of the ventral premamillary nucleus of the hypothalamus (PMVPACAP) project to, and make direct contact with, kisspeptin neurons in the arcuate and AVPV/PeN nuclei and a subset of these neurons respond to PACAP exposure. Targeted deletion of PACAP from the PMV through stereotaxic virally mediated cre- injection or genetic cross to LepR-i-cre mice with Adcyap1fl/fl mice led to delayed puberty onset and impaired reproductive function in female, but not male, mice. We propose a new role for PACAP-expressing neurons in the PMV in the relay of nutritional state information to regulate GnRH release by modulating the activity of kisspeptin neurons, thereby regulating reproduction in female mice.
Disclosure statement:The authors have nothing to disclose. Acknowledgements: This work was supported by NIH R01 HD019938 and R01 HD082314 to U.B.K.; R00 HD071970 and R01 HD090151 to V.M.N.; NHLBI 5T32HL007374-36, NCATS UL1 TR001102, T32 HL007374-36 and Harvard Medical School Dupont Warren Fellowship to R.A.R.; R01 DK075632, R01 DK096010, R01 DK089044, R01 DK111401, P30 DK046200, P30 DK057521 to B.B.L. ABSTRACTPituitary adenylate cyclase activating polypeptide (PACAP) is a neuromodulator implicated in anxiety, metabolism and reproductive behavior. PACAP global knockout mice have decreased fertility and PACAP modulates LH release. However, its source and role at the hypothalamic level remain unknown. We demonstrate that PACAP-expressing neurons of the ventral premamillary nucleus of the hypothalamus (PMV PACAP ) project to, and make direct contact with, kisspeptin neurons in the arcuate and AVPV/PeN nuclei and a subset of these neurons respond to PACAP exposure. Targeted deletion of PACAP from the PMV through stereotaxic virally mediated cre-injection or genetic cross to LepR-i-cre mice with PACAP fl/fl mice led to delayed puberty onset and impaired reproductive function in female, but not male, mice. We propose a new, sex-specific role for PACAP-expressing neurons in the PMV in the relay of nutritional state information to regulate GnRH release by modulating the activity of kisspeptin neurons, thereby regulating reproduction. 43 44 RESULTS 45PACAP release from leptin-responsive neurons is essential for normal timing of puberty 46 onset and fertility. 47An intact PMV is required to transduce metabolic information through leptin signaling to allow for 48 HPG activity 19 . We found that the PMV is the region of the brain with the highest level of co-49 localization of pStat3 (an indirect marker of LepR activity) and PACAP (figure 1a); approximately 50 87% of the PACAP neurons also express pStat3 in that region, though only 70% of the LepR 51 neurons co-express PACAP. There are two other regions, both in the hypothalamus, that also 52 show co-localization of pStat3 and PACAP, although to a lesser extent: the central nucleus of 53 the ventromedial hypothalamus and the supramammillary nucleus (supplemental figure 1). To 54 investigate if PACAP is an important relay for leptin we produced mice with PACAP deleted 55 conditionally from leptin receptor expressing neurons using the leptin receptor cre knock-in 56 mouse 23 crossed to the PACAP fl/fl mouse that we made. The LepR-i-cre mouse expresses cre 57 recombinase in cells that produce the long-form of the leptin receptor, which are found primarily 58 in the brain 23 , thus producing a conditional knockout of PACAP from neurons that express the 59 leptin receptor. We validated this conditional knockout by qPCR and by RNA in situ 60 hybridization (supplemental figure 2 and 3a).61 62 Because this genetic recombination occurs before puberty onset, we were able to address the 63 question of the role that PACAP in LepR neurons may play in puberty, which relies on a normal 64 f...
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.
Impulsivity is a multidimensional heritable phenotype that broadly refers to the tendency to act prematurely and is associated with multiple forms of psychopathology, including substance use disorders. We performed genome-wide association studies (GWAS) of eight impulsive personality traits from the Barratt Impulsiveness Scale and the short UPPS-P Impulsive Personality Scale (N = 123,509–133,517 23andMe research participants of European ancestry), and a measure of Drug Experimentation (N = 130,684). Because these GWAS implicated the gene CADM2, we next performed single-SNP phenome-wide studies (PheWAS) of several of the implicated variants in CADM2 in a multi-ancestral 23andMe cohort (N = 3,229,317, European; N = 579,623, Latin American; N = 199,663, African American). Finally, we produced Cadm2 mutant mice and used them to perform a Mouse-PheWAS (“MouseWAS”) by testing them with a battery of relevant behavioral tasks. In humans, impulsive personality traits showed modest chip-heritability (~6–11%), and moderate genetic correlations (rg = 0.20–0.50) with other personality traits, and various psychiatric and medical traits. We identified significant associations proximal to genes such as TCF4 and PTPRF, and also identified nominal associations proximal to DRD2 and CRHR1. PheWAS for CADM2 variants identified associations with 378 traits in European participants, and 47 traits in Latin American participants, replicating associations with risky behaviors, cognition and BMI, and revealing novel associations including allergies, anxiety, irritable bowel syndrome, and migraine. Our MouseWAS recapitulated some of the associations found in humans, including impulsivity, cognition, and BMI. Our results further delineate the role of CADM2 in impulsivity and numerous other psychiatric and somatic traits across ancestries and species.
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