Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice

The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determine the role of glutamatergic signaling from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces LH release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in LepRb-Cre mice. We collected blood sequentially before and for 1h after iv. clozapine-N-oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of cFos immunoreactive neurons in the PMv. Next, females with deletion of Vglut2 in LepRb neurons (LepR(ΔVGlut2)) showed delayed age of puberty, disrupted estrous cycles, increased GnRH concentration in the axon terminals and disrupted LH responses, suggesting impaired GnRH release. To assess if glutamate is required for PMv actions in pubertal development, we generated a Cre-induced reexpression of endogenous LepRb (LepR(loxTB)) with concomitant deletion of Vglut2 (Vglut2-floxed) mice. Rescue of Lepr and deletion of Vglut2 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control LepR(loxTB) mice with PMv LepRb rescue showed vaginal opening, follicle maturation and became pregnant, while LepR(loxTB);Vglut2(flox) mice showed no pubertal development. Our results indicate that glutamatergic signaling from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.