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

Reviewer #1 (Public Review):

Summary:
In previous work, the Elias group has shown that leptin-sensing PMv neurons make connections with the neuroendocrine reproductive axis and are involved in reproductive function/s. Sáenz de Miera et al. build on this body of work to investigate the sufficiency of leptin sensing PMv neurons to evoke the release of luteinizing hormone. The team further investigates how glutamate signaling from leptin-sensing neurons can influence pubertal timing in females, along with mature estrous cycles. Genetic ablation of Slc17a6 (Vglut2) from LepRb-expressing cells resulted in a delay of the first estrus cycle post-pubertal transition, along with a significantly lengthened estrous cycle in mature females. However, this deficit did not lengthen the latency to the birth of the first litter in experimental dams. Restoration of leptin signaling in LepRb PMv neurons was previously shown to induce puberty and instate reproductive function in LepRb knock-out female mice (Mahany et al., 2018). Here, Sáenz de Miera et al. use a combined genetic and viral strategy to demonstrate that glutamate signaling in LepRb PMv neurons is required for sexual maturation in LepRb knock-out female mice.

Strengths:
Most of the experiments performed in this manuscript are well-justified and rigorously tested. The genetic method to simultaneously remove glutamate signaling and restore the leptin receptor in LepRb PMv neurons was well executed and showed that glutamate signaling in LepRb PMv neurons is necessary for leptin-dependent fertility.

Weaknesses:
Analysis of experimentally induced luteinizing hormone release could be confounded by spontaneous pulses of luteinizing hormone that are independent of LepRb PMv neurons.

Reviewer #2 (Public Review):

Summary:
This is a very well-written manuscript by Saenz de Meira and colleagues on a careful study reporting on the key role of glutamate transporter vGlut2 expression in the neurons of the ventral perimammillary nucleus (PMv) of the hypothalamus expressing the leptin receptor LepRb in energy homeostasis, puberty, and estrous cyclicity. The authors first show using cre-dependent chemogenetic viral tools that the selective activation of the PMv LepRb induces luteinizing hormone (LH) release. Then the authors demonstrate that the selective invalidation of vGlut2 in LepRb-expressing cells in the all body induces obesity and mild alteration of sexual maturation in both sexes and blunted estrous cyclicity in females. Finally, the authors knock out vGlut2 in PMv neurons in which they reintroduce LepRb expression in an otherwise LepRb-null background using an AAV Cre approach. This latter very elegant experiment shows that while the sole re-expression of LepRb in PMv neurons in LepRb-null mice was shown before to restore puberty onset, deleting vGlut2 in LepRb-expressing PMv neurons blunts this effect.

Strengths:
The authors employ state-of-the-art methods and their conclusions are robustly supported by the results.

Weaknesses:
None identified. Only minor comments have been formulated.

Reviewer #3 (Public Review):

Summary:
The authors examined the effects of glutamate release from PMv LepR neurons in the regulation of puberty and reproduction in female mice.

Strengths:
Multiple genetic mouse models were utilized to either manipulate PMv LepR neuron activities, or to delete glutamate vesicle transporters from LepR neurons. The authors have been quite rigorous in validating these models and exploring potential contaminations. Most of the data presented are solid and convincing and support the conclusion.

Weaknesses:
Some results are hard to interpret.