BACKGROUND: Two major populations of kisspeptin-expressing neurons in the hypothalamus synapse onto the gonadotropin-releasing hormone (GnRH) neurons to control the secretion of GnRH and, therefore, the luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The first population is found in the anteroventral periventricular nucleus of the hypothalamus (Kiss1AVPV/PeN) and is characteristic of the female brain. The Kiss1AVPV/PeN population functions as the LH surge generator in females, stimulating a large peak in LH release (called LH surge) mid-cycle to trigger ovulation, by mediating the positive feedback of sex steroids on the hypothalamic-pituitary-gonadal (HPG) axis. The second population is in the arcuate nucleus of the hypothalamus (ARC), where Kisspeptin neurons also co-express Neurokinin B and Dynorphin A (thereafter named KNDy neurons) and act as the pulse generator for GnRH/LH release, essential to normal reproductive function. KNDy neurons mediate the negative feedback of sex steroids on the HPG axis and relay information about the hormonal and neuroendocrine milieu (including metabolic cues) to the reproductive system. In addition to expressing three neuropeptides, KNDy neurons are also glutamatergic, but the role of this fast excitatory amino acid transmitter within KNDy neurons is still unclear. Current research into the potential role of glutamatergic signaling from KNDy neurons, while limited, has found that KNDy neurons send glutamatergic input to (1) Kiss1AVPV/PeN neurons, to (2) other KNDy neurons, and to (3) first-order homeostatic neurons regulating feeding behavior within the ARC. The majority of these findings were based on optogenetic activation of KNDy neurons, an artificial stimulation technique useful for tracing neural connections but unreliable for deducing the functional connection and role of such signaling pathways physiologically.
OBJECTIVE: The aim of this study is to expand on existing optogenetic and electrophysiological studies by using viral CRISPR/Cas9 gene editing technology to examine the physiological impact of glutamatergic signal loss from KNDy neurons (GlutamateKNDy) on reproductive function in vivo.
METHODS: Adult female Kiss1-cre het mice underwent stereotaxic injections of a cre-dependent CRISPR/SaCas9 virus targeting either Slc17a6 (the gene encoding Vglut2, a transporter necessary for glutamate neurotransmitter release), or Rosa-26 as a control. Using this method, we generated mice with a KNDy neuron specific deletion of glutamate release (Vglut2KD) and the impact on reproductive axis function was evaluated. Estrous cycles were tracked for four weeks before, and twelve weeks following Slc17a6 deletion from KNDy neurons. LH pulsatile release parameters were evaluated 10 weeks after viral injections and the females were submitted to an LH surge induced protocol to evaluate potential impairment to the LH surge driving ovulation. Body weight on a regular diet was also followed in the Vglut2KD and the control females to assess for potential significant metabolic impairments.
RESULTS: Loss of glutamatergic signaling from KNDy neurons did not alter estrous cyclicity. Pulsatile LH release was not significantly altered but the frequency and amplitude of LH pulses tended to decrease in the Vglut2KD females as compared to controls. The induced LH surge protocol revealed a significant impairment to the surge mode of LH release in Vglut2KD females. Vglut2KD mice had no significant changes in body weight following Slc17a6 deletion from KNDy neurons.
CONCLUSION: Glutamatergic signaling from KNDy neurons is not necessary to maintain normal pulsatile release of GnRH/LH, or estrous cyclicity, but is critical to the LH surge. This aligns with findings of transgenic Vglut2KO females with regular estrous cycles1 and of optogenetic stimulation of glutamate release from KNDy neurons onto the AVPV, generating a surge-like peak in circulating LH2. The lack of impairment to the pulse mode of LH release contradicts the recent hypothesis that glutamatergic stimulation from KNDy neurons serves as the basis of the population synchronization and pulsatility3. Further research is necessary to determine other potential sources of the noted stochastic glutamate stimulation of the KNDy network.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/48319 |
| Date | 02 March 2024 |
| Creators | Medve, Elizabeth |
| Contributors | Symes, Karen, Talbi, Rajae |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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