Long-term Recordings of Arcuate Nucleus Kisspeptin Neurons Across the Mouse Estrous Cycle.

The arcuate nucleus kisspeptin (ARNKISS) neurons represent the GnRH pulse generator that likely drives pulsatile gonadotropin secretion in all mammals. Using an improved GCaMP fiber photometry system enabling long-term continuous recordings, we aimed to establish a definitive profile of ARNKISS neuronal activity across the murine estrous cycle. As noted previously, a substantial reduction in the frequency of ARNKISS neuron synchronization events (SEs) occurs on late proestrus and extends into estrus. The SE amplitude remains constant throughout the cycle. During metestrus, we unexpectedly detected many multipeak SEs where many SEs occurred rapidly, within 160 seconds of each other. By applying a machine learning-based, k-means clustering analysis, we were further able to detect substantial within-stage variability in the patterns of pulse generator activity. Estrous cycle-dependent changes in SE activity occurred around the time of lights on and off. We also find that a mild stressor such as vaginal lavage reduces ARNKISS neuron SE frequency for up to 3 hours. These observations provide a comprehensive account of ARNKISS neuron activity across the estrous cycle, highlight a new pattern of multipeak SE activity, and introduce a new k-means clustering approach for analyzing ARNKISS neuron population behavior.

Unexpected Plasma Gonadal Steroid and Prolactin Levels Across the Mouse Estrous Cycle.

Despite the importance of the mouse in biomedical research, the levels of circulating gonadal steroids across the estrous cycle are not established with any temporal precision. Using liquid chromatography-mass spectrometry, now considered the gold standard for steroid hormone analysis, we aimed to generate a detailed profile of gonadal steroid levels across the estrous cycle of C57BL/6J mice. For reference, luteinizing hormone (LH) and prolactin concentrations were measured in the same samples by sandwich enzyme-linked immunosorbent assay. Terminal blood samples were collected at 8-hour intervals (10 Am, 6 Pm, 2 Am) throughout the 4 stages of the estrous cycle. As expected, the LH surge was detected at 6 Pm on proestrus with a mean (±SEM) concentration of 11 ± 3 ng/mL and occurred coincident with the peak in progesterone levels (22 ± 4 ng/mL). Surprisingly, estradiol concentrations peaked at 10 Am on diestrus (51 ± 8 pg/mL), with levels on proestrus 6 Pm reaching only two-thirds of this value (31 ± 5 pg/mL). We also observed a proestrus peak in prolactin concentrations (132.5 ± 17 ng/mL) that occurred earlier than expected at 2 Am. Estrone and androstenedione levels were often close to the limit of detection (LOD) and showed no consistent changes across the estrous cycle. Testosterone levels were rarely above the LOD (0.01 ng/mL). These observations provide the first detailed assessment of fluctuating gonadal steroid and reproductive hormone levels across the mouse estrous cycle and indicate that species differences exist between mice and other spontaneously ovulating species.

GnRH pulse generator frequency is modulated by kisspeptin and GABA-glutamate interactions in the posterodorsal medial amygdala in female mice.

Kisspeptin neurons in the arcuate nucleus of the hypothalamus generate gonadotrophin-releasing hormone (GnRH) pulses, and act as critical initiators of functional gonadotrophin secretion and reproductive competency. However, kisspeptin in other brain regions, most notably the posterodorsal subnucleus of the medial amygdala (MePD), plays a significant modulatory role over the hypothalamic kisspeptin population; our recent studies using optogenetics have shown that low-frequency light stimulation of MePD kisspeptin results in increased luteinsing hormone pulse frequency. Nonetheless, the neurochemical pathways that underpin this regulatory function remain unknown. To study this, we have utilised an optofluid technology, precisely combining optogenetic stimulation with intra-nuclear pharmacological receptor antagonism, to investigate the neurotransmission involved in this circuitry. We have shown experimentally and verified using a mathematical model that functional neurotransmission of both GABA and glutamate is a requirement for effective modulation of the GnRH pulse generator by amygdala kisspeptin neurons.