Definition of the estrogen negative feedback pathway controlling the GnRH pulse generator in female mice.

The mechanisms underlying the homeostatic estrogen negative feedback pathway central to mammalian fertility have remained unresolved. Direct measurement of gonadotropin-releasing hormone (GnRH) pulse generator activity in freely behaving mice with GCaMP photometry demonstrated striking estradiol-dependent plasticity in the frequency, duration, amplitude, and profile of pulse generator synchronization events. Mice with Cre-dependent deletion of ESR1 from all kisspeptin neurons exhibited pulse generator activity identical to that of ovariectomized wild-type mice. An in vivo CRISPR-Cas9 approach was used to knockdown ESR1 expression selectively in arcuate nucleus (ARN) kisspeptin neurons. Mice with >80% deletion of ESR1 in ARN kisspeptin neurons exhibited the ovariectomized pattern of GnRH pulse generator activity and high frequency LH pulses but with very low amplitude due to reduced responsiveness of the pituitary. Together, these studies demonstrate that estrogen utilizes ESR1 in ARN kisspeptin neurons to achieve estrogen negative feedback of the GnRH pulse generator in mice.

Increased neuronal activation in sympathoregulatory regions of the brain and spinal cord in type 2 diabetic rats.

Increased cardiac sympathetic nerve activity in type 2 diabetes mellitus (DM) suggests impaired autonomic control of the heart. However, the central regions that contribute to the autonomic cardiac pathologies in type 2 DM are unknown. Therefore, we tested the hypothesis that neuronal activation would be increased in central sympathoregulatory areas in a pre-clinical type 2 DM animal model. Immunohistochemistry in 20-week-old male Zucker diabetic fatty (ZDF) rats revealed an increased number of neurones expressing ΔFosB (a marker of chronic neuronal activation) in the intermediolateral column (IML) of the spinal cord in DM compared to non-diabetic (non-DM) rats (P < 0.05). Rostral ventrolateral medulla (RVLM) neurones activate IML neurones and receive inputs from the hypothalamic paraventricular nucleus (PVN), as well as the nucleus tractus solitarius (NTS) and area postrema (AP), in the brainstem. We observed more ΔFosB-positive noradrenergic RVLM neurones (P < 0.001) and corticotrophin-releasing hormone PVN neurones (P < 0.05) in DM compared to non-DM rats. More ΔFosB-positive neurones were also observed in the NTS (P < 0.05) and AP (P < 0.01) of DM rats compared to non-DM rats. Finally, because DM ZDF rats are obese, we also expected increased activation of pro-opiomelanocortin (POMC) arcuate nucleus (ARC) neurones in DM rats; however, fewer ΔFosB-positive POMC ARC neurones were observed in DM compared to non-DM rats (P < 0.01). In conclusion, increased neuronal activation in the IML of type 2 DM ZDF rats might be driven by RVLM neurones that are possibly activated by PVN, NTS and AP inputs. Elucidating the contribution of central sympathoexcitatory drive in type 2 DM might improve the effectiveness of pharmacotherapies for diabetic heart disease.

Plasticity in Intrinsic Excitability of Hypothalamic Magnocellular Neurosecretory Neurons in Late-Pregnant and Lactating Rats.

Oxytocin and vasopressin secretion from the posterior pituitary gland are required for normal pregnancy and lactation. Oxytocin secretion is relatively low and constant under basal conditions but becomes pulsatile during birth and lactation to stimulate episodic contraction of the uterus for delivery of the fetus and milk ejection during suckling. Vasopressin secretion is maintained in pregnancy and lactation despite reduced osmolality (the principal stimulus for vasopressin secretion) to increase water retention to cope with the cardiovascular demands of pregnancy and lactation. Oxytocin and vasopressin secretion are determined by the action potential (spike) firing of magnocellular neurosecretory neurons of the hypothalamic supraoptic and paraventricular nuclei. In addition to synaptic input activity, spike firing depends on intrinsic excitability conferred by the suite of channels expressed by the neurons. Therefore, we analysed oxytocin and vasopressin neuron activity in anaesthetised non-pregnant, late-pregnant, and lactating rats to test the hypothesis that intrinsic excitability of oxytocin and vasopressin neurons is increased in late pregnancy and lactation to promote oxytocin and vasopressin secretion required for successful pregnancy and lactation. Hazard analysis of spike firing revealed a higher incidence of post-spike hyperexcitability immediately following each spike in oxytocin neurons, but not in vasopressin neurons, in late pregnancy and lactation, which is expected to facilitate high frequency firing during bursts. Despite lower osmolality in late-pregnant and lactating rats, vasopressin neuron activity was not different between non-pregnant, late-pregnant, and lactating rats, and blockade of osmosensitive ΔN-TRPV1 channels inhibited vasopressin neurons to a similar extent in non-pregnant, late-pregnant, and lactating rats. Furthermore, supraoptic nucleus ΔN-TRPV1 mRNA expression was not different between non-pregnant and late-pregnant rats, suggesting that sustained activity of ΔN-TRPV1 channels might maintain vasopressin neuron activity to increase water retention during pregnancy and lactation.

Indirect Suppression of Pulsatile LH Secretion by CRH Neurons in the Female Mouse.

Acute stress is a potent suppressor of pulsatile luteinizing hormone (LH) secretion, but the mechanisms through which corticotrophin-releasing hormone (CRH) neurons inhibit gonadotropin-releasing hormone (GnRH) release remain unclear. The activation of paraventricular nucleus (PVN) CRH neurons with Cre-dependent hM3Dq in Crh-Cre female mice resulted in the robust suppression of pulsatile LH secretion. Channelrhodopsin (ChR2)-assisted circuit mapping revealed that PVN CRH neuron projections existed around kisspeptin neurons in the arcuate nucleus (ARN) although many more fibers made close appositions with GnRH neuron distal dendrons in the ventral ARN. Acutely prepared brain slice electrophysiology experiments in GnRH- green fluorescent protein (GFP) mice showed a dose-dependent (30 and 300 nM CRH) activation of firing in ~20% of GnRH neurons in both intact diestrus and ovariectomized mice with inhibitory effects being uncommon (<8%). Confocal GCaMP6 imaging of GnRH neuron distal dendrons in acute para-horizontal brain slices from GnRH-Cre mice injected with Cre-dependent GCaMP6s adeno-associated viruses demonstrated no effects of 30 to 300 nM CRH on GnRH neuron dendron calcium concentrations. Electrophysiological recordings of ARN kisspeptin neurons in Crh-Cre,Kiss1-GFP mice revealed no effects of 30 -300 nM CRH on basal or neurokinin B-stimulated firing rate. Similarly, the optogenetic activation (2-20 Hz) of CRH nerve terminals in the ARN of Crh-Cre,Kiss1-GFP mice injected with Cre-dependent ChR2 had no effect on kisspeptin neuron firing. Together, these studies demonstrate that PVN CRH neurons potently suppress LH pulsatility but do not exert direct inhibitory control over GnRH neurons, at their cell body or dendron, or the ARN kisspeptin neuron pulse generator in the female mouse.

Highly redundant neuropeptide volume co-transmission underlying episodic activation of the GnRH neuron dendron.

The necessity and functional significance of neurotransmitter co-transmission remains unclear. The glutamatergic 'KNDy' neurons co-express kisspeptin, neurokinin B (NKB), and dynorphin and exhibit a highly stereotyped synchronized behavior that reads out to the gonadotropin-releasing hormone (GnRH) neuron dendrons to drive episodic hormone secretion. Using expansion microscopy, we show that KNDy neurons make abundant close, non-synaptic appositions with the GnRH neuron dendron. Electrophysiology and confocal GCaMP6 imaging demonstrated that, despite all three neuropeptides being released from KNDy terminals, only kisspeptin was able to activate the GnRH neuron dendron. Mice with a selective deletion of kisspeptin from KNDy neurons failed to exhibit pulsatile hormone secretion but maintained synchronized episodic KNDy neuron behavior that is thought to depend on recurrent NKB and dynorphin transmission. This indicates that KNDy neurons drive episodic hormone secretion through highly redundant neuropeptide co-transmission orchestrated by differential post-synaptic neuropeptide receptor expression at the GnRH neuron dendron and KNDy neuron.

Neural Determinants of Pulsatile Luteinizing Hormone Secretion in Male Mice.

The gonadotrophin-releasing hormone (GnRH) pulse generator drives pulsatile luteinizing hormone (LH) secretion essential for fertility. However, the constraints within which the pulse generator operates to drive efficient LH pulsatility remain unclear. We used optogenetic activation of the arcuate nucleus kisspeptin neurons, recently identified as the GnRH pulse generator, to assess the efficiency of different pulse generator frequencies in driving pulsatile LH secretion in intact freely behaving male mice. Activating the pulse generator at 45-minute intervals generated LH pulses similar to those observed in intact male mice while 9-minute interval stimulation generated LH profiles indistinguishable from gonadectomized (GDX) male mice. However, more frequent activation of the pulse generator resulted in disordered LH secretion. Optogenetic experiments directly activating the distal projections of the GnRH neuron gave the exact same results, indicating the pituitary to be the locus of the high frequency decoding. To evaluate the state-dependent behavior of the pulse generator, the effects of high-frequency activation of the arcuate kisspeptin neurons were compared in GDX and intact mice. The same stimulus resulted in an overall inhibition of LH release in GDX mice but stimulation in intact males. These studies demonstrate that the GnRH pulse generator is the primary determinant of LH pulse profile and that a nonlinear relationship exists between pulse generator frequency and LH pulse frequency. This may underlie the ability of stimulatory inputs to the pulse generator to have opposite effects on LH secretion in intact and GDX animals.

GnRH Pulse Generator Activity Across the Estrous Cycle of Female Mice.

A subpopulation of kisspeptin neurons located in the arcuate nucleus (ARN) operate as the GnRH pulse generator. The activity of this population of neurons can be monitored in real-time for long periods using kisspeptin neuron-selective GCaMP6 fiber photometry. Using this approach, we find that ARN kisspeptin neurons exhibit brief (∼50 seconds) periods of synchronized activity that precede pulses of LH in intact female mice. The dynamics and frequency of these synchronization episodes (SEs) are stable at approximately one event every 40 minutes throughout metestrus, diestrus, and proestrus, but slow considerably on estrus to occur approximately once every 10 hours. Evaluation of ARN kisspeptin neuron activity across the light-dark transition, including the time of onset of the proestrus LH surge, revealed no changes in SE frequency. Longer 24-hour recordings across proestrus into estrus demonstrated that an abrupt decrease in SEs occurred ∼4 to 5 hours after the onset of the LH surge to reach the low frequency of SEs observed on estrus. The frequency of SEs was stable across the 24-hour period from metestrus to diestrus. Administration of progesterone to diestrus mice resulted in the abrupt slowing of SEs. These observations show that the GnRH pulse generator exhibits an unvarying pattern of activity from metestrus through to the late evening of proestrus, at which time it slows dramatically, likely in response to postovulation progesterone secretion. The GnRH pulse generator maintains a constant frequency of activity across the time of the LH surge, demonstrating that it is not involved directly in surge generation.

Characterization of GnRH Pulse Generator Activity in Male Mice Using GCaMP Fiber Photometry.

Kisspeptin neurons located in the hypothalamic arcuate nucleus are thought to represent the GnRH pulse generator responsible for driving pulsatile LH secretion. The recent development of GCaMP6 fiber photometry technology has made it possible to perform long-term recordings of the population activity of the arcuate nucleus kisspeptin (ARNKISS) neurons in conscious-behaving mice. Using this approach, we show that ARNKISS neurons in intact male mice exhibit episodes of synchronized activity that last ∼2 minutes and have a mean inter-episode interval of 166 minutes, with a very wide range (43 to 347 minutes). Gonadectomy resulted in dramatic changes in the dynamics of ARNKISS neuron behavior with temporally distinct alterations in synchronization episode (SE) amplitude (sevenfold increase), inter-SE frequency (range, 2 to 58 minutes), and duration (up to 28 minutes), including the frequent appearance of seemingly unstable clusters of doublet and triplet SEs. The combination of photometry with repeated blood sampling revealed a perfect correlation between ARNKISS neuron population SEs and LH pulses in intact and short-term gonadectomized (GDX) mice. No differences were detected in SE frequency across 24 hours in either intact or GDX mice. These observations further support a role for ARNKISS neurons as the GnRH pulse generator and show that it operates in a stochastic manner without diurnal variation in both intact and GDX male mice. The removal of gonadal steroids has multiple time-dependent effects upon ARNKISS neuron synchronizations, indicating their critical role in shaping pulse generator behavior.