Richard Piet

Neural control of ovulation is critical for reproductive success. In spontaneous ovulators, this is achieved through hypothalamic neural circuits relaying ovarian follicle maturity cues to gonadotropin-releasing hormone (GnRH)-secreting neurons to drive the preovulatory luteinizing hormone (LH) surge. Within these circuits, kisspeptin-expressing neurons in the rostral periventricular region of the third ventricle (RP3V ^KISS1 neurons) are thought to stimulate GnRH neurons for the LH surge. In female rodents, the central circadian clock in the suprachiasmatic nucleus (SCN) regulates the timing of RP3V ^KISS1 and GnRH neuron activation, coordinating neuroendocrine control of ovulation and behavior. However, the mechanisms thereof are not fully understood. Here, we examined the potential regulation of RP3V ^KISS1 neuron activity by neuromedin S (NMS)-expressing SCN neurons—a neuronal population essential for circadian rhythms—using anatomical tract-tracing, optogenetics, and electrophysiology in female mice. Our observations indicate that a subset of SCN ^NMS neurons, likely distinct from the canonical arginine vasopressin and vasoactive intestinal peptide SCN populations, projects to RP3V ^KISS1 neurons. Optogenetic activation of these projections evokes monosynaptic release of GABA onto ≈75% of RP3V ^KISS1 neurons. Whereas exogenous NMS only moderately affected RP3V ^KISS1 neuron activity, with no effect on firing and altered intracellular calcium concentration in subsets of cells, GABA release from SCN ^NMS fibers in the RP3V potently suppressed KISS1 neuron action potential firing through activation of GABA _A receptors. Together, these findings reveal additional complexity in the regulation of the GnRH neuronal network by the central circadian clock. Potential implications of these observations for the timing of the LH surge are discussed.

Different populations of hypothalamic kisspeptin (KISS1) neurons located in the rostral periventricular area of the third ventricle (RP3V) and arcuate nucleus (ARC) are thought to generate the sex‐specific patterns of gonadotropin secretion. These neuronal populations integrate gonadal sex steroid feedback with internal and external cues relayed via the actions of neurotransmitters and neuropeptides. The excitatory amino acid neurotransmitter glutamate, the main excitatory neurotransmitter in the brain, plays a role in regulating gonadotropin secretion, at least partially through engaging KISS1 signaling. The expression and function of individual glutamate receptor subtypes in KISS1 neurons, however, are not well characterized. Here, we used GCaMP‐based calcium imaging and patch‐clamp electrophysiology to assess the impact of activating individual ionotropic (iGluR) and group I metabotropic (mGluR) glutamate receptors on KISS1 neuron activity in the mouse RP3V and ARC. Our results indicate that activation of all iGluR subtypes and of group I mGluRs, likely mGluR1, consistently drives activity in the majority of KISS1 neurons within the RP3V and ARC of males and females. Our results also revealed, somewhat unexpectedly, sex‐ and region‐specific differences. Indeed, activating (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) type iGluRs evoked larger responses in female ARC^KISS1 neurons than in their male counterparts whereas activating group I mGluRs induced larger responses in RP3V^KISS1 neurons than in ARC^KISS1 neurons in females. Together, our findings suggest that glutamatergic neurotransmission in KISS1 neurons, and its impact on the activity of these cells, might be sex‐ and region‐dependent in mice.

Background: The neuroendocrine control of ovulation is orchestrated by neuronal circuits that ultimately drive the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus to trigger the preovulatory surge in luteinizing hormone (LH) secretion. While estrogen feedback signals are determinant in triggering activation of GnRH neurons, through stimulation of afferent kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V^KISS1 neurons), many neuropeptidergic and classical neurotransmitter systems have been shown to regulate the LH surge. Among these, several lines of evidence indicate that the monoamine neurotransmitter serotonin (5-HT) has an excitatory, permissive, influence over the generation of the surge, via activation of type 2 5-HT (5-HT₂) receptors. The mechanisms through which this occurs, however, are not well understood. We hypothesized that 5-HT exerts its influence on the surge by stimulating RP3V^KISS1 neurons in a 5-HT₂ receptor-dependent manner.Methods: We tested this using kisspeptin neuron-specific calcium imaging and electrophysiology in brain slices obtained from male and female mice.Results: We show that exogenous 5-HT reversibly increases the activity of the majority of RP3V^KISS1 neurons. This effect is more prominent in females than in males, is likely mediated directly at RP3V^KISS1 neurons and requires activation of 5-HT₂ receptors. The functional impact of 5-HT on RP3V^KISS1 neurons, however, does not significantly vary during the estrous cycle.Conclusion: Taken together, these data suggest that 5-HT₂ receptor-mediated stimulation of RP3V^KISS1 neuron activity might be involved in mediating the influence of 5-HT on the preovulatory LH surge.

Polycystic ovary syndrome (PCOS) is associated with elevated androgen and luteinizing hormone (LH) secretion and with oligo/anovulation. Evidence indicates that elevated androgens impair sex steroid hormone feedback regulation of pulsatile LH secretion. Hyperandrogenemia in PCOS may also disrupt the preovulatory LH surge. The mechanisms through which this might occur, however, are not fully understood. Kisspeptin (KISS1) neurons of the rostral periventricular area of the third ventricle (RP3V) convey hormonal cues to gonadotropin-releasing hormone (GnRH) neurons. In rodents, the preovulatory surge is triggered by these hormonal cues and coincident timing signals from the central circadian clock in the suprachiasmatic nucleus (SCN). Timing signals are relayed to GnRH neurons, in part, via projections from SCN arginine-vasopressin (AVP) neurons to RP3V^KISS1 neurons. Because rodent SCN cells express androgen receptors (AR), we hypothesized that these circuits are impaired by elevated androgens in a mouse model of PCOS. In prenatally androgen-treated (PNA) female mice, SCN Ar expression was significantly increased compared to that found in prenatally vehicle-treated mice. A similar trend was seen in the number of Avp-positive SCN cells expressing Ar. In the RP3V, the number of kisspeptin neurons was preserved. Anterograde tract-tracing, however, revealed reduced SCN^AVP neuron projections to the RP3V and a significantly lower proportion of RP3V^KISS1 neurons with close appositions from SCN^AVP fibers. Functional assessments showed, on the other hand, that RP3V^KISS1 neuron responses to AVP were maintained in PNA mice. These findings indicate that PNA changes some of the neural circuits that regulate the preovulatory surge. These impairments might contribute to ovulatory dysfunction in PNA mice modeling PCOS.

The hormone, oxytocin, is synthesised by magnocellular neurones of the supraoptic and paraventricular nuclei and is released from the posterior pituitary gland into the circulation to trigger uterine contractions during parturition. Kisspeptin fibre density increases around the supraoptic nucleus over pregnancy and intracerebroventricular kisspeptin excites oxytocin neurones only in late pregnancy. However, the mechanism of this excitation is unknown. Here, we found that microdialysis administration of kisspeptin into the supraoptic nucleus consistently increased the action potential (spike) firing rate of oxytocin neurones in urethane‐anaesthetised late‐pregnant rats (gestation day 18–21) but not in non‐pregnant rats. Hazard analysis of action potential firing showed that kisspeptin specifically increased the probability of another action potential firing immediately after each action potential (post‐spike excitability) in late‐pregnant rats. Patch‐clamp electrophysiology in hypothalamic slices showed that bath application of kisspeptin did not affect action potential frequency or baseline membrane potential in supraoptic nucleus neurones. Moreover, kisspeptin superfusion did not affect the frequency or amplitude of excitatory postsynaptic currents or inhibitory postsynaptic currents in supraoptic nucleus neurones. Taken together, these studies suggest that kisspeptin directly activates oxytocin neurones in late pregnancy, at least in part, via increased post‐spike excitability.Key points: Oxytocin secretion is triggered by action potential firing in magnocellular neurones of the hypothalamic supraoptic and paraventricular nuclei to induce uterine contractions during birth. In late pregnancy, kisspeptin expression increases in rat periventricular nucleus neurones that project to the oxytocin system. Here, we show that intra‐supraoptic nucleus administration of kisspeptin increases the action potential firing rate of oxytocin neurones in anaesthetised late‐pregnant rats, and that the increased firing rate is associated with increased oxytocin neurone excitability immediately after each action potential. By contrast, kisspeptin superfusion of hypothalamic slices did not affect the activity of supraoptic nucleus neurones or the strength of local synaptic inputs to supraoptic nucleus neurones. Hence, kisspeptin might activate oxytocin neurons in late pregnancy by transiently increasing oxytocin neuron excitability after each action potential.

For about two decades, recordings of identified gonadotropin‐releasing hormone (GnRH) neurons have provided a wealth of information on their properties. We describe areas of consensus and debate the intrinsic electrophysiologic properties of these cells, their response to fast synaptic and neuromodulatory input, Ca²⁺ imaging correlates of action potential firing, and signaling pathways regulating these aspects. How steroid feedback and development change these properties, functions of GnRH neuron subcompartments and local networks, as revealed by chemo‐ and optogenetic approaches, are also considered.

Coordination of ovulation and behavior is critical to reproductive success in many species. During the female estrous cycle, the preovulatory gonadotropin surge occurs when ovarian follicles reach maturity and, in rodents, it begins just before the daily onset of activity, ensuring that ovulation coincides with sex behavior. Timing of the surge relies on projections from the suprachiasmatic nucleus (SCN), the locus of the central circadian clock, to hypothalamic circuits that regulate gonadotropin secretion. The cellular mechanisms through which the SCN controls these circuits and gates the preovulatory surge to the appropriate estrous cycle stage, however, are poorly understood. We investigated in mice the functional impact of SCN arginine-vasopressin (AVP) neuron projections to kisspeptin (Kiss1) neurons in the rostral periventricular area of the third ventricle (RP3V^Kiss1), responsible for generating the preovulatory surge. Conditional anterograde tracing revealed that SCN^AVP neurons innervate approximately half of the RP3V^Kiss1 neurons. Optogenetic activation of SCN^AVP projections in brain slices caused an AVP-mediated stimulation of RP3V^Kiss1 action potential firing in proestrus, the cycle stage when the surge is generated. This effect was less prominent in diestrus, the preceding cycle stage, and absent in estrus, following ovulation. Remarkably, in estrus, activation of SCN^AVP projections resulted in GABA-mediated inhibition of RP3V^Kiss1 neuron firing, an effect rarely encountered in other cycle stages. Together, these data reveal functional plasticity in SCN^AVP neuron output that drives opposing effects on RP3V^Kiss1 neuron activity across the ovulatory cycle. This might contribute to gating activation of the preovulatory surge to the appropriate estrous cycle stage.

A population of kisspeptin neurones located in the hypothalamic arcuate nucleus (ARN) very likely represent the gonadotrophin‐releasing hormone pulse generator responsible for driving pulsatile luteinising hormone secretion in mammals. As such, it has become important to understand the neural inputs that modulate the activity of ARN kisspeptin (ARN^KISS) neurones. Using a transgenic GCaMP6 mouse model allowing the intracellular calcium levels ([Ca²⁺]_i) of individual ARN^KISS neurones to be assessed simultaneously, we examined whether the circadian neuropeptides vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) modulated the activity of ARN^KISS neurones directly. To validate this methodology, we initially evaluated the effects of neurokinin B (NKB) on [Ca²⁺]_i in kisspeptin neurones residing within the rostral, middle and caudal ARN subregions of adult male and female mice. All experiments were undertaken in the presence of tetrodotoxin and ionotropic amino acid antagonists. NKB was found to evoke an abrupt increase in [Ca²⁺]_i in 95%‐100% of kisspeptin neurones throughout the ARN of both sexes. By contrast, both VIP and AVP were found to primarily activate kisspeptin neurones located in the caudal ARN of female mice. Although 58% and 59% of caudal ARN kisspeptin neurones responded to AVP and VIP, respectively, in female mice, only 0%‐8% of kisspeptin neurones located in other ARN subregions responded in females and 0%‐12% of cells in any subregion in males (P < 0.05). These observations demonstrate unexpected sex differences and marked heterogeneity in functional neuropeptide receptor expression amongst ARN^KISS neurones organised on a rostro‐caudal basis. The functional significance of this unexpected influence of VIP and AVP on ARN^KISS neurones remains to be established.