The Role of Kiss1 Neurons As Integrators of Endocrine, Metabolic, and Environmental Factors in the Hypothalamic-Pituitary-Gonadal Axis.

Kisspeptin-GPR54 signaling in the hypothalamus is required for reproduction and fertility in mammals. Kiss1 neurons are key regulators of gonadotropin-releasing hormone (GnRH) release and modulation of the hypothalamic-pituitary-gonadal (HPG) axis. Arcuate Kiss1 neurons project to GnRH nerve terminals in the median eminence, orchestrating the pulsatile secretion of luteinizing hormone (LH) through the intricate interaction between GnRH pulse frequency and the pituitary gonadotrophs. Arcuate Kiss1 neurons, also known as KNDy neurons in rodents and ruminants because of their co-expression of neurokinin B and dynorphin represent an ideal hub to receive afferent inputs from other brain regions in response to physiological and environmental changes, which can regulate the HPG axis. This review will focus on studies performed primarily in rodent and ruminant species to explore potential afferent inputs to Kiss1 neurons with emphasis on the arcuate region but also considering the rostral periventricular region of the third ventricle (RP3V). Specifically, we will discuss how these inputs can be modulated by hormonal, metabolic, and environmental factors to control gonadotropin secretion and fertility. We also summarize the methods and techniques that can be used to study functional inputs into Kiss1 neurons

Mapping neuronal inputs to Kiss1 neurons in the arcuate nucleus of the mouse.

The normal function of the mammalian reproductive axis is strongly influenced by physiological, metabolic and environmental factors. Kisspeptin neuropeptides, encoded by the Kiss1 gene, are potent regulators of the mammalian reproductive axis by stimulating gonadodropin releasing hormone secretion from the hypothalamus. To understand how the reproductive axis is modulated by higher order neuronal inputs we have mapped the afferent circuits into arcuate (ARC) Kiss1 neurons. We used a transgenic mouse that expresses the CRE recombinase in Kiss1 neurons for conditional viral tracing with genetically modified viruses. CRE-mediated activation of these viruses in Kiss1 neurons allows the virus to move transynaptically to label neurons with primary or secondary afferent inputs into the Kiss1 neurons. Several regions of the brain showed synaptic connectivity to arcuate Kiss1 neurons including proopiomelanocortin neurons in the ARC itself, kisspeptin neurons in the anteroventral periventricular nucleus, vasopressin neurons in the supraoptic and suprachiasmatic nuclei, thyrotropin releasing neurons in the paraventricular nucleus and unidentified neurons in other regions including the subfornical organ, amygdala, interpeduncular nucleus, ventral premammilary nucleus, basal nucleus of stria terminalis and the visual, somatosensory and piriform regions of the cortex. These data provide an insight into how the activity of Kiss1 neurons may be regulated by metabolic signals and provide a detailed neuroanatomical map for future functional studies

Morphological assessment of GABA and glutamate inputs to GnRH neurons in intact female mice using expansion microscopy

Funder: Wellcome TrustFunder: Health Research Council of New Zealand; Id: http://dx.doi.org/10.13039/501100001505Abstract: The roles GABAergic and glutamatergic inputs in regulating the activity of the gonadotrophin‐releasing hormone (GnRH) neurons at the time of the preovulatory surge remain unclear. We used expansion microscopy to compare the density of GABAergic and glutamatergic synapses on the GnRH neuron cell body and proximal dendrite in dioestrous and pro‐oestrous female mice. An evaluation of all synapses immunoreactive for synaptophysin revealed that the highest density of inputs to rostral preoptic area GnRH neurons occurred within the first 45 µm of the primary dendrite (approximately 0.19 synapses µm‐1) with relatively few synapses on the GnRH neuron soma or beyond 45 µm of the dendrite (0.05‐0.08 synapses µm‐1). Triple immunofluorescence labelling demonstrated a predominance of glutamatergic signalling with twice as many vesicular glutamate transporter 2 synapses detected compared to vesicular GABA transporter. Co‐labelling with the GABAA receptor scaffold protein gephyrin and the glutamate receptor postsynaptic density marker Homer1 confirmed these observations, as well as the different spatial distribution of GABA and glutamate inputs along the dendrite. Quantitative assessments revealed no differences in synaptophysin, GABA or glutamate synapses at the proximal dendrite and soma of GnRH neurons between dioestrous and pro‐oestrous mice. Taken together, these studies demonstrate that the GnRH neuron receives twice as many glutamatergic synapses compared to GABAergic synapses and that these inputs preferentially target the first 45 µm of the GnRH neuron proximal dendrite. These inputs appear to be structurally stable before the onset of pro‐oestrous GnRH surge

Kv4.2 channel activity controls intrinsic firing dynamics of arcuate kisspeptin neurons.

KEY POINTS: Neurons in the hypothalamus of the brain which secrete the peptide kisspeptin are important regulators of reproduction, and normal reproductive development. Electrical activity, in the form of action potentials, or spikes, leads to secretion of peptides and neurotransmitters, influencing the activity of downstream neurons; in kisspeptin neurons, this activity is highly irregular, but the mechanism of this is not known. In this study, we show that irregularity depends on the presence of a particular type of potassium ion channel in the membrane, which opens transiently in response to electrical excitation. The results contribute to understanding how kisspeptin neurons generate and time their membrane potential spikes, and how reliable this process is. Improved understanding of the activity of kisspeptin neurons, and how it shapes their secretion of peptides, is expected to lead to better treatment for reproductive dysfunction and disorders of reproductive development. ABSTRACT: Kisspeptin neurons in the hypothalamus are critically involved in reproductive function, via their effect on GnRH neuron activity and consequent gonadotropin release. Kisspeptin neurons show an intrinsic irregularity of firing, but the mechanism of this remains unclear. To address this, we carried out targeted whole-cell patch-clamp recordings of kisspeptin neurons in the arcuate nucleus (Kiss1Arc ), in brain slices isolated from adult male Kiss-Cre:tdTomato mice. Cells fired irregularly in response to constant current stimuli, with a wide range of spike time variability, and prominent subthreshold voltage fluctuations. In voltage clamp, both a persistent sodium (NaP) current and a fast transient (A-type) potassium current were apparent, activating at potentials just below the threshold for spiking. These currents have also previously been described in irregular-spiking cortical interneurons, in which the A-type current, mediated by Kv4 channels, interacts with NaP current to generate complex dynamics of the membrane potential, and irregular firing. In Kiss1Arc neurons, A-type current was blocked by phrixotoxin, a specific blocker of Kv4.2/4.3 channels, and consistent expression of Kv4.2 transcripts was detected by single-cell RT-PCR. In addition, firing irregularity was correlated to the density of A-type current in the membrane. Using conductance injection, we demonstrated that adding Kv4-like potassium conductance (gKv4 ) to a cell produces a striking increase in firing irregularity, and excitability is reduced, while subtracting gKv4 has the opposite effects. Thus, we propose that Kv4 interacting dynamically with NaP is a key determinant of the irregular firing behaviour of Kiss1Arc neurons, shaping their physiological function in gonadotropin release

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

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

Boletín Oficial de la Provincia de Oviedo: Número 252 - 1932 octubre 26

Reproduction and fertility in mammals is critically dependent on the neurosecretion of gonadotrophin-releasing hormone (GnRH) released by the hypothalamic GnRH neurons into the hypophyseal portal system of the anterior pituitary. GnRH binds to GnRH receptors on the pituitary gonadotrophs and directs gonadotrophin secretion into the peripheral circulation. The oestrogen negative feedback mechanism is important in controlling the fluctuating profiles of pulsatile GnRH and luteinizing hormone (LH) release. For the major part of the oestrous cycle, oestrogen exerts negative feedback actions on both GnRH neurons and pituitary gonadotrophs to suppress GnRH release and ultimately restrain LH secretion at its nadir. The precise neurobiological mechanisms underlying oestrogen’s inhibitory effects on GnRH/LH release are largely unknown. It is hypothesised that kisspeptin neurons are required to mediate oestrogen negative feedback actions to suppress pulsatile GnRH/LH secretion. This thesis focused on investigating the role of arcuate nucleus (ARN) kisspeptin neurons in mediating oestrogen negative feedback actions on GnRH neurons in adult female mice. Anterograde tract tracing was conducted to determine the projections of both ARN and rostral periventricular area of the third ventricle (RP3V) kisspeptin neurons. Retrograde tracing was performed to verify the axonal projections of ARN and RP3V kisspeptin neurons into the rostral preoptic area and ARN, respectively. The ARN kisspeptin neurons were found to project widely within both the medial and lateral aspects of the hypothalamus and associated limbic structures, and possibly innervate GnRH neuron cell bodies or dendrites in the rostral preoptic area. The RP3V kisspeptin neurons exhibited a more medial projection pattern throughout the brain and were found to provide substantial inputs into the ARN. The diversity of brain regions targeted by kisspeptin neurons suggests their involvement in regulating multiple circuits in addition to the GnRH neuronal network. Despite the wide projections of kisspeptin neurons into various regions, none of the kisspeptin neuron populations project to the external zone of the median eminence (ME). Together, these data indicate that ARN and RP3V kisspeptin neurons do not innervate GnRH nerve terminals in the external zone of the ME. It is possible that ARN kisspeptin neurons regulate GnRH nerve terminals through a nonsynaptic interaction, whereby the kisspeptin may be released from terminals in the internal zone of the ME and act through volume transmission to eventually reach and control GnRH release in the external zone. Transgenic mouse models harbouring global and neuron-specific deletions of Gpr54 gene were examined for oestrogen negative feedback dynamics to assess the role of kisspeptin-Gpr54 signalling in mediating oestrogen negative feedback. Both animal models exhibited abnormal oestrogen negative feedback profiles with the absence of postovariectomy LH elevations. This implied that kisspeptin neuron input may be required for the generation of high-amplitude GnRH pulses to stimulate rapid LH secretion from the pituitary after ovariectomy. Finally, the role of ARN oestrogen receptor alpha (ERα)-expressing neurons in mediating oestrogen negative feedback was assessed by using adeno-associated virus (AAV)-mediated Cre/loxP recombination to induce ERα deletion specifically in the ARN. The results indicate that ARN ERα-expressing neurons are essential for oestrous cyclicity and appear to be important components of the multimodal negative feedback pathways. Overall, the ARN kisspeptin neurons are unlikely the key neurons conveying oestrogen negative feedback on GnRH pulses/LH secretion. Instead, they are part of the network modulating high amplitude GnRH pulses stimulating LH secretion in the absence of oestrogen inhibitory effects

Mapping neuronal inputs to Kiss1 neurons in the arcuate nucleus of the mouse.

The normal function of the mammalian reproductive axis is strongly influenced by physiological, metabolic and environmental factors. Kisspeptin neuropeptides, encoded by the Kiss1 gene, are potent regulators of the mammalian reproductive axis by stimulating gonadodropin releasing hormone secretion from the hypothalamus. To understand how the reproductive axis is modulated by higher order neuronal inputs we have mapped the afferent circuits into arcuate (ARC) Kiss1 neurons. We used a transgenic mouse that expresses the CRE recombinase in Kiss1 neurons for conditional viral tracing with genetically modified viruses. CRE-mediated activation of these viruses in Kiss1 neurons allows the virus to move transynaptically to label neurons with primary or secondary afferent inputs into the Kiss1 neurons. Several regions of the brain showed synaptic connectivity to arcuate Kiss1 neurons including proopiomelanocortin neurons in the ARC itself, kisspeptin neurons in the anteroventral periventricular nucleus, vasopressin neurons in the supraoptic and suprachiasmatic nuclei, thyrotropin releasing neurons in the paraventricular nucleus and unidentified neurons in other regions including the subfornical organ, amygdala, interpeduncular nucleus, ventral premammilary nucleus, basal nucleus of stria terminalis and the visual, somatosensory and piriform regions of the cortex. These data provide an insight into how the activity of Kiss1 neurons may be regulated by metabolic signals and provide a detailed neuroanatomical map for future functional studies

Sexually dimorphic gene expression and neurite sensitivity to estradiol in fetal arcuate Kiss1 cells

International audienceKiss1 neurons of the arcuate (ARC) nucleus form an interconnected network of cells that communicate via neurokinin B (encoded by Tac2) and its receptor (encoded by Tacr3) and play key roles in the control of the reproductive axis through sex hormone-regulated synthesis and release of kisspeptin peptides (Kp, encoded by Kiss1). The aim of this study was to determine whether the Kiss1 cell population of the ARC already displays sexually dimorphic features at embryonic age E16.5 in mice. At this time of development, Kiss1-GFP- and Kp-immunoreactive cell bodies were restricted to the ARC and not found in the pre-optic area (POA). The Kiss1-GFP cell population was identical in size between sexes but had significantly lower Kiss1, Tac2, and Tacr3 mRNA levels and lower Kp-ir fiber density in the POA in male compared to female fetuses. Receptors for androgen (Ar) and estrogen (Esr1, Esr2, Gpr30) and the Cyp19a1 gene (encoding the estradiol-producing enzyme aromatase) transcripts were also detected in fetal ARC Kiss1-GFP cells with significant sex differences for Ar (higher in males) and Esr1 (higher in females). Functional studies on primary cultures of sorted fetal Kiss1-GFP cells revealed a significant negative effect of estradiol treatment on neurite outgrowth on the fourth day of culture in the female group specifically. We conclude that the ARC Kiss1 cell population is already sexually differentiated at E16.5 and that its morphogenetic development may be particularly vulnerable to estradiol exposure at this early developmental time

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