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The amino acid transmitter γ-aminobutyric acid (GABA) is suspected to play an important role in regulating the activity of the gonadotropin-releasing hormone (GnRH) neurons controlling fertility. Rodent GnRH neurons have a novel dendritic compartment termed the " distal dendron " through which action potentials pass to the axon terminals and where inputs from the kisspeptin pulse generator drive pulsatile GnRH secretion. Combining Gnrh1- Cre mice with the Cre-dependent calcium sensor GCaMP6 and confocal imaging of acute brain slices, we examined whether GABA regulated intracellular calcium concentrations ([Ca2+]) in the GnRH neuron distal dendron. Short puffs of GABA on the dendron evoked either a monophasic sustained suppression of [Ca2+] or a biphasic acute elevation in [Ca2+] followed by the sustained suppression. Application of muscimol to the dendron replicated the acute elevation in [Ca2+] while baclofen generated the sustained suppression. Robust GABAB receptor-mediated inhibition was observed in 80% to 100% of dendrons recorded from females across the estrous cycle and from approximately 70% of dendrons in males. In contrast, the GABAA receptor–mediated excitation was rare in males and varied across the estrous cycle, being most prominent at proestrus. The activation of GABAB receptors potently suppressed the stimulatory effect of kisspeptin on the dendron. These observations demonstrate that the great majority of GnRH neuron distal dendrons are regulated by GABAergic inputs in a sex- and estrous cycle–dependent manner, with robust GABAB receptor-mediated inhibition being the primary mode of signaling. This provides a new, kisspeptin-independent, pathway for the regulation of pulsatile and surge modes of GnRH secretion in the rodent. [ABSTRACT FROM AUTHOR]

The amino acid transmitter γ-aminobutyric acid (GABA) is suspected to play an important role in regulating the activity of the gonadotropin-releasing hormone (GnRH) neurons controlling fertility. Rodent GnRH neurons have a novel dendritic compartment termed the " distal dendron " through which action potentials pass to the axon terminals and where inputs from the kisspeptin pulse generator drive pulsatile GnRH secretion. Combining Gnrh1- Cre mice with the Cre-dependent calcium sensor GCaMP6 and confocal imaging of acute brain slices, we examined whether GABA regulated intracellular calcium concentrations ([Ca2+]) in the GnRH neuron distal dendron. Short puffs of GABA on the dendron evoked either a monophasic sustained suppression of [Ca2+] or a biphasic acute elevation in [Ca2+] followed by the sustained suppression. Application of muscimol to the dendron replicated the acute elevation in [Ca2+] while baclofen generated the sustained suppression. Robust GABAB receptor-mediated inhibition was observed in 80% to 100% of dendrons recorded from females across the estrous cycle and from approximately 70% of dendrons in males. In contrast, the GABAA receptor–mediated excitation was rare in males and varied across the estrous cycle, being most prominent at proestrus. The activation of GABAB receptors potently suppressed the stimulatory effect of kisspeptin on the dendron. These observations demonstrate that the great majority of GnRH neuron distal dendrons are regulated by GABAergic inputs in a sex- and estrous cycle–dependent manner, with robust GABAB receptor-mediated inhibition being the primary mode of signaling. This provides a new, kisspeptin-independent, pathway for the regulation of pulsatile and surge modes of GnRH secretion in the rodent. [ABSTRACT FROM AUTHOR]

The recent use of the tail-tip bleeding approach in mice has enabled researchers to generate detailed pulse and surge profiles of luteinizing hormone (LH) secretion in mice. However, the analysis of pulsatile LH secretion is piecemeal across the field with each laboratory using their own methodology. We have reformulated the once-popular PULSAR algorithm of Merriam and Wachter to operate on contemporary computer systems and provide downloadable and online pulse analysis platforms. As it is now possible to record the activity of the gonadotropin-releasing hormone pulse generator in freely behaving mice, we have been able to unambiguously define LH pulses in intact and gonadectomized male and female mice. These data sets were used to determine the appropriate PULSAR parameter sets for analyzing pulsatile LH secretion in the mouse. This was then used to establish an accurate model of estrogen negative feedback in the mouse. Intact and ovariectomized mice given Silastic capsules containing 1, 2, and 4 μg 17-β-estradiol/20 g body weight were tail-tip bled at 6-min intervals, and the resultant LH profiles were analyzed with PULSAR. Only the 4 μg 17-β-estradiol capsule treatment was found to return LH pulse amplitude and frequency to that of intact diestrous mice. Ultrasensitive mass spectrometry analysis showed that the 4 μg 17-β-estradiol capsule generated circulating estradiol levels equivalent to that of diestrous mice. It is hoped that the reformulation of PULSAR and generation of a realistic model of estrogen-negative feedback will provide a platform for the more uniform assessment of pulsatile hormone secretion in mice. [ABSTRACT FROM AUTHOR]

Chronic stress exerts multiple negative effects on the physiology and health of an individual. In the present study, we examined hypothalamic, pituitary and endocrine responses to 14 days of chronic variable stress (CVS) in male and female C57BL/6J mice. In both sexes, CVS induced a significant decrease in body weight and enhanced the acute corticosterone stress response, which was accompanied by a reduction in thymus weight only in females. However, single‐point blood measurements of basal prolactin, thyroid‐stimulating hormone, luteinising hormone, growth hormone and corticosterone levels taken at the end of the CVS were not different from those of controls. Similarly, pituitary mRNA expression of Fshb, Lhb, Prl and Gh was unchanged by CVS, although Pomc and Tsh were significantly elevated. Within the adrenal medulla, mRNA for Th, Vip and Gal were elevated following CVS. Avp transcript levels within the paraventricular nucleus of the hypothalamus were increased by CVS; however, levels of Gnrh1, Crh, Oxt,Sst, Trh, Ghrh, Th and Kiss1 remained unchanged. Oestrous cycles were lengthened slightly by CVS and ovarian histology revealed a reduction in the number of preovulatory follicles and corpora lutea. Taken together, these observations indicate that 14 days of CVS induces an up‐regulation of the neuroendocrine stress axis and creates a mild disruption of female reproductive function. However, the lack of changes in other neuroendocrine axes controlling anterior and posterior pituitary secretion suggest that most neuroendocrine axes are relatively resilient to CVS. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The neural mechanisms generating pulsatile GnRH release from the median eminence (ME) remain unclear. Studies undertaken in the mouse demonstrate that GnRH neurons extend projections to the ME that have properties of both dendrites and axons, termed "dendrons," and that the kisspeptin neuron pulse generator targets these distal dendrons to drive pulsatile GnRH secretion. It presently remains unknown whether the GnRH neuron dendron exists in other species. We report here the generation of a knock-in Gnrh1-Ires-Cre rat line with near-perfect targeting of Cre recombinase to the GnRH neuronal phenotype. More than 90% of adult male and female GnRH neurons express Cre with no ectopic expression. Adeno-associated viruses were used in adult female Gnrh1-Ires-Cre rats to target mCherry or GCAMP6 to rostral preoptic area GnRH neurons. The mCherry tracer revealed the known unipolar and bipolar morphology of GnRH neurons and their principal projection pathways to the external zone of the ME. Synaptophysin-labeling of presynaptic nerve terminals revealed that GnRH neuron distal projections received numerous close appositions as they passed through the arcuate nucleus and into the median eminence. Confocal GCaMP6 imaging in acute horizontal brain slices demonstrated that GnRH neuron distal projections lateral to the median eminence were activated by kisspeptin. These studies indicate the presence of a dendron-like arrangement in the rat with GnRH neuron distal projections receiving synaptic input and responding to kisspeptin. [ABSTRACT FROM AUTHOR]

A population of kisspeptin-GABA coexpressing neurons located in the rostral periventricular area of the third ventricle (RP3V) is believed to activate gonadotropin-releasing hormone (GnRH) neurons to generate the luteinizing hormone (LH) surge triggering ovulation. Selective optogenetic activation of RP3V kisspeptin (RP3VKISS) neurons in female mice for >30 s and >10 Hz in either a continuous or bursting mode was found to reliably generate a delayed and long-lasting activation of GnRH neuron firing in brain slices. Optogenetic activation of RP3VKISS neurons in vivo at 10 Hz generated substantial increments in LH secretion of similar amplitude to the endogenous LH surge. Studies using GABAA receptor antagonists and optogenetic activation of RP3VGABA (RP3VGABA) neurons in vitro revealed that low-frequency (2 Hz) stimulation generated immediate and transient GABAA receptor-mediated increases in GnRH neuron firing, whereas higher frequencies (10 Hz) recruited the long-lasting activation observed following RP3VKISS neuron stimulation. In vivo, 2 Hz activation of RP3KISS neurons did not alter LH secretion, whereas 10 Hz stimulation evoked a sustained large increase in LH identical to RP3VKISS neuron activation. Optogenetic activation of RP3VKISS neurons in which kisspeptin had been deleted did not alter LH secretion. These studies demonstrate the presence of parallel transmission streams from RP3V neurons to GnRH neurons that are frequency dependent and temporally distinct. This comprises a rapid and transient GABAA receptor-mediated activation and a slower onset kisspeptin-mediated stimulation of long duration. At the time of the LH surge, GABA release appears to be functionally redundant with the neuropeptide kisspeptin being the dominant cotransmitter influencing GnRH neuron output. [ABSTRACT FROM AUTHOR]

Fast cell-to-cell communication in the brain is achieved by action potential-dependent synaptic release of neurotransmitters. The fast kinetics of transmitter release are determined by transient Ca2+ elevations in presynaptic nerve terminals. Neuromodulators have previously been shown to regulate transmitter release by inhibiting presynaptic Ca2+ influx. Few studies to date have demonstrated the opposite, that is, neuromodulators directly driving presynaptic Ca2+ rises and increases in nerve terminal excitability. Here we use GCaMP Ca2+ imaging in brain slices from mice to address how nerve terminal Ca2+ is controlled in gonadotropin-releasing hormone (GnRH) neurons via action potentials and neuromodulators. Single spikes and bursts of action potentials evoked fast, voltage-gated Ca2+ channel-dependent Ca2+ elevations. In contrast, brief exposure to the neuropeptide kisspeptin-evoked long-lasting Ca2+ plateaus that persisted for tens of minutes. Neuropeptide-mediated Ca2+ elevations were independent of action potentials, requiring Ca2+ entry via voltage-gated Ca2+ channels and transient receptor potential channels in addition to release from intracellular store mechanisms. Together, these data reveal that neuromodulators can exert powerful and long-lasting regulation of nerve terminal Ca2+ independently from actions at the soma. Thus, GnRH nerve terminal function is controlled over disparate timescales via both classical spike-dependent and nonclassical neuropeptide-dependent mechanisms [ABSTRACT FROM AUTHOR]

Circulating estradiol exerts a profound influence on the activity of the gonadotropin-releasing hormone (GnRH) neuronal network controlling fertility. Using genetic strategies enabling neuron-specific deletion of estrogen receptor α (Esr1), we examine here whether estradiol-modulated GABA and glutamate transmission are critical for the functioning of the GnRH neuron network in the female mouse. Using Vgat- and Vglut2-ires-Cre knock-in mice and ESR1 immunohistochemistry, we demonstrate that subpopulations of GABA and glutamate neurons throughout the limbic forebrain express ESR1, with ESR1-GABAergic neurons being more widespread and numerous than ESR1-glutamatergic neurons. We crossed Vgat- and Vglut2-ires-Cre mice with an Esr1lox/lox line to generate animals with GABAneuron-specific or glutamate-neuron-specific deletion of Esr1. Vgat-ires-Cre;Esr1lox/lox mice were infertile, with abnormal estrous cycles, and exhibited a complete failure of the estrogen positive feedback mechanism responsible for the preovulatory GnRH surge. However, puberty onset and estrogen negative feedback were normal. Vglut2-ires-Cre;Esr1lox/lox mice were also infertile but displayed a wider range of deficits, including advanced puberty onset, abnormal negative feedback, and abolished positive feedback. Whereasα25% of preoptic kisspeptin neurons expressed Cre in Vgat- and Vglut2-ires-Cre lines,∼70% of arcuate kisspeptin neurons were targeted in Vglut2-ires- Cre;Esr1lox/lox mice, possibly contributing to their advanced puberty phenotype. These observations show that, unexpectedly, ESR1-GABAneurons are only essential for the positive feedback mechanism. In contrast, we reveal the key importance of ESR1 in glutamatergic neurons for multiple estrogen feedback loops within the GnRH neuronal network required for fertility in the female mouse. [ABSTRACT FROM AUTHOR]

The anteroventral periventricular nucleus (AVPV) is thought to play a key role in regulating the excitability of gonadotropin-releasing hormone (GnRH) neurons that control fertility. Using an angled, parahorizontal brain slice preparation we have undertaken a series of electrophysiological experiments to examine how theAVPVcontrolsGnRHneurons in adult male and female mice. More than half (59%) of GnRH neurons located in the rostral preoptic area were found to receive monosynaptic inputs from the AVPV in a sex-dependent manner. AVPV stimulation frequencies <1 Hz generated short-latency action potentials in GnRH neurons with GABA and glutamate mediating>90% of the evoked fast synaptic currents. TheAVPVGABAinput was dominant and found to excite or inhibitGnRHneurons in a cell-dependent manner. Increasing theAVPVstimulation frequency to 5-10Hzresulted in the appearance of additional poststimulus inhibitory as well as delayed excitatory responses in GnRH neurons that were independent of ionotropic amino acid receptors. The inhibition observed immediately following the end of the stimulation period was mediated partly by GABAB receptors, while the delayed activation was mediated by the neuropeptide kisspeptin. The latter response was essentially absent in Gpr54 knock-out mice and abolished by a Gpr54 antagonist. Together, these studies show that AVPV neurons provide direct amino acid and neuropeptidergic inputs to GnRH neurons. Low-frequency activation generates predominant GABA/glutamate release with higher frequency activation recruiting release of kisspeptin. This frequency-dependent release of amino acid and neuropeptide neurotransmitters greatly expands the range of AVPV control of GnRH neuron excitability. [ABSTRACT FROM AUTHOR]

The gonadotropin-releasing hormone (GnRH) neurons exhibit substantial functional, anatomical, and molecular heterogeneity, which has hampered their thorough examination. This study was undertaken in an effort to understand whether the anatomical distribution of GnRH neurons is related to their developmental history, because such an association may help explain differences within the population. Using bromodeoxyuridine pulse labeling of timed pregnant female mice we labeled dividing cells, including GnRH neuron progenitors in the olfactory placode, throughout the window of GnRH neuron differentiation. Our results indicate that cells that become postmitotic early tend to populate the rostral aspects of the adult GnRH neuron continuum, whereas later-generated cells tend to settle more caudally; an inside-out pattern reminiscent of neocortex. These observations suggest that the timing of differentiation influences the ability of postmitotic GnRH neurons to navigate to their adult location, and hence may be important in determining the ultimate wiring of the adult network. Developmental Dynamics 238:524-531, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The amino acid transmitter γ-aminobutyric acid (GABA) is suspected to play an important role in regulating the activity of the gonadotropin-releasing hormone (GnRH) neurons controlling fertility. Rodent GnRH neurons have a novel dendritic compartment termed the "distal dendron" through which action potentials pass to the axon terminals and where inputs from the kisspeptin pulse generator drive pulsatile GnRH secretion. Combining Gnrh1-Cre mice with the Cre-dependent calcium sensor GCaMP6 and confocal imaging of acute brain slices, we examined whether GABA regulated intracellular calcium concentrations ([Ca2+]) in the GnRH neuron distal dendron. Short puffs of GABA on the dendron evoked either a monophasic sustained suppression of [Ca2+] or a biphasic acute elevation in [Ca2+] followed by the sustained suppression. Application of muscimol to the dendron replicated the acute elevation in [Ca2+] while baclofen generated the sustained suppression. Robust GABAB receptor-mediated inhibition was observed in 80% to 100% of dendrons recorded from females across the estrous cycle and from approximately 70% of dendrons in males. In contrast, the GABAA receptor-mediated excitation was rare in males and varied across the estrous cycle, being most prominent at proestrus. The activation of GABAB receptors potently suppressed the stimulatory effect of kisspeptin on the dendron. These observations demonstrate that the great majority of GnRH neuron distal dendrons are regulated by GABAergic inputs in a sex- and estrous cycle-dependent manner, with robust GABAB receptor-mediated inhibition being the primary mode of signaling. This provides a new, kisspeptin-independent, pathway for the regulation of pulsatile and surge modes of GnRH secretion in the rodent., (© The Author(s) 2022.Published by Oxford University Press on behalf of the Endocrine Society.)

The roles of kisspeptin signaling outside the hypothalamus in the brain are unknown. We examined here the impact of Kiss1r-deletion on hippocampus-related behaviors of anxiety and spatial learning in adult male mice using two mouse models. In the first, global Kiss1r-null and control mice were gonadectomized (GDX KISS1R-KO). In the second, KISS1R signalling was rescued selectively in gonadotropin-releasing hormone neurons to generate Kiss1r-null mice with normal testosterone levels (intact KISS1R-KO). Intact KISS1R-KO rescue mice were found to spend twice as much time in the open arms of the elevated plus maze (EPM) compared to controls (P < 0.01). GDX KISS1R-KO mice showed a similar but less pronounced trend. No differences were detected between intact KISS1R-KO mice and controls in the open field test (OFT), although a marked reduction in time spent in the centre quadrant was observed for all GDX mice (P < 0.001). No effects of KISS1R deletion or gonadectomy were detected in the Morris water maze. These observations demonstrate that KISS1R signalling impacts upon anxiogenic neural circuits operative in the EPM, while gonadal steroids appear important for anxiety behaviour observed in the OFT. The potential anxiogenic role of kisspeptin may need to be considered in the development of kisspeptin analogs for the clinic.

Inputs from GABAergic and glutamatergic neurons are suspected to play an important role in regulating the activity of the gonadotropin-releasing hormone (GnRH) neurons. The GnRH neurons exhibit marked plasticity to control the ovarian cycle with circulating estradiol concentrations having profound "feedback" effects on their activity. This includes "negative feedback" responsible for suppressing GnRH neuron activity and "positive feedback" that occurs at mid-cycle to activate the GnRH neurons to generate the preovulatory luteinizing hormone surge. In the present study, we employed brain slice electrophysiology to question whether synaptic ionotropic GABA and glutamate receptor signaling at the GnRH neuron changed at times of negative and positive feedback. We used a well characterized estradiol (E)-treated ovariectomized (OVX) mouse model to replicate negative and positive feedback. Miniature and spontaneous postsynaptic currents (mPSCs and sPSCs) attributable to GABA A and glutamatergic receptor signaling were recorded from GnRH neurons obtained from intact diestrous, OVX, OVX + E (negative feedback), and OVX + E+E (positive feedback) female mice. Approximately 90% of GnRH neurons exhibited spontaneous GABA A -mPSCs in all groups but no significant differences in the frequency or kinetics of mPSCs were found at the times of negative or positive feedback. Approximately 50% of GnRH neurons exhibited spontaneous glutamate mPSCs but again no differences were detected. The same was true for spontaneous PSCs in all cases. These observations indicate that the kinetics of ionotropic GABA and glutamate receptor synaptic transmission to GnRH neurons remain stable across the different estrogen feedback states.

Using a new tail-tip bleeding procedure and a sensitive ELISA, we describe here the patterns of LH secretion throughout the mouse estrous cycle; in ovariectomized mice; in ovariectomized, estradiol-treated mice that model estrogen-negative and -positive feedback; and in transgenic GNR23 mice that exhibit allele-dependent reductions in GnRH neuron number. Pulsatile LH secretion was evident at all stages of the estrous cycle, with LH pulse frequency being approximately one pulse per hour in metestrous, diestrous, and proestrous mice but much less frequent at estrus (less than one pulse per 4 h). Ovariectomy resulted in substantial increases in basal and pulsatile LH secretion with pulses occurring approximately every 21 minutes. Chronic treatment with negative-feedback, estradiol-filled capsules returned LH pulse frequency to intact follicular phase levels, although pulse amplitude remained elevated. On the afternoon of proestrus, the LH surge was found to begin in a highly variable manner over a 4-hour range, lasting for more than 3 hours. In contrast, ovariectomized, estradiol-treated, positive-feedback mice exhibited a relatively uniform surge onset at approximately 0.5 hour prior to lights out. Gonadectomized wild-type and heterozygous GNR23 (∼200 GnRH neurons) male mice exhibited an LH pulse every 60 minutes. Homozygous GNR23 mice (∼80 GnRH neurons) had very low basal LH concentrations but continued to exhibit small amplitude LH pulses every 90 minutes. These studies provide the first characterization in mice of pulse and surge modes of LH secretion across the estrous cycle and demonstrate that very few GnRH neurons are required for pulsatile LH secretion.