Robust GABAergic Regulation of the GnRH Neuron Distal Dendron.

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.

Altered membrane properties but unchanged intrinsic excitability and spontaneous postsynaptic currents in an aged APP swe /PS1dE9 model of Alzheimer's disease.

Neuronal hyperexcitability in Alzheimer's disease (AD) models is thought to either contribute to the formation of amyloid beta plaques or result from their formation. Neuronal hyperexcitability has been shown in the cerebral cortex of the widely used young APPswe/PS1dE9 mice, which have accelerated plaque formation. However, it is currently unclear if hyperexcitability also occurs in CA1 hippocampal neurons of aged animals in this model. In the present work, we have compared intrinsic excitability and spontaneous synaptic inputs from CA1 pyramidal cells of 8-month-old APPswe/PS1dE9 and wildtype control mice. We find no change in intrinsic excitability or spontaneous postsynaptic currents (PSCs) between groups. We did, however, find a reduced input resistance and an increase in hyperpolarization-activated sag current. These results are consistent with findings from other aged AD model mice, including the widely used 5xFAD and 3xTg. Together these results suggest that neuronal hyperexcitability is not a consistent feature of all AD mouse models, particularly at advanced ages.

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.

Innervation of GnRH Neuron Distal Projections and Activation by Kisspeptin in a New GnRH-Cre Rat Model.

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.

Direct inhibition of arcuate kisspeptin neurones by neuropeptide Y in the male and female mouse.

Adverse energy states exert a potent suppressive influence on the reproductive axis by inhibiting the pulsatile release of gonadotrophin-releasing hormone and luteinising hormone. One potential mechanism underlying this involves the metabolic-sensing pro-opiomelanocortin and agouti-related peptide/neuropeptide Y (AgRP/NPY) neuronal populations directly controlling the activity of the arcuate nucleus kisspeptin neurones comprising the gonadotrophin-releasing hormone pulse generator. Using acute brain slice electrophysiology and calcium imaging approaches in Kiss1-GFP and Kiss1-GCaMP6 mice, we investigated whether NPY and α-melanocyte-stimulating hormone provide a direct modulatory influence on the activity of arcuate kisspeptin neurones in the adult mouse. NPY was found to exert a potent suppressive influence upon the neurokinin B-evoked firing of approximately one-half of arcuate kisspeptin neurones in both sexes. This effect was blocked partially by the NPY1R antagonist BIBO 3304, whereas the NPY5R antagonist L152,804 was ineffective. NPY also suppressed the neurokinin B-evoked increase in intracellular calcium levels in the presence of tetrodotoxin and amino acid receptor antagonists, indicating that the inhibitory effects of NPY are direct on kisspeptin neurones. By contrast, no effects of α-melanocyte-stimulating hormone were found on the excitability of arcuate kisspeptin neurones. These studies provide further evidence supporting the hypothesis that AgRP/NPY neurones link energy status and luteinising hormone pulsatility by demonstrating that NPY has a direct suppressive influence upon the activity of a subpopulation of arcuate kisspeptin neurones.

Dynamics of GnRH Neuron Ionotropic GABA and Glutamate Synaptic Receptors Are Unchanged during Estrogen Positive and Negative Feedback in Female Mice.

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 GABAA 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 GABAA-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.

Kisspeptin Regulation of Neuronal Activity throughout the Central Nervous System.

Kisspeptin signaling at the gonadotropin-releasing hormone (GnRH) neuron is now relatively well characterized and established as being critical for the neural control of fertility. However, kisspeptin fibers and the kisspeptin receptor (KISS1R) are detected throughout the brain suggesting that kisspeptin is involved in regulating the activity of multiple neuronal circuits. We provide here a review of kisspeptin actions on neuronal populations throughout the brain including the magnocellular oxytocin and vasopressin neurons, and cells within the arcuate nucleus, hippocampus, and amygdala. The actions of kisspeptin in these brain regions are compared to its effects upon GnRH neurons. Two major themes arise from this analysis. First, it is apparent that kisspeptin signaling through KISS1R at the GnRH neuron is a unique, extremely potent form or neurotransmission whereas kisspeptin actions through KISS1R in other brain regions exhibit neuromodulatory actions typical of other neuropeptides. Second, it is becoming increasingly likely that kisspeptin acts as a neuromodulator not only through KISS1R but also through other RFamide receptors such as the neuropeptide FF receptors (NPFFRs). We suggest likely locations of kisspeptin signaling through NPFFRs but note that only limited tools are presently available for examining kisspeptin cross-signaling within the RFamide family of neuropeptides.

Novel role for anti-Müllerian hormone in the regulation of GnRH neuron excitability and hormone secretion.

Anti-Müllerian hormone (AMH) plays crucial roles in sexual differentiation and gonadal functions. However, the possible extragonadal effects of AMH on the hypothalamic-pituitary-gonadal axis remain unexplored. Here we demonstrate that a significant subset of GnRH neurons both in mice and humans express the AMH receptor, and that AMH potently activates the GnRH neuron firing in mice. Combining in vivo and in vitro experiments, we show that AMH increases GnRH-dependent LH pulsatility and secretion, supporting a central action of AMH on GnRH neurons. Increased LH pulsatility is an important pathophysiological feature in many cases of polycystic ovary syndrome (PCOS), the most common cause of female infertility, in which circulating AMH levels are also often elevated. However, the origin of this dysregulation remains unknown. Our findings raise the intriguing hypothesis that AMH-dependent regulation of GnRH release could be involved in the pathophysiology of fertility and could hold therapeutic potential for treating PCOS.

Kisspeptin regulation of arcuate neuron excitability in kisspeptin receptor knockout mice.

The G protein-coupled receptor 54 (GPR54) is critical for kisspeptin to activate GnRH neurons to modulate fertility. However, the often mismatching distribution of kisspeptin and GPR54 in the brain suggests that kisspeptin may also act on other receptors. The arcuate nucleus (ARN) is one brain region with a very high density of kisspeptin fibers but only limited evidence for the expression of GPR54. Using acute brain slice electrophysiology in combination with Gpr54 knockout (GPR54KO) mouse models, we examined whether actions of kisspeptin in the ARN were dependent upon GPR54. Cell-attached recordings from unidentified ARN neurons in wild-type mice revealed that approximately one third of neurons were either excited or inhibited by kisspeptin in a dose-dependent manner. The responses of ARN neurons to kisspeptin were exactly the same in GPR54KO mice despite effects of kisspeptin on GnRH neurons being abolished. To evaluate whether kisspeptin may be acting through neuropeptide FF receptors, the effects of an agonist RFamide-related peptide 3 (RFRP-3) and antagonists RF9 and BIBP-3226 were evaluated. Both the excitatory and inhibitory effects of kisspeptin were mimicked by the agonist RFRP-3. RF9 itself activated ARN neurons and suppressed only the inhibitory actions of kisspeptin. BIBP-3226 suppressed kisspeptin actions in 50% of neurons. Whole-cell recordings in GPR54KO mice demonstrated that both kisspeptin and RFRP-3 acted directly on the same ARN neurons and activated the same ion channels. Together, these studies demonstrate that kisspeptin can act partly through neuropeptide FF receptors to modulate neuronal activity independent of GPR54 in the mouse brain.

Electrical properties of kisspeptin neurons and their regulation of GnRH neurons.

Kisspeptin neurons are critical components of the neuronal network controlling the activity of the gonadotropin-releasing hormone (GnRH) neurons. A variety of genetically-manipulated mouse models have recently facilitated the study of the electrical activity of the two principal kisspeptin neuron populations located in the rostral periventricular area of the third ventricle (RP3V) and arcuate nucleus (ARN) in acute brain slices. We discuss here the mechanisms and pathways through which kisspeptin neurons regulate GnRH neuron activity. We then examine the different kisspeptin-green fluorescent protein mouse models being used for kisspeptin electrophysiology and the data obtained to date for RP3V and ARN kisspeptin neurons. In light of these new observations on the spontaneous firing rates, intrinsic membrane properties, and neurotransmitter regulation of kisspeptin neurons, we speculate on the physiological roles of the different kisspeptin populations.

RF9 excitation of GnRH neurons is dependent upon Kiss1r in the adult male and female mouse.

The neuropeptide FF receptor antagonist 1-adamantanecarbonyl-Arg-Phe-NH2 trifluoroacetate salt (RF9) has been found to be a remarkably potent activator of gonadotropin secretion in mammals. However, the mechanism of RF9 action on the reproductive axis is unknown. Using acute brain slice electrophysiology in genetically modified mouse models, we have investigated the possibility that RF9 may activate GnRH neurons. In transgenic GnRH-GFP male and female mice, RF9 was found to exert potent, dose-dependent, stimulatory effects on the firing rate of approximately 70% of GnRH neurons. These effects occurred directly on GnRH neurons and were independent of fast amino acid transmission. To assess RF9's action as an neuropeptide FF receptor antagonist at the GnRH neuron, its ability to antagonize the inhibitory effects of RFamide-related peptide-3 on GnRH neuron firing was examined. RF9 exhibited variable ability to prevent the inhibitory effects of RFamide-related peptide-3 on GnRH neurons. Whole-cell recordings from GnRH neurons showed that RF9 generated an inward current in GnRH neurons reminiscent of that evoked by kisspeptin. We therefore examined RF9 actions in kisspeptin receptor knockout mice. RF9 was found to have no effects at all on GnRH neurons in GnRH-GFP;Kiss1r-null mice, although these cells exhibited normal intrinsic electrical properties and remained responsive to GABA and glutamate. This study reveals that RF9 directly activates GnRH neurons in the mouse and that this is dependent upon Kiss1r expression.

Dependence of fertility on kisspeptin-Gpr54 signaling at the GnRH neuron.

Signaling between kisspeptin and its receptor, G-protein-coupled receptor 54 (Gpr54), is now recognized as being essential for normal fertility. However, the key cellular location of kisspeptin-Gpr54 signaling is unknown. Here we create a mouse with a GnRH neuron-specific deletion of Gpr54 to assess the role of gonadotropin-releasing hormone (GnRH) neurons. Mutant mice are infertile, fail to go through puberty and exhibit markedly reduced gonadal size and follicle-stimulating hormone levels alongside GnRH neurons that are unresponsive to kisspeptin. In an attempt to rescue the infertile phenotype of global Gpr54⁻/⁻ mutants, we use BAC transgenesis to target Gpr54 to the GnRH neurons. This results in mice with normal puberty onset, estrous cyclicity, fecundity and a recovery of kisspeptin's stimulatory action upon GnRH neurons. Using complimentary cell-specific knockout and knockin approaches we demonstrate here that the GnRH neuron is the key site of kisspeptin-Gpr54 signaling for fertility.

Dopamine regulation of gonadotropin-releasing hormone neuron excitability in male and female mice.

Numerous in vivo studies have shown that dopamine is involved in the regulation of LH secretion in mammals. However, the mechanisms through which this occurs are not known. In this study, we used green fluorescent protein-tagged GnRH neurons to examine whether and how dopamine may modulate the activity of adult GnRH neurons in the mouse. Bath-applied dopamine (10-80 µm) potently inhibited the firing of approximately 50% of GnRH neurons. This resulted from direct postsynaptic inhibitory actions through D1-like, D2-like, or both receptors. Further, one third of GnRH neurons exhibited an increase in their basal firing rate after administration of SCH23390 (D1-like antagonist) and/or raclopride (D2-like antagonist) indicating tonic inhibition by endogenous dopamine in the brain slice. The role of dopamine in presynaptic modulation of the anteroventral periventricular nucleus (AVPV) γ-aminobutyric acid/glutamate input to GnRH neurons was examined. Exogenous dopamine was found to presynaptically inhibit AVPV-evoked γ-aminobutyric acid /glutamate postsynaptic currents in about 50% of GnRH neurons. These effects were, again, mediated by both D1- and D2-like receptors. Neither postsynaptic nor presynaptic actions of dopamine were found to be different between diestrous, proestrous, and estrous females, or males. Approximately 20% of GnRH neurons were shown to receive a dopaminergic input from AVPV neurons in male and female mice. Together, these observations show that dopamine is one of the most potent inhibitors of GnRH neuron excitability and that this is achieved through complex pre- and postsynaptic actions that each involve D1- and D2-like receptor activation.

Estrous cycle- and sex-dependent changes in pre- and postsynaptic GABAB control of GnRH neuron excitability.

The GnRH neurons are the key neurons controlling fertility in mammals. Although γ-aminobutyric acid (GABA) plays an important role in the regulation of GnRH neurons, the role of GABA(B) receptors is poorly understood. Using GnRH-green fluorescent protein transgenic mice and a parahorizontal brain slice preparation, we have undertaken a series of electrophysiological experiments to examine 1) postsynaptic GABA(B) receptors expressed by GnRH neurons, and 2) presynaptic GABA(B) receptors located on the terminals of an important neural input to GnRH neurons originating from the anteroventral periventricular nucleus (AVPV). The GABA(B) receptor agonist baclofen induced a direct postsynaptic hyperpolarization of GnRH neurons through induction of an outward current blocked by barium. Baclofen also acted presynaptically to suppress AVPV-activated GABA- and glutamate-evoked postsynaptic currents in GnRH neurons. The number of GnRH neurons exhibiting postsynaptic GABA(B) receptors was significantly (P < 0.05) different in males (22%) and females (70%), whereas presynaptic GABA(B) modulation of AVPV afferents was the same in the two sexes. Across the estrous cycle, a striking approximately 70% reduction (P < 0.05) in presynaptic GABA(B) modulation of AVPV afferents to GnRH neurons was found on proestrus compared with diestrus and estrus. In contrast, postsynaptic GABA(B) receptors did not change. Together, these findings show that GABA(B) receptors are active at both pre- and postsynaptic sites to modulate the excitability of GnRH neurons. The balance of this pre- and postsynaptic activity is different between the sexes and changes in a dynamic manner across the estrous cycle.

Gap junctions between neuronal inputs but not gonadotropin-releasing hormone neurons control estrous cycles in the mouse.

The role of gap junctions in the neural control of fertility remains poorly understood. Using acute brain slices from adult GnRH-green fluorescent protein transgenic mice, individual GnRH neurons were filled with a mixture of fluorescent dextran and neurobiotin. No dye transfer was found between any GnRH neurons, although approximately 30% of GnRH neurons exchanged neurobiotin with closely apposed cells. Dual electrophysiological recordings from pairs of GnRH neurons revealed an absence of electrical coupling. Using adult connexin 36 (Cx36)-cyan fluorescent protein transgenic mice, Cx36 was identified in cells within several hypothalamic brain regions, including 64% of preoptic area kisspeptin neurons but not in GnRH neurons. To assess the potential role of Cx36 in non-GnRH neurons within the GnRH neuronal network (i.e. neurons providing afferent inputs to GnRH neurons), a calmodulin kinase IIα-Cre (CKC)-LoxP strategy was used to generate mice with a neuron-specific deletion of Cx36 beginning in the first postnatal week. Mutant female mice exhibited normal puberty onset but disordered estrous cyclicity, although their fecundity was normal as was their estrogen-negative and -positive feedback mechanisms. The effects of adult deletion of Cx36 from neurons were assessed using a tamoxifen-dependent inducible CKC-Cx36 transgenic strategy. Mutant mice exhibited the same reproductive phenotype as the CKC-Cx36 animals. Together these observations demonstrate that there is no gap junctional coupling between GnRH neurons. However, it is apparent that other neurons within the GnRH neuronal network, potentially the preoptic kisspeptin neurons, are dependent on Cx36 gap junctions and that this is critical for normal estrous cyclicity.

Frequency-dependent recruitment of fast amino acid and slow neuropeptide neurotransmitter release controls gonadotropin-releasing hormone neuron excitability.

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 the AVPV controls GnRH neurons 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. The AVPV GABA input was dominant and found to excite or inhibit GnRH neurons in a cell-dependent manner. Increasing the AVPV stimulation frequency to 5-10 Hz resulted 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 GABA(B) 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.

Glutamate regulation of GnRH neuron excitability.

The gonadotropin-releasing hormone (GnRH) neuronal network is the master controller of the reproductive axis. It is widely accepted that the amino acid transmitters GABA and glutamate play important roles in controlling GnRH neuron excitability. However, remarkably few studies have examined the functional role of direct glutamate regulation of GnRH neurons. Dual-labeling investigations have shown that GnRH neurons express receptor subunits required for AMPA, NMDA and kainate signaling in a heterogeneous manner. Electrophysiological and calcium imaging studies have confirmed this heterogeneity and shown that while the majority of adult GnRH neurons express AMPA/kainate receptors, only small sub-populations have functional NMDA or metabotropic glutamate receptors. Accumulating evidence suggests that one important role of direct glutamate signaling at GnRH neurons is for their activation at the time of puberty. Whereas in vivo studies have indicated the importance of NMDA signaling within the whole of the GnRH neuronal network, including afferent neurons and glia, investigations at the level of the GnRH neuron suggest that peripubertal changes in AMPA receptor expression may be dominant in the mouse. The sources of glutamatergic inputs to the GnRH neurons are only just beginning to be examined and include the anteroventral periventricular nucleus as well as the possibility that GnRH neurons may use glutamate as a neurotransmitter in recurrent collateral innervation. It is expected that a full understanding of the glutamatergic regulation of GnRH neurons will provide significant insight into the mechanisms underlying their control of reproductive function.

Neurobiological mechanisms underlying kisspeptin activation of gonadotropin-releasing hormone (GnRH) neurons at puberty.

Studies undertaken in many species indicate that kisspeptin-Gpr54 signaling is essential for the activation of gonadotropin-releasing hormone (GnRH) neurons to bring about puberty. Investigations in transgenic mouse models, in particular, have highlighted the importance of kisspeptin signaling at the level of the GnRH neuron itself in this process. This review aims to highlight current understanding of the neurobiological mechanisms underlying the kisspeptin activation of postnatal GnRH neurons. The three key features of the kisspeptin-Gpr54-GnRH neuron axis leading up to puberty are (i) the expression of adult-like levels of Gpr54 mRNA in GnRH neurons well in advance of puberty, (ii) a modest increase in the electrical response of GnRH neurons to Gpr54 activation across postnatal development and (iii), the "sudden" appearance of kisspeptin fibers surrounding GnRH neuron cell bodies/proximal dendrites just prior to puberty onset. These kisspeptin fibers are likely to originate from the kisspeptin population located in the rostral periventricular region of the third ventricle (RP3V). Together, available data suggest that the key step in the kisspeptin control of puberty lies in the control of kisspeptin synthesis within RP3V kisspeptin neurons that innervate GnRH neurons. This has recently been shown to be dependent upon circulating estradiol concentrations. As such, we propose that RP3V kisspeptin neurons represent a critical estradiol-dependent amplification mechanism brought into play relatively late in pubertal development to activate GnRH neurons and complete the process of puberty onset. Subsequently, in the adult female, this same circuitry is used to activate GnRH neurons to generate the cyclical preovulatory GnRH/LH surge.

Leptin indirectly regulates gonadotropin-releasing hormone neuronal function.

The adipose-derived hormone leptin communicates information about metabolic status to the hypothalamic GnRH neuronal system. It is unclear whether leptin can act directly on GnRH neurons. To examine this, we used three approaches. First, the presence of leptin-induced signal transducer and activator of transcription-3 activation was examined in GnRH neurons in male and female rats. Intracerebroventricular treatment with 4 mug leptin-induced robust signal transducer and activator of transcription-3 expression within the anteroventral periventricular nucleus but not in GnRH neurons. Second, fertility was assessed in male and female CRE-loxP transgenic mice with conditional leptin receptor (Lepr) deletion from either all forebrain neurons or GnRH neurons only. Forebrain neuron LEPR deletion prevented the onset of puberty resulting in infertility in males and females and blocked estradiol-induced LH surge. However, mice with GnRH neuron-selective Lepr deletion exhibited normal fertility apart from a slight puberty delay in males. Lastly, the highly sensitive technique of single-cell nested PCR was used to test for Lepr transcript presence in individual GnRH neurons, identified in situ using GnRH-green fluorescent protein transgenics. Whereas 75% of positive control (proopiomelanocortin) neurons contained Lepr mRNA, no (none of 18) GnRH neurons were Lepr mRNA positive. Collectively, these results show that leptin does not act directly on GnRH neurons in rats and mice. Leptin appears to regulate GnRH function via forebrain neurons that are afferent to GnRH because forebrain neuronal LEPR deletion caused infertility. The location and phenotype of these leptin-responsive neurons remains to be elucidated.