Modulation of Gonadotropin-Releasing Hormone Neuron Activity and Secretion in Mice by Non-peptide Neurotransmitters, Gasotransmitters, and Gliotransmitters.

Gonadotropin-releasing hormone (GnRH) neuron activity and GnRH secretion are essential for fertility in mammals. Here, I review findings from mouse studies on the direct modulation of GnRH neuron activity and GnRH secretion by non-peptide neurotransmitters (GABA, glutamate, dopamine, serotonin, norepinephrine, epinephrine, histamine, ATP, adenosine, and acetylcholine), gasotransmitters (nitric oxide and carbon monoxide), and gliotransmitters (prostaglandin E2 and possibly GABA, glutamate, and ATP). These neurotransmitters, gasotransmitters, and gliotransmitters have been shown to directly modulate activity and/or GnRH secretion in GnRH neurons in vivo or ex vivo (brain slices), from postnatal through adult mice, or in embryonic or immortalized mouse GnRH neurons. However, except for GABA, nitric oxide, and prostaglandin E2, which appear to be essential for normal GnRH neuron activity, GnRH secretion, and fertility in males and/or females, the biological significance of their direct modulation of GnRH neuron activity and/or GnRH secretion in the central regulation of reproduction remains largely unknown and requires further exploration.

Dynamic Network Activation of Hypothalamic MCH Neurons in REM Sleep and Exploratory Behavior.

Most brain neurons are active in waking, but hypothalamic neurons that synthesize the neuropeptide melanin-concentrating hormone (MCH) are claimed to be active only during sleep, particularly rapid eye movement (REM) sleep. Here we use deep-brain imaging to identify changes in fluorescence of the genetically encoded calcium (Ca2+) indicator GCaMP6 in individual hypothalamic neurons that contain MCH. An in vitro electrophysiology study determined a strong relationship between depolarization and Ca2+ fluorescence in MCH neurons. In 10 freely behaving MCH-cre mice (male and female), the highest fluorescence occurred in all recorded neurons (n = 106) in REM sleep relative to quiet waking or non-REM sleep. Unexpectedly, 70% of the MCH neurons had strong fluorescence activity when the mice explored novel objects. Spatial and temporal mapping of the change in fluorescence between pairs of MCH neurons revealed dynamic activation of MCH neurons during REM sleep and activation of a subset of the same neurons during exploratory behavior. Functional network activity maps will facilitate comparisons of not only single-neuron activity, but also network responses in different conditions and disease.SIGNIFICANCE STATEMENT Functional activity maps identify brain circuits responding to specific behaviors, including rapid eye movement sleep (REM sleep), a sleep phase when the brain is as active as in waking. To provide the first activity map of individual neurons during REM sleep, we use deep-brain calcium imaging in unrestrained mice to map the activity of hypothalamic melanin-concentrating hormone (MCH) neurons. MCH neurons were found to be synchronously active during REM sleep, and also during the exploration of novel objects. Spatial mapping revealed dynamic network activation during REM sleep and activation of a subset of the neurons during exploratory behavior. Functional activity maps at the cellular level in specific behaviors, including sleep, are needed to establish a brain connectome.

Neuropeptidergic modulation of GnRH neuronal activity and GnRH secretion controlling reproduction: insights from recent mouse studies.

Gonadotropin-releasing hormone (GnRH) secretion from GnRH neurons and its modulation by neuropeptides are essential for mammalian reproduction. Here, I review the neuropeptides that have been shown to act directly and that may also act indirectly, on GnRH neurons, the reproduction-related processes with which the neuropeptides may be associated or the physiological information they may convey, as well as their cognate receptors, signaling pathways and roles in the modulation of GnRH neuronal firing, [Ca2+]i, GnRH secretion and reproduction. The review focuses on recent research in mice, which offer the most tractable experimental system for studying mammalian GnRH neurons.

Deciphering the Contributions of CRH Receptors in the Brain and Pituitary to Stress-Induced Inhibition of the Reproductive Axis.

Based on pharmacological studies, corticotropin-releasing hormone (CRH) and its receptors play a leading role in the inhibition of the hypothalamic-pituitary-gonadal (HPG) axis during acute stress. To further study the effects of CRH receptor signaling on the HPG axis, we generated and/or employed male mice lacking CRH receptor type 1 (CRHR1) or type 2 (CRHR2) in gonadotropin-releasing hormone neurons, GABAergic neurons, or in all central neurons and glia. The deletion of CRHRs revealed a preserved decrease of plasma luteinizing hormone (LH) in response to either psychophysical or immunological stress. However, under basal conditions, central infusion of CRH into mice lacking CRHR1 in all central neurons and glia, or application of CRH to pituitary cultures from mice lacking CRHR2, failed to suppress LH release, unlike in controls. Our results, taken together with those of the earlier pharmacological studies, suggest that inhibition of the male HPG axis during acute stress is mediated by other factors along with CRH, and that CRH suppresses the HPG axis at the central and pituitary levels via CRHR1 and CRHR2, respectively.

Effects of leptin and melanocortin signaling interactions on pubertal development and reproduction.

Leptin and melanocortin signaling control ingestive behavior, energy balance, and substrate utilization, but only leptin signaling defects cause hypothalamic hypogonadism and infertility. Although GnRH neurons do not express leptin receptors, leptin influences GnRH neuron activity via regulation of immediate downstream mediators including the neuropeptides neuropeptide Y and the melanocortin agonist and antagonist, α-MSH, agouti-related peptide, respectively. Here we show that modulation of melanocortin signaling in female db/db mice through ablation of agouti-related peptide, or heterozygosity of melanocortin 4 receptor, restores the timing of pubertal onset, fertility, and lactation. Additionally, melanocortin 4 receptor activation increases action potential firing and induces c-Fos expression in GnRH neurons, providing further evidence that melanocortin signaling influences GnRH neuron activity. These studies thus establish melanocortin signaling as an important component in the leptin-mediated regulation of GnRH neuron activity, initiation of puberty and fertility.

Kisspeptin inhibits high-voltage activated Ca2+ channels in GnRH neurons via multiple Ca2+ influx and release pathways.

Kisspeptin plays an important role in puberty and subsequent fertility by activating its receptor, G-protein-coupled receptor 54 (GPR54), and increasing cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and gonadotropin-releasing hormone (GnRH) secretion in GnRH neurons. Yet the mechanism by which kisspeptin increases [Ca(2+)](i) in GnRH neurons remains to be fully elucidated. In other neurons, voltage-gated Ca(2+) channel (VGCC) activity has been shown to be inversely related to [Ca(2+)](i). We used whole-cell patch-clamp recording to examine the effects of kisspeptin-10 (KP-10) on VGCC activity evoked by step depolarizations in GnRH neurons in brain slices from pubertal male GnRH-green fluorescent protein transgenic mice. Prolonged (>30 s) KP-10 application inhibited Ca(2+) currents. The GPR54 antagonist peptide 234, chelation of intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, substitution of Ba(2+) for Ca(2+), the calmodulin antagonists calmidazolium and trifluoperazine, the phospholipase C inhibitor edelfosine, the canonical transient receptor potential (TRPC) channel and inositol 1,4,5-trisphosphate receptor (IP(3)R) antagonist 2-APB, the TRPC channel antagonist BTP2 and the endoplasmic reticulum Ca(2+)-ATPase blocker cyclopiazonic acid each prevented inhibition. The IP(3)R antagonists caffeine (10 µM), heparin and intracellular 2-APB prevented inhibition to a lesser extent. The ryanodine receptor (RyR) antagonists ryanodine and dantrolene prevented inhibition, and the RyR agonist caffeine (30 mM) mimicked the effects of KP-10 on Ca(2+) currents. Our results suggest that kisspeptin induces Ca(2+) influx through TRPC channels and Ca(2+) release via IP(3)Rs and RyRs, and that this is followed by Ca(2+)/CaM-dependent inhibition of VGCCs.

Innervation of gonadotropin-releasing hormone neurons by peptidergic neurons conveying circadian or energy balance information in the mouse.

BACKGROUND: Secretion of gonadotropin-releasing hormone (GnRH) produced in neurons in the basal forebrain is the primary regulator of reproductive maturation and function in mammals. Peptidergic signals relating to circadian timing and energy balance are an important influence on the reproductive axis. The aim of this study was to investigate the innervation of GnRH neurons by peptidergic neurons. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemistry and confocal microscopy were used to detect appositions of peptidergic fibers (NPY, beta-endorphin, MCH) associated with energy balance and metabolic status in transgenic mice expressing a green fluorescent protein reporter construct in GnRH neurons. The frequency of these appositions was compared to those of vasoactive intestinal peptide (VIP), a hypothalamic neuropeptide likely to convey circadian timing information to the GnRH secretory system. The majority of GnRH neurons (73-87%) were closely apposed by fibers expressing NPY, beta-endorphin, or MCH, and a significant proportion of GnRH neurons (28%) also had close contacts with VIP-ir fibers. CONCLUSIONS/SIGNIFICANCE: It is concluded that GnRH neurons in the mouse receive a high frequency of direct modulatory inputs from multiple hypothalamic peptide systems known to be important in conveying circadian information and signalling energy balance.

Calcium and small-conductance calcium-activated potassium channels in gonadotropin-releasing hormone neurons before, during, and after puberty.

The pubertal increase in GnRH secretion resulting in sexual maturation and reproductive competence is a complex process involving kisspeptin stimulation of GnRH neurons and requiring Ca(2+) and possibly other intracellular messengers. To determine whether the expression of Ca(2+) channels, or small-conductance Ca(2+)-activated K(+) (SK) channels, whose activity reflects cytoplasmic free Ca(2+) concentration, changes at puberty in GnRH neurons, Ca(2+) and SK currents in GnRH neurons were recorded in brain slices of juvenile [postnatal day (P) 10-21], pubertal (P28-P42), and adult (> or =P56) male GnRH-green fluorescent protein transgenic mice using perforated-patch and whole-cell techniques. Ca(2+) currents were inhibited by the Ca(2+) channel blocker Cd(2+) and showed marked heterogeneity but were on average similar in juvenile, pubertal, and adult GnRH neurons. SK currents, which were inhibited by the SK channel blocker apamin and enhanced by the SK and intermediate-conductance Ca(2+)-activated K(+) channel activator 1-ethyl-2-benzimidazolinone, were also on average similar in juvenile, pubertal, and adult GnRH neurons. These findings suggest that whereas Ca(2+) and SK channels may participate in the pubertal increase in GnRH secretion and there may be changes in Ca(2+) or SK channel subtypes, overall Ca(2+) and SK channel expression in GnRH neurons remains relatively constant across pubertal development. Hence, the expected increase in GnRH neuron cytoplasmic free Ca(2+) concentration required for increased GnRH secretion at puberty appears to be due to mechanisms other than altered Ca(2+) or SK channel expression, e.g. increased membrane depolarization and subsequent activation of preexisting Ca(2+) channels after increased excitatory synaptic input.

Impaired reproductive behavior by lack of GluR-B containing AMPA receptors but not of NMDA receptors in hypothalamic and septal neurons.

The roles of ionotropic glutamate receptors in mammalian reproduction are unknown. We therefore generated mice lacking a major subtype of (S)-alpha-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA) receptors or all N-methyl-d-aspartate (NMDA) receptors in GnRH neurons and other mainly limbic system neurons, primarily in hypothalamic and septal areas. Male mice without NMDA receptors in these neurons were not impaired in breeding and exhibited similar GnRH secretion as control littermates. However, male mice lacking GluR-B containing AMPA receptors in these neurons were poor breeders and severely impaired in reproductive behaviors such as aggression and mounting. Testis and sperm morphology, testis weight, and serum testosterone levels, as well as GnRH secretion, were unchanged. Contact with female cage bedding failed to elicit male sexual behavior in these mice, unlike in control male littermates. Their female counterparts had unchanged ovarian morphology, had bred successfully, and had normal litter sizes but exhibited pronounced impairments in maternal behaviors such as pup retrieval and maternal aggression. Our results suggest that NMDA receptors and GluR-B containing AMPA receptors are not essential for fertility, but that GluR-B containing AMPA receptors are essential for male and female reproduction-related behaviors, perhaps by mediating responses to pheromones or odorants.

Neurotrophic effects of BDNF on embryonic gonadotropin-releasing hormone (GnRH) neurons.

Secretion of gonadotropin-releasing hormone (GnRH) at the median eminence is the essential activator of the reproductive axis. The mechanisms by which embryonic GnRH neurons migrate from the olfactory placode to the preoptic area and then elaborate neurites that course through the hypothalamus to terminate at the median eminence are largely unknown. We investigated the hypothesis that GnRH neurite outgrowth is promoted by brain-derived neurotrophic factor (BDNF) because GnRH neurites course through BDNF-rich areas of the forebrain during their development. Confocal microscopy revealed that most (86%) cultured embryonic GnRH cells tagged with a green fluorescent protein reporter were immunoreactive for TrkB. In primary cultures of E12.5 olfactory tissue, treatment with BDNF induced a dose-dependent increase in neurite outgrowth, but had no discernible effect on branching. BDNF induced phosphorylation of Ca(2+)/cAMP response element-binding protein (pCREB) in both GnRH and non-GnRH cells in these cultures. This was not associated with phosphorylation of ERK in GnRH-immunoreactive cells, though BDNF treatment did stimulate pERK in neighbouring non-GnRH cells. Promotion of neurite outgrowth is unlikely therefore to result from activation of the Ras-MAPK/ERK pathway. We conclude that the developing GnRH secretory system is directly sensitive to BDNF and that this polypeptide functions as a neurotrophic factor for GnRH neurons.

A confocal microscopic study of gonadotropin-releasing hormone (GnRH) neuron inputs to dopaminergic neurons containing estrogen receptor alpha in the arcuate nucleus of GnRH-green fluorescent protein transgenic mice.

The purpose of the present study was to determine whether the septo-preoptico-tuberoinfundibular gonadotropin-releasing hormone (GnRH) pathway comes in close juxtaposition with tyrosine hydroxylase immunoreactive (TH-IR) neurons in the arcuate nucleus of female mice. Immunohistochemical staining with a TH monoclonal antibody coupled with confocal microscopy was employed on vibratome-cut brain sections of female GnRH-green fluorescent protein (GFP) transgenic mice to evaluate possible appositions between GnRH and tuberoinfundibular dopaminergic (TIDA) neurons. TH-IR neurons of the arcuate nucleus received GnRH neuronal appositions in adult female mice at proestrus and estrus stages. In contrast, no GnRH appositions were observed in adult females at diestrus. Subsequently, double immunohistochemical staining for TH and estrogen receptor-alpha (ERalpha) was performed to examine the role of estradiol on this relationship. We found that most TH-IR neurons contacted by GnRH fibers were immunoreactive for ERalpha. Our observations suggest that GnRH neurons communicate directly with TIDA neurons in the adult female. Furthermore, ERalpha activation in TIDA neurons may be involved in the formation of connections between GnRH neurons and TIDA neurons.