GnRH-green Fluorescent Protein Research Articles

Retrograde Endocannabinoid Signaling Reduces GABAergic Synaptic Transmission to Gonadotropin-Releasing Hormone Neurons

Cannabinoids suppress fertility via reducing hypothalamic GnRH output. γ-Aminobutyric acid (GABA)A receptor (GABAA-R)-mediated transmission is a major input to GnRH cells that can be excitatory. We hypothesized that cannabinoids act via inhibiting GABAergic input. We performed loose-patch electrophysiological studies of acute slices from adult male GnRH-green fluorescent protein transgenic mice. Bath application of type 1 cannabinoid receptor (CB1) agonist WIN55,212 decreased GnRH neuron firing rate. This action was detectable in presence of the glutamate receptor antagonist kynurenic acid but disappeared when bicuculline was also present, indicating GABAA-R involvement. In immunocytochemical experiments, CB1-immunoreactive axons formed contacts with GnRH neurons and a subset established symmetric synapses characteristic of GABAergic neurotransmission. Functional studies were continued with whole-cell patch-clamp electrophysiology in presence of tetrodotoxin. WIN55,212 decreased the frequency of GABAA-R-mediated miniature postsynaptic currents (mPSCs) (reflecting spontaneous vesicle fusion), which was prevented with the CB1 antagonist AM251, indicating collectively that activation of presynaptic CB1 inhibits GABA release. AM251 alone increased mPSC frequency, providing evidence that endocannabinoids tonically inhibit GABAA-R drive onto GnRH neurons. Increased mPSC frequency was absent when diacylglycerol lipase was blocked intracellularly with tetrahydrolipstatin, showing that tonic inhibition is caused by 2-arachidonoylglycerol production of GnRH neurons. CdCl2 in extracellular solution can maintain both action potentials and spontaneous vesicle fusion. Under these conditions, when endocannabinoid-mediated blockade of spontaneous vesicle fusion was blocked with AM251, GnRH neuron firing increased, revealing an endogenous endocannabinoid brake on GnRH neuron firing. Retrograde endocannabinoid signaling may represent an important mechanism under physiological and pathological conditions whereby GnRH neurons regulate their excitatory GABAergic inputs.

Electrophysiological Characteristics of Gonadotrophin‐Releasing Hormone 1–3 Neurones: Insights From a Study of Fish Brains

The vertebrate gonadotrophin-releasing hormone (GnRH) neurones are considered to consist of one group of hypothalamic neuroendocrine and two groups of extrahypothalamic neuromodulatory GnRH neurones, and each group of neurones expresses different molecular species of GnRH peptide. Different GnRH peptides are produced by one of the three paralogous GnRH genes, gnrh1, gnrh2 and gnrh3, which are considered to have originated from gene duplications. All three GnRH systems are well developed in teleost brains. By taking advantage of this, and especially the use of GnRH-green fluorescent protein transgenic fish, the anatomical and electrophysiological properties of all three types of GnRH neurones can now be studied. The hypophysiotropic GnRH1 neurones in the preoptic area show episodic spontaneous electrical activities, whereas the extrahypothalamic GnRH2 neurones in the midbrain and GnRH3 neurones in the terminal nerve show regular intrinsic pacemaker activities. It is suggested that these different electrophysiological properties are related to their different functions (i.e. GnRH1 neurones act as hypophysiotropic neuroendocrine regulators and GnRH2 and GnRH3 neurones act as neuromodulators). The present review focuses on recent electrophysiological analyses of GnRH3 neurones, which have revealed the excitatory GABAergic and the inhibitory FMRFamide-like peptidergic regulations acting upon them, as well as gap junctional electrotonic coupling.

Somatostatin Inhibition of Gonadotropin-Releasing Hormone Neurons in Female and Male Mice

Previous studies indicate that somatostatin regulates gonadotropin secretion. We investigated here whether somatostatin has direct effects on GnRH neurons in the adult male and female mice. Dual-labeling immunofluorescence experiments revealed the presence of somatostatin-immunoreactive fibers adjacent to GnRH neurons, and three-dimensional confocal reconstructions demonstrated apparent somatostatin fiber appositions with 50–60% of GnRH neurons located throughout the brain in both male and female mice. Perforated patch-clamp recordings from GnRH-green fluorescent protein neurons revealed that approximately 70% of GnRH neurons responded in a dose-dependent manner to 10–300 nm somatostatin with an acute membrane hyperpolarization and cessation of firing. This effect persisted in the presence of tetrodotoxin and amino acid receptor antagonists, indicating a direct postsynaptic site of action on the GnRH neuron. The identity of the somatostatin receptors underlying this action was assessed using GnRH neuron single-cell RT-PCR. Of the somatostatin receptor subtypes, the sstr2 transcript was the most prevalent and detected in both males and females. The expression of sstr2 by GnRH neurons was confirmed in the sstr2 knockout/LacZ knock-in mouse line. Electrophysiological studies demonstrated that the sstr2-selective agonist seglitide exerted acute hyperpolarizing actions on GnRH neurons identical to those of somatostatin. Together, these studies reveal somatostatin, acting through sstr2, to be one of the most potent inhibitors of electrical excitability of male and female GnRH neurons identified thus far.

Somatostatin Inhibition of Gonadotropin-Releasing Hormone Neurons in Female and Male Mice

Previous studies indicate that somatostatin regulates gonadotropin secretion. We investigated here whether somatostatin has direct effects on GnRH neurons in the adult male and female mice. Dual-labeling immunofluorescence experiments revealed the presence of somatostatin-immunoreactive fibers adjacent to GnRH neurons, and three-dimensional confocal reconstructions demonstrated apparent somatostatin fiber appositions with 50-60% of GnRH neurons located throughout the brain in both male and female mice. Perforated patch-clamp recordings from GnRH-green fluorescent protein neurons revealed that approximately 70% of GnRH neurons responded in a dose-dependent manner to 10-300 nm somatostatin with an acute membrane hyperpolarization and cessation of firing. This effect persisted in the presence of tetrodotoxin and amino acid receptor antagonists, indicating a direct postsynaptic site of action on the GnRH neuron. The identity of the somatostatin receptors underlying this action was assessed using GnRH neuron single-cell RT-PCR. Of the somatostatin receptor subtypes, the sstr2 transcript was the most prevalent and detected in both males and females. The expression of sstr2 by GnRH neurons was confirmed in the sstr2 knockout/LacZ knock-in mouse line. Electrophysiological studies demonstrated that the sstr2-selective agonist seglitide exerted acute hyperpolarizing actions on GnRH neurons identical to those of somatostatin. Together, these studies reveal somatostatin, acting through sstr2, to be one of the most potent inhibitors of electrical excitability of male and female GnRH neurons identified thus far.

Dendro-dendritic bundling and shared synapses between gonadotropin-releasing hormone neurons

The pulsatile release of gonadotropin-releasing hormone (GnRH) is critical for mammalian fertility, but the mechanisms underlying the synchronization of GnRH neurons are unknown. In the present study, the full extent of the GnRH neuron dendritic tree was visualized by patching and filling individual GnRH neurons with biocytin in acute brain slices from adult GnRH-green fluorescent protein (GFP) transgenic mice. Confocal analysis of 42 filled GnRH neurons from male and female adult mice revealed that the dendrites of the great majority of GnRH neurons (86%) formed multiple close appositions with dendrites of other GnRH neurons. Two types of interactions were encountered; the predominant interaction was one of vertical dendritic bundling where dendrites were found to wrap around each other in the same axis. The other interaction was one in which a GnRH neuron dendrite intercepted other GnRH neuron dendrites in a perpendicular fashion. Electron microscopy using pre-embedded, silver-enhanced immunogold labeling for both GnRH and GFP peptides in GnRH-GFP transgenic mice, confirmed that GnRH neuron dendrites were often immediately juxtaposed. Membrane specializations, including punctae and zonula adherens, were found connecting adjacent dendritic elements of GnRH neurons. Remarkably, individual afferent axon terminals were found to synapse with multiple GnRH neuron dendrites at sites of bundling. Together, these data demonstrate that GnRH neurons are not isolated from one another but, rather, interconnected via their long dendritic extensions. The observation of shared synaptic input to bundled GnRH neuron dendrites suggests a mechanism of GnRH neuron synchronization.

Three Types of Gonadotrophin‐Releasing Hormone Neurones and Steroid‐Sensitive Sexually Dimorphic Kisspeptin Neurones in Teleosts

In general, the gonadotrophin-releasing hormone (GnRH) neuronal systems of vertebrates consist of one group of hypothalamic neuroendocrine and one or two group(s) of extrahypothalamic neuromodulatory GnRH neurones. By taking advantage of the brains of dwarf gourami and GnRH-green fluorescent protein transgenic medaka, the spontaneous electrical activities of all three different types of GnRH neurones have now been characterised. The hypophysiotrophic preoptic area-gnrh1 neurones show irregular and episodic spontaneous electrical activities, whereas extrahypothalamic midbrain gnrh2 and terminal nerve-gnrh3 neurones show regular pacemaker potentials. It is suggested that these spontaneous electrical activities are related to their different functions as neuroendocrine hormones (gnrh1 neurones) and neuromodulators (gnrh2 and gnrh3 neurones). On the other hand, recent evidence strongly suggests that the GnRH neurones are regulated by another class of peptidergic neurones, the kisspeptin neurones. The gene encoding kisspeptin (kiss1 gene) has been cloned, and the anatomical distribution of kiss1 mRNA expressing neurones (kiss1 neurones) has recently been studied in brains of several fish species. In medaka, two kiss1 neuronal populations in hypothalamic areas, called the nucleus ventral tuberis (NVT) and nucleus posterioris periventricularis (NPPv), have been identified. The NVT kiss1 neurones are sexually dimorphic in number (male >> female) under breeding conditions and are sensitive to ovarian oestrogens, whereas the NPPv kiss1 neurones are neither sexually dimorphic, nor sensitive to steroids. The steroid-sensitive changes in kiss1 mRNA expression in the NVT occur physiologically, closely linked to the reproductive state. As in the mammalian counterpart, the medaka kiss1 neuronal system is suggested to be involved in the central regulation of reproductive functions. Interestingly, kiss2, another gene paralogous to kiss1, has been cloned in some fish species. The possible regulation of reproduction and other unknown functions by kiss1 and kiss2 neurones may be the focus of future studies.

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.

Classical estrogen receptor alpha signaling mediates negative and positive feedback on gonadotropin-releasing hormone neuron firing.

During the female reproductive cycle, the neuroendocrine action of estradiol switches from negative feedback to positive feedback to initiate the preovulatory GnRH and subsequent LH surges. Estrogen receptor-alpha (ERalpha) is required for both estradiol negative and positive feedback regulation of LH. ERalpha may signal through estrogen response elements (EREs) in DNA and/or via ERE-independent pathways. Previously, a knock-in mutant allele (ERalpha-/AA) that selectively restores ERE-independent signaling onto the ERalpha-/- background was shown to confer partial negative but not positive estradiol feedback on serum LH. The current study investigated the roles of the ERE-dependent and ERE-independent ERalpha pathways for estradiol feedback at the level of GnRH neuron firing activity. The above ERalpha genetic models were crossed with GnRH-green fluorescent protein mice to enable identification of GnRH neurons in brain slices. Targeted extracellular recordings were used to monitor GnRH neuron firing activity using an ovariectomized, estradiol-treated mouse model that exhibits diurnal switches between negative and positive feedback. In wild-type mice, GnRH neuron firing decreased in response to estradiol during negative feedback and increased during positive feedback. In contrast, both positive and negative responses to estradiol were absent in GnRH neurons from ERalpha-/- and ERalpha-/AA mice. ERE-dependent signaling is thus required to increase GnRH neuron firing to generate a GnRH/LH surge. Furthermore, ERE-dependent and -independent ERalpha signaling pathways both appear necessary to mediate estradiol negative feedback on serum LH levels, suggesting central and pituitary estradiol feedback may use different combinations of ERalpha signaling pathways.

The Gonadotropin-Releasing Hormone (GnRH) Neuronal Population Is Normal in Size and Distribution in GnRH-Deficient and GnRH Receptor-Mutant Hypogonadal Mice

Hypothalamic GnRH neurons are essential for initiation and regulation of reproductive function. In addition to pituitary gonadotrope stimulation, activity of GnRH through its receptor (GnRHR) has been suggested to include autocrine regulation of the GnRH neuron. Two hypogonadal mouse strains, the Gnrh1 mutant (hpg) mice and Gnrhr mutant mice were used to investigate the potential role of GnRH signaling in the proper development and maintenance of GnRH neurons. Immunocytochemical analysis of heterozygous hpg mice revealed a GnRH neuron population that was normal in size and distribution, indicating no effect from reduced Gnrh1 gene dosage on the neurons themselves. To visualize GnRH neurons in homozygous GnRH-deficient hpg mice, heterozygous hpg mice were crossed with GnRH-green fluorescent protein (GFP) transgenic mice with targeted expression of the GFP reporter gene in GnRH neurons. Analysis of forebrains of homozygous hpg/GFP-positive mice immunostained for GFP revealed a normal population size and appropriate distribution of GnRH neurons in hpg mice, with immunoreactive neuronal processes present at the median eminence. Similarly, adult mice deficient in functional GnRHR possessed a full complement of GnRH neurons in the basal forebrain that was indistinguishable from the distribution of GnRH neurons in their wild-type counterparts. Moreover, hpg/GFP neurons retained the ability to generate spontaneous bursts of action potential firing activity, suggesting that GnRH peptide is not required for this function. These data establish that autocrine-paracrine GnRH-signaling is not a prerequisite for the developmental migration of GnRH neurons into the brain or for the projection of GnRH neurosecretory axons.

Small-Conductance Calcium-Activated Potassium Channels Control Excitability and Firing Dynamics in Gonadotropin-Releasing Hormone (GnRH) Neurons

The cellular mechanisms determining the firing patterns of GnRH neurons are presently under intense investigation. In this study, we used GnRH-green fluorescent protein transgenic mice and perforated-patch electrophysiology to examine the role of small conductance calcium-activated potassium (SK) channels in determining the electrical excitability and burst-firing characteristics of adult GnRH neurons. After establishing an appropriate protocol for examining the afterhyperpolarization potential (AHP) currents in GnRH neurons, the highly selective SK channel blocker apamin was used to demonstrate that all GnRH neurons express functional SK channels (35.7 +/- 2.7 pA, mean decay time constant = 2167 msec, apamin IC(50) = 9.6 nm) and that this channel underlies approximately 90% of the AHP in these cells. Current-clamp experiments showed that apamin-sensitive SK channels were tonically active in the majority (74%) of GnRH neurons, with apamin (100 nm) administration resulting in a mean 6.9 +/- 0.5 mV membrane depolarization. Apamin also elevated the firing rate of GnRH neurons, including increased burst frequency and duration in spontaneously bursting cells as well as the ability of GnRH neurons to fire action potentials in response to current injection. In GnRH neurons activated by current injection, apamin significantly enhanced the amplitude of the afterdepolarization potential after a single action potential and eliminated spike frequency adaptation. Together, these studies show that apamin-sensitive SK channels play a key role in restraining GnRH neuron excitability. Through direct modulation of the AHP and indirect actions on the afterdepolarization potential, the SK channel exerts a powerful tonic influence upon the firing dynamics of GnRH neurons.

Norepinephrine Suppresses Gonadotropin-Releasing Hormone Neuron Excitability in the Adult Mouse

Norepinephrine (NE) is considered to exert an important modulatory influence upon the activity of GnRH neurons. In the present study, we used a transgenic GnRH-green fluorescent protein mouse model to examine the effects of NE on the electrical excitability of GnRH neurons in male and female mice. Gramicidin-perforated patch recordings demonstrated that NE (10-100 mum) exerted a robust membrane hyperpolarization, with associated suppression of firing, in more than 85% of male prepubertal and adult GnRH neurons (n = 25). The same hyperpolarizing action was observed in female GnRH neurons from diestrous (91%, n = 11), proestrous (50%, n = 14), estrous (77%, n = 13), and ovariectomized (82%, n = 11) mice. A subpopulation (<10%) of silent GnRH neurons in all groups responded to NE with hyperpolarization followed by the initiation of firing upon NE washout. The hyperpolarizing actions of NE were mimicked by alpha1-adrenergic (phenylephrine) and beta-adrenergic (isoproterenol) receptor agonists, but alpha2 receptor activation (guanabenz) had no effect. Approximately 75% of the NE-evoked hyperpolarization was blocked by the alpha1 receptor antagonist prazosin, and 75% of GnRH neurons responded to both phenylephrine and isoproterenol. These findings indicate that NE acts through both alpha1- and beta-adrenergic receptors located on the soma/dendrites of GnRH neurons to directly suppress their excitability throughout the estrous cycle and after ovariectomy. These data force a reanalysis of existing models explaining the effects of NE on gonadotropin secretion.

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.

Postnatal Remodeling of Dendritic Structure and Spine Density in Gonadotropin-Releasing Hormone Neurons

The GnRH neurons represent the output cells of the neuronal network controlling gonadal function. Their activation initiates the onset of puberty, but the underlying mechanisms remain unclear. Using a GnRH-green fluorescent protein mouse model, we have been able to fill individual GnRH neurons with biocytin in the acute brain slice preparation to examine their morphological characteristics across puberty. GnRH neurons in prepubertal male mice [postnatal d 10-15 (PND10-15)] exhibited half as many dendritic and somal spines as adult male mice (>PND60; P < 0.05) but, surprisingly, a much more complex dendritic tree with 5-fold greater branch points (P < 0.05). Experiments examining somal and proximal dendritic spine numbers in vivo, in perfusion-fixed tissue from GnRH-green fluorescent protein mice, revealed the same pattern of approximately twice as many spines on adult GnRH neurons compared with PND10 male mice (P < 0.01). In contrast to the spine density alterations, reflecting changing excitatory input, confocal immunofluorescence studies revealed no differences in the numbers of vesicular gamma-aminobutyric acid transporter-immunoreactive elements adjacent to GnRH soma or proximal dendrites in prepubertal and adult male mice. Experiments evaluating dendritic tree structure in vivo (PND3, -10, and -35 and adult) revealed that GnRH neurons located in the rostral preoptic area, but not the medial septum, exhibited a more complex branching pattern at PND10, but that this was adult-like by PND35. These studies demonstrate unexpected dendritic tree remodeling in the GnRH neurons and provide evidence for an increase in direct excitatory inputs to GnRH neurons across the time of puberty.

Biocytin Filling of Adult Gonadotropin-Releasing Hormone Neuronsin SituReveals Extensive, Spiny, Dendritic Processes

Ultrastructural studies suggest that GnRH neurons receive relatively few synaptic inputs. However, these techniques are biased toward the analysis of portions of the neuron containing GnRH peptide. Using acute brain slices prepared from transgenic GnRH-green fluorescent protein mice, individual fluorescing GnRH neurons were identified, patched, and filled with the small-molecular-weight dye biocytin. Cells were subsequently visualized with an avidin-conjugated fluorophore, and their morphological characteristics were analyzed by confocal microscopy. In total, 45 GnRH neurons from seven adult male and eight diestrus female mice were examined. Unexpectedly, we found that GnRH neurons possess remarkably long dendritic processes, in some cases extending over 1000 microm distal to the cell body. The somata and dendrites of all GnRH neurons were decorated with an assortment of spine-like protrusions, including filopodia, in an heterogeneous manner. Overall, GnRH neurons had a mean dendritic spine density of 0.4 spines/microm, with the highest densities found in the first 50 microm of the dendrite. GnRH neurons with dendrites running in a horizontal orientation had significantly (P < 0.05) more spines than dendrites with a vertical orientation. The comparison of male and female GnRH neurons revealed no sexually differentiated characteristics of somal or dendritic spine density. Using a technique in which the full extent of the GnRH neuron can be visualized, we demonstrate here a previously unrecognized GnRH neuron morphology of long dendrites covered in spines. These observations suggest that GnRH neurons are not poorly innervated and that they receive abundant excitatory synaptic inputs.

A Confocal Microscopic Study of Gonadotropin-Releasing Hormone (GnRH) Neuron Inputs to Dopaminergic Neurons Containing Estrogen Receptor α 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-α (ERα) 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 ERα. Our observations suggest that GnRH neurons communicate directly with TIDA neurons in the adult female. Furthermore, ERα activation in TIDA neurons may be involved in the formation of connections between GnRH neurons and TIDA neurons.