Kisspeptin signaling in astrocytes modulates the reproductive axis.

Reproduction is safeguarded by multiple, often cooperative regulatory networks. Kisspeptin signaling, via KISS1R, plays a fundamental role in reproductive control, primarily by regulation of hypothalamic GnRH neurons. We disclose herein a pathway for direct kisspeptin actions in astrocytes that contributes to central reproductive modulation. Protein-protein-interaction and ontology analyses of hypothalamic proteomic profiles after kisspeptin stimulation revealed that glial/astrocyte markers are regulated by kisspeptin in mice. This glial-kisspeptin pathway was validated by the demonstrated expression of Kiss1r in mouse astrocytes in vivo and astrocyte cultures from humans, rats and mice, where kisspeptin activated canonical intracellular signaling-pathways. Cellular co-expression of Kiss1r with the astrocyte markers, GFAP and S100-ß, occurred in different brain regions, with higher percentage in Kiss1- and GnRH-enriched areas. Conditional ablation of Kiss1r in GFAP-positive cells, in the G-KiRKO mouse, altered gene expression of key factors in PGE2 synthesis in astrocytes, and perturbed astrocyte-GnRH neuronal appositions, as well as LH responses to kisspeptin and LH pulsatility, as surrogate marker of GnRH secretion. G-KiRKO mice also displayed changes in reproductive responses to metabolic stress induced by high-fat diet, affecting female pubertal onset, estrous cyclicity and LH-secretory profiles. Our data unveil a non-neuronal pathway for kisspeptin actions in astrocytes, which cooperates in fine-tuning the reproductive axis and its responses to metabolic stress.

Transcriptional profiling of Kiss1 neurons from arcuate and rostral periventricular hypothalamic regions in female mice.

In brief: The transcriptional profiles of Kiss1 neurons from the arcuate and the rostral periventricular region of the third ventricle of the hypothalamus have been directly compared in diestrous female mice. Differentially expressed genes provide molecular signatures for these two populations of Kiss1 neurons and insights into their physiology. Abstract: The neuropeptide kisspeptin is produced by Kiss1 neurons and is required for normal mammalian fertility. The two main populations of Kiss1 neurons are located in the arcuate (ARC) and the rostral periventricular area of the third ventricle (RP3V) of the hypothalamus. To define the molecular signature of these Kiss1 populations, transcriptomics profiling was performed using purified Kiss1 neurons from diestrous stage female mice. From a data set of 7026 genes, 332 differentially expressed transcripts were identified between the Kiss1ARC and Kiss1RP3V neurons. These data have uncovered novel transcripts and expanded the receptor expression, co-transmitter and transcription factor profiles of Kiss1 neurons. Validation by quantitative RT-PCR confirmed differential expression of Cartpt, Ddc, Gal, Gda, Npy2r, Penk, Rasp18, Rxfp3, Slc18a2, and Th in Kiss1RP3V neurons and Gpr83, Hctr2, Nhlh2, Nmn, Npr3, Nr4a2, Nr5a2, Olfm2, Tac2 and Tacr3 in Kiss1ARC neurons. Enriched pathways common to both Kiss1 populations included the NF-kB, mTor, endocannabinoid, GPCR, Wnt and oestrogen signalling while some pathways (e.g. cytomegalovirus infection, dopaminergic and serotonergic biosynthesis) were specific to Kiss1RP3V neurons. Our gene expression data set augments the existing data sets describing the transcriptional profiles of Kiss1 neuronal populations.

Dax1 modulates ERα-dependent hypothalamic estrogen sensing in female mice.

Coupling the release of pituitary hormones to the developmental stage of the oocyte is essential for female fertility. It requires estrogen to restrain kisspeptin (KISS1)-neuron pulsatility in the arcuate hypothalamic nucleus, while also exerting a surge-like effect on KISS1-neuron activity in the AVPV hypothalamic nucleus. However, a mechanistic basis for this region-specific effect has remained elusive. Our genomic analysis in female mice demonstrate that some processes, such as restraint of KISS1-neuron activity in the arcuate nucleus, may be explained by region-specific estrogen receptor alpha (ERα) DNA binding at gene regulatory regions. Furthermore, we find that the Kiss1-locus is uniquely regulated in these hypothalamic nuclei, and that the nuclear receptor co-repressor NR0B1 (DAX1) restrains its transcription specifically in the arcuate nucleus. These studies provide mechanistic insight into how ERα may control the KISS1-neuron, and Kiss1 gene expression, to couple gonadotropin release to the developmental stage of the oocyte.

GnRH pulse generator frequency is modulated by kisspeptin and GABA-glutamate interactions in the posterodorsal medial amygdala in female mice.

Kisspeptin neurons in the arcuate nucleus of the hypothalamus generate gonadotrophin-releasing hormone (GnRH) pulses, and act as critical initiators of functional gonadotrophin secretion and reproductive competency. However, kisspeptin in other brain regions, most notably the posterodorsal subnucleus of the medial amygdala (MePD), plays a significant modulatory role over the hypothalamic kisspeptin population; our recent studies using optogenetics have shown that low-frequency light stimulation of MePD kisspeptin results in increased luteinsing hormone pulse frequency. Nonetheless, the neurochemical pathways that underpin this regulatory function remain unknown. To study this, we have utilised an optofluid technology, precisely combining optogenetic stimulation with intra-nuclear pharmacological receptor antagonism, to investigate the neurotransmission involved in this circuitry. We have shown experimentally and verified using a mathematical model that functional neurotransmission of both GABA and glutamate is a requirement for effective modulation of the GnRH pulse generator by amygdala kisspeptin neurons.

Estrogen differentially regulates transcriptional landscapes of preoptic and arcuate kisspeptin neuron populations.

Kisspeptin neurons residing in the rostral periventricular area of the third ventricle (KPRP3V) and the arcuate nucleus (KPARC) mediate positive and negative estrogen feedback, respectively. Here, we aim to compare transcriptional responses of KPRP3V and KPARC neurons to estrogen. Transgenic mice were ovariectomized and supplemented with either 17ß-estradiol (E2) or vehicle. Fluorescently tagged KPRP3V neurons collected by laser-capture microdissection were subjected to RNA-seq. Bioinformatics identified 222 E2-dependent genes. Four genes encoding neuropeptide precursors (Nmb, Kiss1, Nts, Penk) were robustly, and Cartpt was subsignificantly upregulated, suggesting putative contribution of multiple neuropeptides to estrogen feedback mechanisms. Using overrepresentation analysis, the most affected KEGG pathways were neuroactive ligand-receptor interaction and dopaminergic synapse. Next, we re-analyzed our previously obtained KPARC neuron RNA-seq data from the same animals using identical bioinformatic criteria. The identified 1583 E2-induced changes included suppression of many neuropeptide precursors, granins, protein processing enzymes, and other genes related to the secretory pathway. In addition to distinct regulatory responses, KPRP3V and KPARC neurons exhibited sixty-two common changes in genes encoding three hormone receptors (Ghsr, Pgr, Npr2), GAD-65 (Gad2), calmodulin and its regulator (Calm1, Pcp4), among others. Thirty-four oppositely regulated genes (Kiss1, Vgf, Chrna7, Tmem35a) were also identified. The strikingly different transcriptional responses in the two neuron populations prompted us to explore the transcriptional mechanism further. We identified ten E2-dependent transcription factors in KPRP3V and seventy in KPARC neurons. While none of the ten transcription factors interacted with estrogen receptor-α, eight of the seventy did. We propose that an intricate, multi-layered transcriptional mechanism exists in KPARC neurons and a less complex one in KPRP3V neurons. These results shed new light on the complexity of estrogen-dependent regulatory mechanisms acting in the two functionally distinct kisspeptin neuron populations and implicate additional neuropeptides and mechanisms in estrogen feedback.

Transcriptome profiling of kisspeptin neurons from the mouse arcuate nucleus reveals new mechanisms in estrogenic control of fertility.

Kisspeptin neurons in the mediobasal hypothalamus (MBH) are critical targets of ovarian estrogen feedback regulating mammalian fertility. To reveal molecular mechanisms underlying this signaling, we thoroughly characterized the estrogen-regulated transcriptome of kisspeptin cells from ovariectomized transgenic mice substituted with 17ß-estradiol or vehicle. MBH kisspeptin neurons were harvested using laser-capture microdissection, pooled, and subjected to RNA sequencing. Estrogen treatment significantly (p.adj. < 0.05) up-regulated 1,190 and down-regulated 1,139 transcripts, including transcription factors, neuropeptides, ribosomal and mitochondrial proteins, ion channels, transporters, receptors, and regulatory RNAs. Reduced expression of the excitatory serotonin receptor-4 transcript (Htr4) diminished kisspeptin neuron responsiveness to serotonergic stimulation. Many estrogen-regulated transcripts have been implicated in puberty/fertility disorders. Patients (n = 337) with congenital hypogonadotropic hypogonadism (CHH) showed enrichment of rare variants in putative CHH-candidate genes (e.g., LRP1B, CACNA1G, FNDC3A). Comprehensive characterization of the estrogen-dependent kisspeptin neuron transcriptome sheds light on the molecular mechanisms of ovary-brain communication and informs genetic research on human fertility disorders.

Sexually Dimorphic Neurosteroid Synthesis Regulates Neuronal Activity in the Murine Brain.

Sex steroid hormones act on hypothalamic kisspeptin neurons to regulate reproductive neural circuits in the brain. Kisspeptin neurons start to express estrogen receptors in utero, suggesting steroid hormone action on these cells early during development. Whether neurosteroids are locally produced in the embryonic brain and impinge onto kisspeptin/reproductive neural circuitry is not known. To address this question, we analyzed aromatase expression, a key enzyme in estrogen synthesis, in male and female mouse embryos. We identified an aromatase neuronal network comprising ∼6000 neurons in the hypothalamus and amygdala. By birth, this network has become sexually dimorphic in a cluster of aromatase neurons in the arcuate nucleus adjacent to kisspeptin neurons. We demonstrate that male arcuate aromatase neurons convert testosterone to estrogen to regulate kisspeptin neuron activity. We provide spatiotemporal information on aromatase neuronal network development and highlight a novel mechanism whereby aromatase neurons regulate the activity of distinct neuronal populations expressing estrogen receptors.SIGNIFICANCE STATEMENT Sex steroid hormones, such as estradiol, are important regulators of neural circuits controlling reproductive physiology in the brain. Embryonic kisspeptin neurons in the hypothalamus express steroid hormone receptors, suggesting hormone action on these cells in utero Whether neurosteroids are locally produced in the brain and impinge onto reproductive neural circuitry is insufficiently understood. To address this question, we analyzed aromatase expression, a key enzyme in estradiol synthesis, in mouse embryos and identified a network comprising ∼6000 neurons in the brain. By birth, this network has become sexually dimorphic in a cluster of aromatase neurons in the arcuate nucleus adjacent to kisspeptin neurons. We demonstrate that male aromatase neurons convert testosterone to estradiol to regulate kisspeptin neuron activity.

Hypermethylation and reduced expression of Gtl2, Rian and Mirg at the Dlk1-Dio3 imprinted locus as a marker for poor developmental potential of mouse embryonic stem cells.

Mouse embryonic stem cells (ESCs) have played a crucial role in biomedical research where they can be used to elucidate gene function through the generation of genetically modified mice. A critical requirement for the success of this technology is the ability of ESCs to contribute to viable chimaeras with germ-line transmission of the genetically modified allele. We have identified several ESC clones that cause embryonic death of chimaeras at mid to late gestation stages. These clones had a normal karyotype, were pathogen free and their in vitro differentiation potential was not compromised. Chimaeric embryos developed normally up to E13.5 but showed a significant decrease in embryo survival by E17.5 with frequent haemorrhaging. We investigated the relationship between the ESCs transcriptional and epigenomic state and their ability to contribute to viable chimaeras. RNA sequencing identified four genes (Gtl2, Rian, Mirg and Rtl1as) located in the Dlk1-Dio3 imprinted locus that were expressed at lower levels in the compromised ESC clones and this was confirmed by qRT-PCR. Bisulphite sequencing analysis showed significant hypermethylation at the Dlk1-Dio3 imprinted locus with no consistent differences in methylation patterns at other imprinted loci. Treatment of the compromised ESCs with 5-azacytidine reactivated stable expression of Gtl2 and rescued the lethal phenotype but only gave low level chimaeras.

Optogenetic stimulation of kisspeptin neurones within the posterodorsal medial amygdala increases luteinising hormone pulse frequency in female mice.

Kisspeptin within the arcuate nucleus of the hypothalamus is a critical neuropeptide in the regulation of reproduction. Together with neurokinin B and dynorphin A, arcuate kisspeptin provides the oscillatory activity that drives the pulsatile secretion of gonadotrophin-releasing hormone (GnRH), and therefore luteinising hormone (LH) pulses, and is considered to be a central component of the GnRH pulse generator. It is well established that the amygdala also exerts an influence over gonadotrophic hormone secretion and reproductive physiology. The discovery of kisspeptin and its receptor within the posterodorsal medial amygdala (MePD) and our recent finding showing that intra-MePD administration of kisspeptin or a kisspeptin receptor antagonist results in increased LH secretion and decreased LH pulse frequency, respectively, suggests an important role for amygdala kisspeptin signalling in the regulation of the GnRH pulse generator. To further investigate the function of amygdala kisspeptin, the present study used an optogenetic approach to selectively stimulate MePD kisspeptin neurones and examine the effect on pulsatile LH secretion. MePD kisspeptin neurones in conscious Kiss1-Cre mice were virally infected to express the channelrhodopsin 2 protein and selectively stimulated by light via a chronically implanted fibre optic cannula. Continuous stimulation using 5 Hz resulted in an increased LH pulse frequency, which was not observed at the lower stimulation frequencies of 0.5 and 2 Hz. In wild-type animals, continuous stimulation at 5 Hz did not affect LH pulse frequency. These results demonstrate that selective activation of MePD Kiss1 neurones can modulate hypothalamic GnRH pulse generator frequency.

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

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

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

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

Sex- and sub region-dependent modulation of arcuate kisspeptin neurones by vasopressin and vasoactive intestinal peptide.

A population of kisspeptin neurones located in the hypothalamic arcuate nucleus (ARN) very likely represent the gonadotrophin-releasing hormone pulse generator responsible for driving pulsatile luteinising hormone secretion in mammals. As such, it has become important to understand the neural inputs that modulate the activity of ARN kisspeptin (ARNKISS ) neurones. Using a transgenic GCaMP6 mouse model allowing the intracellular calcium levels ([Ca2+ ]i ) of individual ARNKISS neurones to be assessed simultaneously, we examined whether the circadian neuropeptides vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) modulated the activity of ARNKISS neurones directly. To validate this methodology, we initially evaluated the effects of neurokinin B (NKB) on [Ca2+ ]i in kisspeptin neurones residing within the rostral, middle and caudal ARN subregions of adult male and female mice. All experiments were undertaken in the presence of tetrodotoxin and ionotropic amino acid antagonists. NKB was found to evoke an abrupt increase in [Ca2+ ]i in 95%-100% of kisspeptin neurones throughout the ARN of both sexes. By contrast, both VIP and AVP were found to primarily activate kisspeptin neurones located in the caudal ARN of female mice. Although 58% and 59% of caudal ARN kisspeptin neurones responded to AVP and VIP, respectively, in female mice, only 0%-8% of kisspeptin neurones located in other ARN subregions responded in females and 0%-12% of cells in any subregion in males (P < 0.05). These observations demonstrate unexpected sex differences and marked heterogeneity in functional neuropeptide receptor expression amongst ARNKISS neurones organised on a rostro-caudal basis. The functional significance of this unexpected influence of VIP and AVP on ARNKISS neurones remains to be established.

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

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

Female sexual behavior in mice is controlled by kisspeptin neurons.

Sexual behavior is essential for the survival of many species. In female rodents, mate preference and copulatory behavior depend on pheromones and are synchronized with ovulation to ensure reproductive success. The neural circuits driving this orchestration in the brain have, however, remained elusive. Here, we demonstrate that neurons controlling ovulation in the mammalian brain are at the core of a branching neural circuit governing both mate preference and copulatory behavior. We show that male odors detected in the vomeronasal organ activate kisspeptin neurons in female mice. Classical kisspeptin/Kiss1R signaling subsequently triggers olfactory-driven mate preference. In contrast, copulatory behavior is elicited by kisspeptin neurons in a parallel circuit independent of Kiss1R involving nitric oxide signaling. Consistent with this, we find that kisspeptin neurons impinge onto nitric oxide-synthesizing neurons in the ventromedial hypothalamus. Our data establish kisspeptin neurons as a central regulatory hub orchestrating sexual behavior in the female mouse brain.

Correction: Mechanistic insights into the more potent effect of KP-54 compared to KP-10 in vivo.

[This corrects the article DOI: 10.1371/journal.pone.0176821.].

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

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

Definition of the hypothalamic GnRH pulse generator in mice.

The pulsatile release of luteinizing hormone (LH) is critical for mammalian fertility. However, despite several decades of investigation, the identity of the neuronal network generating pulsatile reproductive hormone secretion remains unproven. We use here a variety of optogenetic approaches in freely behaving mice to evaluate the role of the arcuate nucleus kisspeptin (ARNKISS) neurons in LH pulse generation. Using GCaMP6 fiber photometry, we find that the ARNKISS neuron population exhibits brief (∼1 min) synchronized episodes of calcium activity occurring as frequently as every 9 min in gonadectomized mice. These ARNKISS population events were found to be near-perfectly correlated with pulsatile LH secretion. The selective optogenetic activation of ARNKISS neurons for 1 min generated pulses of LH in freely behaving mice, whereas inhibition with archaerhodopsin for 30 min suppressed LH pulsatility. Experiments aimed at resetting the activity of the ARNKISS neuron population with halorhodopsin were found to reset ongoing LH pulsatility. These observations indicate the ARNKISS neurons as the long-elusive hypothalamic pulse generator driving fertility.

Mechanistic insights into the more potent effect of KP-54 compared to KP-10 in vivo.

Kisspeptins regulate the mammalian reproductive axis by stimulating release of gonadotrophin releasing hormone (GnRH). Different length kisspeptins (KP) are found of 54, 14, 13 or 10 amino-acids which share a common C-terminal 10-amino acid sequence. KP-54 and KP-10 have been widely used to stimulate the reproductive axis but data suggest that KP-54 and KP-10 are not equally effective at eliciting reproductive hormone secretion after peripheral delivery. To confirm this, we analysed the effect of systemic administration of KP-54 or KP-10 on luteinizing hormone (LH) secretion into the bloodstream of male mice. Plasma LH measurements 10 min or 2 hours after kisspeptin injection showed that KP-54 can sustain LH release far longer than KP-10, suggesting a differential mode of action of the two peptides. To investigate the mechanism for this, we evaluated the pharmacokinetics of the two peptides in vivo and their potential to cross the blood brain barrier (BBB). We found that KP-54 has a half-life of ~32 min in the bloodstream, while KP-10 has a half-life of ~4 min. To compensate for this difference in half-life, we repeated injections of KP-10 every 10 min over 1 hr but failed to reproduce the sustained rise in LH observed after a single KP-54 injection, suggesting that the failure of KP-10 to sustain LH release may not just be related to peptide clearance. We tested the ability of peripherally administered KP-54 and KP-10 to activate c-FOS in GnRH neurons behind the blood brain barrier (BBB) and found that only KP-54 could do this. These data are consistent with KP-54 being able to cross the BBB and suggest that KP10 may be less able to do so.

Transforming growth factor ß receptor inhibition prevents ventricular fibrosis in a mouse model of progressive cardiac conduction disease.

AIMS: Loss-of-function mutations in SCN5A, the gene encoding NaV1.5 channel, have been associated with inherited progressive cardiac conduction disease (PCCD). We have proposed that Scn5a heterozygous knock-out (Scn5a+/-) mice, which are characterized by ventricular fibrotic remodelling with ageing, represent a model for PCCD. Our objectives were to identify the molecular pathway involved in fibrosis development and prevent its activation. METHODS AND RESULTS: Our study shows that myocardial interstitial fibrosis occurred in Scn5a+/- mice only after 45 weeks of age. Fibrosis was triggered by transforming growth factor ß (TGF-ß) pathway activation. Younger Scn5a+/- mice were characterized by a higher connexin 43 expression than wild-type (WT) mice. After the age of 45 weeks, connexin 43 expression decreased in both WT and Scn5a+/- mice, although the decrease was larger in Scn5a+/- mice. Chronic inhibition of cardiac sodium current with flecainide (50 mg/kg/day p.o) in WT mice from the age of 6 weeks to the age of 60 weeks did not lead to TGF-ß pathway activation and fibrosis. Chronic inhibition of TGF-ß receptors with GW788388 (5 mg/kg/day p.o.) in Scn5a+/- mice from the age of 45 weeks to the age of 60 weeks prevented the occurrence of fibrosis. However, current data could not detect reduction in QRS duration with GW788388. CONCLUSION: Myocardial fibrosis secondary to a loss of NaV1.5 is triggered by TGF-ß signalling pathway. Those events are more likely secondary to the decreased NaV1.5 sarcolemmal expression rather than the decreased Na+ current per se. TGF-ß receptor inhibition prevents age-dependent development of ventricular fibrosis in Scn5a+/- mouse.