Neuronal Secretion Research Articles

Mechanistic Evaluation of miRNAs and Their Targeted Genes in the Pathogenesis and Therapeutics of Parkinson's Disease.

MicroRNA (miRNA) are usually 18-25 nucleotides long non-coding RNA targeting post-transcriptional regulation of genes involved in various biological processes. The function of miRNA is essential for maintaining a homeostatic cellular condition, regulating autophagy, cellular motility, and inflammation. Dysregulation of miRNA is responsible for multiple disorders, including neurodegeneration, which has emerged as a severe problem in recent times and has verified itself as a life-threatening condition that can be understood by the continuous destruction of neurons affecting various cognitive and motor functions. Parkinson's disease (PD) is the second most common, permanently debilitating neurodegenerative disorder after Alzheimer's, mainly characterized by uncontrolled tremor, stiffness, bradykinesia or akinesia (slowness in movement), and post-traumatic stress disorder. PD is mainly caused by the demolition of the primary dopamine neurotransmitter secretory cells and dopaminergic or dopamine secretory neurons in the substantia nigra pars compacta of the midbrain, which are majorly responsible for motor functions. In this study, a systematic evaluation of research articles from year 2017 to 2022 was performed on multiple search engines, and lists of miRNA being dysregulated in PD in different body components were generated. This study highlighted miR-7, miR-124, miR-29 family, and miR-425, showing altered expression levels during PD's progression, further regulating the expression of multiple genes responsible for PD.

Perinatal metabolic inflammation in the hypothalamus impairs the development of homeostatic feeding circuitry.

Over the past few decades, there has been a global increase in childhood obesity. This rise in childhood obesity contributes to the susceptibility of impaired metabolism during both childhood and adulthood. The hypothalamus, specifically the arcuate nucleus (ARC), houses crucial neurons involved in regulating homeostatic feeding. These neurons include proopiomelanocortin (POMC) and agouti-related peptide (AGRP) secreting neurons. They play a vital role in sensing nutrients and metabolic hormones like insulin, leptin, and ghrelin. The neurogenesis of AGRP and POMC neurons completes at birth; however, axon development and synapse formation occur during the postnatal stages in rodents. Insulin, leptin, and ghrelin are the essential regulators of POMC and AGRP neurons. Maternal obesity and postnatal overfeeding or a high-fat diet (HFD) feeding cause metabolic inflammation, disrupted signaling of metabolic hormones, netrin-1, and neurogenic factors, neonatal obesity, and defective neuronal development in animal models; however, the mechanism is unclear. Within the hypothalamus and other brain areas, there exists a wide range of interconnected neuronal populations that regulate various aspects of feeding. However, this review aims to discuss how perinatal metabolic inflammation influences the development of POMC and AGRP neurons within the hypothalamus.

Acetylcholine regulation of GnRH neuronal activity: A circuit in the medial septum.

In vertebrates, gonadotropin-releasing hormone (GnRH)-secreting neurons control fertility by regulating gonadotrophs in the anterior pituitary. While it is known that acetylcholine (ACh) influences GnRH secretion, whether the effect is direct or indirect, and the specific ACh receptor (AChR) subtype(s) involved remain unclear. Here, we determined 1) whether ACh can modulate GnRH cellular activity and 2) a source of ACh afferents contacting GnRH neurons. Calcium imaging was used to assay GnRH neuronal activity. With GABAergic and glutamatergic transmission blocked, subtype-specific AChR agonists and antagonists were applied to identify direct regulation of GnRH neurons. ACh and nicotine caused a rise in calcium that declined gradually back to baseline after 5-6min. This response was mimicked by an alpha3-specific agonist. In contrast, muscarine inhibited GnRH calcium oscillations, and blocking M2 and M4 together prevented this inhibition. Labeling for choline acetyltransferase (ChAT) and GnRH revealed ChAT fibers contacting GnRH neurons, primarily in the medial septum (MS), and in greater number in females than males. ChAT positive cells in the MS are known to express p75NGFRs. Labeling for p75NGFR, ChAT and GnRH indicated that ChAT fibers contacting GnRH cells originate from cholinergic cells within these same rostral areas. Together, these results indicate that cholinergic cells in septal areas can directly regulate GnRH neurons.

The influence of estro-progestin therapy on neurohormonal activity in functional hypothalamic amenorrhea

Background: Functional hypothalamic amenorrhea (FHA) is a chronic endocrine disorder caused by the abnormal pulsatile secretion of neurohormones in the hypothalamus. Secretion of GnRH is regulated by kisspeptin/neurokinin B/dynorphin (KNDy) neurons. These neurons produce, among other neurohormones, neurokinin B (NKB) which regulates the coordinated stimulation or inhibition of GnRH secreting neurons. Aim of the study: Assessment and comparison of serum NKB in patients with FHA at baseline, and following 6 months of estrogen-progestagen therapy. Materials and methods: Fifty-five patients with functional hypothalamic amenorrhea were included in the study group. Serum concentrations of neurokinin B (NKB), follicle stimulating hormone (FSH), luteinizing hormone (LH), 17-β-estradiol (E2), prolactin (PRL), cortisol, testosterone (T), dehydroepiandrosterone sulfate (DHEA-S), thyroid-stimulating hormone (TSH), free thyroxine (fT4), fasting glucose and insulin, as well as lipid profile were measured at baseline. At the time of diagnosis, patients with FHA were prescribed a course of 2 mg 17-β-estradiol and 10 mg dydrogesterone for duration of 6 months. Serum NKB was then reassessed following treatment at 6 months. Results: At baseline, the FHA group was found to have a decreased serum NKB concentration when compared to a healthy control group. Following 6 months of sequential estrogen-progestogen hormone therapy, this study did not find any statistically significant difference in serum NKB concentration in the treatment arm compared to baseline. Conclusions: For the first time, NKB secretion has been studied in patients with FHA. A significantly lower level of serum NKB was observed in these patients at baseline, when compared to a control group. After 6 months of combination estrogen-progesterone therapy, no significant changes in NKB levels were observed in these patients. These findings, for the first time in the literature, provide insight into the perceived benefit of HRT, calling into question its benefit in addressing the underlying etiopathogenetic contributors of FHA. These new findings may contribute to more targeted and appropriate treatment of such patients in the future.

Localization of SNARE proteins in the brain and corpus allatum of Bombyx mori.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) make up the core machinery that mediates membrane fusion. SNAREs, syntaxin, synaptosome-associated protein (SNAP), and synaptobrevin form a tight SNARE complex that brings the vesicle and plasma membranes together and is essential for membrane fusion. The cDNAs of SNAP-25, VAMP2, and Syntaxin 1A from Bombyxmori were inserted into a plasmid, transformed into Escherichiacoli, and purified. We then produced antibodies against the SNAP-25, VAMP2, and Syntaxin 1A of Bombyxmori of rabbits and rats, which were used for immunohistochemistry. Immunohistochemistry results revealed that the expression of VAMP2 was restricted to neurons in the pars intercerebralis (PI), dorsolateral protocerebrum (DL), and central complex (CX) of the brain. SNAP-25 was restricted to neurons in the PI and the CX of the brain. Syntaxin 1A was restricted to neurons in the PI and DL of the brain. VAMP2 co-localized with SNAP-25 in the CX, and with Syntaxin 1A in the PI and DL. VAMP2, SNAP-25, and Syntaxin 1A are present in the CA. Bombyxin-immunohistochemical reactivities (IRs) of brain and CA overlapped with VAMP2-, SNAP-25, and Syntaxin 1A-IRs. VAMP2 and Syntaxin 1A are present in the prothoracicotropic hormone (PTTH)-secretory neurons of the brain.

Comparative Expression Profile of RFRP-3 in Hypothalamus and Testis of Adult Rats

Background: The present study focused on the localization and comparative expression analysis of RFRP-3 gene in the hypothalamus and testis of adult male rats. Methods: Brain and testis tissue samples were collected from adult Wister rats and used for immunohistochemistry for identification and localization RFRP-3 producing neurons. The expression level of the RFRP-3 gene was assessed by Real-time PCR and compared with muscle tissue. Result: The results confirmed the presence of RFRP-3 secreting neurons in the hypothalamus and in the testes. Scattered RFRP-3 in cell bodies was identified primarily in the VMH, DMH and surrounding the third ventricle of the brain. In testis, RFRP-3 was detected in Leydig cells, Sertoli cells and germ cells. The expression level of RFRP-3 gene in the hypothalamus was observed to be significantly higher in hypothalamus than in testicular tissue (p less than 0.01) of adult rats.

The Dopamine D4 Receptor Regulates Gonadotropin-Releasing Hormone Neuron Excitability in Male Mice.

Gonadotropin-releasing hormone (GnRH)-secreting neurons control fertility. The release of GnRH peptide regulates the synthesis and release of both luteinizing hormone (LH) and Follicle stimulation hormone (FSH) from the anterior pituitary. While it is known that dopamine regulates GnRH neurons, the specific dopamine receptor subtype(s) involved remain unclear. Previous studies in adult rodents have reported juxtaposition of fibers containing tyrosine hydroxylase (TH), a marker of catecholaminergic cells, onto GnRH neurons and that exogenous dopamine inhibits GnRH neurons postsynaptically through dopamine D1-like and/or D2-like receptors. Our microarray data from GnRH neurons revealed a high level of Drd4 transcripts [i.e., dopamine D4 receptor (D4R)]. Single-cell RT-PCR and immunocytochemistry confirmed GnRH cells express the Drd4 transcript and protein, respectively. Calcium imaging identified changes in GnRH neuronal activity during application of subtype-specific dopamine receptor agonists and antagonists when GABAergic and glutamatergic transmission was blocked. Dopamine, dopamine with D1/5R-specific or D2/3R-specific antagonists or D4R-specific agonists decreased the frequency of calcium oscillations. In contrast, D1/5R-specific agonists increased the frequency of calcium oscillations. The D4R-mediated inhibition was dependent on Gαi/o protein coupling, while the D1/5R-mediated excitation required Gαs protein coupling. Together, these results indicate that D4R plays an important role in the dopaminergic inhibition of GnRH neurons.

Modulation of pulsatile GnRH dynamics across the ovarian cycle via changes in the network excitability and basal activity of the arcuate kisspeptin network.

Pulsatile GnRH release is essential for normal reproductive function. Kisspeptin secreting neurons found in the arcuate nucleus, known as KNDy neurons for co-expressing neurokinin B, and dynorphin, drive pulsatile GnRH release. Furthermore, gonadal steroids regulate GnRH pulsatile dynamics across the ovarian cycle by altering KNDy neurons' signalling properties. However, the precise mechanism of regulation remains mostly unknown. To better understand these mechanisms, we start by perturbing the KNDy system at different stages of the estrous cycle using optogenetics. We find that optogenetic stimulation of KNDy neurons stimulates pulsatile GnRH/LH secretion in estrous mice but inhibits it in diestrous mice. These in vivo results in combination with mathematical modelling suggest that the transition between estrus and diestrus is underpinned by well-orchestrated changes in neuropeptide signalling and in the excitability of the KNDy population controlled via glutamate signalling. Guided by model predictions, we show that blocking glutamate signalling in diestrous animals inhibits LH pulses, and that optic stimulation of the KNDy population mitigates this inhibition. In estrous mice, disruption of glutamate signalling inhibits pulses generated via sustained low-frequency optic stimulation of the KNDy population, supporting the idea that the level of network excitability is critical for pulse generation. Our results reconcile previous puzzling findings regarding the estradiol-dependent effect that several neuromodulators have on the GnRH pulse generator dynamics. Therefore, we anticipate our model to be a cornerstone for a more quantitative understanding of the pathways via which gonadal steroids regulate GnRH pulse generator dynamics. Finally, our results could inform useful repurposing of drugs targeting the glutamate system in reproductive therapy.

In Vitro Treg Immunosuppression Assay of GITR Proteins Could Elucidate the Autoimmune-mediated Mechanism Underlying Type 1 Narcolepsy

Abstract: Type 1 narcolepsy is a hypersomnia sleep disorder characterized by excessive daytime sleep and shallow NREM nighttime sleep. Deficiency of hypocretin-1 secreting neurons in the lateral hypothalamus is the primary cause of the disorder and studies demonstrated that these neurons were solely diminished in a brain region of remarkable heterogeneous neuronal population. Specific destruction of targeted neurons could be achieved via antigen presentation to immune cells, a characteristic of cell-mediated response in the adaptive immune system. Given that hypocretin-1 neurons were exclusively targeted, this cultivated significant interest in searching for an autoimmune-mediated mechanism but studies currently lack adequate knowledge and consistent results. In this research proposal, it is hypothesized that enhanced glucocorticoid-induced TNFR-related (GITR) protein expression in T regulatory (Treg) cells results in defective suppression capacity of CD4+CD25+ T helper cells and defective self-tolerance thereby promoting destruction of hypocretin-1 secreting-neurons in the lateral hypothalamus of narcoleptics. The proposal devises a correlational study to measure cerebrospinal fluid (CSF) hypocretin-1 levels that were inversely proportional to GITR expression levels in Treg cells to assess the autoimmune nature of the disorder. This study is aimed at investigating how defective T regulatory cells respond in type 1 narcolepsy patients via CSF hypocretin-1 measurement and in vitro human T regulatory cell suppression assay.

Central injection of neuropeptide Y modulates sexual behavior in male rats: interaction with GnRH and kisspeptin/neurokinin B/dynorphin

Aims A number of studies have shown that neuropeptide Y (NPY) is considered to be one of the key regulators of hypothalamic-pituitary-gonadal (HPG) axis in the mammals. In addition, kisspeptin (encode by Kiss1 gene), neurokinin B (encode by Tac3 gene) and dynorphin (encode by Pdyn gene) (commonly known as KNDy secreting neurons) are a powerful upstream regulators of GnRH neuron in hypothalamus. Materials and Methods The present study aims to investigate the effects of the intracerebroventricular (icv) injection of NPY and BIBP3226 (NPY receptor antagonist (NPYRA)) on the male sexual behavioral. Additionally, in order to see whether NPY signals can be relayed through the pathway of kisspeptin/neurokinin B/dynorphin, the gene expression of these peptides along with Gnrh1 gene in the hypothalamus were measured. Results The icv injection of NPY decreased the latencies and increase the frequencies of sexual parameters of the male rats in a significant way. In this line, NPYRA antagonized the stimulative effects of NPY. Moreover, data from real-time quantitative PCR indicated that injection of NPY significantly increased the gene expression of Gnrh1, Kiss1 and Tac3 and decrease the Pdyn while treatment with NPYRA controlled the modulative effects of NPY on these gene expression. Conclusions In conclusion based on the results of this study, NPY can exert its impacts on the sexual behavior of male rats via modulation of the KNDy secreting neurons as an interneural pathway to GnRH neurons.

Utility of measuring CSF hypocretin-1 level in patients with suspected narcolepsy

ObjectivesThe patho-aetiology of narcolepsy Type I (NT1) is the loss of hypocretin-1 secreting neurons in the hypothalamus. Diagnostic criteria for NT1 include excessive daytime sleepiness (EDS) for at least three months not explained by any other condition, cataplexy and cerebrospinal fluid (CSF) hypocretin-1 concentrations lower than 110 pg/ml. In this study we evaluated the utility of measuring CSF hypocretin-1 levels in patients with suspected narcolepsy (N). MethodsThe study included 29 consecutively recruited patients at a tertiary sleep centre presenting with EDS for exclusion of N. All patients were examined using an extensive clinical interview followed by two weeks of actigraphy and sleep diary recordings, polysomnography (PSG) and multiple sleep latency testing (MSLT). Additionally, HLA-typing, urinary screening for substances of abuse and a lumbar puncture to measure CSF hypocretin-1 expression using radioimmunoassay were carried out. ResultsIn sum, 19 patients (66%) had a CSF hypocretin-1 level <110 pg/ml, of whom two had current severe depression without any features of narcolepsy except EDS. The predictive potential of hypocretin-1 measurement in diagnosing narcolepsy revealed a positive predictive value (PPV) of 89%, a specificity of 83%, with both negative predictive value (NPV) and sensitivity equal to 100%. ConclusionsDespite a high sensitivity and specificity, the MSLT is not always a reliable diagnostic test for narcolepsy and where this uncertainty exits, CSF hypocretin-1 concentrations <110 pg/ml can be useful. However, due to a lower PPV and specificity at this cut-off, it may also not be entirely reliable as a stand-alone diagnostic test, particularly in the context of severe depression.

Neuropeptide galanin and its effects on metabolic and reproductive disturbances in female rats with estradiol valerate (EV) - Induced polycystic ovary syndrome (PCOS)

A functional role of the neuropeptide galanin, executed through the three G-protein coupled receptor subtypes GAL₁₋₃, has been demonstrated in many biological systems and under pathological circumstances. Galanin is involved in many central and peripheral actions, in particular associated with endocrine mechanisms such as anterior pituitary hormone regulation, reproduction, glucose metabolism and also inflammation. The role of galanin in the pathology of the polycystic ovary syndrome (PCOS) and possible therapeutic effects are unknown. However, based on the well known neuroendocrine changes in PCOS patients, it may be assumed that galanin via effects on gonadotropin-releasing hormone (GnRH) secretory neurons could play a significant role in the development of PCOS. The aim of this study was to examine possible therapeutic effects of galanin on hormonal, metabolic and molecular parameters in PCOS. Accordingly, intraperitoneal injection of galanin in a dose- dependent manner in female PCOS rats induced a significant reduction in inflammatory markers (TNF-α, IL-6), an increase in FSH and a decrease in LH, insulin and testosterone (using ELISA kit) compared to the PCOS groups. Moreover, data from real-time quantitative PCR showed significantly ameliorated changes in the mRNA levels of the steroidogenic acute regulatory protein (StAR) and aromatase cytochrome P450 (CYP19). Taken together, galanin has neuroendocrine, anti- and pro-inflammatory and metabolic effects, and we therefore suggest that treatment with this peptide could represent new therapeutic approach for managing hormonal and metabolic disturbances in the PCOS disease.

Correlation of IL-6 secretion and hyponatremia with the use of CD19+ chimeric antigen receptor T-cells .

Various studies have demonstrated that interleukin-6 (IL-6) activates the central magnocellular arginine vasopressin (AVP)-secreting neurons in the brain to produce non-osmotic, non-volume-mediated increases in AVP. The most common toxicity of CD19+ chimeric antigen receptor (CAR) T-cells is cytokine release syndrome, which is related to increased levels of IL-6. This study will evaluate the correlation of IL-6 levels with hyponatremia in patients receiving CD19+ CAR T-cells. This is a single-center retrospective analysis of adult patients who received CD19+ CAR T-cells for the treatment of relapsed/refractory acute lymphoblastic leukemia (ALL). Hyponatremia, defined as a serum sodium (Na) ≤ 135mEq/L, occurred in 31 (61%) patients. A change in Na >7mEq occurred in 32 (63%) patients, and the median lowest Na was 133mEq/L (interquartile range (IQR): 131-136)). There was an inverse linear relationship between IL-6 levels and lowest Na (p=0.001). Overall, per 10-fold increase in IL-6, Na decreased by an average of 2.68mEq/L. Hyponatremia is common in patients who received CD19+ CAR T-cells. There is an inverse linear relationship between IL-6 levels and nadir Na (p=0.001). Further studies will be needed to confirm a causative relationship between IL-6 levels and hyponatremia following CD19+ CAR T-cell infusion.

Surviving nutritional deprivation during development: neuronal intracellular calcium signaling is critical.

Developing cells and tissues in a growing animal need to sense food quality and integrate this information with on-going time-bound developmental programs. The integration of metabolism with development requires cellular and systemic coordination. Work in our laboratory has focused on Ca2+ signaling arising from the release of Ca2+ stored in the endoplasmic reticulum (ER), which triggers store-operated Ca2+ entry. We describe a role for ER-store Ca2+ that operates at the cellular level in various classes of neurons, and eventually drives the systemic coordination required to survive and complete development under conditions of nutritional deprivation. In the model system Drosophila melanogaster, we have developed a paradigm to induce nutritional stress during the larval stage and used pupariation as a read-out for development. Applying the vast genetic tool kit available in Drosophila to this paradigm, we have uncovered novel roles for intracellular Ca 2+ signaling in regulating neuronal activity, at the level of transcription in glutamatergic neurons, and translation in neuropeptidergic neurons. We find that such regulation of cellular processes is critical for integrating information across a neural circuit at multiple levels, starting from the point of sensing systemic and environmental levels of amino acids to finally connecting with neuropeptide secreting neurons, that communicate with the prothoracic gland, an organ that makes the key developmental hormone, ecdysone. This work underscores the importance of ER-store Ca2+ for neuronal health, with consequences for animal development.

Effect of food deprivation on neuropeptide Y immunoreactivity and concurrent biochemical and gonadal responses in tilapia Oreochromis mossambicus (Peters, 1852)

The effect of short- and long-term fasting on neuropeptide Y (NPY) secreting neurons in the brain and concurrent biochemical and gonadal responses were determined in the cichlid fish Oreochromis mossambicus (Peters, 1852). Exposure of fish to fasting for 6 or 12 days resulted in intensely stained NPY-immunoreactive neurons in the nucleus entopeduncularis and the nucleus preopticus regions of the brain in contrast to moderately stained neurons in control fishes, whereas food supplementation after 6 days starvation restored NPY-immunoreactive profile to the same state as in control groups in both male and female fish. The gonadosomatic index remained significantly lower in female fishes subjected to fasting alone as well as fasting followed by refeedings compared to controls. While the hepatosomatic index (HSI) also showed a significant decrease in fasting male and female fish compared to controls, it increased subsequently in fishes exposed to refeeding after starvation. In addition, a significant decrease in the serum levels of glucose and triglycerides as well as the hepatic tissue protein content in fasting fish compared to those of controls in both sexes suggested gluconeogenesis. Although the serum levels of glucose and triglycerides were reinstated in both males and female fishes subjected to starvation and refeeding, the hepatic tissue protein levels were not completely restored in females. Collectively, these results suggest that food-deprivation, either for short or long term duration, significantly increases the secretory activity of NPY neurons in the brain concomitant with a significant decrease in the serum levels of glucose and triglycerides, the hepatic tissue protein content and the GSI in both sexes. Although males recovered from the effects of starvation following refeeding, females appeared to be more vulnerable to the effects of fasting.

Distribution of Kiss2 receptor in the brain and its localization in neuroendocrine cells in the zebrafish.

Kisspeptin is a hypothalamic neuropeptide, which acts directly on gonadotropin-releasing hormone (GnRH)-secreting neurons via its cognate receptor (GPR54 or Kiss-R) to stimulate GnRH secretion in mammals. In non-mammalian vertebrates, there are multiple kisspeptins (Kiss1 and Kiss2) and Kiss-R types. Recent gene knockout studies have demonstrated that fish kisspeptin systems are not essential in the regulation of reproduction. Studying the detailed distribution of kisspeptin receptor in the brain and pituitary is important for understanding the multiple action sites and potential functions of the kisspeptin system. In the present study, we generated a specific antibody against zebrafish Kiss2-R (=Kiss1Ra/GPR54-1/Kiss-R2/KissR3) and examined its distribution in the brain and pituitary. Kiss2-R-immunoreactive cell bodies are widely distributed in the brain including in the dorsal telencephalon, preoptic area, hypothalamus, optic tectum, and in the hindbrain regions. Double-labeling showed that not all but a subset of preoptic GnRH3 neurons expresses Kiss2-R, while Kiss2-R is expressed in most of the olfactory GnRH3 neurons. In the posterior preoptic region, Kiss2-R immunoreactivity was seen in vasotocin cells. In the pituitary, Kiss2-R immunoreactivity was seen in corticotropes, but not in gonadotropes. The results in this study suggest that Kiss2 and Kiss2-R signaling directly serve non-reproductive functions and indirectly subserve reproductive functions in teleosts.

Neurotensin Enhances Locomotor Activity and Arousal and Inhibits Melanin-Concentrating Hormone Signaling

Background: Hypothalamic neurotensin (Nts)-secreting neurons regulate fundamental physiological processes including metabolism and feeding. However, the role of Nts in modulation of locomotor activity, sleep, and arousal is unclear. We previously identified and characterized Nts neurons in the zebrafish hypothalamus. Materials and Methods: In order to study the role of Nts, nts mutant (nts–/–), and overexpressing zebrafish were generated. Results: The expression of both nts mRNA and Nts protein was reduced during the night in wild-type zebrafish. Behavioral assays revealed that locomotor activity was decreased during both day and night, while sleep was increased exclusively during the nighttime in nts–/– larvae. Likewise, inducible overexpression of Nts increased arousal in hsp70:Gal4/uas:Nts larvae. Furthermore, the behavioral response to light-to-dark transitions was reduced in nts–/– larvae. In order to elucidate potential contenders that may mediate Nts action on these behaviors, we profiled the transcriptome of 6 dpf nts–/– larvae. Among other genes, the expression levels of melanin-concentrating hormone receptor 1b were increased in nts–/– larvae. Furthermore, a portion of promelanin-concentrating hormone 1 (pmch1) and pmch2 neurons expressed the nts receptor. In addition, expression of the the two zebrafish melanin-concentrating hormone (Mch) orthologs, Mch1 and Mch2, was increased in nts–/– larvae. Conclusion: These results show that the Nts and Mch systems interact and modulate locomotor activity and arousal.

SAT-427 Low Chloride Transporter Expression in Vasopressin Neurons

Several membrane proteins function as ion pumps/transporters that work to maintain the relative ionic concentrations necessary for neuronal signaling. The potassium-chloride co-transporter two (KCC2) is a symporter that actively pumps K+ and Cl- out of the cell in order to maintain a low intracellular [Cl-], while the sodium-potassium-chloride cotransporters one and two (NKCC1/NKCC2) are symporters that actively pump Na+, K+, and Cl- into the cell. KCC2 and NKCC1/2, therefore, have opposing actions on Cl- transport. In the adult, high KCC2 expression leads to low intracellular [Cl-], which causes GABAA receptor Cl- channels to flux Cl- into the neuron, causing a membrane hyperpolarization. Early in development however, low KCC2 expression reverses the Cl- gradient and causes GABAA receptors to flux Cl- out of the neuron, leading to membrane depolarization and neuronal excitation. The change in the polarity of GABA signaling during development is, in part, due to a shift in KCC2 expression from low to high with the maturation of synaptic circuits. Our previous studies have demonstrated an excitatory GABAA receptor-mediated response due to a GABA equilibrium potential (EGABA) that is shifted positive, like that seen early in development, in adult vasopressin (VP)-secreting neurons of the rat hypothalamus. In the current study, we used immunohistochemistry to compare expression levels of the main Cl- transporters, KCC2, NKCC1, and NKCC2, between VP- and oxytocin (OT)-secreting neurons of the hypothalamic paraventricular and supraoptic nuclei. We found that, in adult male Wistar rats, expression of all three transporters is uniformly lower in VP neurons than in adjacent OT neurons in the hypothalamus. Because the phosphorylation state of KCC2 affects membrane trafficking and stabilization, we are also using Western blot to analyze phosphorylation of KCC2 under different osmotic conditions. Finally, we are using Cre-dependent viral transduction to express a light-activatable Cl- channel conditionally in vasopressin-secreting neurons to test for the effect of Cl- flux on vasopressin secretion.

Sensory and behavioural control of gonadotrophin secretion during suckling-mediated anovulation in cows

A major limiting factor in the resumption of postpartum ovarian cycles in cattle is the inhibitory influence of the suckling calf on central regulatory elements controlling the release of GnRH from the median eminence. This inhibitory influence occurs only as a consequence of specifically defined behavioural interactions between the cow and calf in the presence of a maternal bond, cannot be simulated experimentally using thermal, electrical or mechanical stimuli, and is not dependent upon sensory ennervation within the udder. Indeed, the identity of the calf, either own or unrelated, appears to define the neuroendocrine events that attend the maintenance of the anovulatory state under controlled experimental conditions. Hence, new hypotheses that focus upon the relationship between physiological correlates of maternal behaviour and hypothalamic regulation of LH secretion are currently being tested. Specific aspects of these relationships remain conjectural, but are postulated to include the regulation of opioid tone, intracerebral oxytocin, and modulation of synthetic or excitatory activity of GnRH secretory neurones. Defining the role of the special senses in transduction of signals from calves that influence these neuronal processes may provide insight for developing practical intervention strategies of the future.

Low wnt/β-catenin signaling determines leaky vessels in the subfornical organ and affects water homeostasis in mice.

The circumventricular organs (CVOs) in the central nervous system (CNS) lack a vascular blood-brain barrier (BBB), creating communication sites for sensory or secretory neurons, involved in body homeostasis. Wnt/β-catenin signaling is essential for BBB development and maintenance in endothelial cells (ECs) in most CNS vessels. Here we show that in mouse development, as well as in adult mouse and zebrafish, CVO ECs rendered Wnt-reporter negative, suggesting low level pathway activity. Characterization of the subfornical organ (SFO) vasculature revealed heterogenous claudin-5 (Cldn5) and Plvap/Meca32 expression indicative for tight and leaky vessels, respectively. Dominant, EC-specific β-catenin transcription in mice, converted phenotypically leaky into BBB-like vessels, by augmenting Cldn5+vessels, stabilizing junctions and by reducing Plvap/Meca32+ and fenestrated vessels, resulting in decreased tracer permeability. Endothelial tightening augmented neuronal activity in the SFO of water restricted mice. Hence, regulating the SFO vessel barrier may influence neuronal function in the context of water homeostasis.