Transient receptor potential vanilloid 5 (TRPV5), a highly Ca2+ -selective TRP channel in the rat brain: relevance to neuroendocrine regulation.

Recent studies suggest an important role for transient receptor potential vanilloid (TRPV) ion channels in neural and neuroendocrine regulation. The TRPV subfamily consists of six members: TRPV1-6. While the neuroanatomical and functional correlates of TRPV1-4 have been studied extensively, relevant information about TRPV5 and TRPV6, which are highly selective for Ca2+ , is limited. We detected TRPV5 mRNA expression in the olfactory bulb, cortex, hypothalamus, hippocampus, midbrain, brainstem and cerebellum of the rat. TRPV5-immunoreactive neurones were conspicuously seen in the hypothalamic paraventricular (PVN), supraoptic (SON), accessory neurosecretory (ANS), supraoptic nucleus, retrochiasmatic part (SOR), arcuate (ARC) and medial tuberal nuclei, hippocampus, midbrain, brainstem and cerebellum. Glial cells also showed TRPV5-immunoreactivity. To test the neuroendocrine relevance of TRPV5, we focused on vasopressin, oxytocin and cocaine- and amphetamine-regulated transcript (CART) as representative candidate markers with which TRPV5 may co-exist. In the hypothalamic neurones, co-expression of TRPV5 was observed with vasopressin (PVN: 50.73±3.82%; SON: 75.91±2.34%; ANS: 49.12±4.28%; SOR: 100%) and oxytocin (PVN: 6.88±1.21; SON: 63.34±5.69%; ANS: 20.4±4.14; SOR: 86.5±1.74%). While ARC neurones express oestrogen receptors, 17ß-oestradiol regulates TRPV5, as well as CART neurones and astrocytes, in the ARC. Furthermore, ARC CART neurones are known to project to the preoptic area, and innervate and regulate GnRH neurones. Using double-immunofluorescence, glial fibrillary acidic protein-labelled astrocytes and the majority of CART neurones in the ARC showed TRPV5-immunoreactivity. Following iontophoresis of retrograde neuronal tracer, cholera toxin ß (CtB) into the anteroventral periventricular nucleus and median preoptic nucleus, retrograde accumulation of CtB was observed in most TRPV5-equipped ARC CART neurones. Next, we determined the response of TRPV5-elements in the ARC during the oestrous cycle. Compared to pro-oestrus, a significant increase (P<.001) in the percentage of TRPV5-expressing CART neurones was observed during oestrus, metoestrus, and dioestrus. TRPV5-immunoreactivity in the astrocytes, however, showed a significant increase during metoestrus and dioestrus. We suggest that the TRPV5 ion channel may serve as an important regulator of neural and neuroendocrine pathways in the brain.

Role of Isotocin in the Regulation of the Hypophysiotropic Dopamine Neurones in the Preoptic Area of the Catfish, Clarias batrachus.

Dopamine (DA) has emerged as a potent inhibitory neuromodulator of luteinsing hormone (LH) secretion and reproduction in teleosts. The DA neurones located in the anterior subdivision of nucleus preopticus periventricularis (NPPa) in the preoptic area (POA) innervate the pituitary gland and regulate LH cells. Although a reduction in the inhibitory DAergic tone is crucial for stimulatory action of gonadotrophin-releasing hormone (GnRH) on LH cells, the role of other hypothalamic factors is suggested but not fully understood. Nonapeptide, isotocin (IST) has emerged as a likely candidate that may also influence the LH cell function. IST neurones reside in the nucleus preopticus and innervate LH cells. While IST treatment dramatically elevated LH secretion, the IST levels in brain peaked during spawning. In a pilot study on the catfish, Clarias batrachus, we observed a dense network of IST-immunoreactive (IST-IR) fibres in the NPPa, the region known to harbour hypophysiotropic DA neurones. Application of the double immunofluorescence method showed a dense IST-IR fibre network around the tyrosine hydroxylase-immunoreactive (TH-IR) neurones in the NPPa region. A great majority of the TH-IR neurones in the NPPa were contacted by IST-IR fibres during the spawning phase. The NPPa therefore appears to be a site for the intense interaction of DA and IST. IST-IR fibre innervation in NPPa showed reproduction phase-dependent changes. The percent fluorescent area of IST-IR fibres showed a gradual increase from the resting through prespawning phases (resting: 7.5 ± 1.04; preparatory: 8.6 ± 0.8; prespawning: 15.5 ± 1.4), reaching a peak in the spawning phase (28 ± 2.3; P < 0.001). Compared to the spawning phase, a drastic reduction in IST-IR fibres in the NPPa was observed during the postspawning phase (8.4 ± 0.9; P < 0.001). Superfused slices of the POA of C. batrachus treated with IST peptide resulted in a significant reduction in TH immunoreactivity in the NPPa (Control: 45.3 ± 4.2; IST peptide, 5 µm: 29.4 ± 4.7; P < 0.05). We suggest that the intense interaction between IST and DA in the NPPa, most probably of an inhibitory nature, may be critical for the regulation of LH cells and reproduction in teleosts.

Tyrosine hydroxylase in the olfactory system, forebrain and pituitary of the Indian major carp, Cirrhinus cirrhosus: organisation and interaction with neuropeptide Y in the preoptic area.

Dopamine (DA) inhibits, whereas gonadotrophin-releasing hormone (GnRH) stimulates, luteinisiing (LH) cells in the pituitary of some but not all teleosts. A reduction in the hypophysiotropic dopaminergic tone is necessary for the stimulatory effect of GnRH on LH cells. Neuropeptide Y (NPY) has emerged as one of the potent, endogenous agent that modulates LH secretion directly or indirectly via GnRH. Involvement of NPY in the regulation of hypophysiotropic DA neurones, however, is not known, but there is good evidence suggesting an interaction in the mammalian hypothalamus. DA neurones, identified by tyrosine hydroxylase (TH)-immunoreactivity, were observed widely throughout the brain of the Indian major carp, Cirrhinus cirrhosus. The granule cells and ganglion cells of terminal nerve in the olfactory bulb, and cells in ventral telencephalon and preoptic area (POA) showed conspicuous TH immunoreactivity. In the POA, the nucleus preopticus periventricularis (NPP), divisible into anterior (NPPa) and posterior (NPPp) components, showed prominent TH-immunoreactivity. The majority of TH neurones in NPPa showed axonal extensions to the pituitary and were closely associated with LH cells. The NPPa also appeared to be the site for intense interaction between NPY and DA because it contains a rich network of NPY fibres and few immunoreactive cells. Approximately 89.7 ± 1.5% TH neurones in NPPa were contacted by NPY fibres. Superfused POA slices treated with a NPY Y2 -receptor agonist, NPY 13-36 resulted in a significant (P < 0.001) reduction in TH-immunoreactivity in NPPa. TH neurones in NPPa did not respond to NPY Y1 -receptor agonist, [Leu(31) , Pro(34) ] Neuropeptide Y treatment. We suggest that, by inhibiting DAergic neurones in NPPa via Y2 -receptors, NPY may contribute to the up-regulation of the GnRH-LH cells axis. The microcircuitry of DA and NPY and their interaction in NPPa might be a crucial component in the central regulation of LH secretion in the teleosts.

Interaction between dopamine- and isotocin-containing neurones in the preoptic area of the catfish, Clarias batrachus: role in the regulation of luteinising hormone cells.

Apart from gonadotrophin-releasing hormone (GnRH) and dopamine (DA), oxytocin has emerged as an important endogenous agent that regulates reproduction. Although the interaction between these factors has been extensively studied in mammals, parallel information in teleosts is much limited. We studied the organisation of tyrosine hydroxylase (TH; a marker for dopamine) and isotocin neurones in the preoptic area (POA) and hypothalamus of the catfish, Clarias batrachus and its implication in the regulation of luteinising hormone (LH) cells in the pituitary. Nucleus preopticus periventricularis (NPP), a major dopaminergic centre in the brain, consists of anterior (NPPa) and posterior (NPPp) subdivisions. Using retrograde neuronal tracing, we found that majority of the DA neurones in NPPa, but none from NPPp, project to the pituitary. The nucleus preopticus (NPO) of C. batrachus contains a conspicuous assemblage of large isotocin-positive neurones. It consists of a paraventricular subdivision (NPOpv) located on either side of the third ventricle and lies roughly sandwiched between the dopaminergic neurones of NPPa and NPPp. An additional subset of isotocin neurones was located above the optic chiasm in the supraoptic subdivision of the NPO (NPOso). Isotocin-containing neurones in both the subdivisions of NPO were densely innervated by DA fibres. Superfusion of the POA-containing brain slices with DA D(1) -like receptor agonist (SKF-38393) resulted in significant increase in isotocin immunoreactivity in the NPOpv neurones; NPOso neurones did not respond. However, treatment with DA D(2) -like receptor agonist (quinpirole) reduced isotocin immunoreactivity in the NPOso, but not in the NPOpv. Thus, DA appears to differentially regulate the components of isotocinergic system. Isotocin fibres extend to the pituitary and terminate on LH cells and the superfused pituitary slices treated with isotocin caused significant reduction in LHß-immunoreactivity. An elaborate interplay between the DA and isotocin systems appears to be an important component of the LH regulatory system.

Activation of anorexigenic pro-opiomelanocortin neurones during refeeding is independent of vagal and brainstem inputs.

After fasting, satiety is observed within 2 h after reintroducing food, accompanied by activation of anorexigenic, pro-opiomelanocortin (POMC)-synthesising neurones in the arcuate nucleus (ARC), indicative of the critical role that α-melanocyte-stimulating hormone has in the regulation of meal size during refeeding. To determine whether refeeding-induced activation of POMC neurones in the arcuate is dependent upon the vagus nerve and/or ascending brainstem pathways, bilateral subdiaphragmatic vagotomy or transection of the afferent brainstem input to one side of the ARC was performed. One day after vagotomy or 2 weeks after brain surgery, animals were fasted and then refed for 2 h. Sections containing the ARC from vagotomised animals or animals with effective transection were immunostained for c-Fos and POMC to detect refeeding-induced activation of POMC neurones. Quantitative analyses of double-labelled preparations demonstrated that sham-operated and vagotomised animals markedly increased the number of c-Fos-immunoreactive (-IR) POMC neurones with refeeding. Furthermore, transection of the ascending brainstem pathway had no effect on diminishing c-Fos-immunoreactivity in POMC neurones on either side of the ARC, although it did diminish activation in a separate, subpopulation of neurones in the dorsomedial posterior ARC (dmpARC) on the transected side. We conclude that inputs mediated via the vagus nerve and/or arising from the brainstem do not have a primary role in refeeding-induced activation of POMC neurones in the ARC, and propose that these neurones may be activated solely by direct effects of circulating hormones/metabolites during refeeding. Activation of the dmpARC by refeeding indicates a previously unrecognised role for these neurones in appetite regulation in the rat.

Central regulation of feeding behavior during social isolation of rat: evidence for the role of endogenous CART system.

OBJECTIVE: Although hyperphagia and body weight gain are well-recognized consequences of social isolation, the underlying mechanisms are not understood. The aim of this work is to test the possibility that the endogenous cocaine- and amphetamine-regulated transcript peptide (CART) may be involved in the process. DESIGN: Socially isolated rats were screened for increase in food intake and body weight, and the modifications of these parameters by CART were evaluated. Furthermore, isolated animals were re-socialized and screened for reversal of these effects. Response of the endogenous CART system, in certain hypothalamic nuclei of the isolated and re-socialized rats, was evaluated with immunohistochemistry. SUBJECTS: Fifty days old naive male Sprague-Dawley rats were used. MEASUREMENTS: The effects of CART/CART antibody on the social isolation and subsequent re-socialization on feeding and body weight changes were monitored. Moreover, the immunohistochemical response of endogenous CART system to social isolation and re-socialization was analyzed morphometrically. RESULTS: While social isolation of rats for a period of 6 weeks caused progressive increase in food consumption and body weight gain, these rats showed a significant reduction in food intake and body weight when injected daily with CART via intracerebroventricular (i.c.v.) route, for the following 7 days. The re-socialization of isolated rats reduced food intake and body weight to the control levels. These effects of re-socialization were attenuated by immunoneutralization of the endogenous CART by i.c.v. CART antibody. Social isolation also resulted in a drastic reduction in CART immunoreactivity in the cells and/or fibers in the hypothalamic areas like dorsomedial, ventromedial, lateral, paraventricular and arcuate nuclei, recognized for their role in feeding. On the other hand, the CART immunoreactivity profile was fully restored following 7 days of re-socialization of the isolation-reared rats. CONCLUSION: Social isolation might down-regulate the hypothalamic CART-containing system, which in turn may lead to increase in food intake and body weight.

Regulation of cocaine- and amphetamine-regulated transcript-synthesising neurons of the hypothalamic paraventricular nucleus by endotoxin; implications for lipopolysaccharide-induced regulation of energy homeostasis.

Infectious diseases and the administration of bacterial lipopolysaccharide (LPS) result in decreased food intake and increased energy expenditure. Because the hypothalamic paraventricular nucleus (PVN) has pivotal roles in the regulation of energy homeostasis and expresses an anorexic peptide, cocaine- and amphetamine-regulated transcript (CART), we hypothesised that increased CART synthesis in this nucleus may contribute to LPS-induced changes in energy homeostasis. Therefore, we studied the effects of intraperitoneal administration of LPS on CART gene expression in the PVN by semiquantitative in situ hybridisation. LPS caused a rapid increase in CART mRNA levels in the PVN. One hour after treatment, the density of silver grains was increased by three-fold in the PVN, and remained elevated 3 h after treatment. Because the dorsal vagal complex, an important vegetative centre in the brainstem, is heavily innervated by CART-containing axons, we determined whether the retrograde tracer, cholera toxin B subunit (CTB), accumulates in CART neurons in the PVN following stereotaxic injection of the tracer into the dorsal vagal complex. One week after injection, CTB accumulated in CART neurons in the ventral, medial, and lateral parvocellular subdivisions of the PVN. In addition, LPS administration induced c-fos expression in a population of CART neurons in the PVN that project to the dorsal vagal complex. These data indicate that increased CART gene expression in neurons of PVN may contribute to LPS-induced anorexia, and suggest that this action may be mediated, at least in part, through a PVN-dorsal vagal complex pathway.

Reproduction phase-related expression of beta-endorphin-like immunoreactivity in the nucleus lateralis tuberis of the female Indian major carp Cirrhinus mrigala: correlation with the luteinising hormone cells-ovary axis.

The present study aimed to determine whether beta-endorphin immunoreactivity (bEP-ir) in the neurones of the nucleus lateralis tuberis (NLT) is linked to the seasonal cycle and shows correlation with the number of luteinising hormone (LH) cells in the pituitary gland and ovaries in the teleost, Cirrhinus mrigala. Although LH cells were moderately immunostained during the resting phase (December to January), the morphological profile suggested increased synthetic and secretory activity during the preparatory (February to April) and prespawning (May to June) phases. However, LH immunoreactivity was greatly reduced (P < 0.001) in the spawning (July to August) phase, suggesting massive discharge of the hormone; this pool was partly replenished in the postspawning (September to November) phase. The ovaries grew rapidly in the preparatory and prespawning phases; maximal size was attained during spawning, when ovulation occurred. Thereafter, the ovaries regressed. The NLT of C. mrigala is divisible into the pars lateralis (NLTl) and medialis (NLTm). During the postspawning and resting phases, bEP-ir was readily detectable in the NLTm as well as NLTl neurones. However, a steady reduction in the immunoreactivity was observed in the NLTm neurones during the preparatory through spawning phases (P < 0.001), suggesting a negative correlation with the LH cells-ovary axis. Thus, the inhibitory influence of beta-endorphin on the gonadotrophin-releasing hormone (GnRH)-LH axis appears to be attenuated during the preparatory through spawning phases. This may be necessary for the rapid stimulation of the axis culminating in spawning. Neurones of the NLTl also showed a gradual reduction in bEP-ir during the preparatory and prespawning phases (P < 0.01) and may therefore play a similar role. However, significant augmentation of the immunoreactivity was noticed in these neurones during the spawning phase (P < 0.001), the physiological significance of which is unknown. Although the present study demonstrated a temporal correlation between the beta-endorphin in the NLT, LH cells and the ovary, we suggest that the peptide in the NLTl and NLTm may show functional duality during the spawning phase.