Uday Singh

CART peptide in the nucleus accumbens shell acts downstream to dopamine and mediates the reward and reinforcement actions of morphine.

The opioid-mesolimbic-dopamine circuitry operates between ventral tegmental area (VTA) and nucleus accumbens (Acb) and serves as a major reward pathway. We hypothesized that the neuropeptide cocaine- and amphetamine-regulated transcript (CART) is involved in the natural reward action mediated by the circuitry. Therefore, the modulation of opioid-mesolimbic-dopamine reward circuitry by CART was investigated using pellet self-administration paradigm in operant chamber. Morphine administered bilaterally in shell region of Acb (AcbSh) significantly increased active lever pressings and pellet self-administration. While CART given bilaterally in the AcbSh significantly increased pellet self-administration, CART antibody produced no effect. Morphine induced pellet self-administration was potentiated by CART, and antagonized by CART antibody administered in AcbSh. A close interaction between dopamine and CART systems was observed. Several tyrosine hydroxylase (marker for dopamine) immunoreactive fibers were seen contacting CART neurons in the AcbSh. Intraperitoneal administration of pramipexole, a dopamine agonist, increased pellet self-administration. The effect was blocked by prior treatment with CART antibody targeted at AcbSh. CART-immunoreactive cells and fibers in the AcbSh, and cells but not fibers in hypothalamic paraventricular nucleus (PVN), were significantly increased in the animals trained in operant chamber. However, CART-immunoreactive profile in the medial forebrain bundle, VTA and arcuate nucleus of hypothalamus did not respond. We suggest that CART, released from the axonal terminals in the framework of AcbSh, may serve as the final output of the endogenous opioid-mesolimbic-dopamine circuitry that processes natural reward.

Alpha-melanocyte stimulating hormone modulates ethanol self-administration in posterior ventral tegmental area through melanocortin-4 receptors

Although the role of alpha‐melanocyte stimulating hormone (α‐MSH) in alcohol seeking behaviour in rats has been demonstrated, the underlying mechanisms are not understood. Herein, we test the hypothesis that α‐MSH might have a permissive effect in promoting the reward action of ethanol. Rats were implanted with cannulae targeted at the posterior ventral tegmental area (pVTA), because the site is sensitive to reinforcing effects of ethanol. These rats were trained to self‐administer ethanol in standard two‐lever (active/inactive) operant chamber test. Each active lever press resulted in self‐administration of 100 nl of ethanol (100–300 mg%) containing solution. Over a period of 7 days, ethanol significantly increased the number of lever presses, which was considered as a measure of reward. Because ethanol at 200 mg% resulted in maximum number of lever presses (∼18–20 lever presses/30‐minute session), the dose was employed in further studies. While prior administration of melanocortin (MC) agonists, α‐MSH or [Nle4,D‐Phe7]‐alpha‐MSH into pVTA, resulted in an 89% increase in lever presses, the response was attenuated following pre‐treatment with MC4 receptors (MC4R) antagonist, HS014. In an immunohistochemical study, the brains of rats that were trained to self‐infuse ethanol showed significantly increased α‐MSH immunoreactivity in the nucleus accumbens shell, bed nucleus of stria terminalis and arcuate nucleus of the hypothalamus. In the pVTA, α‐MSH fibres were found to run close to the dopamine cells, labelled with tyrosine hydroxylase antibodies. We suggest that α‐MSH‐MC4R system in the pVTA might be a part of the neuroadaptive mechanism underlying ethanol addiction.

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 D1‐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 D2‐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 LHβ‐immunoreactivity. An elaborate interplay between the DA and isotocin systems appears to be an important component of the LH regulatory system.

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, [Leu31, Pro34] 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.

Molecular interpretation of ACTH-β-endorphin coaggregation: relevance to secretory granule biogenesis.

Peptide/protein hormones could be stored as non-toxic amyloid-like structures in pituitary secretory granules. ACTH and β-endorphin are two of the important peptide hormones that get co-stored in the pituitary secretory granules. Here, we study molecular interactions between ACTH and β-endorphin and their colocalization in the form of amyloid aggregates. Although ACTH is known to be a part of ACTH-β-endorphin aggregate, ACTH alone cannot aggregate into amyloid under various plausible conditions. Using all atom molecular dynamics simulation we investigate the early molecular interaction events in the ACTH-β-endorphin system, β-endorphin-only system and ACTH-only system. We find that β-endorphin and ACTH formed an interacting unit, whereas negligible interactions were observed between ACTH molecules in ACTH-only system. Our data suggest that ACTH is not only involved in interaction with β-endorphin but also enhances the stability of mixed oligomers of the entire system.

Transient receptor potential vanilloid 6 (TRPV6) in the mouse brain: Distribution and estrous cycle-related changes in the hypothalamus.

Transient receptor potential vanilloid (TRPV) subfamily of cationic channels have emerged as novel players in neural regulation. Unlike other members of TRPV subfamily, TRPV5 and TRPV6 are highly Ca2+-selective. Although TRPV5/TRPV6 transcripts are expressed in mouse brain, understanding the full functional spectrum of these ion channels in the brain is however limited due to the lack of information on their neuroanatomical distribution. We have studied TRPV6 in mouse brain in further detail. In the hypothalamus, while Western blot analysis using TRPV6 specific antiserum showed a distinct ∼95 kDa band corresponding to the molecular weight of TRPV6, transcripts for TRPV6 were detected with RT-PCR. TRPV6-immunoreactive cells/fibers were observed in vascular organ of the lamina terminalis, olfactory bulb, amygdala, hippocampus, septohypothalamic, supraoptic, arcuate (ARC), dorsomedial, and subincertal nuclei. TRPV6-immunoreactive cells/fibers were also observed in the brainstem and cerebellum. Estrogen has emerged as a potential regulator of TRPV6 in peripheral tissues. TRPV6 gene promoter contains estrogen-response element, estrogen activates TRPV6 via estrogen receptor alpha (ERα), and ERα-expressing ARC neurons in mediobasal hypothalamus (MBH) serve as primary site for estradiol feedback. Using double immunofluorescence, co-expression of TRPV6 and ERα was observed in several ARC neurons. MBH of mice during different phases of estrous cycle were subjected to Western blot analysis of TRPV6. Compared to proestrus, a significant reduction (P<0.01) in intensity of TRPV6-immunoreactive band was observed in MBH during metestrus and diestrus phases. While the wide distribution of TRPV6-expressing elements in the brain suggests its role in a range of CNS functions, the ion channel may serve as novel component of the neural pathway mediating effects of estradiol in MBH.

Cocaine- and amphetamine-regulated transcript peptide (CART) in the brain of zebra finch, Taeniopygia guttata: Organization, interaction with neuropeptide Y, and response to changes in energy status.

Cocaine‐ and amphetamine‐regulated transcript (CART) has emerged as a potent anorectic agent. CART is widely distributed in the brain of mammals, amphibians, and teleosts, but the relevant information in avian brain is not available. In birds, CART inhibits food intake, whereas neuropeptide Y (NPY), a well‐known orexigenic peptide, stimulates it. How these neuropeptides interact in the brain to regulate energy balance is not known. We studied the distribution of CART‐immunoreactivity in the brain of zebra finch, Taeniopygia guttata, its interaction with NPY, and their response to dynamic energy states. CART‐immunoreactive fibers were found in the subpallium, hypothalamus, midbrain, and brainstem. Conspicuous CART‐immunoreactive cells were observed in the bed nucleus of the stria terminalis, hypothalamic paraventricular, supraoptic, dorsomedial, infundibular (IN), lateral hypothalamic, Edinger‐Westphal, and parabrachial nuclei. Hypothalamic sections of fed, fasted, and refed animals were immunostained with cFos, NPY, and CART antisera. Fasting dramatically increased cFos‐ and NPY‐immunoreactivity in the IN, followed by rapid reduction by 2 hours and restoration to normal fed levels 6–10 hours after refeeding. CART‐immunoreactive fibers in IN showed a significant reduction during fasting and upregulation with refeeding. Within the IN, double immunofluorescence revealed that 94 ± 2.1% of NPY‐immunoreactive neurons were contacted by CART‐immunoreactive fibers and 96 ± 2.8% NPY‐immunoreactive neurons expressed cFos during fasting. Compared to controls, superfused hypothalamic slices of fasted birds treated with CART‐peptide showed a significant reduction (P < 0.001) in NPY‐immunoreactivity in the IN. As in other vertebrates, CART in the brain of T. guttata may perform several functions, and has a particularly important role in the hypothalamic regulation of energy homeostasis. J. Comp. Neurol. 524:3014–3041, 2016.

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, 17β‐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 β (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.

Transient receptor potential vanilloid 1-6 (Trpv1-6) gene expression in the mouse brain during estrous cycle

In recent years estradiol has emerged as a potential regulator of transient receptor potential vanilloid (TRPV) cationic channels in the peripheral tissues and sensory neurons, however, its analogous role in the CNS is poorly understood. TRPV channels modulate Ca2+ signalling, neurotransmission and behaviour, and expression of these ion channels and estrogen receptors show a great degree of overlap in different brain regions. Herein, we probe if Trpv1-6 genes contain estrogen receptor-binding sites and if their expression in different brain regions is modulated during estrous cycle. Bioinformatics analysis of the mouse Trpv1-6 gene sequences showed presence of putative functional estrogen response element in their promoter regions. Using qRT-PCR, Trpv1-6 mRNA expression was observed in the olfactory bulb, cortex, hypothalamus, hippocampus, brainstem, and cerebellum of mouse. In these regions, compared to estrus, metestrus, and diestrus, reduced levels of Trpv1 and Trpv5 but elevated Trpv2 and Trpv6 mRNA levels were observed during proestrus. Lower levels of Trpv3 and Trpv4 mRNAs were seen during estrus but higher expression of Trpv3 during metestrus and diestrus, and Trpv4 during proestrus was observed. Estradiol seems to regulate Trpv1/Trpv5 and Trpv2/Trpv6 mRNA expression in opposite manner. Except Trpv4 mRNA expression in the hippocampus and Trpv6 expression in the olfactory bulb, hippocampus and brainstem, expression of other members of TRPV subfamily in distinct brain regions of male mice was comparable to those in metestrus and diestrus mice. We suggest that the circulating levels of estradiol during the estrous cycle may differentially regulate the activity of TRPV1-6 ion channels 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.