Shinji Muroya

Orexins (hypocretins) directly interact with neuropeptide Y, POMC and glucose-responsive neurons to regulate Ca2+ signaling in a reciprocal manner to leptin: orexigenic neuronal pathways in the mediobasal hypothalamus

Orexin‐A and ‐B (hypocretin‐1 and ‐2) have been implicated in the stimulation of feeding. Here we show the effector neurons and signaling mechanisms for the orexigenic action of orexins in rats. Immunohistochemical methods showed that orexin axon terminals contact with neuropeptide Y (NPY)‐ and proopiomelanocortin (POMC)‐positive neurons in the arcuate nucleus (ARC) of the rats. Microinjection of orexins into the ARC markedly increased food intake. Orexins increased cytosolic Ca2+ concentration ([Ca2+]i) in the isolated neurons from the ARC, which were subsequently shown to be immunoreactive for NPY. The increases in [Ca2+]i were inhibited by blockers of phospholipase C (PLC), protein kinase C (PKC) and Ca2+ uptake into endoplasmic reticulum. The stimulation of food intake and increases in [Ca2+]i in NPY neurons were greater with orexin‐A than with orexin‐B, indicative of involvement of the orexin‐1 receptor (OX1R). In contrast, orexin‐A and ‐B equipotently attenuated [Ca2+]i oscillations and decreased [Ca2+]i levels in POMC‐containing neurons. These effects were counteracted by pertussis toxin, suggesting involvement of the orexin‐2 receptor and Gi/Go subtypes of GTP‐binding proteins. Orexins also decreased [Ca2+]i levels in glucose‐responsive neurons in the ventromedial hypothalamus (VMH), a satiety center. Leptin exerted opposite effects on these three classes of neurons. These results demonstrate that orexins directly regulate NPY, POMC and glucose‐responsive neurons in the ARC and VMH, in a manner reciprocal to leptin. Orexin‐A evokes Ca2+ signaling in NPY neurons via OX1R–PLC–PKC and IP3 pathways. These neural pathways and intracellular signaling mechanisms may play key roles in the orexigenic action of orexins.

Glucose-sensitive neurons in the rat arcuate nucleus contain neuropeptide Y

Glucose is known to regulate the activity of the hypothalamic feeding centers. Neuropeptide Y (NPY)-containing neurons in the hypothalamic arcuate nucleus (ARC) have been implicated in the stimulation of feeding. We examined the presence of glucose-sensitive neurons in the ARC and their coincidence with NPY-containing neurons. Cytosolic Ca2+ concentration ([Ca2+]i) in single ARC neurons isolated from rat hypothalamus was measured with fura-2 fluorescence imaging; the cells were then stained immunocytochemically with an anti-NPY antiserum. Lowering the glucose concentration from 10 to 1 mM increased [Ca2+]i in 36 out of 180 neurons (20%), the majority of which (34 neurons, 94%) were immunoreactive for NPY. In conclusion, the ARC contains glucose-sensitive NPY-containing neurons. The suggested role of these neurons is to transduce a reduction in the glucose concentration in the brain to the release of NPY and, subsequently, stimulation of feeding.

Orexin-a activates phospholipase C- and protein kinase C-mediated Ca2+ signaling in dopamine neurons of the ventral tegmental area

The orexin–orexin receptor system has been implicated in the regulation of wakefulness/sleep states. Behavioral and psycho-stimulant effects of orexins have also been shown. Mesolimbic dopamine neurons in the ventral tegmental area (VTA) are implicated in the regulation of reward and wakefulness/sleep, In the present study, we examined the effect of orexin-A on cytosolic [Ca2+]i concentration ([Ca2+]) in the isolated rat VTA dopamine neurons. Orexin-A (10−12–10−8 M) concentration dependently increased [Ca2+]i in dopamine-containing neurons. The [Ca2+]i responses to orexin-A were inhibited under Ca2+-free conditions and by blockers of voltage-gated L- and N-type [Ca2+]i channels, nitrendipine and ω-conotoxin, respectively. The [Ca2+]i responses were also abolished by a phosphatidylcholine-specific phospholipase C inhibitor, D609, and a protein kinase C (PKC) inhibitor, calphostin C. A PKC activator, TPA, mimicked orexin-A in increasing [Ca2+]i. These results indicate that orexin-A increases [Ca2+]i in VTA dopamine neurons via phosphatidylcholine-specific PLC- and PKC-mediated activation of L- and N-type Ca2+ channels. This effect may serve as the mechanism by which orexin regulates wakefulness/sleep states and exerts its behavioral and psychostimulant effects.

Lowering glucose concentrations increases cytosolic Ca2+ in orexin neurons of the rat lateral hypothalamus.

Orexin neurons are specifically localized in and around the lateral hypothalamus (LH), a feeding center. Intracerebroventricular administration of orexin-A and -B stimulates feeding as well as arousal. However, little is known regarding the regulators of the orexin neuron activity. The neurons that are activated under low glucose conditions, glucose-sensitive neurons, are located in the LH and have been implicated in the control of feeding. The present study investigated the effect of glucose on the single orexin neurons isolated from the rat LH, by measuring cytosolic Ca(2+) concentration ([Ca(2+)](i)) by fura-2 microfluorometry followed by immunocytochemical staining with anti-orexin antiserum. A shift of glucose concentration form 8.3 to 2.8 mM in the superfusion solution increased [Ca(2+)](i) in 13 out of 32 orexin-immunoreactive LH neurons. The results demonstrate that glucose-sensitive orexin neurons are present in the LH and that these neurons may play a role in linking the metabolic state in the body to the orexigenic, and could also, awakening signaling in the brain.

The effect of leptin on feeding-regulating neurons in the rat hypothalamus

Intense immunoreactivity for the leptin receptor was detected in the hypothalamic arcuate nucleus (ARC), ventromedial nucleus (VMH), and lateral hypothalamus (LH) by immunohistochemistry. Cytosolic Ca2+ concentration ([Ca2+]i) in single neurons isolated from the ARC, VMH and LH was measured with dual wavelength fura-2 fluorescence imaging. A reduction of the superfusate glucose concentration from 10 to 1 mM increased [Ca2+]i in 21% of ARC neurons and 22% of LH neurons. Leptin at 0.1 nM inhibited the [Ca2+]i increase in 66 and 64% of these glucose-sensitive ARC and LH neurons, respectively. Inversely, 10 mM glucose increased [Ca2+]i in 49% of the VMH neurons, and 0.1 nM leptin at 1 mM glucose also increased [Ca2+]i in 84% of these glucose-responsive neurons. These results reveal that leptin inhibits the ARC and LH neurons and stimulates the VMH neurons via the leptin receptor expressed in these cells.

A gene-targeted mouse model for chorea-acanthocytosis

Chorea‐acanthocytosis (CHAC) is a hereditary neurodegenerative disorder with autosomal recessive transmission, in which selective degeneration of striatum has been reported in brain pathology. Clinically, CHAC shows Huntingtons disease‐like neuropsychiatric symptoms and red blood cell acanthocytosis. Recently, we identified the gene, CHAC, encoding a novel protein, chorein, in which a deletion mutation was found in Japanese families with CHAC. In the present study, we have identified the mouse CHAC cDNA sequence and the exon–intron structures of the gene and produced a CHAC model mouse introducing no. 60–61 exon deletion corresponding to a human disease mutation by a gene‐targeting technique. The mice began to show acanthocytosis and motor disturbance in old age. In behavioral observations, locomotor activity was significantly decreased and the contact time at social interaction test was decreased significantly in the model mice. In the brain pathology, many apoptotic cells were observed in the striatum of the mutant mice. In neurochemical determinations, the dopamine metabolite, homovanillic acid, concentration decreased significantly in the portion including the midbrain of the mutant mice. These findings are consistent with the human results reported elsewhere and indicate that the CHAC model mice showed a mild phenotype with late adult onset. The CHAC model mouse therefore provides a good model system to study the human disease.

Clinical and molecular genetic assessment of a chorea-acanthocytosis pedigree

BACKGROUND Chorea-acanthocytosis (ChAc) is an autosomal recessive hereditary disease characterized by neurodegeneration in the striatum and acanthocytosis that is caused by mutations in the VPS13A gene. There are only few reports that studied clinical status of the obligate carriers of ChAc. Clinical courses with follow-up neuroradiological and neuropsychological evaluations in individuals with ChAc have been rarely reported. METHODS We followed an index patient with ChAc and evaluated the clinical features of the pedigree members. Genetic analyses of VPS13A and genes responsible for other neuroacanthocytotic and neurodegenerative diseases were performed. CONCLUSIONS The index patient was homozygous for a 3889C>T nonsense mutation in the VPS13A gene and presented with a typical ChAc phenotype. Neuropsychological evaluation with brain imaging in the patient over 3 years revealed atrophy and a decrease in blood flow at the basal ganglia and frontal lobe, and impairment in cognitive function reflecting frontal lobe dysfunction in progressive manners. Four out of five heterozygous mutation carriers in the pedigree showed signs or symptoms potentially attributable to a heterozygous VPS13A mutation.

Noradrenaline activates vasopressin neurons via α1-receptor-mediated Ca2+ signaling pathway

Noradrenaline (NA) (1-10 microM), dibutyryl-cAMP (1-5 mM), and forskolin (10-20 microM) increased cytosolic Ca2+ concentration ([Ca2+]i) in isolated arginine-vasopressin (AVP)-containing neurons in the hypothalamic supraoptic nucleus (SON). The NA-induced increase in [Ca2+]i in AVP-containing neurons was abolished by a specific alpha1-antagonist, prazosin (1 microM) and was markedly reduced when treated with a protein kinase A (PKA) blocker, H89 (40 microM). The NA-induced [Ca2+]i was not altered by a protein kinase C (PKC) inhibitor, calphostin C (0.1 microM) and a PKC activator, TPA (100 nM). In general, NA, a known neurotransmitter in the SON, activates AVP-containing neurons via alpha1-receptor which is linked to stimulation of cAMP-PKA-regulated Ca2+ signaling pathway.

Methamphetamine induces cytosolic Ca2+ oscillations in the VTA dopamine neurons.

Methamphetamine (METH) induces a schizophrenia-like psychosis. The dopamine neurons in the ventral tegmental area (VTA) have been implicated in schizophrenia and drug abuse. The present study investigated direct effects of METH on VTA dopamine neurons. We treated adult SD rats with METH (5 mg/kg/day) or saline for 7 days, isolated single VTA neurons, and monitored neurona activities by measuring cytosolic Ca2+ concentration ([Ca2+]i) in immunocytochemically identified dopamine neurons. Acutely administered METH increased [Ca2+]i in dopamine neurons from METH-and saline-treated rats and induced oscillations of [Ca2+]i in dopamine neurons only from METH-treated rats. The METH-induced [Ca2+]i oscillations were inhibited by Ca2+-free conditions and Ca2+ channel blockers. The results indicate that acute METH increases [Ca2+]i in VTA dopamine neurons and that subchronic METH treatment sensitizes them to this drug, resulting in induction of [Ca2+]i oscillations. The activation of VTA dopamine neurons may be related to psycho-stimulant effects of METH.

Functional Significance of Colocalization of PACAP and Catecholamine in Nerve Terminals

Abstract: Medullary neurons containing pituitary adenylate cyclase‐activating polypeptide (PACAP) and noradrenalin (NA) project to the hypothalamus and they are involved in the regulation of arginine vasopressin (AVP) neurons. At the ultrastructural level, PACAP immunoreactivity was detected in the granular vesicles in catecholaminergic nerve terminals that made synaptic contact with AVP neurons. Both PACAP (at least 1 nM) and NA (at least 1 μmM) induced large increases in the cytosolic Ca2+ concentration ([Ca2+]i) in isolated AVP cells. PACAP at 0.1 nM and NA at 0.1 μmM had little effects, if any, on [Ca2+]i. However, when 0.1 nM PACAP and 0.1 μM NA were combined, they evoked large increase in [Ca2+]i in AVP neurons. An inhibitor of protein kinase A (PKA) completely inhibited the PACAP‐induced increase in [Ca2+]i, but only partly inhibited the NA‐induced increase in [Ca2+]i. In AVP cells that were prelabeled with quinacrine, PACAP and NA acted synergistically to induce a loss of quinacrine fluorescence, indicating secretion of neurosecretory granules in AVP neurons. The results suggest that PACAP and NA, coreleased from the same nerve terminals, act in synergy to evoke calcium signaling and secretion in AVP neurons, and that the synergism is mediated by the interaction between cAMP‐PKA pathway an as yet unidentified factor “X” linked to L‐type Ca2+ channels. The synergism between PACAP and NA may contribute to the regulation of AVP secretion under physiological conditions.