Kazuhide Uramura

Orexin-induced hyperlocomotion and stereotypy are mediated by the dopaminergic system

We demonstrated involvement of the ventral tegmental area (VTA) dopaminergic system in orexin-induced hyperlocomotion and stereotypy in rats. In double-label immunohistochemical study of rat brain, we found that tyrosine hydroxylase (TH)-immunoreactive cells in the VTA received innervation from orexin immunoreactive-fibers. Orexin-A induced an increase in [Ca(2+)](i) in isolated A10 dopamine neurons in a dose-dependent manner. In behavioral studies, we found that orexin-A induced hyperlocomotion, stereotypy and grooming behavior when administered centrally in rats, and these effects were abolished by dopamine D(2) (haloperidol and sulpiride) or D(1) (SCH23390) antagonists. These results suggest that the orexin-induced hyperlocomotion, stereotypy and grooming behavior are mediated by the dopaminergic system and this pathway might be involved in orexin-induced emotional responses.

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.

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.

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.

Gamma aminobutyric acid regulates glucosensitive neuropeptide Y neurons in arcuate nucleus via A/B receptors

Gamma aminobutyric acid (GABA) is localized in neuropeptide Y (NPY) neurons of the hypothalamic arcuate nucleus (ARC). We examined regulation of ARC NPY neurons by GABA. Light and electron microscopic immunohistochemistry confirmed that GABA-containing nerve terminals contacted NPY-containing neurons in the ARC. Lowering glucose (1 mM) increased cytosolic Ca2+ concentration ([Ca2+]i) in isolated ARC neurons that were immunoreactive to NPY. The [Ca2+]i increases were inhibited by GABA, the &ggr;-aminobutyric acid type A receptor (GABAA) agonist muscimol and the &ggr;-aminobutyric acid type B receptor (GABAB) agonist baclofen. Neither the GABAA antagonist bicuculline nor the GABAB antagonist CGP35348 counteracted the GABA inhibition when applied alone, but did so when applied together. These results indicate that GABA regulates ARC glucose-sensitive NPY neurons via GABAA and GABAB receptors, which could function to attenuate the orexigenic NPY pathway when it is not beneficial.

Chronic phencyclidine treatment induces long-lasting glutamatergic activation of VTA dopamine neurons

Use of phencyclidine (PCP) can mimic some aspects of schizophrenia. However, the underlying mechanism is unclear. Administration of PCP is known to activate mesolimbic dopamine pathway. In this study, we focused on ventral tegmental area (VTA) of mesolimbic dopamine pathway as target of PCP for inducing schizophrenia-like symptoms. Single VTA neuron was isolated and its neural activity was monitored by measuring cytosolic Ca(2+) concentration ([Ca(2+)]i) followed by immunocytochemical identification of dopamine neurons. Administration of glutamate increased [Ca(2+)]i in dopamine neurons from control rats, and the [Ca(2+)]i increase was inhibited in the presence of PCP. In contrast, in VTA dopamine neurons from rats chronically treated with PCP for 7 days, administration of glutamate was able to induce [Ca(2+)]i increase in the presence of PCP. Furthermore, this glutamate-induced [Ca(2+)]i increase in the presence of PCP continued even after washout of glutamate and this effect lasted as long as PCP was present. This long-lasting glutamate-induced [Ca(2+)]i increase in the presence of PCP was not observed or significantly attenuated under Ca(2+) free condition and by N-type Ca(2+) channel blocker ω-conotoxin. The results indicate that chronic treatment with PCP reverses the acute PCP effect on VTA dopamine neurons from inhibitory to stimulatory tone, and consequently induces long-lasting activation of dopamine neurons by glutamate.

Ca2+ Oscillations in Response to Methamphetamine in Dopamine Neurons of the Ventral Tegmental Area in Rats Subchronically Treated with This Drug

Mesolimbic dopamine neurons in the ventral tegmental area (VTA), which project to the nucleus accumbens and prefrontal cortex, play an important role in the regulation of emotion, rewarding, and cognition. The dopamine neurons in the VTA have also been implicated in schizophrenia and drug abuse. Methamphetamine (METH) can induce a schizophrenia‐like psychosis. Thus, the VTA is a likely effector site for the action of METH. However, effects of METH on the mesolimbic dopamine neurons are largely unknown. We treated adult SD rats with METH (5 mg/kg/day) or saline for 7 days, isolated single VTA neurons from these treated rats, and monitored the neuronal activities by measuring cytosolic Ca2+ concentration ([Ca2+]i), which was followed by immunocytochemical identification of dopamine neurons. Acute administration of METH under superfusion conditions concentration‐dependently increased [Ca2+]i in VTA dopamine neurons isolated from METH‐ and saline‐treated rats. Furthermore, acutely administered METH induced oscillations of [Ca2+]i only in the dopamine neurons of the METH‐treated group. The METH‐induced [Ca2+]i oscillations were inhibited by Ca2+‐free conditions and by Ca2+ channel blockers. In conclusion, subchronic METH treatment sensitizes VTA dopamine neurons to this drug, resulting in induction of [Ca2+]i oscillations. This sensitization of VTA dopamine neurons may account, at least in part, for the psycho‐stimulant effects of METH, such as the dependence on and sensitization to METH.

Functional expression of cholecystokinin-A receptor on ventromedial hypothalamic neurons in the immature rat brain

Cholecystokinin-8 (CCK-8) dose-dependently increased the cytosolic Ca2+ concentration ([Ca]i) in ventromedial hypothalamic neurons acutely dissociated from the immature rat brain. The CCK-8 response was mimicked by caerulein, but not by CCK(B) agonists, and was often inhibited by CCK(A) receptor antagonists, but rarely by CCK(B) receptor antagonists. The response was dependent on external Ca2+ and Na+, and was inhibited by voltage-dependent Ca2+ channel blockers. The results suggest that CCK-8-induced depolarization via CCK(A) receptors increased Ca2+ influx through a voltage-dependent Ca2+ channel, which in turn increased [Ca]i.