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Hiroshi Katsuki - One of the best experts on this subject based on the ideXlab platform.
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orexin neurons in Hypothalamic Slice cultures are vulnerable to endoplasmic reticulum stress
Neuroscience, 2011Co-Authors: Shotaro Michinaga, Akinori Hisatsune, Yoichiro Isohama, Hiroshi KatsukiAbstract:Narcolepsy results from disruption of orexin neurons in the hypothalamus that play a key role in maintenance of the arousal state. Underlying mechanisms leading to selective loss of orexin neurons remain unknown. On the other hand, endoplasmic reticulum stress, namely, conditions associated with impairment of endoplasmic reticulum functions such as proper folding and sorting of newly synthesized proteins, is implicated in pathogenesis of several types of neurodegenerative disorders. Here we found that application of endoplasmic reticulum stress inducers such as tunicamycin (that prevents protein N-glycosylation) and thapsigargin (that inhibits Ca²⁺-ATPase) to organotypic Slice cultures of the hypothalamus caused preferential loss of orexin-immunoreactive neurons, as compared to melanin-concentrating hormone- or calcitonin gene-related peptide-immunoreactive neurons. The decrease in orexin-immunoreactive neurons at early time points (6-24 h) was not accompanied by induction of cell death as indicated by the absence of caspase-3 activation and no significant change in the number of NeuN-positive cells, whereas sustained treatment with tunicamycin for 72 h induced cell death. At 24-h treatment, tunicamycin and thapsigargin did not decrease expression of prepro-orexin mRNA, suggesting that post-transcriptional mechanisms were responsible for depletion of orexin peptides. In addition, inhibition of axonal transport by colchicine and inhibition of proteasomal activity by MG132 significantly prevented the decrease in orexin immunoreactivity by tunicamycin. Comparative examinations of expression of unfolded protein response-related proteins revealed that C/EBP-homologous protein (a transcription factor that promotes induction of apoptosis) as well as phosphorylated form of RNA-dependent protein kinase-like endoplasmic reticulum kinase (a protein kinase that mediates inhibition of protein translation) was expressed more prominently in orexin neurons than in melanin-concentrating hormone neurons, in response to tunicamycin. These results indicate that orexin neurons are particularly sensitive to endoplasmic reticulum stress, which may be relevant to pathogenic events in narcolepsy.
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an anti parkinson drug ropinirole depletes orexin from rat Hypothalamic Slice culture
Neuroscience Research, 2010Co-Authors: Shotaro Michinaga, Akinori Hisatsune, Yoichiro Isohama, Hiroshi KatsukiAbstract:Abstract Non-ergot-type dopamine receptor agonists such as ropinirole are used for the treatment of Parkinson disease, but they occasionally show serious side effects including sleep attacks and daytime sleepiness. These symptoms are reminiscent of narcolepsy, a major sleep disorder. Because narcolepsy is thought to result from deficiency of a Hypothalamic neuropeptide orexin, we examined whether ropinirole affected the integrity of orexin-containing neurons, using organotypic Slice culture of rat hypothalamus. Application of ropinirole induced a significant decrease in the number of orexin-immunoreactive neurons. The same treatment showed no significant effect on the number of melanin-concentrating hormone-immunoreactive neurons. The decrease of orexin-immunoreactive neurons was reversible after washout of ropinirole and was not accompanied by induction of cell death. Antagonism of dopamine D 2 receptors and of serotonin 5-HT 1A receptors attenuated the effect of ropinirole, suggesting involvement of these receptors in depletion of orexin. On the other hand, a moderate concentration of N -methyl- d -aspartate that excited orexin neurons counteracted the effect of ropinirole on the number of orexin-immunoreactive neurons. These results suggest that ropinirole can cause deficiency of orexin by inhibiting excitatory activities of orexin neurons, which may be relevant to the adverse actions of this drug on sleep and wakefulness.
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depolarizing stimuli cause persistent and selective loss of orexin in rat Hypothalamic Slice culture
Peptides, 2010Co-Authors: Hiroshi Katsuki, Shotaro Michinaga, Akinori Hisatsune, Yoichiro Isohama, Shinsuke Kurosu, Yasuhiko Izumi, Toshiaki Kume, Akinori AkaikeAbstract:Abstract A Hypothalamic neuropeptide orexin (hypocretin) is a critical regulator of physiological processes including sleep/wakefulness and feeding. Using organotypic Slice culture of rat hypothalamus, we found that exposure to elevated extracellular concentration of K + (+10–30 mM) for 24–72 h led to a substantial decrease in the number of neurons immunoreactive for orexin and a co-existing neuropeptide dynorphin-A. In contrast, the same treatment affected neither the number of melanin-concentrating hormone-immunoreactive neurons nor the number of total neurons. A substantial decrease of orexin-immunoreactive neurons was also induced by 72 h treatment with 1–10 μM veratridine, a Na + channel activator. The effect of elevated K + was only partially reversible, and that of veratridine was virtually irreversible, although the decrease in orexin immunoreactivity was not associated with signs of cell damage assessed by propidium iodide uptake and Hoechst 33342 nuclear staining. In addition, the level of preproorexin mRNA did not decrease during treatment with elevated K + or veratridine. After treatment with elevated K + and veratridine, c-Fos immunoreactivity appeared in orexin-immunoreactive neurons but not in melanin-concentrating hormone-immunoreactive neurons, suggesting selective excitation of orexin neurons. However, the amount of orexin released extracellularly was paradoxically decreased by treatment with elevated K + and veratridine. Overall, these characteristics of orexin neurons may be taken into consideration to understand the behaviors of these neurons under physiological and pathophysiological conditions.
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inhibition of neural activity depletes orexin from rat Hypothalamic Slice culture
Journal of Neuroscience Research, 2010Co-Authors: Shotaro Michinaga, Akinori Hisatsune, Yoichiro Isohama, Hiroshi KatsukiAbstract:Orexins (hypocretins) are neuropeptides produced by a small population of Hypothalamic neurons whose dysregulation may lead to narcolepsy, a neurological disorder characterized by disorganization of sleep and wakefulness. Excessive stimulation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors causes preferential loss of orexin neurons in the hypothalamus, whereas an adequate level of neuronal excitatory activities is generally known to be important for the maintenance of central neurons. By examining the effect of manipulation of neural activity, we found that 24–72 hr application of tetrodotoxin (TTX) caused a substantial decrease in the number of orexin-immunoreactive neurons, but not of melanin-concentrating hormone-immunoreactive neurons, in Hypothalamic Slice culture. Similar results were obtained when neural activity was arrested by added extracellular Mg2+. Reduction of orexin expression by TTX and Mg2+ was also observed at mRNA level. The decrease of orexin-immunoreactive neurons was attributable to depletion of orexin, because it was reversible after washout of TTX or elevated extracellular Mg2+ and was not associated with induction of cell death. Blockers of voltage-dependent Ca2+ channels as well as of NMDA receptors also induced a significant and selective decrease of orexin-immunoreactive neurons. Moreover, TTX-induced decrease of orexin immunoreactivity was largely abrogated by concurrent application of a moderate concentration of NMDA. These results suggest that Ca2+ entry associated with nontoxic levels of spontaneous activity of glutamatergic inputs plays an important role in the maintenance of orexin neurons in a tissue culture model. © 2009 Wiley-Liss, Inc.
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excitotoxic degeneration of Hypothalamic orexin neurons in Slice culture
Neurobiology of Disease, 2004Co-Authors: Hiroshi Katsuki, Akinori AkaikeAbstract:Several lines of evidence indicate that narcolepsy, a sleep disorder, results from the loss of Hypothalamic orexin (hypocretin)-containing neurons, but the mechanisms responsible for selective elimination of this neuronal population are unknown. Using organotypic rat Hypothalamic Slice cultures, we investigated vulnerability of orexin neurons to excitotoxic insults. Twenty-four hours of incubation with N-methyl-D-aspartate (NMDA) followed by a recovery period of 72 h resulted in a marked decrease in the number of orexin-immunoreactive neurons, whereas melanin-concentrating hormone (MCH)-immunoreactive neurons in the same cultures were relatively spared. In contrast, orexin neurons were more resistant to kainic acid cytotoxicity than MCH neurons. Examinations of the effects of several endogenous glutamate receptor agonists as well as a glutamate transporter blocker highlighted quinolinic acid as an endogenous excitotoxin that could cause selective loss of orexin neurons as compared to MCH neurons by activating NMDA receptors. In addition, quinolinic acid-induced decrease of orexin neurons was prevented by an inhibitor of poly(ADP-ribose) polymerases. These results provide the first evidence concerning cytotoxic consequences onto orexin neurons, and indicate that NMDA receptor-mediated injury may contribute to the selective loss of these neurons in the hypothalamus, a prominent neuropathological feature found in narcolepsy patients.
David B Maclean - One of the best experts on this subject based on the ideXlab platform.
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increased atp content production in the hypothalamus may be a signal for energy sensing of satiety studies of the anorectic mechanism of a plant steroidal glycoside
Brain Research, 2004Co-Authors: David B MacleanAbstract:A steroidal glycoside with anorectic activity in animals, termed P57AS3 (P57), was isolated from Hoodia gordonii and found to have homologies to the steroidal core of cardiac glycosides. Intracerebroventricular (i.c.v.) injections of the purified P57AS3 demonstrated that the compound has a likely central (CNS) mechanism of action. There is no evidence of P57AS3 binding to or altering activity of known receptors or proteins, including Na/K-ATPase, the putative target of cardiac glycosides. The studies demonstrated that the compound increases the content of ATP by 50–150% in Hypothalamic neurons. In addition, third ventricle (i.c.v.) administration of P57, which reduces subsequent 24-h food intake by 40–60%, also increases ATP content in Hypothalamic Slice punches removed at 24 h following the i.c.v. injections. In related studies, in pair fed rats fed a low calorie diet for 4 days, the content of ATP in the hypothalami of control i.c.v. injected animals fell by 30–50%, which was blocked by i.c.v. injections of P57AS3. With growing evidence of metabolic or nutrient-sensing by the hypothalamus, ATP may be a common currency of energy sensing, which in turn may trigger the appropriate neural, endocrine and appetitive responses as similar to other fundamental Hypothalamic homeostatic centers for temperature and osmolarity.
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Increased ATP content/production in the hypothalamus may be a signal for energy-sensing of satiety: studies of the anorectic mechanism of a plant steroidal glycoside.
Brain research, 2004Co-Authors: David B MacleanAbstract:A steroidal glycoside with anorectic activity in animals, termed P57AS3 (P57), was isolated from Hoodia gordonii and found to have homologies to the steroidal core of cardiac glycosides. Intracerebroventricular (i.c.v.) injections of the purified P57AS3 demonstrated that the compound has a likely central (CNS) mechanism of action. There is no evidence of P57AS3 binding to or altering activity of known receptors or proteins, including Na/K-ATPase, the putative target of cardiac glycosides. The studies demonstrated that the compound increases the content of ATP by 50-150% in Hypothalamic neurons. In addition, third ventricle (i.c.v.) administration of P57, which reduces subsequent 24-h food intake by 40-60%, also increases ATP content in Hypothalamic Slice punches removed at 24 h following the i.c.v. injections. In related studies, in pair fed rats fed a low calorie diet for 4 days, the content of ATP in the hypothalami of control i.c.v. injected animals fell by 30-50%, which was blocked by i.c.v. injections of P57AS3. With growing evidence of metabolic or nutrient-sensing by the hypothalamus, ATP may be a common currency of energy sensing, which in turn may trigger the appropriate neural, endocrine and appetitive responses as similar to other fundamental Hypothalamic homeostatic centers for temperature and osmolarity.
Akinori Akaike - One of the best experts on this subject based on the ideXlab platform.
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depolarizing stimuli cause persistent and selective loss of orexin in rat Hypothalamic Slice culture
Peptides, 2010Co-Authors: Hiroshi Katsuki, Shotaro Michinaga, Akinori Hisatsune, Yoichiro Isohama, Shinsuke Kurosu, Yasuhiko Izumi, Toshiaki Kume, Akinori AkaikeAbstract:Abstract A Hypothalamic neuropeptide orexin (hypocretin) is a critical regulator of physiological processes including sleep/wakefulness and feeding. Using organotypic Slice culture of rat hypothalamus, we found that exposure to elevated extracellular concentration of K + (+10–30 mM) for 24–72 h led to a substantial decrease in the number of neurons immunoreactive for orexin and a co-existing neuropeptide dynorphin-A. In contrast, the same treatment affected neither the number of melanin-concentrating hormone-immunoreactive neurons nor the number of total neurons. A substantial decrease of orexin-immunoreactive neurons was also induced by 72 h treatment with 1–10 μM veratridine, a Na + channel activator. The effect of elevated K + was only partially reversible, and that of veratridine was virtually irreversible, although the decrease in orexin immunoreactivity was not associated with signs of cell damage assessed by propidium iodide uptake and Hoechst 33342 nuclear staining. In addition, the level of preproorexin mRNA did not decrease during treatment with elevated K + or veratridine. After treatment with elevated K + and veratridine, c-Fos immunoreactivity appeared in orexin-immunoreactive neurons but not in melanin-concentrating hormone-immunoreactive neurons, suggesting selective excitation of orexin neurons. However, the amount of orexin released extracellularly was paradoxically decreased by treatment with elevated K + and veratridine. Overall, these characteristics of orexin neurons may be taken into consideration to understand the behaviors of these neurons under physiological and pathophysiological conditions.
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excitotoxic degeneration of Hypothalamic orexin neurons in Slice culture
Neurobiology of Disease, 2004Co-Authors: Hiroshi Katsuki, Akinori AkaikeAbstract:Several lines of evidence indicate that narcolepsy, a sleep disorder, results from the loss of Hypothalamic orexin (hypocretin)-containing neurons, but the mechanisms responsible for selective elimination of this neuronal population are unknown. Using organotypic rat Hypothalamic Slice cultures, we investigated vulnerability of orexin neurons to excitotoxic insults. Twenty-four hours of incubation with N-methyl-D-aspartate (NMDA) followed by a recovery period of 72 h resulted in a marked decrease in the number of orexin-immunoreactive neurons, whereas melanin-concentrating hormone (MCH)-immunoreactive neurons in the same cultures were relatively spared. In contrast, orexin neurons were more resistant to kainic acid cytotoxicity than MCH neurons. Examinations of the effects of several endogenous glutamate receptor agonists as well as a glutamate transporter blocker highlighted quinolinic acid as an endogenous excitotoxin that could cause selective loss of orexin neurons as compared to MCH neurons by activating NMDA receptors. In addition, quinolinic acid-induced decrease of orexin neurons was prevented by an inhibitor of poly(ADP-ribose) polymerases. These results provide the first evidence concerning cytotoxic consequences onto orexin neurons, and indicate that NMDA receptor-mediated injury may contribute to the selective loss of these neurons in the hypothalamus, a prominent neuropathological feature found in narcolepsy patients.
Kiyotoshi Inenaga - One of the best experts on this subject based on the ideXlab platform.
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damgo suppresses both excitatory and inhibitory synaptic transmission in supraoptic neurones of mouse Hypothalamic Slice preparations
Journal of Neuroendocrinology, 2004Co-Authors: Eiko Honda, K Ono, Kiyotoshi InenagaAbstract:Opioid effects on synaptic transmission in the mouse supraoptic nucleus (SON) were investigated using whole-cell, patch-clamp techniques. The mu-opioid receptor agonist, [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) decreased the amplitude of both evoked excitatory postsynaptic currents (eEPSCs) and inhibitory postsynaptic currents (eIPSCs), and also decreased the frequency of both miniature EPSCs and IPSCs without effect on the amplitude. The selective mu-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2), and the nonselective antagonist naloxone, antagonized these inhibitory effects. The application of DAMGO suppressed the amplitude of both the first and second evoked postsynaptic currents with a paired-pulse stimulus protocol, but increased the paired-pulse ratios (second ePSC/first ePSC). DAMGO induced neither inward nor outward currents, and had no significant changes in either glutamate- or GABA-induced currents. When compared with the relatively selective kappa- and delta-opioid receptor agonists dynorphin and [D-Pen(2), D-Pen(5)]-enkephalin, DAMGO showed the most potent inhibitory effects on evoked and miniature postsynaptic currents. Taken together, these results imply that DAMGO strongly suppresses the release of glutamate and GABA via mu-opioid receptors in the mouse SON, and support the involvement of presynaptic regulation by opioids in the control of magnocellular neurosecretory neurones.
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inhibition by brain natriuretic peptide of vasopressin neurons in the supraoptic nucleus and neurons in the region of the anteroventral third ventricle in rat Hypothalamic Slice preparations
Journal of Neuroendocrinology, 1991Co-Authors: S Yamamoto, Kiyotoshi Inenaga, Hiroshi YamashitaAbstract:Abstract It is not entirely clear whether or not atrial natriuretic peptide (ANP) directly inhibits vasopressin neurons in the supraoptic nucleus (SON) and paraventricular nucleus. Recently, a novel peptide, brain natriuretic peptide (BMP), which has been isolated from the brain, has been shown to have a similar action to ANP on the regulation of vasopressin release. Intracerebroventricular injection of both BNP and ANP inhibits stimulus-evoked increases of plasma vasopressin level. The present study was undertaken: 1) to investigate whether BNP affects the activity of neurons in the region of the anteroventral third ventricle (AV3V) and SON which are involved in the control of body fluid homeostasis and blood pressure regulation, 2) to reassess effects of ANP on SON neurons, and 3) to test whether BNP exerts its effects by mechanisms which are different from those of ANP. Extracellular recordings were made from 213 AV3V and 110 SON spontaneously firing neurons in the rat coronal Hypothalamic Slice preparation. Of the AV3V neurons tested, BNP inhibited 86 (40%) and excited 2 (1%) while 125 neurons remained unaffected. A dose-response relationship was obtained for 7 AV3V neurons at different BNP concentrations ranging from 10(-11) M to 10(-6) M; the firing rates of all 7 neurons decreased. The threshold concentration to evoke inhibitory responses was approximately 10(-10)M in the AV3V. When BNP and ANP were applied to the same neuron, most AV3V neurons which were inhibited by BNP were also inhibited by ANP and the neurons which were unaffected by BNP were also unaffected by ANP. Thus, these two peptides probably have a similar action on AV3V neurons. When BNP and angiotensin II were applied to a group of 60 neurons in the AV3V, most of the responsive neurons showed either inhibitory responses to BNP or excitatory responses to angiotensin II. Both BNP and ANP were applied to a group of 110 SON neurons: BNP (10 (-7) M) inhibited 52 (75%) of 69 phasic (putative vasopressin) neurons, while BNP affected none of the 41 non-phasic (putative oxytocin) neurons. By contrast, ANP inhibited only 20 (29%) of 69 phasic neurons tested but it also had no effect on 41 non-phasic neurons tested. Our results are consistent with the suggestion that BNP is involved in the regulation of vasopressin release by acting on SON neurons and AV3V neurons.
Shotaro Michinaga - One of the best experts on this subject based on the ideXlab platform.
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orexin neurons in Hypothalamic Slice cultures are vulnerable to endoplasmic reticulum stress
Neuroscience, 2011Co-Authors: Shotaro Michinaga, Akinori Hisatsune, Yoichiro Isohama, Hiroshi KatsukiAbstract:Narcolepsy results from disruption of orexin neurons in the hypothalamus that play a key role in maintenance of the arousal state. Underlying mechanisms leading to selective loss of orexin neurons remain unknown. On the other hand, endoplasmic reticulum stress, namely, conditions associated with impairment of endoplasmic reticulum functions such as proper folding and sorting of newly synthesized proteins, is implicated in pathogenesis of several types of neurodegenerative disorders. Here we found that application of endoplasmic reticulum stress inducers such as tunicamycin (that prevents protein N-glycosylation) and thapsigargin (that inhibits Ca²⁺-ATPase) to organotypic Slice cultures of the hypothalamus caused preferential loss of orexin-immunoreactive neurons, as compared to melanin-concentrating hormone- or calcitonin gene-related peptide-immunoreactive neurons. The decrease in orexin-immunoreactive neurons at early time points (6-24 h) was not accompanied by induction of cell death as indicated by the absence of caspase-3 activation and no significant change in the number of NeuN-positive cells, whereas sustained treatment with tunicamycin for 72 h induced cell death. At 24-h treatment, tunicamycin and thapsigargin did not decrease expression of prepro-orexin mRNA, suggesting that post-transcriptional mechanisms were responsible for depletion of orexin peptides. In addition, inhibition of axonal transport by colchicine and inhibition of proteasomal activity by MG132 significantly prevented the decrease in orexin immunoreactivity by tunicamycin. Comparative examinations of expression of unfolded protein response-related proteins revealed that C/EBP-homologous protein (a transcription factor that promotes induction of apoptosis) as well as phosphorylated form of RNA-dependent protein kinase-like endoplasmic reticulum kinase (a protein kinase that mediates inhibition of protein translation) was expressed more prominently in orexin neurons than in melanin-concentrating hormone neurons, in response to tunicamycin. These results indicate that orexin neurons are particularly sensitive to endoplasmic reticulum stress, which may be relevant to pathogenic events in narcolepsy.
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an anti parkinson drug ropinirole depletes orexin from rat Hypothalamic Slice culture
Neuroscience Research, 2010Co-Authors: Shotaro Michinaga, Akinori Hisatsune, Yoichiro Isohama, Hiroshi KatsukiAbstract:Abstract Non-ergot-type dopamine receptor agonists such as ropinirole are used for the treatment of Parkinson disease, but they occasionally show serious side effects including sleep attacks and daytime sleepiness. These symptoms are reminiscent of narcolepsy, a major sleep disorder. Because narcolepsy is thought to result from deficiency of a Hypothalamic neuropeptide orexin, we examined whether ropinirole affected the integrity of orexin-containing neurons, using organotypic Slice culture of rat hypothalamus. Application of ropinirole induced a significant decrease in the number of orexin-immunoreactive neurons. The same treatment showed no significant effect on the number of melanin-concentrating hormone-immunoreactive neurons. The decrease of orexin-immunoreactive neurons was reversible after washout of ropinirole and was not accompanied by induction of cell death. Antagonism of dopamine D 2 receptors and of serotonin 5-HT 1A receptors attenuated the effect of ropinirole, suggesting involvement of these receptors in depletion of orexin. On the other hand, a moderate concentration of N -methyl- d -aspartate that excited orexin neurons counteracted the effect of ropinirole on the number of orexin-immunoreactive neurons. These results suggest that ropinirole can cause deficiency of orexin by inhibiting excitatory activities of orexin neurons, which may be relevant to the adverse actions of this drug on sleep and wakefulness.
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depolarizing stimuli cause persistent and selective loss of orexin in rat Hypothalamic Slice culture
Peptides, 2010Co-Authors: Hiroshi Katsuki, Shotaro Michinaga, Akinori Hisatsune, Yoichiro Isohama, Shinsuke Kurosu, Yasuhiko Izumi, Toshiaki Kume, Akinori AkaikeAbstract:Abstract A Hypothalamic neuropeptide orexin (hypocretin) is a critical regulator of physiological processes including sleep/wakefulness and feeding. Using organotypic Slice culture of rat hypothalamus, we found that exposure to elevated extracellular concentration of K + (+10–30 mM) for 24–72 h led to a substantial decrease in the number of neurons immunoreactive for orexin and a co-existing neuropeptide dynorphin-A. In contrast, the same treatment affected neither the number of melanin-concentrating hormone-immunoreactive neurons nor the number of total neurons. A substantial decrease of orexin-immunoreactive neurons was also induced by 72 h treatment with 1–10 μM veratridine, a Na + channel activator. The effect of elevated K + was only partially reversible, and that of veratridine was virtually irreversible, although the decrease in orexin immunoreactivity was not associated with signs of cell damage assessed by propidium iodide uptake and Hoechst 33342 nuclear staining. In addition, the level of preproorexin mRNA did not decrease during treatment with elevated K + or veratridine. After treatment with elevated K + and veratridine, c-Fos immunoreactivity appeared in orexin-immunoreactive neurons but not in melanin-concentrating hormone-immunoreactive neurons, suggesting selective excitation of orexin neurons. However, the amount of orexin released extracellularly was paradoxically decreased by treatment with elevated K + and veratridine. Overall, these characteristics of orexin neurons may be taken into consideration to understand the behaviors of these neurons under physiological and pathophysiological conditions.
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inhibition of neural activity depletes orexin from rat Hypothalamic Slice culture
Journal of Neuroscience Research, 2010Co-Authors: Shotaro Michinaga, Akinori Hisatsune, Yoichiro Isohama, Hiroshi KatsukiAbstract:Orexins (hypocretins) are neuropeptides produced by a small population of Hypothalamic neurons whose dysregulation may lead to narcolepsy, a neurological disorder characterized by disorganization of sleep and wakefulness. Excessive stimulation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors causes preferential loss of orexin neurons in the hypothalamus, whereas an adequate level of neuronal excitatory activities is generally known to be important for the maintenance of central neurons. By examining the effect of manipulation of neural activity, we found that 24–72 hr application of tetrodotoxin (TTX) caused a substantial decrease in the number of orexin-immunoreactive neurons, but not of melanin-concentrating hormone-immunoreactive neurons, in Hypothalamic Slice culture. Similar results were obtained when neural activity was arrested by added extracellular Mg2+. Reduction of orexin expression by TTX and Mg2+ was also observed at mRNA level. The decrease of orexin-immunoreactive neurons was attributable to depletion of orexin, because it was reversible after washout of TTX or elevated extracellular Mg2+ and was not associated with induction of cell death. Blockers of voltage-dependent Ca2+ channels as well as of NMDA receptors also induced a significant and selective decrease of orexin-immunoreactive neurons. Moreover, TTX-induced decrease of orexin immunoreactivity was largely abrogated by concurrent application of a moderate concentration of NMDA. These results suggest that Ca2+ entry associated with nontoxic levels of spontaneous activity of glutamatergic inputs plays an important role in the maintenance of orexin neurons in a tissue culture model. © 2009 Wiley-Liss, Inc.