Hypocretin/orexin peptide signaling in the ascending arousal system

Elevation of intracellular calcium in the mouse dorsal raphe and laterodorsal tegmentum

Kristi A. Kohlmeier, Takafumi Inoue, Christopher S. Leonard

Research output: Contribution to journalArticle

70 Citations (Scopus)

Abstract

Dysfunction of the hypocretin/orexin (Hcrt/Orx) peptide system is closely linked to the sleep disorder narcolepsy, suggesting that it is also central to the normal regulation of sleep and wakefulness. Indeed, Hcrt/Orx peptides produce long-lasting excitation of arousal-related neurons, including those in the laterodorsal tegmentum (LDT) and the dorsal raphe (DR), although the mechanisms underlying these actions are not understood. Since Hcrt/Orx mobilizes intracellular calcium ([Ca2+]i) in cells transfected with orexin receptors and since receptor-mediated Ca2+ transients are ubiquitous signaling mechanisms, we investigated whether Hcrt/Orx regulates [Ca2+]i in the LDT and DR. Changes in [Ca 2+]i were monitored by fluorescence changes of fura-2 AM loaded cells in young mouse brain slices. We found Hcrt/Orx (Orexin-A, 30-1,000 nM) evoked long-lasting increases in [Ca2+]i with differing temporal profiles ranging from spiking to smooth plateaus. A fragment of Hcrt/Orx (16-33) failed to evoke changes in [Ca2+]i and changes were not blocked by TTX or ionotropic glutamate receptor antagonists, suggesting they resulted from specific activation of postsynaptic orexin receptors. Unlike orexin receptor-transfected cells, Hcrt/Orx-responses were not attenuated by depletion of Ca2+ stores with cyclopiazonic acid (CPA; 3-30 μM), thapsigargin (3 μM), or ryanodine (20 μM), although store-depletion by either CPA or ryanodine blocked Ca2+ mobilization by the metabotropic glutamate receptor agonist (±)-1-aminocyclopentane- trans-1,3-dicarboxylic acid (trans-ACPD; 30 μM). In contrast, Hcrt/Orx responses were strongly attenuated by lowering extracellular Ca2+ (∼20 μM) but were not inhibited by concentrations of KB-R7943 (10 μM) selective for blockade of sodium/calcium exchange. Nifedipine (10 μM), inhibited Hcrt/Orx responses but was more effective at abolishing spiking than plateau responses. Bay K 8644 (5-10 μM), an L-type calcium channel agonist, potentiated responses. Finally, responses were attenuated by inhibitors of protein kinase C (PKC) but not by inhibitors of adenylyl cyclase. Collectively, our findings indicate that Hcrt/Orx signaling in the reticular activating system involves elevation of [Ca2+]i by a PKC-involved influx of Ca2+ across the plasma membrane, in part, via L-type calcium channels. Thus the physiological release of Hcrt/Orx may help regulate Ca 2+-dependent processes such as gene expression and NO production in the LDT and DR in relation with behavioral state. Accordingly, the loss of Hcrt/Orx signaling in narcolepsy would be expected to disrupt calcium-dependent processes in these and other target structures.

Original languageEnglish
Pages (from-to)221-235
Number of pages15
JournalJournal of Neurophysiology
Volume92
Issue number1
DOIs
Publication statusPublished - 2004 Jul
Externally publishedYes

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Arousal
Calcium
Peptides
Orexin Receptors
Narcolepsy
Ryanodine
L-Type Calcium Channels
Orexins
Dorsal Raphe Nucleus
Protein Kinase C
Calcium Channel Agonists
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester
Excitatory Amino Acid Agonists
Ionotropic Glutamate Receptors
Excitatory Amino Acid Antagonists
Dicarboxylic Acids
Thapsigargin
Wakefulness
Fura-2
Nifedipine

ASJC Scopus subject areas

  • Physiology
  • Neuroscience(all)

Cite this

Hypocretin/orexin peptide signaling in the ascending arousal system : Elevation of intracellular calcium in the mouse dorsal raphe and laterodorsal tegmentum. / Kohlmeier, Kristi A.; Inoue, Takafumi; Leonard, Christopher S.

In: Journal of Neurophysiology, Vol. 92, No. 1, 07.2004, p. 221-235.

Research output: Contribution to journalArticle

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