Hypocretin/Orexin Peptide Signaling in the Ascending Arousal System: Elevation of Intracellular Calcium in the Mouse Dorsal Raphe and Laterodorsal Tegmentum

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 ([Ca 2ϩ ] i ) in cells transfected with orexin receptors and since receptor-mediated Ca 2ϩ transients are ubiquitous signaling mechanisms, we investigated whether Hcrt/Orx regulates [Ca 2ϩ ] i in the LDT and DR. Changes in [Ca 2ϩ ] 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 [Ca 2ϩ ] i with differing temporal profiles ranging from spiking to smooth plateaus. A fragment of Hcrt/Orx (16-33) failed to evoke changes in [Ca 2ϩ ] 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 Ca 2ϩ stores with cyclopiazonic acid (CPA; 3-30 M), thapsigargin (3 M), or ryanodine (20 M), although store-depletion by either CPA or ryanodine blocked Ca 2ϩ mobilization by the metabotropic glutamate receptor agonist (Ϯ)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD; 30 M). In contrast, Hcrt/Orx responses were strongly attenuated by lowering extracellular Ca 2ϩ (ϳ20 M) but were not inhibited by concentrations of KB-R7943 (10 M) selective for blockade of sodium/calcium exchange. Nifedipine (10 M), inhibited Hcrt/Orx responses but was more effective at abolishing spiking than plateau responses. Bay K 8644 (5-10 M), 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 [Ca 2ϩ ] i by a PKC-involved influx of Ca 2ϩ across the plasma membrane, in part, via L-type calcium channels. Thus the physiological release of Hcrt/Orx may help regulate Ca 2ϩ -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.

The Orexin OX1Receptor Regulates Ca2+Entry via Diacylglycerol-Activated Channels in Differentiated Neuroblastoma Cells

“…At low agonist concentrations, a measurable Ca 2ϩ release from intracellular stores was essentially absent. This is in agreement with previous studies van den Pol, 1999;Lund et al, 2000;Uramura et al, 2001;Holmqvist et al, 2002;Willie et al, 2003;Kohlmeier et al, 2004) and indicates Ca 2ϩ entry as a primary OX 1 R response. In neuronal cells, a Ca 2ϩ elevation in response to G-protein-coupled receptors could be the consequence of several different mechanisms, including activation of VGCC, reversal of electrogenic Na ϩ /Ca 2ϩ exchange, or phospholipase C-linked Ca 2ϩ store discharge with a consequent activation of storeoperated channels.…”

“…How does our finding relate to other studies performed on neurons? A [Ca 2ϩ ] i elevation in response to orexins has been described in several instances van den Pol, 1999;Uramura et al, 2001;Kohlmeier et al, 2004;Muroya et al, 2004). One of the first reports on orexin action, which was performed on hypothalamic neurons, described an activated Ca 2ϩ entry with no measurable membrane current .…”

“…van den found that the same inhibitor of PKC that we used totally blocked the [Ca 2ϩ ] i increase. In several other studies, PKC has been suggested to be a crucial intermediate effector in orexin action, often with downstream effects linked to specific ion channels (Uramura et al, 2001;Xu et al, 2002;Kohlmeier et al, 2004) or less well defined ion conductances (Yang et al, 2003). PKC has long been known to modulate VGCCs (Yang and Tsien, 1993) and K ϩ conductances (Henry et al, 1996).…”

“…The [Ca 2ϩ ] i can increase in cells by a variety of mechanisms, including depolarization-induced opening of voltagegated calcium channels (VGCCs), opening of nonselective cation channels, release from intracellular stores, as well as reversal of electrogenic Na ϩ /Ca 2ϩ exchanger. An increase in [Ca 2ϩ ] i during OXR stimulation is evident in neurons (van den Uramura et al, 2001;Kohlmeier et al, 2004), and it is also observable in different cell types used for heterologous expression (Sakurai et al, 1998;Smart et al, 1999;Holmqvist et al, 2002).…”

Hypocretin/Orexin Selectively Increases Dopamine Efflux within the Prefrontal Cortex: Involvement of the Ventral Tegmental Area

“…These include basal forebrain (España et al, 2001;Thakkar et al, 2001) and brainstem structures such as the locus coeruleus (Hagan et al, 1999;Horvath et al, 1999;Bourgin et al, 2000), the dorsal raphe (Liu et al, 2002;Kohlmeier et al, 2004), the lateral dorsal tegmental nucleus (Burlet et al, 2002;Kohlmeier et al, 2004) and the tuberomammillary nucleus (Eriksson et al, 2001). DA systems have also been implicated in the modulation of behavioral state and state-dependent processes, particularly under high arousal appetitive and aversive conditions (Trulson, 1985;Smith et al, 1992;Arnsten et al, 1994;Isaac and Berridge, 2003).…”