Cytosolic calcium elevation induced by orexin/hypocretin in granule cell domain cells of the rat cochlear nucleus in vitro
Introduction
Orexin-A (ORX-A) and orexin-B (ORX-B), also called hypocretin-1 and hypocretin-2, respectively, are novel neuropeptides that are synthesized in the perifornical region of the lateral hypothalamic area (LHA). ORX-A and ORX-B bind to ORX 1 (OX1) receptors and ORX 2 (OX2) receptors that belong to the G protein-coupled receptor superfamily [33]. OX1 receptors have a higher affinity for ORX-A than for ORX-B, whereas OX2 receptors have a similar affinity for both ORX-A and ORX-B. The nerve terminals of ORX neurons from the perifornical region of the LHA are distributed throughout almost the entire brain, including the cortex, limbic system, hypothalamus and brainstem [6], [22], [26], [30]. In accordance with the distribution of nerve terminals, OX1 and/or OX2 receptors are also found in these brain regions [5], [10], [20], [21], [41]. These widespread distributions of ORX nerve terminals and receptors in the brain suggest multifunctional roles for the ORX system. Indeed, potential roles for ORX-A and ORX-B have already been demonstrated; these include the regulation of arousal, sensory processing, energy homeostasis, and autonomic functions [27], [36].
The gateway for neural processing in the ascending auditory system is the cochlear nucleus. This nucleus is divided into two parts: a magnocellular core and a microneuronal shell [7], [32]. The microneuronal shell is mainly situated over the medial, dorsal and lateral surface of the ventral cochlear nucleus and expands into layer II of the dorsal cochlear nucleus [23], [24], [32], [45]. The microneuronal shell includes three types of cells – granule, unipolar brush and chestnut cells – and it is sometimes referred to as the granule cell domain (GCD) due to the abundance of granule cells [7], [32], [45]. The GCD receives non-auditory inputs rather than rapidly conducted auditory inputs, and it sends its output to the dorsal cochlear nucleus [7], [32], [45]. The non-auditory inputs include vestibular signals concerning head position and somatic proprioceptive signals that indicate neck muscle position and tension. The level of arousal is also one of the non-auditory inputs to the GCD [32]. Indeed, spontaneous and evoked unitary firing of the cochlear nucleus exhibit changes closely related to stages of sleep and wakefulness [28]. ORX neurons in the LHA also change their discharge rate across the sleep-waking cycle; they increase firing during and preceding active waking, and virtually cease firing during sleep [19]. ORX-immunoreactive nerve terminals project to the cochlear nucleus [9], [22], [26], [30], and neurons in the cochlear nucleus express OX1 and OX2 receptors [5], [9], [10], [21]; this suggests a close relationship between the ORX system and auditory sensory processing.
Alterations of cytosolic Ca2+ concentration ([Ca2+]i) have been shown to regulate many neuronal functions, such as neuronal excitability, transmitter release, gene expression, neuronal plasticity, cell survival, apoptosis and enzyme activity [4], [38]. Sakurai et al. [33] were the first to discover that ORX induces a transient increase in [Ca2+]i in Chinese hamster ovary (CHO) cells which recombinantly express human ORX receptors. Subsequent studies in rodents further demonstrated that ORX elevates [Ca2+]i in neurons in the various brain regions to which ORX fibers project and in which ORX receptors are expressed [11], [17], [42], [43], [44]. Thus, it seems likely that [Ca2+]i in GCD cells of the cochlear nucleus may be also elevated by ORX via ORX receptors. However, the effects of ORX on [Ca2+]i of GCD cells have not been described. Therefore, the aim of the present study was to examine the effects of ORX on [Ca2+]i in GCD cells, using rat brain slice preparations. To investigate the electrophysiological effects of ORX on GCD cells, whole-cell patch clamp recordings were also made.
Section snippets
Animals
Male Wistar rats, 12–16 days old, were used (Sankyo Lab., Shizuoka, Japan). The rats were housed with their mothers in a light-controlled room (light on: 06:00–18:00) at a temperature of 23 ± 1 °C for several days prior to the experiments. Food and water were available ad libitum. The animals and experimental procedures used were approved by the Institutional Animal Care and Use Committee of the University of Toyama.
Slice preparation
After sevoflurane anesthesia, the rats were decapitated and their brains were
TTX-resistant and TTX-sensitive elevations in [Ca2+]i induced by ORX-B
Application of ORX-B (100 nM) in standard ACSF elicited reversible, reproducible and consistent [Ca2+]i elevations in GCD cells. A sample recording is shown in the left panel of Fig. 1A. Application of ORX-B produced an increase in [Ca2+]i, following a latency of about 1.2 min, and the increase in [Ca2+]i returned to the baseline level within 14 min after removal of ORX-B (control). To explore whether the intracellular Ca2+ transient induced by ORX-B was evoked postsynaptically, [Ca2+]i elevation
Discussion
In agreement with previous studies demonstrating that ORX produced cytosolic Ca2+ transients in thalamic, hypothalamic, brainstem and spinal cord neurons [11], [17], [42], [43], [44], the present results revealed that ORX induced reversible, reproducible and consistent increases in [Ca2+]i in GCD cells. In more than half of the GCD cells that responded to ORX, the increase in [Ca2+]i was not blocked by TTX; this suggested that it was mediated by direct and specific activation of postsynaptic
Acknowledgements
This work was partly supported by the Grant-in-Aid for Scientific Research (No. 20590227 to K.S.) from Japan Society for the Promotion of Science. We thank Mrs. K. Mukai, K. Yoshida and M. Ogaya for assistance in electrophysiological experiments. One of the authors (K.S.) offers special thanks to Mr. Chikamitsu Nakayama for his encouragement throughout this work.
References (52)
- et al.
Axon initial segment Ca2+ channels influence action potential generation and timing
Neuron
(2009) - et al.
Protein distribution of the orexin-2 receptor in the rat central nervous system
Regul Pept
(2002) - et al.
Hypocretin receptor protein and mRNA expression in the dorsolateral pons of rats
Brain Res Mol Brain Res
(2001) - et al.
Gene expression and protein distribution of the orexin-1 receptor in the rat brain and spinal cord
Neuroscience
(2001) - et al.
Effects of orexins/hypocretins on neuronal activity in the paraventricular nucleus of the thalamus in rats in vitro
Peptides
(2005) - et al.
A novel isothiourea derivative selectively inhibits the reverse mode of Na+/Ca2+ exchange in cells expressing NCX1
J Biol Chem
(1996) - et al.
Orexin receptors and G-protein coupling: evidence for another “promiscuous” seven transmembrane domain receptor
J Pharmacol Sci
(2003) - et al.
Electrophysiological effects of orexins/hypocretins on pedunculopontine tegmental neurons in rats: an in vitro study
Peptides
(2009) - et al.
Electrophysiological effects of ghrelin on pedunculopontine tegmental neurons in rats: an in vitro study
Peptides
(2009) - et al.
Differential distribution and regulation of OX1 and OX2 orexin/hypocretin receptor messenger RNA in the brain upon fasting
Horm Behav
(2000)
Distribution of hypocretin-(orexin) immunoreactivity in the central nervous system of Syrian hamsters (Mesocricetus auratus)
J Chem Neuroanat
Electrophysiological effects of orexin/hypocretin on nucleus accumbens shell neurons in rats: an in vitro study
Peptides
Distribution of orexin neurons in the adult rat brain
Brain Res
Orexin neuronal circuitry: role in the regulation of sleep and wakefulness
Front Neuroendocrinol
Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior
Cell
Selective enhancement of excitatory synaptic activity in the rat nucleus tractus solitarius by hypocretin 2
Neuroscience
Ca2+ regulation and gene expression in normal brain aging
Trends Neurosci
Electrogenic Na+/Ca2+-exchange of nerve and muscle cells
Prog Neurobiol
Distribution of orexin receptor mRNA in the brain
FEBS Lett
Pharmacology of brain Na+/Ca2+ exchanger: from molecular biology to therapeutic perspectives
Pharmacol Rev
Orexin excites GABAergic neurons of the arcuate nucleus by activating the sodium–calcium exchanger
J Neurosci
The neuronal calcium-sensor proteins
Biochim Biophys Acta (BBA)—Mol Cell Res
Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems
Proc Natl Acad Sci USA
Projections from the ventral cochlear nucleus to the dorsal cochlear nucleus in rats
J Comp Neurol
Orexin/hypocretin excites the histaminergic neurons of the tuberomammillary nucleus
J Neurosci
Ion channels generating complex spikes in cartwheel cells of the dorsal cochlear nucleus
J Neurophysiol
Cited by (15)
The regulation of PKA signaling in obesity and in the maintenance of metabolic health
2022, Pharmacology and TherapeuticsCitation Excerpt :While less direct evidence exists for their putative role in the control of food intake, orexin receptor 2 (OXR2), OXR2 can be regulated downstream by PKA/CREB signaling through altered transcriptional activity in the lateral hypothalamus to regulate sleep patterns and has been associated with control of food intake ((Peyron et al., 1998, Xu et al., 2013). OXR2 is a Gαi-coupled GPCR that inhibits cAMP formation when activated, however orexins increase postsynaptic [Ca2+] via OX2R by activation of voltage-gated R- and T-type Ca2+ channels that is mediated by AC-PKA activation (Nakamura, Miura, Yoshida, Kim, & Sasaki, 2010). GLP1 is a multifaceted hormone with roles in energy balance, glucose homeostasis, cardiovascular health and more, and the regulation of its secretion is similarly complex (Muller et al., 2019).
Forced ethanol ingestion by Wistar rats from a juvenile age increased voluntary alcohol consumption in adulthood, with the involvement of orexin-A
2018, AlcoholCitation Excerpt :The dopaminergic system is known to participate in the locomotor actions of orexins. For instance, tyrosine-hydroxylase immunoreactive cells in the VTA receive innervations from IR-OX fibers, and OX-A is able to increase cytosolic Ca+2 in dopaminergic neuron culture (Nakamura, Miura, Yoshida, Kim, & Sasaki, 2010). Additionally, in vivo experiments have shown that antagonists for D1 and D2 dopaminergic receptors interfere with hyperlocomotion and stereotypical behavior stimulated by the central administration of OX-A (Nakamura et al., 2000).
OX2R activation induces PKC-mediated ERK and CREB phosphorylation
2012, Experimental Cell ResearchCitation Excerpt :Taking our results, along with the evidence of decreased CREB expression in tissues from human major depression patients, OX2R activation-induced CREB phosphorylation seems to be beneficial for depression treatment. Orexin induced signal transduction involves phospholipase C (PLC) [66,67], PKC [67–69], and PKA [38,70,71]. Orexin induces Gq activation, and then Gq activates PLC.
Orexins cause epileptic activity
2012, PeptidesCitation Excerpt :According to in vitro experimental studies, orexins cause depolarization in neurons [33] and increase in firing frequencies of neurons [3,9]. Orexins may form direct excitatory effects on neurons via increasing the influx of sodium [20], activate sodium–calcium exchanger pump [9], increase influx of calcium [26,42] or decrease efflux of potassium [14]. The importance of intracellular calcium increase in terms of neuronal excitability and epileptic activity is known [35].
Orexins increase penicillin-induced epileptic activity
2012, PeptidesCitation Excerpt :According to in vitro experimental studies, orexins cause depolarization in neurons [29] and increase in firing frequencies of neurons [3,7]. Orexins may form direct excitatory effects on neurons via increasing the influx of sodium [17], activate sodium–calcium exchanger pump [7], increase influx of calcium [22,34] or decrease efflux of potassium [12]. The importance of intracellular calcium increase in terms of neuronal excitability and epileptic activity is known [31].