Elsevier

Peptides

Volume 31, Issue 8, August 2010, Pages 1579-1588
Peptides

Cytosolic calcium elevation induced by orexin/hypocretin in granule cell domain cells of the rat cochlear nucleus in vitro

https://doi.org/10.1016/j.peptides.2010.04.029Get rights and content

Abstract

Using rat brain slice preparations, we examined the effect of orexin on cytosolic Ca2+ concentrations ([Ca2+]i) in the granule cell domain (GCD) cells of the cochlear nucleus that carry non-auditory information to the dorsal cochlear nucleus. Application of orexin concentration-dependently increased [Ca2+]i, and in two thirds of GCD cells these increases persisted in the presence of tetrodotoxin. There was no significant difference between the dose–response curve for orexin-A and that for orexin-B. Extracellular Ca2+ removal abolished the [Ca2+]i elevation induced by orexin-B, whereas depletion of intracellular Ca2+ stores had no effect. The orexin-B-induced elevation of [Ca2+]i was not blocked by inhibitors of reverse-mode Na+/Ca2+ exchanger (NCX) and nonselective cation channel, whereas it was blocked by lowering the extracellular Na+ or by applying inhibitors of forward-mode NCX and voltage-gated R- and T-type Ca2+ channels. The ORX-B-induced increase in [Ca2+]i was also blocked by inhibitors of adenylcyclase (AC) and protein kinase A (PKA), but not by inhibitors of phosphatidylcholine-specific and phosphatidylinositol-specific phospholipase C. In electrophysiological experiments using whole-cell patch clamp recordings, half of GCD cells were depolarized by orexin-B, and the depolarization was abolished by a forward-mode NCX inhibitor. These results suggest that orexin increases [Ca2+]i postsynaptically via orexin 2 receptors, and the increase in [Ca2+]i is induced via the AC–PKA–forward-mode NCX-membrane depolarization-mediated activation of voltage-gated R- and T-type Ca2+ channels. The results further support the hypothesis that the orexin system participates in integrating neural systems that are involved in arousal, sensory processing, energy homeostasis and autonomic function.

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.

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