Lowering glucose concentrations increases cytosolic Ca 21 in orexin neurons of the rat lateral hypothalamus Shinji Muroya a,b , Kazuhide Uramura a,b , Takeshi Sakurai c , Morikuni Takigawa b , Toshihiko Yada a,d, * a Department of Physiology, Jichi Medical School, Minamikawachi, Kawachi, Tochigi 329-0498, Japan b Departments of Psychiatry, Kagoshima University School of Medicine, Kagoshima 890-8520, Japan c Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Ibaraki 305-8575, Japan d Laboratory of Intracellular Metabolism, National Institute for Physiological Sciences, Okazaki 444-8585, Japan Received 8 June 2001; received in revised form 26 June 2001; accepted 28 June 2001 Abstract Orexin neurons are specifically localized in and around the lateral hypothalamus (LH), a feeding center. Intracerebro- ventricular administration of orexin-A and -B stimulates feeding as well as arousal. However, little is known regarding the regulators of the orexin neuron activity. The neurons that are activated under low glucose conditions, glucose- sensitive neurons, are located in the LH and have been implicated in the control of feeding. The present study investi- gated the effect of glucose on the single orexin neurons isolated from the rat LH, by measuring cytosolic Ca 21 concen- tration ([Ca 21 ] i ) by fura-2 microfluorometry followed by immunocytochemical staining with anti-orexin antiserum. A shift of glucose concentration form 8.3 to 2.8 mM in the superfusion solution increased [Ca 21 ] i in 13 out of 32 orexin- immunoreactive LH neurons. The results demonstrate that glucose-sensitive orexin neurons are present in the LH and that these neurons may play a role in linking the metabolic state in the body to the orexigenic, and could also, awakening signaling in the brain. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lateral hypothalamus; Orexin; Glucose-sensitive neuron; Glucose; Feeding; Arousal; Cytosolic Ca 21 ; Immunocytochemistry The lateral hypothalamus (LH) has been recognized as the feeding center, an important nucleus for regulation of feeding behavior [8,13,14]. Orexin-A and -B, recently discovered neuropeptides, are specifically localized in the cellbodies of neurons located in and around the LH, while orexin-containing axons innervate to a broad area in the brain [2,16,17]. Moreover, intracerebroventricular adminis- tration of orexins stimulates food consumption [16,17], while that of orexin antibody suppresses it [20]. The expres- sion of orexin messenger RNA in the LH is increased with fasting and normalized after feeding [1,9,21]. These find- ings suggest that orexin neurons may be involved in the orexigenic function of the LH. In the LH, a class of neurons that are activated by lowering glucose concentrations, named glucose-sensitive neurons (GSNs), are located and thought to play a key role in the stimulation of feeding [13,14,18], though their neurochemical features are unknown. Therefore, it is of particular importance to deter- mine the functional relationship between orexin neurons and GSNs in the LH. The present study was undertaken to examine whether the orexin neurons in the LH are activated by lowering glucose concentrations. The effect of glucose on the isolated LH neurons from adult rats was studied by measuring cytosolic Ca 21 concentration ([Ca 21 ] i ) by fura-2 microfluorometry, followed by immunocytochemical stain- ing with an anti-orexin antiserum. Male Sprague–Dawley rats were maintained on a 12-h light/dark cycle and given food and water ad libitum. Single LH neurons were prepared from 8–10 week old rats accord- ing to the previously reported procedures for preparation of hypothalamic neurons [4,11] with slight modifications. Briefly, rats were anesthetized and decapitated, and their brain was removed. Hypothalamic slices containing the LH were prepared and the LH was punched out. The dissected tissues were washed twice with HEPES-buffered and Krebs-Ringer bicarbonate buffer (HKRB) (composed of 129 mM NaCl, 5.0 mM NaHCO 3 , 4.7 mM KCl, 1.2 mM KH 2 PO 4 , 1.8 mM CaCl 2 , 1.2 mM MgSO 4, and 10 mM Neuroscience Letters 309 (2001) 165–168 0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(01)02053-5 www.elsevier.com/locate/neulet * Corresponding author. Tel.: 181-285-58-7319; fax: 181-285- 44-9962. E-mail address: tyada@jichi.ac.jp (T. Yada).