Hypoxia Modulates Cholinergic but Not Opioid Activation of G Proteins in Rat Hippocampus V.S. Hambrecht, 1 P.E. Vlisides, 1 B.W. Row, 2 D. Gozal, 2 H.A. Baghdoyan, 1 and R. Lydic 1 * ABSTRACT: Intermittent hypoxia, such as that associated with ob- structive sleep apnea, can cause neuronal death and neurobehavioral dysfunction. The cellular and molecular mechanisms through which hy- poxia alter hippocampal function are incompletely understood. This study used in vitro [ 35 S]guanylyl-5 0 -O-(c-thio)-triphosphate ([ 35 S]GTPcS) autoradiography to test the hypothesis that carbachol and DAMGO acti- vate hippocampal G proteins. In addition, this study tested the hypothe- sis that in vivo exposure to different oxygen (O 2 ) concentrations causes a differential activation of G proteins in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus. G protein activation was quan- tified as nCi/g tissue in CA1, CA3, and DG from rats housed for 14 days under one of three different oxygen conditions: normoxic (21% O 2 ) room air, or hypoxia (10% O 2 ) that was intermittent or sustained. Across all regions of the hippocampus, activation of G proteins by the cholinergic agonist carbachol and the mu opioid agonist [D-Ala 2 , N- Met-Phe 4 , Gly 5 ] enkephalin (DAMGO) was ordered by the degree of hy- poxia such that sustained hypoxia > intermittent hypoxia > room air. Carbachol increased G protein activation during sustained hypoxia (38%), intermittent hypoxia (29%), and room air (27%). DAMGO also activated G proteins during sustained hypoxia (52%), intermittent hy- poxia (48%), and room air (43%). Region-specific comparisons of G protein activation revealed that the DG showed significantly less activa- tion by carbachol following intermittent hypoxia and sustained hypoxia than the CA1. Considered together, the results suggest the potential for hypoxia to alter hippocampal function by blunting the cholinergic acti- vation of G proteins within the DG. V V C 2007 Wiley-Liss, Inc. KEY WORDS: acetylcholine; neurobehavioral impairment; sleep apnea INTRODUCTION Obstructive sleep apnea (OSA) is characterized by repeated obstruc- tion of the upper airway during sleep and results in episodes of intermit- tent hypoxia and sleep fragmentation (Ryan and Bradley, 2005). An esti- mated 12–18 million people in the United States have sleep apnea, with a higher prevalence in men (4%) than in women (2%) and OSA is asso- ciated with risk factors such as age, excessive weight, and high blood pressure (Young et al., 1993). In OSA patients, hypoxic events can occur 20–30 times per hour with cessation of breathing last- ing for 10–20 s or more (Shamsuzzaman et al., 2003). Daytime symptoms of OSA include excessive sleepiness and neurobehavioral impairment (Engleman and Joffe, 1999; Beebe, 2005; Walker and Stickgold, 2006). Research concerning the effects of intermittent hypoxia has been stimulated by efforts to understand the impact of hypoxia on sleep, learning, and memory (Ninomiya et al., 1989; Greenberg et al., 1999; Gozal et al., 2001a; Ham- rahi et al., 2001; Gozal et al., 2002; Goldbart et al., 2003a,b; Row et al., 2003; Zhao et al., 2005). The hippocampus is vulnerable to hypoxia (Yamaoka et al., 1993; Matsuoka et al., 1997; Gozal et al., 2001a,b) and intermittent hypoxia alters the excitability of hippocampal neurons (Schiff and Somjen, 1985). Cholinergic neurotransmission contributes to hippo- campal function (Colom et al., 1991), and muscarinic cholinergic receptors are expressed by hippocampal neurons (Satoh and Kaneko, 1994; Rouse et al., 1999; van der Zee and Luiten, 1999). Muscarinic cholinergic receptors are G protein coupled (reviewed in Caulfield and Birdsall, 1998), and carbachol activates muscarinic cholinergic receptors and G proteins in brain regions regulating sleep (Capece et al., 1998a; DeMarco et al., 2003; Douglas et al., 2004). Activated G proteins can be labeled using [ 35 S]guanylyl-5 0 -O-(g-thio)-triphos- phate ([ 35 S]GTPgS) (Sim et al., 1995). The hippocam- pus also contains endogenous opioids (Watson et al., 1982) and mu opioid receptors (Crain et al., 1986; Mansour et al., 1987; Tempel and Zukin, 1987; Drake and Milner, 2002). Mu opioid receptors are coupled to G proteins (Corbett et al., 2006), providing a key ra- tionale for the present use of the mu opioid receptor agonist [D-Ala 2 , N-Met-Phe 4 , Gly 5 ] enkephalin (DAMGO). G proteins in brain regions regulating sleep are activated by DAMGO (Capece et al., 1998b; Tanase et al., 2001; Bernard et al., 2006). Many studies have shown that activating mu opioid receptors can have excitatory actions throughout the hippocampus (Zieglgansberger et al., 1979; Dunwiddie et al., 1980; Siggins and Zieglgansberger, 1981; Mayer et al., 1994; Mayer and Henriksen, 1995). Hippocampal mu opioid receptors have a region-specific distribution and con- tribute to normal hippocampal function (Svodoba et al., 1999; Drake and Milner, 2002). No previous studies have quantified the effect of hy- poxia on cholinergic or opioid activation of G proteins 1 Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan; 2 Department of Pediatrics, Kosair Children’s Hospital Research Institute, University of Louisville, Louisville, Kentucky Grant sponsor: National Institutes of Health; Grant numbers: HL40881, HL57120, HL65272, MH45361, HL69932, 2P50HL60296. *Correspondence to: Ralph Lydic, Ph.D., Department of Anesthesiology, University of Michigan, 7433 Medical Sciences Bldg. I, 1150 W Medical Center Drive, Ann Arbor, MI 48109-0615, USA. E-mail: rlydic@umich.edu Accepted for publication 18 April 2007 DOI 10.1002/hipo.20312 Published online 27 June 2007 in Wiley InterScience (www.interscience. wiley.com). HIPPOCAMPUS 17:934–942 (2007) V V C 2007 WILEY-LISS, INC.