NEUROPROTECTION AFFORDED BY NICOTINE AGAINST OXYGEN AND GLUCOSE DEPRIVATION IN HIPPOCAMPAL SLICES IS LOST IN 7 NICOTINIC RECEPTOR KNOCKOUT MICE J. EGEA, a A. O. ROSA, a M. SOBRADO, b L. GANDÍA, a M. G. LÓPEZ a,d AND A. G. GARCÍA a,c,d * a Instituto Teófilo Hernando and Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo, 4, 28029 Madrid, Spain b Instituto de Investigaciones Biomédicas “Alberto Sols,” CSIC-UAM, Madrid, Spain c Servicio de Farmacología Clínica, Hospital Universitario de La Prin- cesa, Madrid, Spain d Instituto Universitario de Investigación Gerontológica y Metabólica, Hospital Universitario de La Princesa, Madrid, Spain Abstract—Although 7-receptors are considered the main target for neuroprotection, other receptor subtypes (42 or 34) have also been implicated. Hence, we have used 7- transgenic mice, to study the hypothesis that 7-receptors play a dominant role in mediating neuroprotection in an in vitro model of ischemia. We have used rat and mouse hippocampal slices to establish the model of nicotinic neu- roprotection against oxygen and glucose deprivation (OGD). Neuronal damage caused by OGD during 1 h plus 3 h re- oxygenation, was quantified by measuring lactate dehydro- genase (LDH) release from hippocampal slices. In rat hip- pocampal slices, OGD increased over twofold basal LDH release. Such increase was reduced when treated with 10 – 100 M nicotine; maximal protection afforded by nicotine amounted to 46%. This neuroprotection was antagonized by the non-selective nicotinic receptor for acetylcholine (nAChR) blocker mecamylamine (10 M). In hippocampal slices from wild-type control mice, nicotine (100 M) decreased by 54.4% LDH release evoked by OGD plus re-oxygenation. In contrast, nicotine failed to exert neuroprotection in 7 knockout mice. This finding reinforces the view that the hippocampal neuro- protective effects of nicotine are predominantly linked to 7 receptors. © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: nicotine, oxygen-glucose deprivation, hippocam- pal slices, 7-knockout mice, neuroprotection. Neuronal nicotinic receptors for acetylcholine (nAChRs) are highly concentrated in the hippocampus, thalamus, and cerebral cortex (Clarke et al., 1985; Wada et al., 1989; Hogg et al., 2003). A great deal of interest has evolved around the hypothesis that several nAChR sub- types might be involved in the neuroprotective mecha- nisms against neuronal damage produced by various noxious stimuli. The clearest data in this direction emerge from studies performed in neuronal cultures, where nicotinic agonists exhibit neuroprotective effects against glutamate (Donnelly-Roberts et al., 1996; Stevens et al., 2003; Gahring et al., 2003; Sun et al., 2004), trophic factor deprivation (Yamashita and Naka- mura, 1996), -amyloid (Gahring et al., 2003; Kihara et al., 1997; Liu and Zhao, 2004; Moore et al., 2004), or hypoxia (Hejmadi et al., 2003). More dubious results emerge from in vivo models of cerebral ischemia. For instance, the 7 nAChR selective agonist GTS-21 (Kem, 2000) protects against neuronal death elicited by cerebral ischemia in gerbils but surpris- ingly, nicotine affords no neuroprotection; this contrasts with the fact that both nicotine and GTS-21 mitigate the memory deficits induced by such ischemic insult (Nanri et al., 1998). On the other hand, nicotine provides pro- tection against CA1 hippocampal neuronal death after transient cerebral ischemia in the rat (Kagitani et al., 2000). Even fewer studies are yet available in hip- pocampal slices subjected to oxygen and glucose depri- vation (OGD) followed by a re-oxygenation period. To our knowledge, the only study where the topic of nico- tinic receptor involvement in neuroprotection was ap- proached was with galantamine (Sobrado et al., 2004), an allosteric potentiating ligand (APL) that augments the effects of acetylcholine (ACh) on nicotinic receptors (Maelicke and Albuquerque, 2000). The question of which nAChR subtype(s) is involved in the neuroprotective effects of nicotinic agonists is highly relevant in the context of the design and devel- opment of selective nicotinic receptor ligands that might eventually reach the clinic for the treatment of stroke. As far as we know, attempts to answer this question have been carried out only in neuronal cultures. For instance, the non-selective blocker of nAChRs mecamylamine and the 2 selective blocker dihydro--erythroidine, re- versed the neurotoxic effects of hypoxia, suggesting that 42 as well as 7 and other receptor subtypes, could be involved in the neuroprotective effects of nicotinic agonists (Hejmadi et al., 2003; Kaneko et al., 1997). Also, two selective 7 receptor blockers, methyllyca- conitine (MLA) and -bungarotoxin mitigate the neuro- protective effects of nicotine against glutamate neuro- toxicity (Donnelly-Roberts et al., 1996; Kaneko et al., 1997) and hypoxia (Hejmadi et al., 2003); this suggests that 7 nAChRs mediate such neuroprotective effects. *Correspondence to: A. G. García, Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo, 4, 28029 Madrid, Spain. Tel: +34-91-497-5380; fax: +34-91-4975397. E-mail address: agg@uam.es (A. G. García). Abbreviations: LDH, lactate dehydrogenase; mOD, mean optical den- sity; nAChR, nicotinic receptor for acetylcholine; OGD, oxygen and glucose deprivation; +/+, wild type; -/-, nAChR knockout. Neuroscience 145 (2007) 866 – 872 0306-4522/07$30.00+0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.12.036 866