Expression and plasticity of galanin systems in cortical neurons, oligodendrocyte progenitors and proliferative zones in normal brain and after spreading depression Pei-Juan Shen, 1 Jari A. Larm 1 and Andrew L. Gundlach 1,2 1 Howard Florey Institute of Experimental Physiology and Medicine and 2 Department of Medicine, Austin and Repatriation Medical Centre, The University of Melbourne, Victoria 3010, Australia Keywords: corpus callosum, galanin receptor-1, neurogenesis, rat, rostral migratory stream, subventricular zone Abstract Neocortex contains very few galanin neurons but receives a moderate galanin innervation from various subcortical loci. Recent data suggest that galanin helps regulate the tonic neuronal excitability of hippocampus and probably cerebral cortex but relatively little is known about the anatomy and functional regulation of cortical galanin systems. Therefore, we examined, in the rat, the effect of the intense but benign stimulus, cortical spreading depression (CSD), on the expression of galanin and galanin receptors (GalR1 and GalR2) in the neocortex and associated regions, revealing complex, multicellular responses. Thus, following acute, unilateral KCl- induced CSD, a delayed and transient induction (onset after 48h, lasting 24h) of galanin mRNA and peptide production occurred across the ipsilateral cerebral cortex in activated oligodendrocyte progenitor cells (OPCs), identi®ed by speci®c NG2 proteoglycan immunostaining. An increase in GalR1 mRNA, immunoreactivity and receptor binding occurred in neurons within layers II and V of neocortex and in piriform cortex at 7±28days after CSD, associated with a long-lasting depletion of galanin-positive nerve ®bres in these regions. In contrast, GalR2 mRNA expression was largely unaltered after CSD. Additional novel ®ndings in normal, adult brain were the detection of galanin mRNA and immunoreactivity in OPCs within the medial corpus callosum and in immature progenitor cells in the subventricular zone and rostral migratory stream. GalR1 and GalR2 mRNA was also present in these latter regions. These ®ndings and the complex modulation of galanin and galanin receptors in multiple cell types (neurons/OPCs) follow- ing acute cortical activation/depression further demonstrate the potential plasticity of neuronal and non-neuronal galanin systems under physiological and pathological conditions and strongly suggest additional functions for this pleiotropic peptide in mammalian brain. Introduction Galanin, a 29±30 amino acid neuropeptide (Tatemoto et al., 1983; Ho Èkfelt et al., 1998), is widely distributed in the central nervous system (Melander et al., 1986; Merchenthaler et al., 1993; Ryan & Gundlach, 1996). Three galanin receptors, GalR1, GalR2 and GalR3, havebeenclonedandbelongtotheG-protein-coupledreceptorsuper- family (Habert-Ortoli et al., 1994; Howard et al., 1997; Wang et al., 1997;seeBranchek etal.,2000forreview).Galaninisthoughttohave important roles in cognition, nociception, metabolism and reproduc- tion(e.g.Gundlach2002;Kinney etal.,2002;Liu&Ho Èkfelt2002)and toactviadirectpost-synapticeffectsandviapre-synapticregulationof classical transmitter release. In this regard, galanin coexists with a variety of transmitters in brain including acetylcholine, dopamine, noradrenaline,serotoninand g-aminobutyricacidandisthoughttobe released at both somatodendritic and nerve terminal sites (Melander etal.,1986;Ho Èkfelt etal.,1998,2000).Forexample,galaninhasbeen shown to inhibit acetylcholine release in hippocampus (Fisone et al., 1987)andinhibitthereleaseofand/orresponsivenesstonoradrenaline inlocuscoeruleus(LC)(Pieribone etal.,1995)andserotoninindorsal raphe (Xu et al., 1998a). Galanin has also been shown to inhibit glutamate release via pre-synaptic mechanisms in rat hypothalamus (Kinney et al., 1998) and more recent studies focused on the hippo- campus further support a role for galanin in the regulation of tonic excitatory transmission. The hippocampus of the rat contains very few galanin-producing neurons [mRNA or immunoreactivity (IR); Cortes et al., 1990] but receives an abundance of galanin-positive nerve terminals projecting from septum and LC (Melander et al., 1985; Merchenthaler et al., 1993).GalaninIRintheseafferentnerve®bresisdepletedbyseizures butadministrationofexogenousgalaninattenuatesseizureactivityand galanin antagonists facilitate it (Mazarati et al., 1998; Saar et al., 2002).Furthermore,galaningeneknockoutmicedisplayanincreased propensity for seizures and mice over-expressing galanin have an increased resistance to seizures (Mazarati et al., 2000). Similarly, the neocortex of the rat normally contains very few galanin neurons (Sko®tsch & Jacobowitz, 1985; Ryan & Gundlach, 1996)butdoesreceivesubstantialgalanin-containingprojectionsfrom the LC, septum and hypothalamus (Senut et al., 1989; Merchenthaler etal.,1993;Gabriel etal.,1995;Xu etal.,1998b).Negligible,speci®c [ 125 I]-galanin binding is reportedly present in neocortex, suggesting thepresenceofrelativelylowlevelsofgalaninreceptorsintheregion comparedwiththehighlevelsinpiriformcortexandsubcorticalareas (Sko®tsch et al., 1986; Melander et al., 1988). In situ hybridization European Journal of Neuroscience, Vol. 18, pp. 1362±1376, 2003 ß Federation of European Neuroscience Societies doi:10.1046/j.1460-9568.2003.02860.x Correspondence: Dr Andrew L. Gundlach, at 1 Howard Florey Institute of Experimental Physiology and Medicine, as above. E-mail: a.gundlach@hfi.unimelb.edu.au Received 23 April 2003, revised 12 June 2003, accepted 23 June 2003