Further insight in how exactly galanin
Further insight in how exactly galanin might inhibit seizures was obtained from galanin transgenic mice. It occurred that the altered susceptibility to seizures was in direct correlation with glutamate release from hippocampal slices obtained from these animals (Fig. 2(b)). While no differences were found between wild type, galanin knockout and galanin-overexpressing mice under resting conditions, depolarization of hippocampal slices with 60 mM K+, which led to 3-fold increase of glutamate release in wild type animals, failed to induce an increase of glutamate release from hippocampal slices of galanin overexpressing mice. In Gambogic Acid hippocampal slices obtained from galanin knockout animals responded with much steeper increase (6–9-fold) of neurotransmitter release, than those from wild type littermates (Mazarati et al., 2000). By association, these data confirmed that anticonvulsant effects of galanin occurred through presynaptic inhibition of excitatory neurotransmission.
Role of galanin receptor subtypes in anticonvulsant effects of galanin Out of three GalR subtypes cloned to date, two subtypes, GalR1 and GalR2 are expressed in the hippocampus (Branchek et al., 1998, Branchek et al., 2000, Burazin et al., 2000, Depczynski et al., 1998, Gustafson et al., 1996, O’Donnel et al., 1999, Xu et al., 1998). Studies on the GalR subtype contribution into anticonvulsant effects of galanin have been complicated by the limited availability of subtype-selective ligands. Galanin itself does not discriminate between GalR1 and GalR2. Only two GalR2-selective agonists currently exist, Galanin (2–11) and D-Trp-2-Galanin (1–29) (Floren et al., 2000, Hua et al., 2004, Kerekes et al., 2003, Liu et al., 2001), while no GalR1 selective agonists are available. Likewise, none of the available GalR antagonists discriminates between GalR1 and GalR2 (Floren et al., 2000). Early studies made attempts to identify receptor subtype-specificity of anticonvulsant effects of galanin. Thus, Zini et al., 1993a, Zini et al., 1993b found that glutamate release from hippocampal slices was inhibited not only by galanin (1–29), but also by galanin (1–16), which later was shown to prefer GalR2 over GalR1 (Floren et al., 2000). Mazarati et al., 1998, Mazarati et al., 2004a) showed that preferential GalR2 agonists Ala-2-Galanin (1–29), D-Trp-2-Galanin (1–29), and Galanin (2–11) were as effective as galanin (1–29) in inhibiting seizures. On the other hand, anticonvulsant effects of non-selective synthetic GalR agonist (see below) were attenuated by in vivo pretreatment with anti-GalR1 antisense (Saar et al., 2002). All the mentioned experiments, however, were far from satisfactory, as that they did not clarify the role of GalR1, and also because available GalR2 agonists still bind to GalR1 in high concentrations (Floren et al., 2000) making it difficult to relate their in vitro subtype-specific concentrations to the in vivo amount which would limit their action to GalR2. The first significant advance in studying GalR subtype involvement in seizure regulation was made possible, when GalR1 knockout mice were developed by Jacoby et al., 2002a, Jacoby et al., 2002b. Interestingly, a combination of GalR1 deletion with C57bl/j6 background resulted in the occurrence of spontaneous seizures in about 25% of the animals. GalR1 knockout mice with spontaneous seizure phenotype showed a cascade of the changes in the expression of neuropeptides, similar to that observed in models of limbic epilepsy (Fetissov et al., 2003). Use of experimental seizure models gave further insight in the role of GalR1 in seizure regulation. Mazarati et al. (2004b) compared patterns of seizures in GalR1 knockout mice which did not exhibit spontaneous seizures, with their wild type littermates in three models of seizures: induced by Li-Pilocarpine, by perforant path stimulation, and by systemic kainic acid. GalR1 knockout mice showed more severe and longer-lasting seizures in Li-Pilocarpine and perforant path stimulation-induced, but not in kainic acid-induced seizures (Fig. 3(a)). The look into the mechanisms which underlie the initiation phase of the three models of SE, may provide further insight in possible mechanisms of anticonvulsant effects of galanin acting through GalR1. The initiation phase of Li-pilocarpine SE depends on the activation M-cholinoreceptors by both pilocarpine directly and though reported increase of acetylcholine content in the brain upon pilocarpine administration (Jope et al., 1987, Morrisett et al., 1987). Galanin, on the other hand coexists with acetylcholine in septum/diagonal band complex, an area which sends cholinergic/galaninergic projections to the hippocampus (Melander et al., 1986, Consolo et al., 1994). In the hippocampus galanin inhibits both acetylcholine release (Dutar et al., 1989, Fisone et al., 1987), and postsynaptic cholinergic functions (Consolo et al., 1991). Inactivation of GalR1 in knockout mice might facilitate both acetylcholine release and postsynaptic effects of pilocarpine and acetylcholine, thus promoting Li-Pilocarpine SE.