However, the causes of sporadic ALS remain obscure. Since the discovery of the genetic linkage of mutations in superoxide

dismutase 1 (SOD1) gene with familial ALS patients, one of the highlighted putative mechanisms is that degeneration of MNs is closely linked to involvement of SOD1 in both sporadic and familial cases (Bosco et al. 2010). It is proposed that the occurrence of misfolded SOD1 triggers a cascade of neurodegeneration by “gains-of-function” through activation of glutamate-mediated excitotoxicity, which induces an uncontrolled increase of intracellular calcium concentration (de Carvalho and Swash 2011). Data regarding Inhibitors,research,lifescience,medical cholinergic activity in animal models carrying SOD1 mutations are mainly reported linked to MN loss in the symptomatic phase (Crochemore et al. 2005; Alves et al. 2011). Nevertheless, a question that remains to be solved is how and when cholinergic function is compromised along the neurodegenerative process. In order to answer these questions, we have analyzed the spatiotemporal expression of ChAT, considering local Inhibitors,research,lifescience,medical cholinergic circuitry, efferences, and afferences, within the spinal cord from early presymptomatic until symptomatic Inhibitors,research,lifescience,medical stages of an ALS mouse model. The results obtained highly the importance of the

performance of longitudinal studies to unravel the etiopathogenesis of ALS. Material and Methods Animals Experiments were performed in transgenic mice carrying the mutation G93A in SOD1 gene and in nontransgenic wild-type (WT) littermates considered Inhibitors,research,lifescience,medical controls. SOD1G93A high copy mice (Tg[SOD1-G93A]1Gur) were obtained from the Jackson Laboratory (Bar Harbor, ME), with B16xSJL background. These mice were bred and maintained as hemizygotes by mating transgenic males with F1 hybrid (B6SJLF1/J) females obtained from Charles River Laboratories (Belgium). Animals were bread at the Animal Supply Services, FHPI cost Unidad Mixta de Investigación, Inhibitors,research,lifescience,medical University of Zaragoza, and were cared for and handled in accordance with the guidelines of the European Union Council (86/609/UE) and Spanish regulations (BOE 67/8509-12; BOE 1201/2005) on the use of Urease laboratory animals. Experimental procedures were approved by the

local Ethics Committee of the Universitat Autònoma de Barcelona. Transgenic mice were identified by polymerase chain reaction amplification of DNA extracted from the tail. Studies were performed in groups of 1-, 2-, and 3-month-old female mice (n = 8 each). One- and 2-month-old SOD1G93A mice are considered to be in early and adult presymptomatic stages of disease, respectively, whereas 3-month-old mice had an early symptomatic phenotype by behavioral (Chiu et al. 1995) and electrophysiological testing (Mancuso et al. 2011). Immunohistochemistry Animals were anesthetized with sodium pentobarbital (50 mg/kg i.p.), and perfused transcardially with phosphate buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 mol/L PB, pH 7.4 at 4°C.