Presynaptic mechanisms involved in the cholinergic and GABAergic transmitter release in the central nervous system of the mdx mouse: analysis of nicotinic modulation in young and adult mice

Abstract

Duchenne muscle dystrophy (DMD) is a recessive and progressive muscle disease caused by mutations of the dystrophin gene, a protein involved in stabilization of the sarcolema, regulation of the muscular nicotinic acetylcholine receptor (nAChR) and in signal transduction. Lack of dystrophin in the central nervous system (CNS) causes brain atrophy, changes in the neuronal structures and synaptic transmission that have been associated with mild mental retardation in DMD patients. Previous studies from this laboratory have shown significant changes in the number of neuronal nAChRs a7 and a4b2 subtypes in the hippocampus of the mutant mdx mouse, a brain region associated with memory processing (Souccar et al., 2007). More recently, we found that the quantitative changes of nAChRs are accompanied by altered ACh release, indicating probable changes of neurotransmission in cholinergic pathways in the mdx mouse brain (Proc. FAPESP 2007/02536-4). In continuation of these studies, this project was aimed to evaluate the presynaptic mechanisms involved in the cholinergic and GABAergic transmitter release and its modulation by nAChRs in brain regions of normal and dystrophic mice, seeking a possible relationship with the cognitive and memory deficits reported in DMD patients and mdx mice. The experiments will be done using synaptossomes prepared with brain regions containing dystrophin (cortex, hippocampus and cerebellum), from young (4 months) and adult (12 months) control and mdx mice, and will consist of: 1) Measurements of the basal and stimulated [3H]-ACh overflow from superfused synaptossomes of all three brain regions from control and mdx groups; 2) Measurements of the basal and evoked [3H]-GABA overflow from synaptosomes preparations of the same brain regions; 3) Analysis of presynaptic nicotinic modulation of [3H]-ACh and [3H]-GABA overflow in the presence of agonists and antagonists of a7 and a4b2 nAChRs subtypes, from the same synaptosomes preparations; 4) Determinations of the ACh and GABA neuronal uptakes by immunoblotting techniques of the vesicular transporters VAChT and VGAT, and synaptic proteins (synapsins, SV2A, synaptophysin) in synaptosomes preparations from control and mdx mice. This project includes MS thesis of two postgraduate students and recipient of a fellowship from FAPESP (Proc. 2008/56023-0) and CAPES, respectively. (AU)