Characterization of synaptic circuits changes in ventral horn of embrionic spinal slices cultures from SOD1 G93A mice

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease characterized by loss of motoneurons. The discovery of mutations in the gene for the cytosolic Cu/Zn superoxide dismutase in a small proportion of familiar ALS patients led to an animal model in which the human mutant SOD1 is overexpressed in mice (G93A). For this study, we employed the long term spinal cord organotypic cultures developed from G93A embryonic mice and their wild type (WT) littermates, starting from the recent findings emerged from a study by Avossa et al. (2006). These authors reported that G93A organotypic spinal cultures exhibited increased vulnerability to AMPA glutamate receptormediated excitotoxic stress, prior to clear disease appearance, besides showing a significantly increased ratio between inhibitory and excitatory synapses, although they did not express evident morphological differences, when compared to WT ones (Avossa et al., 2006). The primary objective of this study was to investigate this early ALS stage to understand how functional changes can predate morphological alterations. To that aim we monitored spontaneous synaptic activity via patch clamping interneurons both in WT and G93A spinal cultures after 7, 14 and 21 days of in vitro (DIV) growth. At 7 DIV, when synchronous episodes of activity are normally detected in cultured spinal circuits, G93A slices displayed bursting with a higher probability (83%) when compared to controls (54%). Between 14 and 21 DIV, when bursting activity disappear, both in G93A and WT slices, pharmacological dissection of glutamate, GABA and glycine mediated post synaptic currents (PSCs), showed, in G93A, a significant reduction in GABAergic PSCs and mPSCs in respect to WT. Upon pharmacological removal of the GABAergic component, fast glycinergic events were unmasked and these events displayed a similar frequency in both culture groups. Along with in vitro growth, we detected a progressive reduction in the decay time constant of glycinergic PSCs, such process was significantly faster in G93A. Thus, a shift in dynamic communication within spinal networks might be involved in ALS progression.