Astrocytic gap junction removal, connexin43 redistribution, and epitope masking at excitatory amino acid lesion sites in rat brain.

Abstract

We previously reported that kainic acid (KA) lesion sites in rat brain exhibit an absence of astrocytic gap junctions at 1 week post-lesion. Loss of immunocytochemical reactivity with a sequence-specific antibody against the astrocytic gap junctional protein connexin43 (Cx43) suggested epitope masking since persistence of Cx43 was observed on Western blots. Here, we determined the fate of Cx43 at various times after thalamic KA and striatal NMDA lesions. In normal tissue and at 6 hr post-KA lesion, Cx43 immunoreactivity predominated at typical astrocytic gap junctions. Immunolabelled junctions were still seen at 3 days, with epitope masking already present, and were virtually absent by 6 days post-lesion. Gap junction remodeling was indicated by the appearance of intracellular immunostained annular profiles and uncharacteristically extensive gap junctions between symmetrically immunolabelled membranes and between labelled astrocytic and unlabelled oligodendrocytic membranes. Labelled multivesicular clusters emerged at 2 days, were numerous at 3 days and constituted the sole Cx43 sequestration site by post-lesion day 6. Ultrastructural disruption and gap junction disassembly progressed more slowly in NMDA-injected tissue where immunoreactivity persisted, albeit at markedly decreasing levels until the final survival time examined (16 days). Intense Cx43 immunolabelling was seen in filopodia of putative reactive astrocytes at the lesion periphery at 6-8 days and was associated at 16 days with an increased number of gap junctions primarily between fine astrocytic processes. These results demonstrate that massive neuronal loss alone or in conjunction with direct actions of excitotoxins on astrocytes precipitates an astrocytic reaction accompanied initially by removal of their gap junctions followed by redistribution of Cx43, and suggest that the astrocytic syncytium may undergo reorganization in a manner leading to isolation of the lesion site.