Immunogold evidence that neuronal gap junctions in adult rat brain and spinal cord contain connexin-36 but not connexin-32 or connexin-43

Physiological and ultrastructural evidence indicates that gap junctions lin k many classes of neurons in mammalian central nervous system (CNS), allowi ng direct electrical and metabolic communication. Among at least six gap ju nction-forming connexin proteins in adult rat brain, connexin- (Cx) 32, Cx3 6, and Cx43 have been reported to occur in neurons. However, no connexin ha s been documented at ultrastructurally defined neuronal gap junctions. To a ddress this question directly, freeze-fracture replica immunogold labeling (FRIL) and immunofluorescence (IF) were used to visualize the subcellular a nd regional localization of Cx36 in rat brain and spinal cord. Three antibo dies were generated against different sequences in Cx36. By Western blottin g, these antibodies detected protein at 36 and 66 kDa, corresponding to Cx3 6 monomer and dimer forms, respectively. After double-labeling for Cx36 and Cx43 by FRIL, neuronal gap junctions in inferior olive. spinal cord, and r etina were consistently immunogold-labeled for Cx36, but none were labeled for Cx43, Conversely. Cx43 but not Cx36 was detected in astrocyte and epend ymocyte gap junctions. In >250 Cx32/Cx43 single- and double-labeled replica s from 10 CNS regions, no neuronal gap junctions were labeled for either Cx 32 or Cx43, Instead, Cx32 and Cx43 were restricted to glial gap junctions. By IF. Cx36 labeling was widely distributed in neuropil, including along de ndritic processes and within neuronal somata, On the basis of FRIL identifi cation of Cx36 in neuronal gap junctions and IF imaging of Cx36 throughout rat brain and spinal cord, neuronal gap junctions containing Cx36 appear to occur in sufficient density to provide widespread electrical and metabolic coupling in adult CNS.