Immunoelectron microscopic localization of the HPC-1 antigen in rat cerebellum

HPC-1 antigen is a neuron-specific 34 kDa protein, identical to p35A (syntaxin), and is thought to play important roles in docking or fusion of synaptic vesicles to presynaptic active zones. In the present study we analyze the distribution of HPC-1 antigen in rat cerebellum by a cryoimmunogold technique using an antibody against the fusion protein of beta-galactosidase and the HPC-1 antigen. HPC-1 antigen was detected at high density on the plasma membranes and synaptic vesicles of presynaptic boutons which formed synapses with dendrites of Purkinje cells, and on the plasma membranes of parallel fibres in the cerebellar molecular layer. In the granule cell layer, gold particles were also detected on the endoplasmic reticulum, nuclear membranes and the plasma membranes of granule cells. Presynaptic membranes and synaptic vesicles in glomeruli were also labelled by gold particles. To determine the topology of HPC-1 antigen on the membranes, the synaptosome fraction prepared from rat cerebellum was embedded in agarose, and processed for the pre-embedding protein A-gold technique. Intact synaptosomes were not labelled by gold particles. However, when fixed in hypotonic fixative to rupture plasma membranes, or when ruptured after fixation in normotonic fixative, the cytoplasmic surfaces of presynaptic membranes and synaptic vesicles were labelled by gold particles. These results suggest that most of the epitopes of HPC-1 antigen are located on the cytoplasmic surface of plasma membranes and synaptic vesicle membranes.