ABSTRACT
Functional evidence suggests that neuronal enriched endosomal protein of 21 kDa (NEEP21) takes part in facilitating transport of AMPA receptors (AMPAR) in the synapse. To explore the anatomical basis for a role in this synaptic trafficking, we investigated the ultrastructural localization of NEEP21 in rodent brain. Using immunogold electron microscopy, we show that NEEP21 is colocalized with the AMPAR subunits GluR2/3 in postsynaptic spines. Quantitative analysis of gold particle distribution along an axis perpendicular to the postsynaptic specialization indicated that NEEP21 occurs in the postsynaptic membrane but also in the interior of the spines. NEEP21 positive endosomes/multivesicular bodies were found throughout cell bodies and dendrites. In light microscopical preparations, the NEEP21 antibody produced a labeling pattern in the neocortex, hippocampus and cerebellum that mimicked that of GluR2/3 and not that of GluR1 or 4. Our findings are consistent with a role for NEEP21 in facilitating vesicular transport of GluR2 between intracellular compartments and the postsynaptic plasma membrane.
Subject(s)
Dendritic Spines/metabolism , Endocytosis/physiology , Nerve Tissue Proteins/metabolism , Receptors, AMPA/metabolism , Synaptic Membranes/metabolism , Animals , Brain/metabolism , Brain/ultrastructure , Cells, Cultured , Dendritic Spines/ultrastructure , Endosomes/metabolism , Endosomes/ultrastructure , Female , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Microscopy, Immunoelectron , Protein Transport/physiology , Rats , Rats, Wistar , Synaptic Membranes/ultrastructure , Synaptic Transmission/physiologyABSTRACT
We describe a technique wherein single muscle fibers of intact mice are made transgenic by intracellular injection of DNA expression vectors for the reporter genes lacZ and green fluorescent protein (GFP). Application of in vivo imaging techniques allowed identification of single cells during the injections, and of the same single cells when the muscle was reexposed days or weeks later. DNA concentration by itself had little effect on fiber survival or expression efficacy, but it seemed crucial to exceed a threshold of about 10(6) injected plasmid molecules in order to obtain expression. On the other hand, experiments with coinjection of two different plasmids suggested that relatively few individual molecules were eventually transcribed in expressing cells. Plasmid DNA remained localized to the injection site, and expression was confined to nuclei in the vicinity. Expression was stable, as reporter was detected 2-61 days after injection.
