There are few neurochemical markers that reliably identify retinal ganglion cells (RGCs), which are a heterogeneous population of cells that integrate and transmit the visual signal from the retina to the central visual nuclei. We have developed and characterized a new set of affinity purified guinea pig and rabbit antibodies against RNA-binding protein with multiple splicing (RBPMS). On Western blots these antibodies recognize a single band at ~24 kDa, corresponding to RBPMS, and they strongly label RGC and displaced RGC (dRGC) somata in mouse, rat, guinea pig, rabbit and monkey retina. RBPMS immunoreactive cells and RGCs identified by other techniques have a similar range of somal diameters and areas. The density of RBPMS cells in mouse and rat retina is comparable to earlier semi-quantitative estimates of RGCs. RBPMS is mainly expressed in medium and large DAPI-, DRAQ5-, NeuroTrace- and NeuN-stained cells in the ganglion cell layer (GCL), and RBPMS is not expressed in syntaxin (HPC-1) immunoreactive cells in the inner nuclear layer (INL) and GCL, consistent with their identity as RGCs, and not displaced amacrine cells. In mouse and rat retina, most RBPMS cells are lost following optic nerve crush or transection at three weeks, and all Brn3a, SMI-32 and melanopsin immunoreactive RGCs also express RBPMS immunoreactivity. RBPMS immunoreactivity is localized to CFP-fluorescent RGCs in the B6.Cg-Tg(Thy1-CFP)23Jrs/J mouse line. These findings show that antibodies against RBPMS are robust reagents that exclusively identify RGCs and dRGCs in multiple mammalian species, and they will be especially useful for quantification of RGCs.

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Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the ,. opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR. Antibody specificity was confirmed by the positive staining ofhuman embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the 6 or ic receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment. Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons. Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR-IR was present in vesiclelike structures, which were identified as endosomes by confocal microscopy. At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles. MOR-IR was still internalized at 120 min. Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.

The termination of GABA synaptic action by high-affinity, Na(+)-dependent, neuronal, and glial plasma membrane transporters plays an important role in regulating neuronal activity in physiological and pathological conditions. We have investigated the cellular localization and distribution in the cerebral cortex of adult rats of one GABA transporter (GAT), GAT-3, by immunocytochemistry with affinity-purified polyclonal antibodies directed to its predicted C terminus that react monospecifically with a protein of approximately 70 kDa. Light microscopic studies revealed specific GAT-3 immunoreactivity (ir) in small punctate structures, and it was never observed in fibers or cell bodies. No changes in immunostaining were observed in sections incubated with GAT-3 antibodies preadsorbed with the related rat GAT-1 or mouse GAT-2/ BGT-1 C-terminal peptides, whereas in sections incubated with GAT-3 antibodies preadsorbed with rat GAT-3 C-terminal peptide, ir was not present. The highest number of GAT-3-positive puncta was in layer IV and in a narrow band corresponding to layer Vb, followed by layers II and III. Many GAT-3-positive puncta were in close association with pyramidal and nonpyramidal neuron cell bodies. Ultrastructural studies showed that GAT-3 ir was localized exclusively to astrocytic processes, which were found in the neuropil and adjacent to axon terminals having either symmetric or asymmetric specializations. In sections processed by both preembedding labeling for GAT-3 and postembedding immunogold labeling for GABA, only some of the GAT-3-positive astrocytic processes were found close to GABAergic profiles. These findings on the localization of GAT-3 in the cerebral cortex indicate that this transporter mediates GABA uptake into glial cells, and suggest that glial GABA uptake may function to limit the spread of GABA from the synapse, as well as to regulate overall GABA levels in the neuropil.