Immunocytochemical evidence of vesicular localization of the orphan transporter Rxt1 in the rat spinal cord

Rxt1, a member of the Na+/Cl- orphan transporter family, exhibits numerous features suggesting a role as plasma membrane transporter. Despite numerous attempts, its substrate has not yet been identified, although immunocytoch emical studies have shown that Rxt1 distribution generally matches that of glutamate or GABA, In order to further characterize Rxt1, its detailed immu nocytochemical distribution in the rat spinal cord and dorsal root ganglia was studied at both light microscope and ultrastructural levels. The widesp read distribution of Rxt1 in spinal cord and ganglia cannot be correlated w ith any known classical or peptidergic transmitter. Rxt1 is expressed in a subpopulation of glutamatergic primary afferent fibers, in large and medium -sized ganglion cells, while small glutamate cells exhibit generally no Rxt 1-like immunoreactivity. In the spinal cord, Rxt1-immunoreactive cell body distribution is quite ubiquitous since Rxt1 is expressed in all laminae in various neuronal types like interneurons, some projection neurons and moton eurons. Some of these neurons are cholinergic. At the electron microscope l evel, the peroxidase labeling was never localized to the plasma membrane, b ut rather associated with different organelles including the outer membrane of small synaptic vesicles and large granular vesicles, This localization resembles that of vesicular transporters detected with the same method and suggests that Rxt1, in contrast to other Na+/Cl- transporters, is expressed on vesicles, This was confirmed using a pre-embedding silver-intensified c olloidal gold method. Indeed, most gold particles appeared to be localized into the axoplasm on synaptic vesicle accumulations; only few gold particle s were observed close to the plasma membrane. These results suggest that Rxt1, despite its molecular characteristics pred icting a plasma membrane localization, might be a vesicular transporter. (C ) 1999 IBRO. Published by Elsevier Science Ltd.