Ontogeny of Rxt1, a vesicular "orphan" Na(+)/Cl(-)-dependent transporter, in the rat.

The developmental expression of the orphan Na(+)/Cl(-)-dependent transporter, Rxt1, was studied in the rat using a specific [(35)S]complementary RNA probe and affinity purified antibodies. Western blotting experiments allowed the detection of Rxt1 in brain as early as on embryonic day 16. After birth, the brain levels of Rxt1 increased dramatically up to a maximum around postnatal day 30 and then decreased slightly to the adult value. In situ hybridization experiments allowed the earliest detection of Rxt1 messenger RNA in the brain and spinal cord at embryonic day 14. In embryonic day 18 embryos, Rxt1 messenger RNA was present not only in the nervous system but also in the pituitary, the thymus and the heart. Immunoautoradiograms of whole embryo at embryonic days 16 and 18 showed high amounts of the Rxt1 protein in the spinal cord and brain. Moreover, at embryonic day 18, the orphan transporter was expressed in the thymus, heart and liver. At these ages, Rxt1 immunolabeling was localized in neurons of the subplate and in the ventricular zone of the brain. During early postnatal stages, Rxt1 messenger RNA expression demonstrated dynamic and complex changes until postnatal day 13. In particular, this transcript was relatively abundant in the striatum at postnatal days 3 and 5 and then decreased to very low levels after postnatal day 10. At the same period, Rxt1 immunostaining in the hippocampus and the cerebral cortex was observed all over the gray matter, in cell bodies as well as in the neuropil. Finally, the adult pattern was reached around postnatal day 13 for Rxt1 messenger RNA, but only at postnatal day 20 for the Rxt1 protein. The presence of Rxt1 messenger RNA and protein at embryonic stages and the high expression of the protein during synaptogenesis suggest that this vesicular "orphan" transporter is involved in the brain maturation process.

The two orphan Na+/Cl(-)-dependent transporters Rxt1 and V-7-3-2 have an overlapping expression pattern in the rat central nervous system.

The messenger RNA expression patterns of two orphan Na+/Cl(-)-dependent transporters, Rxt1 and V-7-3-2, were compared in the adult rat CNS by in situ hybridization histochemistry. The sites of synthesis of both orphan transporters were found to largely overlap. The highest concentrations of Rxt1 and V-7-3-2 mRNAs were present in the olfactory bulb, cerebral cortex, hippocampus, habenular and pontine nuclei, and cerebellum. Low levels of orphan transporters mRNAs were observed in the basal ganglia (caudate-putamen, nucleus accumbens, globus pallidus), septum nuclei, substantia nigra, colliculi and brainstem. Only few regions were found to express only one of these two orphan transporters: Rxt1 mRNA in the thalamic area and the hilus of the dentate gyrus. V-7-3-2 mRNA in motoneurons of the most ventral zone of the spinal cord. Rxt1 mRNA was generally more abundant than V-7-3-2 mRNA, except in few areas (piriform cortex, horizontal limb of the diagonal band, medial habenular nucleus, pyramidal and granular cell layers in the hippocampus) where the levels of both mRNAs were similar. The overall parallelism between both distributions was further confirmed at the cellular level on emulsion-coated sections, especially in the hippocampus where pyramidal and granular neurons very probably synthesize both Rxt1 and V-7-3-2. These data provide a further demonstration of the synthesis of the orphan transporters Rxt1 and V-7-3-2 exclusively in neurons whose locations suggest that they might correspond to glutamatergic neurons and subsets of GABAergic neurons.

Immunolabeling of the Na+/Cl(-)-dependent "orphan" transporter Rxt1 in the rat central nervous system.

Previous studies have shown that the mRNA encoding the Na+/Cl(-)-dependent "orphan" transporter Rxt1 is expressed exclusively in the central nervous system (CNS). In the present study, specific antibodies were raised in rabbits for the detailed mapping of this transporter in the rat. The C-terminal part of Rxt1 was fused with glutathione-S-transferase (Rxt1ct-GST) and the resulting fusion protein was used as antigen. The specificity of the antiserum toward Rxt1 was confirmed by immunofluorescent, Western blot, and immunoautoradiographic experiments. In cerebral cortex membranes, Rxt1-like material recognized by the antiserum is a glycosylated protein of 97-116 kDa. This protein was the most abundant in the caudate-putamen, followed, in decreasing order, by the cerebral cortex approximately hippocampus > cerebellum > brainstem > spinal cord. In contrast, no immunoreactive material could be detected in peripheral tissues (tongue, thymus, heart, lung, spleen, kidney, adrenals, liver, skeletal muscle, intestine, testis). Immunoautoradiographic labeling with affinity-purified anti-Rxt1ct-GST antibodies showed high levels of Rxt1-like material in the olfactory bulb, cerebral cortex, striatal complex, hippocampal formation, superior layer of the anterior colliculus, cortex, and deep nuclei in the cerebellum. The regional distribution of Rxt1-like material generally matched that of GABAergic and glutamatergic projections in agreement with previous in situ hybridization data.

The "orphan" Na+/Cl(-)-dependent transporter, Rxt1, is primarily localized within nerve endings of cortical origin in the rat striatum.

Previous studies have shown that the striatum expresses very low levels of Na+/Cl(-)-dependent "orphan" transporter Rxt1 transcripts but contains high levels of protein. This study investigated the origin of Rxt1 expression in rat striatum. Striatal Rxt1 contents assessed by immunocytochemistry or western blotting were found to be significantly reduced after corticostriatal denervation but not after striatal or thalamic lesion with kainic acid or selective 6-hydroxydopamine-induced nigrostriatal deafferentation. Corticostriatal neurons retrogradely labeled by intrastriatal fluorogold injections were shown to express Rxt1 mRNA. Combination of anterograde biotin-dextran amine labeling of the corticostriatal pathway with Rxt1 immunogold detection at the ultrastructural level demonstrated the presence of Rxt1 in about one-third of the corticostriatal synaptic terminals and in numerous unidentified synaptic terminals. All the Rxt1-positive terminals formed asymmetrical contacts on spines. These data provide evidence that striatal Rxt1 immunoreactivity is mainly of extrinsic origin and more specifically associated with the corticostriatal pathway. Rxt1 appears as a selective presynaptic marker of synapses formed by presumably excitatory amino acid afferents, but it segregates a subclass of these synapses, thereby revealing a functional heterogeneity among excitatory amino acid systems.

Characterization and distribution of Hxt1, a Na(+)/Cl(-)-dependent orphan transporter, in the human brain.

Rxt1, a transporter-like protein structurally related to the large family of Na(+)/Cl(-)-dependent carriers, was isolated from the rat brain. In the present study, Hxt1, the homologue of Rxt1, was isolated from human cortex cDNA. Comparison of their respective nucleotidic sequences revealed a 96% conservation between Hxt1 and Rxt1. Genetic mapping with human genome radiation hybrids allowed the location of the gene coding for Hxt1 between 323ya5 and 084xb3 AFM markers, on a portion of chromosome 1p which spans over 7 cM or 118 cRay. Northern blot analyses demonstrated that Hxt1 mRNA ( approximately 7.5 Kb) is expressed in the human brain but not in peripheral tissues. The immunodistribution of Hxt1 was determined with antibodies raised against the C-terminus of Rxt1. Hxt1 is concentrated in the cerebral cortex, caudate-putamen, substantia nigra, hippocampus, and cerebellum, appearing as a diffuse or a punctate labeling at the light microscope level. This regional and cellular distribution suggests that Hxt1, as its rat homologue, could be present in axon terminals of glutamatergic neurons. The high pressure of selection exerted upon this protein, its strategic anatomical and subcellular distributions suggest that this orphan transporter could be involved in critical functions in the central nervous system.

Unexpected localization of the Na+/Cl--dependent-like orphan transporter, Rxt1, on synaptic vesicles in the rat central nervous system.

Numerous features of its primary structure demonstrate that the orphan transporter Rxt1 belongs to the Na+/Cl--dependent neurotransmitter plasma membrane transporter superfamily, which includes the dopamine, norepinephrine, serotonin and gamma-aminobutyric acid (GABA) transporters. Initial immunocytochemical investigations with affinity-purified antibodies have established that Rxt1 is localized, almost exclusively, in axon terminals of glutamatergic neurons and subsets of GABAergic neurons in the CNS. Further studies were carried out to determine its subcellular distribution. In a first series of experiments, PC-12 cells were transfected with plasmids encoding either the dopamine transporter or Rxt1. Immunofluorescence experiments showed that the dopamine transporter was expressed in these cells, and, as expected, addressed to their plasma membrane. Surprisingly, this was never the case with Rxt1, which was targeted to the same subcellular compartment as synaptophysin, a vesicular protein. In a second set of experiments, subcellular fractionation of rat striatum showed that Rxt1, but not the dopamine transporter, was relatively abundant in the purified synaptic vesicle fraction. Finally, electron microscopic immunocytochemistry with anti-Rxt1 antibodies showed peroxidase as well as pre- and post-embedding immunogold labelling confined to the intracellular compartment in various brain regions. Moreover, quantitative analysis of post-embedding experiments demonstrated that the immunogold particles corresponding to Rxt1 immunoreactivity were mostly localized to small synaptic vesicles. These data indicate that, in contrast with the other members of the Na+/Cl--dependent neurotransmitter transporter superfamily, which are targeted to the plasma membrane, Rxt1 is distributed as a vesicular protein in the CNS.