Localization of VGLUT3, the vesicular glutamate transporter type 3, in the rat brain.

We have recently identified a third subtype of glutamate vesicular transporter (VGLUT) named VGLUT3. In the present study, we provide a detailed account of the regional and cellular distributions of VGLUT3 in the rat brain, using specific nucleotide probes and antisera. The distribution of VGLUT3 protein was compared with that of the other vesicular transporters (VGLUT1 and VGLUT2). All the areas expressing VGLUT3 also contain high levels of VGLUT1 and -2 proteins, but, at a finer level of analysis, the distribution of the three subtypes differs. Unlike VGLUT1 and -2, VGLUT3 expression is limited to discrete cell populations. Neurons containing VGLUT3 transcript are essentially observed in the caudate-putamen, the olfactory tubercle, the nucleus accumbens, the hippocampus, the interpeduncular nucleus and the dorsal and medial raphe nuclei. More scattered populations of VGLUT3 expressing neurons are found in the cerebral cortex. The distribution of the VGLUT3 protein, as determined with specific antisera, overlaps with that of the transcript in the caudate-putamen, olfactory tubercles, hippocampus, cortex, interpeduncular nucleus, and raphe nuclei, suggesting that VGLUT3 is essentially present in local projection neurons in these regions. Microscopic examination reveals staining of terminals and perikarya. Furthermore, co-localization studies indicate that VGLUT3 is present in GABAergic interneurons in the hippocampus, as well as in the interpeduncular nucleus. However, other regions, such as the substantia nigra (pars compacta), the ventral tegmental area, and the parabigeminal nucleus, receive a dense VGLUT3 terminal labeling although they do not contain VGLUT3 expressing neurons. In these regions, VGLUT3 immunoreactivity may be present in terminals of long projecting neurons. This subclass of glutamatergic afferents differs from other "classical" excitatory terminals that express VGLUT1 or VGLUT2. The distribution of VGLUT3 in the rat brain suggests an unsuspected function of vesicular glutamate transport in subsets of interneurons and in neuromodulatory neurons.

Tetanus neurotoxin-insensitive vesicle-associated membrane protein localizes to a presynaptic membrane compartment in selected terminal subsets of the rat brain.

Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicular soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE) that has been implicated in neurite outgrowth. It has previously been reported that TI-VAMP is localised in the somatodendritic compartment of neurons indicating a role in membrane fusion events within dendrites. Using a newly produced monoclonal antibody to TI-VAMP that improves signal/noise immunodetection, we report that TI-VAMP is also present in subsets of axon terminals of the adult rat brain. Four distinctive populations of labelled axon terminals were identified: 1) the hippocampal mossy fibres of the dentate gyrus and of CA3, 2) the striatal peridendritic terminal plexuses in the globus pallidus (GP), substantia nigra pars reticulata (SNr), 3) peridendritic plexuses in the central nucleus of the amygdala, and 4) the primary sensory afferents in the dorsal horn of the spinal cord. The presynaptic localisation of TI-VAMP in these locations was demonstrated by co-localisation with synaptophysin. Ultrastructural studies showed TI-VAMP labelling over synaptic vesicles in the mossy fibres, whereas it was localised in tubulo-vesicular structures and multivesicular bodies in the pyramidal cell dendrites. The presynaptic localisation of TI-VAMP occurred by P15, so relatively late during development. In contrast, dendritic labelling was most prominent during the early post-natal period. Co-localisation with markers of neurotransmitters showed that TI-VAMP-positive terminals are GABAergic in the GP and SNr and glutamatergic in the mossy fibre system and in the dorsal root afferents. Most of these terminals are known to co-localise with neuropeptides. We found met-enkephalin-immunoreactivity in a sizeable fraction of the TI-VAMP positive terminals in the GP, amygdala, and dorsal horn, as well as in a few mossy fibre terminals. The function of TI-VAMP in subsets of mature axon terminals remains to be elucidated; it could participate in the exocytotic molecular machinery and/or be implicated in particular growth properties of the mature axon terminals. Thus, the presence of TI-VAMP in the mossy fibres may correspond to the high degree of plasticity that characterises this pathway throughout adult life.

Abnormal trafficking and subcellular localization of an N-terminally truncated serotonin transporter protein.

We report here that a truncated 5-HTT protein is produced in the neurons of the raphe, in serotonin transporter (5-HTT) knockout (KO) mice. The 5-HTT gene has exon 2 deleted and we found that one main transcript, shortened by 450 bp, is produced in these KO mice. The mutated 5-HTT protein is only recognized by antibodies against the C-terminal portion of 5-HTT. This protein is not functional as there is no high-affinity serotonin uptake in 5-HTT KO mice, in adults or during development. Conversely, low-affinity serotonin uptake was detected in vitro, and in dopaminergic neurons of the substantia nigra in vivo. The truncated 5-HTT, recognized by antibodies to the C-terminus, is present exclusively in the somatodendritic compartment of the raphe neurons instead of being exported to axons. As shown with confocal and electron microscopy, the truncated 5-HTT does not reach the plasma membrane and is essentially retained in the endoplasmic reticulum. However, this does not seem to trigger refolding or degradation responses, as no upregulation of the chaperone BiP or of the degradation signal ubiquitin was detected. Last, as observed in heterozygous mice, the presence of the truncated 5-HTT protein, although produced in large quantities, does not disturb the normal trafficking of the wild-type protein. This study therefore validates the 5-HTT KO model despite the occurrence of an incomplete translation, and brings novel information on the in vivo 5-HT uptake and cellular processing of an abnormal 5-HTT protein.

Early postnatal changes of the dopaminergic mesencephalic neurons in the weaver mutant mouse.

In adult weaver (wv) mutant mice up to 70% of the mesostriatal dopaminergic neurons are lost and major alterations of the dopaminergic dendrites of the substantia nigra have been described. We sought to determine the time of onset of these alterations. Cell counts of the main dopaminergic (DA) mesencephalic cell groups (A8, A9, A10), as labeled with tyrosine hydroxylase immunocytochemistry were done in wild-type and homozygous wv/wv pups. No loss of the DA neurons, was detectable at postnatal day 7 (P7), while reductions in substantia nigra (and retrorubral area) amounted to 35% at P14 and 47% by P21. On the other hand, the severe reduction of dopaminergic dendrites, particularly of their distal compartments was already visible from P3 on. During the first postnatal week, this was associated to abnormal clustering of the dopaminergic neurons. These early neuritic alterations were present, though to a milder degree, in heterozygous (wv/+) mice.