Glutamate transporter 1-expressing glia in the rat substantia nigra-Morphometric analysis and relationships to synapses.

Glial cells have a major role in protecting neurons against various forms of stress. Especially, astrocytes mediate the bulk of glutamate clearance in the brain via specific membrane transporters (GLAST and GLT1), thereby preventing the occurrence of excitotoxic events. Although glutamate-mediated mechanisms are thought to contribute to nigral dopaminergic neuron degeneration in Parkinson's disease, detailed information on the organization of glia in the substantia nigra is still lacking. The present study was performed to provide quantitative information on the organization of astroglia and on the relationships between astrocytes and excitatory synapses in the rat substantia nigra. Using immunolabeling of GLT1 and confocal imaging, we found that the substantia nigra was filled with a dense meshwork of immunoreactive astrocyte processes. Stereological analysis performed on electron microscope images revealed that the density of immunoreactive astrocyte plasma membranes was substantial, close to 1 µm2 /µm3 , in the substantia nigra neuropil, both in the pars compacta and the pars reticulata. Excitatory synapses had on average two thirds of their perimeters free from glia, a disposition that may favor transmitter spillover. The density of glutamatergic synapses, as quantified on confocal images by the simultaneous detection of bassoon and of vesicular glutamate transporter 1 or 2, was very low (0.01 and 0.025 per µm3 in the reticulata and compacta subdivisions, respectively). Thus the ratio of GLT1-expressing glial membrane surface to glutamatergic synapses was very high (40-100 µm2 ), suggesting an efficient regulation of extracellular glutamate concentrations.

Synaptic localization of GLT-1a in the rat somatic sensory cortex.

GLT-1a, the major glutamate transporter, plays an important role in both physiological and pathological conditions. Uncertainty regarding its localization in the cerebral cortex prompted us to re-examine its cellular and subcellular localization in the rat somatic sensory cortex. GLT-1a detection was sensitive to fixation; in optimal conditions approximately 25% of GLT-1a+ profiles were axon terminals. GLT-1a/VGLUT1 double-labeling and pre-embedding electron microscopy studies showed that approximately 50% of GLT-1a+ profiles were in the vicinity of asymmetric synapses. Using pre-embedding electron microscopy, we found that approximately 70% of GLT-1a located in the vicinity of asymmetric synapses was astrocytic and approximately 30% was neuronal. Post-embedding immunogold studies showed that the density of gold particles coding for GLT-1a was much higher in astrocytic processes than in axon terminals, and that in the latter they were never at the active zone. In both astrocytic processes and axon terminals most gold particles were localized in a membrane region extending for about 250 nm from active zone margin, with a peak at 140 nm for astrocytic processes and at 80 for axon terminals. We conclude that, although GLT-1a is expressed by both astrocytes and axon terminals, astrocytic GLT-1a predominates at asymmetric synapses, and that the perisynaptic localization of GLT-1a in cortex is well-suited to modulate Glu concentrations at the cleft and also to restrict Glu spillover.

Glial glutamate transporters and maturation of the mouse somatosensory cortex.

In the adult nervous system, glutamatergic neurotransmission is tightly controlled by neuron-glia interactions through glial glutamate reuptake by the specific transporters GLT-1 and GLAST. Here, we have explored the role of these transporters in the structural and functional maturation of the somatosensory cortex of the mouse. We provide evidence that GLT-1 and GLAST are early and selectively expressed in barrels from P5 to P10. Confocal and electron microscopy confirm that the expression is restricted to the astroglial membrane. By P12, and despite an increased global expression as observed by immunoblotting, the barrel pattern of GLAST and GLT-1 staining is no longer evident. In P10 GLT-1 -/- and GLAST -/- mice, the cytoarchitectural segregation of the barrels is preserved. However, at P9-10, the functional response to whisker stimulation, measured by deoxyglucose uptake, is markedly decreased in GLT-1 -/- and GLAST -/- mice. The role of GLAST is transient since the metabolic response is already restored at P11-12 in GLAST -/- mice and remains unchanged in adulthood. However, deletion of GLT-1 seems to impair the functional metabolic response until adulthood. Our data suggest that astrocyte-neuron interactions via the glial glutamate transporters are involved in the functional maturation of the whisker representation in the somatosensory cortex.

Similar perisynaptic glial localization for the Na+,K+-ATPase alpha 2 subunit and the glutamate transporters GLAST and GLT-1 in the rat somatosensory cortex.

Several isoenzymes of the Na(+),K(+)-ATPase are expressed in brain but their specific roles are poorly understood. Recently, it was suggested that an isoenzyme of the Na(+),K(+)-ATPase containing the alpha(2) subunit, together with the glutamate transporters GLAST and GLT-1, participate in a coupling mechanism between neuronal activity and energy metabolism taking place in astrocytes. To substantiate this hypothesis, we compared the distribution of alpha(2), GLAST and/or GLT-1 in the rat cerebral cortex using double immunofluorescence and confocal microscopy, and immunocytochemistry at the electron microscopic level. We also investigated the relationship between alpha(2), GLAST or GLT-1 and asymmetrical synaptic junctions (largely glutamatergic) and GABAergic nerve terminals. Results show that the alpha(2) subunit has an exclusive astroglial localization, and that it is almost completely co-distributed with GLAST and GLT-1 when evaluated by confocal microscopy. This similar distribution was confirmed at the ultrastructural level, which further showed that the vast majority of the alpha(2) staining (73% of all labelled elements), like that of GLAST and GLT-1, was located in glial leaflets surrounding dendritic spines and the dendritic and/or axonal elements of asymmetrical (glutamatergic) axo-dendritic synapses. Synapses ensheathed by alpha(2), GLAST or GLT-1 virtually never included (