Neuronal, glial, and epithelial localization of gamma-aminobutyric acid transporter 2, a high-affinity gamma-aminobutyric acid plasma membrane transporter, in the cerebral cortex and neighboring structures.

Neuronal and glial high-affinity Na+/Cl(-)-dependent plasma membrane gamma-aminobutyric acid (GABA) transporters (GATs) contribute to regulating neuronal function. We investigated in the cerebral cortex and neighboring regions of adult rats the distribution and cellular localization of the GABA transporter GAT-2 by immunocytochemistry with affinity-purified polyclonal antibodies that react monospecifically with a protein of 82 kDa. Conventional and confocal laser-scanning light microscopic studies revealed intense GAT-2 immunoreactivity (ir) in the leptomeninges, choroid plexus, and ependyma. Weak GAT-2 immunoreactivity also was observed in the cortical parenchyma, where it was localized to puncta of different sizes scattered throughout the radial extension of the neocortex and to few cell bodies. In sections double-labeled with GAT-2 and glial fibrillary acidic protein (GFAP) antibodies, some GAT-2-positive profiles also were GFAP positive. Ultrastructural studies showed GAT-2 immunoreactivity mostly in patches of varying sizes scattered in the cytoplasm of neuronal and nonneuronal elements: GAT-2-positive neuronal elements included perikarya, dendrites, and axon terminals forming both symmetric and asymmetric synapses; nonneuronal elements expressing GAT-2 were cells forming the pia and arachnoid mater; astrocytic processes, including glia limitans and perivascular end feet; ependymal cells; and epithelial cells of the choroid plexuses. The widespread cellular expression of GAT-2 suggests that it may have several functional roles in the overall regulation of GABA levels in the brain.

GAT-3, a high-affinity GABA plasma membrane transporter, is localized to astrocytic processes, and it is not confined to the vicinity of GABAergic synapses in the cerebral cortex.

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.

Type I collagen CNBr peptides: species and behavior in solution.

The properties of type I collagen CNBr peptides in solution were studied to investigate the molecular species formed, their conformation, and factors influencing equilibria between peptide species. Peptides formed homologous trimers, even though the native parent protein is heterotrimeric, [alpha 1(I)]2 alpha 2-(I). Their triple-helical content was found to be high (> 75% for most peptides). Full helical content was not reached mainly because of the presence of monomer species; chain misalignment, if present, and trimer unraveling at terminal ends appeared to play a minor role in reducing helicity. Circular dichroism spectra and resistance to trypsin digestion at 4 and 20 degrees C demonstrated that the conformation of trimers was very similar to the collagen triple-helical conformation. Rotary shadowing of peptide alpha 1(I) CB7 supported this finding. Analytical gel filtration in nondenaturing conditions showed that the trimers of some peptides have the ability to autoaggregate. In the case of peptides alpha 1(I) CB8 and alpha 2(I) CB4, most of the intermolecular interactions between trimeric molecules were disrupted by 0.5 M NaCl, demonstrating that their ionic character is important. Changes in ionic strength also altered the hydrodynamic size of single- and triple-stranded molecules. The different molecular species are in equilibrium. The kinetics of the conversion of trimer to monomer species was determined in a time course experiment using trypsin digestion and found to be a relatively slow process (trimer half-life is a few days at 4 degrees C, about one order of magnitude lower at 20 degrees C) with an activation energy of roughly 4-9 kcal/mol. The circular dichroism profile at increasing temperatures showed that the melting temperature for triple-helical peptides is about 6-10 degrees C lower than that of the parent native type I collagen. The folding of peptides is a spontaneous process (exothermic but with unfavourable entropy change), and the triple-helical conformation originates solely as the result of the collagen sequence because it forms from heat-denatured samples.

Fine structure of isolated fibrils from the cuticle of Lumbricus sp.

A method has been developed to isolate and purify cuticular fibrils of Lumbricus. Polarizing microscopy confirms the collagenous nature of the isolated fibrils. Study in the electron microscope of isolated fibrils, negatively or positively stained, shows that they are cylindrical, unbranching and without periodic structure. Enzymatic treatment of cuticles with alpha-amylase and trypsin results in a more or less complete dissociation of the fibrils which appear clearly to be made up of helically wound bundles of filaments (30-40 A). The biophysical data and compared to the ultrastructural organization of other periodically cross-banded fibrils.

[Cuticular collagen in Lumbricus sp. Ultrastructure of native fibrils and of precipitates obtained from acetic solutions]. / Le collagène cuticulaire chez Lumbricus sp. Ultrastructure des fibrilles natives. Et des precipités obtenus des solutions acétiques.

The paper describes the techniques used to isolate the cuticular collagen fibrils of Lumbricus sp. As revealed by electron microscope examination the isolated fibrils, positively and negatively stained, are aperiodic and appear made up of bundles of filaments (30 A) with an helicoidal arrangement. A filamentous network has been precipitated from the acetic solutions of cuticular collagen. The network exhibits an electrondense cross-striation every 3 000 A.