No abstract

High affinity, GABA plasma membrane transporters influence the action of GABA, the main inhibitory neurotransmitter. The cellular expression of GAT-1, a prominent GABA transporter, has been investigated in the cerebral cortex of adult rats using in situ hybridization with 35S-labeled RNA probes and immunocytochemistry with affinity purified polyclonal antibodies directed to the C-terminus of rat GAT-1. GAT-1 mRNA was observed in numerous neurons and in some glial cells. Double-labeling experiments were performed to compare the pattern of GAT-1 mRNA containing and GAD67 immunoreactive cells. The majority of neurons expressing GAT-1 mRNA also contained GAD67 immunoreactivity (ir), but GAT-1 mRNA was also observed in a few pyramidal neurons. GAT-1-ir was localized to numerous puncta and fibers and to astrocytic processes, was not observed in sections incubated in GAT-1 antibodies preadsorbed with rat GAT-1 C-terminal peptide, and was observed in sections incubated in GAT-1 antibodies preadsorbed with the C-terminal portion of the related peptides rat GAT-3(607-627) or rat glycine transporter-1(625-633). The highest number of GAT-1-ir puncta was in layer IV, followed by layers II-III. GAT-1 positive puncta appeared to have a preferential relationship to the soma and proximal dendrites of unlabeled pyramidal cells. All GAT-1 positive axon terminals formed symmetric synapses. This study demonstrates that (1) GAT-1 is expressed by both neurons and astrocytes, (2) the majority of GAT-1 expressing neurons contain GAD67, and (3) GAT-1 uptake system is more extensive than the GABA synthetizing system. These observations support the hypothesis that, in addition to its role in terminating GABA action by uptake into GABAergic axon terminals, GAT-1 influences both excitatory and inhibitory transmission by modulating the "paracrine" spread of GABA (Isaacson et al., 1993), and suggest that astrocytes may play an important role in this process.

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.

High-affinity uptake of glutamate from the synaptic cleft plays a crucial role in regulating neuronal activity in physiological and pathological conditions. We have used affinity-purified specific polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal region of rabbit and rat EAAC1, a glutamate (Glu) transporter believed to be exclusively neuronal, to investigate its cellular and subcellular localization and whether it is expressed exclusively in glutamatergic cells of infragranular layers, as suggested by previous studies. Light microscopic studies revealed that EAAC1 immunoreactivity (ir) is localized to neurons and punctate elements in the neuropil. EAAC1-positive neurons were more numerous in layers II-III and V-VI, i.e. throughout all projection layers. Most EAAC1-positive neurons were pyramidal, although nonpyramidal cells were also observed. Some EAAC1-positive non-pyramidal neurons stained positively with an antiserum to GAD, thus demonstrating that EAAC1 is not confined to glutamatergic neurons. Non-neuronal EAAC1-positive cells were also observed in the white matter, and some of them stained positively with an antiserum to GFAP. Ultrastructural studies showed that EAAC1-ir was in neuronal cell bodies, dendrites and dendritic spines, but not in axon terminals, i.e. exclusively postsynaptic. Analysis of the type of axon terminals synapsing on EAAC1-ir profiles showed that 97% of them formed asymmetric contacts, thus indicating that EAAC1 is located at the very sites of excitatory amino acid release. Unexpectedly, EAAC1-ir was also found in a few astrocytic processes located in both the gray and the white matter. The localization of EAAC1 may explain the pathological symptoms that follow EAAC knockout (seizures and mild toxicity), as seizures could be due to the loss of EAAC1-mediated fine regulation of neuronal excitability at axodendritic and axospinous synapses, whereas the mild toxicity may be related to the functional inactivation of astrocytic EAAC1.

No abstract

High-affinity gamma-aminobutyric (GABA) plasma membrane transporters (GATs) influence the action of GABA, the main inhibitory neurotransmitter in the human cerebral cortex. In this study, the cellular expression of GAT-1, the main cortical GABA transporter, was investigated in the human cerebral cortex by using immunocytochemistry with affinity-purified polyclonal antibodies directed to the C-terminus of rat GAT-1. In temporal and prefrontal association cortex (Brodmann's areas 21 and 46) and in cingulofrontal transition cortex (area 32), specific GAT-1 immunoreactivity (ir) was localized to numerous puncta and fibers in all cortical layers. GAT-1+ puncta were distributed homogeneously in all cortical layers, although they were slightly more numerous in layers II-IV, and appeared to have a preferential relationship to the somata and proximal dendrites of unlabeled pyramidal cells, even though, in many cases, they were also observed around nonpyramidal cells. Electron microscopic observations showed that GAT-1+ puncta were axon terminals that formed exclusively symmetric synapses. In addition, some distal astrocytic processes also contained immunoreaction product. Analysis of the patterns of GAT-1 labeling in temporal and prefrontal association areas (21 and 46), in cingulofrontal transition areas (32), and in somatic sensory and motor areas (1 and 4) of the monkey cortex revealed that its distribution varies according to the type of cortex examined and indicated that the distribution of GAT-1 is similar in anatomically corresponding areas of different species. The present study demonstrates that, in the human homotypical cortex, GAT-1 is expressed by both inhibitory axon terminals and astrocytic processes. This localization of GAT-1 is compatible with a major role for this transporter in GABA uptake at GABAergic synapses and suggests that GAT-1 may contribute to determining GABA levels in the extracellular space.

No abstract

Light and electron microscopic immunocytochemical techniques and Western blotting were used to investigate the postnatal development of the vesicular GABA transporter (VGAT) in the rat somatic sensory cortex. VGAT immunoreactivity was low at birth, it increased gradually through the first and second weeks of life and achieved the adult pattern during the third week. At postnatal day (P)0-P5, VGAT immunoreactivity was associated exclusively to fibers and puncta. Electron microscopic studies performed at P5 showed that all identified synaptic contacts formed by VGAT-positive axonal swellings were of the symmetric type and that a substantial proportion of the boutons appeared not to have formed synapses. From P10 onward, labeled puncta were both scattered in the neuropil and in apposition to unstained cellular profiles; VGAT was also expressed in few GABAergic cell bodies. Western blottings at the same postnatal ages revealed a 55-kDa band whose intensity was weak at P0 (17% of adult), it increased constantly until P15 (P2: 35%; P5: 44%; P10: 68%; P15: 97%), and then leveled off. Overall, the present results show that during neocortical development the expression of VGAT slightly precedes the complete maturation of inhibitory synaptogenesis and suggest that it may contribute to the formation of neocortical GABAergic circuitry.