Research reportImmunoreactivity for GABA plasma membrane transporter, GAT-1, in the developing rat cerebral cortex: transient presence in the somata of neocortical and hippocampal neurons
Introduction
The γ-aminobutyric acid (GABA) uptake mechanism plays an important role in the regulation of the magnitude and duration of GABA's synaptic action 9, 19, 21, 26. The GABA uptake system may also transport GABA into the extracellular space in a Ca2+-independent, nonvesicular manner 1, 3, 28, 31. The GABA transporters have been cloned and classified into 4 members, GAT-1, GAT-2, GAT-3 and GAT-4, by differential amino acid sequences and pharmacological properties 9, 15, 21, 25, 32, 58, 70. GAT-1 displays pharmacological features typical of a `neuronal transporter': its GABA uptake is strongly inhibited by cis-3-aminocyclohexane carboxylic acid (ACHC) but not by β-alanine. GAT-2 and GAT-3 exhibit pharmacological properties often associated with `glial transporters' whose GABA uptake is inhibited by β-alanine but not ACHC. GABA uptake by GAT-4 is inhibited by neither agent 9, 15, 19, 26, 32. Antibodies directed against these proteins have been applied to study their tissue distribution, and GAT-1 and GAT-3 have been shown to be brain-specific 25, 38, 42, 43, 58.
GABA plays an important role in regulating the activity of principal neurons in the cerebral cortex 27, 29, 48. GABAergic neurons have been well characterized in the neocortex and the hippocampal formation using immunocytochemistry to visualize GABA, and its synthetic enzyme, glutamic acid decarboxylase (GAD) 22, 23, 44, 51, 55, 67. Recent studies indicate that the major GABA transporter in the adult cerebral cortex is GAT-1 which is localized mainly to axon terminals and astroglia 38, 42, 43, 58. We have recently shown that immunolabeling for both GAT-1 and GAT-3 in the adult rat hippocampal formation is detected in glial cells, but only GAT-1 immunoreactivity is found in neurons[47]. Although it was not possible to detect GAT-1 in neuronal somata at the light microscopic level, the Golgi complex of some interneurons displayed sparse immunolabeling at the electron microscopic level [47].
GABA is one of the earliest expressed neurotransmitters in the mammalian cerebral cortex during development. GABA-synthesizing or -containing neurons are present prenatally in rodents and other species 14, 16, 17, 18, 30, 33, 35, 49, 52, 60, 61, 62, 64, 65. GABA-accumulating activity is also detected in early developmental stages in the cerebral cortex 14, 63. Additionally, neighboring cells differ considerably in their GABA uptake capability, but strong and light GABA-accumulating cells do not show differences in their positions, orientations, or fine structure [14]. Moreover, it has been suggested that GABA plays a neurotrophic role during development 4, 14, 20, 27, 30, 37, 40, 56, 60, 61, 65. However, little is known about the GABA transport mechanism used by developing GABA-synthesizing neurons. To obtain a better understanding of the role of the GABAergic system in the developing cerebral cortex, it is necessary to characterize the cellular localization of GABA transporter molecules during cortical development. Therefore, we studied the developmental localization of GAT-1 in the neocortex and hippocampal formation of rats from the day of birth to the adult stage with immunocytochemistry.
Section snippets
Animals
The experiments were performed on male and female albino rats (Sprague-Dawley) at different postnatal ages. The day of birth was referred to as postnatal day 0 (P0) and the time points examined were at P0, P2, P5, P10, P20, P30, P45, P60, and adult stage (around P90). In each of the above groups three or four animals were investigated.
Tissue preparation
The animals were deeply anesthetized with sodium pentobarbital and perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4%
Results
GAT-1 immunolabeling was found in the neocortex and hippocampal formation in all brains examined. The immunostaining in the newborns and the 3 oldest groups was solely associated with neuropil, whereas for the other age groups cell bodies were also labeled. In addition, the pattern of the labeled neuropil and the immunoreactivity of the somata exhibited age-dependent changes. Furthermore, the pattern of the GAT-1 immunolabeling did not exhibit any notable areal difference across the
Discussion
The present study examined the postnatal development of GAT-1 immunoreactivity in the neocortex and hippocampal formation of the rat. One of the major findings of this study is that GAT-1 is strongly expressed in cortical and hippocampal neuronal somata during development. Another interesting observation is that the immunostaining for GAT-1 in layers I and VIb of the neocortex and the supragranular band in the dentate gyrus displays a dramatic developmental regression. In the following
Acknowledgements
This study was supported by the National Science Foundation (IBN 9422392) and National Institute of Health (NS 15669). We thank Marian Shiba-Noz for her excellent technical assistance and Drs. Richard T. Robertson and John J. Hablitz for their constructive comments on the manuscript.
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