Immunocytochemical localization of gamma-aminobutyric acid plasma membrane transporters in the tiger salamander retina.

Abstract

Gamma-aminobutyric acid (GABA) plasma membrane transporters (GATs) play an important role in regulating GABA neurotransmission in the nervous system. The distribution of two GATs, GAT 1 and GAT 3, in salamander retina was investigated by using affinity-purified polyclonal antisera directed to the predicted C-terminals of rat GAT 1 and rat GAT 3. GAT 1-immunoreactivity (-IR) was found in type IB and IIB orthotopic bipolar cells (BCs) located in the distal and middle of the inner nuclear layer (INL), respectively; in type IIA and IA amacrine cells (ACs) located in the middle and proximal INL, respectively; and in interplexiform cells and cells in the ganglion cell layer (GCL). No detectable staining was found in horizontal cells (HCs) or in structures resembling Müller cells. GAT 1-immunoreactive fibers were present in the outer plexiform layer (OPL) and inner plexiform layer (IPL) in three bands corresponding to the three bands previously reported to be GABA-IR. GAT 3 antibodies labeled fewer cells and cell types than GAT 1 antibodies. GAT 3-IR was localized to type IIA and IA ACs and cells in the GCL, but not to BCs, HCs, or Müller cell-like structures. There was weak labeling of the OPL and stronger labeling of the IPL, with three distinct bands at the same depth as observed with GAT 1-IR. Double-labeling showed that the majority of GAT 1-IR BCs (88%), ACs (88%), and cells in the GCL (78%) colocalized with GABA-IR. The present study provides the first direct evidence of the expression of two GAT subtypes in neurons of nonmammalian retinas. These transporters could regulate GABA neurotransmission by reuptake and termination of GABA's action and, perhaps, by GABA release mechanisms. The presence of GAT 1-IR/GABA-IR bipolar cells further supports our earlier observations that a subgroup of orthotopic bipolar cells are likely to be GABAergic.