Localization Of GABA Receptors And Transporters In The Outerplexiform Layer Of The Goldfish Retina

Abstract

Abstract: : Purpose: Immunohistochemical techniques were used to determine the localization of key components of the GABAergic system in the outer plexiform layer (OPL) of goldfish retina: the GABA transporters (GAT) and GABA_A receptors. Methods: Goldfish retinas were fixed in paraformaldehyde 4%, incubated in polyclonal antibodies against GAT1, 2 and 3 and in polyclonal antibodies against the subunits GABA_Aα1 and GABA_Aα3. LM histofluorescence and pre-embedding EM were used to localize GAT-, GABA_Aα1- and GABA_Aα3-immunoreactivity (IR). Results: At LM level GAT1-IR was restricted to the innerplexiform layer (IPL), whereas GAT3-IR could be observed both in the IPL and in the OPL. GAT2-IR could not be detected in the OPL. To determine the localization of GAT3 relative to the synaptic terminal of the photoreceptors, a monoclonal GluR2 antibody was used as marker for invaginating horizontal cell (HC) dendrites (J. Klooster et al, J Comp Neurol, in press). At LM level, using confocal microscopy, almost no co-localization of GluR2-IR and GAT3-IR was found. At EM level GAT3-IR was mainly observed in the neuropyl of the OPL. Occasionally a lateral element of the HC dendrite invaginating the cone terminal, showed GAT3-IR. GABA_Aα1-IR and GABA_Aα3-IR was found in a wide band in the OPL. Double labelling with GABA and GABA_Aα1 and GABA_Aα3 did not result in extensive co-localization in the OPL. Conclusion: Physiological experiments have shown that the main release of GABA from HCs is via GATs. Our data show that the GATs are not predominantly localized in the synaptic complex of the cone/HC/bipolar cell, suggesting that HCs modulate the GABA concentration in a large extracellular compartment in the OPL.

Keywords: 555 retina: distal(photoreceptors, horizontal cells, bipolar cells) • 559 retinal connections, networks, circuitry • 472 microscopy: electron microscopy