The expression of GAD₆₇ isoforms in zebrafish retinal tissue changes over the light/dark cycle

Abstract

We show the levels of glutamic acid decarboxylase (GAD), the enzyme catalyzing the conversion of glutamic acid to GABA, changes in zebrafish retinal tissue during the light/dark cycle. Further, we identify two transcripts of the GAD₆₇ gene, full-length GAD₆₇ and the truncated 25 kDa alternative splice variant (ES), as the major GAD isoforms in this tissue. GAD-positive neurons were identified immunocytochemically by probing retinal sections with K2, an antibody to the GAD₆₇ isoform, and with an antibody specific for the 25 kDa splice variant. For both antibodies, GAD-immunoreactivity was observed in horizontal cells in the distal retina and amacrine cells in the proximal retina, with both cell bodies and processes labeled. No apparent difference in K2 labeling pattern was observed in tissue harvested 8 hrs after light offset or onset, whereas ES label was identified in more structures in dark tissue. Quantification of GAD levels was determined by densitometry of Western Blots. The protein content of GAD₆₇ and ES varied between tissue harvested during the light and the dark. ES expression was up-regulated in dark tissue; whereas, full-length GAD₆₇ expression increased in light tissue. In vivo GABA content, measured with high performance liquid chromatography (HPLC), was found to increase in light tissue, paralleling the expression of full-length GAD₆₇ transcripts. Expression of ES did not correlate with measured GABA levels, suggesting this isoform, which lacks the catalytic domain necessary for enzymatic activity, may have a different physiological role in retinal tissue. The inverse expression patterns of full-length GAD₆₇ and ES suggest that alternative splicing of GAD₆₇ may be triggered by the light and/or dark cycle, resulting in a change in inhibitory neurotransmitter content in retinal tissue.