Abstract
Purpose::
While several studies suggest that GABA is released by a vesicular mechanism in mammalian horizontal cells (HC), other studies fail to detect either GABA or its synthesizing enzyme GAD67 in these cells. To further analyze the role of GABA and GAD67 in HCs, the GAD67-GFP transgenic mouse may be a good model. In this study, we have (1) determined the regional distribution and developmental expression of GAD67-GFP-containing HCs in this transgenic mouse and (2) tested if these HCs show GAD67 immunoreactivity.
Methods::
The GAD67-GFP BAC transgenic mouse line (J. Huang, Cold Spring Harbor, NY) carries a GFP reporter under the control of the GAD67 promoter. In retinal whole mounts, the number of GAD67-GFP-fluorescent HCs, relative to the total number of HCs, identified using a calbindin antibody, was counted in 230x230 µm2 areas in superior, temporal, inferior and nasal retina at 3 eccentricities: near the optic nerve, and 0.7 and 2.0 mm from the optic nerve (2 adjacent areas at each location were averaged). Some retinas of GAD67-GFP transgenic and of C57Bl/6J mice were immunostained with a GAD67 antibody. Retinas were evaluated at postnatal day P5 (n=3), P10 (n=6), P25 (n=3), P50 (n=8) and P370 (n=3), using confocal microscopy.
Results::
(1) At P5, GAD67-GFP-containing HCs were evenly distributed across the retina and they account for 51.7±11.6% of the total number of HCs in the GAD67-GFP transgenic mouse line. The number of GAD67-GFP-containing HCs decreased with age (mean of all regions: P10: 48.0±15.2%; P370: 13.1±15.7%) and there was a differential reduction in GFP-containing HCs with most cells in the superior and temporal retina (superior retina at 0.7 mm; e.g. P25: 54.2±16.3%), and the fewest number of cells in the nasal and inferior retina (inferior retina at 2.0 mm; e.g. P25: 12.3±4.1%). (2) In contrast, GAD67 immunostaining was found in HCs at P5 only, in both GAD67-GFP and C57Bl/6J mice. GAD67 immunoreactivity was present in HCs expressing the GFP transgene (mean in overall retina: 15.4±6.7%) and those that did not express the GFP transgene (mean: 12.5±7.7%).
Conclusions::
The GAD67-GFP BAC transgenic mouse line expresses GFP-containing HCs, with a greater percentage during the postnatal period compared to adults. Interestingly, GAD67 immunoreactive HCs were observed at early postnatal ages only, but not in the adult retina. This pattern of GFP and GAD67 expression suggests that GAD67 promoter activity, synthesis or levels are higher during the postnatal period compared to adulthood.
Keywords: horizontal cells • inhibitory neurotransmitters • development