Colocalization of GAD‐like immunoreactivity and ³H‐GABA uptake in amacrine cells of rabbit retina

Abstract

Rabbit retinas were double labeled to determine the degree of colocalization of glutamic-acid-decarboxylase-like immunoreactivity (GAD-like IR) and 3H-GABA uptake using light (LM) and electron microscopic (EM) autoradiography. Both GAD-like IR and 3H-GABA uptake were found in amacrine cell bodies in the inner nuclear layer (INL) as well as in cell bodies in the ganglion cell layer (GCL), and throughout the inner plexiform layer. GAD-like IR was found in 32% of the amacrine cells in the INL, 86% of which also showed 3H-GABA uptake; 3H-GABA uptake was observed in 38% of the amacrine cells. However, only 72% of these cells showed GAD-like IR. Labeled cells in the GCL were only 10-15% as common as similarly labeled cells in the INL. As in the INL, all GAD-positive cells in the GCL were double labeled, but only 53% of the cells taking up 3H-GABA were double labeled. We suggest that labeled cells in the GCL were ganglion cells rather than displaced amacrine cells. Cells, in both the INL and GCL, that showed 3H-GABA uptake but no GAD-like IR had a higher average grain density than double-labeled cells, indicating that uptake by these cells was specific. The relevance to GABAergic function of 3H-GABA uptake without an indication of GAD-like IR is yet to be determined. Statistical analysis at the EM level showed that one-third of the GAD-positive synaptic terminals of amacrine cells were double labeled after a 4-month exposure. Longer exposures at the EM level should reveal a higher percentage of GAD-positive terminals because at the LM level, one-half of the double-labeled cell bodies were "lightly" labeled with grains. The high degree of colocalization of GAD-like IR and 3H-GABA uptake suggests that both markers may be useful for labeling GABAergic neurons in the rabbit retina.

γ-Aminobutyric acid immunoreactivity in multiple cell types of the developing rabbit retina

“…By postnatal day 20, consistent labeling is seen only in elements of the inner plexiform layer and a limited population of cell bodies which we identified as amacrine and displaced amacrine cells. These results are consistent with other reports of [ 3 H]-GABA accumulation, and GABA and GAD immunohistochemical staining of interplexiform, amacrine, and displaced amacrine cells in the adult rabbit retina (Ehinger & Falck, 1971;Brandon et al, 1979;Mosinger & Yazulla, 1985, 1987Brandon, 1987;Agardh et al, 19876;Brecha et al, 1988;Koontz & Hendrickson, 1990).…”

Cellular distribution and subcellular localization of molecular components of vesicular transmitter release in horizontal cells of rabbit retina

“…For example, GATs have not been localized to rodent and rabbit horizontal cells (Brecha and Weigmann, 1994;Johnson et al, 1996;Hu et al, 1999), and patchclamp recordings of isolated mammalian (rabbit and mouse) horizontal cells have not revealed the presence of GABA-dependent transporter currents (Blanco et al, 1996;Feigenspan and Weiler, 2004). Finally, no highaffinity transport of [ 3 H] GABA or GABA analogs by horizontal cells has been reported for any adult mammalian retina (Ehinger, 1977;Blanks & Roffler-Tarlov, 1982;Mosinger and Yazulla, 1985;Wä ssle & Chun, 1988Löhrke et al, 1994). More recently, alternative mechanisms of horizontal cell feedback involving electrical resistance or ephaptic feedback onto cones in goldfish (Kamer- Kamermans and Fahrenfort, 2004) and a pH-dependent modulation of cone calcium channels in newts (Hirasawa and Kaneko, 2003) have been proposed.…”

“…However, GAT isoforms do not localize to horizontal cells in rodent and rabbit retina Weigmann, 1994, Johnson et al, 1996;Hu et al, 1999), and GABA uptake by adult mammalian horizontal cells has not been reported (Ehinger, 1977;Blanks and Roffler-Tarlov, 1982;Mosinger and Yazulla, 1985;Chun, 1988, 1989;Löhrke et al, 1994). Moreover, electrophysiological analyses of GABA-induced currents in isolated horizontal cells from rabbit and mouse retina have not revealed a transporter current (Blanco et al, 1996;Feigenspan and Weiler, 2004).…”

Cholinergic amacrine cells of the rabbit retina contain glutamate decarboxylase and gamma-aminobutyrate immunoreactivity.

“…A matching group of displaced amacrine cells located in the ganglion cell layer (GCL) have processes that ramify in lamina 4 of the IPL. A large diversity of GABAergic retinal'cell types including interplexiform, amacrine, and displaced amacrine cells have been identified in the rabbit retina by high-affinity uptake of [3H]GABA and immunohistochemical staining for GABA and the GABA-synthesizing enzyme, glutamate decarboxylase (GluDCase) (6,(12)(13)(14)(15)(16). The existence of several types of GABAergic amacrine and displaced amacrine cells has been suggested on the basis of cell body size and the multilaminar distribution of processes in the IPL.…”