Rabbit retinas were isolated from the eye and maintained in vitro. When they were incubated for 60 min in the presence of 3H-GABA, subsequent autoradiography showed radioactivity to be present primarily in amacrine cells. Under these conditions, most of the radioactivity contained in the retinas remained in the chemical form of GABA. Autoradiography and immunohistochemistry of alternate sections showed the amacrine cells that accumulate 3H-GABA to be the same cells that contain endogenous GABA immunoreactivity. These include the starburst cells, the indoleamine-accumulating cells, and other, as yet unidentified amacrine cells. The localization confirms previous immunohistochemical findings. When retinas containing 3H-GABA were expressed to elevated concentrations of K+, their content of 3H-GABA decreased. Autoradiography showed a reduced 3H-GABA content in all of the cells that contained 3H-GABA. Since those include the starburst cells, previously shown to be cholinergic, the finding demonstrates that the starburst cells release both ACh and GABA. Retinas simultaneously labeled with 14C-GABA and 3H-ACh were superfused, and the release of radioactive compounds from the retina was studied. Depolarization by elevated K+ caused an increased recovery of both ACh and GABA in the superfusate, but the predominant mechanisms of their release appeared to be different. The stimulated release of ACh was entirely Ca2+ dependent, while the release of radioactivity originating from GABA was much less so. A concentration-dependent counterflux (homoexchange) of intracellular GABA was demonstrated by raising the extracellular concentration of GABA (or nipecotic acid). These results suggest that a large outward flux of GABA occurs via the GABA transporter, probably by the potential-sensitive mechanism studied by Schwartz (1982, 1987). Stimulation of double-labeled retinas by flashing light or moving bars always increased the release of ACh, and the release was entirely dependent on the presence of extracellular Ca2+. Stimulation with light never caused a detectable release of GABA. This was unexpected, since the two neurotransmitters are present in the same amacrine cells: stimulation adequate to release one neurotransmitter should release both.(ABSTRACT TRUNCATED AT 400 WORDS)

In many calcium-imaging studies, the nuclear envelope appears to maintain a gradient of free calcium between the nucleus and cytosol. This issue was examined by loading amphibian sympathetic neurons with the calcium indicator fluo 3 via whole-cell patch clamping. Confocal optical sectioning allowed acquisition of independent calibration curves for the nucleus and cytoplasm. Cells were loaded with free calcium levels ranging from 10 nM to 50 microM, using 10 mM BAPTA to control free calcium. The nuclear fluorescence was usually about 130% brighter than the cytoplasmic fluorescence. Had the increased nuclear fluorescence been due to a calcium gradient, then, as fluo 3 was saturated with calcium in both compartments, the fluorescence gradient should have gradually disappeared. Instead, with free-calcium in the pipette set at 50 microM, about five times the level required to nearly saturate fluo 3, the nuclear/cytoplasmic (N/C) fluorescence ratio was not decreased but instead increased slightly. Perfusion of the patch pipette was used in conjunction with imaging to confirm that cytoplasmic fluo 3 was saturated with calcium. After loading cells with 10 nM free calcium, the patch pipette was perfused with high calcium (10 microM). Again, the N/C fluorescence ratio increased at high calcium. The effectiveness of patch-pipette perfusion in changing cellular free calcium levels was indicated by the degree of fluorescence increase--both nuclear and cytosolic compartments showed a roughly 20-fold increase in fluorescence, that is, most of the dynamic range observed in test droplets. To confirm further that cytoplasmic fluo 3 was saturated, cells were perfused with manganese, which binds with very high affinity to fluo 3. Manganese rapidly entered the cytoplasm and nucleus, causing a large increase in fluorescence, but the N/C fluorescence ratio remained relatively constant. Because free manganese in the pipette was 50,000 times the amount required to saturate fluo 3, the greater nuclear fluorescence probably results from additional fluo 3 in the nucleus rather than from calcium or manganese gradients. To gauge further the permeability of the nuclear envelope, the diffusion of calcium was visualized. Under voltage clamp, calcium channels were opened for periods ranging from 5 to 200 msec. Peak calcium levels were observed within 2 microns of the plasma membrane, and declined as calcium diffused into the cell. The nuclear fluorescence increased more than cytosolic fluorescence, but this apparent “amplification” was eliminated by correcting for autofluorescence. Use of cells cultured on glass coverslips and a high- NA microscope objective allowed a satisfactory correction.(ABSTRACT TRUNCATED AT 400 WORDS)