GABA-ACTIVATED AND GLYCINE-ACTIVATED CURRENTS IN THE ROD BIPOLAR CELLOF THE RABBIT RETINA

1. Voltage- and ligand-gated currents were recorded from solitary rabb it rod bipolar cells using the whole cell patch-clamp technique. The r od bipolar cell forms a single, stereotypical physiological and morpho logical class of cells that was easily identified from other neurons a nd support cells after enzymatic and mechanical dissociation from isol ated retina. Protein kinase C immunoreactivity confirmed the validity of using a purely morphological identification of this cell type. 2. V oltage steps in 15-mV increments from a holding potential of -45 mV el icited a large outward current activated near -30 mV. These voltage-ga ted currents were eliminated by using equimolar substitutions of Csi a nd tetraethylammonium(+) for K+ in the pipette, indicating that they r epresent a mixture of K+ currents. 3. The putative inhibitory neurotra nsmitters gamma-aminobutyric acid (GABA) and glycine activated inward Cl- currents when pressure-applied from pipettes placed near the axon terminals of rod bipolar cells, which were voltage-clamped at -45 mV. With changes in intracellular or extracellular Cl concentration, the r eversal potential of these ligand-gated currents changed as predicted by the Nernst equation for Cl- activity. The dose-response curves for GABA and glycine were sigmoidal with saturating concentrations of 100 and 300 mu M, respectively. 4. GABA-activated currents were I) reversi bly reduced by the allosteric inhibitor picrotoxin and the competitive antagonist bicuculline; 2) potentiated by the benzodiazepine diazepam and the barbiturate barbital sodium; and 3) indistinguishable from mu scimol-activated currents. There was no response to the GABA(B) agonis t baclofen. Collectively, these data strongly suggest that the GABA-ac tivated currents in rabbit rod bipolar cells are mediated by the GABA( A) receptor. This is similar to the GABA-activated currents in other m ammalian rod bipolar cells. 5. Application of the conformationally res tricted GABA analogue cis-4-aminocrotonic acid (CACA) failed to elicit a response, whereas the conformationally extended GABA analogue trans -4-aminocrotonic acid (TACA) elicited a response similar to that of GA BA. Although bicuculline appeared to suppress the GABA-activated curre nt slightly more than the TACA-activated current (not significant usin g Student's t-distribution), GABA- and TACA-activated currents were eq ually suppressed by picrotoxin and equally enhanced by diazepam and ba rbital sodium. These data, coupled with the inefficacy of CACA, argue against the existence of a GABA(c)-type channel in the rod bipolar cel l of the rabbit and suggest that GABA and TACA were activating the sam e GABA, receptor-channel complex. 6. The rabbit rod bipolar cell, like those of other mammalian retinas, demonstrated the presence of conven tional glycine receptors that were antagonized by nanomolar and low mi cromolar concentrations of strychnine. 7. Focal application of GABA an d glycine was used to examine the regional distribution of chemical se nsitivity across the cell. Ligand-activated currents elicited from the rod bipolar cell terminals were always larger than those generated fr om the soma To correlate the distribution of the sensitivity with a vi sual representation of receptor density, solitary rod bipolar cells we re incubated with BODIPY-labeled receptor-specific neuroactive substan ces. BODIPY Ro-1986, a benzodiazepine receptor ligand used to visualiz e the GABA(A) receptors, demonstrated no discernible focality of recep tor distribution, a result incompatible with the electrophysiological data By contrast, glycine receptors labeled with BODIPY strychnine, th e glycine antagonist, demonstrated the highest density of receptors on the axon terminal, intermediate receptor density on the soma, and vir tually no labeling on the dendrites, a result consistent with predicti ons based on electrophysiology. 8. Dopamine, 5-hydroxytryptamine, and adenosine were applied either individually to evaluate direct current responses or in conjunction with GABA and glycine to determine their a bility to modulate the known Ligand-gated currents. None of these puta tive transmitters, shown by immunocytochemistry to be localized to the region of the inner plexiform layer (IPL) where rabbit rod bipolar ce ll terminals reside, either activated a current or modulated the GABA- or glycine-activated currents. 9. The neuroactive peptides substance P, substance Y, somatostatin, neurotensin, and vasoactive intestinal p eptide (VIP) have also been localized by immunocytochemical studies to the strata of the IPL in which the rod bipolar axon endings terminate . When these substances were applied singly to the rod bipolar cell du ring whole cell recording experiments, they elicited no observable cur rent response at any holding potential. However, the application of a prepulse of VIP before the test pulse of GABA reduced the GABA-activat ed current by an average of 37% in the 45 cells tested; a similar prep ulse had no affect on the glycine-activated currents. The remaining ne uroactive peptides had no modulatory effect on GABA- and glycine-activ ated currents.