Article Figures & Data
Figures
- Figure 1.
Bipolar cell terminals in retinal slices have tonic currents. A, Whole-cell mode voltage-clamp recording from an isolated bipolar cell terminal (severed axon) in the retinal slice shows a standing current. BMI (20 μm) has no effect on this current, but NO-711 (3 μm) increases the standing current severalfold (−18.4 ± 3 pA in control vs −126 ± 15 pA in NO-711). This increase is completely reversed by TPMPA (150 μm), and the standing current is abolished (below control level, −18.4 ± 3 pA in control vs −5.3 ± 0.7 pA in TPMPA). Note also the current deflections that are superimposed on the tonic leak in NO-711. These are large and slow spontaneous GABAC IPSCs that sometimes occur in NO-711, and these TPMPA-sensitive current oscillations can exhibit periodicity (data not shown; see also Fig. 2A). B, TPMPA also abolishes the standing current in the absence of NO-711 (−31.7 ± 6.9 pA in control vs 10.6 ± 3.0 pA in TPMPA) and prevents the NO-711-induced increase in the standing current until the TPMPA is washed out. BMI does not reduce the standing current in the presence of NO-711, but TPMPA still abolishes it. The occasional breaks (flat portions) in the current traces shown here and in Figure 2 are brief periods when data acquisition was interrupted to record Ca2+ currents. In this example, data are reported as ±SD.
- Figure 2.
GABAC-mediated standing currents are enhanced by the selective GAT-1 transporter inhibitor NO-711 (3 μm) and blocked by TPMPA (200 μm). A, After break-in to the whole-cell mode under high internal chloride (125 mm), the terminal exhibited a standing current. This current was decreased slightly with a combination of CNQX, APV, and TTX (25, 50, and 1 μm, respectively) and was increased slightly in SR95531 (25 μm). NO-711 caused a dramatic increase in the standing current, whereas TPMPA completely reversed this increase and also essentially eliminated the original standing current (2 pA leak current in TPMPA). To fully reduce the standing current in this example, TPMPA was elevated from 150 to 200 μm (arrowhead). B, Average standing conductances in each drug condition: control (0.34 ± 0.06 mS/cm2, n = 10), CNQX/APV/TTX (0.27 ± 0.06 mS/cm2, n = 10), CNQX/APV/TTX+SR95531 (0.39 ± 0.14 mS/cm2, n = 5), CNQX/APV/TTX+NO-711 (1.28 ± 0.22 mS/cm2, n = 5), CNQX/APV/TTX+TPMPA (0.056 ± 0.002 mS/cm2, n = 8). Average conductances were calculated using Ohm’s law, and the terminal surface area was calculated using the measured capacitance of each terminal and assuming a specific membrane capacitance of 1 μF/cm2. The average conductance was significantly larger only in NO-711 (p = 0.0006) and significantly smaller in TPMPA (p = 0.0010) compared with control. C, GABAC receptors did not desensitize in the presence of continuous GABA (10 mm). GABA was applied for 3–4 min to outside-out patches with no GABAA receptors (TPMPA blocked all current, no effect of SR95531), and in every case, GABAC currents remained at their peak amplitude throughout the GABA application (5 patches). D, GABAA receptors quickly desensitize in the presence of continuous GABA (10 mm; 9 patches; red line is a double-exponential fit). Note the difference in timescales for C and D.
- Figure 3.
NO-711 selectively enhances GABAC reciprocal feedback. A, Under low internal chloride (15 mm), depolarizing the bipolar terminal from −60 to 0 mV produces reciprocal GABAergic feedback from amacrine cells via both GABAA and GABAC receptors. After bath application of NO-711 (3 μm), this total GABAergic feedback is significantly potentiated. B, Using TPMPA (200 μm) to isolate the pure GABAA receptor-mediated feedback, NO-711 had no effect on feedback currents. C, Using SR95531 (SR; 25 μm) to isolate the GABAC-mediated feedback, NO-711 dramatically potentiated the feedback currents. D, Average potentiation of GABAergic currents in NO-711. Integration of feedback currents during the depolarization revealed a 49.0 ± 7.1% increase in the total feedback after bath application of NO-711 (n = 7). The pure GABAA receptor-mediated feedback was not potentiated (0 ± 1.3% change after NO-711 application; n = 9). The pure GABAC receptor-mediated feedback was potentiated 47.1 ± 1.2% after application of NO-711 (n = 6). Note that the potentiation in control terminals was not significantly different than the potentiation measured in terminals in which the pure GABAC receptor-mediated feedback was isolated (p = 0.86).
- Figure 4.
Blocking GAT-1 transporters decreased the excitability of bipolar cell terminals by reducing their input resistance. A, Voltage response (bottom) of a bipolar cell terminal under the current-clamp mode to current steps (top). Notice the spikes for large positive current injections. B, NO-711 (1 μm) reduced the voltage responses to all current steps, and most spikes were eliminated. C, TPMPA (200 μm) reversed the effect of NO-711. Recordings in A–C are from the same terminal. D, The I–V curve for nine bipolar cell terminals: NO-711 (1–3 μm) reduced the input resistance (slope), whereas TPMPA (200 μm) increased it approximately back to control levels, on average.
