Yue L , Pawlowski M , Dellal SS , Xie A , Feng F , Otis TS , Bruzik KS , Qian H , Pepperberg DR .

AA01973 NIAAA NIH HHS , EY001792 NEI NIH HHS , EY016094 NEI NIH HHS , UL1RR029879 NCRR NIH HHS , UL1 RR029879 NCRR NIH HHS , P30 EY001792 NEI NIH HHS , R01 EY016094 NEI NIH HHS , R21 AA019737 NIAAA NIH HHS

	Figure 2. MPC088 agonist activity in oocytes expressing α1β2γ2 GABAARsa: Representative responses to MPC088 obtained in a single experiment. b: Concentration-response functions (mean ± SD) for trans- and cis-dominant MPC088 (n = 6) and propofol (n = 6). In each of the MPC088 experiments, the cis-dominant form was generated by UV light during static bathing. Amplitudes normalized to the 100 μM GABA response. Inset: Sensitivity (mean ± SD) of the MPC088 (15 μM) response to 100 μM PTX (n = 4), 100 μM bicuculline (n = 4) and 30 μM gabazine (n = 4). c: Photo-regulation of the response to 15 μM MPC088. d: Normalized current amplitudes (mean ± SD) determined in the experiment of c and in 3 others of similar design. Amplitudes in each experiment normalized to that elicited by 3 μM GABA. Numbers beneath the waveform in c denote amplitude determination conditions for the experiment shown in c. Repeated-measures ANOVA conducted on the aggregate data yielded F(6,18) = 68.988, p < 0.001. Post-hoc paired-sample t-tests corrected for multiple comparisons indicated significant differences produced by the initial UV, the initial High-Vis, the second UV, and the second High-Vis illuminations (p ≤ 0.010 for amplitude group 2 vs. group 1, for 4 vs. 3, for 5 vs. 4, and for 6 vs. 5, respectively); amplitude groups 7 and 1 did not differ significantly (p = 0.96). e: Representative waveform obtained with repeated presentation of UV light during continuous High-Vis illumination (static bathing in MPC088-supplemented Ringer).
	Figure 3. Effect of MPC088 on α1β3γ2 and α4β3δ GABAAR-expressing oocytesData in each group obtained from 4 oocytes. a: Aggregate concentration-response data (mean ± SD) obtained from α1β3γ2 (black) and α4β3δ (red) receptors with co-applied GABA at similar ~EC8 dose (i.e., 3 μM for the former and 0.05 μM for the latter receptor type) and varying concentrations of trans-dominant MPC088. Response amplitudes obtained from each oocyte normalized to its saturating GABA response (i.e., responses obtained with 1,000 μM GABA for α1β3γ2 and 100 μM GABA for α4β3δ GABAARs). b: Aggregate concentration-response data (mean ± SD) obtained from α1β3γ2 (black) and α4β3δ (red) receptors with trans-dominant MPC088 alone. Data similarly normalized to the saturating GABA response obtained from each oocyte. Results for α1β2γ2 GABAARs in a and b (blue) reproduced from those of Figures 1c and 2b, respectively.
	Figure 4. Persistent potentiation and activation by MPC100 in γ-79C-expressing oocytesa: GABA responses of a single oocyte before (left) and after (right) MPC100 treatment (100 μM). Right-hand waveforms recorded after ~7-min incubation with MPC100 and subsequent Ringer perfusion. b: GABA response functions showing aggregate data (mean ± SD; 5 oocytes) obtained before MPC100 treatment, and after treatment with 100 μM MPC100 and subsequent washout (~5 min). Amplitudes normalized to that elicited by 100 μM GABA before MPC100 treatment. c: Light-dependence of persistent potentiation. Black trace: response to UV and visible light in the presence of 0.1 μM GABA, following treatment with 100 μM trans-dominant MPC100. Dotted lines i and ii highlight the difference in baseline current before (ii) vs. after (i) UV illumination). Red trace: later phase of the same experiment (responses obtained after a further ~1 min Ringer perfusion). d: Baseline currents obtained in a single experiment. Trace (i): nominal response to PTX (100 μM). Traces ii-iii: PTX responses obtained after treatment with 100 μM MPC100, in the absence (ii) and presence (iii) of UV illumination. e: Responses to UV and visible light following exposure to trans-dominant MPC100 and washout (~5 min) of free MPC100. Data obtained from a single oocyte. A period of Ringer perfusion (~1 min) separated the initial (black trace) and later (red trace) phases of the experiment. f: Aggregate results (mean ± SD) from experiment e and 3 others, each involving amplitude determinations under 6 sequential experimental conditions. Data normalized to the persisting shift in baseline produced by MPC100 treatment. Periods of treatment in each experiment were similar to those illustrated in e. Numbers beneath the waveforms in e denote amplitude determination conditions for the experiment shown in e. Repeated-measures ANOVA conducted on the aggregate data yielded F(6,18) = 65.290, p < 0.001. Post-hoc paired-sample t-tests corrected for multiple comparisons indicated significant differences produced by the initial UV, the initial High-Vis, the second UV, and the second High-Vis illuminations (p ≤ 0.007 for amplitude group 2 vs. group 1, for 3 vs. 2, for 4 vs. 3, and for 6 vs. 5, respectively).
	Figure 5. Action of MPC088 on retinal ganglion cellsa: Potentiation data obtained with trans- and cis-dominant MPC088; the cis-dominant preparation was obtained by pre-treating trans-dominant MPC088 with UV light for 5 min. Representative responses obtained from a single cell treated with 200 μM GABA (black), 2 μM GABA (red), 2 μM GABA co-applied with 10 μM of trans-dominant (blue) or cis-dominant (magenta) MPC088. Inset: Aggregate data (mean ± SD) for potentiation factor determined with trans- and cis-dominant MPC088 (left and right bars, respectively) (n=6, p = 0.003). b: Direct activation data obtained with trans- and cis-dominant MPC088. Representative responses obtained from a cell treated with 200 μM GABA (black), 60 μM of trans-dominant (blue) and 60 μM of cis-dominant (magenta) MPC088. Inset: Aggregate data (mean ± SD) for direct activation by MPC088. Left, middle and right bars show results obtained, respectively, with 30 μM trans-dominant (n=3), 60 μM trans-dominant (n=7), and 60 μM cis-dominant MPC088 (n=4).
	Figure 6. Effect of MPC088 photoactivation on GABA-evoked currents in cerebellar PNsa: Optical configuration. b: Representative trace from a whole-cell voltage-clamp recording from a PN. The cell was held at -70 mV, and 10 μM GABA + 30 μM MPC088 were applied via pressure pipette for 30 s. The cell was flashed with UV light (1 s, shuttered from a mercury arc lamp), then blue light (1 s, from a 470 nm LED), and then again with UV light (1 s). Insets expand the segments of the trace recorded when the light pulses were delivered. c: Trace from the same cell as in b, but with exposure to 10 μM GABA alone. d: Trace from the same cell as in b-c, but with exposure to 10 μM GABA + 300 μM propofol. e: Summary of results described in b-d. The left-hand trace shows the method used to obtain the numerical data shown at the right. The magnitude of the UV-to-blue transition was calculated as (B-UV1)/UV1; that of the blue-to-UV transition was calculated as (UV2-B)/UV2. Here, UV1, B and UV2 are, respectively, the magnitude of the current evoked by the first UV flash, the blue flash and the second UV flash, as referenced to the pre-GABA current level. The number above each condition indicates the number of cells from which data were obtained. For the negative control experiments (i.e., those involving exposure to vehicle or propofol alone) in which no light-evoked current was detectable, current was measured for identical epochs at the terminations of the light pulses. Error bars represent the SEM. For both the blue-to-UV and the UV-to-blue transitions, the Kruskal-Wallis ANOVA on Ranks yielded p < 0.005 (H > 11, df = 2). Post-hoc statistical tests showed that results obtained with MPC088 treatment differed significantly from those obtained under either negative control condition (Mann-Whitney Rank-Sum Test: MPC088 vs. vehicle (blue-to-UV and UV-to-blue), p = 0.002; MPC088 vs. propofol (blue-to-UV and UV-to-blue), p = 0.014; p values adjusted for multiple comparisons). f: Representative trace from a cell exposed to multiple UV/blue light flashes during application of GABA and MPC088.
	Figure 7. Effect of MPC088 photoactivation on action potential firing frequency in PNsa: Representative data obtained by whole-cell current-clamp recording from a PN which was injected with 468 pA to elicit a high-frequency train of action potentials. During this current step, 10 μM GABA + 30 μM trans-dominant MPC088 were applied via pressure pipette for 10 s. The cell was then exposed for 1-s durations to UV light (UV1), then blue light, and then again with UV (UV2). The entire illustrated voltage vs. time trace occurs during application of GABA/MPC088. Below this trace is plotted the instantaneous firing frequency vs. time; the dashed line reflects the average firing frequency during the 1-s epoch that preceded the first UV exposure. b: Summary of results from experiments of the type shown in a. Average spike frequencies were obtained for each cell in each of five 1-s epochs, then normalized to the pre-GABA/MPC088 firing frequency (n = 8 cells from 4 animals). Error bars here and in c represent the SEM. Repeated-measures ANOVA on the aggregate data yielded F(3,21) = 10.213, p < 0.001. Red asterisks above the purple and blue bars indicate significance of the average frequency in that epoch vs.the preceding epoch (e.g., the asterisk above the UV1 bar refers to significance of the average firing rate in the UV1 epoch vs. the GABA/MPC088 pre-UV epoch). Significance was determined from post-hoc paired-sample t-tests corrected for multiple comparisons, which yielded p < 0.02. c: Summary of results from experiments similar to that described in a, but in which the cell was exposed only to the UV/Blue/UV flash sequence (i.e., no GABA/MPC088 applied). Data normalized to the average firing frequency during the 1-s period preceding the first UV flash (n = 7 cells from 3 animals). Repeated-measures ANOVA on the aggregate data yielded F(3,18) = 3.057, p > 0.05. Three post-hoc statistical tests were then performed as described in b; none of these yielded significance at the uncorrected 0.05 level.
	Figure 8. Lack of agonist activity of MPC088 on PNsa: Averages of 10 recordings from a PN exposed to multiple Blue/UV light flashes, in the presence of cis-dominant MPC088 (30 μM) in the external solution, before (black) and after (red) the addition of gabazine (30 μM). There was no exogenous GABA present in the tissue. b: Summary of results from 8 experiments (8 PNs) including that described in a. The data indicate the magnitude of an outward current. The temporal sequence of illumination differed among the experiments (e.g., only the cell in a received multiple blue/UV exposures), but the data plotted are the amplitudes from the blue to UV transition (in the case of the cell in a, this is the blue/UV transition from the first of the three exposures). Yellow-filled data points indicate results from the experiment in a. Each data point is the average obtained from 4-10 consecutive recordings. The light-evoked membrane current was significantly reduced by gabazine (Wilcoxon Signed Rank Test, p=0.008). Error bars represent the SEM.

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