Article Figures & Data
Figures
- Figure 1.
α1-antibody affects receptor distribution over time. A, Staining of cortical-striatal cultures (1:3) with MAP2 (white), VGAT (green), and α1-antibody (red) after 1-h α1-antibody incubation at 15°C and for 24 h at 37°C; scale bar: 20 μm. B, Close-up of boxed area in A, showing synaptic colocalization between VGAT and α1-antibody; scale bar: 10 μm. C, Over time, the α1-antibody leads to reductions in percentage of synapses covered with α1-antibody puncta, t(146.4) = 6.664, p < 0.0001, Welch’s t test, while (D) the α1-antibody can be found equally at synaptic and extrasynaptic sites over time, t(121) = 0.691, p = 0.4907, Welch’s t test. In addition, (E) long antibody incubation (24 h) leads to reductions in overall α1-antibody intensity, t(148.5) = 4.11, p < 0.0001, Welch’s t test, (F) because of reduction in intensity at synaptic sites, t(136.4) = 3.701, p = 0.0003, Welch’s t test, (G) and extrasynaptic sites t(129.8) = 2.329, p = 0.0214, Welch’s t test. H, Overall, VGAT intensity is also decreased over time, t(156.1) = 2.479, p = 0.0142, Welch’s t test. This is also seen in histograms when VGAT intensity is plotted against the α1-antibody intensity at 1 h (I) versus at 24 h (J), where we observed a reduction in the slope of the correlation because of a decrease in α1-antibody intensity. Each data point represents one ROI, except in I and J, where each data point represents a synapse. Error bars represent SEM.
- Figure 2.
α1γ2-antibody stains neurons in a GABAAR-specific pattern. A, Staining of cortical-striatal cultures (1:3) with MAP2 (white), VGAT (green), and α1γ2-antibody (red) after 1-h α1γ2-antibody incubation at 15°C or for 24 h at 37°C; scale bar: 20 μm. B, Close-up of boxed area, showing synaptic colocalization between VGAT and α1γ2-antibody positive puncta; scale bar: 10 μm. C, After incubation with the α1γ2-antibody, we observed that the percentage of VGAT puncta colocalizing with α1γ2-antibody puncta remain similar from 1 to 24 h, t(163) = 1.62, p = 0.1072, unpaired t test, (D) as do the percentage of α1-antibody puncta that colocalize with VGAT puncta over time, t(161) = 0.9327 p = 0.3524, unpaired t test. E, Over time (24 h), the α1γ2-antibody addition is associated with increases in α1γ2-antibody puncta intensity, t(109.8) = 3.702, p = 0.0003, Welch’s t test. When we split α1γ2-puncta intensity based on colocalization with VGAT puncta, we see an increase in both synaptic (F), t(100.7) = 4.442, p < 0.0001, Welch’s t test, and extrasynaptic α1γ2-antibody intensity (G), t(81.46) = 3.243, p = 0.0017, Welch’s t test. H, However, the α1γ2-antibody does not alter VGAT intensity t(158.4) = 0.4497, p = 0.6535, Welch’s t test. In histograms comparing VGAT versus α1γ2-antibody puncta intensity at 1 h (I) versus at 24 h (J), we observe no difference in the slope of the correlation, although auto-antibody intensity increases. Each data point represents one ROI, except in I and J, where each data point represents a synapse. Error bars represent SEM.
- Figure 3.
α1-antibody but not the α1γ2-antinbody reduces GABAergic currents after a 24-h treatment. A, Staining of striatal autaptic neurons with MAP2 (white) and VGAT (green); scale bar: 20 μm. B, Example IPSC traces for each condition. C, Quantification of IPSC amplitude shows that autaptic neurons treated with α1-antibody for 24 h have reduced synaptic IPSC amplitudes, H(3) = 17.78, p = 0.0005, Kruskal–Wallis; untreated 1 ± 0.10 versus α1-antibody 0.56 ± 0.15, p = 0.0009, control 1.04 ± 0.14 versus α1-antibody p = 0.0053, (D) and longer decay times, H(3) = 9.79, p = 0.0205, Kruskal–Wallis; untreated 118.4 ± 5.93 ms versus α1-antibody 186.3 ± 17.68 ms, p = 0.0202. E, Example traces current induced after bath application of 5 μm GABA show (F) that the presence of α1-antibody leads to reductions in GABA currents H(3) = 25.28, p < 0.0001, Kruskal–Wallis; untreated 1 ± 0.06 versus α1-antibody 0.6 ± 0.05 p < 0.0001, control 0.99 ± 0.07 versus α1-antiody p = 0.0002, α1-antibody versus α1γ2-antibody 0.96 ± 0.09 p = 0.0054. G, It does not affect excitatory currents induced by bath application of Kainate, H(3) = 0.52, p = 0.9135, Kruskal–Wallis. H, Representative traces of currents induced following the addition of sucrose show (I) that the ready releasable pool (RRP) is decreased in the presence of the α1-antibody, H(3) = 16.2, p = 0.0003, Kruskal–Wallis; untreated 1 ± 0.15 versus α1-antibody 0.40 ± 0.08 p = 0.0025, control 1.21 ± 0.22 versus α1-antibody p = 0.0009, (J) but that release probability (Pvr) is not affected, H(3) = 1.59, p = 0.4506, Kruskal–Wallis. Example traces of mIPSCs (K) show that (L) frequency was reduced after α1-antibody incubation compared with control-antibody conditions, H(3) = 10.49, p = 0.0149, Kruskal–Wallis; control 1.42 ± 0.32 Hz versus α1-antibody 0.46 ± 0.14 Hz p = 0.0151, (M) but mIPSC amplitude remained the same, H(3) = 6.94, p = 0.0737, Kruskal–Wallis. Incubation with the α1γ2-antibody (K–M) does not lead to any differences in synaptic currents. Each data point represents one cell. Error bars represent SEM.
- Figure 4.
Cortical-striatal neuron network activity increases after a 24 h treatment with α1-antibody but not the α1γ2-antibody. Examples of network activity, based on the frequency of calcium transients, 24 h after antibody treatment (A) and directly after the addition of 30 μm bicuculline (B). C–G, The average spiking frequency per field of view per condition shows that the addition of bicuculline leads to higher average spike frequency for all conditions, untreated 9.30 ± 0.47 Hz versus bicuculline 18.86 ± 1.22 Hz, t(32) = 7.64, p < 0.0001; control-antibody 9.17 ± 0.55 Hz versus bicuculline 20.18 ± 0.99 Hz, t(26) = 10.34, p < 0.0001; α1-antibody 13.76 ± 0.58 Hz versus bicuculline 17.05 ± 0.69, Hz t(32) = 3.29, p = 0.0024; α1γ2-antibody 9.30 ± 7.61 Hz versus bicuculline 21.91 ± 1.08 Hz, t(26) = 11.04, p < 0.0001; α1γ2-antibody 5 μg 9.79 ± 0.54 Hz versus bicuculline 23.9 ± 1.31 Hz, t(20) = 9.80, p < 0.0001; all paired t tests. H, However, under conditions treated with α1-antibody for 24 h (A) spiking starts off with a higher average frequency, H(4) = 29.62, p < 0.0001, Kruskal–Wallis; untreated 9.58 ± 0.47 Hz versus α1-antibody 14.03 ± 0.66 Hz p < 0.0001, control 9.68 ± 0.53 Hz versus α1-antibody p < 0.0001, α1-antibody versus α1γ2-antibody 10.68 ± 0.79 Hz p = 0.0010, α1-antibody versus α1γ2-antibody 5 μg 9.79 ± 0.54 p = 0.0026, (I) and shows a smaller percentage increase after the addition of bicuculline compared with other conditions, F(4,127) = 9.46, p < 0.0001, ANOVA; untreated 123.5 ± 15.01% versus α1-antibody 42.7 ± 9.66% p = 0.0017, control 136.6 ± 16.16% versus α1-antibody p = 0.0002, α1-antibody versus α1γ2-antibody 155 ± 16.01% p < 0.0001, α1-antibody versus α1γ2-antibody 5 μg 160.8 ± 20.02% p < 0.0001. Example images of a low (J) and high (K) network spiking activity; scale bar: 40 μm. Each data point represents the average spiking activity of one ROI. Error bars represent SEM.
- Figure 5.
α1-antibody but not α1γ2-antibody reduce GABA currents after a 1-h incubation. Example traces of mIPSC frequency (A) and amplitude (B). C, mIPSCs recorded 1 h after antibody treatment show reduction in frequency, F(3,116) = 7.10, p = 0.0002, ANOVA; untreated 4.62 ± 0.41 Hz versus α1-antibody 2.10 ± 0.41 Hz p = 0.0002, control 3.90 ± 0.50 Hz versus α1-antibody p = 0.0141, α1-antibody versus α1γ2-antibody 4.18 ± 0.32 Hz p = 0.0033, (D) and amplitude for the α1-antibody compared with other groups, F(3,112) = 5.27 p = 0.0020, ANOVA; untreated 42.97 ± 3.24 pA versus α1-antibody 27.45 ± 2.71 pA p = 0.0058, control 44.03 ± 3.87 pA versus α1-antibody p = 0.0030. E, mIPSC charge only showed a difference between α1 and control-antibody, F(3,112) = 3.52, p = 0.0174, ANOVA; control 707.3 ± 64.83 fC versus α1-antibody 475.5 ± 45.19 fC p = 0.0161. F, However, rise time, H(3) = 4.22, p = 0.2385, Kruskal–Wallis, (G) half width, H(3) = 1.23, p = 0.7452, Kruskal–Wallis, (H) and decay time, even for the α1-antibody, were not affected, H(3) = 2.79, p = 0.425, Kruskal–Wallis. Data points represent one cell each. Error bars represent SEM.
- Figure 6.
α1-antibody elicits rapid effects on cortical-striatal calcium transients. A, Example spike frequency plots and quantification (B) of network spiking activity shows that 1 h after α1-antibody is added, activity is increased compared with other conditions, H(3) = 40.12, p < 0.0001, Kruskal–Wallis; untreated 13.98 ± 1.21 Hz versus α1-antibody 32.46 ± 2.58 Hz p < 0.0001, control 17.64 ± 1.93 Hz versus α1-antibody p = 0.0001, α1-antibody versus α1γ2-antibody 5 μg 12.87 ± 1.13 Hz p < 0.0001. This effect was not reversible by antibody washout (Extended Data Fig. 6-1). When the α1-antibody is added for only 12 min to cortical-striatal cultures (C, example plots), there is a significant increase in spiking frequency, already after 2 min, compared with other conditions (D) that is sustained for the full 12 min, F(1,29) = 10.82, p = 0.0026, repeated measures two-way ANOVA. The dotted vertical line indicates when antibody is added to the culture. Addition of an Fab fragment from the α1-antibody (E) similarly induces an increase in spike frequency that keeps rising up till 4 min after addition, even higher than the full-length antibody, and then slowly declines, although not all the way back to control level over the course of 12 min (F), F(1,33) = 18.27, p = 0.0002, repeated measures two-way ANOVA. Each data point represents the average spiking activity of one field of view. Error bars represent SEM.
- Figure 7.
α1-antibody and α1γ2-antibody do not alter the effect of benzodiazepine. Example traces (A) and quantification (B–F) show that in all conditions, the addition of 1 μm diazepam (DZP) is still able to increase the half width of mIPSCs. C–F, show the individual increase in half width of each cell per condition [untreated (C): t(29) = 7.991, p < 0.0001, paired t test; untreated base 12.27 ± 0.49 ms, untreated benzo 14.46 ± 0.57 ms; control-antibody (D): t(28) = 10.32, p < 0.0001, paired t test; control-antibody base 12.77 ± 0.57 ms, control-antibody benzo 15.74 ± 0.66 ms; α1-antibody (E): t(28) = 4.30, p = 0.0002, paired t test; α1-antibody base 12.69 ± 0.82 ms, α1-antibody benzo 15.08 ± 0.75 ms; α1γ2-antibody (F): t(29) = 8.73, p < 0.0001, paired t test; α1γ2-antibody base 13.14 ± 0.67 ms, α1γ2-antibody benzo 15.8 ± 0.61 ms]. B, Quantitation of half-widths ratio (calculated as halfwidth with diazepine/halfwidth baseline). A value above 1 indicates an increase (H(3) = 5.03, p = 0.1694, Kruskal–Wallis; untreated 1.18 ± 0.02, control-antibody 1.25 ± 0.02, α1-antibody 1.52 ± 0.27, α1γ2-antibody 1.22 ± 0.02). Each data point represents the average half-width of mIPSCs per cell. Error bars represent SEM.
- Figure 8.
Difference in network reaction to diazepam when treated with the α1-antibody but not α1γ2-antibody. Example traces of calcium transient spiking activity 1 h after the addition of α1-antibody, α1γ2-antibody, or control-antibody (A). Example traces of network spiking activity after addition of 1 μm diazepam to the cultures shown in A (B). Example traces of network spiking activity after addition of 30 μm bicuculline to the cultures shown in B (C). D–G, Average spiking activity in each field of view (∼15 cells) in baseline, diazepam, and bicuculline condition for each experimental group shows spiking responses to these drugs [untreated (D): F(2,52) = 129.8, p < 0.0001, repeated measures ANOVA; base 13.94 ± 1.22 Hz vs benzo 2.03 ± 0.53 Hz p < 0.0001, base vs bic 38.93 ± 3.04 Hz p < 0.0001, benzo vs bic <0.0001; control-antibody (E): F(2,52) = 91.27, p < 0.0001 repeated measures ANOVA; base 17.64 ± 1.93 Hz vs benzo 5.78 ± 1.44 Hz p = 0.0003, base vs bic 43.08 ± 3.70 Hz p < 0.0001, benzo vs bic p < 0.0001; α1-antibody (F): F(2,52) = 67.03, p < 0.0001, repeated measures ANOVA; base 32.46 ± 2.58 Hz vs benzo 19.7 ± 3.64 Hz p = 0.0001, base vs bic 52.87 ± 3.14 Hz p < 0.0001, benzo vs bic p < 0.0001; α1γ2-antibody 5 μg (G): F(2,52) = 94.32, p < 0.0001, repeated measures ANOVA; base 12.87 ± 1.16 Hz vs benzo 4.64 ± 1.06 Hz p = 0.0084, base vs bic 39.38 ± 3.34 Hz p < 0.0001, benzo vs bic p < 0.0001]. H, Quantification of percent changes in spiking activity following the addition of 1 μm diazepam. Here, diazepam leads to a ∼75% reduction in spiking activity compared with cultures without diazepam (Fig. 6B), with the exception to the α1-antibody condition where the reduction was significantly smaller (∼50%) H(3) = 14.67, p = 0.0021, Kruskal–Wallis; untreated −86.29 ± 3.37% versus α1-antibody −48.32 ± 7.49% p = 0.0024, control −76.37 ± 5.46% versus α1-antibody p = 0.0148. I, Quantification of percent changes in spiking activity following the addition of 30 μm bicuculline. This leads to an ∼200% increase in network activity compared with baseline (Fig. 6B) for all groups except for α1-antibody condition, where the increase is only 80%, H(3) = 22.77, p < 0.0001, Kruskal–Wallis; untreated versus α1-antibody p = 0.0007, control versus α1-antibody p = 0.0141, α1-antibody versus α1γ2-antibody p < 0.0001. Error bars represent SEM.
Extended Data
Extended Data Figure 6-1
Effects of α1-antibody 6 h after antibody washout. Example of neurons stained for MAP2 (white), α1-antibody (red), VGAT (blue), and Merge (α1-antibody and VGAT) 6 h after antibody washout (A). Example spike plots of untreated (B) and α1-antibody-treated (C) cortical-striatal cultures during calcium imaging experiments. D, Spiking frequency is still increased 6 h after α1-antibody washout t(52) = 11.51, p < 0.0001, t test, untreated 12.24 ± 0.98 Hz, α1-antibody 36.71 ± 1.88 Hz. E, Example mIPSC traces. Even 6 h after α1-antibody removal, we see decreases in frequency t(58) = 3.838, p = 0.0003, t test, untreated 3.35 ± 0.36 Hz, α1-antibody 1.56 ± 0.30 Hz, (F) amplitude t(46.71) = 4.44, p < 0.0001, Welch’s t test, untreated 36.65 ± 2.86 pA, α1-antibody 21.93 ± 1.68 pA, (G) charge t(57) = 4.77, p < 0.0001, t test, untreated 655.8 ± 53.81 fC, α1-antibody 345.6 ± 35.69 fC, (H) half-width t(58) = 2.426, p = 0.0184, t test, untreated 13.35 ± 0.46 ms, α1-antibody 11.26 ± 0.73 ms, (J) and decay time t(58) = 3.414, p = 0.0012, t test, untreated 37.57 ± 1.38 ms, α1-antibody 29.07 ± 2.07 ms (K), and an increase in rise time t(30.38) = 2.976, p = 0.0057, Welch’s t test, untreated 0.72 ± 0.03 ms, α1-antibody 1.2 ± 0.16 ms (I). Download Figure 6-1, TIF file.
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