(a) Electron micrograph of crude CSV fraction (LP2) purified as described in Methods. This fraction served as a starting material for immunoisolation of subsets of synaptic vesicles. Scale bar represents 200 nm. (b) Electron micrograph of CLC-3 immuno-detection on immuno-isolated inhibitory synaptic vesicles. Monoclonal VGAT antibody was conjugated to Spherobeads (diameter = 1–1.4 µm) and used for positive selection of inhibitory synaptic vesicles followed by post-embedding staining with anti-CLC-3 antibody and secondary goat anti-rabbit IgG conjugated to 15 nm gold particles. Scale bar represents 200 nm. Lower panel: Corresponding enlargements of boxed areas in (b) above.

(a) Representative evoked IPSC traces recorded before (black line) and during extracellular application of 20 mM (red trace) and 50 mM (blue trace) of TRIS. (b) Time course of evoked IPSC peak amplitude changes with bath application of 50 mM TRIS (pH 7.4) during 0.2 Hz stimulation. Average of every 10 eIPSC peaks is shown by the smooth red line. (c) Representative evoked IPSC traces recorded before (black line) and during extracellular application of 20 mM (red trace) of TRIS from a representative CA1 pyramidal neuron from the Clcn3^+/+ and Clcn3^−/− animals. (d) Pooled data showing significantly different (*P < 0.05, **P < 0.005 one-way ANOVA) inhibition of evoked IPSCs with 20 mM TRIS in slices from Clcn3^+/+ (n = 7) and Clcn3^−/− (n = 5) animals and 50 mM (n = 6) TRIS in slices from Clcn3^+/+ animals. (e) Representative time course of miniature IPSC peak amplitude (grey dots) changes with bath application of 50 mM TRIS (pH 7.4). Average of every 10,000 mIPSC peaks is shown by the smooth red line. (f) Representative detected and superimposed mIPSC traces recorded from a CA1 pyramidal cell in Clcn3^+/+ slice at 10 min and 40 min of recording in 50 mM TRIS. Average current traces are shown by the smooth red line. (g) Cumulative amplitude distribution of mIPSCs recorded before (filled gray bars) and 30 min after (hatched red bars) perfusion Clcn3^+/+ slices with 50 mM TRIS; 6,000 events from 6 cells were used for analysis for each group. Cumulative amplitude probability graph of mIPSCs peaks during control (black line) and 50 mM TRIS (red line) from Clcn3^+/+ neurons shows that the amplitude distribution is shifted to the left for events recorded in TRIS indicating smaller mIPSC sizes (P < 0.001 Kolmogorov-Smirnov test). Insert: Box plot analysis of average amplitude of mIPSCs in control period (black) and period with TRIS effect (red) on mIPSC. (h) Data summary demonstrating changes in mIPSC frequency in control period and 30 min after 50 mM TRIS application (Control period: 16.1 ± 1.8 Hz, 30 min in TRIS: 14.6 ± 0.9 Hz, n = 7 cells).

(a) Western blot characterization of rat enriched inhibitory synaptic vesicle fraction immuno-isolated by depletion on magnetic beads coated with anti-VGLUT1 antibodies (see Methods). Upper panel: blot probed with anti-VGLUT1 antibody to show the depletion of VGLUT1 (CSV(−)VGLUT1). Middle panel: probed with anti-VGAT antibody to show VGAT enrichment of (CSV(−)VGLUT1) fraction. Lower panel: probed with anti-VAMP antibody as a load control. (b) Representative traces of acidification in arbitrary fluorescence units (a.u.) of inhibitory (CSV(−)VGLUT1) and excitatory (CSV(−)VGAT) synaptic vesicles as a function of various Cl⁻ concentrations and in the presence of 10 mM GABA or glutamate, respectively. (c) Comparison of the degree of [Cl⁻]-dependent acidification (in arbitrary units) calculated as the fraction of the initial level of acridine orange fluorescence at 2 mM ATP and 0 mM Cl⁻ in inhibitory (blue) vs. excitatory (red) synaptic vesicle fractions in the presence of 10 mM of GABA or glutamate, respectively. (d) Comparison of the rate of [Cl⁻]-dependent acidification in inhibitory (blue) and excitatory (red) synaptic vesicle fractions calculated as the decay time constant (τ) determined from exponential fits to individual traces. Data in c and d are presented as the mean ± standard error (n = 3), (*P < 0.05 one-way ANOVA). N represents a number of preparations each consisting of at least 5 animals. For full-length blots, see Supplementary Figure 6.

Immunofluorescent labeling of the CLC-3 in CA1 area of hippocampus. (a) DIC image and fluorescent immunostaining for CLC-3 (red dots) show perisomatic localization of CLC-3 around CA1 pyramidal cell bodies (*). Calibration bar represents 5 µm. (b–d). Double immunostaining with anti-CLC-3_730–744 (green) and anti-VGAT antibody (red) in the CA1 region reveals prominent co-localization of CLC-3 with the vesicular GABA transporter in the CA1 pyramidal cell layer (pcl). (e) Enlarged image (taken from boxed area in (d) specifies their co-localization to a subset of puncta adjacent to cell bodies of pyramidal neurons. Qualitative analysis of colocalization was done using ImageJ plug-in Jacop. Background subtraction was performed to eliminate noise. Spots analyzed were plotted as the normalized intensity of CLC-3 (green) as a function of VGAT (red). Manders’ Coefficients (with threshold) were 0.44 for fraction of VGAT overlapping ClC-3 and 0.8 for the fraction of CLC-3 overlapping VGAT with an overlap correlation coefficient of 0.92. Calibration bar represents 10 µm. Stratum radiatum = sr, pyramidal cell layer = pcl, and stratum oriens = so.

(a) Representative miniature IPSCs (mIPSCs) traces recorded in the presence of TTX (1 µM) and kynurenic acid (3 mM) from a CA1 pyramidal neuron from Clcn3^+/+ and Clcn3^−/− preparations. (b) Data summary demonstrating a significant decrease in IPSC frequency in Clcn3^−/− neurons (Clcn3^+/+ 14 ± 1.3 Hz, n = 13 cells; Clcn3^−/− 8.4 ± 1.0 Hz, n = 9 cells, (**P < 0.005 One way ANOVA). (c) Representative detected and superimposed mIPSC traces recorded from CA1 pyramidal cells in Clcn3^+/+ and Clcn3^−/− slices. Average current traces are shown by the smooth red line. (d) Cumulative amplitude distribution of mIPSCs recorded from Clcn3^+/+ (filled gray bars) and Clcn3^−/− (hatched red bars) neurons; 3934 events from 7 cells were used for analysis for each group. Superimposed cumulative amplitude probability graph of mIPSCs from Clcn3^+/+ (black line) and Clcn3^−/− (red line) neurons shows that amplitude distribution is shifted to the left for the Clcn3^−/− events indicating smaller mIPSCs in the Clcn3^−/− mice (P < 0.001 Kolmogorov-Smirnov test). Insert: Box plot analysis of average amplitude of mIPSCs from Clcn3^+/+ (black) and Clcn3^−/− (red) neurons.

(a) Averaged traces of [Cl⁻]-dependent acidification of CSV obtained from Clcn3^+/+ (black, n = 10) and Clcn3^−/− (red, n = 9) mice normalized against the original level of acridine orange fluorescence at 2 mM ATP and 0 mM Cl⁻. (b) Effect of CLC-3 on the rates of acidification of Clcn3^+/+ versus Clcn3^−/− CSV calculated as tau of decay from exponential fits of individual traces. Data are presented as a mean ± standard error, Clcn3^+/+ (black, n = 10) and Clcn3^−/−, (red, n = 9); (*P < 0.05 one-way ANOVA). Insert: Exponential fits of individual acidification traces at a single Cl⁻ concentration as an example of differential effect of CLC-3 on the rate of acidification of Clcn3^+/+ versus Clcn3^−/− CSV. (c) Importance of CLC-3 for the degree of acidification of inhibitory synaptic vesicles demonstrated by representative traces of acidification of inhibitory synaptic vesicle (CSV(−)VGLUT1) from Clcn3^−/− (red), Clcn3^+/+ (black) and Clcn3^+/+ CLC-3-depleted (blue) synaptic vesicles. Traces represent preparations each consisting of at least 8 animals. (d–f) Western blot characterization of immunoisolated fractions of synaptic vesicles obtained from Clcn3^+/+ and Clcn3^−/− mice. (d) Upper panel: blot probed with anti-VGLUT1 antibody to show the depletion of VGLUT1 (CSV(−)VGLUT1). Middle panel: probed with anti-VGAT antibody to show VGAT enrichment of (CSV(−)VGLUT1) fraction. Lower panel: probed with anti-VAMP antibody as a load control. (e) Western blot of doubly depleted fraction probed with anti-CLC-3 (upper panel) shows that CSV(−)VGLUT1(−)CLC-3 fraction is in fact devoid of CLC-3. Anti-VAMP detection was used as a load control (lower panel). (f) Characterization of VGLUT1 depletion (upper panel) and VGAT enrichment (middle panel) of SV immunoisolated from Clcn3^−/− mouse. Anti-VAMP detection was performed to control for loading of the lanes. For full-length blots, see Supplementary Figure 5.

(a) Western blot characterization of CSVs, inhibitory synaptic vesicle fraction (CSV(−)VGLUT1) and inhibitory synaptic vesicle fraction depleted of CLC-3 on magnetic beads coated with anti-CLC-3 (CSV(−)VGLUT1(−)CLC-3). Western blot was probed with antibodies against VGLUT1, VGLUT2, VGAT, CLC-3 and vATPase. Blot probed with anti-VAMP2 antibody served as a load control. (b) Representative traces of [Cl⁻]-dependent acidification for inhibitory synaptic vesicles in the presence (black) or absence (red) of CLC-3. Acidification was initiated by addition of 2 mM ATP in the presence of 10 mM GABA. (c) Comparison of levels of [Cl⁻]-dependent acidification of inhibitory synaptic vesicles in the presence (black) or absence (red) of CLC-3 calculated as a fraction of the original level at 2 mM ATP and 0 mM Cl⁻. For full-length blots, see Supplementary Figure 7.