Figure 4.
Neurons lacking Ecm29 exhibit a delayed GABAergic excitation-to-inhibition switch and an altered GABA-induced calcium response. (A) Ratiometric fluorescent imaging of hippocampal or cortical neurons transfected with the genetically encoded, membrane-bound chloride indicator PalmPalm-ClopHensor to assess chloride fluxes. (A1) Top: Schematic illustrating Cl−-dependent static quenching of ClopHensor fluorescence upon excitation with blue laser (λex = 445 nm). Kd, dissociation constant. Middle: Representative images depicting chloride levels, as indicated by the PalmPalm-ClopHensor signal calculated as the ratio of cyan to DsRed fluorescence (F(cyan)/F(DsRed)) acquired from a 7-DIV neuron. Bottom: Background substrate images and traces corresponding to the outlined region in the middle panel, indicating inward chloride transitions (arrows) after 20 µM focal GABA application. Scale bar, 20 µm. (A2) Representative traces depicting GABA-induced fluxes in wild-type (Ecm29+/+) and Ecm29 KO (Ecm29−/−) neurons at 5–9 DIV, before and after 20 µM focal GABA application. Blue traces denote chloride influxes; black traces denote chloride effluxes or no response. Arrows indicate time points of focal GABA addition. (A3) Experiments similar to A2, except cells were transfected with plasmid encoding FLAG-tagged Ecm291–1,840 (FLAG-Ecm29FL) or pretreated with the NKCC1 inhibitor BUM (10 µM; for 16 h before experiments) as indicated. (B) Averaged GABA-induced changes in the cyan-to-DsRed ratio (denoted ΔRCl−) at the proximal axon region reveals a prolonged excitatory period in Ecm29 KO (Ecm29−/−) neurons in all experiments performed as described in A. Data represent mean ± SEM (n ≥ 25 cells for each group from at least three independent experiments). (C) Summary of percentages of neurons exhibiting different GABA-induced fluxes in the proximal axon region in cultures described in A. Data points connected by dashed lines differ significantly (mean ± SEM, n = 90 cells; *, P < 0.05; **, P < 0.001, compared with corresponding sets in control wild-type (Ecm29+/+) neurons, based on multiple t tests). (D) Schematic showing GABA-induced chloride fluxes for different cell regions, and table summarizing timing of the switch in GABA response from chloride efflux to influx for each indicated group. (E1 and E2) Young Ecm29−/− hippocampal neurons (5–9 DIV) exhibit a higher Ca2+ influx than wild-type Ecm29+/+ neurons in response to a GABA stimulus (20 µM) as indicated in representative images (E1, 9-DIV cultures) and traces (E2, 5–14-DIV cultures), based on analysis with the calcium-sensitive indicator Fluo-4. n = 25–175 cells; *, P < 0.05; **, P < 0.001, compared with the wild-type (Ecm29+/+) group by multiple t tests. Scale bars, 50 μm. Arrow denotes addition of GABA. (E3) Histograms show average percentages of neurons exhibiting Ca2+ responses to focal GABA application. Data represent mean ± SEM, n = 25–175; **, P < 0.01, t test.

Neurons lacking Ecm29 exhibit a delayed GABAergic excitation-to-inhibition switch and an altered GABA-induced calcium response. (A) Ratiometric fluorescent imaging of hippocampal or cortical neurons transfected with the genetically encoded, membrane-bound chloride indicator PalmPalm-ClopHensor to assess chloride fluxes. (A1) Top: Schematic illustrating Cl−-dependent static quenching of ClopHensor fluorescence upon excitation with blue laser (λex = 445 nm). Kd, dissociation constant. Middle: Representative images depicting chloride levels, as indicated by the PalmPalm-ClopHensor signal calculated as the ratio of cyan to DsRed fluorescence (F(cyan)/F(DsRed)) acquired from a 7-DIV neuron. Bottom: Background substrate images and traces corresponding to the outlined region in the middle panel, indicating inward chloride transitions (arrows) after 20 µM focal GABA application. Scale bar, 20 µm. (A2) Representative traces depicting GABA-induced fluxes in wild-type (Ecm29+/+) and Ecm29 KO (Ecm29−/−) neurons at 5–9 DIV, before and after 20 µM focal GABA application. Blue traces denote chloride influxes; black traces denote chloride effluxes or no response. Arrows indicate time points of focal GABA addition. (A3) Experiments similar to A2, except cells were transfected with plasmid encoding FLAG-tagged Ecm291–1,840 (FLAG-Ecm29FL) or pretreated with the NKCC1 inhibitor BUM (10 µM; for 16 h before experiments) as indicated. (B) Averaged GABA-induced changes in the cyan-to-DsRed ratio (denoted ΔRCl−) at the proximal axon region reveals a prolonged excitatory period in Ecm29 KO (Ecm29−/−) neurons in all experiments performed as described in A. Data represent mean ± SEM (n ≥ 25 cells for each group from at least three independent experiments). (C) Summary of percentages of neurons exhibiting different GABA-induced fluxes in the proximal axon region in cultures described in A. Data points connected by dashed lines differ significantly (mean ± SEM, n = 90 cells; *, P < 0.05; **, P < 0.001, compared with corresponding sets in control wild-type (Ecm29+/+) neurons, based on multiple t tests). (D) Schematic showing GABA-induced chloride fluxes for different cell regions, and table summarizing timing of the switch in GABA response from chloride efflux to influx for each indicated group. (E1 and E2) Young Ecm29−/− hippocampal neurons (5–9 DIV) exhibit a higher Ca2+ influx than wild-type Ecm29+/+ neurons in response to a GABA stimulus (20 µM) as indicated in representative images (E1, 9-DIV cultures) and traces (E2, 5–14-DIV cultures), based on analysis with the calcium-sensitive indicator Fluo-4. n = 25–175 cells; *, P < 0.05; **, P < 0.001, compared with the wild-type (Ecm29+/+) group by multiple t tests. Scale bars, 50 μm. Arrow denotes addition of GABA. (E3) Histograms show average percentages of neurons exhibiting Ca2+ responses to focal GABA application. Data represent mean ± SEM, n = 25–175; **, P < 0.01, t test.

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