Figure 10.
GxTX-594 puncta on hippocampal CA1 neurons are sensitive to voltage stimulus.(A) Two-photon excitation optical section from CA1 pyramidal neurons in a cultured hippocampal slice after incubation with 100 nM GxTX-594. Scale bar, 5 μm. (B) 594 fluorescence from a single two-photon excitation optical section before and during depolarization of a whole-cell patch-clamped neuron. Text labels of holding potential (−70 mV or 0 mV) indicate approximate position of the patch-clamp pipette. Red arrows indicate stimulus-sensitive puncta; white arrows indicate stimulus-insensitive puncta. Left panel is the average fluorescence of the three frames at −70 mV before depolarization, while the right panel is the average fluorescence of three frames after holding potential was stepped to 0 mV. Scale bar, 5 μm. Video 1 contains time-lapse images from this experiment. (C) ROIs used in analysis for D–F. Same slice as B. ROI 1 contains the apparent plasma membrane of the cell body of the patch-clamped neuron; it was generated by drawing a path tracing the apparent plasma membrane and then expanding to an ROI containing 15 pixels on either side of the path (1.2 μm total width). ROI 2 contains the area 15–45 pixels outside the membrane path (1.2 μm total width). RO1 3 contains the area >45 pixels outside the membrane path. ROI 0 contains the area >15 pixels inside the membrane path. Scale bar, 5 μm. (D) Fluorescence from each ROI shown in C. Squares represent ROI 0, circles represent ROI 1, up triangles represent ROI 2, and down triangles represent ROI 3. Background was defined as the mean fluorescence of ROI 0 during the experiment. Finit was defined as the mean fluorescence of ROI 1 during the first six frames, after subtraction of background. Dotted lines represent the average fluorescence of the first six frames of each ROI. The voltage protocol is shown above the graph. (E) Change in fluorescence during a 0-mV step for different ROIs in three hippocampal slices from three separate rats. ROIs for each slice were defined by methods described in C. Circles represent mean fluorescence from six frames during 0-mV stimulus (F), normalized to mean fluorescence from the same ROI in six frames before stimulus (Finit). Circle color is consistent between ROIs for each hippocampal slice; red circles are from the slice shown in D. Black bars are arithmetic mean ± SE from three hippocampal slices. A statistical difference between ROIs was detected by ANOVA (P = 0.0003) followed by the Tukey’s post hoc test, where ROI 1 versus ROI 2 (***, P < 0.001), ROI 1 versus ROI 3 (***, P < 0.001), and ROI 2 versus ROI 3 (n.s., P = 0.98). (F) Kinetics of fluorescence change from ROI 1 during a 0-mV step in three hippocampal slices. ROI 1 for each slice was defined by the methods described in C. Lines are monoexponential fits (Eq. 1): kΔF = 6.8 × 10−2 ± 2.6 × 10−2 s−1 (yellow), 6.8 × 10−2 ± 4.6 × 10−2 s−1 (red), and 4.9 × 10−2 ± 2.10−2 s−1 (green). The voltage protocol is shown above the graph. Colors of individual circles indicate the same slices as E. (G) ROI used in analysis for H–J. Same image as C. The shaded ROI was generated by drawing a path tracing the apparent plasma membrane and then expanding to an ROI containing 5 pixels on either side of the path (0.4 μm total width). Numbers indicate puncta that appear as peaks in H. Scale bar, 5 μm. (H) A plot of the fluorescence intensity along the ROI shown in G before (red trace) and during (black trace) 0-mV stimulus. Numbers above peaks correspond to puncta labeled in G. Red trace: Mean fluorescence intensity during the three frames immediately before the stimulus, normalized to mean intensity of entire ROI (black dotted line), plotted against distance along path. Black trace: Mean fluorescence intensity during three frames at 0 mV, normalized by the same Finit value as the red trace. (I) Kinetics of fluorescence change of individual puncta from G. Puncta intensity are average intensity of points extending to half maximum of each peak in H. Asterisks indicate mean fluorescence intensity of puncta 1 (pink), 4 (red), and 5 (dark red). Lines are monoexponential fits (Eq. 1): kΔF = 7.10−2 ± 2.9 × 10−2 s−1 (pink), 6.7 × 10−2 ± 2.5 × 10−2 s−1 (red), and 1.2 × 10−1 ± 5.6 × 10−2 s−1 (dark red). Fits to other puncta had SDs larger than kΔF values and were excluded. Finit was defined as the mean background-subtracted fluorescence of the puncta during the six frames before stimuli. The voltage protocol is displayed above the graph. Blue line is prediction from Scheme 1. (J) Comparison of fluorescence change of puncta (above average) and interpuncta (below average) regions in response to 0-mV stimulus. The regions before stimulus shown by the red line of H that had F/Finit ≥1 (above average) were binned separately from regions with F/Finit <1. The mean fluorescence of each region during 0-mV stimulus (H, black trace) was compared with the fluorescence before stimulus (H, red trace). Circles indicate values from three independent hippocampal slices; colors indicate same slices as E and F. A weak statistical difference in fluorescence was detected between interpuncta and puncta regions by Student’s t test (*, P = 0.046). Black bars are arithmetic mean ± SE. (K) Rate of fluorescence change of GxTX-594 after a 0-mV stimulus from puncta (as in I), ROI 1 (as in F), or Kv2.1-CHO cells in 100 nM GxTX-594. Kv2.1-CHO cells were imaged at the same temperature as neurons (30°C) using neuronal intracellular solution. CHO CE solution was used for Kv2.1-CHO cell experiments. Kv2.1-CHO measurements were made by Airy disk confocal imaging. Black bars are mean ± SEM from data shown. Blue dotted line is kΔF = 6.31 × 10−2 s−1 prediction of Scheme 1. No statistical difference was detected between groups by ANOVA (P = 0.11).

GxTX-594 puncta on hippocampal CA1 neurons are sensitive to voltage stimulus. (A) Two-photon excitation optical section from CA1 pyramidal neurons in a cultured hippocampal slice after incubation with 100 nM GxTX-594. Scale bar, 5 μm. (B) 594 fluorescence from a single two-photon excitation optical section before and during depolarization of a whole-cell patch-clamped neuron. Text labels of holding potential (−70 mV or 0 mV) indicate approximate position of the patch-clamp pipette. Red arrows indicate stimulus-sensitive puncta; white arrows indicate stimulus-insensitive puncta. Left panel is the average fluorescence of the three frames at −70 mV before depolarization, while the right panel is the average fluorescence of three frames after holding potential was stepped to 0 mV. Scale bar, 5 μm. Video 1 contains time-lapse images from this experiment. (C) ROIs used in analysis for D–F. Same slice as B. ROI 1 contains the apparent plasma membrane of the cell body of the patch-clamped neuron; it was generated by drawing a path tracing the apparent plasma membrane and then expanding to an ROI containing 15 pixels on either side of the path (1.2 μm total width). ROI 2 contains the area 15–45 pixels outside the membrane path (1.2 μm total width). RO1 3 contains the area >45 pixels outside the membrane path. ROI 0 contains the area >15 pixels inside the membrane path. Scale bar, 5 μm. (D) Fluorescence from each ROI shown in C. Squares represent ROI 0, circles represent ROI 1, up triangles represent ROI 2, and down triangles represent ROI 3. Background was defined as the mean fluorescence of ROI 0 during the experiment. Finit was defined as the mean fluorescence of ROI 1 during the first six frames, after subtraction of background. Dotted lines represent the average fluorescence of the first six frames of each ROI. The voltage protocol is shown above the graph. (E) Change in fluorescence during a 0-mV step for different ROIs in three hippocampal slices from three separate rats. ROIs for each slice were defined by methods described in C. Circles represent mean fluorescence from six frames during 0-mV stimulus (F), normalized to mean fluorescence from the same ROI in six frames before stimulus (Finit). Circle color is consistent between ROIs for each hippocampal slice; red circles are from the slice shown in D. Black bars are arithmetic mean ± SE from three hippocampal slices. A statistical difference between ROIs was detected by ANOVA (P = 0.0003) followed by the Tukey’s post hoc test, where ROI 1 versus ROI 2 (***, P < 0.001), ROI 1 versus ROI 3 (***, P < 0.001), and ROI 2 versus ROI 3 (n.s., P = 0.98). (F) Kinetics of fluorescence change from ROI 1 during a 0-mV step in three hippocampal slices. ROI 1 for each slice was defined by the methods described in C. Lines are monoexponential fits (Eq. 1): kΔF = 6.8 × 10−2 ± 2.6 × 10−2 s−1 (yellow), 6.8 × 10−2 ± 4.6 × 10−2 s−1 (red), and 4.9 × 10−2 ± 2.10−2 s−1 (green). The voltage protocol is shown above the graph. Colors of individual circles indicate the same slices as E. (G) ROI used in analysis for H–J. Same image as C. The shaded ROI was generated by drawing a path tracing the apparent plasma membrane and then expanding to an ROI containing 5 pixels on either side of the path (0.4 μm total width). Numbers indicate puncta that appear as peaks in H. Scale bar, 5 μm. (H) A plot of the fluorescence intensity along the ROI shown in G before (red trace) and during (black trace) 0-mV stimulus. Numbers above peaks correspond to puncta labeled in G. Red trace: Mean fluorescence intensity during the three frames immediately before the stimulus, normalized to mean intensity of entire ROI (black dotted line), plotted against distance along path. Black trace: Mean fluorescence intensity during three frames at 0 mV, normalized by the same Finit value as the red trace. (I) Kinetics of fluorescence change of individual puncta from G. Puncta intensity are average intensity of points extending to half maximum of each peak in H. Asterisks indicate mean fluorescence intensity of puncta 1 (pink), 4 (red), and 5 (dark red). Lines are monoexponential fits (Eq. 1): kΔF = 7.10−2 ± 2.9 × 10−2 s−1 (pink), 6.7 × 10−2 ± 2.5 × 10−2 s−1 (red), and 1.2 × 10−1 ± 5.6 × 10−2 s−1 (dark red). Fits to other puncta had SDs larger than kΔF values and were excluded. Finit was defined as the mean background-subtracted fluorescence of the puncta during the six frames before stimuli. The voltage protocol is displayed above the graph. Blue line is prediction from Scheme 1. (J) Comparison of fluorescence change of puncta (above average) and interpuncta (below average) regions in response to 0-mV stimulus. The regions before stimulus shown by the red line of H that had F/Finit ≥1 (above average) were binned separately from regions with F/Finit <1. The mean fluorescence of each region during 0-mV stimulus (H, black trace) was compared with the fluorescence before stimulus (H, red trace). Circles indicate values from three independent hippocampal slices; colors indicate same slices as E and F. A weak statistical difference in fluorescence was detected between interpuncta and puncta regions by Student’s t test (*, P = 0.046). Black bars are arithmetic mean ± SE. (K) Rate of fluorescence change of GxTX-594 after a 0-mV stimulus from puncta (as in I), ROI 1 (as in F), or Kv2.1-CHO cells in 100 nM GxTX-594. Kv2.1-CHO cells were imaged at the same temperature as neurons (30°C) using neuronal intracellular solution. CHO CE solution was used for Kv2.1-CHO cell experiments. Kv2.1-CHO measurements were made by Airy disk confocal imaging. Black bars are mean ± SEM from data shown. Blue dotted line is kΔF = 6.31 × 10−2 s−1 prediction of Scheme 1. No statistical difference was detected between groups by ANOVA (P = 0.11).

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