Article Figures & Data
Figures
- Figure 1.
The AP threshold is substantially lowered in CA3 pyramidal cells during conditions of chloride loading. A, Representative slice used for experiments as seen three weeks following viral delivery of AAV-hSyn-NpHR3.0-EYFP into the hippocampus. A biocytin-filled CA3 pyramidal cell is shown within the same slice. B, In ACSF conditions and during 5-min light application, spontaneously APs arises at a much lowered AP threshold. Periods (1–2) from the same trace are magnified and shown below. C, AP frequency during 5-min light for ACSF (n = 6 slices from three animals) and ACSF + NBQX + AP5 (n = 8 slices from three animals) conditions shown in 60-s bins. D, The same cell as depicted in B before light application, displaying APs evoked by current injection. Top trace (1) shows a 500-pA ramp depolarization, whereas bottom trace (2) shows a step current depolarization from the same cell. E, Examples of APs elicited spontaneously when a cell was recorded with a pipette containing high concentration of chloride. Periods (1-2) from the same trace are magnified and shown below. F, Basic properties of APs evoked by current ramp injection via patch pipette (current), elicited spontaneously during light application (light), and elicited spontaneously during high-chloride loading via patch pipette (chloride). All three groups were compared by one-way ANOVA with Dunnett’s post hoc test and significance is denoted by #, with current serving as the control group, #p < 0.05, ###p < 0.001; threshold F(2,17) = 23.62, p < 0.0001; amplitude F(2,17) = 4.77, p = 0.02; overshoot F(2,17) = 3.50, p = 0.053; duration F(2,17) = 0.66, p = 0.53. A paired t test (denoted by *) directly compares APs induced by current and light within the same cells; *p < 0.05, **p < 0.01. G, Phase plot showing the slope trajectory during the entire AP cycle of the average APs elicited by current, light, and chloride conditions. Current and light group, n = 6 neurons from four animals; chloride group, n = 8 neurons from four animals. H, Synaptically evoked AP in the presence of NBQX and AP5. Trace 1, AP threshold determined by step current injection. Trace 2, AP threshold determined by stimulating putative perisomatic GABAergic neurons. Trace 3, Magnified from trace 2, showing two repetitive stimulations with a ∼2.5-s delay from the stimulation artifact to the onset of depolarization. One stimulation give rise to AP (black trace) whereas the other give rise to an EPSP (blue trace). The AP threshold determined for current injection and electrical stimulation is summarized on the right graph, **p < 0.01. In B, D, E, H, red colored text/circle denotes critical values/points of the membrane potential. In magnified traces in B, E, H, the top part of the AP is truncated. In C, F, H, data are shown as mean ± SEM.
- Figure 2.
Long-term hyperpolarization using eNpHR3.0 renders the CA3 network into a hyperexcitable condition. A, Whole-cell patch-clamp (WC) recordings of individual CA3 pyramidal cells were performed in physiologic ACSF. Top trace shows the entire recording of a representative cell at its RMP in current-clamp mode (0 pA). Light was applied for 5 min. Various periods (1-4) from the same trace are magnified and shown below. The top part of the APs is truncated in magnified trace 2. PSPs are shown in bins (10 and 60 s) and quantified by number (F(2,13) = 5.17, p = 0.02), amplitude (F(2,13) = 7.64, p = 0.006), and strength (F(2,13) = 4.81, p = 0.03), n = 6 slices from three animals. B, For field recordings, the placement of the electrode in stratum pyramidale CA3 was guided by electrical stimulation before the light experiment. C, Same as A, but for field recordings in eNpHR3.0-transfected slices, F(2,24) = 5.20, p = 0.01, n = 9 slices from five animals. D, Same as A, but performed in slices from naïve animals (i.e., no eNpHR3.0 expression), number: F(2,12) = 0.55, p = 0.59; amplitude: F(2,12) = 1.97, p = 0.18; strength: F(2,12) = 2.00, p = 0.18, n = 5 slices from two animals. E, Same as C, but performed in slices from naïve animals, F(2,9) = 1.0, p = 0.41, n = 4 slices from two animals. All data are shown as mean ± SEM for 10- and 60-s bins. All comparisons for 60-s bins were made by one-way ANOVA with Dunnett’s post hoc test with prelight period serving as the control, *p < 0.05, **p < 0.01. Yellow bar indicates the period when light was applied, and red line in A, D shows the detection threshold.
- Figure 3.
GABAergic neurotransmission is a main trigger of the hyperexcitable condition caused by eNpHR3.0 activation. A, Whole-cell patch-clamp recording of CA3 pyramidal cells were performed in physiologic ACSF together with TTX, and light was applied for 5 min. Top, Representative trace with magnified traces (1-2) shown on the right. PSPs are shown in bins (10 and 60 s) and quantified by number (F(2,24) = 8.87, p = 0.0013), amplitude (F(2,20) = 3.29, p = 0.058), and strength (F(2,24) = 7.57, p = 0.0028); n = 9 slices from four animals. B, Same as A, but performed in physiologic ACSF together with NBQX + AP5, number: F(2,21) = 34.12, p < 0.0001; amplitude: F(2,17) = 33.1, p < 0.0001; strength: F(2,21) = 32.47, p < 0.0001, n = 8 slices from three animals. C, Same as A, but performed in physiologic ACSF together with NBQX + AP5 + TTX, number: F(2,10) = 4.77, p = 0.035; amplitude: F(2,6) = 1.01, p = 0.42; strength: F(2,10) = 2.68, p = 0.12, n = 5 slices from two animals. D, Same as A, but performed in physiologic ACSF together with NBQX + AP5 + PTX, number: F(2,18) = 4.52, p = 0.027; amplitude: F(2,9) = 1.67, p = 0.24; strength: F(2,18) = 5.82, p = 0.011, n = 7 slices from three animals. All data are shown as mean ± SEM in 10- and 60-s bins, as indicted, and analyzed using one-way ANOVA with Dunnett’s post hoc test with prelight period serving as the control, *p < 0.05, **p < 0.01, ***p < 0.001. Yellow bar indicates the period when light was applied, whereas red line shows the detection threshold.
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