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. 2023 Jun 2;17:1166480. doi: 10.3389/fncel.2023.1166480

FIGURE 3.

FIGURE 3

Simultaneous measurement of norepinephrine using GRABNE fluorescent sensor and electrophysiological recording in CA1 neurons. (A) Schematic drawing of the design of simultaneous imaging and electrophysiological recording experiments in cultured rat hippocampal slices. The red arrow indicates the patch electrode of control cell and green arrow indicates that of sensor-expressing cell. The white squares indicating regions of interest (ROIs). (B) Left, simultaneous fluorescence (upper panel) and electrophysiological (lower panel) responses of control non-expressing and GRABNE-expressing CA1 neurons to a brief puff application of NE. Right, the responses in the left rectangle box are shown again on an expanded time scale. (C) Values for the fluorescence responses of GRABNE-expressing CA1 neurons compared to non-expressing CA1 neurons (GRABNE: 8.68 ± 1.41%; Ctrl: 0.12 ± 0.01%; p < 0.0010; n = 7). (D) Values for the amplitudes of adrenergic currents in GRABNE expressing CA1 neurons compared to non-expressing CA1 neurons (GRABNE: 23.80 ± 2.95 pA; Ctrl: 23.99 ± 3.59 pA; p > 0.99; n = 9). (E) Values for the signal-to-noise ratio (SNR) of fluorescence responses of GRABNE-expressing CA1 neurons compared to non-expressing CA1 neurons (GRABNE: 92.61 ± 19.87; Ctrl: 2.43 ± 0.39; p < 0.0010; n = 9), and that of adrenergic currents of GRABNE-expressing CA1 neurons compared to non-expressing CA1 neurons (GRABNE: 9.78 ± 1.80; Ctrl: 10.15 ± 2.80; p = 0.67; n = 9). Values for the SNR of adrenergic currents in GRABNE-expressing CA1 neurons (9.78 ± 1.80; p < 0.0010; n = 9) compared to those of the fluorescence responses of GRABNE-expressing CA1 neurons (92.61 ± 19.87; n = 9). (F) Values for the latencies of adrenergic currents in non-expressing (Ctrl: 0.65 ± 0.14 s; p = 0.0091; n = 9) and GRABNE-expressing (GRABNE: 0.69 ± 0.15 s; p = 0.0051; n = 9; Ctrl vs. GRABNE current: p = 0.96; n = 9) CA1 neurons compared to those of fluorescence responses of GRABNE-expressing CA1 neurons (GRABNE: 0.12 ± 0.02 s; n = 9). (G) Values for rise time of the adrenergic currents in non-expressing (Ctrl: 4.31 ± 0.82 s; p = 0.014; n = 8) and GRABNE-expressing (GRABNE: 3.94 ± 0.55 s; p = 0.016; n = 8) CA1 neurons compared to those of fluorescence responses of GRABNE-expressing CA1 neurons (GRABNE: 1.37 ± 0.17 s; n = 8). (H) Values for decay time of the adrenergic currents in non-expressing (Ctrl: 17.08 ± 2.27 s; p = 0.21; n = 9) and GRABNE-expressing (GRABNE: 18.90 ± 2.50 s; p = 0.086; n = 9) neurons compared to those of fluorescence responses of GRABNE-expressing CA1 neurons (GRABNE: 14.50 ± 1.38 s; n = 9). Data are shown as mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05, two-tailed Student’s unpaired t-test in panels (C,D,G,H), ###p < 0.001, ##p < 0.01, one-way ANOVA with Dunnett’s post-hoc test in panels (E,F).