Figure 1.
Distributions of GABA receptors in CA1 pyramidal neurons investigated by 2P uncaging of GABA.
A, Representative traces for IPSCs evoked by 2P uncaging of BCMACM-GABA. Each current trace was evoked at the corresponding numbers on the fluorescence images in (B, D). B, D, E, Mapping of functional GABA receptors. The upper, middle and lower panels represent the fluorescence images, maps of GABA-induced currents, and their overlays, respectively. Scale bar represents 5 µm. (B) Two-dimensional (2D) map at the soma. BCMACM-GABA (6 mM) was uncaged with the mode-locked laser at 800 nm (8 mW, 1 ms). The interval between pixels was 0.9 µm. (D, E) Three-dimensional (3D) maps of apical dendrites. The interval between pixels was 1.6 µm (D) or 1.3 µm (E). We obtained 2D maps at three consecutive z-axis planes with an interval of 5 µm (D) or 1.5 µm (E), and represented with the maximal intensity projection in the 3D maps. C, Spatial resolution of 2P mapping. The smooth line represents Gaussian fitting of the data from a GABA hot spot in (B). The FWHM lateral resolution was determined to be 0.85 µm. F, Clustering of GABA receptors at the branch points of the major dendritic trunks. Amplitudes of IPSCs along the dendrites were normalized by the maximal amplitude of IPSCs at the branch points. Horizontal bars represent the mean values. Filled and open circles represent data obtained from dendritic locations distal and proximal to the branch point, respectively. The normalized values differ from 1.0 with ** P<0.01 (t-test, n = 8–16).
Figure 2.
Distributions of GABA receptors investigated with 1P uncaging of GABA.
A, Representative 1pIPSCs. Numbers correspond to those in the fluorescence image (B). B, Two-dimensional map of functional GABA receptors at the apical dendrite. Upper, middle and lower images represent the fluorescence image, the map, and their overlay, respectively. Scale bar represents 10 µm. RuBi-GABA (0.2 mM) was uncaged by the 473 nm laser (0.5 mW, 4 ms) at each pixel. The interval of pixels was 2.7 µm. C, Spatial resolution of 1P mapping. The smooth line represents Gaussian fitting of the data shown in (B). The lateral FWHM resolution was estimated as 7.2 µm.
Figure 3.
Inhibition of bAP-induced Ca2+ transients by uncaging of RuBi-GABA at oblique dendrites.
A, Fluorescence image (Alexa-594) of a dendrite investigated in (B,C). The red circle in the branch (Br) 1 represents uncaging point. Line scanning was performed along the yellow line for Ca2+ measurements. B, Current trace of 1pIPSC evoked at −20 mV. Uncaging of RuBi-GABA was applied to the red circle in (A). The red vertical line represents the time of GABA uncaging (0.5 mW, 4 ms). C, Normalized changes (ΔF/F) in fluorescence of OGB-1 (0.1 mM) evoked by a single bAP. Inhibition of the Ca2+ transients occurred at branch 1, but not at branch 2. Uncaging was applied 10 ms after bAP. Blue and red vertical lines denote the times for bAP induction and GABA uncaging, respectively. We masked the traces during 473 nm laser irradiation for GABA uncaging. D, Inhibition of bAP-induced Ca2+ transients in the branches which were applied with GABA uncaging or not. The amplitudes of the Ca2+ transients with GABA uncaging were normalized by those without. Bars represent the mean ± SEM. The relative values are different from 1.0 with ** P<0.01 (t-test, n = 8). E, Time dependence of Ca2+ inhibition by GABA uncaging. Uncaging was applied 0 ms, 10 ms, or 20 ms after bAP induction. Ca2+ imaging was performed at the site of uncaging. The relative values differ from 1.0 with ** P<0.01 (t-test, n = 6–10). F, Example of the effect of GABA uncaging on spike after-depolarization. G, Laser-power dependence of the amplitudes of 1pIPSCs induced by GABA uncaging at the soma. The correlation is significant (P<0.001). H, Laser-power dependence of inhibition of the Ca2+ transients by GABA uncaging at oblique dendrites. The correlation is significant (P<0.001).
Figure 4.
Spatial confinement of GABA inhibition on the Ca2+ transients evoked by single bAP.
A, Fluorescence image (Alexa-594) of the cell where imaging (yellow line) and uncaging (red pints) were applied to an oblique branch. B, Distance dependence of inhibition of the Ca2+ transients by GABA uncaging. Uncaging of RuBi-GABA was applied at 0 µm, 10 µm, and 20 µm proximal to the imaging site at 0 ms after bAP induction. Bars represent mean ± SEM. The relative values differ from 1.0 with ** P<0.01 and n.s. P>0.05 (t-test, n = 5–7). C, Fluorescence image of the cell where imaging (yellow line) was performed at an oblique branch, while uncaging (red pints) were applied either at the imaging site (IS) or the branch point (BP) of the major apical dendritic trunk. D, Inhibition of the Ca2+ transients by GABA uncaging at imaging site (IS) or branch point (BP). Uncaging was applied at IS (a) or BPs which were 20 µm (b, c) or 30–40 µm (d) proximal to the imaging site. Inhibition was applied at 0 ms after AP induction. Laser powers were reduced to 50% in (c) relative to (b), which resulted in significant reduction of inhibition (P<0.05 with paired t-test, n = 6). The relative values differ from 1.0 with ** P<0.01 and n.s. P>0.05 (t-test, n = 6–17).