HALO 1 TERMINALS FREE

HALO 1 TERMINALS FREE

О”F/F was calculated based on baseline fluorescence in L-lactate free medium (g) Average О”F/F during baseline and light-on periods shown in f. (f) Time course of О”F/F in neurons recorded in TTX (1 ОјM, red, n = 10) or in TTX and L-lactate (50 mM, pH = 7.3 orange, n = 8). (e) Quantification of average EPSC rates during baseline and light-on periods ( n = 5, P = 0.24). (d) Time course of EPSC rates recorded in Ca 2+-free medium.

(c) Quantification of average О”F/F during basline and light-on periods shown in b ( n = 10, P = 6.21Г—10 вЂ“3). (b) Time course of О”F/F in Ca 2+-free medium. AF/F was calculated based on baseline fluorescence in 2 mM Ca 2+. Representative images depict average fluorescence during a 5 min baseline period followed by 5 min of 590 nm illumination. (a) Experimental time course: neurons expressing eArch3.0 and SyGCaMP6s were recorded in Ca 2+-free medium, followed by reintroduction of Ca 2+ into the extracellular medium. (* P < 0.05 see Supplementary Table 1 for statistics). (l,n) Quantification of EPSC and IPSC rates from k and m. Insets depict sample eArch3.0 traces from baseline (top), light (middle) and post-light (bottom) periods (scale bars, 40 pA, 200 ms). (k,m) Effect of constant illumination on spontaneous EPSC (k) and IPSC (m) rates (z-scores). (j) Quantification of average fluorescence during baseline, light and post-light periods shown in i. Paired pulse ratio: n = 14) (i) SyGCaMP6s imaging of presynaptic boutons during the same light application protocol. ((e) eEPSC amplitude: n = 10 Paired pulse ratio: n = 9. (e,h) eEPSC amplitude relative to baseline and paired-pulse ratio during 5 min constant illumination. (c,d,f,g) Quantification of eEPSCs before and during brief light application (200 ms, 590 nm, 2 mW mm вЂ“2 (c) n = 24, P = 0.019. Black ticks indicate time of electrical stimulation. Individual traces are overlaid with their mean. (b) Example traces of EPSCs evoked by electrical microstimulation (eEPSCs) with and without light application to the presynaptic terminals. Yellow circle indicates area of light application for opsin acivation. The bipolar stimulation electrode was placed in the thalamocortical fiber tract, while whole-cell voltage clamp recordings were performed in layer 4 of barrel cortex.

(a) Schematic illustration of the experimental setup. (* P < 0.01 see Supplementary Table 1 for statistics).Д¬haracterization of presynaptic terminal inhibition in acute brain slices using the microbioal ion transporters eArch3.0 and eNpHR3.0. Error bars and shaded regions indicate s.e.m.

(h) Peak EPSC rates during illumination relative to peak EPSC rates during baseline. (g) Average EPSC rates during baseline, light and post-light periods, as depicted in f (control, n = 11 eNpHR3.0, n = 12 eArch3.0, n = 12). Inset depicts representative voltage clamp recording traces of an eArch3.0 expressing culture before (top), during (middle) and after (bottom) 590 nm illumination (scale bar, 30 pA, 150 ms). (f) Average EPSC rates recorded in neuronal cultures expressing the indicated constructs. (e) Peak SyGCaMP6s fluorescence during 590 nm illumination relative to peak SyGCaMP6s fluorescence during baseline activity of neurons expressing SyGCaMP6s (control) or co-expressing SyGCaMP6s with eArch3.0 or eNpHR3.0. Neurons co-expressing eArch3.0 ( n = 12) showed increased SyGCaMP6s signal compared with neurons expressing eNpHR3.0 ( n = 12) and controls ( n = 8). (d) Average О”F/F during baseline, light (590 nm, 2 mW mm вЂ“2) and post-light periods depicted in c. (cвЂ“d) Relative fluorecence change from baseline ( О”F/F) of spontaneously active neuronal cultures expressing SyGCaMP6s. (b) Representative time-averaged images acquired during the indicated time periods from a hippocampal neuron culture co-expressing SyGCaMP6s and eArch3.0. Left: cultured neurons expressing SyGCaMP6s center: anti-Synapsin I labeling right: merge. (a) Evaluation of SyGCaMP6s targeting to the presynaptic terminal.

Halo 1 terminals