Journal: Nature Communications

Article Title: Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling

doi: 10.1038/s41467-022-35373-4

Figure Lengend Snippet: A Design of the AAV-expression vector with YFP fused to the opsins. B Whole-cell voltage-clamp experiments allowed direct perfusion of the Cal-630 indicator, with high loading efficiency in the soma. C Exemplary activation spectra of CapChR2, the excitation spectrum of Cal-630 and the band-pass filtered light for ChR and Cal-630 excitation. CapChR2 action spectra reproduced from Supplementary Fig. , with n = 6, the excitation spectrum of Cal-630 provided by the manufacturer. D Exemplary live-cell wide-field images of neurons expressing C2-LC-YFP or CapChR2-YFP, overlaid with a maximum intensity projection of the Cal-630 signal obtained during the recording session (scale bars: 10 µm). E Fluorescent images of the Cal-630 signal before (−2 s), after 1 or 4 s of blue light flashes, or 18 s after blue-light illumination from the cells shown in C (scale bars: 10 µm). F Single-trial intensity profiles of somatic Ca 2+ signals evoked by 20 50-ms flashes of 470 nm (1 mW/mm 2 , saturating conditions) at 5 Hz in a C2-LC (red) or CapChR2 (yellow)-expressing neuron. Simultaneously recorded currents are shown underneath. Insets show the current decay after the last light flash. G Comparison of the maximum Δ F / F 0 evoked by blue-light flashes (C2-LC, n = 6; CapChR2, n = 7; unpaired, two-tailed t -test: p = 0.012). H Quantification of the total charge transfer evoked by the opsin stimulation (C2-LC, n = 6; CapChR2, n = 7 unpaired, two-tailed t -test: p = 0.063, no adjustment for multiple comparisons). I Time course of peak currents evoked by 470 nm flashes in C2-LC and CapChR2-expressing neurons (C2-LC, n = 6; CapChR2, n = 7). J Scatter plot of the light-evoked charge transfer against the maximum Ca 2+ signal within single cells. Graphs depict the mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. n = X biologically independent cells. AU arbitrary units.

Article Snippet: For neuronal expression, coding sequences of C2-LC, CapChR 1 and 2 were fused to either mScarlet or YFP and inserted into an AAV expression vector (backbone of Addgene Plasmid #58806) between the neuron-specific human synapsin promoter and the Woodchuck-Hepatitis-Virus Posttranscriptional Response Element (WPRE).

Techniques: Expressing, Plasmid Preparation, Activation Assay, Comparison, Two Tailed Test

Journal: Nature methods

Article Title: Machine learning-guided channelrhodopsin engineering enables minimally-invasive optogenetics

doi: 10.1038/s41592-019-0583-8

Figure Lengend Snippet: Validation of high-performance ChRger2 for minimally-invasive optogenetic behavioral modulation. ( a ) Systemic delivery of rAAV-PHP.eB packaged CAG-DIO ChRger2-TS-eYFP or ChR2(H134R)-TS-eYFP (3×10 11 vg/mouse) into Dat -Cre animals coupled with fiber optic implantation above the VTA enabled blue light-induced intracranial self-stimulation (ten 5 ms laser pulses) exclusively with ChRger2 and not ChR2(H134R) with varying light power and varying stimulation frequencies. ChRger2, n = 4 animals; ChR2(H134R), n = 4 animals. Images show fiber placement and opsin expression for ChR2(H134R) (top) and ChRger2 (bottom). ( b ) Minimally-invasive, systemic delivery of rAAV-PHP.eB packaged CaMKIIa ChRger2-TS-eYFP or ChR2(H134R)-TS-eYFP (5×10 11 vg/mouse) into wild type (WT) animals coupled with surgically secured fiber-optic cannula guide to the surface of the skull above the right M2 that had been thinned to create a level surface for the fiber-skull interface. Three weeks later, mice were trained to walk on a linear-track treadmill at fixed velocity. Coronal slices show expression throughout cortex with higher magnification image of M2 (inset) for ChR2(H134R) (left) and ChRger2 (right). Unilateral blue light stimulation of M2 induced turning behavior exclusively with ChRger2 and not ChR2(H134R) (10 Hz stimulation with 5 ms 447 nm light pulses at 20 mW). ChRger2, n = 5 animals; ChR2(H134R), n = 5 animals. No turning behavior was observed in any animal with 10 Hz stimulation with 5 ms 671 nm light pulses (20 mW). Plotted data are mean±SEM. vg, viral genomes.

Article Snippet: For characterization in neurons, selected ChR variants [ChRger1, ChRger2, ChRger3, CoChR 11 , and hChR2(H134R)] were inserted into a pAAV-hSyn vector backbone [Addgene plasmid #26973], a pAAV-CamKIIa vector backbone [Addgene plasmid #51087], and a pAAV-CAG-DIO vector backbone [Addgene plasmid #104052].

Techniques: Expressing