Journal: Nature Communications

Article Title: Engineering of a bona fide light-operated calcium channel

doi: 10.1038/s41467-020-20425-4

Figure Lengend Snippet: Data are shown as mean ± s.e.m. a Schematic depiction of photo-switchable Ca 2+ influx through an engineered ORAI1 Ca 2+ channel. The LOV2 domain is inserted into the intracellular loop of a constitutively active ORAI1 (caORAI1), which maintains the hybrid channel in a largely closed state in the dark. Upon photosimulation at 470 nm, conformational changes within LOV2 trigger allosteric activation of engineered ORAI1 to evoke Ca 2+ flux across the plasma membrane. b The fold-change of photo-induced Ca 2+ responses reported by GCaMP6m in HeLa cells expressing LOV2–ORAI1 hybrid variants. LOV2 was inserted between residues R167 and M168. The mutant P245T showed the most notable light-induced changes in intracellular Ca 2+ (designated LOCa1). n = 24–52 cells. c Snake-like diagram of the ORAI1 Ca 2+ channel. The LOV2 insertion sites tested in the study are indicated as arrowheads. TM3 and the second extracellular loop regions targeted for randomized mutagenesis are highlighted in green. d Comparison of light-induced Ca 2+ changes after inserting LOV2 into the indicated positions of the ORAI1-P245T variant. S6 showed the highest light-dependent changes and was named as LOCa2. n = 22-66 cells from three independent assays. e Light-induced changes in cytosolic Ca 2+ reported by GCaMP6m in HeLa cells transfected with LOCa2. Two cycles of light stimulation were applied. The domain architecture of the construct is shown above the curve. Blue bar, photo-illumination at 470 nm with a power density of 40 µW/mm 2 . n = 24 cells from three independent assays. f Blue light modulated Ca 2+ entry (top) and NFAT nuclear translocation (bottom) in HeLa cells expressing LOCa2. Scale bar, 10 μm. g The experimental flow for high-throughput screening of evolved LOCa constructs. h Biplot showing the degrees of NFAT nuclear translocation in HeLa cells expressing evolved mutants, either in the dark ( x -axis) or under photo-illumination ( y -axis). The construct showing the least dark activation and high Ca 2+ response in the lit-condition was designated LOCa3, which bears two mutations in ORAI1: H171D and P245T. i Representative confocal images showing blue light-triggered Ca 2+ response and changes in subcellular localization of NFAT 1-460 -GFP. Scale bar, 10 μm.

Article Snippet: Images were acquired every 1 min for 15 min under blue light stimulation (470 nm, 40 μW/mm 2 , ThorLabs, Inc.).

Techniques: Activation Assay, Clinical Proteomics, Membrane, Expressing, Mutagenesis, Comparison, Variant Assay, Transfection, Construct, Translocation Assay, High Throughput Screening Assay

Journal: Nature Communications

Article Title: Engineering of a bona fide light-operated calcium channel

doi: 10.1038/s41467-020-20425-4

Figure Lengend Snippet: Data are shown as mean ± s.e.m. a A typical reversible influx of Ca 2+ (blue), but not Sr 2+ (black), reported by a red genetically encoded Ca 2+ indicator (jRCaMP1b) in ORAI-null HEK293 cells transfected with LOCa3. Cells were cultured in an imaging buffer containing 2 mM Sr 2+ or Ca 2+ and then subjected to three repeated light–dark cycles of stimulation. The activation and deactivation half-lives were 48.7 ± 4.5 s and 56.8 ± 3.8 s, respectively. ORAI1 channel is known to uniquely prohibit Sr 2+ influx because of its strict ion selectivity toward Ca 2+ . n = 14-16 cells from three independent biological replicates. b Spatial control of Ca 2+ influx in two neighboring HeLa cells. Cells in the two indicated regions (dashed lines) were sequentially subjected to photo-illumination using a 488-nm laser (0.5% input). Scale bar, 10 μm. Also see Supplementary Movie . c Comparison of light-induced Ca 2+ influx among the indicated LOCa3 variants. The channel-inactivating mutations R91W and E106A completely blocked photo-triggered Ca 2+ entry. The quantification of the relative change of GCaMP6m signals after light stimulation is shown in the bar graph on the right. n = 48-52 cells from three biological replicates. d . BTP2 as a CRAC channel blocker effectively suppressed blue-light-induced Ca 2+ influx. Left, the time-course of GCaMP6 fluorescence. Right, quantification of the GCaMP6 signals before and after BTP2 treatment (5 μM). n = 10 cells. e . The mean time-courses of whole-cell currents in HEK cells expressing LOCa3 or a control vector following exposure to blue light illumination. n = 8–9 cells. f . Mean current-voltage relationships at the peak of light-induced currents ( n = 8 cells).

Article Snippet: Images were acquired every 1 min for 15 min under blue light stimulation (470 nm, 40 μW/mm 2 , ThorLabs, Inc.).

Techniques: Transfection, Cell Culture, Imaging, Activation Assay, Control, Comparison, Fluorescence, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: Engineering of a bona fide light-operated calcium channel

doi: 10.1038/s41467-020-20425-4

Figure Lengend Snippet: Data are shown as mean ± s.e.m. Blue light was delivered using pulsed LEDs emitting at 470 nm with a power density of 40 µW/mm . Flies were subjected to a total of 10 min photostimulation per hour for up to two months. a Diagram showing the generation of AD flies expressing Aβ 42 and /or LOCa3 by using the GAL4-UAS expression system, with the driver stain bearing GAL4 under the control of a Drosophila neuron-specific Elav (embryonic lethal abnormal visual system) promoter to enable pan-neuronal expression of target genes. b Statistics showing jRCaMP1b fluorescence within the same regions of the brain from LOCa3-expressing AD Drosophila before and after exposure to blue light ( n = 24 from 5 flies). c . Quantification of changes of jRCaMP1b fluorescence before and after light stimulation in the brains of AD flies and AD flies co-expressing LOCa3 ( n = 5 flies each). d Graphs showing the effects of photostimulation on the climbing ability during aging in WT (left panel), Aβ 42 (middle), and Aβ 42 + LOCa3 (right) flies (5 independent replicates; 10 files per repeat). ** P = 0.0046; **** P < 0.0001 (compared to the dark group; two-tailed unpaired Student’s t -test). Also see Supplementary Movies – .

Article Snippet: Images were acquired every 1 min for 15 min under blue light stimulation (470 nm, 40 μW/mm 2 , ThorLabs, Inc.).

Techniques: Expressing, Staining, Control, Fluorescence, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: An endocannabinoid-regulated basolateral amygdala–nucleus accumbens circuit modulates sociability

doi: 10.1172/JCI131752

Figure Lengend Snippet: (A) Three-chamber SI task and optogenetic stimulation protocol. Animals were continuously stimulated during the 5-minute test using a 20-Hz pattern (10 mW, 5 seconds on and 5 seconds off). (B) Representative heatmaps of chamber time in a ChR2-expressing and YFP-expressing control mouse. (C) Optogenetic stimulation of the BLA-NAc circuit did not affect distance traveled (NS, P = 0.3224). (D) Animals that express ChR2 showed reduced social preference (**P = 0.0010, ****P < 0.0001, and NS, P = 0.5565), (E) decreased close interaction time (**P = 0.0011, ****P < 0.0001, and NS, P = 0.9353), and (F) reduced time investigating, or sniffing, the target mouse compared with animals that express YFP (M-M **P = 0.0026, E-M ****P < 0.001, and ##P = 0.0071). YFP n = 17, and ChR2 n = 13 (C–F). Data analyzed via 2-way mixed-effects ANOVA followed by Holm-Šídák multiple-comparisons test (D–F) or unpaired, 2-tailed t test (C). P and F values for chamber × virus interaction shown in D–F.

Article Snippet: In blue light stimulation studies, animals received 20-Hz blue light stimulation (Plexon) in a 5 seconds on, 5 seconds off, pattern at 10–13 mW.

Techniques: Expressing, Control, Virus

Journal: The Journal of Clinical Investigation

Article Title: An endocannabinoid-regulated basolateral amygdala–nucleus accumbens circuit modulates sociability

doi: 10.1172/JCI131752

Figure Lengend Snippet: (A) Optogenetic stimulation of the BLA-NAc circuit increased fleeing and withdrawal behaviors (***P = 0.0007) and (B) decreased social sniffing behaviors (**P = 0.0041) compared with YFP-expressing controls. There was no effect of BLA-NAc activation on (C) following (P = 0.1573) or (D) passive social behavior (P = 0.2604). Animals expressing ChR2 were significantly more (E) immobile (***P = 0.0007) and (F) explored less (*P = 0.0049) than YFP controls. (G) Animals that expressed ChR2 self-groomed significantly less than YFP-expressing controls (*P = 0.0184). YFP n = 15, and ChR2 n = 19 (A–G). (H) Social CPP paradigm. (I) Bilateral activation of the BLA-NAc circuit significantly decreased time in the social-paired chamber during the posttest (**P = 0.0013) (J) and reduced CPP score relative to YFP controls (**P = 0.0041). (K) Representative heatmaps for the social CPP experiment. (L) No pretest preference to either chamber was detected (YFP dots vs. stripes, P = 0.1777; ChR2 dots vs. stripes, P = 0.4619). YFP n = 9, and ChR2 n = 10 (I, J, L). Data analyzed via unpaired, 2-tailed t test (A–G, I, and J) or 2-way mixed-effects ANOVA with Holm-Šídák multiple-comparisons post hoc test (L), with P and F values for chamber × virus interaction shown in L.

Article Snippet: In blue light stimulation studies, animals received 20-Hz blue light stimulation (Plexon) in a 5 seconds on, 5 seconds off, pattern at 10–13 mW.

Techniques: Expressing, Activation Assay, Virus

Journal: The Journal of Clinical Investigation

Article Title: An endocannabinoid-regulated basolateral amygdala–nucleus accumbens circuit modulates sociability

doi: 10.1172/JCI131752

Figure Lengend Snippet: (A–C) Effects of BLA-NAc stimulation in the RtPP assay. (A) Representative heatmaps of RtPP results. (B and C) Animals expressing ChR2 spent significantly more time in the stimulation-paired (On) compared with nonpaired (Off) side in the RtPP assay (NS, P = 0.9083, and ****P < 0.0001) without any effect on total distance traveled (unpaired, 2-tailed t test, P = 0.0568). YFP n = 5, and ChR2 n = 9 (B and C). (D–G) Effects of BLA-NAc stimulation on Ensure-seeking behavior. (D) In a modified 3-chamber task a sipper bottle of Ensure was added to 1 chamber while stimulation was delivered to animals. (E) Activation of the BLA-NAc circuit did not alter time spent in the chamber with (***P = 0.0003, and ****P < 0.0001), (F) close to (*P = 0.0102, and **P = 0.0037), or (G) drinking (****P < 0.0001, and *P = 0.0128) the nutrition shake in ChR2-expressing animals compared to YFP-expressing animals. YFP n = 12, and ChR2 n = 9 (E–G). Data analyzed via unpaired, 2-tailed t test (C) or 2-way mixed-effects ANOVA with Holm-Šídák multiple-comparison post hoc tests (B, E–G), with P and F values for chamber × virus interaction shown in B, and E–G.

Article Snippet: In blue light stimulation studies, animals received 20-Hz blue light stimulation (Plexon) in a 5 seconds on, 5 seconds off, pattern at 10–13 mW.

Techniques: Expressing, Modification, Activation Assay, Comparison, Virus

Journal: The Journal of Clinical Investigation

Article Title: An endocannabinoid-regulated basolateral amygdala–nucleus accumbens circuit modulates sociability

doi: 10.1172/JCI131752

Figure Lengend Snippet: (A) The cannabinoid receptor agonist CP55940 decreased oEPSC amplitude in the NAc; N = 3. (B) JZL184 reduced aEPSC frequency in D1 receptor+ (D1R+; *P = 0.0171) and D1 receptor– (D1R–) cells (#P = 0.0197) (C) but did not affect aEPSC amplitude after BLA-NAc optogenetic stimulation; D1+ n = 3, and D1– n = 3 (B, C). aEPSC, asynchronous excitatory postsynaptic current; BL, baseline. (D) WIN55212-2 (Win55), a cannabinoid receptor agonist, uniformly decreased oEPSC amplitude in D1+ and D1– NAc cell types; D1+ n = 4, and D1– n = 4. (E) DSE was present in both D1+ and D1– NAc cells, (F) although DSE magnitude was increased in D1+ compared with D1– cells (*P = 0.0450); D1+ n = 4, and D1– n = 5 (E, F). (G) sEPSC frequency in NAc recordings were unaffected by JZL184, (H) but there was a significant effect of JZL184 on sIPSC frequency (*P = 0.0400). (I) There were no effects of JZL184 on sIPSC amplitude; D1+ n = 4, D1– n = 4. Data analyzed via 2-way mixed-effects ANOVA followed by Holm-Šídák multiple-comparisons test (B, C, G–I) or unpaired, 2-tailed t test (F). P and F values for drug effect shown in B, C, and G–I.

Article Snippet: In blue light stimulation studies, animals received 20-Hz blue light stimulation (Plexon) in a 5 seconds on, 5 seconds off, pattern at 10–13 mW.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: An endocannabinoid-regulated basolateral amygdala–nucleus accumbens circuit modulates sociability

doi: 10.1172/JCI131752

Figure Lengend Snippet: (A) Shank3B–/– mice expressing NpHR in the BLA with bilateral orange light stimulation delivered to the NAc spent significantly more time in the mouse chamber during orange light delivery (On) compared with light Off conditions (*P = 0.0230). YFP animals did not show a preference for the mouse chamber (NS, P = 0.2529). (B) Shank3B–/– mice expressing YFP under light On conditions did not exhibit social preference (NS, P = 0.2831), while animals that express NpHR had a preference for the mouse chamber (****P < 0.0001). (C) No effect on distance traveled was observed (NS, P = 0.9057) in the light On paradigm. (D) Mice that express NpHR had a significant increase in time investigating the mouse cup under light On conditions (****P < 0.0001). (E) Representative heatmaps under light On conditions. (F) Neither YFP (NS, P = 0.7166) nor NpHR (NS, P = 0.8731) Shank3B–/– mice showed mouse chamber preference (H) or preference for time investigating the mouse target (YFP NS, P = 0.1617; NpHR NS, P = 0.6193) under light Off conditions. (G) There were no effects on distance traveled under light Off conditions (P = 0.7959). (I) Representative heatmaps under light Off conditions. YFP n = 9, and NpHR n = 9 (B–D, F–H). Data analyzed via 2-way mixed-effects ANOVA with Holm-Šídák multiple-comparisons test (A, B, D, F, H) or unpaired, 2-tailed t test (C, G). P and F values for light × virus interaction shown in A and chamber × virus interaction shown in B, D, F, and H.

Article Snippet: In blue light stimulation studies, animals received 20-Hz blue light stimulation (Plexon) in a 5 seconds on, 5 seconds off, pattern at 10–13 mW.

Techniques: Expressing, Virus

Journal: The Journal of Clinical Investigation

Article Title: An endocannabinoid-regulated basolateral amygdala–nucleus accumbens circuit modulates sociability

doi: 10.1172/JCI131752

Figure Lengend Snippet: (A) sIPSC frequency on the NAc MSNs was significantly increased in Shank3B–/– compared with WT mice (**P = 0.0060) and was restored by JZL184 (##P = 0.0033). (B) There was no effect of Shank3B–/– on sIPSC amplitude. (C) sEPSC frequency was significantly increased in Shank3B–/– mice relative to WT animals (**P = 0.0084) and was restored by JZL184 (##P = 0.0060). (D) There was no effect of genotype or drug on sEPSC amplitude; WT + Veh n = 3, WT + JZL184 n = 3, KO + Veh n = 5, and KO + JZL184 n = 4 (A–D). (E) There was no difference between WT and Shank3B–/– BLA FF oIPSCs onto NAc cells. (F) However, at maximal stimulation (1.85 mW), JZL184 significantly reduced BLA FF oIPSCs onto NAc MSNs (*P = 0.0101). (G and H) There was no effect of JZL184 or genotype on BLA oEPSCs in the NAc across intensities; WT + Veh n = 3, WT + JZL184 n = 3, KO + Veh n = 4, and KO + JZL184 n = 4 (E–H). Data analyzed via 2-way ANOVA with Holm-Šídák multiple-comparisons test (A–H). P and F values for drug effect shown in A–H. M, males; F, females.

Article Snippet: In blue light stimulation studies, animals received 20-Hz blue light stimulation (Plexon) in a 5 seconds on, 5 seconds off, pattern at 10–13 mW.

Techniques: