Fig 1: Functional assessment of OPN4-mGluR1 Variants by in vitro Ca2+-Imaging in PCs(A, E, and I) Scheme of experimental procedure for the 2-photon imaging in vitro and expression of OPN4-mGluR1a.(B) OPN4-mGluR1b.(F and J) or mCherry (J) together with GCaMP6m in the PC of cerebellar slices. Scale bar, 120µm.(C and G) Exemplary transient Ca2+ signal during light stimulation. Time-course of light-activated Ca2+ increase by OPN4-mGluR1a (C, right) or OPN4-mGluR1b (G, right) at the soma, the proximal and distal dendrite. Scale bar, 25µm.(D and H) Population data of the time to 2/3 peak at the different regions are shown as boxplot (mean ± SEM).(K) Time-course of mean light-activated Ca2+ signaling of the respective constructs at the soma (OPN4-mGluR1a: n = 8; OPN4-mGluR1b: n = 9; mCherry: n = 10). Lines indicate the means, shading ±SEM.(L) Population data of the responses shown in k are shown as boxplot (mean ± SEM). Maximal fluorescence changes are compared between optogenetic chimeras and the mCherry-control and showed statistically significant differences (Kruskal-Wallis test; p < 0.0001; 1a/mCherry, p < 0.0001; 1b/mCherry, p = 0.015).
Fig 2: OPN4-mGluR1a elevates Purkinje cell simple spike firing rate in vivo(A) Scheme of experimental procedure for single-cell recordings in cerebellum in anesthetized mice. Recordings were made two weeks after AAV8-OPN4-mGluR1a injection, DHPG was locally applied during recordings.(B) Pharmacological activation of mGluR1 (DHPG - mGluR1 agonist, purple trace) or blue light activation OPN4-mGluR1a (488 nm; blue trace) lead to a rise in simple spike firing frequency (DHPG: n = 30 PCs in n = 3 mice; OPN4-mGluR1a: n = 12 PCs in n = 4 mice). Lines indicate the means, shading ±SEM.(C) Exemplary Purkinje cell recordings (identified by characteristic simple and complex spike firing pattern, marked with *) over 5 min with 60 s baseline recoding and subsequent optogenetic activation using 488 nm wavelength (top) or pharmacological activation (DHPG - mGluR1 agonist, bottom).
Fig 3: Design and characterization of OPN4-mGluR1 variants(A) Design of OPN4-mGluR1a (blue) and OPN4-mGluR1b (orange) constructs. Chimeras comprise OPN4, mCherry in the third intracellular loop and mGluR1a or mGluR1b fused c-terminally.(B) Scheme of OPN4-mGluR1 design and Gq-coupled Ca2+ increase following photostimulation.(C) Top, exemplary OPN4-mGluR1a expression in two HEK293 cells. The construct is localized in the cell membrane. Bottom, transient Ca2+ signal of both cells during light stimulation. Scale bar, 10µm.(D) Time-course of light-activated Ca2+ signaling of OPN4-mGluR1a (blue, n = 15 dishes) and OPN4-mGluR1b (orange, n = 8 dishes) over a stimulation period of 90 seconds. Shading indicates ± SEM.(E) Pharmacological activation of mGluR1a in HEK293 cells shows comparable increase and delayed peak of Ca2+ signaling (purple, n = 13 dishes). Pharmacological block of Gq/11 protein signaling abolishes OPN4-mGluR1a Ca2+ signals (green).(F) Ca2+ signal components ton, toff and time to peak were compared between optogenetic chimeras and showed no differences. Population data of the responses shown in d are shown as boxplot (mean ± SEM).
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