Journal: bioRxiv

Article Title: Calcium dynamics tune developmental tempo to generate evolutionarily divergent axon tract lengths

doi: 10.1101/2024.12.28.630576

Figure Lengend Snippet: a. Axon tract of human (day 70) brain organoid slices in micropatterned devices expressing GCaMP7f and treated with DMSO (control), nimodipine (L-Type VGCC antagonist) or BayK8644 (L-Type VGCC agonist). Arrowheads mark axons showing calcium activity in a five minute timelapse, indicating nearly complete perturbation of calcium dynamics upon L-Type VGCC blockage. Zooms show representative timelapse of individual axons, with ΔF/F0 fluorescent GCaMP7f intensity plots of axons in zooms, showing the increase in calcium dynamics upon L-Type VGCC activation. b. Quantifications of number of axons firing in an ROI during a five minutes timelapse in axon tracts of human brain organoid slices (day 70) in micropatterned devices expressing GCaMP7f and treated with DMSO (control), nimodipine (L-Type VGCC antagonist) or BayK8644 (L-Type VGCC agonist). Control: n=13 regions of interest, L-Type VGCC antagonist: n=11, L-Type VGCC agonist: n=10. c. Quantifications of average mean amplitudes of spontaneous calcium transients in human brain organoid slice cultures (day 70) in micropatterned devices expressing GCaMP7f and treated with DMSO (control) or BayK8644 (L-Type VGCC agonist). Control: n=137 axons, L-Type VGCC agonist: n=100. d. Quantifications of average frequencies of spontaneous calcium transients in human brain organoid slice cultures (day 70) in micropatterned devices expressing GCaMP7f and treated with DMSO (control) or BayK8644 (L-Type VGCC agonist). Control: n=137 axons, L-Type VGCC agonist: n=100. e. Quantifications of average event durations of spontaneous calcium transients in human brain organoid slice cultures (day 70) in micropatterned devices expressing GCaMP7f and treated with DMSO (control) or BayK8644 (L-Type VGCC agonist). Control: n=137 axons, L-Type VGCC agonist: n=100. f. Representative images of axon tracts of human (day 90) brain organoid slices in micropatterned devices immunostained for pan-axonal neurofilaments and treated with DMSO (control), nimodipine (L-Type VGCC antagonist), BayK8644 (L-Type VGCC agonist), or cAMP. g. Quantifications of axon tract length of human brain organoid slice cultures in micropatterned devices, treated with DMSO (control), nimodipine (L-Type VGCC antagonist) or BayK8644 (L-Type VGCC agonist), at different timepoints. n=8-32 organoids. h. Quantifications of axon tract length of human brain organoid slice cultures in micropatterned devices, treated with DMSO (control) or BayK8644 (L-Type VGCC agonist), at day 55+7 and 55+28. Control: n=15 organoids (55+7) and n=14 (55+28), L-Type VGCC agonist: n=11 (55+7) and n=8 (55+28). i. Quantifications of axon tract length of human brain organoid slice cultures in micropatterned devices, treated with DMSO (control) or cAMP, at different timepoints. n=4-5 organoids. j. Quantifications of axon tract length of human brain organoid slice cultures in micropatterned devices, treated with DMSO (control) or cAMP, at day 55+7 and 55+28. Control: n=10 organoids (55+7) and n=10 (55+28), cAMP: n=9 (55+7) and n=9 (55+28). k. Schematic of model. Increase of L-type VGCC-mediated calcium, and downstream cAMP, levels accelerate developmental tempo during axonogenesis, resulting in shorter axon tract lengths. treatm., treatment. antag., antagonist. ag., agonist. All data are shown as mean ± SEM; ** P < 0.1, **** P < 0.0001 as determined by unpaired t tests.

Article Snippet: For drug treatments, one day after brain organoid slices were placed in the devices, and during subsequent medium changes, medium was replaced with SFSCM or BrainPhys (at similar timepoints as with ALI-CO cultures described above) supplemented with indicated final concentrations of either 1 μM or 10 μM BayK8644 (Stratech Scientific, S7924-SEL), 1 μM or 10 μM Nimodipine (Selleckchem, S1747) or 100 μM 8-Br-cAMP (BioLog, B007).

Techniques: Expressing, Control, Activity Assay, Activation Assay