Extended Data Fig. 8: MISCOs display functional dopaminergic innervation.
a-c, Representative GCAMP recordings of 130-day-old MISCOs with Syn-GCAMP expression either in the VM, or striatal, or cortical tissue as well as selected traces of individual neurons (bottom) (similar results in n = 7/7 organoids per region) d, Ctx-Str-VMSyn-GCAMP MISCOs displayed calcium network events in VM derived projections in forebrain (Fb) tissue (d’: no synchronous event, d”: calcium network event) (similar results in n = 4/4 organoids of 2 batches). e, Cumulative fluorescence intensity over time of the recording from (d) displaying VM derived network events. Inserts indicate selected time points for panel d’ and d”. f, Photography of a silicon neural probe set-up with optogenetic stimulation with mounted fused organoid. Organoids in this experiment were transduced with the optogenetic vector AAV-RG.AAV-CAG-hChR2-H134R-tdTomato. g, Widefield images before (left) and during stimulation (right) of the VM tissue while recording from striatal tissue. SiPro…Silicon Probe. h, Stimulation set-up. After baseline recording, the VM side of a MISCO was stimulated for 500 ms every 10 sec. i, Representative active channel raster plots in forebrain tissue with baseline (left), VM stimulation (middle) and VM stimulation after application of synaptic blockers (right). 460-nm LED light pulses set at an interval of 10 seconds with a 500-millisecond duration (blue). After a 5 min incubation with a cocktail of synaptic blockers (D-AP5, CNQX, Gabazine, SCH-23390, Sulpiride−/−), population responses in forebrain were absent during stimulation of VM. j, Normalized mean firing rate in MISCOs before and after light application, and repeated with synaptic blocker application (n = 127 active channels from 2 organoids). Padj = <0.0001|< 0.0001. Samples were analyzed with one-way ANOVA followed by Tukey’s multiple comparisons test. k-l, AAV2−pAAVss-hSyn-GRAB-DA2m transduces striatal (DARPP32+ in striatal tissue, yellow arrows) as well as cortical (TBR1+ in cortical tissue) neurons (yellow arrows: double-positive cells). m-n, Heatmap of activity regulated response genes and dopamine signaling response genes. The majority of activity-regulated genes were upregulated in RNA-seq of striatal and cortical tissues of separated MISCOs on day 60 in comparison to day 60 individual organoids. PRGs… primary response genes. SRGs… secondary response genes.
