Fig. 7: Functional assembloids can be made from conjoined spheroids to model neural circuitry. | Communications Biology

Fig. 7: Functional assembloids can be made from conjoined spheroids to model neural circuitry.

From: Functional brain region-specific neural spheroids for modeling neurological diseases and therapeutics screening

Fig. 7

a Schematic showing that spheroids can be transfected with DREADDs viruses tagged with mCherry and activity can be recorded with a FLIPR at 3 weeks using a cal6 dye. Schematic was created with Biorender.com. b Activity from single-neuron spheroids (SNSs) comprised of 90% neuron and 10% astrocytes can be recorded and displays similar phenotypes in spheroids transfected with stimulatory (hM3Dq) or inhibitory (hM4Di) DREADDs viruses. Top panel: vehicle control-treated spheroids expressing no DREADDs virus; middle panel: 60-min after treatment with CNO to activate DREADDs virus and induce stimulatory activity; bottom panel: 60-min after treatment with CNO to activate DREADDs virus and induce inhibitory activity. c Radar plots showing multiparametric peak alterations across 10 peak parameters for both stimulatory (teal) and inhibitory (red) DREADDs in SNSs with dopaminergic, glutamatergic, and GABAergic neurons, respectively. d–f is from confocal recordings. d Representative images of two assembloids where one component expresses GCaMP6f and the other expresses either stimulatory (hM3Dq) or inhibitory (hM4Di) DREADDs viruses; Left: VTA-like expressing GCaMP6f, PFC-like expressing hM4Di. Right: PFC-like expressing GCaMP6f, VTA-like expressing hM4Di. Scale bar = 200 µm. e, f Quantification of baseline vs 90-min activity for peak count (hM4Di: t(6) = 3.15, P = 0.019; hM3Dq: t(4) = 1.6, P = 0.19), amplitude (hM4Di: t(6) = 0.04, P = 0.97; hM3Dq: t(4) = 7.6, P = 0.002), width (hM4Di: t(6) = 1.93, P = 0.1; hM3Dq: t(4) = 3.29, P = 0.03), and synchronicity (hM4Di: t(6) = 0.35, P = 0.74; hM3Dq: t(4) = 2.7, P = 0.05) of the VTA-like component (e) or PFC-like (f) component (peak count: hM4Di: t(8) = 1.94, P = 0.09, hM3Dq: t(4) = 0.65, P = 0.55; amplitude: hM4Di: t(8) = 0.8, P = 0.45, hM3Dq: t(4) = 7.3, P = 0.002; width: hM4Di: t(8) = 1.003, P = 0.35, hM3Dq: t(4) = 2.11, P = 0.103; synchronicity: hM4Di: t(8) = 2.28, P = 0.052, hM3Dq: t(4) = 4.95, P = 0.008) of assembloids before (yellow) and after (red) CNO was added to media to activate inhibitory (hM4Di) or stimulatory (hM3Dq) DREADDs in the PFC-like (e) or VTA-like (f) component of assembloids. b, c n = 8–12 per group over three separate experiments; d–f n = 4–9 per group over two experiments. Data from (c) are represented as mean and from (e, f) are represented as mean plus individual values before and after CNO was used to activate DREADDs. Data from (e, f) were analyzed with paired t tests *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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