Journal: bioRxiv

Article Title: Human neuromodulatory assembloids to study serotonin signaling and disease

doi: 10.64898/2026.03.08.710407

Figure Lengend Snippet: (A) RT-qPCR of marker gene expression comparing hCO and hMHO. Day 19-22: GATA2 (**p = 0.0012), GATA3 (*p = 0.0286); day 50-60: FOXG1 (***p = 0.0002), TPH2 (***p = 0.0006), FEV (**p = 0.0047), SLC6A4 (**p = 0.0070); (Mann-Whitney test for all genes, 3 hiPS cell lines, 1-4 differentiation experiments, Mann-Whitney test for all genes).

Article Snippet: The following antibodies were used for immunostaining: anti-TPH2 antibody (1:500 dilution, rabbit, Novus Biologicals, NB100-74555), anti-5-HT antibody (1:1000 dilution, rabbit, Immunostar, 20080), and anti-GFP antibody (1:1000 dilution, rabbit, Invitrogen, A-21311).

Techniques: Quantitative RT-PCR, Marker, Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: Human neuromodulatory assembloids to study serotonin signaling and disease

doi: 10.64898/2026.03.08.710407

Figure Lengend Snippet: (A) Canonical markers of 5-HT neurons in single-cell RNA-seq data of hMHO. (B) Proportions of brain regions mapping to 5-HT neurons based on label transfer of PCW 6.9 primary developing human brain. (C) Tph2 expression in mouse E13 spatial atlas and projection of 5-HT neurons onto mouse reference using VoxHunt . (D) Immunohistochemical staining of TPH2 in hMHO (day 84, scale bar = 250 µ m (top) and 40 µ m (bottom)). (E) Immunohistochemical staining of 5-HT in hMHO (day 83). (F) HPLC measurements of 5-HT in hMHO and hCO (hMHO: 20 samples, 13 differentiation experiments, hCO: 9 samples, 4 differentiation experiments; 3 hiPS cell lines, day 91-183. Mann-Whitney test, ****p<0.0001). (G) Biocytin labeling of AAV-TPH2::EGFP labeled 5-HT neurons (day 157, one iPS cell line, one differentiation experiment). (H) Schematic of patch clamp recording of 5-HT neurons (TPH2::EGFP + cells) in hMHO at baseline and during addition of 5-HT and NAN-190. (I) Example trace of action potentials during addition of 5-HT (100 µ m) and NAN-190 (50 µ m, 5-HT1A receptor blocker). (J) Action potential frequencies of TPH2::EGFP + neurons during baseline and addition of 5-HT and NAN-190 (14 cells, 1 hiPS cell line, 3 differentiation experiments. Friedman test *p = 0.0124, with Dunn’s multiple comparison testing: baseline vs. 5-HT **p = 0.0092, baseline vs. NAN-190 p = 0.7902, ns: non-significant). (K) Structure of serotonin sensor PsychLight2 (adapted from Dong et al with permission from Elsevier publishing, License #: 6152850897994) and schematic of serotonin sensor imaging. (L) Example images of PsychLight2 imaging in hCO and hMHO during KCl stimulation at baseline (10s) and during KCl stimulation (60s) (day 172, scale bar = 200 µ m). (M) Quantification of temporal dynamics of serotonin sensor fluorescence changes in hMHO and hCO (hCO: 9 organoids, hMHO: 8 organoids, 2 hiPS cell lines, 3 differentiation experiments). (N) Change in fluorescence intensity for PsychLight2 in hCO and hMHO after 30 s of KCl stimulation across different iPS cell lines (hCO: n = 135 ROIs (left), 9 organoids (right), hMHO: 120 ROIs (left), 8 organoids (right), 2 hiPS cell lines, 3 differentiation experiments for hCO and hMHO. Mann-Whitney test, ****p<0.0001).

Article Snippet: The following antibodies were used for immunostaining: anti-TPH2 antibody (1:500 dilution, rabbit, Novus Biologicals, NB100-74555), anti-5-HT antibody (1:1000 dilution, rabbit, Immunostar, 20080), and anti-GFP antibody (1:1000 dilution, rabbit, Invitrogen, A-21311).

Techniques: Single Cell, RNA Sequencing, Expressing, Immunohistochemical staining, Staining, MANN-WHITNEY, Labeling, Patch Clamp, Comparison, Imaging, Fluorescence

Journal: bioRxiv

Article Title: Human neuromodulatory assembloids to study serotonin signaling and disease

doi: 10.64898/2026.03.08.710407

Figure Lengend Snippet: (A, B) Representative immunohistochemical stainings of TPH2 in hMHO (top scale bar = 250 µ m; bottom scale bar = 40 µ m). (C) Representative immunohistochemical staining of 5-HT in hMHO (top scale bar = 250 µ m; bottom scale bar = 40 µ m). (D) IHC staining for TPH2 and EGFP in hMHO (d197) infected with AAV-TPH2::EGFP. Yellow arrows: EGFP + /TPH2 + cells, white arrows: EGFP + /TPH2 - cells (scale bar = 40 µ m).

Article Snippet: The following antibodies were used for immunostaining: anti-TPH2 antibody (1:500 dilution, rabbit, Novus Biologicals, NB100-74555), anti-5-HT antibody (1:1000 dilution, rabbit, Immunostar, 20080), and anti-GFP antibody (1:1000 dilution, rabbit, Invitrogen, A-21311).

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry, Infection

Journal: bioRxiv

Article Title: Human neuromodulatory assembloids to study serotonin signaling and disease

doi: 10.64898/2026.03.08.710407

Figure Lengend Snippet: (A) Schematic of patch clamp recordings in TPH2::EGFP + neurons in hMHO and SYN1::mCherry + neurons in hCO. (B) Representative traces of action potentials in hMHO and hCO. (C) Example action potential traces in hMHO during current injection. (D) Example action potential traces in hCO during current injection. (E) Action potential half width of labeled cells in hMHO and hCO (hMHO: 62 cells, 6 differentiation experiments, 3 hiPS cell lines, day 133 - 205; hCO: 33 cells, 3 differentiation experiments, 3 hiPS cell lines, day 152 - 208. Mann Whitney test, ***p = 0.0002). (F) Action potential threshold of labeled cells in hMHO and hCO (hMHO: 62 cells, 6 differentiation experiments, 3 hiPS cell lines day 133 - 205; hCO: 33 cells, 3 differentiation experiments, 3 hiPS cell lines, day 152 - 208. Mann Whitney test, *p = 0.0355). (G) Peak action potential amplitude of labeled cells in hMHO and hCO (hMHO: 62 cells, 6 differentiation experiments, 3 hiPS cell lines, day 133 - 205; hCO: 33 cells, 3 differentiation experiments, 3 hiPS cell lines, day 152 - 208. Mann Whitney test, p = 0.3802). (H) Resting membrane potential of labeled cells in hMHO and hCO (hMHO: 62 cells, 6 differentiation experiments, 3 hiPS cell lines, day 133 - 205; hCO: 33 cells, 3 differentiation experiments, 3 hiPS cell lines, day 152 - 208. Mann Whitney test, p = 0.9829). (I) Capacitance of labeled cells in hMHO and hCO (hMHO: 62 cells, 6 differentiation experiments, 3 hiPS cell lines, day 133 - 205; hCO: 33 cells, 3 differentiation experiments, 3 hiPS cell lines, day 152 - 208. Mann Whitney test, p = 0.1197). All error bars are shown as mean ± SD.

Article Snippet: The following antibodies were used for immunostaining: anti-TPH2 antibody (1:500 dilution, rabbit, Novus Biologicals, NB100-74555), anti-5-HT antibody (1:1000 dilution, rabbit, Immunostar, 20080), and anti-GFP antibody (1:1000 dilution, rabbit, Invitrogen, A-21311).

Techniques: Patch Clamp, Injection, Labeling, MANN-WHITNEY, Membrane

Journal: bioRxiv

Article Title: Human neuromodulatory assembloids to study serotonin signaling and disease

doi: 10.64898/2026.03.08.710407

Figure Lengend Snippet: (A) Schematic of imaging of bilateral projections in hNMA. (B) Example image of TPH2::EGFP projections from hMHO to hCO in hNMA after ~4 weeks of fusion (scale bar = 500 µ m). (C) Quantification of TPH2-EGFP + projections into hCO (n = 41 assembloids, 3 hiPS cell lines, 4 differentiation experiments. Kruskal-Wallis test, p** = 0.0033 with Dunn’s multiple comparison testing: 2 wks vs. 8 wks **p = 0.0025, 4 wks vs. 8 wks p > 0.9999). (D) Quantification of SYN1-mCherry + projections into hMHO (n = 41 assembloids, 3 hiPS cell lines, 4 differentiation experiments. Kruskal-Wallis test, p**** < 0.0001 with Dunn’s multiple comparison testing: 2 wks vs. 8 wks ****p < 0.0001, 4 wks vs. 8 wks p = 0.2911). (E) Schematic of serotonin sensor imaging in hNMA and unassembled hCO and hMHO. (F) Example images of serotonin sensor imaging of fused and unfused hNMA at base line (10s) and after KCl stimulation (60s) (scale bar = 200 µ m). (G) Temporal dynamics of serotonin sensor fluorescence signal in unassembled hCO and hCO in hNMA (assembled: n = 11 assembloids, unassembled n = 10 pairs of organoids, 2 hiPS cell lines, 3 differentiation experiments). (H) Quantification of serotonin sensor signal at maximum fluorescence intensity across multiple hiPS cell lines (assembled: n = 165 ROIs (left), 11 assembloids (right); unassembled: 150 ROIs (left), 10 pairs of organoids (right); 2 hiPS cell lines, 3 differentiation experiments. Mann-Whitney test, *p=0.0159). (I) Example patch clamp traces and response types of SYN1::mCherry + cells in hCO during 5-HT addition. (J) Quantification of response types in hCO during 5-HT administration (20 cells, 2 hiPS cell lines, 3 differentiation experiments, 157-180d). (K) Example patch clamp traces and response types in SYN1::mCherry + cells in hCO in hNMA during optogenetic stimulation of hMHO. (L) Quantification of response types in hCO during optogenetic stimulation in hNMA (46 cells, one hiPS cell line, 3 differentiation experiments, 142-200d). (M) Schematic of calcium imaging in hCO and hNMA. (N) Example calcium imaging traces in unassembled hCO and hCO in hNMA. (O) SCA correlograms of unassembled hCO and hCO in hNMA. (P) Peak correlations from SCA analysis in hCO and hCO in hNMA in multiple hiPS cell lines (hCO: 318 cells (left), 30 organoids (right); hCO in hNMA: 463 cells (left), 44 organoids (right), 3 hiPS cell lines, 3 differentiation experiments. Mann-Whitney-U test ***p = 0.0004).

Article Snippet: The following antibodies were used for immunostaining: anti-TPH2 antibody (1:500 dilution, rabbit, Novus Biologicals, NB100-74555), anti-5-HT antibody (1:1000 dilution, rabbit, Immunostar, 20080), and anti-GFP antibody (1:1000 dilution, rabbit, Invitrogen, A-21311).

Techniques: Imaging, Comparison, Fluorescence, MANN-WHITNEY, Patch Clamp

Journal: bioRxiv

Article Title: Human neuromodulatory assembloids to study serotonin signaling and disease

doi: 10.64898/2026.03.08.710407

Figure Lengend Snippet: (A) Patient-derived 22q11.2DS and control hiPS cell lines for generating hCO, hMHO and hNMA. (B) Example SNP array of one control and 22q11.2DS hiPS cell line (22q11.2DS: 6303-5, Ctrl: 1208-2). (C) PsychLight2 imaging in hCO from 22q11.2DS and control hNMA during KCl stimulation (Ctrl: 200 ROIs (left), 40 assembloids (middle), 4 hiPS cell lines (right), 3 differentiation experiments; 22q11.2DS: 175 ROIs (left), 35 assembloids (middle), 4 hiPS cell lines (right), 3 differentiation experiments. Unpaired t -test with Welch’s test, ****p<0.0001). (D) PsychLight2 imaging in 22q11.2DS and control hCO during KCl stimulation (Ctrl: 225 ROIs (left), 27 organoids (middle), 4 hiPS cell lines (right), 3 differentiation experiments; 22q11.2DS: 300 ROIs (left), 36 organoids (middle), 5 hiPS cell lines (right), 3 differentiation experiments. Unpaired t-test with Welch’s test, p = 0.9690). (E) PsychLight2 imaging in 22q11.2DS and control hMHO during KCl stimulation (Ctrl: 220 ROIs (left), 28 assembloids (middle), 4 hiPS cell lines (right), 3 differentiation experiments; 22q11.2DS: 280 ROIs (left), 32 assembloids (middle), 5 hiPS cell lines (right), 3 differentiation experiments. Unpaired t-test with Welch’s test, p = 0.7334). (F) Imaging of TPH2::EGFP + projections from hMHO to hCO in hMNA and examples of TPH2::EGFP + signals in control and 22q11.2DS hNMA (scale bar = 500 µ m). (G) Quantification of TPH2::EGFP + projections from hMHO to hCO in hMNA after 30-40 days of fusion (Ctrl: 20 assembloids, 2 differentiation experiments, 4 hiPS cell lines; 22q11.2DS: 19 assembloids, 2 differentiation experiments, 4 hiPS cell lines. Mann-Whitney test, p = 0.4955). (H) PsychLight2 imaging in hCO from control hNMA with SSRI treatment during KCl stimulation (without SSRI: 18 assembloids, 4 hiPS cell lines, 3 differentiation experiments; with SSRI: 20 assembloids, 4 hiPS cell lines, 3 differentiation experiments). (I) Representative images of PsychLight2 imaging in control hNMA with and without SSRI addition. (J) PsychLight2 imaging in hCO 22q11.2DS hNMA with SSRI treatment during KCl stimulation (without SSRI: 19 assembloids, 4 hiPS cell lines, 3 differentiation experiments; with SSRI: 19 assembloids, 4 hiPS cell lines, 3 differentiation experiments). (K) Representative images of PsychLight2 imaging in 22q11.2DS hNMA with and without SSRI addition.

Article Snippet: The following antibodies were used for immunostaining: anti-TPH2 antibody (1:500 dilution, rabbit, Novus Biologicals, NB100-74555), anti-5-HT antibody (1:1000 dilution, rabbit, Immunostar, 20080), and anti-GFP antibody (1:1000 dilution, rabbit, Invitrogen, A-21311).

Techniques: Derivative Assay, Control, Imaging, MANN-WHITNEY

Journal: Journal of Neurochemistry

Article Title: Dysregulated Plasticity in Serotonin, Galanin, and Opioid Systems Contributes to Limbic Seizure Recruitment in Wistar Audiogenic Rat

doi: 10.1111/jnc.70397

Figure Lengend Snippet: Effect of audiogenic kindling (AK) on messenger RNA (mRNA) expression in Dorsal Raphe Nucleus (DRN) of Tryptophan hydroxylase 2 ( Tph2 ), Tryptophan hydroxylase 1 ( Tph1 ), solute carrier family 6 (serotonin neurotransporter— Slc6a4 ) and serotonin receptor 1A ( Htr1a ) in Wistar and Wistar Audiogenic rats (WAR) (A1, B1, C1, D1). These data were analyzed by Two‐way ANOVA followed by Tukey's post‐test. We also analyzed whether AK stimuli trigger limbic recruitment (LiR) or not (n‐LiR) in WARs under AK versus naive rats on the mRNA expression of Tph2, Tph1, Slc6a4 and Htr1a (A2, B2, C2, D2) in the DRN, analyzed by One‐way followed by the Tukey post‐test. # p < 0.05 WARs versus Wistars; *AK versus naive; +AK versus control; @WARs under AK versus Wistars. Box plots display the median (center line), interquartile range (box), and minimum/maximum values (whiskers). All individual data points are shown.

Article Snippet: For TaqMan assays, pre‐designed probes were used for Actb (Rn00667869_m1), Htr1a (Rn00561409_s1), Htr1b (Rn01637747_S1), Htr2a (Rn00568473_m1) and Htr2c (Rn00562748_m1), Slc6a4 (Rn00564737_m1), Gal (Rn00583681_m1), Galr1 (Rn02132426_s1), Galr2 (Rn00695901_g1), Tph1 (Rn01476867_m1), Tph2 (Rn00598017_m1), (Applied Biosystems, Beverly, MA, USA).

Techniques: Expressing, Control