Synaptic plasticity in human thalamocortical assembloids

Abstract

Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA sequencing revealed that >80% of cells in thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.

Keywords: CP: Neuroscience; LTD; LTP; brain organoid; cortical organoid; hiPSC; synaptic plasticity; synaptic transmission; thalamic organoid; thalamocortical.

Figure 1.. hThOs contain functional glutamatergic thalamic neurons

(A) Reporter cell line validation for hThOs. Top: schematic of TCF7L2 exon 1 in the TP-190a-TCF7L2-tdTomato reporter line. Bottom: VoxHunt deconvolution of bulk RNA-seq data from D69-D70 hThOs using E13 mouse brain data from the Allen Brain Atlas as a reference. TPMs: transcripts per million. n = 27 bulk RNA-seq samples, with each sample derived from 2 to 3 pooled hThOs. (B) Immunofluorescence images of TCF7L2, TUBB3, OTX2, and SOX2 labeling in D60 hThOs. Nuclei are indicated by DAPI (cyan). TCF7L2-tdTomato fluorescence is indicated in magenta. Images were acquired from serial sections of the same organoid. Scale bars: 200 μm (whole section), 100 μm (insets). (C) UMAP plot with cluster annotations indicated by color. (D) VoxHunt mapping of snRNA-seq clusters to BrainSpan reference (human fetal tissue, 13–24 pcw). Excitatory neuron (ExN) clusters exhibit the highest correlations with mediodorsal nucleus of the thalamus (MD). A1C, primary auditory cortex; AMY, amygdala; CB, cerebellum; CBC, cerebellar cortex; DFC, dorsolateral prefrontal cortex; HIP, hippocampus; IPC, posteroventral (interior) parietal cortex; ITC, inferolateral temporal cortex; M1C, primary motor cortex; M1C-S1C, primary motor-sensory cortex; MFC, anterior (rostral) cingulate (medial prefrontal) cortex; OFC, orbital frontal cortex; S1C, primary somatosensory cortex; STC, posterior (caudal) superior temporal cortex; STR, striatum; V1C, primary visual cortex; VFC, ventrolateral prefrontal cortex. (E) Bar plot showing the number of nuclei per cluster, with clusters indicated by fill color. (F) UMAP plots of glutamatergic markers SLC17A6 (VGLUT2) and SLC17A7 (VGLUT1). Color indicates normalized transcript level. (G) VoxHunt correlation analysis mapping clusters ExN1–4 onto the E15 mouse brain. (H) VoxHunt correlation analysis mapping the PT/ZLI/rTh cluster onto the E15 mouse brain. (I) UMAP plots of the PT/ZLI/rTh cluster, demonstrating the expression of markers associated with the PT, ZLI, and rTh. Transcript information is indicated by color. The relative locations of these structures within the developing diencephalon are shown in the schematic. cTh, caudal thalamus; eTh, epithalamus; preTh, prethalamus; PT, pretectum; ZLI, zona limitans intrathalamica; rTh, rostral thalamus. (J) Example traces showing voltage and AP responses to current injections in an hThO cell. (K) Pseudotime ordering of cells. (L) UMAP plot of the neural progenitor marker TNC. Color indicates the normalized transcript level. (M and N) UMAP plots of cell cycle analysis results for the cycling progenitor, radial glia, and glia cell clusters. Color indicates S score (M) or G2M score (N). (O) UMAP plot of the astrocyte marker GFAP in the cycling progenitor, radial glia, and glia cell clusters. Color indicates the normalized transcript level. Data in (C)–(I) and (K)–(O) were produced by snRNA-seq analysis of 15,363 nuclei from D90 hThOs. See Figures S1–S3 for additional data validating hiPSC lines and hThOs. See Figure S4 for additional data related to electrophysiological properties and synapses.

Figure 2.. Fusing hThOs and hCOs produces assembloids that form reciprocal synapses

(A) Reporter line validation for hCOs. Top: schematic of SLC17A7 (VGLUT1) exon 12 in the TP-190a-VGLUT1-tdTomato reporter line. Bottom: VoxHunt deconvolution of bulk RNA-seq data from D70 hCOs using E13 mouse brain data from the Allen Brain Atlas as a reference. Organoids were visually categorized as positive or negative for tdTomato fluorescence prior to sequencing. The tdTomato RNA level for each sample is indicated in TPMs. Each stacked bar indicates one bulk RNA-seq sample derived from 2 to 3 pooled hCOs. (B) The snRNA-seq analysis of hCOs. Left: UMAP plot with cluster annotations. ExN, excitatory neuron; DL, deep layer; UL, upper layer; Un., unknown. Right: dot plot showing subplate marker expression by cluster. Avg Exp, normalized average expression; % cells, percentage of cells expressing a marker within a cluster. (C) VoxHunt analysis mapping hCOs (all clusters) onto the E15 mouse brain. (D) Pseudotime analysis of the neural cell trajectory (cycling progenitors to UL ExNs, DL ExNs, and subplate/DL ExNs) from hCOs. (E) UMAP plots of glutamatergic markers SLC17A6 (VGLUT2) and SLC17A7 (VGLUT1). Color indicates normalized transcript level. (F) Traces showing the voltage and AP responses to current injections in an hCO cell. (G) Fluorescence and bright-field image of a TC assembloid at 5 days post-fusion (dpf). (H) Representative fluorescence images for 2-dimensional fusion assay. Thalamic neurons (magenta, right) and cortical neurons (green, left) extend processes from their respective chambers, across the barrier region (dashed yellow lines), and into the opposite chamber starting at D9. Elaborate processes extending from the opposite sides are seen in both halves by D61. (I) Fluorescence image of an hCO co-transduced with hSyn-GFP and hSyn-V5-Mito-APEX2 lentiviruses. (J) Schematic and TEM image of an APEX2⁺ mitochondrion (circled in magenta) in a TC synapse. Pre, presynaptic compartment; post, postsynaptic compartment. (K) Schematic and TEM image of an APEX2⁺ mitochondrion (circled in green) in a CT synapse. APEX2^– mitochondria are indicated by asterisks (*). Data in (B)–(E) were produced by snRNA-seq analysis of 12,008 nuclei from D90 hThOs. See Figures S1 and S5 for additional data validating the TP-190a-VGLUT1-tdTomato reporter line and hCOs. See Figure S4 for additional data related to electrophysiological properties and synapses. See Figure S6 for NeuronChat analysis.

Figure 3.. Assembloids contain glutamatergic TC and CT synapses

(A) Left: schematic of the recording configuration for the TC pathway. Right: bar graph of the percentage of responsive (green) and unresponsive (gray) cells in 11 assembloids. The numbers of cells recorded per assembloid are shown in white. (B) Left: schematic of the recording configuration for the CT pathway. Middle: the percentages of hThO cells that responded (magenta) or did not respond (gray) to hCO stimulation across 10 assembloids. Right: bar graph of the average percentage of responsive cells for TC and CT synapses, based on (A) and (B). (C) Line graph of paired-pulse ratios (PPRs) across five interstimulus intervals (ISIs) in CT (magenta) and TC (green) synapses (one-sample t test: μ = 1, #p < 0.05, ##p < 0.01, n = 18–23 cells/9–13 assembloids [TC], n = 8–24/7–12 [CT]). Differences between CT and TC synapses were evaluated by unpaired t test (**p < 0.01). Inset: sample traces depicting PPRs in CT and TC synapses. (D) Average TC EPSC amplitude (holding potential [V_h] −70 mV) in the presence of NBQX (3 mM) is decreased compared to control aCSF conditions (paired t test: **p = 0.009, n = 5/2). (E) The average TC EPSP amplitude (V_h +40 mV) in NBQX and AP5 (50 mM) is lower than in aCSF (paired t test: *p = 0.038, n = 5/3). (F) Traces of evoked TC AMPAR- and NMDAR-mediated currents in aCSF and in NBQX or NBQX and AP5, respectively. (G) Average CT EPSC amplitude (V_h −70 mV) in NBQX is decreased compared to aCSF conditions (paired t test: *p = 0.012, n = 5/3). (H) The average CT EPSC amplitudes (V_h +40 mV) are reduced in NBQX and AP5 compared to aCSF (paired t test: ***p = 0.0006, n = 5/3). (I) Example traces of evoked CT AMPAR- and NMDAR-mediated currents in aCSF and in NBQX or NBQX and AP5, respectively. (J) Schematics of two-photon Ca²⁺ imaging in postsynaptic dendritic spines of hCO cells upon hThO stimulation. Alexa Fluor 594: AF-594 (R), magenta; Fluo-5F (G), green. (K) Image of an hCO dendrite. Line scans (white line) were performed across a dendritic spine (Sp) and parent dendritic shaft (Sh). (L) Left: representative changes in G/R of Sp and Sh responses over time to a single synaptic stimulation (arrowhead and black line). Right: representative line scans of Sp (light) and Sh (dark). (M) Average changes in synaptically evoked G/R (paired t test: **p = 0.002, n = 9/4). (N) Average changes in synaptically evoked Sp G/R in aCSF and in AP5 (paired t test: *p = 0.018, n = 7/5). Data in (D), (E), (G), (H), (M), and (N) are shown as the mean values with individual responses overlaid. Grouped data (C) are shown as mean ± SEM. n = cells/assembloids. Circles in (C), (F), and (I) represent stimulus artifacts. See Figure S7 for snRNA-seq data supporting glutamatergic communication.

Figure 4.. TC synapses in assembloids undergo LTP

(A) Left: schematic of the recording configuration. Right, top: 40-Hz electrical stimulation LTP-induction protocol. Right, bottom: representative trace of a response. (B) Left: time course data demonstrating that 40-Hz stimulation repeated three times (arrows) induces LTP in TC assembloids (n = 9 cells/9 assembloids). Right: representative traces from the first 5 min (1, dark) and final 5 min (2, light) of the experiment. (C) Bar graph of group data after 40-Hz induction from (B) shows EPSC amplitudes differ from baseline values (one-sample t test, μ = 100 versus full postinduction time period, ##p = 0.0077). (D) Top: spike-timing-dependent plasticity (STDP) was induced by stimulating presynaptic hThO inputs (Pre) then delivering four current injections (2 nA) to the postsynaptic cell (Post), 50 times. Bottom: representative trace of a response. (E) Left: time course data demonstrating that the short STDP protocol (arrow) in TC assembloids induces LTP (n = 7/3). Right: representative traces from the first (dark) and final (light) 5 min of the experiment. (F) Bar graph of group data following the ×1 STDP induction from (E) shows that EPSC amplitudes differ from baseline values (one-sample t test, μ = 100 versus full postinduction time period, #p = 0.04). (G) Top: long STDP-induction protocol, as in (D) but repeated ×3 every 5 min. Bottom: representative trace of a response. (H) Bar graph showing the average responses of nine cells from six assembloids after TC LTP induction. Shades of gray indicate different batches of assembloids; vertical lines denote separate assembloids. (I) Time course of series resistance (Rs) normalized to the 5-min baseline period demonstrating TC LTP is not due to changes in Rs. (J) Time course demonstrating the 33 STDP protocol (arrows) induces LTP in TC synapses (black, n = 9/6). MPEP (blue, n = 6/5) or iBAPTA blocked LTP (orange, n = 6/4). AP5 did not block TC LTP (green, n = 6/3). Shaded area depicts the presence of bath-applied drugs. (K) Bar graph of group data following ×3 STDP induction from (J). Differences from baseline were evaluated by one-sample t test (μ = 100 versus full postinduction time period, ##p < 0.01). Differences between treatments and aCSF were evaluated by one-way ANOVA, p < 0.0001. Dunnett’s test: ***p = 0.0001, ****p < 0.0001. (L) Example traces from the first (1) and final (2) 5 min of the experiment across conditions. Data shown are mean ± SEM in (B), (E), (I), and (J), with individual data points overlaid as dots in (C), (F), and (K). n = cells/assembloids. Circles in (B), (E), and (L) represent stimulus artifacts. See Figure S7 for additional supporting data.

Figure 5.. CT synapses in assembloids undergo LTP

(A) Left: schematic of the recording configuration. Right: the long (33) STDP-induction protocol and example response. (B) Bar graph showing the average responses in 14 cells from 9 assembloids after CT LTP induction in aCSF. Colors indicate different batches of assembloids; vertical lines denote separate assembloids. (C) Time course demonstrating that ×3 STDP delivery (arrows) induces LTP in CT synapses (black, n = 14 cells/9 assembloids). MPEP (blue, n = 8/6), AP5 (green, n = 15/7), or iBAPTA (orange, n = 7/3) blocked LTP. Shaded area depicts the presence of bath-applied drugs. The first (1) and final (2) 5 min of the experiment are noted. (D) Time course of Rs. (E) Bar graph of group data from (C). Differences from baseline were evaluated by one-sample t test (μ = 100 versus full postinduction time period, ##p < 0.01). Differences between treatments and aCSF were evaluated by one-way ANOVA, p = 0.0053. Dunnett’s test: **p < 0.01. (F) Example traces from the first (1) and final (2) 5 min of the experiment across conditions. Data shown are mean ± SEM in (C), (D), and (E) with individual data points overlaid in (E). n = cells/assembloids. See Figure S7 for additional supporting data.

Figure 6.. TC synapses in assembloids undergo LTD

(A) Left: schematic of the recording configuration to induce TC LTD. Right, top: electrical stimulation was delivered at 1 Hz for 900 pulses. Bottom: example responses to a subset of the 900 pulses; dark-to-light traces depict the responses as the number of pulses progressed (from pulse [p] 1 to p900). (B) Bar graph showing the average responses of 10 cells from 10 assembloids after TC LTD induction in aCSF. Colors indicate different batches of assembloids; vertical lines denote separate assembloids. (C) Time course data demonstrating that 1-Hz electrical stimulation (thick dashed line) induces LTD in TC synapses (black, n = 10 cells/10 assembloids). MPEP (blue, n = 7/5), AP5 (green, n = 6/5), or iBAPTA (orange, n = 5/4) blocked LTD. Shaded area depicts the presence of bath-applied drugs. The first (1) and final (2) 5 min of the experiment are noted. (D) Time course of Rs normalized to the 5-min baseline period. (E) Bar graph of group data after 1-Hz stimulation from (C). Differences from baseline were evaluated by one-sample t test (μ = 100 versus full postinduction time period, ###p < 0.005). Differences between treatments and aCSF were evaluated by one-way ANOVA, p = 0.0046. Dunnett’s test: **p < 0.01. (F) Example traces from the first (1) and final (2) 5 min of the experiment across conditions. Circles indicate electrical stimulation. Data shown are mean ± SEM in (C), (D), and (E), with individual data points overlaid in (E). n = cells/assembloids. See Figure S7 for additional supporting data.

Figure 7.. CT synapses in assembloids undergo LTD

(A) Schematic of the experimental condition for CT LTD induction, the 1-Hz LTD-induction protocol, and an example response. (B) Bar graph of the average responses of 10 cells from 9 assembloids after CT LTD induction in aCSF. Colors indicate different batches of assembloids; vertical lines denote separate assembloids. (C) Time course data show that 1-Hz stimulation (thick dashed line) induced LTD in CT synapses (black, n = 10 cells/9 assembloids). MPEP (blue, n = 6/6), AP5 (green, n = 5/4), or iBAPTA (orange, n = 4/4) blocked LTD. Shaded area depicts the presence of bath-applied drugs. The first (1) and final (2) 5 min of the experiment are noted. (D) Time course of Rs normalized to the 5-min baseline period. (E) Bar graph of group data after 1-Hz stimulation from (C). Differences from baseline were evaluated by one-sample t test (μ = 100 versus full postinduction time period, ###p < 0.001). Differences between treatments and aCSF were evaluated by one-way ANOVA, p = 0.0004. Dunnett’s test: ***p < 0.001. (F) Example traces from the first (1) and final (2) 5 min of the experiment across conditions. Circles indicate electrical stimulation. Data shown are mean ± SEM in (C), (D), and (E), with individual data points overlaid in (E). n = cells/assembloids. See Figure S7 for additional supporting data.