Schizophrenia-associated 22q11.2 deletion elevates striatal acetylcholine and disrupts thalamostriatal projections to produce amotivation in mice

Abstract

Schizophrenia is a complex neurodevelopmental disorder characterized by cognitive dysfunction, hallucinations, and negative symptoms such as amotivation. Negative symptoms are largely resistant to current antipsychotic treatments, and the neural circuits underlying amotivational states remain poorly defined. Here, using a mouse model of schizophrenia-associated 22q11.2 deletion syndrome (22q11DS), we report amotivation and weakened glutamatergic synaptic transmission between the thalamic parafascicular nucleus (Pf) and the dorsomedial striatum (DMS). Thalamostriatal disruption is attributed to hyperactivity of striatal cholinergic interneurons (CHIs), which is associated with enhanced Trpc3 and Pex51 (Trip8b) gene expression. Elevated acetylcholine levels in the DMS act on presynaptic M2 muscarinic receptors to weaken Pf-DMS glutamatergic transmission. Importantly, disruption of Pf-DMS synaptic transmission or hyperactivation of CHIs are each sufficient to cause amotivation in wild-type mice. These results identify a striatal hypercholinergic state and subsequent thalamostriatal disruption as core pathogenic events causing amotivation in 22q11DS, providing potential therapeutic targets.

Keywords: 22q11DS; TRIP8b; TRPC3; acetylcholine; amotivation; cholinergic interneuron; dorsomedial striatum; negative symptoms; parafascicular nucleus; schizophrenia.

Figure 1.. 22q11DS mice show amotivation and weakened synaptic transmission at the parafascicular nucleus (Pf) to medium spiny neuron (MSN) synapse.

(A) Diagram depicting the genes in the 22q11.2 syntenic region of mouse chromosome 16. Individual genes are represented by rectangles. Horizontal red bar represents the region of the hemizygous deletion in the Df(16)1/+ mouse model of 22q11 deletion syndrome (22q11DS). (B) Timeline of training and reinforcement schedules for behavioral studies. (C) Left: In the progressive ratio (PR) +1 task, Df(16)1/+ mice (red) lever pressed fewer times than WT littermates (black; two-way ANOVA, *p = 0.02). Right: In the PR+4 task, Df(16)1/+ mice lever pressed fewer times than WT littermates (two-way ANOVA, **p = 0.004). (D) The average ratio of the difference in performance between Df(16)1/+ mice and WT littermates was −0.33 for PR+1 and −0.46 for PR+4. (E) There was no difference in lever presses on the fixed ratio 1 (FR1) task between genotypes (two-way ANOVA, p = 0.37). (F) The average number of training days needed to reach criterion to progress to the operant tasks was similar between genotypes (two-way ANOVA, p = 0.2). (G) Free feeding intake of sweetened condensed milk (SCM) was similar between genotypes (two-way ANOVA, p = 0.62). (H) Both WT and Df(16)1/+ mice preferred SCM over standard rodent chow when both were freely available (mixed-design ANOVA, ****p < 0.0001). There was no difference in the amount of SCM consumed between genotypes (Tukey’s multiple comparisons, p > 0.99). The animal’s sex significantly affected food preference (mixed-design ANOVA, *p = 0.048). (I) There was no difference in the amplitude (left; unpaired t test, p = 0.41) or interevent interval (center; unpaired t test, p = 0.95) of spontaneously occurring excitatory postsynaptic currents (sEPSCs) in nucleus accumbens (NAc) medium spiny neurons (MSNs) between genotypes. Right: Example traces of sEPSCs from NAc MSNs from WT and Df(16)1/+ mice. (J) There was no difference in the amplitude of sEPSCs in dorsomedial striatal (DMS) MSNs between genotypes (left; unpaired t test, p = 0.39). The interevent interval was significantly higher between sEPSC events in MSNs from Df(16)1/+ mice (center; unpaired t test, *p = 0.04). Right: Example traces of sEPSCs from DMS MSNs from WT and Df(16)1/+ mice. (K) Electrically evoked EPSC amplitudes at the cortical (Ctx)-MSN synapse in response to increasing stimulus intensity were similar between genotypes (mixed-design ANOVA, p = 0.98). Inset shows the recording configuration. (L) Left: The average paired pulse ratio (PPR) of evoked EPSCs at the Ctx-MSN synapse was similar between genotypes (unpaired t test, p = 0.6). Right: Example traces of evoked Ctx-MSN EPSCs from WT and Df(16)1/+ mice. (M) There was a significant decrease in the amplitude of electrically evoked EPSCs at the Pf-MSN synapse in Df(16)1/+ mice compared to WT littermates (mixed-design ANOVA, ****p < 0.001). Inset shows the recording configuration. (N) There was a significant decrease in the amplitude of optogenetically evoked EPSCs (oEPSC) at the Pf-MSN synapse in Df(16)1/+ mice (mixed-design ANOVA, ***p < 0.0003). Inset shows the recording configuration. (O) Left: There was a significant difference in the PPR of electrically evoked EPSCs at the Pf-MSN synapse between genotypes (unpaired t test, **p = 0.003). Right: Example traces of electrically evoked Pf-MSN EPSCs from both genotypes. (P) Left: There was a significant difference in the PPR of optogenetically evoked EPSCs at the Pf-MSN synapse between genotypes (unpaired t test, **p = 0.005). Right: Example traces of optogenetically evoked Pf-MSN EPSCs from both genotypes. All data shown are mean ± SEM with individual data points overlaid in (C–J), (L), (O), (P). Unless noted, there were no sex differences. n = the number of cells/number of mice. Lightning bolts (K, M) represent electrical stimulation. Circles (L, O) represent stimulus artifacts. Blue star (N) represents optical stimulation. Blue lines (P) represent the optical stimulus. See Figure S1 for additional behavioral controls and for additional electrophysiology measures.

Figure 2.. Dampening Pf-DMS synaptic transmission causes amotivation.

(A) Schematic of the injection paradigm. (B) Histologic verification of injection sites in the DMS (AP +0.74 to +0.14 mm from bregma) and Pf (AP −2.18 to −2.46). Circles are virus expression from individual mice. (C) Mice injected with the DREADD agonist Compound 21 (C21; blue) lever pressed less than vehicle-injected mice (black) in the PR+1 task (left; two-way repeated measure (RM) ANOVA, **p = 0.009) and in the PR+4 task (right; two-way RM ANOVA, *p = 0.03). (D) The average difference ratio between vehicle and C21 injection was −0.22 ± 0.07 in the PR+1 task performance and −0.19 ± 0.07 in the PR+4 performance. These ratios were significantly different from a theoretical mean of 0 (one-sample t test for PR+1, **p = 0.007; for PR+4, *p = 0.01). (E) The number of rewards earned on the PR+1 (left) and PR+4 (right) tasks was lower with C21 injection compared to vehicle (two-way RM ANOVA for PR+1, **p = 0.006; for PR+4, **p = 0.009). (F) Left: There were significantly fewer lever presses in the FR1 choice task after C21 injection compared to vehicle (two-way RM ANOVA, *p = 0.02). Right: There was significantly higher ingestion of freely available standard rodent chow in the FR1 choice task after C21 injection compared to vehicle (two-way RM ANOVA, *p = 0.02). (G) There was no difference in the number of lever presses (left) or the rewards earned (right) on the RR task between C21 and vehicle injection (two-way RM ANOVA for lever presses, p = 0.16; for rewards earned, p = 0.07). (H) Mice preferred SCM over standard rodent chow when both were freely available regardless of injection type (three-way ANOVA, ****p < 0.0001). There was no difference in the amount of SCM consumed based on injection type (Tukey’s multiple comparisons, p = 0.97). (I) Schematic of the recording configuration. (J) In mice expressing hM4Di in the Pf-DMS pathway, bath application of C21 decreased the evoked amplitude of Pf-MSN EPSCs (blue), whereas bath application of artificial cerebrospinal fluid (ACSF) did not (black). Shaded area depicts the time of drug or ACSF wash. (K) Left: The average Pf-MSN EPSCs in response to ACSF and C21 wash were significantly different (unpaired t test, **p = 0.005). The data analyzed are from the final 5 min of the experiment. The response to ACSF wash was not different from baseline (one-sample t test, μ = 100, p = 0.89). The response to C21 wash was different from baseline (one-sample t test, μ = 100, ##p = 0.003). Right, top: Representative traces showing the decrease in amplitude after C21 application (2) compared to baseline (1). Right, bottom: Representative traces showing no difference in EPSC amplitude after ACSF application and at baseline (4 and 3, respectively). The numbers 1–4 correspond to the timepoints labeled in (J). (L) Schematic of the experimental design depicting a WT mouse with no intracranial viral injections. (M) In the absence of DREADDs, injection of C21 did not alter performance on PR+1 (left; two-way RM ANOVA, p = 0.33) or PR+4 (right; two-way RM ANOVA, p = 0.74). (N) The average difference ratio in performance between vehicle and C21 injection was 0.09 ± 0.13 for PR+1 and 0.006 ± 0.09 for PR+4. These ratios were not different from a theoretical mean of 0 (one-sample t test for PR+1, p = 0.51; for PR+4, p = 0.94). (O) Mice injected with vehicle or C21 preferred SCM over food chow when both were readily available (three-way ANOVA, ****p < 0.0001). There was no difference in the amount of SCM consumed based on injection type (Tukey’s multiple comparisons, p = 0.11). The animal’s sex affected food preference (three-way ANOVA, *p = 0.02). All data shown are mean ± SEM with individual data points overlaid in (C–H), (K), (M–O). Unless noted, there were no sex differences. n = the number of cells/number of mice. Lightning bolt (I) represents electrical stimulation. Circles (K, right) represent stimulus artifacts. n.s.: not significant. See Figure S2 for additional electrophysiology measures of Pf neurons.

Figure 3.. Hypercholinergic state in the DMS of 22q11DS mice.

(A) Left: Inclusion of the muscarinic acetylcholine (ACh) M2 receptor antagonist AF-DX-116 in the ACSF (black and red) restored Df(16)1/+ synaptic transmission at the Pf-MSN synapse compared to control ACSF (gray and pink; mixed-design ANOVA, ****p < 0.0001). Inset: Schematic of the recording configuration. Right: Example traces of optogenetically evoked Pf-MSN EPSCs in the presence of AF-DX-116. (B) Left: Bath application of AF-DX-116 rescued Df(16)1/+ Pf-DMS oEPSC amplitude in response to a 12-mW stimulus intensity (two-way ANOVA, ***p = 0.0002; Fisher’s LSD test between genotypes, ##p = 0.001; between genotypes in the presence of AF-DX-116, #p = 0.03; WT AF-DX-116 compared to ACSF, #p = 0.02; Df(16)1/+ AF-DX-116 compared to ACSF, ##p = 0.002). Right: Bath application of AF-DX-116 restored Df(16)1/+ Pf-MSN PPR to WT levels (two-way ANOVA, *p = 0.03; Fisher’s LSD test between genotypes, ##p = 0.008; between genotypes in the presence of AF-DX-116, p = 0.58; WT AF-DX-116 compared to ACSF, p = 0.57; Df(16)1/+ AF-DX-116 compared to ACSF, #p = 0.02). (C) Left: Immunofluorescence of tdTomato expression under the choline acetyltransferase (ChAT) promoter in WT mice at lower (left) and higher (right top, bottom) magnifications. Right: Immunofluorescence of ChAT-driven tdTomato in Df(16)1/+ mice at lower (left) and higher (right top, bottom) magnifications. Scale bars for left images: 1 mm. Scale bars for right top: 0.5 mm. Scale bars for right bottom: 0.25 mm. White boxes show the area of higher magnification. (D) The total number of DMS tdTomato⁺ cholinergic interneurons (CHI) was similar between genotypes (unpaired t test, p = 0.11). (E) Left: Schematic of the recording configuration. Right: There were more tonically active CHIs in Df(16)1/+ mice than in WT littermates (Chi-square test, *p = 0.04). n = number of cells; N = number of mice. (F) Left: The evoked firing rate in response to a depolarizing current injection of CHIs from Df(16)1/+ mice was higher than that from WT littermates (mixed-design ANOVA, **p = 0.004). Center: The average number of evoked AP in response to a +240-pA current injection was higher in Df(16)1/+ mice compared to WTs (unpaired t test, *p = 0.03). Left: Example traces showing the response of CHIs from both genotypes to a depolarizing current injection (+150 pA). (G) Schematic showing the imaging and experimental configuration. (H) There was a greater change in fluorescence (dF/F) of the ACh sensor GRAB-ACh in response to increasing stimulation frequency in Df(16)1/+ mice compared to WT littermates (mixed-design ANOVA, **p = 0.009). n = number of fields of view or number of mice. (I) One example region of interest from WT and Df(16)1/+ DMS expressing GRAB-ACh during no stimulation (left) and 20 Hz electrical stimulation (right) is highlighted in the white circle. Scale bar: 35 μm. (J) Schematic representing the Pf-CHI-MSN trisynaptic circuit in WT mice. (K) Schematic representing disruption in synaptic transmission at the Pf-CHI-MSN trisynaptic circuit in Df(16)1/+ mice. Data shown are mean ± SEM with individual data points overlaid in (B), (D), (F). n = the number of cells/number of mice, unless otherwise noted. Blue star (A) represents optical stimulation. Blue lines (A) represent the optical stimulus n.s.: not significant. See Figure S3 for additional electrophysiologic measurements.

Figure 4.. Chemogenetic enhancement of CHI activity produces amotivation.

(A) Schematic of the injection paradigm. A Cre-dependent virus containing the excitatory DREADD receptor hM3Dq was bilaterally injected into the DMS of ChAT^Cre mice. (B) Histologic verification of injection sites in the DMS (AP +0.74 to +0.14 mm from bregma). Circles are virus expression from individual mice. (C) Mice injected with the DREADDs agonist C21 lever pressed less than vehicle-injected mice in the PR+1 task (left; two-way RM ANOVA,* p = 0.04) and in the PR+4 task (right; two-way RM ANOVA, *p = 0.03). (D) The average difference ratio between vehicle and C21 injection was −0.32 ± 0.11 in PR+1 task performance and −0.13 ± 0.11 in PR+4 performance. The PR+1 difference ratio was significantly different from a theoretical mean of 0 (one-sample t test, **p = 0.007), but the PR+4 difference ratio was not (one-sample t test, p = 0.28). (E) The number of rewards earned on the PR+1 (left) and PR+4 (right) tasks was lower after C21 injection than vehicle injection (two-way RM ANOVA for PR+1, **p = 0.005; for PR+4, *p = 0.02). (F) Left: There were significantly fewer lever presses in the FR1 choice task after C21 injection compared to vehicle (two-way RM ANOVA, *p = 0.02). Right: There was no difference in the amount of freely available rodent chow ingested in the FR1 choice task between C21 and vehicle injection (two-way RM ANOVA, p = 0.89). (G) Both lever presses (left) and the rewards earned (right) on the RR task after C21 injection were significantly different from vehicle injection (two-way RM ANOVA for lever presses, ***p = 0.0001; for rewards earned, ***p = 0.0001). (H) Mice preferred SCM over standard rodent chow when both were freely available regardless of injection type (three-way ANOVA, ****p < 0.0001). There was no difference in SCM consumption based on injection type (Tukey’s multiple comparison, p = 0.56). (I) Schematic of the recording configuration. Green receptors represent hM3Dq expression. (J) Left: The rheobase current required to induce AP firing in CHIs was lower after application of C21 compared to vehicle (paired t test, **p = 0.008). Right: Example traces of CHIs in the presence of ACSF or C21 responding to a depolarizing current injection ramp, from 0 pA to +200 pA. (K) The membrane potential of CHIs expressing hM3Dq was depolarized after application of C21 compared to vehicle (paired t test, *p = 0.02). All data shown are mean ± SEM with individual data points overlaid in (C–K). Unless noted, there were no sex differences. n.s.: not significant.

Figure 5.. Pf-MSN synaptic transmission is bidirectionally altered by cholinergic transmission.

(A) Schematic of the recording configuration. Green receptors represent hM3Dq expression. (B) Left: Activation of DMS CHIs with bath application of C21 decreased Pf-MSN synaptic transmission over time (filled circles). Bath application of C21 in the presence of AF-DX-116 enhanced Pf-MSN synaptic transmission (open circles). Shaded region depicts the bath application of 1–5 μM C21. Right, closed circles: Representative traces demonstrating the decrease in amplitude following C21 application (2) compared to baseline (1). Right, open circles: Representative traces demonstrating an increase in EPSC amplitude following C21 application in the presence of AF-DX-116 relative to baseline (4 and 3, respectively). (C) There was a significant difference in Pf-MSN synaptic transmission in the presence or absence of AF-DX-116 after C21 application (unpaired t test of min 25–30, ****p < 0.0001). The changes in synaptic transmission following C21 application with and without AF-DX-116 present were different from their baseline values (one-sample t test with μ = 100 of min 25–30, ###p = 0.0009 for ACSF; #p = 0.028 for AF-DX-116). (D) There was a significant difference in the PPR between electrically evoked EPSCs at the Pf-MSN synapse after C21 wash in the presence or absence of AF-DX-116 (paired t test of min 25–30, #p = 0.039 for ACSF; ##p = 0.004 for AF-DX-116). There was a significant difference in the change in PPR at min 25–30 between groups (data analyzed by unpaired t test, *p = 0.01). (E) Schematic of the recording configuration. (F) Left: Bath application of AF-DX-116 temporarily enhanced Pf-MSN synaptic transmission in WT mice. In Df(16)1/+ mice, bath application of AF-DX-116 led to a prolonged increase in Pf-MSN synaptic transmission. Shaded region depicts bath application of 1 μM AF-DX-116. Right: Representative traces showing no change in WT or the increase in Df(16)1/+ mice in Pf-MSN synaptic transmission after AF-DX-116 application. (G) There was a significant difference in the response to AF-DX-116 application based on genotype and time following application (two-way RM ANOVA for min 1–5 (5), 6–11 (11), and 25–30 (30), **p = 0.009; Tukey’s multiple comparison test min 5 vs min 11 for wildtype, #p = 0.046; for Df(16)1/+ ###p = 0.0006. For Df(16)1/+ min 5 vs min 30 ###p = 0.0005). (H) There was a significant difference in the PPR in response to AF-DX-116 application based on genotype and time following application (two-way RM ANOVA, *p = 0.034; Tukey’s multiple comparison test min 5 vs 11 for wildtype, ###p = 0.0001; for min 11 vs 30 for wildtype ##p = 0.006). Data shown are mean ± SEM. n = the number of cells/number of mice. Lightning bolts (A, E) represent electrical stimulation. Circles (B, F), right, represent stimulus artifacts.

Figure 6.. Gene expression is altered in CHIs from the dorsal striatum of 22q11DS mice.

(A) Overview of single-nucleus RNA-sequencing (snRNA-seq) analysis. (B) Uniform Manifold Approximation and Projection (UMAP) plot with cluster annotations indicated by color. (C) UMAP plot of the CHI marker, Chat. Color indicates the normalized transcript level. (D) Dot plot showing CHI marker expression among neuronal clusters. (E) Bar plot of the number of CHI nuclei detected per mouse. Color indicates mouse identity. (F) Volcano plot showing differential gene expression in Df(16)1/+ CHIs compared to WT littermates. Red circles indicate genes within the Df(16)1/+ hemizygous region. (G) Heatmap showing cellular compartment (CC) terms overrepresented among transcripts differentially expressed in Df(16)1/+ CHIs. Color indicates log2(Fold Change) of each transcript. (H) Dot plot showing normalized expression of each transcript from (G) within each cell type found in our dorsal striatal data. Red box highlights the CHI cluster. expression: normalized average expression; proportion: proportion of cells expressing a marker within a cluster. Data in (A–H) were produced by snRNA-seq analysis of 35,167 nuclei derived from the dorsal striatum of 4 Df(16)1/+ mice and 4 WT littermates. Nuclei derived from cortex and thalamus were removed before analysis. See Figure S4 for additional snRNA-seq analyses and Tables S1 and S2 for supporting data.

Figure 7.. Manipulating CHI activity and Pf-DMS synaptic transmission in 22q11DS mice is not sufficient to rescue amotivation.

(A) Schematic of the injection paradigm. A Cre-dependent virus containing the inhibitory DREADD receptor hM4Di was bilaterally injected into the DMS of Df(16)1/+;ChAT^Cre mice. (B) Histologic verification of injection sites into the DMS (AP +0.5 to −0.10 mm from bregma). Circles are virus expression from individual mice. (C) Injection of C21 did not affect performance on PR+1 (left) or PR+4 (right) in Df(16)1/+ mice (two-way RM ANOVA for PR+1, p = 0.98; for PR+4, p = 0.45). (D) The difference ratio in the PR+1 and PR+4 task performance between vehicle and C21 injection was not significantly different from 0 (one-sample t test for PR+1, −0.11 ± 0.12, p = 0.37; for PR+4, 0.18 ± 0.21, p = 0.41). (E) The number of rewards earned on the PR+1 (left) and PR+4 (right) tasks were not significantly different between vehicle and C21 injection (two-way RM ANOVA for PR+1, p = 0.38; for PR+4, p = 0.21). (F) Schematic of the injection paradigm. A retrogradely-transported virus containing Cre was bilaterally injected into the DMS of Df(16)1/+ mice. A Cre-dependent virus containing the excitatory DREADD receptor hM3Dq was bilaterally injected into the Pf of the same mouse. (G) Histologic verification of the injection sites in the DMS (AP +0.74 to +0.14 mm) and Pf (AP −2.18 to −4.26 mm). Circles are virus expression from individual mice. (H) Injection of C21 did not alter performance on the PR+1 (left) or PR+4 (right) tasks in Df(16)1/+ mice (two-way RM ANOVA for PR+1, p = 0.49; for PR+4, p = 0.49). (I) The difference ratio in the PR+1 and PR+4 task performance between vehicle and C21 injection is not different from 0 (one-sample t test for PR+1, −0.11 ± 0.11, p = 0.36; for PR+4, −0.1 ± 0.09, p = 0.3). (J) There was no difference in the number of rewards earned for PR+1 (left) or PR+4 (right) in Df(16)1/+ mice after C21 injection compared to vehicle (two-way RM ANOVA for PR+1, p = 0.23; for PR+4, p = 0.24). (K) Schematic of the injection paradigm. A retrogradely-transported Cre virus and a virus containing the Cre-dependent inhibitory DREADD receptor hM4Di were bilaterally injected into the DMS of Df(16)1/+;ChAT^Cre mice. In the same mice, a virus containing a Cre-dependent excitatory DREADD receptor hM3Dq was bilaterally injected into the Pf. (L) Histologic verification of the injection sites in the DMS (AP +0.5 to −0.1 mm) and Pf (AP −2.18 to −2.46 mm). Circles are virus expression from individual mice. (M) Injection of C21 did not affect Df(16)1/+ mouse performance on the PR+1 task (left; two-way RM ANOVA, p = 0.08) or the PR+4 (right; two-way RM ANOVA, p = 0.08). (N) The difference ratio for PR+1 and PR+4 task performance between vehicle and C21 injection was not different from 0 (one-sample t test for PR+1, −0.17 ± 0.11, p = 0.14; for PR+4, −0.1 ± 0.12, p = 0.39). (O) C21 injection did not affect the number of rewards earned during the PR+1 or PR+4 tasks (left and right, respectively; two-way RM ANOVA for PR+1, p = 0.08; for PR+4, p = 0.18). Data shown are mean ± SEM with individual data points overlaid in (C–E), (H–J), (M–O). Unless noted, there were no sex differences. n.s.: not significant. See Figure S5 for additional behavioral measurements.