Behavioral abnormalities and circuit defects in the basal ganglia of a mouse model of 16p11.2 deletion syndrome

Abstract

A deletion on human chromosome 16p11.2 is associated with autism spectrum disorders. We deleted the syntenic region on mouse chromosome 7F3. MRI and high-throughput single-cell transcriptomics revealed anatomical and cellular abnormalities, particularly in cortex and striatum of juvenile mutant mice (16p11(+/-)). We found elevated numbers of striatal medium spiny neurons (MSNs) expressing the dopamine D2 receptor (Drd2(+)) and fewer dopamine-sensitive (Drd1(+)) neurons in deep layers of cortex. Electrophysiological recordings of Drd2(+) MSN revealed synaptic defects, suggesting abnormal basal ganglia circuitry function in 16p11(+/-) mice. This is further supported by behavioral experiments showing hyperactivity, circling, and deficits in movement control. Strikingly, 16p11(+/-) mice showed a complete lack of habituation reminiscent of what is observed in some autistic individuals. Our findings unveil a fundamental role of genes affected by the 16p11.2 deletion in establishing the basal ganglia circuitry and provide insights in the pathophysiology of autism.

Figure 1. A Mouse Model for the Human 16p11.2 Microdeletion

(A) Top: the region on human chromosome 16p11.2 is flanked by segmental duplications (SD), which likely mediate CNVs of the locus by nonhomologous recombination. Bottom: the syntenic region on mouse chromosome 7F3, in which LoxP sites (blue arrowheads) were inserted at positions indicated. (B) Sequential recombination steps yield deletion of 440 kb containing the mouse 16p11 genes. pA, poly A; int, intron; CAG, chicken β-actin enhanced CMV promoter; Neor and Puror, neomycin and puromycin resistance cassettes; STOP, translational stop codon; Che, mCherry. Scale bar, 1 kb. Inset, Genotyping of F1 offspring from floxed (16p11^flx/+) chimeras and HPRT-Cre^tg/+ females shows germline transmission. Arrows in scheme show PCR primer positions. (C) Red fluorescence in E16.5 16p11+/− embryo confirms mCherry expression and deletion of 16p11 genes. (D) At birth (P0): qPCR analysis shows global downregulation of 16p11 genes (red) in the brain upon deletion of one allele, whereas neighboring genes are unaffected. (E) Adult animals (4 months): 16p11+/− females lack abdominal fat pads (white arrowheads) typical for this age. (F) Adult animals (3 months): relative body length (n = 12). (G) Relative body weight (n ≥ 6 per time point) across development normalized to the average of gender-matched wild-type littermates. Inset: independent analysis of body weight performed at NIMH at P6. (H) At 6 weeks of age: increased juvenile mortality of 16p11+/− mice is rescued by improved nutritional regimen and alleviated sibling competition. (I) Adult animals (3 months): food intake is comparable between 16p11+/− and wild-type animals. (J) Postnatal developmental trajectory of brain weight as a measure for global brain growth (n ≥ 6). Data are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. Inset in (G), NIMH.

Figure 2. Anatomical Abnormalities in Juvenile 16p11+/− Mice

MRI analysis of mouse brains at P7 (n = 26 for each genotype). (A–C) Volume and shape difference are displayed for the BG regions, namely, the striatum (A), GP (B), and NAc (C). Volumes in (A)–(C) are shown as both absolute (in mm³) and relative volumes. Shape differences in (A)–(C) show 3D surface renderings of the given region of interest. Highlighted on that surface are significant shape differences (q < 0.05) between the 16p11+/− mouse and control. Orange, outward movement; blue, inward movement. (D) Coronal flythrough highlighting significant differences in the relative volume of the 16p11+/− mouse and control (red: larger, blue: smaller); only highly significant areas are shown (q < 0.01). Error bars represent SEM. *q < 0.05, **q < 0.01.

Figure 3. Single-Cell Gene Expression Profiling of Cell Types in the Neonate Brain

(A) Experimental workflow: brain dissection, single-cell sorting by FACS, reverse transcription (RT), and preamplification of cDNAs of interest. A total of 190 genes were profiled by multiplex qPCR using two 96.96 dynamic arrays per sample plate. ctx, cortex; hc, hippocampus; spa, subpallium; ms-dienc, mesodiencephalon. (B and C) Identification of cell-type-specific gene clusters for the subpallium (B) and cortex (C) by coexpression mapping (n > 500 cells). The dendrogram shows the proximity of genes based on their coexpression with all other genes. Listed genes are known to be expressed in a cell-type- and/or developmental-stage-specific manner and used accordingly for identification of cell-type-specific gene clusters. (D and E) Separate clustering for 16p11+/− and wild-type samples (n ≈ 250 cells each) of subpallium (D) and cortex (E). Color code matches (C) and (D), respectively. LVW, lateral ventricular wall; LMS, lateral migratory stream; SVZ, subventricular zone; VZ, ventricular zone.

Figure 4. Altered DA Signaling in the Neonate 16p11+/− Brain

(A) Numbers of subpallial cells expressing genes specific to GABAergic neurons, striatal MSNs, and striatopallidal MSNs suggest an increase in the number of indirect pathway MSNs and total MSNs in 16p11+/− mice. (B) Striatal MSNs as defined by DA receptor gene expression only. Note that the increase in Drd2⁺ MSNs is not at the expense of Drd1+ cells, but is likely due to a total increase in the number of MSNs consistent with (A). (C) Abnormalities in deeper layer cortical excitatory neurons and Pax6⁺ VZ progenitors as well as callosal projection neurons (CPN). Reduction of Darpp32 and Drd1 expression indicate a loss of dopamine-sensitive cells. (D) Combined consideration of Ctip2 and Darpp32 expression suggests a lower number of dopamine-sensitive cells in the deeper cortical layers but no major reduction in deeper layer corticofugal cell types. n.s., not significant. (E) Decreased expression of Th in ventral midbrain DA cells. (F) Top: coexpression of the 16p11 genes in striatopallidal MSNs as defined by Drd2 expression. Framed 16p11 genes have been reported in ASD individuals with a smaller deletion in the 16p11.2 locus. Bottom: fraction of Drd2⁺ cells expressing each of the 16p11 genes. Error bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 where not otherwise indicated.

Figure 5. Excess Numbers of Striatopallidal MSNs and Hypodopaminergia in Cortex of Juvenile 16p11+/− Mice

(A) Coronal cryosections show the expression of a Drd2-GFP BAC transgene in the mouse striatum at P7. (B) Magnification of boxed regions of the dorsal striatum from (A). (C) Quantification of GFP⁺ cells (n = 3 animals per genotype). (D) The GP, the output structure of striatopallidal projecting (Drd2⁺) MSNs, is enlarged in 16p11+/− brains. (E) GPm, the striatonigral (Drd1⁺ MSNs) output structure contains Drd2-GFP⁺ fibers in 16p11+/− not found in wild-type. (F) Magnification of boxed regions in (E). (G) Somatosensory cortex in sagittal sections of P7 brains shows downregulation of DARRP32 expression. CTIP2 was used for visualization of layer V (large, bright CTIP2⁺ pyramidal neurons), and layer VI (smaller, less bright CTIP⁺ neurons). Blue: Hoechst nuclear stain. (H) Magnification of boxed area in (G). Although DARPP32 expression is much weaker in 16p11+/− cortex, some DARPP32⁺ cells can still be identified (yellow arrowheads). (I) Quantification of DARPP32⁺ cells in cortical layers V and VI (irrespective of expression level). Error bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6. Deficits at Excitatory Synapses onto NAc MSNs in 16p11+/− Mice

Electrophysiological recordings in NAc MSNs at 4–8 weeks. (A and B) Comparable I/V relationships (A) AMPAR rectification index (B) in 16p11+/− and wild-type mice (n = 9 cells for each genotype). (C) Increased AMPAR/NMDAR ratio in 16p11+/− mice. (D) Decreased paired-pulse ratios (PPRs) across multiple interstimulus intervals (ISIs) in 16p11+/− mice. (E) Consistent with a higher presynaptic release probability, significant increase in mEPSC frequency in 16p11^+/− mice (n = 12 wild-type cells, n = 14 16p11+/− cells). (F) Comparable mEPSC amplitude between genotypes. (G–I) Morphological analysis (8 weeks of age) of MSN dendrites (n = 10 cells per genotype). (G) Representative Golgi-stained MSN dendrites covered with dendritic spines. (H and I) No change in spine density (H) or number of primary dendrites (I) was detected. (J) Recording of synaptic events in Drd2-EGFP⁺ MSNs of the 16p11+/− NAc (4–6 weeks) show a significant increase in sEPSC frequency, whereas the amplitude remained unchanged (n = 9 wild-type cells, n = 10 16p11+/− cells). Error bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 7. Behavioral Deficits of Adult 16p11+/− Animals

(A) Adult 16p11+/− mice (2–3 months) display a significantly reduced startle response at increasing decibels (NIMH: wild-type n = 17, 16p11+/− n = 15) and repeated 20-stimulus presentations (inset, SBFNL). (B and C) Movement control: 16p11+/− mice show lack of gait fluidity (B) and frequent tremor (C). (D) Hyperactivity of 16p11+/− mice in a home-cage environment. (E) In a novel empty-cage environment, 16p11+/− mice exhibited significantly more hanging, less self-grooming, and less resting than wild-type littermates. A fraction of 16p11+/− mice showed continuous circling. (F) Follow-up quantification of circling behavior in a rotational assay in a cylindrical cage (inset, total number of rotations over the 30 min period). (G) Adult 16p11+/− mice exhibited initial hypoactivity and abnormal dishabituation to novel environment in the open field. Inset, similar behaviors of the 16p11+/− mice in the open-field test independently reproduced at NIMH. (H and I) Adult 16p11+/− mice display altered performance in a novel object recognition test (H) and a six-trial novel object recognition assay (I) compared to wild-type littermates. 16p11+/− mice did not show a significant preference for the novel object, compared to wild-type mice (H). This effect was due to a lack of habituation to the familiar object in the 16p11+/− mice. This was further corroborated in a six-trial novel object recognition test, where the 16p11+/− mice spent longer time in trials 2–4 and 6 sniffing the first object that had been presented as a novel object in trial 1 and also longer time sniffing the second novel object in trial 5 than the control mice (I). (J) Adult 16p11+/− mice showed hyperactivity in the activity chamber during a 10 min period. (K) Acute administration of risperidone had no significant effect on the activity level of 16p11+/− mice in the activity chamber, whereas wild-type littermates exhibited a dramatic decrease in activity level after risperidone administration. For all panels, mean ± SEM is presented for each data point; *p < 0.05, – **p < 0.01, and ***p < 0.001. (A), (E), (G) inset, and (H): NIMH; (A) inset, (B) (D), (F), (G), and (I–K): SBFNL.