Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice

Abstract

Obsessive-compulsive disorder (OCD) is an anxiety-spectrum disorder characterized by persistent intrusive thoughts (obsessions) and repetitive actions (compulsions). Dysfunction of cortico-striato-thalamo-cortical circuitry is implicated in OCD, although the underlying pathogenic mechanisms are unknown. SAP90/PSD95-associated protein 3 (SAPAP3; also known as DLGAP3) is a postsynaptic scaffolding protein at excitatory synapses that is highly expressed in the striatum. Here we show that mice with genetic deletion of Sapap3 exhibit increased anxiety and compulsive grooming behaviour leading to facial hair loss and skin lesions; both behaviours are alleviated by a selective serotonin reuptake inhibitor. Electrophysiological, structural and biochemical studies of Sapap3-mutant mice reveal defects in cortico-striatal synapses. Furthermore, lentiviral-mediated selective expression of Sapap3 in the striatum rescues the synaptic and behavioural defects of Sapap3-mutant mice. These findings demonstrate a critical role for SAPAP3 at cortico-striatal synapses and emphasize the importance of cortico-striatal circuitry in OCD-like behaviours.

Figure 1. Facial lesions, excessive grooming and anxiety-like behaviors in SAPAP3 mutant mice

a, SAPAP3^-/- mice have facial and neck skin lesions (arrows). b, c, SAPAP3^-/- mice (KO) showed more grooming bouts (b) and spent more time in self-grooming (c) than wildtype mice (WT) at all times examined. d, Pre-lesion and post-lesion groups of SAPAP3^-/- mice showed similar degrees of increased grooming. e,f, In the open field test, SAPAP3^-/- mice spent less time in the center (e), whereas locomotion along the perimeter was comparable to wildtype controls (f). g-i, In the dark-light emergence test, SAPAP3^-/- mice took longer to cross from the dark to the brightly lit chamber (g) and spent less time in the brightly lit chamber (h), while total activity in both chambers was similar (i). j-l, In the elevated zero maze, SAPAP3^-/- mice took longer to cross into the open areas (j) and spent less time in these areas than wildtype controls (k), while total activity in both open and closed areas was similar (l). *p < 0.05, **p < 0.01, ***p < 0.001, repeated measures ANOVA for b-e and two-tailed t-test for f-l; all data are presented as means ± SEM from 8-9 mice per genotype, Cohen’s kappa for intra-observer agreement exceeded 0.92.

Figure 2. Fluoxetine treatment alleviates excessive grooming and anxiety-like behavior

a, Daily fluoxetine treatment (5 mg/kg, i.p.) over 6 days reduced grooming behavior in SAPAP3^-/- mice. b, c, Given over 6 days, fluoxetine alleviated anxiety-like behavior of SAPAP3^-/- mice in the dark-light emergence test. d, A single injection of fluoxetine (5 mg/kg, i.p.) had no effect on the grooming behavior of SAPAP3^-/- mice. **p < 0.01, ***p < 0.001, repeated measures ANOVA; all data are presented as means ± SEM from 9-11 mice per group, Cohen’s kappa for intra-observer agreement exceeded 0.92.

Figure 3. Altered cortico-striatal synaptic transmission in SAPAP3 mutant mice

a, Only SAPAP3 mRNA is highly expressed in the striatum (STR). b, Cortico-striatal field potential recordings of acute brain slices show decreased fEPSPs in SAPAP3^-/- mice. p< 0.001, repeated measures ANOVA. Inset shows a typical field recording (black trace) and sensitivity to NBQX (red trace). NP1, negative peak 1. c, Paired-pulse ratios (slope fEPSP#2/slope fEPSP#1) are similar between wildtype and SAPAP3^-/- mice. d, Example traces of NMDAR-dependent fEPSPs recorded in the presence of 50 μM NBQX, 0 mM Mg²⁺ and 2 mM glycine. Sensitivity to APV indicated by red trace. e, NMDAR fEPSP area is increased in SAPAP3^-/- mice. *p < 0.05, two-tailed t-test; number of recordings in parentheses.

Figure 4. Structural and biochemical analyses of cortico-striatal synapses in SAPAP3 mutant mice

a, b, The levels of PSD95, PSD93 and Shank in striatal PSD fractions are not affected in SAPAP3^-/- mice. The levels of NR1 and NR2B subunits are increased, whereas that of NR2A is decreased. β-actin and β-tubulin were used as loading controls. *p < 0.05, two-tailed t test. c, d, Electron micrographs show the presence of synaptic vesicles (arrowheads), postsynaptic densities (arrows) and dendritic spines (stars). e, The length of the PSD is not significantly different in wildtype and SAPAP3^-/- mice. f, g, The thickness of the dense layer (f) of the PSD in SAPAP3^-/- mice, but not the light layer (g), is reduced. p< 0.001, two-tailed t test; n=94 for wildtype and n=92 for SAPAP3^-/- mice.

Figure 5. Lentiviral-mediated rescue of behavioral and synaptic defects in SAPAP3 mutant mice

a, Diagram showing the approximate locations of microinjections in the striatum of SAPAP3^-/- mice. Injection site 1, locations 1-3 are more anterior than injection site 2, locations 4-8. b-c’, Brain sections from a SAPAP3^-/- mouse injected with GFP lentivirus show GFP fluorescence (b, c) and an absence of SAPAP3 staining (b’, c’). d-e’, Brain sections from a SAPAP3^-/- mouse injected with GFP-SAPAP3 lentivirus show both GFP fluorescence (d, e) and SAPAP3 immunostaining (d’, e’). f, Compared to SAPAP3^-/- mice injected with GFP lentivirus, SAPAP3^-/- mice injected with GFP-SAPAP3 lentivirus showed significantly reduced over-grooming behavior. **p < 0.01, two-tailed t-test; n=8 mice/group for f, i-k. g-h, SAPAP3^-/- mice injected with GFP-SAPAP3 lentivirus (h) had reduced severity of facial lesions when compared to SAPAP3^-/- mice injected with GFP lentivirus (g). i, Semi-quantitative lesion scores. **p < 0.01, Mann-Whitney U test. j-k, Reduced anxiety-like behaviors in SAPAP3^-/- mice injected with GFP-SAPAP3 lentivirus in the dark-light emergence test, including decreased latency to cross from the dark to light chamber (j) and increased time in the light chamber (k). *p < 0.05, two-tailed t-test. l, m, Field recordings from infected striatal area of P21-P25 SAPAP3^-/- mice injected with GFP-SAPAP3 lentivirus showed an increase in cortico-striatal fEPSP amplitude (l) and a reduction of NMDAR-dependent fEPSP area (m). p < 0.001, repeated measures ANOVA for l; *p < 0.05, two-tailed t-test for m; n is 12 and 10 for l, and 10 and 9 for m for GFP injected and GFP-SAPAP3 injected, respectively .