Shank2/3 double knockout-based screening of cortical subregions links the retrosplenial area to the loss of social memory in autism spectrum disorders

Abstract

Members of the Shank protein family are master scaffolds of the postsynaptic architecture and mutations within the SHANK genes are causally associated with autism spectrum disorders (ASDs). We generated a Shank2-Shank3 double knockout mouse that is showing severe autism related core symptoms, as well as a broad spectrum of comorbidities. We exploited this animal model to identify cortical brain areas linked to specific autistic traits by locally deleting Shank2 and Shank3 simultaneously. Our screening of 10 cortical subregions revealed that a Shank2/3 deletion within the retrosplenial area severely impairs social memory, a core symptom of ASD. Notably, DREADD-mediated neuronal activation could rescue the social impairment triggered by Shank2/3 depletion. Data indicate that the retrosplenial area has to be added to the list of defined brain regions that contribute to the spectrum of behavioural alterations seen in ASDs.

Fig. 1. Shank2/3 dKO mice show severe autistic-like behaviours.

A Representative immunoblot showing the loss of Shank2A/E and Shank3a/c/d/e isoforms in cortical homogenates of dKO mice. B dKO did not bury any marble in comparison to WT mice; WT = 13, dKO = 10. C Impaired nest building in dKO mice; WT n = 16, KO n = 16. D Representative heatmaps of the three-chamber sociability trial (left) and dKO mice showed social deficits (right); WT = 11, dKO = 11. E Representative heatmaps of the three-chamber social novelty trial (left) and dKO mice displayed social memory deficits (right); WT = 11, dKO = 11. F Frequency spectrogram of typical ultrasonic vocalizations (USVs) emitted by WT and dKO male mice during female urine presentation. G, H dKO male mice emitted reduced USVs in the presence of female urine (G), as well as during direct interaction with a female (H); WT = 8, dKO = 8. I, J dKO mice showed social interaction deficits (I) and increased aggressive acts (J); WT = 7, dKO = 8. K dKO mice detected odour cues; WT = 8, dKO = 5. L, M dKO mice displayed skin lesions (L) due to increased self-grooming (M); WT n = 14, dKO n = 11. N–Q A significant number of dKO mice showed increased time spent jumping (N, O) and upright scrabbling (P, Q); WT n = 12, dKO n = 11 (N, O); WT n = 14, dKO n = 11 (P, Q). See Materials and Methods, as well as Supplementary Table 2 for detailed statistical analysis.

Fig. 2. Shank2/3 loss in the nucleus accumbens leads to repetitive behaviours.

A Schematic of bilateral injections of AAV9-CMV-GFP or AAV9-CMV-GFP-Cre into nucleus accumbens (ACB) of dKO^fx/fx. B Representative images showing GFP (left) and GFP-Cre (right) expression in the ACB. C, D Immunohistochemistry staining of the ACB using the Shank2 (C) and Shank3 (D) markers; GFP n = 4, GFP-Cre n = 5. E Cre-expressing mice displayed repetitive behaviours measured as time spent grooming; GFP n = 5, GFP-Cre n = 6. F Nest building behaviour; GFP n = 5, GFP-Cre n = 6. G Representative heatmaps of the three-chamber sociability trial. H–J GFP- and Cre-expressing mice spent more time exploring the S1 instead of the empty cage; GFP n = 5, GFP-Cre n = 6. K Representative heatmaps of the three-chamber social novelty trial. L–N GFP- and Cre-expressing mice spent more time together with the S2 instead of the S1 (L, M), but Cre-expressing mice displayed reduced preference for the novel stimulus in comparison to control animals (N); GFP n = 5, GFP-Cre n = 6. Scale bars: 500 μm in (B) and 5 μm in (C, D). See Materials and Methods, as well as Supplementary Table 2 for detailed statistical analysis.

Fig. 3. Cortical screening.

A Schematic of bilateral injections of AAV9-CMV-GFP-Cre into 10 cortical coordinates (coordinate a-j). B–K Representative images showing GFP-Cre expression (left) and volumetric measurements of targeted single brain areas (right). ACA Anterior cingulate area; MOs, secondary motor area; SSp Primary somatosensory area, MOp Primary motor area, OLF Olfactory areas, VIS Visual areas, PTLp Posterior parietal association areas, RSP Retrosplenial area. L–O Time spent self-grooming (L), nesting score (M), preference index for sociability (N) and preference index for the novel stimulus (O) of dKO^fx/fx mice injected with AAV9-CMV-GFP-Cre in the different cortical coordinates. Nucleus accumbens (ACB) was used as a reference brain area for the screening; ACB (GFP) n = 5, ACB (GFP-Cre) n = 6, screening (GFP-Cre) n = 3 per each coordinate. Scale bar: 1 mm. See Materials and Methods, as well as Supplementary Table 2 for detailed statistical analysis.

Fig. 4. Lack of Shank2/3 in the retrosplenial area leads to social memory deficits and excitatory synapses loss, which cannot be rescued by acute DREADD-mediated neuronal activation.

A Representative images showing GFP (left) and GFP-Cre (right) expression in the retrosplenial area (RSP). B GFP- and Cre-expressing mice preferred to investigate S1 instead of the empty cage; GFP n = 5, GFP-Cre n = 4. C Cre-expressing mice had no preference between the S1 and the S2; GFP n = 5, GFP-Cre n = 4. D Immunohistochemistry (IHC) and quantification of excitatory synapses in the RSP using the Homer1b/c and VGlut1 markers; GFP n = 3, GFP-Cre n = 3. E Immunohistochemistry (IHC) and quantification of neurons positive for c-Fos after three-chamber test; GFP n = 3, GFP-Cre n = 3. F Representative images showing GFP (left) and GFP-Cre/Gq (right) expression in the RSP. G GFP- and Cre-expressing mice treated with either vehicle (veh) or CNO are sociable; GFP + veh n = 4, GFP-Cre + veh n = 5, GFP + CNO[1x] n = 3, GFP-Cre + CNO[1x] n = 4, GFP + CNO[5x] n = 5, GFP-Cre + CNO[5x] n = 5. H Cre-expressing mice treated with CNO still showed social memory deficits; GFP + veh n = 4, GFP-Cre + veh n = 5, GFP + CNO[1x] n = 3, GFP-Cre + CNO[1x] n = 4, GFP + CNO[5x] n = 5, GFP-Cre + CNO[5x] n = 5. I IHC showing that 93% of Cre-positive neurons also expressed Gq; GFP-Cre + CNO n = 3. J IHC showing that CNO administration increased c-Fos levels in the RSP; GFP-Cre + Veh[5x] n = 3, GFP-Cre + CNO[5x] n = 3. K IHC showing that repeated increase of neuronal firing did not rescue the loss of excitatory synapses in the RSP of Cre-expressing mice; GFP-Cre + Veh[5x] n = 3, GFP-Cre + CNO[5x] n = 3. Scale bars: 500 μm in (A, F), 5 μm in (D–K), 20 μm in (E, I, and J). See Materials and Methods, as well as Supplementary Table 2 for detailed statistical analysis.

Fig. 5. Single brain regions causally linked to ASD-related behaviours.

In this schematic overview, identified subregions of the brain are highlighted and associated behavioural alterations seen in ASD models are listed [–81].