Characterization of the genetic mutation of shank3a and its effects on protein transcription and shank3a expression. (a) The shank3a paralogue is found on chromosome 18, where mutation to Exon 2 precedes the localisation of the four typical SHANK3 domains, ankyrin repeats (ANK), SRC Homology 3 (SH3), PDZ and sterile α motif (SAM). (b) The genetic mutation induced in our study included a five base pair deletion that elicits (c) transcription of a markedly truncated protein structure and (d) significant decreases in shank3a expression

Experimental assessment of shank3a mutation effects on social contagion and underlying recognition and attention components. (a) The set-up included a corridor separated in three equal parts by removable transparent dividers, a camera with a birds-eye-view of the arena and monitors on either side of the tank for displaying demonstrator videos. (b) The experiment included three phases. Acclimation to the arena and background videos for 10 min, where baseline mobility during the last 5 min could be assessed. Observation of two contrasting videos from the central compartment for 5 min, where the demonstrator exhibited either neutral behaviour (control) or periodic distress (stimulus: erratic and freezing). Test of local preferences for 10 min, following removal of dividers and access to the whole tank, while both videos displayed the demonstrator in a neutral state. During the observation phase, fish mobility, orientation towards the distress behaviour (heading: 0–180°) and repetition of the observed erratic and freezing behaviour were measured. During the test phase, discrimination based on the local preference for either video was used to assess the recall of recognised differences between distress and neutral state. (c) Mobility in terms of total distance travelled, was lower in Shank3a mutants only during observation phase, suggesting no motor deficits are present. (d) Temporal changes in the directional changes exceeded baseline thresholds, and together with the proportion time erratic response was exhibited, following analogous behaviour in the stimulus video, were markedly lower in mutants, compared to wild-type animals. (e) Immobility, used to measure freezing, was greater in mutants, but this related to temporal differences in velocity that revealed an overall low activity in mutants compared to the freezing bouts in wild types. (f) Attention towards the distressed stimulus, compared to the neutral control, was greater in the wild-type animals but not in the Shank3a mutants. (g) Local preference scores revealed recognition deficits in the Shank3a mutants compared to wild types. Heat maps are representative examples with the least deviation from the mean. [*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001]

Quantification of changes in genetic neuroplasticity markers derived by the shank3 mutation and analysis of their interrelated effects on attention. (a) Compared to wild types, shank3a mutants presented decreases in the expression of two neuroligin genes, nlgn1 and nlgn2, and increases in the expression of the synaptic strengthening genes npas4 and bdnf, and the neurogenesis genes wnt3 and neurod. (b) Based on absolute correlations between their expression levels (|r|), neuroligins clustered with shank3a, which (c) was quantified by PCA as a functionality-based composite synaptogenesis component, while all other genes clustered separately and quantified as a broad neuronal plasticity component. (d) Only the synaptogenesis component presented significant associations to the levels of attention towards the distressed conspecific exhibited during the social contagion test. [*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001]