The Sapap3-/- mouse reconsidered as a comorbid model expressing a spectrum of pathological repetitive behaviours

Abstract

Symptom comorbidity is present amongst neuropsychiatric disorders with repetitive behaviours, complicating clinical diagnosis and impeding appropriate treatments. This is of particular importance for obsessive-compulsive disorder (OCD) and Tourette syndrome. Here, we meticulously analysed the behaviour of Sapap3 knockout mice, the recent rodent model predominantly used to study compulsive-like behaviours, and found that its behaviour is more complex than originally and persistently described. Indeed, we detected previously unreported elements of distinct pathologically repetitive behaviours, which do not form part of rodent syntactic cephalo-caudal self-grooming. These repetitive behaviours include sudden, rapid body and head/body twitches, resembling tic-like movements. We also observed that another type of repetitive behaviour, aberrant hindpaw scratching, might be responsible for the flagship-like skin lesions of this mouse model. In order to characterise the symptomatological nature of observed repetitive behaviours, we pharmacologically challenged these phenotypes by systemic aripiprazole administration, a first-line treatment for tic-like symptoms in Tourette syndrome and trichotillomania. A single treatment of aripiprazole significantly reduced the number of head/body twitches, scratching, and single-phase grooming, but not syntactic grooming events. These observations are in line with the high comorbidity of tic- and compulsive-like symptoms in Tourette, OCD and trichotillomania patients.

Fig. 1. Behavioural assessment of Sapap3^−/− mice.

A Photographs of custom-made apparatus for behavioural assessment, consisting of four acrylic chambers, each equipped with top and side cameras, connected to a digital video-recording system. B Detailed graphic illustration of a single video chamber with ad libitum water and food access. C Time scale of behavioural assessment. Mice were video-recorded in the behavioural apparatus for 24 h. Four intermittent time bins of 30 min each (i.e., a total of 2 h) were manually analysed offline for repetitive behaviours including self-grooming, head-body twitches and hindpaw scratching. The scored time bins were distributed regularly across the light/dark circadian following previous protocols (Welch et al., 2007).

Fig. 2. Sapap3^−/− mice express aberrant head/body twitches and scratching behaviours.

A Sapap3^−/− mice execute a significant amount of head/body twitches, which are nearly absent in wildtype mice (n = 9 mice per genotype; Mann–Whitney U, p < 0.01). B Sapap3^−/− mice show a significant amount of hindpaw scratching compared to wildtype control mice (n = 9 mice per genotype; Mann–Whitney U test, p < 0.01). C The duration of hindpaw scratching is significantly elevated in Sapap3^−/− in comparison to wildtype mice (n = 9 mice per genotype; Mann–Whitney U test, p < 0.001). D The number of head/body twitches and scratching bouts correlate positively in both wildtype (Spearman correlation, p < 0.05) and Sapap3^−/− mice (n = 9 mice per genotype; Spearman correlation, p < 0.001). E Photographs of three individual mice with representative lesions before, and 2 days or 2 weeks after hindpaw nail clipping treatment. F Lesions, assessed through a lesion score ranging from no lesions (score = 1) to severe lesions (score = 4), significantly improved already 2 days after clipping the hindpaw claws (n = 17 Sapap3^−/− mice; Wilcoxon signed-rank test, paired, p < 0.001). G Lesions are further improved 2 weeks after clipping the hindpaw claws as assessed through a significantly lowered lesion score (n = 16 Sapap3^−/− mice; Wilcoxon signed-rank test, paired, p < 0.001). Box plots illustrate the first and third quartiles; whiskers indicate the minimum and the maximal value of each dataset at no further than 1.5 interquartile range. The indicated average is the median. Quartiles of Sapap3^−/− and wildtype mice are plotted in grey or white, and individual data points are in filled black and empty black dots, respectively. **p < 0.01, ***p < 0.001.

Fig. 3. Short, single-phase grooming events are more exaggerated than syntactic grooming in Sapap3^−/− mice.

A Sapap3^−/− mice show significantly more grooming events compared to wildtype controls (Mann–Whitney U test, p < 0.001). B Total grooming duration is comparable between Sapap3^−/− and wildtype mice (Mann–Whitney U test, p = ns). C Self-grooming behaviour of Sapap3^−/− mice compared to wildtype mice is characterised by a large proportion of grooming events of short duration. The x-axis is depicted on a log₁₀ scale. D Both short grooming events (<3 s duration) as well as long grooming events (>3 s duration) were significantly enhanced in Sapap3^−/− mice compared to wildtype controls (Mann–Whitney U, p < 0.001 and p < 0.01, respectively). Self-grooming behaviour depended both on genotype and bout length (ART ANOVA, p_{genotype*grooming type} < 0.01). All plots illustrate data from n = 9 Sapap3^−/− and n = 9 wildtype mice; box whisker plots were designed as described in the legend of Fig. 2. **p < 0.01; ***p < 0.001; ns non-significant.

Fig. 4. Excessive head/body twitches, scratching and short grooming events are associated in Sapap3^−/− mice.

A The proportion of novel detected repetitive behaviours in Sapap3^−/− mice outweighs previously reported syntactic self-grooming behaviour (Pearson’s χ² test, p < 0.0001). B Head/body twitches positively correlate with short, single-phase grooming but not long, syntactic grooming bouts in Sapap3^−/− mice (Spearman correlation, p < 0.05, p = ns, respectively). C Scratching bouts also correlate positively with short, single-phase grooming but not long, syntactic grooming bouts in Sapap3^−/− mice (Spearman correlation, p < 0.01, p = ns, respectively). Correlation estimates are plotted in a grey solid line or a dotted black line for wildtype or Sapap3^−/− mice (n = 9 animals per genotype), respectively.

Fig. 5. Short grooming bouts, head/body twitches and scratching were reduced by aripiprazole.

A Acute treatment with aripiprazole (1.5 mg/kg) significantly reduced the number of single-phase grooming, head/body twitches and scratching (Wilcoxon signed-rank test: all p < 0.01; non-parametric, paired permutation test: all p < 0.01), but not the number of syntactic grooming events (Wilcoxon signed-rank test: p = 0.08 and non-parametric, paired permutation test: p = 0.09). Plotted are the proportions of number of RB events under aripiprazole treatment and the sum of the number of RB events (vehicle + aripiprazole) of individual mice. B Aripiprazole in particular shorter grooming events in Sapap3^−/− mice. The x-axis is depicted on a log₁₀ scale. Box whisker plots were designed as described in the legend of Fig. 2. Vehicle and aripiprazole conditions are colour-coded in blue and red, respectively. *p < 0.05, **p < 0.01, ns non-significant.