Social and emotional alterations in mice lacking the short dystrophin-gene product, Dp71

Abstract

Background: The Duchenne and Becker muscular dystrophies (DMD, BMD) are neuromuscular disorders commonly associated with diverse cognitive and behavioral comorbidities. Genotype-phenotype studies suggest that severity and risk of central defects in DMD patients increase with cumulative loss of different dystrophins produced in CNS from independent promoters of the DMD gene. Mutations affecting all dystrophins are nevertheless rare and therefore the clinical evidence on the contribution of the shortest Dp71 isoform to cognitive and behavioral dysfunctions is limited. In this study, we evaluated social, emotional and locomotor functions, and fear-related learning in the Dp71-null mouse model specifically lacking this short dystrophin.

Results: We demonstrate the presence of abnormal social behavior and ultrasonic vocalization in Dp71-null mice, accompanied by slight changes in exploratory activity and anxiety-related behaviors, in the absence of myopathy and alterations of learning and memory of aversive cue-outcome associations.

Conclusions: These results support the hypothesis that distal DMD gene mutations affecting Dp71 may contribute to the emergence of social and emotional problems that may relate to the autistic traits and executive dysfunctions reported in DMD. The present alterations in Dp71-null mice may possibly add to the subtle social behavior problems previously associated with the loss of the Dp427 dystrophin, in line with the current hypothesis that risk and severity of behavioral problems in patients increase with cumulative loss of several brain dystrophin isoforms.

Keywords: Anxiety; Dp71; Dystrophin; Dystrophinopathies; Fear memory; Social behavior; Ultrasonic vocalizations.

Fig. 1

DMD gene and dystrophin isoforms. A Diagram showing the DMD gene (79 exons) and its internal promoters (angled arrows) that give rise to several dystrophin proteins expressed in different tissues/regions: The full-length dystrophin Dp427 that exists as 3 isoforms is expressed in muscle, brain and cerebellar Purkinje cells, Dp260 in retina, Dp140 in brain and kidney, Dp116 in peripheral nerves and Dp71 in brain, retina and liver. The dystrophins expressed in brain are represented as hatched bars. The thick vertical arrows and dotted lines indicate the three main regions where disease-causing mutations lead to the unique loss of Dp427 (a), or a deficiency in Dp427, Dp260 and Dp140 (b), or a deficiency in all dystrophins (c). B Table showing the expression pattern of dystrophins in DMD mouse models. The mouse model analyzed in the present study is highlighted in grey. +: normal expression; − deficient

Fig. 2

Social motivation and social interactions. A, B Histograms represent exploratory behavior expressed as percent time spent sniffing the tubes containing either a mouse (black bars) or an object (white bars) during the sociability trial (A), and percent time spent sniffing towards the familiar (black bars) and novel mouse (white bars) during the trial assessing their preference for social novelty (B). C Number of sniffing episodes, dominant acts and pursuits initiated by WT and Dp71-null resident mice in the resident-intruder paradigm. D–F Total time spent by WT and Dp71-null male residents sniffing awake females (D), anesthetized females (E) and males (F). G–I Temporal evolution of social exploration initiated by residents exposed to awake females (G), anesthetized females (H) and males (I) across three 60-s time bins. The time spent sniffing the anesthetized females was significantly higher in Dp71-null than in WT mice during the first 120 s of the trial (*p < 0.05). White points represent individual data points for each mouse

Fig. 3

Time spent vocalizing during social interactions. A–C Mean percent time vocalizing during interaction with awake females (A), anesthetized females (B) and awake males (C) (t-test, *p < 0.05). D–F Percent time spent vocalizing across 60-s time bins during interaction with awake females (D), anesthetized females (E) and awake males (F). Time spent sniffing (%) was significantly longer in Dp71-null than in WT mice across all time bins during interaction with anesthetized females (p < 0.05) and awake males (Significant during the first 60 s; Tukey posthoc test; *p < 0.05). White points represent individual data points for each mouse

Fig. 4

Call duration and distribution of call durations. A–C Mean duration of calls emitted during interactions with awake females (A), anesthetized females (B) and awake males (C). The mean call duration of Dp71-null mice was particularly increased during interactions with males (*p < 0.05). White points represent individual data points for each mouse. D–F Frequency distribution (%) of ultrasonic vocalizations as a function of the call duration (ms) during exposure to freely-moving females (D), anesthetized females (E) and freely-moving males (F). Dp71-null mice emitted a greater proportion of longer calls as compared to controls in all test conditions (KS-Tests; all p < 0.001)

Fig. 5

Repertoire of socially-induced ultrasonic vocalizations. A–C Relative proportion of distinct call shapes emitted during interactions with awake females (A), anesthetized females (B) and awake males (C). Note that Dp71-null mice emitted a larger proportion of peak calls compared to WT mice in all experimental conditions (*t-tests, p < 0.05), while they produced a significantly higher proportion of other specific calls in distinct social contexts, i.e., more sinusoidal calls during the investigation of anesthetized females (B) (*t-test, p < 0.05) and more U-shape calls during the interaction with freely-moving males (*t-test, p < 0.05). White points represent individual data points for each mouse

Fig. 6

Anxiety and unconditioned fear. A Open-field test. From left to right are shown the total distance travelled and percent distance travelled in central area as a function of time, then the mean number of entries in central area and time spent expressing thigmotaxis. B Light–dark test. Plots show the number of head dips towards the light compartment (left) and time spent in light compartment (right). C Plus-maze test. The plots show the number of visits to open and closed arms as a function of time (left), the percent time spent in open arms and central area (middle), and the number of head dips into the central area and open arms (right). D Unconditioned fear-related tonic immobility. Percent freezing in 5 min following brief scruff restraint. *p < 0.05. White points in bar plots represent individual data points for each mouse

Fig. 7

Auditory brainstem responses (ABR). A ABR thresholds for clicks and pure tone frequencies demonstrate normal hearing in Dp71-null mice. B Sample ABR to a click at 70 dB, showing the successive waves labeled I to IV (positive peaks) and I’ to IV’ (negative peaks). Wave I arises from the auditory nerve and waves II to IV reflect evoked activity at different relays of the auditory brainstem. Gray arrow: click presentation

Fig. 8

Associative fear learning and memory. A, B Delayed cued fear conditioning. Performance is expressed as the percent time spent freezing during acclimatization periods (A), and in response to the CS during the acquisition session, consisting in five (A) or three (B) CS-US pairings, and in response to CS during the two retention sessions in a distinct context (blocks of four CS alone presentations) performed at 24 h intervals. C Trace Fear conditioning. Performance is expressed as the percent time spent freezing to the CS during the acquisition session, consisting in six CS-US pairings, and the two retention sessions (cued and contextual). Cued-retention session was performed 24 h post-acquisition by presenting CS alone in a different context. Contextual-retention session was performed by placing the animal in the same context without CS presentation 3 h after the cued-retention. H: habituation to apparatus before acquisition. A: Pre-session acclimatization. C: Contextual retention