Altered ultrasonic vocalizations in a tuberous sclerosis mouse model of autism

Abstract

Tuberous sclerosis (TSC) is an autosomally dominant neurocutaneous disease notable for its high comorbidity with autism in human patients. Studies of murine models of tuberous sclerosis have found defects in cognition and learning, but thus far have not uncovered deficits in social behaviors relevant to autism. To explore social communication and interaction in TSC2 heterozygous mice, we recorded ultrasonic vocalizations (USV) and found that although both wild-type (WT) and heterozygous pups born to WT dams showed similar call rates and patterns, baseline vocalization rates were elevated in pups born to heterozygous dams. Further analysis revealed several robust features of maternal potentiation in all but WT pups born to heterozygous dams. This lack of potentiation is suggestive of defects in mother-pup social interaction during or before the reunion period between WT pups and heterozygous dams. Intriguingly, male pups of both genotypes born to heterozygous dams showed particularly heightened call rates and burst patterns. Because our maternal retrieval experiments revealed that TSC2(+/-) dams exhibited improved defensive reactions against intruders and highly efficient pup retrieval performance, the alterations in their pups' USVs and maternal potentiation do not appear to result from poor maternal care. These findings suggest that a pup's interaction with its mother strongly influences the pup's vocal communication, revealing an intriguing dependence of this social behavior on TSC2 gene dosage of both parties involved. Our study of this murine model thus uncovers social abnormalities that arise from TSC haploinsufficiency and are suggestive of autism.

Fig. 1.

Vocalization rates were elevated in pups born to heterozygous dams. (A) Offspring from both wild type (WT) and heterozygous (het) dams (blue and red, respectively) were isolated, reunited, and reisolated for 5 min per session. Although WT and heterozygous pups from WT dams did not differ significantly in call rate or temporal pattern, pups from heterozygous dams were more vocal than pups from WT dams. (B) All groups but WT pups born from heterozygous dams exhibited maternal potentiation. Note the overall increase in calls from the heterozygous dam group (P < 0.001), and a three-way statistical interaction among isolation period × dam genotype × pup genotype (P = 0.015). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Maternal potentiation was exhibited in call duration and latency. (A) Representative spectrogram of a multicomponent call and its segmentation. (Left) Call with segment lines, calculated from amplitude envelope shown in overlay. (Center) Extracted instantaneous frequencies. (Right) Histogram of frequency counts. (B) Mean duration of each call rose on average from isolation 1 to isolation 2 (P < 0.001), and call durations were longer on average across isolation periods from pups born to heterozygous dams (P = 0.012). (C) Call latency strongly decreased following reunion on average across all genotypes (P < 0.001). Only the WT pup, heterozygous dam group did not statistically significantly increase in call duration or decrease in latency.

Fig. 3.

Proportion of low-frequency calls was elevated in pups from heterozygous dams. (A) Probability density plots of median sound frequencies of individual calls overlaid from before and after reunion (purple and green, respectively) reveal a bimodal distribution of call frequencies. (B) Fraction of calls above and below 75 kHz became almost equal to one another in pups from heterozygous dams after reunion. These pups’ fraction of high frequency calls was significantly lower than that of pups from WT dams across isolation period (P < 0.001).

Fig. 4.

Multicomponent calls were selectively elevated. (A) There were no significant differences in numbers of simple calls across isolation period or genotype group. (B) Multicomponent call numbers increased after reunion in both WT (P < 0.001) and TSC2^+/− (P = 0.014) pups from WT dams as well as TSC2^+/− pups from TSC2^+/− dams (P = 0.001), but not from WT pups born to TSC2^+/− dams (P = 0.242). Pups from heterozygous dam emitted a significantly higher fraction of multicomponent calls across both periods (P = 0.007). (C) Multivariate statistics summary shows a significant main effect of dam genotype on both multisyllabic and stacked subgroupings of multicomponent calls.

Fig. 5.

Males from heterozygous dams were particularly susceptible to changes in rate. (A) Male pups born to heterozygous dams were found to vocalize at significantly higher rates than either male (P = 0.002) or female pups (P = 0.003) born to WT dams. (B) Burst analysis revealed that this same group also vocalized at higher burst rates than did male (P = 0.023) or female pups from WT dams (P = 0.021).

Fig. 6.

TSC2^+/− dams exhibited improved maternal care. (A) Heterozygous dams demonstrated more efficient retrieval scores, bringing all three pups back to the nest in less than half the time as WT dams (P = 0.006 for dam effect). (B) Total time for final collection of all pups back to the nest was significantly lower for TSC2^+/− dams (P = 0.011). (C) WT dams exhibited significantly higher failure rate to deposit pup in nest or to keep pup there (P = 0.012). (D) Resident intruder tests showed no significant difference in total duration of sniffs or attacks and time over nest. (E) Although numbers of sniffs or attacks did not differ between dam groups, heterozygous dams entered nest significantly more often (P = 0.003).