Unusual repertoire of vocalizations in the BTBR T+tf/J mouse model of autism

Abstract

BTBR T+ tf/J (BTBR) is an inbred mouse strain that displays social abnormalities and repetitive behaviors analogous to the first and third diagnostic symptoms of autism. Here we investigate ultrasonic vocalizations in BTBR, to address the second diagnostic symptom of autism, communication deficits. As compared to the commonly used C57BL/6J (B6) strain, BTBR pups called more loudly and more frequently when separated from their mothers and siblings. Detailed analysis of ten categories of calls revealed an unusual pattern in BTBR as compared to B6. BTBR emitted high levels of harmonics, two-syllable, and composite calls, but minimal numbers of chevron-shaped syllables, upward, downward, and short calls. Because body weights were higher in BTBR than B6 pups, one possible explanation was that larger thoracic size was responsible for the louder calls and different distribution of syllable categories. To test this possibility, we recorded separation calls from FVB/NJ, a strain with body weights similar to BTBR, and 129X1/SvJ, a strain with body weights similar to B6. BTBR remained the outlier on number of calls, displaying low numbers of complex, upward, chevron, short, and frequency steps calls, along with high harmonics and composites. Further, developmental milestones and growth rates were accelerated in BTBR, indicating an unusual neurodevelopmental trajectory. Overall, our findings demonstrate strain-specific patterns of ultrasonic calls that may represent different lexicons, or innate variations in complex vocal repertoires, in genetically distinct strains of mice. Particularly intriguing is the unusual pattern of vocalizations and the more frequent, loud harmonics evident in the BTBR mouse model of autism that may resemble the atypical vocalizations seen in some autistic infants.

Figure 1. Ultrasonic vocalizations (USVs) in BTBR, B6, 129X1 and FVB/NJ pups.

A) Number and B) Duration of vocalizations on postnatal day (pnd) 2, 4, 6, 8 and 12 in response to social separation during a five minute session. Significant strain differences were detected across five days of testing. C) Peak frequency and D) amplitude of USVs analyzed on each day of testing. Data are expressed as mean±SEM of calls. Figures 1– 6 present results from Cohort 1, N = 20 mice per strain, representing one male and one female from each of 10 litters per strain. *p<.05 and **p<.01 for the comparisons between strains.

Figure 2. Typical sonograms of ultrasonic vocalizations, classified into ten distinct categories of calls emitted by (a–l) B6, (a'–l') BTBR, (a''–l'') 129X1, and (a'''–l''') FVB/NJ mice.

Descriptive statistics (mean±SEM) are given for the duration of each call type, as well as the beginning and ending dominant frequency.

Figure 3. Production of ultrasonic vocalizations by call category at postnatal day 8.

A) Frequency and B) Duration of ultrasonic vocalizations, during a total of twenty sonograms per strain, each of one minute duration. Harmonic and composite calls frequency and harmonics duration: **p<.01 BTBR compared to B6, 129X1 and FVB/NJ pups; Two-syllables frequency and duration and composite call duration: **p<.01 BTBR compared to B6 pups.

Figure 4. Production of calls within strain.

Probability of producing calls from each of the ten categories of USV. ^#Data were expressed by angular transformation. Number of calls analyzed: B6 = 2333; BTBR = 3633; 129X1 = 1806; FVB/NJ = 2575 collected from 20 sonograms per strain representative of each pup tested at pnd 8. *p<.05; **p<.01 and ***p<.001 for the comparisons between strains.

Figure 5. Strain profiles.

Pie graphs show the percentages of the different call categories within strain. Percentages were calculated in each strain as number of calls in each category for each subject/total number of calls analyzed in each subject. Number of total calls analyzed: B6 = 2333; BTBR = 3633; 129X1 = 1806; FVB/NJ = 2575.

Figure 6. A) Body weights and B) Righting reflex latencies in animals that were tested for ultrasonic vocalizations (cohort 1).

Body weights were higher in BTBR and FVB/NJ compared to B6 and 129X1 through pnd 4 to 12. **p<.01. Pups acquired the righting reflex response at different rates, with BTBR, 129X1 and FVB/NJ showing shorter latencies than B6 starting at pnd 2. **p<.01.

Figure 7. Somatic growth.

Representative photograph of the body size differences between a pnd 2 BTBR (upper pup) and a pnd 2 B6 (lower pup). Analysis of the markers of somatic growth revealed that BTBR displayed accelerated development as compared to B6 on A) body weight, B) body and C) tail length, D) pinnae detachment, E) opening of the eyes and F) incisor eruption. Figures 7– 10 present results from Cohort 2, N = 20 B6 and N = 20 BTBR, representing one male and one female from each of 10 litters per strain. ** p<.01 and *p<.05, for B6 vs. BTBR.

Figure 8. Somatosensory reflexes.

Accelerated development was seen in BTBR as compared to B6 on A) righting reflex, B) negative geotaxis, C) forelimb grasping, D) screen climbing, E) bar holding, F) cliff aversion, G) forelimb placing and H) auditory startle. **p<.01 and *p<.05 for B6 vs. BTBR.

Figure 9. Pup homing test performed at pnd 9.

Strains differed on A) latency to reach the area containing nest litter from their own home cages, B) Time spent in the nest area and C) general locomotor activity. *p<.05 for B6 vs. BTBR on latency; no significant difference for time spent in the nest area; **p<.01 for locomotor activity.

Figure 10. Open field activity was assayed at pnd 18.

BTBR was initially more active higher than B6 on A) distance travelled and B) horizontal activity. There were no significant strain differences on C) center time and D) vertical activity in the open field arena. **p<.01 for B6 vs. BTBR.