Unusual Repertoire of Vocalizations in the BTBR T+tf/J Mouse Model of Autism methods
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Classification of ultrasonic vocalizations into distinct categories
Descriptive statistics (mean±SEM) are given for the duration of each call type, as well as the beginning and ending dominant frequency.
Classification of ultrasonic vocalizations into distinct categories
illustrates strain-dependent effects on both frequency [F(3,76) = 29.62, p<.0001] and duration of calls [F(3,76) = 19.33, p<.0001]. BTBR emitted a significantly higher number of harmonic-modulated calls [strain × calls subtype interaction: F(27,684) = 8.87, p<.0001; Newman-Keuls p<.001 for BTBR vs all strains], two-syllable calls [Newman-Keuls p<.05 for BTBR vs B6], and composite calls [Newman-Keuls p<.001 for BTBR vs all strains].
Classification of ultrasonic vocalizations into distinct categories
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.
Classification of ultrasonic vocalizations into distinct categories
BTBR pups also emitted longer harmonic calls than the other strains [strain × calls subtype interaction: F(27,684) = 4.67, p<.0001; Newman-Keuls p<.001]. The two-syllable and composite calls emitted by BTBR pups were of longer duration in comparison to the same subtypes emitted by age-matched B6 [p<.01].
Open field test
Locomotor activity of 18-day-old pups in a non-social empty novel open field was initially higher in BTBR than in B6 [strain × minutes interaction: F(14,252) = 3.3, p<.0001; Newman-Keuls p<.001 for BTBR versus B6 in the first 4 minutes of the test] but subsequently, the two strains showed similar activity levels after habituation ( ). The same profile was seen for horizontal activity where the BTBR mice showed a higher activity level [strain effect, F(1,18) = 8.7, p<.01; Newman-Keuls p<.001 for BTBR versus B6 in the first, third and fourth minute of the test].
Developmental Milestones
Negative geotaxis: pup turns approximately 180° to either side when placed head down on a wire mesh screen (4 × 4 mm) held at a 45° angle.
Materials and Methods
BTBR, B6, FVB/NJ, and 129X1 breeding pairs were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in standard wire-topped Plexiglas cages (42 cm × 27 cm × 14 cm) in a temperature and humidity controlled vivarium, with food and water available ad libitum, with lights on from 19:00 to 7:00. Behavioral testing was conducted between 9.30 and 14.00 h, during the dark phase of the circadian cycle. All procedures followed NIH guidelines for the care and use of laboratory animals, and were approved by the NIMH Animal Care and Use Committee. Ten days after pairing for breeding, the females were individually housed and subsequently inspected daily at 9:30 for pregnancy and delivery. The day of birth was considered as postnatal day (pnd) 0.
Ultrasonic vocalizations in separated pups
Litters chosen for testing contained more than seven pups for BTBR (7.8±1.05), B6 (7.1±0.58) and FVB/NJ (7.5±0.34), and more than five pups for 129X1 (5.5±0.40; a strain known for small litters). One female and one male from each litter of BTBR, B6, FVB/NJ and 129X1 mice (n = 10 litters each strain) were used for baseline measurements of the ultrasonic vocalizations from pnd 2 to 12. Body weights and body temperatures of pups were measured after the ultrasonic vocalization test on pnd 2, 4, 6, 8 and 12. On each day of testing, each pup was placed into an empty plastic container (diameter, 5 cm; height 10 cm), located inside a sound-attenuating styrofoam box, and assessed for USVs during a five minute test. At the end of the five minute recording session, each pup was weighed and its axillary temperature measured by gentle insertion of the thermal probe...
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Descriptive statistics (mean±SEM) are given for the duration of each call type, as well as the beginning and ending dominant frequency.
illustrates strain-dependent effects on both frequency [F(3,76) = 29.62, p<.0001] and duration of calls [F(3,76) = 19.33, p<.0001]. BTBR emitted a significantly higher number of harmonic-modulated calls [strain × calls subtype interaction: F(27,684) = 8.87, p<.0001; Newman-Keuls p<.001 for BTBR vs all strains], two-syllable calls [Newman-Keuls p<.05 for BTBR vs B6], and composite calls [Newman-Keuls p<.001 for BTBR vs all strains].
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.
BTBR pups also emitted longer harmonic calls than the other strains [strain × calls subtype interaction: F(27,684) = 4.67, p<.0001; Newman-Keuls p<.001]. The two-syllable and composite calls emitted by BTBR pups were of longer duration in comparison to the same subtypes emitted by age-matched B6 [p<.01].
Locomotor activity of 18-day-old pups in a non-social empty novel open field was initially higher in BTBR than in B6 [strain × minutes interaction: F(14,252) = 3.3, p<.0001; Newman-Keuls p<.001 for BTBR versus B6 in the first 4 minutes of the test] but subsequently, the two strains showed similar activity levels after habituation ( ). The same profile was seen for horizontal activity where the BTBR mice showed a higher activity level [strain effect, F(1,18) = 8.7, p<.01; Newman-Keuls p<.001 for BTBR versus B6 in the first, third and fourth minute of the test].
Negative geotaxis: pup turns approximately 180° to either side when placed head down on a wire mesh screen (4 × 4 mm) held at a 45° angle.
BTBR, B6, FVB/NJ, and 129X1 breeding pairs were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in standard wire-topped Plexiglas cages (42 cm × 27 cm × 14 cm) in a temperature and humidity controlled vivarium, with food and water available ad libitum, with lights on from 19:00 to 7:00. Behavioral testing was conducted between 9.30 and 14.00 h, during the dark phase of the circadian cycle. All procedures followed NIH guidelines for the care and use of laboratory animals, and were approved by the NIMH Animal Care and Use Committee. Ten days after pairing for breeding, the females were individually housed and subsequently inspected daily at 9:30 for pregnancy and delivery. The day of birth was considered as postnatal day (pnd) 0.
Litters chosen for testing contained more than seven pups for BTBR (7.8±1.05), B6 (7.1±0.58) and FVB/NJ (7.5±0.34), and more than five pups for 129X1 (5.5±0.40; a strain known for small litters). One female and one male from each litter of BTBR, B6, FVB/NJ and 129X1 mice (n = 10 litters each strain) were used for baseline measurements of the ultrasonic vocalizations from pnd 2 to 12. Body weights and body temperatures of pups were measured after the ultrasonic vocalization test on pnd 2, 4, 6, 8 and 12. On each day of testing, each pup was placed into an empty plastic container (diameter, 5 cm; height 10 cm), located inside a sound-attenuating styrofoam box, and assessed for USVs during a five minute test. At the end of the five minute recording session, each pup was weighed and its axillary temperature measured by gentle insertion of the thermal probe in the skin pocket between upper foreleg and chest of the animal for about 30 seconds (Microprobe digital thermometer with mouse probe, Stoelting Co., Illinois, USA). No differences in patterns of calling were detected in a comparison of male and female pups, therefore data were collapsed across sex.
Machine-readable layer
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