Unusual Repertoire of Vocalizations in the BTBR T+tf/J Mouse Model of Autism methods
Aim. Evidence-backed execution summary for Unusual Repertoire of Vocalizations in the BTBR T+tf/J Mouse Model of Autism methods from Unusual Repertoire of Vocalizations in the BTBR T+tf/J Mouse Model of Autism.
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This experiment, in seven questions
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Open field test
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.
- Use
- 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.
Open field test
Center time and vertical activity in the open field arena did not differ between strains [center time: strain × minutes interaction, F(14,252) = 1.2, p =.29; vertical activity: F(14,252) = 1.4, p =.17 ].
- Use
- Center time and vertical activity in the open field arena did not differ between strains [center time: strain × minutes interaction, F(14,252) = 1.2, p =.29; vertical activity: F(14,252) = 1.4, p =.17 ].
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 acti...
- Use
- 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 acti...
Ultrasonic vocalizations in separated pups
An Ultrasound Microphone (Avisoft UltraSoundGate condenser microphone capsule CM16, Avisoft Bioacoustics, Berlin, Germany) sensitive to frequencies of 10-180 kHz, recorded the pup vocalizations in the sound-attenuating chamber. The microphone was placed through a hole in the middle of the cover of the styrofoa...
- Use
- An Ultrasound Microphone (Avisoft UltraSoundGate condenser microphone capsule CM16, Avisoft Bioacoustics, Berlin, Germany) sensitive to frequencies of 10-180 kHz, recorded the pup vocalizations in the sound-attenuating chamber. The microphone was placed through a hole in the middle of the cover of the styrofoa...
Developmental Milestones
To avoid potential confounds from using previously handled animals, a second cohort of B6 and BTBR pups was bred for the assays of developmental milestones, homing, and open field activity. One female and one male from each litter of BTBR and B6 mice (n = 10 litters each strain) were tested from pnd 2 to...
- Use
- To avoid potential confounds from using previously handled animals, a second cohort of B6 and BTBR pups was bred for the assays of developmental milestones, homing, and open field activity. One female and one male from each litter of BTBR and B6 mice (n = 10 litters each strain) were tested from pnd 2 to...
Developmental Milestones
On pnd 9, the litter was separated from the dam and kept for 30 min in one holding cage. The cage was placed on a heating pad set at a temperature of 35°C to maintain normal body temperature of the pups in the nest. Individual pups were then transferred to a Plexiglas cage (36 cm × 22.5 cm, walls 10 cm high)...
- Use
- On pnd 9, the litter was separated from the dam and kept for 30 min in one holding cage. The cage was placed on a heating pad set at a temperature of 35°C to maintain normal body temperature of the pups in the nest. Individual pups were then transferred to a Plexiglas cage (36 cm × 22.5 cm, walls 10 cm high)...
Open field test (pnd 18)
One male and one female from each litter, which were not used in the developmental milestones and homing assessments, were tested for locomotor activity on pnd 18. General exploratory locomotion in a novel environment was tested by placing pups in a VersaMax Animal Activity Monitoring System (AccuScan Instruments, C...
- Use
- One male and one female from each litter, which were not used in the developmental milestones and homing assessments, were tested for locomotor activity on pnd 18. General exploratory locomotion in a novel environment was tested by placing pups in a VersaMax Animal Activity Monitoring System (AccuScan Instruments, C...
Statistical analysis
A mixed-model Analysis of Variance (ANOVA) with Repeated Measures was used to analyze a) neonatal USVs and developmental milestone responses, with the strain as factor and postnatal day as the repeated measures, b) homing responses, frequency and duration, c) open field activity, with session minutes as the repeated...
- Use
- A mixed-model Analysis of Variance (ANOVA) with Repeated Measures was used to analyze a) neonatal USVs and developmental milestone responses, with the strain as factor and postnatal day as the repeated measures, b) homing responses, frequency and duration, c) open field activity, with session minutes as the repeated...
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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...
Measurement outputs
What raw and processed outputs should exist?
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...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The righting reflex, measured as latency to turn back onto all four paws when placed on the back, was tested at pnd 2 to 14 in all four strains in the first cohort used for ultr...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis of markers of somatic growth ( ) revealed that BTBR pups had an accelerated development compared to B6 pups on a) body weight, F(1,18) = 22.9, p<.0001; b) b...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
A significant strain effect was found on latency to reach the area containing the nest litter [Mann-Whitney, p =.03]. BTBR pups did not spend significantly more tim...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Descriptive statistics (mean±SEM) are given for the duration of each call type, as well as the beginning and ending dominant frequency.
from paperScoring or quantification
Quantify the primary readouts for this experiment: 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...; The righting reflex, measured as latency to turn back onto all four paws when placed on the back, was tested at pnd 2 to 14 in all four strains in the first cohort used for ultr...; Analysis of markers of somatic growth ( ) revealed that BTBR pups had an accelerated development compared to B6 pups on a) body weight, F(1,18) = 22.9, p<.0001; b) b...; A significant strain effect was found on latency to reach the area containing the nest litter [Mann-Whitney, p =.03]. BTBR pups did not spend significantly more tim....
from paperStatistical comparison
Descriptive statistics (mean±SEM) are given for the duration of each call type, as well as the beginning and ending dominant frequency.; 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...; Body weights were also measured in a second cohort of mice used for the full set of developmental milestones. ANOVA revealed a significant difference in body weight of BTBR in c...; The righting reflex, measured as latency to turn back onto all four paws when placed on the back, was tested at pnd 2 to 14 in all four strains in the first cohort used for ultr...
from paperReporting output
Report representative outputs alongside summary comparisons for 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..., The righting reflex, measured as latency to turn back onto all four paws when placed on the back, was tested at pnd 2 to 14 in all four strains in the first cohort used for ultr..., Analysis of markers of somatic growth ( ) revealed that BTBR pups had an accelerated development compared to B6 pups on a) body weight, F(1,18) = 22.9, p<.0001; b) b..., A significant strain effect was found on latency to reach the area containing the nest litter [Mann-Whitney, p =.03]. BTBR pups did not spend significantly more tim....
inferred from protocolStructured statistical methods
Descriptive statistics (mean±SEM) are given for the duration of each call type, as well as the beginning and ending dominant frequency.; 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...; Body weights were also measured in a second cohort of mice used for the full set of developmental milestones. ANOVA revealed a significant difference in body weight of BTBR in c...; The righting reflex, measured as latency to turn back onto all four paws when placed on the back, was tested at pnd 2 to 14 in all four strains in the first cohort used for ultr...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
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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