Mice with Deficient BK Channel Function Show Impaired Prepulse Inhibition and Spatial Learning, but Normal Working and Spatial Reference Memory methods
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Hearing measurements/auditory brainstem responses (ABR)
Auditory brainstem responses (ABR) were recorded from 10 WT (5/5 male/female) and 8 BKα -/- mice (6/2), as described previously. Each mouse was tested at several time points between the age of 4-15 weeks (9±3.5 weeks) to evaluate the development of the hearing thresholds. Briefly, recordings were performed while mice were under ketamine (75 mg/kg body weight) and xylazine (5 mg/kg body weight) anesthesia. ABRs to free field click (100 µs), and pure tone (3 ms, 1 ms ramp) acoustic stimuli were recorded using sub-dermal silver wire electrodes at the ear (active), the vertex (reference) and the back (ground) of the animals. Signals were amplified (50-100-fold), band-pass filtered (200 Hz high-pass and 5 kHz low-pass), and averaged for 64-256 repetitions at each sound pressure level (SPL) presented (0 -100 dB in steps of 5 dB). Hearing threshold was defin...
Methods
All mice were generated and genotyped at the Pharmaceutical Institute, University of Tübingen, Germany. In order to ensure proper tracking each mouse got an individual ear-tag when collecting the DNA sample for the genotyping. The mice were shipped to Canada at the age of 1.5-3 months and subsequently allowed to acclimate for 2 weeks before behavioural testing started. Mice were group housed with mixed genetic background within groups, with a 12 hour light-dark cycle and with ad libitum food and water. Testing only occurred during the light cycle from 7am to 7pm. If animals were tested repeatedly over several days, the time of testing for each animal was kept constant.
Grip force test
In order to assess muscular strength, the grip force test was used. The front limb strength of 16 animals of each genotype (WT: 9 males/ 7 females, BKα +/-: 10/6, BKα -/-: 9/7) was measured in 5 trials per animal. The animals were between 7 and 9 months (34±2 weeks) old at the time of testing. A force gauge (Grip Strength Meter, Columbus Instruments, Columbus, Ohio, USA) was connected to a metal grid which allowed the animal to hold on. The animal was held by the base of its tail and lowered towards the grid until it gripped it with both front limbs. Then the animal was gently pulled back until it lost the grip. Between trials the animals were allowed to recover in their home cages for about 10 minutes. For subsequent analyses the results from the 5 trials were averaged for each animal.
Cat walk
To evaluate gait 11 WT (6 males/5 females), 11 BKα +/- (7/4), and 10 BKα -/- mice (7/3) aged 7 to 9 month (33±2 weeks) were tested on the CatWalk (Noldus Information Technology Inc, Leesburg, VA, USA). Animals traversed a transparent walkway through which they were video-taped. Animals were allowed to walk back and forth until a minimum of 5 consecutive steps could be recorded. Using the CatWalk™software (Version 7.0, Noldus Information Technology Inc, Leesburg, VA, USA) the paw prints of the animals were tracked. We analyzed stride length and paw print area of each individual paw, as well as inter-limb coordination using the regularity index (RI) as introduced by Hamers et al.. The regularity index grades the inter-limb coordination as the ratio between the number of normal step sequence patterns and the total number of paw placements. A RI of...
Open field locomotor activity
General locomotor activity was measured in 16 mice of each genotype (WT: 9 males/ 7 females, BKα +/-: 10/6, BKα -/-: 9/7) at the age of 6 to 9 month (30±2.5 weeks). Each animal was placed in a square open field box (Versamax Animal Activity Monitor, AccuScan Instruments, Colombus, OH, USA) for 2 hours. During the whole time of testing the room was moderately lit (200 lux). Using the VersMax™ software (AccuScan Instruments) we analyzed the distance traveled during the 2 hours, the number of rearing movements, as well as the time spent in the center versus the time spent at the margins as a measure of anxiety.
Acoustic startle reflex/prepulse inhibition (PPI)
Prepulse inhibition of the acoustic startle reflex was used to measure sensorimotor gating. 18 WT (10 males/8 females), 17 BKα +/- (10/7), and 19 BKα -/- mice (9/10) were tested as described previously,. Only animals aged 4-6 months (21.5±2 weeks) were tested to avoid possible effects of age induced hearing loss. Testing was conducted in sound attenuated startle boxes from MED Associates (MED-ASR-PRO1, St Albans, VT, USA) using the associated software for stimulus presentation and recordings (Startle Reflex, Version 6.0, MED Associates, Inc.).
Acoustic startle reflex/prepulse inhibition (PPI)
Sound was presented by two loud speakers in each startle box. The first speaker presented a constant background noise (65 dB SPL white noise), whereas the second speaker presented the startle and prepulse stimuli. The sound was calibrated with the sound pressure level measurement package provided by MED Associates (ANL-929A-PC) placing the microphone into the animal holder at approximately the same distance to the speaker as the animals' head during the test sessions. To record the startle response, the animals were placed into a tube like plastic animal holder which was then mounted on top of a piezo driven transducer which translated vertical movements into a voltage signal. The voltage signal in response to a given stimulus was amplified, digitalized and stored on a computer. We started the recording 50 ms before any stimulus was given and recorded for a total of 500 ms per s...
Acoustic startle reflex/prepulse inhibition (PPI)
Before the actual testing the animals were acclimatized to the startle boxes for 3 days to reduce anxiety. During these 5 minute long acclimation phases the background noise was presented. On the third day, these 5 minutes were followed by a short input/output (I/O) test to determine the appropriate gain setting for the transducer signal for each individual animal. The I/O test consisted of 12 acoustic stimuli with increasing intensity (65-120 dB SPL, 20 ms duration). Once set, the gain was kept constant for all recordings of a given animal. In order to reduce variability we tested each animal on 5 consecutive days using the following protocol.
Measurement outputs
What raw and processed outputs should exist?
Gait recovery and locomotor function
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary readouts for this experiment: Gait recovery and locomotor function.
from paperStatistical comparison
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inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function.
inferred from protocolSource and audit
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Evidence quotes (8)
Auditory brainstem responses (ABR) were recorded from 10 WT (5/5 male/female) and 8 BKα -/- mice (6/2), as described previously. Each mouse was tested at several time points between the age of 4-15 weeks (9±3.5 weeks) to evaluate the development of the hearing thresholds. Briefly, recordings were performed while mice were under ketamine (75 mg/kg body weight) and xylazine (5 mg/kg body weight) anesthesia. ABRs to free field click (100 µs), and pure tone (3 ms, 1 ms ramp) acoustic stimuli were recorded using sub-dermal silver wire electrodes at the ear (active), the vertex (reference) and the back (ground) of the animals. Signals were amplified (50-100-fold), band-pass filtered (200 Hz high-pass and 5 kHz low-pass), and averaged for 64-256 repetitions at each sound pressure level (SPL) presented (0 -100 dB in steps of 5 dB). Hearing threshold was defined as the lowest SPL that produced a potential visually distinct from background noise.
All mice were generated and genotyped at the Pharmaceutical Institute, University of Tübingen, Germany. In order to ensure proper tracking each mouse got an individual ear-tag when collecting the DNA sample for the genotyping. The mice were shipped to Canada at the age of 1.5-3 months and subsequently allowed to acclimate for 2 weeks before behavioural testing started. Mice were group housed with mixed genetic background within groups, with a 12 hour light-dark cycle and with ad libitum food and water. Testing only occurred during the light cycle from 7am to 7pm. If animals were tested repeatedly over several days, the time of testing for each animal was kept constant.
In order to assess muscular strength, the grip force test was used. The front limb strength of 16 animals of each genotype (WT: 9 males/ 7 females, BKα +/-: 10/6, BKα -/-: 9/7) was measured in 5 trials per animal. The animals were between 7 and 9 months (34±2 weeks) old at the time of testing. A force gauge (Grip Strength Meter, Columbus Instruments, Columbus, Ohio, USA) was connected to a metal grid which allowed the animal to hold on. The animal was held by the base of its tail and lowered towards the grid until it gripped it with both front limbs. Then the animal was gently pulled back until it lost the grip. Between trials the animals were allowed to recover in their home cages for about 10 minutes. For subsequent analyses the results from the 5 trials were averaged for each animal.
To evaluate gait 11 WT (6 males/5 females), 11 BKα +/- (7/4), and 10 BKα -/- mice (7/3) aged 7 to 9 month (33±2 weeks) were tested on the CatWalk (Noldus Information Technology Inc, Leesburg, VA, USA). Animals traversed a transparent walkway through which they were video-taped. Animals were allowed to walk back and forth until a minimum of 5 consecutive steps could be recorded. Using the CatWalk™software (Version 7.0, Noldus Information Technology Inc, Leesburg, VA, USA) the paw prints of the animals were tracked. We analyzed stride length and paw print area of each individual paw, as well as inter-limb coordination using the regularity index (RI) as introduced by Hamers et al.. The regularity index grades the inter-limb coordination as the ratio between the number of normal step sequence patterns and the total number of paw placements. A RI of 100% indicates perfect inter-limb coordination.
General locomotor activity was measured in 16 mice of each genotype (WT: 9 males/ 7 females, BKα +/-: 10/6, BKα -/-: 9/7) at the age of 6 to 9 month (30±2.5 weeks). Each animal was placed in a square open field box (Versamax Animal Activity Monitor, AccuScan Instruments, Colombus, OH, USA) for 2 hours. During the whole time of testing the room was moderately lit (200 lux). Using the VersMax™ software (AccuScan Instruments) we analyzed the distance traveled during the 2 hours, the number of rearing movements, as well as the time spent in the center versus the time spent at the margins as a measure of anxiety.
Prepulse inhibition of the acoustic startle reflex was used to measure sensorimotor gating. 18 WT (10 males/8 females), 17 BKα +/- (10/7), and 19 BKα -/- mice (9/10) were tested as described previously,. Only animals aged 4-6 months (21.5±2 weeks) were tested to avoid possible effects of age induced hearing loss. Testing was conducted in sound attenuated startle boxes from MED Associates (MED-ASR-PRO1, St Albans, VT, USA) using the associated software for stimulus presentation and recordings (Startle Reflex, Version 6.0, MED Associates, Inc.).
Sound was presented by two loud speakers in each startle box. The first speaker presented a constant background noise (65 dB SPL white noise), whereas the second speaker presented the startle and prepulse stimuli. The sound was calibrated with the sound pressure level measurement package provided by MED Associates (ANL-929A-PC) placing the microphone into the animal holder at approximately the same distance to the speaker as the animals' head during the test sessions. To record the startle response, the animals were placed into a tube like plastic animal holder which was then mounted on top of a piezo driven transducer which translated vertical movements into a voltage signal. The voltage signal in response to a given stimulus was amplified, digitalized and stored on a computer. We started the recording 50 ms before any stimulus was given and recorded for a total of 500 ms per stimulus.
Before the actual testing the animals were acclimatized to the startle boxes for 3 days to reduce anxiety. During these 5 minute long acclimation phases the background noise was presented. On the third day, these 5 minutes were followed by a short input/output (I/O) test to determine the appropriate gain setting for the transducer signal for each individual animal. The I/O test consisted of 12 acoustic stimuli with increasing intensity (65-120 dB SPL, 20 ms duration). Once set, the gain was kept constant for all recordings of a given animal. In order to reduce variability we tested each animal on 5 consecutive days using the following protocol.
Machine-readable layer
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