3.8. Behavioural assays
Several comparative behavioural assays have been developed in the field of zebrafish neuroscience research, which along with the presence of several standardized and automated behavioural analysis system and tools, have made it possible to model relevant behavioural domains (Colwill and Creton, 2011). The following tests were used: prepulse inhibition, locomotor activity, light-dark transition, thigmotaxis and shoaling.
3.8.1. Prepulse inhibition of the acoustic startle response
In paper II, and parts of papers III and IV of this thesis, we used the automated live video tracking system called the ZebraBox Revo (ZebraLab, ViewPoint Life Sciences, France) and the automated video analysis software EthoVision version 14 (Noldus, Netherlands) to perform the PPI of the ASR test in larval zebrafish. The ZebraBox Revo uses a high-speed infrared camera that records videos at 1000 frames per second at 2048 × 500 resolution. The other components of the system include the following: a test cabinet, a stereo amplifier (Dynavox; CS-PA1 MK) and a dB meter (PCE instruments; PCE-MSM 4) to calibrate sound intensity, input and output cables, and the software (PPI generator) to generate and deliver acoustic stimuli. The ZebraBox Revo system allows for delivering acoustic stimulus with timing and waveforms generated by the PPI software.
The intensity of the stimulus was calibrated and measured using the volume knob of the stereo amplifier and a decibel meter respectively. To determine the exact onset of stimuli, an infrared light source of 10000 Hz was coupled to the entire duration of the stimulus. The infrared light is not visible to the larvae; therefore, it does not interfere with the behaviour of the larvae.
35 Next, we used EthoVision (Noldus, Netherlands), which is an automated tracking software to analyze the videos previously acquired using the ZebraBox Revo system. Tracking of the larvae was performed in a non-live mode with static subtraction of the background, which reduced artifacts. The tracked features were the center-point, nose-point, and tail-base of each larva. The absolute bend angle of the body was calculated for each larva using the tracked features. Various bend angle thresholds were set between 20 – 80o averaged over 5 ms (i.e. 5-time frames). Once the tracking parameters were optimized, EthoVision software was used for the analysis of the larval response to the various acoustic stimuli. A change in body angle of >25o with a cut-off latency of 100 ms after stimulus onset was considered a positive response. The C-start threshold of >25o was determined after preliminary analysis of larvae not presented with any stimuli. To define the intensity of the startle stimulus, we presented 660 Hz tones of different stimulus intensities between 40 – 80 dB in increments of 5 dB and calculated using the following formula;
% ݎ݁ݏ݊݀݁ݎݏ=(݊ݑܾ݉݁ݎ ݂ ݈ܽݎݒܽ݁ ݎ݁ݏ݊݀݅݊݃) (ݐݐ݈ܽ ݊ݑܾ݉݁ݎ ݂ ݈ܽݎݒܽ݁) כ100
Any stimulus intensity that was capable of eliciting a C-start response in >70% of the larvae were considered a suitable startle stimulus as used in previous studies (Burgess and Granato, 2007b).
We excluded larvae that responded <60% to the startle stimulus. In all PPI experiments, a pseudorandom order was used to interleave prepulse trials with pulse alone trials. Where multiple stimuli were presented in an experiment, an inter-trial interval (ITI) of 30 sec was used. We calculated % PPI using the formula below:
% PPI=(percentage responding to startle stimulus) - (percentage responding to prepulse + startle stimulus)
(percentage responding to startle stimulus) *100
All PPI experiments were performed on individual larvae at 6 dpf, in a custom-made plexiglass plate of 96-well format. We used the plexiglass plate to reduce interference from shadows that we observed initially when using commercial plates. Larvae were placed individually in a well (16 larvae per plate in total) and acclimated to a 100 Lux illuminated ZebraBox Revo for 5 minutes before the experiments were started. Burgess & Granato (2007) reported an influence on the startle response by temperature. Hence, we performed all experiments in a temperature-controlled setting
36 (27±1oC). To reduce the background white noise of the room, the ZebraBox Revo was insulated with a custom-built sound booth. In all experiments, we used a 100 ms startle stimulus (pulse) of 660 Hz, and all prepulse stimuli were given for 5 ms at 440 Hz.
3.8.2. Locomotor-based analysis
In papers III and IV of this thesis, we used the automated live video tracking system called the ZebraBox (ZebraLab, ViewPoint Life Sciences, France) to perform the following locomotor based tests in larval zebrafish: locomotor activity, light-dark transition, thigmotaxis, and shoaling. The ZebraBox system consists of an infrared light source, a high-resolution digital video camera to capture larval movements within a defined period, and the ZebraLab software to analyze larval locomotor activity (Colwill and Creton, 2011). For all experiments, 6 dpf larvae were used except in the shoaling tests where we used 7 dpf larvae. On the day of the experiments, the larvae were transported from the incubator, allowed to acclimate to the conditions of the recording room for a minimum of one hour. All experiments were performed between 10:00 and 18:00. We used custom-made 48 well plates made from plexiglass for the locomotor activity and light-dark transition tests. A 24 well plate (#83.3922.500, Sarstedt, Germany) and a round dish [of diameter 60 mm, height 15 mm, and volume 30 ml (#82.1194.500, Sarstedt)] were used for the thigmotaxis and shoaling tests respectively. In paper IV, we did not use homozygous mutants in the thigmotaxis and shoaling tests because the reliability of the tests depend on the mobility of the animals.
However, cacna1c homozygous mutants have reduced mobility.
3.8.2.1. Locomotor activity test
Habituation and tracking of larvae were performed under dark conditions for 15 and 10 min respectively. The total movement was defined as the average of the sum of large + small movements expressed in the mm.
37 3.8.2.2. Light-dark transition test
Larvae were first habituated in the dark chamber for 15 min, then the light switched on to begin tracking for 10 min followed by a light switch off (darkness) for another 10 min of tracking. Total movement in each illumination state was calculated as the average of the sum of large + small movements expressed in the mm per time bin.
3.8.2.3. Thigmotaxis test
Each well was divided into an outer and inner zone and thigmotaxis was calculated as the % total distance moved in the outer zone as described by (Schnörr et al., 2012; Liu et al., 2016). The preference of each fish to remain at the periphery or explore the center of the arena was monitored for 10 min spontaneous behaviour in either light or dark conditions.
݄ܶ݅݃݉ݐܽݔ݅ݏ (% ݀݅ݏݐܽ݊ܿ݁ ݅݊ ݑݐ݁ݎ ݖ݊݁) = (௦௧ ௨௧ ௭)
(௦௧ ௨௧ ା ௭)כ100
3.8.2.4. Shoaling test
Larvae (5 larvae/arena) were placed in the center of the testing arena. The testing arena was then placed in the dark ZebraBox. After 15 minutes of habituation in the dark, a 20-minute recording session was performed also in the dark. The unit of measurement in this case is a shoal made up of 5 larvae. Except for homozygous mutants, it was difficult to sort WT and heterozygous mutant larvae without genotyping and since genotyping larvae would result in either the injury or death of larvae we had to come out with a testing paradigm described previously (Maaswinkel, Zhu and Weng, 2013). Whereas the control group comprised WT siblings, the mutant group was typically a mixed population of heterozygous and wild type siblings of the same line. This means that there was variation in the ratio of heterozygous to WT in each independent shoaling population. The shoaling characteristics we measured were nearest neighbor distance (NND), and inter-individual distance (IID) as described elsewhere (Miller and Gerlai, 2012).
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3.9. Local field potential recordings
7-dpf larvae were prepared and handed over to two post-doctoral researchers in the Esguerra lab (Dr. Kinga Gawel and Dr. Wietske van der Ent) for electrode local field potential recordings (LFP) from the optic tecta of larvae as previously described (Gawel, Kukula-Koch, et al., 2020; Gawel, Turski, et al., 2020; Tiraboschi et al., 2020). The aforementioned researchers performed the analysis of seizure-like activity. Afterwards, the LFP recordings were sent to Dr. Tuomo Mäki-Marttunen for spectral power analysis.
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