Materials and Reagents

Paper towels
Note: Paper towels and ethanol are used to clean the cage from excrements after habituation phase in the days before the proper testing.
Gloves
Adhesive paper tape
Note: The tape is useful to mark the position of the cage and the camera in the experimental room.
Laboratory mice: Each mouse strain which models a neurological disorder can be tested. Different life phases of mice can be considered to assess tactile hypersensitivity: young (1-3 months old), mature adult (3-6 months old), middle-aged (10-14 months) and old (18-24 months).
Note: All mice tested should be of equal or similar age. Male mice should be tested separately in advance or in a different day since the smell of female mice could affect male mice behavior.
70% ethanol (v/v)
Note: Spray your hands with ethanol before and after handling of each mouse.
Odor-free disinfectant
Note: Spray all working surfaces with disinfectant after each testing session. Air dry for 2-3 min.

Equipment

Transparent standard mouse cage
Note: In our laboratory we use standard individual ventilated cage (Sealsafe Plus GM500, Tecniplast, Italy) with 501 cm² floor area (W x D x H dimensions of 391 x 199 x 160 mm). Any other transparent plastic cage with comparable measure can be used.
Video camera (LifeCam Studio 1080p HD Sensor/computer, Microsoft, CA), equivalent models from other companies can be used
Note: The quality of video recording should be higher enough (ideally 1,080p HD) to clearly assess single behaviors offline. The camera should be positioned 20-30 cm away from the cage in a position that allow to record the whole cage minimizing blind angles and reflexes (for reference see Figure 1). If recording light is present, cover it with paper tape. The raw data are stored in a computer for successive analysis.

Figure 1. Representation of how the experimenters should position the camera and the experimental cage
Wooden stick (length: 20 cm; diameter: 3 mm) bought in any drug store
Note: Blunt ends must be preferred to pointed ones in order to avoid possible physical harm.
Digital chronometer (Thermo Fisher Scientific–US, Fisherbrand^TM Traceable^TM Jumbo-Digit Stopwatch), equivalent chronometer can be used
Note: Beeping noise from the chronometer should be avoided since could cause anxiety and fear in mice.

Software

Prism 8.0 (GraphPad, https://www.graphpad.com/) or similar software to perform statistical analysis

Procedure

See Figure 2 for the steps of the experimental procedure.

Figure 2. Graphic representation of the behavioral testing steps. The behavioral testing consists of a habituation phase, in which the mice get used to the experimental cage, the novel environment and the experimenter. Habituation is then followed by sham-stimulation trial, where the wooden stick is introduced in the cage avoiding tactile contact with the animal. This will provide a baseline score of the animal anxious behavior (animals with high anxious response to the sham condition should be excluded from the WN test). Finally, the actual test phase will consist in bilateral stimulation of mice facial vibrissae.

Notes:

Each experimental mouse must be single-housed in a cage at least 1 week prior to beginning of behavioral testing. These cages are referred to as home cages.
Behavioral experiments must be performed in a dedicated room. Ideally, the behavioral room should have black curtains all around the experiment area in order to minimize the interference with environmental stimuli. The room must also be free of conspecific and heterospecific odors, since they can affect mouse behavior.
Noise should also be reduced to the minimum or to a comparable level of housing room. The experimenter must avoid making loud noises or sudden movements during both the habituation phase than the experimental phase.
The light should be reduced or at least indirect, however attention should be paid to the quality of video recording.
We recommend always running a pilot experiment to establish position of the camera and the cage and standardizing the experimental environment before starting the proper experiment.
Once the experimental conditions are standardized the experimenters can proceed with the behavioral testing.

Preparation of experimental cages (Day 0)
Prepare several clean cages equal to the number of mice you intend to test, these cages are referred to as experimental cages. Be sure to label all the cages properly since each mouse will have its own experimental cage.
Transfer a handful of bedding from the home cage to the experimental cage, close it and leave it for all the night. This step is necessary in order to facilitate the habituation to the novel environment. Be sure to clean your hands with alcohol each time you change cage.
Note: At any point during the experiment the investigator performing both stimulation and scoring of the test must be blinded from the experimental conditions. This procedure is easily applicable by preventing the experimenter from knowing the mice group of belonging.
Habituation phase (Days 1-3)
Choose a time for the habituation phase and the behavioral testing (i.e., morning), and then keep it for the duration of the whole experimental procedure. It is very important to be consistent in testing time parameters.
1. Remove the bedding from the experimental cage.
2. Transfer the mouse from its home cage to its experimental cage, without lid (Figure 3).
  Note: The cage should be placed in the same position every day; use adhesive tape to keep track of the correct position.
  
  Figure 3. Mouse in the experimental cage during habituation phase
3. Leave the animal in the experimental cage for 30 min. Be there, the animal should habituate also to your presence.
  Note: The first day of habituation is the trickiest, the animals could be scared and jump out of the cage, pay attention and be patient.
4. At the end of the habituation phase transfer the mouse from the experimental cage to its home cage.
5. Remove all the urine and fecal boli from the experimental cage and clean with 70% ethanol.
6. Place a handful of bedding from the home cage to the experimental cage, close it and leave it for all the night.
7. On Day 2 repeat Steps B1 to B6.
8. On Day 3 repeat Steps B1 to B6.
  Note: The habituation phase should be performed for at least 3 days, less days could have a negative impact on the behavioral assessment. A successful habituation is when the animals show no sign of fear, stress and/or anxiety (e.g., freezing, evasiveness, excessive grooming) due to the novel environment and to the presence of the experimenters.
Experimental phase (Day 4)
The experimental phase consists in the sham stimulation and in three trials of bilateral whisker stimulation. Both sham phase and stimulation phase last 5 min with 1 min of inter-trial interval.
1. Remove the bedding from the experimental cage.
2. Transfer the mouse from its home cage to its experimental cage, close the lid and position the cage in front of the camera.
3. Leave the animal in the experimental cage for 30 min.
4. Open the lid of the cage and start recording
5. Start sham stimulation for 5 min (Video 1).
  Note: In the sham stimulation, the animal is presented with the wooden stick. Pretend to stimulate the whisker of the mouse without touching them.
  
  Video 1. Example of sham stimulation. (All animal procedures described in this protocol were approved by the University of Trento animal care committee and the Italian Ministry of Health (protocol 949/2015-PR).)
6. Start directly with trial 1 and continuously stimulate the whiskers for 5 min (Video 2).
  Note. Use rhythmic movements to bend the middle of the whiskers and alternate the whisker stimulation (about 3 seconds in duration) for each side of the mouse’s snout.
  
  Video 2. Example of whisker stimulation. (All animal procedures described in this protocol were approved by the University of Trento animal care committee and the Italian Ministry of Health (protocol 949/2015-PR).)
7. Wait 1 min.
8. Repeat Steps C6 and C7 other 2 times.
9. Stop the recording, save the video on a computer and go on with another mouse.

Data analysis

The behavioral assessment is based on manual video scoring of single behaviors. The behavioral assessment is made following Table 1. Five different categories are scored; the behavioral responses analyzed are fearful behavior (freezing, Video 3), stance (Video 4), breathing (hyperventilation, Video 5), aggressive response to stick presentation (Video 6) and evasiveness (Video 7) on a 0-2 points qualitative scale (0, absent; 1, scarcely present during the observation period; 2, present for most of the observation period).

Video 3. Example of freezing. (All animal procedures described in this protocol were approved by the University of Trento animal care committee and the Italian Ministry of Health (protocol 949/2015-PR).)

Video 4. Example of stance. (All animal procedures described in this protocol were approved by the University of Trento animal care committee and the Italian Ministry of Health (protocol 949/2015-PR).)

Video 5. Example of hyperventilation. (All animal procedures described in this protocol were approved by the University of Trento animal care committee and the Italian Ministry of Health (protocol 949/2015-PR).)

Video 6. Example of aggressive response to stick presentation. (All animal procedures described in this protocol were approved by the University of Trento animal care committee and the Italian Ministry of Health (protocol 949/2015-PR).)

Video 7. Example of evasiveness. (All animal procedures described in this protocol were approved by the University of Trento animal care committee and the Italian Ministry of Health (protocol 949/2015-PR).)

Normal behavioral responses to stimulation are assigned a zero value, conversely meaningful abnormal behavioral responses (i.e., for most of the observation period) are assigned a value of 2. The maximum score is 10. This 10-point scale represents a modified version of the WN scoring scale by McNamara et al. (2010). High scores (8-10) indicate abnormal responses to the stimulation, in which the mouse freezes, becomes agitated or is aggressive. Low scores (0-3) indicate normal responses, in which the mouse is either curious or indifferent to the stimulation.

Table 1. The table shows the quantification of behavioral responses in the WN test. Five different parameters (fearful behavior, stance, breathing, response to stick, evasion) were monitored across test sessions (sham and trials 1-3). The total score indicates three major categories of behavioral responses (0 to 3, curious/restful; 4 to 7, annoyed/bothered; 8 to 10, scared/worried).

Scoring should be performed at least by two trained independent operators to ensure a reliable scoring consistency. Statistical analysis can be conducted using GraphPad Prism 6.0 or any other statistical program and should be preferentially performed using analysis of variance (ANOVA), followed by appropriate post-hoc tests, with significance level set at P < 0.05. We recommend using a minimum of 8-10 mice per genotype/group to reach statistical significance as well as to minimize the effects of inter-individual variation, although more may be necessary.
We recently provided a sample analysis of behavioral response to whisker stimulation in mice lacking the ASD-related gene Engrailed-2 (En2^-/-) mice (Chelini et al., 2019). En2^-/- mice displayed a significantly higher score across the 3 trials, as compared with WT controls. When we analyzed the behavioral response of WT and En2^-/- mice for each of the five categories used to calculate the WN score, En2^-/- mice displayed a significantly higher score in freezing and breathing, indicating a predominant fearful behavior of En2 mutants in response to repeated whisker stimulation. The reader is referred to Figure 5 of our original publication (Chelini et al., 2019) for details.

Acknowledgments

This behavioral protocol has been adapted from a previous study (McNamara et al., 2010), which succeeded in identifying sensory hypersensitivity to whisker stimulation in rats following diffuse brain injury. We thank the technical and administrative staff of CIMeC and CIBIO for excellent assistance. G.P. and Y.B. are currently supported by the University of Trento 2018-2020 Strategic Project “Trentino Autism Initiative–TRAIN”.

Competing interests

The authors declare no conflict of interest.

Ethics

All animal procedures described in this protocol were approved by the University of Trento animal care committee and the Italian Ministry of Health (protocol 949/2015-PR).

References

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