Materials and Reagents

1. 50-ml tube (Corning, France, catalog number: 430828) with two custom-made 5 mm-diameter holes in the lid
2. Laboratory-bred mouse pups from 6-8 days after birth
   Notes: 
   
   1. Put in a single cage each pregnant female one week before the timed day of birth. 
   2. For the test, male and female pups can be used. We only tested Oncins France 1 (OF1) mouse line from Charles Rivers, France with this behavioral protocol. This mouse strain is productive and widely used for teratology. Pups of other mouse strains such as C57Bl/6J emit similar USV (Castellucci et al., 2018).
   3. Infected-pups can be used as previously described (Lazarini et al., 2018). OF1 mother mice and its litter from Charles Rivers, France were individually housed in two isolators, one for the control (CTL) group and the second for the CMV group, kept in a BSL-2 room with controlled temperature (22 °C) and humidity (range: 40%-70%), under 12 h light/dark cycle (lights on at 8:00 AM) in the Pasteur Institute animal facilities accredited by the French Ministry of Agriculture for performing experiments on live rodents. Mice were manipulated in class II safety cabinets.
   4. You can identify the pups at Day 1 after birth using long-lasting paw tattoos, subcutaneously injected with a 0.3 mm x 13 mm needle.
3. Male scent (10 g soiled bedding from a group of 6 unfamiliar male adult OF1 mice)
4. Mineral oil (Sigma, France catalog number: M5904) 
5. Citral (Sigma, France catalog number: W230316)
   Note: Citral has lemon scent.
6. 70% Ethanol solution
7. Citral solution (see Recipes)
   

Equipment

1. Class II safety cabinets
2. Custom-made sono-olfactometer (depicted in Figure 1)
   
   1. The audio recording system is composed of:
      
      1. A sound card recorder (PreSonus AudioBox iTwo) (Figure 1). The sound card can be replaced by any other commercial model. The recording potentiometer is adjusted to maximize the signal-to-noise ratio and avoid overloading.
      2. A heterodyne bat detector (whose microphone has been moved into the chamber isolator using a BNC cable) (Magenta Bat5 Digital Bat Detector, RSPB, UK) (Figures 1 and 2). The heterodyne bat detector is set to the center frequency of the mouse vocalization: The volume control of the bat detector is adjusted in the middle to avoid background noise.
         
         

      
      Figure 1. The sono-olfactometer. The sono-olfactometer is a system that delivers odors while recording the ultrasound emitted by mice. It is composed of four sub-systems: 1) An audio recording system; 2) Two identical isolated chambers (only one chamber will be presented); 3) An odor dispenser (olfactometer); 4) A computer with its own software.

      
      
   2. The isolated chamber (Figure 2):
      
      1. Exhaust air pump (Schego, catalog number: 850)
      2. Non-return (check) valve (composed of two elements from Colder Products, catalog numbers: PLCD220-04 and PLCD10004)
      3. Exhaust air flow meter (Brooks Instrument, catalog number: FR2A13BVBN)
         
         
      
      Figure 2. The isolated chamber. The isolated chamber (inside size 18 x 12 x 12 cm) is sealed and airtight when closed. The isolated chamber was made with components (8 mm-thick black PVC walls for the ceiling, the floor and sides, 8 mm-thick transparent plexiglass for the door, 5 mm-diameter rubber seal for the door, 0.1 mm-thick aluminum coating plate with 2 mm diameter holes) easily available in DIY stores such as Lacrylic shop, Bonneuil sur Marne, France. The odor port and the ultrasonic microphone are on the right side of the chamber. The odor dispenser releases the Odor/Air mixture in a controlled manner through the odor port (Figure 3). A drain hole in the center of the left side is connected to a HEPA exhaust air filter (Millex-FG, 0.20 µm, PTFE hydrophobe, 50 mm). The air is evacuated by an exhaust air pump, a non-return (check) valve (composed of two elements from Colder Products, and an exhaust air flow meter (Figure 3), thus avoiding microbial contamination of the environment. The exhaust air containing odorants is directly diverted to the air exhaust of the animal facility. The HEPA exhaust air filter is changed at the end of the experiment, after the testing of all the pups. The ultrasonic microphone is offset inside the box.
      
      
   3. The odor dispenser (olfactometer) (Figure 3):
      
      1. Emitting air pump (Schego, catalog number: M2K3)
      2. Air flow meter (Key Instrument, catalog number: FR2A14BVBN)
      3. Odor flow meter (Brooks Instrument, catalog number: FR2A13BVBN)
      4. Odor valves (e.g., ASCO, catalog number: SCH284A005.12/DC or Bio-Chem Fluidics, catalog number: 100P2NC12-05B or equivalent normally closed solenoid pinch valves)
      5. Exhaust air pump (Schego, catalog number: 850)
      6. C-Flex^® Standard Tubing (ID: 0.125 OD: 0.250)
         Note: The tubing is GMP Compliant.
Note: All electronics used to control the system is a custom-made device but it could easily be replaced by a commercialized version of Arduino card: https://www.arduino.cc/en/Main/Products.


Figure 3. The odor dispenser. The odor dispenser distributes the Odor/Air mixture in a controlled manner. The air from the emitting air pump is transferred to the air flow meter and the odor flow meter. This device allows one to adjust the Odor/Air ratio (0.3 L/min for odorant and 2 L/min for air). If odor valves (can be “ASCO”, “Bio-Chem Fluidics” or equivalent normally closed solenoid pinch valves) are closed, then clean air will be diffused into the chamber, otherwise the odor will be diffused homogeneously in the chamber. The air is evacuated into the isolated chamber by the exhaust air pump at a flow rate of 1.5 L/min. C-Flex^® Standard Tubing (ID: 0.125 OD: 0.250) is used to connect the different devices.


3. Personal protective equipment (PPE)
   May include (but is not limited to) scrubs, a sterile combination, latex gloves, bouffant cap, ventilation mask, protective glasses, and shoe covers, depending on the regulation of the animal facility in which the work is taking place.
   

Software

1. The software (Figure 4):
   Pups emitted ultrasonic vocalizations at 40-120 kHz that were detected using an ultrasonic microphone connected to a bat detector (frequency range 10-130 kHz) that converts ultrasonic sounds to the audible frequency range. Using the broadband 60 kHz output of the detector, ultrasonic calls were sampled, recorded and analyzed using Audacity open software (www.audacityteam.org).
   
   
   Figure 4. The audio recording software. This figure shows the computer screen with the open images of the audio recording and the odor diffusion software. The control of the diffusion of odors is ensured by a custom-made software. This software can be replaced by the Node-RED software coupled with an Arduino. As the Arduino appears as a Serial device, the Serial in/out nodes can be used to communicate with it (https://nodered.org/docs/hardware/arduino).
   
   
2. GraphPad Prism software (GraphPad Software, USA) is used for data analysis
   

Procedure