We here provide a detailed protocol for assessing the rewarding or aversive properties of the genetic stimulation of a specific brain region. The technique can answer rapidly if stimulation of specific cells is rewarding or aversive. We describe setup for stimulating the ventral tegmental area but our protocols can easily be adapted to change specific parameters in terms of different regions studied.
To begin, use single-board microcontrollers to control the laser source. Load the script on the microcontroller board using the appropriate connection cable to the computer. The script includes external modulation coming from the tracking software through a TTL box, and an output to the laser to control stimulation parameters.
Use a network cable to connect the TTL box to the board. Ensure the laser is set to control by external modulation, and connect the laser to the board using an FC/PC cable. Connect the appropriate pins to ground parts of the board.
To connect the laser source to the optic fiber, first connect the laser source to a rotary joint. Connect a patch cord to the rotary joint. Stabilize the rotary joint above the apparatus, but outside the recording area.
Make sure the length of the fiber-optic patch cord is appropriate to allow the mouse to move without difficulties in the arena. To calibrate the arena setup, use a ruler to measure a specific part of the physical apparatus. In the software, draw a line corresponding to the part measured.
Under the Draw Scale to Calibrate tab, enter the already known value. Design the arena, draw the area where the movement of the mice will be recorded. Create the zones, draw the zones that will eventually be assigned as laser-paired, laser-unpaired, and neutral.
Press the Validate Setup button to validate the setup to confirm that there are no conflicting parameters, for example, zones outside the arena. Make sure hardware control is enabled, and set the time of the experiment to 30 minutes, to the repeat til option in the reference box settings. Assign a compartment as laser-paired, in which entry of the mouse will trigger a TTL signal through the tracking software to the microcontroller board under the settings of condition.
To modify the setup for the neutral compartment preference approach, after arena and time setup, assign both A and B zones as laser-paired by adding when center point is in any of zone A and zone B for the condition box related to the settings for A and B compartments. To set up the detection settings, use a dummy to resemble the mouse. Place the dummy in one compartment of the apparatus, and use automated setup with dynamic subtraction.
Remove the dummy and place it in the opposite compartment. Make sure the dummy is fully detected. Place the dummy in the laser-paired compartment.
To check if the stimulation works, start acquisition using the previously configured trial control settings and see if the stimulation is triggered as it should. Then place the dummy in the unpaired or the neutral compartment, and see if the stimulation is stopped. Use the knob on the laser to set the laser power to 10 milliwatts.
Connect the fiber-optic implant to the fiber-optic patch cord using a ceramic sleeve, and place the mouse in the neutral compartment of the three-compartment apparatus. Wait until the mouse is detected by the software. Remove the vertical sliding doors restricting the animal from entering the main compartments.
Allow the animal to explore freely without any disturbances. The entire real-time place preference experiment takes place throughout eight sessions. DAT-Cre mice injected with AAV channelrhodopsin EYFP virus in the ventral tegmental area were tested to target dopaminergic neurons.
The mice spent on average about 70%of their time in the laser-paired compartment, as opposed to the unpaired compartment at 20%and the neutral compartment at 10%An opposite behavioral phenotype was observed with Vglut2-Cre mice injected with AAV channelrhodopsin EYFP in the ventral tegmental area. The mice avoided the compartment paired to the stimulation, and spent more time in the unpaired. During the conditioned responses days five and eight, the mice did not show a clear avoidance of the previously paired compartment.
To further demonstrate the rewarding or aversive role of specific neurons, the described method can be complimented with other methods such as operon paradigms, or photogenetic substimulation. Pay attention while handling viruses and when you use laser sources.
