This method provides a preclinical behavioral surrogate for photophobia, which is a chief complaint of migraine patients. The main advantage of this technique is that the detection is automatic thus very convenient. Also, light aversion and anxiety can be distinguished by conducting these tests.
On day one, turn the light/dark assay apparatus on. Set the light intensity to 27, 000 lux. Put the dark insert into light/dark chamber.
Open the tracking software and set up a new protocol. In the new protocol setting, set the duration to 30 minutes. In the new analysis setting, set data bins by duration to 300 seconds.
In the new zone setting, choose predefined zones. Choose two, and then horizontal. Check if the chamber is divided into two equal sized zones horizontally for recording.
Prior to the testing, habituate the mice to the testing room for one hour, keeping the room light on so as not to disturb the mouses circadian rhythm. Ensure all the equipment for the light/dark assay is turned on allowing the mice to fully acclimate to the testing room environment. Select acquire data, enter the mouse IDs and start the protocol.
Pull the drawer outside of the sound attenuating cubicle to access the light/dark chamber and the dark insert. Gently place a mouse in the light zone of the chamber and push the drawer inside the cubicle. Ensure that the software detects the mouse immediately and begins to record activity.
Wait for the recording to automatically stop after 30 minutes. Return the mouse to its home cage. Clean the chamber and dark insert using alcohol odor germicidal disposable wipes to eradicate any olfactory cues left by the previous mouse.
On day four, repeat all the steps that were performed on day one. On day seven, repeat the steps performed on day one, but only until the habituation of the mice. After the habituation, administer the mice with CGRP intraperitoneal.
Then return mice to their home cages. After 30 minutes, resume the protocol and run the mouse in the light/dark chamber as mentioned earlier. Clean the chamber and dark insert as described previously.
On day 10, repeat all the steps performed on day one. Turn the apparatus on and set the light intensity to 27, 000 lux. Open the tracking software and set up a new protocol as demonstrated previously.
In the new zone settings, choose one followed by center. Set the periphery as 3.97 centimeters and the center as 19.05 by 19.05 centimeters. Habituate the mice to the testing room as demonstrated previously.
Administer the mice with CGRP intraperitoneal. Return the mice to their home cages. After 30 minutes, start the protocol.
Pull the pullout drawer outside of the sound attenuating cubicle and gently place a mouse in the middle of the open field chamber. Push the drawer inside of the cubicle. Track the behavior of the mouse for 30 minutes, then return mice to their home cages.
Clean the apparatus as demonstrated previously. Results revealed that intraperitoneal injection of CGRP significantly decreased the duration of time spent in the light zone in the light/dark assay in CD1 and C57 black 6J mice, but did not affect the time mice spent in the center in the open field assay in CD1 and C57 black 6J mice. Treatment with CGRP also increased the amount of time mice rested in the dark zone, but not in the light zone in both CD1 and C57 black 6J mice.
The optical stimulation of neurons in the posterior thalamic nuclei, led to a corresponding decrease in the duration mice spent in the light zone in the light/dark assay in channelrhodopsin-2 injected mice compared to control virus injected mice. An increase in the resting time in the dark zone but not in the light zone was also observed in the case of the channelrhodopsin-2 injected mice, as compared to the control virus injected mice. It's important to remember that when putting the mouse into the light/dark chamber, the mouse should be handled gently to avoid as much stress as possible.
This technique will pave the way to explore mechanisms underlying migraine and other photophobia related disorders, and test the preclinical efficacy for potential therapeutics.
