This work was supported by grants from the NIH NS R01 NS075599 and&#160;RF1 NS113839. The contents do not represent the views of VA or the United States Government.

Animal procedures were approved by the University of Iowa Animal Care and Use Committee and performed in compliance with the standards set by the National Institutes of Health.
1. Light/dark assay
<ol>
	<li>Light/dark chamber apparatus (see Table of Materials) setup. All the equipment in this section is commercially available.
	<ol>
		<li>On a shelf, place the sound-attenuating cubicle (interior: 59.7 x 38 x 35.6 cm in W x H x D) containing a pull-out drawer for easy access ...

The light/dark assay is widely used to assess anxiety-like behavior12. The assay relies on the innate aversion of mice to light and their drive to explore when placed into a novel environment (light zone). However, as we report here, this assay can also be used to assess light-aversive behavior as well.
It is critical to consider the number and necessity of pre-exposures prior to testing. This depends on the mouse strain or model. For example, in our light/dark assay pr...

Migraine is a prevalent neurological disease, affecting approximately 17% of Americans1 and is the second leading cause of disability globally2,3. Patients experience headache that lasts 4-72 hours accompanied with at least one of the following symptoms: nausea and/or vomiting, or photophobia and phonophobia4. Recent advances in the development of calcitonin gene-related peptide (CGRP) antibodies that are now FDA approved have begun a new era for migraine treatment5,6,7. These antibodies block either CGRP or its receptor and prevent migraine symptoms in approximately 50% of migraine patients7. Within the past year, two small-molecule antagonists of the CGRP receptor have also been FDA approved for abortive treatment of migraine, and two more are in the pipeline8. Despite this therapeutic progress, mechanisms by which migraine attacks occur still remain elusive. For example, the sites of CGRP action are not known. The efficacy of therapeutic antibodies that do not appreciably cross the blood-brain barrier suggests that CGRP acts at peripheral sites, such as the meninges and/or trigeminal ganglia. However, we cannot rule out central actions at circumventricular organs, which lack a blood-brain barrier9. At least for photophobia, we think this is less likely given our results with light aversion using transgenic nestin/hRAMP1 mice in which hRAMP1 is overexpressed in the nervous tissue10. Understanding mechanisms of migraine pathophysiology will provide new avenues to the development of migraine therapeutics.
Preclinical animal models are critical to understanding disease mechanisms and the development of new drugs. However, migraine assessment in animals is challenging since animals cannot verbally report their sensations of pain. Given the fact that 80-90% of migraine patients exhibit photophobia11, light aversion is considered to be an indicator of migraine in animal models. This led to the need to develop an assay to assess light aversion in mice.
The light/dark assay contains a light zone and a dark zone. It is widely used for measuring anxiety in mice based on their spontaneous exploration of novel environments that is countered by their innate aversion to light12. Some studies set 1/3 of the chamber as the dark zone, while others set 1/2 of the chamber as the dark zone. The former setting is often used to detect anxiety13. While we initially chose equally sized light/dark chambers, we have not compared the two relative sizes. We can comment that the overall size of both chambers is not a major factor since the initial testing box14 was considerably larger than the subsequent apparatus15, yet results were essentially the same.
Two critical modifications to this light/dark assay to assess light aversion were: the testing condition and the light intensity (Figure 1). First, mice are pre-exposed to the light/dark chamber to reduce exploratory drive16 (Figure 1A). The necessity and times of pre-exposures depend on mouse strains and models. Wildtype C57BL/6J mice usually require two pre-exposures10, while only one pre-exposure for CD1 mice is sufficient17. In this manner, light-aversive behavior can be unmasked in these two mouse strains. Second, the chamber lighting has been adapted to include an adjustable range of light intensities from dim (55 lux) to bright (27,000 lux) where 55 lux is comparable to a dark overcast day, and 27,000 lux is comparable to a bright sunny day in the shade10. We have found that the required light intensity varies with the strain and genetic model. For this reason, individuals should first assess the minimum light intensity for their experimental paradigm.
Even with these modifications to the assay, which can reveal a light-aversive phenotype, it is necessary to test anxiety-like behavior to distinguish between light aversion due to light alone versus due to anxiety. The open field assay is a traditional way to measure anxiety based on the spontaneous exploration of novel environments. It differs from the light/dark assay in that the exploratory drive is countered by the innate aversion to unprotected open spaces. Both the center and edges of the chamber are in the light, so the open field assay is a light-independent anxiety assay. Thus, the combination of the light/dark and open field assays enables us to distinguish between light aversion due to an avoidance of light versus an overall increase in anxiety.
CGRP is a multifunctional neuropeptide that regulates vasodilation, nociception, and inflammation18. It is widely expressed in the peripheral and central nervous systems. It plays an important role in migraine pathophysiology18. However, the mechanism underlying CGRP action in migraine is unclear. By utilizing the light/dark and open field assays with this modified test paradigm, we were able to identify light-aversive behavior in mice following peripheral10,16 (Figure 2) and central14,15,16,19 CGRP administration. In addition to neuropeptides, the identification of brain regions involved in light aversion is also important in understanding migraine pathophysiology. The posterior thalamic nuclei are an integrative brain region for pain and light processing19, and the thalamus is activated during migraine20. Thus, we targeted posterior thalamic nuclei by injecting adeno-associated virus (AAV) containing channelrhodopsin-2 (ChR2) or eYFP into this region. By combining this optogenetic approach with these two assays, we demonstrated that optical stimulation of ChR2-expressing neurons in the posterior thalamic nuclei induced light aversion19 (Figure 3). In this experiment, given the dramatic effect on the evoked light aversion in these optogenetically manipulated mice, pre-exposures to the chamber were skipped.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" fo:font-size="6px">
	<tbody>
		<tr>
			<td>Activity monitor</td>
			<td>Med Assoc. Inc</td>
			<td></td>
			<td>Software tracking mouse behavior</td>
		</tr>
		<tr>
			<td>Customized acrylic shelf</td>
			<td></td>
			<td></td>
			<td>For adjusting the height of the LED panel</td>
		</tr>
		<tr>
			<td>Dark box insert</td>
			<td>Med Assoc. Inc</td>
			<td>ENV-511</td>
			<td></td>
		</tr>
		<tr>
			<td>DC power supply</td>
			<td>Med Assoc. Inc</td>
			<td>SG-500T</td>
			<td></td>
		</tr>
		<tr>
			<td>DC regulated power supply</td>
			<td>Med Assoc. Inc</td>
			<td>SG-506</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-optic cannula</td>
			<td>Doric</td>
			<td>MFC_200/ 240-0.22_4.5mm_ZF1.25_FLT</td>
			<td></td>
		</tr>
		<tr>
			<td>Germicidal disposable wipes</td>
			<td>Sani-Cloth</td>
			<td>SKU # Q55172</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Sink</td>
			<td>Wakefield</td>
			<td>490-6K</td>
			<td>Connecting to LED panel</td>
		</tr>
		<tr>
			<td>IR controller power cable</td>
			<td>Med Assoc. Inc</td>
			<td>SG-520USB-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IR USB controller</td>
			<td>Med Assoc. Inc</td>
			<td>ENV-520USB</td>
			<td></td>
		</tr>
		<tr>
			<td>Mating sleeve</td>
			<td>Doric</td>
			<td>SLEEVE_ZR_1.25</td>
			<td></td>
		</tr>
		<tr>
			<td>Modified LED light panel</td>
			<td>Genaray Spectro</td>
			<td>SP-E-360D</td>
			<td>Daylight-balanced color (5600K)</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>MEAN WELL USA</td>
			<td>SP-320-12</td>
			<td>Connecting to LED panel</td>
		</tr>
		<tr>
			<td>Seamless open field chamber</td>
			<td>Med Assoc. Inc</td>
			<td>ENV-510S</td>
			<td></td>
		</tr>
		<tr>
			<td>Sound-attenuating cubicle</td>
			<td>Med Assoc. Inc</td>
			<td>ENV-022MD-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Stand and clamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Three 16-beam IR arrays</td>
			<td>Med Assoc. Inc</td>
			<td>ENV-256</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating migraine-like behavior using light aversion in mice

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

This behavioral test paradigm is designed to test light-aversive behavior. It can be performed using both na&#239;ve wildtype mice and optogenetic mice to investigate light aversion in real time during the stimulation of a targeted neuronal population.
This procedure has been used to study the effect of peripheral CGRP treatment in CD1 and C57BL/6J mice10,16 and optical stimulation of neurons in the posterior thalamic nuclei in C57BL/6...

Watch this Scientific Journal Video about Investigating Migraine-Like Behavior Using Light Aversion in Mice at JoVE.com

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as ...

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.

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Abstract