Name: Investigating Migraine-Like Behavior Using Light Aversion in Mice (Video) | JoVE Journal
Uploaded: 2021-08-11T15:00:00.000+00:00
Duration: 5 min 23 s
Description: Learn a light/dark assay method to assess light aversion in mice as a preclinical model for migraine photophobia. This technique distinguishes light aversion from anxiety using automatic detection.

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5 minutes

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 to the chamber and dark insert.</li>
		<li>Connect the DC power supply and a DC-regulated power supply to the sound-attenuating cubicle.</li>
		<li>Place the transparent seamless open field chamber (27.31 x 27.31 x 20.32 cm in L x W x H) on the pull-out drawer of the cubicle.</li>
		<li>Place the black, infrared (IR)-transparent plastic dark insert (28.7 X 15 X 20.6 cm in L x W x H) in the open field chamber. Ensure that the chamber is divided into two zones of an equal size: a dark zone and a light zone.</li>
		<li>Connect three sets of 16-beam IR arrays on the X, Y and Z axes of the open field chamber to the IR USB controller via cables.</li>
		<li>Connect the IR USB controller to a computer.</li>
		<li>Install the tracking software on the computer which can record and collect mouse location and activity.</li>
		<li>For the light panel setup, first remove the light-emitting diode (LED) light panel (27.70 x 27.70 cm in L x W; 360 LEDs, daylight-balanced color, 5600K, 60&#176; flood beam spread) from its original housing.</li>
		<li>Assemble the light panel with the LED driver, the heat sink, and the power supply. Multiple LED light panels can be connected to one power supply, heat sink, and LED driver to achieve uniform light panel control.</li>
		<li>Construct a customized acrylic platform (29.77 x 27.70 x 8.10 cm in L x W x H) comprising of 7 identical shelves at 0.53 cm intervals (Figure 1B). Permanently affix the customized acrylic shelf to the ceiling inside the cubicle above the chamber.</li>
		<li>Insert the LED light panel into the slot between the bottom two shelves. Adjust the light panel to different heights (Figure 1B,C), if necessary (e.g., if using optogenetic mice. Details are discussed in Section 3).</li>
		<li>Turn on the heat sink, LED driver, and power supply. Confirm that the LED driver can dictate the LED light intensity by measuring the light intensity on the chamber floor and confirm that the floor is lit evenly.</li>
	</ol>
	</li>
	<li>Behavioral test procedure 
	NOTE: Mice are housed on a 12 h light cycle. All behavioral experiments are performed during the light cycle. Mice, including both males and females, aged 10-20 weeks old, are used. In this protocol, na&#239;ve wildtype CD1 and C57BL/6J mice experience two pre-exposures to the light/dark chamber followed by exposure with treatment and a post-treatment exposure. There is a three-day interval between each exposure to allow mice to recover (Day 1, 4, 7 and 10 as described below and Figure 1A). However, CD1 mice do not require the 2nd pre-exposure and can be tested in dim light.
	<ol>
		<li>On day 1 (pretreatment 1), turn the light/dark assay apparatus on and set the light intensity to 27,000 lux.</li>
		<li>Open the tracking software and set up a new protocol. In the New Protocol setting, set the Duration to 30 min. In the New Analysis setting, set Data Bins by Duration to 300 s.</li>
		<li>In the New Zone setting, choose Pre-Defined Zones. Choose 2 and then Horizontal. Check if the chamber is divided into two equal-size zones horizontally for recording.</li>
		<li>Habituate mice to the testing room for 1 h prior to the testing. During habituation, keep the room light on to not disrupt the mouse&#39;s circadian rhythm. Make sure all the equipment for the light/dark assay is turned on, allowing the mice to fully acclimate to the testing room environment.</li>
		<li>Select Acquire Data. Enter mouse IDs. Start the protocol.</li>
		<li>Pull the drawer outside of the sound-attenuating cubicle to access the light/dark chamber and the dark insert. Gently pick up the mouse by the base of the tail, place it&#160;in the light zone of the chamber, and push the drawer inside of the cubicle. Ensure that the software detects the mouse immediately and begins to record activity.</li>
		<li>Wait for the recording to automatically stop after 30 min. Return the mouse to its home cage.</li>
		<li>Clean the chamber and dark insert using alcohol-odor germicidal disposable wipes containing 55.0% isopropyl alcohol, 0.25% alkyl C12-18 dimethyl ethylbenzyl ammonium, and 0.25% alkyl C12-18 dimethyl benzyl ammonium chloride as anti-microbial active ingredients to eradicate any olfactory cues left by the previous mouse.</li>
		<li>On day 4 (pretreatment 2), repeat steps 1.2.1 to 1.2.8.</li>
		<li>On day 7 (the treatment day), repeat step 1.2.1 and 1.2.4. After the habituation, administer CGRP (0.1 mg/kg, 10 &#181;l/g based on mouse body weight, intraperitoneal injection (i.p.)), tilting the mouse&#8217;s head forward and injecting in the lower right quadrant. Return the mouse to the home cage.</li>
		<li>After 30 min, start the protocol and run the mouse in the light/dark chamber&#160;as mentioned in steps 1.2.5 to 1.2.7. The recovery time in home cages after injections can be shortened or lengthened depending on the treatment21.</li>
		<li>Clean the chamber and dark insert as described in step 1.2.8.</li>
		<li>On day 10 (post-treatment day), repeat steps 1.2.1 to 1.2.8. The experiment can be paused at step 1.2.13&#160;before starting the open field assay.</li>
	</ol>
	</li>
</ol>
2. Open field assay
<ol>
	<li>The apparatus setup
	<ol>
		<li>Open field chamber setup: Use the same sound-attenuating cubicle and open field chamber used in the light/dark assay, without using the dark insert.</li>
		<li>Light panel setup: Use the same setup used in the light/dark assay. Ensure that the light intensity is the same as used in the light/dark assay.</li>
	</ol>
	</li>
	<li>Behavioral test procedure
	<ol>
		<li>Turn the apparatus on. Set the light intensity to 27,000 lux.</li>
		<li>Open the tracking software.</li>
		<li>Set up a new protocol, the same as is used in the light/dark assay except for the New Zone settings. Choose 1 followed by the Center in the New Zone settings. Set the periphery as 3.97 cm from the perimeter and the center as 19.05 &#215; 19.05 cm.</li>
		<li>Habituate mice to the testing room as described in step 1.2.4.</li>
		<li>Administer CGRP (0.1 mg/kg, 10 &#181;l/g based on mouse body weight, i.p.), tilting the mouse&#8217;s head forward and injecting in the lower right quadrant. Return the mouse to the home cage.</li>
		<li>After 30 min, start the protocol. Pull the pull-out drawer outside of the sound-attenuating cubicle and&#160;gently&#160;place the mouse&#160;in the middle of the open field chamber. Push the drawer inside of the cubicle.</li>
		<li>Track behavior for 30 min. Then return mice to their home cages.</li>
		<li>Clean the apparatus as described in step 1.2.8.</li>
	</ol>
	</li>
</ol>
3. Modified light/dark assay for optogenetic mice
<ol>
	<li>The apparatus setup
	<ol>
		<li>Make two modifications to the dark insert.
		<ol>
			<li>Modify the opening of the dark insert to 5.08 x 5.08 cm (W x H) with a small slit 0.95 x 10.16 cm (W X H) between the top and the opening of the dark insert (Figure 1D top left). 
			NOTE: This modification allows a mouse to go to the dark zone without difficulty when the fiber-optic cannula on the mouse head is attached to the patch cord.</li>
			<li>Extend the top of the dark insert over the light area as a triangular porch (H=6.5 cm) (Figure 1D top right and bottom left). Cut a circular hole (D=1.7 cm) out of the porch and insert a holder into the hole to place and stabilize the rotary joint, which connects the laser and the fiber-optic patch cords (Figure 1D top left and bottom left). 
			NOTE: The modifications result in small change in the light intensity reaching the floor of the dark zone (17 lux with modifications vs 14 lux without modifications, measured on the back-right corner of the dark zone under 27,000 lux).</li>
		</ol>
		</li>
		<li>Insert the rotary joint into the holder on the dark insert.</li>
		<li>Connect the 30.5 cm fiber-optic patch cord to the rotary joint. Confirm that the rotary joint can rotate smoothly so that the patch cord can rotate without difficulty as the mouse traverses the chamber.</li>
		<li>For the rest of the setup, use the same apparatus setup as used in section 1 (light/dark assay).</li>
	</ol>
	</li>
	<li>Behavioral test procedure 
	NOTE: Unlike the wildtype mice, the optogenetic mice do not receive pre-exposures (pretreatment 1 and 2).
	<ol>
		<li>On the test day, insert the LED light panel into the second lowest slot (28.23 cm from the floor of the camber) to allow space for connecting the patch cord. Turn the light/dark assay apparatus on and set the light intensity to 55 lux.</li>
		<li>Use the same protocol setup as that in 1.2.2 and 1.2.3 except that Data Bins By Duration is set to 60 s in the New Analysis setting to be congruent with the laser stimulation protocol in Step 3.2.3.</li>
		<li>Turn the laser power button on. Set the laser pulse controller to stimulate for 1 min followed by 1 min without stimulation over 30 min.</li>
		<li>Habituate mice to the testing room with the light on for 1 h prior to the testing.</li>
		<li>Start the protocol. Pull the pull-out drawer outside of the sound-attenuating cubicle to access the light/dark chamber and the dark insert.</li>
		<li>Gently restrain the mouse and couple the optic-fiber cannula on the mouse head to the fiber-optic patch cord via a mating sleeve (Figure 1D bottom right). Place the mouse gently in the light zone and push the drawer inside of the cubicle. Make sure that the protocol will begin to record mouse behavior automatically.</li>
		<li>At 1 min, switch on the pulse controller and then turn the failsafe key to ON. Make sure laser stimulation of the targeted brain region is occurring every other minute.</li>
		<li>After 30 min when the protocol stops automatically, turn the failsafe key to OFF. Then turn the pulse controller off.</li>
		<li>Uncouple the mouse and the fiber-optic patch cord. Return the mouse to the home cage.</li>
		<li>Clean the chamber and dark insert as described in step 1.2.8.</li>
	</ol>
	</li>
</ol>
4. Modified open field assay for optogenetic mice
<ol>
	<li>The apparatus setup
	<ol>
		<li>Stabilize the rotary joint above the chamber using a stand and a clamp (Figure 1E).</li>
		<li>Connect the fiber-optic patch cord with a length of 50 cm to the rotary joint. Check if the rotary joint can rotate smoothly.</li>
		<li>Set the rotary joint to the appropriate height on the stand: ensure that the fiber-optic patch cord can only just reach every corner of the chamber, which will help avoid any interference with mouse movement.</li>
		<li>For the rest of the setup, use the same apparatus setup as used in section 1 (light/dark assay), but without the dark insert.</li>
	</ol>
	</li>
	<li>Behavioral test procedure
	<ol>
		<li>Turn the light/dark assay apparatus on and set the light intensity to 55 lux.</li>
		<li>Use the same protocol setup as that in the modified light/dark assay (section 3) except for the New Zone settings. Choose 1 following by Center in New Zone settings. Set the periphery as 3.97 cm from the perimeter and the center as 19.05 &#215; 19.05 cm.</li>
		<li>Turn the laser power button on. Set laser pulse controller to stimulate for 1 min followed by 1 min without stimulation over 30 min.</li>
		<li>Perform habituation and the rest of the test as described in steps 3.2.4 to 3.2.10 except for two changes to step 3.2.6: place the mouse gently in the middle of the chamber instead of the light zone; keep the pull-out drawer outside of the cubicle due to the patch cord connecting to the mouse&#39;s head.</li>
	</ol>
	</li>
</ol>

<ol><li>Loder, S., Sheikh, H. U., Loder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+prevalence%2C+burden%2C+and+treatment+of+severe%2C+frequent%2C+and+migraine+headaches+in+US+minority+populations%3A+statistics+from+National+Survey+studies%5BTitle%5D)+AND+%22Headache%22%5BJournal%5D)">The prevalence, burden, and treatment of severe, frequent, and migraine headaches in US minority populations: statistics from National Survey studies.</a> Headache. 55 (2), 214-228 (2015).</li>
<li>Collaborators, G. B. D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global%2C+regional%2C+and+national+burden+of+migraine+and+tension-type+headache%2C+1990-2016%3A+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2016%5BTitle%5D)+AND+%22Lancet+Neurology%22%5BJournal%5D)">Global, regional, and national burden of migraine and tension-type headache, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016.</a> Lancet Neurology. 17 (11), 954-976 (2018).</li>
<li>GBD 2017 Disease and Injury Incidence and Prevalence Collaborators. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global%2C+regional%2C+and+national+incidence%2C+prevalence%2C+and+years+lived+with+disability+for+354+diseases+and+injuries+for+195+countries+and+territories%2C+1990-2017%3A+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2017%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017.</a> Lancet. 392 (10159), 1789-1858 (2018).</li>
<li>international headache society. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Headache+classification+committee+of+the+international+headache+society+%28IHS%29.+The+international+classification+of+headache+disorders%2C+3rd+edition%5BTitle%5D)+AND+%22Cephalalgia%22%5BJournal%5D)">Headache classification committee of the international headache society (IHS). The international classification of headache disorders, 3rd edition.</a> Cephalalgia. 38 (1), 1(2018).</li>
<li>Edvinsson, L., Haanes, K. A., Warfvinge, K., Krause, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CGRP+as+the+target+of+new+migraine+therapies+-+successful+translation+from+bench+to+clinic%5BTitle%5D)+AND+%22Nature+Reviews+Neurology%22%5BJournal%5D)">CGRP as the target of new migraine therapies - successful translation from bench to clinic.</a> Nature Reviews Neurology. 14 (6), 338-350 (2018).</li>
<li>Rapoport, A. M., McAllister, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+headache+pipeline%3A+Excitement+and+uncertainty%5BTitle%5D)+AND+%22Headache%22%5BJournal%5D)">The headache pipeline: Excitement and uncertainty.</a> Headache. 60 (1), 190-199 (2020).</li>
<li>Maasumi, K., Michael, R. L., Rapoport, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CGRP+and+Migraine%3A+The+role+of+blocking+calcitonin+gene-related+peptide+ligand+and+receptor+in+the+management+of+Migraine%5BTitle%5D)+AND+%22Drugs%22%5BJournal%5D)">CGRP and Migraine: The role of blocking calcitonin gene-related peptide ligand and receptor in the management of Migraine.</a> Drugs. 78 (9), 913-928 (2018).</li>
<li>Caronna, E., Starling, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Update+on+calcitonin+gene-related+peptide+antagonism+in+the+treatment+of+migraine%5BTitle%5D)+AND+%22Neurologic+Clinics%22%5BJournal%5D)">Update on calcitonin gene-related peptide antagonism in the treatment of migraine.</a> Neurologic Clinics. 39 (1), 1-19 (2021).</li>
<li>Eftekhari, S., Edvinsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Possible+sites+of+action+of+the+new+calcitonin+gene-related+peptide+receptor+antagonists%5BTitle%5D)+AND+%22Therapeutic+Advances+in+Neurological+Disorders%22%5BJournal%5D)">Possible sites of action of the new calcitonin gene-related peptide receptor antagonists.</a> Therapeutic Advances in Neurological Disorders. 3 (6), 369-378 (2010).</li>
<li>Mason, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+migraine-like+photophobic+behavior+in+mice+by+both+peripheral+and+central+cgrp+mechanisms%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Induction of migraine-like photophobic behavior in mice by both peripheral and central cgrp mechanisms.</a> Journal of Neuroscience. 37 (1), 204-216 (2017).</li>
<li>Russell, M. B., Rasmussen, B. K., Fenger, K., Olesen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Migraine+without+aura+and+migraine+with+aura+are+distinct+clinical+entities%3A+A+study+of+four+hundred+and+eighty-four+male+and+female+migraineurs+from+the+general+population%5BTitle%5D)+AND+%22Cephalalgia%22%5BJournal%5D)">Migraine without aura and migraine with aura are distinct clinical entities: A study of four hundred and eighty-four male and female migraineurs from the general population.</a> Cephalalgia. 16 (4), 239-245 (1996).</li>
<li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exploratory+behavior+models+of+anxiety+in+mice%5BTitle%5D)+AND+%22Neuroscience+and+Biobehavioral+Reviews%22%5BJournal%5D)">Exploratory behavior models of anxiety in mice.</a> Neuroscience and Biobehavioral Reviews. 9 (1), 37-44 (1985).</li>
<li>Crawley, J., Goodwin, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preliminary+report+of+a+simple+animal+behavior+model+for+the+anxiolytic+effects+of+benzodiazepines%5BTitle%5D)+AND+%22Pharmacology%2C+Biochemistry+and+Behavior%22%5BJournal%5D)">Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines.</a> Pharmacology, Biochemistry and Behavior. 13 (2), 167-170 (1980).</li>
<li>Recober, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+calcitonin+gene-related+peptide+in+light-aversive+behavior%3A+implications+for+migraine%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Role of calcitonin gene-related peptide in light-aversive behavior: implications for migraine.</a> Journal of Neuroscience. 29 (27), 8798-8804 (2009).</li>
<li>Recober, A., Kaiser, E. A., Kuburas, A., Russo, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+multiple+photophobic+behaviors+in+a+transgenic+mouse+sensitized+to+CGRP%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">Induction of multiple photophobic behaviors in a transgenic mouse sensitized to CGRP.</a> Neuropharmacology. 58 (1), 156-165 (2010).</li>
<li>Kaiser, E. A., Kuburas, A., Recober, A., Russo, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+CGRP-induced+light+aversion+in+wild-type+mice+by+a+5-HT%281B%2FD%29+agonist%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Modulation of CGRP-induced light aversion in wild-type mice by a 5-HT(1B/D) agonist.</a> Journal of Neuroscience. 32 (44), 15439-15449 (2012).</li>
<li>Kuburas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PACAP+induces+light+aversion+in+mice+by+an+inheritable+mechanism+independent+of+CGRP%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">PACAP induces light aversion in mice by an inheritable mechanism independent of CGRP.</a> Journal of Neuroscience. , (2021).</li>
<li>Russo, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcitonin+gene-related+peptide+%28CGRP%29%3A+a+new+target+for+migraine%5BTitle%5D)+AND+%22Annual+Review+of+Pharmacology+and+Toxicology%22%5BJournal%5D)">Calcitonin gene-related peptide (CGRP): a new target for migraine.</a> Annual Review of Pharmacology and Toxicology. 55, 533-552 (2015).</li>
<li>Sowers, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stimulation+of+Posterior+Thalamic+Nuclei+Induces+Photophobic+Behavior+in+Mice%5BTitle%5D)+AND+%22Headache%22%5BJournal%5D)">Stimulation of Posterior Thalamic Nuclei Induces Photophobic Behavior in Mice.</a> Headache. 60 (9), 1961-1981 (2020).</li>
<li>Afridi, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+positron+emission+tomographic+study+in+spontaneous+migraine%5BTitle%5D)+AND+%22Archives+of+Neurology%22%5BJournal%5D)">A positron emission tomographic study in spontaneous migraine.</a> Archives of Neurology. 62 (8), 1270-1275 (2005).</li>
<li>Mason, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascular+actions+of+peripheral+CGRP+in+migraine-like+photophobia+in+mice%5BTitle%5D)+AND+%22Cephalalgia%22%5BJournal%5D)">Vascular actions of peripheral CGRP in migraine-like photophobia in mice.</a> Cephalalgia. 40 (14), 1585-1604 (2020).</li>
<li>Guo, S., Vollesen, A. L. H., Olesen, J., Ashina, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Premonitory+and+nonheadache+symptoms+induced+by+CGRP+and+PACAP38+in+patients+with+migraine%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Premonitory and nonheadache symptoms induced by CGRP and PACAP38 in patients with migraine.</a> Pain. 157 (12), 2773-2781 (2016).</li>
<li>Christensen, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Migraine+induction+with+calcitonin+gene-related+peptide+in+patients+from+erenumab+trials%5BTitle%5D)+AND+%22Journal+of+Headache+and+Pain%22%5BJournal%5D)">Migraine induction with calcitonin gene-related peptide in patients from erenumab trials.</a> Journal of Headache and Pain. 19 (1), 105(2018).</li>
<li>Younis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Investigation+of+distinct+molecular+pathways+in+migraine+induction+using+calcitonin+gene-related+peptide+and+sildenafil%5BTitle%5D)+AND+%22Cephalalgia%22%5BJournal%5D)">Investigation of distinct molecular pathways in migraine induction using calcitonin gene-related peptide and sildenafil.</a> Cephalalgia. 39 (14), 1776-1788 (2019).</li>
<li>Asghar, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+a+vascular+factor+in+migraine%5BTitle%5D)+AND+%22Annals+of+Neurology%22%5BJournal%5D)">Evidence for a vascular factor in migraine.</a> Annals of Neurology. 69 (4), 635-645 (2011).</li>
<li>Ueno, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+repetitive+gentle+handling+of+male+C57BL%2F6NCrl+mice+on+comparative+behavioural+test+results%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Effects of repetitive gentle handling of male C57BL/6NCrl mice on comparative behavioural test results.</a> Scientific Reports. 10 (1), 3509(2020).</li>
<li>Campos, A. C., Fogaca, M. V., Aguiar, D. C., Guimaraes, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+anxiety+disorders+and+stress%5BTitle%5D)+AND+%22Revista+Brasileira+De+Psiquiatria%22%5BJournal%5D)">Animal models of anxiety disorders and stress.</a> Revista Brasileira De Psiquiatria. 35, 101-111 (2013).</li>
<li>Kuburas, A., Thompson, S., Artemyev, N. O., Kardon, R. H., Russo, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photophobia+and+abnormally+sustained+pupil+responses+in+a+mouse+model+of+bradyopsia%5BTitle%5D)+AND+%22Investigative+Ophthalmology+and+Visual+Science%22%5BJournal%5D)">Photophobia and abnormally sustained pupil responses in a mouse model of bradyopsia.</a> Investigative Ophthalmology and Visual Science. 55 (10), 6878-6885 (2014).</li>
<li>Goadsby, P. J., Edvinsson, L., Ekman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vasoactive+peptide+release+in+the+extracerebral+circulation+of+humans+during+migraine+headache%5BTitle%5D)+AND+%22Annals+of+Neurology%22%5BJournal%5D)">Vasoactive peptide release in the extracerebral circulation of humans during migraine headache.</a> Annals of Neurology. 28 (2), 183-187 (1990).</li>
<li>Lassen, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CGRP+may+play+a+causative+role+in+migraine%5BTitle%5D)+AND+%22Cephalalgia%22%5BJournal%5D)">CGRP may play a causative role in migraine.</a> Cephalalgia. 22 (1), 54-61 (2002).</li>
<li>Cernuda-Morollon, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interictal+increase+of+CGRP+levels+in+peripheral+blood+as+a+biomarker+for+chronic+migraine%5BTitle%5D)+AND+%22Neurology%22%5BJournal%5D)">Interictal increase of CGRP levels in peripheral blood as a biomarker for chronic migraine.</a> Neurology. 81 (14), 1191-1196 (2013).</li>
<li>Chanda, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+evidence+for+photophobia+and+stress-related+ipsilateral+head+pain+in+transgenic+Cacna1a+mutant+mice%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Behavioral evidence for photophobia and stress-related ipsilateral head pain in transgenic Cacna1a mutant mice.</a> Pain. 154 (8), 1254-1262 (2013).</li>
<li>Mahmoudi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cerebrolysin+attenuates+hyperalgesia%2C+photophobia%2C+and+neuroinflammation+in+a+nitroglycerin-induced+migraine+model+in+rats%5BTitle%5D)+AND+%22Brain+Research+Bulletin%22%5BJournal%5D)">Cerebrolysin attenuates hyperalgesia, photophobia, and neuroinflammation in a nitroglycerin-induced migraine model in rats.</a> Brain Research Bulletin. 140, 197-204 (2018).</li>
<li>Farajdokht, F., Babri, S., Karimi, P., Mohaddes, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ghrelin+attenuates+hyperalgesia+and+light+aversion-induced+by+nitroglycerin+in+male+rats%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">Ghrelin attenuates hyperalgesia and light aversion-induced by nitroglycerin in male rats.</a> Neuroscience Letters. 630, 30-37 (2016).</li>
<li>Jacob, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endocannabinoids+render+exploratory+behaviour+largely+independent+of+the+test+aversiveness%3A+Role+of+glutamatergic+transmission%5BTitle%5D)+AND+%22Genes%2C+Brain%2C+and+Behavior%22%5BJournal%5D)">Endocannabinoids render exploratory behaviour largely independent of the test aversiveness: Role of glutamatergic transmission.</a> Genes, Brain, and Behavior. 8 (7), 685-698 (2009).</li>
<li>Thiels, E., Hoffman, E. K., Gorin, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+reliable+behavioral+assay+for+the+assessment+of+sustained+photophobia+in+mice%5BTitle%5D)+AND+%22Current+Eye+Research%22%5BJournal%5D)">A reliable behavioral assay for the assessment of sustained photophobia in mice.</a> Current Eye Research. 33 (5), 483-491 (2008).</li>
<li>Ramachandran, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+Toll-like+receptor+4+signaling+in+mast+cell-mediated+migraine+pain+pathway%5BTitle%5D)+AND+%22Molecular+Pain%22%5BJournal%5D)">Role of Toll-like receptor 4 signaling in mast cell-mediated migraine pain pathway.</a> Molecular Pain. 15, 1744806919867842(2019).</li>
<li>Marek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implication+of+Melanopsin+and+Trigeminal+Neural+Pathways+in+Blue+Light+Photosensitivity+in+vivo%5BTitle%5D)+AND+%22Frontiers+in+Neuroscience%22%5BJournal%5D)">Implication of Melanopsin and Trigeminal Neural Pathways in Blue Light Photosensitivity in vivo.</a> Frontiers in Neuroscience. 13, 497(2019).</li>
<li>Christensen, S. L. T., Petersen, S., Sorensen, D. B., Olesena, J., Jansen-Olesen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infusion+of+low+dose+glyceryl+trinitrate+has+no+consistent+effect+on+burrowing+behavior%2C+running+wheel+activity+and+light+sensitivity+in+female+rats%5BTitle%5D)+AND+%22Journal+of+Pharmacological+and+Toxicological+Methods%22%5BJournal%5D)">Infusion of low dose glyceryl trinitrate has no consistent effect on burrowing behavior, running wheel activity and light sensitivity in female rats.</a> Journal of Pharmacological and Toxicological Methods. 80, 43-50 (2016).</li>
<li>De Vera Mudry, M. C., Kronenberg, S., Komatsu, S., Aguirre, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blinded+by+the+light%3A+retinal+phototoxicity+in+the+context+of+safety+studies%5BTitle%5D)+AND+%22Toxicologic+Pathology%22%5BJournal%5D)">Blinded by the light: retinal phototoxicity in the context of safety studies.</a> Toxicologic Pathology. 41 (6), 813-825 (2013).</li>
<li>White, D. A., Fritz, J. J., Hauswirth, W. W., Kaushal, S., Lewin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+sensitivity+to+light-induced+damage+in+a+mouse+model+of+autosomal+dominant+retinal+disease%5BTitle%5D)+AND+%22Investigative+Ophthalmology+and+Visual+Science%22%5BJournal%5D)">Increased sensitivity to light-induced damage in a mouse model of autosomal dominant retinal disease.</a> Investigative Ophthalmology and Visual Science. 48 (5), 1942-1951 (2007).</li>
<li>Song, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinal+pre-conditioning+by+CD59a+knockout+protects+against+light-induced+photoreceptor+degeneration%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Retinal pre-conditioning by CD59a knockout protects against light-induced photoreceptor degeneration.</a> PloS One. 11 (11), 0166348(2016).</li>
<li>Matynia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Light+aversion+and+corneal+mechanical+sensitivity+are+altered+by+intrinscally+photosensitive+retinal+ganglion+cells+in+a+mouse+model+of+corneal+surface+damage%5BTitle%5D)+AND+%22Experimental+Eye+Research%22%5BJournal%5D)">Light aversion and corneal mechanical sensitivity are altered by intrinscally photosensitive retinal ganglion cells in a mouse model of corneal surface damage.</a> Experimental Eye Research. 137, 57-62 (2015).</li>
</ol>

The authors have no conflicts of interest to report.

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.

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investigating migraine-like behavior using light aversion in mice

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 naïve 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/6J mic...

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 ...

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JoVE Journal

Behavior

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Light/dark Transition Test for Mice

The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Elevated Plus Maze for Mice

Source: Zhang, L., et al. Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium. J. Vis. Exp. (2015)This video demonstrates the process of applying NIr light to a shaved mouse scalp to activate vestibular neurons and stimulate superoxide dismutase-1 (SOD-1) production, supporting neuronal function. The procedure highlights NIr therapy as a neuroprotective strategy.

Modulating Sensory Neuronal Signaling in a Mouse Model Using Near Infrared Light

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Here we present two easy-to-follow step-by-step protocols for place preference paradigms using optogenetics in mice. Using these two different setups, preference and avoidance behaviors can be solidly assessed within the same apparatus with high spatial and temporal selectivity, and in a straightforward manner.

Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

The most notable symptom of migraine is severe head pain, and it is hypothesized that this is mediated by sensory neurons innervating the meninges. Here, we present a method to locally apply substances to the dura in a minimally invasive manner while using facial hypersensitivity as an output.

Dural Stimulation and Periorbital von Frey Testing in Mice As a Preclinical Model of Headache

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

This protocol provides a method for tracking automated eye squint in rodents over time in a manner compatible with time-locking to neurophysiological measures. This protocol is expected to be useful to researchers studying mechanisms of pain disorders such as migraine.

An Automated Squint Method for Time-syncing Behavior and Brain Dynamics in Mouse Pain Studies

Learn a light/dark assay method to assess light aversion in mice as a preclinical model for migraine photophobia. This technique distinguishes light aversion from anxiety using automatic detection.

Investigating Migraine-Like Behavior Using Light Aversion in Mice

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