Investigating Migraine-Like Behavior Using Light Aversion in Mice

Summary

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.

Abstract

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.

Introduction

Migraine is a prevalent neurological disease, affecting approximately 17% of Americans¹ and is the second leading cause of disability globally^2,3. 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 treatment^5,6,7. 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 peripheral^10,16 (Figure 2) and central^14,15,16,19 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.

Protocol

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

1. Light/dark chamber apparatus (see Table of Materials) setup. All the equipment in this section is commercially available.
   1. 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.
   2. Connect the DC power supply and a DC-regulated power supply to the sound-attenuating cubicle.
   3. 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.
   4. 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.
   5. 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.
   6. Connect the IR USB controller to a computer.
   7. Install the tracking software on the computer which can record and collect mouse location and activity.
   8. 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° flood beam spread) from its original housing.
   9. 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.
   10. 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.
   11. 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).
   12. 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.
2. 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, naïve 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 2^nd pre-exposure and can be tested in dim light.
   1. On day 1 (pretreatment 1), turn the light/dark assay apparatus on and set the light intensity to 27,000 lux.
   2. 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.
   3. 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.
   4. 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'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.
   5. Select Acquire Data. Enter mouse IDs. Start the protocol.
   6. 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 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.
   7. Wait for the recording to automatically stop after 30 min. Return the mouse to its home cage.
   8. 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.
   9. On day 4 (pretreatment 2), repeat steps 1.2.1 to 1.2.8.
   10. 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 µl/g based on mouse body weight, intraperitoneal injection (i.p.)), tilting the mouse’s head forward and injecting in the lower right quadrant. Return the mouse to the home cage.
   11. After 30 min, start the protocol and run the mouse in the light/dark chamber 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.
   12. Clean the chamber and dark insert as described in step 1.2.8.
   13. 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 before starting the open field assay.

2. Open field assay

1. The apparatus setup
   1. 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.
   2. 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.
2. Behavioral test procedure
   1. Turn the apparatus on. Set the light intensity to 27,000 lux.
   2. Open the tracking software.
   3. 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 × 19.05 cm.
   4. Habituate mice to the testing room as described in step 1.2.4.
   5. Administer CGRP (0.1 mg/kg, 10 µl/g based on mouse body weight, i.p.), tilting the mouse’s head forward and injecting in the lower right quadrant. Return the mouse to the home cage.
   6. After 30 min, start the protocol. Pull the pull-out drawer outside of the sound-attenuating cubicle and gently place the mouse in the middle of the open field chamber. Push the drawer inside of the cubicle.
   7. Track behavior for 30 min. Then return mice to their home cages.
   8. Clean the apparatus as described in step 1.2.8.

3. Modified light/dark assay for optogenetic mice

1. The apparatus setup
   1. Make two modifications to the dark insert.
      1. 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.
      2. 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).
   2. Insert the rotary joint into the holder on the dark insert.
   3. 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.
   4. For the rest of the setup, use the same apparatus setup as used in section 1 (light/dark assay).
2. Behavioral test procedure
   NOTE: Unlike the wildtype mice, the optogenetic mice do not receive pre-exposures (pretreatment 1 and 2).
   1. 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.
   2. 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.
   3. 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.
   4. Habituate mice to the testing room with the light on for 1 h prior to the testing.
   5. Start the protocol. Pull the pull-out drawer outside of the sound-attenuating cubicle to access the light/dark chamber and the dark insert.
   6. 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.
   7. 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.
   8. After 30 min when the protocol stops automatically, turn the failsafe key to OFF. Then turn the pulse controller off.
   9. Uncouple the mouse and the fiber-optic patch cord. Return the mouse to the home cage.
   10. Clean the chamber and dark insert as described in step 1.2.8.

4. Modified open field assay for optogenetic mice

1. The apparatus setup
   1. Stabilize the rotary joint above the chamber using a stand and a clamp (Figure 1E).
   2. Connect the fiber-optic patch cord with a length of 50 cm to the rotary joint. Check if the rotary joint can rotate smoothly.
   3. 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.
   4. For the rest of the setup, use the same apparatus setup as used in section 1 (light/dark assay), but without the dark insert.
2. Behavioral test procedure
   1. Turn the light/dark assay apparatus on and set the light intensity to 55 lux.
   2. 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 × 19.05 cm.
   3. Turn the laser power button on. Set laser pulse controller to stimulate for 1 min followed by 1 min without stimulation over 30 min.
   4. 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's head.

Results

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 mice^10,16 and optical stimulation of neurons in the posterior thalamic nuclei in C57BL/6...

Discussion

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...

Materials

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