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>

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.

<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 ...

investigating-migraine-like-behavior-using-light-aversion-in-mice

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Behavior

3.6K Views.  University of Iowa. 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. 

Video: Investigating Migraine-Like Behavior Using Light Aversion in Mice

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, sex, condition, and genetic background of the emitting animal. Although many studies have characterized these differences, whether receiver mice can discriminate among objectively different USVs and show preferences for particular sound traits remains to be elucidated. To determine whether mice can discriminate between different characteristics of USVs, a playback experiment was developed recently, in which preference responses of mice to two different USVs could be evaluated in the form of a place preference.
First, USVs from mice were recorded. Then, the recorded USVs were edited, trimmed accordingly, and exported as stereophonic sound files. Next, the USV amplitudes generated by the two ultrasound emitters used in the experiment were adjusted to the same sound pressure level. Nanocrystalline silicon thermo-acoustic emitters were used to play the USVs back. Finally, to investigate the preference of subject mice to selected USVs, pairs of two differing USV signals were played back simultaneously in a two-choice test box. By repeatedly entering a defined zone near an ultrasound emitter and searching the wire mesh in front of the emitter, the mouse reveals its preference for one sound over another. This model allows comparing the attractiveness of the various features of mouse USVs, in various contexts.

Ultrasonic vocalizations (USVs) in mice differ depending on age, sex, condition, and genetic background. Using two ultrasound emitters broadcasting simultaneously in different locations, this two-choice test can evaluate murine recognition and preference responses to different characteristics of USVs.

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

A Method for Investigating Change Blindness in Pigeons (Columba Livia)

Change blindness is a phenomenon of visual attention, whereby changes to a visual display go unnoticed under certain specific circumstances. While many laboratory procedures have been developed that produce change blindness in humans, the flicker paradigm has emerged as a particularly effective method. In the flicker paradigm, two visual displays are presented in alternation with one another. If successive displays are separated by a short inter-stimulus interval (ISI), change detection is impaired. The simplicity of the procedure and the clear, performance-based operational definition of change blindness make the flicker paradigm well-suited to comparative research using nonhuman animals. Indeed, a variant has been developed that can be implemented in operant chambers to study change blindness in pigeons. Results indicate that pigeons, like humans, are worse at detecting the location of a change if two consecutive displays are separated in time by a blank ISI. Furthermore, pigeons&#39; change detection is consistent with an active, location-by-location search process that requires selective attention. The flicker task thus has the potential to contribute to investigations of the dynamics of pigeons&#39; selective spatial attention in comparison to humans. It also illustrates that the phenomenon of change blindness is not exclusive to humans&#39; visual perception, but may instead be a general consequence of selective attention. Finally, while the useful aspects of attention are widely appreciated and understood, it is also important to acknowledge that they may be accompanied by specific imperfections such as change blindness, and that these imperfections have consequences across a wide range of contexts.

A Method for Investigating Change Blindness in Pigeons Columba Livia

Change blindness is a phenomenon of visual attention, whereby changes to a visual display go unnoticed under certain specific circumstances. This protocol describes a variation on the flicker paradigm for investigating change blindness that is appropriate and effective for research with pigeons.

Change blindness is a phenomenon of visual attention, whereby changes to a visual display go unnoticed under certain specific circumstances. While many ...

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...