This research was supported by the Chungnam National University and the National Research Foundation of Korea (NRF) grant funded by the government of Korea (NRF-2019R1A6A3A01093963 and NRF-2021R1F1A1062509).

All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Chungnam National University in South Korea, and then conducted based on the ethical guidelines of the International Association for the Study of Pain18.
1. Induction of scalding burn injury on the hind paw
<ol>
	<li>House the male ICR mice weighing 20-25 g in a light and temperature-controlled room (12/12 h light-dark cycle, 22.5 &#176;C &#177; 2.5 &#176;C) with a humidity of 40%-60%. 
	NOTE: Both male and female mice can be used for this protocol.</li>
	<li>Allow the animal free access to food and water, and acclimatize for at least 1 week before starting the experiment. 
	NOTE: All the animals were group-housed to exclude variables such as isolation stress.</li>
	<li>Assign the mice randomly to the experimental or control group and conduct blind experiments using animal numbers as codes.</li>
	<li>On the day of burn induction, anesthetize the mouse by intraperitoneal (i.p.) injection of 300 &#181;L of alfaxalone at a dose of 100 mg/kg. Wear a surgical gown, gloves, and mask while performing the burn induction.</li>
	<li>After deeply anesthetizing the mouse, disinfect around the right hind paw with 70% ethanol. 
	NOTE: Check the lack of response to pinch stimulation applied to the hind toes or tail to confirm the state of deep anesthesia.</li>
	<li>Apply an ophthalmic ointment to the eyes to prevent corneal drying after induction of anesthesia.</li>
	<li>Immerse the right hind paw of the deeply anesthetized mouse in hot water at 65 &#176;C &#177; 0.5 &#176;C for 3 s. Make a mark on the ankle of each mouse before immersing the hind paw in hot water to maintain consistency in the burned area.</li>
	<li>After induction of burns, bring the mice in a clean home cage and place them on a heating pad until the animals recover from anesthesia. 
	&#8203;NOTE: The analgesic agent, acetaminophen (200 mg/kg), was administered intraperitoneally once daily for 7 days starting from the day of burn injury (Only Burn + Acetaminophen group). The Burn group was treated with saline as vehicle control. The experiment was performed according to the method described in a previous study4.</li>
</ol>
2. Measurement of mechanical allodynia
<ol>
	<li>Bring the mice to the behavioral testing room and let them acclimatize at least for 30 min prior to the test. Wear a surgical gown, gloves, and mask while performing the test.</li>
	<li>Place the mice into a square box (diameter: 13 cm, height: 12 cm) on a metal mesh floor (mesh size: 0.7 cm x 0.7 cm) and let them acclimatize for at least 30 min.</li>
	<li>Assess the mechanical threshold of the hind paw using the ascending stimulus method19,20.</li>
	<li>Gently poke a series of von Frey filaments with 5-8 s intervals to stimulate the hind plantar. Obtain the baseline values on the day before burn induction. 
	NOTE: The 0.16-1.2 g von Frey filaments were used in the test to measure the paw withdrawal threshold in all animals, respectively. The paw withdrawal response test was started with the lowest bending force of von Frey filament (0.16 g in this protocol). If there was no response, then a filament with the next bending force was applied.</li>
	<li>Perform five trials to evaluate mechanical thresholds for each ipsilateral (injured) hind paw. 
	NOTE: The bending force of von Frey filament that produces response more than three times of the five trials in each animal was expressed as paw withdrawal threshold (PWT, g). Mechanical thresholds were measured a day before and at 1, 3, 5, and 7 days after burn injury. Analgesic effect was assessed 1 h after administration of the acetaminophen in the animal.</li>
</ol>
3. Automated gait analysis
<ol>
	<li>Acclimate the mice in the gait analysis system once daily for 10-15 min from 5 days before the burn injury. Wear a surgical gown, gloves, and mask while performing gait analysis.</li>
	<li>On the day of the test, bring the mice to the behavioral test room and acclimatize them for at least 30 min before the test. 
	NOTE: Perform acclimation and gait analysis tests in a dark environment. Set the conditions of the program menu as follows.
	<ol>
		<li>After running the program, click on the Create New Experiment menu to designate the folder to save the data.</li>
		<li>After designation, set the maximum running time to 5 s and maximum allowed speed variations to 50%.</li>
		<li>Select a registered camera and set the walkway length to 30 cm in the Setup tab of the program.</li>
		<li>On the Acquire tab of the program menu, select Open Acquisition.</li>
		<li>Based on the status messages, click on the Snap Background button to acquire a background image of an empty walkway.</li>
	</ol>
	</li>
	<li>Click on the Start Acquisition button, and then place the mouse at the entrance of the left-right traversable walkway. The recording will automatically start following the free movement of the mouse. 
	NOTE: If the animal&#39;s gait has been successfully recorded and all footsteps have been detected, it will be marked as Compliant Run with a green icon. If the software does not detect any footsteps, a red icon is displayed, in which case it is recommended to perform the recording again. The authors recommend collecting and analyzing at least five successful compliant runs performed with similar running speeds.</li>
	<li>On the Acquire tab of the program menu, select Classify Runs. 
	NOTE: After selecting the data obtained from the successful compliance run above, go to the video analysis screen where the gait patterns of the mice were recorded.</li>
	<li>Select the run to be analyzed and click on the Auto Classify button.</li>
	<li>After performing automatic classification, remove nose, genital recognition, and the misrecognition of paws to junk data in each run, and then analyze the data. 
	&#8203;NOTE: All statistical parameters are automatically analyzed and saved in the program, and raw data values can be found in the experimenter&#39;s analysis menu. Automatic gait analysis was performed before and at 1, 3, 5, and 7 days after burn injury. The evaluation was performed 30 min after acetaminophen administration in the Burn + Acetaminophen group and 30 min after saline treatment in the Burn group. This experiment was performed according to the method described in the previous studies4,21,22.</li>
</ol>
4. Measurement of depression-like behavior
NOTE: Despair-based behavior, immobility time in the water was measured by the forced swimming test.
<ol>
	<li>Bring the mice to the behavioral test room and acclimatize them for at least 30 min before the test. Wear a surgical gown, gloves, and mask while performing the forced swimming test.</li>
	<li>Put the mouse into a clear plexiglass cylinder (10 cm x 25 cm) containing 15 cm of water (25 &#176;C &#177; 0.5 &#176;C) for 15 min.</li>
	<li>After 24 h, put the mouse into the cylinder of the same conditions and measure the immobility time. 
	NOTE: Immobility time was measured for 5 min of test time, and the time whenever mice stopped climbing or swimming and just floated to keep their head above the water surface was recorded. The forced swimming test was performed on day 7 after the burn injury. The evaluation was performed 1 h after acetaminophen administration in the Burn + Acetaminophen group, and 1 h after saline treatment in the Burn group. The experiment was performed according to the method described in previous studies23,24.</li>
</ol>
5. Measurement of normal motor function
NOTE: The rota-rod test was performed to differentiate the abnormal motor function after burn injury.
<ol>
	<li>Bring the mice to the behavioral test room and acclimatize them for at least 30 min before the test. Wear a surgical gown, gloves, and mask while performing the forced swimming test.</li>
	<li>Place the animals on a rolling cylindrical platform (5.7 cm wide; 3 cm diameter) suspended 16 cm above the bottom of the apparatus.</li>
	<li>Allow each animal to train once a day on a rota-rod for at least 5 days prior to induction of burn injury.</li>
	<li>Perform the rota-rod test every 20 min for 2 h after drug administration. Set the cut-off time to 2 min.</li>
	<li>Measure the duration of time the mouse runs on a rotating rod at the constant speed of 15 revolutions per minute without falling. 
	NOTE: The rota-rod test was performed 7 days after the induction of burn injury. The evaluation was performed immediately after acetaminophen administration in the Burn + Acetaminophen group and after saline treatment in the Burn group. Alfaxalone was used as a positive control for experimentally treated drugs in this test. During the rotarod test, the duration of time the mouse runs on the rotating rod without falling is measured. The experiment was performed according to the method described in previous studies22,25.</li>
</ol>

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The authors have nothing to disclose.

The scalding burn is a kind of thermal burn that is caused by heated liquids. It has been suggested that first- or second-degree burns occur in most cases, but long-term contact with heat sources can cause third-degree burns26. In the present study, burn injury was induced by exposing the right hind paw of mice into hot water at 65 &#176;C for 3 s4,26. Tissue damage was detected in the burn-injured paw, which shows common symptoms of burns...

Tissue damage, such as burn and trauma, is generally associated with the co-occurrence of acute pain. Burn injuries and trauma-related symptoms are an estimated 1,80,000 deaths every year are caused by burns-the vast majority occur in low- and middle-income countries from different types of burns1. According to a worldwide report, burns are common in children and account for about 40%-60% of hospitalized patients2,3. These specific injuries are even more serious as they can occur in everyday life, such as boiling or bathing water4,5. Although acute pain can be resolved spontaneously after recovery from tissue damage in most cases, it may be possible to become chronic due to abnormal changes in the nervous system6,7.
Recently, it has been suggested that acute pain can induce a depressed mood, and chronic pain can cause anxiety and depression8,9,10,11. The coexistence of pain and depression makes it more difficult to treat the patient. Depression also tends to increase pain sensitivity, which is likely to induce more intense depression and pain12. Complications of pain and depression are shown in animal models of peripheral inflammation13,14,15,16. The detailed mechanisms underlying pain-induced depression are not well known until now17. Thus, it is necessary to develop more effective treatments for burns to alleviate the side effects and symptoms.
Thus, the present study was designed to develop an animal model to study burn injury-induced acute pain and depression-like behavior in mice. For this, burn injury-related abnormal tactile sensitivity, altered gait pattern, and depression-like behavior were measured. In addition, this study attempts to validate the model using NSAIDs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>BD</td>
			<td>307809</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetaminophen BioXtra, &#8805;99.0%</td>
			<td>Sigma-Aldrich</td>
			<td>A7085-100G</td>
			<td>Positive control (The analgesic agent, acetaminophen (200 mg/kg) was administered intraperitoneally once-daily for 7 days starting from the day of after burn injury (Only Burn + Acetaminophen group. (von-Frey test, gait analysis, and forced swimming test: Used for drug-dependent behavioral testing after burn injury), (Rota-rod test: It was used to investigate the motor and functional impairments of the drug in animals after burn injury).</td>
		</tr>
		<tr>
			<td>Alfaxan multidose (Alfaxalone)</td>
			<td>JUROX Pty.Limited</td>
			<td></td>
			<td>In this experiment, this material used for animal anesthesia, and was used as a positive control for experimentally treated drugs in the rota-rod test.</td>
		</tr>
		<tr>
			<td>CatWalk automated gait analysis system</td>
			<td>Noldus</td>
			<td>CatWalk XT</td>
			<td>Gait analysis in freely walking rodents is used to study the changes in limb movement and positioning in models with sensory-motor dysfunction</td>
		</tr>
		<tr>
			<td>OPTISHIELD (Cyclosporin ophthalmic ointment)</td>
			<td>Ashish Life Science</td>
			<td></td>
			<td>In this experiment, this material was used for an ointment to prevent corneal drying after induction of anesthesia.</td>
		</tr>
		<tr>
			<td>Plexiglass cylinder</td>
			<td>SCITECH KOREA</td>
			<td>custom made products</td>
			<td>Used in forced swimming test</td>
		</tr>
		<tr>
			<td>Rota-rod system</td>
			<td>SCITECH KOREA</td>
			<td>Accelerating rota rod</td>
			<td>Used in the measurement of Normal Motor Function</td>
		</tr>
		<tr>
			<td>von Frey filaments</td>
			<td>North Coast Medical</td>
			<td>NC12775</td>
			<td>Used in the measurement of Mechanical Allodynia</td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>CHANGSHIN SCIENCE</td>
			<td>C-WBE</td>
			<td>Used in the burn injury induction</td>
		</tr>
	</tbody>
</table>

burn injury-induced pain and depression-like behavior in mice

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the high mortality and sequelae in low- and middle-income countries. Burns frequently induce intense spontaneous pain and persistent allodynia as well as life-threatening problem. More importantly, excessive pain is often accompanied by depression, which may significantly decrease the quality of life. This article shows how to develop an animal model for the study of burn-induced pain and depression-like behavior. After anesthesia, burn injury was induced by dipping one hind paw of the mouse into hot water (65 &#176;C &plusmn; 0.5 &#176;C) for 3 s. The von Frey test and automated gait analysis were performed every 2 days after burn injury. In addition, depression-like behavior was examined using the forced swimming test, and the rota-rod test was performed to differentiate the abnormal motor function after burn injury. The main purpose of this study is to describe the development of an animal model for the study of burn injury-induced pain and depression-like behavior in mice.

In order to minimize animal suffering and reduce the number of animals used per the Three Rs (Replacement, Reduction, and Refinement) guidelines, this study was designed with the minimum number of animals for the collection of significant data established through preliminary experiment. In this study, behavioral experiments were independently conducted twice as follows. The gait analysis, mechanical allodynia, and depression-like behavior tests were conducted with Control (n = 5), Burn (n = 7; vehicle control; saline), a...

Watch this Scientific Journal Video about Burn Injury-Induced Pain and Depression-Like Behavior in Mice at JoVE.com

Burn Injury-Induced Pain and Depression-Like Behavior in Mice

A transient scald injury (65 &#176;C &plusmn; 0.5 &#176;C, 3 s) of one hind paw decreases the threshold (g) to von Frey filament stimulation of the ipsilateral side and alters gait pattern. Besides, burn injury induces depression-like behavior in the forced swimming test.

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the ...

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3.0K Views.  Chungnam National University. A transient scald injury (65 °C ± 0.5 °C, 3 s) of one hind paw decreases the threshold (g) to von Frey filament stimulation of the ipsilateral side and alters gait pattern. Besides, burn injury induces depression-like behavior in the forced swimming test.

Video: Burn Injury-Induced Pain and Depression-Like Behavior in Mice

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Low-stress Route Learning Using the Lashley III Maze in Mice

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...

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

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system. For example, they protect the brain from the skull and anchor/organize the vascular and neuronal supply of the cortex. However, the meninges are also implicated in nervous system disorders such as migraine, where the pain experienced during a migraine is attributed to local sterile inflammation and subsequent activation of local nociceptive afferents. Of the layers in the meninges, the dura mater is of particular interest in the pathophysiology of migraines. It is highly vascularized, harbors local nociceptive neurons, and is home to a diverse array of resident cells such as immune cells. Subtle changes in the local meningeal microenvironment may lead to activation and sensitization of dural perivascular nociceptors, thus leading to migraine pain. Studies have sought to address how dural afferents become activated/sensitized by using either in vivo electrophysiology, imaging techniques, or behavioral models, but these commonly require very invasive surgeries. This protocol presents a method for comparatively non-invasive application of compounds on the dura mater in mice and a suitable method for measuring headache-like tactile sensitivity using periorbital von Frey testing following dural stimulation. This method maintains the integrity of the dura and skull and reduces confounding effects from invasive techniques by injecting substances through a 0.65 mm modified cannula at the junction of unfused sagittal and lambdoid sutures. This preclinical model will allow researchers to investigate a wide range of dural stimuli and their role in the pathological progression of migraine, such as nociceptor activation, immune cell activation, vascular changes, and pain behaviors, all while maintaining injury-free conditions to the skull and meninges.

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.

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system.  ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.