We would like to thank Connor J. Smith for his help in building the customized mouse heat chamber. We acknowledge the help of O&#39;Dowd lab members, Lisha Zeng and Andrew Salgado for standardizing the heating protocol during the early stages of the assay development. We also thank Danny Benavides and Kumar Perinbam for video recording parts of the experimental procedure for the manuscript. This work was supported by the NIH grant (NS083009) awarded to D.O.D.

All animal procedures were performed in accordance with the guidelines of Institutional Animal Care and Use Committee (IACUC) at University of California, Irvine.
1. Preparation for the heat-induced seizure assay
<ol>
	<li>Switch on the Power On button on the heat chamber, followed by the Heat On button.</li>
	<li>Set the temperature of the heat chamber at 50 &#176;C using the keypad on the digital temperature controller.</li>
	<li>Wait for a minimum of 1 h to preheat the chamber at 50 &#176;C before introducing the first mouse into the chamber. Preheating ensures uniform heating inside the chamber.</li>
	<li>Line the floor of the mouse heat chamber with cob bedding.</li>
	<li>Mount a video recording camera in front of the heat chamber for recording each heat-induced seizure assay trial.</li>
	<li>Line a 140 mm diameter Petri dish with thick layers of tissue paper and place it on ice to serve as a cooling pad. 
	&#8203;NOTE: At the end of the assay, individual mouse will be transferred on the prechilled cooling pad to help bring down their elevated body temperature.</li>
</ol>
2. Preparing the mouse for heat-induced seizure assay
<ol>
	<li>Select 10 adult mice (P30-P40), 5 that carry the epilepsy causing mutation and 5 of the wild-type litter mates for heat-induced seizure screening assay. 
	NOTE: Wild-type mice, not harboring any epilepsy causing mutation do not exhibit heat-induced seizures at temperatures below 44 &#176;C and serve as the control group.</li>
	<li>Weigh each mouse to be used for the screening assay and record its body weight. Only mice weighing 15 g or more should be used for the assay.</li>
	<li>Screen one mouse at a time in the mouse heat chamber.</li>
	<li>Briefly anesthetize the mouse for 10-15 s using a few drops of isoflurane at the bottom of a bell jar.</li>
	<li>Take the animal out of the bell jar and place it on a paper towel.</li>
	<li>Ensure that the mouse is completely anesthetized by checking that the mouse is unresponsive to a noxious toe pinch.</li>
	<li>Coat the metal tip of the rectal temperature probe with a lubricant (such as petroleum jelly) and gently insert it into the mouse at a depth of less than or equal to 2 cm.</li>
	<li>Secure the rectal probe to the mouse&#39;s tail with tape, so the probe does not come out during the assay. 
	NOTE: Alternatively, place the animal in a mouse restrainer cone and insert the rectal temperature probe. Secure it by taping to the tail.</li>
	<li>Ensure the rectal probe is connected to a multimeter that displays internal body temperature of the mouse.</li>
	<li>Place the animal in a fresh cage lined with cob bedding, i.e., the recovery cage.</li>
	<li>Start a timer and wait for 5 min. Observe the mouse until it has completely recovered from anesthesia and the mouse is active and grooming.
	<ol>
		<li>Simultaneously, monitor the core body temperature of the mouse till it stabilizes at 35-36 &#176;C.</li>
	</ol>
	</li>
	<li>At the end of 5 min, note the body temperature of the mouse. This is the initial body temperature at time &#34;0&#34; min. 
	NOTE: If the core body temperature of the mouse is below 35 &#176;C, wait for additional time for the animal to recover from anesthesia-induced hypothermia.</li>
	<li>Quickly, transfer the individual mouse into the preheated mouse chamber. This marks the START of the experiment trial. Only one mouse is screened at a given time.</li>
</ol>
3. Heat-induced seizure assay
<ol>
	<li>After gently placing the mouse on the floor of the pre-heated mouse heat chamber, close the plexiglass door&#160;and&#160;start the camera for video recording the experiment.</li>
	<li>Start the stopwatch. Record the body temperature of the mouse from the rectal thermometer at 1 min intervals for the duration of the experiment.</li>
	<li>At regular intervals, increase the temperature of the mouse heat chamber such that the body temperature of the mouse increases at a rate of 0.25-0.5 &#176;C /min. 
	NOTE: Rapid increases in body temperature can lead to heat stroke or death and should be avoided.</li>
	<li>Following this protocol, increase the temperature of the mouse heat chamber by 5 &#176;C every 10 min as shown in Figure 2A.</li>
	<li>At 9.5 min, set the temperature of the heat chamber to 55 &#176;C, to stabilize the temperature of the heat chamber to 55 &#176;C by the 10th min as shown on the digital temperature display.</li>
	<li>Similarly, increase the temperature to 60 &#176;C at 19.5 min to stabilize the temperature of the heat chamber to 60 &#176;C by the 20th min. Each seizure screening trial lasts for 30 min.</li>
	<li>If the mouse has a seizure (vocalizes, shows head nodding, forelimb clonus, hindlimb extension, falls on its side, or experiences generalized tonic/clonic convulsions), record the following information.
	<ol>
		<li>Record the body temperature of the mouse during the seizure (seizure threshold temperature) from the rectal temperature thermometer.</li>
		<li>Record the seizure behavior characteristics such as head nodding, forelimb clonus, hind limb extension, falling on the side, and/or generalized tonic/clonic seizures (GTCS) displayed by the mouse.</li>
	</ol>
	</li>
	<li>Quickly but gently pick up the mouse from the chamber and place it on the cooling pad prepared in step 1.6. 
	NOTE: If a mouse is experiencing Racine scale 5 seizures and exhibiting uncontrolled jumping, it can be difficult to pick up the animal from the heat chamber and transferred to the cooling pad outside. However, a typical heat-induced seizure lasts between 30 s to 60 s.Thus, the mouse should be taken out of the heat-chamber and put on the cooling pad within 60 s of start of heat-induced seizure episode.</li>
	<li>Wait for the mouse body temperature to come down to 36-37 &#176;C, before transferring it to a recovery cage. Only one mouse is placed in a recovery cage at a time. 
	NOTE: Do not mix mice that are yet to be used for heat-induced screening with the mouse that has already experienced the heat-induced seizure experiment trial.</li>
	<li>Gently and carefully, cut the tape between the mouse tail and rectal probe wire with a pair of scissors to remove the rectal probe from the mouse.</li>
	<li>Wipe clean the metal tip of the rectal probe with 70% alcohol and soft tissue wipes to keep it ready for the next trial.</li>
	<li>Continue to observe the mouse in the recovery cage until it resumes normal activity (walking, grooming, etc.), before returning the mouse to its home cage. This marks the END of the experiment trial for this mouse.</li>
	<li>Record the animal status after the assay-alive and recovered from the test session or dead. High intensity seizures involving uncontrolled jumping and generalized tonic/clonic seizures can sometimes result in the death of the mouse.</li>
	<li>If a mouse does not experience heat-induced seizures within the 30 min observation period or the body temperature of the mouse reaches 44 &#176;C, remove the mouse from the heat chamber and place on the cooling pad till the body temperature of the mouse returns to 36-37 &#176;C.</li>
	<li>Reset the temperature of the mouse heat chamber to 50 &#176;C and allow it to equilibrate till the display temperature on the digital temperature controller shows 50 &#176;C.</li>
	<li>Change cob bedding between individual mouse trials.</li>
	<li>Prepare the next mouse for screening trial as described in section 2 and repeat the steps from section 3.</li>
</ol>
4. Euthanizing the animals
<ol>
	<li>Although most animals recover post heat-induced seizures, in our experience, a few of the mice undergo SUDEP (Sudden Unexplained Death in EPilepsy) in their home cage within 24-48 hrs of heat-induced seizures. After concluding the screening on all the mice individually for heat-induced seizures following the 30 min trial, euthanize all the mice as per the institution&#39;s IACUC guidelines.</li>
</ol>
5. Analyzing the heat-induced seizure data
<ol>
	<li>After completing the screening of a cohort of animals, calculate the percentage of mice in a given genotype showing seizures using the following formula: 
	<img alt="Equation 1" src="/files/ftp_upload/62846/62846eq01.jpg" /></li>
	<li>Estimate the mean seizure threshold temperature of mice within a given genotype by averaging the seizure threshold temperature of all mice (noted in step 3.7) in that genotype that exhibit heat-induced seizures.</li>
	<li>While still being blind to the identity and genotype, replay the video recordings of each of the mouse during the heat-induced seizure assay screening on a computer screen to score severity of seizure bouts.</li>
	<li>Give scores to individual mouse exhibiting heat-induced seizure behavior by using the modified Racine scale13 as described by previous studies13,14. See Table 1 for details.</li>
	<li>If a mouse, while experiencing heat-induced seizures, only shows head nodding, give it a score of 2. If a mouse starts a seizure episode with head nodding but also exhibits forelimb clonus, falling over, and/or jumping give it a score of 5.</li>
	<li>Record the maximum score for each mouse using the modified Racine scale13 as described above.</li>
	<li>Plot a scatter graph of maximum Racine scores exhibited by all mice in a given genotype.</li>
	<li>Statistically compare maximum Racine scores among different mouse groups as a method to determine the severity of behavioral seizures such as heat-induced seizures. 
	NOTE: Racine scores are helpful to compare seizure characteristics between different mutant mice groups or genotypes. It is expected that the wild-type mice would not undergo heat-induced seizures and would not have to be considered for Racine score comparisons.</li>
	<li>Based on experimental design, perform appropriate statistical analysis to determine if the percentage of mice exhibiting seizures between wild-type and mutant mice, and their mean seizure threshold temperature values are significantly different from each other.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Racine Score</td>
			<td colspan="3">Seizure characteristics</td>
		</tr>
		<tr>
			<td>0</td>
			<td colspan="3">No seizures</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="3">Mouth and facial movements</td>
		</tr>
		<tr>
			<td>2</td>
			<td colspan="3">Head nodding</td>
		</tr>
		<tr>
			<td>3</td>
			<td colspan="3">Forelimb clonus, usually one limb</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="3">Forelimb clonus with rearing</td>
		</tr>
		<tr>
			<td>5</td>
			<td colspan="3">Generalized tonic-clonic seizure, rearing, jumping, falling over</td>
		</tr>
	</tbody>
</table>
Table 1: Racine scores.

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The authors declare no conflicts of interest.

We describe a simple and effective protocol to screen for occurrence of heat-induced seizures in mice, the behavioral equivalent of febrile seizures in human patients. The assay evaluates several parameters -&#160;including the percentage of mice showing seizures, seizure threshold, severity of seizures on a Racine scale, in order to compare the sensitivity of control and test mice groups to increases in body temperature.
A critical step in this protocol involves increasing the heat in the chamber while continuously monitoring the body temperature of the mouse. It is imperative that the maximum body temperature the mice will experience in these assays is 44 &#176;C because wild-type animals can undergo heat-induced seizures at body temperatures &#62;44 &#176;C. Pretreatment with general anesthesia or analgesics might reduce core body temperature of animals or interfere with thermoregulation, which in turn would confound seizure threshold temperature data collection. Thus, mice under this screening protocol could not be provided with these agents during the 30 min trial window. All procedures must&#160;be approved by the institution&#39;s IACUC committee. To ensure continuous monitoring of core body temperature of the mouse during the assay, securely tape the rectal temperature probe to the tail of the mice. If during the assay, the mouse body temperature is found to remain unchanged for prolonged periods of time even after increasing the temperature of the mouse chamber, ensure that the rectal temperature probe has not come out of the mouse or is attached loosely to the tail.
Genetic background of mouse models can affect sensitivity to the SCN1A mutation and pharmacologically induced seizures18,25,26,27. As discussed in the results above, the genetic background of the mice can influence their susceptibility to heat-induced seizures. Scn1a K1270T GEFS+ mutant mice were tested in two genetic backgrounds - 129X1 and B6NJ, and a small percentage of wild-type mice (33%) in the seizure sensitive B6NJ background, were also observed to undergo heat-induced seizures. However, in comparison to the heterozygous mutant Scn1aKT/+ mice, the B6NJ wild-type mice experienced heat-induced seizures at a significantly higher temperature threshold. This confirms that the genetic mutation (Scn1a K1270T) that was introduced by CRISPR knock-in makes the mutant mice more susceptible to hyperthermia-induced seizures.
There are several advantages of adopting this protocol, which are summarized below. First, unlike the use of stream of dry air or heated lamps, a temperature-controlled forced air set up within an enclosed space provides the experimenter more control over heating up the test arena at a desired rate. The steps in the heating protocol can be easily modified to increase/decrease the starting temperature, duration of each step, etc. to screen older mice that are heavier or larger rodents such as rats. Second, continuous monitoring of mouse body temperature via the attached rectal probe, gives valuable information about the rate of body temperature change in individual mouse, throughout the assay. This allows the experimenter to closely observe that the rate of temperature change in the mouse does not exceed 0.25-0.5 &#176;C/min (which might be stressful for the animals), when adapting this protocol to other test arenas. Importantly, the rate of change of body temperature across time in different mice groups can shed light on their ability to thermoregulate and could be helpful to understand whether febrile seizure causing mutations also alter thermoregulation in mice. Third, continuous body temperature monitoring ensures that the seizure threshold temperature measurements using this protocol are accurate, since they are recorded concurrently with the first bout of seizure experienced by the mouse. If the body temperature of the animal is not continuously monitored or seizure threshold temperature is measured after taking the animal out of the test arena, seizure threshold values can vary due to the time taken to handle the mice post seizures. Finally, this method circumvents the need to use invasive methods to induce fever (by injecting pathogens) in mice to mimic febrile seizures in human patients.
One of the limitations of this protocol is that it is difficult to screen juvenile (less than P30 in age) mice for heat-induced seizures. The protocol was developed to screen for sensitivity of adult mice (P30-P40 and above) to heat- or hyperthermia-induced seizures. In our experience, the younger wild-type mice, especially those weighing below 15 g, are more likely to undergo heat-induced seizures, which could be due to underdeveloped thermoregulation mechanisms, physiological thermal stress, or a combination of both. Hence, it is not ideal to perform the heat-induced seizure screen on juvenile mice using this protocol.
Future studies that combine EEG monitoring while subjecting the mouse to heat-induced seizures can shed light on EEG seizure patterns of heat-induced seizures, similar to a previous study19. Neuronal activity in specific areas in the mouse brain can be traced by combining optogenetic approaches and immunohistochemistry-based studies after harvesting the brain tissue. Also, the effects of restrictive diets such as keto diet on reducing febrile seizures can be evaluated by subjecting keto-fed mice and normal chow-fed mice to heat-induced seizure protocol. Similarly, epilepsy drug screening paradigms can be developed to test and identify candidate anti-epileptic drugs that ameliorate or suppress heat-induced seizures in drug-fed or treated mice when compared to vehicle-fed or control mice.

Epilepsy, the fourth most common family of neurological disorders in the United States1, are characterized by an imbalance of excitatory and inhibitory drive in the CNS that leads to recurrent seizures. Febrile seizures (FS) or fever associated seizures can occur in the general population, most often in children as early as 3 months up through 6 -7 years of age. However, in some individuals with genetic mutations, most often in a sodium channel gene, FS can persist beyond the age of 7 years into adulthood. This condition is referred to as febrile seizures plus or FS+. Rapid advances in genome sequencing has identified over 1,300 mutations in the human sodium ion channel gene SCN1A, making it a hotspot for epilepsy mutations. SCN1A mutations have been linked to a wide spectrum of seizure disorders, including febrile seizures (FS), genetic epilepsy with febrile seizures plus (GEFS+), and Dravet Syndrome (DS)2,3,4,5,6. About 20% of SCN1A missense mutations leads to GEFS+5,7,8. Pediatric history of complex or prolonged FS in childhood can subsequently develop into more debilitating forms of epilepsy such as temporal lobe epilepsy (TLE)9,10,11. Dravet Syndrome arises due to truncation mutations or loss of function mutations in SCN1A and is a severe form of intractable epilepsy, with childhood onset of febrile seizures that develop into refractory seizures, and is often associated with cognitive, developmental, and motor impairments2,5,12. Since many individuals with GEFS+ and/or DS exhibit febrile seizures, it becomes imperative to develop novel therapies to better combat these seizure disorders.
Animal models of SCN1A associated epilepsy have proven invaluable in characterizing different types of seizures (febrile vs generalized) and dissecting the neuronal mechanism of seizure generation13,14,15,16,17,18. While the study of spontaneous seizures via EEG/EMG recordings in rodent brains is well established and is a very useful tool, only a few studies have attempted to mimic febrile seizures in mouse models14,16,19,20,21,22,23. Previous studies have used a jet of heated dry air, or a methacrylate cylinder fitted with a thermal system, or heat lamps with a temperature controller in enclosed test arenas9,16,21,22,23,24 to induce seizures via hyperthermia. In order to increase body temperature in a more controlled environment, the protocol described here uses a custom-built chamber with a temperature-controlled heating system that allowed reproducible rates of increase in the body temperature of a mouse inside the chamber. The heat chamber was constructed from wood (length 40 cm x width 34 cm x height 31 cm) and was fitted with a digital temperature controller with a K thermocouple. A small axial fan equipped with a heater at the back panel of the chamber directs heated air into the chamber regulated by a digital temperature controller. This forced air heating system enables one to control the rate at which the chamber temperature increases. (Figure 1A,B). The K thermocouple located inside the wooden heat chamber sends feedback to the digital temperature controller, to maintain constant temperatures inside of the box during the assay. Setting the temperature on the digital temperature controller, enables the electric fan to send heated forced air through vents to uniformly heat the chamber (Figure 1A). The front panel of the heat chamber is a clear plexiglass sheet to enable easy video recording of the trials.
Adult (P30-P40) mice, heterozygous for a missense mutation in SCN1A that causes GEFS+ and an equal number of wild-type litter mates to serve as the control group, were selected for each experiment. Animals, both male and female, used in these studies weighed at least 15 g as wild-type mice weighing less were more sensitive to heat-induced seizures than heavier animals of the same age. In the pilot study, both mutant and wild-type mice were observed to seek out the cooler corners of the chamber at the back and remained there for prolonged periods of time. To circumvent this, effective floor size inside the heat chamber test arena was reduced to length 16.5 cm x width 21.5 cm x height 27.5 cm by placing a wooden block B (dimensions 20 cm x 8cm x 7.2 cm) at the right side of the chamber (Figure 1A). The heat chamber was constructed from 1.9 cm thick plywood (length 40 cm x width 34 cm x height 31 cm) covered with white laminate&#160;and fitted with a digital temperature controller with a K thermocouple. The laminate surface of the chamber walls is impermeable and can be easily sanitized between trials by wiping down with 70% ethanol. The temperature of the heat chamber was initially set at 50 &#176;C and preheated for at least 1 h before the start of the experiment, to ensure uniform heating inside the chamber. Each mouse was fitted with a rectal thermometer for continuous monitoring of body temperature throughout the experiment. A single mouse was placed in the chamber at a time and the temperature was kept at 50 &#176;C between 1st-10th minute. The temperature was then raised to 55 &#176;C for 11th-20th minute, and finally raised to 60 &#176;C for 21st-30th minute. This resulted in a reproducible rate of increase in the mouse body temperature (Figure 2A). Each trial was video-taped and behavioral analysis was conducted offline.
The heating protocol can be easily modified to change the initial temperature of the heat chamber and the rate that the chamber is heated, which in turn changes how quickly the body temperature of the mouse is elevated during the assay. Thus, this method provides more flexibility over traditional methods in setting up the behavioral screens involving heat-induced seizures. The heat-induced seizure protocol can also be used to screen for anti-epileptic drugs that make mutant mice more resistant to heat-induced seizures or increase the threshold temperature at which seizures are observed. Similarly, beneficial effects of restrictive diet regimes such as keto diet on heat-induced seizures can be examined in normal chow-fed vs keto-fed mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62846/62846fig1v2.jpg" /> 
Figure 1: Description of the custom-built mouse heat chamber. (A) The front panel of the wooden mouse heat chamber shows the side control panel containing Power ON/OFF switch that turns on digital temperature controller, K thermocouple, fan heater&#39;s ON/OFF switch and heat indicator. The outer dimensions of the box and the inner test arena are shown in cm. A wooden block B used to effectively reduce test arena surface is also shown. The bottom of the test arena is covered with cob&#160;bedding to prevent mice from directly coming in contact with heated wooden surfaces. (B) The back panel of the heat chamber shows the fan mounted on the top air vent and the power cord to supply electricity to the chamber. This figure is modified from Figure 3 in Das et al., 2021, eNeuro14. <a href="https://www.jove.com/files/ftp_upload/62846/62846fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Axial fan</td><td>Farnam</td><td>AF20-200-120-xx10-3.1</td><td>Farnam custom products -Axial Fan Heater with Fan</td></tr><tr><td>Digital temperature controller</td><td>Inkbird</td><td>ITC-100RH</td><td>Inkbird digital PID temperature controller ITC-100RH with K thermocouple</td></tr><tr><td>Mouse rectal temperature probe</td><td>ThermoWorks, Braintree Scientific, Inc</td><td>RET-3</td><td>Mouse rectal temperature probe with thermometer</td></tr></tbody></table>

a behavioral screen for heat-induced seizures in mouse models of epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

Animal models with febrile seizure mutations are expected to undergo heat-induced seizures at elevated body temperatures that do not induce seizures in the wild-type&#160;litter mates. SCN1A mutations have been linked with febrile seizures, including K1270T GEFS+ patients, who display both febrile and afebrile generalized seizures7. We screened CRISPR generated SCN1A K1270T GEFS+ mutant mice recently described in a study14 for the occurrence of heat seizures in two genetic backgrounds - seizure resistant 129X1/SvJ (129X1) and seizure susceptible C57BL/NJ (B6N) backgrounds. Age matched wild-type litter mates&#160;in&#160;the&#160;mouse&#160;heat chamber which do not harbor any GEFS+&#160;mutations and thus, are not expected to exhibit heat-induced seizures, served as the control group. The rate of the body temperature change over time was evaluated by plotting mean body temperature of mice recorded every minute during the assay. There was no difference in the rate of change of body temperature between heterozygous mutant mice and wild-type&#160;litter mates tested in respective 129X1 and B6N genetic backgrounds (Figure 2B,C). This suggests that thermoregulation is not altered in K1270T GEFS+ heterozygous mutant mice.
All heterozygous mutant mice from 129X1 (n = 15) or B6N (n = 9) genetic backgrounds exhibited heat-induced seizures (Figure 2D). None of the wild-type mice in the 129X1 enriched background (n = 13) exhibited heat-induced seizures (Figure 2D). In contrast, a third of the mice tested (n = 3 out of the 9 mice) in the seizure sensitive B6N background exhibited heat-induced seizures. Statistical comparison shows that percentage of heterozygous mutant mice exhibiting heat-induced seizures was significantly higher than their respective wild-type counterpart mice in both the 129X1and B6N genetic backgrounds (Figure 2D, Fisher&#39;s exact test, 129X1 p &#60; 0.0001; B6NJ p = 0.009). The average seizure threshold temperature between the heterozygous mutant mice in 129X1 and B6N genetic backgrounds was similar. 129X1 mutant mice have a mean seizure threshold temperature of 42.6 &#177; 0.20 &#176;C, which was not significantly different from the mean seizure threshold temperature of 42.7 &#177; 0.06 &#176;C seen in B6N mice (Figure 2E; two-tailed unpaired Student&#39;s t-test, p = 0.782). It is important to note that the mean seizure threshold temperature of three B6N wild-type mice that exhibited heat-induced seizures was 43.7 &#177; 0.08 &#176;C and significantly higher than the mean seizure threshold of 42.7 &#177; 0.06 &#176;C displayed by B6N heterozygous mutant mice (Figure 2E, two-tailed unpaired Student&#39;s t-test, p &#60; 0.0001).
The plexiglass front of the chamber makes it possible to do continuous video recordings during the assay that can be used later to score for seizure severity in each mouse on a modified Racine scale as described previously14,20. During a typical assay, heterozygous mutant mice would display heat-induced seizures with vocalization and/or head-nodding (Racine score 2), and rapidly transition to forelimb clonus, falling on side, jumping, hindlimb extension, and/or generalized tonic/clonic seizures (Racine scores 3-5) when body temperature reached about 42 &#176;C. The maximum Racine score represents the most severe heat-induced seizure behavior among the mutant mice. The maximum Racine score of heterozygous mutant mice in 129X1 enriched background (n = 15) is not different from heterozygous mutant mice in B6N (n = 9) genetic background (Figure 2F; Mann-Whitney test, p &#62; 0.9999). This suggests that heat-induced seizure behavior characteristics in K1270T GEFS+ mutant mice are independent of strain background.
Taken together, the data demonstrate&#160;that all mutant mice exhibit heat-induced seizures with similar frequency, seizure threshold temperature, and behavioral seizure severity in a strain-independent manner. The majority of wild-type litter mates do not exhibit such seizures at or below 44 &#176;C. About one-third of the wild-type control mice in a seizure sensitive B6N background did display heat-induced seizures (possibly due to genetic background effects) but the seizure threshold temperature was significantly higher compared to mutant mice in the same background. These results suggest that mutant mice in B6N genetic background are susceptible to heat-induced seizures at lower temperature thresholds due to the SCN1A GEFS+ mutation they harbor. Thus, using this protocol, one can evaluate heat-induced seizures in epilepsy mutant mice and distinguish from wild-type litter mate mice, which either do not undergo heat-induced seizures or display heat seizures at significantly higher temperatures.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62846/62846fig02.jpg" /> 
Figure 2: Mutant mice exhibit heat-induced seizures. (A) The heating protocol for behavioral screening of heat-induced seizures in mice. (B-C) Mean body temperature of mice across time in wild-type (Scn1a+/+ - black triangles) and heterozygous mutant (Scn1aKT/+ - orange circles) mice in two genetic backgrounds 129X1 and B6N, respectively. (D) Percentage of mice showing heat-induced seizures in both genetic backgrounds. Wild-type (Scn1a+/+) and heterozygous (Scn1aKT/+) mice are represented by black and orange bars, respectively. Heterozygous mutants in 129X1 and B6N backgrounds are shown in orange solid bars and orange bars with black stripes, respectively. (E) Seizure temperature threshold to heat-induced seizures in wild-type (Scn1a+/+) and heterozygous mutant (Scn1aKT/+) mice in both strains. (F) Scatter distribution of maximum Racine scores of heat-induced seizures exhibited by heterozygous (Scn1aKT/+) mice in both genetic backgrounds. Each dot represents maximum Racine score in a single mouse. Number of animals in each genotype is shown within parentheses. Data shown in panels B-F are mean &#177; S.E.M. This figure is modified from Figure 3 in Das et al., 2021, eNeuro14. <a href="https://www.jove.com/files/ftp_upload/62846/62846fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy at JoVE.com

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The ...

a-behavioral-screen-for-heat-induced-seizures-mouse-models

Research

JoVE Journal

Neuroscience

4.8K Views.  University of California. GEFS+mutaties veroorzaken koortsstuipen bij patiënten. Het vermogen om door hypothermie geïnduceerde aanvallen bij GEFS + muizen te bestuderen, stelt ons in staat om de relatie tussen specifieke mutaties en het aanvalsprofiel te onderzoeken. De digitale temperatuurregelaar helpt bij het reproduceren van de temperatuurgradiënt in de verwarmingsbox.Zo kan de lichaamstemperatuur van de muis langzaam en gestaag worden verhoogd. Dit protocol kan worden gebruikt om potentiële therapieën...

Video: A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

Genetics

Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of direct mechanical forces, sometimes penetrating through the cranium, creating a direct lesion in the brain tissue. These are visible during brain imaging as areas with contusion, laceration, and hemorrhage. Focal lesions induce neuronal death and glial scar formation and are present in 20%&minus;25% of all people who incur a TBI. However, in the majority of TBI cases, injury is caused by acceleration-deceleration forces and subsequent tissue shearing, resulting in nonfocal, diffuse damage. A subpopulation of TBI patients continues to develop post-traumatic epilepsy (PTE) after a latency period of months or years. Currently, it is impossible to predict which patients will develop PTE, and seizures in PTE patients are challenging to control, necessitating further research. Until recently, the field was limited to only two animal/rodent models with validated spontaneous post-traumatic seizures, both presenting with large focal lesions with massive tissue loss in the cortex and sometimes subcortical structures. In contrast to these approaches, it was determined that diffuse TBI induced using a modified weight drop model is sufficient to initiate development of spontaneous convulsive and non-convulsive seizures, even in the absence of focal lesions or tissue loss. Similar to human patients with acquired post-traumatic epilepsy, this model presents with a latency period after injury before seizure onset. In this protocol, the community will be provided with a new model of post-traumatic epilepsy, detailing how to induce diffuse non-lesional TBI followed by continuous long-term video-electroencephalographic animal monitoring over the course of several months. This protocol will detail animal handling, the weight drop procedure, the electrode placement for two acquisition systems, and the frequent challenges encountered during each of the steps of surgery, postoperative monitoring, and data acquisition.

This systematic protocol describes a new animal model of post-traumatic epilepsy after repetitive mild traumatic brain injury. The first part details steps for traumatic brain injury induction using a modified weight drop model. The second part provides instructions on the surgical approach for single- and multi-channel electroencephalographic data acquisition systems.

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of ...

Behavior

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status epilepticus (SE) is frequently selected to recapitulate spontaneous recurrent seizures (SRS). Here we present a protocol of SE induction by intraperitoneal (i.p.) injection of pilocarpine and monitoring of chronic recurring seizures in live animals using a wireless telemetry video and electroencephalogram (EEG) system. We demonstrated notable behavioral changes that need attention after pilocarpine injection and their correlation with hippocampal neuronal loss at 7 days and 6 weeks post-pilocarpine. We also describe the experimental procedures of electrode implantation for video and EEG recording, and analysis of the frequency and duration of chronic recurrent seizures. Finally, we discuss the possible reasons why the expected results are not achieved in each case. This provides a basic overview of modeling chronic epilepsy in mice and guidelines for troubleshooting. We believe this protocol can serve as a baseline for suitable models of chronic epilepsy and epileptogenesis.

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute seizures.

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...

Medicine

Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after kindling and a likely mechanism - insertion of Cre into the VGAT gene - disrupted its expression and reduced GABAergic tone. The present study extends these observations to a larger cohort of mice, focusing on key issues such as how long the SRS continues after kindling and the effect of the animal's sex and age. This report describes the protocols for the following key steps: making headsets with hippocampal depth electrodes for electrical stimulation and for reading the electroencephalogram; surgery to affix the headset securely on the mouse's skull so that it does not fall off; and key details of the electrical kindling protocol such as duration of the pulse, frequency of train, duration of train, and amount of current injected. The kindling protocol is robust in that it reliably leads to epilepsy in most VGAT-Cre mice, providing a new model to test for novel antiepileptogenic drugs.

This report describes the methods to generate a model of temporal lobe epilepsy based on the electrical kindling of transgenic VGAT-Cre mice. Kindled VGAT-Cre mice may be useful in determining what causes epilepsy and for screening novel therapies.

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique ...

A Model for Epilepsy of Infectious Etiology using Theiler's Murine Encephalomyelitis Virus

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop epilepsy as a consequence, with rates being significantly higher in less economically developed countries. This work provides an overview of modeling epilepsy of infectious etiology and using it as a platform for novel antiseizure compound testing. A protocol of epilepsy induction by non-stereotactic intracerebral injection of Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice is presented, which replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. The clinical evaluation of mice during encephalitis to monitor seizure activity and detect the potential antiseizure effects of novel compounds is described. Furthermore, histopathological consequences of viral encephalitis and seizures such as hippocampal damage and neuroinflammation are shown, as well as long-term consequences such as spontaneous epileptic seizures. The TMEV model is one of the first translational, infection-driven, experimental platforms to allow for the investigation of the mechanisms of epilepsy development as a consequence of CNS infection. Thus, it also serves to identify potential therapeutic targets and compounds for patients at risk of developing epilepsy following a CNS infection.

Intracerebral infection with the Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. This paper describes the virus infection, symptoms, and histopathology of the TMEV model.

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop ...

A Preclinical Model of Exertional Heat Stroke in Mice

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, whereas exertional heat stroke (EHS) occurs during physical activity. EHS differs from CHS in etiology, clinical presentation, and sequelae of multi-organ dysfunction. Until recently, only models of CHS have been well established. This protocol aims to provide guidelines for a refined preclinical mouse model of EHS that is free from major limiting factors such as the use of anesthesia, restraint, rectal probes, or electric shock. Male and female C57Bl/6 mice, instrumented with core temperature (Tc) telemetric probes were utilized in this model. For familiarization with the running mode, mice undergo 3 weeks of training using both voluntary and forced running wheels. Thereafter, mice run on a forced wheel inside a climatic chamber set at 37.5 &#176;C and 40%-50% relative humidity (RH) until displaying symptom limitation (e.g., loss of consciousness) at Tc of 42.1-42.5 &#176;C, although suitable results can be obtained at chamber temperatures between 34.5-39.5 &#176;C and humidity between 30%-90%. Depending on the desired severity, mice are removed from the chamber immediately for recovery in ambient temperature or remain in the heated chamber for a longer duration, inducing a more severe exposure and a higher incidence of mortality. Results are compared with sham-matched exercise controls (EXC) and/or na&#239;ve controls (NC). The model mirrors many of the pathophysiological outcomes observed in human EHS, including loss of consciousness, severe hyperthermia, multi-organ damage as well as inflammatory cytokine release, and acute phase responses of the immune system. This model is ideal for hypothesis-driven research to test preventative and therapeutic strategies that may delay the onset of EHS or reduce the multi-organ damage that characterizes this manifestation.

The protocol describes the development of a standardized, repeatable, preclinical model of exertional heat stroke (EHS) in mice free from adverse external stimuli such as electric shock. The model provides a platform for mechanistic, preventative, and therapeutic studies.

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, ...

Triggering and Monitoring Heat-Induced Seizures in a Transgenic Mouse Model

Source: Das, A., et al. <a href="https://app.jove.com/v/62846">A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure to induce heat-induced seizures in a transgenic mouse with epilepsy-related gene mutations by gradually increasing body temperature in a controlled chamber. This protocol is used to study seizure mechanisms and potential therapies for epilepsy.

Source: Das, A., et al. A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy. J. Vis. Exp. (2021)
This video demonstrates the ...

JoVE Encyclopedia of Experiments

Neuropathology

Epilepsy and Seizure Disorders

Systemic Pilocarpine Treatment: Mouse Model of Temporal Lobe Epilepsy

Source: Kim, J. E. and Cho, K. O. <a href="https://www.jove.com/video/56831/the-pilocarpine-model-temporal-lobe-epilepsy-eeg-monitoring-using">The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice</a>. J. Vis. Exp. (2018).
This video presents the procedure for inducing status epilepticus (SE) in mice by systemic administration of pilocarpine to model temporal lobe epilepsy.

Source: Kim, J. E. and Cho, K. O. The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice. J. Vis. Exp. ...

Biology

Rodent Models

Adult

Handling-Induced Seizures in a Mouse Model

First, bring all the cages to the bench and observe the animals for seizures twice daily during the light phase. Score the seizure activity by a modified Racine scale and report the number and intensity of seizures. Next, slide a pen across the cage to make some noise and transfer each animal to another box and back. For animals that have not seized spontaneously or after gentle cage shaking, trigger seizures by more intense handling while carefully turning over the mouse by flipping it at its tail from left to right. Observe each animal for seizure behavior again and repeat the process for subsequent cages.

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

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