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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

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JoVE Journal

Neuroscience

4.7K Views.  University of California. 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.

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

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...