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

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation for the heat-induced seizure assay

1. Switch on the Power On button on the heat chamber, followed by the Heat On button.
2. Set the temperature of the heat chamber at 50 °C using the keypad on the digital temperature controller.
3. Wait for a minimum of 1 h to preheat the chamber to 50 °C before introducing the first mouse into the chamber. Preheating ensures uniform heating inside the chamber.
4. Line the floor of the mouse heat chamber with cob bedding.
5. Mount a video recording camera in front of the heat chamber to record each heat-induced seizure assay trial.
6. Line a 140 mm diameter Petri dish with thick layers of tissue paper and place it on ice to serve as a cooling pad.
   ​NOTE: At the end of the assay, individual mice will be transferred to the prechilled cooling pad to help bring down their elevated body temperature.

2. Preparing the mouse for heat-induced seizure assay

1. 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 °C and serve as the control group.
2. 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.
3. Screen one mouse at a time in the mouse heat chamber.
4. Briefly anesthetize the mouse for 10-15 s using a few drops of isoflurane at the bottom of a bell jar.
5. Take the animal out of the bell jar and place it on a paper towel.
6. Ensure that the mouse is completely anesthetized by checking that the mouse is unresponsive to a noxious toe pinch.
7. 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.
8. Secure the rectal probe to the mouse'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 it to the tail.
9. Ensure the rectal probe is connected to a multimeter that displays the internal body temperature of the mouse.
10. Place the animal in a fresh cage lined with cob bedding, i.e., the recovery cage.
11. 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.
    1. Simultaneously, monitor the core body temperature of the mouse till it stabilizes at 35-36 °C.
12. At the end of 5 min, note the body temperature of the mouse. This is the initial body temperature at time "0" min.
    NOTE: If the core body temperature of the mouse is below 35 °C, wait for additional time for the animal to recover from anesthesia-induced hypothermia.
13. 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.

3. Heat-induced seizure assay

1. After gently placing the mouse on the floor of the pre-heated mouse heat chamber, close the plexiglass door and start the camera for video recording the experiment.
2. Start the stopwatch. Record the body temperature of the mouse from the rectal thermometer at 1 min intervals for the duration of the experiment.
3. 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 °C /min.
   NOTE: Rapid increases in body temperature can lead to heat stroke or death and should be avoided.
4. Following this protocol, increase the temperature of the mouse heat chamber by 5 °C every 10 min as shown in Figure 1A.
5. At 9.5 min, set the temperature of the heat chamber to 55 °C, to stabilize the temperature of the heat chamber to 55 °C by the 10th min as shown on the digital temperature display.
6. Similarly, increase the temperature to 60 °C at 19.5 min to stabilize the temperature of the heat chamber to 60 °C by the 20th min. Each seizure screening trial lasts for 30 min.
7. 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.
   1. Record the body temperature of the mouse during the seizure (seizure threshold temperature) from the rectal temperature thermometer.
   2. 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.
8. 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.
9. Wait for the mouse body temperature to come down to 36-37 °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.
10. 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.
11. Wipe clean the metal tip of the rectal probe with 70% alcohol and soft tissue wipes to keep it ready for the next trial.
12. 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.
13. 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.
14. If a mouse does not experience heat-induced seizures within the 30 min observation period or the body temperature of the mouse reaches 44 °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 °C.
15. Reset the temperature of the mouse heat chamber to 50 °C and allow it to equilibrate till the display temperature on the digital temperature controller shows 50 °C.
16. Change cob bedding between individual mouse trials.

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Results

Figure 1: 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 the maximum Racine score in a single mouse. Number of animals in each genotype is shown in parentheses. Data shown in panels B-F are mean ± standard error mean (S.E.M).

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Materials

Name	Company	Catalog Number	Comments
Axial fan	Farnam	AF20-200-120-xx10-3.1	Farnam custom products -Axial Fan Heater with Fan
Digital temperature controller	Inkbird	ITC-100RH	Inkbird digital PID temperature controller ITC-100RH with K thermocouple
Mouse rectal temperature probe	ThermoWorks, Braintree Scientific, Inc	RET-3	Mouse rectal temperature probe with thermometer