Materials and Reagents

18 G needle
3 x 3 in. medium gauze pads (CVS, catalog number: 893120 )
C57BL/6J male mice (6-7 weeks on arrival) (THE JACKSON LABORATORY, catalog number: 000664 )
Aquarium sealant
Flurothyl (Bis(2,2,2-trifluoroethyl) ether or 2,2,2-trifluoroethyl ether) (Sigma-Aldrich, catalog number: 287571 )
IMPORTANT: Perform all flurothyl exposures in a certified chemical fume hood with exhaust out of the laboratory, since the inhalation of flurothyl will result in seizures in humans.
95% ethanol (ethyl alcohol 190 proof) (PHARMCO-AAPER, catalog number: 111000190 )
Petroleum jelly
10% flurothyl solution (see Recipes)

Equipment

All-clear vacuum Plexiglas desiccator chamber (Ted Pella, model: 2240-1 )
20 ml glass syringe (Sigma-Aldrich, catalog number: Z101079 )
Syringe pump (Kent Scientific, model: GENIE Plus )
Forceps (for removing the flurothyl saturated gauze pad from the chamber)
Wire mesh colander with at least ¼” square mesh
Chemical fume hood

Procedure

Construction of flurothyl chamber
1. The Plexiglas chamber needs to have a small hole drilled in the top of the chamber to allow for the fixing of an 18 G needle in this hole (the 18 G needle is cut at the luer lock end of the plastic edge of the needle and at the beveled edge of the needle resulting in a short 18 G blunted steel tube). The 18 G needle tube, upon fixing it to the top of the chamber, is connected to small diameter tubing on the outside of the chamber. At this point, seal the connection with the tubing and the top of the chamber with aquarium sealant to ensure that the chamber is air-tight. The no-tubing end of the 18 G tube should now be extended into the top of the chamber. Also, be sure to seal any other openings in the top half of the chamber with aquarium sealant (this is usually where the vacuum valve would be attached).
2. Next, tape a screen support to hold a gauze pad below where the 18 G needle tubing is hanging. The top and bottom of the chamber should be regularly greased with petroleum jelly to ensure an adequate seal between the top, bottom and O-ring of the chamber. The other end of the tubing is connected to a 20 ml glass syringe that is attached to a syringe pump (Figure 1).
  
  Figure 1. The repeated flurothyl seizure model set-up. Black arrows are pointing to the tubing with slack running from the syringe to the 18 G steel tube in the top of the chamber.

Repeated flurothyl seizure model

Mice are allowed ad libitum access to food and water, and are maintained on a standard 12 h light-dark cycle with lights on at 7:00 AM. Mice are allowed to acclimate to the animal facility for ~1 week before seizure testing. Individual mice (7-8 weeks old) are exposed to 10% flurothyl (see Recipes section). Flurothyl is delivered to mice in a closed Plexiglas chamber. Flurothyl binds at the GABA_A receptor where it acts as a noncompetitive antagonist (Krasowski, 2000). Since a receptor bound by a non-competitive antagonist will not be activated by binding of an agonist, and since ethanol is a positive allosteric modulator of the GABA_A receptor, ethanol is unlikely to exert a major effect at the GABA_A receptor in the presence of flurothyl.
Be sure to perform all flurothyl exposures in a certified chemical fume hood with exhaust out of the laboratory, since the inhalation of flurothyl will result in seizures in humans.
Ten percent flurothyl is infused via a syringe pump and glass syringe at a flow rate of 6 ml/h onto a gauze pad (folded in half) suspended from the top of the chamber by a screen (Figure 1). Since flurothyl is highly volatile, it rapidly vaporizes, leading to inhalation and subsequent seizure expression. The generalized seizure threshold (GST) is defined as the latency from the commencement of the infusion of flurothyl to the occurrence of an animal’s loss of postural control.
Once the animal losses its posture (i.e., expresses a generalized clonic seizure; grades 1-2 [see Table 1]), the chamber is opened to fresh air, resulting in the rapid elimination of flurothyl (and the flurothyl infusion pump is turned off). Mice with different genetic backgrounds will respond with different latencies to loss of postural control (i.e., GSTs). In different mouse strains, trial 1 GSTs can range from 200-500 sec (Papandrea et al., 2009). However, the variability within strains is quite small given that this is a behavioral analysis. It is good practice to place a wire mesh colander over the bottom of the chamber, when the top is removed, to prevent the mouse from jumping out of the chamber. This is particularly important following the 28-day flurothyl incubation period and flurothyl rechallenge, since mice will often have brainstem seizures which can result in the mouse having wild running and bouncing seizures in which they can escape from the bottom half of the chamber. The wire mesh colander helps to contain the mouse in the bottom half of the chamber, while also exposing the animal to room air.

The latency to the first myoclonic jerk (behaviorally, myoclonic jerks are brief, but significant, contractions of the neck and body musculature, while maintaining postural control [Applegate et al., 1997; Samoriski and Applegate, 1997]), the number of myoclonic jerks expressed before the onset of a generalized seizure, the latency to the loss of postural control (GST), the time to regain posture, the duration of the seizure (calculated as the time from the start of the generalized seizure to the time when the animal regains its posture and stops showing clonus of the limbs), and the type of seizure is recorded (Table 1) for each trial.

Table 1. Behavioral seizure classification grading scale

Seizure grade	Description of corresponding seizure behavior*
Grade 1	Loss of posture associated with facial clonus, including chewing, and clonus of forelimbs and/or hindlimbs
Grade 2	Grade 1 seizure followed by recovery of the righting reflex and low intensity bouncing
Grade 3	Grade 1 and/or 2 features with recovery of the righting reflex followed by wild running and popcorning
Grade 4	Grade 3 followed by forelimb and/or hindlimb treading
Grade 5	Grades 3 and/or 4 followed by bilateral tonic extension of the forelimbs
Grade 6	Grade 5 followed by bilateral tonic extension of the hindlimbs
Grade 7	Grade 6 followed by immediate death

*In general, grades 1 and 2 are clonic-forebrain seizures comparable to a grade 5 seizure on the Racine scale for electrical kindling (Racine, 1972). Grades 3-7 are brainstem seizures. Importantly, mice recover their righting reflex before transitioning into grades 3-7 (Kreindler et al., 1958; Browning and Nelson, 1986). We categorize mice having seizure grades 3-7 as forebrainbrainstem seizures to denote the progression of this type of seizure.

Upon seizure resolution, the animal is returned to its home cage until the next day. One mouse is tested at a time in the flurothyl chamber using a new gauze pad each time. Mice are given one 10% flurothyl-induced seizure per day for 8 days (the flurothyl induction phase) followed by a 4 week rest period (the flurothyl incubation period). We routinely try to keep the time intervals between induction trials as close to 24 h as possible (24 ± 4 h). Previous work has demonstrated the necessity of 8 induction trials and a 28-day incubation period for the effects observed (Samoriski and Applegate, 1997). The gauze pad, saturated in flurothyl, is removed from the top half of the chamber from the screening, using forceps, so as to not contaminate one’s gloves with flurothyl. The gauze pads are laid out under the fume hood to allow for flurothyl evaporation. Gauze pads can be reused the next day, since flurothyl will have evaporated completely from the gauze pad by this time.
Following the 28-day incubation period, mice are again given a flurothyl exposure (retest/rechallenge) and the criteria described above are recorded. Representative examples from C57BL6/J mice of the observed changes in the latency to the first myoclonic jerk, the number of myoclonic jerks, and generalized seizure thresholds are shown (Figure 2). C57BL6/J mice typically do not express forebrainbrainstem seizures during the 8 flurothyl induction trials, but upon a 28-day incubation period and flurothyl retest, a significant percentage of mice now express this new seizure behavior (Figure 3).

Figure 2. Myoclonic jerk threshold, the number of myoclonic jerks, and the generalized seizure threshold (GST) in C57BL6/J mice. The latency to the first myoclonic jerk (myoclonic jerk threshold in seconds), the number of myoclonic jerks expressed before the onset of a generalized seizure, and the latency to a generalized seizure (generalized seizure threshold) on each seizure trial in C57BL6/J mice. The retest trial is a rechallenge to flurothyl following a 28-day incubation period.

Figure 3. Flurothyl-induced seizure behaviors in C57BL6/J mice during 8 induction phase seizures, and a final flurothyl rechallenge (retest) after a 28-day incubation phase. While none of the C57BL6/J mice expressed a complex forebrain→brainstem seizure during the flurothyl induction trials (these mice only expressed clonic-forebrain seizures), 75-100% of C57BL6/J mice will express forebrain→brainstem seizures following a rechallenge to flurothyl following a 28-day incubation period.
Additionally, local field potential recordings from the cerebral cortex and hippocampus of C57BL6/J mice, during the 28-day incubation period, reveals that flurothyl-exposed mice develop spontaneous seizures following the 8^th flurothyl induction trial with relatively high seizure occurrences (Kadiyala et al., 2016). However, these seizures begin to remit over the 28-day incubation phase (Kadiyala et al., 2016). Interestingly, DBA/2J mice similarly treated also develop spontaneous seizures with fewer seizure occurrences than C57BL6/J mice, but the spontaneous seizures observed in DBA/2J mice do not appear to remit (Kadiyala and Ferland, 2017).

Data analysis

Repeated measures analyses of variance (ANOVA) are employed for comparisons between flurothyl-exposed and control groups across trials followed by post-hoc tests. Student’s t-tests are used to determine differences in seizure characteristics between trial 8 of the induction phase and the retest/rechallenge that followed the 28-day incubation period. Evaluation of differences (percentage) in the numbers of mice exhibiting clonic–forebrain seizures and the numbers of mice exhibiting forebrainbrainstem seizures, after flurothyl exposure, utilize Chi-square or Fisher Exact tests.

Notes

Be sure to perform ALL flurothyl exposures in a functional and annually certified chemical fume hood to ensure safety of laboratory personnel, given that flurothyl is an inhalant chemoconvulsant. Moreover, do not remove any materials that were exposed to flurothyl (i.e., gauze pads, etc.) in a liquid state, for at least 24 h, to ensure evaporation of flurothyl in the chemical fume hood.
As mice get older than 7-8 weeks of age, some mice do not have an obvious loss of posture with flurothyl exposure. They can present with very severe myoclonic jerks and even some bilateral forelimb clonus in the absence of loss of posture. Mice experiencing these behaviors often result in brainstem seizure expression, since the mice are continually exposed to flurothyl because the top of the chamber was not removed. Missing the clonic seizure (as defined by loss of posture) results in the chamber top not being removed and exposing the mice to room air. Continual flurothyl exposure will always result in a brainstem seizure, therefore, it is important to detect the generalized clonic seizure to terminate the trial, which may involve having to record EEGs to detect generalized seizures.

Recipes

Preparing the 10% flurothyl solution
10% flurothyl is made by diluting a 5 g ampule (~5 ml) of liquid flurothyl into 45 ml of 95% ethanol in a glass container
The flurothyl/ethanol mixture is then shaken to mix the flurothyl
Note: Flurothyl is always prepared and stored in a chemical fume hood (with the bottle and cap sealed with Parafilm).Be sure that the glass container is clearly labeled as a potential hazard as an inhalant chemoconvulsant.

Acknowledgments

This work was supported by an NIH/NINDS R01NS064283 grant to RJF. This work was adapted from previous publications (Samoriski and Applegate, 1997; Kadiyala et al., 2016).

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