Pentylenetetrazole-Induced Kindling Mouse Model

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.

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This method can help the deep analysis of epilepsy. Seizure will induce histological changes in the brain which may be involved in the neuropsychiatric developmental disorders. The main advantage with this technique is that self-conversive dose of PTZ can induce controllable seizures better than the other current methods.

Begin preparing the injection by dissolving 2 milligrams per milliliter PTZ in sterile 0.9 percent sodium chloride. Weigh the animal, then place it in an observation chamber for a 3-minute habituation period. Next, inject PTZ interperitoneally with a 1 milliliter syringe attached to a 27 gauge needle into the left or right quadrant of the abdomen of the animal.

Finally, observe the animal after PTZ injection for abnormal behavior. Obtain at least two two-to-six month old 129 SV mice who are of the same sex as the subject mice to be the stranger mouse. Each stranger mouse must be kept separately.

For each training session, leave the mouse in the cage for habituation for 15 minutes. Clean the entire apparatus in both cages by wiping the surface with 70%ethanol. Then, place the empty cages in the two side chambers of the three-chamber apparatus and open the doors between the chambers.

To habituate the subject mouse, place the subject mouse in the center chamber and allow the mouse to move freely throughout the apparatus until the mouse has investigated both cages, plus an additional five minutes. After the animal moves into the center chamber, close the doors and let the mouse move freely in the center chamber for five minutes. During this period, put one of the 129 SV mice in a cage to habituate to the cage.

Immediately after the habituation period of the subject mouse, place the cage containing the 129 SV mouse, termed the Stranger One cage, in one of the side chambers and place the empty cage, termed the Object cage, in the other side chamber. Open the door and monitor the behavior of the subject mouse. Allow the subject mouse to move freely for 10 minutes and record four behavioral parameters.

Number one, the time spent in each chamber, the Stranger One chamber, the Object chamber and the center chamber. Number two, time spent investigating each cage, the Stranger One cage and the Object cage. Number three, the number of entrances into each chamber and Number four, the total distance traveled.

After the mouse moves into the center chamber, close the doors. After wiping the cages, begin the social novelty analysis by placing another 129 SV mouse in one of the cages, termed the Stranger Two cage, and place the Stranger Two cage in one of the chambers. Place the Stranger One mouse in the other cage, termed the Stranger One cage and place the Stranger One cage in the other chamber.

Finally, open the door and monitor the behavior of the subject mouse. Allow the subject mouse to move freely for 10 minutes and measure the same behavioral parameters described for the sociality analysis. Begin by placing a mouse in a conditioning apparatus with specific conditions.

For example, a square apparatus with clear plexiglass walls, a metal grid floor, an ethanol odor, 100 lux brightness and 65 decibel background white noise. Condition the mouse by foot shock with pseudo-random timing of a 0.1 milliamp electrical shocks for two seconds three times over the course of five minutes. Then, return the mouse to the home cage after conditioning.

The day after conditioning, perform a memory assessment by placing the mouse into the same apparatus as the shock condition and measuring the freezing time over the course of five minutes. Later the same day, place the mouse in a novel apparatus and measure the freezing time during a five-minute assessment. Finally, compare the freezing time between the same condition and novel condition.

Results indicated that the seizure score gradually increased with PTZ injections, whereas no seizures or abnormal behaviors were evoked by saline injections. Further, repetitive seizure promoted aberrant axonal branch formation or mossy fiber sprouting and abnormal migration of granule cells in the hippocampus. PTZ-treated mice showed normal sociality.

Mice spent more time in the Stranger One chamber than in the Object chamber and investigated the Stranger One cage more than they investigated the Object cage. PTZ-treated mice showed abnormal social novelty, indicative of impaired social memory. PTZ-treated mice also showed impaired memory in the contextual fear test.

Following this procedure, other methods like Morris water maze or the other memory tests can be performed to answer the additional questions about the impaired memory ability affected by epileptic seizure. After its development, this technique paves the way for the researchers in the field of epilepsy to explore as a mechanism of seizure-induced neuropsychiatric developmental disorders by using model animals.

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Journal (Neuroscience)

Journal (Neuroscience)

Pentylenetetrazole-Induced Kindling Mouse Model

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