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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Pentylenetetrazole (PTZ)
<ol>
	<li>Dissolve 2 mg/mL PTZ in sterile 0.9% (w/v) NaCl. Prepare PTZ on the day of use.</li>
</ol>
2. Injection of PTZ
<ol>
	<li>Perform all experiments between 9:00 a.m. and 12:00 p.m. (noon).</li>
	<li>Measure the animal&#39;s body weight.</li>
	<li>Place the animal in an observation chamber for habituation.</li>
	<li>During the habituation period (3 min), calculate the volume of PTZ solution for the injection based on the body weight of the animal and the previously determined injection dose. 
	NOTE: For example, when 35 mg/kg PTZ is used, 0.875 mg of PTZ is required for a mouse weighing 25 g (35 mg/kg x 0.025 kg = 0.875 mg). Therefore, 437.5 &#956;L of a 2 mg/mL PTZ solution should be injected (0.875 mg / 2 mg/mL = 0.4375 mL). The injection dose of PTZ depends on the mouse genotype and strain. For wild-type C57BL/6 mice, a dose of 30-35 mg PTZ/kg body weight is recommended for the first trial. Sensitivity to PTZ depends on the mouse strain used. The injection dose varies according to the purpose of the experiment.</li>
	<li>Inject PTZ intraperitoneally with a 1-mL syringe attached to a 27-gauge needle into the left or right quadrant of the abdomen of the animal. Avoid injecting at the midline.
	<ol>
		<li>Avoid repeated injections at the same position.</li>
	</ol>
	</li>
	<li>Observe animal behaviors for 30 min after PTZ administration (Figure 1A) and classify and score the abnormal behavior as shown below. If possible, keep the animals under observation for 24 h, or at least an additional 6-10 h post-injection, especially once the seizure severity score reaches 3 or above.
	<ol>
		<li>Note any mild seizures or behavioral changes in the animals beyond the 30-min observation period. This prolonged observation period may address a particularly important and complete effect of a subconvulsive dose of PTZ in the generation of chronic unprovoked seizures in epilepsy.</li>
		<li>In addition, measure the seizure duration of each observed seizure as changes in seizure duration relate to the seizure severity. Moreover, the latency to the first seizure after the PTZ injection is another important measure to collect. Monitoring the seizure frequency, duration, and latency is critical for any post-seizure molecular studies.</li>
	</ol>
	</li>
	<li>Inject PTZ every other day (Figure 1A). The number of injections depends on the objective of the experiment. Representative examples are as follows.
	<ol>
		<li>To generate completely kindled animals, once an animal experiences a seizure with a score of 5 (tonic-clonic seizure), finish the injection within an additional three administrations. In this case, increase the injection dose if the seizure score does not increase for three consecutive administrations. Each animal can receive a different number and dose of PTZ injections.</li>
		<li>Fix the number and dose of PTZ injection in every condition to evaluate the vulnerability to PTZ, including in assessments of anti-epilepsy drugs and studies of the phenotype of genetically modified mice. Give all animals the same number of PTZ injections; a total of 8 to 12 injections is recommended. If an animal dies, the seizure score should be denoted as 6.</li>
		<li>Determine the injection dose necessary to maintain a high seizure score (4 or 5) for 10 or more injections to study the histopathological changes that occur epileptic seizures. In this case, the total injection number should range from 25 to 30. Decrease the injection dose if the seizure score reaches 5. If the seizure score decreases to 3 or lower, increase the injection dose.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Pentylenetetrazole</td>
			<td>Sigma-Aldrich</td>
			<td>P6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>MANAC</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>CLEA Japan</td>
			<td></td>
			<td>C57Bl/6NJcl, postnatal 8 week, male</td>
		</tr>
		<tr>
			<td>Syringe (1mL)</td>
			<td>Terumo</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle(27G x 3/4&#34;) (0.40 x 19 mm)</td>
			<td>Terumo</td>
			<td>NN-2719S</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing scale</td>
			<td>Mettler</td>
			<td>PE2000</td>
			<td>This item is a discontinued product. Almost equivalent to FX-2000i with FXi-12-JA from A&#38;D company.</td>
		</tr>
	</tbody>
</table>

establishing a pentylenetetrazole-induced epileptic seizure model in mice

Source: Shimada, T., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56573">Pentylenetetrazole-Induced Kindling Mouse Model.</a> J. Vis. Exp. (2018).
This video demonstrates the process of inducing epilepsy in mice using pentylenetetrazole (PTZ) injections, a GABA-A receptor antagonist. Repeated intraperitoneal administration of PTZ progressively lowers the seizure threshold by inhibiting chloride ion influx, disrupting inhibitory neurotransmission, and leading to hyperexcitability in neurons that trigger seizures. This process results in kindling and the development of spontaneous seizures, mimicking chronic epilepsy.

<img alt="Figure 1" src="/files/ftp_upload/23114/23114_Figure1.jpg" /> 
Figure 1: Brief description of the protocol. (A) Schematic illustration of PTZ-mediated kindling. (B) Illustrations of representative animal behaviors for respective seizure scores.

Establishing a Pentylenetetrazole-Induced Epileptic Seizure Model in Mice

Source: Shimada, T., et al. Pentylenetetrazole-Induced Kindling Mouse Model. J. Vis. Exp. (2018).
This video demonstrates the process of inducing epilepsy ...

Begin by intraperitoneally injecting a low dose of pentylenetetrazole or PTZ, a seizure-inducing agent, into an adult mouse.
PTZ is absorbed into the bloodstream and crosses the blood-brain barrier, reaching the brain.
In the brain, gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter, binds to GABA-A receptors on neurons.
This binding facilitates chloride ion influx, hyperpolarizing the neurons and decreasing their excitability.
PTZ acts as a GABA-A receptor antagonist, inhibiting chloride ion influx and increasing neuronal excitability.
Excitatory neurotransmitters then bind to these neurons and induce prolonged depolarization and action potential generation.
This heightened excitability affects nearby neurons, leading to synchronized and repetitive firing, resulting in seizures.
Administer low-dose PTZ injections every other day.
With each repeated injection, neuronal hyperexcitability progressively increases, causing seizures to intensify and spread across larger brain areas.
Over time, this leads to spontaneous seizures without PTZ administration, resulting in epilepsy.

establishing-pentylenetetrazole-induced-epileptic-seizure-model

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Epilepsy and Seizure Disorders

42 Views. Source: Shimada, T., et al. Pentylenetetrazole-Induced Kindling Mouse Model. J. Vis. Exp. (2018). This video demonstrates the process of inducing epilepsy in mice using pentylenetetrazole (PTZ) injections, a GABA-A receptor antagonist. Repeated intraperitoneal administration of PTZ progressively lowers the seizure threshold by inhibiting chloride ion influx, disrupting inhibitory neurotransmission, and leading to hyperexcitability in neurons that trigger seizures. This process results in kindli...

Video: Establishing a Pentylenetetrazole-Induced Epileptic Seizure Model in Mice - Concept

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Valutazione della funzione di memoria nei topi epilettici indotti da pilocarpina

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

Ricerca

JoVE Journal

Comportamento

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Un modello di epileptogenesi nelle culture delle fette organotipiche della corteccia rinale-ippocampo

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

Neuroscienze

Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few studies have described the experimental protocol in detail. This paper illustrates the whole process of amygdala electrical kindling epilepsy model making, with the introduction of a method of bipolar electrode fabrication. This electrode can both stimulate and record, reducing brain injury caused by implanting separate electrodes for stimulation and recording. For long-term electroencephalogram (EEG) recording purposes, slip rings were used to eliminate the record interruption caused by cable tangles and falling off.
After periodic stimulation (60 Hz, 1 s every 15 min) of the basolateral amygdala (AP: 1.67 mm, L: 2.7 mm, V: 4.9 mm) for 19.83 &plusmn; 5.742 times, full kindling was observed in six mice (defined as induction of three continuous grade V episodes classified by Racine's scale). An intracranial EEG was recorded throughout the entire kindling process, and an epileptic discharge in the amygdala lasting 20-70 s was observed after kindling. Therefore, this is a robust protocol for modeling epilepsy originating from the amygdala, and the method is suitable for revealing the role of the amygdala in temporal lobe epilepsy. This research contributes to future studies on the mechanisms of mesial temporal lobe epilepsy and novel antiepileptogenic drugs.

The amygdala plays a key role in temporal lobe epilepsy, which originates in and propagates from this structure. This article provides a detailed description of the fabrication of deep brain electrodes with both recording and stimulating functions. It introduces a model of medial temporal lobe epilepsy originating from the amygdala.

Utilizzo di un elettrodo bipolare per creare un modello murino per l'epilessia del lobo temporale mediante accensione elettrica dell'amigdala

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few ...

Establishing a Pentylenetetrazole-Induced Epileptic Seizure Model in Mice

Trascrizione

Establishing a Pentylenetetrazole-Induced Epileptic Seizure Model in Mice

Valutazione della funzione di memoria nei topi epilettici indotti da pilocarpina

Un modello di epileptogenesi nelle culture delle fette organotipiche della corteccia rinale-ippocampo

Utilizzo di un elettrodo bipolare per creare un modello murino per l'epilessia del lobo temporale mediante accensione elettrica dell'amigdala