Name: Behavioral Characterization of Pentylenetetrazole-induced Seizures: Moving Beyond the Racine Scale (Video) | JoVE Journal
Uploaded: 2025-07-08T13:30:00.000+00:00
Duration: 7 min 35 s
Description: Discover a computer-assisted video analysis protocol for quantifying ictal behaviors in pentylenetetrazole-induced seizures. This method captures spasms and myoclonus overlooked by traditional Racine scale scoring.

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This work was partly supported by Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq, grants numbers 308110/2020-0 and 316441/2023-6), Funda&#231;&#227;o de Amparo e Promo&#231;&#227;o da Ci&#234;ncia, Tecnologia e Inova&#231;&#227;o do Rio Grande de Norte (FAPERN, grant number 042/2022) and Funda&#231;&#227;o Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES, grant number #385/2019), and Propesq-UFRN (grant number PVP18039-2020).

All experimental procedures were approved by the local ethical committee of the Federal University of Rio Grande do Norte (Protocol number 043/2022, License number 307.043/2022). Male C57BL/6 mice ranging from 3 to 4 months old were used in this protocol, with a recommended sample size of 6-10 animals according to established PTZ induction protocols16.
1. Preparation of the observational arena
<ol>
	<li>Use a square or circular arena with at least 10 x 10 cm surface area and 30 cm of wall height. Ensure that the arena is well-illuminated. If necessary, use infrared lighting to guarantee saturated image acquisition (Figure 2A).</li>
	<li>Place at least one video camera over the arena, with a top-down view of the entire floor of the arena and at least 10 cm of the wall (to capture rearing behavior without cropping the mouse head, Figure 2B).</li>
	<li>If desired, place a second video camera with a frontal view of the arena. In this case, ensure that one of the arena walls is transparent.</li>
	<li>Ensure that the temperature (21-24 °C) and humidity (50-70%) match the conditions of the animal facility, monitoring them with a thermo-hygrometer and adjusting as needed.</li>
	<li>Place the necessary bedding to cover the bottom of the arena and ensure that the animal is visible from the background. Make water and food pellets freely available during the entire duration of the experiment.</li>
	<li>Start experiments preferably at the beginning of the light phase (e.g., between 7:00 and 10:00 am). Maintain consistent illumination in the experimental room (900-1,100 lux) with a 12/12 h light/dark cycle, with lights on at 6 a.m. Use a lux meter to verify lighting conditions close to the recording arena, ensuring uniform video recording quality.</li>
	<li>Connect the camera to the video recording device to acquire the video with at least 24 frames per second (fps).</li>
</ol>
2. Preparation of the PTZ solution
NOTE: Ictal behaviors (i.e., spasms) can be induced with doses starting from 40 mg/kg. Limbic seizures are observed at 60 mg/kg and higher doses. Tonic seizures associated with respiratory arrest and death are commonly observed from a dose of 80 mg/kg or higher. For the kindling protocol, a dosage of 40 mg/kg was used.
The dose may vary for different mouse strains. It is recommended to conduct a pilot experiment to define the PTZ dose that best applies to the research question.
<ol>
	<li>Administer 10 mL/kg via systemic injections to the mice. Therefore, dissolve the PTZ powder in deionized water to reach the final concentration of 4 or 6 mg/mL (for the 40 and 60 mg/kg doses, respectively). 
	NOTE: To reduce variability, prepare a single PTZ solution for the entire experimental batch.</li>
</ol>
3. Administration of PTZ
<ol>
	<li>Before placing the animal in the arena, measure its body weight using a scale.</li>
	<li>Calculate the volume of PTZ solution necessary for each animal based on their body weight. 
	NOTE: For a dose of 60 mg/kg and a body weight of 30 g, inject the animal with 0.3 mL of a 6 mg/mL PTZ solution.</li>
	<li>Record the behavior for at least 1 h before the PTZ injection. 
	NOTE: This habituation period decreases novelty-induced ambulation and anxiety, while it can also be used to extract baseline activity.</li>
	<li>Administer PTZ either intraperitoneally (i.p.) or subcutaneously (s.c.)15,16. For s.c. administration, secure the animal with one hand, gently pinching the skin between its shoulder blades (Figure 2C).</li>
	<li>Using a 1 mL syringe attached to a 26 ½ G needle, insert the needle inside the subcutaneous space, parallel to the orientation of the body, and slowly inject the solution into the subcutaneous space. Gently remove the needle when the injection is complete, holding the skin at the needle exit to avoid the solution being spilled out due to cavity pressure (Figure 2C). 
	​NOTE: Injections must not be administered too quickly, as rapid accumulation of the solution can lead to spillage from the injection site or leakage upon needle removal.</li>
	<li>Make a note of any leakage, as it will affect the expression of ictal behaviors. Consider the possibility of immediate injection of an additional 50 µL of the same PTZ solution.</li>
	<li>Position the animal back in the arena for 1 h of continuous video recording.</li>
	<li>One hour after PTZ injection (or more), stop the recordings and copy the file for postprocessing (video tracking and behavioral coding).</li>
</ol>
<img alt="Mouse tracking setup in behavioral experiment; diagrams show maze, monitoring system, data analysis." class="xfigimg" src="/files/ftp_upload/68112/68112fig2v2.jpg" data-altupdatedat="1762509930000" data-alt="Figure 2"> 
Figure 2: Experimental setup and injection procedure. (A) Four transparent square arenas with top-down view cameras placed over the center were used to record behaviors and track animal positions before and after the PTZ administration. (B) Snapshot of one representative video obtained with the setup described in A. (C) Example of the subcutaneous injection of PTZ. The needle is placed between the shoulder blades of the animal in the subcutaneous space. (D) Schematic representation of the behavioral data acquisition and analyses used in the present study. <a href="https://www.jove.com/files/ftp_upload/68112/68112fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Animal welfare
<ol>
	<li>Remove and euthanize animals if a seizure lasts more than 5 min to prevent cell death and brain tissue damage, especially during repeated-injections protocols.</li>
	<li>Monitor the weight of the animals daily throughout the experimental period.</li>
	<li>Monitor the general condition of the animals, including food intake, locomotion, posture, grooming, and signs of pain or distress.</li>
	<li>Administer anti-inflammatory treatment (Ibuprofen, 5 mg/kg) and pain management as necessary. Provide supplementary food or water if required.</li>
	<li>Remove animals if they exhibit a weight loss greater than 20% that cannot be recovered within 2 days of individual supplementation, epidermal lesions, or any signs of chronic pain or discomfort unresponsive to pharmacological treatment.</li>
	<li>Euthanize all animals according to international and institutional ethical guidelines.</li>
</ol>
5. Software installation, configuration, and use
<ol>
	<li>For the analysis described in this study, use MouseLabTrack19 and Solomon Coder. Download them and follow the installation procedure (see Supplemental File 1 and Supplemental File 2). 
	NOTE: While MouseLabTrack runs under the MATLAB framework, Solomon Coder is an executable for the Windows operating system. Both software are open source.</li>
	<li>Click on Solomon in the Windows Start Menu or search for Solomon Coder and open the software.
	<ol>
		<li>If a configuration file is available (see Supplemental File 3), go to File | Load Configuration. The screen will load the predefined settings.</li>
		<li>If no configuration file is available,
		<ol>
			<li>Right-click on the free area to create a new button. Right-click on the newly created button to open the configuration screen.</li>
			<li>In the Button Name field, rename the button to represent the desired behavior.</li>
			<li>In the Button Face Color field, select a color.</li>
			<li>In the Categories field, rename Default to Behaviors (this will be the column name).</li>
			<li>In the Hotkey field, assign a keyboard key to mark the behavior during video analysis (e.g., key "1").</li>
			<li>(Optional) Check the box above Hotkey if the event is instantaneous, meaning it will register a single event when the key is pressed and released. If unchecked, the behavior will remain recorded until the key is released.</li>
		</ol>
		</li>
		<li>To open a video, go to File | Open Video.</li>
		<li>Press Play to start the video.</li>
		<li>As behaviors are observed, click the left mouse button or press the assigned keyboard key to mark them.</li>
		<li>To save the data as a comma-separated values (.csv) file go to Analyze | Save Output As. To save the Solomon coding sheet (.arch), go to File | Save Coding Sheet.</li>
	</ol>
	</li>
	<li>To open and load MouseLabTracker.
	<ol>
		<li>Open MATLAB, in the console, type `main()` and press Enter.</li>
		<li>To load the video, click the open button. After loading, the adjacent screen will display the video with a green background and the animal in white.</li>
		<li>Specify the time interval to be analyzed in the video. Click Start to set the beginning of the analysis. Click End to set the end of the analysis. 
		NOTE: If no interval is set, the software analyzes the entire video.</li>
		<li>To configure the tracking settings, go to the Tracking Settings tab.
		<ol>
			<li>Select Dark Objects / Light Background to indicate that the animal is darker than the background.</li>
			<li>To adjust the number of animals, go to Subjects and change the number to 1.</li>
			<li>To adjust the detection parameters, change the Threshold parameter setting so that the radius values are between 30 and 40.</li>
			<li>Define the color of the animal by clicking on Color and selecting the appropriate color.</li>
			<li>To define the arena, click on Define Arena, then select the top-left corner of the arena, then select the bottom-right corner of the arena.</li>
		</ol>
		</li>
		<li>To start the tracking process, click on Track. Wait until the progress bar reaches 100%.</li>
		<li>Once tracking is complete, save the output file of the analysis by clicking on Save results.</li>
	</ol>
	</li>
</ol>
6. Coding behaviors
<ol>
	<li>First, tag the PTZ administration time when the mouse reappears in the video frame after being placed in the observational arena. This moment serves as time zero, serving as the temporal reference for all other behaviors, including latency calculations.</li>
	<li>Observe spasms and myoclonus a few minutes after PTZ injection (Figure 3), followed by possible limbic seizures. Spasms are brief, transient, and fast contractions of axial muscles, including bilateral ear flexion, back muscle contraction, tail raising (Straub's tail), and rarely, a startle jump response (Figure 3A). Code spasms as discrete events, associating them with a single video frame without duration. 
	NOTE: Quantify the diversity and combination of different body parts in the expression of spasms (see Representative Results section).</li>
	<li>To code myoclonus, observe events longer than 200 ms; they are characterized by an abrupt, strong myoclonic jerk involving the head, neck, and paws. It may be associated with tremors and loss of body posture (Figure 3B). Subclassification includes myoclonus with tremors or with forward, leftward, or rightward posture loss. Duration typically ranges from 1 to 2 s. Due to their length, myoclonus requires coding across multiple time units for a single event.</li>
	<li>To code limbic seizures, observe events that resemble the one described by Racine and can be coded using the Racine scale. It typically begins with behavioral arrest or a staring spell, followed by olfactory and gustatory automatisms, orofacial movements, forelimb and/or head myoclonus, rearing with or without falling, wild running or jumping, and tonic seizure with respiratory arrest. Use the modified Racine scale18 to subclassify limbic seizures, which last more than 10-15 s and are distinguishable from spasms and myoclonus.</li>
	<li>Include additional behavioral classes based on the experimental protocol, allowing for a more comprehensive screening of behaviors.</li>
	<li>Mark the end of the recording session at a single time frame (1 h after PTZ injection). If the animal dies after a tonic seizure, identify the time of death with the ending mark. 
	​NOTE: The coding sheet can be saved at any point during the coding process and reopened within the software as needed for continued coding or modifications.</li>
</ol>
<img alt="Mouse behavior analysis during seizures; frames show spasm, myoclonus, limbic seizure stages." class="xfigimg" src="/files/ftp_upload/68112/68112fig3v2.jpg" data-altupdatedat="1762509930000" data-alt="Figure 3"> 
Figure 3: Snapshots of ictal behaviors observed after PTZ administration. (A) This is a representative example of one of the first spasms observed after PTZ injection. All frames are shown (sampling 30 fps). Note that the spasm lasts less than 200 ms and moves in the rostrocaudal direction. In this example, no overt tail lifting occurred. (B) A representative example of myoclonus associated with forward leaning. In this example, the myoclonus lasted more than 1 s, where multiple tail and forelimb clonus occurred. (C) Representative example of a limbic seizure. Before Racine stage 1, the mouse showed sustained limb myoclonus, including loss of body posture. The sequence shows all five traditional stages of the conventional Racine scale. In this case, the mouse would receive a score of 5. <a href="https://www.jove.com/files/ftp_upload/68112/68112fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
7. Data analysis and visualization
<ol>
	<li>Calculate the latency of the first occurrence of each of the behavior categories (e.g., Spasm, myoclonus, Seizure, etc.) by subtracting the PTZ injection time from its recorded time in the coding sheet.</li>
	<li>Calculate the duration of each myoclonus and seizure event, then sum them to obtain the total duration for each category.</li>
	<li>Calculate the rate of occurrence for each behavior by dividing the total number of events by the recording duration.</li>
	<li>Calculate the inter-event interval for each behavior by finding the time difference between consecutive events.</li>
	<li>Define episodes according to the temporal distribution of ictal behaviors in each recording session when applicable. Calculate the above parameters for each episode. 
	NOTE: The end of seizures leads to a postictal suppression period, marking the conclusion of an episode for this protocol. The reappearance of ictal behaviors marks the start of a new episode. 
	NOTE: In the absence of seizures, classify the recording session based on the temporal distribution of spasms and myoclonus.</li>
	<li>Test for normality using the Shapiro-Wilk test for each group.</li>
	<li>For parametric comparisons between multiple groups, use one-way analysis of variance (ANOVA) test. For paired data, use a paired t-test or repeated-measures ANOVA, depending on the data structure.</li>
	<li>Set statistical significance to p-value of &lt; 0.05. Additionally, round p-values below 0.0001 and report them as &lt;0.0001.</li>
</ol>

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<li>McIntyre, D. C., Poulter, M. O., Gilby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kindling%3A+some+old+and+some+new%5BTitle%5D)+AND+%22Epilepsy+Res%22%5BJournal%5D)">Kindling: some old and some new.</a> Epilepsy Res. 50 (1-2), 79-92 (2002).</li>
<li>Yuskaitis, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factors+influencing+the+acute+pentylenetetrazole-induced+seizure+paradigm+and+a+literature+review%5BTitle%5D)+AND+%22Ann+Clin+Transl+Neurol%22%5BJournal%5D)">Factors influencing the acute pentylenetetrazole-induced seizure paradigm and a literature review.</a> Ann Clin Transl Neurol. 8 (7), 1388-1397 (2021).</li>
<li>da Silva, L. F., Pereira, P., Elisabetsky, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Neuropharmacological+analysis+of+PTZ-induced+kindling+in+mice%5BTitle%5D)+AND+%22Gen+Pharmacol%22%5BJournal%5D)">A Neuropharmacological analysis of PTZ-induced kindling in mice.</a> Gen Pharmacol. 31 (1), 47-50 (1998).</li>
<li>Copping, N. A., Adhikari, A., Petkova, S. P., Silverman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+backgrounds+have+unique+seizure+response+profiles+and+behavioral+outcomes+following+convulsant+administration%5BTitle%5D)+AND+%22Epilepsy+Behav%22%5BJournal%5D)">Genetic backgrounds have unique seizure response profiles and behavioral outcomes following convulsant administration.</a> Epilepsy Behav. 101, 106547(2019).</li>
<li>Medina, A. E., Manhães, A. C., Schmidt, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sex+differences+in+sensitivity+to+seizures+elicited+by+pentylenetetrazol+in+mice%5BTitle%5D)+AND+%22Pharmacol+Biochem+Behav%22%5BJournal%5D)">Sex differences in sensitivity to seizures elicited by pentylenetetrazol in mice.</a> Pharmacol Biochem Behav. 68 (3), 591-596 (2001).</li>
<li>Pollo, M. L. M., Gimenes, C., Covolan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Male+rats+are+more+vulnerable+to+pentylenetetrazole-kindling+model+but+females+have+more+spatial+memory-related+deficits%5BTitle%5D)+AND+%22Epilepsy+Behav%22%5BJournal%5D)">Male rats are more vulnerable to pentylenetetrazole-kindling model but females have more spatial memory-related deficits.</a> Epilepsy Behav. 129, 108632(2022).</li>
</ol>

The authors declare no conflicts of interest.

This study employed a categorization of behaviors based on motor expressions to provide a more comprehensive framework for analyzing seizure dynamics. This approach enables quantification of key parameters, including latency, duration, and frequency of spasms, myoclonus, and limbic seizures. Results from weekly PTZ injections demonstrated that repeated exposure significantly alters the timing and frequency of limbic seizures, with a notable reduction in latency to the first spasms and limbic seizures, alongside an increa...

Behavioral characterization of seizures is the most employed readout in studies of the pathophysiology of epilepsy and seizure activity, and it is widely used for the screening of new antiseizure medication1,2. Most of the work published in the last 50 years used the progressive score system developed by Ronald J. Racine to describe behavioral seizure severity during amygdala and hippocampal kindling3. The Racine scale showed a strong correlation between behavior and electrophysiological paroxysmal activity in rats3 and cats4. It classifies the behavioral expression of the seizure into five progressive stages: "(i) mouth and facial movements, (ii) head nodding, (iii) forelimb clonus, (iv) rearing, and (v) rearing and falling"3. Due to its simplicity, ease of use and interpretation, velocity of scoring, and reproducibility, the use of the Racine scale in epilepsy and seizure studies continues to grow even nowadays (Figure 1).
<img alt="Yearly citation count graph; shows increasing trend from 1970 to 2024; publication analysis." class="xfigimg" src="/files/ftp_upload/68112/68112fig1.jpg" data-altupdatedat="1762509930000" data-alt="Figure 1"> 
Figure 1: Number of yearly citations received by Racine's 1972 study. Note the steady increase in the number of citations in the last 50 years. A Web of Science search performed on November 27th, 2024 revealed 6,040 citations, and 91 records did not contain information in the 'year of publication' field. <a href="https://www.jove.com/files/ftp_upload/68112/68112fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The scale was then applied to other animal models of epilepsy and seizure, including those using drugs to induce seizures. The early descriptions of kainic acid-induced seizures5 and status epilepticus (SE)6, as well as local7 and systemic pilocarpine-induced SE8, used the Racine scale to quantify both induced and spontaneous seizures. Interestingly, these drugs did not induce limbic seizures only, as observed in the electrically stimulated animals and described by Racine, but they also elicited a different set of behaviors that were poorly or less often described9.
The limitations of the Racine scale to grasp the diversity of ictal behaviors have been identified early by researchers using seizure models in which the seizure focus lies in other brain regions than the limbic system. For example, Garcia-Cairasco was one of the first to use ethological tools to evaluate the behavior in audiogenic seizures10, revealing the complexity and temporal progression of ictal behaviors10,11. Recently, Ivan Soltesz used sophisticated video-depth analysis and detailed temporal coding to reveal 'hidden behavioral fingerprints' in electrically kindled rats12. Building on these approaches, a novel coding system is introduced here for pentylenetetrazole-induced acute seizures in mice.
Pentylenetetrazole (PTZ) leads to seizures by inhibiting GABAergic neurotransmission13, resulting in increased neuronal excitability and synchronous activation of neuronal populations14,15. After a single high dose (40-80 mg/kg) of systemic PTZ, rodents exhibit reduced mobility, non-specific whisker movements, whole-body spasms and myoclonic jerks, orofacial and paw clonus, and full-blown limbic seizures16. Unlike the status epilepticus models, abnormal behavior after PTZ lasts up to 1 h and remits spontaneously16,17,18. Previous authors have proposed a revised version of the Racine scale for rats17 and mice18. Despite their attempt to provide new insights into the diversity of ictal behaviors, their new scale incorporates one or two new levels, associated with wild-running and/or jumping (level 6, van Erum et al.18) and tonic extension leading to respiratory arrest (level 7, van Erum et al.18).
This protocol presents a new approach for quantifying and representing PTZ-induced ictal behaviors, with a specific focus on spasms and myoclonus. These behaviors are among the most prevalent following systemic PTZ administration, yet they are often overlooked in seizure and epilepsy studies. Thus, this approach addresses a critical methodological gap by providing specific criteria for identifying spasms and myoclonus and distinguishing them behaviorally from limbic seizures. It demonstrates that spasms and myoclonus exhibit distinct temporal dynamics following both single and repeated PTZ administrations. This coding system is easy to implement, integrates novel parameters of abnormal activity into existing methodologies, and offers fresh insights into seizure generation mechanisms. Finally, this new strategy has the potential to standardize ictal behavior quantification across laboratories, enhance reproducibility, and broaden perspectives on seizure mechanisms.

<table><tbody><tr><td>0.9% Sodium Chloride Solution</td><td>Fresenius Kabi Brasil Ltda</td><td>74MK3168</td><td>Closed system for intravenous use; injectable solution, 250 mL.</td></tr><tr><td>Analytical Scale</td><td>Shimadzu</td><td>ATY224</td><td>Maximum capacity: 220 g, Minimum capacity: 10 mg, Readability: 0.1 mg. Serial No: 1311070096</td></tr><tr><td>Camera</td><td>Jortan</td><td>Jortan-8816AHD</td><td>IR CCD camera, NTSC system, 2.0 MP resolution, 3.6 mm lens, DC 12 V power supply.</td></tr><tr><td>Digital Lux Meter</td><td>Minipa</td><td>MLM-1011</td><td>Measures light intensity in lux units.</td></tr><tr><td>Hypodermic Needle</td><td>Descarpack</td><td>353101</td><td>Size 26.5 G.</td></tr><tr><td>Mice</td><td>-</td><td>-</td><td>Male C57BL/6 wild type mice. Two-three months old.</td></tr><tr><td>Pentylenetetrazole</td><td>Sigma-Aldrich (Merck)</td><td>P6500-25G</td><td>Powder, CNS stimulant. Purchased: 25 g. Storage: -25 &amp;deg;C.</td></tr><tr><td>Software: MouseLabTracker</td><td>&amp;nbsp;Giuliano Vilela</td><td>-</td><td>Open-source software, reference paper: DOI: 10.1016/j.jneumeth.2006.04.005.&lt;br/&gt; Site: https://neuro.ufrn.br/softwares/mouselabtracker</td></tr><tr><td>Software: Solomon Coder</td><td>Andr&amp;aacute;s P&amp;eacute;ter</td><td>-</td><td>Open-source software, v.19.08.02.&lt;br/&gt; Site:&amp;nbsp; https://solomon.andraspeter.com/</td></tr><tr><td>Thermo-Hygrometer</td><td>Hikari</td><td>HK-T240</td><td>Measures temperature and humidity.</td></tr></tbody></table>

behavioral characterization of pentylenetetrazole-induced seizures: moving beyond the racine scale

Behavioral Characterization of Pentylenetetrazole-induced Seizures: Moving Beyond the Racine Scale

Detailed behavioral characterization of seizures is essential in animal models of epilepsy. Although various protocols exist for inducing seizures, the Racine scale remains the most widely adopted method for its quantification. Initially developed to score the progressive recruitment of behaviors during electrical kindling of limbic structures, the scale has since become the standard for classifying seizures across models with varying etiologies.
However, careful examination of behavior following pentylenetetrazole (PTZ) administration, a commonly used seizure model, reveals that the Racine scale cannot capture the full spectrum of behavioral alterations induced by this GABAergic antagonist. After a single subcutaneous high-dose PTZ injection, mice first exhibit non-convulsive behaviors such as hypotonia, reduced ambulation, behavioral arrest, and tremors. These are succeeded by ictal behaviors, including spasms, myoclonic jerks, and limbic seizures, though only the latter is quantified by the Racine scale.
To overcome these limitations, a refined approach is employed that combines video tagging with animal tracking to distinguish spasms and myoclonus as distinct behavioral expressions from limbic seizures. These early behaviors may facilitate the recruitment of limbic structures, eventually triggering limbic seizures. The method is straightforward to implement and enables a more comprehensive assessment of PTZ-induced acute seizures. Its utility is further evident in the PTZ kindling protocol, in which repeated low doses of PTZ result in temporal changes in spasms, myoclonus, and limbic seizure severity. This approach holds potential for improving reproducibility in seizure and epilepsy research.

To evaluate the coding strategy and its ability to detail ictal behaviors during PTZ-induced seizures, three doses of PTZ were first tested and their effects on C57BL/6 mice observed (40, 60, and 80 mg/kg, N = 7, 5, and 5, respectively). Behavioral outcomes observed across three doses of PTZ revealed clear dose-dependent differences after a single injection. Animals treated with 40 mg/kg PTZ displayed spasms but no limbic seizures or myoclonus. In contrast, animals treated with 60 and 80 mg/kg consistently expressed spas...

The protocol introduces an innovative method for analyzing and visualizing behaviors associated with pentylenetetrazole-induced seizures in mice. This approach extends beyond traditional Racine's scores, providing a more comprehensive evaluation of the diversity and temporal dynamics of ictal behaviors.

Detailed behavioral characterization of seizures is essential in animal models of epilepsy. Although various protocols exist for inducing seizures, the ...

Our research focuses on characterizing and quantifying two types of ictal behaviors, the spasm and myoclonus, after administering pentylenetetrazole. We're trying to unveil how these behaviors contribute to the onset of limbic seizures and how the patterns evolve over time. Lately, there's been a lot of progress in understanding how focal seizures spread through neural networks and eventually generalize.With the help of computer-assisted video analysis, we're now able to capture much finer details about how seizures unfold. Most seizure studies rely on the Racine scale, but that approach often misses important behaviors triggered by PTZ. Our protocol helps fill that gap by identifying and quantifying events that are usually overlooked, both before and after the limbic seizure actually appears.Our protocol allows for a much more detailed analysis of behavior. Instead of relying on a single score, like the Racine scale, we can measure multiple parameters per each ictal event, giving us a deeper understanding of what's actually happening during seizures. Right now, our lab is focused on exploring whether alternative anti-seizure medications can prevent, delay, or lessen spasms and myoclonus, and how that might affect the development of limbic seizures.To begin, use a scale to measure the body weight of the animal. Calculate the volume of pentylenetetrazole solution needed for each animal based on their body weight. Record the animal's behavior inside the arena for at least one hour prior to the pentylenetetrazole injection.This habituation period reduces novelty-induced ambulation and anxiety and allows extraction of baseline activity. Prepare the pentylenetetrazole injection. For subcutaneous administration, use one hand to secure the animal and gently pinch the skin between its shoulder blades.Using a 1-milliliter syringe attached to a 26-1/2-gauge needle, insert the needle into the subcutaneous space in parallel with the body orientation and inject the solution slowly. Once the injection is complete, gently remove the needle while holding the skin at the exit point to prevent leakage due to internal cavity pressure. Observe and make a note of any solution leakage from the injection site.If leakage is present, consider administering an additional 50 microliters of the same pentylenetetrazole solution. Now, place the animal back into the arena and record continuously for one hour. One hour or more after pentylenetetrazole injection, stop the recording and copy the video file for post-processing.Use this for video tracking and behavioral coding. Open the Solomon Coder software. If a configuration file is available, select File and click Load Configuration to load predefined settings.To open a video, select File and click Open Video. Press play to begin video playback. Click the left mouse button or press the assigned keyboard key to mark observed behaviors during playback.To save the data, go to Analyze and select Save Output As to create a comma-separated values file. For the coding sheet, go to File and choose Save Coding Sheet. Tag the pentylenetetrazole administration time at the moment the mouse reappears in the video frame after being returned to the observational arena.Use this moment as time zero to serve as the temporal reference point for all subsequent behaviors and latency calculations. Observe for spasms and myoclonus a few minutes after pentylenetetrazole injection. Identify spasms as fast, transient contractions of axial muscles, including bilateral ear flexion, back muscle contraction, tail raising, and rarely, a startle jump.Code these spasms as discreet, instantaneous events, associating them with a single video frame. To code myoclonus, identify events longer than 200 milliseconds marked by a strong, abrupt myoclonic jerk of the head, neck, and paws. Classify subtypes based on associated tremors or directional posture loss, including forward, leftward, or rightward.Code the event across multiple frames, as the duration typically ranges from one to two seconds. For limbic seizures, identify behaviors aligned with the Racine scale, starting with behavioral arrest or staring. Follow with automatisms, orofacial movements, forelimb or head myoclonus, and escalation to rearing, jumping, or tonic seizures.Use the modified Racine scale to assign subcategories to events lasting longer than 10 to 15 seconds. Finally, mark the end of the recording session on a single video frame exactly one hour after pentylenetetrazole injection. The figure details parameters that can be extracted from this coding system, including latency to each ictal behavior, number of events, event duration, inter-event interval, and severity of limbic seizures.Spasms typically involved trunk stiffening in majority of events, while only 6%of spasms exclusively activated the tail. Myoclonus presented predominantly as tremors in 64%of cases. Forward, rightward, and leftward motions were also noted.The latency to onset was significantly different among spasms, myoclonus, and limbic seizures, with spasms occurring earliest. The total number of spasms was significantly higher than that of myoclonus or limbic seizures. The median inter-event interval did not differ significantly between spasms and myoclonus, but the limbic seizures lasted significantly longer than myoclonus.In the daily PTZ injection protocol, spasm latency and inter-event interval significantly decreased by day eight, whereas the overall spasm events increased.

behavioral-characterization-pentylenetetrazole-induced-seizures

Research

JoVE Journal

Neuroscience

This video demonstrates electroencephalographic (EEG) recording of epileptic seizures in epilepsy-induced rats. A rat with electrodes implanted in the ventral hippocampus is connected to the EEG recording system and placed in a plexiglass chamber. The rat is allowed to move freely while the brain activity is recorded to monitor the occurrence of seizures.

Electroencephalographic Recording of Epileptic Seizures in Epilepsy-Induced Rats

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.

Establishing a Pentylenetetrazole-Induced Epileptic Seizure Model in Mice

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

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.

Pentylenetetrazole-Induced Kindling Mouse Model

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute seizures.

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

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.

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

This systematic protocol describes a new animal model of post-traumatic epilepsy after repetitive mild traumatic brain injury. The first part details steps for traumatic brain injury induction using a modified weight drop model. The second part provides instructions on the surgical approach for single- and multi-channel electroencephalographic data acquisition systems.

Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury

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.

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

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Discover a computer-assisted video analysis protocol for quantifying ictal behaviors in pentylenetetrazole-induced seizures. This method captures spasms and myoclonus overlooked by traditional Racine scale scoring.

Behavioral Characterization of Pentylenetetrazole-induced Seizures: Moving Beyond the Racine Scale

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