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

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.