Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Summary

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Abstract

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg²⁺ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Introduction

Acute seizure models can successfully reproduce electrographic signatures reminiscent of ictal events observed in the electroencephalogram (EEG) of individuals experiencing a seizure. Researchers use these ictal-like events (herein referred to as 'ictal events') as surrogates for the seizure event¹. Clinically, ictal events serve as a reliable proxy for seizure events since seizures are a neurological disorder which originates from the brain. In the epilepsy monitoring unit, neurologists rely upon the detection of ictal events to confirm the brain's epileptogenic region and isolate it for resection². In the i....

Protocol

All research involving patients was performed under a protocol approved by the University Health Network Research Ethics Board in accordance with the Declaration of Helsinki. Procedures involving animals were in accordance with guidelines by the Canadian Council on Animal Care and approved by the Krembil Research Institute Animal Care Committee.

1. Protocol I: Acute In vitro Seizure Model

1. Preparation of dissection solutions and artificial cerebrospinal fluid
   1. Carbogenate ultrapure water (which is water filtered with a resistivity of 18.2 MΩ·cm) with carbogen (95% O₂/5% CO....

Results

The application of 100 µM 4-AP to good-quality (undamaged) 450 µm-sized cortical brain slices from a juvenile VGAT-ChR2 mouse reliably induced recurrent ictal events (> 5 s) within 15 min (Figure 1Ai). The application of 100 µM 4-AP to slices of poor-quality resulted in bursting events or spiking activity (Figure 1Aii). On average, 40% of the slices from each dissected mouse brain successfully generated ictal events. Moreover,.......

Discussion

The brain slices are treated with a proconvulsant drug or an altered ACSF perfusate to increase the neural network's excitability and promote a precipitation of ictal events (electrographic seizure-like events). For mice, the preferred coronal slices of the somatosensory-motor area should contain the cingulate cortex, area 2 (CG), but not the retrosplenial area (RS); these anatomical markers help identify the range of coronal slices that are best for inducing ictal events. An optional modification for mice tissue is .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium pentobarbital	N/A	N/A	Purchased through the Toronto Western Hospital's Suppliers
1 mm syringe	N/A	N/A	Purchased through UT Med Store
25G 5/8” sterile needle	N/A	N/A	Purchased through UT Med Store
Single edge razor blade (2x)	N/A	N/A	Purchased through UT Med Store
Instant adhesive glue	N/A	N/A	Purchased through UT Med Store
Lens paper	N/A	N/A	Purchased through UT Med Store
Glass petri dish (2x)	N/A	N/A	Purchased through UT Med Store
Splinter forceps (2x)	N/A	N/A	Purchased through UT Med Store
PVC handle micro spatula	N/A	N/A	Purchased through UT Med Store
Micro spoon with flat end	N/A	N/A	Purchased through UT Med Store
Detailing brush 5/0	N/A	N/A	Purcahsed from a boutique art store
Wide bore transfer pipette	N/A	N/A	Purchased through UT Med Store
Dental Tweezer	N/A	N/A	Purchased through UT Med Store
Thermometer (digital)	N/A	N/A	Purchased on Amazon.ca
Check carbogen tank (95%O2/5%CO2)	N/A	N/A	Purchased through the Toronto Western Hospital's Suppliers
Vibratome	Leica	N/A	Purchased through the Toronto Western Hospital's Suppliers
brain slice incubation chamber (a.k.a. brain slice keeper)	Scientific Systems Design Inc	N/A
Sodium Chloride (NaCl)	N/A	N/A	Purchased through UT Med Store
Sodium Bicarbonate	N/A	N/A	Purchased through UT Med Store
Dextrose	N/A	N/A	Purchased through UT Med Store
Potassium Chloride (KCl)	N/A	N/A	Purchased through UT Med Store
Magnesium Sulfate (MgSO4 H2O)	N/A	N/A	Purchased through UT Med Store
Sodium phosphate monobasic monohydrate (HNaPO4·H2O)	N/A	N/A	Purchased through UT Med Store
Calcium Chloride (CaCl2·2H2O)	N/A	N/A	Purchased through UT Med Store
Sucrose	N/A	N/A	Purchased through UT Med Store