Planarian as an Animal Model for Experimental Acute Seizure

Summary

Seizures negatively impact various functions and life quality. Planaria worms were exposed to varying concentrations of chemoconvulsants to evaluate their seizure phenotypes and disruptive motility. This study proposes using planaria worms as a model for acute seizures in humans and holds significance in drug development for epilepsy.

Abstract

Epilepsy is among the most prevalent neurological disorders characterized by recurring spontaneous seizures. Seizures represent a clinical manifestation of uncontrolled, excessively synchronized neural cell activity. The extent of brain damage from seizures depends on their duration and intensity. Regrettably, there is no effective remedy for epilepsy. The aim of this investigation is to assess whether the planaria worm Dugesia dorotocephala could serve as a model to aid in identifying and developing treatments for epilepsy that can target acute seizures. Currently, various models, such as marine models, are used to evaluate antiseizure medications (ASM). However, they are very expensive, and there are ethical concerns. Alternatively, invertebrate models offer a cost-effective research opportunity in the drug discovery process for ASM. Planaria belong to the flatworm family and inhabit marine freshwater and terrestrial environments. Dugesia dorotocephala is the dominant species of aquatic planaria across North America. D. dorotocephala presents as a viable invertebrate model for epilepsy studies due to its cost-effectiveness, vertebrate-like neurons, and quantifiable behaviors, unlike other invertebrates or larger animals. They have been used in various pharmacology and environmental toxicology studies related to age, memory, and regeneration. In this study, planaria were exposed to different concentrations of pilocarpine, a common chemoconvulsant to study their behavior upon exposure. Following the observation, planaria were euthanized and preserved in either formaldehyde or Golgi solution for neurohistological assessment. Six distinct behavioral phenotypes were observed in planaria: dorsal oscillations, head oscillations, tail dorsal expansion, C-shape, head flick, and tail flick. Dorsal oscillation frequencies were significantly increased among experimental groups compared to the control and exhibited dose dependence. Additionally, pilocarpine disrupted the motility of the planaria. Pilocarpine-induced seizures in planaria can serve as a model to evaluate acute seizures and antiseizure medication, which is essential in developing therapeutic interventions for human patients suffering from epilepsy.

Introduction

Epilepsy, characterized by two or more seizures within 24 h without an apparent cause, impacts ~50 million people globally¹. Among them, 10-15 million individuals are reported to have drug-resistant epilepsy². Therefore, epilepsy drug investigation is crucial. The condition entails brief episodes of either partial or generalized involuntary movement, ranging from blank staring to body stiffening and shaking, and is linked with a surge of electrical activity in the brain³.

Historically, epilepsy research has relied on rodents and other mammals due to their evolutionary s....

Protocol

NOTE: The overall experimental design is described in Figure 1.

1. Behavior phenotype assay

1. Prepare concentrations of 1 mM and 2 mM pilocarpine dissolved in spring water as well as control with Springwater only.
2. Pipette 3 mL of each solution into a 4 x 3 well plate, with each row representing a different concentration of pilocarpine.
3. Place one lab-reared Dugesia dorotocephala that is about 2 weeks old in each of the 12 wells using a transfer pipette with the tip cut off.
   NOTE: Cut the tip so the end is large enough to fit the planaria without....

Discussion

This study demonstrated pilocarpine-induced behavior in planaria at different concentrations. The most pertinent behavior was oscillating dorsal expansion, as this behavior has not been documented in other studies regarding seizure-like behavior in planaria^9,10,11,12. The mechanism by which pilocarpine induces seizure-like behaviors in planaria is still unknown. However, this study demonstrates.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL centrifuge tubes
4% paraformaldehyde solution	Himedia	TCL119
Aqueous mounting media	Clini Sciences	NB-47-02240-30ML
Beakers (one for each concentration tested)
Carolina Springwater	carolina	132450
Cryostat
Diluted primary and secondary antibodies
Ethanol (100%)	sigma Aldrich
EthosVision XT 16	noldus
Fiji Version 2.9.0
Gelatin-coated slides	sigma Aldrich	643203
Golgi Antibody 1H6	DSHB	AB_2619608
Golgi stain kit_	Neuroscience Associate	PK 401/401A
Hydrogen Peroxide
Methanol
Mounting media	thermo fischer scientific
OCT compound
PBS buffer	sigma Aldrich	P4417
Powdered Milk
Tin foil
Transfer pipettes
Xylene