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 similarities to humans. However, these methods can be time-consuming and expensive, necessitating an alternative approach^4,5. Non-mammalian creatures like fruit flies, leeches, tadpoles, zebrafish, and roundworms have been utilized in studies and have shown promising outcomes⁶. Furthermore, it was shown that planaria could provide a comparative genomic study model between invertebrate and human genomes alongside the capability to test pro-convulsant, anti-seizure medications (ASM), and behavioral patterns⁶. Planaria (Phylum Platyhelminthes), known as flatworms and members of the Turbellaria class, are primarily renowned for their regenerative abilities; however, this investigation concentrates on their response to seizure-inducing substances.

Planaria share fundamental neurological mechanisms with humans, such as responsiveness to serotonin and dopamine, showing a 95% similarity to the nervous system-related genes in the mammalian brain and possessing a recognizable brain structure⁷. Additionally, they exhibit observable motions in laboratory conditions and are cost-effective, time-efficient, and ethical compared to rodents or other mammals. These observable behaviors, such as screw-like, C-like, and walnut-shaped motions, have been extensively documented for decades and are associated with substances like cocaine, nicotine, dopamine, and pilocarpine^{7,8,9,10,11,12,13,14}. Hence, planaria emerge as a viable model for epilepsy drug research in humans.

This method aims to characterize neurons of planaria that have been exposed to pilocarpine using a Golgi stain. The Golgi stain is used to visualize neurons under light microscopy and has been used to investigate whether a change in morphology is related to seizures^15,16,17. Current literature has no evidence of Golgi staining being performed on planarian brains. Although previous studies have documented pharmacological effects by observing behavioral phenotypes, this manuscript is the first to characterize the neurons of planaria exposed to pilocarpine using Golgi staining^11,18. This technique proves valuable in visualizing and understanding the morphological changes associated with seizures. This study noted a significant increase in the frequency of oscillating dorsal oscillation behavior in planarian worms as the concentration of pilocarpine was increased.

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 damaging it.
4. Record the behavior of the planaria for 1 h using any camera that records the behaviors well and is observable to the human eye, positioned over the 4 x 3 well plate in normal indoor lighting conditions.
5. Repeat steps 1.1-1.4 with concentrations of 3 mM, 4 mM, and 6 mM pilocarpine dissolved in spring water.

2. Motility analysis

NOTE: Planaria behaviors were recorded in 2.5 cm diameter wells. The 1 h long video recordings were split into 30 min parts and cropped using commercial software to analyze planaria individually.

1. Begin a new experiment using automated behavior analysis software. Open the enhanced automated behavior tracking system. Click New under New experiment. Name the experiment.
2. Set up the experimental settings as described below.
   1. Click Experiment Settings in the menu prompt. Ensure that settings are default: video source (from video file), Number of Arenas (1), Tracked Features (Center-point detection), Body Point Detection Technique (Contour-based), Analysis Options (none), Units (cm, s, deg).
3. Set up the arena settings as described below.
   1. Click Arena Settings under the Arena Settings tab in the top left Setup panel. When prompted to upload a video, select recorded footage of planaria from the stored file location. Once the video is selected, click Grab.
   2. Follow the prompts on the top right panel of the screen. Click on 1. Draw Scale to Calibrate. On the video image of the well, drag a scale line from edge to edge of the specimen well. Enter the real-world distance of the well (2.5 cm).
   3. Click on 2. Select Shape and Draw Arena. Ensure that Arena 1 is selected in the right panel. Move the mouse cursor to the top middle of the screen and click the Circle Icon.
   4. Create a circle in the uploaded video image by dragging from one perimeter of the arena to another. Ensure the arena is in the orange zone. Adjust the shape so that it fits the arena. Ensure that the Arena 1 label is within the bounds of the arena.
   5. At the bottom of the right panel, change the Shape, Size, and Position (Width:2.75, Height:2.75, X:0, Y:0) to the desired measurements.
   6. Click 3. Select Shape and Draw Zones (Optional). Zone Group 1 within the right panel will now be selected. Move the mouse cursor to the top middle of the screen and select the Circle Icon. Create a smaller circle in the arena image by dragging it within.
   7. In the bottom right panel, change Shape, Size, and Position to desired measurements (Width:1.75, Height:1.75, X:0, Y:0).
   8. Click the Add Zone Label button in the same menu bar as the circle. Click Within the Center of the Smaller Circle. Right-click the Zone 1 tag in the right panel to rename the Zone Center.
   9. Click the Add Zone Label button again. Click on the Perimeter of the Arena, Outside of the Center Zone. Right click Zone 2 in the right panel to rename the zone Perimeter.
   10. Click 4. Validate Setup to ensure Valid Arena Settings.
4. Set up the Trial control settings as described below.
   1. Click Trial Control Settings under the Trial Control Settings tab in the top left Setup panel. Six component boxes will appear. Click Settings within the fourth box labeled Condition, which includes Time (1) and Infinite time (condition never met).
   2. Click the Bubble beside After, then enter the duration (30 min) in the right text box. There will also be a drop-down menu to the right of the time text box to select between h, min, and s.
      NOTE: Detection settings for recorded footage will depend on footage quality. The following methods can be manipulated to observe the best results, specific to individual footage.
5. Set up the detection settings as described below.
   1. Click Detection Settings under the Detection Settings tab in the top left Setup panel. Click the Select Video button in the top right panel. Upload the desired video to base detection settings on.
   2. Click the Advanced tab in the same right panel. Now, under the Method tab, ensure it is set to Gray Scaling. Change the range (0 - 100).
   3. Under the Smoothing tab of the right panel, ensure Video pixel smoothing is None, Dropped frames correction is Off, and Track noise reduction is Off.
   4. Under the Subject Contour tab of the right panel, adjust the sequence setting and change Erosion, Dilation, and Erosion to 1, 1, and 0, respectively.
   5. Under the Subject Size tab, change Minimum (0) and Maximum (125000). Click Save in the bottom right.
6. Set up a sequence of video trials for observation as described below.
   1. Click Trial List in the top left Setup panel. Upload video footage of specimens in the desired sequence by clicking the Ellipses under the System Video file column. To add more trials, click Add Trials near the top left of the Trial List Panel.
7. Set up Acquisition settings.
   1. Click Acquisition under the top left Acquisition tab. Under Acquisition settings in the right panel, Click Track all planned trials.
   2. To begin acquisition, click on the Red Button in the lower middle of the screen next to Ready for Start. Video acquisition will now begin and take a certain amount of time, depending on the quantity and length of video trials uploaded.
8. Set up the analysis settings as described below.
   1. Click Analysis Profile under the left panel Analysis tab. Click on the default selected dependent variables, Velocity and Distance Moved, and delete them.
   2. Within the dependent variables panel, under the Location tab, click In Zone. Click the Center and Perimeter Zone boxes so that they are check marked.
   3. Click the Trial Statistics tab within the In Zone box. Click to uncheck Latency to First. Click Add.
   4. Under the Body tab, click the Rotation button. Click Clockwise and leave the Threshold (50.00 degrees) and minimum distance moved (2.00 cm) at default. Click Add.
   5. Click the Rotation button again and click Counterclockwise. Click Add.
9. Analyze tracks as described below.
   1. Under the Results tab, click Track Visualization. On the right panel, under Filter, uncheck Last. The playback control bar in the bottom middle panel of the screen will depict track visualization of specimens during specific times of observation.
10. Analyze and export results. Results can be found in the remainder of the bottom left panel tabs. To export data, click Raw Data or Statistics under Export within the left panel.

3. Euthanasia

1. Prepare a 22 mM solution of eugenol in spring water. Place planaria into Petri dishes with 9 mL of 22 mM eugenol solution using the cut transfer pipette, keeping each concentration separate. Euthanize for 3 min or until all movement has ceased.
2. Place planaria into a fresh Petri dish filled with 9 mL of spring water to rinse 1x-2x, and place into either the Golgi solution described below (Golgi stain) or 4% paraformaldehyde (immunofluorescence stain).

4. Histological analysis

1. Golgi staining
   1. After planaria are euthanized, place them into a beaker or Petri dish filled with a 1:1 mixture of Solutions A and B from the Golgi Stain Kit. Replace solutions A and B the following day by placing planaria into a new beaker filled with a mixture of solutions A and B.
   2. Allow planaria to sit for 1 week in solutions A and B.
   3. Remove planaria from solutions A and B and place it into solution C in another beaker. Replace solution C the following day. Allow planaria to sit for a minimum of 72 h and up to 1 week in solution C.
   4. Remove planaria from solution C with a transfer pipette and embed by placing a small amount of OCT compound on a chuck, adding the planaria, and then surrounding the planaria with OCT compound. Transversally cut planaria into 5 µM sections with the cryostat machine at -24° C.
   5. Mount planaria onto gelatin-coated slides using a transfer pipette and solution C and allow it to dry in the dark at RT for up to 3 days.
   6. Stain slides using the staining solution (1 part solution D, 1 part solution E, 2 parts Springwater) by submerging the slides in beakers with the solutions as follows: Staining solution for 10 min, then in Spring water 2x for 4 min each, then 50% ethanol for 4 min, then 75% ethanol for 4 min, then 95% ethanol for 4 min, then in 100% ethanol 4x for 4 min, and finally in xylene 3x for 4 min.
   7. Coverslip slides with histological mounting media and observe tissue under Bright field Microscopy.
2. Immunofluorescence staining
   1. After planaria are euthanized, place into 4% paraformaldehyde for at least 24 h. It can also be stored in a fridge after this step.
   2. Transfer planaria to 20% sucrose from 4% PFA and leave in it for 1 day. Remove planaria from sucrose and place it into PBS buffer for 5 min.
   3. Rinse with 3-4 washes of PBS buffer and transfer to 20% sucrose for storage until ready to stain.
   4. Place into 70%, 95%, and 100% alcohol solutions for 1 min each. Place in xylene for 1 min.
   5. Place in 95% and 70% alcohol for 1 min each. Place into PBS buffer 3x for 5 min each. Place into a 60:40 methanol and hydrogen peroxide solution for 10-15 min.
   6. Rinse in PBS 3x for 5 min each. Add the volume of 4% paraformaldehyde used previously in 4.2.1 into this PBS buffer.
   7. Rinse in PBS 3x for 5 min each. Block for 1 h with powdered milk, by covering the planaria with it.
   8. Dilute primary antibody (1H6) to 5 µg/mL with PBS and incubate the slides overnight at 4 °C in the antibody solution. The following day, rinse planaria in PBS for 10 min.
   9. Dilute secondary antibody (goat anti-mouse IgG) to 1 µg/mL in PBS and incubate the slides overnight at RT in the antibody solution. The following day, rinse planaria in PBS for 15 min.
      NOTE: Different antibodies have different optimal concentrations; determine this before starting the experiment.
   10. Mount whole planaria on the slide, cover with a coverslip, seal with aqueous mounting media, and observe the tissue under a fluorescent microscope.

5. Image analysis

1. Open the open-source image analysis software application. Install the colour_deconvolution2.jar plugin to the open-source image analysis software application by dragging the plugin to the application.
2. Select the image to analyze and drag it to the open-source image analysis software application. Go to the Image tab. Click color. Click Colour Deconvolution2 v2.1.
3. In the vectors option, click H DAB. In the output option click 8bit_Transmittance. Make sure the Simulated LUTs, Cross product for Colour 3, Show matrices, and Hide legend are all selected. Click OK.
4. Select the Image in Black and White to ensure the open-source image analysis software application can effectively analyze the image.
5. Go to the Image tab. Click Adjust. Click Threshold. Adjust the threshold so that the DAB staining or the foreground is red in color with a white background. Make sure the threshold is the same for each image if multiple images are being quantified.
6. Click Analyze. Click Set Measurements. Select Area, Min & max gray value, Limit to threshold, Display label, Median, and Mean gray value. Click OK. Click Analyze. Click Measure. Obtain the result.
7. Repeat steps 1.2 to 1. U6ntil all the images are quantified. Click the Results Image. Click File. Click Save As. Save file as a spreadsheet and view the data on the spreadsheet software.

6. Statistical Analysis

1. Calculate the mean and standard errors of the mean (SEM) for the planaria's behavior using Analysis of Variance (ANOVA) and Student's t-test for statistical significance (p < 0.05).

Results

The following behavior was observed by the planaria exposed to varying concentrations of pilocarpine:
Dorsal oscillations: a bubble-like formation that travels from the cranial end of the planarian's body to the caudal end.
Head oscillations: bubble-like formation by the head of the planarian that forms a tadpole-like appearance.
C-Shape: head moving clockwise and tail moving counterclockwise to form a C.
Head flick: the head of the planarian abruptly jerks to the left or the right.
Tail...

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