We want to thank EVMS Research Incentive Fund (PI: A.E. Musto) and Dr. Jorge Jacot for his helpful suggestions with optimizing the immunofluorescence protocol.

NOTE: The overall experimental design is described in Figure 1.
1. Behavior phenotype assay
<ol>
	<li>Prepare concentrations of 1 mM and 2 mM pilocarpine dissolved in spring water as well as control with Springwater only.</li>
	<li>Pipette 3 mL of each solution into a 4 x 3 well plate, with each row representing a different concentration of pilocarpine.</li>
	<li>Place one lab-reared Dugesia&#160;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.</li>
	<li>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.</li>
	<li>Repeat steps 1.1-1.4 with concentrations of 3 mM, 4 mM, and 6 mM pilocarpine dissolved in spring water.</li>
</ol>
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.
<ol>
	<li>Begin a new experiment using automated behavior analysis software. Open the enhanced automated behavior tracking system. Click New under New experiment. Name the experiment.</li>
	<li>Set up the experimental settings as described below.
	<ol>
		<li>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).</li>
	</ol>
	</li>
	<li>Set up the arena settings as described below.
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>In the bottom right panel, change Shape, Size, and Position to desired measurements (Width:1.75, Height:1.75, X:0, Y:0).</li>
		<li>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.</li>
		<li>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.</li>
		<li>Click 4. Validate Setup to ensure Valid Arena Settings.</li>
	</ol>
	</li>
	<li>Set up the Trial control settings as described below.
	<ol>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Set up the detection settings as described below.
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>Under the Subject Size tab, change Minimum (0) and Maximum (125000). Click Save in the bottom right.</li>
	</ol>
	</li>
	<li>Set up a sequence of video trials for observation as described below.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Set up Acquisition settings.
	<ol>
		<li>Click Acquisition under the top left Acquisition tab. Under Acquisition settings in the right panel, Click Track all planned trials.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Set up the analysis settings as described below.
	<ol>
		<li>Click Analysis Profile under the left panel Analysis tab. Click on the default selected dependent variables, Velocity and Distance Moved, and delete them.</li>
		<li>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.</li>
		<li>Click the Trial Statistics tab within the In Zone box. Click to uncheck Latency to First. Click Add.</li>
		<li>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.</li>
		<li>Click the Rotation button again and click Counterclockwise. Click Add.</li>
	</ol>
	</li>
	<li>Analyze tracks as described below.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
3. Euthanasia
<ol>
	<li>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.</li>
	<li>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).</li>
</ol>
4. Histological analysis
<ol>
	<li>Golgi staining
	<ol>
		<li>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.</li>
		<li>Allow planaria to sit for 1 week in solutions A and B.</li>
		<li>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.</li>
		<li>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 &#181;M sections with the cryostat machine at -24&#176; C.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Coverslip slides with histological mounting media and observe tissue under Bright field Microscopy.</li>
	</ol>
	</li>
	<li>Immunofluorescence staining
	<ol>
		<li>After planaria are euthanized, place into 4% paraformaldehyde for at least 24 h. It can also be stored in a fridge after this step.</li>
		<li>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.</li>
		<li>Rinse with 3-4 washes of PBS buffer and transfer to 20% sucrose for storage until ready to stain.</li>
		<li>Place into 70%, 95%, and 100% alcohol solutions for 1 min each. Place in xylene for 1 min.</li>
		<li>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.</li>
		<li>Rinse in PBS 3x for 5 min each. Add the volume of 4% paraformaldehyde used previously in 4.2.1 into this PBS buffer.</li>
		<li>Rinse in PBS 3x for 5 min each. Block for 1 h with powdered milk, by covering the planaria with it.</li>
		<li>Dilute primary antibody (1H6) to 5 &#181;g/mL with PBS and incubate the slides overnight at 4 &#176;C in the antibody solution. The following day, rinse planaria in PBS for 10 min.</li>
		<li>Dilute secondary antibody (goat anti-mouse IgG) to 1 &#181;g/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.</li>
		<li>Mount whole planaria on the slide, cover with a coverslip, seal with aqueous mounting media, and observe the tissue under a fluorescent microscope.</li>
	</ol>
	</li>
</ol>
5. Image analysis
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Select the Image in Black and White to ensure the open-source image analysis software application can effectively analyze the image.</li>
	<li>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.</li>
	<li>Click Analyze. Click Set Measurements. Select Area, Min &#38; max gray value, Limit to threshold, Display label, Median, and Mean gray value. Click OK. Click Analyze. Click Measure. Obtain the result.</li>
	<li>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.</li>
</ol>
6. Statistical Analysis
<ol>
	<li>Calculate the mean and standard errors of the mean (SEM) for the planaria&#39;s behavior using Analysis of Variance (ANOVA) and Student&#39;s t-test for statistical significance (p &#60; 0.05).</li>
</ol>

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The authors have nothing to declare.

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 planaria9,10,11,12. The mechanism by which pilocarpine induces seizure-like behaviors in planaria is still unknown. However, this study demonstrates...

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 approach4,5. 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 pilocarpine7,8,9,10,11,12,13,14. 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 seizures15,16,17. 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 staining11,18. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL centrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde solution</td>
			<td>Himedia</td>
			<td>TCL119</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting media</td>
			<td>Clini Sciences</td>
			<td>NB-47-02240-30ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Beakers (one for each concentration tested)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carolina Springwater</td>
			<td>carolina</td>
			<td>132450</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diluted primary and secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (100%)</td>
			<td>sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EthosVision XT 16</td>
			<td>noldus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji Version 2.9.0</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin-coated slides</td>
			<td>sigma Aldrich</td>
			<td>643203</td>
			<td></td>
		</tr>
		<tr>
			<td>Golgi Antibody 1H6</td>
			<td>DSHB</td>
			<td>AB_2619608</td>
			<td></td>
		</tr>
		<tr>
			<td>Golgi stain kit_</td>
			<td>Neuroscience Associate</td>
			<td>PK 401/401A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>thermo fischer scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OCT compound</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>sigma Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Powdered Milk</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tin foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

planarian as an animal model for experimental acute seizure

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&#160;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.

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&#39;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...

Planarian as an Animal Model for Experimental Acute Seizure

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.

Epilepsy is among the most prevalent neurological disorders characterized by recurring spontaneous seizures. Seizures represent a clinical manifestation ...

planarian-as-an-animal-model-for-experimental-acute-seizure

Research

JoVE Journal

Neuroscience

645 Views.  Old Dominion University. 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.

Video: Planarian as an Animal Model for Experimental Acute Seizure

Whole Animal Perfusion Fixation for Rodents

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7. Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3. It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the spinal cord, tumor resections, or tissue defects resulting from traumatic accidents can aid in facilitating general tissue repair as well as regenerative growth of nerve fibers into and beyond the affected area. Two experimental treatment strategies are presented: (1) implantation of a novel microconnector device into an acutely and completely transected thoracic rat spinal cord to readapt severed spinal cord tissue stumps, and (2) polyethylene glycol filling of the SCI site in chronically lesioned rats after scar resection. The chronic spinal cord lesion in this model is a complete spinal cord transection which was inflicted 5 weeks before treatment. Both methods have recently achieved very promising outcomes and promoted axonal regrowth, beneficial cellular invasion and functional improvements in rodent models of spinal cord injury.
The mechanical microconnector system (mMS) is a multi-channel system composed of polymethylmethacrylate (PMMA) with an outlet tubing system to apply negative pressure to the mMS lumen thus pulling the spinal cord stumps into the honeycomb-structured holes. After its implantation into the 1 mm tissue gap the tissue is sucked into the device. Furthermore, the inner walls of the mMS are microstructured for better tissue adhesion.
In the case of the chronic spinal cord injury approach, spinal cord tissue - including the scar-filled lesion area - is resected over an area of 4 mm in length. After the microsurgical scar resection the resulting cavity is filled with polyethylene glycol (PEG 600) which was found to provide an excellent substratum for cellular invasion, revascularization, axonal regeneration and even compact remyelination in vivo.

Severe spinal cord injuries often result in tissue defects. Two possibilities are described to successfully bridge such gaps to promote tissue adaptation, regenerative responses and functional improvement in rats via implantation of a mechanical microconnector system after acute injury and five weeks after complete spinal cord transection.

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...