The authors thank John Williamson for helpful discussions on this protocol. This work was supported by NIH/NINDS grant NS112549.

Animal use followed ARRIVE12 guidelines and was approved by the Animal Care and Use Committee of the University of Virginia.
1. Making headsets with two bipolar electrodes (Figure 1)
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62929/62929fig01.jpg" /> 
Figure 1: Key steps in EEG headset fabrication. (A) Appearance of the electrodes at the various steps in the protocol (numbers in the left match step). (B) A picture of the final product mounted in a homemade holder that fits the stereotaxic frame. Note, the holder ends with a collar pin assembly that fits into the headset pedestal. <a href="https://www.jove.com/files/ftp_upload/62929/62929fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Cut 3.5 cm of stainless polytetrafluoroethylene-coated stainless-steel wire.</li>
	<li>Strip about 1 mm of the insulation coat off the wire on both ends. Do not have too much of the wire stripped.</li>
	<li>Put two pins on a vise holder with the bottom part of the pin that has a longer slit facing down.</li>
	<li>Apply flux onto the stripped ends of the wire and onto the tops of the pins.</li>
	<li>Tin the stripped part of the wire with just enough solder to coat it.</li>
	<li>Add a minimal amount of solder to the top of the pin without overflowing onto the sides.</li>
	<li>Place one end of the stripped ends of the wire into the pin as deep as it will allow while the solder is melted. 
	NOTE: There is a side hole where the wire can come out - do not let the wire come out of the pin. All of the stripped wire must stay within the pin.</li>
	<li>Repeat steps 1.6-1.7 for the second pin, now with the other stripped ends of the wire.</li>
	<li>Let the pins sit for 30 s&#160;to set, remove them from the vise holder, and then pull on them to make sure that the connection between the wire and the pins is strong and holds.</li>
	<li>Rinse the pins in cold water, and then dry.</li>
	<li>Verify conductance between pin 1 and pin 2 using an ohmmeter.</li>
	<li>Bring the pins at the ends of the wire together; hold them parallel and close. Clamp a hemostat to the center of the wire. Then, rotate the hemostat, so the wire twists around itself fairly tight. Remove the hemostat.</li>
	<li>Clamp a forceps onto the twisted wire 2 mm below the pins and bend the wire at 90&#730;.</li>
	<li>Push the same wire again 90&#730; back over the forceps creating another bend 1 mm from the first.</li>
	<li>Cut the twisted wire at a 45&#176; angle below the bend at 3.5 mm with small sharp scissors.</li>
	<li>Prepare two of these bipolar (double-pin twisted) electrodes for each headset (optional, second is back-up in case of electrical problems with the first).</li>
	<li>Prepare one single reference electrode by cutting a wire with pins soldered on both ends into two (steps 1.1-1.17, Figure 1A).</li>
	<li>Cut the wire at 7 mm.</li>
	<li>Bend the end 1 mm below the tip.</li>
	<li>Then, cover the bent 1 mm tip of the wire with forceps and rotate the wire tight around the forceps to create a small loop (1 mm in diameter).</li>
	<li>Bend the loop perpendicular to the straight part of the wire to make the wire tip point outward again.</li>
	<li>Assemble the two bipolar electrodes and the single reference electrode into the six-pin pedestal in a way that the bipolar electrodes are side by side with 6 mm distance between them, and that the reference electrode is placed in the middle outer hole (Figure 1B). 
	NOTE: An alternative method is to implant the electrodes, cement them in place, and then insert their pins into the pedestal.</li>
</ol>
2. Stereotaxic electrode implantation
<ol>
	<li>Sterilize all surgical tools and the six-pin electrode assembly by autoclaving before the surgery. A sterile surgical field must be maintained during the surgery, and sterile surgical gloves must be used. Sterile drapes (e.g., Press n&#8217; Seal) are recommended to cover the animal except for the surgical area.</li>
	<li>Use 8-week-old VGAT-Cre mice (age-matched, both male and female) for surgeries 4 weeks after weaning. Record the weight of the animal before surgery to allow the measurement of post-surgical weight loss.</li>
	<li>Use a certified isoflurane vaporizer or a low-flow anesthesia system equipped with a precision syringe pump, integrated digital vaporizer, and feedback heat pad. 
	NOTE: Low-flow systems are capable of delivering anesthesia at low flow rates proportionate to the animal&#39;s size into either an induction chamber or through a nose cone on the stereotaxic frame (70 mL/min, isoflurane concentration in air is 4% for induction and 2% for surgery). Using less anesthesia not only benefits the animal during surgeries but also reduces the risk of lab personnel&#39;s exposure to isoflurane.</li>
	<li>Place the anesthetized animal on a heated pad warmed to 37 &#176;C to keep it warm during surgery. If using a feedback-controlled temperature system, insert the lightly lubricated temperature probe into the rectum of the animal for temperature monitoring during surgery.</li>
	<li>Mount the animal onto the stereotaxic frame by gently placing ear bars into the ears and the front upper teeth into the incisor bar. Place the nose cone over the nose for proper anesthesia delivery. Make sure that the head is leveled and centered and cannot be moved when slightly probed.</li>
	<li>Subcutaneously inject 0.5 mL of normosol for hydration.</li>
	<li>Apply ocular lubricant to prevent corneal drying.</li>
	<li>Monitor for the depth of anesthesia by the absence of the withdrawal reflex after pinching a hindlimb toe, and then decrease isoflurane to 1.5%-2.0% during surgery.</li>
	<li>Remove hair at and around the surgical area by plucking or using clippers (shaving) or depilatory cream, and disinfect the skin with three cycles of alternating application of iodine and ethanol, finishing with iodine. Removing hair away from the surgical site is recommended only if there are means to maintain anesthesia at that location. If necessary, use alcohol-dipped cotton tip applicators to remove hair from the immediate area surrounding the head. Inject 0.05 mL of the local analgesic bupivacaine (0.25%) subcutaneously.</li>
	<li>Make an incision on the skull using a scalpel, and then cut out a part of the skin with sharp surgical scissors, exposing the skull. Pushing the skin aside, using a cotton swab, clean the skull from all the muscles and underlying tissues obstructing the view. 
	NOTE: To stop accidental bleeding, apply pressure over the bleeding site with a sterile cotton swab until it stops.</li>
	<li>Clean the skull with hydrogen peroxide using sterile cotton swabs to make the skull sutures and both bregma and lambda visible.</li>
	<li>Dry the skull thoroughly, and then apply one drop of self-etching dental adhesive using its applicator. Brush it into the skull, wait 60 s, and cure with a dental UV light for 40 s. A glossy surface indicates that the adhesive has been effectively cross-linked with the skull. 
	NOTE: This step is critical for secure attachment of the headset.</li>
	<li>Use a 0.031&#34; drill bit (0.79 mm) to drill two burr holes bilaterally for implantation of hippocampal depth electrodes (approximately 5,000 rpm). Drill one extra burr hole for the reference electrode above the cerebellum behind the lambda. 
	&#8203;NOTE: When drilling, take care to lower the drill slowly and avoid drilling into the brain.</li>
	<li>The coordinates for the electrodes are as follows (from bregma in mm): hippocampal electrodes at 3 mm posterior, 3 mm lateral, and 3 mm depth; and cerebellar reference electrode at 6 mm posterior, 0 mm lateral, and 0 mm depth (subdural).</li>
	<li>To increase the accuracy, use a stereotaxically mounted drill, zero the stereotaxic manipulator X/Y axis when touching bregma - this is the point of reference for the coordinates.</li>
	<li>Assemble a headset by inserting all the electrodes in the six-pin pedestal, making sure the pins are pushed all the way into the pedestal. Mount the pedestal into the electrode holder on a stereotaxic frame (Figure 1B).</li>
	<li>Align electrodes above the corresponding burr holes. Stereotaxically implant twisted bipolar stainless-steel wire electrodes in the right and left hippocampus and reference electrode into the cerebellum by slowly lowering the headset and guiding the electrodes into the burr holes.</li>
	<li>When the hippocampal twist electrode is right above the hole, zero the Z axis and slowly lower to -3.0 mm.</li>
	<li>Cover the skull surface and electrodes with dental cement and fill in the space between the skull surface and the bottom of the pedestal. The skin edges will be adjacent to the dental cement such that no underlying tissue will remain exposed. Wait for the cement to dry and harden. Then detach the electrode holder from the stereotaxic arm and remove the holder from the pedestal.</li>
	<li>Inject 0.1 mL of ketoprofen (1 mg/mL, SC) for analgesia and a second dose of 0.5 mL of normosol (SC) for hydration and remove the animal from the stereotaxic frame.</li>
	<li>Place an isothermal pad preheated to 37 &#176;C inside an empty vivarium cage covered with a paper towel. Once it is fully awake, place the animal in a clean cage with bedding and soft food, and return it to the vivarium. Animals are housed singly from this point on to prevent chewing on each other&#8217;s headsets. Wire bar hoppers are not used to avoid headsets getting stuck, and instead, water bottles are tied to the underside of the cage tops, and chow is present in the bedding.</li>
	<li>Feed the animal some soft food for 72 h after surgery, and monitor for weight loss and body condition score. Dehydrated animals may be administered 0.5 mL of normosol subcutaneously if dehydrated (weight loss, increased skin turgor, sunken eyes). Ketoprofen can be given subcutaneously once daily for 2 more days following surgery (follow the analgesia regimen according to the local IACUC guidelines). Allow animals to fully recover in their cages for 4-7 days before transferred to the EEG recording system.</li>
</ol>
3. Electrical kindling protocol
<ol>
	<li>Hook-up the mice to the EEG recording system using a flexible cable that fits the sockets on the mouse head and commutator (see the Table of Materials list). Allow the animals to acclimate for one day before proceeding with the electrical stimulation protocol below. Monitor their overall health and acclimation on a daily basis, and remove them from the system when there are signs of illness, distress, and/or continuous body weight loss exceeding 25%</li>
	<li>Connect both leads from the stimulating electrode to the output of a constant current stimulator. 
	NOTE: It is very helpful to have a circuit board that switches these electrodes away from the EEG recorder and to the stimulator.</li>
	<li>Set the stimulator to deliver 1 ms pulses at 50 Hz for a 2 s train duration.</li>
	<li>Set the output of the stimulator at 20 microamps (&#181;A) and deliver the 1st pulse.</li>
	<li>Monitor the EEG for a characteristic after-discharge of high-frequency spikes that outlast the electrical stimulation pulse.</li>
	<li>If no discharge is observed, then increase the amount of current injected in 20 &#181;A increments until an after-discharge is triggered. The amount of current required is the After-Discharge Threshold (ADT).</li>
	<li>Typical ADTs are 20-50 &#181;A. If no discharge is observed even after increasing to 200 &#181;A, then troubleshooting electrical connections and headset wiring with a high sensitivity ohmmeter is required. If the problem is in the stimulating electrode, then try stimulating with the other depth electrodes.</li>
	<li>Animals are kindled by stimulating either 2x or 6x per day using a current that is 1.5x the ADT value for that mouse.</li>
	<li>Monitor the behavioral response to the stimulation, which rises from the change of state to bilateral tonic-clonic seizures with falling. Score using a modified Racine class system11. To avoid evoked fatal tonic seizures, kindling should be paused if successive stimuli lead to escalating severity and length of seizures up to modified Racine score 6 (running and jumping)</li>
</ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+new+epilepsy+treatments:+issues+in+preclinical+methodology.">Identification of new epilepsy treatments: issues in preclinical methodology.</a> Epilepsia. 53 (3), 571-582 (2012).</li><li>Kehne, J. H., Klein, B. D., Raeissi, S., Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+National+Institute+of+Neurological+Disorders+and+Stroke+(NINDS)+Epilepsy+Therapy+Screening+Program+(ETSP).">The National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP).</a> Neurochemical Research. 42 (7), 1894-1903 (2017).</li><li>Buckmaster, P. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postictal+death+is+associated+with+tonic+phase+apnea+in+a+mouse+model+of+sudden+unexpected+death+in+epilepsy.">Postictal death is associated with tonic phase apnea in a mouse model of sudden unexpected death in epilepsy.</a> Annals of Neurology. 89 (5), 1023-1035 (2021).</li><li>Morimoto, K., Fahnestock, M., Racine, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kindling+and+status+epilepticus+models+of+epilepsy:+rewiring+the+brain.">Kindling and status epilepticus models of epilepsy: rewiring the brain.</a> Progress in Neurobiology. 73 (1), 1-60 (2004).</li></ol>

The authors have no conflicts to disclose.

This report describes a protocol where electrical kindling of mice leads to epilepsy. Since the stimulating electrode is placed in the hippocampus, this is a focal limbic epilepsy that models temporal lobe epilepsy (TLE) in patients. A critical step in this protocol is to use VGAT-Cre mice, which due to insertion of an IRES-Cre recombinase cassette in the Vgat gene, shows impaired inhibitory GABA currents11. C57BL/6 do not develop epilepsy after kindling with this protocol, although it is...

Epilepsy is a major neurological disorder with significant economic and human burdens. NINDS estimates there are 3 million Americans with epilepsy. Approximately 0.6 million of these patients have temporal lobe epilepsy (TLE)1. Unfortunately, medical treatment of TLE fails in one-third of the patients because of ineffectiveness, development of drug resistance, or intolerance to side effects2. Clearly, there is a significant need to develop novel therapies for TLE, a conclusion shared by the American Epilepsy Society Basic Science Committee, the International League Against Epilepsy Working Group for Preclinical Epilepsy Drug Discovery, and the National Advisory Neurological Disorders and Stroke Council3,4.
Current animal models of temporal lobe epilepsy use either chemoconvulsants (e.g., kainate, pilocarpine) or prolonged electrical stimulation to induce a long-lasting status epilepticus5,6,7. Many animals die during the procedure (10%-30% in rats, up to 90% in mice8). Animals that survive and develop epilepsy show extensive neuronal death throughout the brain9,10. This death triggers a cascade of responses, beginning with the activation of microglia, astrocytes, and infiltrating monocytes. Neuronal responses include circuit reorganization (e.g., mossy fiber sprouting), birth of new neurons that fail to integrate properly into circuits (e.g., ectopic granule cells), and intrinsic changes that lead to hyperexcitability (e.g., upregulation of Na+ channels). An epilepsy model without significant neuronal death will facilitate the search for new antiepileptic drugs.
While testing the GABA hypothesis of epilepsy, it was discovered that treating VGAT-Cre mice with a mild electrical kindling protocol led to the spontaneous motor and electrographic seizures11. In general, the electrical kindling of rodents does not lead to spontaneous seizures that define epilepsy, although it can, in cases of over-kindling11. VGAT-Cre mice express Cre recombinase under the control of the vesicular GABA transporter (VGAT) gene, which is specifically expressed in GABAergic inhibitory neurons. It was found that insertion of Cre disrupted the expression of VGAT at the mRNA and protein levels, thus impairing GABAergic synaptic transmission in the hippocampus. It was concluded that kindled VGAT-Cre mice could be useful to study the mechanisms involved in epileptogenesis and for screening novel therapeutics11. The present report provides the methods used in generating the model in detail.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16 Channel Extracellular Differential AC Amplifier (115V/60Hz)</td>
			<td>AD Instruments</td>
			<td>AM3500-115-60</td>
			<td>Alternate EEG amplifier</td>
		</tr>
		<tr>
			<td>363/CP PLUG COLLAR, PINS SLEEVE</td>
			<td>P1 Technologies</td>
			<td>363SLEEVPIN0NL</td>
			<td>For electrode holder</td>
		</tr>
		<tr>
			<td>Cable, 363-363 5CM - 100CM W/MESH 6TCM</td>
			<td>P1 Technologies</td>
			<td>363363XXXXCM004</td>
			<td>mouse-to-commutator cable</td>
		</tr>
		<tr>
			<td>CCTV cameras Qcwox HD Sony IR LED</td>
			<td>Sony</td>
			<td>QC-SP316</td>
			<td></td>
		</tr>
		<tr>
			<td>Commutator SL6C/SB (single brush)</td>
			<td>P1 Technologies</td>
			<td>8BSL6CSBC0MT</td>
			<td>formerly Plastics One, Inc.</td>
		</tr>
		<tr>
			<td>Current amplifier</td>
			<td>A-M Systems</td>
			<td>Model 2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Stoelting</td>
			<td>51459</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill bits, #75, OD&#160; 0.310&#34; LOC 130 PT</td>
			<td>Kyocera</td>
			<td>105-0210.310</td>
			<td></td>
		</tr>
		<tr>
			<td>E363/0 SOCKET CONTACT SKEWED</td>
			<td>P1 Technologies</td>
			<td>8IE3630XXXXE</td>
			<td>pins for connector</td>
		</tr>
		<tr>
			<td>iBond Self Etch glue</td>
			<td>Kulzer</td>
			<td>CE0197</td>
			<td></td>
		</tr>
		<tr>
			<td>MS363 PEDESTAL 2298 6 PIN WHITE</td>
			<td>P1 Technologies</td>
			<td>8K000229801F</td>
			<td>EEG headset connector</td>
		</tr>
		<tr>
			<td>Ohmeter</td>
			<td>Simpson</td>
			<td>260</td>
			<td>High sensitivity</td>
		</tr>
		<tr>
			<td>PowerLab 16/35 and LabChart Pro</td>
			<td>AD Instruments</td>
			<td>PL3516/P</td>
			<td>Alternate EEG software</td>
		</tr>
		<tr>
			<td>SomnoSuite</td>
			<td>Kent Scientific Corp.</td>
			<td>SS-01</td>
			<td>anesthesia unit &#38; RightTemp monitoring</td>
		</tr>
		<tr>
			<td>Stereotactic drill and micromotor kit</td>
			<td>Foredom Electric Co.</td>
			<td>K.1070</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>David Kopf Instruments</td>
			<td>Model 940</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon-coated wire for depth electrode, OD 0.008&#39;</td>
			<td>A-M Systems</td>
			<td>791400</td>
			<td></td>
		</tr>
		<tr>
			<td>VGAT-Cre mice on congenic C57BL/6J background</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and implantation of electrodes for electrically kindling vgat-cre mice to generate a model for temporal lobe epilepsy

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after kindling and a likely mechanism - insertion of Cre into the VGAT gene - disrupted its expression and reduced GABAergic tone. The present study extends these observations to a larger cohort of mice, focusing on key issues such as how long the SRS continues after kindling and the effect of the animal's sex and age. This report describes the protocols for the following key steps: making headsets with hippocampal depth electrodes for electrical stimulation and for reading the electroencephalogram; surgery to affix the headset securely on the mouse's skull so that it does not fall off; and key details of the electrical kindling protocol such as duration of the pulse, frequency of train, duration of train, and amount of current injected. The kindling protocol is robust in that it reliably leads to epilepsy in most VGAT-Cre mice, providing a new model to test for novel antiepileptogenic drugs.

Animals 
The model was originally developed using VGAT-Cre mice (Slc32a1tm2(cre)Lowl/J)13 on a mixed background. However, it has also been applied to the VGAT-Cre strain that is congenic with C57BL/6J. No difference has been observed in epilepsy that develops between the strains. Both strains express Cre recombinase under the control of the vesicular GABA transporter promoter. These mice were generated by knocking in an IRES-Cre cassette after the stop codon in the...

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Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy

This report describes the methods to generate a model of temporal lobe epilepsy based on the electrical kindling of transgenic VGAT-Cre mice. Kindled VGAT-Cre mice may be useful in determining what causes epilepsy and for screening novel therapies.

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique ...

preparation-implantation-electrodes-for-electrically-kindling-vgat

Research

JoVE Journal

Neuroscience

2.2K Views.  University of Virginia. This report describes the methods to generate a model of temporal lobe epilepsy based on the electrical kindling of transgenic VGAT-Cre mice. Kindled VGAT-Cre mice may be useful in determining what causes epilepsy and for screening novel therapies.

Video: Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

A Model for Epilepsy of Infectious Etiology using Theiler's Murine Encephalomyelitis Virus

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop epilepsy as a consequence, with rates being significantly higher in less economically developed countries. This work provides an overview of modeling epilepsy of infectious etiology and using it as a platform for novel antiseizure compound testing. A protocol of epilepsy induction by non-stereotactic intracerebral injection of Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice is presented, which replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. The clinical evaluation of mice during encephalitis to monitor seizure activity and detect the potential antiseizure effects of novel compounds is described. Furthermore, histopathological consequences of viral encephalitis and seizures such as hippocampal damage and neuroinflammation are shown, as well as long-term consequences such as spontaneous epileptic seizures. The TMEV model is one of the first translational, infection-driven, experimental platforms to allow for the investigation of the mechanisms of epilepsy development as a consequence of CNS infection. Thus, it also serves to identify potential therapeutic targets and compounds for patients at risk of developing epilepsy following a CNS infection.

Intracerebral infection with the Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. This paper describes the virus infection, symptoms, and histopathology of the TMEV model.

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop ...

Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few studies have described the experimental protocol in detail. This paper illustrates the whole process of amygdala electrical kindling epilepsy model making, with the introduction of a method of bipolar electrode fabrication. This electrode can both stimulate and record, reducing brain injury caused by implanting separate electrodes for stimulation and recording. For long-term electroencephalogram (EEG) recording purposes, slip rings were used to eliminate the record interruption caused by cable tangles and falling off.
After periodic stimulation (60 Hz, 1 s every 15 min) of the basolateral amygdala (AP: 1.67 mm, L: 2.7 mm, V: 4.9 mm) for 19.83 &plusmn; 5.742 times, full kindling was observed in six mice (defined as induction of three continuous grade V episodes classified by Racine's scale). An intracranial EEG was recorded throughout the entire kindling process, and an epileptic discharge in the amygdala lasting 20-70 s was observed after kindling. Therefore, this is a robust protocol for modeling epilepsy originating from the amygdala, and the method is suitable for revealing the role of the amygdala in temporal lobe epilepsy. This research contributes to future studies on the mechanisms of mesial temporal lobe epilepsy and novel antiepileptogenic drugs.

The amygdala plays a key role in temporal lobe epilepsy, which originates in and propagates from this structure. This article provides a detailed description of the fabrication of deep brain electrodes with both recording and stimulating functions. It introduces a model of medial temporal lobe epilepsy originating from the amygdala.

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few ...

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using interneurons to replace or mitigate the effects of altered neuronal circuits is an attractive therapeutic avenue. To this end, more knowledge is needed about how human stem cell-derived GABAergic interneuron-like cells (hdINs) mature, integrate, and function over time in the host circuitry. Of particular importance in neurodevelopmental disorders is a better understanding of whether these processes in transplanted cells are affected by an evolving and maturing host brain. The present protocol describes a fast and highly efficient generation of hdINs from human embryonic stem cells based on the transgenic expression of the transcription factors Ascl1 and Dlx2. These neuronal precursors are transplanted unilaterally, after 7 days in vitro, to the hippocampus of neonatal 2-day-old mice. The transplanted neurons disperse in the ipsi- and contralateral hippocampus of a mouse model of cortical dysplasia-focal epilepsy syndrome and survive for up to 9 months after transplantation. This approach allows for investigating the cellular identity, integration, functionality, and therapeutic potential of transplanted interneurons over an extended time in developing healthy and diseased brains.

Transplantation of human pluripotent stem cell-derived GABAergic neurons generated by neuronal programming could be a potential treatment approach for neurodevelopmental disorders. This protocol describes the generation and transplantation of human stem cell-derived GABAergic neuronal precursors into the brains of neonatal mice, allowing long-term investigation of grafted neurons and evaluation of their therapeutic potential.

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using ...

Monitoring Epileptiform Events in Drosophila via Calcium Imaging

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Author Spotlight: Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...

Precise Urodynamic Measurement in Awake Mice Using Cyanoacrylate Skin Barriers

Optimizing Mouse Urodynamic Techniques for Improved Accuracy 

Accurate measurement of urinary parameters in awake mice is crucial for understanding lower urinary tract (LUT) dysfunction, particularly in conditions like neurogenic bladder post-traumatic spinal cord injury (SCI). However, conducting cystometry recordings in mice presents notable challenges. When mice are in a prone and restricted position during recording sessions, urine tends to be absorbed by the fur and skin, leading to an underestimation of voided volume (VV). The goal of this study was to enhance the accuracy of cystometry and external urethral sphincter electromyography (EUS-EMG) recordings in awake mice. We developed a unique method utilizing cyanoacrylate adhesive to create a waterproof skin barrier around the urethral meatus and abdomen, preventing urine absorption and ensuring precise measurements. Results show that after applying the cyanoacrylate, the sum of VV and RV remained consistent with the infused saline volume, and there were no wet areas observed post-experiment, indicating successful prevention of urine absorption. Additionally, the method simultaneously stabilized the electrodes connected with the external urethral sphincter (EUS), ensured stable electromyography (EMG) signals, and minimized artifacts caused by the movement of the awakened mouse and manipulation of the experimenter. Methodological details, results, and implications are discussed, highlighting the importance of improving urodynamic techniques in preclinical research.

Author Spotlight: Enhanced Urodynamic Method for Precise Urine Measurement in Awake Mice with Neurogenic Bladder

This protocol provides a guide for waterproofing the skin with cyanoacrylate to prevent urine absorption by fur and skin. It includes instructions for applying the glue to the skin, implanting a bladder catheter, and electrodes for cystometry and external urethral sphincter electromyography recordings in awake mice.

Accurate measurement of urinary parameters in awake mice is crucial for understanding lower urinary tract (LUT) dysfunction, particularly in conditions ...