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.remotexs.ntu.edu.sg/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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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 possible that other strains of mice will.
The protocol was developed using placement of the stimulating depth electrodes in the hippocampus. The coordinates provided the target with the perforant path projections from the entorhinal cortex as they enter the CA1 subfield. The electric field induced by electrical stimulation has not been defined, therefore, the precise location of the electrodes is not critical. In fact, rodents can be electrically kindled anywhere in the limbic circuit, e.g., amygdala and piriform cortex19. The following are critical steps in the protocol: one, proper soldering of the EEG headsets to ensure low resistance to the stimulating current; two, using a dental adhesive that is in use in dental clinics to bind to the skull and provide a surface for adhesion of the dental cement; and three, the use of a constant current amplifier to deliver the described electrical pulses.
Trouble-shooting is typically limited to checking electrical connections, which include the connections in the headset, the headset to the cable, the cable to the commutator, and the commutator to the recording device. Using an ohmmeter with high sensitivity is critical.
Limitations to the technique include the requirement of appropriate expertise and equipment to perform the surgeries and record the EEG.
An advantage of the model over existing animal models of TLE is that there is minimal death of neurons11. The other TLE models are post-status epilepticus models, which trigger extensive neuronal death10. This death leads to extensive activation of microglia, astrocytes, and infiltration by circulating monocytes. Taken together, it becomes difficult to distinguish mechanisms that cause epilepsy from mechanisms triggered by prolonged status epilepticus. It is anticipated that kindled VGAT-Cre mice will be useful for developing novel therapies that are both anti-seizure and anti-epileptic. This report provides key metrics and power analysis that can guide future drug development efforts using these mice.
Another advantage to the kindled VGAT-Cre model is the high percentage of animals that develop epilepsy (90%) and the regularity of the spontaneous seizures. Disadvantages of the model are a relatively low seizure frequency (1.5/day) and that seizure frequency declines after 3 weeks, and in some cases seizures seem to stop. Efforts are underway to address these issues.

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><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&amp;nbsp; 0.310&quot; 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 &amp;amp; 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&#039;</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 Vgat gene. These mice breed normally, so they are maintained as homozygotes. To prevent genetic drift of the colony, purchase breeders and only use F1 generation mice for experiments. All mice were housed in an AAALAC-accredited vivarium. Mice were given free access to food and water, 12 h light/dark cycles, and an enriched environment. For electroencephalographic (EEG) recording, mice were individually housed in clear plastic cages (homemade), allowing for concurrent video monitoring. Use both male and female mice for experiments. No difference in terms of kindling rate or seizure frequency has been observed between the sexes. The majority of these studies used 8-week-old mice at the time of headset surgery. However, one can also use mice that are between 4-20 weeks old. At 4-weeks, the mouse skull is ~90% of its final size14, so affixing a headset has minimal effect on growth. The time between surgery and commencement of the kindling protocol is not critical, although mice do need to be observed carefully during recovery from surgery for 72 h.
Kindling parameters 
The electrical stimulation protocol was developed by Lothman and coworkers and described in detail in their 1989 paper15. In brief, the hippocampal electrode is connected to a constant current stimulator, and a 1 ms biphasic square wave pulse is delivered at 50 Hz over 2 s. Various parameters of the kindling protocol have been tested16. Use a current intensity that is 1.5x the minimal amount of current required to trigger an electrographic seizure for that mouse (After-Discharge Threshold, ADT). Mice are electrically stimulated twice a day every other day (2x, mid-morning and early afternoon). Figure 2A shows an overview of the kindling protocol and key seizure properties. However, a fast-kindling protocol is also effective, herein mice are stimulated six times a day delivered every other day (6x, one-hour between stimulations). Interestingly, kindling rates are similar between 2x and 6x protocols in terms of the number of stimulations required to achieve the kindled state (Figure 2B: 2x, 15 &#177; 1, n = 46; 6x, 13 &#177; 1, n = 12, mean &#177; SEM, P = 0.3). The mice are considered fully kindled when a total of five stimulations evoke tonic-clonic seizures with loss of postural control, which is a behavioral score of 5 on a modified Racine scale17. Kindling beyond this level, so-called over-kindling, can be performed but carries the risk of a fatal tonic seizure or SUDEP18. Mortality in this VGAT-Cre protocol is ~13% (15 out of 119 mice); this includes mice of either sex that died after either evoked or spontaneous seizures (8 male, 7 female).
Seizure properties 
The recording system used in these studies has been discontinued. Alternative suppliers of EEG recording set-ups that are in use at the University of Virginia Rodent Epilepsy Monitoring Unit are provided in the Table of Materials. Figure 2A shows an overview of the kindling protocol and key seizure properties.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62929/62929fig02.jpg" /> 
Figure 2: Seizure properties of kindled VGAT-Cre mice. (A) Schematic diagram of the time course of an experiment. (B) The distribution of the number of electrical stimulations required to reach the fully kindled state. The kindled state is achieved when stimulations evoke five bilateral tonic-clonic motor seizures. (C) The distribution of latency to first spontaneous seizure after the last electrical stimulation. (D) The distribution of observed seizure frequencies, which is calculated as the total number of seizures divided by the number of days recorded. (E) The distribution of how long mice were epileptic as defined by spontaneous seizures occurring with an inter-seizure interval below 5 days. All the graphs use violin plots where the median is shown with a dark line and 25th and 75th quartiles are shown with light lines. The number of animals in each plot is shown on the X-axis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62929/62929fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
VGAT-Cre mice typically reach the kindled state criterion after 15 stimulations (pooled 2x and 6x protocols, mean &#177; SD of 7.4 stimulations, Figure 2B). As can be seen by the violin plot of the data, many animals require fewer stimulations (10) and many require more (18). It has not been possible to find a factor that determines the difference, excluding sex, age, ADT value, or electrode placement. Within a few weeks of being kindled, most mice develop multiple spontaneous seizures (90%), which defines epilepsy. The latency to spontaneous recurring seizures (SRS) is 10.7 &#177; 6.3 days (Figure 2C). A fraction of mice develop SRS before reaching the kindled state. As noted previously11, VGAT-Cre mice are not spontaneously epileptic and require electrical stimulation to develop epilepsy. Once seizures begin, they occur at a frequency of 1.3 &#177; 0.6 seizures per day (Figure 2D). All electrographic seizures are accompanied by tonic-clonic motor seizures. The initial studies were short-term (spontaneous seizure frequency measured 1-2 weeks), but when the recording period was extended it was discovered that seizure frequency diminished with time. Using an arbitrary cut-off of 5 consecutive days without a seizure, reliable seizures occur for 23 &#177; 11 days. Taken together, this defines the period kindled VGAT-Cre mice are useful for drug screening campaigns. Power analysis using these SD and an effect size of 50% reduction shows 16 mice per group are required for statistically significant effects on stimulations to kindle, seizure frequency, and epilepsy duration.
A feature of the kindled VGAT-Cre model that distinguishes it from post-status epilepticus models is the absence of neuronal death. This was assayed using two accepted methods (Figure. 3): one, by counting nuclei in the CA1 layer stained with anti-NeuN antibody; and two, by measuring the amount of Fluoro-Jade C staining in hippocampal subfields that are vulnerable to chemoconvulsant-induced cell death (dentate, CA1, and entorhinal cortex).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62929/62929fig03.jpg" /> 
Figure 3: Neuronal death as assayed by either anti-NeuN staining or Fluoro-Jade C staining. (A) Plot of anti-NeuN-positive neurons in various sub-fields of the hippocampus and entorhinal cortex. Abbreviations are as follows: DGC, dentate granule cell layer; hilus refers to the dentate hilus, CA1, cornus ammonis pyramidal layer I; and EC, entorhinal cortex; L2, layer 2; and L3, layer 3. Two horizontal brain slices corresponding to -4 mm below bregma were stained from each animal (Control naive mice, n = 7; epileptic VGAT-Cre, n = 13; see Straub et al. for details11). Data were normalized to average neuronal counts determined in naive VGAT-Cre mice in the respective subfield. Images of Fluoro-Jade C staining from an epileptic VGAT-Cre mouse (B) and from a post-status rat (C) (Li/pilocarpine model, see Dey et al. for details and analysis10). Analysis of the Fluoro-Jade C staining of VGAT-Cre mice was presented in Straub et al.11). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62929/62929fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy at JoVE.com

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 ...

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Neuroscience

2.3K 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

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

Genetics

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for various neurological and psychiatric disorders. HFS in the deep region of the brain, also named deep-brain stimulation (DBS), is becoming increasingly important in clinical trials. Recent progress in the field of high-frequency DBS (HF-DBS) surgery has begun to spread the possibility of utilizing this invasive technique to other situations, such as treatment for major depression disorder (MDD), obsessive-compulsive disorder (OCD), and so on. 
Despite these expanding indications, the underlying mechanisms of the beneficial effects of HF-DBS remain enigmatic. To address this question, one approach is to use implanted electrodes that sparsely activate distributed subpopulations of neurons by HFS. It has been reported that HFS in the anterior nucleus of the thalamus could be used for the treatment of refractory epilepsy in the clinic. The underlying mechanisms might be related to the increased neurogenesis and altered neuronal activity. Therefore, we are interested in exploring the physiological alterations by the detection of neuronal activity as well as neurogenesis in the mouse dentate gyrus (DG) before and after HFS treatment. 
In this manuscript, we describe methodologies for HFS to target the activation of the DG in mice, directly or indirectly and in an acute or chronic manner. In addition, we describe a detailed protocol for the preparation of brain slices for c-fos and Notch1 immunofluorescent staining to monitor the neuronal activity and signaling activation and for bromodeoxyuridine (BrdU) labeling to determine the neurogenesis after the HF-DBS induction. The activation of the neuronal activity and neurogenesis after the HF-DBS treatment provides direct neurobiological evidence and potential therapeutic benefits. Particularly, this methodology can be modified and applied to target other interested brain regions such as the basal ganglia and subthalamic regions for specific brain disorders in the clinic.

This protocol shows how to set up a reliable HFS method in mice. Neurons throughout the hippocampal dentate gyrus are electrically stimulated by HFS directly and indirectly in vivo. Neuronal activity and molecular signaling are examined by c-fos and Notch1 immunofluorescent staining, respectively; neurogenesis is quantified by bromodeoxyuridine labeling assay.

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status epilepticus (SE) is frequently selected to recapitulate spontaneous recurrent seizures (SRS). Here we present a protocol of SE induction by intraperitoneal (i.p.) injection of pilocarpine and monitoring of chronic recurring seizures in live animals using a wireless telemetry video and electroencephalogram (EEG) system. We demonstrated notable behavioral changes that need attention after pilocarpine injection and their correlation with hippocampal neuronal loss at 7 days and 6 weeks post-pilocarpine. We also describe the experimental procedures of electrode implantation for video and EEG recording, and analysis of the frequency and duration of chronic recurrent seizures. Finally, we discuss the possible reasons why the expected results are not achieved in each case. This provides a basic overview of modeling chronic epilepsy in mice and guidelines for troubleshooting. We believe this protocol can serve as a baseline for suitable models of chronic epilepsy and epileptogenesis.

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute seizures.

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...

Medicine

Electrophoretic Delivery of &#x3B3;-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice

Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the cases, serious side effects affect the quality of life of patients. Moreover, a high percentage of epileptic patients are drug resistant; in their case, neurosurgery or neurostimulation are necessary. Therefore, the major goal of epilepsy research is to discover new therapies which are either capable of curing epilepsy without side effects or preventing recurrent seizures in drug-resistant patients. Neuroengineering provides new approaches by using novel strategies and technologies to find better solutions to cure epileptic patients at risk.
As a demonstration of a novel experimental protocol in an acute mouse model of epilepsy, a direct in situ electrophoretic drug delivery system is used. Namely, a neural probe incorporating a microfluidic ion pump (&#181;FIP) for on-demand drug delivery and simultaneous recording of local neural activity is implanted and demonstrated to be capable of controlling 4-aminopyridine-induced (4AP-induced) seizure-like event (SLE) activity. The &#947;-aminobutyric acid (GABA) concentration is kept in the physiological range by the precise control of GABA delivery to reach an antiepileptic effect in the seizure focus but not to cause overinhibition-induced rebound bursts. The method allows both the detection of pathological activity and intervention to stop seizures by delivering inhibitory neurotransmitters directly to the epileptic focus with precise spatiotemporal control.
As a result of the developments to the experimental method, SLEs can be induced in a highly localized manner that allows seizure control by the precisely tuned GABA delivery at the seizure onset.

Electrophoretic Delivery of &#947;-aminobutyric Acid GABA into Epileptic Focus Prevents Seizures in Mice

The challenge of epilepsy research is to develop novel treatments for patients where classical therapy is inadequate. Using a new protocol&#8212;with the help of an implantable drug delivery system&#8212;we are able to control seizures in anesthetized mice by the electrophoretic delivery of GABA into the epileptic focus.

Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the ...

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 ...

Flexible Multi-Region Neural Recording Device for Epilepsy Research

An Integrated Method for Crafting Flexible and Convenient Electrophysiological Optrodes for Multi-Region In Vivo Recording

Epilepsy is a neurological disorder characterized by synchronized abnormal discharges involving multiple brain regions. Focal lesions facilitate the propagation of epileptic signals through associated neural circuits. Therefore, in vivo recording of local field potential (LFP) from the critical brain regions is&#160;essential for deciphering the circuits involved in seizure propagation. However, current methods for electrode fabrication and implantation lack flexibility. Here, we present a handy device designed for electrophysiological recordings (LFPs and electroencephalography [EEG]) across multiple regions. Additionally, we seamlessly integrated optogenetic manipulation and calcium signaling recording with LFP recording. Robust after-discharges were observed in several separate regions during epileptic seizures, accompanied by increasing&#160;calcium signaling. The approach used in this study offers a convenient and flexible strategy for synchronous neural recordings across diverse regions of the brain. It holds the potential for advancing research on neurological disorders by providing insights into the neural profiles of multiple regions involved in these disorders.

Author Spotlight: Unraveling Neural Communication and Circuit Interactions in Health and Disease

We have developed a simplified and cost-effective approach for electrode fabrication and conducted recordings of signals across multiple regions in freely moving mice. Utilizing optogenetics, alongside multi-region electrophysiology and calcium signal recording, enabled the revelation of neuronal activities across regions in the seizure kindling model.

Epilepsy is a neurological disorder characterized by synchronized abnormal discharges involving multiple brain regions. Focal lesions facilitate the ...

Implantation of Electrodes into a Mouse Cortex for Electrocorticographic Recordings

Source: Dufort-Gervais, et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61976">Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice.</a> J. Vis. Exp. (2021)
This video demonstrates a protocol for implanting electrocorticographic electrodes in the mouse cerebral cortex to record electrical signals in response to adeno-associated viruses that target and manipulate cofilin expression.

1. ECoG/EMG electrode implantation

	Using straight Kelly forceps, slowly screw one ECoG electrode (with straight gold wire) in the vertical axis in the ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Implanting an On-Head Electrode System in a Rat Model for Real-Time Seizure Detection

Source: Koz&#225;k, G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56669">Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats.</a> J. Vis. Exp. (2018).
This video demonstrates the method of implanting an on-head electrode system in an anesthetized rat for real-time seizure detection. The procedure includes placing stimulation and recording electrodes, securing them with dental cement, and connecting them to a head-mounted amplifier.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...