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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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><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...

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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 ...

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2.3K Views.  University of Virginia. Dit protocol genereert een nieuw model voor temporale kwab epilepsie waarbij muizen spontane aanvallen ontwikkelden, en het is zeer nuttig geweest voor het onderzoeken van de mechanismen van epileptogenese en ook voor het screenen van nieuwe therapieën. Deze procedure bij VGAT-Cre-muizen leidt op betrouwbare wijze tot spontane terugkerende elektrografische en motorische en motorische composietaanvallen zonder uitgebreide hippocampale celdood te veroorzaken die vaak wordt gezien na status epi...

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

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 ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

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 ...

Implantation of Electrodes into a Mouse Cortex for Electrocorticographic Recordings

Source: Dufort-Gervais, et al. <a href="https://app.jove.com/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

Electroencephalographic Recording of Epileptic Seizures in Epilepsy-Induced Rats

Source: Soukupov&#225;, M., et al. <a href="https://app.jove.com/v/58455">Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats.</a> J. Vis. Exp. (2018)
This video demonstrates electroencephalographic (EEG) recording of epileptic seizures in epilepsy-induced rats. A rat with electrodes implanted in the ventral hippocampus is connected to the EEG recording system and placed in a plexiglass chamber. The rat is allowed to move freely while the brain activity is recorded to monitor the occurrence of seizures.

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

Stimulation and Modulation 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/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.

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

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