This work was supported by the Canadian Institutes of Health Research (MOP 119603 to Peter L. Carlen and Taufik A. Valiante), the Ontario Brain Institute (to Taufik A. Valiante), and the Mightex Student Research Grant (to Michael Chang). We would like to thank Liam Long for his assistance in filming the video manuscript. We would like to acknowledge Paria Baharikhoob, Abeeshan Selvabaskaran, and Shadini Dematagoda for their assistance in compiling the figures and tables in this manuscript. Figures 1A, 3A, 4A, and 6A are all original figures made from data published in Chang et al.16.

All research involving patients was performed under a protocol approved by the University Health Network Research Ethics Board in accordance with the Declaration of Helsinki. Procedures involving animals were in accordance with guidelines by the Canadian Council on Animal Care and approved by the Krembil Research Institute Animal Care Committee.
1. Protocol I: Acute In vitro Seizure Model
<ol>
	<li>Preparation of dissection solutions and artificial cerebrospinal fluid
	<ol>
		<li>Carbogenate ultrapure water (which is water filtered with a resistivity of 18.2 M&#937;&#183;cm) with carbogen (95% O2/5% CO2) for 5 min using air stone bubble diffusers (aerators for aquariums). 
		Note: Air stones can be connected to the carbogen tank&#8217;s regulator via standard silicone tubing. If air stones are not available, seal shut the end of the silicone tubing and poke small holes in the seal to allow the carbogen gas to bubble out and carbogenate the solution.</li>
		<li>Prepare a dissection solution by dissolving the solutes from Table 1 in ultrapure water. Add CaCl2 to a solution that has been carbogenated for at least 5 min to help it dissolve. 
		Note: Alternatively, add CaCl2 first, so that it can dissolve easily in an unsaturated solution. The solution, in liquid form, should be 300 - 320 mOsm/L and have a pH of 7.4 after being saturated with carbogen.
		<ol>
			<li>Chill the dissection solution to 0 - 4 &#176;C. Leave the solution in the fridge overnight or place the solution in the freezer for 1 h and continually monitor the temperature. 
			Note: The dissection solution can be stored for up to 3 nights; discard afterward.</li>
			<li>Carbogenate the dissection solution for 5 - 10 min before use. 
			Note: Ideally, the solution should either appear as slush or be transitioning to slush prior to use.</li>
		</ol>
		</li>
		<li>Prepare rodent artificial cerebrospinal fluid (ACSF) by dissolving the solutes from Table 2 (or Table 3 for human ACSF) in ultrapure water. Carbogenate the ACSF solution while it is in a water bath heated to 35 &#176;C until use. Add CaCl2 to a solution that has been carbogenated for at least 5 min to help it dissolve. 
		Note: Alternatively, add CaCl2 first so that it can dissolve easier in an unsaturated solution. The solution, in liquid form, should be 290 - 320 mOsm/L and have a pH of 7.4 after being saturated with carbogen. Solutions should be made in 1, 2, or 4 L volumes depending on the duration of the experiments. Make 4 L for a full day (8 h) of experiments. The ACSF should be made fresh each day; do not store it for longer than 1 d.</li>
	</ol>
	</li>
	<li>Mouse dissection to collect brain tissue 
	Note: For human tissue, proceed to the next step (step 1.3) on making brain slices with the vibratome. The cube-shaped blocks (1 cm3) of brain tissue should be obtained from the neurosurgeon using procedures previously described26,27.
	<ol>
		<li>Collect all the tools and materials necessary for animal dissections. Set up the workspace to prepare for animal dissections.
		<ol>
			<li>Check the carbogen tank (95%O2/5%CO2) to ensure it is not empty. Replace the gas cylinder prior to experiments if there is less than 500 psi of pressure remaining.</li>
			<li>Calibrate the vibratome (vibrating blade tissue slicer) according to the manufacturer&#8217;s instruction manual. Adjust the vibratome setting to have a cutting amplitude of 1 mm and a cutting speed of 0.12 mm/s. 
			Note: The optimal settings may differ for each individual vibrating tissue blade cutter; however, in general, the amplitude should be high and the cutting speed should be low.</li>
			<li>Fill the brain slice incubation chamber (i.e., the brain slice keeper) with ACSF and bubble it with carbogen. Then place the incubation chamber in a water bath set at 35 &#176;C. 
			Note: Use rodent ACSF for rodent brain slices and human ACSF for human brain slices.</li>
		</ol>
		</li>
		<li>Obtain a juvenile mouse of either sex, between the age of 13 d (p13) to 21 d (p21, where p0 is the date of birth).</li>
		<li>Anesthetize the mouse with an intraperitoneal injection of sodium pentobarbital (55 mg/kg of body weight). Once the mouse is deeply anesthetized, as indicated by the absence of the toe pinch reflex, promptly use a&#160;vet-approved guillotine instrument to decapitate the mouse in one swift movement. 
		Note: Prepare the sodium pentobarbital injection according to the procedures recommended by institutional guidelines.</li>
		<li>Hold the mouse&#8217;s nose to stabilize the decapitated head and gently make a midline incision starting between the mouse&#8217;s eyes and along the entire length of the scalp to expose the skull. Ensure the skull is not compressed by the pressure applied by the razor. Use the corner of the razor&#8217;s edge to increase cut precision.</li>
		<li>Spread apart the flaps of the mouse&#8217;s scalp using fingers to expose the skull. Then, use the fresh corner of a razor&#8217;s edge to make an incision along the midline of the skull; pay careful attention to avoid any contact with the cerebral cortex.</li>
		<li>Insert splinter forceps into the mouse&#8217;s eye sockets to stabilize the decapitated head and place it in a Petri dish filled with cold (0 - 4&#160;&#176;C) dissection solution. Ensure that the head is fully submerged in the dissection solution.</li>
		<li>Use a second set of splinter forceps to gently peel away the mouse&#8217;s scalp, remove the nose bone by peeling it, and remove the back (caudal side) of the skull. 
		Note: The nose bone of juvenile mice is very soft and easily broken.</li>
		<li>Use a micro spatula to cut the optic nerves, trigeminal nerves, and spinal cord. Then, use the micro spatula to gently separate the brain from the cranium. Leave the brain fully submerged in the Petri dish filled with dissection solution.</li>
	</ol>
	</li>
	<li>Preparation of cortical slices from the somatosensory-motor cortex
	<ol>
		<li>Fill the vibratome&#8217;s buffer tray with ~150 mL of cold (0 - 4&#176;C) dissection solution. 
		Note: Optionally, keep the buffer tray in the freezer until needed to maintain a low temperature during the slicing procedure.</li>
		<li>Glue the brain tissue from mouse or human onto the vibratome stage (specimen holder/tray) using instant adhesive glue. For mice, cut off a small caudal portion of the brain (i.e., the cerebellum) so that it can be easily glued flat onto the specimen holder (allowing the rostral side of the brain to face the ceiling). 
		Note: Alternatively, horizontal mouse brain slices can be prepared by gluing the dorsal side of the brain onto the specimen holder (the ventral side of the brain should face the ceiling)28.</li>
		<li>Gently place the specimen holder (with brain tissue) into the buffer tray. Ensure that the dorsal portion of the brain is facing the vibratome&#8217;s blade. 
		Note: It is critical to minimize the amount of time the brain is exposed to air.</li>
	</ol>
	</li>
	<li>Brain tissue sectioning and collection 
	<ol>
		<li>Slice the brain into 450 &#956;m thick slices using the vibratome in the dorsal to ventral direction. Ensure that the brain remains completely anchored to the specimen tray while slicing it.
		<ol>
			<li>Make the first cut in the mouse brain to remove the olfactory bulb. Then, make subsequent cuts until the somatosensory-motor area is observed (located approximately halfway between the olfactory bulb and bregma). 
			Note: Optionally, slice the brain in thinner (200 &#956;m) slices until the corpus callosum appears in the coronal view of the brain, which indicates that the somatosensory-motor area is close.</li>
		</ol>
		</li>
		<li>Use a wide-bore transfer pipette to collect coronal slices (450 &#956;m) that contain the somatosensory-motor area and submerge them in a Petri dish containing cold (0 - 4 &#176;C) dissection solution.</li>
		<li>Use a new razor blade and cut off any excess tissue from the slices. For coronal slices from mice, perform a transverse cut just below the neocortical commissure (i.e., corpus callosum). Do not cut in a sawing motion; simply apply pressure on the blade into the tissue and use a detailing brush to gently separate the tissue. Make sure to minimize the movement of the coronal slice.</li>
		<li>Use a wide-bore transfer pipette to transfer the dorsal portion of the coronal slices that contain the neocortex (layer 1 - 6) to a second Petri dish filled with warm (35 &#176;C) ACSF for a moment (~1 s). Then, promptly transfer the slices to an incubation chamber containing warm (35 &#176;C) carbogenated ACSF. 
		Note: The purpose of the transfer into the Petri dish with ACSF is to minimize the transfer of dissection solution to the incubation chamber while transferring brain slices with the wide-bore pipette. Utilize the incubation chamber&#8217;s multiple wells to keep different brain slices organized.</li>
		<li>Discard the rest of the brain and animal carcass according to the institutional guidelines.</li>
	</ol>
	</li>
	<li>Incubation and maintenance 
	<ol>
		<li>Leave the brain slices slightly submerged in the incubation chamber at 35 &#176;C for 30 min. Then, remove the incubation chamber from the water bath and allow it to return to room temperature (20 - 25 &#176;C). Wait 1 h for the brain slices to recover before performing electrophysiological recordings. 
		Note: Ensure there are no air bubbles that collect underneath the brain slices in the incubation chamber. Air bubbles are air interfaces that cause tissue damage. The mouse brain slices can be maintained for 6 - 8 h, while human brain tissue can be stored for up to 24 h in a well-carbogenated incubation chamber with the appropriate ACSF.</li>
	</ol>
	</li>
	<li>Electrophysiological recordings of the superficial cortical layer 
	Note: Ictal events are observed in the extracellular local field potential (LFP) recordings from brain slices. The LFP of the brain slice can be observed and recorded with either an interface type chamber29 or a submerged multi-electrode array (MEA) system16. The procedure has been previously described16 and additional details are described below.
	<ol>
		<li>Use a wide-bore transfer pipette or a detailing brush to move a brain slice onto slightly larger pre-cut lens paper that is held in place using a dental tweezer. Transfer the lens paper (that the brain slice is resting upon) to the recording chamber and secure it in position with a harp screen.</li>
		<li>Run warm (35 &#176;C), carbogenated ACSF perfusate through the recording chamber over the brain slice at a rate of 3 mL/min (~1 drip/s). Use a digital thermometer to ensure the recording chamber is 33 - 36&#176;C.</li>
		<li>Pull glass electrodes with an impedance of 1 - 3 M&#8206;&#937; from borosilicate glass tubing (with an outer diameter of 1.5 mm) using a puller. Backfill the glass electrodes with ACSF (~10 &#181;L) using a Hamilton syringe. Immediately discard the electrode if the tip is damaged. 
		Note: Bleach the silver wire (5 min) and only allow a minimal portion (i.e., the tip) of the silver wire to be submerged in the ACSF back-filled glass electrodes to minimize noise and drift during the recordings. Remove any excess ACSF from the glass electrode with a Hamilton syringe if necessary.</li>
		<li>Use a 20X stereo microscope to accurately guide the recording glass electrode into the superficial cortical layer (2/3) using manual manipulators. Record/view the electrical activity of the brain slice on a computer with standard software. 
		Note: Viable (high-quality) brain slices will exhibit a robust evoked potential in response to the applied electrical stimuli (100 &#181;s, 30 - 300 &#956;A) or light pulses (30 ms, 10 mW/mm2) for optogenetic tissue.</li>
	</ol>
	</li>
	<li>Induction of seizure-like activities
	<ol>
		<li>Perfuse ACSF containing 100 &#181;M 4-aminopyrimidine (4-AP) over the brain slice. Dissolve 80 mg of 4-AP in 8.5 mL of water to make a stock solution of 100 mM 4-AP. Add 100 &#181;L of 100 mM 4-AP stock solution to 100 mL of ACSF to achieve an ACSF perfusate with 100 &#181;M 4-AP. 
		Note: Alternatively, use Zero-Mg2+ ACSF (Table 4), a modified ACSF solution containing no added Mg2+, to perfuse the brain slice. For human tissue, use a combination of 100 &#181;M 4-AP in Zero-Mg2+ Human ACSF (Table 5) to achieve optimal results. The average time for ictal events to appear is 15 min; however, it may take up to 40 min for some brain slices.</li>
	</ol>
	</li>
	<li>On-demand seizure generation: an optogenetic strategy for optogenetic mice
	<ol>
		<li>Apply a brief (30 ms) pulse of blue (470 nm) light (with a minimum 1 mW/mm2 output intensity) to initiate an ictal event. Use a manual manipulator to position a 1,000 &#181;m core diameter optical fiber (0.39 NA) directly above the recording region. 
		Note: Set the rate of the photo-stimulation to match the desired rate of the ictal event occurrence (i.e., 1 pulse every 50 s). However, the rate cannot be overly exaggerated from the intrinsic rate at which ictal events occur.</li>
	</ol>
	</li>
	<li>On-demand seizure generation: a non-optogenetic strategy for human tissue
	<ol>
		<li>Position the brain slice so that the superficial layers are upstream, and the deep layers are downstream to the flow of the perfusate through the recording chamber.</li>
		<li>Pull a glass electrode (the same type as used for the LFP recordings) and gently touch its tip with a delicate task wiper to create a small opening. Backfill the electrode with ~25 &#181;L of 100 mM GABA (dissolved in water) and attach it to the picospritzer. Alternatively, backfill the electrode with 200 &#181;M glutamate (dissolved in water). 
		Note: To estimate the volume being puffed from the picospritzer (~50 &#181;L), apply a test puff onto a plastic plate (preferably gridded). Then, apply a droplet of known volumes (i.e., 10 &#181;L, 20 &#181;L, 50 &#181;L, or 100 &#181;L) with a pipette for a comparison with the test puff.</li>
		<li>Apply puffs of the neurotransmitter (GABA or glutamate) to the human tissue with a picospritzer (10 - 20 psi for 30 - 100 ms) to initiate an ictal event. Apply a single puff of 100 mM GABA onto the deep layer (5) of the brain tissue to generate ictal events in the superficial layer (2/3). Alternatively, apply a single puff of 200 &#181;M glutamate directly onto the superficial layer to generate ictal events in the superficial layer. 
		Note: Set the rate of the picospritzer puff to match the desired rate of the ictal event occurrence (i.e., 1 pulse every 50 s). However, the rate cannot be overly exaggerated from the intrinsic rate at which ictal events occur.</li>
	</ol>
	</li>
</ol>
2. Protocol II: Acute In vivo Seizure Model
<ol>
	<li>Surgical preparation and surgery
	<ol>
		<li>Obtain an adult mouse (p35 - p60) of either sex.</li>
		<li>Deeply anesthetize mouse with Ketamine (95 mg/kg) and Xylazine (5 mg/kg). Perform the toe-pinch reflex test to ensure the mouse is sedated. Alternatively, use isoflurane (4% for induction, 1.5 - 2% for surgery) to anesthetize the mouse. 
		Note: The choice of anesthetics can impact the seizure activity30,31.</li>
		<li>Mount the mouse into a stereotaxic frame by securing its head with ear bars. Place a heated pad underneath the mouse for the duration of the surgery.</li>
		<li>Shave the top of the mouse&#8217;s head using a rodent trimmer to expose the scalp. Inject 0.3 mL of lidocaine with a 25 G 5/8 in needle into the periosteum to avoid excessive bleeding or pain. Apply eye ointment to the mouse&#8217;s eyes to prevent them from drying out during the experiment.</li>
		<li>Pinch and lift the center of the mouse&#8217;s scalp to assess if its reflexes are gone. Subsequently, perform a horizontal cut to expose the bregma to lambda region of the skull. Gently scrape the entire exposed area of the skull with a cotton swab to create a dry surface.</li>
		<li>Perform a craniotomy of 4 mm in diameter at coordinates 2.0 mm lateromedial and -2.0 mm rostrocaudal to expose the somatosensory cortex. Mark the location with a circle (4 mm in diameter) using a permanent marker. Drill around the circle with a pneumatic dental drill until the remaining layer of the craniotomy bone easily gives way when pushed down. Apply saline solution onto the layer of bone before removing the craniotomy with splinter forceps. Subsequently, apply warm saline solution to the exposed region.</li>
	</ol>
	</li>
	<li>Recording methods and chemical induction of seizures
	<ol>
		<li>Pull glass electrodes with an impedance of 1 - 3 M&#8206;&#937; from borosilicate glass tubing (with an outer diameter of 1.5 mm) using a puller. Backfill the glass electrodes with ~10 &#181;L of ACSF or saline using a Hamilton syringe. Immediately discard the electrode if the tip is damaged. 
		Note: Bleach the silver wire (5 min) and only allow a minimal portion (i.e., the tip) of the silver wire to be submerged in the ACSF back-filled glass electrodes to minimize noise and drift during the recordings. Remove any excess ACSF from the glass electrode with a Hamilton syringe if necessary.</li>
		<li>Use a manual manipulator to guide the glass electrode into the superficial cortical layer (2/3) in the somatosensory-motor area. Record/view the electrical activity of the brain slice on a computer with standard software. 
		Note: Layer 2/3 is approximately 0.3 mm deep from the surface32.</li>
		<li>Topically apply ~0.5 mL 1.5 mM 4-AP saline onto the exposed cortex of the mouse with a syringe until it completely covers the craniotomy. Dissolve 14 mg of 4-AP in 100 mL of isotonic saline to make a stock solution of 1.5 mM 4-AP saline. 
		Note: Prepare the 4-AP saline before the experiment begins. On average, ictal events appear 15 - 30 min after topical application.</li>
	</ol>
	</li>
	<li>On-demand generation of seizures in optogenetic mice
	<ol>
		<li>Apply a brief (30 ms) pulse of blue (470 nm) light (with a minimum 10 mW/mm2 output intensity) to initiate an ictal event. Use a manual manipulator to position a 1,000 &#181;m core diameter optical fiber (0.39 NA) directly above the recording region. 
		Note: Set the rate of the photo-stimulation to match the desired rate of the ictal event occurrence (i.e., 1 pulse every 300 s). However, the rate cannot be overly exaggerated from the intrinsic rate at which ictal events occur.</li>
	</ol>
	</li>
</ol>

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(12), e680 (2008).</li><li>Ritter, L. M., 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=WONOEP+appraisal:+optogenetic+tools+to+suppress+seizures+and+explore+the+mechanisms+of+epileptogenesis.">WONOEP appraisal: optogenetic tools to suppress seizures and explore the mechanisms of epileptogenesis.</a> Epilepsia. 55 (11), 1693-1702 (2014).</li><li>Zhao, 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=Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nature Methods. 8 (9), 745-752 (2011).</li><li>Arenkiel, B. R., 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=In+vivo+light-induced+activation+of+neural+circuitry+in+transgenic+mice+expressing+channelrhodopsin-2.">In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2.</a> Neuron. 54 (2), 205-218 (2007).</li><li>Heinemann, U., Pitk&#228;nen, A., Buckmaster, P., Galanopoulou, A. S., Mosh&#233;, 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=Brain+slices+from+human+resected+tissue.">Brain slices from human resected tissue.</a> Models of Seizures and Epilepsy. , 285-299 (2017).</li><li>Florez, 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=In+vitro+recordings+of+human+neocortical+oscillations.">In vitro recordings of human neocortical oscillations.</a> Cerebral Cortex. 25 (3), 578-597 (2015).</li><li>Lein, P. J., Barnhart, C. D., Pessah, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+hippocampal+slice+preparation+and+hippocampal+slice+cultures.">Acute hippocampal slice preparation and hippocampal slice cultures.</a> Methods in Molecular Biology. , 115-134 (2011).</li><li>Haas, H. L., Schaerer, B., Vosmansky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+perfusion+chamber+for+the+study+of+nervous+tissue+slices+in+vitro.">A simple perfusion chamber for the study of nervous tissue slices in vitro.</a> Journal of Neuroscience Methods. 1 (4), 323-325 (1979).</li><li>Poulton, T. J., Ellingson, 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=Seizure+associated+with+induction+of+anesthesia+with+isoflurane.">Seizure associated with induction of anesthesia with isoflurane.</a> Anesthesiology: The Journal of the American Society of Anesthesiologists. 61 (4), 471-476 (1984).</li><li>Borris, D. J., Bertram, E. H., Kapur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ketamine+controls+prolonged+status+epilepticus.">Ketamine controls prolonged status epilepticus.</a> Epilepsy Research. 42 (2-3), 117-122 (2000).</li><li>DeFelipe, J., Alonso-Nanclares, L., Arellano, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructure+of+the+neocortex:+comparative+aspects.">Microstructure of the neocortex: comparative aspects.</a> Journal of Neurocytology. 31 (3-5), 299-316 (2002).</li><li>Velasco, A. L., Wilson, C. L., Babb, T. L., Engel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+anatomic+correlates+of+two+frequently+observed+temporal+lobe+seizure-onset+patterns.">Functional and anatomic correlates of two frequently observed temporal lobe seizure-onset patterns.</a> Neural Plasticity. 7 (1-2), 49-63 (2000).</li><li>Vlachos, A., Reddy-Alla, S., Papadopoulos, T., Deller, T., Betz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostatic+regulation+of+gephyrin+scaffolds+and+synaptic+strength+at+mature+hippocampal+GABAergic+postsynapses.">Homeostatic regulation of gephyrin scaffolds and synaptic strength at mature hippocampal GABAergic postsynapses.</a> Cerebral Cortex. 23 (11), 2700-2711 (2012).</li><li>Kirmse, K., 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=GABA+depolarizes+immature+neurons+and+inhibits+network+activity+in+the+neonatal+neocortex+in+vivo.">GABA depolarizes immature neurons and inhibits network activity in the neonatal neocortex in vivo.</a> Nature Communications. 6, 7750 (2015).</li><li>Stein, V., Hermans-Borgmeyer, I., Jentsch, T. J., H&#252;bner, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+KCl+cotransporter+KCC2+parallels+neuronal+maturation+and+the+emergence+of+low+intracellular+chloride.">Expression of the KCl cotransporter KCC2 parallels neuronal maturation and the emergence of low intracellular chloride.</a> Journal of Comparative Neurology. 468 (1), 57-64 (2004).</li><li>Wong, B. Y., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lateral+spread+of+ictal+discharges+in+neocortical+brain+slices.">The lateral spread of ictal discharges in neocortical brain slices.</a> Epilepsy Research. 7 (1), 29-39 (1990).</li><li>Trevelyan, A. J., Sussillo, D., Watson, B. O., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+propagation+of+epileptiform+activity:+evidence+for+an+inhibitory+veto+in+neocortex.">Modular propagation of epileptiform activity: evidence for an inhibitory veto in neocortex.</a> Journal of Neuroscience. 26 (48), 12447-12455 (2006).</li><li>Brahma, B., Forman, R., Stewart, E., Nicholson, C., Rice, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbate+inhibits+edema+in+brain+slices.">Ascorbate inhibits edema in brain slices.</a> Journal of Neurochemistry. 74 (3), 1263-1270 (2000).</li><li>MacGregor, D. G., Chesler, M., Rice, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEPES+prevents+edema+in+rat+brain+slices.">HEPES prevents edema in rat brain slices.</a> Neuroscience Letters. 303 (3), 141-144 (2001).</li><li>Ting, J. T., Daigle, T. L., Chen, Q., Feng, G., Martina, M., Taverna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+brain+slice+methods+for+adult+and+aging+animals:+Application+of+targeted+patch+clamp+analysis+and+optogenetics.">Acute brain slice methods for adult and aging animals: Application of targeted patch clamp analysis and optogenetics.</a> Patch-clamp Methods and Protocols. , 221-242 (2014).</li><li>Swartzwelder, H. S., Lewis, D., Anderson, W., Wilson, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seizure-like+events+in+brain+slices:+suppression+by+interictal+activity.">Seizure-like events in brain slices: suppression by interictal activity.</a> Brain Research. 410 (2), 362-366 (1987).</li><li>Lees, G., St&#246;hr, T., Errington, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereoselective+effects+of+the+novel+anticonvulsant+lacosamide+against+4-AP+induced+epileptiform+activity+in+rat+visual+cortex+in+vitro.">Stereoselective effects of the novel anticonvulsant lacosamide against 4-AP induced epileptiform activity in rat visual cortex in vitro.</a> Neuropharmacology. 50 (1), 98-110 (2006).</li></ol>

The authors have nothing to disclose.

The brain slices are treated with a proconvulsant drug or an altered ACSF perfusate to increase the neural network&#39;s excitability and promote a precipitation of ictal events (electrographic seizure-like events). For mice, the preferred coronal slices of the somatosensory-motor area should contain the cingulate cortex, area 2 (CG), but not the retrosplenial area (RS); these anatomical markers help identify the range of coronal slices that are best for inducing ictal events. An optional modification for mice tissue is ...

Acute seizure models can successfully reproduce electrographic signatures reminiscent of ictal events observed in the electroencephalogram (EEG) of individuals experiencing a seizure. Researchers use these ictal-like events (herein referred to as &#39;ictal events&#39;) as surrogates for the seizure event1. Clinically, ictal events serve as a reliable proxy for seizure events since seizures are a neurological disorder which originates from the brain. In the epilepsy monitoring unit, neurologists rely upon the detection of ictal events to confirm the brain&#39;s epileptogenic region and isolate it for resection2. In the intensive care unit, physicians monitor ictal activity to assess if any seizure activity persists in sedated patients3. Controlling seizures remains to be a challenging issue for the medical community, as 30% of epilepsy patients are drug resistant to the available medication4,5, and 10% of medical cases involving drug-induced seizures are unresponsive to the standard treatment3. This presents a serious concern for the society, as 10% of the American population is prospected to experience one seizure event in their lifetime and 3% are expected to develop epilepsy6.
Studying seizures in chronic epilepsy models is expensive, laborious, and often take months to prepare7. It is also difficult to perform electrophysiological recordings in freely moving animals. Human clinical trials face similar issues, as well as additional complexities related to patient consent, variability in participants&#39; backgrounds, and the moral and ethical considerations involved8. Acute seizure models, on the other hand, are favorable because they are relatively convenient to prepare, cost-efficient, and capable of generating large volumes of ictal events for study9. Additionally, the tissue is fixed in a stable position, so the conditions are ideal for performing the electrophysiological recordings necessary to study seizure dynamics and the related underlying pathophysiology. Acute seizure models remain favorable over in silico (computer) models because they are based on biological material comprised of the brain&#39;s constituent neuronal network with all its inherent factors and synaptic connectivity, that may not be captured by even the most detailed computer models10. These features make acute seizure models poised to be efficient at screening for potential anti-seizure therapies and making preliminary findings before advancing them for further investigation in chronic epilepsy models and clinical trials.
Typically, acute seizure models are derived from the normal brain tissue that has been subjected to hyper-excitable conditions. To induce clinically relevant ictal events in healthy brain tissue, it is important to understand that the brain functions optimally in a critical state11 where excitation (E) and inhibition (I) are balanced12. A disruption of the E-I balance can lead to the hyper-excitable seizure state in which ictal events precipitate. Accordingly, within this conceptual framework, there are two major strategies to generate ictal events in brain slices (in vitro) or in whole-brain (in vivo) preparations: either decreased inhibition (&#34;disinhibition&#34;) or increased excitation (&#34;non-disinhibition&#34;). However, ictal events are highly ordered and synchronized events that require the influence of GABAergic interneurons to orchestrate the neural network activity13,14. For this reason, non-disinhibition models are the most effective for generating ictal events in isolated neural networks, such as in an in vitro brain slice15, whereas in vitro disinhibition models commonly lead to spiking activity reminiscent of interictal-like spiking. Furthermore, within this conceptual framework, a momentary synchronizing event can also reliably trigger an ictal event16. In fact, an ictal event can be triggered by any minor perturbation applied to the neural system17 when it is at a critical state transition (&#34;bifurcation&#34;) point18. Traditionally, these perturbations were induced by electrical stimulation. The recent development of optogenetics in neuroscience, however, now offers a more elegant strategy to induce critical state transitions16.
The methods described in this paper demonstrate how to generate ictal events on-demand in acute seizure models for both in vitro (step 1 of the Protocol) and in vivo studies (step 2 of the Protocol). They involve the choice of brain region, seizure induction method, study type, and species; however, the focus will be on the recommended choice of an acute 4-AP cortical seizure model because of its versatility in a wide variety of study types. The acute in vitro 4-AP seizure model is based on the standard protocol to prepare high-quality brain slices for electrophysiological recordings and imaging studies19. These protocols have already been used to make in vitro coronal brain slices from the somatosensory-motor cortex of mice16,20 and humans21. Modifications to generate ictal events in these types of brain slices have been previously demonstrated16 and the full details are described in the Protocol below. The acute in vivo 4-AP cortical seizure model is based on the standard protocol to prepare a craniotomy for imaging studies22. The modification is that no (glass slide) window is installed following the craniotomy. Instead, proconvulsant agents (4-AP) are topically applied to the exposed cortex to induce ictal events while the animal is under general anesthesia. To our knowledge, our group was the first to develop this acute in vivo cortical seizure model in mice16,23. The acute in vivo 4-AP cortical seizure model prepared from adult mice was developed to complement the in vitro slice model from juvenile tissue. The replication of findings in the adult in vivo seizure model helps to generalize the findings from slice models by addressing the inherent concerns regarding the non-physiological conditions of a 2D brain slice (versus a 3-D whole-brain structure) and the physiological differences between juvenile and adult tissue.
The method of on-demand ictal event initiation is demonstrated using either puffs of neurotransmitters with a picospritzer or optogenetic strategies. To the best of our knowledge, our group is the first to initiate ictal events in human tissue using neurotransmitters via a picospritzer16. For optogenetic strategies, the C57BL/6 mice strain is the conventional strain used for expressing transgenes. The expression of channelrhodopsin-2 (ChR2) in either GABAergic interneurons or glutamatergic pyramidal cells will provide the optional ability to generate ictal events on-demand with brief light pulses. Suitable optogenetic mice strains include the commercially available C57BL/6 variant that expresses ChR2 in either interneurons, using the mouse vesicular GABA transporter promotor (VGAT)24, or pyramidal cells, using the mouse thymus cell antigen 1 promotor (Thy1)25. These commercially available VGAT-ChR2 and Thy1-ChR2 mice offer the opportunity to activate GABAergic neurons or glutamatergic neurons, respectively, in the neocortex with blue (470 nm) light. The ability to generate ictal events on demand in acute seizure models can offer novel opportunities to study seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

<table><tbody><tr><td>Sodium pentobarbital</td><td>N/A</td><td>N/A</td><td>Purchased through the Toronto Western Hospital&#039;s Suppliers</td></tr><tr><td>1 mm syringe</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>25G 5/8&amp;rdquo; sterile needle</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Single edge razor blade (2x)</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Instant adhesive glue</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Lens paper</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Glass petri dish (2x)</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Splinter forceps (2x)</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>PVC handle micro spatula</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Micro spoon with flat end</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Detailing brush 5/0</td><td>N/A</td><td>N/A</td><td>Purcahsed from a boutique art store</td></tr><tr><td>Wide bore transfer pipette</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Dental Tweezer</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Thermometer (digital)</td><td>N/A</td><td>N/A</td><td>Purchased on Amazon.ca</td></tr><tr><td>Check carbogen tank (95%O&lt;sub&gt;2&lt;/sub&gt;/5%CO&lt;sub&gt;2&lt;/sub&gt;)&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>Purchased through the Toronto Western Hospital&#039;s Suppliers</td></tr><tr><td>Vibratome</td><td>Leica</td><td>N/A</td><td>Purchased through the Toronto Western Hospital&#039;s Suppliers</td></tr><tr><td>brain slice incubation chamber (a.k.a. brain slice keeper)&amp;nbsp;</td><td>Scientific Systems Design Inc</td><td>N/A</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Sodium Bicarbonate</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Dextrose</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Potassium Chloride (KCl)</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Magnesium Sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt; H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Sodium phosphate monobasic monohydrate (HNaPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr><tr><td>Sucrose</td><td>N/A</td><td>N/A</td><td>Purchased through UT Med Store</td></tr></tbody></table>

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.

The application of 100 &#181;M 4-AP to good-quality (undamaged) 450 &#181;m-sized cortical brain slices from a juvenile VGAT-ChR2 mouse reliably induced recurrent ictal events (&#62; 5 s) within 15 min (Figure 1Ai). The application of 100 &#181;M 4-AP to slices of poor-quality resulted in bursting events or spiking activity (Figure 1Aii). On average, 40% of the slices from each dissected mouse brain successfully generated ictal e...

Watch this Scientific Journal Video about Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue at JoVE.com

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

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 ...

generation-on-demand-initiation-acute-ictal-activity-rodent-human

Research

JoVE Journal

Neuroscience

8.9K Views.  Krembil Research Institute. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de studie van aanvallenmechanismen, zoals, welke neurale subpopulaties verantwoordelijk zijn voor het begin en de beëindiging van de elektrografische aanval. Het belangrijkste voordeel van deze techniek is dat het elektrografische aanvallen kan reproduceren, op aanvraag, en in vivo en in vitro modellen die doen denken aan die klinisch waargenomen. Deze techniek kan ook worden gebruikt om te zoeken naar mogelijke anti-aanv...

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

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Medicine

Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test interventions at various stages of the disease process. These animal models are particularly difficult to implement in immature rodents, a classic model organism for laboratory study of these disorders. Recording continuous EEG in young animal models of seizures and other neurological disorders presents a technical challenge due to the small physical size of young rodents and their dependence on the dam prior to weaning. Therefore, there is not only a clear need for improving pre-clinical research that will better identify those therapies suitable for translation to the clinic but also a need for new devices capable of recording continuous EEG in immature rodents. Here, we describe the technology behind and demonstrate the use of a novel miniature telemetry system, specifically engineered for use in immature rats or mice, which is also effective for use in adult animals.

Here we demonstrate the use of a wireless enabling technology for electroencephalogram (EEG) in neonatal rodent models of human disease. With telemetry, there are no encumbering connections, thus allowing natural behaviors.

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test ...

Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with focal epilepsy. This methodology relies on the assumption that seizures originate from the same regions of the brain from which interictal epileptiform discharges (IEDs) emerge. Preclinical models are very useful to find correlates between IED locations and the actual regions underlying seizure initiation in focal epilepsy. Rats have been commonly used in preclinical studies of epilepsy1; hence, there exist a large variety of models for focal epilepsy in this particular species. However, it is challenging to record multichannel EEG and to perform brain source imaging in such a small animal. To overcome this issue, we combine a patented-technology to obtain 32-channel EEG recordings from rodents2 and an MRI probabilistic atlas for brain anatomical structures in Wistar rats to perform brain source imaging. In this video, we introduce the procedures to acquire multichannel EEG from Wistar rats with focal cortical dysplasia, and describe the steps both to define the volume conductor model from the MRI atlas and to uniquely determine the IEDs. Finally, we validate the whole methodology by obtaining brain source images of IEDs and compare them with those obtained at different time frames during the seizure onset.

This video introduces the preparation, recording, and source analysis procedures of high-resolution EEG on sedated rats with a particular preclinical model of focal epilepsy under noninvasive conditions.

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

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) ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

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

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

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

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

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.

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 ...

Consistent Induction and Recording of Seizure-Like Activity Using CMOS-HD-MEAs

High-Quality Seizure-Like Activity from Acute Brain Slices Using a Complementary Metal-Oxide-Semiconductor High-Density Microelectrode Array System

Complementary metal-oxide-semiconductor high-density microelectrode array (CMOS-HD-MEA) systems can record neurophysiological activity from cell cultures and ex vivo brain slices in unprecedented electrophysiological detail. CMOS-HD-MEAs were first optimized to record high-quality neuronal unit activity from cell cultures but have also been shown to produce quality data from acute retinal and cerebellar slices. Researchers have recently used CMOS-HD-MEAs to record local field potentials (LFPs) from acute, cortical rodent brain slices. One LFP of interest is seizure-like activity. While many users have produced brief, spontaneous epileptiform discharges using CMOS-HD-MEAs, few users reliably produce quality seizure-like activity. Many factors may contribute to this difficulty, including electrical noise, the inconsistent nature of producing seizure-like activity when using submerged recording chambers, and the limitation that 2D CMOS-MEA chips only record from the surface of the brain slice. The techniques detailed in this protocol should enable users to consistently induce and record high-quality seizure-like activity from acute brain slices with a CMOS-HD-MEA system. In addition, this protocol outlines the proper treatment of CMOS-HD-MEA chips, the management of solutions and brain slices during experimentation, and equipment maintenance.

Author Spotlight: Unraveling Seizure Dynamics and Novel Therapeutics for Status Epilepticus Using CMOS High-Density Microelectrode Array Systems

Here, we outline a protocol for using complementary metal-oxide-semiconductor high-density microelectrode array systems (CMOS-HD-MEAs)&#160;to record seizure-like activity from ex vivo brain slices.

Complementary metal-oxide-semiconductor high-density microelectrode array (CMOS-HD-MEA) systems can record neurophysiological activity from cell cultures ...

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

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