The research was supported by the National Natural Science Foundation of China (No. 82030037, 81871009) and Beijing Municipal Health Commission (11000022T000000444685). We thank TopEdit (www.topeditsci.com) for its linguistic assistance during the preparation of this manuscript.

This experiment was approved by the Experimental Animal Ethics Committee of Xuanwu Hospital, Capital Medical University. All mice were kept in the animal laboratory of Xuanwu Hospital, Capital Medical University. This protocol is divided into four parts. The first two parts introduce the method of building the electrode and the electric circuit using a slip ring to connect the electrodes and the EEG recording/stimulation equipment. The third part describes the operation method of electrode implantation, and the fourth part presents the EEG recording and stimulation parameters used for the amygdala epilepsy model.
1. Fabrication of electrodes
<ol>
	<li>Keep the following previously prepared materials ready: two 3 cm long pieces of Teflon-coated tungsten wire (bare diameter: 76.2 &#181;m), one piece of silver wire (bare diameter: 127 &#181;m) of the same length, and one set of 2 x 2 gauged row pins.</li>
	<li>Use a lighter to burn one end of each tungsten wire to remove 5 mm of the insulation coating. 
	NOTE: The tungsten wire with the insulation removed turns black; this part of the tungsten wire is referred to as the upper end.</li>
	<li>Peel a section of ultrafine multi-strand wire and wrap it from the bottom to the upper end where it starts to get dark, continuing to the top. Combine this superfine wire (has a soft texture) with the tungsten wire by pinching one end and gently twisting the other end, which enables the two materials to be easily entwined together.</li>
	<li>Gently pull to ensure that they are tightly wrapped and cut off excess superfine wire. Try to keep the tungsten wire straight throughout the process.</li>
	<li>Fix the row pin to the clamp on the welding table with the longer side of the pins facing outward. Use the syringe needle to pick up some solder paste and apply it to the pins. Heat the welding torch to 320 &#176;C; melt and smear some lead-free tin wire with the torch tip.</li>
	<li>Overlap the upper end of the tungsten wire with one needle of the row pins and use the solder on the torch to bond the tungsten wire to the pin. 
	NOTE: It would be very difficult to weld the tungsten wire with the pins directly without the help of the superfine wire.</li>
	<li>Weld another tungsten wire and another silver wire to the row pin in the same way so that each wire corresponds to a needle (see Figure 1,i).</li>
	<li>Cut two heat-shrinkable tubes slightly longer than the upper end of the tungsten wire. Put them on the solder joint of two tungsten wires, ensuring that the conductive part is fully covered in the tube so that the circuit of the two tungsten wires is not placed in series. 
	NOTE: Although there are three wires, if two of them are insulated, the three wires will not be in series; a tube can also be added to the silver wire.</li>
	<li>Remove the electrode from the welding table clamp and hold the electrode gently with large pliers, as it is easy for electrodes to lose their shape when heating the shrinkable tube, using a good thermal conductivity clamp with slightly more force.</li>
	<li>Turn on the air duct and heat until a temperature of 320 &#176;C is reached. Blow the heat-shrinkable tube for several seconds until it is tightened (see Figure 1,ii).</li>
	<li>If the needles separate from the plastic body during the welding process, splice the welding part and the plastic body with a hot melt adhesive (see Figure 1,iii). Be careful not to smear it on the interface as this would affect the interface insertion.</li>
	<li>Hold the two tungsten wires and twist them together, keeping their ends apart (see Figure 1,iv). Trim the twisted tungsten wires to approximately 10 mm in length so that the separation at the ends does not exceed 0.5 mm. 
	&#8203;NOTE: This step can also be performed before electrode implantation to allow flexible adjustment of electrode length.</li>
	<li>Heat the glue gun and apply the glue evenly around the electrode.</li>
	<li>Check the electrodes with a multimeter: place one bar of the multimeter on the unwelded side of the row pins, and gently touch the end of tungsten wire or silver wire to the other bar, checking whether the circuit is smooth. Ensure that the lines are not placed in series.</li>
</ol>
2. Slip ring connection and circuit description
NOTE: When the electrodes on the mice are plugged into an EEG device via cables in a free-moving condition, the cables can become tangled as the mice move and turn around. This causes the cables to become shorter, eventually hindering the mice from moving or causing the cables to fall off their heads. In the method described here, a four-channel slip ring is introduced to prevent the cables from falling off. The four channels are represented in four colors in Figure 1B.
<ol>
	<li>Peel off 5 mm of the insulation skin at each end to expose the metal wire inside.</li>
	<li>Add a section of heat-shrinkable tube to each stator wire.</li>
	<li>Weld each wire with the EEG device connector plug.</li>
	<li>Shrink the heat-shrinkable tube with hot air.</li>
	<li>Add a section of heat-shrinkable tube to each rotor wire.</li>
	<li>Screw the conducting parts of the red and orange wires together and weld them to a joint in the header to fit the row pin.</li>
	<li>Weld the other two wires on the header to each joint. 
	&#8203;NOTE: The brown channel that corresponds to the silver wire is connected to the EEG device for grounding. The red and orange channels receive signals from the same tungsten wire, and the orange channel serves as a reference for the EEG device. The signals in the red channel are meaningless, but they must coexist with the black channel to form a current stimulus. The signals in the black channel are the real electrical signals in the brain. Different circuits can be designed with multi-channel slip rings to suit different devices.</li>
</ol>
3. Surgery for implantation
<ol>
	<li>Animals
	<ol>
		<li>Use 8-week-old C57BL/6 wild-type male mice, weighing 24-26 g, for surgeries.</li>
		<li>House them with a 12 h light-dark cycle (light time: 8:00-20:00) in a temperature-controlled environment (22 &#177; 1 &#176;C) and provide water and feed ad libitum.</li>
		<li>Use an extra heat mat to keep the animals warm during the surgery.</li>
		<li>After the surgery, inject meloxicam subcutaneously (10 mg/kg) as the first administration of analgesics. Then, place the animals in separate cages to optimize recovery. Add meloxicam to the animal&#39;s diet for the first week after surgery.</li>
		<li>After the experiment, infuse the left ventricles of the mice with 4% paraformaldehyde under anesthesia, and collect the brain tissues for histological verification of the electrode target.</li>
	</ol>
	</li>
	<li>Weigh the mouse and anesthetize it by intraperitoneal injection of 1% pentobarbital solution. Sterilize all the surgical instruments and consumables to be used, including drill bits, electrodes, dental cement, etc., by autoclaving.</li>
	<li>When the mouse is completely anesthetized, shave the hair from the eye to the ear area with a razor.</li>
	<li>Fix the mouse on the stereotaxic frame. Put the front upper teeth into the incisor bar and insert both ear bars equally deeply into the ears. Apply erythromycin eye ointment to the eyes to prevent dryness and blindness caused by a bright light during surgery.</li>
	<li>Disinfect the surgical area with three alternating swabs of iodophor and 75% alcohol in a circular motion. Then, make a sagittal incision forward from the middle of this incision, and cut off the skin on either side of the incision to create a triangular window.</li>
	<li>Roll a small piece of cotton into a ball and wet it with 3% hydrogen peroxide. Remove the soft tissue attached to the skull by gently rubbing the exposed area with a small cotton ball until the anterior and posterior fontanelle are clearly seen.</li>
	<li>Adjust the anterior and posterior heights so that the anterior and posterior fontanelle is in the horizontal position. Consider the position of the anterior fontanelle to be the origin of the axes.</li>
	<li>Fix a stainless-steel screw to the left cerebellar skull, using a drill to create a flat surface. Ensure that the screw protrudes halfway out of the skull.</li>
	<li>Ensure the coordinates for the amygdala kindling are &#8722;1.67 mm posterior, &#8722;2.7 mm lateral, and &#8722;4.9 mm ventral from the bregma. Adjust the stereotaxic device to locate this spot and mark it.</li>
	<li>Drill a hole on the marked spot with a 0.5 mm diameter skull drill.</li>
	<li>Fix the electrodes to the locating rod of the stereotaxic device, place the electrode vertically above the hole, and drop the position to &#8722;4.9 mm slowly. Wrap the silver wire around the screw three times, taking care not to shake the electrode body during operation.</li>
	<li>Mix the dental cement and gently apply it to the electrode and skull surface. When the dental cement hardens, modify the outside until the cement that encloses the fixed electrode turns into a cone.</li>
	<li>When the cement has hardened, release the electrode from the stereotaxic device. Subcutaneously administer meloxicam 10mg/kg to relieve discomfort caused by pain in animals. Administer meloxicam to animal food for analgesic effect in the first week after surgery. Remove the mouse and place it back in the cage, keeping it separate from the other mice.</li>
</ol>
4. Electrical kindling
<ol>
	<li>Allow the mice to rest for at least 1 week after surgery before kindling to allow for postoperative recovery and to allow the inflammation to subside. 
	NOTE: In general, mice that have not recovered adequately do not respond well to kindling.</li>
	<li>Put the mouse in a customized box with slip-ring cables connecting the electrode on the mouse&#39;s head and the EEG device. Run the cable through a hole in the lid of the box, and adjust the length left in the box to allow the mouse to move freely.</li>
	<li>Turn on the EEG device and check whether it is working properly. Set the stimulator to deliver 1 ms monophasic square-wave pulses at 60 Hz for a 1 s train duration.</li>
	<li>Start with a current intensity of 50 &#181;A for the first stimulation; monitor the EEG for after-discharge, which is characterized by high-frequency spikes. If no after-discharge is observed, add 25 &#181;A to the next stimulus, and continue this process every 10 min until an after-discharge is observed and lasts 5 s. 
	NOTE: If the experiment does not require a discharge, step 4.4 can be skipped; 300 &#181;A is strong enough for kindling.</li>
	<li>Stimulate the mouse with the current intensity determined in step 4.3 every 15 min, no more than 20 times a day.</li>
	<li>Monitor the behavioral responses to the stimulus. 
	NOTE: The occurrence of three consecutive grade V episodes is considered full kindling, combined with Racine rank standard17.</li>
</ol>

<ol><li>Kurita, T., Sakurai, K., Takeda, Y., Horinouchi, T., Kusumi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Very+long-term+outcome+of+non-surgically+treated+patients+with+temporal+lobe+epilepsy+with+hippocampal+sclerosis%3A+A+retrospective+study%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Very long-term outcome of non-surgically treated patients with temporal lobe epilepsy with hippocampal sclerosis: A retrospective study.</a> PLoS One. 11 (7), 0159464(2016).</li>
<li>Choy, M., Duffy, B. A., Lee, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optogenetic+study+of+networks+in+epilepsy%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Research%22%5BJournal%5D)">Optogenetic study of networks in epilepsy.</a> Journal of Neuroscience Research. 95 (12), 2325-2335 (2017).</li>
<li>Aroniadou-Anderjaska, V., Fritsch, B., Qashu, F., Braga, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathology+and+pathophysiology+of+the+amygdala+in+epileptogenesis+and+epilepsy%5BTitle%5D)+AND+%22Epilepsy+Research%22%5BJournal%5D)">Pathology and pathophysiology of the amygdala in epileptogenesis and epilepsy.</a> Epilepsy Research. 78 (2-3), 102-116 (2008).</li>
<li>Smith, P. D., McLean, K. J., Murphy, M. A., Turnley, A. M., Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Seizures%2C+not+hippocampal+neuronal+death%2C+provoke+neurogenesis+in+a+mouse+rapid+electrical+amygdala+kindling+model+of+seizures%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Seizures, not hippocampal neuronal death, provoke neurogenesis in a mouse rapid electrical amygdala kindling model of seizures.</a> Neuroscience. 136 (2), 405-415 (2005).</li>
<li>Reyes, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amygdala+enlargement%3A+Temporal+lobe+epilepsy+subtype+or+nonspecific+finding%5BTitle%5D)+AND+%22Epilepsy+Research%22%5BJournal%5D)">Amygdala enlargement: Temporal lobe epilepsy subtype or nonspecific finding.</a> Epilepsy Research. 132, 34-40 (2017).</li>
<li>Fan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnosis+and+surgical+treatment+of+non-lesional+temporal+lobe+epilepsy+with+unilateral+amygdala+enlargement%5BTitle%5D)+AND+%22Neurological+Sciences%22%5BJournal%5D)">Diagnosis and surgical treatment of non-lesional temporal lobe epilepsy with unilateral amygdala enlargement.</a> Neurological Sciences. 42 (6), 2353-2361 (2021).</li>
<li>Dhir, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pentylenetetrazol+%28PTZ%29+kindling+model+of+epilepsy%5BTitle%5D)+AND+%22Current+Protocols+in+Neuroscience%22%5BJournal%5D)">Pentylenetetrazol (PTZ) kindling model of epilepsy.</a> Current Protocols in Neuroscience. , Chapter 9, Unit 9 37(2012).</li>
<li>Van Erum, J., Van Dam, D., De Deyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PTZ-induced+seizures+in+mice+require+a+revised+Racine+scale%5BTitle%5D)+AND+%22Epilepsy+%26+Behavior%22%5BJournal%5D)">PTZ-induced seizures in mice require a revised Racine scale.</a> Epilepsy & Behavior. 95, 51-55 (2019).</li>
<li>Carriero, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+guinea+pig+model+of+mesial+temporal+lobe+epilepsy+following+nonconvulsive+status+epilepticus+induced+by+unilateral+intrahippocampal+injection+of+kainic+acid%5BTitle%5D)+AND+%22Epilepsia%22%5BJournal%5D)">A guinea pig model of mesial temporal lobe epilepsy following nonconvulsive status epilepticus induced by unilateral intrahippocampal injection of kainic acid.</a> Epilepsia. 53 (11), 1917-1927 (2012).</li>
<li>Levesque, M., Avoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+kainic+acid+model+of+temporal+lobe+epilepsy%5BTitle%5D)+AND+%22Neuroscience+Biobehavioral+Reviews%22%5BJournal%5D)">The kainic acid model of temporal lobe epilepsy.</a> Neuroscience Biobehavioral Reviews. 37, 10 Pt 2 2887-2899 (2013).</li>
<li>Fujita, A., Ota, M., Kato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Urinary+volatile+metabolites+of+amygdala-kindled+mice+reveal+novel+biomarkers+associated+with+temporal+lobe+epilepsy%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Urinary volatile metabolites of amygdala-kindled mice reveal novel biomarkers associated with temporal lobe epilepsy.</a> Scientific Reports. 9 (1), 10586(2019).</li>
<li>Li, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+spatiotemporal+dynamics+of+phase+synchronization+during+epileptogenesis+in+amygdala-kindling+mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The spatiotemporal dynamics of phase synchronization during epileptogenesis in amygdala-kindling mice.</a> PLoS One. 11 (4), 0153897(2016).</li>
<li>Wang, Y., Wei, P., Yan, F., Luo, Y., Zhao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+epilepsy%3A+A+phenotype-oriented+review%5BTitle%5D)+AND+%22Aging+and+Disease%22%5BJournal%5D)">Animal models of epilepsy: A phenotype-oriented review.</a> Aging and Disease. 13 (1), 215-231 (2022).</li>
<li>Fallah, M. S., Dlugosz, L., Scott, B. W., Thompson, M. D., Burnham, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antiseizure+effects+of+the+cannabinoids+in+the+amygdala-kindling+model%5BTitle%5D)+AND+%22Epilepsia%22%5BJournal%5D)">Antiseizure effects of the cannabinoids in the amygdala-kindling model.</a> Epilepsia. 62 (9), 2274-2282 (2021).</li>
<li>Chipika, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amygdala+pathology+in+amyotrophic+lateral+sclerosis+and+primary+lateral+sclerosis%5BTitle%5D)+AND+%22Journal+of+the+Neurological+Sciences%22%5BJournal%5D)">Amygdala pathology in amyotrophic lateral sclerosis and primary lateral sclerosis.</a> Journal of the Neurological Sciences. 417, 117039(2020).</li>
<li>Kuchukhidze, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emotional+recognition+in+patients+with+mesial+temporal+epilepsy+associated+with+enlarged+amygdala%5BTitle%5D)+AND+%22Frontiers+in+Neurology%22%5BJournal%5D)">Emotional recognition in patients with mesial temporal epilepsy associated with enlarged amygdala.</a> Frontiers in Neurology. 12, 803787(2021).</li>
<li>Soper, C., Wicker, E., Kulick, C. V., N'Gouemo, P., Forcelli, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optogenetic+activation+of+superior+colliculus+neurons+suppresses+seizures+originating+in+diverse+brain+networks%5BTitle%5D)+AND+%22Neurobiology+of+Disease%22%5BJournal%5D)">Optogenetic activation of superior colliculus neurons suppresses seizures originating in diverse brain networks.</a> Neurobiology of Disease. 87, 102-115 (2016).</li>
<li>Devinsky, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epilepsy%5BTitle%5D)+AND+%22Nature+Reviews+Disease+Primers%22%5BJournal%5D)">Epilepsy.</a> Nature Reviews Disease Primers. 4, 18024(2018).</li>
<li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+between+thalamus+and+hippocampus+in+termination+of+amygdala-kindled+seizures+in+mice%5BTitle%5D)+AND+%22Computational+and+Mathematical+Methods+in+Medicine%22%5BJournal%5D)">Interaction between thalamus and hippocampus in termination of amygdala-kindled seizures in mice.</a> Computational and Mathematical Methods in Medicine. 2016, 9580724(2016).</li>
<li>Ghotbedin, Z., Janahmadi, M., Mirnajafi-Zadeh, J., Behzadi, G., Semnanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrical+low+frequency+stimulation+of+the+kindling+site+preserves+the+electrophysiological+properties+of+the+rat+hippocampal+CA1+pyramidal+neurons+from+the+destructive+effects+of+amygdala+kindling%3A+the+basis+for+a+possible+promising+epilepsy+therapy%5BTitle%5D)+AND+%22Brain+Stimulation%22%5BJournal%5D)">Electrical low frequency stimulation of the kindling site preserves the electrophysiological properties of the rat hippocampal CA1 pyramidal neurons from the destructive effects of amygdala kindling: the basis for a possible promising epilepsy therapy.</a> Brain Stimulation. 6 (4), 515-523 (2013).</li>
<li>Hristova, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Medial+septal+GABAergic+neurons+reduce+seizure+duration+upon+optogenetic+closed-loop+stimulation%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">Medial septal GABAergic neurons reduce seizure duration upon optogenetic closed-loop stimulation.</a> Brain. 144 (5), 1576-1589 (2021).</li>
</ol>

The authors have no conflicts of interest to disclose.

Epilepsy is a group of diseases with multiple manifestations and diverse causes18; it should be noted that no single model can be used for all types of epilepsy, and researchers must select an appropriate model for their specific study. The present study introduces one of the most accessible methods of electrode fabrication. Various parts of this method can be adjusted to adapt to different experimental conditions.
This method utilizes electrodes with both stimulating and recording functions, which reduces the injury to the animal's brain caused by implanting separate electrodes for stimulation and EEG recording. When fabricating the electrodes, different sizes of row pins can be chosen. Jumbo row pins can connect to the slip ring the most firmly. However, multiple objects may need to be implanted in the animal's head; in this case, small row pins can be selected because they take up less space and are easier to operate, and a multi-channel slip ring can be used to connect all implanted electrodes. Slip rings can weld different types of interfaces to meet the needs of different laboratory EEG devices. In addition, they allow the animal to move freely without the cables becoming tangled.
To ensure that the electrodes do not fall off over a long period, it is necessary to apply dental cement after the skull is completely dry. A few horizontal and vertical cuts on the skull surface in advance can also increase firmness. After surgery, animals must recover for at least a week to allow the inflammation to subside, and anti-inflammatory drugs can be used as appropriate to aid recovery. Conducting other experiments is not recommended during this week.
Despite the merits of this approach, the method has several limitations. Because of the small size of the mouse brain, the electrode may not be accurately embedded in the target location during stereotactic surgery13. Compared with other modeling methods, this method requires the animal to carry the implanted object for a long time; this inevitably has an impact on the animals. For example, we found that animals often scratched their heads because they were uncomfortable.
This method can be used in combination with a variety of technologies, such as electrophysiology19, patch clamp20 and optogenetic techniques; however, it is not suitable for experiments using closed-loop stimulation21. Methods using the same stimulus parameters may not be representative of a natural spontaneous seizure, which means that they are not suited for machine learning. In conclusion, this electrical kindling method excludes the influence of drug metabolism on the experiment and is accessible, stable, reliable, and widely applicable to many studies.

Temporal lobe epilepsy (TLE) is the most prevalent type of epilepsy and has a high risk of conversion into drug-resistant epilepsy. Surgery, such as selective amygdalohippocampectomy, is an effective treatment for TLE, and the epileptogenesis and ictogenesis of the disease are still under investigation1,2. Pathogenesis of TLE has been shown to occur not only in the hippocampus but also extensively in the amygdala3,4. For example, both amygdala sclerosis and amygdala enlargement have been frequently reported as the origins of TLE seizures5,6. The importance of the amygdala cannot be underestimated; an amygdala model is essential for the study of epileptogenesis, and a clear illustration of this model is urgently needed.
Several approaches have been proposed to induce seizures in animal models. In the past, convulsant drugs were injected intraperitoneally at an early stage7. Although this method was convenient, the location of epileptic foci was uncertain. With the development of stereotactic technology and a detailed animal brain atlas, intracranial drug injection was applied to solve the problem of localization8. However, a lack of intervention for severe seizures during the acute stage resulted in a high mortality rate, and chronic spontaneous seizures were accompanied by the problem of unstable interictal and seizure frequency9,10. Finally, the electrical kindling method was developed; this method periodically stimulates specific brain regions several times, allowing seizures to be induced with definite control of both the location and the onset time11.
An advantage of this method is that the intracranial implantation of electrodes is minimally invasive12. Furthermore, the severity of the seizure is controllable by the termination of the stimuli, reducing the mortality caused by the seizures. These changes solved the shortcomings of the previous approaches. Notably, this model can adequately mimic human seizures and is especially suitable for the study of status epilepticus (SE) because of its ability to induce SE quickly13. It can also be used for anti-epileptic drug screening14 and in studies on the mechanism of epilepsy. Finally, it is well known that the amygdala is closely associated with memory modulation, reward processing, and emotion15. Disorders of these mental functions are often encountered in epileptic patients and, thus, the amygdala epilepsy model may be a better choice for studying emotional problems in epilepsy16.

<table><tbody><tr><td>Alexa Fluor 488-conjugated Donkey anti-Rabbit IgG</td><td>invitrogen</td><td>A-21206</td><td></td></tr><tr><td>c-Fos antibody</td><td></td><td>ab222699</td><td></td></tr><tr><td>Cranial drill</td><td>SANS</td><td>SA302</td><td></td></tr><tr><td>dental cement</td><td>NISSIN</td><td></td><td></td></tr><tr><td>EEG recording and stimulation equipment</td><td>Neuracle Technology (Changzhou) Co., Ltd</td><td>NSHHFS-210803</td><td></td></tr><tr><td>lead-free tin wire</td><td>BAKON</td><td></td><td></td></tr><tr><td>Pin header/Female header</td><td>XIANMISI</td><td></td><td>spacing of 1.27 mm</td></tr><tr><td>Silver wire</td><td>A-M systems</td><td>786000</td><td></td></tr><tr><td>Slip ring</td><td>Senring Electronics Co.,Ltd</td><td>SNM008-04</td><td></td></tr><tr><td>Tungsten wire</td><td>A-M systems</td><td>796000</td><td></td></tr><tr><td>ultrafine multi-stand wire</td><td>Shenzhen Chengxing wire and cable</td><td>UL10064-FEP</td><td></td></tr><tr><td>welding equipment</td><td>BAKON</td><td>BK881</td><td></td></tr></tbody></table>

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 electrode and circuit enable the EEG to be recorded and function as a stimulation (Figure 1); this setup avoids the complexity of implanting recording and stimulating electrodes separately and minimizes damage to the brain tissue. The application of slip rings allows electrode connection with all types of devices.
We performed electrode implantation surgery on six healthy adult male C57BL/6 mice, and electrical stimulation was performed 2 weeks after surgery. The behavioral seizure level gradually increased with the number of stimuli increasing, grading is based on Racine&#39;s scale: 1 = mouth or facial automatisms; 2 = two or less myoclonic jerks; 3 = three or more myoclonic jerks and/or forelimb clonus; 4 = tonic-clonic forelimb and back extension; 5 = tonic-clonic forelimb and back extension with rearing and collapsing; 6 = tonic-clonic forelimb and back extension with wild running or jumping14. The number of stimuli required for complete kindling was recorded (Table 1).
The representative results of an EEG for stimulation after complete kindling are illustrated in Figure 2. The after-discharges last 5-15 s; then, the intracranial spontaneous discharges intensify, and behavioral symptoms begin. Seizure duration is usually less than 1 min, which reduces the risk of death from severe convulsions resulting in apnea.
The expression of c-Fos in the brain tissue was detected by immunohistochemistry 2 h after complete kindling (Figure 3); c-Fos antibody and Alexa Fluor 488-conjugated donkey anti-rabbit IgG were used. The results showed that the expression of c-Fos in the ipsilateral amygdala was significantly increased, verifying the feasibility of this model.
All animals underwent histological verification at the end of the experiment to ensure that the stimulation target was accurate, the electrode path is shown in Figure 4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64113/64113fig01.jpg" /> 
Figure 1: Key steps in electrode fabrication. (A) Appearance of electrodes at different steps; corresponding steps are marked on the diagrams. (B) The slip ring connects to the interface plugs; the female header circuit is shown in the inset (top right). Scale bars = 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64113/64113fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64113/64113fig02.jpg" /> 
Figure 2: Representative results of the electroencephalography. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64113/64113fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64113/64113fig03.jpg" /> 
Figure 3: c-Fos expression in amygdala. c-Fos (green) in amygdala neurons; DAPI (blue) labels the nucleus; scale bar = 100 &#181;m. (A) c-Fos in ipsilateral amygdala; (B) c-Fos in contralateral amygdala. Abbreviation: DAPI = 4&#39;,6-diamidino-2-phenyindole. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64113/64113fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64113/64113fig04.jpg" /> 
Figure 4: Histological verification of electrode path. The red arrows point to the electrode track, the white dashed oval is the amygdala. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64113/64113fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
			<td>6</td>
		</tr>
		<tr>
			<td>Number of stimuli</td>
			<td>24</td>
			<td>12</td>
			<td>18</td>
			<td>21</td>
			<td>16</td>
			<td>28</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="6">Average: 19.83 Standard deviation: 5.742</td>
		</tr>
	</tbody>
</table>
Table 1: The number of stimuli required for each of the six mice to be fully kindled.

Watch this Scientific Journal Video about Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala at JoVE.com

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

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 ...

using-bipolar-electrode-to-create-temporal-lobe-epilepsy-mouse-model

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.4K Views.  Xuanwu Hospital Capital Medical University. 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.

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

Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of direct mechanical forces, sometimes penetrating through the cranium, creating a direct lesion in the brain tissue. These are visible during brain imaging as areas with contusion, laceration, and hemorrhage. Focal lesions induce neuronal death and glial scar formation and are present in 20%&minus;25% of all people who incur a TBI. However, in the majority of TBI cases, injury is caused by acceleration-deceleration forces and subsequent tissue shearing, resulting in nonfocal, diffuse damage. A subpopulation of TBI patients continues to develop post-traumatic epilepsy (PTE) after a latency period of months or years. Currently, it is impossible to predict which patients will develop PTE, and seizures in PTE patients are challenging to control, necessitating further research. Until recently, the field was limited to only two animal/rodent models with validated spontaneous post-traumatic seizures, both presenting with large focal lesions with massive tissue loss in the cortex and sometimes subcortical structures. In contrast to these approaches, it was determined that diffuse TBI induced using a modified weight drop model is sufficient to initiate development of spontaneous convulsive and non-convulsive seizures, even in the absence of focal lesions or tissue loss. Similar to human patients with acquired post-traumatic epilepsy, this model presents with a latency period after injury before seizure onset. In this protocol, the community will be provided with a new model of post-traumatic epilepsy, detailing how to induce diffuse non-lesional TBI followed by continuous long-term video-electroencephalographic animal monitoring over the course of several months. This protocol will detail animal handling, the weight drop procedure, the electrode placement for two acquisition systems, and the frequent challenges encountered during each of the steps of surgery, postoperative monitoring, and data acquisition.

This systematic protocol describes a new animal model of post-traumatic epilepsy after repetitive mild traumatic brain injury. The first part details steps for traumatic brain injury induction using a modified weight drop model. The second part provides instructions on the surgical approach for single- and multi-channel electroencephalographic data acquisition systems.

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of ...

Behavior

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 ...

Pentylenetetrazole-Induced Kindling Mouse Model

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

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

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

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) ...

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

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

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 ...

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 ...

Establishing a Pentylenetetrazole-Induced Epileptic Seizure Model in Mice

Source: Shimada, T., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56573">Pentylenetetrazole-Induced Kindling Mouse Model.</a> J. Vis. Exp. (2018).
This video demonstrates the process of inducing epilepsy in mice using pentylenetetrazole (PTZ) injections, a GABA-A receptor antagonist. Repeated intraperitoneal administration of PTZ progressively lowers the seizure threshold by inhibiting chloride ion influx, disrupting inhibitory neurotransmission, and leading to hyperexcitability in neurons that trigger seizures. This process results in kindling and the development of spontaneous seizures, mimicking chronic epilepsy.

Source: Shimada, T., et al. Pentylenetetrazole-Induced Kindling Mouse Model. J. Vis. Exp. (2018).
This video demonstrates the process of inducing epilepsy ...

JoVE Encyclopedia of Experiments

Neuropathology

Epilepsy and Seizure Disorders

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

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

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