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 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 ± 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.
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.
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
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.
3. Surgery for implantation
4. Electrical kindling
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'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.
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. Please click here to view a larger version of this figure.
Figure 2: Representative results of the electroencephalography. Please click here to view a larger version of this figure.
Figure 3: c-Fos expression in amygdala. c-Fos (green) in amygdala neurons; DAPI (blue) labels the nucleus; scale bar = 100 µm. (A) c-Fos in ipsilateral amygdala; (B) c-Fos in contralateral amygdala. Abbreviation: DAPI = 4',6-diamidino-2-phenyindole. Please click here to view a larger version of this figure.
Figure 4: Histological verification of electrode path. The red arrows point to the electrode track, the white dashed oval is the amygdala. Please click here to view a larger version of this figure.
1 | 2 | 3 | 4 | 5 | 6 | |
Number of stimuli | 24 | 12 | 18 | 21 | 16 | 28 |
Average: 19.83 Standard deviation: 5.742 |
Table 1: The number of stimuli required for each of the six mice to be fully kindled.
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.
The authors have nothing to disclose.
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.
Alexa Fluor 488-conjugated Donkey anti-Rabbit IgG | invitrogen | A-21206 | |
c-Fos antibody | ab222699 | ||
Cranial drill | SANS | SA302 | |
dental cement | NISSIN | ||
EEG recording and stimulation equipment | Neuracle Technology (Changzhou) Co., Ltd | NSHHFS-210803 | |
lead-free tin wire | BAKON | ||
Pin header/Female header | XIANMISI | spacing of 1.27 mm | |
Silver wire | A-M systems | 786000 | |
Slip ring | Senring Electronics Co.,Ltd | SNM008-04 | |
Tungsten wire | A-M systems | 796000 | |
ultrafine multi-stand wire | Shenzhen Chengxing wire and cable | UL10064-FEP | |
welding equipment | BAKON | BK881 |