마우스 뇌 조각 준비에서 발작 같은 활동의 직류 자극 및 다중 전극 배열 기록
Summary
Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.
Abstract
Cathodal 두개 직류 자극 (tDCS)의 약제 내성에 발작 억제 효과를 유도한다. 효율적인 동작을 수행하기 위해, 상기 자극 파라미터 (예를 들면, 방향 전계 강도와 자극 시간)을 마우스 뇌 슬라이스 제제에서 검사 될 필요가있다. 테스트 및 마우스 뇌 슬라이스의 위치에 전극의 상대적 배향을 정렬이 가능하다. 본 발명의 방법은 앞쪽에 cingulate 피질 발작 같은 활동에 대한 DCS의 효과를 평가하기 thalamocingulate 경로를 유지합니다. 다 채널 어레이 레코딩 결과 cathodal DCS 크게 -4- 아미노 피리딘과 bicuculline 유도 발작 같은 활성의 자극 – 유발 반응의 크기 및 지속 시간을 감소 것으로 나타났다. 이 연구는 또한 15 분에서 cathodal DCS 응용 프로그램이 thalamocingulate 경로에 장기 우울증의 원인 것으로 나타났습니다. 본 연구는 thalamocingulat에 DCS의 효과를 조사전자 시냅스 가소성 및 급성 발작 같은 활동. 현재의 방법은 시험 관내 마우스 모델에서 방향 전계 강도와 자극 시간을 포함하여 최적의 자극 파라미터를 테스트 할 수있다. 또한,이 방법은 두 셀룰러 네트워크 레벨에서 대뇌 발작 같은 활동 DCS의 효과를 평가할 수있다.
Introduction
Epilepsy is a common neurological disorder. Thirty percent of patients with epilepsy suffer from drug-resistant seizures1. Transcranial direct-current stimulation (tDCS) provides a noninvasive approach to control or alter network activities across large brain areas, such as seizures. Clinical studies have shown that tDCS effectively treats intractable seizures2 and can produce both short- and long-term suppressive effects on seizures3-5. However, the therapeutic mechanism of tDCS actions is still unclear. The brain slice model presented is an in vitro method to investigate how the therapeutic mechanism of tDCS actions alters the symptoms of seizure-like brain activities. Accordingly, to achieve its optimal effects, specific stimulation parameters including orientation, field strength, and stimulation duration need to be tested in an experimental model. Previous studies have shown that the orientation of the electric field is important to obtain therapeutic effects6. Thus, testing and arranging the orientation of electrodes relative to the position of the tested brain slice are feasible.
Frontal lobe epilepsy and anterior cingulate cortex (ACC) seizures are often drug-resistant7,8. Some studies have reported the application of tDCS in the cingulate cortex9-11. tDCS is shown to affect vigilance, decision making and emotion through alteration of ACC activities, and can modulate neuronal excitability and seizure activity in this brain region12. Therefore, suppressive effects of tDCS on ACC seizures might be helpful for clinical treatment and the evaluation of alternative treatments.
The present protocol describes the preparation of an electrode in the recording chamber for DCS of a brain slice and its effect on seizure-like activity recording with a multielectrode array (MEA).
Protocol
Representative Results
Discussion
본 연구에서 ACC 간질 유사 활성에 DCS 기간 및 배향의 영향을 시험 하였다. 마우스 뇌 조각에서 안정적인 데이터를 얻으려면 어떻게 MT-ACC 경로의 무결성을 유지하고,이 각도 복부 상처와 피질의 지느러미를 잘라 만든되는, 특히 단계 핵심 손상을 방지 할 수 있습니다. 또한, 뇌 조각을 준비하는 시간은 신선하고 강한 뇌를 유지할 수 짧은 시간이어야 뇌 슬라이스의 활성에 영향을 미칠 수있다. 이전…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We are grateful for the technical support from the Neural Circuit Electrophysiology Core at Academia Sinica. This work was supported by the National Science Council (102-2320-B-001-026-MY3 and 100-2311-B-001-003-MY3) and Neuroscience Program of Academia Sinica.
Materials
| Anesthetic: | |||
| Isoflurane | Halocarbon Products Corporation | NDC 12164-002-25 | 4% |
| Name | Company | Catalog Number | Comments |
| aCSF (total:1L): | |||
| D(+)-Glucose | MERCK | 1.08337.1000 | 10 mM |
| Sodium hydrogen carbonate | MERCK | 1.06329.0500 | 25 mM |
| Sodium chloride | MERCK | 1.06404.1000 | 124 mM |
| (+)-Sodium L-ascorbate, >=98% | SIGMA | A4034-100G | 0.15 g / 2 c.c |
| Magnesium sulfate, anhydrous,ReagentPlus | SIGMA | M7506-500G | 2 mM |
| Calcium chloride dihydrate | MERCK | 1.02382.1000 | 2 mM |
| Sodium dihydrogen phosphate monohydrate | MERCK | 1.06346.1000 | 1 mM |
| Potassium chloride | May & Baker LTD Dagenham England | MS 7616 | 4.4 mM |
| Name | Company | Catalog Number | Comments |
| Drugs: | |||
| (+)-Bicuculline | TOCRIS | 0130 | 5 µM in aCSF |
| 4-Aminopyridine | TOCRIS | 0940 | 250 µM in aCSF |
| Name | Company | Catalog Number | Comments |
| Brain slice Preparation: | |||
| Vibratome | Vibratome | Series 1000 | Block slicing into 500 µm thick slices |
| Name | Company | Catalog Number | Comments |
| MEA system: | |||
| Multielectrode array (MEA) probes: 6 x 10 planar MEA | Multi Channel Systems | 60MEA500/30iR-Ti-pr MEAS 6×10 | electrode diameter, 30 µm; electrode spacing, 500 µm; impedance, 50 kΩ at 200 Hz |
| Multielectrode array (MEA) probes: 8 x 8 MEA | Ayanda Biosystems | 60MEA200/10iR-Ti-pr MEAS 8×8 | pyramidal-shaped electrode; diameter, 40 µm; tip height, 50 µm; electrode spacing, 200 µm; impedance, 1000 kΩ at 200 Hz |
| A 60-channel amplifier was used with a band-pass filter set between 0.1 Hz and 3 KHz at 1200X amplification | Multi-Channel Systems | MEA-1060-BC | |
| MC Rack software at a 10 KHz sampling rate | Multi-Channel Systems | Software for data collect and recordings | |
| control of a pulse generator | Multi-Channel Systems | STG 1002 | |
| slice anchor kits and hold-downs | Warner Instruments | SHD-26H/10; WI64-0250 | |
| Peristaltic Pump-minipuls3 | Gilsom | MINIPULS3 | perfusion rate : 8 ml/min |
| Name | Company | Catalog Number | Comments |
| Stimulation system: | |||
| Isolated stimulator | A-M Systems | Model 2100 | intensity of ±350 μA , duration of 200 μs |
| Tungsten electrode | A-M Systems | 575300 | placed in thalamus |
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