Summary

マウス脳スライスの準備中に発作のような活動の直流刺激とマルチ電極アレイ録音

Published: June 07, 2016
doi:

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

陰極経頭蓋直流刺激(TDCの)は、薬剤耐性発作の抑制効果を誘導します。効果的なアクションを実行するために、刺激パラメータ( 例えば 、配向、磁界強度、および刺激期間)は、マウスの脳スライス標本で検査する必要があります。試験マウスの脳切片の位置に電極の向きを配置も可能です。本発明の方法は、前帯状皮質の発作のような活動にDCSの効果を評価するthalamocingulate経路を保持します。マルチチャンネル・アレイ・レコーディングの結果は陰極DCSが大幅に4-アミノピリジン及びビククリン誘発性発作様活性の刺激誘発応答の振幅および持続時間を減少することが示されました。この研究はまた15分で陰極DCSアプリケーションがthalamocingulate経路の長期不況を引き起こしたことがわかりました。本研究は、thalamocingulat上のDCSの影響を調査します電子シナプス可塑性および急性発作のような活動。現在の手順は、in vitroマウスモデルにおける配向、磁界強度、および刺激期間を含む最適な刺激パラメータをテストすることができます。また、この方法は、両方の携帯電話やネットワークレベルでの皮質発作のような活動にDCSの効果を評価することができます。

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

動物被験体が関与する手順は、施設内動物管理と活用委員会、中央研究院、台北、台湾によって承認されました。 1.多電極アレイ記録のための実験的なソリューションと機器を準備人工脳脊髄液(aCSFの調製95%のO 2を通気124ミリモルのNaCl、4.4ミリモルのKCl、1mMののNaH 2 PO 3、2mMのMgSO 4を、2ミリモルのCaCl 2、25mMのNaHCO 3</…

Representative Results

ThalamocingulateスライスとMEA記録システムのセットアップの準備マウスからのMT-ACCスライスはthalamocingulate経路の電気生理学的特性の探査を可能にする特殊なスライス標本である。 図1Aは、MT-ACCスライスが用意された方法を示しています。マウスの脳を速やかに除去し、( 図1A、B)クール酸素aCSFの中…

Discussion

本研究では、ACC発作様活性にDCSの持続時間と方向の効果を試験しました。マウス脳切片で安定したデータを取得するには、どのようにMT-ACC経路の整合性を保つために、それは、2角度を付け腹カットと皮質の背側カットが行われている、特にステップキーである損傷を避けるために。また、脳スライスを準備する時間も新鮮で強い脳を維持するための最短時間であるべき脳切片の活性に影響を…

Disclosures

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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Cite This Article
Lu, H., Chang, W., Chang, W., Shyu, B. Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation. J. Vis. Exp. (112), e53709, doi:10.3791/53709 (2016).

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