Summary

A estimulação de corrente contínua e Multi-eletrodo de gravação matriz de perda de consciência em Ratos Cérebro Slice Preparação

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

Catódica transcraniana estimulação de corrente contínua (ETCC) induz efeitos supressores sobre as apreensões resistentes aos medicamentos. Para executar ações efetivas, os parâmetros de estimulação (por exemplo, orientação, força de campo, e duração de estimulação) precisam ser examinadas em preparações fatia ratos cerebrais. Testes e providenciar a orientação do eléctrodo em relação à posição da fatia de cérebro ratinhos são viáveis. O presente método preserva a via thalamocingulate para avaliar o efeito de DCS sobre as actividades do tipo convulsivas córtex cingulado anterior. Os resultados das gravações de matriz multicanal indicou que catódica DCS diminuiu significativamente a amplitude das respostas evocadas por estimulação e a duração de 4-aminopiridina e perda de consciência induzida por bicuculina. Este estudo também descobriu que as aplicações DCS catódica em 15 min causou depressão a longo prazo na via thalamocingulate. O presente estudo investiga os efeitos da DCS em thalamocingulate plasticidade sináptica e atividades agudas apreensão-like. O processo actual pode testar os parâmetros de estimulação óptima incluindo a orientação, intensidade de campo, e a duração de uma estimulação in vitro no modelo de ratinho. Além disso, o método pode avaliar os efeitos da DCS sobre as atividades do tipo convulsivas corticais em ambos os níveis celulares e de rede.

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

Procedimentos que envolvem indivíduos animais foram aprovados pelo Cuidado Institucional Animal e Comité de Utilização, Academia Sinica, Taipei, Taiwan. 1. Preparar Solução Experimental e Equipamentos para multieletrodo gravação matriz Prepare artificial fluido cerebrospinal (aCSF; NaCI 124 mM, KCl 4,4 mM, 1 mM de NaH 2 PO 3, MgSO 2 mM de 4, 2 mM de CaCl2, mM de NaHCO 25, 3, e 10 mM de glucose, borbulhado com 95% de O …

Representative Results

Preparação do Setup Thalamocingulate Fatia e MEA Sistema de Gravação A fatia MT-ACC de ratinhos é uma preparação fatia especial que permite a exploração das propriedades electrofisiológicas da via thalamocingulate. A Figura 1A mostra a maneira na qual a fatia MT-ACC foi preparado. O cérebro de rato foi rapidamente removido e mantido em aCSF oxigenado fresco (Figura 1A, a, b). Para r…

Discussion

No presente estudo, foram testados os efeitos da duração e orientação da DCS na perda de consciência ACC. Para obter dados estáveis ​​em fatias de cérebro de rato, como para manter a integridade da via-MT ACC e para evitar danos que é fundamental, em especial os passos no qual dois cortes angulares ventral e dorsal de um corte do córtex são feitas. Além disso, o tempo necessário para preparar a fatia de cérebro também pode afectar a actividade da fatia do cérebro, o que deve ser o menor tempo possíve…

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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