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
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
Representative Results
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 |
References
- Schiller, Y., Najjar, Y. Quantifying the response to antiepileptic drugs: effect of past treatment history. Neurology. 70 (1), 54-65 (2008).
- Fregni, F., et al. A controlled clinical trial of cathodal DC polarization in patients with refractory epilepsy. Epilepsia. 47 (2), 335-342 (2006).
- Auvichayapat, N., et al. Transcranial direct current stimulation for treatment of refractory childhood focal epilepsy. Brain Stimul. 6 (4), 696-700 (2013).
- Chung, M. G., Lo, W. D. Noninvasive brain stimulation: the potential for use in the rehabilitation of pediatric acquired brain injury. Arch Phys Med Rehabil. 96 (4 Suppl), S129-S137 (2015).
- Del Felice, A., Magalini, A., Masiero, S. Slow-oscillatory Transcranial Direct Current Stimulation Modulates Memory in Temporal Lobe Epilepsy by Altering Sleep Spindle Generators: A Possible Rehabilitation Tool. Brain Stimul. 8 (3), 567-573 (2015).
- Garnett, E. O., Malyutina, S., Datta, A., den Ouden, D. B. On the Use of the Terms Anodal and Cathodal in High-Definition Transcranial Direct Current Stimulation: A Technical Note. Neuromodulation. , (2015).
- Biraben, A., et al. Fear as the main feature of epileptic seizures. J. Neurol. Neurosurg. Psychiatry. 70 (2), 186-191 (2001).
- Zaatreh, M. M., et al. Frontal lobe tumoral epilepsy: clinical, neurophysiologic features and predictors of surgical outcome. Epilepsia. 43 (7), 727-733 (2002).
- Karim, A. A., et al. The truth about lying: inhibition of the anterior prefrontal cortex improves deceptive behavior. Cereb. Cortex. 20 (1), 205-213 (2010).
- Keeser, D., et al. Prefrontal transcranial direct current stimulation changes connectivity of resting-state networks during fMRI. J. Neurosci. 31 (43), 15284-15293 (2011).
- Nelson, J. T., McKinley, R. A., Golob, E. J., Warm, J. S., Parasuraman, R. Enhancing vigilance in operators with prefrontal cortex transcranial direct current stimulation (tDCS). Neuroimage. 85 (Pt 3), 909-917 (2014).
- Chang, W. P., Lu, H. C., Shyu, B. C. Treatment with direct-current stimulation against cingulate seizure-like activity induced by 4-aminopyridine and bicuculline in an in vitro mouse model. Exp. Neurol. 265, 180-192 (2015).
- Lee, C. M., Chang, W. C., Chang, K. B., Shyu, B. C. Synaptic organization and input-specific short-term plasticity in anterior cingulate cortical neurons with intact thalamic inputs. Eur. J. Neurosci. 25 (9), 2847-2861 (2007).
- Chang, W. P., Shyu, B. C., Pandalai, S. G. Involvement of the thalamocingulate pathway in the regulation of cortical seizure activity. Recent Research Developments in Neuroscience. 4, 1-27 (2013).
- Brummer, S. B., Turner, M. J. Electrochemical considerations for safe electrical stimulation of the nervous system with platinum electrodes. IEEE Trans. Biomed. Eng. 24 (1), 59-63 (1977).
- Durand, D. M., Bikson, M. Suppression and control of epileptiform activity by electrical stimulation: a review. Proc. IEEE. 89 (7), 1065-1082 (2001).
- Fritsch, B., et al. Direct current stimulation promotes BDNF-dependent synaptic plasticity: potential implications for motor learning. Neuron. 66 (2), 198-204 (2010).
