Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

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 transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

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 seizures^3-5. 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 t....

Protocol

Procedures that involve animal subjects were approved by the Institutional Animal Care and Utilization Committee, Academia Sinica, Taipei, Taiwan.

1. Preparing Experimental Solution and Equipment for Multielectrode Array Recording

1. Prepare artificial cerebral spinal fluid (aCSF; 124 mM NaCl, 4.4 mM KCl, 1 mM NaH₂PO₃, 2 mM MgSO₄, 2 mM CaCl₂, 25 mM NaHCO₃, and 10 mM glucose, bubbled with 95% O₂ and 5% CO₂).
2. Use two types of MEA probes: 6 x 10 planar MEA and 8 x 8 MEA. The former probe covers the region that comprises the cortex, striatum, and tha....

Results

Preparation of the Thalamocingulate Slice and MEA Recording System Setup

The MT-ACC slice from mice is a special slice preparation that allows exploration of the electrophysiological properties of the thalamocingulate pathway. Figure 1A shows the way in which the MT-ACC slice was prepared. The brain of the mouse was quickly removed and kept in cool oxygenated aCSF (Figure 1A, a, b). To reveal su.......

Discussion

In the present study, the effects of the duration and orientation of DCS on ACC seizure-like activity were tested. To obtain stable data in mouse brain slices, how to keep the integrity of the MT-ACC pathway and to avoid damage it is key, especially the steps in which two angled ventral cuts and a dorsal cut of the cortex are made. Moreover, the time to prepare the brain slice can also affect the activity of the brain slice, which should be the shortest time possible to keep the brain fresh and strong. A previous study s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anesthetic:
Isoflurane	Halocarbon Products Corporation	NDC 12164-002-25	4%
Name	Company	Catalog Number	Comments
aCSF (total:1 L):
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 6x10	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 8x8	pyramidal-shaped electrode; diameter, 40 µm; tip height, 50 µm; electrode spacing, 200 µm; impedance, 1,000 kΩ at 200 Hz
A 60-channel amplifier was used with a band-pass filter set between 0.1 Hz and 3 KHz at 1,200X 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 μsec
Tungsten electrode	A-M Systems	575300	placed in thalamus