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 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-resistant^7,8. Some studies have reported the application of tDCS in the cingulate cortex^9-11. 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

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 thalamus. The latter probe covers only the cortical region.
3. Use a 60-channel amplifier with a band-pass filter set between 0.1 Hz and 3 kHz at 1,200 amplification. Acquire data at a 10 kHz sampling rate.
4. Place two AgCl-coated silver wires inside the MEA chamber for DCS. Use the AgCl-coated silver wires to produce electric fields that are generated by an isolated stimulator.
5. Place a tungsten electrode (diameter, 127 µm; length, 7.62 cm; 8° AC tapered tip; resistance, 5 MΩ) for thalamic stimulation, and place the reference electrode in the MEA chamber. Deliver the tungsten electrode's currents using an isolated stimulator that is controlled by a pulse generator.

2. Brain Slice Preparation

1. Use male C57BL/6J mice, 4-8 weeks old. House the animals in an air-conditioned room (21-23 °C; 50% humidity; 12 hr/12 hr light /dark cycle, lights on at 8:00 AM) with free access to food and water.
2. Take a 250 ml aliquot of the aCSF that was prepared in Step 1.1, and place it in a beaker that contains ice. At the same time, supply continuous gas that is composed of 95% O₂ and 5% CO₂.
3. Surgery
   1. Anesthetize the animal with 4% isoflurane in a glass box for approximately 3 min. Once the animal reaches a surgical depth of anesthesia (indicated by the lack of a response to toe pinch), place it on a shallow tray that is filled with crushed ice, and remove the head using scissors.
   2. Expose the skull, and trim off the remaining muscle. Next, using rongeurs, peel away the dorsal surface of the skull from the brain. Trim away the sides of the skull using rongeurs. Sterilize all of the surgical instruments with a 75% ethanol solution.
   3. Using a spatula, cut the olfactory bulbs and nerve connections along the ventral surface of the brain, and remove the brain. After decapitation, quickly transfer the brain to a beaker filled with ice-cold oxygenated aCSF.
4. Preparation of Medial Thalamus (MT)-ACC Brain Slice
   Note: Prepare slices that contain the pathway from the MT to ACC¹³.
   1. Hand-cut the brain block with two sagittal cuts 2.0 mm lateral to the midline in each hemisphere to display the subcortical anatomy. Then make two angled cuts. Make the first cross-cut parallel to the visible fiber tract in the striatum.
   2. Make the second cross-cut from the connection between the cerebellum and visual cortex to the midpoint between the anterior commissure and optic tract that are ventral and parallel to the thalamocingulate pathway.
   3. Attach the brain block to an angular plate (~120°) with cyanoacrylate adhesive, and make a cut just above the turning point of the pathway. Unfold the plate, flatten it, and glue it onto the chamber stage of a vibratome.
   4. Make medial thalamus-ACC brain slices (500 µm thick) and then immerse them in ice-cold oxygenated aCSF.Transfer slices to the recording chamber, and keep at 32 °C under continuous perfusion (12 ml/min) with oxygenated aCSF for 1 hr.

3. Preparation of Perfusion Chamber for Multielectrode Array Recording

1. Preparation of Perfusion Chamber
   1. Place a MEA probe on a multi-channel system, and use two separate polyethylene tubes to connect the probe to a peristaltic pump. Use one tube to guide the aCSF into the MEA chamber and the other tube to guide the aCSF out of the chamber. Finally, continuously perfuse the preparation with warm (29-30 °C) oxygenated aCSF (8 ml/min).
2. Transfer brain slice to MEA. Hold down the brain slice on the MEA using a wet cotton swab. Carefully move the brain slice to ensure the ACC is oriented above the electrodes.
3. Use slice anchor kits and hold-downs to press the brain slice. This step ensures a good electrical connection between the slice and electrodes.

4. Generation of Electric Fields by DCS

Note: The definition of the electric field orientation was based on the direction of the axodendritic axis in the ACC. The orientations of dendrite and soma compartments were confirmed using Golgi staining¹².

1. Place the AgCl electrode (defined as the anode) proximal to the ACC, and place the other electrode (defined as the cathode) distal to the ACC. Record the field strength that is generated by the two field orientations (parallel and perpendicular to the ACC axodendritic fibers) by the MEA, and deliver the currents of the electric fields using a stimulator.
2. Fix the distance of the AgCl electrodes (about 1.5-2 cm), and adjust the stimulator's current strength to make the DCS between 0.5 and 2 mA.

5. Electrically-induced Cortical Synaptic Responses

Note: Induce synaptic responses in the ACC by electrical stimulation in the MT, in which a programmable electrical stimulus generator produces rectangular biphasic current pulses.

1. Repeat Section 3 above.
2. Place a tungsten electrode in the MT, and deliver pulses from the stimulator to the thalamic region of the slices via bipolar tungsten electrodes.
3. Use various current intensities to determine the threshold that elicits an ACC response. Here, use an intensity of ±150 µA and duration of 200 µsec, which elicited an 80% maximal response in the ACC in most slices.
4. Move the tungsten electrode along the thalamocingulate pathway (from MT to corpus callosum) in the MT-ACC slice to obtain the optimal response profiles.
5. Make 10-20 sweeps of ACC responses, and use the software to automatically average all of the ACC evoked by MT stimulation. The result iss the synaptic responses in ACC induced from MT stimulation by MT-ACC pathway.

6. Electrically-induced Seizure-like Activity

Note: Seizure-like activity was induced by the application of 4-aminopyridine (4-AP; 250 µM) and bicuculline (5 µM). Previous time-control studies showed that maximal and stable responses appeared 2-3 hr after drug application¹⁴.

1. Repeat Section 5 above.
2. Add drugs to the perfusion solution. Use 4-AP (250 µM) and bicuculline (5 µM). Mix the drugs uniformly, and continue perfusion for 2-3 hr.
3. To facilitate seizure-like activity, maintain the perfusion pump at a relatively fast perfusion rate (8 ml/min), which can also help prevent the build-up of a pH gradient.
4. Place a tungsten electrode in the MT, and deliver electrical stimulation (150 µA, 200 µsec duration) to obtain ACC response profiles.
5. Make 10-20 sweeps and average the responses.
6. Replace the perfusion solution with fresh aCSF to wash out the drugs. Repeat Step 6.5.

7. Testing Effect of DCS on Evoked Cortical Responses

1. Repeat Sections 3 and 4. Ensure that uniform electric fields are generated by passing currents between two parallel AgCl-coated silver wires that are placed inside the MEA chamber. If there are no issues, the DCS stays between 0.5 and 2 mA.
2. Turn off the DCS, and place a tungsten electrode to stimulate the thalamus (±150 µA, 200 µsec duration). To obtain maximal synaptic responses in the ACC, make 10-20 sweeps and average the responses.
3. Simultaneously turn on the DCS (2 mV/mm DCS strength) and thalamic stimulation (350 µA, 200 µsec duration). Evaluate the changes of amplitude of the thalamic stimulation-evoked ACC response during DCS.
4. Turn off the DCS, and add 4-AP (250 µM) and bicuculline (5 µM) to the perfusion solution. Then wait 2-3 hr. When the drugs affect the brain slice, the slice produces cortical seizure responses.
5. Make 10-20 sweeps of ACC responses, and then measure the amplitude and duration of electrical evoked cortical seizure responses.
6. After step 7.5, simultaneously turn on the DCS (2 mV/mm DCS strength) and thalamic stimulation (150 µA, 200 duration µsec). Evaluate changes in the amplitude and duration of evoked cortical seizure responses during DCS application.
7. Replace the perfusion solution with fresh aCSF to wash out the drugs, and repeat steps 7.2 and 7.3.
8. Collect all of the recording data, and group the data into the different experimental conditions. Evaluate the amplitude and duration of cortical seizure responses under different experimental conditions.

8. Data Analysis

1. Use software (e.g., MC Rack software) to automatically average the recorded responses, and export the raw data to a spreadsheet. Analyze the amplitude and duration of the raw data and generate color figures.
2. To detect oscillatory seizure events, use software to measure the baseline value and standard deviations (SD). Set 3 SD of the noise level as the threshold. Amplitudes of the peaks during an oscillation event that surpass this threshold are automatically detected.
3. Perform the statistical analysis using Student's t-test.
4. Express measurements and one-way analysis of variance (ANOVA) results in the text as mean ± SE, with n indicating the number of slices studied¹².

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