小鼠脑切片准备扣押样活性的直接电流刺激和多电极阵列记录

The overall goal of this experimental procedure, is to access the arrangement of electrodes in the recording chamber for direct current stimulation of a brain slice and it's effect on seizure-like activity recorded with a multi-electrode array. This better can help answer key questions in direct current stimulation, and a multi-electrode array recorded seizure-like activity in mouse brain slice. An advantage of this technique is the effects of direct current stimulation can be evaluated in the specific pathway of the mouse brain slice.

Demonstrating the procedure will be Hsiang Chin, and Wei-Jen Chang, technicians from my laboratory. In this procedure, sterilize all of the surgical instruments with 75%ethanol solution before the experiment. Then, expose the skull of the animal and trim off the remaining muscle.

Next, peel away the dorsal surface of the skull using rongeurs, and trim away the sides. Subsequently, using a spatula, cut the olfactory bulbs and nerve connections along the ventral surface of the brain before removing it. Quickly transfer the brain to a beaker filled with ice cold, oxygenated, ACSF.

To prepare slices containing the MT-ACC pathway, make a sagittal cut, 2 mm lateral to the midline in each hemisphere, to display the subcortical anatomy. Then, make an angled cross cut parallel to the visible fiber tract in the striatum. Make the second angled cross cut from the connection between the cerebellum and visual cortex, to the midpoint between the anterior commissure and the optic tract, that are ventral and parallel to the thalamus cingulate pathway.

After that, attach the brain block to an angular plate with a cyanoacrylate adhesive. Make a cut just above the turning point of the pathway. After that, unfold the plate, flatten it, and glue it on to the chamber stage of a vibratome.

Subsequently section the brain slices at 500 micrometers thickness, and keep them in ice cold oxygenated ACSF. Transfer a slice to the recording chamber at 32 degrees Celsius with continuous profusion of oxygenated ACSF for one hour. Confirm the placement of a MEA probe on the multichannel system.

Use one tube to guide the ACSF into the MEA chamber and the other tube to guide the ACSF out of the chamber. Continuously perfuse the preparation with warm, oxygenated ACSF at 30 degrees Celsius. Using a wet cotton swab, transfer a brain slice to the MEA.

Carefully move the brain slice to orient the ACC above the electrodes, then, stabilize the brain slice with a slice anchor to ensure a good electrical connection between the slice and the electrodes. Afterward, place the anode electrode proximal to the ACC and the cathode electrode distal to the ACC. Record the field strength by the two field orientations of MEA.

Then deliver the electric currents using a stimulator. Adjust the distance between the two silver chloride electrodes to about 1.5 to 2 cm and adjust the stimulators current strength to make the DCS between 0.5 and 2 milliamps. In this procedure, place a tungsten electrode in MT, and deliver pulses from the stimulator to the thalamic region of the slice.

Next, use various current intensities, to determine the threshold that elicits an ACC response. Move the tungsten electrode along the thalamus cingulate pathway to obtain the optimal response profile. Record 1-20 ACC responses and use the software to automatically average all of the ACC responses evoked my MT stimulation.

To induce seizure-like activity, add 250 micromolar 48P and 5 micromolar bicuculline to the perfusion solution, and continue to profuse the slice for two to three hours. Maintain the pump at a relatively fast perfusion rate, which can help prevent the build up of a pH gradient. Next, place the tungsten electrode at MT and deliver electrical stimulation to obtain an ACC response profile.

Record 10-20 sweeps and average the responses. Afterward, replace the prefusion solution with fresh ACSF to wash out the drugs. In this procedure, ensure the generation of uniform electric fields, by passing currents between two parallel silver chloride coated silver wires that are placed inside the MEA chamber.

If there are no issues, the DCS should stay between 0.5 and 2 milliamps. Next, turn off the DCS and stimulate the thalamus with a tungsten electrode. To obtain maximal synaptic responses in ACC record 10-20 responses and then average them.

Then, turn on the DCS and stimulate the thalamus simultaneously. Evaluate the amplitude changes of the thalamic stimulation evoked ACC response during DCS. Now, turn off the DCS, add 250 micromolar 48P, and 5 micromolar bicuculline to the perfusion solution, and wait 2-3 hours.

If the drugs effect the brain slice, the slice should produce cortical seizure responses. Subsequently, collect 10-20 anterior cingulate cortical responses, and measure the amplitude and duration of the electrically evoked cortical seizure responses. Nest, turn on the DCS and stimulate the thalamus simultaneously.

Evaluate the changes in the amplitude and duration of the evoked cortical seizure responses during DCS application. After that, replace the perfusion solution with fresh ACSF to wash out the drugs. This figure shows different evoked responses, which include the thalamic stimulation evoked responses in ACC, drug-induced, seizure-like activity, and both the thalamic stimulation and drug induced seizure-like activity.

This figure shows the affect of different orientations in DCS, such as different orientations in the electric field, thalamic stimulation evoked responses with DCS, and the effect of cathodal DCS on seizure-like activity. And in this figure, 15 minutes of cathodal DCS is shown the have effectively induced long term depression and depressed evoked responses. The seizure duration was significantly decresed after 15 minutes of cathodal DCS compared with no DCS application.

faster, this technically can be done in four hours if it is performed properly. Following this procedure, other matters like transcranial magnetic stimulation can be performed, in order to answer additional questions like providing noninvasive approaches for controlling resistance seizures. After this development, this technique paved the way for researchers in the field of noninvasive therapy to explore seizure treatments in of animal model.

After watching this video, you should have a good understanding of how to use the current stimulation to assess seizure-like activity in brain slices.