뇌 조각에서 전체 셀 패치 클램프 녹음

The overall goal of this procedure is to describe how to perform whole-cell patch-clamp recordings in freshly dissected brain slices. This method can help answer key questions in the field of neuroscience, such has how experimental control manipulations alter the activity of specific neurons in specific brain regions. The main advantage of this technique is that it provides the medium to identify an X vivo preparation long lasting changes in neuron functions that have developed in intact awake animals.

Though this method can provide insight into neuronal functions in brain slices, it can also be applied to other systems, such as cells in culture and neurons in vivo. Demonstrating the procedure will be Francisco Garcia-Oscos, a student from my lab. To begin this procedure, using a plastic trim-tipped transfer pipette, gently draw up one brain slice from the recovery chamber.

Place the transfer pipette in the recording chamber, and gently squeeze the slice out of the pipette on to the cover slip lining at the bottom of the chamber. Next, using the microscope 4x objective, position the slice so that the desired area is placed exactly in the center of the recording chamber. After the desired position has been achieved, secure the brain slice position with the slice hold down, also known as a harp.

After that, switch to the 40x objective, and lower the lens gently until it contacts the ACSF in the chamber. Then, use the fine adjustment wheel to bring the tissue into focus. When the focus is at tissue level, observe the cells in the targeted region.

Now, look for a target cell. Mark it on the computer screen in order to help guide the recording micropipette. Raise the objective lens to allow for sufficient space for the placement of the recording micropipette.

In this step using a one milliliter syringe, a non-metallic microsyringe needle, and a dedicated filter, fill a micropipette with the internal solution prepared in advance. Make sure there are no air bubbles in the micropipette. Then place the micropipette in an electrode holder, so the solution comes in contact with the silver chloride coated wire electrode.

Tighten the pipette cap, so that the cone washer forms a seal around the micropipette. Next, apply positive pressure with the air filled syringe connected to the pipette holder before immersing he micropipette in the ACSF to prevent debris from entering it. Using the micro-manipulator, guide the pipette down to the chamber so that it is roughly under the center of the immersed objective.

While moving the micropipette with the micro-manipulator at medium to high speed, locate the micropipette on the computer screen, and guide the micropipette toward the location of the cell on the XY axis. In the mean time, measure the micropipette resistance by applying a voltage step. Apply positive pressure in order to remove any air bubbles or other foreign objects blocking the micropipette.

After clearing the micropipette, perform a voltage offset to reduce pipette current to zero. Using the fine focus wheel of the microscope, start focusing down while lowering the micropipette gradually. Always focus down first and then lower the micropipette to the plane of focus to ensure that the micropipette tip does not abruptly penetrate into the slice.

When the micropipette comes in contact with the surface of the slice, slow down the micro-manipulator speed to medium low mode. Gently apply light positive pressure to clear any debris on the path. Then approach the cell, either by alternating with the XYZ control knobs or by approaching diagonally where both XZ axis are changed with rotation of the Z-axis knob.

When the micropipette is close enough to the cell, a dimple will appear on the cell surface. Now apply a weak and brief suction through the tube that is connected to the pipette holder suction tube in order to create a seal. While a giga-seal is forming, use the computer controlled amplifier commander to bring the cells holding potential to the physiological resting potential in order to prevent sudden changes, once the membrane is ruptured.

After the giga-seal has formed, compensate for the fast or slow capasitons. Here it is very important that the suction applied is not too strong. Otherwise the membrane may rupture before establishing the seal.

If the seal remains stable and above one gigaom, apply a brief and strong suction to rupture the plasma membrane. The suction must be brief and stronger than the pressure applied when establishing a seal. In order to properly rupture the membrane, and achieve a stable whole-cell configuration.

Switching to cell mode in the membrane test, view different parameters of the cell, such as input resistance, series resistance, and membrane capacitons. After achieving the whole-cell configuration, continue to monitor these parameters during recording. Shown here, is an example of the evoked EPSCs slope from a single nucleus accumbens shell MSN.

Increasing the temperature from 24 to 28 and to 32 degrees Celsius, increases the slope of evoked EPSCs. Here the evoked EPSCs amplitude is assessed in the voltage clamp mode at minus 80 millivolts. When series resistance increased, the amplitude of evoked EPSCS decreases.

And here, is a example of the traces from two neurons showing the effect of input resistance on the neuron capability to generate spikes. Neurons are current clamped and held at minus 80 millivolts. When input resistance increases, the number of action potentials increases as well.

While attempting this procedure, it's important to remember that generating healthy brain slices is critical. It is also important to monitor the parameters that may influence the electrical signal waveform, such as the series resistance, input resistance, and the temperature. After watching this video, you should have a good understanding of the basic aspect of whole-cell recording technique in brain slices.