Intracellular recording experiments

Summary

Source : Practical Laboratory Techniques in Neurobiology

Operation method

basic program

Principle

The recording of membrane potential requires the placement of an electrode on each side of the cell membrane to form a loop, so when an electrode is inserted into the cell membrane to record the corresponding electrical characteristics, this recording method is called intracellular recording method. With this method, the absolute value of membrane potential can be measured accurately, and excitatory postsynaptic potentials, inhibitory postsynaptic potentials, and action potentials can also be determined.

Materials and Instruments

Cells
Microelectrode Amplifier Glass Microelectrodes . Micro-propellers

Move

1. Prepare physiological salt solutions and electrode internal solutions required for experiments.

2. Prepare in vitro or ex vivo specimens according to specific experimental requirements.

3. Fix the specimen so that it is in a certain mechanical stable state.

4. Draw the recording electrode with suitable shape and tip, and fill the inner liquid.

5. Approach and insert the fixed microelectrode inside the cell gradually by using a step-by-step approach.

6 Stabilize the recording membrane potential, and carry out the stimulus-response test and other related indexes for observation.

7The typical membrane potential of a cell is -80 to 60 mV, and during the action potential, this value can reach 40 mV (Figure 3-2).

Caveat

1 microelectrode amplifier field effect tube is a thousand highly sensitive devices, the use of the process must be to avoid the accumulation of electrostatic charge so that its breakdown. Record the required electrodes should be hard silicone glass tube, built-in fine core, pulled made of electrode tip up to a certain fineness, but also has a considerable hardness, enough to pierce the cell. Recording process, the electrode reaches the tissue or cell surface, will see the impedance change, at this time, the record should be zeroed (equivalent to the extracellular electrode zeroing), in order to puncture the electrode into the cell membrane to accurately respond to changes in membrane potential.

Common Problems

1. Intracellular recordings require an extremely high degree of stability of the specimen, a requirement that is 'even more difficult to meet in the in vivo situation'. In experiments, the following measures can be taken to make the recordings as stable as possible:

① Strengthen the animal fixation, depending on the site of recording, the head and spine and other bone structures should be fixed accordingly;

② animals in the natural state, its random movement will cause the relative displacement of the electrode and the recording cell, so that the record can not be stabilized for a long time, in order to reduce the instability caused by the movement of the animal, you can use muscle relaxants (such as arrow venom) to reduce the movement of the animal;

③Artificial respiration must be given to the animal after the use of muscle relaxants to ensure that its respiratory and circulatory status is good, and at the same time, the tidal volume of respiration should be adjusted appropriately.

2. During the penetration of the electrode into the lipid bilayer cell membrane, it is sometimes necessary to slightly adjust its position so that the relative position of the electrode and the cell is in an optimal state to ensure stable recording for a long time. At the same time, after the electrode enters the cell, it should not be depolarizing stimulation immediately, but need to give a certain intensity of hyperpolarizing current, so that the membrane potential is stabilized at a slightly hyperpolarized range compared with the resting value for a few minutes, the so-called "feed the cell".

3. Because the electrode resistance of the microelectrode is very large, the electrode resistance compensation must be carried out in the experiment in order to truly respond to the changes in the membrane potential.

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