This experimental method was obtained from the official website of the Fourth Military Medical University
Operation method
Experiments on the recording of sodium channel currents in rat hippocampal nerve cells
Principle
Sodium channels are widely found in a variety of cells especially in excitable cells such as nerves and muscles. The sodium current (INa) is the most important depolarizing ion current on fast-responding cells and is closely related to cellular excitability. Sodium channels start to activate at a membrane potential of -70 to -65 mV, generating a rapidly activating and rapidly inactivating inward current, with a maximum peak current at a membrane potential of -40 to -30 mV and a reversal potential of about +30 mV. In the parameter design, the membrane potential should be clamped below -80 mV, at this time, given different degrees of depolarizing step voltage, then the sodium current can be obtained, due to the sodium channel activation and inactivation are very fast, belongs to the fast channel, therefore, the stimulation pulse wavewidth is often 20 ~ 50 ms. When the resting membrane potential rises to -50 mV or so, it can make the Na+ channel inactivated completely, and can't cause the sodium channel to open. To confirm that the measured current is a sodium current, the sodium channel is often selectively blocked with the help of instrumental drugs, such as tetrodotoxin (TTX) in the extracellular fluid or class I antiarrhythmic drugs.
Materials and Instruments
Adult rats Move 1. Acute isolation methods for hippocampal nerve cells Caveat 1. When separating the cells, pay attention to adjusting the amount of trypsin and digestion time to prevent cell damage caused by excessive digestion. 2. Select the cells with good adherence to the wall, strong stereoscopic sense, good refractive index and smooth surface for the experiments. 3. Polishing the tip of microelectrode will be favorable to the formation of high impedance sealing and improve the success rate of the experiment. For more product details, please visit Aladdin Scientific website.
Artificial Cerebrospinal Fluid Composition: NaCl KCl NaH2PO4 NaHCO3 CaCl2 MgSO4 HEPES Glucose TEA 4-AP Intra-Electrode Fluid Composition CsF EGTA HEPES TEA
Diaphragm Clamp Amplifier Microelectrode Puller Inverted Microscope 3D Manipulator Electrode Polisher Glass Microelectrodes Thermostatic Bath Slicer Mammalian Surgical Instruments Oxygen Cylinder
Rats were anesthetized with 20% urethane 1g/kg (5ml/kg) and then decapitated, the brain tissues were immediately removed and rapidly placed into low temperature artificial cerebrospinal fluid (0℃～4℃) for 10～20s, then the hippocampus was separated from the ventral part of the cerebral hemisphere, and the hippocampus was sliced into 400 μm slices, and then placed in the artificial cerebrospinal fluid for 30 minutes, and then replaced by the artificial cerebrospinal fluid with 1g/L trypsin to be enzymatically digested for 40mins. After rinsing the slices with artificial cerebrospinal fluid for 3 times, the slices were incubated in the artificial cerebrospinal fluid for use, and the solution was kept at 32°C and continuously gassed with 5% CO2+95% O2 during the above incubation and enzymatic digestion. Finally, part of the brain slices were transferred into a centrifuge tube containing oxygen-saturated artificial cerebrospinal fluid, and were gently blown with pipettes with heat-treated tips of about 400 μm and 150 μm in diameter until individual hippocampal neurons were separated; the upper part of the cell suspension was taken and added into a culture dish, and the cells were adhered to the wall after about 20 min, and then the morphology of the cells could be observed under an inverted microscope, and the membrane clamp was performed for recordings.
2. Production of glass microelectrodes
With a microelectrode puller, the glass capillary is pulled into a microelectrode with a tip diameter of about lμm in two steps; in order to improve the success rate of sealing, the tip of the microelectrode can be polished under the microscope close to the heat source of the polisher. Then a syringe needle was used to fill the electrode from the end of the electrode with the electrode liquid into the microelectrode for backup.
3. Connection of the instrument (see Figure 1) 
4. Formation of high-impedance seals
Isolated rat hippocampal single cells were placed in the bath of an inverted microscope, and after the cells were adhered to the wall, the tip of the microelectrode, which was charged with the internal fluid of the internal charging electrode, was brought into the bath under the advancement of a three-dimensional hydraulic manipulator. A square wave pulse signal with a voltage of 10 mV and a wave width of 40 ms was delivered to the microelectrode by a membrane clamp amplifier to observe the seal formation process. When the microelectrode tip was in contact with the cell surface, the response current was seen to decrease, and then a negative pressure was applied to the microelectrode tip to further reduce the response current to zero, and a G-ohm seal was formed.
5. Recording of single-channel sodium currents in rat hippocampal nerve cells
After the sealing resistance of the microelectrode and the cell membrane reaches the G ohm level, the cell-adhesive recording mode is formed. At this time, if the membrane is given a stimulus with a retention potential of -120 mV and a command potential of -50 mV, the single-channel currents of hippocampal neuronal cells can be recorded (Figure 2). 
6. Recording of whole-cell sodium currents in rat hippocampal nerve cells
After recording the single-channel current, negative pressure suction can be given via the microelectrode or an electric pulse can break the diaphragm at the electrode tip, so that the intra-electrode fluid is connected to the intracellular fluid, then the whole-cell recording mode is formed, and the fast capacitance compensation is adjusted to offset the capacitive spikes, and the slow capacitance compensation of the amplifier and the series resistance compensation are adjusted to offset the transient current. In voltage-clamp mode, whole-cell sodium channel currents can be directed by giving the cell a stimulus with the following parameters: a holding potential of -120 mV, a command voltage of -90 to -25 mV, a pulse step of 10 mV, a stimulus frequency of 0.5 Hz, and a duration of 40 ms (Fig. 22-14). If 50 μmol/L of tetrodotoxin (TTX) or 20 μmol/L of propafenone, a class I antiarrhythmic drug, is given in the bath solution, and the cells are perfused for 10 min and then given the stimuli described above, changes in the amplitude of the sodium currents can be observed.