Nerve tissue block membrane clamp whole-cell recording experiments
Nerve tissue block membrane clamp whole-cell recording experiments
Source : Practical Laboratory Techniques in Neurobiology
Operation method
basic program
Principle
Detection of neuronal excitability and its firing activity by whole-cell membrane-clamp technique is a basic method for electrophysiological experiments. The neural tissue block membrane clamp technique is closer to the original physiological environment than the cultured cell membrane clamp technique, and the cells have a better physiological state, which can be maintained for a longer period of time after sealing.
Materials and Instruments
Cells Move 1. The rats were anesthetized by intraperitoneal injection of 0.5ml/100g of 1% pentobarbital sodium, their backs were depilated and prepared for skinning, and they were executed by cervical dislocation method. 2.Rapidly cut the skin on its back, transect the spine from L5 and L3, cut the muscles, quickly remove the L5-3 spine and place it in an oxygen-saturated ACSF in a mixed state of ice and water, and the above process was required to be completed quickly, within 1 min, to ensure the neuronal activity.3.Carefully remove the muscle tissues around the spine. 4 Dissect the spine longitudinally along the sagittal plane, divide it into two, and use a free wire ingestion to peel off the spinal cord. If a unilateral pain model is used, e.g., CCD, CCI, SNL, etc., the right and left dorsal root ganglion (DRG) need to be separated, and the differentiation is made by the spinal cord being dissected along the median sagittal plane and the diameter of its vertebral canal being larger than the caudal end of the spinal cord. 5 Along the vertebral junction gently splintered, you can see the dorsal root ganglion under the vertebral plate, carefully separated from the surrounding dura mater, gently clamped the dorsal root ganglion axonal bundle deep with a swimmer's camera, gently lifted it up, extracted the L5, L3 dorsal root ganglion, and placed it on an oxygen-saturated room temperature ACSF in a 35-mm Petri dish (note that this step requires the experimenter to be gentle, and do not use a swimmer's camera to clamp the ganglion directly, which may cause damage to it). (Note that this step requires the experimenter to move gently, do not clip the ganglion directly with the swimmer's capsule and cause damage to it). 6 Under a stereomicroscope, carefully peel off the connective tissue and peritoneum around the DRG. 7. Put the tissue block and digestive enzyme concentrate (see above) into a centrifuge tube, dilute it with oxygen-saturated ACSF to 2 ml, and incubate it for 45 min at 37℃ in a constant temperature water bath. 8 Remove the tissue block, put it into oxygen-saturated room temperature ACSF, and let it stand for 1-2h before starting the experiment. 9. The digested cells are shown in Fig. 3-4 under 40X objective microscope. 10. The above steps can be used for the preparation of other tissues, the key point is to ensure that the activity of the target cells and try to remove the adhesion of the cell surface of the adhesion protein, cell debris and other impurities, the preparation of the digestive solution and digestion time can be adjusted according to the different specimens. 11. Choose 1.4mm hard glass microtubes (domestic glass microtubes need to be soaked in ethanol, cleaned with ultrapure water and ultrasonic microwave vibration to remove grease and dust, and dried for spare parts). 12 With an ethanol lamp, the ends of the glass microtubes were slightly burnished to make the corners rounded. 13. In the P-97 pulling instrument with a three-step method to pull out the tip diameter of 1µm or so of the glass microelectrode (test method for filling the electrode into the inner liquid into the water resistance of 5-8MΩ). 14. The electrode is drawn before the experiment, not overnight. 15 Fill the electrode liquid, containing glass capillary electrode can be filled from the end with a syringe with a suitable diameter of the syringe. 16 DRG after enzymatic digestion was placed in the oxygen-saturated ACSF recording tank with cyclic perfusion, fixed with a nylon mesh grid of "U"-shaped lead wire, so that the specimen was completely immersed in ACSF, and to ensure that a certain perfusion rate so that the neurons can be adequately replenished, and adjust the fluid flow to a steady state, then begin to seal and other operations. 17 Experiments were performed with an Olympus orthostatic microscope with a 40X immersion objective to observe the morphology of individual DRG neurons and visualize the sealing. A clear image of the cell was visible when the cell was located under high magnification (the lever at the Camera was in the pulled-out state, and a thousand pushes in at the lens lever) and the field of view was gradually dried up and brightened. Under the infrared visualization condition, the cells with smooth surface, clear outline, soft appearance and elasticity were selected for sealing, and the electrode tip contacting the cell surface was easy to form a depression. In contrast, neurons in poor condition have wrinkled or swollen surfaces, blurred outlines, and nuclei can be seen. 18. The glass microelectrode filled with intracellular fluid is mounted on the electrode holder and fixed with a screw nut (note that the rubber ring used to fix the glass microelectrode must be tightly combined with it, and no air leakage can be generated, otherwise the sealing will be affected). 19. Give the electrode tip proper positive pressure filling through the silicone tube connected with the electrode holder to prevent contamination of the glass microelectrode tip. 20. The glass microelectrode tip is lowered to below the perfusate level by the micro-operating system. At this time, the pClamp software saltest can measure the resistance of the glass microelectrode to ensure that it is 5-8Ω. 21 The baseline of the test waveform is not in the zero position when the electrode is first introduced to the water, and the baseline must be compensated back to zero. Auto button is used on the Multiclamp700B interface, in the case of Multiclamp200B, manual adjustment is required. 22 Locate the electrode under the low magnification objective and place it in the center or slightly to the left of the field of view. 23.Put the high magnification lens into the water so that it is in the lowest state, gradually raise the high magnification lens up to find the electrode tip, medium magnification Manipulation speed down to the visible outline of the cell, turn the Manipulation to slow speed, adjust the electrode tip position relative to the cell, slow contact, when the electrode tip is in contact with the cell membrane, the sealtest indicates that the Rm will rise. rises, and when the electrode is pressed down further, Rm will rise further. 24 A slight negative pressure was applied to the electrode through the plastic tube connected to the electrode gripper, and when the cell was in good condition, the Rm value would rise rapidly until a high-resistance seal (GΩ seal) was formed. 25 Electrode capacitance compensation, when using the Multiclamp 700B amplifier, directly click the Auto button for fast and slow capacitance compensation in sequence (Figure 3-5). 26 A greater negative pressure suction is used to break the cell membrane, allowing the electrode to penetrate into the cell and become a whole-cell membrane clamp recording mode. This step tends to cause instability of the seal and requires extra care. For beginners, it takes a little more time to get the hang of it, and can be done by mouth suction, usually with pulsed negative pressure suction, which is more effective. Some amplifiers offer a "Zap" function to break the membrane. 27 Series resistance compensation. Check "Wholecell", check "RsCompensation", increase "correction" and "Prediction" under "RsCompensation" to more than 70%, and gradually adjust the capacitance and resistance values under ''Wholecell" to meet the requirements, see the results of the interpretation section (Figure 3-6). See the results interpretation section (Fig. 3-6). 28. Electrophysiological experiments were performed on neurons by parameters set by Clampex software. After the membrane was broken to form the whole-cell recording mode, the I1,.k value could be read out on the Multiclamp700B software interface, and the value, and the most Cm, Rm, and Ra values could be measured using Membranetest in the Clampex software to ensure that the 18,.k < 200pA, Vm < -50mV, Ra < 20mfl, rm> ;SOMO. rm should preferably be 10 times or more than Ra, otherwise series resistance compensation is required. Figure 3-8 shows a typical current clamp record obtained by applying Clampex recording, and Figure 3-9 shows a typical voltage clamp record obtained by applying Clampex recording. Caveat 1. Ensure that the neurons are in oxygen-saturated ACSF or in a state of thousands of perfused blood flow at all times during the sampling process. 2. The pH and osmolality of the intracellular and extracellular fluids should be in accordance with the physiological state. 3. When clamping the cells, choose an orientation with a smooth surface. Common Problems 1 When preparing the electrode internal fluid, we should ensure that the temperature of the whole process is around O℃, and the internal fluid should be filtered more than 3 times with a membrane to ensure that there are no impurities in the internal fluid. 2. The degree of tearing the membrane in the experimental process determines the success or failure of the experiment, after tearing off the dorsal root ganglion membrane (dura mater), it should be noted that there is also a layer of membrane (arachnoid membrane and soft spinal cord), it should try to remove it, and after removing the ganglion neuron there is a slightly fluffy feeling. The ganglion neurons are slightly fluffy after removal. Be careful not to damage the neurons. For more product details, please visit Aladdin Scientific website.
Artificial cerebrospinal fluid (ACSF) Digestive enzyme concentrate ACSF pass-through mixture Nylon mesh preparation Electrode internal fluid
Amplifier (e.g., Multiclamp 700B) Digital-to-analog converter (e.g., Digidata 1322series) Microscope (e.g., Olympus BXSI) Micromanipulator