Source : Practical Laboratory Techniques in Neurobiology
Operation method
basic program
Principle
Whole-cell membrane-clamp technique is used to detect phenomena such as postsynaptic potentials, postsynaptic currents, and plastic changes in such postsynaptic potentials or currents of neurons in tissue thin slices (spinal cord slices or brain slices). The advantage of the tissue slice membrane clamp technique over the dispersed cell membrane clamp technique is that the cells in the tissue slice are closer to the original in vivo environment, and the synaptic connections between neurons are well maintained.
Materials and Instruments
Nerve cells Move 1 Preparation of brain slices (1) Animals were anesthetized, then severed and the brain tissue was removed into a frozen incubating extracellular solution which was pre-saturated with 95% 02 and 5% CO2, and frozen to the extent of ice-water mixture. (2) The brain tissue blocks were glued to a sectioning table and 200-300 µm thick brain slices were cut with a vibrating slicer. (3) The cut brain slices were transferred to an incubation tank for incubation, and a mixture of 95% C02 and 5% C02 was continuously passed through, and the recordings were started after 1h. 2. Preparation of spinal cord slices (1) Animals were anesthetized by intraperitoneal injection of urethane at a dose of 1.2-1.5 mg/kg. (2) The skin and muscles of the back were incised along the midline and laminectomy was performed to expose the spinal cord. (3) The spinal cord was transected at the thoracic segment, the stump was lifted, and the paramedullary dorsal and ventral roots were cut one by one with Venus scissors, so that the dorsal roots of the target segment were retained at a length of more than 3-5 mm, and the lumbar bulging spinal cord was removed. (4) Perform operations such as tearing the membrane under the microscope, retaining the L4 or L5 dorsal root of one side of the spinal nerve, tearing off all other anterior and dorsal roots, and carefully removing the dura mater and arachnoid membrane on the surface of the spinal cord. (5) The spinal cord with L4 or L5 dorsal roots was placed on an agar block with a 90° groove. (6) The agar block was adhered to the sectioning groove, taking care to keep the caudal side of the spinal cord upward. Incubation extracellular fluid mixed with ice water was poured into the sectioning slot, ensuring that the spinal cord was upright and the dorsal root of the target segment was suspended on the spinal cord side. (7) With the Vibrotom speed set to 1-2 and the amplitude set to 7-8, the knife was fed above and below the root of the dorsal root of the target segment. When slicing above, the dorsal root was lightly pressed with a regent to keep it under the blade; when slicing below, the underside of the dorsal root was gently lifted with forceps so that the blade passed under the root, and the thickness of the slices was kept at 350-450 µm. (8) Cut the unsegmented dorsal root that had been held by the regents in the slices. (9) Using a pipette, the cut spinal cord thin section was placed in the incubation extracellular fluid with a mixture of 95% 02 and 5% CO2, incubated for 1h, and then transferred to the recording extracellular fluid for membrane clamping experiments. 3. Observation and recording of tissue thin section: (1) The continuous perfusion of the perfusate in the recording tank may cause fluttering of the tissue thin section, so it is necessary to fix the tissue thin section in order to ensure the stability of the recording. A lead-gold wire is usually made into an open rectangular frame, on which the nylon wire is tautly glued at intervals with 502 glue. Such a lead and gold press net pressed on the tissue sheet can be very good to fix the sheet. (2) Under infrared visualization, select neurons with smooth surfaces and clear contours for sealing and whole-cell recording (Figure 3-10). (3) Recordings on a thin slice of the spinal cord are used as an example to show the recording methods for different forms of postsynaptic currents. Excitatory postsynaptic currents evoked by stimulation of the dorsal root (evokedEPSC, abbreviated as eEPSC). Under the condition of clamp voltage of -70mV and the addition of Strychnin (Glycine receptor blocker), Bicuculin (GAB vanilloid receptor blocker) and AP-5 (NMDA receptor blocker) to the recording solution, a square wave stimulus of 0.2Hz was applied to the dorsal root through the suction electrode (DurationI00µs) until the evokedEPSC was recorded. Based on the stimulus intensity threshold and conduction velocity, we determined which type of primary afferent fibers mediated the eEPSCs. eEPSCs recorded were mainly mediated by Aδ or C fibers because the superficial dorsal horn of the spinal cord mainly receives afferents of injurious information. eEPSCs induced by A& fibers generally had stimulus intensity thresholds ranging from 10-60 µA, and conduction velocities ranging from 2-13 m/s; while eEPSCs induced by A& fibers usually had stimulus intensity thresholds ranging from 10-60 µA and conduction velocities ranging from 2 to 13 m/s. A& fiber-evoked eEPSC had a stimulus intensity threshold of 10-60 µA and a transmission speed of 2-13 m/s, while C fiber-evoked eEPSC had a stimulus intensity threshold of 160-530 µA and a transmission speed of less than 0.8 m/s. Whether eEPSC were mediated by monosynaptic transmission of primary afferent fibers or multisynaptic transmission mediated by intermediate neurons in the dorsal horn of the spinal cord was determined according to whether eEPSC were induced to fail to be triggered by high-frequency stimulation (20 Hz or 2 Hz) or whether latency was the same. For eEPSC evoked by Ao fibers, we usually used 20 Hz stimulation for 20 consecutive stimulations, and if each eEPSC was successfully evoked (i.e., no evoked failure) and the latency was consistent, we considered the eEPSC to be monosynaptically mediated; otherwise, it was polysynaptically mediated; for eEPSC evoked by C fibers, we usually used 2 Hz stimulation for 20 consecutive stimulations to determine whether the eEPSC was monosynaptically mediated via primary transmitter fibers or multisynaptically mediated via interneurons in the dorsal horn of the spinal cord (Figures 3-11). For C-fiber-evoked eEPSCs, we usually used 2 Hz stimuli for 20 consecutive stimulations, and if each eEPSC was successfully evoked (i.e., there was no evoked failure) and the latencies were consistent, we considered the eEPSCs to be monosynaptically mediated; otherwise, they were polysynaptically mediated. Spontaneous excitatory postsynaptic current (sEPSC) and minute excitatory postsynaptic current (mEPSC). Spontaneously generated excitatory postsynaptic currents recorded from spinal cord dorsal horn neurons without external stimuli at a clamp voltage of -70 mV and with the addition of Strychnin (a glycine receptor blocker), Bicuculin (a GAB vanilloid receptor blocker), and AP-5 (an NMDA receptor blocker) to the recording solution are referred to as sEPSCs. TTX blocked the action potential transmission between cells, a small excitatory postsynaptic current (i.e., mEPSC) could be recorded. Inhibitory postsynaptic current (IPSC). Inhibitory postsynaptic currents (IPSC) were recorded at a clamp voltage of -70 mV and with the addition of CNQX (an AMPA receptor blocker) and AP-5 (an NMDA receptor blocker) to the recording solution.The IPSC consisted of GABfan receptor-mediated IPSC, which could be isolated by the addition of Strychnin to the recording solution, and glycine receptor-mediated IPSC, which could be isolated by the addition of Strychnin to the recording solution. The former can be isolated by adding Strychnin to the extracellular fluid, and the latter can be isolated by adding Bicuculin to the extracellular fluid. Caveat 1. Note that the temperature of the whole system should be maintained at about O℃ when slicing, i.e., crushed ice should be used to cool down the blades and the area around the slicing tank. 2. Selecting healthy cells for clamping is a key factor to ensure efficient experiments and accurate results. 3. Be careful not to pull the dorsal root when tearing the membrane of the spinal cord, and use scissors to cut off the dorsal root of the non-purpose segment. Common Problems 1 The preparation of tissue slices is very critical in the whole process of the experiment, because the quality of the slices directly determines the state of the cells and the success of the experiment. Especially in the process of making thin slices of the spinal cord with posterior roots, we must pay attention to the gentle action, do not make a big cut, especially pay attention to the protection of the posterior roots and the posterior root entry area, because this area is very fragile, once damaged, even if the neuron is in good condition, it will not be able to record the response induced by stimulating the posterior roots. 2. Solution preparation: 1. Incubate the extracellular solution containing NaCl 95mM, KCl 1.8mM, KH2P04 1.2mM, CaCl2 0.5mM, MgS04 7mM , NaHC03 26mM, glucose 15mM, sucrose 50mM, pass through a gas mixture containing 95% 02 and 5% CO, pH adjusted to 7.4, osmolarity adjusted to 310 The osmolality was adjusted to 310-320mOsm. 2. Record the extracellular fluid, containing NaCl 127mM, KCI 1.8mM, KH2P04 I.2mM, CaC12 2 .4mM, MgS04 1.3mM, NaHC03 26mM, glucose 15mM, sucrose 0mM, through a gas mixture of 95% 02 and 5% C02, pH adjusted to 7.4, osmolarity adjusted to 310-320m0sm. 310-320m0sm. 3. Intracellular solution containing potassium gluconate 120mM,KCl 20mM, MgCl2 2mM, Na2ATP 2mM,NaGTP 0.5mM,HEPES 20mM,EGTA 0.5mM,pH was adjusted to 7.28 with KOH,osmolality was adjusted to 300m0sm. 4. Incubate the extracellular fluid first through the gas mixture (95% 02, 5% C02 ) for half an hour to the state of oxygen saturation, take out 200 ml of the refrigerator frozen to the state of ice-water mixing, and the rest of the liquid continue to maintain the gas mixture ventilation. For more product details, please visit Aladdin Scientific website.
Incubate extracellular fluid, record extracellular fluid, intracellular fluid.
2 ice packs, some crushed ice, 2 Venus scissors, scissors, camera, 2 filament cameras, 2 Venus scissors, scissors, camera, 2 filament cameras.