How to Read Pipette Resistance on the Oscilloscope

How to... Whole Cell Patch Clamp

Whole Cell Patch Clamp Recordings for Characterizing Neuronal Electric Properties of iPSC-derive neurons

Kindly Provided Past Ana González Rueda, Ole Paulsen Lab at the University of Cambridge

Introduction

Patch-clamp is the gold standard technique for high-fidelity analysis of the electrical backdrop and functional connectivity of neurons. Several patch clamp configurations can be used depending on the research interests, just in all cases, electrophysiological recordings are produced using a drinking glass micropipette in contact with a patch of the neuron's membrane. In the cell-attached way, the membrane patch is left intact allowing the recording of ion channels within the patch likewise as action potentials. Applying a pore-forming amanuensis, such every bit amphotericin, in the pipette results in a perforated patch, which establishes electrical continuity whilst preventing the dialysis of intracellular proteins. However, the most commonly used patch-clamp manner is the whole-cell mode where the membrane patch is disrupted past briefly applying potent suction to establish electric and molecular admission to the intracellular space. This has two main configurations: the voltage-clench mode, in which the voltage is held constant allowing the written report of ionic currents, and the current-clamp mode, in which the current is controlled enabling the report of changes in membrane potential.

Several books have been written describing this technique in detail. Described here, is a simplified protocol of the whole-jail cell patch clamp technique, for use in neuronal cultures. This protocol has been used to generate the results described beneath. The solutions and voltage and current steps used are specific for these recordings and tin be modified co-ordinate to the scientist'due south requirements.

Materials Required

Solutions

Artificial cerebrospinal fluid (aCSF)

Composition: 126 mM NaCl, iii mM KCl, 2 mM MgSO4, two mM CaCl2, 1.25 mM NaH2PO4, 26.4 mM NaHCO3 and ten mM glucose.

Preparation: Make upward 1 50 10X stock solution containing but NaHCO3, another 1 L 10X stock solution containing the residuum of the components and store for up to one calendar week at 4 °C. Before recording, prepare 1X solution; adjust osmolarity to 290 mOsm (+/- ten mOsm) and bubble with carbogen (95% O2 – 5% CO2).

IMPORTANT: This aCSF recipe uses a bicarbonate buffer; alternatively, HEPES (ten-fifteen mM) tin can be used to buffer the pH of the solution. If a HEPES buffered solution is used, NaHCO3 and CO2 bubbles are not required.

Intracellular solution

Limerick: 115 mM K-Gluconate, 4 mM NaCl, 0.3 mM GTP-NaCl, 2 mM ATP-Mg, 40 mM.

Training: Brand up 1X solution; adjust the pH to 7.2 with KOH and the osmolarity at 270 mOsm L-1 (+/- 10 mOsm L-one). Aliquot the solution and store at -xx °C. Earlier recording, thaw an aliquot and filter it using a standard 0.2 μm filter. Use a 1 mL syringe with a microloader tip to load the recording pipettes. If analysis of cellular morphology post hoc is required, include an intracellular dye or characterization in the internal solution (e.g. biocytin, neurobiotin, etc) and reduce the concentration of K-Gluconate correspondingly.

Electrophysiology Equipment Setup

· Anti-vibration table (optionally with a Faraday muzzle)
· Microscope
· Micromanipulator
· Borosilicate glass capillary holder
· Pressure level control system
· Headstage
· Amplifier
· Data interface
· Computer
· Recording software
· Camera
· Brandish screen
· Perfusion pump
· Carbogen (95% O2 and 5% CO2) input

Consumables

· Borosilicate glass capillaries: 0.68 mm inner diameter (ID) and 1.20 mm outer bore (OD) with filament

· 0.2 µm pore filters
· twenty µL Micro-loader tips
· 3-manner valve
· Tubes for perfusion organization, pressure arrangement and carbogen system.
· Carbogen (95% O2 and five% CO2) tank with regulator
· Air stone to deliver carbogen to the aCSF

Other Equipment Required

· Drinking glass capillary puller
· Osmometer (optional)

Figure 1. Whole-cell patch clamp set up.

The carmine arrows show the direction of the electrical signal recorded

Whole Cell Patch Clench Procedure

Preparation

1. Plate the neurons a few days prior to recording onto coverslips.

two. Plough on all the equipment and set up the pump to perfuse aCSF through the recording bedchamber (a unremarkably used speed for whole-prison cell patch clamp in cultures is one.v mL per minute).

IMPORTANT: a perfusion speed over 2 mL per minute might atomic number 82 to movements of the recording pipette and lifting of the cells from the coverslip.

iii. Place the coverslip with cells in the recording chamber with the cells facing up.

iv. Use a glass capillary puller to make two recording pipettes from a borosilicate glass capillary with a resistance of betwixt 3 and 7 MΩ when filled with K-Gluconate based internal solution.

Important: 5 MΩ is the standard resistance used for most recordings. Lower resistance pipettes (of 3-4 MΩ) will give a lower series resistance and thus, will be meliorate for voltage-clamp recordings. However, the tip of the glass pipette will be larger than for higher resistance pipettes and, as a consequence, the seal formation will exist challenging and the seal volition exist more hard to keep stable over time. Higher resistance drinking glass pipettes (of half-dozen-7 MΩ, with a thinner tip) are easier to form a seal with and tin can be more than suitable for prolonged current-clamp recordings; even so, they are slightly more than difficult to break through the membrane into whole-cell way and volition give a college series resistance, sometimes too high for reliable voltage-clamp recordings.

5. Fill a 1 mL syringe with 200 µL of intracellular solution, connect a 0.two µm pore filter to the syringe and attach a micro-loader tip to the filter. Alternatively, filter 200 µL of intracellular solution with a 0.2 µm pore filter, fill a ane mL syringe with the filtered solution and attach a micro-loader tip to the syringe butt.

Primary process

ane. Detect a cell to patch. Do not move the microscope stage for the rest of the procedure. If using an upright microscope, move the objective exterior the bath.

2. Utilise the syringe linked to the filter and micro-loader tip to make full halfway a borosilicate pipette with intracellular solution. Tap the pipette a few times to eliminate any air bubbles that might be present in the tip of the pipette.

3. Place the drinking glass pipette in the pipette holder. Place the pipette tip in the bath and focus the tip.

four. In one case the pipette is in the bathroom, use very light positive pressure through the force per unit area command arrangement and agree the pressure in the pipette by closing the three-way valve.

5. Arroyo the coverslip past moving the micromanipulator and monitoring the pipette superlative on the screen. Always focus at or below the tip of the glass pipette.

half dozen. Cease moving the glass pipette just before approaching the cell layer on the coverslip, making sure that the cell bodies are not in focus at this stage. Change the settings of the micromanipulator to smooth and slow motion.

vii. Fix the amplifier to voltage-clamp and right the pipette offset and then the currents measured at that point are considered as 0 pA. Employ a seal test (a 10 mV test pulse at 100 Hz) through the recording electrode.

IMPORTANT: The oscilloscope should show the square current response to the seal examination. Following Ohm's law (electric current (I)= voltage (Eastward) / resistance(R)), the amplitude of the response will depend on the resistance of the pipette.

8. Arroyo the cell body moving the drinking glass pipette forth its long axis until the tip touches the prison cell and a very small dimple is seen on the cell's membrane.

IMPORTANT: Practice non patch the nucleus of the cell. Approach the cell abroad from the nucleus.

ix. Release the positive pressure level to obtain a GΩ seal. The pressure should be and then light that a seal could form spontaneously simply by budgeted the cell.

Important: A GΩ seal is characterised past a resistance that reaches at least i GΩ. The current response to the seal exam should be very modest and if the scale of the oscilloscope is kept unchanged, the response should appear virtually flat.

10. Once a GΩ seal has been formed, change the voltage clamp to a negative voltage close to the expected jail cell resting potential (- lx to -70 mV) and correct for fast capacitance.

eleven. To break through the membrane, apply lite and curt suction pulses using a syringe (or by rima oris only if it is prophylactic to practise so). If the membrane does not break, try applying stronger suction or apply brief electrical pulses through the pipette if the patch clench amplifier has a 'zap' function.

Of import: Since the membrane of the jail cell acts as a capacitor, when the glass pipette has gained admission to the intracellular space, the current response to the seal test should show an exponential decay.

12. Acquire and analyse recordings using the appropriate software.

a) For current clamp experiments, alter to current-clamp way, read the resting membrane potential at zero current, and conform the membrane potential to -lx to -70 mV by applying electric current if required. To analyse the passive membrane properties and the spiking properties of the cells, apply alternate steps of negative and positive electric current. To confirm the spikes seen are sodium spikes, tetrodotoxin (TTX) can be used to block voltage-dependent sodium channels.

b) For voltage-clench recordings, go on in voltage-clamp manner and remove the seal exam. Concord the cell at -70 mV to tape spontaneous excitatory postsynaptic currents (EPSCs) and at 0 mV to tape spontaneous inhibitory postsynaptic currents (IPSCs). Blockers of AMPA receptors, NMDA receptors or GABA receptors can exist applied to ameliorate isolate the currents of interest or to confirm the presence of a specific current.

Figure 2. Three principal steps on whole-prison cell patch clamp procedure.

The readout on the oscilloscope is shown for all 3 steps.

Anticipated Results

Whole-cell patch clamp can be used to narrate the maturation of neuronal cultures, both at the level of private cells and at the network'due south connectivity level. As neurons derived from AxolNSCs mature over fourth dimension, the number of cells spiking increased upwardly to 100% of the total number of neurons recorded at 1 calendar month afterward plating (Figure 3A). The maturation stage of neurons is reflected by their spiking contour. Over the course of maturation, neurons express more voltage-dependent Na+ channels causing higher amplitude action potentials and more G+ channels producing a reduction in the spike's width (Figure 3B and C). Along with a modification in the spiking profile, synaptic connections start to appear afterwards one month in culture (Figure 3E). The enrichment on excitatory and inhibitory connections to cells after 45 days in culture suggests a fully mature neuronal network (Figure 3F).

Figure iii. Electrophysiological characterisation of neurons derived from Axol Cortical NSCs.

A. Number of cells recorded that showed evoked action potentials compared to the number of total cells recorded. 3 different developmental stages were analysed: x to xv days after plating (DAP) in coverslips, 25 to 30 DAP and twoscore to 45 DAP. B. Representative traces of evoked action potentials. C. Developmental profile of the spike properties of neurons derived from NSCs. D. Voltage clamp recording at -70 mV from NSCs at ten to 15 DAP. No synaptic currents were detected. E. 25 to thirty DAP, some synaptic currents were observed. These currents were excitatory postsynaptic currents (EPSCs) and were blocked past CNQX (10 µM), an AMPA and kainate receptor blocker. F. Fully mature neurons at 40 to 45 days post-plating showed both EPSCs and inhibitory postsynaptic currents (IPSCs), which could be blocked using a GABAA receptor blocker (gabazine, 2 µM). Inhibitory postsynaptic currents (IPSCs) were recorded at 0 mV.

Many thanks to Ana González Rueda for her difficult work and dedication that made this protocol possible and for kindly sharing the data she generated with us.

Axol'south Neural Cell Product Range

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Source: https://www.axolbio.com/page/whole-cell-patch-clamp-protocol