Patch-clamp is the industry recognized method for high-fidelity investigation of the electrical characteristics and functional connectivity of neurons. Many patch clamp configurations can be utilized depending on the research application, but in every case, electrophysiological recordings are created utilizing a glass micropipette in contact with a patch of the neuron’s membrane.
The membrane patch is left intact in the cell-attached mode, which enables the recording of ion channels within the patch along with action potentials. Adding a pore-forming agent, for example amphotericin, in the pipette creates a perforated patch, which initiates electrical continuity along with stopping the dialysis of intracellular proteins.
However, the most frequently employed patch-clamp mode is the whole-cell mode where the membrane patch is interrupted by applying strong suction for a brief amount of time to allow electrical and molecular access to the intracellular space.
There are two main configurations: the voltage-clamp mode, where the voltage is consistently held enabling the study of ionic currents, and the current-clamp mode, where the current is managed, allowing for the study of changes in membrane potential.
Many books have been written outlining this method in detail. Outlined in this article is a simplified protocol of the whole-cell patch clamp method to be applied in neuronal cultures. This protocol has been utilized to create the results described below. The solutions, voltage and current steps employed are particular to these recordings and can be changed depending on the scientist’s needs.
Artificial cerebrospinal fluid (aCSF)
Composition: 126 mM NaCl, 3 mM KCl, 2 mM MgSO4, 2 mM CaCl2, 1.25 mM NaH2PO4, 26.4 mM NaHCO3 and 10 mM glucose.
Preparation: Create 1 L 10X stock solution made up of only NaHCO3, an additional 1 L 10X stock solution containing the remaining components and keep for up to one week at 4 °C. Before recording, create 1X solution; change osmolarity to 290 mOsm (+/- 10 mOsm) and bubble with carbogen (95% O2 – 5% CO2).
IMPORTANT: This aCSF recipe uses a bicarbonate buffer. As an alternative method, HEPES (10-15 mM) can be utilized to buffer the pH of the solution. If a HEPES buffered solution is utilized, NaHCO3 and CO2 bubbling are not necessary.
Composition: 115 mM K-Gluconate, 4 mM NaCl, 0.3 mM GTP-NaCl, 2 mM ATP-Mg, 40 mM.
Preparation: Create the 1X solution; change the pH to 7.2 with KOH and the osmolarity to 270 mOsm L-1 (+/- 10 mOsm L-1). Aliquot the solution and keep at -20 °C. Prior to recording, thaw an aliquot and filter it using a standard 0.2 μm filter.
Utilize a 1 mL syringe with a microloader tip to load the recording pipettes. If an examination of cellular morphology post hoc is necessary, include an intracellular dye or label in the internal solution (e.g., biocytin, neurobiotin, etc.) and reduce the concentration of K-Gluconate accordingly.
Electrophysiology Equipment Setup
- Anti-vibration table (Faraday cage can be additionally used)
- Borosilicate glass capillary holder
- Pressure control system
- Data interface
- Recording software
- Display screen
- Perfusion pump
- Carbogen (95% O2 and 5% CO2) input
- Borosilicate glass capillaries: 0.68 mm inner diameter (ID) and 1.20 mm outer diameter (OD) with filament
- 0.2 µm pore filters
- 20 µL Micro-loader tips
- Three-way valve
- Tubes for pressure system, perfusion system, and carbogen system.
- Carbogen (95% O2 and 5% CO2) tank with regulator
- Air stone to transport carbogen to the aCSF
Other Equipment Required
- Glass capillary puller
- Osmometer (optional)
Figure 1. Whole-cell patch clamp set up.
The red arrows show the direction of the electrical signal recorded
Whole Cell Patch Clamp Procedure
- Plate the neurons a few days before recording onto coverslips.
- Activate all the equipment and configure the pump to perfuse aCSF through the recording chamber (a frequently utilized speed for whole-cell patch clamp in cultures is 1.5 mL per minute).
IMPORTANT: A perfusion speed of over 2 mL per minute might cause movements of the recording pipette and elevation of the cells from the coverslip.
- Put the coverslip with cells in the recording chamber with the cells facing upwards.
- Utilize a glass capillary puller to produce two recording pipettes from a borosilicate glass capillary with a resistance of between 3 and 7 MΩ when filled with K-Gluconate based internal solution.
IMPORTANT: 5 MΩ is the standard resistance employed for most recordings. Lower resistance pipettes (of 3-4 MΩ) will give a smaller series resistance and therefore, will be best when used in voltage-clamp recordings.
However, glass pipette’s tip will be larger than for higher resistance pipettes and, as a result, the seal formation will be challenging and the seal will be harder to keep consistent over time.
Higher resistance glass pipettes (of 6-7 MΩ, with a thinner tip) are simpler to form a seal with and can be more adapted to lengthier current-clamp recordings. Although, they are slightly harder to break through the membrane into whole-cell mode and will give a greater series resistance, sometimes too high for trustworthy voltage-clamp recordings.
- Fill a 1 mL syringe with 200 µL of intracellular solution, fix a 0.2 µm pore filter to the syringe and connect a micro-loader tip to the filter. As an alternative method, filter 200 µL of intracellular solution with a 0.2 µm pore filter, fill a 1 mL syringe with the filtered solution and connect a micro-loader tip to the syringe barrel.
- Locate a cell to patch. Do not adjust the microscope stage for the duration of the process. If an upright microscope is utilized, move the objective outside of the bath.
- Utilize the syringe connected to the filter and micro-loader tip to fill a borosilicate pipette halfway with intracellular solution. Tap the pipette several times to take away any air bubbles that might be seen in the pipette’s tip.
- Place the glass pipette in the pipette holder. Focus the tip once the pipette has been placed in the bath.
- When the pipette is in the bath, apply very light positive pressure through the pressure control system and keep the pressure in the pipette by closing the three-way valve.
- Approach the coverslip by adjusting the micromanipulator and observing the pipette height on the screen. Consistently focus at or below the tip of the glass pipette.
- Halt movements with the glass pipette immediately before approaching the cell layer on the coverslip, ensuring that the cell bodies are not in focus at this stage. Adjust the configurations of the micromanipulator to smooth and reduce motion.
- Configure the amplifier to voltage-clamp and correct the pipette offset so the currents analyzed at that point are considered as 0 pA. Apply a seal test (a 10 mV test pulse at 100 Hz) through the recording electrode.
IMPORTANT: The oscilloscope should exhibit the square current response to the seal test. In adherence to Ohm’s law, (current (I)= voltage (E) / resistance(R)), the amplitude of the response will be subject to the resistance of the pipette.
- Approach the cell body moving the glass pipette along its long axis until the tip comes in contact with the cell and a very small dimple is observed on the cell’s membrane.
IMPORTANT: The nucleus of the cell should not be patched. Approach the cell away from the nucleus.
- Release the positive pressure to obtain a GΩ seal. The pressure should be so small that a seal could form randomly just by approaching the cell.
IMPORTANT: A GΩ seal is known as having a resistance that reaches 1 GΩ at minimum. The current response to the seal test should be very small and if the scale of the oscilloscope is kept consistent, the response should appear almost flat.
- Once a GΩ seal has been formed, adjust the voltage clamp to a negative voltage close to the expected cell resting potential (- 60 to -70 mV) and correct for fast capacitance.
- To impact the membrane, light and short suction pulses should be applied utilizing a syringe (or by mouth, if it is safe to do so). Try applying greater suction if the membrane does not break, or apply quick electrical pulses through the pipette if the patch clamp amplifier has a ‘zap’ function.
IMPORTANT: As the cell membrane acts as a capacitor, when the glass pipette has reached the intracellular space, the current response to the seal test should exhibit an exponential decay.
- Collect and analyze recordings utilizing the appropriate software.
- For current clamp experiments, adjust to current-clamp mode, read the resting membrane potential at zero current, and change the membrane potential to -60 to -70 mV by the application of current if necessary.
To investigate the passive membrane characteristics and the spiking features of the cells, alternate steps of negative and positive current should be applied. Tetrodotoxin (TTX) can be utilized to block voltage-dependent sodium channels and confirm that the spikes observed are sodium spikes.
- Continue in voltage-clamp mode for voltage-clamp recordings and remove the seal test. Hold the cell at -70 mV to track spontaneous excitatory postsynaptic currents (EPSCs) and at 0 mV to record spontaneous inhibitory postsynaptic currents (IPSCs).
Blockers of NMDA receptors, AMPA receptors or GABA receptors can be applied to better isolate the currents of interest or to verify the presence of a specific current.
Figure 2. Three main steps on whole-cell patch clamp procedure.
The readout on the oscilloscope is shown for all 3 steps.
Whole-cell patch clamp can be utilized to define the maturation of neuronal cultures, both at the individual cell level and at the network’s connectivity level. As neurons provided by AxolNSCs mature over time, the amount of spiking cells increased by up to 100% of the total number of neurons observed at one month after plating (Figure 3A).
The maturation stage of neurons is shown by their spiking profile. Over the course of maturation, neurons exhibit more voltage-dependent Na+ channels creating higher amplitude action potentials and more K+ channels causing a reduction in the spike’s width (Figure 3B and C).
Along with a modification in the spiking profile, synaptic connections begin to appear after one month in culture (Figure 3E). The enrichment on excitatory and inhibitory connections to cells after 45 days in culture shows a fully mature neuronal network (Figure 3F).
Figure 3. Electrophysiological characterisation of neurons derived from Axol Cortical NSCs.
A. Number of cells recorded that exhibited evoked action potentials in comparison with the number of total cells recorded. Three different developmental stages were observed: 10 to 15 days after plating (DAP) in coverslips, 25 to 30 DAP and 40 to 45 DAP.
B. Representative traces of evoked action potentials.
C. Developmental profile of the spike characteristics of NSC derived neurons.
D. Voltage clamp recording at -70 mV from NSCs at 10 to 15 DAP. No synaptic currents were observed.
E. 25 to 30 DAP, some synaptic currents were recorded. These currents were excitatory postsynaptic currents (EPSCs) and were stopped by CNQX (10 µM), an AMPA and kainate receptor blocker.
F. Fully mature neurons at 40 to 45 days post-plating gave both EPSCs and inhibitory postsynaptic currents (IPSCs), which could be blocked utilizing a GABAA receptor blocker (gabazine, 2 µM). Inhibitory postsynaptic currents (IPSCs) were recorded at 0 mV.
About AXOL Biosciences
Axol specializes in human cell culture.
Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.
Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.
The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.
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