Whole-cell patch-clamping of cultured human neurons

Pull borosilicate glass pipettes using a Sutter P-97 Flaming Brown puller. The pulling programme is set at 470 (i.e. Ramp) Heat, 0 Pull, 150 Velocity and 500 Pressure.

Intrinsic membrane properties:

1. Use the Intracellular Solution (ICS) for current-clamp and channel current recordings (see Materials ).

2. Automatically report whole-cell capacitance and input resistance from Clampex membrane test in response to small (5 mV) and brief (30 ms) voltage steps.

3. Set sampling rate at 33 kHz and holding potential at -70 mV.

4. Acquire and note down resting membrane potentials (RMP) immediately upon successful break-in in current-clamp with zero current injection.

Voltage-gated channels:

1. Use the Intracellular Solution (ICS) for current-clamp and channel current recordings (see Materials ).

2. Record simultaneously NaV and KV currents in voltage clamp.

3. Set the protocol of a series of 400 ms square voltage steps of 10 mV increments from -70 mV to +70 mV.

4. Set signal sampling and filter rate at 10 kHz and 2 kHz, respectively.

5. Pre-set leak subtraction with 4 sub-sweeps and a settling time of 250 ms.

Induced action potential:

1. Use the Intracellular Solution (ICS) for current-clamp and channel current recordings (see Materials ).

2. Record evoked action potentials in current clamp.

3. Set the protocol of a series of 500 ms current steps in 10 pA increments from -10 pA to +130 pA.

Spontaneous excitatory (EPSC) and inhibitory postsynaptic potential (IPSC):

1. Use the Intracellular Solution (ICS) for post-synaptic recording (see Materials ).

2. Record spontaneous EPSC and IPSC in voltage-clamp mode at reversal potential for inward inhibitory post-synaptic current (i.e. mostly via chloride-based channel) and inward excitatory post-synaptic current (i.e. mostly via AMPA receptors) i.e. -70 mV and 0 mV, respectively.

3. Record 1-4 mins in duration with 20x gain.

Pre-warm the external solution to 27°C in a water bath.

Turn on controllers, digitiser, commander, camera and microscope.

Open HCImageLive (for live image), Clampex 10.6 (and open membrane test) and MultiClamp 700B Commander.

Use a 1 ml syringe and needle, suck up internal solution (thawed on ice) and replace the needle with filter and tip.

Inject some solution into a small tube to place the glass tubings.

Wash the recording chamber with water first, and then with ECS.

Adjust the position of the reference electrode towards the edge of the slope of the recording chamber to avoid direct contact with the cell layer.

Go to Configure and choose the right digitiser and scan. Make sure security key (as a USB drive) is inserted.

Place the coverslip into empty plate, rinse with some external solution.

Transfer the coverslip onto the recording platform.

Search for a healthy cell to patch under the microscope and elevate the objective along y axis.

Take the pulled glass pipette, inject sufficient internal solution into it, and flick it to get rid of air bubbles at the tip.

Secure the glass pipette into the pipette holder on the manipulator.

Submerge the glass pipette into the recording platform with ICS to check for intact circuit and pipette resistance.

Switch to “Bath" mode on membrane test and press play icon.

Dip the glass pipette into the solution at the platform.

Apply (and maintain) some positive pressure before dipping (with syringe) to keep small particles away from the tip of the glass tubing.

Lower the objective into the solution at the platform.

Find the tip.

Move the tip down, then chase by moving objective down to focus. Continue until you are close to the cell.

Set the amplifier to voltage-clamp and press Pipette Offset Auto.

Switch to slow movement for the tip and objective operator.

Repeat steps 24 and 25 , in finer increments until you can see the top layer of the cell and the glass electrode tip stay directly above, but not touching, the cells.

Start blowing while approaching the cell, observe increase in membrane resistance.

As the tip makes contact the cell membrane, wait for the pipette resistance shown on the membrane test panel to reach 1 GΩ.

As the tip touches the cell, a very small dimple is often seen on the cell’s membrane.

Upon contact, release the positive pressure and the seal should be formed spontaneously or slightly suck up to create negative pressure so as to facilitate the formation of the GΩ seal.

Following successful formation of a tight GΩ seal, snap-suck up to rupture the cell membrane.

Check for good access resistance (<50 MΩ).

Select and run desired recording programmes as described in Section: Recording Programmes .

Capture brightfield images of the patched neurons for posthoc identification.

Replace ECS in the recording chamber with fresh ECS after recordings of each cell.

Patch each coverslip for no longer than 2 hours.

Transfer the patched coverslip to 12-well plate and fix with 2% PFA for 20 mins.

Keep fixed coverslips in PBS at 4°C till immunostaining.

Upon recording completion, switch to "Bath’" mode and very slowly and gently pull the tip and objective away from the cell.

Simultaneously monitor the cell capacitance and input resistance using the membrane test to visualize the loss of capacitive transients and the collapse of the current responses to a straight line, indicating the re-sealing of the cell and the establishment of an outside-out-patch at the pipette tip.

After successful detachment of the pipette from the cell, retain the coverslip in the recording platform for a little while longer for even distribution of the neurobiotin along the processes.

Replace ECS in the recording chamber with fresh ECS after recordings of each cell.

Patch each coverslip for no longer than 2 hours.

If you intend to perform immunocytochemistry on these cells, transfer the patched coverslip to a 12-well plate and fix with 2% PFA (diluted in PBS) for 20 mins.

Keep fixed coverslips in PBS at 4°C till immunostaining.

All analysis is performed on Clampfit 10.7.

Intrinsic membrane properties

1. Record down whole-cell capacitance and input resistance from Clampex membrane test in response to small (5 mV) and brief (30 ms) voltage steps in voltage-clamp mode.

2. Acquire resting membrane potentials (RMP) immediately upon successful break-in in current-clamp with zero current injection.

EPSC and IPSC:

1. All traces are automatically low-pass Bessel 8-pole filtered.

2. Spontaneous post-synaptic events and event magnitude are automatically detected based on absolute magnitude difference from the baseline (>10 pA for EPSCs and >15 pA for IPSCs) using threshold search function on Clampfit.

3. Putative EPSC or IPSC events are excluded based on template-dependent criteria including rise time and half-width (<4 ms and <1.2 ms, respectively) and manually validated to reject false-positive events.

Ionic current densities:

1. Perform analysis of Ionic current densities on leak-subtracted traces.

2 . Na_vpeak currents are identified as the maximum negative current at each membrane potential within the first 50 ms.

3. Fast A-type K_vpeak currents are identified as the maximum positive current at each membrane potential within the first 50 ms.

4. Slow activating K_vpeak currents are identified as the average positive current at each membrane potential of the last 50 ms.

5. Current density at each membrane potential is calculated by diving the peak current to the cell capacitance.

Induced Action Potential:

1 . At each current injection, an action potential is determined as a significant upward deflection of at least 20 mV that overshoot 0.

2. Rheobase current is determined as the minimal current amplitude that results in the depolarization of the cell membranes and generation of an action potential.