TITLE: Whole-cell patch-clamping of cultured human neurons
AUTHORS: Quyen  Do, Dayne Beccano-Kelly, Richard Wade-Martins

[DESCRIPTION]
This protocol describes procedures of the whole-cell patch clamping of human neurons derived from induced pluripotent stem cells and cultured in adherent monolayer. 

[STEPS]
SECTION: Pulling of glass electrodes
1. 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.
 

SECTION: Recording Programmes
2. 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. 
SECTION: Recording Programmes
3. 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. 
SECTION: Recording Programmes
4. 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.

 

SECTION: Recording Programmes
5. 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. 


 


SECTION: Preparation of the RIG
6. Pre-warm the external solution to 27°C in a water bath.
SECTION: Preparation of the RIG
7. Turn on controllers, digitiser, commander, camera and microscope.
SECTION: Preparation of the RIG
8. Open HCImageLive (for live image), Clampex 10.6 (and open membrane test) and MultiClamp 700B Commander.
SECTION: Preparation of the RIG
9. Use a 1 ml syringe and needle, suck up internal solution (thawed on ice) and replace the needle with filter and tip.
SECTION: Preparation of the RIG
10. Inject some solution into a small tube to place the glass tubings.

 

SECTION: Preparation of the RIG
11. Wash the recording chamber with water first, and then with ECS. 
SECTION: Preparation of the RIG
12. 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.
SECTION: Preparation of the RIG
13. Go to Configure and choose the right digitiser and scan. Make sure security key (as a USB drive) is inserted. 
SECTION: Whole-cell patch clamping measurements using Clampex
14. Place the coverslip into empty plate, rinse with some external solution.
SECTION: Whole-cell patch clamping measurements using Clampex
15. Transfer the coverslip onto the recording platform.
SECTION: Whole-cell patch clamping measurements using Clampex
16. Search for a healthy cell to patch under the microscope and elevate the objective along y axis.
SECTION: Whole-cell patch clamping measurements using Clampex
17. Take the pulled glass pipette, inject sufficient internal solution into it, and flick it to get rid of air bubbles at the tip.
SECTION: Whole-cell patch clamping measurements using Clampex
18. Secure the glass pipette into the pipette holder on the manipulator.
SECTION: Whole-cell patch clamping measurements using Clampex
19. Submerge the glass pipette into the recording platform with ICS to check for intact circuit and pipette resistance.

 

SECTION: Whole-cell patch clamping measurements using Clampex
20. Switch to “Bath" mode on membrane test and press play icon.
SECTION: Whole-cell patch clamping measurements using Clampex
21. Dip the glass pipette into the solution at the platform.
SECTION: Whole-cell patch clamping measurements using Clampex
22. Apply (and maintain) some positive pressure before dipping (with syringe) to keep small particles away from the tip of the glass tubing.
SECTION: Whole-cell patch clamping measurements using Clampex
23. Lower the objective into the solution at the platform.
SECTION: Whole-cell patch clamping measurements using Clampex
24. Find the tip.
SECTION: Whole-cell patch clamping measurements using Clampex
25. Move the tip down, then chase by moving objective down to focus. Continue until you are close to the cell.
SECTION: Whole-cell patch clamping measurements using Clampex
26. Set the amplifier to voltage-clamp and press Pipette Offset Auto.
SECTION: Whole-cell patch clamping measurements using Clampex
27. Switch to slow movement for the tip and objective operator. 
SECTION: Whole-cell patch clamping measurements using Clampex
28. 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.
SECTION: Whole-cell patch clamping measurements using Clampex
29. Start blowing while approaching the cell, observe increase in membrane resistance. 
SECTION: Whole-cell patch clamping measurements using Clampex
30. As the tip makes contact the cell membrane, wait for the pipette resistance shown on the membrane test panel to reach 1 GΩ.
SECTION: Whole-cell patch clamping measurements using Clampex
31. As the tip touches the cell, a very small dimple is often seen on the cell’s membrane.
SECTION: Whole-cell patch clamping measurements using Clampex
32. 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.
SECTION: Whole-cell patch clamping measurements using Clampex
33. Following successful formation of a tight GΩ seal, snap-suck up to rupture the cell membrane.

SECTION: Whole-cell patch clamping measurements using Clampex
34. Check for good access resistance (<50 MΩ).


 

SECTION: Whole-cell patch clamping measurements using Clampex
35. Select and run desired recording programmes as described in Section: Recording Programmes.


 

SECTION: Whole-cell patch clamping measurements using Clampex
36. Capture brightfield images of the patched neurons for posthoc identification. 
SECTION: Whole-cell patch clamping measurements using Clampex
37. Upon recording completion, switch to "Bath’" mode and very slowly and gently pull the tip and objective away from the cell. 

 

SECTION: Whole-cell patch clamping measurements using Clampex
38. 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. 

 


SECTION: Whole-cell patch clamping measurements using Clampex
39. 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. 
SECTION: Whole-cell patch clamping measurements using Clampex
40. Replace ECS in the recording chamber with fresh ECS after recordings of each cell.
SECTION: Whole-cell patch clamping measurements using Clampex
41. Patch each coverslip for no longer than 2 hours. 
SECTION: Whole-cell patch clamping measurements using Clampex
42. 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.
SECTION: Whole-cell patch clamping measurements using Clampex
43. Keep fixed coverslips in PBS at 4°C till immunostaining. 

 

SECTION: Analysis
44. 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. Nav peak currents are identified as the maximum negative current at each membrane potential within the first 50 ms.
3. Fast A-type Kv peak currents are identified as the maximum positive current at each membrane potential within the first 50 ms.
4. Slow activating Kv peak 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.



SECTION: Whole-cell patch clamping measurements using Clampex
36.1. Replace ECS in the recording chamber with fresh ECS after recordings of each cell.
SECTION: Whole-cell patch clamping measurements using Clampex
36.2. Patch each coverslip for no longer than 2 hours.
SECTION: Whole-cell patch clamping measurements using Clampex
36.3. Transfer the patched coverslip to 12-well plate and fix with 2% PFA for 20 mins.
SECTION: Whole-cell patch clamping measurements using Clampex
36.4. Keep fixed coverslips in PBS at 4°C till immunostaining.