Overview

Ex vivo electrophysiology is otherwise known as whole-cell patching (or just patching, most of the time).

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Experiment Process

In general, the process to start a patch experiment start the same and is detailed below. Some important points to know/remember are that during the slice preparation stage, it's critically important that you keep the brain/slices as 'alive' as possible. The longer the brain spends without oxygen, the more cells will die and make the actual experiment more difficult.

This protocol assumes you are patching slices from a mouse brain and taking coronal slices from the cerebrum.

Slicing

Prep

To prep the space where the brain extraction and mounting onto the vibratome block will be done, make sure you have out:

• Forceps and spatulas
• Razor blade for sectioning off the cerebellum or frontal cortex for mounting
• Plastic petri dish for where you'll manipulate the brain, fill with ice
• Filter paper to put onto the petri dish stage to protect the brain
• Vibratome mounting block (where you'll superglue the brain)
• Block of agar superglued onto mounting block (personal preference)
• aCSF pot/slice hotel
• Ice
• Slushy cutting solution
• Petri dish filled with aCSF for bifurcating the slices

Getting the brain

1. Sac the mouse and remove the brain as quickly (and carefully) as possible. Make sure to take special care near the areas that you are attempting to patch

It's helpful to place the head on the ice-filled, filter paper covered petri dish as a place to extract the brain. This gives you a surface to do this as well as keeping the brain as cold as possible

2. Carefully remove the brain from the skull using spatulas and place in bubbling cutting solution. You never want to squeeze the brain.
3. Prep anything you need for the following steps while the brain is in the cutting solution. Continue on as quickly as you can.
4. Remove brain from cutting solution, place onto filter paper and cut off the cerebellum so you have a flat surface to mount the brain on.
5. Add superglue to mounting block (in front of agar if using).
6. Carefully transfer the brain to the superglue and get block into the cutting solution as quickly as possible.

If the brain isn't mounted flat, still place into the cutting solution promptly so the superglue hardens and is easier to scrape off with a razor blade. Repeat Steps 5 and 6.

7. Set the bounds of the vibratome's working space, adjust the height of the stage, and start slicing.

Setting up the rig

Setting up the computer

1. Turn on the amplifier, acquisition system, power supplies for the Scientifica manipulators, and heater on the top of the server rack
2. Switch on the power and light to the microscope
3. Open Ocular
4. Open Multiclamp
5. Open Clampex and set file path for saving recordings
6. Open membrane test in Clampex and set to desired current (typically 10 or -10)
7. Open protocol file in Clampex

Setting up the rig space

• Get pot of aCSF and start bubbling it
• Realign tubing in pump and start the pump
• Make sure that the ground in the bath is well placed and stays within contact of the aCSF. The aCSF should fill the bath to nearly the top but not overflow. You may have to adjust the inflow and outflow to achieve this.
• Once aCSF has run through the bath for 3-5 minutes, start recycling aCSF

Getting a slice & and pipette in the bath!

• Place slice in bath, and harp on top of slice to hold it in place
• Pull patch pipettes by running Step 2 on pipette puller

Try to make them equal in length since that requires less adjustment over time of the headstage and frankly makes your life easier

• Fill pipette with enough internal solution to make contact with the silver ground in the headstage, making sure to get rid of pockets of air at the tip

Small amounts of air at the tip can be removed by using the 10mL syringe to add positive pressure once the pipette is situated in the holder

• Situate the headstage with the pipette over the bath in position, and focus camera to move the pipette into place above the slice

Remember to move down in the focal plane with the camera before the pipette so you don't accidentally crash the pipette into the slice!

• Once you have the pipette tip just above the slice in your target region, switch the manipulator into its slower mode (-), pull the objective up a bit and switch to 40x magnification.

To make finding the pipette under 40x easier, make sure your target region and the pipette are in the center of the camera frame.

• Carefully lower the 40x to near the top of the bath and encourage water immersion by adding more aCSF until it touches.
• Slowly move the camera up and down in the Z direction to find the pipette. Repeat earlier step of moving down in the Z with the camera and following with the pipette to keep the pipette from ripping through the slice.
• Before touching the slice with the pipette, add ~0.4mL of positive pressure using the 1mL syringe. This will allow you to move through the slice more effectively.
• Congrats! You're in the slice, with your patch pipette. Now all you have to do is search around for a healthy cell.

Patching a cell

Once you've found a cell that looks patchable (not a fried egg, not a circle with a dark ring around the outside, not a crumpled mess), you're probably wanting to patch it. There's a few steps to doing so, and within the lab there is some variability to how people patch. So here let's just outline the basic steps, with some tips and tricks.

Approaching the cell

• With a pipette that has positive pressure and isn't visibly blocked, bring the pipette tip close to the cell from above (in the Z).
• As the pipette is brought down, you should see a dimple appear on the surface of the cell - this tells you that you have made contact and have some positive pressure.
• The next few steps need to be taken quickly.
• Auto the pipette offset in Multiclamp
• Run the membrane test in Clampex
• Apply negative pressure by steadily pulling back on the 1mL syringe. You should see the membrane test become flat and the resistance increase into 1+ GΩ range. If you do, you've created a seal.

Rarely, applying even more negative pressure by cutting off the pressure supply using the stopcock on the tubing, removing the syringe to empty it, and applying more negative pressure works to create a seal on the cell.

• Once you have a GΩ seal, Auto the Whole Cell on Multiclamp and hold the cell at -70mV.
• Open the tubing stopcock to the side that has your mouth pipetting tubing in it and open the cell by applying quick, strong bursts of negative pressure. This may take several attempts.
• If the cell opens, and you achieve a whole cell patch, the membrane test should look something like this:
• Turn off the membrane test and continue on with your experiment!
• If you've applied negative pressure and failed to get a seal or open the cell, replace the pipette with a new one and start again.