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Recording.md

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Checklist before recording

The idea of this checklist is to go over the steps in multiclamp and symphony in order to record.

A detailed explanation on how to make the membrane tearing for CA, and seal and break the membrane for patch, is better done in person.

  1. Mark the mouse as deceased on AI

Coordinates and condenser focus

  1. Inspect the whole retina on 10x
  2. Find the ventral (biggest) cut and go up
  3. Find the optic nerve and set XY coordinates to 0. Not Z!
  4. Look for a flat area and anotate the XY coordinates
  5. Focus on the photorreceptors.
  6. Get out of the retina and focus the condenser on the photorreceptors height and set C to 0.
    1. In B, after setting C to zero, focus C to the coordinate you previously registered.
  7. You are ready to record now.

Electrode in

  1. Put the electrode in, and lock in positive pressure (~1.3-1.4)
  2. Set pippete offset (you should do this everytime a new pipette enters the solution).
  1. Press home in. Move forward in X, then in Y and find the electrode.

Tearing the inner limiting membrane

  1. Once you have identified a flat area susceptible to recording, you need peel off glial cells end feet (inner limiting membrane) with the pipette without hurting the ganglion cells.
  • Image Alt Text

  • An explanatory video can be found here

Cell Attached Recording

  1. Once you have identified a cell, play play lightstep in symphony.
    • Press Ctrl P to load protocol presets. Check that R* intensity is reasonable (~200), then you can play the light step.
    • This needs to be done the first time you are presenting a stimuli to the retina. Afterwards, the parameters are saved.
  2. Approach the electrode to the cell while constantly giving short pulses of possitve pressure.
  3. Once in the cell, apply constant negative pressures until you see spike trains. Apply a little more to improve the resolution of the spikes without breaking in the cell.
  4. If you are sucessfully getting spikes, play the receptive field 1d protocol to center your stimuli.

Whole cell patch clamp (work in progress)

Current clamp

  1. In symphony, run the Seal and leak protocol. It should start running the seal step. The osciloscope should looke like this (but with a single current step, will change the image later.)
  1. Approach to your cell and make the seal, change to leak.

In multiclamp run the following commands (color coded with fruitmojis)

In Voltage Clamp (VC) mode:

  1. Compensate the capacitive currents: Press auto in Cp fast. (🍏)

  2. Break the membrane with a gentle fast negative pressure pulse.

  3. Check your access resistance ( 🥭).

    • Should be low (20-40 mOhms). A high resistance icreases the series resistance compensation error and decreases the time resolution of current changes.

If doing a current clamp recording (the most common type of recording we do):

  1. Change from VC to I=0 and then to IC (Current Clamp) (🍇)
  2. In symphony, run multipulse (or your desired protocol. )

Note: different instructions are needed for voltage clamp recordings.