Electrophysiology

Detailed electrophysiology protocol for Grassmeyer et al. 

Electrophysiology

Rods and cones were recorded in flatmount retinal preparations and cones were also recorded using vertical retinal slices prepared similarly to salamander slices (Van Hook and Thoreson, 2013; https://pubmed.ncbi.nlm.nih.gov/23770753/ ). The chamber design is shown in Fig. 1 of Van Hook and Thoreson (2013) [ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724563/ ].  The techniques we use for mouse retina are very similar so we recommend checking out this earlier article. 

Initial set up:

Fill an aspirator bottle with Ames’ solution and bubble with 95% O₂/5% CO₂.  Draw Ames’ from the aspirator bottle into a syringe at the end of multi-valve stopcock. Push Ames’ solution through the Teflon perfusion tubing to ensure fluid is flowing freely then close stopcock near aspirator bottle base. 

Turn on nitrogen for air table.

Preparation of retinal slices:

For vertical slices, attach a microscope slide to the chamber with vacuum grease (re-use old slides after washing with alcohol because grease does not adhere well to new slides).  Place two beads of grease across the chamber to form a channel in which you will embed the slices.  You may want to embed one end of a small triangular piece of Kimwipe to the bead of grease near the tip of the suction needle with another end of the Kimwipe piece anchored with grease to the chamber above the reference electrode. This wick will help smooth fluid flow and ensure electrical continuity between reference electrode and bath.  Flatten a piece of Millipore nitrocellulose filter paper (0.8 micron pores, cut to approx. 4x7 mm) onto the slide with two strips of grease at either edge of the filter strip, a few millimeters away from the existing beads of grease. 

Euthanize the mouse and enucleate an eye. Puncture the cornea with a scalpel blade.  Place the eye on linoleum block, with the eye submerged in a few drops of Ames medium. Using fine iris scissors, extend incision to the outer edge of the iris.  Cut around the front of the eye to remove the cornea and iris.  Pull away the lens, carefully cutting attachments to the retina. Isolate the retina from the retinal pigment epithelium and cut the optic nerve insertion to free it entirely.  Make four small cuts at each quadrant of the retina.  Gently nudge the isolated retina, photoreceptors down, onto a small square of the blue paper that separates individual pieces of Millipore filter paper.  Remove the blue paper from the Ames medium and then flatten the retina with a fine hair. After it is flat, place the blue paper with retinal ganglion cells facing downward, onto the Millipore filter paper in the chamber. Gently press the edges of the blue paper to adhere the retina to the white filter paper below.  Submerge the retina and filter paper with Ames. Peel away the blue paper; the retina should remain stuck to the filter paper.  Fill the chamber with oxygenated Ames’ medium.

Slice the retina and underlying filter paper into 125 micron thick pieces using a razor blade tissue chopper (Stoelting; https://www.stoeltingco.com/stoelting-tissue-slicer.html) mounted with a double-edged razor blade that has been broken in half.  After slicing, move the chamber to a dissecting microscope.  While keeping the tissue submerged, use fine forceps to transfer the slices of filter paper with attached retina to the channel formed by the two parallel beads of grease at the center of the chamber.  Embed one edge of the filter paper in one of the two beads of grease and the other edge in the other bead of grease.  As you do so, rotate the filter paper towards you so that the cut face is upward.  Press the edges of the filter paper down onto the slide so that the cut face is flattened to the bottom of the slide.  When done properly, you should be able to see layering of the retina under a dissecting microscope. Mount as many slices as you can.  If you run out of slices, use pieces of filter paper to fill in any gaps in the perfusion channel.  Long gaps in the perfusion chamber between slices can make the fluid flow irregular. Place the chamber on the stage of an upright fixed stage microscope, lower the water immersion objective, and begin superfusion with Ames’ medium. Fluid can be removed by suction through the outflow line as described in the chamber design.  Monitor for short time to make sure flow is smooth and even. 

For flatmount experiments, after isolated the retina, transfer it into the recording chamber on a small piece of blue paper.  Gently slide the retina off the paper and place it photoreceptors up in the center of the chamber. Anchor the retina in place with a tissue slice harp (Warner Instruments).  Place the chamber on an upright, fixed stage microscope and lower the water immersion objective.  Begin perfusion with Ames’ medium.  Using a broken patch pipette, suction away the rod outer segments from a small area of retina to expose photoreceptor cell bodies for recording.   

Whole cell recordings are typically performed at room temperature. Response rundown is much more rapid at higher temperatures. Preparations were constantly superfused at ~1 mL/min with Ames solution (US Biological) bubbled with 95% O₂/5% CO₂. 

The intracellular pipette solution for I_Ca measurements contained (in mM): 120 CsGluconate, 10 TEACl, 10 HEPES, 2 EGTA, 1 CaCl₂, 1 MgCl₂, 0.5 NaGTP, 5 MgATP, 5 phosphocreatine, pH 7.2-7.3. For I_A(Glu) measurements, KSCN replaced CsGluconate in the intracellular solution and EGTA was raised to 5 mM. We aim for an osmolarity in the pipette solutions of around 272 mOsm, slightly below that of the Ames’ medium. 

Membrane capacitance, membrane resistance, and access resistance values for cones in slices using the CsGluconate pipette solution averaged 9.4 ± 0.4 pF, 1.1 ± 0.07 GΩ, and 74 ± 4.0 MΩ (n = 30); and for cones in flatmounts using the KSCN solution were 6.2 ± 0.2 pF, 0.53 ± 0.05 GΩ, and 77 ± 6.5 MΩ (n = 27). For rods using the CsGluconate pipette solution these values averaged 3.3 ± 0.2 pF, 2.0 ± 0.4 GΩ, and 55 ± 12 MΩ (n = 8); and for the KSCN solution were 3.6 pF ± 0.2, 1.9 ± 0.2 GΩ, and 56 ± 7.9 MΩ (n = 12).

Slice and flatmount experiments were performed on an upright fixed-stage microscope (Nikon E600FN) under a water-immersion objective (60×, 1.0 NA). Cell bodies were identified morphologically for rods or using tdTomato fluorescence for cones. Recording electrodes were positioned with Huxley-Wall micromanipulators (Sutter Instruments). For rod recordings, we use electrodes with fine tips and resistance values of 12-18 MOhms when filled with CsGluconate pipette solution. After obtaining a gigaohm seal, the patch is ruptured with gentle suction. 

Photoreceptor recordings were performed in voltage clamp using an Axopatch 200B (Axon Instruments/Molecular Devices) amplifier. Cone membrane currents from the Axopatch were filtered at 2 kHz. Some membrane currents were low-pass filtered post-hoc at 600 Hz to facilitate data presentation. Signals were digitized with a Digidata 1322A (Axon Instruments, Molecular Devices) and acquired with pClamp 10 software (Molecular Devices). Passive membrane resistance was subtracted from I_Ca and I_A(Glu) currents using P/8 subtraction. Voltages were not corrected for liquid junction potentials (CsGluconate pipette solution: 12.3 mV, KSCN pipette solution: 3.9 mV). For V_0.5 calculations, I_Ca and Q_Ca measurements were fit with Boltzmann functions adjusted for Ca²⁺ driving force assuming a Ca²⁺ reversal potential of +50 mV. I_A(Glu) charge transfer was measured from the end of the test step until the current returned to baseline.

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