Single-cell Suction Recordings from Mouse Cone Photoreceptors

Summary

We will show how to record flash responses from single mouse cones using a suction electrode.

Abstract

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane ¹. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette ^2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

Protocol

Making electrodes

1. Make the recording electrodes using a micropipette puller and polish the electrodes with heating filament under microscope. For mouse cone single-cell suction recording, the inner diameter on the electrode tip is about 6-7 μm.
2. Prepare reference electrodes. Weigh 1% agar in distilled water. Melt the agar solution in hot water bath. Fill the glass pipettes (L = 100 mm, OD/ID = 1/0.75 mm) with 1% agar using syringe. Solidify the agar at room temperature for 10 minutes.
3. Cut the pipettes into halves using a diamond knife.
4. Soak the reference electrodes in reference solution for at least 24 hours at 4 &d....

Discussion

Single-cell suction recording from photoreceptor cells was developed 3 decades ago. It enables us to record trans-membrane current change induced by light stimulation without penetrating the cell membrane. Because of high cell-cell adhesion, it is difficult to isolate healthy single rod and cone from mouse retina like amphibian retina and it is hard to find individual cones due to the low percentage (3%) and small size. The inner-segment-in (IS-in) recording method overcomes this difficulty by recording current from the .......