Patch clamp recording from bipolar cells in the wholemount mouse retina

Abstract

Bipolar cells are the second-order neurons in the retina that are less accessible for investigating their synaptic responses. Here, we present a protocol to conduct patch clamp recordings from bipolar cells in the wholemount retina from Ai32 mutant mice. We detail whole-cell patch-clamp recording from bipolar cells to examine their light-evoked responses to optogenetic stimulation, followed by imaging terminals of recorded cells to determine bipolar cell type. We describe light stimulus information to activate channelrhodopsin-2 (ChR2). For complete details on the use and execution of this protocol, please refer to Hellmer et al. (2021).

Keywords: Cell Biology; Microscopy; Model Organisms; Neuroscience; Single Cell.

Graphical abstract

Figure 1

Dissection preparation for the mouse retina (A) A dark box for dark adaptation of mice. A CO₂ tank is attached to a cage lid for euthanasia of a mouse before dissection. (B) The curved forceps and dish used for enucleation of the eyeballs after euthanasia. Included is a cage card holder used for the cervical dislocation to assure euthanasia. (C) The dissection station used for this protocol. 1) Bubbling HEPES solution used in dissection. 2) An oxygenated dark box used to store the retinal tissue during the experiment. 3) Dissection tools necessary for this protocol. 4) The dissection chamber used for isolation of the retinal tissue.

Figure 2

The patch clamp electrophysiology rig (A) An image of the full patch clamp recording microscope with the rack mount housing all necessary hardware external to the microscope. (B) The gravity perfusion setup using an ALA MV4 to control solution channels during the whole-cell recordings. (C) A magnified view of the patch clamp recording chamber including: 1) The head of recording microscope and a 10× objective 2) A pipette holder connecting to an amplifier head with a microelectrode. 3) A perfusion inlet for fresh oxygenated and warmed Ames’ solution during the experiment 4) A suction outlet to remove old Ames’ solution. 5) A thermo probe to measure the bath temperature. This communicates with the in-line heater attached to the perfusion tube just prior to the outlet to control the bath temperature.

Figure 3

The retinal dissection in the HEPES solution-filled chamber (A) An enucleated eye held in place with #5 forceps. (B) Initial corneal cut with a surgical knife. (C) Inserting a blade of scissors into the incision, cut the cornea toward the sclera. (D) Cut out the cornea and the edge of sclera by a circumferential cut. (E) Removing the lens with forceps. (F) A small transfer pipette blowing fresh solution into the eyecup. (G) Detach the retina from the sclera using two sets of forceps. One to hold the sclera of the eyeball and the other to carefully peel off the retina from the pigment epithelium. (H) A piece of isolated retina in the dissecting dish. (I) Cut the retinal tissue into quarters. (J) A piece of quarter retina. (K) A horseshoe net and a coverslip with two grease rails prior to mounting of the retina. (L) A wholemount retinal preparation is mounted between the coverslip and the horseshoe net, ready for patch clamp recordings. Scale bars for panels A-J represent 5 mm.

Figure 4

Whole cell recordings of bipolar cells (A) The surface of the retina under a 60× objective DIC imaging prior to puncture with a microelectrode. (B) The ganglion cell layer is exposed after puncturing of the ILM with a microelectrode and applying positive pressure. (C) An image of the bipolar cells layer with a microelectrode. (D) After physiological recording, an image of the bipolar cell was viewed under sulforhodamine imaging. The image shows a soma with the electrode. The vasculature is sometimes labeled by sulforhodamine in the whole mount retina. (E) Example recordings of ChR2-evoked EPSC responses in a bipolar cell under control, HEX, and MLA solution. The recordings show a cell that had MLA sensitive ACh currents and were not sensitive to HEX. Scale bars for main panels A-D are 30 μm. The inset for panel C scale bar is 5 μm.

Figure 5

Identification of bipolar cell types Representative images of different bipolar cell types are shown under sulforhodamine and YFP fluorescence. The soma with dendrites verifies that the recorded cell was a bipolar cell. Axon terminal ramification in relation to ChAT band allows cell type identification. Panels are multiple cells from Type 1 or 2, Type 3, Type 5, and Type 7 cone bipolar cells.