We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.
One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells – the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor’s membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon – a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 – and how it contributes to the unique signaling properties of this first retinal synapse.
The retinal slice preparation has proven very useful for analyzing the circuitry and mechanisms employed by the retina to process visual information. The ability to obtain whole cell recordings simultaneously from pre- and post-synaptic neurons has been particularly helpful in this endeavor. Paired whole cell recordings are much easier to achieve with slices than with flat-mount retina preparations because the different retina layers are exposed. Moreover, because of their large retinal neurons, salamanders have a long history as a retinal preparation and therefore provide a particularly well-characterized model system.
With practice, healthy slices of salamander retina can be prepared regularly. A few key steps can make the difference between success and failure. 1) Make sure the razor blade is mounted on the tissue slicer so that it lays flat against the glass surface and slices cleanly though both the tissue and underlying nitrocellulose membrane. If you have made a clean cut through the nitrocellulose membrane, you should hear a faint click as the razor blade strikes the surface of the glass slide. 2) Make sure the retina has adhered to the nitrocellulose membrane. Otherwise, the retina can float away from the membrane during any step of the procedures. 3) Do not expose cut slices to air, as this will damage many of the superficial cells. 4) Make sure the slices and nitrocellulose membrane lie flat against the glass slide so that the retinal layers are apparent under the dissecting microscope. 5) Balance the rates of superfusate inflow and outflow in order to avoid overflowing the recording chamber. This prevents sudden changes in solution levels, which can cause abrupt tissue movements. 6) Select a healthy pair of cells close to one another. Cells with smooth cytoplasm are healthier than cells with grainy cytoplasm. Cells deeper in the slice are more likely to retain intact synaptic contacts. 7) Make sure the pipette tip has not broken or brushed against other tissue or debris on the way down to the cells. 8) Check the pipette resistance to ensure that it is not clogged with debris or a bubble, both of which can make it difficult to obtain a quality whole-cell recording.
Rather than attaching the retina to nitrocellulose filter paper, some investigators embed retinas in a block of agar and use a vibratome to cut retinal slices. Although we have not tried this approach, Kim et al.11 discuss advantages of both approaches. In their experience, the agar-based approach provides a more consistent yield of flat slices with well-delineated retinal layers but the filter paper-based approach yields healthier photoreceptors.
Rods and cones are responsible for transducing light into changes in membrane potential. With paired recordings, the membrane potential of rods or cones can be manipulated directly and so the ability to generate light responses, while helpful for identifying cell types, may not be essential. We therefore often prepare slices in white light. However, even when prepared under bright illumination, salamander retinal neurons can generate large light responses as illustrated by the responses in Fig. 3. This is partly due to a relatively large reservoir of chromophore in the large outer segment volume but may also reflect the ability of Müller cells to regenerate 11-cis-retinal for cones12. To obtain fully dark-adapted light responses, one can prepare the slices under infrared illumination. For dissections under infrared light, we attach GenIII image intensifiers (Nitemate NAV3, Litton Industries, Tempe, AZ) to the oculars of the dissecting microscope and illuminate the tissue with an infrared LED flashlight. For slicing and other procedures that are not conducted under the dissecting microscope, we employ a head-mounted image intensifier. For placement of the patch pipettes, we visualize slices using an infrared-sensitive CCD camera (e.g. Watec 502H, Watec Inc., Middletown, NY) mounted to the upright, fixed-stage microscope. With these precautions, one can obtain rod responses exhibiting single photon sensitivity6, 13.
One limitation of working in retinal slices is that long cellular processes of large field retinal neurons may lose many of their dendrites during the slicing procedure. Retinal slice preparations are therefore more useful for studying the physiology of cells in which the synaptic contacts involve processes close to the cell body. Amphibian and mammalian retinas share many of the same cell types and utilize similar physiological mechanisms14-16. While salamander retina is a good model for many aspects of mammalian retina, one important difference appears to be the presence of a dedicated rod pathway in mammals that involves contacts of specialized rod bipolar cells onto AII amacrine cells14. An additional limitation of the salamander retina is the small number of genetic tools developed specifically for this species. However, antibodies and shRNA reagents that target well-conserved regions in other species can be used successfully in salamander, as can many small molecule inhibitors and peptide reagents. Additionally, with a few modifications in technique, retinal slices can be prepared from other species in which some of these tools are more readily available.
Beyond its utility for paired whole-cell recording, the salamander retinal slice preparation is also amenable to a variety of other approaches. As discussed above, retinal slices can be used to study light responses in combination with various voltage clamp protocols17. Retinal neurons can also be loaded with fluorescent dyes sensitive to Ca2+, Cl–, or Na+ introduced through the patch pipette or by bath-application15,18-20. A fluorescent peptide that binds to the synaptic ribbon21 can be introduced through the patch pipette and used for imaging the ribbon10 or, when conjugated to fluorescein, for acutely and selectively damaging the ribbon22. We have also used retinal slices in combination with quantum dots to monitor the movements of individual calcium channels at rod and cone synaptic terminals23. Thus, the vertical retinal slice is a versatile experimental preparation for studying basic synaptic mechanisms and the unique processing functions performed at the first synapse in the visual signaling pathway.
The authors have nothing to disclose.
This work was funded by Research to Prevent Blindness and National Institutes of Health grant EY10542.
Name of the reagent/material | Company | Catalogue number | Comments (optional) |
Tissue slicer | Stoelting | 51425 | |
Double edge razor blades | Ted Pella, Inc | 121-6 | |
Nitrocellulose membranes | Millipore | AAWP02500 | Type AAWP 0.8 mm pore |
Borosilicate glass pipettes | World Precision Instruments | TW120F-4 | 1.2mm OD 0.95 mm ID |
Ag/AgCl pellet | Warner | E206 | |
MicroFil | World Precision Instruments | MF34G-5 | 34 ga. Filling needle, 67 mm long |