Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Our group is interested in the neuronal circuitry and synaptic mechanisms underlying visual processing in the retina. The Morgans Lab is investigating molecular mechanisms of light adaptation in bipolar cells. And the Sivyer Lab is interested in how inner retinal neurons contribute to ganglion cell function.

Accessing inner nuclear layer neurons in whole mount retina is a challenge for both anatomical and physiological studies. Inner nuclear layer neurons are accessible in vertical sections, but there are very few neurons in the field of view. Furthermore, slicing severus lateral processes and connections which can impact physiological studies.

The removal of the photoreceptors in the split retina technique dramatically improves antibody diffusion into the inner retina, which makes immuno labeling of inner retinal targets over 20 times faster when compared to traditional whole mount retinas. The split retina also greatly improves access to inner nuclear layer neurons during patch-clamp electrophysiology, The split retina technique will open doors to new approaches and accelerate the pace of our experiments. For example, we plan to use this technique to study bipolar cell inputs to melanopsin ganglion cells by expressing channel rhodopsin in the bipolar cells.

With a wide mouth transfer pipette, transfer one mouse eye to a new Petri dish with ice cold, fresh Ames media. Using a pair of forceps, pin its extra connective tissue to the dish bottom to stabilize the eye. Now, puncture the eye along the ora serrata line with a 25 gauge needle to create an entry point for the vannas scissors.

Use the vannas scissors to cut along the ora serrata, until the cornea separates. With a pair of forceps, remove the lens from the eye cup, then use the wide mouth transfer pipette to move the eye cup into a dish containing a large volume of actively carbogenated Ames. Transfer one eye cup to a Petri dish filled with fresh carbogenated Ames solution.

With vannas scissors, make a small incision inward from the sclera's edge. With two pairs of forceps, carefully peel the sclera away from the retina. Now, cut the optic nerve connecting the sclera and retina, then use scissors to gently pry the retina from the sclera to isolate it.

Next, with a scalpel, cut the retina into halves or quarters. Using a wide mouth transfer pipette, place the retina pieces in a large volume of continuously carbogenated Ames media. Repeat the retinal dissection for the other eye before retinal splitting.

Start by replacing the Ames media from the Petri dish that contained the mouse retina with a fresh supply of carbogenated Ames. Use a wide mouth transfer pipette to place a piece of dissected mouse retina onto a glass slide, ganglion cell side up. Remove the excess liquid with a delicate task wipe to flatten it.

If necessary, use a fine tip paintbrush to gently pull the retinal ledges under a dissection microscope. Use forceps to place a dry piece of nitrocellulose membrane on the retina, adhering it to the ganglion cell side. Flip the retina over to lay the nitrocellulose on the glass slide and place another dry membrane piece on the retina's photoreceptor side.

Now, wet the tip of the paintbrush with Ames media. Apply gentle downward pressure to the upper membrane to aid uniform adherence. Next, pin the lower membrane to the glass with one pair of forceps, then carefully use another pair to peel off the upper membrane in a slow, steady motion.

Dispose of the upper membrane, which contains the photoreceptors. Finally, return the lower membrane containing the inner retina to the carbogenated Ames media. Begin by immersing the split mouse retina with attached nitrocellulose membranes in 4%paraformaldehyde on ice for 30 minutes.

Next, replace the paraformaldehyde with five to 10 milliliters of room temperature PBS to wash the split retinas Before removing the split retina from the nitrocellulose, use a hydrophobic barrier pen to prepare circular wells on a microscope slide. Let the wells air dry for five to 10 minutes. Afterward, place the split retinas in the prepared wells and add enough PBS to immerse them.

Under a dissection microscope, gently lift the edges of the tissue with a fine paint brush and circle around the retina to separate it from the membrane. Use forceps and the paintbrush to remove the membrane from beneath the floating piece of the retina. Then carefully aspirate away the remaining PBS so that the retinal tissue rests on the microscope slide, ganglion cell side down.

Immunofluorescence labeling of synaptophysin across the top of the split retina indicated that the photoreceptor synaptic terminals were retained. However, photoreceptor nuclei were absent, confirming the retina split through the photoreceptor axons. Synaptic integrity was assessed by labeling RGS11 in non-BC dendrites and CtBP2 in photoreceptor synaptic ribbons.

Splitting did not damage the morphology of photoreceptor terminals in the outer plexiform layer. RBC viability in the split retina was assessed using a near-infrared nuclear dye. Regional variability in cell viability across the tissue was observed with higher cell death in some areas with most RBCs remaining viable after splitting.

Immunofluorescence labeling of RBCs against PKC alpha and horizontal cells against calbindin-D showed rapid antibody diffusion in the split retina after one hour incubation.