Examination of anole retina AxJ:L6

Open science experiment: This webpage is not a presentation of final results. I thought it could be interesting to share the exploration as we go. We acquired the images on 8/26/2024 and uploaded them on 8/27/2024.

Angled slice through the central fovea of a green anole.

Summary

We examined the retina of a green anole with a new combination of techniques we are testing. The off-angle sectioning of the retina provided some interesting views of the central fovea.

Background

The goal of this experiment: We are interested in using anoles to study vision and neural regeneration. We therefore need techniques for labeling their visual system. In this experiment, we are working on a protocol for labeling retinal ganglion cells in the context of non-specific stains.

Tissue preparation: Aldehyde fixed retina cut into 100-micrometer thick vibratome sections.

Green: To label retinal ganglion cells, we injected the eye of a live, deeply anesthetized, green anole with fluorescently labeled cholera toxin B. This toxin is taken up by retinal ganglion cells, is transported down their axons, and brightly labels their synapses within the brain. The injection also allows us to see retinal ganglion cells, Mueller glial cells, and some other structures in the retina.

Red: We used DiI to label the cell membranes of all cells in the tissue. DiI is a carbocyanine dye that is strongly hydrophobic. In live tissue, DiI is used to trace neurons because it will spread through the membrane of a neuron without jumping from cell to cell. It took some trial and error to figure out the right solution for turning the DiI into a non-specific stain for our tissue, but I’m happy with the result.

Blue: We used DAPI to label the cell nuclei. DAPI is a standard fluorescent label used to count cells. At high resolution, the distribution of chromatin within the nucleus can reveal information about the type and condition of the labeled cells.

Imaging: Images were acquired with an FV1000 confocal microscope. Confocal microscopy uses a pinhole to filter out out-of-focus light. Information can, therefore, be acquired from a single depth within the tissue. This optical section can be used to reconstruct 3D volumes or examine single slices (as shown below) of the tissue. The brightness, contrast, and gamma of the images have been adjusted for clarity.

Results

We are starting at the far peripheral retina. In the peripheral retina, the layers are flat and easy to identify. The density of the retinal ganglion cells (blue circles at the top) is relatively low in the periphery. The bright green at the top of the image is the cholera toxin-labeled axons of the retinal ganglion cells.
Here, we are zooming in on the photoreceptor layer of the retina. The nuclei of the photoreceptors are the double row of blue dots in the middle.
We are now looking at the central fovea of the green anole. The fovea itself is symmetrical. The angled slice produces the avocado shape.
The outer plexiform layer under the thickest regions of the fovea (red top right) has washboard ridges. I’m not yet sure if the ridges are a fixation artifact or a property of the retina.
At this depth, the angled slice approaches the bottom of the foveal pit (lower right of the black empty space). Retinal ganglion cell nucleus density is approaching zero at this point. The inner nuclear layer has also thinned to almost nothing.
This section is slightly deeper than the previous sections. Note that the inner plexiform layer (yellow) has nearly disappeared from the bottom right side of the foveal pit.
We have now moved deeper to the surface of the foveal pit (green spot on the right). The nuclei visible in this position are those of photoreceptors. Under the foveal pit, there is a concentration of choleratoxin-B labeled processes (green dots left side). These are likely the apical processes of Mueller glial cells.
A closer look at the choleratoxin-labeled processes in the photoreceptor layer. These processes appear to be outside the outer limiting membrane of the retina. they could be filopodia of Mueller cells running along the inner segments of photoreceptors.
At the bottom of the foveal pit, there are round structures that were initially mysterious. At first examination, they look like cell bodies without nuclei. They have taken up some of the choleratoxin (green). Perhaps these are normal outer plexiform layer structures that have taken up the choleratoxin at the fovea simply because the other layers are shifted out of the way.
Further comparison with the outer plexiform layer elsewhere in the retina supports the idea that these structures represent the foveal pit outer plexiform layer. Performing electron microscopy or doing some reading should clear it up.

Conclusions

Labeling: The experiment was a technical success. The lab now has a labeling protocol working in lizards that we can use to reveal retinal ganglion cells and rich background anatomy. We should be able to combine this protocol with immunolabeling and electron microscopy.

Mueller cells: I was surprised by the density and distribution of the apparent Mueller cell processes in the photoreceptor layer. I need to dive into the literature to see how well the Mueller cell distribution in the central foveal pit is understood. Is it possible for these processes to effect the optics of the foveal pit?

Foveal pit: I was surprised to see what looks like continuity in the outer plexiform layer at the bottom of the foveal pit. I wonder how many bipolar cells are innervated by the centermost photoreceptor terminal?

Credits

Julie A. Hodges and Josh L. Morgan