Horizontal cell connectivity in the anchovy retina-a 3D electron microscopic study

Abstract

Background: Block-face scanning electron microscopy has opened a new era of connectomics research, in which it is possible to make dense reconstructions of all cells in a clipping of a neuronal network, such as the retina, resolving synaptic contacts. Anchovies, exceptionally abundant marine teleosts, have retinae with regions for triple cone-based color vision and a region with specialized cone photoreceptors, so-called polycones, made of long and short cones with axially oriented outer segment lamellae for polarization contrast vision. This modality, discovered in the 1970s, is unique in vertebrates, but the neural wiring for contrast generation in deeper retinal layers is unknown so far.

Results: To elucidate the retinal connectomics of the European anchovy Engraulis encrasicolus (Linnaeus, 1758), in a first project, we investigated the shapes and cone-specific wiring rules of 3 horizontal cell types using volume electron microscopy and subsequent computer-aided reconstruction: H1 cells contact both cone types of the polycone, H2 cells contact only the short cones, and H3 cells are exclusively connected to rods. In addition, a distinctive double band of Müller fibers and a layer of H1 axon terminals were structurally clarified.

Conclusions: The findings suggest that (1) the monochromatic polarization contrast system based on fine structure specializations in the outer retina is connected to an inherited (bichromatic) color contrast mechanism in the inner retina, (2) the anchovy polycones arose from red (now long) and green (now short) cones, and (3) the blue single cones disappeared in the relevant retinal region.

Keywords: Neural network; Polarization vision; Vertebrate retina; Volume electron microscopy.

Fig. 1

EM volume data from the ventro-temporal anchovy retina. A Cropped volume of Apreo scan 2 from horizontal cell dendrites (top) to scleral-most bipolar cell nuclei (bottom) represented by three orthogonal planes: XY front (cutting plane), YZ left, XZ top, inverted grayscale. Levels of digital reslices C–F indicated by c–f. B Volume rendering of A, highlighting less electron-dense structures, conveys the three-dimensionality of the structural data, especially at the level of the H3 cells. C XZ plane at the level of H1 cells. Nuclei surrounded by electron light cytoplasm. Between the lobed H1 profiles dendrite bundles of H2, H3, and BCs are seen. Arrowheads: neighboring H1 cells are connected by gap junctions. D XZ plane at the level of H2 cells. The space between the lobed H2 profiles is filled by dendrite bundles of H3 and BCs. Arrowheads: neighboring H2 cells are connected by gap junctions. E XZ plane at the level of H3 cells. F XZ plane (slightly oblique) at the level of horizontal cell axon terminals and bipolar nuclei. Arrowheads: neighboring H1 ATs are connected by gap junctions. # cone pedicles, B bipolar cell nucleus, b dendrite bundles of BCs, H1/2/3 horizontal cell type 1/2/3, * horizontal cell type 3, H1 AT horizontal cell type 1 axon terminal, mb Müllerband. Scale bars: A 10 µm (also B); F 10 µm (also C–E)

Fig. 2

Layering and morphology of horizontal cells. A–C Surface renderings of horizontal cell somata and nuclei in radial view from FIB scan 5, D and E from Apreo scan 1. A Horizontal cell type 1 (yellow), H1–H3 horizontal cell nuclei. B Horizontal cell type 2 (H2, orange), H1 nuclei omitted. C Horizontal cell type 3 (H3, orange), H3 nucleus obscured. D Radial view of H1 cell with axon and axon terminal (yellow), H2 cell (orange), H3 cell (red), and H cell nuclei (H1–H3) in shades of blue. cp cone pedicle, HAT horizontal cell (type 1) axon terminal, mb Müllerband. E Horizontal view of a H1, H2, and H3 cell, nuclei not shown, * H1 axon terminal, B bipolar cell nucleus. Scale bars: C 10 µm (also A, B); D, E 10 µm

Fig. 3

H1 axons with axon terminals, H2 axons. A Oblique view of the H1 axon terminal layer (in blue and green colors, by chance with a central hole formed by slightly displaced b-cells) with two reconstructed H1 cell somata (Apreo scan 1). * Axon terminal of yellow soma (see also Fig. 2D, E), B bipolar cell nucleus, cp cone pedicle, H1/2/3 horizontal cell type 1/2/3. B Horizontal view of the dense H1 axon terminal layer (with tiny holes for bipolar dendrite bunches) overlayed by H1 nuclei (semitransparent blue), ¼ of the stitched area of scan 4b, i.e., 1 tile of 4. C Apreo scan 2. H1 axons in warm colors (yellow to red, two completely reconstructed, the rest as skeletons, five reaching the H1 somata) and H2 axons in cold colors (green, blue, pink; skeletons with indicated varicosities). Labels as in A + HAT horizontal cell (type 1) axon terminal, OPL outer plexiform layer. D Volume rendering of the same data clipped by an oblique cutting plane just below the H2 somata showing H2 axons (arrowheads), partly as surface renderings. B bipolar cell, H3 horizontal cell type 3, HAT horizontal cell (type 1) axon terminal. Scale bars: A 20 µm; B–D 10 µm

Fig. 4

OPL connectivity of horizontal cells. A–F surface renderings from FIB scan 5, G–I segmentations from FIB scan 5, J + K STEM images. A H1 cell dendrites (yellow) contacting both, ribbons of short cone pedicles (green) and long cone pedicles (blue). B H2 cell dendrites (turquoise) contacting the ribbons of short cone pedicles (green) only. C H3 cell dendrites (red, pink) contacting the ribbons of rod spherules (#). D H1 dendrites (yellow) at the level of long cone ribbons (blue) and a bipolar dendrite. E H1 dendrites (yellow, with some artificial swellings *) and H2 dendrites (turquoise) at the level of short cone ribbons (green). F H3 dendrites (red, blue) at the level of rod spherules. G Selected EM plane showing the synaptic triad from D. H EM plane from E. I EM plane from F. J H cell dendrites with synaptic spinules invaginating a long cone pedicle (horizontal plane). K Radial plane showing two bunches of synaptic invaginations of a long cone pedicle. Arrows: spinules with postsynaptic densities, arrowheads: ribbons. Scale bars: A 5 µm (also B, C); D, E, G–J 1 µm; F, K 2 µm

Fig. 5

Schematic drawing of HC connectivity in the anchovy retina. H1, H2, H3 tree types of horizontal cells. lc long cones, sc short cones, r rods