Glial Cells in the Fish Retinal Nerve Fiber Layer Form Tight Junctions, Separating and Surrounding Axons

Abstract

In the retina of teleost fish, cell addition continues throughout life involving proliferation and axonal growth. To study how this is achieved in a fully functioning retina, we investigated the nerve fiber layer (NFL) of the cichlid fish Astatotilapia burtoni for components that might regulate the extracellular environment. We hypothesized that growing axons are surrounded by different cell structures than signal conducting axons. Using immunohistochemistry and freeze fracture electron microscopy we found that the endfeet of Müller cells (MCs) expressed aquaporin-4 but not in high densities as in mammals. The presence of this water channel indicates the involvement of MCs in water homeostasis. Remarkably, we discovered conspicuous tight junctions in the retinal NFL. These tight junctions formed branching strands between myelin-like wrappings of ganglion cell axons that differed morphologically from any known myelin, and also an elaborate meshwork on large membrane faces between axons. We speculated that these tight junctions have additional functions than solely facilitating nerve conductance. Immunostainings against the adaptor protein ZO-1 labeled the NFL as did antibodies against the mammalian claudin-1, 3, and 19. Performing PCR analysis, we showed expression of claudin-1, 3, 5a, 5b, 9, 11, and 19 in the fish retina, claudins that typically occur at brain barriers or myelin. We could show by immunostains for doublecortin, a marker for differentiating neurons, that new axons are not surrounded by the myelin-like wrappings but only by the endfeet of MCs. We hypothesize that the tight junctions in the NFL of fish might contribute to the separation of an extracellular space around axons facilitating conductance, from a growth-promoting environment. For a functional test we applied Evans Blue dye to eye cup preparations which showed a retention of the dye in the NFL. This indicates that these remarkable tight junctions can indeed act as a diffusion barrier.

Organization of radial glia reveals growth pattern in the telencephalon of a percomorph fish Astatotilapia burtoni

“…A comparative stain for glial markers did not reveal a contorted course of the radial glia fibers in the zebrafish brain (SOM Figure S2). Unfortunately, the DCX antibody does not show reactivity in the zebrafish brain, possibly due to the expression of doublecortin‐like protein kinase but not DCX in zebrafish (Shimomura et al, 2007), although DCX has been reported in other fish groups including the telencephalon of sharks (Docampo‐Seara et al, 2020) and the retina of cichlids (Garcia‐Pradas et al, 2018).…”

“…In particular, the doublecortin (C‐18) sc‐8066 antibody is an affinity purified antibody raised against a peptide at the C‐terminus of the human doublecortin protein. It has been shown to label differentiating neurons next to the peripheral growth zone in the retina of A. burtoni (Garcia‐Pradas et al, 2018). The islet1 antibody is known to bind to a subset of neurons in the zebrafish CNS (Johnson et al, 2016).…”

Relief of Cystoid Macular Edema-Induced Focal Axonal Compression with Anti-Vascular Endothelial Growth Factor Treatment

“…37 These glial cells establish tight junctions and prevent fluid ingress into the axons. 38 Breakdown of this barrier, although possible, should manifest itself with symmetric swelling of the RNFL on both sides of the peak point along with a decrease in RNFL reflectivity. However, thickening of the RNFL occurs focally at and nasal to the peak site of compression, and accompanied by an increase in RNFL reflectance.…”

Decline in Constitutive Proliferative Activity in the Zebrafish Retina with Ageing

“…This idea emerges in relevant articles on this topic during the last decades: “Fish retinas differ fundamentally from those of other vertebrates because they continue to grow throughout the life of the animal, both by adding new neurons and by stretching existing retinal tissue” [ 32 ]; “In fish and amphibia, retinal stem cells located in the periphery of the retina, the ciliary marginal zone (CMZ), produce new neurons in the retina throughout life” [ 33 ]; “The retina of many fish and amphibians grows throughout life, roughly matching the overall growth of the animal. The new retinal cells are continually added at the anterior margin of the retina, in a circumferential zone of cells […]” [ 34 ]; “The retinas of lower vertebrates grow throughout life from retinal stem cells (RSCs) and retinal progenitor cells (RPCs) at the rim of the retina” [ 35 ]; “In the retina of teleost fish, cell addition continues throughout life involving proliferation and axonal growth” [ 36 ], to name a few. However, studies from our group in the sea lamprey, Petromyzon marinus , and the catshark, Scyliorhinus canicula , revealed the loss of proliferative activity in the retina of adult individuals of these ancient vertebrate groups [ 37 , 38 ].…”