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.
Highlights
In the vertebrate nervous system, neurons are usually separated from circulating cells and compounds by structures such as the blood-brain barrier and blood-cerebrospinal fluid barrier
We focused on the glial elements in the nerve fiber layer (NFL) in the cichlid fish retina known for its life-long growth
The growth of the fish retina is reflected by the thickening of the NFL: In larger fish, the NFL close to the optic nerve head is thicker than the rest of the retinal layers together due to the addition of new ganglion cells (GCs) in the retinal periphery, but the NFL is relatively thinner in smaller conspecifics
Summary
In the vertebrate nervous system, neurons are usually separated from circulating cells and compounds by structures such as the blood-brain barrier and blood-cerebrospinal fluid barrier. The inner limiting membrane is the border of the inner retina to the vitreous and consists of a basal lamina paved with the endfeet of Müller cells (MCs) but does not contain a junctional barrier. In the innermost cell layer of the vertebrate retina, the axons of ganglion cells (GCs) give rise to the optic nerve projecting to the brain. These axons are surrounded by myelin formed by oligodendrocytes. Myelination begins mostly at the lamina cribrosa, and GC axons running in the nerve fiber layer (NFL) within the retina are accompanied only by the processes of MCs or astrocytes
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