Retinal and lenticular ultrastructure in the aestivating salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) with special reference to a new type of photoreceptor mosaic

The salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) is endemic to southwestern Australia and inhabits shallow, freshwater pools which evaporate during the hot summer months. Burrowing into the substrate in response to falling water levels allows these fish to aestivate for extended periods of time while encapsulated in a mucous cocoon even when the pools contain no water. Only a few minutes after a major rainfall, these fish emerge into relatively clear water which subsequently becomes laden with tannin, turning the water black and reducing the pH to approximately 4.3. As part of a large study of the visual adaptations of this unique species, the retinal and lenticular morphology of the aestivating salamanderfish is examined at the level of the light and electron microscopes. The inner retina is highly vascularised by a complex system of vitreal blood vessels, while the outer retina receives a blood supply by diffusion from a choriocapillaris. This increased retinal blood supply may be an adaptation for reducing the oxygen tension during critical periods of aestivation. Large numbers of Muller cells traverse the thickness of the retina from the inner to the outer limiting membranes. The ganglion cells are arranged in two ill-defined layers, separated from a thick inner nuclear layer containing two layers of horizontal cells by a soma-free inner plexiform layer. The photoreceptors can be divided into three types typical of many early actinopterygian representatives; equal double cones, small single cones and large rods (2:1:1). These photoreceptors are arranged into a unique regular square mosaic comprising a large rod bordered by four equal double cones with a small single cone located at the corner of each repeating unit. The double cones may optimise perception of mobile prey which it tracks by flexion of its head and neck and the large rods may increase sensitivity in the dark tannin-rich waters in which it lives. Each single cone also possesses a dense collection of polysomes and glycogen (a paraboloid) beneath its ellipsoid, the first such finding in teleosts. The retinal pigment epithelium possesses melanosomes, pha,oocytes and a large number of mitochondria. The anatomy of the retina and the photoreceptor mosaic is discussed in relation to the primitive phylogeny of this species and its unique life history.

Light and electron microscopic analysis of aquaporin 1-like-immunoreactive amacrine cells in the rat retina

Aquaporin 1 (AQP1; also known as CHIP, a channel-forming integral membrane protein of 28 kDa) is the first protein to be shown to function as a water channel and has been recently shown to be present in the rat retina. We previously showed (Kim et al. [1998] Neurosci Lett 244:52-54) that AQP1-like immunoreactivity is present in a certain population of amacrine cells in the rat retina. This study was conducted to characterize these cells in more detail, With immunocytochemistry using specific antisera against AQP1, whole-mount preparations and 50-mum-thick vibratome sections were examined by light and electron microscopy. These cells were a class of amacrine cells, which had symmetric bistratified dendritic trees ramified in stratum 2 and in the border of strata 3 and 4 of the inner plexiform layer (IPL). Their dendritic field diameters ranged from 90 to 230 mum. Double labeling with antisera against AQP1 and gamma-aminobutyric acid or glycine demonstrated that these AQP1-like-immunoreactive amacrine cells were immunoreactive for glycine. Their most frequent synaptic input was from other amacrine cell processes in both sublaminae a and b of the IPL, followed by a few cone bipolar cells. Their primary targets were other amacrine cells and ganglion cells in both sublaminae a and b of the IPL. In addition, synaptic output Onto bipolar cells was rarely observed in sublamina b of the IPL. Thus, the AQP1 antibody labels a class of glycinergic amacrine cells with small to medium-sized dendritic fields in the rat retina. (C) 2002 Wiley-Liss, Inc.

Distribution of two splice variants of the glutamate transporter GLT-1 in the developing rat retina

The distributions of a carboxyl terminal splice variant of the glutamate transporter GLT-1, referred to as GLT-1B, and the carboxyl terminus of the originally described variant of GLT-1, referred to hereafter as GLT-1alpha, were examined using specific antisera. GLT-1B was present in the retina at very early developmental stages. Labelling was demonstrable at embryonic day 14, and strong labelling was evident by embryonic day 18. Such labelling was initially restricted to populations of cone photoreceptors, the processes of which extended through the entire thickness of the retina and appeared to make contact with the retinal ganglion cells. During postnatal development the GLT-1B-positive photoreceptor processes retracted to form the outer plexiform layer, and around postnatal day 7, GLT-1B-immunoreactive bipolar cells appeared. The pattern of labelling of bipolar cell processes within the inner plexiform layer changed during postnatal development. Two strata of strongly immunoreactive terminals were initially evident in the inner plexiform layer, but by adulthood these two bands were no longer evident and labelling was restricted to the somata and processes (but not synaptic terminals) of the bipolar cells, as well as the somata, processes, and terminals of cone photoreceptors. By contrast, GLT-1alpha appeared late in postnatal development and was restricted mainly to a population of amacrine cells, although transient labelling was also associated with punctate elements in the outer plexiform layer, which may represent photoreceptor terminals, (C) 2002 Wiley-Liss, Inc.

Identification of cholinoceptive glycinergic neurons in the mammalian retina

The light-evoked release of acetylcholine (ACh) affects the responses of many retinal ganglion cells, in part via nicotinic acetylcholine receptors (nAChRs). nAChRs that contain beta2alpha3 neuronal nicotinic acetylcholine receptors have been identified and localized in the rabbit retina; these nAChRs are recognized by the monoclonal antibody mAb210. We have examined the expression of beta2alpha3 nAChRs by glycinergic amacrine cells in the rabbit retina and have identified different subpopulations of nicotinic cholinoceptive glycinergic cells using double and triple immunohistochemistry with quantitative analysis. Here we demonstrate that about 70% of the cholinoceptive amacrine cells in rabbit retina are glycinergic cells. At least three nonoverlapping subpopulations of mAb210 glycine-immunoreactive cells can be distinguished with antibodies against calretinin, calbindin, and gamma-aminobutyric acid (GABA)(A) receptors. The cholinergic cells in rabbit retina are thought to synapse only on other cholinergic cells and ganglion cells. Thus, the expression of beta2alpha3 nAChRs on diverse populations of glycinergic cells is puzzling. To explore this finding, the subcellular localization of beta2alpha3 was studied at the electron microscopic level. mAb210 immunoreactivity was localized on the dendrites of amacrines and ganglion cells throughout the inner plexiform layer, and much of the labeling was not associated with recognizable synapses. Thus, our findings indicate that ACh in the mammalian retina may modulate glycinergic circuits via extrasynaptic beta2alpha3 nAChRs. (C) 2002 Wiley-Liss, Inc.

Type 1 nitrergic (ND1) cells of the rabbit retina: Comparison with other axon-bearing amacrine cells

NADPH diaphorase (NADPHd) histochemistry labels two types of nitrergic amacrine cells in the rabbit retina. Both the large ND1 cells and the small ND2 cells stratify in the middle of the inner plexiform layer, and their overlapping processes produce a dense plexus, which makes it difficult to trace the morphology of single cells. The complete morphology of the ND1 amacrine cells has been revealed by injecting Neurobiotin into large round somata in the inner nuclear layer, which resulted in the labelling of amacrine cells whose proximal morphology and stratification matched those of the ND1 cells stained by NADPHd histochemistry. The Neurobiotin-injected ND1 cells showed strong homologous tracer coupling to surrounding ND1 cells, and double-labelling experiments confirmed that these coupled cells showed NADPHd reactivity. The ND1 amacrine cells branch in stratum 3 of the inner plexiform layer, where they produce a sparsely branched dendritic tree of 400-600 mum diameter in ventral peripheral retina. In addition, each cell gives rise to several fine beaded processes, which arise either from a side branch of the dendritic tree or from the tapering of a distal dendrite. These axon-like processes branch successively within the vicinity of the dendritic field before extending, with little or no further branching, for 3-5 mm from the soma in ventral peripheral retina. Consequently, these cells may span one-third of the visual field of each eye, and their spatial extent appears to be greater than that of most other types of axon-bearing amacrine cells injected with Neurobiotin in this study. The morphology and tracer-coupling pattern of the ND1 cells are compared with those of confirmed type 1 catecholaminergic cells, a presumptive type 2 catecholaminergic cell, the type 1 polyaxonal. cells, the long-range amacrine cells, a novel type of axon-bearing cell that also branches in stratum 3, and a type of displaced amacrine cell that may correspond to the type 2 polyaxonal cell. (C) 2004 Wiley-Liss, Inc.

The type 1 polyaxonal amacrine cells of the rabbit retina: A tracer-coupling study

The type 1 polyaxonal (PA1) cell is a distinct type of axon-bearing amacrine cell whose soma commonly occupies an interstitial position in the inner plexiform layer; the proximal branches of the sparse dendritic tree produce 1-4 axon-like processes, which form an extensive axonal arbor that is concentric with the smaller dendritic tree (Dacey, 1989; Famiglietti, 1992a,b). In this study, intracellular injections of Neurobiotin have revealed the complete dendritic and axonal morphology of the PA1 cells in the rabbit retina, as well as labeling the local array of PA1 cells through homologous tracer coupling. The dendritic-field area of the PA1 cells increased from a minimum of 0.15 mm(2) (0.44-mm equivalent diameter) on the visual streak to a maximum of 0.67 mm(2) (0.92-mm diameter) in the far periphery; the axonal-field area also showed a 3-fold variation across the retina, ranging from 3.1 mm(2) (2.0-mm diameter) to 10.2 mm(2) (3.6-mm diameter). The increase in dendritic- and axonal-field size was accompanied by a reduction in cell density, from 60 cells/mm(2) in the visual streak to 20 cells/mm(2) in the far periphery, so that the PA1 cells showed a 12 times overlap of their dendritic fields across the retina and a 200-300 times overlap of their axonal fields. Consequently, the axonal plexus was much denser than the dendritic plexus, with each square millimeter of retina containing similar to100 mm of dendrites and similar to1000 mm of axonal processes. The strong homologous tracer coupling revealed that similar to45% of the PA1 somata were located in the inner nuclear layer, similar to50% in the inner plexiform layer, and similar to5% in the ganglion cell layer. In addition, the Neurobiotin-injected PA1 cells sometimes showed clear heterologous tracer coupling to a regular array of small ganglion cells, which were present at half the density of the PA1 cells. The PA1 cells were also shown to contain elevated levels of gamma-aminobutyric acid (GABA), like other axon-bearing amacrine cells.

The fountain amacrine cells of the rabbit retina

We have characterized a distinctive type of bistratified amacrine cell in the rabbit retina at both the single cell and population levels. These cells correspond to the fountain amacrine cells recently identified by MacNeil and Masland (1998). The fountain cells can be distinguished in superfused retinal wholemounts labeled with nuclear dyes, thus enabling them to be targeted for intracellular injection with Neurobiotin. This revealed that the primary dendrites ascend steeply to sublamina b of the inner plexiform layer, where they form an irregular arbor at the border of strata 4 and 5. These dendrites then give rise to multiple varicose processes that descend obliquely to sublamina a, where they form a more extensive arbor in stratum 1. The fountain amacrine cells show strong homologous tracer coupling when injected with Neurobiotin, and this has enabled us to map their density distribution across the retina and to examine the dendritic relationships between neighboring cells. The fountain amacrine cells range in density from 90 to 360 cells/mm(2) and they account for 1.5% of the amacrine cells in the rabbit retina. The thick tapering dendrites in sublamina b form highly territorial arbors that tile the retina with minimal overlap, whereas the thin varicose processes intermingle in sublamina a. The fountain cells are immunopositive for gamma-aminobutyric acid and immunonegative for glycine. We further propose that these cells are homologous to the substance P-immunoreactive (SP-IR) amacrine cells in the cat retina and that they may account for a subset of the SP-IR amacrine cells in the rabbit retina.

The eyes of deep-sea fish II. Functional morphology of the retina

Three different aspects of the morphological organisation of deep-sea fish retinae are reviewed: First, questions of general cell biological relevance are addressed with respect to the development and proliferation patterns of photoreceptors, and problems associated with the growth of multibank retinae, and with outer segment renewal are discussed in situations where there is no direct contact between the retinal pigment epithelium and the tips of rod outer segments. The second part deals with the neural portion of the deep-sea fish retina. Cell densities are greatly reduced, yet neurohistochemistry demonstrates that all major neurotransmitters and neuropeptides found in other vertebrate retinae are also present in deep-sea fish. Quantitatively, convergence rates in unspecialised parts of the retina are similar to those in nocturnal mammals. The differentiation of horizontal cells makes it unlikely that species with more than a single visual pigment are capable of colour vision. In the third part. the diversity of deep-sea fish retinae is highlighted. Based on the topography of ganglion cells, species are identified with areae or foveae located in various parts of the retina, giving them a greatly improved spatial resolving power in specific parts of their visual fields. The highest degree of specialisation is found in tubular eyes. This is demonstrated in a case study of the scopelarchid retina, where as many as seven regions with different degrees of differentiation can be distinguished, ranging from an area giganto cellularis, regions with grouped rods to retinal diverticulum. (C) 1998 Elsevier Science Ltd. All rights reserved.

The foveal photoreceptor mosaic in the pipefish, Corythoichthyes paxtoni (Syngnathidae, Teleostei)

The foveal and non-foveal retinal regions of the pipefish, Corythoichthyes paxtoni (Syngnathidae, Teleostei) are examined at the level of the light and electron microscopes. The pipefish possesses a deep, pit (convexiclivate) fovea which, although lacking the displacement of the inner retinal layers as described in other vertebrate foveae, is characterised by the exclusion of rods, a marked increase in the density of photoreceptors and a regular square mosaic of four double cones surrounding a central single cone. In the perifoveal and peripheral retinal regions, the photoreceptor mosaic is disrupted by the insertion of large numbers of rods, which reduce spatial resolving power but may uniformly increase sensitivity for off-axis rays. In addition to a temporal fovea subtending the frontal binocular field, there is also a central area centralis subtending the monocular visual field. Based on morphological comparisons with other foveate teleosts, four foveal types are characterised and foveal function discussed with respect to the theoretical advantage of a regular square mosaic.

Amino acids and their transporters in the retina

This review provides an overview of the distributions, properties and roles of amino acid transport systems in normal and pathological retinal tissues and discusses the roles of specific identified transporters in the mammalian retina. The retina is used in this context as a vehicle for describing neuronal and glial properties. which are in semi, but not all cases comparable to those found elsewhere an the brain. Where significant departures are noted, these are discussed in the context of functional specialisations of the retina and its relationship to adjacent supporting tissues such as the retinal pigment epithelium. Specific examples are given where immunocytochemical labelling for amino acid transporters may yield inaccurate results, possibly because of activity-dependent conformation changes of epitopes in these proteins which render the epitopes more or less accessible to antibodies. (C) 2001 Elsevier Science Ltd. All rights reserved.

Combined effect of cyclosporine and sirolimus on improving the longevity of recombinant adenovirus-mediated transgene expression in the retina

Objectives: To reevaluate the longevity and intraocular safety of recombinant adenovirus (rAd)-mediated gene delivery after subretinal injection, and to prolong transgene expression through the combination of 2 synergistic immunosuppressants. Methods: An rAd vector carrying green fluorescent protein (GFP) gene was delivered subretinally in the rat eye. The GFP expression was monitored in real time by fundus fluorescent photography. Intraocular safety was examined by observation of changes of retinal pigmentation, cell infiltration in virus-contacted area, immunophenotyping for CD4(+) and CD8(+) cytotoxic T lymphocytes, and CD68(+) macrophages, histologic findings, and dark-adapted electroretinography. Two synergistic immunosuppressants, cyclosporine and sirolimus, were used alone or in combination to prolong transgene expression by temporary immunosuppression. Results: The GFP expression peaked on day 4, dramatically decreased on day 10, and was not detectable on day 14. The decreased GFP expression was coincident with cell infiltration in virus-contacted area. Immunostaining showed that the infiltrating cells were CD4(+) and CD8(+) cytotoxic T lymphocytes and CD68(+) macrophages. Clumped retinal pigmentation and decreased b wave of dark-adapted electroretinogram were observed at 3 to 4 weeks after injection. Histologic examination confirmed rAd-induced retinal degeneration. Transient immunosuppression by cyclosporine and sirolimus, either alone or in combination, improved transgene expression, with the combination being the most efficient. The combined immunosuppression attenuated but did not retard the rAd-induced retinal damage. Conclusions: Transgene expression mediated by rAd after subretinal delivery is short-term and toxic to the retina. Combination of cyclosporine and sirolimus may act as an immunosuppressive adjunct to prolong rAd-mediated gene transfer. Clinical Relevance: The intraocular safety of rAd should be carefully considered before clinical trials are performed.

The expression of fibroblast growth factors and their receptors in the embryonic and neonatal mouse inner ear

Four different fibroblast growth factor receptors (FGFR) are known, three of which have splice variants (known as the b and c variants) in the FGF-binding domain, to give different patterns of sensitivity to the different FGFs. The expression of the b and c variants of the FGF receptors. together with the expression of the ligands FGF1. FGF2, FGF3, FGF7, FGF8b and FGF8c, was determined by quantitative reverse transcription-polymerase chain reaction in developing whole mouse inner ears, and in dissected components of the postnatal mouse inner ear. At embryonic age (E)10.5 days, when the otocyst is a simple closed sac, the receptor most heavily expressed was FGFR2b, relative to the postnatal day 0 level. Over the period E10.5-E12.5. during which the structures of the inner ear start to form, the expression of the different FGF receptors increased 10(2)-10(4) fold per unit of tissue, and there was a gradual switch towards expression of the 'c' splice variants of FGFR2 and FGFR3 rather than the 'b' variants. At E10.5, the ligands most heavily expressed, relative to the postnatal day 0 level, were FGF3, FGF8b and FGF8c. In the postnatal inner eat. the patterns of expression of receptors and ligands tended to be correlated, such that receptor variants were expressed in the same regions as the ligands that are known to activate them effectively. The neural/sensory region expressed high levels of FGFR3c, and high levels of the ligand FGF8b. The same area also expressed high levels of FGFR1b and FGFR2b, and high levels of FGF3. The lateral wall of the cochlea (including the stria vascularis and the spiral ligament) expressed high levels of FGFR1c and FGF1. 11 is suggested that the different FGF receptors and ligands are expressed in a spatially coordinated pattern to selectively program cochlear development. (C) 2001 Elsevier Science B.V. All rights reserved.