Spatial and temporal patterns of proliferation and differentiation in the developing turtle eye

Here we show for the first time different aspects of the pattern of neurogenesis in the developing turtle retina by using different morphological and molecular clues. We show the chronotopographical fashion of occurrence of three major aspects of retinal development: (1) morphogenesis of the optic primordia and emergence of the different retinal layers, (2) the temporal progression of neurogenesis by the cessation of proliferative activity, and (3) the apparition and cellular localization of different antigens and neuroactive substances. Retinal cells were generated in a conserved temporal order with ganglion cells born first, followed by amacrine, photoreceptor, horizontal and bipolar/Müller cells. While eventually expressed in many types of retinal neurons, Islet1 was permanently expressed in differentiating and mature ganglion cells. Calbindin-immunoreactive elements were found in the ganglion cell layer and the inner nuclear layer. Interestingly, at later stages the amount of expressing cells in these layers was reduced dramatically. On the contrary, the number of calbindin-immunoreactive photoreceptors increased as development proceeded. In addition, calretinin expressing cells were prominent in the horizontal cell bodies, and their processes extending into the outer plexiform layer were also strongly labeled. Finally, the synthesis of gamma-aminobutyric acid (GABA) was detected in developing and matured horizontal and amacrine cells. All these maturational features began in the dorso-central area, in a region slightly displaced towards the temporal retina.     

Distribution of muscarinic acetylcholine receptors on processes of isolated retinal cells

Binding of propylbenzilylcholine mustard, a muscarinic acetylcholine receptor antagonist, to isolated retinal cells was examined with light microscopic autoradiography. Dissociation of the adult tiger salamander retina yielded identifiable rod, cone, horizontal, bipolar, amacrine/ganglion, and Müller cells. Preservation of fine structure was assessed with conventional electron microscopy. For all cell types, the plasmalemma was intact and free of adhering debris; in addition, presynaptic ribbon complexes were present in photoreceptor and bipolar axon terminals indicating that synaptic structures were retained. Specific binding to cell bodies and processes was analyzed separately by using morphometric and statistical techniques. The highest grain densities occurred on processes of amacrine/ganglion cells and axons and 2 degrees and 3 degrees dendrites of bipolar neurons. Bipolar cells, however, seemed to be a heterogeneous population because there was great variation in the density of binding sites on both their axons and distal dendrites. Intermediate levels of binding were found on bipolar 1 degree dendrites and horizontal cells. No specific binding was detected on Müller cells and most parts of photoreceptors. Comparisons between cells showed that grain densities were similar for bipolar axons and amacrine/ganglion cell processes but bipolar dendrites were richer in binding sites than horizontal cell dendrites. Thus, muscarinic receptors in the salamander retina are located on amacrine/ganglion, bipolar, and horizontal cells and primarily confined to the processes which compose the two synaptic layers. In the inner plexiform layer, muscarinic receptors reside on processes from all three inner retinal neurons: in the outer synaptic layer, receptors are only on second-order cells and are more numerous along bipolar than horizontal cell dendrites.     

Pax6-positive Müller glia cells express cell cycle markers but do not proliferate after photoreceptor injury in the mouse retina

In lower vertebrates, such as fish, Müller glia plays an essential role in the restoration of visual function after retinal degeneration by transdifferentiating into photoreceptors and other retinal neurons. During this process, Müller cells re-enter the cell cycle, proliferate, and migrate from the inner nuclear layer (INL) to the photoreceptor layer where they express photoreceptor-specific markers. This process of Müller cell transdifferentiation is absent in mammals, and the loss of photoreceptors leads to permanent vision deficits.The mechanisms underlying the failure of mammalian Müller cells to behave as stem cells after photoreceptor degeneration are poorly understood. In the present study, we show that photoreceptor injury induces migration of PAX6-positive Müller cell nuclei toward the outer part of the INL and into the inner part of the outer nuclear layer. These cells express markers of the cell cycle, suggesting an attempt to re-enter the cell cycle similarly to lower vertebrates.However, mouse Müller cells do not proliferate in response to photoreceptor injury implying a blockade of the S-phase transition. Our results suggest that a release of the S-phase blockade may be crucial for Müller cells to successfully transdifferentiate and replace injured photoreceptors in mammals.     

Cannabinoid receptor type 1 expression during postnatal development of the rat retina

Cannabinoid receptor type 1 (CB1R) participates in developmental processes in the central nervous system (CNS). The rodent retina represents an interesting and valuable model for studying CNS development, because it contains well-identified cell types with clearly established and distinct developmental timelines. Very little is known about the distribution or function of CB1R in the developing retina. In this study, we investigated the expression pattern of CB1R in the rat retina during all stages of postnatal development. Western blots were performed on retinal tissue at different time points between P1 and adulthood. In order to identify the cells expressing the receptor and the age at which this expression started, immunohistochemical co-staining was carried out for CB1R and markers of the different cell types comprising the retina. CB1R was already present at P1 in various cell types, i.e., ganglion, amacrine, horizontal, and mitotic cells. In the course of development, it appeared in cone photoreceptors and bipolar cells. For some cell types (bipolar, Müller, and some amacrine cells), CB1R was transiently expressed, suggesting a potential role of this receptor in developmental processes, such as migration, morphological changes, sub-identity acquisition, and patterned retinal spontaneous activity. Our results also indicated that CB1R is largely expressed in the adult retina (cone photoreceptors and horizontal, most amacrine, and retinal ganglion cells), and may therefore contribute to retinal functions. Overall these results indicate that, as shown in other structures of the brain, CB1R could play an instrumental role in the development and function of the retina.     

Biphasic retinal neurogenesis in the brush-tailed possum, Trichosurus vulpecula: further evidence for the mechanisms involved in formation of ganglion cell density gradients.

We investigated cell generation in the retina of the brush-tailed possum (Trichosurus vulpecula) by using tritiated (3H)-thymidine labelling of newly generated cells. Animals aged between postnatal day (P) 5 and 85 each received a single injection of 3H-thymidine. Following autoradiographic processing, maps of labelled cells were constructed from retinal sections. Retinal cell generation takes place in two phases, the first is concluding in the retinal periphery at P53 as the second is seen to commence in midtemporal retina. In the first phase, cells in central retina are generated earlier than those in peripheral regions. In the second phase, cells complete their final division in midtemporal retina first and in the periphery last. Cells generated in the first phase comprise virtually all cells in the ganglion cell layer, amacrine cells, horizontal cells, and cones. Ganglion cells are produced at a slightly earlier stage than displaced amacrine cells, horizontal cells, or cones. Amacrine cells in the inner nuclear layer are the final cells produced in the first phase. When ganglion cells and amacrine cells are pooled, their combined rate of production matches that of the other cell types. These data indicate that the ratio of displaced amacrine cells: horizontal cells: cones: combined ganglion cells and amacrine cells does not change throughout development. However, the ratio of ganglion cells:macrines changes steadily as development proceeds to favour amacrine cells. In the second phase, sparse numbers of nonganglion cells in the ganglion cell layer and large numbers of bipolar and Müller cells are produced along with all rods. The two phases in the possum are similar to those seen in the wallaby, the quokka. However, fewer cells are added in central retina in the possum than in the quokka and cell addition continues for a more extended period in the periphery in the possum. We suggest that this difference in cell addition could account for the development of a more pronounced visual streak of retinal ganglion cells in the possum than in the quokka. A comparison of possum retinal cell generation with that of other marsupials adds support for the "homochrony theory."     

Identification of molecular markers of bipolar cells in the murine retina

Retinal bipolar neurons serve as relay interneurons that connect rod and cone photoreceptor cells to amacrine and ganglion cells. They exhibit diverse morphologies essential for correct routing of photoreceptor cell signals to specific postsynaptic amacrine and ganglion cells. The development and physiology of these interneurons have not been completely defined molecularly. Despite previous identification of genes expressed in several bipolar cell subtypes, molecules that mark each bipolar cell type still await discovery. In this report, novel genetic markers of murine bipolar cells were found. Candidates were initially generated by using microarray analysis of single bipolar cells and mining of retinal serial analysis of gene expression (SAGE) data. These candidates were subsequently tested for expression in bipolar cells by RNA in situ hybridization. Ten new molecular markers were identified, five of which are highly enriched in their expression in bipolar cells within the adult retina. Double-labeling experiments using probes for previously characterized subsets of bipolar cells were performed to identify the subtypes of bipolar cells that express the novel markers. Additionally, the expression of bipolar cell genes was analyzed in Bhlhb4 knockout retinas, in which rod bipolar cells degenerate postnatally, to delineate further the identity of bipolar cells in which novel markers are found. From the analysis of Bhlhb4 mutant retinas, cone bipolar cell gene expression appears to be relatively unaffected by the degeneration of rod bipolar cells. Identification of molecular markers for the various subtypes of bipolar cells will lead to greater insights into the development and function of these diverse interneurons.     

Structure and functional connections of presynaptic terminals in the vertebrate retina revealed by activity-dependent dyes and confocal microscopy

The fluorescent dyes sulforhodamine 101 (SR 101) and FM1-43 were used as activity-dependent dyes (ADDs) to label presynaptic terminals in the retinas of a broad range of animals, including amphibians, mammals, fish, and turtles. The pattern of dye uptake was studied in live retinal preparations by using brightfield, fluorescence, and confocal microscopy. When bath-applied to the retina-eyecup, these dyes were avidly sequestered by the presynaptic terminals of virtually all rods, cones, and bipolar and amacrine cells; ganglion cell dendrites and horizontal cells lacked significant dye accumulation. Other structures stained with these dyes included pigment epithelial cells, cone outer segments, and Müller cell end-feet. Studies of dye uptake in dark- and light-adapted preparations showed significant differences in the dye accumulation pattern in the inner plexiform layer (IPL), suggesting a dynamic, light-modulated control of endocytotic activity. Presynaptic terminals in the IPL could be segregated on the basis of volume: bipolar varicosities in the IPL were typically larger than those of amacrine cells. The combination of retrograde labeling of ganglion cells and presynaptic terminal labeling with ADDs served as the experimental preparation for three-dimensional reconstruction of both structures, based on dual detector, confocal microscopy. Our results demonstrate a new approach for studying synaptic interactions in retinal function. These findings provide new insights into the likely number and position of functional connections from amacrine and bipolar cell terminals onto ganglion cell dendrites.     

Sequential course of uptake of intravitreal 5,7-dihydroxytryptamine by carp retinal cells

The sequential course of uptake by retinal cells of intravitreally injected 5,7-dihydroxytryptamine (5,7-DHT) together with dopamine (DA) was investigated in juvenile carp retinas, which were removed at various intervals (1-24 h) after injection. The cells taken up 5,7-DHT were visualized immunohistochemically with anti-serotonin (5-HT) antibody and FITC-conjugated IgG. After a mixture of 5,7-DHT and DA (2.5, 10 or 20 micrograms each) was given, large-sized indoleamine (IA) amacrine cells first (1-4 h), and then small-sized indoleamine-accumulating amacrine amacrine (IAA) cells (4-12 h), bipolar cells (8-12 h) and in some cases photoreceptor cells (12-24 h) were sequentially observed, and finally the immunoreactive structures almost disappeared around 24 h after injection. When the mixture of 5,7-DHT and DA (10 micrograms each) was injected into the eyes of reserpinized fish, the same sequential uptake of 5,7-DHT was seen in a faster time course, but additionally various classes of retinal cells (horizontal, ganglion and Müller cells) became visible as irregular clusters. However, DA cells were never visualized at any stages of all the experiments, indicating that DA cells do not take up 5,7-DHT in the carp retina, which was further confirmed by double labeling of 5-HT- and tyrosine hydroxylase-like immunoreactive cells. Double labeling also revealed that 5,7-DHT-accumulating bipolar cells appear to represent a subclass different from that of protein kinase C-like immunoreactive bipolar cells.     

The inner plexiform layer of the vertebrate retina: a quantitative and comparative electron microscopic analysis

The inner plexiform layer of human, monkey, cat, rat, rabbit, ground squirrel, frog and pigeon retinas was studied by electron microscopy. All showed the same qualitative synaptic arrangements: bipolar cells made dyad ribbon synapses onto amacrine and ganglion cells; amacrine cells made conventional synaptic contacts onto bipolar, ganglion cells; amacrine cells montage of electron micrographs through the full thickness of the inner plexiform layer were made for each species and were scored for synaptic contacts. Both absolute and relative quantitative differences were found between species. The ratio of amacrine cell (conventional) synapses to bipolar cell (ribbon) synapses, the absolute number of amacrine cell synapses and the number of inter-amacrine cell synapses were all found to be higher in those animals which are known to have relatively complex retinal ganglion cell receptive field properties. It is suggested that the amacrine cell is involved in mediating complex visual transformations in certain vertebrate retinas.     

Analysis of a glycinergic inhibitory pathway in the cat retina

Incubation of cat retinas with 3H-glycine in vitro, followed by horizontal sectioning and autoradiography, showed labeling of 10-12% of bipolar cells and 45% of amacrine cells. To ascertain the effects of glycine-accumulating bipolar and amacrine cells on the response properties of retinal ganglion cells, in vivo iontophoretic studies were performed in the cat eye. Glycine inhibited all ganglion cells, and this action was blocked by strychnine. Aminophosphonobutyric acid (APB) suppressed ON-ganglion cells, but activated OFF-ganglion cells. The influence of APB upon OFF-ganglion cells could be completely blocked by strychnine. In the mudpuppy, APB suppressed ON-bipolar cells without affecting OFF-bipolar cells and without direct effects on other retinal neurons (Slaughter and Miller, 1981, 1985). If APB has the same mode of action in the cat retina, the present results suggest a glycinergic inhibitory input of the ON-channel upon OFF-ganglion cells.     

Oxidative stress promotes proliferation and dedifferentiation of retina glial cells in vitro

Oxidative damage is involved in triggering neuronal death in several retinal neurodegenerative diseases. The recent finding of stem cells in the retina suggests that both preventing neuronal death and replacing lost neurons might be useful strategies for treating these diseases. We have previously shown that oxidative stress induces apoptosis in cultured retinal neurons. We now investigated the response of Müller cells, proposed as retina stem cells, to this damage. Treatment of glial cell cultures prepared from rat retinas with the oxidant paraquat (PQ) did not induce glial cell apoptosis. Instead, PQ promoted their rapid dedifferentiation and proliferation. PQ decreased expression of a marker of differentiated glial cells, simultaneously increasing the expression of smooth muscle actin, shown to increase with glial dedifferentiation, the levels of cell-cycle markers, and the number of glial cells in the cultures. In addition, glial cells protected neurons in coculture from apoptosis induced by PQ and H(2)O(2). In pure neuronal cultures, PQ induced apoptosis of photoreceptors and amacrine neurons, simultaneously decreasing the percentage of neurons preserving mitochondrial membrane potential; coculturing neurons with glial cells completely prevented PQ-induced apoptosis and preserved mitochondrial potential in both neuronal types. These results demonstrate that oxidative damage activated different responses in Müller glial cells; they rapidly dedifferentiated and enhanced their proliferation, concurrently preventing neuronal apoptosis. Glial cells might not only preserve neuronal survival but also activate their cell cycle in order to provide a pool of new progenitor cells that might eventually be manipulated to preserve retinal functionality.     

Retinal amacrine cell involvement in Tay-Sachs disease

Summary Ultrastructural study of the retina from a patient with Tay-Sachs disease disclosed that amacrine cells as well as ganglion cells were loaded with numerous membranous cytoplasmic bodies, suggesting an accumulation of GM₂ ganglioside, whereas the horizontal cells, bipolar cells, and photoreceptor cells were intact. Chromatography of lipids from the retina showed a prominent spot of GM₂ ganglioside. These facts suggest that lipid metabolism in amacrine cells may be different from that in other retinal cells.     

Retinal neurons of the California ground squirrel,Spermophilus beecheyi: A Golgi study

Although the optic nerve fibers of the cone-dominant ground squirrel retina have been well studied physiologically, the morphological details of the retinal neurons have not. To that end, retinal neurons of the California ground squirrel have been studied in Golgi-impregnated wholemounts. Two types of horizontal cell have been identified: H1 has an axon and axon terminal, whereas H2 is axonless. The dendritic field of H1 cells enlarges in a nonuniform manner with increasing displacement from the central retina. The smallest examples lie centrally in the visual streak, and the largest occur in the superior periphery. Eight types of bipolar cell are distinguished by morphological differences in dendritic branching pattern and field size in the outer plexiform layer, cell body size, and layering within the inner nuclear layer and by the morphology and stratification of axon terminals in the inner plexiform layer. A large bistratified bipolar cell (B8) is introduced here; the other 7 types closely resemble those in the retinas of other sciurid species described by R.W. West (1976, J. Comp. Neurol. 168:355–378; 1978, Vision Res. 18:129–136). The B1 type is proposed as a blue cone bipolar cell. Amacrine cells are classified into 27 cell types. Six of these occur as mirror-image pairs across the inner plexiform layer, the soma of one of each pair being “displaced” to the ganglion cell layer. The best described of these pairs is the very elaborate starburst amacrine cell, A5, which stains regularly in these wholemounted retinas. Changes in dendritic field size of both A5 subtypes with retinal location are quantified. The morphology of three amacrine cell types identified in Spermophilus beecheyi suggests that their possible counterparts in S. mexicanus (West, 1976) were, as displaced amacrine cells, misidentified as ganglion cells. Amacrine cell types that may play roles in the rod pathway, the blue cone pathway, and ganglion cell directional selectivity are discussed. No type of interplexiform cell was observed. Ganglion cells are classified into 19 cell types, 9 of which probably correspond to the ganglion cells described by West (1976) in the Mexican ground squirrel. The bistratified G11 cell is proposed as an ON-OFF directionally selective type. © 1996 Wiley-Liss, Inc.     

Differential expression of inwardly rectifying K+ channels and aquaporins 4 and 5 in autoimmune uveitis indicates misbalance in Müller glial cell-dependent ion and water homeostasis

Reactive gliosis is a well-established response to virtually every retinal disease. Autoimmune uveitis, a sight threatening disease, is characterized by recurrent relapses through autoaggressive T-cells. The purpose of this study was to assess retinal Müller glial cell function in equine recurrent uveitis (ERU), a spontaneous disease model resembling the human disease, by investigating membrane proteins implicated in ion and water homeostasis. We found that Kir2.1 was highly expressed in diseased retinas, whereas Kir4.1 was downregulated in comparison to controls. Distribution of Kir2.1 appeared Müller cell associated in controls, whereas staining of cell somata in the inner nuclear layer was observed in uveitis. In contrast to other subunits, Kir4.1 was evenly expressed along equine Müller cells, whereas in ERU, Kir4.1 almost disappeared from Müller cells. Hence, we suggest a different mechanism for potassium buffering in the avascular equine retina and, moreover, an impairment in uveitis. Uveitic retinas showed significantly increased expression of AQP4 as well as a displaced expression from Müller cells in healthy specimens to an intense circular expression pattern in the outer nuclear layer in ERU cases. Most interestingly, we detected the aquaporin family member protein AQP5 to be expressed in Müller cells with strong enrichments in Müller cell secondary processes. This finding indicates that fluid regulation within the equine retina may be achieved by an additional aquaporin. Furthermore, AQP5 was significantly decreased in uveitis. We conclude that the Müller cell response in autoimmune uveitis implies considerable changes in its potassium and water physiology.     

The new targets of ouabain in retinal interneurons of Sprague-Dawley rats

Ouabain is both a cardiac glycoside used in therapy of congestive heart failure and an endogenous steroid hormone. It specifically binds to Na⁺, K⁺-ATPase (NKA) and blocks its activity. Overdose of ouabain induces retinal damage. In different species ouabain-induced retinal degeneration affects different cell types. In fish and rabbit ouabain induces retinal cell death preferentially in the ganglion cell layer and outer photoreceptor segments respectively. In rats, the pattern of NKA expression has been studied with most detail among retinal neurons. In addition, ouabain selectively destroyed some types of neurons in rodents. However, ouabain-sensitive retinal neurons remain unclear in rats. We show here that injection of ouabain into the rat vitreous body induced dramatic cell death in the inner nuclear layer (INL). The cell death was time- and dose-dependent. Ouabain-induced dying cells in the INL were TUNEL-positive. Immunohistochemistry analysis revealed that there was a significant decrease in the number of calbindin D-28K- and syntaxin-1-positive horizontal and amacrine cells in the INL of ouabain-treated rat retinas. Thus our results revealed that the horizontal and amacrine cells are the most sensitive cell types to ouabain in the retina of Sprague-Dawley rat.     

Synaptic connections of starburst amacrine cells and localization of acetylcholine receptors in primate retinas

Starburst amacrine cells in the macaque retina were studied by electron microscopic immunohistochemistry. We found that these amacrine cells make a type of synapse not described previously; they are presynaptic to axon terminals of bipolar cells. We also confirmed that starburst amacrine cells are presynaptic to ganglion cell dendrites and amacrine cell processes. In order to determine the functions of these synapses, we localized acetylcholine receptors using a monoclonal antibody (mAb210) that recognizes human alpha3- and alpha5-containing nicotinic receptors and also antisera against the five known subtypes of muscarinic receptors. The majority of the mAb210-immunoreactive perikarya were amacrine cells and ganglion cells, but a subpopulation of bipolar cells was also labeled. A subset of bipolar cells and a subset of horizontal cells were labeled with antibodies to M3 muscarinic receptors. A subset of amacrine cells, including those that contain cholecystokinin, were labeled with antibodies to M2 receptors. Taken together, these results suggest that acetylcholine can modulate the activity of retinal ganglion cells by multiple pathways.     

Scanning electron microscopy and light microscopy studies on the retina of chick embryos in cell culture

Histological and scanning electron microscopy studies (SEM) suggest a classification of neural elements into five main types: receptor cells, bipolar cells, horizontal, amacrine, and ganglion cells in dissociated cell cultures of the retina from 7 to 10 days old chick embryos. By use of SEM the development of the receptor cell inner segments was observed. At the same time many protrusions were noticed at the receptor cell surface. Specific synaptic contacts between the axons of the receptor cells and the dendrites of the bipolar neurons, as well as unspecific contacts between the pericarya of the receptor cells and other dendrites were demonstrated. The bipolar neurons showed smooth cell surfaces, however, the horizontal cells appeared rough on the cell surfaces. Müller cells were always found adjacent to the photoreceptor cells.     

Plasmalemmal and vesicular gamma-aminobutyric acid transporter expression in the developing mouse retina

Plasmalemmal and vesicular gamma-aminobutyric acid (GABA) transporters influence neurotransmission by regulating high-affinity GABA uptake and GABA release into the synaptic cleft and extracellular space. Postnatal expression of the plasmalemmal GABA transporter-1 (GAT-1), GAT-3, and the vesicular GABA/glycine transporter (VGAT) were evaluated in the developing mouse retina by using immunohistochemistry with affinity-purified antibodies. Weak transporter immunoreactivity was observed in the inner retina at postnatal day 0 (P0). GAT-1 immunostaining at P0 and at older ages was in amacrine and displaced amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), respectively, and in their processes in the inner plexiform layer (IPL). At P10, weak GAT-1 immunostaining was in Müller cell processes. GAT-3 immunostaining at P0 and older ages was in amacrine cells and their processes, as well as in Müller cells and their processes that extended radially across the retina. At P10, Müller cell somata were observed in the middle of the INL. VGAT immunostaining was present at P0 and older ages in amacrine cells in the INL as well as processes in the IPL. At P5, weak VGAT immunostaining was also observed in horizontal cell somata and processes. By P15, the GAT and VGAT immunostaining patterns appear similar to the adult immunostaining patterns; they reached adult levels by about P20. These findings demonstrate that GABA uptake and release are initially established in the inner retina during the first postnatal week and that these systems subsequently mature in the outer retina during the second postnatal week.     

Appearance of glutamate-like immunoreactivity during retinal regeneration in the adult newt

The appearance of endogenous glutamate during retinal regeneration in the newt was examined by immunohistochemistry. Glutamate-like immunoreactivity (Glu-LI) first appeared in prospective ganglion cells along the vitreal margin of retinas that were about six cells thick, in prospective photoreceptors immediately before segregation of retinal plexiform layers and then in prospective bipolar cells immediately after the initial appearance of thin plexiform layers. In retinas nearing complete regeneration, Müller cells showed immunoreactivity. The appearance of glutamatergic phenotypes during retinal regeneration seemed to follow the order of cell differentiation [T. Saito, Y. Kaneko, F. Maruo, M. Niino, Y. Sakaki, Study of the regenerating newt retina by electrophysiology and immunohistochemistry (bipolar- and cone-specific antigen localization), J. Exp. Zool. 270 (1994) 491–500]. However, changes in the amount of endogenous glutamate during retinal regeneration were more complex. On the one hand, Glu-LI at the prospective ganglion cell layer temporarily increased during the initial period of segregation of the inner plexiform layer. On the other hand, immunoreactivity in the photoreceptor layer declined during segregation of the outer plexiform layer. The transient expression of immunoreactivity may represent a function of glutamate in events such as cell survival or neurite extension during retinal regeneration.     

Amino acid signatures in the developing mouse retina

This study characterizes the developmental patterns of seven key amino acids: glutamate, γ-amino-butyric acid (GABA), glycine, glutamine, aspartate, alanine and taurine in the mouse retina. We analyze amino acids in specific bipolar, amacrine and ganglion cell sub-populations (i.e. GABAergic vs. glycinergic amacrine cells) and anatomically distinct regions of photoreceptors and Müller cells (i.e. cell bodies vs. endfeet) by extracting data from previously described pattern recognition analysis. Pattern recognition statistically classifies all cells in the retina based on their neurochemical profile and surpasses the previous limitations of anatomical and morphological identification of cells in the immature retina. We found that the GABA and glycine cellular content reached adult-like levels in most neurons before glutamate. The metabolic amino acids glutamine, aspartate and alanine also reached maturity in most retinal cells before eye opening. When the overall amino acid profiles were considered for each cell group, ganglion cells and GABAergic amacrine cells matured first, followed by glycinergic amacrine cells and finally bipolar cells. Photoreceptor cell bodies reached adult-like amino acid profiles at P7 whilst Müller cells acquired typical amino acid profiles in their cell bodies at P7 and in their endfeet by P14. We further compared the amino acid profiles of the C57Bl/6J mouse with the transgenic X-inactivation mouse carrying the lacZ gene on the X chromosome and validated this animal model for the study of normal retinal development. This study provides valuable insight into normal retinal neurochemical maturation and metabolism and benchmark amino acid values for comparison with retinal disease, particularly those which occur during development.