Cells In Inner Nuclear Layer Research Articles

Rhythms of glycerophospholipid synthesis in retinal inner nuclear layer cells

The present study demonstrates that the biosynthesis of phospholipids in the inner nuclear layer cells of the chicken retina displays daily rhythms under constant illumination conditions. The vertebrate retina contains circadian oscillators and photoreceptors (PRCs) that temporally regulate its own physiology and synchronize the whole organism to the daily environmental changes. We have previously reported that chicken photoreceptors and retinal ganglion cells (RGCs) present significant daily variations in their phospholipid biosynthesis under constant illumination conditions. Herein, we demonstrate that cell preparations highly enriched in inner nuclear layer cells also exhibit a circadian-regulated phospholipid labeling after the in vivo administration of [ 32P]phosphate or [ 3H]glycerol both in animals maintained under constant darkness or light for at least 48 h. In constant darkness, there was a significant incorporation of both precursors into phospholipids with the highest levels of labeling around midday and dusk. In constant light, the labeling of 32P-phospholipids was also significantly higher during the day and early night whereas the incorporation of [ 3H]glycerol into phospholipids, that indicates de novo biosynthesis, was greater during the day but probably reflecting a higher precursor availability at those phases. We also measured the in vitro activity of phosphatidate phosphohydrolase and diacylglycerol lipase in preparations obtained from the dark condition. The two enzymes exhibited the highest activity levels late in the day. When we assessed the in vitro incorporation of [ 14C]oleate into different lysophospholipids from samples collected at different phases in constant darkness, reaction catalyzed by lysophospholipid acyltransferases II, labeling showed a complex pattern of daily activity. Taken together, these results demonstrate that the biosynthesis of phospholipids in cells of the chicken retinal inner nuclear layer exhibits a daily rhythmicity under constant illumination conditions, which is controlled by a circadian clock.

Endogenous tissue type plasminogen activator facilitates NMDA-induced retinal damage

To investigate the role of tissue plasminogen activator (tPA) in retinal damage, tPA-deficient and wild-type mice were employed. Two different retinal neuron insult models were used in the present study. One is an excitotoxin-treated retinal model, created by direct intravitreal injection of glutamate analogs, NMDA or kainic acid (KA), and the other is an ischemia–reperfusion model induced by transient elevation of intraocular pressure. TdT-dUTP terminal nick-end labeling (TUNEL) method was used to examine the retinal cell nuclear damage. The number of TUNEL-positive cells in ganglion cell layer (GCL) and inner nuclear layer (INL) in tPA-deficient mice after low-, but not high-dose NMDA was significantly less compared to wild type. In contrast, neither intravitreal KA or transient ischemia produced significant difference in retinal damage in tPA vs. wild-type mice. These data show that tPA-deficient mice are resistant to retinal damage by intravitreal injection of NMDA, and indicate that tPA plays a role in the retinal cell damage induced by excitotoxins, especially NMDA.

Endogenous tissue type plasminogen activator facilitates NMDA-induced retinal damage

To investigate the role of tissue plasminogen activator (tPA) in retinal damage, tPA-deficient and wild-type mice were employed. Two different retinal neuron insult models were used in the present study. One is an excitotoxin-treated retinal model, created by direct intravitreal injection of glutamate analogs, NMDA or kainic acid (KA), and the other is an ischemia–reperfusion model induced by transient elevation of intraocular pressure. TdT-dUTP terminal nick-end labeling (TUNEL) method was used to examine the retinal cell nuclear damage. The number of TUNEL-positive cells in ganglion cell layer (GCL) and inner nuclear layer (INL) in tPA-deficient mice after low-, but not high-dose NMDA was significantly less compared to wild type. In contrast, neither intravitreal KA or transient ischemia produced significant difference in retinal damage in tPA vs. wild-type mice. These data show that tPA-deficient mice are resistant to retinal damage by intravitreal injection of NMDA, and indicate that tPA plays a role in the retinal cell damage induced by excitotoxins, especially NMDA.

Correlation of retinal function with retinal histopathology following ischemia-reperfusion in rat eyes

Purpose. To investigate the relationship between retinal function and histopathology following retinal ischemia-reperfusion in rats. Methods. Retinal ischemia was induced in 27 Sprague-Dawley rats by raising the intraocular pressure for 60- or 90-minutes. Two weeks after the retinal ischemia-reperfusion, ganzfeld stimuli of different luminances (maximum 0.51 log cd-s/m2) were used to elicit full-field ERGs from the right (experimental) and left (control) eyes. The amplitudes of the scotopic b-waves were measured, and intensity-response curves were plotted. The b-wave amplitudes at the initial peak (VbIP), the dip (Vbdip), and the maximum stimulus intensity (Vbmax) were measured. The animals were sacrificed after the ERG recordings to determine the surviving retinal cells in the inner nuclear layer (INL). Results. The log values of the VbIP, Vbdip and Vbmax were linearly correlated with the number of surviving cells in the INL (r = 0.928, 0.905, and 0.913 for VbIP, Vbdip and Vbmax, respectively; p < 0.0001 for each). The slope of the regression line for the Vbdip was significantly steeper than that for the VbIP and Vbmax (p < 0.005). The regression line for the Vbdip reached undetectable level of the b-wave (<1 µV) even though approximately 25% of the cells still remained in the INL. Conclusions. The amplitude of the scotopic b-wave is a sensitive index of the surviving INL cells. However, if one selects the stimulus intensity at the dip, the functional loss would be exaggerated in spite of the presence of cells in the INL.

Dopaminergic amacrine cells in the inner nuclear layer and ganglion cell layer comprise a single functional retinal mosaic.

Many types of retinal neuron are distributed in an orderly manner across the surface of the retina. Indeed, the existence of such regularity amongst a population of neurons, termed a retinal mosaic, may be a defining feature of functionally independent types of retinal neuron. We have examined the spatial distribution of dopaminergic amacrine cells in the ferret retina both in the inner nuclear layer (INL) and in the ganglion cell layer (GCL) to determine whether the cells in each layer form an independent retinal mosaic as evidence of whether they should be considered as two separate types. Ferret retinas contain approximately 1,900 dopaminergic amacrine cells, of which 27% are located in the GCL, and the rest in the INL. Based on analysis of their Voronoi domains as well as autocorrelation analysis and tests for complete spatial randomness, we found that the distribution of INL cells was statistically regular, while that of the GCL cells was not. However, by using cross-correlation analysis, these two groups of cells were found to be spatially dependent: an exclusion zone was detected in the cross-correlogram of roughly the same size as that found in the autocorrelograms of both INL and GCL cells. Such a pattern would be expected if dopaminergic amacrine cells in the INL and GCL were members of a single regular population differing only in their somatic depth. By using computer simulations, we tested this hypothesis directly, confirming that a random assignment of 27% from the total population produces cross-correlograms that are indistinguishable from those of the biological mosaics. We conclude, therefore, that the cells in the two layers form a single functional population; those in the GCL appear to be misplaced. Somatic positioning with respect to depth within the retina is not, by itself, a reliable guide for functional classification.

Enhanced expression of TNF-R1 protein in NMDA-mediated cell death in the retina

Multiple apoptosis-related genes are expressed in the retina after exposure to N-methyl- d-aspartic acid (NMDA). For example, the mRNAs for TNF-R1, FasL, and Nur77 are up-regulated between 2.8 and 7-fold [Mol. Brain Res. 91 (2001) 34–42]. The purpose of the present study is to examine prospective changes in protein expression for these genes and to determine their cellular localizations subsequent to NMDA stimulation. Following anesthesia, a single intravitreal injection of 4 mM NMDA was administered into the right eye of anesthetized rats. The left eye was injected with phosphate-buffered saline. Retinae were harvested at 2 and 24 h postinjection. Western-blot and immunocytochemical techniques were used to detect changes in protein expression levels, and to localize their distributions within the retina. Analyses of Western blots demonstrated a significant increase in TNF-R1 (100 and 80%) compared to the sham-controls at 2 and 24 h postinjection with NMDA. Immunolabeling of TNF-R1 was observed in the inner nuclear layer (INL) at 2 h postinjection with NMDA. TNF-R1 was also clearly evident in cells within the INL and ganglion cell layers (GCL) at 24 h post-injection with NMDA. In contrast to these changes in TNF-R1 there were no significant changes in the levels or distributions of FasL or Nur77 in NMDA-stimulated animals at either 2 or 24 h when compared to the sham-controls. These results implicate the TNF-R1 signal transduction pathway in NMDA-induced cell death in the INL and GCL of the retina.

The 23-kDa polypeptide from Common Frog lens: isolation, characteristics and cellular localization

The major water-soluble polypeptide with molecular weight of approximately 23 kDa (the 23-kDa polypeptide) was identified in the lens of common frog Rana temporaria L. According to the gel filtration data, the peptide is a part of an oligomeric protein with molecular weight of more than 300 kDa (alpha-crystallin fraction). A highly pure fraction of the 23-kDa polypeptide was isolated by two-step ion-exchange chromatography and SDS electrophoresis and the specific antibodies were obtained. Immunohistochemistry showed the presence of the 23-kDa polypeptide in the cytoplasm of lens epithelial cells (including its central region) and in the zones neighbouring the plasma membranes in cortical fibers. The 23-kDa polypeptide was not found in the lens central zone (nucleus). It was also present in the retina (in the cells of inner nuclear layers), but not in the other tissues and organs of adult frog. Immunochemical analysis showed that the 23-kDa polypeptide was different from all known crystallins of frogs and other animals (bull, mouse, rat, and chicken). The nature of the 23-kDa polypeptide and the relation of its expression with lens cell differentiation are discussed.

Two phases of increased cell death in the inner retina following early elimination of the ganglion cell population.

Neurons in the inner nuclear layer (INL) of the vertebrate retina undergo considerable programmed cell death during development, but the determinants of this cell death remain largely unknown. The present study examines the role of retinal ganglion cells in support of INL neurons in the developing ferret retina. The retinal ganglion cell population was eliminated by optic nerve transection at postnatal day (P) 2, and the incidence of cell death was examined using terminal deoxytransferase dUTP nick-end labelling (TUNEL) at various ages during the first 3 postnatal weeks. Significant increases in TUNEL-positive cells were observed in the neuroblast layer (NBL) as early as P3, prior to synapse formation within the inner plexiform layer (IPL), and again in the INL at P22, the normal peak of naturally occurring cell death within the ferret's INL. A decrease in TUNEL-positive cells was found in the NBL at P8. These results show three phases of response to the loss of retinal ganglion cells and suggest that cells in the NBL/INL are normally dependent on retinal ganglion cells for their survival. Recent studies have shown that certain populations of retinal neurons are reduced in adult animals that had lost the population of ganglion cells during early development, so the present study also examined when this reduction could first be detected. The number of parvalbumin-immunoreactive amacrine cells was decreased significantly in the NBL of the manipulated eye as early as P8, when we could first label this population, and this difference persisted through adulthood. The fact that cell death in the NBL has already increased within 24 hours of ganglion cell elimination, coupled with the specificity of this effect on the adult complement of INL cell types, shows that cell-cell interactions controlling survival are already highly specific for particular types of retinal neuron early in development

RINX(VSX1), a Novel Homeobox Gene Expressed in the Inner Nuclear Layer of the Adult Retina

The locus control region (LCR) of the human red and green visual pigment genes is critical for the formation of functional red and green cones in the retina. A 37-bp core of the LCR is perfectly conserved among mammals and binds specific retinal nuclear proteins. Here, we employed a yeast one-hybrid screen of an adult retinal cDNA library to clone and characterize these proteins. We identified clones encoding homeodomain (HD) transcription factors Pax6, Rx, and Chx10 and a novel paired-like HD protein, RINX. In the adult retina, RINX is exclusively expressed in a subset of cells (likely to be bipolar cells) of the retinal inner nuclear layer (INL). RINX is closely related to Chx10, which is also exclusively expressed in the INL of the adult retina and is critical for retinal development. The RINX gene is expressed in two classes of mRNA. One class encodes proteins that lack either part of or all of the HD, but retain the transcriptional activation domain. The RINX gene maps to chromosome 20p11.2 to which no retinal disease has been assigned. In conclusion, the LCR contains two adjacent motifs that are targets for binding of HD proteins that may specify the development and differentiation of cone photoreceptors and a subset of INL bipolar cells. Mutations in the related human CHX10 gene cause microphthalmia in a subset of families, and, therefore, the RINX gene is a candidate for this phenotype in another subset of patients. Since the RINX gene is likely an ortholog of the goldfish Vsx1 gene, it has been named VSX1 by the Human Gene Nomenclature Committee.

Calorie restriction modulates age-dependent changes in the retinas of Brown Norway rats

The present study examined the effect of a 40% reduction in caloric intake (CR) versus ad libitum (AL) feeding on retinal aging. CR- and AL-fed Brown Norway (BN) rats were obtained at 12, 24 and 30 months of age from the National Center for Toxicological Research (NCTR). Age-dependent declines in outer nuclear layer (ONL=photoreceptor) cell densities, ONL height, inner nuclear layer (INL) cell densities, and thicknesses of the inner retina and whole retina were quantified in thick sections at six loci across the circumference of the sensory retina (four peripheral, two central). Data were analyzed by repeated measures, general linear models. Aging in both diet groups was associated with declines in ONL cell density, ONL height, peripheral INL cell density and total retinal thickness ( P≤0.05). However, ONL cell densities, ONL height and retinal thickness were significantly greater in the CR versus AL diet group at all three ages ( P≤0.005). CR was also associated with a trend for greater peripheral INL cell density ( P=0.06) and with greater INL thickness at 30 months (Bonferroni P=0.03). Elevated ONL cell densities in the CR-12 cohort relative to the AL-12 cohort could be explained by diet-associated differences in retinal length, i.e. delayed retinal growth in response to CR. Enhanced ONL cell density, ONL height, INL cell density, INL thickness and total retinal thickness in the CR-30 cohort appear to be as a result of reduced rates of retinal cell loss between 24 and 30 months. However, the protective effect of CR in retinas of older animals may also reflect the initial growth-associated enhancements which were observed in 12 month-old animals. The rat retina may provide a useful model for elucidating the neuroprotective mechanism(s) of CR.

The birthdates of GABA-immunoreactive amacrine cells in the rat retina.

The birthdates of GABAergic amacrine cells in the rat retina were investigated by immunocytochemistry using anti-GABA and anti-bromodeoxyuridine (BrdU) antisera. The ratio of co-localization of GABA to BrdU increased gradually from embryonic-day 13 (E13) and showed a peak value on E18 in the central retina and on E20 in the periphery. After birth, until postnatal-day 3 (P3), a few co-localized cells were observed in the inner nuclear layer (INL). However, in the peripheral retina, co-localized cells were observed in the INL and ganglion cell layer until P5. Our results suggest that the birthdates of GABA-immunoreactive cells vary, depending on cell-type and that there is a temporal lag in the GABA-immunoreactive cell production in the peripheral retina relative to the central retina.

Dopamine D1-receptor immunolocalization in goldfish retina.

Dopamine, a neuromodulator in the vertebrate retina, is involved in numerous functions related to light adaptation. However, unlike in mammals, localization of retinal D1-dopamine receptors in nonmammalian vertebrates has been hampered due to a lack of antisera. To address this problem, an antiserum against the 18 C-terminal amino acids of the goldfish D1 receptor (gfD1r) was generated in chicken eggs and tested in retinae of goldfish and rat, and rat caudate putamen, by using immunoblots and light microscopic immunocytochemistry. No labeling was observed in any tissue or immunoblots with preabsorbed gfD1r antiserum. Immunoblot analysis of goldfish retina revealed a single band at about 101 kDa. The patterns of gfD1r immunoreactivity (gfD1r-IR), found in rat caudate putamen and rat retina were virtually identical to that previously reported with other D1-receptor ligands and antisera. In goldfish retina, gfD1r-IR was most intense over cell bodies in the ganglion cell layer, amacrine cells in the proximal inner nuclear layer (INL), and bipolar cells in the distal INL. Weaker gfD1r-IR was observed over horizontal cell bodies and both plexiform layers. Müller cells and axons of cone photoreceptors were labeled as well. Double labeling showed that all protein kinase C-immunoreactive bipolar cells (ON type) were gfD1r-IR on the soma, axon terminal, and dendrites. All glutamate decarboxylase-immunoreactive (i.e., gamma-aminobutyric acid utilizing) amacrine cells and horizontal cells were gfD1r-IR. Retinal D1r distribution is more extensive than dopamine neuron innervation, but is consistent with physiologic estimates of dopamine function, suggestive of both wiring and volume transmission of dopamine in the retina. The gfD1r antiserum displays cross-reactivity to dopamine receptors in a mammal and a nonmammal and should prove useful in future studies of dopaminergic systems.

Birth and fate of proliferative cells in the inner nuclear layer of the mature fish retina

Birth and fate of proliferative cells in the inner nuclear layer of the mature fish retina

Transient, high levels of SNAP-25 expression in cholinergic amacrine cells during postnatal development of the mammalian retina.

In the present study, we have examined the development of cholinergic amacrine cells in the retina of the Brazilian opossum, Monodelphis domestica. An antibody directed against choline acetyltransferase (ChAT) revealed that ChAT-like immunoreactivity (ChAT-IR) was first observed at 15 days postnatal (15PN). By 25PN, ChAT-IR identified two matching populations of amacrine cells in the inner nuclear and ganglion cell layer. Bromodeoxyuridine birth-dating analysis coupled with immunolabeling with the anti-ChAT antibody revealed that the cholinergic amacrine cells are born postnatally, between 2PN and 15PN. In addition, we have examined the differentiation of the cholinergic amacrine cells by using an antibody directed against a presynaptic terminal-associated protein, synaptosomal-associated protein of 25 kDa (SNAP-25). Double-labeling analysis revealed that relatively high levels of SNAP-25-IR were selectively present in cholinergic amacrine cells prior to eye opening. However, in the mature retina, high levels of SNAP-25-IR were no longer observed in the ChAT-IR amacrine cells. These results reveal a distinct period in development, prior to eye opening, when high levels of SNAP-25-IR are selectively expressed in cholinergic amacrine cells. The specificity and time course of the high levels of SNAP-25 in cholinergic amacrine cells may be critical in mediating the transient properties of these cells during visual system development.

Immunocytochemical localization of basic fibroblast growth factor and glial fibrillary acidic protein after laser photocoagulation in the Royal College of Surgeons rat

Argon laser photocoagulation slows photoreceptor degeneration in the Royal College of Surgeons (RCS) rat, as does intravitreal injection of basic fibroblast growth factor (bFGF). We hypothesize that up-regulation of retinal bFGF is a consequence of laser lesioning in RCS rats. Therefore, we examined the localization of bFGF after laser and correlated this with Mailer cell glial fibrillary acidic protein (GFAP) expression, which is known to increase after injury. A total of 34 RCS rats at postnatal day 23 were anaesthetized (ketamine 40 mg/kg) and their retinas were irradiated with a grid pattern of 40 non-overlapping argon green lesions with a power of 120 mW for 0.2 s using a 50 microm spot size. At 0, 6, 12, 24 and 48 h and 7, 14 and 21 days post-lesion, rats were anaesthetized and their eyes were enucleated and cryostat sectioned and the sections were processed using either an antibody to bFGF or GFAP using the standard avidin-biotinylated peroxidase complex method. Five age-matched RCS rats without laser lesions served as controls. Basic fibroblast growth factor immunoreactivity (IR) was normally located within cells in the ganglion cell layer inner nuclear layer and in retinal pigment epithelium cells and in the extracellular matrix/cell membranes of the outer nuclear layer (ONL). In lasered retinas, there was elevated bFGF-IR in the coagulated outer segments for the first 24 h. Retinal blood vessels/Müller cells/astrocytes were moderately labelled in and near each lesion immediately after lesion and became more intense after 48 h and persisted for at least 21 days. There was an elevation of bFGF-IR in the ONL on the lesion flanks at 14 days. Muller cell GFAP-IR was first detected at 6 h post-lesion and spread for a considerable distance beyond the lesion site. At 7 and 14 days, Müller cells at the lesion site had sprouted, while those on the flanks were still GFAP-IR. Following laser lesion there was an increase in bFGF at the lesion core only for the first 24 h. However, elevated levels of bFGF were observed in the ONL at 14 days, which extended into the lesion flanks for a similar distance to that over which increased photoreceptor survival is found. These results provide support for the hypothesis that laser lesions induce bFGF and this may be the mechanism whereby photoreceptors are spared. Müller cell activation is consistent with growth factor stimulation, but was more widespread than the bFGF changes in ONL. However, blood vessel labelling was similarly widespread and so the responses may be linked between Müller cell GFAP reaction and blood vessel bFGF localization after laser lesions.

Fos expression in the retina of rd/ rd mice during the light/dark cycle

An anti-Fos protein antiserum was used to elucidate the diurnal expression of Fos protein in the normal and degenerate rd/ rd mice retina. We have found that Fos expression is stimulated in cells of both inner nuclear layer (INL) and ganglion cell layer (GCL) at the onset of light period and reaches its maximum after 2 h. After which, the number of stained nuclei decreases along the light/dark cycle until almost no reaction is observed at the end of dark period. This expression pattern was similar in both normal and rd/ rd mice although degenerate retinas showed a much lower number of stained nuclei. Aged rd animals also show Fos expression in GCL and INL in response to light stimuli suggesting that severely degenerate retinas are still able to transduce light stimulus.

Localisation of neuronal nitric oxide synthase-immunoreactivity in rat and rabbit retinas.

The distribution of neuronal nitric oxide synthase (NOS) immunoreactivity was examined in rat and rabbit retinas and was compared with the distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase reactivity and vasoactive intestinal peptide (VIP) immunoreactivity. An antibody raised against a C-terminal fragment of a cloned rat cerebellar NOS was used to localise NOS immunoreactivity. NOS immunoreactive cells were not detected in rat retinas at postnatal day 1 or 4, but were seen from postnatal day 7 onwards. NOS immunolabelling was seen in a small population of cells in the proximal inner nuclear layer. Most of the labelled cells had the position of amacrine cells and were seen to send processes into the inner plexiform layer. A few labelled cells were at times also seen in the ganglion cell layer, which are likely to correspond to displaced amacrine cells. The same NOS-labelling pattern was seen in rat and rabbit retinas. NADPH-diaphorase staining was observed in both species, in photoreceptor inner segments, in cells with the position of horizontal cells, in a subset of amacrine and displaced amacrine cells, in large cell bodies in the ganglion cell layer, in both plexiform layers, and in endothelium. Colocalisation of NOS immunoreactivity and NADPH-diaphorase staining was only observed among amacrine cells. However, not all NADPH-diaphorase-reactive amacrine cells were found to be NOS immunoreactive. VIP immunoreactivity was also localised in rat retinas in a subpopulation of amacrine cells, but no colocalisation of NOS and VIP immunoreactivity was observed. Our observations indicate that only amacrine cells contain the NOS form recognisable by the antibody used, and suggest that different isoforms of neuronal NOS may be present in retinal cells. Further, the onset of NOS expression in rat amacrine cells appears to occur independently of neuronal activity.

The organization of the inner nuclear layer of the rabbit retina

The initial goal of this study was to establish an accounting of the major classes of cells present in the inner nuclear layer (INL) of the rabbit's retina. Series of 80-100 radial sections 1 micron thick were cut from retinal blocks dissected at intervals along the vertical meridian. They were photographed at high magnification in the light microscope. By visualizing the initial segments of processes leaving the somata, we could identify each cell as a bipolar, amacrine, horizontal, or Müller cell. The identifications made by light microscopy were confirmed by electron microscopy of alternating ultrathin sections. On average, bipolar cells made up 41% of the total INL cells, amacrine cells 32%, horizontal cells 1.5%, and Müller cells 24%. These fractions varied relatively little across the retina or among different animals. We next immunolabeled the rod bipolar cells of whole-mounted retinas with antibodies against protein kinase C, using FITC as the visualizing agent. The same retinas were counterstained with a DNA-binding probe that fluoresces at longer wavelengths. Serial optical horizontal sections of the double-labeled wholemounts were made by confocal microscopy. On average, rod bipolars accounted for 10% of the total INL cells. By subtraction, the cone bipolars made up 31% of the total cells. We conclude that cone bipolars substantially outnumber rod bipolars, even in a retina in which rods outnumber cones by more than 20:1. Using the base of reference created here, a similar analysis can be carried out for other subclasses of retinal neuron. Because the analysis does not depend on absolute cell densities or corrections for shrinkage, data acquired by different histochemical techniques may be combined.

Glutamate-, GABA-, and GAD-immunoreactivities co-localize in bipolar cells of tiger salamander retina.

The presence of inhibitory bipolar cells in salamander retina was investigated by a comparative analysis of the distribution of glutamate- and GABA-immunoreactivities (GLU-IR; GABA-IR) using a postembedding immunocytochemical method. GLU-IR was found in virtually all photoreceptors, bipolar cells and ganglion cells, neuronal elements that transfer information vertically through the retina. GLU-IR also was found in numerous amacrine cells in the mid and proximal inner nuclear layer as well as in the cytoplasm of horizontal cells, while the nucleus of horizontal cells was either lightly labeled or not labeled at all. GLU-IR was found in the outer plexiform layer and intensely in the inner plexiform layer, in which there was no apparent sublamination. Forty-seven percent of Type IB bipolar cells in the distal inner nuclear layer and 13% of the displaced bipolar cells were GABA-IR. All bipolar cells were also GLU-IR, indicating that GABA-IR bipolar cells were a subset of GLU-IR bipolar cells rather than a separate population. About 12% of the Type IB bipolar cells were moderately GABA-IR and likely comprised a GABAergic subtype. GLU-IR levels in the presumed GABAergic bipolar cells were higher than in other purely GLU-IR bipolar cells suggesting that these GABA-IR bipolar cells are glutamatergic as well. All of the displaced bipolar cells were only lightly GABA-IR, indicating that displaced bipolar cells comprise a more homogeneous class of glutamatergic cell than orthotopic bipolar cells. GAD-IR co-localized with GABA-IR in orthotopic but not displaced bipolar cells, further supporting the idea that some orthotopic bipolar cells are GABAergic. A small proportion of bipolar cells in salamander retina contain relatively high levels of both GABA and glutamate. Co-release of these substances by bipolar cells could contribute to the "push-pull" modulation of ganglion cell responses.

Retinal growth and cell addition during embryogenesis in the teleost, Haplochromis burtoni.

Neurogenesis of the developing embryonic retina is described for the African cichlid fish, Haplochromis burtoni, from 4 days post fertilization until all cell phenotypes are generated (day 7). Cell addition and differentiation both begin at the same absolute location which later becomes the central retina. As observed in most other vertebrates, cones and ganglion cells differentiate first, followed by amacrine and bipolar cells. Rod photoreceptors, which are added late, differentiate last. Changes in retinal thickness, retinal stretching, cell size, and cell density were measured during development. From day 4 through 7, there is an increase in retinal thickness largely due to the expansion of the inner plexiform layer (IPL) and outer nuclear layer (ONL). The inner nuclear layer (INL) decreases in thickness and there is a transient decrease in the density of cells in the scleral portion of the INL. Cells increase in size in the ganglion cell layer (GCL) and the vitread INL, decrease in size in the sclerad INL, and remain the same in the ONL. Changes in the density of the cell layers were observed: the density of ONL cells increased, the density of GCL cells decreased, and INL cells increased then decreased. From day 4 to day 6, eye growth is entirely due to cell addition because no retinal stretching was observed in the ONL or the horizontal layer. During this same developmental period, the pattern and rate of neurogenesis were measured in the differentiated portion of the retina by means of 3H-thymidine labeling. A small number of cell divisions within the differentiated INL precede the onset of cell divisions in the ONL. The number of 3H-thymidine labeled cells within the INL increases at a low rate consistent with an asymmetric pattern of cell division characteristic of stem cells. In contrast, cell divisions in the ONL increase exponentially, consistent with a symmetric pattern of cell division characteristic of progenitor cells. Double-label experiments (3H-thymidine and a rod specific opsin antibody) show that some of the symmetrically dividing cells in the ONL express the rod specific opsin within 2 days, suggesting that these dividing cells are rod progenitors. Although we do not hae conclusive evidence, these developmental processes support the hypothesis that stem cells within the INL could be the source of rod precursors in the embryonic teleost retina.