Histochemical demonstration of glutamate dehydrogenase and phosphate-activated glutaminase activities in semithin sections of the rat retina.

The activities of the glutamate metabolizing enzymes phosphate-activated glutaminase (PAG) and glutamate dehydrogenase (Gldh) are demonstrated in semithin sections of the rat retina. Highest activities of both enzymes are found in the photoreceptor inner segments, PAG additionally in the outer plexiform layer and Gldh in the inner plexiform layer and in mueller glial cells. Although their non randomly distribution makes a role in neurotransmitter metabolism possible, their high activities in inner segments point towards the general problem of the functional interpretation of both molecules.     

Distribution of Three Enzymes of γ-Aminobutyric Acid Metabolism in Monkey Retina

Abstract: The distributions of glutamate decarboxylase (EC 4.1.1.15), γ-aminobutyric acid transaminase (EC 2.6.1.19), and succinate semialdehyde dehydrogenase (EC 1.2.1.24) were determined in monkey retina. The decarboxylase was almost restricted to the inner plexiform layer. The transaminase was also highest in this layer, but activities were 40% as high in the adjacent third of the inner nuclear layer and in the ganglion cell and fiber layers. Succinate semialdehyde dehydrogenase was distributed very differently. Although it also showed a peak of activity in the inner plexiform layer, there was a second equal peak in the photoreceptor inner segment layer and a smaller peak in the outer plexiform layer, regions where both γ-aminobutyric acid transaminase and glutamate decarboxylase were essentially absent.     

Distribution of Glycine, γ-Aminobutyric Acid, Glutamate Decarboxylase, and γ-Aminobutyric Acid Transaminase in Rabbit and Mudpuppy Retinas

Abstract: The distributions of glycine, γ-aminobutyric acid (GABA), glutamate decarboxylase (EC 4.1.1.15), and GABA transaminase (EC 2.6.1.19) were determined in rabbit and mudpuppy retinas. In both species, peak levels of the amino acids and the enzymes occurred in the inner plexiform layer. Glutamate decarboxylase was almost entirely confined to the inner plexiform layer. Determinations were also made of the GABA content of 107 individual putative amacrine cell somas from mudpuppy retina. About 30% of those somas were found to have high endogenous GABA levels.     

Distribution of activities of aspartate aminotransferase isoenzymes and malate dehydrogenase in guinea pig retinal layers

Distributions of activity of the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase and of malate dehydrogenase (MDH) were determined in guinea pig retinal layers. The distribution of total AAT activity (tAAT = cAAT + mAAT) and of mAAT activity correlated well (r = 0.88-0.91) with the distribution of MDH activity. mAAT activity was highest in the inner segments of the photoreceptors; there was a greater than twelve-fold difference between activity in that layer and in the inner retinal layers. cAAT activity was also highest in the inner segments, but the difference between the activity in the inner segments and the other layers was not nearly as great as with mAAT. cAAT activity was also relatively high in the outer nuclear layer, outer plexiform layer, and part of the inner plexiform layer. The high activity of cAAT, mAAT, and MDH in the inner segments indicates that all of these enzymes are involved in metabolic reactions related to energy production and/or to photoreceptive processes in the outer segments and, therefore, that the enzymes are probably involved in energy-related metabolism at synapses. However, other functions, including those related to neurotransmission, are not excluded.     

Distributions of aspartate aminotransferase and malate dehydrogenase activities in rat retinal layers

Aspartate aminotransferase (AAT), an enzyme interconverting glutamate and aspartate, has been suggested to be a marker for glutamatergic and/or aspartatergic neurons. However, AAT, glutamate, and aspartate are also involved in cellular metabolism, e.g., the malate-aspartate shuttle. To investigate the extent to which AAT might be involved in these several functions in retina, the distribution of AAT activity in rat retinal layers was compared to that of malate dehydrogenase (MDH), an enzyme of aerobic metabolism proposed to be physically complexed with AAT in the malate-aspartate shuttle mechanism. The distribution of AAT activity in retinal layers closely paralleled that of MDH (correlation coefficient AAT versus MDH = 0.93). AAT activity was proportionately higher than MDH in the photoreceptor inner segments, containing a high density of mitochondria, and in the outer plexiform layer (OPL), containing photoreceptor terminals and bipolar and horizontal cell processes. The amount of total AAT activity in the inner segments related to the mitochondrial isoenzyme is almost twice that in the other layers tested, including the OPL. The correlation between AAT and MDH activities is consistent with AAT involvement in retinal energy metabolism, although other functions, such as neurotransmission, are possible.     

In-vivo labeling of (3H)D-aspartate uptake sites in monkey retina

Summary Following prolonged topical application of (³H)D-aspartate in vivo, selective labeling of three distinct cell classes was observed in light-microscopic radioautographs from squirrel monkey retina. Müller (glial) cell bodies and their processes were intensely and consistently labeled in all preparations. Moderately labeled perikarya were occasionally detected in the area of bipolar cells, within the inner nuclear layer. These were particularly numerous in sections from the central retina where an intense diffuse labeling of the inner plexiform layer was also prominent. Finally, moderate to dense accumulations of label were observed over the cell bodies, internal segments and fiber processes of cone photoreceptors. These results strongly suggest that cones, as well as a sub-population of bipolar cells, use glutamate and/or aspartate as neurotransmitter(s) in monkey retina.     

Microphotometric determination of enzymes in brain sections. III. Glutamate dehydrogenase

An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM L-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN3), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN3 as a blocker of the respiratory chain was indispensible, because without NaN3 most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities.     

Laminar Distributions of Choline Acetyltransferase and Acetylcholinesterase Activities in the Inner Plexiform Layer of Rat Retina

Choline acetyltransferase and acetylcholinesterase activities were measured in samples taken at 7-micron increments through the inner plexiform layer of rat retina. These enzyme activities were not uniformly distributed through the depth of the inner plexiform layer. Peaks of choline acetyltransferase activity occurred at about one-third and peaks of acetylcholinesterase activity at about one-fifth of the depth into the inner plexiform layer from either side. The positions of the two peaks of choline acetyltransferase activity most likely correspond to the locations of processes from cholinergic amacrine somata in the inner nuclear layer, which spread in sublamina a, and processes from cholinergic amacrine somata "displaced" in the ganglion cell layer which spread in sublamina b of the inner plexiform layer. The peaks of acetylcholinesterase activity may in addition correspond to the processes of cholinoceptive amacrine and ganglion cells. The magnitudes of choline acetyltransferase and acetylcholinesterase activities are as high as found anywhere in rat brain, emphasizing the important role of cholinergic mechanisms in visual processing through the rat inner plexiform layer.     

Aspartate aminotransferase and glutaminase activities in rat olfactory bulb and cochlear nucleus; Comparisons with retina and with concentrations of substrate and product amino acids

The quantitative distributions of aspartate aminotransferase and glutaminase were mapped in subregions of olfactory bulb and cochlear nucleus of rat, and were compared with similar data for retina and with the distributions of their substrate and product amino acids aspartate, glutamate, and glutamine. The distributions of both enzymes paralleled that of aspartate in the olfactory bulb and that of glutamate in the cochlear nucleus. In retina (excluding inner segments), there were similarities between aspartate aminotransferase and both glutamate and aspartate distributions. The distribution of -aminobutyrate (GABA) was similar to those of both enzymes in olfactory bulb, to aspartate aminotransferase in cochlear nucleus, and to glutaminase in retina (excluding inner segments). The results are consistent with significant involvement of aspartate aminotransferase, especially the cytosolic isoenzyme, and glutaminase in accumulation of the neurotransmitter amino acids glutamate, aspartate, and GABA, although with preferential accumulation of different amino acids in different brain regions.     

Ageing transition of superoxide dismutase in rat retina

To examine the relationship between retinal ageing and superoxide dismutase, the distribution and expression of the dismutase was studied in the retina of 2-year-old Sprague--Dawley albino rats with immunohistochemistry and immunochemical quantitative analysis. Eight-week-old Sprague--Dawley albino rats were used as controls. In 2-year-old rats, manganese superoxide dismutase (Mn-SOD) immunoreactivities in the photoreceptor inner segments, the outer nuclear layer and the inner plexiform layer were stronger than those in 8-week-old rats. Copper--zinc superoxide dismutase (CuZn-SOD) immunoreactivities in the outer nuclear layer and inner plexiform layer of 2-year-old rats were stronger than those in 8-week-old rats. Faint CuZn-SOD immunoreactivity became visible in the photoreceptor inner segments of 2-year-old rats, whereas no CuZn-SOD immunoreactivity was observed in 8-week-old rats. Our immunochemical quantitative analysis also showed an increase in the immunoreactivities of superoxide dismutases in the sensory retina with age. The transition of the dismutases may have some relationship with retinal ageing. © 1998 Chapman & Hall     

Microphotometric determination of enzymes in brain sections

Summary An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM l-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN₃), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. — The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN₃ as a blocker of the respiratory chain was indispensible, because without NaN₃ most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities. Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-2)     

Human retinal development: ultrastructure of the inner retinal layers

Studies of the developing human retina from 6.5 to 18 weeks' gestational age (16–156 mm) by light and electron microscopy are concerned with the morphogenesis of neuroblast cells, plexiform layers, and inner limiting membrane. The transient layer of Chievitz is formed posteriorly by 20 mm (7 weeks), inner plexiform by 48 mm (9 weeks), outer plexiform layer by 83 mm (12 weeks), identifiable cones by 83 mm, and rods by 120 mm (15 weeks). Mitotic activity continues posteriorly until 120 mm and was seen in inner layers of the retina until 103 mm (13 weeks). Outer neuroblastic differentiation is marked by diversification from a uniform cell population to one containing at least three cell types differing in their nuclear shape, chromatin pattern, and cytoplasmic characteristics. Differentiating ganglion cells accumulate polysomes, rough endoplasmic reticulum, Golgi complexes, microtubules, and dense bodies. Müller cell bodies in ganglion and inner nuclear layers extend processes between ganglion cells, and radial fibers, containing extensive smooth endoplasmic reticulum, to the vitreal surface. Synapses appear in the inner and outer plexiform layers by 83 mm (12 weeks), and by 120 mm (15 weeks) demonstrate a variety of conventional and ribbon forms similar to those found in the adult. Synaptogenesis therefore begins considerably before the development of photoreceptor outer segments.     

Intrinsic organization of the medial cerebral cortex of the lizard Lacerta pityusensis: A golgi study

The morphology of cells and the organization of axons were studied in Golgi-Colonnier and toluidine blue stained preparations from the medial cerebral cortex of the lizard Lacerta pityusensis. In the medial cortex, six strata were distinguished between the superficial glial membrane and the ependyma. Strata I and II formed the outer plexiform layer, stratum III formed the cellular layer, and strata IV go VI the inner plexiform layer. The outer plexiform layer contained smooth bipolar neurons; their dendrites were oriented anteroposteriorly and their axons were directed towards the posterior zone of the brain. Five neuronal types were observed in the cellular layer. The spinous pyramidal neurons had well-developed apical dendrites and poorly developed basal ones. Their axons entered the inner plexiform layer and gave off collaterals oriented anteroposteriorly. The small, sparsely spinous pyramidal neurons had poorly developed dendrites and their axons entered the inner plexiform layer. The spinous bitufted neurons had well-developed apical and basal dendritic tufts. Their axons gave off collaterals that reached the outer and inner plexiform layers of both the dorsomedial and dorsal cortices. The sparsely spinous horizontal neurons had dendrites restricted to the outer plexiform layer. Their axons entered the inner plexiform layer. The sparsely spinous, multipolar neurons had their soma close to stratum IV and their axons entered the outer plexiform layer. In stratum V of the inner plexiform layer were large, spiny polymorphic neurons; they had dendrites with long spines, and their axons reached the cellular layer. On the basis of these results, we have subdivided the medial cortex into two subregions: the superficial region, which contains the neurons of the cellular layer and their dendritic domains, and the deep region, strata V and VI, which contains the large, spiny polymorphic neurons. The neurons in the medial cortex of these lizards resembles those in the area dentata of mammals. On this basis, the superficial region may be compared to the dentate gyrus and the deep region to the hilar region of the hippocampus of mammals.     

Adenylate Cyclases in the Vertebrate Retina: Distribution and Characteristics in Rabbit and Ground Squirrel

Adenylate cyclase activity and the effects of EGTA, 5'-guanylylimidodiphosphate (GPP(NH)P), and dopamine were measured in microdissected layers of rod-dominant (rabbit) and cone-dominant (ground squirrel) retinas, The distribution of basal enzyme activity was similar in both species, with the highest levels found in the inner plexiform and photoreceptor cell inner segment layers, EGTA inhibited adenylate cyclase in the inner retina of both species and stimulated activity in rabbit outer and inner segment layers, but had no effect in these layers from ground squirrel. Enzyme activity was stimulated in all regions by GPP(NH)P, except in the outer segments of the photoreceptors. Dopamine stimulated the enzyme in the outer and inner plexiform and inner nuclear layers in rabbit, but only in the inner plexiform layer in ground squirrel. These data demonstrate that the enzymatic characteristics of adenylate cyclase vary extensively from region to region in vertebrate retina and suggest that cyclic AMP may have multiple roles in this tissue. A model for the distribution of the different forms of adenylate cyclase in retina is proposed.     

Differential expression of syntaxin-1 and synaptophysin in the developing and adult human retina

Synaptophysin and syntaxin-1 are membrane proteins that associate with synaptic vesicles and presynaptic active zones at nerve endings, respectively. The former is known to be a good marker of synaptogenesis; this aspect, however, is not clear with syntaxin-1. In this study, the expression of both proteins was examined in the developing human retina and compared with their distribution in postnatal to adult retinas, by immunohistochemistry. In the inner plexiform layer, both were expressed simultaneously at 11–12 weeks of gestation, when synaptogenesis reportedly begins in the central retina. In the outer plexiform layer, however, the immunoreactivities were prominent by 16 weeks of gestation. Their expression in both plexiform layers followed a centre-to-periphery gradient. The immunoreactivities for both proteins were found in the immature photoreceptor, amacrine and ganglion cells; however, synaptophysin was differentially localized in bipolar cells and their axons, and syntaxin was present in some horizontal cells. In postnatal-to-adult retinas, synaptophysin immunoreactivity was prominent in photoreceptor terminals lying in the outer plexiform layer; on the contrary, syntaxin-1 was present in a thin immunoreactive band in this layer. In the inner plexiform layer, however, both were homogeneously distributed. Our study suggests that (i) syntaxin-1 appears in parallel with synapse formation; (ii) synaptogenesis in the human retina might follow a centre-to-periphery gradient; (iii) syntaxin-1 is likely to be absent from ribbon synapses of the outer plexiform layer, but may occur at presynaptic terminals of photoreceptor and horizontal cells, as is apparent from its localization in these cells, which is hitherto unreported for any vertebrate retina.     

Morphology of calretinin and tyrosine hydroxylase-immunoreactive neurons in the pig retina

The morphology of calretinin- and tyrosine hydroxylase-immunoreactive (IR) neurons in adult pig retina was studied. These neurons were identified using antibody immunocytochemistry. Calretinin immunoreactivity was found in numerous cell bodies in the ganglion cell layer. Large ganglion cells, however, were not labeled. In the inner nuclear layer, the regular distribution of calretinin-IR neurons, the inner marginal location of their cell bodies in the inner nuclear layer, and the distinctive bilaminar morphologies of their dendritic arbors in the inner plexiform layer suggested that these calretinin-IR cells were AII amacrine cells. Calretinin immunoreactivity was observed in both A-and B-type horizontal cells. Neurons in the photoreceptor cell layer were not labeled by this antibody. The great majority of tyrosine hydroxylase-IR neurons were located at the innermost border of the inner nuclear layer (conventional amacrines). The processes were monostratified and ran laterally within layer 1 of the inner plexiform layer. Some of the tyrosine hydroxylase-IR neurons were located in the ganglion cell layer (displaced amacrines). The processes of displaced tyrosine hydroxylase-IR amacrine cells were also located within layer 1 of the inner plexiform layer. Some processes of a few neurons were located in the outer plexiform layer. A very low density of neurons had additional bands of tyrosine hydroxylase-IR processes in the middle and deep layers of the inner plexiform layer. The processes of tyrosine hydroxylase-IR neurons extended radially over a wide area and formed large, moderately branched dendritic fields. These processes occasionally had varicosities and formed "dendritic rings". These results indicate that calretinin- and tyrosine hydroxylase-IR neurons represent specific neuronal cell types in the pig retina.     

Distribution of Proteins Providing Homeostasis of Iron Ions in Bovine Retina

Distribution of proteins providing homeostasis of iron ions in bovine retina was studied by methods of indirect immunohistochemistry, which allowed detection of localization of transferrin, ferritin, and transferrin receptor. In bovine retina, transferrin is revealed in the region of outer and inner segments of photoreceptors and in the external plexiform layer. Distributions of ferritin and transferrin receptor are identical; they are revealed in all layers of retina, the maximal immunoreactivity against these proteins is found in pigment epithelium, in the region of inner segments of photoreceptors, in the external plexiform and internal nuclear layers. The obtained results are discussed from the point of view of mechanisms providing with iron the cells of the outer and inner retina.     

Histochemical responses in the retina after acute blood loss

Adult albino mice were bleed through the hearts by cardiac puncture under Nembutal anesthetic. 0.3 ml of blood was withdrawn form every animal. The retinae were then studied on a timed basis with succinic dehydrogenase histochemistry and alkaline phosphatase histochemistry. In control retinae, high SDH activities were localized in the inner segments, outer plexiform, inner plexiform, and ganglion cells layers and high alkaline phosphatase activities were localized in the ganglion cell layers and the vessels of the plexiform layers. Decrease in the enzymatic activities of both SDH and alkaline phosphatase in these layers were most evident 5h after bleeding. 9 to 24 h after bleeding, a compensatory increase was detected. 48 to 72 h after, the enzymatic activities decreased again. Reperfusion of experimental animals with 5% dextrose would increase the retinal enzymatic activities back to normal, even if the reperfusion was carried out as late as 48 h after bleeding.     

Somatostatin and VIP neurons in the retina of different species

Neurons displaying somatostatin or vasoactive intestinal polypeptide (VIP) immunoreactivity were detected among the amacrine cells in the retina of baboon, cynomolgus monkey, squirrel monkey, cow, pig, cat, rabbit, guinea-pig, rat, mouse, frog and goldfish. Generally, immunoreactive cell bodies were located in the inner nuclear layer with processes ramifying in three more or less well-defined sublayers in the inner plexiform layer. The density of the sublayers and their location varied with the peptide and species investigated. In most cases there was a sublayer in the outermost part (Ramon y Cajal's sublamina 1) of the inner plexiform layer and this sublayer was usually the best developed. In some species a few somatostatin fibres were also detected in the outer plexiform layer, suggesting that some interplexiform cells contain somatostatin. In the baboon VIP was found exclusively in interstitial amacrine cells which have their cell bodies and processes entirely within the inner plexiform layer.     

Somatostatin and VIP neurons in the retina of different species

Summary Neurons displaying somatostatin or vasoactive intestinal polypeptide (VIP) immunoreactivity were detected among the amacrine cells in the retina of baboon, cynomolgus monkey, squirrel monkey, cow, pig, cat, rabbit, guinea-pig, rat, mouse, frog and goldfish. Generally, immunoreactive cell bodies were located in the inner nuclear layer with processes ramifying in three more or less well-defined sublayers in the inner plexiform layer. The density of the sublayers and their location varied with the peptide and species investigated. In most cases there was a sublayer in the outermost part (Ramon y Cajal's sublamina 1) of the inner plexiform layer and this sublayer was usually the best developed. In some species a few somatostatin fibres were also detected in the outer plexiform layer, suggesting that some interplexiform cells contain somatostatin. In the baboon VIP was found exclusively in interstitial amacrine cells which have their cell bodies and processes entirely within the inner plexiform layer.