Phospholipase Cγ1 in Bovine Rod Outer Segments: Immunolocalization and Light-Dependent Binding to Membranes

Abstract: We have investigated the isozymes of a phosphoinositide-specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCβ₁, γ₁, and δ₁. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCγ₁ was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCγ₁ showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light-adapted in vitro had more PLCγ₁ on immunoblots than ROS from dark-adapted retinas. PLC enzyme activity in ROS from light-adapted retinas was 69 and 46% higher than ROS from dark-adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca²⁺]_free between 0.32 and 100 µ M . These results demonstrate the presence of PLCγ₁ in bovine ROS and show that ROS prepared from light-adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.     

Target cells of vitamin D in the vertebrate retina

Using PAP technique, cellular localization of vitamin D-dependent calcium-binding protein (D-CaBP) was investigated in vertebrate retina with monospecific antisera against chick duodenal D-CaBP. In the chick retina, the receptor cells were positive. In the inner nuclear layer, horizontal cells and some bipolar cells were also positive. Some amacrine cells as well as different levels of the inner plexiform layer were also positive for D-CaBP. A few interspersed ganglion cells were positive but their axons forming the optic tract were negative. Müller's cells were negative. In 1-day-old chicks and 4-week-old rachitic chicks there was paucity and absence, respectively, of D-CaBP staining in horizontal cells. In the mouse, rat, and rabbit the receptors had only trace amounts of reaction product in their outer segment and pedicle. Horizontal cells were densely positive throughout their cellular body and processes. Some amacrine cells in the inner nuclear layer were positive. In the mouse and rat three horizontal levels of the outer plexiform layer were very prominent because of their dense staining for D-CaBP. Many ganglion cells were also positive along with their axons forming the optic nerve. In the rabbit, no positive layers were seen in the inner plexiform layer, and ganglion cells with their fibers were negative. In the frog retina there were smaller amounts of D-CaBP in the receptor cells and horizontal cells than that of the chick retina. Also, the fibers of the ganglionic cells were positive for D-CaBP. In all species studied, some amacrine cells were stained for D-CaBP. Because of its possible roles in membrane calcium transport and intracellular Ca++ regulation, it has perhaps similar functions in these positive cells. The synthesis of D-CaBP is dependent upon vitamin D. These positive cells are thus target cells of vitamin D.     

GABA concentration and GAD activity levels in normal and degenerated retinas from mice

Freeze-dried sections (14 m thick) were prepared from mice with normal (C57BL strain) and degenerated (C3H strain) retinas. GABA concentration and GAD activity were determined in the microsamples (1.8–20 ng dry weight) of retinal layers and sublayers, using an enzymatic amplication reaction, NADP cycling. 1) GABA was distributed over all layers of normal retina with a broad concentration peak covering both inner nuclear and plexiform layers. In contrast, GAD activity was mostly localized in the inner plexiform layer. 2) GABA concentration was similar in one-fourth of the sublayers of each inner nuclear or plexiform layer. GAD activity was highest in the innermost sublayer of the inner nuclear layer. An increasing gradient of GAD activity was present in the inward direction in the inner plexiform layer. 3) In the degenerated retina, lacking in photoreceptors, the inner nuclear and plexiform layers remained, and GABA and GAD levels in these layers were similar to those in normal retina.Special Issue dedicated to Dr. O. H. Lowry.     

Adrenergic retinal neurons of some new world monkeys

Summary The retina of Aotes monkeys, Cebus monkeys, squirrel monkeys, and marmosets were investigated. Adrenergic perikarya were found in the innermost cell rows of the inner nuclear layer of all the investigated species. In addition, the Cebus monkey was found to have a special type of adrenergic neurons in the inner nuclear layer. This cell type was called the adrenergic pleomorph cell. Its processes ramify in the inner nuclear and inner plexiform layers. Adrenergic terminals occur in three more or less well developed sublayers of the inner plexiform layer, the layers being best developed in the Cebus monkey. Adrenergic terminals were also found around the cells of the inner nuclear layer and at the horizontal cells, where a scant sublayer is formed. More than one adrenergic sublayer of the inner plexiform layer has not been observed in primates previously, nor have the adrenergic terminals in the inner nuclear layer been observed previously in any species. The adrenergic pleomorph cells of the Cebus monkey also seem to be unique. The marked differences even between animals as closely related as some platyrhine monkeys call for caution when comparing the detailed function of the retina in different animals.This study was supported by grants from the Swedish Medical Research Council (B69-14X-2321-02) and the Faculty of Medicine, University of Lund, and was carried out within a research group sponsored by the Swedish Medical Research Council (projects No. B69-14X-56-05C and B69-14X-712-04C).     

γ-Aminobutyric Acid System in Developing and Degenerating Mouse Retina

Freeze-dried sections (14 microns thick) of retinal layers were prepared from mice with retinal degeneration (C3H strain) and control mice (C57BL strain). The weighed sections (2-30 ng dry weight) were analyzed using our microassay methods. In the control retina, gamma-aminobutyric acid (GABA) concentration and glutamate decarboxylase (GAD) activity, on a dry weight basis, increased from birth to 9 weeks of age and decreased slightly at 20 weeks. In the degenerated retina, the levels of GABA and GAD activity were higher at birth than in the control retina, and continued to increase until 20 weeks of age, at which time the GAD activity reached a markedly high level. This increase was found when the total GABA and GAD levels per retina were determined. In the normal retinal layers, GABA and GAD were confined primarily to the inner plexiform layer. In the degenerated retina, GAD activity gradually increased in the inner layers during postnatal development, but by 20 weeks the increase was most prominent in the inner part of inner nuclear layer and in the outer part of inner plexiform layer. GABA transaminase activity and its distribution were not much different in both normal and degenerated retinas during development.     

Spatiotemporal distribution of 1P1 antigen expression in the plexiform layers of developing chick retina

Changes in the distribution of 1P1-antigen in the developing chick retina have been examined by indirect immunofiuorescence staining technique using the novel monoclonal antibody (MAb) 1P1. Expression of the 1P1 antigen was found to be regulated in radial as well as in tangential dimension of the retina, being preferentially or exclusively located in the inner and outer plexiform layers of the neural retina depending on the stages of development. With the onset of the formation of the inner plexiform layer 1P1 antigen becomes expressed in the retina. With progressing differentiation of the inner plexiform layer 1P1 immunofiuorescence revealed 2 subbands at E9 and 6 subbands at E18. At postnatal stages (after P3) immunoreactivity was reduced in an inside-outside sequence leading to the complete absence of the 1P1 antigen in adulthood. 1P1 antigen expression in the outer plexiform layer was also subject to developmental regulation. The spatio-temporal pattern of 1P1 antigen expression was correlated with the time course of histological differentiation of chick retina, namely the synapse rich plexiform layers. Whether the 1P1 antigen was functionally involved in dendrite extension and synapse formation was discussed.     

IMMUNOHISTOCHEMICAL LOCALIZATION OF GABA IN CHAMELEON RETINA (CHAMALEO CHAMALEO)

We used a policlonal antiserum against GABA and demonstated GABA-immunoreactivity (GABA-IR) in several populations of amacrine cells in the inner nuclear layer (INL), and other cells in the inner plexiform layer (IPL) of the central and peripheral retina of the chameleon. Horizontal cells do not contain GABA-IR and the chameleon retina is therefore an exception among non-mammals. GABA-IR was not seen in cell bodies in the position of photoreceptor, bipolar and interplexiform cells suggesting that GABA is not involved in synaptic transmission in the outer plexiform layer of chameleon retina.     

Microscopic investigation of the comparative morphology of the retina

Morphological differences in the architectonics (the relations and composition of the layers and sublayers) of the retina are described in various vertebrates: pike, frog, and cat. These differences apply to both cellular and plexiform layers. The differences are particularly marked in the composition of the sublayers of the inner nuclear layer. In the frog the greatest degree of subdivision into layers of processes of the ganglion and amacrine cells is observed to correspond to the particularly complex differentiation of the inner plexiform layer of the retina (about 10 sublayers). In all the animals studied the ganglion cells can be divided into two principal types: symmetrical and asymmetrical, with many varieties. Asymmetrical amacrine cells are found in the pike and frog retina. The presence of vertical processes branching in the outer plexiform layer is confirmed for amacrine cells in the cat retina. The structural features of the retina are discussed in connection with physiological findings.     

Localization of 2-[125I]Iodomelatonin Binding Sites in Mammalian Retina

Specific melatonin binding sites were localized in the mammalian retina using the selective radioligand 2-[125I]iodomelatonin. Frozen sections obtained from both pigmented and albino rabbit eyes and albino mouse eyes were incubated with 2-[125I]iodomelatonin in the absence and presence of competing agents. In eyecups from albino rabbits, the highest density of specific 2-[125I]iodomelatonin binding sites was localized over the inner plexiform layer. Approximately 40-60% of the binding was specific, as determined with both the agonist 6-chloromelatonin and the antagonist luzindole. A high density of binding sites was observed over the choroid and retinal pigmented epithelium, but no statistical difference between total and nonspecific binding was detected. Results were similar with eyecups from pigmented rabbits. Albino mice showed a significant extent of 2-[125I]iodomelatonin binding in both the inner plexiform and the outer and inner segment layers. The specific binding of 2-[125I]iodomelatonin in retinas from albino rabbits maintained in the light for 24 h before decapitation was increased in the inner retina compared with the control. The distribution of 2-[125I]iodomelatonin binding sites in the various layers of the mammalian retina is consistent with the described functions for this hormone in retinal physiology.     

Neuropeptide Y (NPY) immunoreactive neurons in the retina of different species

Summary Neurons displaying Neuropeptide Y (NPY) immunoreactivity were found among amacrine cells in the retina of baboon, pig, cat, pigeon, chicken, frog, trout, carp and goldfish. The immunoreactive cell bodies were located in the middle and the innermost cell rows of the inner nuclear layer with processes forming one, two or three more or less well-defined sublayers in the inner plexiform layer. The location and the density of the sublayers varied with the species investigated. In the frog retina, bipolar-like cell bodies were found in the middle of the inner nuclear layer as well as sparsely occurring ovoid cell bodies in the ganglion cell layer. Like the amacrine cells, these cells emitted processes ramifying in three sublayers in the inner plexiform layer.     

Light- and electron-microscopic study of substance P-immunoreactive neurons in the guinea pig retina

Substance P (SP) immunoreactivity in the guinea pig retina was studied by light and electron microscopy. The morphology and distribution of SP-immunoreactive neurons was defined by light microscopy. The SP-immunoreactive neurons formed one population of amacrine cells whose cell bodies were located in the proximal row of the inner nuclear layer. A single dendrite emerged from each soma and descended through the inner plexiform layer toward the ganglion cell layer. SP-immunoreactive processes ramified mainly in strata 4 and 5 of the inner plexiform layer. SP-immunoreactive amacrine cells were present at a higher density in the central region around the optic nerve head and at a lower density in the peripheral region of the retina. The synaptic connectivity of SP-immunoreactive amacrine cells was identified by electron microscopy. SP-labeled amacrine cell processes received synaptic inputs from other amacrine cell processes in all strata of the inner plexiform layer and from bipolar cell axon terminals in sublamina b of the same layer. The most frequent postsynaptic targets of SP-immunoreactive amacrine cells were the somata of ganglion cells and their dendrites in sublamina b of the inner plexiform layer. Amacrine cell processes were also postsynaptic to SP-immunoreactive neurons in this sublamina. No synaptic outputs onto the bipolar cells were observed.     

Neuropeptide Y (NPY) immunoreactive neurons in the retina of different species

Neurons displaying Neuropeptide Y (NPY) immunoreactivity were found among amacrine cells in the retina of baboon, pig, cat, pigeon, chicken, frog, trout, carp and goldfish. The immunoreactive cell bodies were located in the middle and the innermost cell rows of the inner nuclear layer with processes forming one, two or three more or less well-defined sublayers in the inner plexiform layer. The location and the density of the sublayers varied with the species investigated. In the frog retina, bipolar-like cell bodies were found in the middle of the inner nuclear layer as well as sparsely occurring ovoid cell bodies in the ganglion cell layer. Like the amacrine cells, these cells emitted processes ramifying in three sublayers in the inner plexiform layer.     

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.     

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.     

THE DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE ACTIVITY IN VERTEBRATE RETINA1

Abstract— Choline acetyltransferase (ChAc) activity was determined in retinal layers from 10 vertebrates. In all animals, the highest activity was in the inner plexiform layer, intermediate activity in the inner nuclear and ganglion cell layers, and very low activity in the photoreceptor and outer plexiform layers and optic nerve. The pattern of distribution of enzyme activity within the inner nuclear layer corresponds quantitatively to the distribution of amacrine cells within that layer. A species difference of almost 90-fold was found between the lowest and highest values for ChAc activity in inner plexiform layer. The variation in enzyme activity found among homeotherms in inner nuclear and inner plexiform layers is related to the number of amacrine cell synapses in the inner plexiform layer. But the differences in enzyme activity are generally greater than those which have been found in numbers of amacrine cell synapses between species. The data suggest that cholinergic neurons in retina are to be found predominantly among the amacrine cell types and that not all amacrine cells will be found to be cholinergic.     

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.     

NADPH-Diaphorase Activity in Normally Developing and Intracranially Transplanted Retinas

The activity and distribution of nicotinamide dinucleotide phosphate diaphorase (NADPH-d), an enzyme that is widely distributed in the central nervous system and involved in the production of the free radical nitric oxide, were investigated histochemically in the normal developing and intracranially transplanted retinas. In the normal rat retina, NADPH-d activity was first detected in cells in the ganglion cells layer (GCL) and blood vessels on the first postnatal day (P0). A small but distinct population of NADPH-d positive cells were observed along the inner border of the inner nuclear layer at P7. NADPH-d positive sublaminae began to appear in the inner plexiform layer during the second postnatal week, and several strongly reactive sublaminae resembling those observed in the adult were observed by the fourth postnatal week. The overall spatio- temporal sequence of development of NADPH-d positive cells in the transplanted retina was similar to that of the normal retina, except a lack of reactive in the inner plexiform layer in more mature transplants as compared with normal retinas of corresponding ages. These results indicate that the time course of development and distribution of NADPH-d cells in early postnatal retina requires signals mainly of intraretinal origin and is independent of influence from the surroundings. While this finding is supportive to the notion that neurons that are rich in NADPH-d are resistant to injury or perturbation, the observation of a lack of well organized NADPH-d reactive sublaminae in the inner plexiform layer in older transplants suggests a possible alteration in the synaptic circuitry in the inner retina with increasing postgrafting survival time.     

Developmental Alteration of the Expression and Kinase Activity of Cyclin-Dependent Kinase 5 (Cdk5)/p35nck5a in the Rat Retina

Abstract: Neuronal Cdc2-like kinase has been purified from the bovine brain as a proline-directed serine/threonine kinase. This kinase is a heterodimer of Cdk5 and p35^nck5a and influences neuronal maturation or sprouting in the normal brain. In this study, we showed the expression of Cdk5/p35^nck5a kinase in the developing rat retina. The expression of Cdk5 and p35^nck5a increased between 1 week and 3 weeks after birth. These expression levels were most prominent from 2 weeks to 3 weeks after birth and decreased after 4 weeks. The developmental change of Cdk5/p35^nck5a kinase activity coincided with those of the expression of p35^nck5a and Cdk5. An immunohistochemical study showed that Cdk5 was expressed in the ganglion cells and in some cells in the inner nuclear layer at an early stage. With retinal development, Cdk5 was expressed in the inner plexiform layer also. In the adult, the expression of Cdk5 was restricted to the inner plexiform layer and to some cells in the inner nuclear layer. These changes of localization in the developing retina were very close to those of B-50/GAP-43. On the other hand, the expression of p35^nck5a was restricted to the soma of the neuron in the developing retina. This subcellular localization in the developing retina agreed with that in the developing rat brain. The expression levels of Cdk5 and p35^nck5a in retina of rats raised from fetus to 3 weeks after birth in darkness were 36% and 40% respectively, of the baseline for control rats. Moreover, the kinase activity in rats raised in darkness was lower than that in control rats. These data suggest that Cdk5/p35^nck5a may play a role in neuronal plasticity in the developing rat retina.     

Studies of the developing chick retina using monoclonal antibody 8A2 that recognizes a novel set of gangliosides

A monoclonal antibody, Mab 8A2, that recognizes a novel set of gangliosides was produced by immunizing a mouse with Embryonic Day 14 chick optic nerve. Immunohistochemical studies of the developing chick retina revealed a complex pattern of Mab 8A2 immunoreactivity. Initially, staining is concentrated in the optic fiber layer in the central retina. Later in development, the most intense staining is seen at the periphery of the retina and 8A2 immunoreactivity appears in other retina layers. In the adult retina, 8A2 immunoreactivity is lost from the optic fiber layer but persists in the inner plexiform layer, inner nuclear layer, and outer plexiform layer. Cell culture experiments showed intense staining of neurites from retinal ganglion cells but no staining of Muller cells. Biochemical characterization of the epitope recognized by Mab 8A2 suggests that it includes a 9-O-acetyl group that is present on five different gangliosides. The 8A2 immunoreactive gangliosides are distinct from and have slower mobilities on thin-layer chromatographs than those recognized by Mab D1.1 which recognizes 9-O-acetyl GD3.     

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.