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.     

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.     

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.     

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.     

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.     

Immunocytochemical and autoradiographic localization of GABA system in the vertebrate retina

Summary The localization of -aminobutyric acid (GABA) neurons in the goldfish and the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (GAD, L-glutamate 1-carboxy-lase, EC 4.1.1.15) and by [³H] GABA uptake autoradiography. In the goldfish retina, GAD is localized in some horizontal cells (H1 type), a few amacrine cells and sublamina b of the inner plexiform layer. Results from immunocytochemical studies of GAD-containing neurons and autoradiographic studies of GABA uptake reveals a marked similarity in the labeling pattern suggesting that in goldfish retina, the neurons which possess a high-affinity system for GABA uptake also contain significant levels of GAD. In the rabbit retina, when Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In autoradiographic studies by intravitreal injection of [³H] GABA a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer was observed. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.     

Histochemical and cytochemical localization of acetylcholinesterase in retina and optic tectum of teleost fish.

The activity of cholinesterase and its cellular and subcellular localization were investigated in the retina and optic tectum of Eugerres plumieri and in the retina of Carassius carassius by means of radiometric, histochemical, and cytochemical procedures. In both fishes only the presence of acetylcholinesterase could be demonstrated. This study, besides confirming previous findings that acetylcholinesterase is located in the ganglion and amacrine cells of the retina as well as in the inner plexiform layer, in addition provides evidence that the enzyme is also present at the region of photoreceptor synapses between the cell bodies and apposing extensions of the horizontal cells of the same layer. The latter localization may indicate the involvement of a cholinergic mechanism at the functional contacts (transferapses) between the horizontal cells. In the optic tectum of Eugerres plumieri, histochemistry reveals fine distinguishable bands of acetylcholinesterase activity; two of the bands are quite sharply defined, whereas three others have rather a more diffuse appearance. The presence of these bands and their distribution may suggest a widespread distribution of cholinergic elements in the optic tectum.     

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.     

Light-and electron-microscopic studies of the somatostatin-immunoreactive plexus in the cat retina

Summary Two monoclonal antibodies directed against somatostatin 14 were used to study immunoreactive neurons, their processes and their synapses in the cat retina. In retinal whole-mounts, a sparse population of wide-field displaced amacrine cells was observed predominantly in the ventral retina and near the retinal margin. Processes of these cells ramified mainly in two distinct strata within the inner plexiform layer: one near the inner nuclear layer (INL), and the other near the ganglion cell layer (GCL). The length of immunoreactive fibres within each plexus was measured: 232±32 mm/mm² near the INL and 230±74 mm/mm² near the GCL in all retinal regions. The immunoreactive processes were studied using electron-microscopic techniques; conventional and some ribbon-containing synapses (dyads) were found. Immunolabelled processes received input synapses from other amacrine cell processes. These investigations provide further evidence that this cell population has a diffuse, regulatory or modulatory role for visual-information processing in the inner plexiform layer.     

Monoaminergic neurons of the mudpuppy retina

Summary The mudpuppy retina was investigated with the histofluorescence method of Falck and Hillarp in normal animals and in animals injected intraocularly with -methylnoradrenaline, 5,6-dihydroxytryptamine, or a combination of the two drugs. Catecholaminergic amacrine cells were found to form a thin layer of terminals at the border between the inner nuclear and the inner plexiform layers. Catecholaminergic interplexiform cells were not found. Indoleamine-accumulating amacrine cells were also observed. They are fifteen to twenty times more numerous than the catecholaminergic cells, and their terminals occur diffusely throughout the inner plexiform layer. In a number of eyes the majority of the indoleamine-accumulating terminals were eliminated with intraocular injections of the neurotoxin, 5,7-dihydroxytryptamine, but the reproducibility of this effect was not consistent. Intravitreal injections of 5,6-dihydroxytryptamine were used to label both types of neurons for electron microscopy. They were found to make conventional type synapses on amacrine cells and, less frequently, on bipolar cells.     

Distribution of calcitonin gene-related peptide immunoreactivity in the central nervous system of the forg,Rana esculenta

Summary Tyrosine hydroxylase (TH) immunocytochemistry was utilized to quantify dopaminergic synapses in the inner plexiform layer of the retina of Bufo marinus. Since dopaminergic cells have bistratified dendritic arborisation in the inner plexiform layer, attention was given to the segregation of synapses between the scleral and the vitreal sublaminae. Light-microscopically, a more elaborate dendritic branching was observed in the scleral than in the vitreal sublamina. In contrast, about 55% of synapses occurred in the vitreal one fifth of the inner plexiform layer, 30% in the scleral fifth, and 15% in the intermediate laminae. Input sources and output targets showed only minor quantitative differences between sublaminae 1 and 5. TH-immunoreactive processes were found in presynaptic (62.8%) and postsynaptic (37.2%) positions. Synapses to the stained dendrites derived from bipolar (40.4%) and amacrine (59.6%) cells, whereas outputs from the TH-positive processes were directed to amacrine cells (56.8%) and to small and medium-sized dendrites (35.4%); at least some of these can be considered as ganglion cell dendrites. TH-positive profiles neither formed synapses with each other nor were presynaptic to bipolar cell terminals. Junctional appositions of the immunoreactive profiles were occasionally seen on non-stained amacrine and ganglion cell dendrites in the scleral sublamina of the inner plexiform layer and on optic axons in the optic fibre layer. Although dopaminergic cells are mainly involved in amacrine-amacrine interactions, inputs from bipolar terminals and outputs to ganglion cell dendrites were also substantial, suggestive of a role also in vertical information processing.     

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.     

Colocalization of GLUT3 and choline acetyltransferase immunoreactivity in the rat retina

Toward elucidating the functional aspects ofGLUT3, a primary neuronal glucose transporter isoform in the vertebrate central nervous system, this study examined its expression in cholinergic amacrine cells made identifiable by the presence of acetylcholine-synthesizing enzyme, choline acetyltransferase (ChAT), in the rat retina. Double-immunofluorescence staining of adult rat retinal tissue with anti-GLUT3 and anti-ChAT antibodies revealed characteristic stratified GLUT3 immunoreactivity (GLUT3-IR) in the inner plexiform layer (IPL) that was identical to the arborization pattern of ChAT-positive neuronal processes there. In addition, approximately 30-50% of intensely GLUT3-immunoreactive cell bodies in the inner nuclear layer and ganglion cell layer showed ChAT-IR, while the majority of ChAT-positive cell bodies were also intensely GLUT3 immunoreactive. Analysis at the cellular level using retinal cells in culture revealed similar findings. These results collectively indicate that cholinergic amacrine cells constitute the major component of GLUT3-expressing cells in the rat retina. It is expected that the link demonstrated here between GLUT3 expression and cholinergic amacrine cell population will provide clues for further analyzing GLUT3 function in the retina.     

Neurofibrillar long-range amacrine cells in mammalian retinae

A distinct population of wide-field, unistratified amacrine cells are shown to be selectively stained by using neurofibrillar methods in rabbit and cat retinae. Their cell bodies may be located in the inner nuclear, inner plexiform or ganglion cell layers and they branch predominantly in stratum 2 of the inner plexiform layer. Characteristically, each cell has two or more long-range distal processes which extend for 2-3 mm beyond a more symmetrical, proximal dendritic field of 0.6-0.8 mm diameter. Although the neurofibrillar long-range amacrines account for less than 1 amacrine in 500, they achieve effective coverage of the retina by both the proximal and distal dendrites.     

Electron microscopic observation of pituitary adenylate cyclase-activating polypeptide (PACAP)-containing neurons in the rat retina

The distribution and localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the rat retina were studied by immunocytochemistry with both light and electron microscopy. PACAP-like immunoreactivity (PACAP-LI) was detected in the amacrine and horizontal cells as well as in the inner plexiform layer, the ganglion cell layer and the nerve fiber layer. PACAP-LI seemed to be concentrated predominantly in the neuronal perikarya and their processes, but not in other cells in the retina. At the ultrastructural level, PACAP-LI was visible in the plasma membranes, rough endoplasmic reticulum, and cytoplasmic matrix in the PACAP-positive neurons in the inner nuclear layer. In the inner plexiform layer, PACAP-positive amacrine cell processes made synaptic contact with immunonegative amacrine cell processes, bipolar cell processes, and ganglion cell terminals. These findings suggest that PACAP may function as a neurotransmitter and/or neuromodulator.     

Immunohistochemical and quantitative analysis of 5-hydroxytryptamine in the retina of some vertebrates

5-Hydroxytryptamine immunoreactive neurons were found in retinae from chicken, pigeon, frog and goldfish. They were localized among the amacrine cells with a distribution of cell bodies and nerve fibres that varied with the species. In chicken and pigeon, bipolar-like cell bodies were also found in the middle of the inner nuclear layer, sending processes inwards to the inner plexiform layer and outwards to the horizontal cells. The signalling direction of these cells is doubtful. No 5-hydroxytryptamine immunoreactivity was found in retinae from cow, pig, cat, rabbit, guinea-pig, rat or mouse.Quantitative analyses were performed with HPLC on extracts from chicken, pigeon, frog and goldfish retinae. High concentrations were found in goldfish and frog whereas less, about 100 ng/g, was observed in chicken and pigeon.The results suggest that 5-hydroxytryptamine is the transmitter of a set of amacrine cells in cold-blooded vertebrates and perhaps also in birds. The transmitter of the indoleamine accumulating neurons of mammals remains to be further elucidated.     

Electron microscopy of the indoleamine-accumulating neurons in the retina of the rabbit

Summary The recently discovered indoleamine-accumulating retinal neurons were studied electron microscopically after destruction of the dopaminergic retinal neurons and subsequent labeling with 5,6-dihydroxytryptamine. These observations confirm earlier fluorescence microscopical studies on the distribution of the indoleamine-accumulating neurons in the rabbit retina. Their perikarya are known to be located in the inner nuclear layer (INL) among the amacrine cell bodies. Their processes are found only in the inner plexiform layer (IPL), most of them in the innermost third part of that layer. The indoleamine-accumulating terminals are pre- and postsynaptic to bipolar neurons in the innermost sublayer of the IPL. Reciprocal synapses are probably the rule. The synaptic vesicles of indoleamine-accumulating synapses onto bipolar cells are arranged in globular clusters around a central electron dense, round body. A number of synapses formed by unlabeled amacrine neurons with postsynaptic indoleamine-accumulating elements were also detected. These synapses were mainly found in the outermost third of the IPL. Synaptic contacts between presynaptic indoleamine-accumulating neurons and postsynaptic unlabeled processes of amacrine cells are very rare.     

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.     

Some horizontal cells of the bovine retina receive input synapses in the inner plexiform layer

Bovine retinae were stained immunocytochemically with antibodies against the calcium-binding protein, calbindin. Horizontal cells in the outer plexiform layer were heavily labelled. The processes of most horizontal cells were confined to the level of the outer plexiform layer, and the tips of their dendrites were positioned as the lateral elements of the cone triads, viz. the usual mammalian arrangement. However, some of the horizontal cells had additional thick processes descending to branch within the inner plexiform layer, where they were postsynaptic at bipolar cell dyads and where they also received input from amacrine cells. No output synapses of horizontal cells were observed in the inner plexiform layer.