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.     

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.     

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.     

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.     

Developmental study of the expression of B50/GAP-43 in rat retina

B50/GAP-43 has been implicated in neural plasticity, development, and regeneration. Several studies of axonally transported proteins in the optic nerve have shown that this protein is synthesized by developing and regenerating retinal ganglion cells in mammals, amphibians, and fish. However, previous studies using immunohistochemistry to localize B50/GAP-43 in retina have shown that this protein is found in the inner plexiform layer in adults. Since the inner plexiform layer contains the processes of amacrine cells, ganglion cells, and bipolar cells to determine which cells in the retina express B50/GAP-43, we have now used in situ hybridization to localize the mRNA that codes for this protein in the developing rat retina. We have found that B50/GAP-43 is expressed primarily by cells in the retinal ganglion cell layer as early as embryonic day 15, and until 3 weeks postnatal. Some cells in the inner nuclear layer, possibly a subclass of amacrine cells, also express B50/GAP-43 protein and mRNA; however, the other retinal neurons–bipolar cells, photoreceptors, and horizontal cells express little, if any, B50/GAP-43 at any stage in their development. Early in development, the protein appears in the somata and axons of ganglion cells, while later in development, B50/GAP-43 becomes concentrated in the inner plexiform layer, where it continues to be expressed in adult animals. These results are discussed in terms of previous proposals as to the functions of this molecule. © 1993 John Wiley & Sons, Inc.     

The spatial frequency sensitivity of bipolar cells

Retinal bipolar cells constitute the output stage of the outer layer of the retina. There are several constraints on the ability of the bipolar cell array to respond to the different spatial frequency components of the visual image, including (i) electrical coupling in the dendritic tree receiving receptor input; (iii) the "lateral inhibition" mediated by horizontal cells. Using simple mathematical models, we derive analytical expressions for the spatial frequency response of the bipolar cell array for the case in which horizontal cells are presynaptic to bipolar cells (feedforward model) and also for the case in which horizontal cells are presynaptic to receptors (feedback model). The results illustrate the importance of the three factors mentioned in determining the bipolar cells' properties. The optimal spatial frequency for stimulating the bipolar cell array, and the range of spatial frequencies transmitted onward to the inner plexiform layer, are thus related to the anatomical and electrical properties of the cells in the outer plexiform layer.     

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.     

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.     

Fine structure of the retina of black bass, Micropterus salmoides (Centrarchidae, Teleostei).

The structure of light- and dark-adapted retina of the black bass, Micropterus salmoides has been studied by light and electron microscopy. This retina lacks blood vessels at all levels. The optic fiber layer is divided into fascicles by the processes of Müller cells and the ganglion cell layer is represented by a single row of voluminous cells. The inner nuclear layer consists of two layers of horizontal cells and bipolar, amacrine and interplexiform cells. In the outer plexiform layer we observed the synaptic terminals of photoreceptor cells, rod spherules and cone pedicles and terminal processes of bipolar and horizontal cells. The spherules have a single synaptic ribbon and the pedicles possess multiple synaptic ribbons. Morphologically, we have identified three types of photoreceptors: rods, single cones and equal double cones which undergo retinomotor movements in response to changes in light conditions. The cones are arranged in a square mosaic whereas the rods are dispersed between the cones.     

Reorganization of horizontal cell processes in the developing FVB/N mouse retina

We investigated the morphological changes of horizontal cells after postnatal photoreceptor degeneration in the developing FVB/N mouse retina, using immunocytochemistry with anti-calbindin D-28K. From postnatal day 14 (P14) onwards, processes emerging from horizontal cells descend into the inner plexiform layer (IPL) and ramify mainly in stratum 1 of the IPL. Electron microscopy revealed that the descending processes make synaptic contacts with bipolar cells in the outer plexiform layer. Our results clearly demonstrate that loss of photoreceptor cells induces the reorganization of horizontal cell processes in the retinas of FVB/N mice as they mature.     

A model for the mechanisms of sensitivity control in the submammalian vertebrate retina

A model is proposed for the mechanisms of sensitivity control at the outer and inner plexiform layers in the submammalian vertebrate retina on the basis of Werblin's results and other physiological results. The model is especially based on the following suggestions: The signal that acts to shift the bipolar curves is probably carried by horizontal cell processes extending from the surround to the center of the receptive field. Furthermore, amacrine cells carry a lateral antagonistic signal across the inner plexiform layer that affects the response properties of ganglion cells. The simulations of the model were made and the results of the ones considerably coincided with the experimental results of Werblin.     

Morphology and synaptic connectivity of nitric oxide synthase-immunoreactive neurons in the guinea pig retina

Immunocytochemical methods with an antiserum against neuronal nitric oxide synthase (NOS) were applied to identify the morphology and synaptic connectivity of NOS-like immunoreactive neurons in the guinea pig retina. In the present study, two types of amacrine cells were labeled with anti-NOS antisera. Type 1 cells had large somata located in the inner nuclear layer (INL) with long, sparsely branched processes ramifying mainly in stratum 3 of the inner plexiform layer (IPL). The somata of type 2 cells (smaller diameters) were located in the INL. Some displaced amacrine cells in the ganglion cell layer were labeled. The soma size of the displaced amacrine cells was similar to that of the type 2 amacrine cells. However, processes originating from type 2 amacrine cells and displaced amacrine cells stratified mainly in strata 1 and 5, respectively. Some cone bipolar cells were weakly NOS-immunoreactive. The synaptic connectivity of NOS-like immunoreactive amacrine cells was identified in the IPL by electron microscopy. NOS-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in all strata of the IPL. The most frequent postsynaptic targets of NOS-immunoreactive amacrine cells were other amacrine cell processes. Cone bipolar cells were postsynaptic to NOS-labeled amacrine cells in all strata of the IPL. Labeled amacrine cells synapsing onto ganglion cells were found only in sublamina b. A few synaptic contacts were observed between labeled cell processes. In the outer plexiform layer, dendrites of labeled bipolar cells made basal contact with cone pedicles or formed a synaptic triad opposed to a synaptic ribbon of cone pedicles.     

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.     

Calbindin and calretinin immunoreactivities in the retina of a chondrostean,Acipenser baeri

The distribution of calbindin and calretinin in the retina of the sturgeon Acipenser baeri was studied with immunocytochemistry. Western blot analysis of brain extracts, together with immunocytochemical results in the retina and brain, indicated the presence of the two calcium-binding proteins in sturgeon. Calbindin immunocytochemistry revealed only a large displaced bipolar cell type with narrowly stratified axons, similar to some mixed rod and cones bipolar cells described in teleosts. The plexus formed by the axons of these cells in the inner plexiform sublayer was similar to that formed by calbindin-immunoreactive diffuse bipolar cells of some mammals. Calretinin immunocytochemistry also stained these displaced bipolar cells, most ganglion cells including displaced ganglion cells (Dogiel cells), and some amacrine cells of the inner nuclear layer. The distribution of calbindin and calretinin immunoreactivities in the retina of a primitive bony fish indicates that these proteins are highly specific to the cell type.     

Transient synaptic ribbons in the mammalian retina at unusual sites

In the vertebrate retina the presence of synaptic ribbons (SRs) is well documented in two sites only, viz., in photoreceptor axon terminals in the outer plexiform layer and in bipolar cell axons in the inner plexiform layer. The present paper reports the presence of non-photoreceptor SRs in the outer plexiform layer of cattle and mouse, where they were seen in small numbers in thin cell processes near cone pedicles of light-adapted animals. They were never seen near rod spherules. Quantitative data obtained in mice killed at different time-points revealed that the SRs under consideration increased in number during day time and were absent during the dark phase. Moreover, under high light intensity of 10000 lux they were more frequent in number compared to 100-lux-exposed animals. It is concluded that the cell processes revealing the temporary presence of SRs are processes of flat bipolar cells which may provide a feedback to cones during the light phase.     

Synaptic relationships in the plexiform layers of carp retina

Summary The synaptic contacts made by carp retinal neurons were studied with electron microscopic techniques. Three kinds of contacts are described: (1) a conventional synapse in which an accumulation of agranular vesicles is found on the presynaptic side along with membrane densification of both pre- and postsynaptic elements; (2) a ribbon synapse in which a presynaptic ribbon surrounded by a halo of agranular vesicles faces two postsynaptic elements; and (3) close apposition of plasma membranes without any vesicle accumulation or membrane densification.In the external plexiform layer, conventional synapses between horizontal cells are described. Horizontal cells possess dense-core vesicles about 1,000 Å in diameter. Membranes of adjacent horizontal cells of the same type (external, intermediate or internal) are found closely apposed over broad regions.In the inner plexiform layer ribbon synapses occur only in bipolar cell terminals. The postsynaptic elements opposite the ribbon may be two amacrine processes or one amacrine process and one ganglion cell dendrite. Amacrine processes make conventional synaptic contacts onto bipolar terminals, other amacrine processes, amacrine cell bodies, ganglion cell dendrites and bodies. Amacrine cells possess dense-core vesicles. Ganglion cells are never presynaptic elements. Serial synapses between amacrine processes and reciprocal synapses between amacrine processes and bipolar terminals are described. The inner plexiform layer contains a large number of myelinated fibers which terminate near the layer of amacrine cells.This work was supported by an N.I.H. grant NB 05404-05 and a Fight for Sight grant G-396 to P.W. and N.I.H. grant NB 05336 to J.E.D. The authors wish to thank Mrs. P. Sheppard and Miss B. Hecker for able technical assistance. P.W. is grateful to Dr. G. K. Smelser, Department of Ophthalmology, Columbia University, for the use of his electron microscope facilities.     

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.     

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.     

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.     

Immunohistochemical localization of GABAA receptors in the scotopic pathway of the cat retina

The distribution of GABA_A receptors in the inner plexiform layer of cat retina was studied using monoclonal antibodies against the 2/3 subunits. A dense band of receptor labeling was found in the inner region of the inner plexiform layer where the rod bipolar axons terminate. Three forms of evidence indicate that the GABA_A receptor labeling is on the indoleamine-accumulating, GABAergic amacrine cell that is synaptically interconnected with the rod bipolar cell terminal. (1) Electron microscopy showed that the anti-GABA_A receptor antibody (62-3G1) labeled profiles that were postsynaptic to rod bipolar axons and made reciprocal synapses. (2) Indoleamine uptake (and the subsequent autofluorescence) combined with GABA_A receptor immunohistochemistry showed co-localization of the two markers in half of the receptor-positive amacrine cells. (3) Double labeling demonstrated that half of the receptor-positive somata also contained GABA. These results indicate that a GABAergic amacrine cell interconnected with the rod bipolar cell, most likely the so-called A17 amacrine cell, itself bears GABA_A receptors.