Dopaminergic interplexiform cells and centrifugal fibres in the Xenopus retina.

Summary Putative dopaminergic neurons in theXenopus retina were identified using an immunoreaction against tyrosine hydroxylase. A single class of cell was stained whose perikaryon (12–15 m in diameter) was located at the border of the inner nuclear and inner plexiform layers. About 2% of the stained cell bodies were located in the ganglion cell layer, but the distribution of the processes of displaced cells had the same geometry as for the majority of stained cells. Tyrosine hydroxylase-like immunoreactive perikarya gave rise to one to four stout processes that descended to the most proximal level of the inner plexiform layer, within which they branched repeatedly to generate a diffuse network of fine processes. Secondary branches ascended to the most distal sublayer of the inner plexiform layer where they ramified into fine processes that joined other fibres arising horizontally from the cell body and confined to the distal inner plexiform layer throughout their course. The diameter of the dendritic arbor of stained cells was in the range of 350–600 m. The dense network of fine fibres within the distal inner plexiform layer was arrayed in rings that surrounded other amacrine cells; using an antiserum against glycine we found that at least some of these were glycinergic neurons. Most tyrosine hydroxylase-positive neurons emitted one or two fine ascending processes that arose from the perikaryon, traversed the inner plexiform layer and arborized within the outer plexiform layer. Additionally, fine varicose fibres arising from the sublayer 1 of the inner plexiform layer and running to the outer retina were observed. Thus, based on light microscopic criteria, dopaminergic neurons in theXenopus retina appeared to be interplexiform cells. A few tyrosine hydroxylase-immunoreactive fibres were observed in the optic nerve, some of which entered the inner retina where they ramified, thus indicating that they were centrifugal axons. In addition, a small number of stout smooth processes were observed to traverse the entire inner nuclear layer and course laterally at the level of the photoreceptor bases. Whether this second class of ascending process arises from the tyrosine hydroxylase-like immunoreactive efferents remains to be determined. The total number of dopaminergic neurons per retina was 750–800, equivalent to an average density of 30 cells mm^–2. The dendritic fields of adjacent cells strongly overlapped, with an estimated coverage factor of 4.8.     

Synaptic organization of type 2 catecholamine amacrine cells in the rhesus monkey retina

Summary Two types of amacrine cell immunoreactive for tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthetic pathway, are present in the retina of the rhesus monkey,Macaca mulatta. The well-known dopaminergic, or type 1 catecholamine amacrine cells have relatively large cell bodies almost exclusively in the inner nuclear layer with processes that densely arborize in the outermost stratum of the inner plexiform layer and fine, radially-oriented fibres in the inner nuclear layer. Type 2 catecholamine amacrine cells, in contrast, have smaller cell bodies in the inner nuclear layer, the inner plexiform layer and the ganglion cell layer, and have sparsely-branching processes ramifying in the centre of the inner plexiform layer. Although type 2 catecholamine cells are more numerous than type 1 catecholamine amacrines, type 2 cells contain less than one-third the amount of tyrosine hydrolase as the type 1 cells. Electron microscopy of retinal tissue immunoreacted for tyrosine hydrolase by the peroxidase-antiperoxidase method revealed synaptic input from amacrine cells at conventional synapses, and bipolar cells at ribbon synapses onto the type 2 catecholamine amacrine cells. Curiously, although the synaptic input is comparatively easily found, the output synapses, or synapses of the type 2 catecholamine amacrine cells onto other neuronal elements, are rarely found. Some synapses of the type 2 catecholamine cells onto non-immunoreactive amacrine cells have been identified, however. This unusual pattern of synaptic organization, with many identifiable input synapses but few morphologically characterizable output synapses, suggests a paracrine function for the dopamine released by the type 2 catecholamine amacrine cells in the primate retina.     

Morphology and retinal distribution of tyrosine hydroxylase-like immunoreactive amacrine cells in the retina of developingXenopus laevis

Summary The development of neurons immunoreactive to tyrosine hydroxylase (TH-IR) in the retina ofXenopus laevis was investigated from stage 53 tadpoles to adult, by using an antibody against tyrosine hydroxylase. At all developmental stages, most of the immunoreactive somata were located in the inner nuclear layer, and a few in the ganglion cell layer. Immunoreactive processes arborised in the scleral and vitreal sublaminae of the inner plexiform layer, indicating that these cells were bistratified amacrine cells. However, occasionally a few immunoreactive processes were observed projecting to the outer plexiform layer, suggesting the presence of THIR interplexiform cells. The number of immunoreactive amacrine cells in the inner nuclear layer per retina increased from 204 at stage 53 tadpole to 735 in adult, while the number of immunoreactive amacrine cells in the ganglion cell layer did not change significantly over the same period. Retinal area increased from 1.95 mm² at stage 53 to 23.40 mm² in the adult, and correspondingly cell density in the inner nuclear layer decreased from 104/mm² to 31/mm². At all stages there was an increasing density towards the ciliary margin, but this gradient decreased with age. The soma size of immunoreactive amacrine cells increased with age, and was consistently larger in the central than in the peripheral retina. Dendritic field size was estimated to increase 13-fold, from stage 53 to adult. This study shows that tyrosine hydroxylase-like immunoreactive amacrine cells are generated continuously throughout life, that after metamorphosis the retina grows more by stretching than by cell generation at the ciliary margin, and that the increase of dendritic field size is proportional to the increase in retinal surface area.On leave from Department of Anatomy, Zhanjiang Medical College, Guangdong, People's Republic of China     

Synapsin I expression in the rat retina during postnatal development

Summary The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.Abbreviations GCL ganglion cell layer - INB inner neuroblast layer - INL inner nuclear layer - IPL inner plexiform layer - ONB outer neuroblast layer - ONL outer nuclear layer - OPL outer plexiform layer     

Putative dopamine-containing cells in the retina of seven species demonstrated by tyrosine hydroxylase immunocytochemistry

Immunocytochemistry with antibodies to catecholamine synthesizing enzymes has revealed cells in the retina of chick, mouse, hamster, rat, guinea-pig, piglet and marmoset which contain tyrosine hydroxylase but not dopamine beta-hydroxylase. These findings suggest that the cells in question produce dopamine but that catecholamine synthesis does not proceed further to noradrenaline. Tyrosine hydroxylase-containing amacrine cells, located in the innermost part of the inner nuclear layer, were present in all the species studied. Some species showed atypically located amacrine cells in the inner plexiform or ganglion cell layer. In the rodents, the existence of tyrosine hydroxylase-containing interplexiform cells was suggested by the presence of a few short immunoreactive ascending processes. Three different morphological types of putative dopamine-containing cells were classified according to the level of ramification.     

Cholinergic amacrine cells of the chicken retina: a light and electron microscope immunocytochemical study

Cholinergic amacrine cells of the chicken retina were detected by immunohistochemistry using an antiserum against affinity-purified chicken choline acetyltransferase. Three populations of cells were detected: type I cholinergic amacrine cells had cell bodies on the border of the inner nuclear and inner plexiform layers and formed a prominent laminar band in sublamina 2 of the inner plexiform layer, while type II cholinergic amacrine cells had cell bodies in the ganglion cell layer, and formed a prominent laminar band in sublamina 4 of the inner plexiform layer. Type III cholinergic amacrine cell bodies were located towards the middle of the inner nuclear layer, and their processes were more diffusely distributed in sublaminas 1 and 3-5 of the inner plexiform layer. Type I and type II cells were present at densities of over 7000 cells/mm2 in central areas declining to less than 2000 cells/mm2 in the temporal retinal periphery. The cells were organized locally in a non-random mosaic, with regularity indices ranging from 3 peripherally to over 5 centrally. Neither at the light nor electron microscopic levels was a lattice of cholinergic dendrites of the kind reported by Tauchi and Masland [J. Neurosci. 5, 2494-2501 (1985)] detectable. Within the two prominent dendritic plexuses, a major feature of the synaptic interactions of the type I and type II cholinergic cells was extensive synaptic interaction between cholinergic processes. Apart from this, there was little, if any, input to cholinergic processes from non-cholinergic amacrine cells, but there was input from bipolar cells. Output from the cholinergic amacrine cell processes was directed towards non-cholinergic amacrine cells as well as other cholinergic amacrine cells, and ganglion cells.     

Enzyme histochemical identification of microglial cells in the retina of a fish (Tinca tinca)

Histochemistry for nucleoside diphosphatase was used to study the microglial cells in the adult tench retina. An abundant population of microglial cells was located in the vascular membrane, nerve fibre layer, inner and outer plexiform layers and scattered cells were observed in the inner nuclear layer. Rounded and amoeboid cells could be seen close to the vessel in the vascular membrane, bipolar cells in the nerve fibre layer and ramified cells in the rest of the layers. Several microglial forms could correspond to developing cells. The pattern of distribution was similar to that described in other vertebrates, but with several differences, such as the presence of microglial cells in the vascular membrane and inner nuclear layer and the overlap of processes in the plexiform layers.     

Peptide histidine-isoleucine (PHI)-immunoreactive amacrine cells in the retina of the rat

Peptide histidine-isoleucine (PHI) immunoreactivity was located in amacrine-like cells in adult rat retina by use of immunohistochemical techniques. Immunoreactive somata were found in the proximal part of the inner nuclear layer. From these somata, processes could be followed into the inner plexiform layer. The terminals of these processes were mainly found in the sublayers, 1, 2, and 3, but a few terminals were also present in the other inner plexiform sublaminae. The distribution of PHI-immunoreactive somata and processes corresponds with the cellular distribution of vasoactive intestinal peptide (VIP) and indicates a possible co-localization with this peptide.     

Dopaminergic transmission in the rat retina: evidence for volume transmission

The study was designed to determine whether dopaminergic neurotransmission in the retina can operate via volume transmission. In double immunolabelling experiments, a mismatch as well as a match was demonstrated in the rat retina between tyrosine hydroxylase (TH) and dopamine (DA) immunoreactive (ir) terminals and cell bodies and dopamine D2 receptor-like ir cell bodies and processes. The match regions were located in the inner nuclear and plexiform layers (D2 ir cell bodies plus processes). The mismatch regions were located in the ganglion cell layer, the outer plexiform layer, and the outer segment of the photoreceptor layer, where very few TH ir terminals can be found in relation to the D2 like ir processes. In similar experiments analyzing D1 receptor like ir processes versus TH ir nerve terminals, mainly a mismatch in their distribution could be demonstrated, with the D1 like ir processes present in the outer plexiform layer and the outer segment where a mismatch in D2 like receptors also exists. The demonstration of a mismatch between the localization of the TH terminal plexus and the dopamine D2 and D1 receptor subtypes in the outer plexiform layer, the outer segment and the ganglion cell layer (only D2 immunoreactivity (IR)) suggests that dopamine, mainly from the inner plexiform layer, may reach the D2 and D1 mismatch receptors via diffusion in the extracellular space. After injecting dopamine into the corpus vitreum, dopamine diffuses through the retina, and strong catecholamine (CA) fluorescence appears in the entire inner plexiform layer and the entire outer plexiform layer, representing the match and mismatch DA receptor areas, respectively. The DA is probably bound to D1 and D2 receptors in both plexiform layers, since the DA receptor antagonist chlorpromazine fully blocks the appearance of the DA fluorescence, while only a partial blockade is found after haloperidol treatment which mainly blocks D2 receptors. These results indicate that the amacrine and/or interplexiform DA cells, with sparse branches in the outer plexiform layer, can operate via volume transmission in the rat retina to influence the outer plexiform layer and the outer segment, as well as other layers of the rat retina such as the ganglion cell layer.     

Histology of the ferret retina

The retina of the adult ferret, Mustelo furo, was studied with light and transmission electron microscopy to provide an anatomical basis for use of the ferret as a model for retinal research. The pigment epithelium is a simple cuboidal layer of cells characterized by a zone of basal folds, apical microvilli, and pigment granules at various stages of maturation. The distinction between rod and cone photoreceptor cells is based on their location, morphology, heterochromatin pattern and the electron density of their inner segments. The round, light-staining cone cell nuclei occupy the layer of perikarya along the apical border of the outer nuclear layer. The remainder of the outer nuclear layer consists of oblong, deeply-stained rod cell nuclei. Ribbon type synaptic complexes involving photoreceptor cell axons, horizontal cell processes, and bipolar cell dendrites characterize the outer plexiform layer. The inner nuclear layer is comprised of horizontal, bipolar, and amacrine cell perikarya as well as the perikarya of the Müller cells. The light-staining horizontal cell nuclei are prominent along the apical border of the inner nuclear layer. The light-staining amacrine cell nuclei form a more or less continuous layer along the basal border of the inner nuclear layer. Both conventional and ribbon-type synapses characterize the inner plexiform layer. The ganglion cells form a single cell layer. The optic fiber layer contains bundles of axons surrounded by Müller cell processes. Small blood vessels and capillaries are present in the basal portion of the retina throughout the region extending from the internal limiting membrane to the outer plexiform layer. The adult one-year-old retina is compared with the retina at the time of eye opening.     

Dopamine- and adenosine-3':5'-monophosphate (cAMP)-regulated phosphoprotein of Mr 32,000 (DARPP-32) in the retina of cat, monkey and human.

The cellular localization of a dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) was investigated in cat, monkey and human retina by immunohistochemistry. In cat, DARPP-32-immunoreactive cell bodies identified as Müller cells were demonstrated in the inner nuclear layer (INL) with processes closely surrounding the cell soma of photoreceptors in the outer nuclear layer. Some DARPP-32-IR cells were also seen in the nerve fiber layer (NFL) sending processes to the inner plexiform layer. In monkey and human retina, DARPP-32-IR cell bodies were also demonstrated in the INL, with few cells located in the NFL.     

Demonstration of a substance P-like immunoreactivity in retinal cells of the rat

The distribution of substance P (SP)-like immunoreactivity in the rat retina was investigated by immunohistochemistry. SP-positive cells were found throughout the retina. The majority of them were located in the proximal portion of the inner nuclear layer and the processes from these cells directed to the inner plexiform layer where they ramified, suggesting that SP-positive cells located in this region probably are amacrine cells. Few SP-positive cells were seen within the ganglion cell layer. They were considered displaced amacrine cells.     

Electron microscopy of glutamate decarboxylase (GAD) immunoreactivity in the inner plexiform layer of the rhesus monkey retina

Summary With indirect immunofluorescence, glutamate decarboxylase (GAD), the GABA synthesizing enzyme, was localized to cell bodies in the inner half of the inner nuclear layer and a few in the outer tier of the ganglion cell layer in the rhesus monkey retina. In the inner plexiform layer there were three strongly GAD-immunoreactive laminae separated by two less immunoreactive laminae. Electron microscopy demonstrated that the GAD was contained in amacrine cells and these GAD-immunoreactive amacrines were primarily pre- and postsynaptic to biopolar cell axon terminals. The GAD-containing processes possessed small synaptic vesicles and formed synapses that could be characterized as symmetrical. Large, dense-cored vesicles were often found in the cell bodies and synaptic processes of the GAD-immunoreactive amacrine cells. As the vast majority of the synaptic input and output of the GAD-containing amacrine cells was to and from bipolar cells and the strongest GAD-immunoreactivity correlated with the endings of bipolar cells that connect with a single cone, the functional effects of GABA in the primate retina are likely to be found in the responses of single cone pathways in the inner plexiform layer.     

Discrete distributions of putative cholinergic and somatostatinergic amacrine cell dendrites in chicken retina

The distributions of putative cholinergic and somatostatinergic amacrine cells of the chicken retina were compared. Acetylcholinesterase-positive amacrine cell bodies were concentrated at the border between the inner nuclear and plexiform layers. Similar amacrine cell bodies were detected in a displaced position in the ganglion cell layer. Both populations had dendrites joining the 4 bands of acetylcholinesterase activity in the inner plexiform layer. The cell bodies of somatostatin-immunoreactive amacrine cells were distinct from the intensely acetylcholinesterase-positive cell bodies. The immunoreactive terminal bands did not overlap the acetylcholinesterase-positive bands, except in the inner parts of the inner plexiform layer.     

Demonstration of a neuropeptide Y (NPY)-like immunoreactivity in the pigeon retina

The distribution of neuropeptide Y (NPY)-like immunoreactivity in the pigeon retina was investigated by fluorescence immunohistochemistry. NPY-positive cells were found in central and peripheral retina. NPY somata were located in the proximal portion of the inner nuclear layer and their processes directed to the inner plexiform layer where they ramified in 3 immunoreactive bands. NPY might play a role as a neurotransmitter or neuromodulator in the pigeon retina.     

Immunohistochemical evidence for epinephrine-containing retinal amacrine cells

The enzyme for the synthesis of epinephrine, phenylethanolamine-N-methyltransferase, has been localized, by an indirect immunofluorescent staining method, to a subpopulation of amacrine cells in the rat retina. The immunoreactive cells are located primarily in the inner nuclear layer and send a single process to the inner plexiform layer. Most of the immunoreactivity is found in the center of the inner plexiform layer. A small percentage of immunoreactive cell bodies were found in the inner plexiform layer and occasionally cells were observed in the ganglion cell layer. These epinephrine-containing amacrine cells are morphologically distinct from the dopamine-containing amacrine cells previously described by formaldehyde fluorescence and we speculate from reports in the literature that epinephrine-containing amacrine cells may play a role in modulating the activity of dopamine-containing amacrine cells.     

Localization of substance P-like immunoreactivity in the adult and developing goldfish retina

Substance P-like immunoreactivity was localized to amacrine cells in both adult and developing goldfish retina using immunohistochemical techniques. These studies utilized a well-characterized monoclonal antiserum directed to substance P. Specificity was established by absorption of the anti-serum with 10 μm synthetic substance P. Specific substance P-like immunoreactivity was localized within a seemingly distinct population of unistratified amacrine cells which were distributed in both central and peripheral retinal regions. The immunoreactive somata were located at the border of the inner nuclear layer and inner plexiform layer and were characterized by a round or ovoid somata which measured about 9μm in diameter. These immunoreactive amacrine cells typically had a single process which descended to and ramified within lamina 3 of the inner plexiform layer.Specific substance P-like immunoreactivity first appeared 60 h after hatching (stage 27) within both somata and processes located in differentiated retinal regions. No substance P-like immunoreactive somata or processes were observed in undifferentiated retinal regions. In retinas from stage 27 to 14 days after hatching, the immunoreactive somata were characterized by an ellipsoidal soma and a large nucleus devoid of immunoreactivity. These immunoreactive cells were also characterized by a single process that descended to and ramified within lamina 3 of the differentiated inner plexiform layer. At 30 days after hatching, the substance P-containing cells were identical in appearance to these same cell types observed within the adult retina.     

Neuronal and glial localization of homocysteate-like immunoreactivity in the rat retina

Summary To study the distribution ofl-homocysteate in the rat retina, specific polyclonal and monoclonal anti-homocysteate antibodies have been used in combination with a highly sensitive postembedding method for light microscopic immunocytochemistry. In central and peripheral retina, the most strongly immunoreactive cell bodies lay in the inner nuclear layer. They represented about 17% of the total neuronal cell population of the layer and were identified as bipolar cells (19–20% of cells in the outer half of the inner nuclear layer) and amacrine cells (15% of cells in the inner half of the inner nuclear layer). A third cell type showing heavy homocysteate-like immunoreactivity was identified as Müller glial cells. Characteristically, their descending processes formed three immunoreactive bands in the inner plexiform layer. Furthermore, the outer and inner limiting membranes as well as glia around and between ganglion cell axons and in the vicinity of blood vessels were labelled intensely. Photoreceptors and their terminals, and ganglion cells, were not immunostained. These findings indicate the presence of homocysteate in some bipolar and amacrine cells of the inner nuclear layer and support a role for this sulphur-containing excitatory amino acid as a neurotransmitter candidate in the retina.