Colocalization of GAD-like immunoreactivity and 3H-GABA uptake in amacrine cells of rabbit retina

Rabbit retinas were double labeled to determine the degree of colocalization of glutamic-acid-decarboxylase-like immunoreactivity (GAD-like IR) and 3H-GABA uptake using light (LM) and electron microscopic (EM) autoradiography. Both GAD-like IR and 3H-GABA uptake were found in amacrine cell bodies in the inner nuclear layer (INL) as well as in cell bodies in the ganglion cell layer (GCL), and throughout the inner plexiform layer. GAD-like IR was found in 32% of the amacrine cells in the INL, 86% of which also showed 3H-GABA uptake; 3H-GABA uptake was observed in 38% of the amacrine cells. However, only 72% of these cells showed GAD-like IR. Labeled cells in the GCL were only 10-15% as common as similarly labeled cells in the INL. As in the INL, all GAD-positive cells in the GCL were double labeled, but only 53% of the cells taking up 3H-GABA were double labeled. We suggest that labeled cells in the GCL were ganglion cells rather than displaced amacrine cells. Cells, in both the INL and GCL, that showed 3H-GABA uptake but no GAD-like IR had a higher average grain density than double-labeled cells, indicating that uptake by these cells was specific. The relevance to GABAergic function of 3H-GABA uptake without an indication of GAD-like IR is yet to be determined. Statistical analysis at the EM level showed that one-third of the GAD-positive synaptic terminals of amacrine cells were double labeled after a 4-month exposure. Longer exposures at the EM level should reveal a higher percentage of GAD-positive terminals because at the LM level, one-half of the double-labeled cell bodies were "lightly" labeled with grains. The high degree of colocalization of GAD-like IR and 3H-GABA uptake suggests that both markers may be useful for labeling GABAergic neurons in the rabbit retina.     

Glycinergic neurons in the human retina

Neurotransmitter-specific properties of glycinergic neurons in the human retina were studied using 11 pairs of eyes from donors ranging from 2 1/2 to 54 years in age. A mean endogenous level of 10.3 nmoles glycine per mg protein was measured by amino acid analysis in retinas isolated within 1 hour postmortem. When retinas were incubated with 3H-glycine (2 microM) and processed for autoradiography, label was found associated with neurons whose somata reside within the inner nuclear layer. Some heavily labeled neurons located at the vitread border of the inner nuclear layer were identified as amacrine cells based on ultrastructural verification of the conventional synaptic contacts made by their processes in distal regions of the inner plexiform layer. In proximal regions of the inner plexiform layer, dendrites of glycine-accumulating amacrine cells were postsynaptic to both ribbon and conventional synaptic contacts, suggesting input from bipolar and other, nonglycinergic amacrine cells. Their density (30 +/- 11 S.D. cells/mm linear retinal expanse) tended to be greater toward the central fundus. A second population of lightly labeled, probable bipolar cells was present in the middle of the inner nuclear layer; the density of this second set of glycine-accumulating cells approximated that of the heavily labeled population from the fovea, centrally, to the ora serrata, peripherally. Release of either accumulated or endogenous glycine was elicited by K+-depolarization in a Ca2+-dependent manner. Tissue fragments exposed for 6 minutes to normal medium, 40 mM K+-substituted medium, or K+-substituted medium with Co2+, release endogenous glycine into each bathing solution in average amounts of 0.6, 2.6, and 0.7 nmoles per mg protein, respectively. Together these data strongly implicate glycine as a neurotransmitter in the human retina.     

Synaptic organization of substance P-like immunoreactive amacrine cells in goldfish retina

A class of amacrine cells in the goldfish retina displays substance P-like immunoreactivity (SPIR). We studied the synaptic organization of SPIR amacrine cells by electron microscopical immunocytochemistry. Amacrine cells showing SPIR have processes which ramify in a very narrow band in layer 3 of the inner plexiform layer. SPIR is restricted to large dense-cored vesicles (DCVs), which are distributed throughout the dendrites. Processes labeled with SPIR contain a mixture of DCVs and numerous small agranular vesicles. Of 88 synaptic contracts analyzed, SPIR processes occurred as the presynaptic element 57 times and as the postsynaptic element 31 times. SPIR processes made synapses upon amacrine and ganglion cell dendrites with equal frequency and received synaptic input from both amacrine and bipolar cells. The stratification of SPIR amacrine cells in proximal sublamina a suggests that their synaptic interactions are restricted to "off" and "on-off" neurons. However, this is in contrast to published electrophysiological data. Possible explanations for this discrepancy are discussed in detail.     

Midget ganglion cells of the parafovea of the human retina: a study by electron microscopy and serial section reconstructions.

In this study we used serial section electron microscopy and three-dimensional reconstructions to examine four midget ganglion cells of the human retina. The four cells were located in the parafoveal retina 2.5 mm or 8 degrees from the foveal center. Both type a (with dendritic trees in distal inner plexiform layer) and type b (with dendritic trees in proximal inner plexiform layer) midget ganglion cells have been studied. These cells have dendritic trees of 7-9 microns diameter, and their complete dendritic trees in the neuropil of the inner plexiform layer can be analyzed, as well as the bipolar cell axon terminals having synaptic input, by a study of 100-150 serial ultrathin sections. Type a midget ganglion cells appear to be in a one-to-one relationship with flat midget bipolar cell axon terminals ending in distal inner plexiform layer. Type b midget ganglion cells are in a one-to-one synaptic relationship with invaginating midget bipolar cell axon terminals in proximal inner plexiform layer. The midget bipolar cells primarily involved with the midget ganglion cells do not contact other ganglion cell dendrites. In other words, midget bipolar cells appear to be in exclusive contact with single midget ganglion cells in the human retina. The midget ganglion cells receive most of their input from their associated midget bipolar cells in the form of ribbon synapses at dyads or monads (55-81 ribbons total), although ribbonless synapses are seen occasionally. In all four midget ganglion cells reconstructed, one or two other bipolar cell axon terminals, presumed to be from wide-field bipolar types, provide 1-3 ribbon synapses each. The number of amacrine synapses upon a midget ganglion cell's dendritic tree is approximately equal to the number of bipolar ribbon inputs (43%-56% bipolar ribbons: 44%-57% amacrine synapses). We assume from our knowledge of response characteristics of ganglion cells in other mammalian retinas (Nelson et al., '78: J. Neurophysiol. 41:427-483), that the type a midget ganglion cell and its exclusive connectivity with a flat midget bipolar cell forms a single cone connected OFF-center pathway, whereas the type b midget ganglion cell with its exclusive connectivity to an invaginating midget bipolar cell forms a single cone connected ON-center pathway, through the retina to the brain.     

Stimulation with N-methyl-D-aspartate or kainic acid increases cyclic guanosine monophosphate-like immunoreactivity in turtle retina: Involvement of nitric oxide synthase

In brain and retina, stimulation with excitatory amino acids (EAA) can generate nitric oxide (NO) and increase levels of cyclic guanosine monophosphate (cGMP). Because nitric oxide synthase (NOS) has been found in retinas of all species examined to date, an NO signal-transduction pathway is likely to be present in all retinas. We tested the hypothesis that stimulation of ionotropic glutamate receptors in turtle retina would result in increases in cGMP through an NOS/NO/cGMP pathway. Following in vitro incubations of turtle eye cups with the glutamate receptor agonists, N-methyl-D-aspartate (NMDA) or kainic acid (KA), we quantified the increases in cGMP-like immunoreactivity (cGMP-LI) by using enzyme-linked immunosorbant assay (ELISA) and localized the increased cGMP-LI by using an antibody against cGMP. Stimulation with NMDA or KA increased cGMP-LI in bipolar and amacrine cells as well as in some somata in the ganglion cell layer. Either KA or NMDA produced statistically significant increases in total retinal cGMP-LI by ELISA. To test the involvement of NO, we used the NOS inhibitors 7-nitroindazole and L-nitroarginine. Both inhibitors blocked virtually all of the KA- or NMDA-stimulated increases in cGMP-LI. These results indicate that activation of ionotropic glutamate receptors can increase cGMP in select retinal neurons. Differences between the agonist-evoked increases of retinal cGMP-LI suggest that there can be specificity in the activation of the NOS/NO/cGMP signal-transduction pathway by glutamate. This suggests that, in addition to short-term electrical changes, activation of ionotropic glutamate receptors also may produce longer term modulatory or metabolic effects involving NO/cGMP. J. Comp. Neurol. 404:75–85, 1999. © 1999 Wiley-Liss, Inc.     

Catecholaminergic and cholinergic neurons in the developing retina of the rat

We have examined the development of catecholaminergic and cholinergic neurons in the retina of the rat by using antibodies against the enzymes tyrosine hydroxylase (TH) and choline acetyl transferase (ChAT), respectively. TH-immunoreactivity was first detected at P (postnatal day) 3 in somata located in the inner part of the cytoblast layer (CBL) and in fine dendrites extending toward the middle of the inner plexiform layer (IPL). These cells were similar in shape and soma size to the class 2 TH-immunoreactive (TH-IR) cells of the adult rat. At P6, TH-immunoreactivity was expressed by a second population of cells. Their somata were in the inner part of the inner nuclear layer (INL), but were distinctly larger, with short thick dendrites extending into the outer and/or middle parts of the IPL. Over subsequent days, the dendrites of these larger cells spread profusely in the outer part of the IPL, making it likely that they are the class 1 TH-IR cells of the adult. ChAT-immunoreactive (ChAT-IR) cells were not detected until P15, when ChAT-IR somata were observed in the ganglion cell layer (GCL) and INL, and their dendrites were observed already segregated into the distinct strata of the IPL in which they are found in the adult. The subsequent growth of TH-IR somata of both classes was uneven, persisting longer in temporal than in nasal retina. This extended growth of temporal cells establishes the marked nasotemporal differences in soma diameter apparent among TH-IR cells in the adult (Mitrofanis and Stone, '86; Mitrofanis et al., '88b). The growth and adult size of ChAT-IR somata, on the other hand, did not vary with retinal position; their diameters were similar to those of the adult cells from the time they first appeared. The distribution of ChAT-IR cells at P15 shared several features of the distribution of ganglion cells. The density of ChAT-IR cells was greatest at the area of peak ganglion cell density and declined toward the periphery. In contrast, TH-IR cells concentrated from the time they first appeared at the superior temporal margin, peripheral to the area of peak density of ganglion and ChAT-IR cells.     

Dendritic co-stratification of ON and ON-OFF directionally selective ganglion cells with starburst amacrine cells in rabbit retina.

The morphology, dendritic branching patterns, and dendritic stratification of retinal ganglion cells have been studied in Golgi-impregnated, whole-mount preparations of rabbit retina. Among a large number of morphological types identified, two have been found that correspond to the morphology of ON and ON-OFF directionally selective (DS) ganglion cells identified in other studies. These cells have been characterized in the preceding paper in terms of their cell body size, dendritic field size, and branching pattern. In this paper, the two kinds of DS ganglion cell are compared in terms of their levels of dendritic stratification. They are compared with each other and also with examples of class III.1 cells, defined in the preceding paper with reference to our previous studies. Studies employing computer-aided, 3D reconstruction of dendritic trees, as well as analysis of a pair of ON DS and ON-OFF DS ganglion cells with overlapping dendritic trees show that the two types of DS ganglion cell partly co-stratify in the middle of sublamina b (stratum 4). The report that some ON DS ganglion cells extend a few dendrites into sublamina a is confirmed. The study of pairs of ON-OFF DS ganglion cells and starburst amacrine cells with overlapping dendritic trees reveals a precise co-stratification of these two cell types, and many points of close apposition of starburst boutons with ON-OFF DS ganglion cell dendrites in both sublaminae of the inner plexiform layer (IPL). This is confirmed by high-resolution light microscopy and by electron microscopy. It is possible to conclude, therefore, that ON DS are also partly co-stratified with type b starburst (cholinergic) amacrine cells, and are apparently also partly co-stratified with type a starburst amacrine cells, when occasional dendrites rise to that level. The co-stratification of the two kinds of DS ganglion cell is consistent with the sharing of some inputs in common, including some cone bipolar cell inputs. The co-stratification of both with starburst amacrine cells agrees with the physiological demonstration of the powerful pharmacological effects upon ON and ON-OFF DS ganglion cells reported for cholinergic agonists. The major difference in the dendritic stratification of bistratified ON-OFF DS ganglion cells and generally unistratified ON DS ganglion cells is consistent with the bisublaminar organization of ON and OFF pathways in the IPL. The problem of occasional branches of ON DS cells in sublamina a is discussed in terms of a threshold for OFF responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell-by-cell reconstruction in reaggregates from neonatal gerbil retina begins from the inner retina and is promoted by retinal pigmented epithelium

For future retinal tissue engineering, it is essential to understand formation of retinal tissue in a 'cell-by-cell' manner, as can be best studied in retinal reaggregates. In avians, complete laminar spheres can be produced, with ganglion cells internally and photoreceptors at the surface; a similar degree of retinal reconstruction has not been achieved for mammals. Here, we have studied self-organizing potencies of retinal cells from neonatal gerbil retinae to form histotypic spheroids up to 15 days in culture (R-spheres). Shortly after reaggregation, a first sign of tissue organization was detected by use of an amacrine cell (AC)-specific calretinin (CR) antibody. These cells sorted out into small clusters and sent unipolar processes towards the centre of each cluster. Thereby, inner cell-free spaces developed into inner plexiform layer (IPL)-like areas with extended parallel CR(+) fibres. Occasionally, IPL areas merged to combine an 'inner half retina', whereby ganglion cells (GCs) occupied the outer sphere surface. This tendency was much improved in the presence of supernatants from retinal pigmented cells (RPE-spheres), e.g. cell organization and proliferation was much increased, and cell death shortened. As shown by several markers, a perfect outer ring was formed by GCs and displaced ACs, followed by a distinct IPL and 1-2 rows of ACs internally. The inner core of RPE spheres consisted of horizontal and possibly bipolar cells, while immunostaining and RT-PCR analysis proved that photoreceptors were absent. This shows that (1) mammalian retinal histogenesis in reaggregates can be brought to a hitherto unknown high level, (2) retinal tissue self-organizes from the level of the IPL, and (3) RPE factors promote formation of almost complete retinal spheres, however, their polarity was opposite to that found in respective avian spheroids.     

(3H) glycine-accumulating neurons of the human retina

Isolated human retinas were incubated in physiological saline containing micromolar (3H) glycine. The types, distributions, and synaptologies of glycine-accumulating neurons were determined by light and electron microscope autoradiography. Two types of amacrine cells were discriminated on the bases of number of processes descending into the inner plexiform layer, density of label in light-microscope autoradiographs, size, and synaptic features: (1) Gly1 amacrine cells have moderate labeling, several oblique dendrites arising from the soma, and electron lucent synaptic terminals containing large presynaptic specializations, nd (2) Gly2 amacrine cells have dense labeling, a single proximal dendrite, and moderately electron-dense terminals with small presynaptic specializations. Gly1 amacrine cells constitute approximately 15% and Gly2 amacrine cells approximately 38% of all cells in the amacrine cell layer. The laminar distribution of label in the inner plexiform layer was measured by scanning microdensitometry, which provided a format for categorizing types of synaptic contacts. Many features of glycine-accumulating amacrine cell contacts were similar to those of cat AII/Gly2 amacrine cells: a diffuse yet bisublaminar distribution of label, concentration of synaptic output in sublamina a, rod bipolar cell input in sublamina b and gap junctions in mid-inner plexiform layer involving labeled cells. The evidence seems to indicate that human Gly2 amacrine cells and cat AII/Gly2 amacrine cells are homologous cell types. finally, some cone bipolar cells were labeled.     

Serotonin and dopamine in the retina of a lizard

The objective of this paper is to report the presence and localization of serotonin and dopamine in the retina of the lizard Uta stansburiana. High performance liquid chromatography and electrochemical detection were used to identify and quantitate the two amines. Both compounds are present as endogenous molecules in this retina and are found in concentrations similar to those reported in other non-mammalian retinas. The same methods were employed to confirm, in the isolated retina, the synthesis of serotonin from precursor, tryptophan. Immunocytochemical methods were used to localize, in the neural retina, serotonin and the rate-limiting enzyme of dopamine synthesis, tyrosine hydroxylase. Serotonin immunoreactivity was observed in bistratified amacrine cells (ca. 7 micron dia.) with processes ramifying in sublayers 1, 4, and 5 of the inner plexiform layer. Immunoreactivity to tyrosine hydroxylase was observed in a different population of bistratified amacrine cells (ca. 11 micron dia.) that had processes ramifying in sublayers 1 and 5 (and perhaps 3) of the inner plexiform layer. The enzymes for further metabolism of dopamine were not found in the retina of this lizard by immunocytochemical methods. The results of this research suggest that only single classes of serotoninergic and dopaminergic neurons are present in the retina of U. stansburiana. This retina might, therefore be an appropriate place in which to investigate the functioning of these amines in visual information processing.     

Neuropeptide Y-immunoreactive amacrine cells in the retina of the turtle Pseudemys scripta elegans

Antiserum directed against neuropeptide Y selectively labeled certain amacrine cells in the turtle retina. The cell types, sizes, dendritic stratification, regional distribution, and degrees of immunolabeling were examined. The results indicated that three morphologically distinct cell types were labeled: types A, B, and C. Computer rotation of digitized data from camera lucida drawings was used to study dendritic stratification. The type A somata were large (11.5 micron in diameter), well-stained, and located in the third tier of the inner nuclear layer. Type A somata gave rise to well-stained processes which arborized within the inner plexiform layer in strata 1 and 3 and at the border between strata 4 and 5. Processes in stratum 1 were sparse and delicate with small boutons. Processes in stratum 3 were numerous and often coarse, with many large and small boutons. At the border between strata 4 and 5 the processes were frequently numerous but slender, with many small boutons. Occasional immunolabeled processes were found in the ganglion cell layer. The somata of the type B cells were smaller (9.0 micron in diameter) and gave rise to single labeled processes which descended into the inner plexiform layer and divided quickly into two secondary processes. These secondary processes gave rise to lightly labeled dendritic fields which arborized primarily in strata 2 and 4. The type C cells were usually observed at the periphery of the retina and had large somata (12.0 micron in diameter) with simple, but very elongated, dendritic arborizations in strata 1, 4, and 5. Observations also showed that type A and B cells were often found in close proximity to each other and suggested that dendrites of these cells made contact with each other. The labeled neurons were distributed relatively evenly throughout the retina except for the visual streak where they were sparse. This study indicates that neuropeptide Y-like immunoreactivity is found in more than one anatomically distinct class of amacrine cells in the turtle retina.     

Accumulation of (3H)glycine by cone bipolar neurons in the cat retina

Cone bipolar neurons in the cat retina were studied in serial sections prepared as electron microscope autoradiograms following intravitreal injection of (3H)glycine. The goal was to learn whether the cone bipolar types that accumulate glycine correspond to the types thought on other grounds to be inhibitory. About half of the cone bipolars in a given patch of retina showed specific accumulation of silver grains. The specificity of accumulation was similar to that shown by glycine-accumulating amacrines. All of the cone bipolars arborizing in sublamina b accumulated glycine but none of the cone bipolars arborizing in sublamina a did so. The types of cone bipolars accumulating glycine did not match the types thought to be inhibitory. Cone bipolar types CBb1 and CBb2 both form gap junctions with the glycine-accumulating AII amacrine, thus raising the possibility that glycine might accumulate in these cone bipolars by diffusion from the AII cell or vice versa. Thus it is logically impossible to tell which of these three cells contains a high-affinity uptake mechanism for glycine and consequently which of the three might actually use glycine as a neurotransmitter.     

Synaptic analysis of amacrine cells with neuropeptide Y-like immunoreactivity in turtle retina

Neuropeptide Y-like immunoreactivity has been localized previously within three classes of amacrine cells in the turtle retina. We have used the avidin-biotin with horseradish peroxidase technique to label these neurons for examination at the ultrastructural level to answer the following questions. Where are the synaptic contacts of these neurons made? What types of neurons are involved pre- and postsynaptically? What is the intracellular distribution of the immunoreactivity? Processes with neuropeptide Y-like immunoreactivity were located primarily within three regions of the inner plexiform layer: stratum 1, stratum 3, and at the border between strata 4 and 5. In all three regions the processes with neuropeptide Y-like immunoreactivity received synaptic contacts from both unlabeled amacrine and bipolar cells, but the majority of the synaptic input in all three regions was from unlabeled amacrine cells. Processes with neuropeptide Y-like immunoreactivity were presynaptic to unlabeled amacrine cells in all three regions, but also formed contacts onto unlabeled bipolar cells in the region between strata 4 and 5. The immunoreactivity within these cells gave rise to a diffuse reaction product that was distributed throughout the cytoplasm and within large vesicles. This localization of neuropeptide Y-like immunoreactivity within large vesicles suggests that this peptide may play a neuromodulatory role. Such a role would be consistent with previous studies of neuropeptides in the turtle retina.     

Synaptic organization of the cone horizontal cells in the catfish retina

Horizontal cells of the vertebrate retina are known to contribute to the formation of the receptive field surrounds of photoreceptor and bipolar cells. However, few synapses have been described anatomically that might mediate these interactions. We have observed in the catfish retina that cone horizontal cell perikarya and dendrites make conventional chemical synapses onto photoreceptor terminal telodendria and onto bipolar cell dendrites, while horizontal cell axon terminals make chemical synapses onto the perikarya and processes of amacrine cells. The synapses are characterized by clusters of round vesicles aggregated close to the site of contact, as well as by electron-dense material associated with both pre- and postsynaptic membranes. The three kinds of synapses observed anatomically correspond to the synaptic pathways involving cone horizontal cells that have been suggested by the physiology of these cells.     

Amacrine and ganglion cells with corticotropin-releasing-factor-like immunoreactivity in the turtle retina

This study, which uses immunocytochemical methods at the light microscopical, level, examines the cell types in the turtle retina that contain corticotropin-releasing factor (CRF)-like immunoreactivity. Two anatomically distinct amacrine cell types are labeled when antiserum directed against ovine CRF is used to label the turtle retina. These cell types each have a different dendritic arborization pattern and regional distribution. Type A cells are found only in the visual streak and have elongated dendritic arborizations that run parallel to the visual streak. These cells arborize primarily in stratum 1 and near the border of strata 2 and 3, with some processes extending into stratum 5. Type B amacrine cells are found only ventral to the visual streak and arborize primarily in a wide band in strata 4 and 5 with sparse dendritic arborizations in stratum 1. No labeled amacrine cells of any type were found dorsal to the visual streak. The asymmetric dendritic arborizations of the type A amacrine cells and the different regional distributions of the A and B cell types suggest that these two amacrine cell types perform distinct physiological functions. In addition to these labeled amacrine cells, there are also some immunoreactive cell bodies in the ganglion cell layer. Rhodamine crystals were applied to the optic tectum to retrogradely label the ganglion cell bodies. Double label studies indicate that some of the rhodamine-labeled ganglion cells also contain CRF-like immunoreactivity. The localization of CRF-like immunoreactivity in two distinct amacrine cell types and in ganglion cells suggests that it may play multiple roles in visual processing in the turtle retina.     

Spatial density and immunoreactivity of bipolar cells in the macaque monkey retina.

The anatomical substrates of spatial and color vision in the primate retina are investigated by measuring the immunoreactivity and spatial density of bipolar, amacrine and horizontal cells in the inner nuclear layer of the macaque monkey retina. Bipolar cells can be distinguished from amacrine and horizontal cells by their differential immunoreactivity to antisera against glutamate, glycine, GABA, parvalbumin, calbindin (CaBP D-28K), and the L7 protein from mouse cerebellum. The spatial density of bipolar cells is compared to the densities of photoreceptors and ganglion cells at different retinal eccentricities. In the centralmost 2 mm, cone bipolar cells outnumber ganglion cells by about 1.4:1. The density of cone bipolar cells is thus high enough to allow for input to different (parasol and midget) ganglion cell classes by different (diffuse and midget) bipolar cell classes. The density gradient of cone bipolar cells follows closely that of ganglion cells in central retina but falls less steeply in peripheral retina. This suggests that the convergence of cone signals to the receptive fields of ganglion cells in the peripheral retina occurs in the inner plexiform layer. The density of cone bipolar cells is 2.5-4 times that of cones at all eccentricities studied, implying that cone connectivity to bipolar cells remains constant throughout the retina. Different subgroups of bipolar cells are distinguished by their relative immunoreactivity to the different antisera. All rod and cone bipolar cells show moderate to strong glutamate-like immunoreactivity. The bipolar cells that show weak to moderate GABA-like immunoreactivity are also labeled with the antiserum to the L7 protein and are thus identified as rod bipolar cells. Nearly half of all cone bipolar cells showed glycine-like immunoreactivity. The results suggest that the inhibitory neurotransmitter candidates GABA and glycine are segregated respectively in rod and cone bipolar cell pathways. A diffuse, cone bipolar cell type can be identified by the anti-parvalbumin and the anti-calbindin antisera. All horizontal cells show parvalbumin-like immunoreactivity. Nearly all amacrine cells show GABA-like or glycine-like immunoreactivity; a variety of subpopulations also show immunoreactivity to one or more of the other markers used.     

Serotoninergic neurons in the retina of Xenopus laevis: selective staining, identification, development, and content

Uptake of 3H-serotonin followed by autoradiography, and uptake of the serotonin analog 5,7-dihydroxytryptamine (5,7-DHT), with subsequent staining, were each used to define a unique set of neurons in the retina of the African clawed frog, Xenopus laevis. Both techniques demonstrated the same population of neurons, on the basis of perikaryal size, shape, and position within the retina. Two classes of amacrine cells accumulated 5,7-DHT at the proximal (vitread) margin of the inner nuclear layer; the two classes were distinguished by the size of their perikarya. Two similar populations of cells, observed in the ganglion cell layer with lower frequency, may represent "displaced" counterparts of these two amacrine cell types. A class of bipolar cells whose perikarya were located in middle-to-distal regions of the inner nuclear layer also accumulated 5,7-DHT and 3H-serotonin. Processes of these cells contributed to a dense plexus of fine fibers that appeared evenly distributed throughout the inner plexiform layer. 3H-Serotonin-accumulating cells first appeared in the developing retina at stage 35/36, a time immediately after retinal stratification but before elaboration of either plexiform layer. Electron microscopic analysis permitted an identification of 3H-serotonin-accumulating terminals in the inner plexiform layer. Serotonin-labeled terminals containing conventional contacts, suggestive of amacrine cells, were presynaptic to unidentified processes and postsynaptic to bipolar cells. Labeled terminals containing ribbon contacts, indicative of bipolar cells, were postsynaptic to amacrine cells. The amount of serotonin contained in isolated retinas was 15 pmol/mg protein as measured by HPLC with electrochemical detection. We attempted to stimulate the release of accumulated 3H-serotonin from mature retinas by increasing the K+-concentration in the bathing medium. Although preloaded glycine is readily released from 14C-glycine-accumulating neurons, from the same retinas there was no calcium-dependent, K+-stimulated release of 3H-serotonin. This finding suggests that serotonin and glycine are processed differently by retinal neurons, the consequence of which results in differing responses to 40 mM K+.     

GABA-like immunoreactivity in the cat retina: light microscopy

Semithin sections of the cat retina were stained with antibodies against GABA conjugated to bovine serum albumin with glutaraldehyde. Labelled cells were visualized by means of the peroxidase-antiperoxidase method. In the outer plexiform layer both A- and B-type horizontal cells and the B-type axon terminal system expressed GABA-like immunoreactivity. Approximately 25-30% of all amacrine cells and the whole inner plexiform layer were heavily labelled. Two types of putative GABA-ergic interplexiform cells could be distinguished. One of them also expressed tyrosine-hydroxylase-like immunoreactivity. A few bipolar cells were also GABA-immunolabelled. GABA-like immunoreactivity and 3H-muscimol uptake were colocalized in 90% of the amacrine cells labelled. However, horizontal cells did not accumulate 3H-muscimol.