Synaptic connections of amacrine cells with substance P-like or neuropeptide Y-like immunoreactivity (SP-LI or NPY-LI) in the retina of the cane toad, Bufo marinus, were investigated using ultrastructural immunocytochemistry. The perikarya of SP-LI or NPY-LI amacrine cells were located in the innermost row of the inner nuclear layer. The synapses associated with SP-LI amacrine cells were distributed mainly in sublaminae 3 and 4 with about 10% in sublamina 1 of the inner plexiform layer. The synapses formed by NPY-LI amacrine cells were found in sublaminae 1, 2, and 4 with approximately equal frequency. Of a total of 175 SP-LI profiles, 56% were in presynaptic positions and 44% in postsynaptic positions. The synaptic inputs to SP-LI profiles predominantly derived from other unlabeled amacrine cell dendrites, and to a lesser extent, from bipolar cell terminals. The majority of synaptic outputs from SP-LI amacrine cell dendrites were directed onto unlabeled amacrine cell processes. The SP-LI profiles also made synapses onto bipolar cell terminals and formed synapses onto presumed ganglion cell dendrites. Of a total of 200 NPY-LI profiles, 48% were in presynaptic positions and 52% in postsynaptic positions. The profiles of NPY-LI amacrine cells mainly received their synaptic inputs from other unlabeled amacrine cell processes, and to a lesser extent, from bipolar cell terminals. The majority of NPY-LI amacrine cell profiles gave their synaptic outputs onto unlabeled amacrine cell dendrites, and others formed synapses onto presumed ganglion cell processes. These results suggest that these two populations of neuropeptide-containing amacrine cells in the Bufo retina are involved in different synaptic circuits.

We found that a single intravitreal injection of 6-hydroxy dopamine (6-OHDA) is highly efficient in blocking the development of deprivation-induced myopia in young chickens. To investigate the effects of 6-OHDA on retinal function, we studied electroretinograms (ERGs) in chickens aged 15-25 days, 4 days subsequent to the injection. Both spectral sensitivity and oscillatory potentials were tested. In addition, a histological examination was performed of dopaminergic amacrine cells labeled by a monoclonal antibody against tyrosine hydroxylase. We found that, at doses of 6-OHDA sufficient to suppress deprivation myopia entirely, no effect could be detected on either the ERGs or on the density and appearance of dopaminergic amacrine cells. For higher doses, spectral sensitivity and the number of dopaminergic amacrine cells declined gradually. In contrast, as doses increased, oscillatory potentials 1 and 2 grew in amplitude only to decline at the highest doses. The results indicate that (1) development of deprivation myopia requires normal retinal function and that (2) slight changes in the gains of dopaminergic pathways are sufficient to block the development of deprivation myopia.

There are many regional differences in cell morphology and neurochemistry in the retina. This study examined a specialized population of neuropeptide Y- and glucagon-like immunoreactive amacrine cells in the peripheral retina of the turtle. Some of the dendritic processes from these peptidergic amacrine cells formed a dense circumferentially oriented nerve fiber plexus which ran parallel to the ora serrata. Collaterals from this plexus projected into and innervated the nonpigmented ciliary epithelium in the pars plana region of the ciliary body. Electron microscopy revealed that the neuropeptide Y- and glucagon-like immunoreactive processes in the ciliary epithelium contained many labeled, large dense-cored vesicles. Small crystals of lipid-soluble fluorescent dye were implanted in the retina near the ora serrata in fixed retinal tissue to search for other peripheral retinal specializations. Numerous thick and thin cell processes oriented parallel to the ora serrata were labeled in the retina by the dye. In addition, many dye-labeled somata with circumferentially oriented dendritic arborizations were seen in the extreme periphery of the retina. Many of these dye-labeled cells and processes were clearly not associated with the neuropeptide Y- and glucagon-like immunoreactive cells described above. This study has shown that some peptidergic neurons in the peripheral retina have a unique morphology in comparison to more centrally located cells. The function of these specialized peripheral cells is not established, but the innervation of the ciliary epithelium by peptidergic amacrine cells suggests that they may be involved in control of aqueous inflow.

During the first postnatal week in rodents, cholinergic retinal waves initiate in starburst amacrine cells (SACs), propagating to retinal ganglion cells (RGCs) and visual centers, essential for visual circuit refinement. By modulating exocytosis in SACs, dynamic changes in the protein kinase A (PKA) activity can regulate the spatiotemporal patterns of cholinergic waves. Previously, cysteine string protein-α (CSPα) is found to interact with the core exocytotic machinery by PKA-mediated phosphorylation at serine 10 (S10). However, whether PKA-mediated CSPα phosphorylation may regulate cholinergic waves via SACs remains unknown. Here, we examined how CSPα phosphorylation in SACs regulates cholinergic waves. First, we identified that CSPα1 is the major isoform in developing rat SACs and the inner plexiform layer during the first postnatal week. Using SAC-specific expression, we found that the CSPα1-PKA-phosphodeficient mutant (CSP-S10A) decreased wave frequency, but did not alter the wave spatial correlation compared to control, wild-type CSPα1 (CSP-WT), or two PKA-phosphomimetic mutants (CSP-S10D and CSP-S10E). These suggest that CSPα-S10 phosphodeficiency in SACs dampens the frequency of cholinergic waves. Moreover, the level of phospho-PKA substrates was significantly reduced in SACs overexpressing CSP-S10A compared to control or CSP-WT, suggesting that the dampened wave frequency is correlated with the decreased PKA activity. Further, compared to control or CSP-WT, CSP-S10A in SACs reduced the periodicity of wave-associated postsynaptic currents (PSCs) in neighboring RGCs, suggesting that these RGCs received the weakened synaptic inputs from SACs overexpressing CSP-S10A. Finally, CSP-S10A in SACs decreased the PSC amplitude and the slope to peak PSC compared to control or CSP-WT, suggesting that CSPα-S10 phosphodeficiency may dampen the speed of the SAC-RGC transmission. Thus, via PKA-mediated phosphorylation, CSPα in SACs may facilitate the SAC-RGC transmission, contributing to the robust frequency of cholinergic waves.

There are more than 30 distinct types of mammalian retinal ganglion cells, each sensitive to different features of the visual environment. In rabbit retina, they can be grouped into four classes according to their morphology and stratification of their dendrites in the inner plexiform layer (IPL). The goal of this study was to describe the synaptic inputs to one type of Class IV ganglion cell, the third member of the sparsely branched Class IV cells (SB3). One cell of this type was partially reconstructed in a retinal connectome developed using automated transmission electron microscopy (ATEM). It had slender, relatively straight dendrites that ramify in the sublamina a of the IPL. The dendrites of the SB3 cell were always postsynaptic in the IPL, supporting its identity as a ganglion cell. It received 29% of its input from bipolar cells, a value in the middle of the range for rabbit retinal ganglion cells studied previously. The SB3 cell typically received only one synapse per bipolar cell from multiple types of presumed OFF bipolar cells; reciprocal synapses from amacrine cells at the dyad synapses were infrequent. In a few instances, the bipolar cells presynaptic to the SB3 ganglion cell also provided input to an amacrine cell presynaptic to the ganglion cell. There was apparently no crossover inhibition from narrow-field ON amacrine cells. Most of the amacrine cell inputs were from axons and dendrites of GABAergic amacrine cells, likely providing inhibitory input from outside the classical receptive field.

The synaptic connections of amacrine cells synthesizing or accumulating serotonin in the retina of the cane toad, Bufo marinus, were studied by using preembedding double-labeling electron-microscopic immunocytochemistry. The binding sites of an anti-serotonin antibody were revealed by the diaminobenzidine reaction, whilst a colloidal gold-conjugated secondary antibody was used to detect an antibody to phenylalanine hydroxylase. Since the latter antibody recognizes tryptophan 5-hydroxylase, one of the synthesizing enzymes for serotonin, as well as tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis, the double labeling of the present study enabled us to identify three groups of labeled profiles at the ultrastructural level. The profiles of serotonin-synthesizing amacrine cells contained both diaminobenzidine reaction product and colloidal gold particles, whilst those of serotonin-accumulating and dopaminergic amacrine cells contained only diaminobenzidine reaction product or colloidal gold particles, respectively. The synapses of serotonin-synthesizing or serotonin-accumulating amacrine cells were distributed all through the inner plexiform layer of the retina. The profiles of serotonin-synthesizing amacrine cells predominantly received synapses from, and made synapses onto, unlabeled amacrine cell dendrites. They also received synapses from, and made synapses onto, bipolar cell terminals. They also made synapses onto presumed ganglion cell dendrites. However, the profiles of serotonin-accumulating cells made synapses only with unlabeled amacrine cell processes. There were close contacts between the profiles of serotonin-synthesizing and serotonin-accumulating amacrine cells. No synaptic relationships were observed between dopaminergic and serotonin-synthesizing or serotonin-accumulating amacrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive intestinal polypeptide (VIP) immunoreactive (IR) neurons in the rabbit retina constitute a population of wide-field amacrine cells. To better define this cell population, we examined the coexpression of VIP with other putative retinal transmitters or their biosynthetic enzymes, including gamma-aminobutryic acid (GABA), tyrosine hydroxylase (TH), and somatostatin (SRIF). Colchicine-treated retinas were immersion fixed in 4% paraformaldehyde. The retinas were cut either perpendicular or parallel to the vitreal surface and processed by double-label immunofluorescence techniques using antibodies directed to VIP, GABA, TH, and SRIF. The immunoreactive staining patterns obtained with these antibodies were the same as those described in previous studies. GABA-IR neurons were localized to the proximal inner nuclear layer (INL) and ganglion cell layer (GCL) and processes were distributed throughout the inner plexiform layer (IPL). TH- and SRIF-IR neurons were sparsely distributed to the proximal INL and GCL, respectively. TH-IR processes ramified in laminae 1, 3, and 5, and SRIF-IR processes in laminae 1 and 5 of the IPL. Colocalization experiments showed that all VIP-IR neurons contain GABA immunoreactivity. In contrast, colocalization of VIP and TH or SRIF immunoreactivities was never observed. These results demonstrate that VIP-IR wide-field amacrines of the rabbit retina make up a neurochemically and morphologically distinct subpopulation of the GABA-IR amacrine cell population. Furthermore, VIP-IR amacrine cells constitute a distinct group with respect to the TH- and SRIF-IR amacrine cells.

Both local cell-cell interactions and lineage bias have roles in determining the different retina cell phenotypes. In this study, subpopulations of amacrine cells that dually express GABA or serotonin (5-HT) and dopamine (DA) are identified in the early Xenopus tadpole (stages 42-48) retina. GABA is first detected by immunocytochemistry in amacrine cells at stage 35/36, 5-HT at stage 39, and DA at stage 41. As the number of these subtypes of amacrine cells increases by differentiation, a subset of them transiently express two neurotransmitters. GABA/DA double-labeled amacrine cells are detected first at stage 42, at which time they constitute 52% of the DA-containing population; this percentage decreases to only 3% by stage 48. 5-HT/DA amacrine cells are detected only at stage 44, constituting about 20% of the DA-containing cells and 4% of the small-dim 5-HT-containing cells. Regional location does not differentially affect the differentiation of these three types of amacrine cells (DA only, GABA/DA, and 5-HT/DA cells); each type is found more in the anterior and dorsal than the posterior and ventral quadrants, and their overall distribution patterns are statistically indistinguishable. However, these subtypes of amacrine cells reside in different sublamina of the inner nuclear layer. DA-only amacrine cells are located predominantly in the inner sublayer of the 2-3 cell thick amacrine cell layer, closest to the inner plexiform and the ganglion cell layers. Both types of double-labeled cells are located mostly in the outer sublayer of the amacrine cell layer, closest to other interneurons in the inner nuclear layer. This distinct sublaminar location of different neurotransmitter phenotypes suggests that local laminar cues influence the coexpression of neurotransmitters in amacrine cells.

In a recent study, visual signals were recorded for the first time in starburst amacrine cells of the macaque retina, and, as for mouse and rabbit, a directional bias observed in calcium signals was recorded from near the dendritic tips. Stimulus motion from the soma toward the tip generated a larger calcium signal than motion from the tip toward the soma. Two mechanisms affecting the spatiotemporal summation of excitatory postsynaptic currents have been proposed to contribute to directional signaling at the dendritic tips of starbursts: (1) a "morphological" mechanism in which electrotonic propagation of excitatory synaptic currents along a dendrite sums bipolar cell inputs at the dendritic tip preferentially for stimulus motion in the centrifugal direction; (2) a "space-time" mechanism that relies on differences in the time-courses of proximal and distal bipolar cell inputs to favor centrifugal stimulus motion. To explore the contributions of these two mechanisms in the primate, we developed a realistic computational model based on connectomic reconstruction of a macaque starburst cell and the distribution of its synaptic inputs from sustained and transient bipolar cell types. Our model suggests that both mechanisms can initiate direction selectivity in starburst dendrites, but their contributions differ depending on the spatiotemporal properties of the stimulus. Specifically, the morphological mechanism dominates when small visual objects are moving at high velocities, and the space-time mechanism contributes most for large visual objects moving at low velocities.

The vertebrate retina contains a large number of different types of neurons that can be distinguished by their morphological properties. Assuming that no location should be without a contribution from the circuitry and function linked to a specific type of neuron, it is expected that the dendritic trees of neurons belonging to a type will cover the retina in a regular manner. Thus, for most types of neurons, the contribution to visual processing is thought to be independent of the exact location of individual neurons across the retina. Here, we have investigated the distribution of AII amacrine cells in rat retina. The AII is a multifunctional amacrine cell found in mammals and involved in synaptic microcircuits that contribute to visual processing under both scotopic and photopic conditions. Previous investigations have suggested that AIIs are regularly distributed, with a nearest-neighbor distance regularity index of ~4. It has been argued, however, that this presumed regularity results from treating somas as points, without taking into account their actual spatial extent which constrains the location of other cells of the same type. When we simulated random distributions of cell bodies with size and density similar to real AIIs, we confirmed that the simulated distributions could not be distinguished from the distributions observed experimentally for AIIs in different regions and eccentricities of the retina. The developmental mechanisms that generate the observed distributions of AIIs remain to be investigated.

One subpopulation of amacrine interneurons in the turtle retina was shown to contain met-enkephalin by means of immunocytochemistry, and another was demonstrated to have a high-affinity uptake system for [3H]-dopamine by means of autoradiography. Although the amacrine soma size, density, and distribution of their neurites in IPL substrata was similar in retinas in which met-enkephalin and dopamine were localized, combined light microscope immunocytochemistry-autoradiography demonstrated that these two neurotransmitter systems did not coexist in the same cells. Because the two amacrine cell subtypes ramify in the same IPL substrata, neuronal interaction between them is possible. Release experiments showed that the potassium-induced release of [3H]-dopamine from the superfused turtle retina was reduced by 40% when enkephalin was added to the superfusate. The inhibition of [3H]-dopamine release could be blocked by the addition of naloxone. The addition of enkephalin had no effect of the potassium-induced release of [3H]-GABA from the superfused retina. These findings suggest that an enkephalinergic modulation of the dopaminergic amacrine cell system exists in the turtle retina.

Nitric oxide (NO) is a novel neuronal messenger that likely influences retinal function by activating retinal guanylyl cyclase to increase levels of cGMP. In the present study, the localization of neuronal nitric oxide synthase (nNOS, Type I NOS) in the cone-dominant tree shrew retina was studied using NADPH-d histochemistry and nNOS immunocytochemistry. Both NADPH-d and nNOS-immunoreactivity (IR) labeled the inner segments of rods and the myoids of a regular subpopulation of cones, with their corresponding nuclei outlined. The labeled cone myoids were co-localized with a marker for short-wave-sensitive (SWS) cones (S-antigen) and also displayed the regular triangular packing and density (7%) characteristic of SWS cones in tree shrew and other mammalian retinas. These measures confirmed the identity of the labeled cones as SWS cones. Photoreceptor ellipsoids of all cones were strongly labeled by NADPH-d reactivity, but lacked nNOS-IR. Another novel finding in tree shrew retina was that both NADPH-d and nNOS-IR labeled Muller cells, which have not been labeled by nNOS-IR in other mammalian retinas. Consistent with findings in rod-dominant retinas, two types of amacrine cells at the vitreal edge of the inner nuclear layer and a subpopulation of displaced amacrine cells at the scleral edge of the ganglion cell layer were labeled by both NADPH-d and nNOS-IR. Processes of these labeled cells were seen to extend into the inner plexiform layer, where dense punctate label was seen, especially in the central sublamina. These results show that localization of NOS in the cone-dominant tree shrew retina shares some common properties with rod-dominant mammalian retinas, but also shows some species-specific characteristics. The new finding of nNOS localization in tree shrew SWS cones and rods, but not in other cones, raises interesting questions about the roles of NO in the earliest level of visual processing.

The retinas of adult teleost fish can regenerate neurons following a chemical or mechanical injury. Previous studies have demonstrated that mechanical excision of fish retina induces a hyperplasia within the retinal sheet, including the formation of a proliferative blastema from whence new retinal cells are produced to fill the excision site. The current study was designed to address two issues regarding injury-induced retinal hyperplasia: (1) Retinas of adult zebrafish can regenerate following a surgical excision, but compared to other fish they contain very few proliferative cells: Might retinal injury in adult zebrafish therefore induce minimal, or perhaps no, hyperplasia? (2) The fate of injury-induced, proliferative retinal cells outside surgical excision sites has yet to be determined. Do such cells produce retinal neurons? Evidence is presented that mechanical injury to the adult zebrafish retina induces a dramatic increase in the number of proliferative cells both within and external to the lesion site, and some of these cells apparently migrate within the radial dimension of the retina. Evidence is also presented that injury-induced proliferative cells outside a lesion site can produce retinal neurons--including cone photoreceptors, interplexiform cells, and amacrine cells--that are incorporated into the extant retina. The results suggest that the adult zebrafish retina contains a latent population of cells that is induced to proliferate following retinal injury, and that these cells might represent a novel avenue for pluripotent neurogenesis within the intact adult teleost retina.

Our previous research showed that increased phosphorylation of connexin (Cx)36 indicated extended  coupling of AII amacrine cells (ACs) in the rod-dominant mouse myopic retina. This research will determine whether phosphorylation at serine 276 of Cx35-containing gap junctions increased in the myopic chicken, whose retina is cone-dominant. Refractive errors and ocular biometric dimensions of 7-days-old chickens were determined following 12 h and 7 days induction of myopia by a -10D lens. The expression pattern and size of Cx35-positive plaques were examined in the early (12 h) and compensated stages (7 days) of lens-induced myopia (LIM). At the same time, phosphorylation at serine 276 (functional assay) of Cx35 in strata 5 (S5) of the inner plexiform layer was investigated. The axial length of the 7 days LIM eyes was significantly longer than that of non-LIM controls (P < 0.05). Anti-phospho-Ser276 (Ser276-P)-labeled plaques were significantly increased in LIM retinas at both 12 h and 7 days. The density of Ser276-P of Cx35 was observed to increase after 12 h LIM. In the meanwhile, the areas of existing Cx35 plaques did not change. As there was more phosphorylation of connexin35 at Ser276 at both the early and late stages (12 h) and 7 days of LIM chicken retinal activity, the coupling with ACs could be increased in myopia development of the cone-dominated chicken retina.