Synaptic organization of serotonin-like immunoreactive amacrine cells in the larval tiger salamander retina

Immunoelectron microscopy was used to investigate the ultrastructural features and synaptic relationships of serotonin-like immunoreactive amacrine cells in the larval tiger salamander retina. Serotonin-positive somas exhibited an evenly distributed peroxidase reaction product throughout their cytoplasm. Their nuclei were unstained and possessed indented nuclear membranes. Serotonin-immunoreactive processes were generally stained throughout with the exception of their mitochondria, whose morphology was often disrupted by the staining reaction. They were further characterized by an occasional dense-cored vesicle/s in addition to a generally homogeneous population of small, round, clear synaptic vesicles. Serotonin-immunoreactive amacrine cell processes formed conventional synapses that were characterized by symmetrical synaptic membrane densities. A total of 222 synaptic arrangements were observed that involved the immunostained processes of serotonin-amacrine cells. As presynaptic elements, they primarily contacted amacrine cells processes (37.8%). They also provided substantial synaptic input to processes that lacked synaptic vesicles (16.2%) and whose origin was unidentified. Serotonin-processes provided a far fewer number of synaptic contacts onto the processes of bipolar cells (1.4%) and the somas of cells in the amacrine cell layer (0.5%). As postsynaptic elements, they received synaptic inputs from amacrine cells (27.9%) and bipolar cells (16.2%). With the exception of their synapses onto bipolar cells and the somas of cells in the amacrine cell layer, each of the synaptic relationships of serotonin-amacrine cells was observed in each of sublayers 1-5 of the inner plexiform layer.     

Receptive field organization of bipolar and amacrine cells in the goldfish retina

1. Intracellular recordings were made from bipolar and amacrine cells in the isolated goldfish retina. Cells were identified mainly from their response patterns to a spot and an annulus in reference to the knowledge obtained from the previous work of intracellular Procion Yellow injection. Using white light and monochromatic lights receptive field organization of recorded cells were analysed.2. All bipolar cells had a centre-surround organization in their receptive fields. The field centre was estimated to be 100-200 mum in diameter, and the surround 1-1.5 mm.3. Bipolar cells were classified into two types according to the response properties to monochromatic lights. Opponent colour cells received inputs from red and green cones, responding with red on-centre, red and green off-surround or vice versa. Cells without colour coding received input from red cones both in the field centre and the surround. In these cells the centre and the surround were well balanced.4. Amacrine cells were also classified into two types, a sustained type and a transient type. The sustained type amacrine cells responded with a steady potential change and were colour coded. They were hyperpolarized by red and depolarized by green light. The transient type amacrine cells responded with transient depolarization at on and off of light flashes. They received input chiefly from red cones and were not colour coded. Both types of amacrine cells showed a large spatial summation in an area over 2.5 mm; centre-surround antagonism was not seen.5. Comparing the size of the receptive field with anatomy, especially with the size of dendritic spread, the field centre of bipolar cells agreed in size with their dendritic spread. Bipolar cell surround clearly exceeded its dendritic field. Since the response properties of the bipolar cell surround was mimicked most closely by the receptive field of external horizontal cells, the input to the bipolar cell surround is thought to be mediated by external horizontal cells.6. By comparing receptive field properties of various retinal cells it is suggested that both the opponent colour bipolar cells and the colour coded amacrine cells converge on to the double opponent ganglion cells.     

Synaptic regulation of the light-dependent oscillatory currents in starburst amacrine cells of the mouse retina

Responses of on-center starburst amacrine cells to steady light stimuli were recorded in the dark-adapted mouse retina. The response to spots of dim white light appear to show two components, an initial peak that correspond to the onset of the light stimulus and a series of oscillations that ride on top of the initial peak relaxation. The frequency of oscillations during light stimulation was three time higher than the frequency of spontaneous oscillations recorded in the dark. The light-evoked responses in starburst cells were exclusively dependent on the release of glutamate likely from presynaptic bipolar axon terminals and the binding of glutamate to AMPA/kainate receptors because they were blocked by 6-cyano-7-nitroquinoxalene-2,3-dione. The synaptic pathway responsible for the light responses was blocked by AP4, an agonist of metabotropic glutamate receptors that hyperpolarize on-center bipolar cells on activation. Light responses were inhibited by the calcium channel blockers cadmium ions and nifedipine, suggesting that the release of glutamate was calcium dependent. The oscillatory component of the response was specifically inhibited by blocking the glutamate transporter with d-threo-beta-benzyloxyaspartic acid, suggesting that glutamate reuptake is necessary for the oscillatory release. GABAergic antagonists bicuculline, SR 95531, and picrotoxin increased the amplitude of the initial peak while they inhibit the frequency of oscillations. TTX had a similar effect. Strychnine, the blocker of glycine receptors did not affect the initial peak but strongly decreased the oscillations frequency. These inhibitory inputs onto the bipolar axon terminals shape and synchronize the oscillatory component.     

Concealed colour opponency in ganglion cells of the rhesus monkey retina.

Criteria for distinguishing colour-opponent from spectrally non-opponent cells and identifying colour-opponent subtypes on the basis of the cone inputs they receive, have been examined in ganglion cells of the macaque retina using threshold and suprathreshold stimuli, with and without chromatic adaptation. 2. Criteria based on suprathreshold responses were found to be insufficient for distinguishing between opponent and non-opponent cells in one-third of the sample. Criteria based on a 560 nm neutral point were found to be insufficient for distinguishing between colour-opponent subtypes in one-half of the remaining cells. 3. The neutral point of colour-opponent ganglion cells varies with the geometry and intensity of the stimulus, as well as with the amount of centre-surround interaction and the receptive-field location of a cell. As a result, the neutral point is often an ambiguous criterion for identifying colour-opponent subtypes on the basis of their cone inputs. 4. About one third of the colour-opponent ganglion cells did not show colour opponency in the presence of neutral backgrounds, and only revealed this behaviour in the presence of chromatic adaptation (concealed colour opponency). 5. The proportion of these concealed colour-opponent cells increased towards the peripheral areas of the retina.     

Trichromatic colour opponency in ganglion cells of the rhesus monkey retina.

Two hundred and eleven colour-opponent ganglion cells were studied in the central 10 degrees of the retina of the rhesus monkey, to determine the inputs which they were receiving from different cone mechanisms. Spectral-sensitivity measurements in the presence of neutral and coloured back-grounds showed that 24% of these cells appeared to receive input from all three cone mechanisms. 2. In 3% of the cells, the red-sensitive cone mechanism opposed the blue- and green-sensitive ones. In 18% of the cells, the blue-sensitive cone mechanism opposed the green- and red-sensitive ones. In 3% of the cells, the green-sensitive cone mechanism opposed the blue- and red-sensitive ones. 3. In 12% of the cells receiving opponent green- and red-sensitive cone inputs, responses from the beta-band of the red-sensitive cone mechanism could be detected and distinguished from blue-sensitive cone input. 4. All cells receiving blue-sensitive cone input appeared to be trichromatic. The retinal distribution of cells with trichromatic input and that of cells with beta-band responses seemed to parallel the availability of blue-sensitive cones in the retinal area being considered. 5. The results indicate that trichromatic interactions in the macaque visual system begin in the retina.     

The synaptic organization of the dopaminergic amacrine cell in the cat retina

Summary The dopaminergic amacrine cells of the cat retina have been stained by immunocytochemistry using an antibody to tyrosine hydroxylase (Toh). The complete population of Toh+cells has been studied by light microscopy of retinal wholemounts to evaluate morphological details of dendritic structure and branching patterns. Selected Toh+amacrine cells have been studied by serial-section electron microscopy to analyse synaptic input and output relationships. The majority of Toh+amacrine cells occur in the amacrine cell layer of the retina and have their dendrites ramifying and forming the characteristic rings in stratum 1 of the inner plexiform layer. A minority of Toh+cells have cell bodies displaced to the ganglion cell layer but their dendrites also stratify in stratum 1. All Toh+cells have some dendritic branches running in stratum 2 as well as in stratum 1, and frequently they have long axon-like processes (500–1000 m long) dipping down to run in stratum 5 before passing up to rejoin the major dendritic arbors in stratum 1. In addition Toh+stained processes follow blood vessels in the inner plexiform layer and in the ganglion cell layer. A population of Toh+cells found in the inferior retina appears to give rise to stained processes that pass to the outer plexiform layer and therein to run for as far as one millimeter.Electron microscopy reveals that Toh+amacrine cells are postsynaptic to amacrine cells and a few bipolar cell terminals in stratum 1 of the inner plexiform layer and are primarily presynaptic to All amacrine cell bodies and lobular appendages, and to another type of amacrine cell body and amacrine dendrites hypothesized to be the A17 amacrine cell. The Toh+dendrites in stratum 2 are presynaptic to All lobular appendages primarily. Stained axon-like processes running in stratum 5 prove to be presynaptic to All amacrine dendrites as they approach the rod bipolar axon terminals and they may also be presynaptic to the rod bipolar terminal itself. The Toh+stained dendrites that have been followed in the outer plexiform layer run along the top of the B-type horizontal cell somata and may have small synapses upon them. The only clear synapses seen in the outer plexiform layer are from the Toh+profiles upon vesicle filled amacrine-like profiles that are in turn presynaptic to bipolar cell dendrites in the outer plexiform layer. We presume the cells postsynaptic to the Toh+dendrites in the outer plexiform layer are interplexiform cells. Finally the Toh+profiles that course along blood vessel walls and in the ganglion cell layer appear to end either against the basal lamina of the blood vessel or at intercellular channels of vesicle-laden Muller cell end-feet.     

Somatostatin-like immunoreactivity in amacrine cells of the chicken retina

Somatostatin-like immunoreactivity was detected in chicken retina by radioimmunoassay. The levels of somatostatin-like immunoreactivity decreased after intra-ocular injection of kainic acid, but were not affected by destruction of the ganglion cells. By immunohistochemistry, somatostatinimmunoreactive amacrine cells were found in the inner nuclear layer. These cells were destroyed by kainic acid. At least some of the cells projected to all three sub-layers of the inner plexiform layer in which there were diffuse bands of fluorescence. Specific immunofluorescence was also detected at the level of the outer limiting membrane and the optic nerve fibre layer, but the outer nuclear and plexiform layers, horizontal, bipolar and ganglion cells did not show specific immunofluorescence.It is suggested that other amacrine cell sub-classes, defined in terms of their putative transmitter, may show specific patterns of cell body location and size, and terminal arborisation.     

GABA-Mediated inhibition between amacrine cells in the goldfish retina

Retinal amacrine cells have abundant dendro-dendritic synapses between neighboring amacrine cells. Therefore an amacrine cell has both presynaptic and postsynaptic aspects. To understand these synaptic interactions in the amacrine cell, we recorded from amacrine cells in the goldfish retinal slice preparation with perforated- and whole cell-patch clamp techniques. As the presynaptic element, 19% of the cells recorded (15 of 78 cells) showed spontaneous tetrodotoxin (TTX)-sensitive action potentials. As the postsynaptic element, all amacrine cells (n = 9) were found to have GABA-evoked responses and, under perforated patch clamp, 50 microM GABA hyperpolarized amacrine cells by activating a Cl(-) conductance. Bicuculline-sensitive spontaneous postsynaptic currents, carried by Cl(-), were observed in 82% of the cells (64 of 78 cells). Since the source of GABA in the inner plexiform layer is amacrine cells alone, these events are likely to be inhibitory postsynaptic currents (IPSCs) caused by GABA spontaneously released from neighboring amacrine cells. IPSCs were classified into three groups. Large amplitude IPSCs were suppressed by TTX (1 microM), indicating that presynaptic action potentials triggered GABA release. Medium amplitude IPSCs were also TTX sensitive. Small amplitude IPSCs were TTX insensitive (miniature IPSCs; n = 26). All of spike-induced, medium amplitude, and miniature IPSCs were Ca(2+) dependent and blocked by Co(2+). Blocking of glutamatergic inputs by DL-2-amino-phosphonoheptanoate (AP7; 30 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 2 microM) had almost no effect on spontaneous GABA release from presynaptic amacrine cells. We suggest that these dendro-dendrotic inhibitory networks contribute to receptive field spatiotemporal properties.     

Regional density of monoamine-accumulating amacrine cells in the rabbit retina

The spatial distribution of noradrenaline (NA)- and indoleamine-accumulating (IA) amacrine cells was studied by fluorescence microscopy in flatmounts and tangential cryosections (15 μm thickness) of albino rabbit retinas. The two classes of cells were found to be distributed over the retinal field in a mixed and random fashion. The regional density (mean ± S.D. cells/mm²) of monoamine-accumulating cells was highest in the visual streak (NA cells, 88 ± 5; IA cells, 1507 ± 92), and lowest (41 ± 7; 625 ± 105, respectively) in the inferior periphery. The density ratio of NA:IA cells was 1:15 on average. Among cells located in the amacrine cell layer, NA and IA cells accounted for 0.3 and 3.9% of the total cell population, respectively. Monoamine-accumulating amacrine cells displaced into the ganglion cell layer were few; these displaced cells were only 2% and 0.6% of the cell number of normally situated cells in the amacrine cell layer for NA and IA cells, respectively.     

Dye coupling between amacrine cells in carp retina

Amacrine cells in isolated retinas of the carp (Cyprinus carpio) were intracellularly recorded and marked with a fluorescent dye, Lucifer yellow. On occasion, dye coupling was found to occur between amacrine cells when the dye was iontophoretically injected into an amacrine cell, generating one of the transient or sustained types of photoresponse. Dye-coupled cells in the vicinity of the marked cell were very similar to the marked cell in soma shape and dendritic stratification. Dye diffusion is assumed to take place at gap junctions between dendrities of amacrine cells which belong to a population of similar type cells in morphology and possibly in function.     

The birthdates of GABA-immunoreactive amacrine cells in the rat retina

The birthdates of GABAergic amacrine cells in the rat retina were investigated by immunocytochemistry using anti-GABA and anti-bromodeoxyuridine (BrdU) antisera. The ratio of co-localization of GABA to BrdU increased gradually from embryonic-day 13 (E13) and showed a peak value on E18 in the central retina and on E20 in the periphery. After birth, until postnatal-day 3 (P3), a few co-localized cells were observed in the inner nuclear layer (INL). However, in the peripheral retina, co-localized cells were observed in the INL and ganglion cell layer until P5. Our results suggest that the birthdates of GABA-immunoreactive cells vary, depending on cell-type and that there is a temporal lag in the GABA-immunoreactive cell production in the peripheral retina relative to the central retina. Received: 11 January 1999 / Accepted: 20 April 1999     

Urotensin I-like immunoreactivity in amacrine cells of the goldfish retina

Urotensin I-like immunoreactivity (UILI), in different localization from that of corticotropin releasing factor-like immunoreactivity (CRFLI), in the goldfish retina has been demonstrated by means of radioimmunoassay, high-performance liquid chromatography (HPLC) and immunohistochemistry. Radioimmunoassay showed 350 +/- 40 pg/mg prot. of UILI in goldfish retina extracts. The immunoreactive material present in the retina was also characterized by reversed phase HPLC. Some of the UILI co-eluted with synthetic carp UI, though the HPLC experiments suggested the existence of other UILI substance(s) with less hydrophobicity than synthetic UI. By immunohistochemistry, UILI and CRFLI were seen in different amacrine cells of the goldfish retina. It is suggested that UI may be involved in the fish visual transmission system together with CRF and other neuropeptides.     

Preclinical evaluation of a phosphorothioate oligonucleotide in the retina of rhesus monkey

Overexpression of vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization (CNV) in patients with age-related macular degeneration. In this study, a phosphorothioate oligonucleotide (PS-oligo) targeting both human and rat VEGF(165) genes upstream of the translation initiation code, named DS135 in this study, was evaluated for its uptake dynamics and retinal tolerance after intravitreal (IV) and subretinal (SR) injections in the rhesus monkey. Intravitreal and SR injections of a fluorescent-labeled DS135 (FL-DS135) resulted in both dose- and time-dependent uptake and persistence, and FL-DS135 remained detectable in the retina for at least 3 weeks after injection. Ophthalmic examination showed transient vitreous haze after IV delivery of a high dose but not with a low dose of FL-DS135. Histologic examination showed no evidence of retinal degeneration with respect to IV delivery. After SR delivery, however, dose-related cellular infiltration, transient residual fluid, and slight distortion of the neuroretina were observed. The biologic efficacy of DS135 was further assessed in a laser-induced CNV model, and development of CNV was determined by fluorescein angiography and histologic examination. Incomplete inhibition of CNV formation was observed after IV and SR injection of DS135, but no statistically significant difference was achieved when compared with dose-matched control of PS-oligo. Analysis of fluorescein angiogram and histologic examination showed less than 30% incidence of CNV development in this monkey model. Our study demonstrated that PS-oligos can be successfully introduced into the retina, although with potential limitations, after SR delivery. DS135, a PS-oligo targeting the VEGF gene upstream of the translation initiation code, partially inhibited CNV formation. An improved CNV model is necessary for further confirmation of the full therapeutic potency of DS135 before clinical application.     

Identification of amacrine and ganglion cells in the carp retina.

1. Amacrine and ganglion cells in the carp retina were identified from such criteria as photoresponses, intracellular dye staining, responses to optic nerve stimulation and behaviour to a synapse blocking agent. 2. Responses of ganglion cells were accompanied by spike discharges., either facilitated or suppressed by photic stimulation. The cells were also invaded by antidromic impulses, which survived after chemical synapses had been blocked by application of atomized CoCl2 solution. In subsequent histology of the Procion-stained neurones, the cell bodies were found in the ganglion cell layer and the axons were often traced. 3. Amacrine cells were subdivided into two types. The first type gave rise to transient depolarizations at both on- and offsets of spot and annulus illuminations, usually being associated with spike discharges of which the amplitudes varied in different cells. In histology, the cell bodies of this type were situated in the inner nuclear layer and dendrites ramified in two or more discrete sublayers of the inner plexiform layer (the stratified amacrine cell of Cajal). 4. The second type of amacrine cells produced sustained responses during illumination, being associated with no spike but with small oscillatory wavelets. The cell bodies were situated in the inner nuclear layer and the dendrites ramified in a single sublayer of the inner plexiform layer (the monolayered amacrine cell). 5. An attempt was made to see the effect of activation of centrifugal fibres on amacrine cells, but almost all of about 200 cells examined did not respond to optic nerve stimulation. Only two cells produced, with long latency, a small post-synaptic depolarization which disappeared after chemical synapses in the retina had been blocked. It is considered that the physiological role of the centrifugal system is insignificant in the carp retina.     

Spontaneous oscillatory activity of starburst amacrine cells in the mouse retina

Using patch-clamp techniques, we investigated the characteristics of the spontaneous oscillatory activity displayed by starburst amacrine cells in the mouse retina. At a holding potential of -70 mV, oscillations appeared as spontaneous, rhythmic inward currents with a frequency of approximately 3.5 Hz and an average maximal amplitude of approximately 120 pA. Application of TEA, a potassium channel blocker, increased the amplitude of oscillatory currents by >70% but reduced their frequency by approximately 17%. The TEA effects did not appear to result from direct actions on starburst cells, but rather a modulation of their synaptic inputs. Oscillatory currents were inhibited by 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), an antagonist of AMPA/kainate receptors, indicating that they were dependent on a periodic glutamatergic input likely from presynaptic bipolar cells. The oscillations were also inhibited by the calcium channel blockers cadmium and nifedipine, suggesting that the glutamate release was calcium dependent. Application of AP4, an agonist of mGluR6 receptors on on-center bipolar cells, blocked the oscillatory currents in starburst cells. However, application of TEA overcame the AP4 blockade, suggesting that the periodic glutamate release from bipolar cells is intrinsic to the inner plexiform layer in that, under experimental conditions, it can occur independent of photoreceptor input. The GABA receptor antagonists picrotoxin and bicuculline enhanced the amplitude of oscillations in starburst cells prestimulated with TEA. Our results suggest that this enhancement was due to a reduction of a GABAergic feedback inhibition from amacrine cells to bipolar cells and the resultant increased glutamate release. Finally, we found that some ganglion cells and other types of amacrine cell also displayed rhythmic activity, suggesting that oscillatory behavior is expressed by a number of inner retinal neurons.     

Synaptic organization of immunocytochemically identified GABA neurons in the monkey sensory-motor cortex

Neurons in the monkey somatic sensory and motor cortex were labelled immunocytochemically for the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD), and examined with the electron microscope. The somata and dendrites of many large GAD-positive neurons of layers III-VI receive numerous asymmetric synapses from unlabelled terminals and symmetric synapses from GAD-positive terminals. Comparisons with light and electron microscopic studies of Golgi-impregnated neurons suggest that the large labelled neurons are basket cells. Small GAD-positive neurons generally receive few synapses on their somata and dendrites, and probably conform to several morphological types. GAD-positive axons from symmetric synapses on many neuronal elements including the somata, dendrites and initial segments of pyramidal cells, and the somata and dendrites of non-pyramidal cells. Synapses between GAD-positive terminals and GAD-positive cell bodies and dendrites are common in all layers. Many GAD-positive terminals in layers III-VI arise from myelinated axons. Some of the axons form pericellular terminal nests on pyramidal cell somata and are interpreted as originating from basket cells while other GAD-positive myelinated axons synapse with the somata and dendrites of non-pyramidal cells. These findings suggest either that the sites of basket cell terminations are more heterogeneous than previously believed or that there are other classes of GAD-positive neurons with myelinated axons. Unmyelinated GAD-positive axons synapse with the initial segments of pyramidal cell axons or form en passant synapses with dendritic spines and small dendritic shafts and are interpreted as arising from the population of small GAD-positive neurons which appears to include several morphological types.     

Form and function of ON-OFF amacrine cells in the amphibian retina

ON-OFF amacrine cells were studied with whole cell recording techniques and intracellular staining methods using intact retina-eyecup preparations of the tiger salamander (Ambystoma tigrinum) and the mudpuppy (Necturus maculosus). Morphological characterization of these cells included three-dimensional reconstruction methods based on serial optical sections obtained with a confocal microscope. Some cells had their detailed morphology digitized with a computer-assisted tracing system and converted to compartmental models for computer simulations. The dendrites of ON-OFF amacrine cells have spines and numerous varicosities. Physiological recordings confirmed that ON-OFF amacrine cells generate both large- and small-amplitude impulses attributed, respectively, to somatic and dendritic generation sites. Using a multichannel model for impulse generation, computer simulations were carried out to evaluate how impulses are likely to propagate throughout these structures. We conclude that the ON-OFF amacrine cell is organized with multifocal dendritic impulse generating sites and that both dendritic and somatic impulse activity contribute to the functional repertoire of these interneurons: locally generated dendritic impulses can provide regional activation, while somatic impulse activity results in rapid activation of the entire dendritic tree.