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.     

Functional and morphological correlates of amacrine cells in carp retina

Experiments were conducted on isolated retinas of adult carp (Cyprinus carpio) to investigate correlations between photoresponses and morphological features of amacrine cells. The fluorescence dye Lucifer Yellow CH was iontophoretically injected into single cells which had been characterized electrophysiologically. Photoresponses were classified into two main types (transient ON-OFF and sustained), which were further subgrouped into “fast” and “slow” ON-OFF types and into ON-center and OFF-center types, respectively. In the spectral response curve, all the types dealt with showed a maximum response at 621 nm, indicating that main input signals derive from red-sensitive cones. The cells marked by intracellular injection of the dye showed a great variety in morphology. Cells were classified into 8 subtypes, based on soma shape (fusiform or pyriform), dendritic field area (narrow,< 0.3 mm²; medium,0.3–0.8mm²; wide, 0.8 mm²), and dendritic stratification in the inner plexiform layer (restricted to sublamina a or b, or distributed diffusely). In certain cases a given response type was correlated with a specific morphological type, while receptive field size was not strictly correlated with dendritic field areas. Long and fine peripheral “axon-like” processes were found to arise from the primary dentrites of most fusiform cells. Dye-coupling was found among cells which appear to belong to the same cell category.     

Centrifugal actions on amacrine and ganglion cells in the retina of the turtle.

1. An electrophysiological investigation of efferent synapses in the retina of the turtle was conducted by recording intracellularly from amacrine cells. These cells have been selected because in birds they have been shown to have direct anatomical connexions with centrifugal fibre terminals. 2. Amacrine cells could be easily distinguished from most other retinal cells, except ganglion cells, by their different photo-responses. Because both amacrine and ganglion cells may generate action potentials they were distinguished by their responses to optic nerve stimulation. 3. The response of ganglion cells to single shock stimulation of the optic nerve consists of an antidromic action potential followed by a late synaptic potential. 4. Cells which did not show antidromic responses but were electrically excitable, by passing direct current through the recording electrode, were considered to be amacrine cells. 5. Amacrine cells generate an e.p.s.p. in response to optic nerve stimulation. An analysis of the e.p.s.p. suggests that it may be due to a single afferent fibre terminating in the proximity of the cell soma. By analogy to the bird, it is concluded that the amacrine cells e.p.s.p.s result from the activation of centrifugal fibres.     

Dendritic relationship between starburst amacrine cells and direction-selective ganglion cells in the rabbit retina

We investigated the dendritic relationship between starburst amacrine cells (SAs) and morphologically and physiologically characterized ON and ON-OFF direction-selective ganglion cells (DSGCs) in the rabbit retina. ON and ON-OFF DSGCs were found to exhibit tight dendritic cofasciculation with the SA plexus, visualized by immunolabelling of the vesicular acetylcholine transporter (VAChT). The degree of cofasciculation of both types of DSGC dendrites and SA plexus was found to be significant, unlike the relationship between non-DS cells and the SA plexus, which was close to chance distribution. No difference in the degree of cofasciculation in different regions of the DS dendritic field was observed. Individual SAs intracellularly injected both on the 'preferred' and 'null' side of the DSGCs showed the same degree of cofasciculation with the DSGCs. Therefore, the computation of motion direction is unlikely to result from apparent asymmetry in geometric proximity between SAs and DSGCs. Highly selective synaptic connections between SAs and DSGCs are necessary.     

Cell populations of the ganglion cell layer: displaced amacrine and matching amacrine cells in the pigeon retina

Summary The proportion and size distribution of ganglion and non-ganglion cells in the ganglion cell layer of different areas of the pigeon retina was examined in whole-mounts of the retina by retrograde axonal transport of horseradish peroxidase (HRP) from large brain injections. A maximum of 98% of cells were labelled in the red field and a maximum of 77% in the peripheral yellow field. Unlabelled cell bodies were 30% smaller than labelled ganglion cells and had a mean diameter of 6.2 m and a size range of 4 to 9 m. The morphology of cells in the ganglion cell layer was examined by Golgi staining of retinal whole-mounts. Small glia, displaced amacrine and ganglion cells were found. Displaced amacrine cell bodies were about 30% smaller than ganglion cells and their size distribution was similar to the unlabelled cells in HRP preparations. Displaced amacrine cells had small rounded cell bodies (mean diameter 6.2 m) increasing in size with eccentricity, and a unistratified dendritic tree of fine, nearly radial, varicose dendrites in sublamina 4 of the inner plexiform layer. They had elliptical dendritic fields (mean diameter 66 m) aligned parallel to the retina's horizontal meridian. A population of amacrine cells was found with somas at the inner margin of the inner nuclear layer and soma and dendritic morphology matching those of displaced amacrines. These amacrine cells had unistratified dendritic trees at the junction of sublaminae 1 and 2 of the inner plexiform layer. Pigeon displaced amacrine cells and their matching amacrines are similar to starburst cells of the rabbit retina. They may participate in on and off pathways to ganglion cells and their lamination suggests that they are cholinergic.     

Shift of intracellular chloride concentration in ganglion and amacrine cells of developing mouse retina

GABA and glycine provide excitatory action during early development: they depolarize neurons and increase intracellular calcium concentration. As neurons mature, GABA and glycine become inhibitory. This switch from excitation to inhibition is thought to result from a shift of intracellular chloride concentration ([Cl-]i) from high to low, but in retina, measurements of [Cl-]i or chloride equilibrium potential (ECl) during development have not been made. Using the developing mouse retina, we systematically measured [Cl-]i in parallel with GABA's actions on calcium and chloride. In ganglion and amacrine cells, fura-2 imaging showed that before postnatal day (P) 6, exogenous GABA, acting via ionotropic GABA receptors, evoked calcium rise, which persisted in HCO3- -free buffer but was blocked with 0 extracellular calcium. After P6, GABA switched to inhibiting spontaneous calcium transients. Concomitant with this switch we observed the following: 6-methoxy-N-ethylquinolinium iodide (MEQ) chloride imaging showed that GABA caused an efflux of chloride before P6 and an influx afterward; gramicidin-perforated-patch recordings showed that the reversal potential for GABA decreased from -45 mV, near threshold for voltage-activated calcium channel, to -60 mV, near resting potential; MEQ imaging showed that [Cl-]i shifted steeply around P6 from 29 to 14 mM, corresponding to a decline of ECl from -39 to -58 mV. We also show that GABAergic amacrine cells became stratified by P4, potentially allowing GABA's excitatory action to shape circuit connectivity. Our results support the hypothesis that a shift from high [Cl-]i to low causes GABA to switch from excitatory to inhibitory.     

Identification and characterization of SMI32-immunoreactive amacrine cells in the mouse retina

Mammalian neurons express the neural intermediate filament protein neurofilament (NF). In the retina, NFs have been detected primarily in the axons and processes of retinal ganglion and horizontal cells. We found an amacrine cell type that was immunolabeled with an antibody against SMI32, a non-phosphorylated epitope on neurofilament proteins of high molecular weight, in the mouse retina. This type of amacrine cell was non-randomly distributed, and these cells exhibited a central-peripheral density gradient. Most of these cells co-expressed GABA and ChAT, but not glycine or any other amacrine cell marker. These results suggest that some SMI32-immunoreactive amacrine cells belong to a GABAergic population, and that SMI32 can therefore be used as a marker for a subset of amacrine cells in addition to ganglion cells and horizontal cells in the mouse retina.     

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.     

Identification of ON–OFF direction-selective ganglion cells in the mouse retina

We identified the ON–OFF direction-selective ganglion cells (DSGCs) in the mouse retina and characterized their physiological, morphological and pharmacological properties. These cells showed transient responses to the onset and termination of a stationary flashing spot, and strong directional selectivity to a moving rectangle. Application of various pharmacological reagents demonstrated that the ON–OFF DSGCs in the mouse retina utilize a similar array of transmitters and receptors to compute motion direction to their counterparts in the rabbit retina. Voltage clamp recording showed that ON–OFF DSGCs in the mouse retina receive a larger inhibitory input when the stimulus is moving in the null direction and a larger excitatory input when the stimulus is moving in the preferred direction. Finally, intracellular infusion of neurobiotin revealed a bistratified dendritic field with recursive dendrites forming loop-like structures, previously classified as RG_D2 by morphology. Overall, the ON–OFF DSGCs in the mouse retina exhibit almost identical properties to their counterparts in the rabbit retina, indicating that the mechanisms for computing motion direction are conserved from mouse to rabbit, and probably also to higher mammals. This first detailed characterization of ON–OFF DSGCs in the mouse retina provides fundamental information for further study of maturation and regulation of the neuronal circuitry underlying computation of direction.     

Contrast rectification and distributed encoding By ON-OFF amacrine cells in the retina.

The encoding of luminance contrast by ON-OFF amacrine cells was investigated by intracellular recording in the retina of the tiger salamander (Ambystoma tigrinum). Contrast flashes of positive and negative polarity were applied at the center of the receptive field while the entire retina was light adapted to a background field of 20 cd/m(2). Many amacrine cells showed remarkably high contrast gain: Up to 20-35% of the maximum response was evoked by a contrast step of only 1%. In the larger signal domain, C50, the contrast required to evoke a response 50% of the maximum, was often remarkably low: 24 of 25 cells had a C50 value of < or =10% for at least one contrast polarity. Across cells and contrast polarity, the dynamic ranges varied from extremely narrow to broad, thereby blanketing the range of reflectances associated with objects in natural environments. Although some cells resembled "contrast rectifiers," by showing similar responses to contrasts of opposite polarity, many did not. Thus for contrast gain and C50, individual cells could show a strong preference for either negative or positive contrast. In the time domain, the preference was strong and unidirectional: for equal contrast steps, the latency of the response to negative contrast was 20-45 ms shorter than that for positive contrast. The present results, when compared with those for bipolar cells, suggest that, on average, amacrine cells add some amplification, particularly for negative contrast, to the high contrast gain already established by bipolar cells. In the time domain, our data reveal a striking transformation from bipolar to amacrine cells in favor of negative contrast. These and further observations have implications for the input and output of amacrine cell circuits. The present finding of substantial differences between cells reveals a potential substrate for distributed encoding of luminance contrast within the ON-OFF amacrine cell population.     

Reciprocal synaptic interactions between rod bipolar cells and amacrine cells in the rat retina.

Reciprocal synaptic transmission between rod bipolar cells and presumed A17 amacrine cells was studied by whole cell voltage-clamp recording of rod bipolar cells in a rat retinal slice preparation. Depolarization of a rod bipolar cell evoked two identifiable types of Ca2+ current, a T-type current that activated at about -70 mV and a current with L-type pharmacology that activated at about -50 mV. Depolarization to greater than or equal to -50 mV also evoked an increase in the frequency of postsynaptic currents (PSCs). The PSCs reversed at approximately ECl (the chloride equilibrium potential), followed changes in ECl, and were blocked by gamma-aminobutyric acidA (GABAA) and GABAC receptor antagonists and thus were identified as GABAergic inhibitory PSCs (IPSCs). Bipolar cells with cut axons displayed the T-type current but lacked an L-type current and depolarization-evoked IPSCs. Thus L-type Ca2+ channels are placed strategically at the axon terminals to mediate transmitter release from rod bipolar cells. The IPSCs were blocked by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, indicating that non-NMDA receptors mediate the feed-forward bipolar-to-amacrine excitation. The NMDA receptor antagonist 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid had no consistent effect on the depolarization-evoked IPSCs, indicating that activation of NMDA receptors is not essential for the feedforward excitation. Tetrodotoxin (a blocker of voltage-gated Na+ channels) reversibly suppressed the reciprocal response in some cells but not in others, indicating that graded potentials are sufficient for transmitter release from A17 amacrine cells, but suggesting that voltage-gated Na+ channels, under some conditions, can contribute to transmitter release.     

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.     

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.     

Effects of light and darkness on cell deaths in damaged retinal ganglion cells of the carp retina

Effects of light and darkness on the apoptosis of retinal ganglion cells (RGCs) in young carp were measured by TUNEL method after transection of the optic nerve. Following the operation, the fish were kept under one of four regimens; constant darkness (DD), constant light (LL), 12 hr light and 12 hr dark (LD) and 3 hr of flickering light followed by 21 hr in the dark (FL). On day 3, the highest ratio of apoptotic RGCs was seen under conditions of DD, followed by LL, LD, and FL. On day 6, the percentages of apoptotic RGCs were lower under every experimental condition than what they had been earlier on day 3, but the same ranking order was maintained. Immunohistochemically it could be shown that phosphorylated ERKs were more intensively localized in FL rather than DD retinas. The results suggest that illumination regimens, and in particular cyclic diurnal light/dark changes, have an influence on the degree of apoptosis of damaged RGCs, and that inhibition of apoptosis is correlated with the higher expression of phosphorylated ERKs.     

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.