Gap junctions between AII amacrine cells and calbindin-positive bipolar cells in the rabbit retina

Electrical synapses or gap junctions occur between many retinal neurons. However, in most cases, the gap junctions have not been visualized directly. Instead, their presence has been inferred from tracer spread throughout the network of cells. Thus, tracer coupling is taken as a marker for the presence of gap junctions between coupled cells. AII amacrine cells are critical interneurons in the rod pathway of the mammalian retina. Rod bipolar cell output passes to AII amacrine cells, which in turn make conventional synapses with OFF cone bipolar cells and gap junctions with ON cone bipolar cells. Injections of biotinylated tracers into AII amacrine cells reveals coupling between the AII amacrine cell network and heterologous coupling with a variety of ON cone bipolar cells, including the calbindin-positive cone bipolar cell. To directly visualize gap junctions in this network, we prepared material for electron microscopy that was double labeled with antibodies to calretinin and calbindin to label AII amacrine cells and calbindin-positive cone bipolar cells, respectively. AII amacrine cells were postsynaptic to large vesicle-laden rod bipolar terminals, as previously reported. Gap junctions were identified between AII amacrine cells and calbindin-positive cone bipolar cell terminals identified by the presence of immunostaining and ribbon synapses. This represents direct confirmation of gap junctions between two different yet positively identified cells, which are tracer coupled, and provides additional evidence that tracer coupling with Neurobiotin indicates the presence of gap junctions. These results also definitively establish the presence of gap junctions between AII amacrine cells and calbindin bipolar cells which can therefore carry rod signals to the ON alpha ganglion cell.     

The AII Amacrine Network: Coupling can Increase Correlated Activity

Retinal ganglion cells in the cat respond to single rhodopsin isomerizations with one to three spikes. This quantal signal is transmitted in the retina by the rod bipolar pathway: rod→rod bipolar→AII→cone bipolar→ganglion cell. The two-dimensional circuit underlying this pathway includes extensive convergence from rods to an AII amacrine cell, divergence from a rod to several AII and ganglion cells, and coupling between the AII amacrine cells. In this study we explored the function of coupling by reconstructing several AII amacrine cells and the gap junctions between them from electron micrographs; and simulating the AII network with and without coupling. The simulation showed that coupling in the AII network can: (1) improve the signal/noise ratio in the AII network; (2) improve the signal/noise ratio for a single rhodopsin isomerization striking in the periphery of the ganglion cell receptive field center, and therefore in most ganglion cells responding to a single isomerization; (3) expand the AII and ganglion cells' receptive field center; and (4) expand the “correlation field”. All of these effects have one major outcome: an increase in correlation between ganglion cell activity. Well correlated activity between the ganglion cells could improve the brain's ability to discriminate few absorbed external photons from the high background of spontaneous thermal isomerizations. Based on the possible benefits of coupling in the AII network, we suggest that coupling occurs at low scotopic luminances. Copyright © 1996 Elsevier Science Ltd.     

Gap junctional regulatory mechanisms in the AII amacrine cell of the rabbit retina

Gap junctions are commonplace in retina, often between cells of the same morphological type, but sometimes linking different cell types. The strength of coupling between cells derives from the properties of the connexins, but also is regulated by the intracellular environment of each cell. We measured the relative coupling of two different gap junctions made by AII amacrine cells of the rabbit retina. Permeability to the tracer Neurobiotin was measured at different concentrations of the neuromodulators dopamine, nitric oxide, or cyclic adenosine monophosphate (cAMP) analogs. Diffusion coefficients were calculated separately for the gap junctions between pairs of AII amacrine cells and for those connecting AII amacrine cells with ON cone bipolar cells. Increased dopamine caused diffusion rates to decline more rapidly across the AII-AII gap junctions than across the AII-bipolar cell gap junctions. The rate of decline at these sites was well fit by a model proposing that dopamine modulates two independent gates in AII-AII channels, but only a single gate on the AII side of the AII-bipolar channel. However, a membrane-permeant cAMP agonist modulated both types of channel equally. Therefore, the major regulator of channel closure in this network is the local cAMP concentration within each cell, as regulated by dopamine, rather than different cAMP sensitivity of their respective gates. In contrast, nitric oxide preferentially reduced AII-bipolar cell permeabilities. Coupling from AII amacrine cells to the different bipolar cell subtypes was differentially affected by dopamine, indicating that light adaptation acting via dopamine release alters network coupling properties in multiple ways.     

Intrinsic properties and functional circuitry of the AII amacrine cell

Amacrine cells represent the most diverse class of retinal neuron, comprising dozens of distinct cell types. Each type exhibits a unique morphology and generates specific visual computations through its synapses with a subset of excitatory interneurons (bipolar cells), other amacrine cells, and output neurons (ganglion cells). Here, we review the intrinsic and network properties that underlie the function of the most common amacrine cell in the mammalian retina, the AII amacrine cell. The AII connects rod and cone photoreceptor pathways, forming an essential link in the circuit for rod-mediated (scotopic) vision. As such, the AII has become known as the rod-amacrine cell. We, however, now understand that AII function extends to cone-mediated (photopic) vision, and AII function in scotopic and photopic conditions utilizes the same underlying circuit: AIIs are electrically coupled to each other and to the terminals of some types of ON cone bipolar cells. The direction of signal flow, however, varies with illumination. Under photopic conditions, the AII network constitutes a crossover inhibition pathway that allows ON signals to inhibit OFF ganglion cells and contributes to motion sensitivity in certain ganglion cell types. We discuss how the AII's combination of intrinsic and network properties accounts for its unique role in visual processing.     

Rod vision: pathways and processing in the mammalian retina

Bipolar cells in the mammalian retina are postsynaptic to either rod or cone photoreceptors, thereby segregating their respective signals into parallel vertical streams. In contrast to the cone pathways, only one type of rod bipolar cell exists, apparently limiting the routes available for the propagation of rod signals. However, due to numerous interactions between the rod and cone circuitry, there is now strong evidence for the existence of up to three different pathways for the transmission of scotopic visual information. Here we survey work over the last decade or so that have defined the structure and function of the interneurons subserving the rod pathways in the mammalian retina. We have focused on: (1) the synaptic ultrastructure of the interneurons; (2) their light-evoked physiologies; (3) localization of specific transmitter receptor subtypes; (4) plasticity of gap junctions related to changes in adaptational state; and (5) the functional implications of the existence of multiple rod pathways. Special emphasis has been placed on defining the circuits underlying the different response components of the AII amacrine cell, a central element in the transmission of scotopic signals.     

左旋精氨酸对兔眼AⅡ无长突细胞与开放性视锥型双极细胞间示踪剂藕连的调控研究

目的　探讨兔眼视网膜AⅡ无长突细胞 (AⅡamacrinecell,AⅡ AC)和开放性视锥型双极细胞 (onconebipolarcell,ON CB)间缝隙连接通道的相对通透性及左旋精氨酸对通道的调节。方法　单个AⅡ AC显微注射神经生物素 (neurobiotin ,NB)后 ,采用共焦显微镜测定以上两类异源型细胞群中NB的分布 ,并用 4mmol/L左旋精氨酸对其进行调节。然后用兔抗Calretinin抗体对注射后的视网膜进行免疫组织化学染色。结果　藕连的ON CB中的NB的浓度低于藕连的AⅡ AC中NB的浓度。与藕连的AⅡ AC比较 ,左旋精氨酸选择性的减少了与AⅡ AC藕连的ON CB中的NB的浓度(t=2 5 11,P <0 0 5 )。AⅡ AC被Calretinin抗体染色阳性。结论　相对于AⅡ AC和ON CB间可能的异源型缝隙连接 ,NB较易通过AⅡ AC间的同源型缝隙连接。左旋精氨酸可能使cGMP浓度升高而作用于双极细胞侧的缝隙连接 ,选择性的减少了这种双极细胞的示踪剂标记。     

Unique functional properties of the APB sensitive and insensitive rod pathways signaling light decrements in mouse retinal ganglion cells

Light decrements are mediated by two distinct groups of rod pathways in the dark-adapted retina that can be differentiated on the basis of their sensitivity to the glutamate agonist DL-2-amino-phosphonobutyric (APB). By means of the APB sensitive pathway, rods transmit light decrements via rod bipolar cells to AII amacrine cells, then to Off cone bipolar cells, which in turn innervate the dendrites of Off ganglion cells. APB hyperpolarizes rod bipolar cells, thus blocking this rod pathway. With APB insensitive pathways, rods either directly synapse onto Off cone bipolar cells, or rods pass light decrement signal to cones by gap junctions. In the present study, whole-cell patch-clamp recordings were made from ganglion cells in the dark-adapted mouse retina to investigate the functional properties of APB sensitive and insensitive rod pathways. The results revealed several clear-cut differences between the APB sensitive and APB insensitive rod pathways. The latency of Off responses to a flashing spot of light was significantly shorter for the APB insensitive pathways than those for the APB sensitive pathway. Moreover, Off responses of the APB insensitive pathways were found to be capable of following substantially higher stimulus frequencies. Nitric oxide was found to selectively block Off responses in the APB sensitive rod pathway. Collectively, these results provide evidence that the APB sensitive and insensitive rod pathways can convey different types of information signaling light decrements in the dark-adapted retina.     

Slow and fast rod ERG pathways in patients with X-linked complete stationary night blindness carrying mutations in the NYX gene

PURPOSE: To study the slow and fast rod signals of the scotopic 15-Hz flicker ERG in patients carrying mutations in the NYX gene, which has been recently identified as the cause of the complete form of congenital stationary night blindness, CSNB1. METHODS: Twenty eyes of 11 patients with CSNB1 who had nondetectable standard ERG rod b-waves were involved in the study. Scotopic ERG response amplitudes and phases to flicker intensities ranging from -3.37 to -0.57 log scotopic trolands. sec (scot td. sec) were measured at a flicker frequency of 15 Hz. ERG signals to flicker intensities between -3.37 and -1.97 and between -1.17 and -0.57 log scot td. sec were considered to represent primarily the slow and fast rod ERG pathway, respectively. Additionally, standard ERGs were performed. Twenty-two normal volunteers served as control subjects. RESULTS: For the slow rod ERG pathway, all patients exhibited ERG signals that were indistinguishable from noise. Accordingly, there was no systematic phase behavior for the slow rod signals. For the fast rod ERG pathway, the signals were significantly above noise, but they were significantly reduced in amplitude and advanced in phase. CONCLUSIONS: There is evidence that the slow and the fast rod ERG signals can be attributed to the rod bipolar-AII cell pathway and the rod-cone-coupling pathway, respectively. The current study provides evidence to suggest that a defective NYX gene product (nyctalopin) prevents detectable signal transmission through ON rod bipolar cells, but there is a residual transmission through rod-cone gap junctions in CSNB1, possibly through the OFF cone pathway.     

Comparison of the responses of AII amacrine cells in the dark- and light-adapted rabbit retina.

We studied the light-evoked responses of AII amacrine cells in the rabbit retina under dark- and light-adapted conditions. In contrast to the results of previous studies, we found that AII cells display robust responses to light over a 6-7 log unit intensity range, well beyond the operating range of rod photoreceptors. Under dark adaptation, AII cells showed an ON-center/OFF-surround receptive-field organization. The intensity-response profile of the center-mediated response component followed a dual-limbed sigmoidal function indicating a transition from rod to cone mediation as stimulus intensities were increased. Following light adaptation, the receptive-field organization of AII cells changed dramatically. Light-adapted AII cells showed both ON- and OFF-responses to stimulation of the center receptive field, but we found no evidence for an antagonistic surround. Interestingly, the OFF-center response appeared first following rapid light adaptation and was then replaced gradually over a 1-4 min period by the emerging ON-center response component. Application of the metabotropic glutamate receptor agonist APB, the ionotropic glutamate blocker CNQX, 8-bromo-cGMP, and the nitric oxide donor SNAP all showed differential effects on the various center-mediated responses displayed by dark- and light-adapted AII cells. Taken together, these pharmacological results indicated that different synaptic circuits are responsible for the generation of the different AII cell responses. Specifically, the rod-driven ON-center responses are apparently derived from rod bipolar cell synaptic inputs, whereas the cone-driven ON-center responses arise from signals crossing the gap junctions between AII cells and ON-center cone bipolar cells. Additionally, the OFF-center response of light-adapted AII cells reflects direct synaptic inputs from OFF-center cone bipolar cells to AII dendritic processes in the distal inner plexiform layer.     

Synaptic patterns and response properties of bipolar and ganglion cells in the cat retina

After intracellular recording, bipolar cells of the cat retina have been stained with HRP and their contacts in the outer and inner plexiform layers examined by electron microscopy. Rod bipolars and cone bipolar cb6 make invaginating, ribbon related contacts with photoreceptors, hyperpolarize in response to light, and have axons terminating in layer b of the IPL. The axon terminal of cb2 ends in layer a of the IPL and its basal contacts with cones mediate hyperpolarizing light-responses. Cone bipolar cb5 is a center-depolarizing type with an axon ending in layer b but its cone contacts are at semi-invaginating basal junctions. Except for the amacrine-contacting rod bipolar cell, all cone bipolar types synapse with both amacrine and ganglion cells in the inner plexiform layer. In addition cb5 contacts AII amacrine cells with large gap junctions, and is physiologically rod dominated.     

Processing of scotopic increments and decrements

Rod and cone photoreceptors send their signals to ON- and OFF-retinal ganglion cells through different pathways in the primate retina. We hypothesized that increments and decrements of light may be processed differently by the rod-bipolar pathway because of the funneling of the rod signal through the rod bipolar cell. We tested this hypothesis using a psychophysical adaptation paradigm, which has provided evidence that photopic increments and decrements of light are processed by ON- and OFF-pathways in the human visual system. We had observers adapt to either a rapid-on or rapid-off sawtooth waveform, under both photopic and scotopic conditions. We then measured detection thresholds for one cycle of a rapid-on or rapid-off sawtooth stimulus. For photopic stimuli, sawtooth adaptation asymmetrically raised thresholds for test stimuli in a manner that depended on the polarity of the adaptation stimulus. For scotopic stimuli, thresholds were raised, but no significant selective adaptation effect was found. By repeating the photopic condition with sawtooth stimuli which had been filtered using an impulse response function derived for the rod system, we demonstrated that the lack of selective adaptation was not a consequence of the sluggish temporal response of the rod-bipolar pathway. We conclude instead that the reduced effectiveness of sawtooth adaptation is due to channeling of rod photoreceptor signals through the rod bipolar cell before reaching ON- and OFF-ganglion cells.     

The effect of dark adaptation on the responses of cat retinal ganglion cells to eyeball deformation

Eyeball deformation in total darkness leads to an activation of on-center ganglion cells and an inhibition of off-center ganglion cells. After "deformation off" most on-center ganglion cell activity decreased slowly to the normal spontaneous dark level, while in off-center ganglion cells some returned according to an exponential function to normal dark activity, while others had a transient postinhibitory activation period. In general, the response type of latency class I and latency class II neurons was the same. Dark adaptation of 30-45 min duration only changed this neuronal response pattern slightly, if at all. A detailed statistical analysis is provided for the four classes of retinal ganglion cells recorded: latency class I on-center and off-center neurons and latency class II on-center and off-center neurons. The missing effects of dark adaptation on neuronal responses evoked by eyeball deformation are explained by three possible models. The more plausible one assumes that horizontal cells are depolarized by retinal stretch. Their interaction with cone on-bipolars or cone off-bipolars is fairly independent of photoreceptor adaptation or transmitter release at the cone pedicles and is still effective when all molecular receptor sites at cone/bipolar cell synapses are occupied during scotopic states of dark adaptation. In psychophysical experiments (two subjects), as in the neuronal responses, we also could not find any indication that the "pressure phosphenes" evoked by lateral eyeball indentation are altered during dark adaptation.     

Alterations of slow and fast rod ERG signals in patients with molecularly confirmed Stargardt disease type 1

PURPOSE: To investigate the slow and fast rod signals of the scotopic 15-Hz flicker ERG in patients with molecularly confirmed Stargardt disease type I (STGD1). There is evidence that these slow and the fast rod ERG signals can be attributed to the rod bipolar-AII cell pathway and the rod-cone coupling pathway, respectively. METHODS: Twenty-seven patients with STGD1 with mutations in both alleles of the ABCA4 gene were included. Scotopic ERG response amplitudes and phases to flicker intensities ranging from -3.37 to -0.57 log scotopic troland x sec (log scot td x sec) were measured at a flicker frequency of 15 Hz. In addition, scotopic standard ERGs were obtained. Twenty-two normal subjects served as controls. RESULTS: The amplitudes of both the slow and fast rod ERG signals were significantly reduced in the STGD1 group. The phases of the slow rod signals lagged significantly, whereas those of the fast rod signals did not. The standard scotopic ERG did not reveal significant alterations. CONCLUSIONS: The results provide evidence that a defective ABCA4 transporter can functionally affect both the rod bipolar-AII cell pathway and the rod-cone coupling pathway. In STGD1, the scotopic 15-Hz flicker ERG may reveal subtle abnormalities at different sites within the rod system that remain undetected by standard ERG techniques.     

Clinical electrophysiology of two rod pathways: normative values and clinical application

BACKGROUND: The scotopic 15-Hz flicker electroretinogram (ERG) has two limbs (slow and fast ERG rod signals), and these have been attributed to two retinal rod pathways (the ON rod bipolar and AII amacrine pathway and the rodcone gap-junction pathway). The aim of this study was to provide normative values of the scotopic 15-Hz flicker ERG, to estimate the inter-individual variability, and to apply this method to a clinical setting. METHODS: Twenty-two normal subjects, one patient with retinitis pigmentosa (RP), and two patients with Stargardt's mascular dystrophy (SMD) participated in the study. The SMD patients were screened for mutations in the 50 exons of the ABCA4 (formerly ABCR) gene. We measured ERG response amplitudes and phases to flicker intensities ranging from -3.37 to -0.57 log scotopic trolands s at a flicker frequency of 15 Hz. RESULTS: The normal scotopic 15-Hz flicker ERG showed a biphasic amplitude pattern with a minimum at about-1.57 log scotopic trolands s, where there was an abrupt phase shift of about 180 deg. The inter-individual variability in ERG amplitude ranged from 47% to 67% for the slow and from 41% to 64% for the fast rod signal. Both the RP patient and the SMD patients (who were compound heterozygotes for mutations in the ABCA4 gene) showed reduced amplitudes for the two rod ERG pathways. CONCLUSION: The inter-individual variability might be explained by anatomical differences between individual retinae. In the RP patient, the amplitude reductions corresponded well with the standard rod ERG. In the SMD patients, however, the scotopic 15-Hz flicker ERG revealed rod dysfunction, whereas the standard rod ERG was within normal limits. The scotopic 15-Hz flicker method may be more sensitive than the standard rod ERG.     

Dopaminergic modulation of tracer coupling in a ganglion-amacrine cell network

Many retinal ganglion cells are coupled via gap junctions with neighboring amacrine cells and ganglion cells. We investigated the extent and dynamics of coupling in one such network, the OFF alpha ganglion cell of rabbit retina and its associated amacrine cells. We also observed the relative spread of Neurobiotin injected into a ganglion cell in the presence of modulators of gap junctional permeability. We found that gap junctions between amacrine cells were closed via stimulation of a D(1) dopamine receptor, while the gap junctions between ganglion cells were closed via stimulation of a D(2) dopamine receptor. The pairs of hemichannels making up the heterologous gap junctions between the ganglion and amacrine cells were modulated independently, so that elevations of cAMP in the ganglion cell open the ganglion cell hemichannels, while elevations of cAMP in the amacrine cell close its hemichannels. We also measured endogenous dopamine release from an eyecup preparation and found a basal release from the dark-adapted retina of approximately 2 pmol/min during the day. Maximal stimulation with light increased the rate of dopamine release from rabbit retina by 66%. The results suggest that coupling between members of the OFF alpha ganglion cell/amacrine cell network is differentially modulated with changing levels of dopamine.     

Amacrine cells in scotopic vision

Signals from rod bipolar cells of cat retina are processed by a variety of rod amacrine cells before finally arriving at ganglion cells. Three of these rod amacrine cells (AII, A13, and A17 ) have been studied at the physiological and anatomical levels; the results suggest that each carries out a unique visual function: AII cells appear to quicken the response time of the rod system in the mid-scotopic range, while A17 cells may increase the light-gathering area of rod bipolars near visual threshold. Stimulation of A13 cells may disinhibit ganglion cells, thus heightening their responsiveness at low levels of illumination.     

Localization of AMPA-selective glutamate receptor subunits in the cat retina: a light- and electron-microscopic study

The distribution of AMPA-selective glutamate receptor subunits was studied in the cat retina using antisera against GluR1 and GluR2/3. Both antisera were localized in postsynaptic sites in the outer plexiform layer (OPL) as well as the inner plexiform layer (IPL). Immunoreactivity for GluR1 was seen in a subpopulation of OFF cone bipolar cells and a number of amacrine and ganglion cells. Within the IPL, processes staining for GluR1 received input from OFF and ON cone bipolar cells but not from rod bipolars. Labeling for GluR2/3 was seen in horizontal cells, an occasional cone bipolar cell, and numerous amacrine and ganglion cells. In the IPL, GluR2/3 staining was postsynaptic to cone bipolar cells in both sublaminae. AII amacrine cells which receive rod bipolar input were also labeled for GluR2/3. With both antisera, staining was limited to a single member of the bipolar dyad complex, providing morphological evidence for functional diversity in glutamatergic pathways.     

The rod circuit in the rabbit retina

Mammalian retinae have a well-defined neuronal pathway that serves rod vision. In rabbit retina, the different populations of interneurons in the rod pathway can be selectively labeled, either separately or in combination. The rod bipolar cells show protein kinase C immunoreactivity; the rod (AII) amacrine cells can be distinguished in nuclear-yellow labeled retina; the rod reciprocal (S1 & S2) amacrine cells accumulate serotonin; and the dopaminergic amacrine cells show tyrosine-hydroxylase immunoreactivity. Furthermore, intracellular dye injection of the microscopically identified interneurons enables whole-population and single-cell studies to be combined in the same tissue. Using this approach, we have been able to analyze systematically the neuronal architecture of the rod circuit across the rabbit retina and compare its organization with that of the rod circuit in central cat retina. In rabbit retina, the rod interneurons are not organized in a uniform neuronal module that is simply scaled up from central to peripheral retina. Moreover, peripheral fields in superior and inferior retina that have equivalent densities of each neuronal type show markedly different rod bipolar to AII amacrine convergence ratios, with the result that many more rod photoreceptors converge on an AII amacrine cell in superior retina. In rabbit retina, much of the convergence in the rod circuit occurs in the outer retina whereas, in central cat retina, it is more evenly distributed between the inner and outer retina.     

Unusual coupling patterns of a cone bipolar cell in the rabbit retina

In mammals, gap junctions between retinal bipolar cells are generally small and tracer coupling has not been previously demonstrated. In this study, Neurobiotin was injected into the Ba3-type cone bipolar cell, a medium-field cone bipolar cell that ramifies in sublamina a of the rabbit retina. Tracer spread to many other Ba3 bipolar cells, presumably through gap junctions. It also spread to a smaller field bipolar cell called the Ba1 that ramifies at the same depth of the inner plexiform layer. Injection of Neurobiotin into Ba1 bipolar cells did not produce staining beyond the injected cell. Tracer coupling from the Ba3 was therefore both heterologous, in that different cell types were stained, and asymmetric. The unusual properties of this bipolar cell suggest that its function may differ from that of most cone bipolar cells, which are narrow-field, do not overlap, and are poorly coupled to one another.     

A decay of gap junctions associated with ganglion cell differentiation during retinal regeneration of the adult newt

Changes in the gap junctional coupling and maturation of voltage-activated Na(+) currents during regeneration of newt retinas were examined by whole-cell patch-clamping in slice preparations. Progenitor cells in regenerating retinas did not exhibit Na(+) currents but showed prominent electrical and tracer couplings. Cells identified by LY-fills were typically slender. Na(+) currents were detected in premature ganglion cells with round somata in the 'intermediate-II' regenerating retina. No electrical and tracer couplings were observed between these cells. Mature ganglion cells did not exhibit electrical coupling, but showed tracer coupling. On average, the maximum Na(+) current amplitude recorded from premature ganglion cells was roughly 2.5-fold smaller than that of mature ganglion cells. In addition, the activation threshold of the Na(+) current was nearly 11 mV more positive than that of mature cells. We provide morphological and physiological evidence showing that loss of gap junctions between progenitor cells is associated with ganglion cell differentiation during retinal regeneration and that new gap junctions are recreated between mature ganglion cells. Also we provide evidence suggesting that the loss of gap junctions correlates with the appearance of voltage-activated Na(+) currents in ganglion cells.