Application of transretinal current stimulation for the study of bipolar-amacrine transmission

Transretinal current flowing from the receptor side to the vitreous side depolarizes the axon terminals of retinal cells and facilitates the release of transmitter. Such current elicited a depolarizing response in off-center bipolar cells and a hyperpolarizing response in on-center bipolar cells. It also elicited a response of relatively complex waveform in amacrine cells. The responses elicited in bipolar cells were suppressed in the presence of 5-10 mM glutamate in the perfusing Ringer solution, while the responses of amacrine cells persisted, although their waveform changed to a simple one that showed monotonic depolarization irrespective of the type of amacrine cell and were accompanied by a decrease in the membrane resistance. The results indicate excitatory synaptic transmission from bipolar cells to amacrine cells. Since the response elicited by current in ON-OFF cells was almost identical to those elicited in ON or OFF amacrine cells, the transient nature of their light response cannot be due to their membrane properties. ON-OFF cells responded to transretinal current flowing in the opposite direction with a small hyperpolarization accompanied by a resistance increase. The hyperpolarizing response was suppressed by the addition of GABA in glutamate Ringer solution. The results suggest an activation by the current of GABA-ergic feedback pathways from amacrine cells to bipolar cells.     

Visual stimulation is required for refinement of ON and OFF pathways in postnatal retina

ON and OFF pathways separately relay increment and decrement luminance signals from retinal bipolar cells to cortex. ON-OFF retinal ganglion cells (RGCs) are activated via synaptic inputs onto bistratified dendrites localized in the ON and OFF regions of the inner plexiform layer. Postnatal maturational processes convert bistratifying ON-OFF RGCs to monostratifying ON and OFF RGCs. Although visual deprivation influences refinement of higher visual centers, no previous studies suggest that light regulates either the development of the visual-evoked signaling in retinal ON and OFF pathways, nor pruning of bistratified RGC dendrites. We find that dark rearing blocks both the maturational loss of ON-OFF responsive RGCs and the pruning of dendrites. Thus, in retina, there is a previously unrecognized, pathway-specific maturation that is profoundly affected by visual deprivation.     

Axonal Synapses Utilize Multiple Synaptic Ribbons in the Mammalian Retina

In the mammalian retina, bipolar cells and ganglion cells which stratify in sublamina a of the inner plexiform layer (IPL) show OFF responses to light stimuli while those that stratify in sublamina b show ON responses. This functional relationship between anatomy and physiology is a key principle of retinal organization. However, there are at least three types of retinal neurons, including intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells, which violate this principle. These cell types have light-driven ON responses, but their dendrites mainly stratify in sublamina a of the IPL, the OFF sublayer. Recent anatomical studies suggested that certain ON cone bipolar cells make axonal or ectopic synapses as they descend through sublamina a, thus providing ON input to cells which stratify in the OFF sublayer. Using immunoelectron microscopy with 3-dimensional reconstruction, we have identified axonal synapses of ON cone bipolar cells in the rabbit retina. Ten calbindin ON cone bipolar axons made en passant ribbon synapses onto amacrine or ganglion dendrites in sublamina a of the IPL. Compared to the ribbon synapses made by bipolar terminals, these axonal ribbon synapses were characterized by a broad postsynaptic element that appeared as a monad and by the presence of multiple short synaptic ribbons. These findings confirm that certain ON cone bipolar cells can provide ON input to amacrine and ganglion cells whose dendrites stratify in the OFF sublayer via axonal synapses. The monadic synapse with multiple ribbons may be a diagnostic feature of the ON cone bipolar axonal synapse in sublamina a. The presence of multiple ribbons and a broad postsynaptic density suggest these structures may be very efficient synapses. We also identified axonal inputs to ipRGCs with the architecture described above.     

Ionic mechanisms underlying the responses of off-center bipolar cells in the carp retina. II. Studies on responses evoked by transretinal current stimulation

Transretinal current pulses flowing from the receptor side to the vitreous side of the retina cause transient release of transmitter from the photoreceptor terminals, and in off-center bipolar cells they evoke transient depolarizations with a brief (less than 1 ms) synaptic delay. Since it is known that the presence of Na+ in the external medium is not essential for this type of transmitter release, we used this procedure to examine the role of [Na+]o in the generation of light- evoked responses (hyperpolarizing to spot illumination in the receptive field center and depolarizing to an annulus in the surround) of this type of bipolar cell. When the cell membrane was steadily depolarized by current injection through the recording microelectrode, the depolarizing response evoked by the transretinal current pulses decreased in amplitude and reversed its polarity at above +45 mV. Conversely, the response amplitude increased when the cell was steadily hyperpolarized. The reversal potential seems to be lowered in low [Na+]o (28 mM). Removal of Na+ from the superfusate hyperpolarized the cell and both the light-evoked and current-evoked responses disappeared. From these observations, it is hypothesized that the hyperpolarizing center response of the off-center bipolar cells is a result of removal of sustained depolarization produced by sodium permeability increase.     

Morphological and functional organization of ON and OFF pathways in the adult newt retina

Morphological and functional organization of ON and OFF pathways in the adult newt retina were examined by intracellular recording and staining techniques and immunohistochemistry. Synaptotagmin immunoreactivity discriminated three broad bands within the IPL: the distal band (sublamina I), the middle band (sublamina II) consisting of two dense punctate bands (sublaminae II(a) and II(b)), and proximal band (sublamina III). The Lucifer-yellow labeled OFF amacrine and ganglion cells send their processes mainly in sublamina I and/or II(a) where OFF bipolar cells extend their axon terminals, while ON amacrine and ganglion cells send their processes in sublamina III and/or II(b) where ON bipolar cells extend their axon terminals. Processes of ON-OFF amacrine and ganglion cells ramify broadly in the whole thickness of the IPL. Many bipolar cells responded to light spot with a transient hyperpolarization at both light onset and offset. They are probably subtypes of ON bipolar cells, because their axon terminals branch mainly in sublaminae III and/or II(b), although a few cells ramified the axon at both sublaminae II(a) and III. Two immunohistochemical markers for bipolar cells, PKC and RB-1, identified axon terminals in sublaminae III and/or II(b). From the ramification pattern of axon terminal, they are probably subtypes of ON bipolar cells. ChAT-ir amacrine cells ramified their dendrites in either sublamina I or II(b). Altogether, present studies support the general idea of segregation of ON and OFF pathways in sublaminae a and b of the IPL.     

Response dynamics of bullfrog ON-OFF RGCs to different stimulus durations

Stimulus duration is an important feature of visual stimulation. In the present study, response properties of bullfrog ON-OFF retinal ganglion cells (RGCs) in exposure to different visual stimulus durations were studied. By using a multi-electrode recording system, spike discharges from ON-OFF RGCs were simultaneously recorded, and the cells’ ON and OFF responses were analyzed. It was found that the ON response characteristics, including response latency, spike count, as well as correlated activity and relative latency between pair-wise cells, were modulated by different light OFF intervals, while the OFF response characteristics were modulated by different light ON durations. Stimulus information carried by the ON and OFF responses was then analyzed, and it was found that information about different light ON durations was more carried by transient OFF response, whereas information about different light OFF intervals were more carried by transient ON response. Meanwhile, more than 80 % information about stimulus durations was carried by firing rate. These results suggest that ON-OFF RGCs are sensitive to different stimulus durations, and they can efficiently encode the information about visual stimulus duration by firing rate.     

Synaptic connections of calbindin-immunoreactive cone bipolar cells in the inner plexiform layer of rabbit retina

In the mammalian retina, information concerning various aspects of an image is transferred in parallel, and cone bipolar cells are thought to play a major role in this parallel processing. We have examined the synaptic connections of calbindin-immunoreactive (IR) ON cone bipolar cells in the inner plexiform layer (IPL) of rabbit retina and have compared these synaptic connections with those that we have previously described for neurokinin 1 (NK1) receptor-IR cone bipolar cells. A total of 325 synapses made by calbindin-IR bipolar axon terminals have been identified in sublamina b of the IPL. The axons of calbindin-IR bipolar cells receive synaptic inputs from amacrine cells through conventional synapses and are coupled to putative AII amacrine cells via gap junctions. The major output from calbindin-IR bipolar cells is to amacrine cell processes. These data resemble our findings for NK1 receptor-IR bipolar cells. However, the incidences of output synapses to ganglion cell dendrites of calbindin-IR bipolar cells are higher compared with the NK1-receptor-IR bipolar cells. On the basis of stratification level and synaptic connections, calbindin-IR ON cone bipolar cells might thus play an important role in the processing of various visual aspects, such as contrast, orientation, and approach sensing, and in transferring rod signals to the ON cone pathway.     

Electrophysiological evidence for push-pull interactions in the inner retina of turtle

The responses of the inner retinal neurons of turtle to light spots of sizes were studied in an attempt to reveal characteristics that may reflect possible interactions of the neural circuits underlying the center and surround responses. For the ON-OFF cells, the responses were also analyzed to observe whether interference or augmentation of these responses occur. The intracellular recordings revealed several such interactions, observed either in the form of altered spike activity or as changes in the transiency of the light responses. The ON-responding amacrine cell presented in this study became more sustained, while for the ON-OFF amacrine cells larger light spots tended to make the responses more transient and both the ON and OFF components became more pronounced. The spiking activity of the OFF-type ganglion cell shifted in relation to the light stimulus and the number of spikes observed upon presentation of larger spots increased. We suggest that the surround circuits activated by increasing light spots may substantially influence and reorganize not only the overall center-surround balance, but also the center response of the cells. Although it cannot be excluded that intrinsic membrane properties also influence these processes to some extent, it is more likely that lateral inhibition and disinhibitory mechanisms play the leading role in this process.     

Crossover inhibition in the retina: circuitry that compensates for nonlinear rectifying synaptic transmission

In the mammalian retina, complementary ON and OFF visual streams are formed at the bipolar cell dendrites, then carried to amacrine and ganglion cells via nonlinear excitatory synapses from bipolar cells. Bipolar, amacrine and ganglion cells also receive a nonlinear inhibitory input from amacrine cells. The most common form of such inhibition crosses over from the opposite visual stream: Amacrine cells carry ON inhibition to the OFF cells and carry OFF inhibition to the ON cells (”crossover inhibition”). Although these synapses are predominantly nonlinear, linear signal processing is required for computing many properties of the visual world such as average intensity across a receptive field. Linear signaling is also necessary for maintaining the distinction between brightness and contrast. It has long been known that a subset of retinal outputs provide exactly this sort of linear representation of the world; we show here that rectifying (nonlinear) synaptic currents, when combined thorough crossover inhibition can generate this linear signaling. Using simple mathematical models we show that for a large set of cases, repeated rounds of synaptic rectification without crossover inhibition can destroy information carried by those synapses. A similar circuit motif is employed in the electronics industry to compensate for transistor nonlinearities in analog circuits.     

Network Oscillations Drive Correlated Spiking of ON and OFF Ganglion Cells in the rd1 Mouse Model of Retinal Degeneration

Following photoreceptor degeneration, ON and OFF retinal ganglion cells (RGCs) in the rd-1/rd-1 mouse receive rhythmic synaptic input that elicits bursts of action potentials at ∼10 Hz. To characterize the properties of this activity, RGCs were targeted for paired recording and morphological classification as either ON alpha, OFF alpha or non-alpha RGCs using two-photon imaging. Identified cell types exhibited rhythmic spike activity. Cross-correlation of spike trains recorded simultaneously from pairs of RGCs revealed that activity was correlated more strongly between alpha RGCs than between alpha and non-alpha cell pairs. Bursts of action potentials in alpha RGC pairs of the same type, i.e. two ON or two OFF cells, were in phase, while bursts in dissimilar alpha cell types, i.e. an ON and an OFF RGC, were 180 degrees out of phase. This result is consistent with RGC activity being driven by an input that provides correlated excitation to ON cells and inhibition to OFF cells. A2 amacrine cells were investigated as a candidate cellular mechanism and found to display 10 Hz oscillations in membrane voltage and current that persisted in the presence of antagonists of fast synaptic transmission and were eliminated by tetrodotoxin. Results support the conclusion that the rhythmic RGC activity originates in a presynaptic network of electrically coupled cells including A2s via a Na⁺-channel dependent mechanism. Network activity drives out of phase oscillations in ON and OFF cone bipolar cells, entraining similar frequency fluctuations in RGC spike activity over an area of retina that migrates with changes in the spatial locus of the cellular oscillator.     

Mouse Ganglion-Cell Photoreceptors Are Driven by the Most Sensitive Rod Pathway and by Both Types of Cones

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are depolarized by light by two mechanisms: directly, through activation of their photopigment melanopsin; and indirectly through synaptic circuits driven by rods and cones. To learn more about the rod and cone circuits driving ipRGCs, we made multielectrode array (MEA) and patch-clamp recordings in wildtype and genetically modified mice. Rod-driven ON inputs to ipRGCs proved to be as sensitive as any reaching the conventional ganglion cells. These signals presumably pass in part through the primary rod pathway, involving rod bipolar cells and AII amacrine cells coupled to ON cone bipolar cells through gap junctions. Consistent with this interpretation, the sensitive rod ON input to ipRGCs was eliminated by pharmacological or genetic disruption of gap junctions, as previously reported for conventional ganglion cells. A presumptive cone input was also detectable as a brisk, synaptically mediated ON response that persisted after disruption of rod ON pathways. This was roughly three log units less sensitive than the rod input. Spectral analysis revealed that both types of cones, the M- and S-cones, contribute to this response and that both cone types drive ON responses. This contrasts with the blue-OFF, yellow-ON chromatic opponency reported in primate ipRGCs. The cone-mediated response was surprisingly persistent during steady illumination, echoing the tonic nature of both the rod input to ipRGCs and their intrinsic, melanopsin-based phototransduction. These synaptic inputs greatly expand the dynamic range and spectral bandpass of the non-image-forming visual functions for which ipRGCs provide the principal retinal input.     

Synaptic inputs to the ganglion cells in the tiger salamander retina

The postsynaptic potentials (PSPs) that form the ganglion cell light response were isolated by polarizing the cell membrane with extrinsic currents while stimulating at either the center or surround of the cell's receptive field. The time-course and receptive field properties of the PSPs were correlated with those of the bipolar and amacrine cells. The tiger salamander retina contains four main types of ganglion cell: "on" center, "off" center, "on-off", and a "hybrid" cell that responds transiently to center, but sustainedly, to surround illumination. The results lead to these inferences. The on-ganglion cell receives excitatory synpatic input from the on bipolars and that synapse is "silent" in the dark. The off-ganglion cell receives excitatory synaptic input from the off bipolars with this synapse tonically active in the dark. The on-off and hybrid ganglion cells receive a transient excitatory input with narrow receptive field, not simply correlated with the activity of any presynaptic cell. All cell types receive a broad field transient inhibitory input, which apparently originates in the transient amacrine cells. Thus, most, but not all, ganglion cell responses can be explained in terms of synaptic inputs from bipolar and amacrine cells, integrated at the ganglion cell membrane.     

Extrasynaptic release of GABA and dopamine by retinal dopaminergic neurons

In the mouse retina, dopaminergic amacrine (DA) cells synthesize both dopamine and GABA. Both transmitters are released extrasynaptically and act on neighbouring and distant retinal neurons by volume transmission. In simultaneous recordings of dopamine and GABA release from isolated perikarya of DA cells, a proportion of the events of dopamine and GABA exocytosis were simultaneous, suggesting co-release. In addition, DA cells establish GABAergic synapses onto AII amacrine cells, the neurons that transfer rod bipolar signals to cone bipolars. GABA_A but not dopamine receptors are clustered in the postsynaptic membrane. Therefore, dopamine, irrespective of its site of release—synaptic or extrasynaptic—exclusively acts by volume transmission. Dopamine is released upon illumination and sets the gain of retinal neurons for vision in bright light. The GABA released at DA cells'' synapses probably prevents signals from the saturated rods from entering the cone pathway when the dark-adapted retina is exposed to bright illumination. The GABA released extrasynaptically by DA and other amacrine cells may set a ‘GABAergic tone’ in the inner plexiform layer and thus counteract the effects of a spillover of glutamate released at the bipolar cell synapses of adjacent OFF and ON strata, thus preserving segregation of signals between ON and OFF pathways.     

Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina

Cellular mechanisms underlying the precision by which neurons target their synaptic partners have largely been determined based on the study of projection neurons. By contrast, little is known about how interneurons establish their local connections in vivo. Here, we investigated how developing amacrine interneurons selectively innervate the appropriate region of the synaptic neuropil in the inner retina, the inner plexiform layer (IPL). Increases (ON) and decreases (OFF) in light intensity are processed by circuits that are structurally confined to separate ON and OFF synaptic sublaminae within the IPL. Using transgenic zebrafish in which the majority of amacrine cells express fluorescent protein, we determined that the earliest amacrine-derived neuritic plexus formed between two cell populations whose somata, at maturity, resided on opposite sides of this plexus. When we followed the behavior of individual amacrine cells over time, we discovered that they exhibited distinct patterns of structural dynamics at different stages of development. During cellular migration, amacrine cells exhibited an exuberant outgrowth of neurites that was undirected. Upon reaching the forming IPL, neurites extending towards the ganglion cell layer were relatively more stable. Importantly, when an arbor first formed, it preferentially ramified in either the inner or outer IPL corresponding to the future ON and OFF sublaminae, and maintained this stratification pattern. The specificity by which ON and OFF amacrine interneurons innervate their respective sublaminae in the IPL contrasts with that observed for projection neurons in the retina and elsewhere in the central nervous system.     

The temporal structure of transient ON/OFF ganglion cell responses and its relation to intra-retinal processing

A subpopulation of transient ON/OFF ganglion cells in the turtle retina transmits changes in stimulus intensity as series of distinct spike events. The temporal structure of these event sequences depends systematically on the stimulus and thus carries information about the preceding intensity change. To study the spike events' intra-retinal origins, we performed extracellular ganglion cell recordings and simultaneous intracellular recordings from horizontal and amacrine cells. Based on these data, we developed a computational retina model, reproducing spike event patterns with realistic intensity dependence under various experimental conditions. The model's main features are negative feedback from sustained amacrine onto bipolar cells, and a two-step cascade of ganglion cell suppression via a slow and a fast transient amacrine cell. Pharmacologically blocking glycinergic transmission results in disappearance of the spike event sequence, an effect predicted by the model if a single connection, namely suppression of the fast by the slow transient amacrine cell, is weakened. We suggest that the slow transient amacrine cell is glycinergic, whereas the other types release GABA. Thus, the interplay of amacrine cell mediated inhibition is likely to induce distinct temporal structure in ganglion cell responses, forming the basis for a temporal code. Action Editor: Jonathan D. Victor     

神经毒素ECMA对家鸽视网膜神经节单元反应特性的影响

从家鸽视差表层总共记录了１０１个视网膜神经节单元,并定量分析研究ＥＣＭＡ损伤对其反应特性的影响,在对照组中,神经节单元都没有自发放电,而需要视觉刺激才能引起反应,对闪光刺激的反应,分别为ＯＮ—ＯＦＦ,ＯＮ,ＯＦＦ三种,其反应均是瞬变的,而且也都对在感受野内运动的小条纹起反应。４２个单元中有１４个是方向选择性单元。其它的则为运动敏感单元。方向选择性单元的无效方向不是均等分布的,其中有８个单元的无效是从前向后的,但没有发现其无效方向是从后向前的单元。与对照组相比,经ＥＣＭＡ损伤后的实验组中只记录到ＯＮ－ＯＦＦ反应和ＯＮ反应单元,未能找到单独的ＯＦＦ反应单元。神经节单元的ＯＮ反应部分为持续成分。所有的单元对运动条纹刺激都失掉了方向选择性,这些现象的机理可能是由于ＥＣＭＡ去除了胆碱能无足细胞所致。     

Transient requirement for ganglion cells during assembly of retinal synaptic layers

The inner plexiform layer (IPL) of the vertebrate retina comprises functionally specialized sublaminae, representing connections between bipolar, amacrine and ganglion cells with distinct visual functions. Developmental mechanisms that target neurites to the correct synaptic sublaminae are largely unknown. Using transgenic zebrafish expressing GFP in subsets of amacrine cells, we imaged IPL formation and sublamination in vivo and asked whether the major postsynaptic cells in this circuit, the ganglion cells, organize the presynaptic inputs. We found that in the lak/ath5 mutant retina, where ganglion cells are never born, formation of the IPL is delayed, with initial neurite outgrowth ectopically located and grossly disorganized. Over time, the majority of early neurite projection errors are corrected, and major ON and OFF sublaminae do form. However, focal regions of disarray persist where sublaminae do not form properly. Bipolar axons, which arrive later, are targeted correctly, except at places where amacrine stratification is disrupted. The lak mutant phenotype reveals that ganglion cells have a transient role organizing the earliest amacrine projections to the IPL. However, it also suggests that amacrine cells interact with each other during IPL formation; these interactions alone appear sufficient to form the IPL. Furthermore, our results suggest that amacrines may guide IPL sublamination by providing stratification cues for other cell types.     

Effect of 2-amino-4-phosphonobutyrate on the OFF responses of frog retinal ganglion cells and local ERG after glycinergic blockade

Perfusion with the ON channel blocker 2-amino-4-phosphonobutyrate (APB) of dark adapted frog eyecups not only abolished the ganglion cells' (GC) ON responses and the ERG b-wave, but markedly potentiated the OFF responses of ON-OFF and phasic OFF-GCs and the d-wave amplitude of simultaneously recorded local ERG. Glycinergic blockade by strychnine prevented this potentiating effect in 31 out of 69 GCs, but did not change it at all in the other cells. At the same time the d-wave potentiation was preserved during the glycinergic blockade in all eyecups. The results indicate that glycinergic transmission is involved in the inhibition exerted from ON upon OFF channel in some but not all frog retinal GCs.     

The oscillation-like activity in bullfrog ON–OFF retinal ganglion cell

Oscillatory activity of retinal ganglion cell (RGC) has been observed in various species. It was reported such oscillatory activity is raised within large neural network and involved in retinal information coding. In the present research, we found an oscillation-like activity in ON–OFF RGC of bullfrog retina, and studied the mechanisms underlying the ON and OFF activities respectively. Pharmacological experiments revealed that the oscillation-like activity patterns in both ON and OFF pathways were abolished by GABA receptor antagonists, indicating GABAergic inhibition is essential for generating them. At the meantime, such activities in the ON and OFF pathways showed different responses to several other applied drugs. The oscillation-like pattern in the OFF pathway was abolished by glycine receptor antagonist or gap junction blocker, whereas that in the ON pathway was not affected. Furthermore, the blockade of the ON pathway by metabotropic glutamate receptor agonist led to suppression of the oscillation-like pattern in the OFF pathway. These results suggest that the ON pathway has modulatory effect on the oscillation-like activity in the OFF pathway. Therefore, the mechanisms underlying the oscillation-like activities in the ON and OFF pathways are different: the oscillation-like activity in the ON pathway is likely caused by GABAergic amacrine cell network, while that in the OFF pathway needs the contributions of GABAergic and glycinergic amacrine cell network, as well as gap junction connections.     

Suppression of sustained and transient ON signals of amacrine cells by GABA is mediated by different receptor subtypes

Intracellular recordings were made from amacrine cells in the isolated, superfused carp retina, and the effects of γ-aminobutyric acid (GABA) on sustained and transient ON signals of these cells were studied. Exogenous GABA application partially suppressed the sustained response of ON amacrine cells, which could be completely reversed by picrotoxin (PTX), a chloride channel blocker, and by bicuculline (BCC), a specific GABA_A receptor antagonist. On the other hand, suppression by GABA of the ON response which was predominantly driven by rod signals in a certain portion of transient ON-OFF amacrine cells was completely blocked by PTX, but not by BCC, indicating that GABA_C recepton may be involved in the effect. These results suggest that GABA_A, and GABA_C receptors may be respectively involved in mediating the transmission of sustained and transient signals in the carp inner retina.