Features and functions of nonlinear spatial integration by retinal ganglion cells

Ganglion cells in the vertebrate retina integrate visual information over their receptive fields. They do so by pooling presynaptic excitatory inputs from typically many bipolar cells, which themselves collect inputs from several photoreceptors. In addition, inhibitory interactions mediated by horizontal cells and amacrine cells modulate the structure of the receptive field. In many models, this spatial integration is assumed to occur in a linear fashion. Yet, it has long been known that spatial integration by retinal ganglion cells also incurs nonlinear phenomena. Moreover, several recent examples have shown that nonlinear spatial integration is tightly connected to specific visual functions performed by different types of retinal ganglion cells. This work discusses these advances in understanding the role of nonlinear spatial integration and reviews recent efforts to quantitatively study the nature and mechanisms underlying spatial nonlinearities. These new insights point towards a critical role of nonlinearities within ganglion cell receptive fields for capturing responses of the cells to natural and behaviorally relevant visual stimuli. In the long run, nonlinear phenomena of spatial integration may also prove important for implementing the actual neural code of retinal neurons when designing visual prostheses for the eye.     

Activity-induced long-term potentiation of excitatory synapses in developing zebrafish retina in vivo

Neural activity-induced long-term potentiation (LTP) of synaptic transmission is believed to be one of the cellular mechanisms underlying experience-dependent developmental refinement of neural circuits. Although it is well established that visual experience and neural activity are critical for the refinement of retinal circuits, whether and how LTP occurs in the retina remain unknown. Using in?vivo perforated whole-cell recording and two-photon calcium imaging, we find that both repeated electrical and visual stimulations can induce LTP at excitatory synapses formed by bipolar cells on retinal ganglion cells in larval but not juvenile zebrafish. LTP induction requires the activation of postsynaptic N-methyl-D-aspartate receptors, and its expression involves arachidonic acid-dependent presynaptic changes in calcium dynamics and neurotransmitter release. Physiologically, both electrical and visual stimulation-induced LTP can enhance visual responses of retinal ganglion cells. Thus, LTP exists in developing retinae with a presynaptic locus and may serve for visual experience-dependent refinement of retinal circuits.     

Spatial Distribution of Excitatory Synapses on the Dendrites of Ganglion Cells in the Mouse Retina

Excitatory glutamatergic inputs from bipolar cells affect the physiological properties of ganglion cells in the mammalian retina. The spatial distribution of these excitatory synapses on the dendrites of retinal ganglion cells thus may shape their distinct functions. To visualize the spatial pattern of excitatory glutamatergic input into the ganglion cells in the mouse retina, particle-mediated gene transfer of plasmids expressing postsynaptic density 95-green fluorescent fusion protein (PSD95-GFP) was used to label the excitatory synapses. Despite wide variation in the size and morphology of the retinal ganglion cells, the expression of PSD95 puncta was found to follow two general rules. Firstly, the PSD95 puncta are regularly spaced, at 1–2 µm intervals, along the dendrites, whereby the presence of an excitatory synapse creates an exclusion zone that rules out the presence of other glutamatergic synaptic inputs. Secondly, the spatial distribution of PSD95 puncta on the dendrites of diverse retinal ganglion cells are similar in that the number of excitatory synapses appears to be less on primary dendrites and to increase to a plateau on higher branch order dendrites. These observations suggest that synaptogenesis is spatially regulated along the dendritic segments and that the number of synaptic contacts is relatively constant beyond the primary dendrites. Interestingly, we also found that the linear puncta density is slightly higher in large cells than in small cells. This may suggest that retinal ganglion cells with a large dendritic field tend to show an increased connectivity of excitatory synapses that makes up for their reduced dendrite density. Mapping the spatial distribution pattern of the excitatory synapses on retinal ganglion cells thus provides explicit structural information that is essential for our understanding of how excitatory glutamatergic inputs shape neuronal responses.     

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.     

An analogue model of the luminosity-channel in the vertebrate cone retina

A model of the vertebrate cone retina was tested with physiological stimuli. Results confirm previous findings that, except for photoreceptors, the spatial and temporal properties of simulated retinal elements conform to a linear system. The model is consistent with known physiological correlates. Tonic units detect intensity when the light spot is within the center field, while phasic units detect movement across borders of contrast. There is a dynamic balance between the tonic and phasic channels: the tonic channel is favored by a center field input voltage, while the phasic channel is favored by a surround field input voltage to bipolar cells. The ON discharge of the phasic ganglion cell is developed by the excitatory center field input to the depolarizing-center bipolar cell, which has the shortest delay, while the OFF discharge is the result of the excitatory surround field input voltage to the hyperpolarizing-center bipolar cell, which has the longest delay.     

Regulation of retinal cone bipolar cell differentiation and photopic vision by the CVC homeobox gene Vsx1

Cone bipolar cells of the vertebrate retina connect photoreceptors with ganglion cells to mediate photopic vision. Despite this important role, the mechanisms that regulate cone bipolar cell differentiation are poorly understood. VSX1 is a CVC domain homeoprotein specifically expressed in cone bipolar cells. To determine the function of VSX1, we generated Vsx1 mutant mice and found that Vsx1 mutant retinal cells form but do not differentiate a mature cone bipolar cell phenotype. Electrophysiological studies demonstrated that Vsx1 mutant mice have defects in their cone visual pathway, whereas the rod visual pathway was unaffected. Thus, Vsx1 is required for cone bipolar cell differentiation and regulates photopic vision perception.     

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.     

Bipolar cells use kainate and AMPA receptors to filter visual information into separate channels

Unlike cone photoreceptors, whose light responses have a uniform time course, retinal ganglion cells are tuned to respond to different temporal components in a changing visual scene. The signals in a mammalian cone flow to three to five morphologically distinct "OFF" bipolar cells at a sign-conserving, glutamatergic synapse. By recording simultaneously from pairs of synaptically connected cones and OFF bipolar cells, I now show that each morphological type of OFF bipolar cell receives its signal through a different AMPA or kainate receptor. The characteristic rate at which each receptor recovers from desensitization divides the cone signal into temporal components. Temporal processing begins at the first synapse in the visual system.     

Taurine deficiency damages retinal neurones: cone photoreceptors and retinal ganglion cells

In 1970s, taurine deficiency was reported to induce photoreceptor degeneration in cats and rats. Recently, we found that taurine deficiency contributes to the retinal toxicity of vigabatrin, an antiepileptic drug. However, in this toxicity, retinal ganglion cells were degenerating in parallel to cone photoreceptors. The aim of this study was to re-assess a classic mouse model of taurine deficiency following a treatment with guanidoethane sulfonate (GES), a taurine transporter inhibitor to determine whether retinal ganglion cells are also affected. GES treatment induced a significant reduction in the taurine plasma levels and a lower weight increase. At the functional level, photopic electroretinograms were reduced indicating a dysfunction in the cone pathway. A change in the autofluorescence appearance of the eye fundus was explained on histological sections by an increased autofluorescence of the retinal pigment epithelium. Although the general morphology of the retina was not affected, cell damages were indicated by the general increase in glial fibrillary acidic protein expression. When cell quantification was achieved on retinal sections, the number of outer/inner segments of cone photoreceptors was reduced (20?%) as the number of retinal ganglion cells (19?%). An abnormal synaptic plasticity of rod bipolar cell dendrites was also observed in GES-treated mice. These results indicate that taurine deficiency can not only lead to photoreceptor degeneration but also to retinal ganglion cell loss. Cone photoreceptors and retinal ganglion cells appear as the most sensitive cells to taurine deficiency. These results may explain the recent therapeutic interest of taurine in retinal degenerative pathologies.     

Birth of Cone Bipolar Cells,but Not Rod Bipolar Cells,Is Associated with Existing RGCs

Retinal ganglion cells (RGCs) play important roles in retinogenesis. They are required for normal retinal histogenesis and retinal cell number balance. Developmental RGC loss is typically characterized by initial retinal neuronal number imbalance and subsequent loss of retinal neurons. However, it is not clear whether loss of a specific non-RGC cell type in the RGC-depleted retina is due to reduced cell production or subsequent degeneration. Taking advantage of three knockout mice with varying degrees of RGC depletion, we re-examined bipolar cell production in these retinas from various aspects. Results show that generation of the cone bipolar cells is correlated with the existing number of RGCs. However, generation of the rod bipolar cells is unaffected by RGC shortage. Results report the first observation that RGCs selectively influence the genesis of subsequent retinal cell types.     

Programmed cell death of retinal cone bipolar cells is independent of afferent or target control

Programmed cell death contributes to the histogenesis of the nervous system, and is believed to be modulated through the sustaining effects of afferents and targets during the period of synaptogenesis. Cone bipolar cells undergo programmed cell death during development, and we confirm that the numbers of three different types are increased when the pro-apoptotic Bax gene is knocked out. When their cone afferents are selectively eliminated, or when the population of retinal ganglion cells is increased, however, cone bipolar cell number remains unchanged. Programmed cell death of the cone bipolar cell populations, therefore, may be modulated cell-intrinsically rather than via interactions with these synaptic partners.     

Nitric Oxide Modulates the Temporal Properties of the Glutamate Response in Type 4 OFF Bipolar Cells

Nitric oxide (NO) is involved in retinal signal processing, but its cellular actions are only partly understood. An established source of retinal NO are NOACs, a group of nNOS-expressing amacrine cells which signal onto bipolar, other amacrine and ganglion cells in the inner plexiform layer. Here, we report that NO regulates glutamate responses in morphologically and electrophysiologically identified type 4 OFF cone bipolar cells through activation of the soluble guanylyl cyclase-cGMP-PKG pathway. The glutamate response of these cells consists of two components, a fast phasic current sensitive to kainate receptor agonists, and a secondary component with slow kinetics, inhibited by AMPA receptor antagonists. NO shortened the duration of the AMPA receptor-dependent component of the glutamate response, while the kainate receptor-dependent component remained unchanged. Application of 8-Br-cGMP mimicked this effect, while inhibition of soluble guanylate cyclase or protein kinase G prevented it, supporting a mechanism involving a cGMP signaling pathway. Notably, perfusion with a NOS-inhibitor prolonged the duration of the glutamate response, while the NO precursor L-arginine shortened it, in agreement with a modulation by endogenous NO. Furthermore, NO accelerated the response recovery during repeated stimulation of type 4 cone bipolar cells, suggesting that the temporal response properties of this OFF bipolar cell type are regulated by NO. These results reveal a novel cellular mechanism of NO signaling in the retina, and represent the first functional evidence of NO modulating OFF cone bipolar cells.     

Development and degeneration of cone bipolar cells are independent of cone photoreceptors in a mouse model of retinitis pigmentosa

Retinal photoreceptors die during retinal synaptogenesis in a portion of retinal degeneration. Whether cone bipolar cells establish regular retinal mosaics and mature morphologies, and resist degeneration are not completely understood. To explore these issues, we backcrossed a transgenic mouse expressing enhanced green fluorescent protein (EGFP) in one subset of cone bipolar cells (type 7) into rd1 mice, a classic mouse model of retinal degeneration, to examine the development and survival of cone bipolar cells in a background of retinal degeneration. Our data revealed that both the development and degeneration of cone bipolar cells are independent of the normal activity of cone photoreceptors. We found that type 7 cone bipolar cells achieved a uniform tiling of the retinal surface and developed normal dendritic and axonal arbors without the influence of cone photoreceptor innervation. On the other hand, degeneration of type 7 cone bipolar cells, contrary to our belief of central-to-peripheral progression, was spatially uniform across the retina independent of the spatiotemporal pattern of cone degeneration. The results have important implications for the design of more effective therapies to restore vision in retinal degeneration.     

N-Methyl-d-aspartate receptors in the retina

The vertebrate retina is a “genuine neural center” (Ramón y Cajal), in which glutamate is a major excitatory neurotransmitter. Both N-methyl-d-aspartate (NMDA) and non-NMDA receptors are expressed in the retina. Although non-NMDA receptors and/or metabotropic glutamate receptors are generally thought to be responsible for mediating the transfer of visual signals in the outer retina, there is recent evidence suggesting that NMDA receptors are also expressed in photoreceptors, as well as horizontal and bipolar cells. In the inner retina, NMDA receptors, in addition to other glutamate receptor subtypes, are abundantly expressed to mediate visual signal transmission from bipolar cells to amacrine and ganglion cells, and could be involved in modulation of inhibitory feedback from amacrine cells to bipolar cells. NMDA receptors are extrasynaptically expressed in ganglion cells (and probably amacrine cells) and may play physiological roles in a special mode. Activity of NMDA receptors may be modulated by neuromodulators, such as d-serine and others. This article discusses retinal excitotoxicity mediated by NMDA receptors.     

Colour coding in the primate retina: diverse cell types and cone-specific circuitry

How is the trichromatic cone mosaic of Old World primates sampled by retinal circuits to create wavelength opponency? Red-green (L versus M cone) opponency appears to be mediated largely by the segregation of L versus M cone signals to the centre versus the surround of the midget ganglion cell receptive field, implying a complex cone type-specific wiring, the basis of which remains mysterious. Blue-yellow (S versus L+M cone) opponency is mediated by a growing family of low-density ganglion types that receive either excitatory or inhibitory input from S cones. Thus, the retinal circuits that underlie colour signalling in primates may be both more complex and more diverse then previously appreciated.     

Synaptic organization and ionic basis of on and off channels in mudpuppy retina. I. Intracellular analysis of chloride-sensitive electrogenic properties of receptors, horizontal cells, bipolar cells, and amacrine cells

Intracellular recordings from receptors, horizontal cells, bipolars, and amacrines have been carried out in the perfused mudpuppy eyecup. The introduction of a chloride-free (c-f) medium results in initial transient potential changes in many cells followed by a slow loss of light-evoked activity of the depolarizing bipolar, the horizontal cell, and the on depolarization of amacrine cells. The hyperpolarizing bipolar remains responsive to light stimulation in a c-f medium, but the antagonistic surround mechanism is abolished. These effects are reversible after returning to a normal ionic medium. The results of this study provide insight into the retinal connections which underlie ganglion cell receptive field organization. It is concluded that the depolarizing bipolar is excitatory to on ganglion cells and is also the pathway for on-excitation of on-off cells. The hyperpolarizing bipolar mediates the off discharge of off and on-off cells. Amacrine cells receive input from both depolarizing and hyperpolarizing bipolar cells. These findings raise the possibility that transmembrane movements of chloride ions are critical for the light responsiveness of horizontal and depolarizing bipolar cell activity.     

Synaptic organization and ionic basis of on and off channels in mudpuppy retina. III. A model of ganglion cell receptive field organization based on chloride-free experiments

A chloride-free environment produces selective changes in the retinal network which include a separation of on and off channels. The identification of chloride-sensitive and insensitivie neuronal activity permits identification of some of the connections and intervening polarities of synaptic interactions which are expressed in ganglion cell receptive field organization. These experiments support previous suggestions that surround antagonism is dependent on horizontal cell activity. In addition they suggest a model of the neuronal connections which subserve on-center, off-center, and on-off ganglion cells. Experimental tests of the on-off ganglion cell model favor the idea that this type of ganglion cell receives inhibitory input from amacrine cells and excitatory activation from depolarizing and hyperpolarizing bipolar cells.     

Expression of alpha 7 nicotinic acetylcholine receptors by bipolar, amacrine, and ganglion cells of the rabbit retina.

Cholinergic agents affect the light responses of many ganglion cells (GCs) in the mammalian retina by activating nicotinic acetylcholine receptors (nAChRs). Whereas retinal neurons that express beta2 subunit-containing nAChRs have been characterized in the rabbit retina, expression patterns of other nAChR subtypes remain unclear. Therefore, we evaluated the expression of alpha7 nAChRs in retinal neurons by means of single-, double-, and triple-label immunohistochemistry. Our data demonstrate that, in the rabbit retina, several types of bipolar cells, amacrine cells, and cells in the GC layer express alpha7 nAChRs. At least three different populations of cone bipolar cells exhibited alpha7 labeling, whereas glycine-immunoreactive amacrine cells comprised the majority of alpha7-positive amacrine cells. Some GABAergic amacrine cells also displayed alpha7 immunoreactivity; alpha7 labeling was never detected in rod bipolar cells or rod amacrine cells (AII amacrine cells). Our data suggest that activation of alpha7 nAChRs by acetylcholine (ACh) or choline may affect glutamate release from several types of cone bipolar cells, modulating GC responses. ACh-induced excitation of inhibitory amacrine cells might cause either inhibition or disinhibition of other amacrine and GC circuits. Finally, ACh may act on alpha7 nAChRs expressed by GCs themselves.     

Quantitative morphology of the primate peripheral retina (Macaca irus)

By using electron microscopy to study the quantitative morphology of the retina, it was possible to determine the spatial density of all principal retinal cells at a defined retinal location. In two retinas of cynomolgus monkeys at a position of 30 degrees nasal of the fovea centralis, the following cell densities were determined from composite electron micrographs: retinal pigment epithelium: 3,400 cell/mm2; rod cells: 115,000 and 168,000 cells/mm2; cone cells: 8,200/mm2; horizontal cells: 7,000/mm2; bipolar cells: 50,000/mm2; amacrine cells: 11,500/mm2; Müller cells: 16,000/mm2; and ganglion cells: 5,350 and 6,750/mm2.