OFF Bipolar Cells Research Articles

Pivotal roles of Fezf2 in differentiation of cone OFF bipolar cells and functional maturation of cone ON bipolar cells in retina

During development of the retina, common retinal progenitor cells give rise to six classes of neurons that subsequently further diversify into more than 55 subtypes of neuronal subtypes. Here, we have investigated the expression and function of Fezf2, Fez zinc finger family of protein, in the developing mouse retina. Expression of Fezf2 transcripts was strongly observed in the embryonic retinal progenitors at E14.5 and declined quickly in subsequent development of retina. Then, in postnatal stage at around day 8, Fezf2 was transiently expressed then declined again. Loss-of-function analysis using retinas from mice in which Fezf2 coding region was substituted with β-galactosidase showed that Fezf2 is expressed in a subset of cone OFF bipolar cells and required for their differentiation. Using electroretinogram, we found that Fezf2 knockout retina exhibited significantly reduced photopic b-wave, suggesting functional abnormality of cone ON bipolar cells. Furthermore, reduced expression of synaptic protein Trpm1 and structural alteration of ON bipolar cell invagination, both of which affected cone photoreceptor terminal synaptic activity, was identified by transmission electron microscopy and immunohistochemistry, respectively. Taken together, our results show that Fezf2 is indispensable in differentiation of bipolar precursors into cone OFF bipolar cells and in functional maturation of cone ON bipolar cells during development of mouse retina. These results contribute to our understanding of how diversity of neuronal subtypes and hence specificity of neuronal connections are established in the retina by intrinsic cues.

Lrit1, a Retinal Transmembrane Protein, Regulates Selective Synapse Formation in Cone Photoreceptor Cells and Visual Acuity

In the vertebrate retina, cone photoreceptors play crucial roles in photopic vision by transmitting light-evoked signals to ON- and/or OFF-bipolar cells. However, the mechanisms underlying selective synapse formation in the cone photoreceptor pathway remain poorly understood. Here, we found that Lrit1, a leucine-rich transmembrane protein, localizes to the photoreceptor synaptic terminal and regulates the synaptic connection between cone photoreceptors and cone ON-bipolar cells. Lrit1-deficient retinas exhibit an aberrant morphology of cone photoreceptor pedicles, as well as an impairment of signal transmission from cone photoreceptors to cone ON-bipolar cells. Furthermore, we demonstrated that Lrit1 interacts with Frmpd2, a photoreceptor scaffold protein, and with mGluR6, an ON-bipolar cell-specific glutamate receptor. Additionally, Lrit1-null mice showed visual acuity impairments in their optokinetic responses. These results suggest that the Frmpd2-Lrit1-mGluR6 axis regulates selective synapse formation in cone photoreceptors and is essential for normal visual function.

Exo- and Endocytosis at a Retinal Inhibitory Synapse during Crossover Inhibition

The AII amacrine cell (AII) receives chemical and electrical synapses from rod bipolar cells (BC) and ON cone BCs. AIIs form glycinergic synapses at lobular varicosities near the soma, providing inhibitory output to the OFF pathway; this is called crossover inhibition. We performed whole-cell voltage clamp recordings and used the “sine + DC” method to make measurements of membrane capacitance (Cm) with high temporal resolution. With this approach we were able to investigate the cell membrane dynamics of this inhibitory synapse. AIIs were held at −80 mV and L-type Ca2+ currents were evoked with a 100 ms, 70 mV depolarizing step, triggering Ca2+ dependent vesicle fusion (exocytosis). Our observed Cm jumps ranged from 14.81-152.3 fF, corresponding to the fusion of 370-3800 vesicles (n=38). Presynaptic potentiation was also seen at this synapse after 4 minutes of treatment of 1 mM cAMP (n=22). Following a 50 ms depolarizing step, we saw a gradual decay in Cm, indicative of vesicular membrane retrieval (endocytosis). Tau ranged from 3.90 to 7.11 s (n=10), suggesting that AIIs are suited for fast membrane recovery. We propose that the variability in membrane dynamics of these interneurons is due to their diverse cell-to-cell lobular morphologies. In order to test this hypothesis, we performed Cm recordings in conjunction with wide-field optical sectioning of dye filled AIIs. We were able to compare Cm jumps to 3D reconstructions of the lobular varicosities from individual AIIs. Our results show that the magnitude of glycine release positively correlates to the number and overall size of these lobules (n=10). These results suggest that AIIs with larger lobules have more active zones and make more synaptic connections with OFF BCs and/or ganglion cells.

Functional and Morphological Analysis of OFF Bipolar Cells.

Retinal first-order neurons, photoreceptors, receive visual inputs and convert them to neural signals. The second-order neurons, bipolar cells then sort out the visual signals and encode them through multiple neural streams. Approximately 15 morphological types of bipolar cells have been identified, which are thought to encode different aspects of visual signals such as motion and color (Ichinose et al. J Neurosci 34(26):8761-8771, 2014; Euler et al. Nat Rev Neurosci 15(8):507-519, 2014). To investigate functional aspects of OFF bipolar cells, single cell recordings are preferred; however, bipolar cells in the mouse retina are small and hard to distinguish from other types of cells. Here, we describe our methodology and tips for immunohistochemistry and patch clamp recordings for analyzing light-evoked responses in each type of OFF bipolar cell.

Differential Distribution of Retinal Ca2+/Calmodulin-Dependent Kinase II (CaMKII) Isoforms Indicates CaMKII-β and -δ as Specific Elements of Electrical Synapses Made of Connexin36 (Cx36).

AII amacrine cells are essential interneurons of the primary rod pathway and transmit rod-driven signals to ON cone bipolar cells to enable scotopic vision. Gap junctions made of connexin36 (Cx36) mediate electrical coupling among AII cells and between AII cells and ON cone bipolar cells. These gap junctions underlie a remarkable degree of plasticity and are modulated by different signaling cascades. In particular, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized as an important regulator of Cx36, capable of potentiating electrical coupling in AII cells. However, it is unclear which CaMKII isoform mediates this effect. To obtain a more detailed understanding of the isoform composition of CaMKII at retinal gap junctions, we analyzed the retinal distribution of all four CaMKII isoforms using confocal microscopy. These experiments revealed a differential distribution of CaMKII isoforms: CaMKII-α was strongly expressed in starburst amacrine cells, which are known to lack electrical coupling. CaMKII-β was abundant in OFF bipolar cells, which form electrical synapses in the outer and the inner retina. CaMKII-γ was diffusely distributed across the entire retina and could not be assigned to a specific cell type. CaMKII-δ labeling was evident in bipolar and AII amacrine cells, which contain the majority of Cx36-immunoreactive puncta in the inner retina. We double-labeled retinas for Cx36 and the four CaMKII isoforms and revealed that the composition of the CaMKII enzyme differs between gap junctions in the outer and the inner retina: in the outer retina, only CaMKII-β colocalized with Cx36-containing gap junctions, whereas in the inner retina, CaMKII-β and -δ colocalized with Cx36. This finding suggests that gap junctions in the inner and the outer retina may be regulated differently although they both contain the same connexin. Taken together, our study identifies CaMKII-β and -δ as Cx36-specific regulators in the mouse retina with CaMKII-δ regulating the primary rod pathway.

DSCAM-mediated control of dendritic and axonal arbor outgrowth enforces tiling and inhibits synaptic plasticity

Mature mammalian neurons have a limited ability to extend neurites and make new synaptic connections, but the mechanisms that inhibit such plasticity remain poorly understood. Here, we report that OFF-type retinal bipolar cells in mice are an exception to this rule, as they form new anatomical connections within their tiled dendritic fields well after retinal maturity. The Down syndrome cell-adhesion molecule (Dscam) confines these anatomical rearrangements within the normal tiled fields, as conditional deletion of the gene permits extension of dendrite and axon arbors beyond these borders. Dscam deletion in the mature retina results in expanded dendritic fields and increased cone photoreceptor contacts, demonstrating that DSCAM actively inhibits circuit-level plasticity. Electrophysiological recordings from Dscam-/- OFF bipolar cells showed enlarged visual receptive fields, demonstrating that expanded dendritic territories comprise functional synapses. Our results identify cell-adhesion molecule-mediated inhibition as a regulator of circuit-level neuronal plasticity in the adult retina.

Classification of Mouse Retinal Bipolar Cells: Type-Specific Connectivity with Special Reference to Rod-Driven AII Amacrine Pathways.

We confirmed the classification of 15 morphological types of mouse bipolar cells by serial section transmission electron microscopy and characterized each type by identifying chemical synapses and gap junctions at axon terminals. Although whether the previous type 5 cells consist of two or three types was uncertain, they are here clustered into three types based on the vertical distribution of axonal ribbons. Next, while two groups of rod bipolar (RB) cells, RB1, and RB2, were previously proposed, we clarify that a half of RB1 cells have the intermediate characteristics, suggesting that these two groups comprise a single RB type. After validation of bipolar cell types, we examined their relationship with amacrine cells then particularly with AII amacrine cells. We found a strong correlation between the number of amacrine cell synaptic contacts and the number of bipolar cell axonal ribbons. Formation of bipolar cell output at each ribbon synapse may be effectively regulated by a few nearby inhibitory inputs of amacrine cells which are chosen from among many amacrine cell types. We also found that almost all types of ON cone bipolar cells frequently have a minor group of midway ribbons along the axon passing through the OFF sublamina as well as a major group of terminal ribbons in the ON sublamina. AII amacrine cells are connected to five of six OFF bipolar cell types via conventional chemical synapses and seven of eight ON (cone) bipolar cell types via electrical synapses (gap junctions). However, the number of synapses is dependent on bipolar cell types. Type 2 cells have 69% of the total number of OFF bipolar chemical synaptic contacts with AII amacrine cells and type 6 cells have 46% of the total area of ON bipolar gap junctions with AII amacrine cells. Both type 2 and 6 cells gain the greatest access to AII amacrine cell signals also share those signals with other types of bipolar cells via networked gap junctions. These findings imply that the most sensitive scotopic signal may be conveyed to the center by ganglion cells that have the most numerous synapses with type 2 and 6 cells.

Local synaptic integration enables ON-OFF asymmetric and layer-specific visual information processing in vGluT3 amacrine cell dendrites

A basic scheme of neuronal organization in the mammalian retina is the segregation of ON and OFF pathways in the inner plexiform layer (IPL), where glutamate is released from ON and OFF bipolar cell terminals in separate inner (ON) and outer (OFF) sublayers in response to light intensity increments and decrements, respectively. However, recent studies have found that vGluT3-expressing glutamatergic amacrine cells (GACs) generate ON-OFF somatic responses and release glutamate onto both ON and OFF ganglion cell types, raising the possibility of crossover excitation in violation of the canonical ON-OFF segregation scheme. To test this possibility, we recorded light-evoked Ca2+ responses from dendrites of individual GACs infected with GCaMP6s in mouse. Under two-photon imaging, a single GAC generated rectified local dendritic responses, showing ON-dominant responses in ON sublayers and OFF-dominant responses in OFF sublayers. This unexpected ON-OFF segregation within a small-field amacrine cell arose from local synaptic processing, mediated predominantly by synaptic inhibition. Multiple forms of synaptic inhibition compartmentalized the GAC dendritic tree and endowed all dendritic varicosities with a small-center, strong-surround receptive field, which varied in receptive field size and degree of ON-OFF asymmetry with IPL depth. The results reveal a form of short-range dendritic autonomy that enables a small-field, dual-transmitter amacrine cell to process diverse dendritic functions in a stratification level- and postsynaptic target-specific manner, while preserving the fundamental ON-OFF segregation scheme for parallel visual processing and high spatial resolution for small object motion and uniformity detection.

Selective synaptic connections in the retinal pathway for night vision.

The mammalian retina encodes visual information in dim light using rod photoreceptors and a specialized circuit: rods→rod bipolar cells→AII amacrine cell. The AII amacrine cell uses sign-conserving electrical synapses to modulate ON cone bipolar cell terminals and sign-inverting chemical (glycinergic) synapses to modulate OFF cone cell bipolar terminals; these ON and OFF cone bipolar terminals then drive the output neurons, retinal ganglion cells (RGCs), following light increments and decrements, respectively. The AII amacrine cell also makes direct glycinergic synapses with certain RGCs, but it is not well established how many types receive this direct AII input. Here, we investigated functional AII amacrine→RGC synaptic connections in the retina of the guinea pig (Cavia porcellus) by recording inhibitory currents from RGCs in the presence of ionotropic glutamate receptor (iGluR) antagonists. This condition isolates a specific pathway through the AII amacrine cell that does not require iGluRs: cone→ON cone bipolar cell→AII amacrine cell→RGC. These recordings show that AII amacrine cells make direct synapses with OFF Alpha, OFF Delta and a smaller OFF transient RGC type that co-stratifies with OFF Alpha cells. However, AII amacrine cells avoid making synapses with numerous RGC types that co-stratify with the connected RGCs. Selective AII connections ensure that a privileged minority of RGC types receives direct input from the night-vision pathway, independent from OFF bipolar cell activity. Furthermore, these results illustrate the specificity of retinal connections, which cannot be predicted solely by co-stratification of dendrites and axons within the inner plexiform layer.

LRIT3 Differentially Affects Connectivity and Synaptic Transmission of Cones to ON- and OFF-Bipolar Cells.

PurposeMutations in LRIT3 lead to complete congenital stationary night blindness (cCSNB). Using a cCSNB mouse model lacking Lrit3 (nob6), we recently have shown that LRIT3 has a role in the correct localization of TRPM1 (transient receptor potential melastatin 1) to the dendritic tips of ON-bipolar cells (BCs), contacting both rod and cone photoreceptors. Furthermore, postsynaptic clustering of other mGluR6 cascade components is selectively eliminated at the dendritic tips of cone ON-BCs. The purpose of this study was to further define the role of LRIT3 in structural and functional organization of cone synapses.MethodsExhaustive electroretinogram analysis was performed in a patient with LRIT3 mutations. Multielectrode array recordings were performed at the level of retinal ganglion cells in nob6 mice. Targeting of GluR1 and GluR5 at the dendritic tips of OFF-BCs in nob6 retinas was assessed by immunostaining and confocal microscopy. The ultrastructure of photoreceptor synapses was evaluated by electron microscopy in nob6 mice.ResultsThe patient with LRIT3 mutations had a selective ON-BC dysfunction with relatively preserved OFF-BC responses. In nob6 mice, complete lack of ON-pathway function with robust, yet altered signaling processing in OFF-pathways was detected. Consistent with these observations, molecules essential for the OFF-BC signaling were normally targeted to the synapse. Finally, synaptic contacts made by ON-BC but not OFF-BC neurons with the cone pedicles were disorganized without ultrastructural alterations in cone terminals, horizontal cell processes, or synaptic ribbons.ConclusionsThese results suggest that LRIT3 is likely involved in coordination of the transsynaptic communication between cones and ON-BCs during synapse formation and function.

Dendritic stratification differs among retinal OFF bipolar cell types in the absence of rod photoreceptors.

Retinal OFF bipolar cells show distinct connectivity patterns with photoreceptors in the wild-type mouse retina. Some types are cone-specific while others penetrate further through the outer plexiform layer (OPL) to contact rods in addition to cones. To explore dendritic stratification of OFF bipolar cells in the absence of rods, we made use of the ‘cone-full’ Nrl-/- mouse retina in which all photoreceptor precursor cells commit to a cone fate including those which would have become rods in wild-type retinas. The dendritic distribution of OFF bipolar cell types was investigated by confocal and electron microscopic imaging of immunolabeled tissue sections. The cells’ dendrites formed basal contacts with cone terminals and expressed the corresponding glutamate receptor subunits at those sites, indicating putative synapses. All of the four analyzed cell populations showed distinctive patterns of vertical dendritic invasion through the OPL. This disparate behavior of dendritic extension in an environment containing only cone terminals demonstrates type-dependent specificity for dendritic outgrowth in OFF bipolar cells: rod terminals are not required for inducing dendritic extension into distal areas of the OPL.

A Cre Mouse Line for Probing Irradiance- and Direction-Encoding Retinal Networks.

Cell type-specific Cre driver lines have revolutionized the analysis of retinal cell types and circuits. We show that the transgenic mouse Rbp4-Cre selectively labels several retinal neuronal types relevant to the encoding of absolute light intensity (irradiance) and visual motion. In the ganglion cell layer (GCL), most marked cells are wide-field spiking polyaxonal amacrine cells (ACs) with sustained irradiance-encoding ON responses that persist during chemical synaptic blockade. Their arbors spread about 1 mm across the retina and are restricted to the inner half of the ON sublamina of the inner plexiform layer (IPL). There, they costratify with dendrites of M2 intrinsically photosensitive retinal ganglion cells (ipRGCs), to which they are tracer coupled. We propose that synaptically driven and intrinsic photocurrents of M2 cells pass through gap junctions to drive AC light responses. Also marked in this mouse are two types of RGCs. R-cells have a bistratified dendritic arbor, weak directional tuning, and irradiance-encoding ON responses. However, they also receive excitatory OFF input, revealed during ON-channel blockade. Serial blockface electron microscopic (SBEM) reconstruction confirms OFF bipolar input, and reveals that some OFF input derives from a novel type of OFF bipolar cell (BC). R-cells innervate specific layers of the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC). The other marked RGC type (RDS) is bistratified, transient, and ON-OFF direction selective (DS). It apparently innervates the nucleus of the optic tract (NOT). The Rbp4-Cre mouse will be valuable for targeting these cell types for further study and for selectively manipulating them for circuit analysis.

Changes in Retinal Function and Cellular Remodeling Following Experimental Retinal Detachment in a Rabbit Model.

Purpose. To explore functional electroretinographic (ERG) changes and associated cellular remodeling following experimental retinal detachment in a rabbit model. Methods. Retinal detachment was created in ten rabbits by injecting 0.1 ml balanced salt solution under the retina. Fundus imaging was performed 0, 3, 7, 14, and 21 days postoperatively. ERGs were recorded pre- and 7 and 21 days postoperatively. Eyes were harvested on day 21 and evaluated immunohistochemically (IHC) for remodeling of second- and third-order neurons. Results. Retinal reattachment occurred within two weeks following surgery. No attenuation was observed in the photopic or scotopic a- and b-waves. A secondary wavefront on the descending slope of the scotopic b-wave was the only ERG result that was attenuated in detached retinas. IHC demonstrated anatomical changes in both ON and OFF bipolar cells. Bassoon staining was observed in the remodeled dendrites. Amacrine and horizontal cells did not alter, but Muller cells were clearly reactive with marked extension. Conclusion. Retinal detachment and reattachment were associated with functional and anatomical changes. Exploring the significance of the secondary scotopic wavefront and its association with the remodeling of 2nd- and 3rd-order neurons will shade more light on functional changes and recovery of the retina.

Electrophysiological fingerprints of OFF bipolar cells in rat retina

Retinal bipolar cells (BCs) divide photoreceptor output into different channels for the parallel extraction of temporal and chromatic stimulus properties. In rodents, five types of OFF BCs have been differentiated, based on morphological and functional criteria, but their electrophysiological characterization remains incomplete. This study analyzed OFF BCs with the patch clamp technique in acute slices of rat retina. Their specific voltage-dependent currents and glutamate responses are shown to represent individual fingerprints which define the signal processing and filtering properties of each cell type and allow their unequivocal identification. Two additions to the rat BC repertoire are presented: OFF BC-2′, a variation of BC-2 with wider axonal arbours and prominent Na+ currents, is described for the first time in rodents, and OFF BC-3b, previously identified in mouse, is electrophysiologically characterized in rat. Moreover, the glutamate responses of rat OFF BCs are shown to be differentially sensitive to AMPA- and kainate-receptor blockers and to modulation by nitric oxide (NO) through a cGMP-dependent mechanism. These results contribute to our understanding of the diversity and function of bipolar cells in mammals.

Glutamatergic Monopolar Interneurons Provide a Novel Pathway of Excitation in the Mouse Retina

Excitatory and inhibitory neurons in the CNS aredistinguished by several features, including morphology, transmitter content, and synapse architecture [1]. Such distinctions are exemplified in the vertebrate retina. Retinal bipolar cells are polarized glutamatergic neurons receiving direct photoreceptor input, whereas amacrine cells are usually monopolar inhibitory interneurons with synapses almost exclusively in the inner retina [2]. Bipolar but not amacrine cell synapses have presynaptic ribbon-like structures at their transmitter release sites. We identified a monopolar interneuron in the mouse retina that resembles amacrine cells morphologically but is glutamatergic and, unexpectedly, makes ribbon synapses. These glutamatergic monopolar interneurons (GluMIs) do not receive direct photoreceptor input, and their light responses are strongly shaped by both ON and OFF pathway-derived inhibitory input. GluMIs contact and make almost as many synapses as type 2 OFF bipolar cells onto OFF-sustained A-type (AOFF-S) retinal ganglion cells (RGCs). However, GluMIs and type 2 OFF bipolar cells possess functionally distinct light-driven responses and may therefore mediate separate components of the excitatory synaptic input to AOFF-S RGCs. The identification of GluMIs thus unveils a novel cellular component of excitatory circuits in the vertebrate retina, underscoring the complexity in defining cell types even in this well-characterized region of the CNS.

Multiple cone pathways are involved in photic regulation of retinal dopamine.

Dopamine is a key neurotransmitter in the retina and plays a central role in the light adaptive processes of the visual system. The sole source of retinal dopamine is dopaminergic amacrine cells (DACs). We and others have previously demonstrated that DACs are activated by rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) upon illumination. However, it is still not clear how each class of photosensitive cells generates light responses in DACs. We genetically isolated cone function in mice to specifically examine the cone-mediated responses of DACs and their neural pathways. In addition to the reported excitatory input to DACs from light-increment (ON) bipolar cells, we found that cones alternatively signal to DACs via a retrograde signalling pathway from ipRGCs. Cones also produce ON and light-decrement (OFF) inhibitory responses in DACs, which are mediated by other amacrine cells, likely driven by type 1 and type 2/3a OFF bipolar cells, respectively. Dye injections indicated that DACs had similar morphological profiles with or without ON/OFF inhibition. Our data demonstrate that cones utilize specific parallel excitatory and inhibitory circuits to modulate DAC activity and efficiently regulate dopamine release and the light-adaptive state of the retina.

Crossover Inhibition Generates Sustained Visual Responses in the Inner Retina

SummaryIn daylight, the input to the retinal circuit is provided primarily by cone photoreceptors acting as band-pass filters, but the retinal output also contains neuronal populations transmitting sustained signals. Using in vivo imaging of genetically encoded calcium reporters, we investigated the circuits that generate these sustained channels within the inner retina of zebrafish. In OFF bipolar cells, sustained transmission was found to depend on crossover inhibition from the ON pathway through GABAergic amacrine cells. In ON bipolar cells, the amplitude of low-frequency signals was regulated by glycinergic amacrine cells, while GABAergic inhibition regulated the gain of band-pass signals. We also provide the first functional description of a subset of sustained ON bipolar cells in which synaptic activity was suppressed by fluctuations at frequencies above ∼0.2 Hz. These results map out the basic circuitry by which the inner retina generates sustained visual signals and describes a new function of crossover inhibition.

An Ultrastructural and Immunohistochemical Analysis of the Outer Plexiform Layer of the Retina of the European Silver Eel (Anguilla anguilla L)

Here we studied the ultrastructural organization of the outer retina of the European silver eel, a highly valued commercial fish species. The retina of the European eel has an organization very similar to most vertebrates. It contains both rod and cone photoreceptors. Rods are abundantly present and immunoreactive for rhodopsin. Cones are sparsely present and only show immunoreactivity for M-opsin and not for L-, S- or UV-cone opsins. As in all other vertebrate retinas, Müller cells span the width of the retina. OFF-bipolar cells express the ionotropic glutamate receptor GluR4 and ON-bipolar cells, as identified by their PKCα immunoreactivity, express the metabotropic receptor mGluR6. Both the ON- and the OFF-bipolar cell dendrites innervate the cone pedicle and rod spherule. Horizontal cells are surrounded by punctate Cx53.8 immunoreactivity indicating that the horizontal cells are strongly electrically coupled by gap-junctions. Connexin-hemichannels were found at the tips of the horizontal cell dendrites invaginating the photoreceptor synapse. Such hemichannels are implicated in the feedback pathway from horizontal cells to cones. Finally, horizontal cells are surrounded by tyrosine hydroxylase immunoreactivity, illustrating a strong dopaminergic input from interplexiform cells.

Differentiation of murine models of "negative ERG" by single and repetitive light stimuli.

Marked attenuation of the single-flash electroretinographic (ERG) b-wave in the presence of a normal-amplitude or less-attenuated a-wave is commonly referred to as the "negative ERG." The purpose of this study was to investigate whether the disparate origins of the negative ERG in three murine models can be discriminated using flickering stimuli. Three models were selected: (1) the Nyx (nob) mouse model of complete congenital stationary night blindness, (2) the oxygen-induced retinopathy (OIR) rat model of retinopathy of prematurity (ROP), and (3) the Rs1 knockout (KO) mouse model of X-linked juvenile retinoschisis. Directly after a dark-adapted, single-flash ERG luminance series, a flicker ERG frequency series (0.5-30 Hz) was performed at a fixed luminance of 0.5 log cd s/m(2). This series includes frequency ranges that are dominated by activity in (A) the rod pathways (below 5 Hz), (B) the cone ON-pathway (5-15 Hz), and (C) the cone OFF-pathway (above 15 Hz). All three models produced markedly attenuated single-flash ERG b-waves. In the Nyx (nob) mouse, which features postsynaptic deficits in the ON-pathways, the a-wave was normal and flicker responses were attenuated in ranges A and B, but not C. The ROP rat is characterized by inner-retinal ischemia which putatively affects both ON- and OFF-bipolar cell activity; flicker responses were reduced in all ranges (A-C). Notably, the choroid supplies the photoreceptors and is thought to be relatively intact in OIR, an idea supported by the nearly normal a-wave. Finally, in the Rs1 KO mouse, which has documented abnormality of the photoreceptor-bipolar synapse affecting both ON- and OFF-pathways, the flicker responses were attenuated in all ranges (A-C). The a-wave was also attenuated, likely as a consequence to schisms in the photoreceptor layer. Consideration of both single-flash and flickering ERG responses can discriminate the functional pathology of the negative ERG in these animal models of human disease.

Light adaptation alters inner retinal inhibition to shape OFF retinal pathway signaling.

The retina adjusts its signaling gain over a wide range of light levels. A functional result of this is increased visual acuity at brighter luminance levels (light adaptation) due to shifts in the excitatory center-inhibitory surround receptive field parameters of ganglion cells that increases their sensitivity to smaller light stimuli. Recent work supports the idea that changes in ganglion cell spatial sensitivity with background luminance are due in part to inner retinal mechanisms, possibly including modulation of inhibition onto bipolar cells. To determine how the receptive fields of OFF cone bipolar cells may contribute to changes in ganglion cell resolution, the spatial extent and magnitude of inhibitory and excitatory inputs were measured from OFF bipolar cells under dark- and light-adapted conditions. There was no change in the OFF bipolar cell excitatory input with light adaptation; however, the spatial distributions of inhibitory inputs, including both glycinergic and GABAergic sources, became significantly narrower, smaller, and more transient. The magnitude and size of the OFF bipolar cell center-surround receptive fields as well as light-adapted changes in resting membrane potential were incorporated into a spatial model of OFF bipolar cell output to the downstream ganglion cells, which predicted an increase in signal output strength with light adaptation. We show a prominent role for inner retinal spatial signals in modulating the modeled strength of bipolar cell output to potentially play a role in ganglion cell visual sensitivity and acuity.