In Vivo Efficacy and Safety Evaluations of Therapeutic Splicing Correction Using U1 snRNA in the Mouse Retina.

Efficacy and safety considerations constitute essential steps during development of in vivo gene therapies. Herein, we evaluated efficacy and safety of splice factor-based treatments to correct mutation-induced splice defects in an Opa1 mutant mouse line. We applied adeno-associated viruses to the retina. The viruses transduced retinal cells with an engineered U1 snRNA splice factor designed to correct the Opa1 splice defect. We found the treatment to be efficient in increasing wild-type Opa1 transcripts. Correspondingly, Opa1 protein levels increased significantly in treated eyes. Measurements of retinal morphology and function did not reveal therapy-related side-effects supporting the short-term safety of the treatment. Alterations of potential off-target genes were not detected. Our data suggest that treatments of splice defects applying engineered U1 snRNAs represent a promising in vivo therapeutic approach. The therapy increased wild-type Opa1 transcripts and protein levels without detectable morphological, functional or genetic side-effects in the mouse eye. The U1 snRNA-based therapy can be tailored to specific disease gene mutations, hence, raising the possibility of a wider applicability of this promising technology towards treatment of different inherited retinal diseases.

Integration and evaluation of magnetic stimulation in physiology setups.

A large number of behavioral experiments have demonstrated the existence of a magnetic sense in many animal species. Further, studies with immediate gene expression markers have identified putative brain regions involved in magnetic information processing. In contrast, very little is known about the physiology of the magnetic sense and how the magnetic field is neuronally encoded. In vivo electrophysiological studies reporting neuronal correlates of the magnetic sense either have turned out to be irreproducible for lack of appropriate artifact controls or still await independent replication. Thus far, the research field of magnetoreception has little exploited the power of ex vivo physiological studies, which hold great promise for enabling stringent controls. However, tight space constraints in a recording setup and the presence of magnetizable materials in setup components and microscope objectives make it demanding to generate well-defined magnetic stimuli at the location of the biological specimen. Here, we present a solution based on a miniature vector magnetometer, a coil driver, and a calibration routine for the coil system to compensate for magnetic distortions in the setup. The magnetometer fits in common physiology recording chambers and has a sufficiently small spatial integration area to allow for probing spatial inhomogeneities. The coil-driver allows for the generation of defined non-stationary fast changing magnetic stimuli. Our ex vivo multielectrode array recordings from avian retinal ganglion cells show that artifacts induced by rapid magnetic stimulus changes can mimic the waveform of biological spikes on single electrodes. However, induction artifacts can be separated clearly from biological responses if the spatio-temporal characteristics of the artifact on multiple electrodes is taken into account. We provide the complete hardware design data and software resources for the integrated magnetic stimulation system.

Electrical Coupling of Heterotypic Ganglion Cells in the Mammalian Retina.

Electrical coupling has been reported to occur only between homotypic retinal ganglion cells, in line with the concept of parallel processing in the early visual system. Here, however, we show reciprocal correlated firing between heterotypic ganglion cells in multielectrode array recordings during light stimulation in retinas of adult guinea pigs of either sex. Heterotypic coupling was further confirmed via tracer spread after intracellular injections of single cells with neurobiotin. Both electrically coupled cell types were sustained ON center ganglion cells but showed distinct light response properties and receptive field sizes. We identified one of the involved cell types as sustained ON α-ganglion cells. The presence of electrical coupling between heterotypic ganglion cells introduces a network motif in which the signals of distinct ganglion cell types are partially mixed at the output stage of the retina.SIGNIFICANCE STATEMENT The visual information is split into parallel pathways, before it is sent to the brain via the output neurons of the retina, the ganglion cells. Ganglion cells can form electrical synapses between dendrites of neighboring cells in support of lateral information exchange. To date, ganglion-to-ganglion cell coupling is thought to occur only between cells of the same type. Here, however, we show that electrical coupling between different types of ganglion cells exists in the mammalian retina. We provide functional and anatomical evidence that two different types of ganglion cells share information via electrical coupling. This new network motif extends the impact of the heavily studied coding benefits of homotypic coupling to heterotypic coupling across parallel neuronal pathways.

Pathway-Specific Asymmetries between ON and OFF Visual Signals.

Visual processing is largely organized into ON and OFF pathways that signal stimulus increments and decrements, respectively. These pathways exhibit natural pairings based on morphological and physiological similarities, such as ON and OFF α-ganglion cells in the mammalian retina. Several studies have noted asymmetries in the properties of ON and OFF pathways. For example, the spatial receptive fields (RFs) of OFF α-cells are systematically smaller than ON α-cells. Analysis of natural scenes suggests that these asymmetries are optimal for visual encoding. To test the generality of ON/OFF asymmetries, we measured the spatiotemporal RF properties of multiple RGC types in rat retina. Through a quantitative and serial classification, we identified three functional pairs of ON and OFF RGCs. We analyzed the structure of their RFs and compared spatial integration, temporal integration, and gain across ON and OFF pairs. Similar to previous results from the cat and primate, RGC types with larger spatial RFs exhibited briefer temporal integration and higher gain. However, each pair of ON and OFF RGC types exhibited distinct asymmetric relationships between RF properties, some of which were opposite to the findings of previous reports. These results reveal the functional organization of six RGC types in the rodent retina and indicate that ON/OFF asymmetries are pathway specific.SIGNIFICANCE STATEMENT Circuits that process sensory input frequently process increments separately from decrements, so-called ON and OFF responses. Theoretical studies indicate that this separation, and associated asymmetries in ON and OFF pathways, may be beneficial for encoding natural stimuli. However, the generality of ON and OFF pathway asymmetries has not been tested. Here we compare the functional properties of three distinct pairs of ON and OFF pathways in the rodent retina and show that their asymmetries are pathway specific. These results provide a new view on the partitioning of vision across diverse ON and OFF signaling pathways.

Eliminating Glutamatergic Input onto Horizontal Cells Changes the Dynamic Range and Receptive Field Organization of Mouse Retinal Ganglion Cells.

In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however, how horizontal cell signals also affect the temporal, spatial, and contrast tuning in retinal output neurons, the ganglion cells. To study this, we generated a genetically modified mouse line in which we eliminated the light dependency of feedback by deleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actual mode of feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-α retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse.SIGNIFICANCE STATEMENT Horizontal cells represent a major neuronal class in the mammalian retina and provide lateral feedback and feedforward signals to photoreceptors and bipolar cells, respectively. The mode of signal transmission remains controversial and, moreover, the contribution of horizontal cells to visual processing is still elusive. To address the question of how horizontal cells affect retinal output signals, we recorded the light responses of transient OFF-α retinal ganglion cells in a newly generated mouse line. In this mouse line, horizontal cell signals were no longer modulated by light. With light response recordings, we show that horizontal cells increase the dynamic range of retinal ganglion cells for contrast and temporal changes and contribute to the center/surround organization of their receptive fields.

Identification of a Retinal Circuit for Recurrent Suppression Using Indirect Electrical Imaging.

Understanding the function of modulatory interneuron networks is a major challenge, because such networks typically operate over long spatial scales and involve many neurons of different types. Here, we use an indirect electrical imaging method to reveal the function of a spatially extended, recurrent retinal circuit composed of two cell types. This recurrent circuit produces peripheral response suppression of early visual signals in the primate magnocellular visual pathway. We identify a type of polyaxonal amacrine cell physiologically via its distinctive electrical signature, revealed by electrical coupling with ON parasol retinal ganglion cells recorded using a large-scale multi-electrode array. Coupling causes the amacrine cells to fire spikes that propagate radially over long distances, producing GABA-ergic inhibition of other ON parasol cells recorded near the amacrine cell axonal projections. We propose and test a model for the function of this amacrine cell type, in which the extra-classical receptive field of ON parasol cells is formed by reciprocal inhibition from other ON parasol cells in the periphery, via the electrically coupled amacrine cell network.

Mapping nonlinear receptive field structure in primate retina at single cone resolution.

The function of a neural circuit is shaped by the computations performed by its interneurons, which in many cases are not easily accessible to experimental investigation. Here, we elucidate the transformation of visual signals flowing from the input to the output of the primate retina, using a combination of large-scale multi-electrode recordings from an identified ganglion cell type, visual stimulation targeted at individual cone photoreceptors, and a hierarchical computational model. The results reveal nonlinear subunits in the circuity of OFF midget ganglion cells, which subserve high-resolution vision. The model explains light responses to a variety of stimuli more accurately than a linear model, including stimuli targeted to cones within and across subunits. The recovered model components are consistent with known anatomical organization of midget bipolar interneurons. These results reveal the spatial structure of linear and nonlinear encoding, at the resolution of single cells and at the scale of complete circuits.

Anatomical identification of extracellularly recorded cells in large-scale multielectrode recordings.

This study combines for the first time two major approaches to understanding the function and structure of neural circuits: large-scale multielectrode recordings, and confocal imaging of labeled neurons. To achieve this end, we develop a novel approach to the central problem of anatomically identifying recorded cells, based on the electrical image: the spatiotemporal pattern of voltage deflections induced by spikes on a large-scale, high-density multielectrode array. Recordings were performed from identified ganglion cell types in the macaque retina. Anatomical images of cells in the same preparation were obtained using virally transfected fluorescent labeling or by immunolabeling after fixation. The electrical image was then used to locate recorded cell somas, axon initial segments, and axon trajectories, and these signatures were used to identify recorded cells. Comparison of anatomical and physiological measurements permitted visualization and physiological characterization of numerically dominant ganglion cell types with high efficiency in a single preparation.

A polyaxonal amacrine cell population in the primate retina.

Amacrine cells are the most diverse and least understood cell class in the retina. Polyaxonal amacrine cells (PACs) are a unique subset identified by multiple long axonal processes. To explore their functional properties, populations of PACs were identified by their distinctive radially propagating spikes in large-scale high-density multielectrode recordings of isolated macaque retina. One group of PACs exhibited stereotyped functional properties and receptive field mosaic organization similar to that of parasol ganglion cells. These PACs had receptive fields coincident with their dendritic fields, but much larger axonal fields, and slow radial spike propagation. They also exhibited ON-OFF light responses, transient response kinetics, sparse and coordinated firing during image transitions, receptive fields with antagonistic surrounds and fine spatial structure, nonlinear spatial summation, and strong homotypic neighbor electrical coupling. These findings reveal the functional organization and collective visual signaling by a distinctive, high-density amacrine cell population.

Retinal representation of the elementary visual signal.

The propagation of visual signals from individual cone photoreceptors through parallel neural circuits was examined in the primate retina. Targeted stimulation of individual cones was combined with simultaneous recording from multiple retinal ganglion cells of identified types. The visual signal initiated by an individual cone produced strong responses with different kinetics in three of the four numerically dominant ganglion cell types. The magnitude and kinetics of light responses in each ganglion cell varied nonlinearly with stimulus strength but in a manner that was independent of the cone of origin after accounting for the overall input strength of each cone. Based on this property of independence, the receptive field profile of an individual ganglion cell could be well estimated from responses to stimulation of each cone individually. Together, these findings provide a quantitative account of how elementary visual inputs form the ganglion cell receptive field.

Efficient coding of spatial information in the primate retina.

Sensory neurons have been hypothesized to efficiently encode signals from the natural environment subject to resource constraints. The predictions of this efficient coding hypothesis regarding the spatial filtering properties of the visual system have been found consistent with human perception, but they have not been compared directly with neural responses. Here, we analyze the information that retinal ganglion cells transmit to the brain about the spatial information in natural images subject to three resource constraints: the number of retinal ganglion cells, their total response variances, and their total synaptic strengths. We derive a model that optimizes the transmitted information and compare it directly with measurements of complete functional connectivity between cone photoreceptors and the four major types of ganglion cells in the primate retina, obtained at single-cell resolution. We find that the ganglion cell population exhibited 80% efficiency in transmitting spatial information relative to the model. Both the retina and the model exhibited high redundancy (~30%) among ganglion cells of the same cell type. A novel and unique prediction of efficient coding, the relationships between projection patterns of individual cones to all ganglion cells, was consistent with the observed projection patterns in the retina. These results indicate a high level of efficiency with near-optimal redundancy in visual signaling by the retina.

Cone photoreceptor contributions to noise and correlations in the retinal output.

Transduction and synaptic noise generated in retinal cone photoreceptors determine the fidelity with which light inputs are encoded, and the readout of cone signals by downstream circuits determines whether this fidelity is used for vision. We examined the effect of cone noise on visual signals by measuring its contribution to correlated noise in primate retinal ganglion cells. Correlated noise was strong in the responses of dissimilar cell types with shared cone inputs. The dynamics of cone noise could account for rapid correlations in ganglion cell activity, and the extent of shared cone input could explain correlation strength. Furthermore, correlated noise limited the fidelity with which visual signals were encoded by populations of ganglion cells. Thus, a simple picture emerges: cone noise, traversing the retina through diverse pathways, accounts for most of the noise and correlations in the retinal output and constrains how higher centers exploit signals carried by parallel visual pathways.

Correlated firing among major ganglion cell types in primate retina.

Retinal ganglion cells exhibit substantial correlated firing: a tendency to fire nearly synchronously at rates different from those expected by chance. These correlations suggest that network interactions significantly shape the visual signal transmitted from the eye to the brain. This study describes the degree and structure of correlated firing among the major ganglion cell types in primate retina. Correlated firing among ON and OFF parasol, ON and OFF midget, and small bistratified cells, which together constitute roughly 75% of the input to higher visual areas, was studied using large-scale multi-electrode recordings. Correlated firing in the presence of constant, spatially uniform illumination exhibited characteristic strength, time course and polarity within and across cell types. Pairs of nearby cells with the same light response polarity were positively correlated; cells with the opposite polarity were negatively correlated. The strength of correlated firing declined systematically with distance for each cell type, in proportion to the degree of receptive field overlap. The pattern of correlated firing across cell types was similar at photopic and scotopic light levels, although additional slow correlations were present at scotopic light levels. Similar results were also observed in two other retinal ganglion cell types. Most of these observations are consistent with the hypothesis that shared noise from photoreceptors is the dominant cause of correlated firing. Surprisingly, small bistratified cells, which receive ON input from S cones, fired synchronously with ON parasol and midget cells, which receive ON input primarily from L and M cones. Collectively, these results provide an overview of correlated firing across cell types in the primate retina, and constraints on the underlying mechanisms.

Time-resolved and time-scale adaptive measures of spike train synchrony.

A wide variety of approaches to estimate the degree of synchrony between two or more spike trains have been proposed. One of the most recent methods is the ISI-distance which extracts information from the interspike intervals (ISIs) by evaluating the ratio of the instantaneous firing rates. In contrast to most previously proposed measures it is parameter free and time-scale independent. However, it is not well suited to track changes in synchrony that are based on spike coincidences. Here we propose the SPIKE-distance, a complementary measure which is sensitive to spike coincidences but still shares the fundamental advantages of the ISI-distance. In particular, it is easy to visualize in a time-resolved manner and can be extended to a method that is also applicable to larger sets of spike trains. We show the merit of the SPIKE-distance using both simulated and real data.

Functional connectivity in the retina at the resolution of photoreceptors.

To understand a neural circuit requires knowledge of its connectivity. Here we report measurements of functional connectivity between the input and ouput layers of the macaque retina at single-cell resolution and the implications of these for colour vision. Multi-electrode technology was used to record simultaneously from complete populations of the retinal ganglion cell types (midget, parasol and small bistratified) that transmit high-resolution visual signals to the brain. Fine-grained visual stimulation was used to identify the location, type and strength of the functional input of each cone photoreceptor to each ganglion cell. The populations of ON and OFF midget and parasol cells each sampled the complete population of long- and middle-wavelength-sensitive cones. However, only OFF midget cells frequently received strong input from short-wavelength-sensitive cones. ON and OFF midget cells showed a small non-random tendency to selectively sample from either long- or middle-wavelength-sensitive cones to a degree not explained by clumping in the cone mosaic. These measurements reveal computations in a neural circuit at the elementary resolution of individual neurons.

Receptive field mosaics of retinal ganglion cells are established without visual experience.

A characteristic feature of adult retina is mosaic organization: a spatial arrangement of cells of each morphological and functional type that produces uniform sampling of visual space. How the mosaics of visual receptive fields emerge in the retina during development is not fully understood. Here we use a large-scale multielectrode array to determine the mosaic organization of retinal ganglion cells (RGCs) in rats around the time of eye opening and in the adult. At the time of eye opening, we were able to reliably distinguish two types of ON RGCs and two types of OFF RGCs in rat retina based on their light response and intrinsic firing properties. Although the light responses of individual cells were not yet mature at this age, each of the identified functional RGC types formed a receptive field mosaic, where the spacing of the receptive field centers and the overlap of the receptive field extents were similar to those observed in the retinas of adult rats. These findings suggest that, although the light response properties of RGCs may need vision to reach full maturity, extensive visual experience is not required for individual RGC types to form a regular sensory map of visual space.

High-sensitivity rod photoreceptor input to the blue-yellow color opponent pathway in macaque retina.

Small bistratified cells (SBCs) in the primate retina carry a major blue-yellow opponent signal to the brain. We found that SBCs also carry signals from rod photoreceptors, with the same sign as S cone input. SBCs exhibited robust responses under low scotopic conditions. Physiological and anatomical experiments indicated that this rod input arose from the AII amacrine cell-mediated rod pathway. Rod and cone signals were both present in SBCs at mesopic light levels. These findings have three implications. First, more retinal circuits may multiplex rod and cone signals than were previously thought to, efficiently exploiting the limited number of optic nerve fibers. Second, signals from AII amacrine cells may diverge to most or all of the approximately 20 retinal ganglion cell types in the peripheral primate retina. Third, rod input to SBCs may be the substrate for behavioral biases toward perception of blue at mesopic light levels.

Receptive fields in primate retina are coordinated to sample visual space more uniformly.

In the visual system, large ensembles of neurons collectively sample visual space with receptive fields (RFs). A puzzling problem is how neural ensembles provide a uniform, high-resolution visual representation in spite of irregularities in the RFs of individual cells. This problem was approached by simultaneously mapping the RFs of hundreds of primate retinal ganglion cells. As observed in previous studies, RFs exhibited irregular shapes that deviated from standard Gaussian models. Surprisingly, these irregularities were coordinated at a fine spatial scale: RFs interlocked with their neighbors, filling in gaps and avoiding large variations in overlap. RF shapes were coordinated with high spatial precision: the observed uniformity was degraded by angular perturbations as small as 15 degrees, and the observed populations sampled visual space with more than 50% of the theoretical ideal uniformity. These results show that the primate retina encodes light with an exquisitely coordinated array of RF shapes, illustrating a higher degree of functional precision in the neural circuitry than previously appreciated.

Uniform signal redundancy of parasol and midget ganglion cells in primate retina.

The collective representation of visual space in high resolution visual pathways was explored by simultaneously measuring the receptive fields of hundreds of ON and OFF midget and parasol ganglion cells in isolated primate retina. As expected, the receptive fields of all four cell types formed regular mosaics uniformly tiling the visual scene. Surprisingly, comparison of all four mosaics revealed that the overlap of neighboring receptive fields was nearly identical, for both the excitatory center and inhibitory surround components of the receptive field. These observations contrast sharply with the large differences in the dendritic overlap between the parasol and midget cell populations, revealing a surprising lack of correspondence between the anatomical and functional architecture in the dominant circuits of the primate retina.

The structure of large-scale synchronized firing in primate retina.

Synchronized firing among neurons has been proposed to constitute an elementary aspect of the neural code in sensory and motor systems. However, it remains unclear how synchronized firing affects the large-scale patterns of activity and redundancy of visual signals in a complete population of neurons. We recorded simultaneously from hundreds of retinal ganglion cells in primate retina, and examined synchronized firing in completely sampled populations of approximately 50-100 ON-parasol cells, which form a major projection to the magnocellular layers of the lateral geniculate nucleus. Synchronized firing in pairs of cells was a subset of a much larger pattern of activity that exhibited local, isotropic spatial properties. However, a simple model based solely on interactions between adjacent cells reproduced 99% of the spatial structure and scale of synchronized firing. No more than 20% of the variability in firing of an individual cell was predictable from the activity of its neighbors. These results held both for spontaneous firing and in the presence of independent visual modulation of the firing of each cell. In sum, large-scale synchronized firing in the entire population of ON-parasol cells appears to reflect simple neighbor interactions, rather than a unique visual signal or a highly redundant coding scheme.