PyOKR: A Semi-Automated Method for Quantifying Optokinetic Reflex Tracking Ability.

The study of behavioral responses to visual stimuli is a key component of understanding visual system function. One notable response is the optokinetic reflex (OKR), a highly conserved innate behavior necessary for image stabilization on the retina. The OKR provides a robust readout of image tracking ability and has been extensively studied to understand visual system circuitry and function in animals from different genetic backgrounds. The OKR consists of two phases: a slow tracking phase as the eye follows a stimulus to the edge of the visual plane and a compensatory fast phase saccade that resets the position of the eye in the orbit. Previous methods of tracking gain quantification, although reliable, are labor intensive and can be subjective or arbitrarily derived. To obtain more rapid and reproducible quantification of eye tracking ability, we have developed a novel semi-automated analysis program, PyOKR, that allows for quantification of two-dimensional eye tracking motion in response to any directional stimulus, in addition to being adaptable to any type of video-oculography equipment. This method provides automated filtering, selection of slow tracking phases, modeling of vertical and horizontal eye vectors, quantification of eye movement gains relative to stimulus speed, and organization of resultant data into a usable spreadsheet for statistical and graphical comparisons. This quantitative and streamlined analysis pipeline, readily accessible via PyPI import, provides a fast and direct measurement of OKR responses, thereby facilitating the study of visual behavioral responses.

A semi-automated method for quantifying optokinetic reflex tracking acuity.

The study of murine behavioral responses to visual stimuli is a key component of understanding mammalian visual circuitry. One notable response is the optokinetic reflex (OKR), a highly conserved innate behavior necessary for image stabilization on the retina. The OKR provides a robust readout of image tracking ability and has been extensively studied to understand the logic of visual system circuitry and function in mice from different genetic backgrounds. The OKR consists of two phases: a slow tracking phase as the eye follows a stimulus to the edge of the visual plane, and a compensatory fast phase saccade that maintains the image within the visual field. Assessment of the OKR has previously relied on counting individual compensatory eye saccades to estimate tracking speed. To obtain a more direct quantification of tracking ability, we have developed a novel, semi-automated analysis program that allows for rapid and reproducible quantification of unidirectional tracking gains, in addition to being adaptable to any video-oculography equipment. Our analysis program allows for the selection of slow tracking phases, modeling of the vertical and horizontal eye vectors, quantification of eye movement relative to the stimulus, and organization of resultant data into a usable spreadsheet for statistical and graphical comparisons. This quantitative and streamlined analysis pipeline provides a faster and more direct measurement of OKR responses, thereby facilitating further study of visual behavior responses. SUMMARY: We describe here a semi-automated quantitative analysis method that directly measures eye tracking resulting from murine visual system responses to two-dimensional image motion. A Python-based user interface and analysis algorithm allows for higher throughput and more quantitative measurements of eye tracking parameters than previous methods.

Asymmetric retinal direction tuning predicts optokinetic eye movements across stimulus conditions.

Across species, the optokinetic reflex (OKR) stabilizes vision during self-motion. OKR occurs when ON direction-selective retinal ganglion cells (oDSGCs) detect slow, global image motion on the retina. How oDSGC activity is integrated centrally to generate behavior remains unknown. Here, we discover mechanisms that contribute to motion encoding in vertically tuned oDSGCs and leverage these findings to empirically define signal transformation between retinal output and vertical OKR behavior. We demonstrate that motion encoding in vertically tuned oDSGCs is contrast-sensitive and asymmetric for oDSGC types that prefer opposite directions. These phenomena arise from the interplay between spike threshold nonlinearities and differences in synaptic input weights, including shifts in the balance of excitation and inhibition. In behaving mice, these neurophysiological observations, along with a central subtraction of oDSGC outputs, accurately predict the trajectories of vertical OKR across stimulus conditions. Thus, asymmetric tuning across competing sensory channels can critically shape behavior.

Inhibition, but not excitation, recovers from partial cone loss with greater spatiotemporal integration, synapse density, and frequency.

Neural circuits function in the face of changing inputs, either caused by normal variation in stimuli or by cell death. To maintain their ability to perform essential computations with partial inputs, neural circuits make modifications. Here, we study the retinal circuit's responses to changes in light stimuli or in photoreceptor inputs by inducing partial cone death in the mature mouse retina. Can the retina withstand or recover from input loss? We find that the excitatory pathways exhibit functional loss commensurate with cone death and with some aspects predicted by partial light stimulation. However, inhibitory pathways recover functionally from lost input by increasing spatiotemporal integration in a way that is not recapitulated by partially stimulating the control retina. Anatomically, inhibitory synapses are upregulated on secondary bipolar cells and output ganglion cells. These findings demonstrate the greater capacity for inhibition, compared with excitation, to modify spatiotemporal processing with fewer cone inputs.

Impact of Photoreceptor Loss on Retinal Circuitry.

Our sense of sight relies on photoreceptors, which transduce photons into the nervous system's electrochemical interpretation of the visual world. These precious photoreceptors can be disrupted by disease, injury, and aging. Once photoreceptors start to die, but before blindness occurs, the remaining retinal circuitry can withstand, mask, or exacerbate the photoreceptor deficit and potentially be receptive to newfound therapies for vision restoration. To maximize the retina's receptivity to therapy, one must understand the conditions that influence the state of the remaining retina. In this review, we provide an overview of the retina's structure and function in health and disease. We analyze a collection of observations on photoreceptor disruption and generate a predictive model to identify parameters that influence the retina's response. Finally, we speculate on whether the retina, with its remarkable capacity to function over light levels spanning nine orders of magnitude, uses these same adaptational mechanisms to withstand and perhaps mask photoreceptor loss.

Disassembly and rewiring of a mature converging excitatory circuit following injury.

Specificity and timing of synapse disassembly in the CNS are essential to learning how individual circuits react to neurodegeneration of the postsynaptic neuron. In sensory systems such as the mammalian retina, synaptic connections of second-order neurons are known to remodel and reconnect in the face of sensory cell loss. Here we analyzed whether degenerating third-order neurons can remodel their local presynaptic connectivity. We injured adult retinal ganglion cells by transiently elevating intraocular pressure. We show that loss of presynaptic structures occurs before postsynaptic density proteins and accounts for impaired transmission from presynaptic neurons, despite no evidence of presynaptic cell loss, axon terminal shrinkage, or reduced functional input. Loss of synapses is biased among converging presynaptic neuron types, with preferential loss of the major excitatory cone-driven partner and increased connectivity with rod-driven presynaptic partners, demonstrating that this adult neural circuit is capable of structural plasticity while undergoing neurodegeneration.

Mature Retina Compensates Functionally for Partial Loss of Rod Photoreceptors.

Loss of primary neuronal inputs inevitably strikes every neural circuit. The deafferented circuit could propagate, amplify, or mitigate input loss, thus affecting the circuit's output. How the deafferented circuit contributes to the effect on the output is poorly understood because of lack of control over loss of and access to circuit elements. Here, we control the timing and degree of rod photoreceptor ablation in mature mouse retina and uncover compensation. Following loss of half of the rods, rod bipolar cells mitigate the loss by preserving voltage output. Such mitigation allows partial recovery of ganglion cell responses. We conclude that rod death is compensated for in the circuit because ganglion cell responses to stimulation of half of the rods in an unperturbed circuit are weaker than responses after death of half of the rods. The dominant mechanism of such compensation includes homeostatic regulation of inhibition to balance the loss of excitation.

Partial Cone Loss Triggers Synapse-Specific Remodeling and Spatial Receptive Field Rearrangements in a Mature Retinal Circuit.

Resilience of neural circuits has been observed in the persistence of function despite neuronal loss. In vision, acuity and sensitivity can be retained after 50% loss of cones. While neurons in the cortex can remodel after input loss, the contributions of cell-type-specific circuits to resilience are unknown. Here, we study the effects of partial cone loss in mature mouse retina where cell types and connections are known. At first-order synapses, bipolar cell dendrites remodel and synaptic proteins diminish at sites of input loss. Sites of remaining inputs preserve synaptic proteins. Second-order synapses between bipolar and ganglion cells remain stable. Functionally, ganglion cell spatio-temporal receptive fields retain center-surround structure following partial cone loss. We find evidence for slower temporal filters and expanded receptive field surrounds, derived mainly from inhibitory inputs. Surround expansion is absent in partially stimulated control retina. Results demonstrate functional resilience to input loss beyond pre-existing mechanisms in control retina.

Synaptogenesis and synaptic protein localization in the postnatal development of rod bipolar cell dendrites in mouse retina.

Retinal responses to photons originate in rod photoreceptors and are transmitted to the ganglion cell output of the retina through the primary rod bipolar pathway. At the first synapse of this pathway, input from multiple rods is pooled into individual rod bipolar cells. This architecture is called convergence. Convergence serves to improve sensitivity of rod vision when photons are sparse. Establishment of convergence depends on the development of a proper complement of dendritic tips and transduction proteins in rod bipolar cells. How the dendrites of rod bipolar cells develop and contact the appropriate number of rods is unknown. To answer this question we visualized individual rod bipolar cells in mouse retina during postnatal development and quantified the number of dendritic tips, as well as the expression of transduction proteins within dendrites. Our findings show that the number of dendritic tips in rod bipolar cells increases monotonically during development. The number of tips at P21, P30, and P82 exceeds the previously reported rod convergence ratios, and the majority of these tips are proximal to a presynaptic rod release site, suggesting more rods provide input to a rod bipolar cell. We also show that dendritic transduction cascade members mGluR6 and TRPM1 appear in tips with different timelines. These finding suggest that (a) rod bipolar cell dendrites elaborate without pruning during development, (b) the convergence ratio between rods and rod bipolar cells may be higher than previously reported, and (c) mGluR6 and TRPM1 are trafficked independently during development.

Synaptic Convergence Patterns onto Retinal Ganglion Cells Are Preserved despite Topographic Variation in Pre- and Postsynaptic Territories.

Sensory processing can be tuned by a neuron's integration area, the types of inputs, and the proportion and number of connections with those inputs. Integration areas often vary topographically to sample space differentially across regions. Here, we highlight two visual circuits in which topographic changes in the postsynaptic retinal ganglion cell (RGC) dendritic territories and their presynaptic bipolar cell (BC) axonal territories are either matched or unmatched. Despite this difference, in both circuits, the proportion of inputs from each BC type, i.e., synaptic convergence between specific BCs and RGCs, remained constant across varying dendritic territory sizes. Furthermore, synapse density between BCs and RGCs was invariant across topography. Our results demonstrate a wiring design, likely engaging homotypic axonal tiling of BCs, that ensures consistency in synaptic convergence between specific BC types onto their target RGCs while enabling independent regulation of pre- and postsynaptic territory sizes and synapse number between cell pairs.

Photoreceptor ablation initiates the immediate loss of glutamate receptors in postsynaptic bipolar cells in retina.

Structural changes underlying neurodegenerative diseases include dismantling of synapses, degradation of circuitry, and even massive rewiring. Our limited understanding of synapse dismantling stems from the inability to control the timing and extent of cell death. In this study, selective ablation of cone photoreceptors in live mouse retina and tracking of postsynaptic partners at the cone-to-ON cone bipolar cell synapse reveals that early reaction to cone loss involves rapid and local changes in postsynaptic glutamate receptor distribution. Glutamate receptors disappear with a time constant of 2 h. Furthermore, binding of glutamate receptors by agonists and antagonists is insufficient to rescue glutamate receptor loss, suggesting that receptor allocation depends on the physical presence of cones. These findings demonstrate that the initial step in synapse disassembly involves postsynaptic receptor loss rather than dendritic retraction, providing insight into the early stages of neurodegenerative disease.

Wiring patterns in the mouse retina: collecting evidence across the connectome, physiology and light microscopy.

The visual system has often been thought of as a parallel processor because distinct regions of the brain process different features of visual information. However, increasing evidence for convergence and divergence of circuit connections, even at the level of the retina where visual information is first processed, chips away at a model of dedicated and distinct pathways for parallel information flow. Instead, our current understanding is that parallel channels may emerge, not from exclusive microcircuits for each channel, but from unique combinations of microcircuits. This review depicts diagrammatically the current knowledge and remaining puzzles about the retinal circuit with a focus on the mouse retina. Advances in techniques for labelling cells and genetic manipulations have popularized the use of transgenic mice. We summarize evidence gained from serial electron microscopy, electrophysiology and light microscopy to illustrate the wiring patterns in mouse retina. We emphasize the need to explore proposed retinal connectivity using multiple methods to verify circuits both structurally and functionally.

Sensory experience shapes the development of the visual system's first synapse.

Specific connectivity patterns among neurons create the basic architecture underlying parallel processing in our nervous system. Here we focus on the visual system's first synapse to examine the structural and functional consequences of sensory deprivation on the establishment of parallel circuits. Dark rearing reduces synaptic strength between cones and cone bipolar cells, a previously unappreciated effect of sensory deprivation. In contrast, rod bipolar cells, which utilize the same glutamate receptor to contact rods, are unaffected by dark rearing. Underlying the physiological changes, we find the localization of metabotropic glutamate receptors within cone bipolar, but not rod bipolar, cell dendrites is a light-dependent process. Furthermore, although cone bipolar cells share common cone partners, each bipolar cell type that we examined depends differentially on sensory input to achieve mature connectivity. Thus, visual experience differentially affects maturation of rod versus cone pathways and of cell types within the cone pathway.

The spatial structure of a nonlinear receptive field.

Understanding a sensory system implies the ability to predict responses to a variety of inputs from a common model. In the retina, this includes predicting how the integration of signals across visual space shapes the outputs of retinal ganglion cells. Existing models of this process generalize poorly to predict responses to new stimuli. This failure arises in part from properties of the ganglion cell response that are not well captured by standard receptive-field mapping techniques: nonlinear spatial integration and fine-scale heterogeneities in spatial sampling. Here we characterize a ganglion cell's spatial receptive field using a mechanistic model based on measurements of the physiological properties and connectivity of only the primary excitatory circuitry of the retina. The resulting simplified circuit model successfully predicts ganglion-cell responses to a variety of spatial patterns and thus provides a direct correspondence between circuit connectivity and retinal output.

Diverse strategies engaged in establishing stereotypic wiring patterns among neurons sharing a common input at the visual system's first synapse.

Sensory circuits use common strategies, such as convergence and divergence, typically at different synapses, to pool or distribute inputs. Inputs from different presynaptic cell types converge onto a common postsynaptic cell, acting together to shape neuronal output (Klausberger and Somogyi, 2008). Also, individual presynaptic cells contact several postsynaptic cell types, generating divergence of signals. Attaining such complex wiring patterns relies on the orchestration of many events across development, including axonal and dendritic growth and synapse formation and elimination (reviewed by Waites et al., 2005; Sanes and Yamagata, 2009). Recent work has focused on how distinct presynaptic cell types form stereotypic connections with an individual postsynaptic cell (Morgan et al., 2011; Williams et al., 2011), but how a single presynaptic cell type diverges to form distinct wiring patterns with multiple postsynaptic cell types during development remains unexplored. Here we take advantage of the compactness of the visual system's first synapse to observe development of such a circuit in mouse retina. By imaging three types of postsynaptic bipolar cells and their common photoreceptor targets across development, we found that distinct bipolar cell types engage in disparate dendritic growth behaviors, exhibit targeted or exploratory approaches to contact photoreceptors, and adhere differently to the synaptotropic model of establishing synaptic territories. Furthermore each type establishes its final connectivity patterns with the same afferents on separate time scales. We propose that such differences in strategy and timeline could facilitate the division of common inputs among multiple postsynaptic cell types to create parallel circuits with diverse function.

Spontaneous olfactory receptor neuron activity determines follower cell response properties.

Noisy or spontaneous activity is common in neural systems and poses a challenge to detecting and discriminating signals. Here we use the locust to answer fundamental questions about noise in the olfactory system: Where does spontaneous activity originate? How is this activity propagated or reduced throughout multiple stages of neural processing? What mechanisms favor the detection of signals despite the presence of spontaneous activity? We found that spontaneous activity long observed in the secondary projection neurons (PNs) originates almost entirely from the primary olfactory receptor neurons (ORNs) rather than from spontaneous circuit interactions in the antennal lobe, and that spontaneous activity in ORNs tonically depolarizes the resting membrane potentials of their target PNs and local neurons (LNs) and indirectly tonically depolarizes tertiary Kenyon cells (KCs). However, because these neurons have different response thresholds, in the absence of odor stimulation, ORNs and PNs display a high spontaneous firing rate but KCs are nearly silent. Finally, we used a simulation of the olfactory network to show that discrimination of signal and noise in the KCs is best when threshold levels are set so that baseline activity in PNs persists. Our results show how the olfactory system benefits from making a signal detection decision after a point of maximal information convergence, e.g., after KCs pool inputs from many PNs.

Single-photon absorptions evoke synaptic depression in the retina to extend the operational range of rod vision.

Adaptation or gain control allows sensory neurons to encode diverse stimuli using a limited range of output signals. Rod vision exemplifies a general challenge facing adaptational mechanisms-balancing the benefits of averaging to create a reliable signal for adaptation with the need to adapt rapidly and locally. The synapse between rod bipolar and AII amacrine cells dominates adaptation at low light levels. We find that adaptation occurs independently at each synapse and completes in <500 ms. This limited spatial and temporal integration suggests that the absorption of a single photon modulates gain. Indeed, responses to pairs of brief dim flashes showed directly that synaptic gain was depressed for 100-200 ms following transmission of a single-photon response. Presynaptic mechanisms mediated this synaptic depression. Thus, the division of light into discrete photons controls adaptation at this synapse, and gain varies with the irreducible statistical fluctuations in photon arrival.

Light adaptation in cone vision involves switching between receptor and post-receptor sites.

We see over an enormous range of mean light levels, greater than the range of output signals retinal neurons can produce. Even highlights and shadows within a single visual scene can differ approximately 10,000-fold in intensity-exceeding the range of distinct neural signals by a factor of approximately 100. The effectiveness of daylight vision under these conditions relies on at least two retinal mechanisms that adjust sensitivity in the approximately 200 ms intervals between saccades. One mechanism is in the cone photoreceptors (receptor adaptation) and the other is at a previously unknown location within the retinal circuitry that benefits from convergence of signals from multiple cones (post-receptor adaptation). Here we find that post-receptor adaptation occurs as signals are relayed from cone bipolar cells to ganglion cells. Furthermore, we find that the two adaptive mechanisms are essentially mutually exclusive: as light levels increase the main site of adaptation switches from the circuitry to the cones. These findings help explain how human cone vision encodes everyday scenes, and, more generally, how sensory systems handle the challenges posed by a diverse physical environment.

Synaptic influences on rat ganglion-cell photoreceptors.

The intrinsically photosensitive retinal ganglion cells (ipRGCs) provide a conduit through which rods and cones can access brain circuits mediating circadian entrainment, pupillary constriction and other non-image-forming visual functions. We characterized synaptic inputs to ipRGCs in rats using whole-cell and multielectrode array recording techniques. In constant darkness all ipRGCs received spontaneous excitatory and inhibitory synaptic inputs. Light stimulation evoked in all ipRGCs both synaptically driven ('extrinsic') and autonomous melanopsin-based ('intrinsic') responses. The extrinsic light responses were depolarizing, about 5 log units more sensitive than the intrinsic light response, and transient near threshold but sustained to brighter light. Pharmacological data showed that ON bipolar cells and amacrine cells make the most prominent direct contributions to these extrinsic light responses, whereas OFF bipolar cells make a very weak contribution. The spatial extent of the synaptically driven light responses was comparable to that of the intrinsic photoresponse, suggesting that synaptic contacts are made onto the entire dendritic field of the ipRGCs. These synaptic influences increase the sensitivity of ipRGCs to light, and also extend their temporal bandpass to higher frequencies. These extrinsic ipRGC light responses can explain some of the previously reported properties of circadian photoentrainment and other non-image-forming visual behaviours.

The impact of photoreceptor noise on retinal gain controls.

Multiple retinal mechanisms preserve visual sensitivity as the properties of the light inputs change. Rapid gain controls match the effective signaling range of retinal neurons to the local image statistics. Such gain controls trade an increased sensitivity for some aspects of the inputs for a decreased sensitivity to others. Rapid, local gain control comes at another cost: noise in the signal controlling gain (e.g. from the photoreceptors) will cause gain itself to vary even when the statistics of the light input are constant. Recent advances in identifying retinal pathways and the sites and mechanisms of mean and contrast adaptation have begun to clarify the tradeoffs associated with different gain control locations and how these tradeoffs differ for rod and cone vision.