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SUMMARYComplete congenital stationary night blindness (cCSNB) is a heterogeneous disorder characterized by poor dim light vision, myopia, and nystagmus, that is caused by mutations in genes critical for signal transmission between photoreceptors and depolarizing bipolar cells (DBCs). One such gene, LRIT3, is required for assembly of the post-synaptic signaling complex (signalplex) at the dendritic tips of DBCs, although the number of signalplex components impacted is greater in cone DBCs than in rod bipolar cells. This difference raises the possibility that LRIT3 is expressed both pre- and post-synaptically at cone terminals. Here we show that rAAV-mediated expression of LRIT3 in cones results in robust rescue of cone DBC signalplex components and restores downstream visual function, as measured by the light adapted ERG b-wave and electrophysiological recordings of BCs and RGCs. These data show that LRIT3 successfully restores function to cone DBCs in a trans-synaptic manner, potentially paving the way for therapeutic intervention in LRIT3-associated cCSNB.

Behavioral interactions with moving objects are challenged by response latencies within the sensory and motor nervous systems. In vision, the combined latency from phototransduction and synaptic transmission from the retina to central visual areas amounts to 50–100 ms, depending on stimulus conditions. Time required for generating appropriate motor output adds to this latency and further compounds the behavioral delay. Neuronal adaptations that help counter sensory latency within the retina have been demonstrated in some species, but how general these specializations are, and where in the circuitry they originate, remains unclear. To address this, we studied the timing of object motion-evoked responses at multiple signaling stages within the mouse retina using two-photon fluorescence calcium and glutamate imaging, targeted whole-cell electrophysiology, and computational modeling. We found that both ON-type and OFF-type ganglion cells, as well as the bipolar cells that innervate them, temporally advance the position encoding of a moving object and so help counter the inherent signaling delay in the retina. Model simulations show that this predictive capability is a direct consequence of the spatial extent of the cells’ linear visual receptive field, with no apparent specialized circuits that help predict beyond it.

Neural circuits in the adult nervous system are characterized by stable, cell type-specific patterns of synaptic connectivity. In many parts of the nervous system these patterns are established during development through initial over-innervation by multiple pre- or postsynaptic targets, followed by a process of refinement that takes place during development and is in many instances activity dependent. Here we report on an identified synapse in the mouse retina, the cone photoreceptor➔type 4 bipolar cell (BC4) synapse, and show that its development is distinctly different from the common motif of over-innervation followed by refinement. Indeed, the majority of cones are contacted by single BC4 throughout development, but are contacted by multiple BC4s through ongoing dendritic elaboration between 1 and 6 months of age-well into maturity. We demonstrate that cell density drives contact patterns downstream of single cones in Bax null mice and may serve to maintain constancy in both the dendritic and axonal projective field.

The neural retina is organized along central-peripheral, dorsal-ventral, and laminar planes. Cellular density and distributions vary along the central-peripheral and dorsal-ventral axis in species including primates, mice, fish, and birds. Differential distribution of cell types within the retina is associated with sensitivity to different types of damage that underpin major retinal diseases, including macular degeneration and glaucoma. Normal variation in retinal distribution remains unreported for multiple cell types in widely used research models, including mouse. Here we map the distribution of all known OFF bipolar cell (BC) populations and horizontal cells. We report significant variation in the distribution of OFF BC populations and horizontal cells along the dorsal-ventral and central-peripheral axes of the retina. Distribution patterns are much more pronounced for some populations of OFF BC cells than others and may correspond to the cell type's specialized functions.

A persistent change in illumination causes light-adaptive changes in retinal neurons. Light adaptation improves visual encoding by preventing saturation and by adjusting spatiotemporal integration to increase the signal-to-noise ratio (SNR) and utilize signaling bandwidth efficiently. In dim light, the visual input contains a greater relative amount of quantal noise, and vertebrate receptive fields are extended in space and time to increase SNR. Whereas in bright light, SNR of the visual input is high, the rate of synaptic vesicle release from the photoreceptors is low so that quantal noise in synaptic output may limit SNR postsynaptically. Whether and how reduced synaptic SNR impacts spatiotemporal integration in postsynaptic neurons remains unclear. To address this, we measured spatiotemporal integration in retinal horizontal cells and ganglion cells in the guinea pig retina across a broad illumination range, from low to high photopic levels. In both cell types, the extent of spatial and temporal integration changed according to an inverted U-shaped function consistent with adaptation to low SNR at both low and high light levels. We show how a simple mechanistic model with interacting, opponent filters can generate the observed changes in ganglion cell spatiotemporal receptive fields across light-adaptive states and postulate that retinal neurons postsynaptic to the cones in bright light adopt low-pass spatiotemporal response characteristics to improve visual encoding under conditions of low synaptic SNR. NEW & NOTEWORTHY The mammalian retina is capable of functioning under an extraordinary (~1 billion fold) range of light intensities, from starlight conditions to bright sunlight. This is achieved primarily through light-adaptive mechanisms that adjust visual sensitivity at the level of the rod and cone photoreceptors. Although it is well established that visual function improves with increasing light, our results suggest that there is an apparent limit to this improvement, set at the stage of photoreceptor synaptic release. Our data provide a new, quantitative account of visual signaling at light levels at the high end of the naturally occurring range and further our insight into light-adaptive mechanisms in the mammalian retina.

Retinal ganglion cells (GCs) are a functionally diverse neuron population that encodes and transmits distinct representations of the visual image on the retina to target nuclei in the brain. Independent studies of visually-evoked responses, cell morphology, and gene expression each suggest that GCs in mouse may comprise as many as forty distinct cell types. To date, only a subset of these types have been characterized in detail, and for most genetic access is still lacking. Thus, the majority of identified GC types remains inaccessible for targeted electrophysiology and functional imaging, precluding efficient studies of their response properties, and the cell-intrinsic mechanisms and presynaptic circuits that generate these properties. Here we show that an existing mouse line that is commonly used for studies of cortical inhibitory circuits – Somatostatin-IRES-Cre (Sst-Cre), consistently labels an understudied subset of four GC types with distinct visual responses. We characterized these types both anatomically and functionally using Cre-dependent reporter mouse lines and confocal fluorescence imaging, calcium imaging, and whole-cell electrophysiology. We show that one of the labeled GC types is suppressed by luminance contrast, while another matches a recently described orientation-selective GC type. Our results give new information about these two identified GC types, and establish the utility of the Sst-Cre transgenic mouse line for studies of recently identified GC circuits in the mouse retina.

Under optimal conditions, just 3&ndash, 6 ms of visual stimulation suffices for humans to see motion. Motion perception on this timescale implies that the visual system under these conditions reliably encodes, transmits, and processes neural signals with near-millisecond precision. Motivated by in vitro evidence for high temporal precision of motion signals in the primate retina, we investigated how neuronal and perceptual limits of motion encoding relate. Specifically, we examined the correspondence between the time scale at which cat retinal ganglion cells in vivo represent motion information and temporal thresholds for human motion discrimination. The timescale for motion encoding by ganglion cells ranged from 4.6 to 91 ms, and depended non-linearly on temporal frequency, but not on contrast. Human psychophysics revealed that minimal stimulus durations required for perceiving motion direction were similarly brief, 5.6&ndash, 65 ms, and similarly depended on temporal frequency but, above ~10%, not on contrast. Notably, physiological and psychophysical measurements corresponded closely throughout (r = 0.99), despite more than a 20-fold variation in both human thresholds and optimal timescales for motion encoding in the retina. The match in absolute values of the neurophysiological and psychophysical data may be taken to indicate that from the lateral geniculate nucleus (LGN) through to the level of perception little temporal precision is lost. However, we also show that integrating responses from multiple neurons can improve temporal resolution, and this potential trade-off between spatial and temporal resolution would allow for loss of temporal resolution after the LGN. While the extent of neuronal integration cannot be determined from either our human psychophysical or neurophysiological experiments and its contribution to the measured temporal resolution is unknown, our results demonstrate a striking similarity in stimulus dependence between the temporal fidelity established in the retina and the temporal limits of human motion discrimination.

In this issue of Neuron, Serbe et al. (2016) use cell-type-specific genetic tools to record and manipulate all major inputs to directionally selective neurons in Drosophila. Their results localize the site of motion computation and reveal unexpected complexity of temporal tuning in the underlying neural circuit.

Visual processing in the retina depends on coordinated signaling by interneurons. Photoreceptor signals are relayed to ∼20 ganglion cell types through a dozen excitatory bipolar interneurons, each responsive to light increments (ON) or decrements (OFF). ON and OFF bipolar cell pathways become tuned through specific connections with inhibitory interneurons: horizontal and amacrine cells. A major obstacle for understanding retinal circuitry is the unknown function of most of the ∼30–40 amacrine cell types, each of which synapses onto a subset of bipolar cell terminals, ganglion cell dendrites, and other amacrine cells. Here, we used a transgenic mouse line in which vasoactive intestinal polypeptide-expressing (VIP + ) GABAergic interneurons express Cre recombinase. Targeted whole-cell recordings of fluorescently labeled VIP + cells revealed three predominant types: wide-field bistratified and narrow-field monostratified cells with somas in the inner nuclear layer (INL) and medium-field monostratified cells with somas in the ganglion cell layer (GCL). Bistratified INL cells integrated excitation and inhibition driven by both ON and OFF pathways with little spatial tuning. Narrow-field INL cells integrated excitation driven by the ON pathway and inhibition driven by both pathways, with pronounced hyperpolarizations at light offset. Monostratified GCL cells integrated excitation and inhibition driven by the ON pathway and showed center-surround spatial tuning. Optogenetic experiments showed that, collectively, VIP + cells made strong connections with OFF δ, ON-OFF direction-selective, and W3 ganglion cells but weak, inconsistent connections with ON and OFF α cells. Revealing VIP + cell morphologies, receptive fields and synaptic connections advances our understanding of their role in visual processing. SIGNIFICANCE STATEMENT The retina is a model system for understanding nervous system function. At the first stage, rod and cone photoreceptors encode light and communicate with a complex network of interneurons. These interneurons drive the responses of ganglion cells, which form the optic nerve and transmit visual information to the brain. Presently, we lack information about many of the retina9s inhibitory amacrine interneurons. In this study, we used genetically modified mice to study the light responses and intercellular connections of specific amacrine cell types. The results show diversity in the shape and function of the studied amacrine cells and elucidate their connections with specific types of ganglion cell. The findings advance our understanding of the cellular basis for retinal function.

Calcium imaging is commonly used to measure the neural activity of large groups of neurons in mice. Genetically encoded calcium indicators (GECIs) can be delivered for this purpose using non-invasive genetic methods. Compared to viral gene transfer, transgenic targeting of GECIs provides stable long-term expression and obviates the need for invasive viral injections. Transgenic mice expressing the green GECI GCaMP6 are already widely used. Here we present the generation and characterizarion of transgenic mice expressing the sensitive red GECI jRGECO1a, driven by theThy1promoter. Four transgenic lines with different expression patterns showed sufficiently high expression for cellularin vivoimaging. We used two-photon microscopy to characterize visual responses of individual neurons in the visual cortexin vivo. The signal-to-noise ratio in transgenic mice was comparable to, or better than, for mice transduced with adeno-associated virus. We also show thatThy1-jRGECO1a transgenic mice are useful for transcranial population imaging and functional mapping using widefield fluorescecnce microscopy. We also demonstrate imaging of visual responses in retinal ganglion cells.Thy1-jRGECO1a transgenic mice are therefore a useful addition to the toolbox for imaging activity in intact neural networks.

International audience; Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

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.

At its first synapse, the retina establishes two parallel channels that encode light increments (ON) or decrements (OFF). At the same synapse, changes in photoreceptor glutamate release are sensed by ON bipolar cells (BCs) via the metabotropic glutamate receptor 6 (mGluR6), and OFF BCs via ionotropic BCs, which differ in their synaptic configuration with the photoreceptor terminal. ON BCs form invaginating synapses that bring them in close proximity to presynaptic ribbons and the presumed sole source of glutamate release. OFF bipolar cells form flat contacts distal to the ribbon synapse. We investigated the role of LRIT3 in normal assembly and function of the mGlur6 signaling cascade present in ON BCs. We demonstrate that LRIT3 is required for nyctalopin expression and thus TRPM1 expression and function. Using glutamate imaging, whole-cell electrophysiology, and multi-electrode array extracellular recordings we demonstrate that the loss of LRIT3 impacts both the ON and OFF pathways at the level of the BCs. The effect on ON pathway signaling, a lack of ON BC response, is shared by mutants lacking mGluR6, TRPM1 GPR179 or nyctalopin. The effects on the OFF pathway are unique to LRIT3, and include a decrease in response amplitude of both OFF BC and GCs. Based on these results, we propose a working model where LRIT3 is required for either efficient glutamate release or reuptake from the first retinal synapse.SIGNIFICANCE STATEMENTAt the first visual synapse, photoreceptor cells signal to two distinct bipolar cell (BC) populations, one characterized by a depolarizing response to light onset (ON or DBCs), the other by a hyperpolarizing response (OFF or HBCs). The DBC light response depends on a G protein-coupled receptor and associated protein complex, known as the signalplex. Mutations in signalplex proteins lead to DBC pathway-specific loss of visual function. Here we show how loss of LRIT3, a previously identified signalplex protein, prevents functional assembly of the DBC signalplex and alters visual function in both ON and OFF signaling pathways. Thus, our results indicate that the function of LRIT3 at this first synapse extends beyond assembly of the DBC signalplex.

Direction selectivity represents a fundamental visual computation. In mammalian retina, On-Off direction-selective ganglion cells (DSGCs) respond strongly to motion in a preferred direction and weakly to motion in the opposite, null direction. Electrical recordings suggested three direction-selective (DS) synaptic mechanisms: DS GABA release during null-direction motion from starburst amacrine cells (SACs) and DS acetylcholine and glutamate release during preferred direction motion from SACs and bipolar cells. However, evidence for DS acetylcholine and glutamate release has been inconsistent and at least one bipolar cell type that contacts another DSGC (On-type) lacks DS release. Here, whole-cell recordings in mouse retina showed that cholinergic input to On-Off DSGCs lacked DS, whereas the remaining (glutamatergic) input showed apparent DS. Fluorescence measurements with the glutamate biosensor intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) conditionally expressed in On-Off DSGCs showed that glutamate release in both On- and Off-layer dendrites lacked DS, whereas simultaneously recorded excitatory currents showed apparent DS. With GABA-A receptors blocked, both iGluSnFR signals and excitatory currents lacked DS. Our measurements rule out DS release from bipolar cells onto On-Off DSGCs and support a theoretical model suggesting that apparent DS excitation in voltage-clamp recordings results from inadequate voltage control of DSGC dendrites during null-direction inhibition. SAC GABA release is the apparent sole source of DS input onto On-Off DSGCs.

Rod photoreceptors contribute to vision over an ∼ 6-log-unit range of light intensities. The wide dynamic range of rod vision is thought to depend upon light intensity-dependent switching between two parallel pathways linking rods to ganglion cells: a rod → rod bipolar (RB) cell pathway that operates at dim backgrounds and a rod → cone → cone bipolar cell pathway that operates at brighter backgrounds. We evaluated this conventional model of rod vision by recording rod-mediated light responses from ganglion and AII amacrine cells and by recording RB-mediated synaptic currents from AII amacrine cells in mouse retina. Contrary to the conventional model, we found that the RB pathway functioned at backgrounds sufficient to activate the rod → cone pathway. As background light intensity increased, the RB's role changed from encoding the absorption of single photons to encoding contrast modulations around mean luminance. This transition is explained by the intrinsic dynamics of transmission from RB synapses.

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systemsin vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery ofin vitroassays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, andin vivoinCaenorhabditischemosensory neurons,Drosophilalarval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combiningin vivoimaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activityin vivoand may find widespread applications for cellular imaging in general.

The photopigment melanopsin confers photosensitivity upon a minority of retinal output neurons. These intrinsically photosensitive retinal ganglion cells (ipRGCs) are more diverse than once believed, comprising five morphologically distinct types, M1 through M5. Here, in mouse retina, we provide the first in-depth characterization of M4 cells, including their structure, function, and central projections. M4 cells apparently correspond to ON α cells of earlier reports, and are easily distinguished from other ipRGCs by their very large somata. Their dendritic arbors are more radiate and highly branched than those of M1, M2, or M3 cells. The melanopsin-based intrinsic photocurrents of M4 cells are smaller than those of M1 and M2 cells, presumably because melanopsin is more weakly expressed; we can detect it immunohistochemically only with strong amplification. Like M2 cells, M4 cells exhibit robust, sustained, synaptically driven ON responses and dendritic stratification in the ON sublamina of the inner plexiform layer. However, their stratification patterns are subtly different, with M4 dendrites positioned just distal to those of M2 cells and just proximal to the ON cholinergic band. M4 receptive fields are large, with an ON center, antagonistic OFF surround and nonlinear spatial summation. Their synaptically driven photoresponses lack direction selectivity and show higher ultraviolet sensitivity in the ventral retina than in the dorsal retina, echoing the topographic gradient in S- and M-cone opsin expression. M4 cells are readily labeled by retrograde transport from the dorsal lateral geniculate nucleus and thus likely contribute to the pattern vision that persists in mice lacking functional rods and cones.

Fluorescent calcium indicator proteins, such as GCaMP3, allow imaging of activity in genetically defined neuronal populations. GCaMP3 can be expressed using various gene delivery methods, such as viral infection or electroporation. However, these methods are invasive and provide inhomogeneous and nonstationary expression. Here, we developed a genetic reporter mouse, Ai38, which expresses GCaMP3 in a Cre-dependent manner from theROSA26locus, driven by a strongCAGpromoter. Crossing Ai38 with appropriate Cre mice produced robust GCaMP3 expression in defined cell populations in the retina, cortex, and cerebellum. In the primary visual cortex, visually evoked GCaMP3 signals showed normal orientation and direction selectivity. GCaMP3 signals were rapid, compared with virally expressed GCaMP3 and synthetic calcium indicators. In the retina, Ai38 allowed imaging spontaneous calcium waves in starburst amacrine cells during development, and light-evoked responses in ganglion cells in adult tissue. Our results show that the Ai38 reporter mouse provides a flexible method for targeted expression of GCaMP3.

In the version of this paper originally published, important figure labels in Fig. 3d were not visible. An image layer present in the authors' original figure that included two small dashed outlines and text labels indicating ROI 1 and ROI 2, as well as a scale bar and the name of the cell label, was erroneously altered during image processing. The figure has been corrected in the HTML and PDF versions of the paper.

Retinal ganglion cells that respond selectively to a dark spot on a brighter background (OFF cells) have smaller dendritic fields than their ON counterparts and are more numerous. OFF cells also branch more densely, and thus collect more synapses per visual angle. That the retina devotes more resources to processing dark contrasts predicts that natural images contain more dark information. We confirm this across a range of spatial scales and trace the origin of this phenomenon to the statistical structure of natural scenes. We show that the optimal mosaics for encoding natural images are also asymmetric, with OFF elements smaller and more numerous, matching retinal structure. Finally, the concentration of synapses within a dendritic field matches the information content, suggesting a simple principle to connect a concrete fact of neuroanatomy with the abstract concept of information: equal synapses for equal bits.

A retinal ganglion cell receptive field is made up of an excitatory center and an inhibitory surround. The surround has two components: one driven by horizontal cells at the first synaptic layer and one driven by amacrine cells at the second synaptic layer. Here we characterized how amacrine cells inhibit the center response of on- and off-center Y-type ganglion cells in the in vitro guinea pig retina. A high spatial frequency grating (4-5 cyc/mm), beyond the spatial resolution of horizontal cells, drifted in the ganglion cell receptive field periphery to stimulate amacrine cells. The peripheral grating suppressed the ganglion cell spiking response to a central spot. Suppression of spiking was strongest and observed most consistently in off cells. In intracellular recordings, the grating suppressed the subthreshold membrane potential in two ways: a reduced slope (gain) of the stimulus-response curve by approximately 20-30% and, in off cells, a tonic approximately 1-mV hyperpolarization. In voltage clamp, the grating increased an inhibitory conductance in all cells and simultaneously decreased an excitatory conductance in off cells. To determine whether center response inhibition was presynaptic or postsynaptic (shunting), we measured center response gain under voltage-clamp and current-clamp conditions. Under both conditions, the peripheral grating reduced center response gain similarly. This result suggests that reduced gain in the ganglion cell subthreshold center response reflects inhibition of presynaptic bipolar terminals. Thus amacrine cells suppressed ganglion cell center response gain primarily by inhibiting bipolar cell glutamate release.

Sensorimotor delays decouple behaviors from the events that drive them. The brain compensates for these delays with predictive mechanisms, but the efficacy and timescale over which these mechanisms operate remain poorly understood. Here, we assess how prediction is used to compensate for prey movement that occurs during visuomotor processing. We obtained high-speed video records of freely moving, tongue-projecting salamanders catching walking prey, emulating natural foraging conditions. We found that tongue projections were preceded by a rapid head turn lasting ∼130 ms. This motor lag, combined with the ∼100 ms phototransduction delay at photopic light levels, gave a ∼230 ms visuomotor response delay during which prey typically moved approximately one body length. Tongue projections, however, did not significantly lag prey position but were highly accurate instead. Angular errors in tongue projection accuracy were consistent with a linear extrapolation model that predicted prey position at the time of tongue contact using the average prey motion during a ∼175 ms period one visual latency before the head movement. The model explained successful strikes where the tongue hit the fly, and unsuccessful strikes where the fly turned and the tongue hit a phantom location consistent with the fly's earlier trajectory. The model parameters, obtained from the data, agree with the temporal integration and latency of retinal responses proposed to contribute to motion extrapolation. These results show that the salamander predicts future prey position and that prediction significantly improves prey capture success over a broad range of prey speeds and light levels.SIGNIFICANCE STATEMENTNeural processing delays cause actions to lag behind the events that elicit them. To cope with these delays, the brain predicts what will happen in the future. While neural circuits in the retina and beyond have been suggested to participate in such predictions, few behaviors have been explored sufficiently to constrain circuit function. Here we show that salamanders aim their tongues by using extrapolation to estimate future prey position, thereby compensating for internal delays from both visual and motor processing. Predictions made just before a prey turn resulted in the tongue being projected to a position consistent with the prey's pre-turn trajectory. These results define the computations and operating regimen for neural circuits that predict target motion.

Temporal interactions in direction-sensitive complex cells in area 18 and the posteromedial lateral suprasylvian cortex (PMLS) were studied using a reverse correlation method. Reverse correlograms to combinations of two temporally separated motion directions were examined and compared in the two areas. A comparison to the first-order reverse correlograms allowed us to identify nonlinear suppression or facilitation due to pairwise combinations of motion directions. Results for area 18 and PMLS were very different. Area 18 showed a single type of nonlinear behavior: similar directions facilitated and opposite directions suppressed spike probability. This effect was most pronounced for motion steps that followed each other immediately and decreased with increasing delay between steps. In PMLS, the picture was much more diverse. Some cells exhibited nonlinear interactions, that were opposite to those in area 18 (facilitation for opposite directions and suppression for similar ones), while the majority did not show a systematic interaction profile. We conclude that nonlinear second-order reverse correlation characteristics reveal different functional properties, despite similarities in the first-order reverse correlation profiles. Directional interactions in time revealed optimal integration of similar directions in area 18, but motion opponency—at least in some cells—in PMLS.

We studied receptive field organization of motion-sensitive neurons in macaque middle temporal cortical area (MT), by mapping direction selectivity in space and in time. Stimuli consisted of pseudorandom sequences of single motion steps presented simultaneously at many different receptive field locations. Spatio-temporal receptive field profiles were constructed by cross-correlating stimuli and spikes. The resulting spike-triggered averages revealed centre-surround organization. The temporal dynamics of the receptive fields were generally biphasic with increased probability for the preferred direction at short latency (50-70 ms) and decreased probability at longer latency (80-100 ms). The response latency of the receptive field surround was on average 16 ms longer than that of the centre. Our results show that surround input and biphasic behaviour reflect two different mechanisms, which make MT cells specifically sensitive to motion contrast in space and time.

Visual latencies and temporal dynamics of area 18 and PMLS direction-selective complex cells were defined with a reverse correlation method. The method allowed us to analyze the time course of responses to motion steps, without confounding temporal integration effects. Several measures of response latency and direction tuning dynamics were quantified: optimal latency (OL), latency of first and last significant responses (FSR, LSR), the increase and decrease of direction sensitivity in time, and the change of direction tuning in time. FSR, OL and LSR values for PMLS and area 18 largely overlapped. The small differences in mean latencies (3-4 ms for FSR and OL and 11.9 ms for the LSR) were not statistically significant. All cells in area 18 and the vast majority of cells in PMLS showed no systematic changes in preferred direction (monophasic neurons). In PMLS 5 out of 41 cells showed a reversal of preferred direction after approximately 56 ms relative to their OL (biphasic neurons). Monophasic cells showed no systematic changes in direction tuning width during the interval from FSR to LSR. In both areas, development of direction sensitivity was significantly faster than return to the non-direction sensitive state, but no significant difference was found between the two areas. We conclude that, for the monophasic type of direction-selective complex cells, the dynamics of primary motion processing are highly comparable for area 18 and PMLS. This suggests that motion information is predominantly processed in parallel, presumably based on input from the fast conducting thalamocortical Y-pathway.

We introduce the motion reverse correlation method (MRC), a novel stimulus paradigm based on a random sequence of motion impulses. The method is tailored to investigate the spatio-temporal dynamics of motion selectivity in cells responding to moving random dot patterns. Effectiveness of the MRC method is illustrated with results obtained from recordings in both anesthetized cats and an awake, fixating macaque monkey. Motion tuning functions are computed by reverse correlating the response of single cells with a rapid sequence of displacements of a random pixel array (RPA). Significant correlations between the cell's responses and various aspects of stimulus motion are obtained at high temporal resolution. These correlations provide a detailed description of the temporal dynamics of, for example, direction tuning and velocity tuning. In addition, with a spatial array of independently moving RPAs, the MRC method can be used to measure spatial as well as temporal receptive field properties. We demonstrate that MRC serves as a powerful and time-efficient tool for quantifying receptive field properties of motion selective cells that yields temporal information that cannot be derived from existing methods.

Alpha/Y-type retinal ganglion cells encode visual information with a receptive field composed of nonlinear subunits. This nonlinear subunit structure enhances sensitivity to patterns composed of high spatial frequencies. The Y-cell's subunits are the presynaptic bipolar cells, but the mechanism for the nonlinearity remains incompletely understood. We investigated the synaptic basis of the subunit nonlinearity by combining whole-cell recording of mouse Y-type ganglion cells with two-photon fluorescence imaging of a glutamate sensor (iGluSnFR) expressed on their dendrites and throughout the inner plexiform layer. A control experiment designed to assess iGluSnFR's dynamic range showed that fluorescence responses from Y-cell dendrites increased proportionally with simultaneously recorded excitatory current. Spatial resolution was sufficient to readily resolve independent release at intermingled ON and OFF bipolar terminals. iGluSnFR responses at Y-cell dendrites showed strong surround inhibition, reflecting receptive field properties of presynaptic release sites. Responses to spatial patterns located the origin of the Y-cell nonlinearity to the bipolar cell output, after the stage of spatial integration. The underlying mechanism differed between OFF and ON pathways: OFF synapses showed transient release and strong rectification, whereas ON synapses showed relatively sustained release and weak rectification. At ON synapses, the combination of fast release onset with slower release offset explained the nonlinear response of the postsynaptic ganglion cell. Imaging throughout the inner plexiform layer, we found transient, rectified release at the central-most levels, with increasingly sustained release near the borders. By visualizing glutamate release in real time, iGluSnFR provides a powerful tool for characterizing glutamate synapses in intact neural circuits.

The use of neurotropic viruses as transsynaptic tracers was first described in the 1960s, but only recently have such viruses gained popularity as a method for labeling neural circuits. The development of retrograde monosynaptic tracing vectors has enabled visualization of the presynaptic sources onto defined sets of postsynaptic neurons. Here, we describe the first application of a novel viral tracer, based on vesicular stomatitis virus (VSV), which directs retrograde transsynaptic viral spread between defined cell types. We use this virus in the mouse retina to show connectivity between starburst amacrine cells (SACs) and their known synaptic partners, direction-selective retinal ganglion cells, as well as to discover previously unknown connectivity between SACs and other retinal ganglion cell types. These novel connections were confirmed using physiological recordings. VSV transsynaptic tracing enables cell type-specific dissection of neural circuitry and can reveal synaptic relationships among neurons that are otherwise obscured due to the complexity and density of neuropil.

We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.

Catfish detect and identify invisible prey by sensing their ultra-weak electric fields with electroreceptors. Any neuron that deals with small-amplitude input has to overcome sensitivity limitations arising from inherent threshold non-linearities in spike-generation mechanisms. Many sensory cells solve this issue with stochastic resonance, in which a moderate amount of intrinsic noise causes irregular spontaneous spiking activity with a probability that is modulated by the input signal. Here we show that catfish electroreceptors have adopted a fundamentally different strategy. Using a reverse correlation technique in which we take spike interval durations into account, we show that the electroreceptors generate a supra-threshold bias current that results in quasi-periodically produced spikes. In this regime stimuli modulate the interval between successive spikes rather than the instantaneous probability for a spike. This alternative for stochastic resonance combines threshold-free sensitivity for weak stimuli with similar sensitivity for excitations and inhibitions based on single interspike intervals.

Decoding the wiring diagram of the retina requires simultaneous observation of activity in identified neuron populations. Available recording methods are limited in their scope: electrodes can access only a small fraction of neurons at once, whereas synthetic fluorescent indicator dyes label tissue indiscriminately. Here, we describe a method for studying retinal circuitry at cellular and subcellular levels combining two-photon microscopy and a genetically encoded calcium indicator. Using specific viral and promoter constructs to drive expression of GCaMP3, we labeled all five major neuron classes in the adult mouse retina. Stimulus-evoked GCaMP3 responses as imaged by two-photon microscopy permitted functional cell type annotation. Fluorescence responses were similar to those measured with the small molecule dye OGB-1. Fluorescence intensity correlated linearly with spike rates >10 spikes/s, and a significant change in fluorescence always reflected a significant change in spike firing rate. GCaMP3 expression had no apparent effect on neuronal function. Imaging at subcellular resolution showed compartment-specific calcium dynamics in multiple identified cell types.

Retinal ganglion cells of a given type overlap their dendritic fields such that every point in space is covered by three to four cells. We investigated what function is served by such extensive overlap. Recording from pairs of ON or OFF brisk-transient ganglion cells at photopic intensities, we confirmed that this overlap causes the Gaussian receptive field centers to be spaced at approximately 2 SDs (sigma). This, together with response nonlinearities and variability, was just sufficient to provide an ideal observer with uniform contrast sensitivity across the retina for both threshold and suprathreshold stimuli. We hypothesized that overlap might maximize the information represented from natural images, thereby optimizing retinal performance for many tasks. Indeed, tested with natural images (which contain statistical correlations), a model ganglion cell array maximized information represented in its population responses with approximately 2sigma spacing, i.e., the overlap observed in the retina. Yet, tested with white noise (which lacks statistical correlations), an array maximized its information by minimizing overlap. In both cases, optimal overlap balanced greater signal-to-noise ratio (from larger receptive fields) against greater redundancy (because of larger receptive field overlap). Thus, dendritic overlap improves vision by taking optimal advantage of the statistical correlations of natural scenes.

Retinal ganglion cells fire spikes to an appropriate contrast presented over their receptive field center. These center responses undergo dynamic changes in sensitivity depending on the ongoing level of contrast, a process known as “contrast gain control.” Extracellular recordings suggested that gain control is driven by a single wide-field mechanism, extending across the center and beyond, that depends on inhibitory interneurons: amacrine cells. However, recordings in salamander suggested that the excitatory bipolar cells, which drive the center, may themselves show gain control independently of amacrine cell mechanisms. Here, we tested in mammalian ganglion cells whether amacrine cells are critical for gain control over the receptive field center. We made extracellular and whole-cell recordings of guinea pig Y-type cellsin vitroand quantified the gain change between contrasts using a linear–nonlinear analysis. For spikes, tripling contrast reduced gain by ∼40%. With spikes blocked, ganglion cells showed similar levels of gain control in membrane currents and voltages and under conditions of low and high calcium buffering: tripling contrast reduced gain by ∼20–25%. Gain control persisted under voltage-clamp conditions that minimize inhibitory conductances and pharmacological conditions that block inhibitory neurotransmitter receptors. Gain control depended on adequate stimulation, not of ganglion cells but of presynaptic bipolar cells. Furthermore, horizontal cell measurements showed a lack of gain control in photoreceptor synaptic release. Thus, the mechanism for gain control over the ganglion cell receptive field center, as measured in the subthreshold response, originates in the presynaptic bipolar cells and does not require amacrine cell signaling.

How cone synapses encode light intensity determines the precision of information transmission at the first synapse on the visual pathway. Although it is known that cone photoreceptors hyperpolarize to light over 4-5 log units of intensity, the relationship between light intensity and transmitter release at the cone synapse has not been determined. Here, we use two-photon microscopy to visualize release of the synaptic vesicle dye FM1-43 from cone terminals in the intact lizard retina, in response to different stimulus light intensities. We then employ electron microscopy to translate these measurements into vesicle release rates. We find that from darkness to bright light, release decreases from 49 to approximately 2 vesicles per 200 ms; therefore, cones compress their 10,000-fold operating range for phototransduction into a 25-fold range for synaptic vesicle release. Tonic release encodes ten distinguishable intensity levels, skewed to most finely represent bright light, assuming release obeys Poisson statistics.

We studied the temporal dynamics of motion direction sensitivity in macaque area MT using a motion reverse correlation paradigm. Stimuli consisted of a random sequence of motion steps in eight different directions. Cross-correlating the stimulus with the resulting neural activity reveals the temporal dynamics of direction selectivity. The temporal dynamics of direction selectivity at the preferred speed showed two phases along the time axis: one phase corresponding to an increase in probability for the preferred direction at short latencies and a second phase corresponding to a decrease in probability for the preferred direction at longer latencies. The strength of this biphasic behavior varied between neurons from weak to very strong and was uniformly distributed. Strong biphasic behavior suggests optimal responses for motion steps in the antipreferred direction followed by a motion step in the preferred direction. Correlating spikes to combinations of motion directions corroborates this distinction. The optimal combination for weakly biphasic cells consists of successive steps in the preferred direction, whereas for strongly biphasic cells, it is a reversal of directions. Comparing reverse correlograms to combinations of stimuli to predictions based on correlograms for individual directions revealed several nonlinear effects. Correlations for successive presentations of preferred directions were smaller than predicted, which could be explained by a static nonlinearity (saturation). Correlations to pairs of (nearly) opposite directions were larger than predicted. These results show that MT neurons are generally more responsive when sudden changes in motion directions occur, irrespective of the preferred direction of the neurons. The latter nonlinearities cannot be explained by a simple static nonlinearity at the output of the neuron, but most likely reflect network interactions.