An Annotated Journey through Modern Visual Neuroscience.

Recent advances in microscopy, genetics, physiology, and data processing have expanded the scope and accelerated the pace of discovery in visual neuroscience. However, the pace of discovery and the ever increasing number of published articles can present a serious issue for both trainees and senior scientists alike: with each passing year the fog of progress thickens, making it easy to lose sight of important earlier advances. As part of this special issue of the Journal of Neuroscience commemorating the 50th anniversary of SfN, here, we provide a variation on Stephen Kuffler's Oldies but Goodies classic reading list, with the hope that by looking back at highlights in the field of visual neuroscience we can better define remaining gaps in our knowledge and thus guide future work. We also hope that this article can serve as a resource that will aid those new to the field to find their bearings.

Dynamic tuning of electrical and chemical synaptic transmission in a network of motion coding retinal neurons.

Recently, we demonstrated that gap junction coupling in the population of superior coding ON-OFF directionally selective ganglion cells (DSGCs) genetically labeled in the Hb9::eGFP mouse retina allows the passage of lateral anticipatory signals that help track moving stimuli. Here, we examine the properties of gap junctions in the DSGC network, and address how interactions between electrical and chemical synapses and intrinsic membrane properties contribute to the dynamic tuning of lateral anticipatory signals. When DSGC subtypes coding all four cardinal directions were individually loaded with the gap junction-permeable tracer Neurobiotin, only superior coding DSGCs exhibited homologous coupling. Consistent with these anatomical findings, gap junction-dependent feedback spikelets were only observed in Hb9(+) DSGCs. Recordings from pairs of neighboring Hb9(+) DSGCs revealed that coupling was reciprocal, non-inactivating, and relatively weak, and provided a substrate for an extensive subthreshold excitatory receptive field around each cell. This subthreshold activity appeared to boost coincident light-driven chemical synaptic responses. However, during responses to moving stimuli, gap junction-mediated boosting appeared to be dynamically modulated such that upstream DSGCs primed downstream cells, but not vice versa, giving rise to highly skewed responses in individual cells. We show that the asymmetry in priming arises from a combination of spatially offset GABAergic inhibition and activity-dependent changes in intrinsic membrane properties of DSGCs. Thus, dynamic interactions between electrical and chemical synapses and intrinsic membrane properties allow the network of DSGCs to propagate anticipatory responses most effectively along their preferred direction without leading to runaway excitation.

An intrinsic neural oscillator in the degenerating mouse retina.

The loss of photoreceptors during retinal degeneration (RD) is known to lead to an increase in basal activity in remnant neural networks. To identify the source of activity, we combined two-photon imaging with patch-clamp techniques to examine the physiological properties of morphologically identified retinal neurons in a mouse model of RD (rd1). Analysis of activity in rd1 ganglion cells revealed sustained oscillatory (∼10 Hz) synaptic activity in ∼30% of all classes of cells. Oscillatory activity persisted after putative inputs from residual photoreceptor, rod bipolar cell, and inhibitory amacrine cell synapses were pharmacologically blocked, suggesting that presynaptic cone bipolar cells were intrinsically active. Examination of presynaptic rd1 ON and OFF bipolar cells indicated that they rested at relatively negative potentials (less than -50 mV). However, in approximately half the cone bipolar cells, low-amplitude membrane oscillation (∼5 mV, ∼10 Hz) were apparent. Such oscillations were also observed in AII amacrine cells. Oscillations in ON cone bipolar and AII amacrine cells exhibited a weak apparent voltage dependence and were resistant to blockade of synaptic receptors, suggesting that, as in wild-type retina, they form an electrically coupled network. In addition, oscillations were insensitive to blockers of voltage-gated Ca(2+) channels (0.5 mm Cd(2+) and 0.5 mm Ni(2+)), ruling out known mechanisms that underlie oscillatory behavior in bipolar cells. Together, these results indicate that an electrically coupled network of ON cone bipolar/AII amacrine cells constitutes an intrinsic oscillator in the rd1 retina that is likely to drive synaptic activity in downstream circuits.

Vsx1 regulates terminal differentiation of type 7 ON bipolar cells.

Although retinal bipolar cells represent a morphologically well defined population of retinal interneurons, very little is known about the developmental mechanisms that regulate their processing. Furthermore, the identity of specific bipolar cell types that function in distinct visual circuits remains poorly understood. Here, we show that the homeobox gene Vsx1 is expressed in Type 7 ON bipolar cells. In the absence of Vsx1, Type 7 bipolar cells exhibit proper morphological specification but show defects in terminal gene expression. Vsx1 is required for the repression of bipolar cell-specific markers, including Calcium-binding protein 5 and Chx10. This contrasts its genetic requirement as an activator of gene expression in OFF bipolar cells. To assess possible ON signaling defects in Vsx1-null mice, we recorded specifically from ON-OFF directionally selective ganglion cells (DSGCs), which cofasciculate with Type 7 bipolar cell terminals. Vsx1-null ON-OFF DSGCs received more sustained excitatory synaptic input, possibly due to Type 7 bipolar cell defects. Interestingly, in Vsx1-null mice, the directionally selective circuit is functional but compromised. Together, these findings indicate that Vsx1 regulates terminal gene expression in Type 7 bipolar cells and is necessary for proper ON visual signaling within a directionally selective circuit.

Intrinsic oscillatory activity arising within the electrically coupled AII amacrine-ON cone bipolar cell network is driven by voltage-gated Na+ channels.

In the rd1 mouse model for retinal degeneration, the loss of photoreceptors results in oscillatory activity (∼10­20 Hz) within the remnant electrically coupled network of retinal ON cone bipolar and AII amacrine cells. We tested the role of hyperpolarization-activated currents (I(h)), voltage-gated Na(+) channels and gap junctions in mediating such oscillatory activity. Blocking I(h) (1 mm Cs(+)) hyperpolarized the network and augmented activity, while antagonizing voltage-dependent Na(+) channels (1 µm TTX) abolished oscillatory activity in the AII amacrine-ON cone bipolar cell network. Voltage-gated Na(+) channels were only observed in AII amacrine cells, implicating these cells as major drivers of activity. Pharmacologically uncoupling the network (200 µm meclofenamic acid (MFA)) blocked oscillations in all cells indicating that Na(+) channels exert their influence over multiple cell types within the network. In wt retina, occluding photoreceptor inputs to bipolar cells (10 µm NBQX and 50 µm l-AP4) resulted in a mild (∼10 mV) hyperpolarization and the induction of oscillatory activity within the AII amacrine-ON cone bipolar cell network. These oscillations had similar properties to those observed in rd1 retina, suggesting that no major degeneration-induced network rewiring is required to trigger spontaneous oscillations. Finally, we constructed a simplified computational model that exhibited Na(+) channel-dependent network oscillations. In this model, mild heterogeneities in channel densities between individual neurons reproduced our experimental findings. These results indicate that TTX-sensitive Na(+) channels in AII amacrine cells trigger degeneration-induced network oscillations, which provide a persistent synaptic drive to downstream remnant neurons, thus appearing to replace photoreceptors as the principal drivers of retinal activity.

Flexible cue anchoring strategies enable stable head direction coding in both sighted and blind animals.

Vision plays a crucial role in instructing the brain's spatial navigation systems. However, little is known about how vision loss affects the neuronal encoding of spatial information. Here, recording from head direction (HD) cells in the anterior dorsal nucleus of the thalamus in mice, we find stable and robust HD tuning in rd1 mice, a model of photoreceptor degeneration, that go blind by approximately one month of age. In contrast, placing sighted animals in darkness significantly impairs HD cell tuning. We find that blind mice use olfactory cues to maintain stable HD tuning and that prior visual experience leads to refined HD cell tuning in blind rd1 adult mice compared to congenitally blind animals. Finally, in the absence of both visual and olfactory cues, the HD attractor network remains intact but the preferred firing direction of HD cells drifts over time. These findings demonstrate flexibility in how the brain uses diverse sensory information to generate a stable directional representation of space.

The effect of aminosulfonate buffers on the light responses and intracellular pH of goldfish retinal horizontal cells.

Retinal horizontal cell feedback acts as a gain control at the first synapse in the visual system and generates center-surround receptive fields in the outer retina. One model of feedback proposes that elevation of protons in the photoreceptor synaptic cleft produces feedback. Most evidence supporting the proton model has depended on the effect of proton buffers, in particular aminosulfonates, but these agents could potentially have effects other than external pH regulation. We therefore determined if the effects of aminosulfonates on horizontal cell rollback, an indicator of feedback, were consistent with external proton buffering. Intracellular recording from horizontal cells in isolated goldfish retina revealed that rollback was blocked only by aminosulfonates with an acid dissociation constant suited for buffering at the pH (7.5) of the Ringer's solution. In isolated goldfish horizontal cells, aminosulfonates, even those that did not block rollback, altered intracellular pH. This suggests that the effect of aminosulfonates on rollback is not because of changing intracellular pH. Measures of both intracellular and extracellular pH revealed that treatment with either glutamate or kainate resulted in acidification. As glutamate produced both internal and external acidification, intracellular and extracellular horizontal cell pH would be expected to increase in response to light, a change consistent with the proton model of feedback.

Optogenetic Stimulation for Restoring Vision to Patients Suffering From Retinal Degenerative Diseases: Current Strategies and Future Directions.

Optogenetic strategies for vision restoration involve photosensitizing surviving retinal neurons following retinal degeneration, using emerging optogenetic techniques. This approach opens the door to a minimally-invasive retinal vision restoration approach. Moreover, light stimulation has the potential to offer better spatial and temporal resolution than conventional retinal electrical prosthetics. Although proof-of-concept studies in animal models have demonstrated the possibility of restoring vision using optogenetic techniques, and initial clinical trials are underway, there are still hurdles to pass before such an approach restores naturalistic vision in humans. One limitation is the development of light stimulation devices to activate optogenetic channels in the retina. Here we review recent progress in the design and implementation of optogenetic stimulation devices and outline the corresponding technological challenges. Finally, while most work to date has focused on providing therapy to patients suffering from retinitis pigmentosa, we provide additional insights into strategies for applying optogenetic vision restoration to patients suffering from age-related macular degeneration.

Magnetically guided virus stamping for the targeted infection of single cells or groups of cells.

To understand and control complex tissues, the ability to genetically manipulate single cells is required. However, current delivery methods for the genetic engineering of single cells, including viral transduction, suffer from limitations that restrict their application. Here we present a protocol that describes a versatile technique that can be used for the targeted viral infection of single cells or small groups of cells in any tissue that is optically accessible. First, cells of interest are selected using optical microscopy. Second, a micropipette-loaded with magnetic nanoparticles to which viral particles are bound-is brought into proximity of the cell of interest, and a magnetic field is applied to guide the viral nanoparticles into cellular contact, leading to transduction. The protocol, exemplified here by stamping cultured neurons with adeno-associated viruses (AAVs), is completed in a few minutes and allows stable transgene expression within a few days, at success rates that approach 80%. We outline how this strategy is applied to single-cell infection in complex tissues, and is feasible both in organoids and in vivo.

Whole-Brain Functional Ultrasound Imaging Reveals Brain Modules for Visuomotor Integration.

Large numbers of brain regions are active during behaviors. A high-resolution, brain-wide activity map could identify brain regions involved in specific behaviors. We have developed functional ultrasound imaging to record whole-brain activity in behaving mice at a resolution of ∼100 µm. We detected 87 active brain regions during visual stimulation that evoked the optokinetic reflex, a visuomotor behavior that stabilizes the gaze both horizontally and vertically. Using a genetic mouse model of congenital nystagmus incapable of generating the horizontal reflex, we identified a subset of regions whose activity was reflex dependent. By blocking eye motion in control animals, we further separated regions whose activity depended on the reflex's motor output. Remarkably, all reflex-dependent but eye motion-independent regions were located in the thalamus. Our work identifies functional modules of brain regions involved in sensorimotor integration and provides an experimental approach to monitor whole-brain activity of mice in normal and disease states.

Virus stamping for targeted single-cell infection in vitro and in vivo.

Genetic engineering by viral infection of single cells is useful to study complex systems such as the brain. However, available methods for infecting single cells have drawbacks that limit their applications. Here we describe 'virus stamping', in which viruses are reversibly bound to a delivery vehicle-a functionalized glass pipette tip or magnetic nanoparticles in a pipette-that is brought into physical contact with the target cell on a surface or in tissue, using mechanical or magnetic forces. Different single cells in the same tissue can be infected with different viruses and an individual cell can be simultaneously infected with different viruses. We use rabies, lenti, herpes simplex, and adeno-associated viruses to drive expression of fluorescent markers or a calcium indicator in target cells in cell culture, mouse retina, human brain organoid, and the brains of live mice. Virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types, both in vitro and in vivo.

Causal evidence for retina-dependent and -independent visual motion computations in mouse cortex.

How neuronal computations in the sensory periphery contribute to computations in the cortex is not well understood. We examined this question in the context of visual-motion processing in the retina and primary visual cortex (V1) of mice. We disrupted retinal direction selectivity, either exclusively along the horizontal axis using FRMD7 mutants or along all directions by ablating starburst amacrine cells, and monitored neuronal activity in layer 2/3 of V1 during stimulation with visual motion. In control mice, we found an over-representation of cortical cells preferring posterior visual motion, the dominant motion direction an animal experiences when it moves forward. In mice with disrupted retinal direction selectivity, the over-representation of posterior-motion-preferring cortical cells disappeared, and their responses at higher stimulus speeds were reduced. This work reveals the existence of two functionally distinct, sensory-periphery-dependent and -independent computations of visual motion in the cortex.

Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity.

Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease.

Origins of spontaneous activity in the degenerating retina.

Sensory deafferentation resulting from the loss of photoreceptors during retinal degeneration (rd) is often accompanied by a paradoxical increase in spontaneous activity throughout the visual system. Oscillatory discharges are apparent in retinal ganglion cells in several rodent models of rd, indicating that spontaneous activity can originate in the retina. Understanding the biophysical mechanisms underlying spontaneous retinal activity is interesting for two main reasons. First, it could lead to strategies that reduce spontaneous retinal activity, which could improve the performance of vision restoration strategies that aim to stimulate remnant retinal circuits in blind patients. Second, studying emergent network activity could offer general insights into how sensory systems remodel upon deafferentation. Here we provide an overview of the work describing spontaneous activity in the degenerating retina, and outline the current state of knowledge regarding the cellular and biophysical properties underlying spontaneous neural activity.

PRESYNAPTIC NETWORKS. Single-cell-initiated monosynaptic tracing reveals layer-specific cortical network modules.

Individual cortical neurons can selectively respond to specific environmental features, such as visual motion or faces. How this relates to the selectivity of the presynaptic network across cortical layers remains unclear. We used single-cell-initiated, monosynaptically restricted retrograde transsynaptic tracing with rabies viruses expressing GCaMP6s to image, in vivo, the visual motion-evoked activity of individual layer 2/3 pyramidal neurons and their presynaptic networks across layers in mouse primary visual cortex. Neurons within each layer exhibited similar motion direction preferences, forming layer-specific functional modules. In one-third of the networks, the layer modules were locked to the direction preference of the postsynaptic neuron, whereas for other networks the direction preference varied by layer. Thus, there exist feature-locked and feature-variant cortical networks.

Specific wiring of distinct amacrine cells in the directionally selective retinal circuit permits independent coding of direction and size.

Local and global forms of inhibition controlling directionally selective ganglion cells (DSGCs) in the mammalian retina are well documented. It is established that local inhibition arising from GABAergic starburst amacrine cells (SACs) strongly contributes to direction selectivity. Here, we demonstrate that increasing ambient illumination leads to the recruitment of GABAergic wide-field amacrine cells (WACs) endowing the DS circuit with an additional feature: size selectivity. Using a combination of electrophysiology, pharmacology, and light/electron microscopy, we show that WACs predominantly contact presynaptic bipolar cells, which drive direct excitation and feedforward inhibition (through SACs) to DSGCs, thus maintaining the appropriate balance of inhibition/excitation required for generating DS. This circuit arrangement permits high-fidelity direction coding over a range of ambient light levels, over which size selectivity is adjusted. Together, these results provide novel insights into the anatomical and functional arrangement of multiple inhibitory interneurons within a single computational module in the retina.

Cell-type-specific electric stimulation for vision restoration.

In this issue of Neuron, Jepson et al. (2014) demonstrate that electric stimulation of primate ON parasol ganglion cells evokes spiking patterns similar to those elicited by visual motion. This work represents progress in the development of cell-type-specific retinal prosthetics for vision restoration.

Nonlinear dendritic integration of electrical and chemical synaptic inputs drives fine-scale correlations.

Throughout the CNS, gap junction-mediated electrical signals synchronize neural activity on millisecond timescales via cooperative interactions with chemical synapses. However, gap junction-mediated synchrony has rarely been studied in the context of varying spatiotemporal patterns of electrical and chemical synaptic activity. Thus, the mechanism underlying fine-scale synchrony and its relationship to neural coding remain unclear. We examined spike synchrony in pairs of genetically identified, electrically coupled ganglion cells in mouse retina. We found that coincident electrical and chemical synaptic inputs, but not electrical inputs alone, elicited synchronized dendritic spikes in subregions of coupled dendritic trees. The resulting nonlinear integration produced fine-scale synchrony in the cells' spike output, specifically for light stimuli driving input to the regions of dendritic overlap. In addition, the strength of synchrony varied inversely with spike rate. Together, these features may allow synchronized activity to encode information about the spatial distribution of light that is ambiguous on the basis of spike rate alone.

Rods in daylight act as relay cells for cone-driven horizontal cell-mediated surround inhibition.

Vertebrate vision relies on two types of photoreceptors, rods and cones, which signal increments in light intensity with graded hyperpolarizations. Rods operate in the lower range of light intensities while cones operate at brighter intensities. The receptive fields of both photoreceptors exhibit antagonistic center-surround organization. Here we show that at bright light levels, mouse rods act as relay cells for cone-driven horizontal cell-mediated surround inhibition. In response to large, bright stimuli that activate their surrounds, rods depolarize. Rod depolarization increases with stimulus size, and its action spectrum matches that of cones. Rod responses at high light levels are abolished in mice with nonfunctional cones and when horizontal cells are reversibly inactivated. Rod depolarization is conveyed to the inner retina via postsynaptic circuit elements, namely the rod bipolar cells. Our results show that the retinal circuitry repurposes rods, when they are not directly sensing light, to relay cone-driven surround inhibition.

Lag normalization in an electrically coupled neural network.

Moving objects can cover large distances while they are processed by the eye, usually resulting in a spatially lagged retinal response. We identified a network of electrically coupled motion-coding neurons in mouse retina that act collectively to register the leading edges of moving objects at a nearly constant spatial location, regardless of their velocity. These results reveal a previously unknown neurophysiological substrate for lag normalization in the visual system.