Efficient coding of natural scenes improves neural system identification.

Neural system identification aims at learning the response function of neurons to arbitrary stimuli using experimentally recorded data, but typically does not leverage normative principles such as efficient coding of natural environments. Visual systems, however, have evolved to efficiently process input from the natural environment. Here, we present a normative network regularization for system identification models by incorporating, as a regularizer, the efficient coding hypothesis, which states that neural response properties of sensory representations are strongly shaped by the need to preserve most of the stimulus information with limited resources. Using this approach, we explored if a system identification model can be improved by sharing its convolutional filters with those of an autoencoder which aims to efficiently encode natural stimuli. To this end, we built a hybrid model to predict the responses of retinal neurons to noise stimuli. This approach did not only yield a higher performance than the "stand-alone" system identification model, it also produced more biologically plausible filters, meaning that they more closely resembled neural representation in early visual systems. We found these results applied to retinal responses to different artificial stimuli and across model architectures. Moreover, our normatively regularized model performed particularly well in predicting responses of direction-of-motion sensitive retinal neurons. The benefit of natural scene statistics became marginal, however, for predicting the responses to natural movies. In summary, our results indicate that efficiently encoding environmental inputs can improve system identification models, at least for noise stimuli, and point to the benefit of probing the visual system with naturalistic stimuli.

Spike desensitisation as a mechanism for high-contrast selectivity in retinal ganglion cells.

In the vertebrate retina, several dozens of parallel channels relay information about the visual world to the brain. These channels are represented by the different types of retinal ganglion cells (RGCs), whose responses are rendered selective for distinct sets of visual features by various mechanisms. These mechanisms can be roughly grouped into synaptic interactions and cell-intrinsic mechanisms, with the latter including dendritic morphology as well as ion channel complement and distribution. Here, we investigate how strongly ion channel complement can shape RGC output by comparing two mouse RGC types, the well-described ON alpha cell and a little-studied ON cell that is EGFP-labelled in the Igfbp5 mouse line and displays an unusual selectivity for stimuli with high contrast. Using patch-clamp recordings and computational modelling, we show that a higher activation threshold and a pronounced slow inactivation of the voltage-gated Na+ channels contribute to the distinct contrast tuning and transient responses in ON Igfbp5 RGCs, respectively. In contrast, such a mechanism could not be observed in ON alpha cells. This study provides an example for the powerful role that the last stage of retinal processing can play in shaping RGC responses.

Super-resolution STED imaging in the inner and outer whole-mount mouse retina.

Since its invention, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in deeper layers (30 - 50 µm) of biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the image acquisition settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. By measuring the full width at half maximum of the thinnest filamentous structures in our preparation, we achieved a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed visualisation of putative GABAergic horizontal cell synapses in the outer retina. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina at depths between 30 and 50 µm, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease.

Center-surround interactions underlie bipolar cell motion sensitivity in the mouse retina.

Motion sensing is a critical aspect of vision. We studied the representation of motion in mouse retinal bipolar cells and found that some bipolar cells are radially direction selective, preferring the origin of small object motion trajectories. Using a glutamate sensor, we directly observed bipolar cells synaptic output and found that there are radial direction selective and non-selective bipolar cell types, the majority being selective, and that radial direction selectivity relies on properties of the center-surround receptive field. We used these bipolar cell receptive fields along with connectomics to design biophysical models of downstream cells. The models and additional experiments demonstrated that bipolar cells pass radial direction selective excitation to starburst amacrine cells, which contributes to their directional tuning. As bipolar cells provide excitation to most amacrine and ganglion cells, their radial direction selectivity may contribute to motion processing throughout the visual system.

TTC30A and TTC30B Redundancy Protects IFT Complex B Integrity and Its Pivotal Role in Ciliogenesis.

Intraflagellar transport (IFT) is a microtubule-based system that supports the assembly and maintenance of cilia. The dysfunction of IFT leads to ciliopathies of variable severity. Two of the IFT-B components are the paralogue proteins TTC30A and TTC30B. To investigate whether these proteins constitute redundant functions, CRISPR/Cas9 was used to generate single TTC30A or B and double-knockout hTERT-RPE1 cells. Ciliogenesis assays showed the redundancy of both proteins while the polyglutamylation of cilia was affected in single knockouts. The localization of other IFT components was not affected by the depletion of a single paralogue. A loss of both proteins led to a severe ciliogenesis defect, resulting in no cilia formation, which was rescued by TTC30A or B. The redundancy can be explained by the highly similar interaction patterns of the paralogues; both equally interact with the IFT-B machinery. Our study demonstrates that a loss of one TTC30 paralogue can mostly be compensated by the other, thus preventing severe ciliary defects. However, cells assemble shorter cilia, which are potentially limited in their function, especially because of impaired polyglutamylation. A complete loss of both proteins leads to a deficit in IFT complex B integrity followed by disrupted IFT and subsequently no cilia formation.

Suppression without inhibition: how retinal computation contributes to saccadic suppression.

Visual perception remains stable across saccadic eye movements, despite the concurrent strongly disruptive visual flow. This stability is partially associated with a reduction in visual sensitivity, known as saccadic suppression, which already starts in the retina with reduced ganglion cell sensitivity. However, the retinal circuit mechanisms giving rise to such suppression remain unknown. Here, we describe these mechanisms using electrophysiology in mouse, pig, and macaque retina, 2-photon calcium imaging, computational modeling, and human psychophysics. We find that sequential stimuli, like those that naturally occur during saccades, trigger three independent suppressive mechanisms in the retina. The main mechanism is triggered by contrast-reversing sequential stimuli and originates within the receptive field center of ganglion cells. It does not involve inhibition or other known suppressive mechanisms like saturation or adaptation. Instead, it relies on temporal filtering of the inherently slow response of cone photoreceptors coupled with downstream nonlinearities. Two further mechanisms of suppression are present predominantly in ON ganglion cells and originate in the receptive field surround, highlighting another disparity between ON and OFF ganglion cells. The mechanisms uncovered here likely play a role in shaping the retinal output following eye movements and other natural viewing conditions where sequential stimulation is ubiquitous.

Minimal genetically encoded tags for fluorescent protein labeling in living neurons.

Modern light microscopy, including super-resolution techniques, has brought about a demand for small labeling tags that bring the fluorophore closer to the target. This challenge can be addressed by labeling unnatural amino acids (UAAs) with bioorthogonal click chemistry. The minimal size of the UAA and the possibility to couple the fluorophores directly to the protein of interest with single-residue precision in living cells make click labeling unique. Here, we establish click labeling in living primary neurons and use it for fixed-cell, live-cell, dual-color pulse-chase, and super-resolution microscopy of neurofilament light chain (NFL). We also show that click labeling can be combined with CRISPR/Cas9 genome engineering for tagging endogenous NFL. Due to its versatile nature and compatibility with advanced multicolor microscopy techniques, we anticipate that click labeling will contribute to novel discoveries in the neurobiology field.

Retinal horizontal cells use different synaptic sites for global feedforward and local feedback signaling.

In the outer plexiform layer (OPL) of the mammalian retina, cone photoreceptors (cones) provide input to more than a dozen types of cone bipolar cells (CBCs). In the mouse, this transmission is modulated by a single horizontal cell (HC) type. HCs perform global signaling within their laterally coupled network but also provide local, cone-specific feedback. However, it is unknown how HCs provide local feedback to cones at the same time as global forward signaling to CBCs and where the underlying synapses are located. To assess how HCs simultaneously perform different modes of signaling, we reconstructed the dendritic trees of five HCs as well as cone axon terminals and CBC dendrites in a serial block-face electron microscopy volume and analyzed their connectivity. In addition to the fine HC dendritic tips invaginating cone axon terminals, we also identified "bulbs," short segments of increased dendritic diameter on the primary dendrites of HCs. These bulbs are in an OPL stratum well below the cone axon terminal base and make contacts with other HCs and CBCs. Our results from immunolabeling, electron microscopy, and glutamate imaging suggest that HC bulbs represent GABAergic synapses that do not receive any direct photoreceptor input. Together, our data suggest the existence of two synaptic strata in the mouse OPL, spatially separating cone-specific feedback and feedforward signaling to CBCs. A biophysical model of a HC dendritic branch and voltage imaging support the hypothesis that this spatial arrangement of synaptic contacts allows for simultaneous local feedback and global feedforward signaling by HCs.

Neural circuits in the mouse retina support color vision in the upper visual field.

Color vision is essential for an animal's survival. It starts in the retina, where signals from different photoreceptor types are locally compared by neural circuits. Mice, like most mammals, are dichromatic with two cone types. They can discriminate colors only in their upper visual field. In the corresponding ventral retina, however, most cones display the same spectral preference, thereby presumably impairing spectral comparisons. In this study, we systematically investigated the retinal circuits underlying mouse color vision by recording light responses from cones, bipolar and ganglion cells. Surprisingly, most color-opponent cells are located in the ventral retina, with rod photoreceptors likely being involved. Here, the complexity of chromatic processing increases from cones towards the retinal output, where non-linear center-surround interactions create specific color-opponent output channels to the brain. This suggests that neural circuits in the mouse retina are tuned to extract color from the upper visual field, aiding robust detection of predators and ensuring the animal's survival.

Type-specific dendritic integration in mouse retinal ganglion cells.

Neural computation relies on the integration of synaptic inputs across a neuron's dendritic arbour. However, it is far from understood how different cell types tune this process to establish cell-type specific computations. Here, using two-photon imaging of dendritic Ca2+ signals, electrical recordings of somatic voltage and biophysical modelling, we demonstrate that four morphologically distinct types of mouse retinal ganglion cells with overlapping excitatory synaptic input (transient Off alpha, transient Off mini, sustained Off, and F-mini Off) exhibit type-specific dendritic integration profiles: in contrast to the other types, dendrites of transient Off alpha cells were spatially independent, with little receptive field overlap. The temporal correlation of dendritic signals varied also extensively, with the highest and lowest correlation in transient Off mini and transient Off alpha cells, respectively. We show that differences between cell types can likely be explained by differences in backpropagation efficiency, arising from the specific combinations of dendritic morphology and ion channel densities.

The temporal structure of the inner retina at a single glance.

The retina decomposes visual stimuli into parallel channels that encode different features of the visual environment. Central to this computation is the synaptic processing in a dense layer of neuropil, the so-called inner plexiform layer (IPL). Here, different types of bipolar cells stratifying at distinct depths relay the excitatory feedforward drive from photoreceptors to amacrine and ganglion cells. Current experimental techniques for studying processing in the IPL do not allow imaging the entire IPL simultaneously in the intact tissue. Here, we extend a two-photon microscope with an electrically tunable lens allowing us to obtain optical vertical slices of the IPL, which provide a complete picture of the response diversity of bipolar cells at a "single glance". The nature of these axial recordings additionally allowed us to isolate and investigate batch effects, i.e. inter-experimental variations resulting in systematic differences in response speed. As a proof of principle, we developed a simple model that disentangles biological from experimental causes of variability and allowed us to recover the characteristic gradient of response speeds across the IPL with higher precision than before. Our new framework will make it possible to study the computations performed in the central synaptic layer of the retina more efficiently.

Systematic spatiotemporal mapping reveals divergent cell death pathways in three mouse models of hereditary retinal degeneration.

Calcium (Ca2+ ) dysregulation has been linked to neuronal cell death, including in hereditary retinal degeneration. Ca2+ dysregulation is thought to cause rod and cone photoreceptor cell death. Spatial and temporal heterogeneities in retinal disease models have hampered validation of this hypothesis. We examined the role of Ca2+ in photoreceptor degeneration, assessing the activation pattern of Ca2+ -dependent calpain proteases, generating spatiotemporal maps of the entire retina in the cpfl1 mouse model for primary cone degeneration, and in the rd1 and rd10 models for primary rod degeneration. We used Gaussian process models to distinguish the temporal sequences of degenerative molecular processes from other variability sources.In the rd1 and rd10 models, spatiotemporal pattern of increased calpain activity matched the progression of primary rod degeneration. High calpain activity coincided with activation of the calpain-2 isoform but not with calpain-1, suggesting differential roles for both calpain isoforms. Primary rod loss was linked to upregulation of apoptosis-inducing factor, although only a minute fraction of cells showed activity of the apoptotic marker caspase-3. After primary rod degeneration concluded, caspase-3 activation appeared in cones, suggesting apoptosis as the dominant mechanism for secondary cone loss. Gaussian process models highlighted calpain activity as a key event during primary rod photoreceptor cell death. Our data suggest a causal link between Ca2+ dysregulation and primary, nonapoptotic degeneration of photoreceptors and a role for apoptosis in secondary degeneration of cones, highlighting the importance of the spatial and temporal location of key molecular events, which may guide the evaluation of new therapies.

A retinal model of cerebral malaria.

Malaria is a causative factor in about 500.000 deaths each year world-wide. Cerebral malaria is a particularly severe complication of this disease and thus associated with an exceedingly high mortality. Malaria retinopathy is an ocular manifestation often associated with cerebral malaria, and presumably shares a substantial part of its pathophysiology. Here, we describe that indeed murine malaria retinopathy reproduced the main hallmarks of the corresponding human disease. In the living animal, we were able to follow the circulation and cellular localization of malaria parasites transgenically labelled with GFP via non-invasive in vivo retinal imaging. We found that malaria parasites cross the blood-retinal-barrier and infiltrate the neuroretina, concomitant with an extensive, irreversible, and long-lasting retinal neurodegeneration. Furthermore, anti-malarial treatment with dihydroartemisinin strongly diminished the load of circulating parasites but resolved the symptoms of the retinopathy only in part. In summary, we introduce here a novel preclinical model for human cerebral malaria that is much more directly accessible for studies into disease pathophysiology and development of novel treatment approaches. In vivo retinal imaging may furthermore serve as a valuable tool for the early diagnosis of the human disease.

Neural Circuits: When Neurons 'Remember' Their Connectivity.

Loss of neurons due to injury or neurodegeneration can lead to dramatically altered neural circuits, resulting in reduced or lost function. One mechanism to preserve function could be to re-establish the stereotypic connectivity among the remnant neurons. In the mammalian retina, such a selective re-wiring has now been described.

Local Signals in Mouse Horizontal Cell Dendrites.

The mouse retina contains a single type of horizontal cell, a GABAergic interneuron that samples from all cone photoreceptors within reach and modulates their glutamatergic output via parallel feedback mechanisms. Because horizontal cells form an electrically coupled network, they have been implicated in global signal processing, such as large-scale contrast enhancement. Recently, it has been proposed that horizontal cells can also act locally at the level of individual cone photoreceptors. To test this possibility physiologically, we used two-photon microscopy to record light stimulus-evoked Ca2+ signals in cone axon terminals and horizontal cell dendrites as well as glutamate release in the outer plexiform layer. By selectively stimulating the two mouse cone opsins with green and UV light, we assessed whether signals from individual cones remain isolated within horizontal cell dendritic tips or whether they spread across the dendritic arbor. Consistent with the mouse's opsin expression gradient, we found that the Ca2+ signals recorded from dendrites of dorsal horizontal cells were dominated by M-opsin and those of ventral horizontal cells by S-opsin activation. The signals measured in neighboring horizontal cell dendritic tips varied markedly in their chromatic preference, arguing against global processing. Rather, our experimental data and results from biophysically realistic modeling support the idea that horizontal cells can process cone input locally, extending the classical view of horizontal cell function. Pharmacologically removing horizontal cells from the circuitry reduced the sensitivity of the cone signal to low frequencies, suggesting that local horizontal cell feedback shapes the temporal properties of cone output.

How do horizontal cells 'talk' to cone photoreceptors? Different levels of complexity at the cone-horizontal cell synapse.

The first synapse of the retina plays a fundamental role in the visual system. Due to its importance, it is critical that it encodes information from the outside world with the greatest accuracy and precision possible. Cone photoreceptor axon terminals contain many individual synaptic sites, each represented by a presynaptic structure called a 'ribbon'. These synapses are both highly sophisticated and conserved. Each ribbon relays the light signal to one ON cone bipolar cell and several OFF cone bipolar cells, while two dendritic processes from a GABAergic interneuron, the horizontal cell, modulate the cone output via parallel feedback mechanisms. The presence of these three partners within a single synapse has raised numerous questions, and its anatomical and functional complexity is still only partially understood. However, the understanding of this synapse has recently evolved, as a consequence of progress in understanding dendritic signal processing and its role in facilitating global versus local signalling. Indeed, for the downstream retinal network, dendritic processing in horizontal cells may be essential, as they must support important functional operations such as contrast enhancement, which requires spatial averaging of the photoreceptor array, while at the same time preserving accurate spatial information. Here, we review recent progress made towards a better understanding of the cone synapse, with an emphasis on horizontal cell function, and discuss why such complexity might be necessary for early visual processing.

Connectomics of synaptic microcircuits: lessons from the outer retina.

Photoreceptors form a sophisticated synaptic complex with bipolar and horizontal cells, transmitting the signals generated by the phototransduction cascade to downstream retinal circuitry. The cone photoreceptor synapse shows several characteristic anatomical connectivity motifs that shape signal transfer: typically, ON-cone bipolar cells receive photoreceptor input through invaginating synapses; OFF-cone bipolar cells form basal synapses with photoreceptors. Both ON- and OFF-cone bipolar cells are believed to sample from all cone photoreceptors within their dendritic span. Electron microscopy and immunolabelling studies have established the robustness of these motifs, but have been limited by trade-offs in sample size and spatial resolution, respectively, constraining precise quantitative investigation to a few individual cells. 3D-serial electron microscopy overcomes these limitations and has permitted complete sets of neurons to be reconstructed over a comparatively large section of retinal tissue. Although the published mouse dataset lacks labels for synaptic structures, the characteristic anatomical motifs at the photoreceptor synapse can be exploited to identify putative synaptic contacts, which has enabled the development of a quantitative description of outer retinal connectivity. This revealed unexpected exceptions to classical motifs, including substantial interaction between rod and cone pathways at the photoreceptor synapse, sparse photoreceptor sampling and atypical contacts. Here, we summarize what was learned from this study in a more general context: we consider both the implications and limitations of the study and identify promising avenues for future research.

Inhibition decorrelates visual feature representations in the inner retina.

The retina extracts visual features for transmission to the brain. Different types of bipolar cell split the photoreceptor input into parallel channels and provide the excitatory drive for downstream visual circuits. Mouse bipolar cell types have been described at great anatomical and genetic detail, but a similarly deep understanding of their functional diversity is lacking. Here, by imaging light-driven glutamate release from more than 13,000 bipolar cell axon terminals in the intact retina, we show that bipolar cell functional diversity is generated by the interplay of dendritic excitatory inputs and axonal inhibitory inputs. The resulting centre and surround components of bipolar cell receptive fields interact to decorrelate bipolar cell output in the spatial and temporal domains. Our findings highlight the importance of inhibitory circuits in generating functionally diverse excitatory pathways and suggest that decorrelation of parallel visual pathways begins as early as the second synapse of the mouse visual system.

Connectivity map of bipolar cells and photoreceptors in the mouse retina.

In the mouse retina, three different types of photoreceptors provide input to 14 bipolar cell (BC) types. Classically, most BC types are thought to contact all cones within their dendritic field; ON-BCs would contact cones exclusively via so-called invaginating synapses, while OFF-BCs would form basal synapses. By mining publically available electron microscopy data, we discovered interesting violations of these rules of outer retinal connectivity: ON-BC type X contacted only ~20% of the cones in its dendritic field and made mostly atypical non-invaginating contacts. Types 5T, 5O and 8 also contacted fewer cones than expected. In addition, we found that rod BCs received input from cones, providing anatomical evidence that rod and cone pathways are interconnected in both directions. This suggests that the organization of the outer plexiform layer is more complex than classically thought.

Calcium dynamics change in degenerating cone photoreceptors.

Cone photoreceptors (cones) are essential for high-resolution daylight vision and colour perception. Loss of cones in hereditary retinal diseases has a dramatic impact on human vision. The mechanisms underlying cone death are poorly understood, and consequently, there are no treatments available. Previous studies suggest a central role for calcium (Ca2+) homeostasis deficits in photoreceptor degeneration; however, direct evidence for this is scarce and physiological measurements of Ca2+ in degenerating mammalian cones are lacking.Here, we took advantage of the transgenic HR2.1:TN-XL mouse line that expresses a genetically encoded Ca2+ biosensor exclusively in cones. We cross-bred this line with mouse models for primary ("cone photoreceptor function loss-1", cpfl1) and secondary ("retinal degeneration-1", rd1) cone degeneration, respectively, and assessed resting Ca2+ levels and light-evoked Ca2+ responses in cones using two-photon imaging. We found that Ca2+ dynamics were altered in cpfl1 cones, showing higher noise and variable Ca2+ levels, with significantly wider distribution than for wild-type and rd1 cones. Unexpectedly, up to 21% of cpfl1 cones still displayed light-evoked Ca2+ responses, which were larger and slower than wild-type responses. In contrast, genetically intact rd1 cones were characterized by lower noise and complete lack of visual function.Our study demonstrates alterations in cone Ca2+ dynamics in both primary and secondary cone degeneration. Our results are consistent with the view that higher (fluctuating) cone Ca2+ levels are involved in photoreceptor cell death in primary (cpfl1) but not in secondary (rd1) cone degeneration. These findings may guide the future development of therapies targeting photoreceptor Ca2+ homeostasis.