The dynamics of pattern matching in camouflaging cuttlefish.

Many cephalopods escape detection using camouflage1. This behaviour relies on a visual assessment of the surroundings, on an interpretation of visual-texture statistics2-4 and on matching these statistics using millions of skin chromatophores that are controlled by motoneurons located in the brain5-7. Analysis of cuttlefish images proposed that camouflage patterns are low dimensional and categorizable into three pattern classes, built from a small repertoire of components8-11. Behavioural experiments also indicated that, although camouflage requires vision, its execution does not require feedback5,12,13, suggesting that motion within skin-pattern space is stereotyped and lacks the possibility of correction. Here, using quantitative methods14, we studied camouflage in the cuttlefish Sepia officinalis as behavioural motion towards background matching in skin-pattern space. An analysis of hundreds of thousands of images over natural and artificial backgrounds revealed that the space of skin patterns is high-dimensional and that pattern matching is not stereotyped-each search meanders through skin-pattern space, decelerating and accelerating repeatedly before stabilizing. Chromatophores could be grouped into pattern components on the basis of their covariation during camouflaging. These components varied in shapes and sizes, and overlay one another. However, their identities varied even across transitions between identical skin-pattern pairs, indicating flexibility of implementation and absence of stereotypy. Components could also be differentiated by their sensitivity to spatial frequency. Finally, we compared camouflage to blanching, a skin-lightening reaction to threatening stimuli. Pattern motion during blanching was direct and fast, consistent with open-loop motion in low-dimensional pattern space, in contrast to that observed during camouflage.

Molecular diversity and evolution of neuron types in the amniote brain.

The existence of evolutionarily conserved regions in the vertebrate brain is well established. The rules and constraints underlying the evolution of neuron types, however, remain poorly understood. To compare neuron types across brain regions and species, we generated a cell type atlas of the brain of a bearded dragon and compared it with mouse datasets. Conserved classes of neurons could be identified from the expression of hundreds of genes, including homeodomain-type transcription factors and genes involved in connectivity. Within these classes, however, there are both conserved and divergent neuron types, precluding a simple categorization of the brain into ancestral and novel areas. In the thalamus, neuronal diversification correlates with the evolution of the cortex, suggesting that developmental origin and circuit allocation are drivers of neuronal identity and evolution.

A thalamocortical top-down circuit for associative memory.

The sensory neocortex is a critical substrate for memory. Despite its strong connection with the thalamus, the role of direct thalamocortical communication in memory remains elusive. We performed chronic in vivo two-photon calcium imaging of thalamic synapses in mouse auditory cortex layer 1, a major locus of cortical associations. Combined with optogenetics, viral tracing, whole-cell recording, and computational modeling, we find that the higher-order thalamus is required for associative learning and transmits memory-related information that closely correlates with acquired behavioral relevance. In turn, these signals are tightly and dynamically controlled by local presynaptic inhibition. Our results not only identify the higher-order thalamus as a highly plastic source of cortical top-down information but also reveal a level of computational flexibility in layer 1 that goes far beyond hard-wired connectivity.

Progressive Characterization of Visual Phenotype in Bardet-Biedl Syndrome Mutant Mice.

Purpose: Bardet-Biedl syndrome (BBS) is an archetypical ciliopathy caused by defective ciliary trafficking and consequent function. Insights gained from BBS mouse models are applicable to other syndromic and nonsyndromic retinal diseases. This progressive characterization of the visual phenotype in three BBS mouse models sets a baseline for testing therapeutic interventions. Methods: Longitudinal acquisition of electroretinograms, optical coherence tomography scans, and visual acuity using the optomotor reflex in Bbs6/Mkks, Bbs8/Ttc8, and Bbs5 knockout mice. Gene and protein expression analysis in vivo and in vitro. Results: Complete loss of BBS5, BBS6, or BBS8 leads to different rates of retinal degeneration and visual function over time. BBS8-deficient mice showed the fastest rate of degeneration, and BBS8 seems to be required for cone photoreceptors to reach functional maturity. In contrast, the loss of BBS5 (a further BBSome component) showed very little degeneration. Loss of BBS8 versus BBS5 resulted in different physiologic responses both in vivo and in vitro. BBS6-deficient mice show a slower rate of degeneration with both rod and cone function reducing at a similar rate. Conclusions: The mouse models analyzed show distinct and diverging courses of degeneration upon loss of BBS5, BBS6, or BBS8, which can be used as a benchmark to test therapeutic interventions. Close consideration of the different phenotypes reveal subtle but important differences relating to their function. Because we also see differences in terms of phenotype depending on the type of visual assessment used, our data highlight the importance of using a combinatorial approach for assessment of visual function.

Elucidating the control and development of skin patterning in cuttlefish.

Few animals provide a readout that is as objective of their perceptual state as camouflaging cephalopods. Their skin display system includes an extensive array of pigment cells (chromatophores), each expandable by radial muscles controlled by motor neurons. If one could track the individual expansion states of the chromatophores, one would obtain a quantitative description-and potentially even a neural description by proxy-of the perceptual state of the animal in real time. Here we present the use of computational and analytical methods to achieve this in behaving animals, quantifying the states of tens of thousands of chromatophores at sixty frames per second, at single-cell resolution, and over weeks. We infer a statistical hierarchy of motor control, reveal an underlying low-dimensional structure to pattern dynamics and uncover rules that govern the development of skin patterns. This approach provides an objective description of complex perceptual behaviour, and a powerful means to uncover the organizational principles that underlie the function, dynamics and morphogenesis of neural systems.

Molecular codes for cell type specification in Brn3 retinal ganglion cells.

Visual information is conveyed from the eye to the brain by distinct types of retinal ganglion cells (RGCs). It is largely unknown how RGCs acquire their defining morphological and physiological features and connect to upstream and downstream synaptic partners. The three Brn3/Pou4f transcription factors (TFs) participate in a combinatorial code for RGC type specification, but their exact molecular roles are still unclear. We use deep sequencing to define (i) transcriptomes of Brn3a- and/or Brn3b-positive RGCs, (ii) Brn3a- and/or Brn3b-dependent RGC transcripts, and (iii) transcriptomes of retinorecipient areas of the brain at developmental stages relevant for axon guidance, dendrite formation, and synaptogenesis. We reveal a combinatorial code of TFs, cell surface molecules, and determinants of neuronal morphology that is differentially expressed in specific RGC populations and selectively regulated by Brn3a and/or Brn3b. This comprehensive molecular code provides a basis for understanding neuronal cell type specification in RGCs.

Comparison of optomotor and optokinetic reflexes in mice.

During animal locomotion or position adjustments, the visual system uses image stabilization reflexes to compensate for global shifts in the visual scene. These reflexes elicit compensatory head movements (optomotor response, OMR) in unrestrained animals or compensatory eye movements (optokinetic response, OKR) in head-fixed or unrestrained animals exposed to globally rotating striped patterns. In mice, OMR are relatively easy to observe and find broad use in the rapid evaluation of visual function. OKR determinations are more involved experimentally but yield more stereotypical, easily quantifiable results. The relative contributions of head and eye movements to image stabilization in mice have not been investigated. We are using newly developed software and apparatus to accurately quantitate mouse head movements during OMR, quantitate eye movements during OKR, and determine eye movements in freely behaving mice. We provide the first direct comparison of OMR and OKR gains (head or eye velocity/stimulus velocity) and find that the two reflexes have comparable dependencies on stimulus luminance, contrast, spatial frequency, and velocity. OMR and OKR are similarly affected in genetically modified mice with defects in retinal ganglion cells (RGC) compared with wild-type, suggesting they are driven by the same sensory input (RGC type). OKR eye movements have much higher gains than the OMR head movements, but neither can fully compensate global visual shifts. However, combined eye and head movements can be detected in unrestrained mice performing OMR, suggesting they can cooperate to achieve image stabilization, as previously described for other species.NEW & NOTEWORTHY We provide the first quantitation of head gain during optomotor response in mice and show that optomotor and optokinetic responses have similar psychometric curves. Head gains are far smaller than eye gains. Unrestrained mice combine head and eye movements to respond to visual stimuli, and both monocular and binocular fields are used during optokinetic responses. Mouse OMR and OKR movements are heterogeneous under optimal and suboptimal stimulation and are affected in mice lacking ON direction-selective retinal ganglion cells.

Tamoxifen Provides Structural and Functional Rescue in Murine Models of Photoreceptor Degeneration.

Photoreceptor degeneration is a cause of irreversible vision loss in incurable blinding retinal diseases including retinitis pigmentosa (RP) and atrophic age-related macular degeneration. We found in two separate mouse models of photoreceptor degeneration that tamoxifen, a selective estrogen receptor modulator and a drug previously linked with retinal toxicity, paradoxically provided potent neuroprotective effects. In a light-induced degeneration model, tamoxifen prevented onset of photoreceptor apoptosis and atrophy and maintained near-normal levels of electroretinographic responses. Rescue effects were correlated with decreased microglial activation and inflammatory cytokine production in the retina in vivo and a reduction of microglia-mediated toxicity to photoreceptors in vitro, indicating a microglia-mediated mechanism of rescue. Tamoxifen also rescued degeneration in a genetic (Pde6brd10) model of RP, significantly improving retinal structure, electrophysiological responses, and visual behavior. These prominent neuroprotective effects warrant the consideration of tamoxifen as a drug suitable for being repurposed to treat photoreceptor degenerative disease.SIGNIFICANCE STATEMENT Photoreceptor degeneration is a cause of irreversible blindness in a number of retinal diseases such as retinitis pigmentosa (RP) and atrophic age-related macular degeneration. Tamoxifen, a selective estrogen receptor modulator approved for the treatment of breast cancer and previously linked to a low incidence of retinal toxicity, was unexpectedly found to exert marked protective effects against photoreceptor degeneration. Structural and functional protective effects were found for an acute model of light-induced photoreceptor injury and for a genetic model for RP. The mechanism of protection involved the modulation of microglial activation and the production of inflammatory cytokines, highlighting the role of inflammatory mechanisms in photoreceptor degeneration. Tamoxifen may be suitable for clinical study as a potential treatment for diseases involving photoreceptor degeneration.

Loss of MACF1 Abolishes Ciliogenesis and Disrupts Apicobasal Polarity Establishment in the Retina.

Microtubule actin crosslinking factor 1 (MACF1) plays a role in the coordination of microtubules and actin in multiple cellular processes. Here, we show that MACF1 is also critical for ciliogenesis in multiple cell types. Ablation of Macf1 in the developing retina abolishes ciliogenesis, and basal bodies fail to dock to ciliary vesicles or migrate apically. Photoreceptor polarity is randomized, while inner retinal cells laminate correctly, suggesting that photoreceptor maturation is guided by polarity cues provided by cilia. Deletion of MACF1 in adult photoreceptors causes reversal of basal body docking and loss of outer segments, reflecting a continuous requirement for MACF1 function. MACF1 also interacts with the ciliary proteins MKKS and TALPID3. We propose that a disruption of trafficking across microtubles to actin filaments underlies the ciliogenesis defect in cells lacking MACF1 and that MKKS and TALPID3 are involved in the coordination of microtubule and actin interactions.

Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina.

Microglia, the principal resident immune cell of the CNS, exert significant influence on neurons during development and in pathological situations. However, if and how microglia contribute to normal neuronal function in the mature uninjured CNS is not well understood. We used the model of the adult mouse retina, a part of the CNS amenable to structural and functional analysis, to investigate the constitutive role of microglia by depleting microglia from the retina in a sustained manner using genetic methods. We discovered that microglia are not acutely required for the maintenance of adult retinal architecture, the survival of retinal neurons, or the laminar organization of their dendritic and axonal compartments. However, sustained microglial depletion results in the degeneration of photoreceptor synapses in the outer plexiform layer, leading to a progressive functional deterioration in retinal light responses. Our results demonstrate that microglia are constitutively required for the maintenance of synaptic structure in the adult retina and for synaptic transmission underlying normal visual function. Our findings on constitutive microglial function are relevant in understanding microglial contributions to pathology and in the consideration of therapeutic interventions that reduce or perturb constitutive microglial function. SIGNIFICANCE STATEMENT: Microglia, the principal resident immune cell population in the CNS, has been implicated in diseases in the brain and retina. However, how they contribute to the everyday function of the CNS is unclear. Using the model of the adult mouse retina, we examined the constitutive role of microglia by depleting microglia from the retina. We found that in the absence of microglia, retinal neurons did not undergo overt cell death or become structurally disorganized in their processes. However, connections between neurons called synapses begin to break down, leading to a decreased ability of the retina to transmit light responses. Our results indicate that retinal microglia contribute constitutively to the maintenance of synapses underlying healthy vision.

A system to measure the Optokinetic and Optomotor response in mice.

BACKGROUND: Visually evoked compensatory head movements (Optomotor responses) or eye movements (Optokinetic responses) are extensively used in experimental mouse models for developmental defects, pathological conditions, and testing the efficacy of therapeutic manipulations. NEW METHOD: We present an automated system to measure Optomotor and Optokinetic responses under identical stimulation conditions, enabling a direct comparison of the two reflexes. A semi-automated calibration procedure and a commercial eye tracker are used to record angular eye velocity in the restrained animal. Novel video tracking algorithms determine the location of the mouse head in real time and allow repositioning of the stimulus relative to the mouse head. RESULTS: Optomotor and Optokinetic responses yield comparable results with respect to determining visual acuity in mice. Our new head tracking algorithms enable a far more accurate analysis of head angle determination, and reveal individual head retractions, analogous to saccadic eye movements observed during Optokinetic Nystagmus. COMPARISON WITH EXISTING METHODS: To our knowledge this is the first apparatus allowing the direct comparison of Optomotor and Optokinetic responses in mice. Our tracking algorithms, which allow an objective determination of head movements are a significant increment over existing systems which rely on subjective human observation. The increased accuracy of the novel algorithms increases the robustness of automated Optomotor response determinations and reveals novel aspects of this reflex. CONCLUSIONS: We provide the blueprints for inexpensive hardware, and release open source software for our system, and describe an accurate and accessible method for Optomotor and Optokinetic response determination in mice.

OMR-arena: automated measurement and stimulation system to determine mouse visual thresholds based on optomotor responses.

Measurement of the optomotor response is a common way to determine thresholds of the visual system in animals. Particularly in mice, it is frequently used to characterize the visual performance of different genetically modified strains or to test the effect of various drugs on visual performance. Several methods have been developed to facilitate the presentation of stimuli using computer screens or projectors. Common methods are either based on the measurement of eye movement during optokinetic reflex behavior or rely on the measurement of head and/or body-movements during optomotor responses. Eye-movements can easily and objectively be quantified, but their measurement requires invasive fixation of the animals. Head movements can be observed in freely moving animals, but until now depended on the judgment of a human observer who reported the counted tracking movements of the animal during an experiment. In this study we present a novel measurement and stimulation system based on open source building plans and software. This system presents appropriate 360° stimuli while simultaneously video-tracking the animal's head-movements without fixation. The on-line determined head gaze is used to adjust the stimulus to the head position, as well as to automatically calculate visual acuity. Exemplary, we show that automatically measured visual response curves of mice match the results obtained by a human observer very well. The spatial acuity thresholds yielded by the automatic analysis are also consistent with the human observer approach and with published results. Hence, OMR-arena provides an affordable, convenient and objective way to measure mouse visual performance.

Genetic interactions between Brn3 transcription factors in retinal ganglion cell type specification.

BACKGROUND: Visual information is conveyed from the retina to the brain via 15-20 Retinal Ganglion Cell (RGC) types. The developmental mechanisms by which RGC types acquire their distinct molecular, morphological, physiological and circuit properties are essentially unknown, but may involve combinatorial transcriptional regulation. Brn3 transcription factors are expressed in RGCs from early developmental stages, and are restricted in adults to distinct, partially overlapping populations of RGC types. Previously, we described cell autonomous effects of Brn3b (Pou4f2) and Brn3a (Pou4f1) on RGC axon and dendrites development. METHODS AND FINDINGS: We now have investigated genetic interactions between Brn3 transcription factors with respect to RGC development, by crossing conventional knock-out alleles of each Brn3 gene with conditional knock-in reporter alleles of a second Brn3 gene, and analyzing the effects of single or double Brn3 knockouts on RGC survival and morphology. We find that Brn3b loss results in axon defects and dendritic arbor area and lamination defects in Brn3a positive RGCs, and selectively affects survival and morphology of specific Brn3c (Pou4f3) positive RGC types. Brn3a and Brn3b interact synergistically to control RGC numbers. Melanopsin positive ipRGCs are resistant to combined Brn3 loss but are under the transcriptional control of Isl1, expanding the combinatorial code of RGC specification. CONCLUSIONS: Taken together these results complete our knowledge on the mechanisms of transcriptional control of RGC type specification. They demonstrate that Brn3b is required for the correct development of more RGC cell types than suggested by its expression pattern in the adult, but that several cell types, including some Brn3a, Brn3c or Melanopsin positive RGCs are Brn3b independent.

High speed coding for velocity by archerfish retinal ganglion cells.

BACKGROUND: Archerfish show very short behavioural latencies in response to falling prey. This raises the question, which response parameters of retinal ganglion cells to moving stimuli are best suited for fast coding of stimulus speed and direction. RESULTS: We compared stimulus reconstruction quality based on the ganglion cell response parameters latency, first interspike interval, and rate. For stimulus reconstruction of moving stimuli using latency was superior to using the other stimulus parameters. This was true for absolute latency, with respect to stimulus onset, as well as for relative latency, with respect to population response onset. Iteratively increasing the number of cells used for reconstruction decreased the calculated error close to zero. CONCLUSIONS: Latency is the fastest response parameter available to the brain. Therefore, latency coding is best suited for high speed coding of moving objects. The quantitative data of this study are in good accordance with previously published behavioural response latencies.

A system for the model based emergency detection and communication for the telerehabilitation training of cardiopulmonary patients.

Cardiopulmonary diseases affect millions of people and cause high costs in health care systems worldwide. Patients should perform regular endurance exercises to stabilize their health state and prevent further impairment. However, patients are often uncertain about the level of intensity they should exercise in their current condition. The cost of continuous monitoring for these training sessions in clinics is high and additionally requires the patient to travel to a clinic for each single session. Performing the rehabilitation training at home can raise compliance and reduce costs. To ensure safe telerehabilitation training and to enable patients to control their performance and health state, detection of abnormal events during training is a critical prerequisite. Therefore, we created a model that predicts the heart rate of cardiopulmonary patients and that can be used to detect and avoid abnormal health states. To enable external feedback and an immediate reaction in case of a critical situation, the patient should have the possibility to configure the system to communicate warnings and emergency events to clinical and non-clinical actors. To fulfill this task, we coupled a personal health record (PHR) with a new component that extends the classic home emergency systems. The PHR is also used for a training schedule definition that makes use of the predictive HR model. We used statistical methods to evaluate the prediction model and found that our prediction error of 3.2 heart beats per minute is precise enough to enable a detection of critical states. The concept for the communication of alerts was evaluated through focus group interviews with domain experts who judged that it fulfills the needs of potential users.