Multi-layered maps of neuropil with segmentation-guided contrastive learning.

Maps of the nervous system that identify individual cells along with their type, subcellular components and connectivity have the potential to elucidate fundamental organizational principles of neural circuits. Nanometer-resolution imaging of brain tissue provides the necessary raw data, but inferring cellular and subcellular annotation layers is challenging. We present segmentation-guided contrastive learning of representations (SegCLR), a self-supervised machine learning technique that produces representations of cells directly from 3D imagery and segmentations. When applied to volumes of human and mouse cortex, SegCLR enables accurate classification of cellular subcompartments and achieves performance equivalent to a supervised approach while requiring 400-fold fewer labeled examples. SegCLR also enables inference of cell types from fragments as small as 10 µm, which enhances the utility of volumes in which many neurites are truncated at boundaries. Finally, SegCLR enables exploration of layer 5 pyramidal cell subtypes and automated large-scale analysis of synaptic partners in mouse visual cortex.

High-precision automated reconstruction of neurons with flood-filling networks.

Reconstruction of neural circuits from volume electron microscopy data requires the tracing of cells in their entirety, including all their neurites. Automated approaches have been developed for tracing, but their error rates are too high to generate reliable circuit diagrams without extensive human proofreading. We present flood-filling networks, a method for automated segmentation that, similar to most previous efforts, uses convolutional neural networks, but contains in addition a recurrent pathway that allows the iterative optimization and extension of individual neuronal processes. We used flood-filling networks to trace neurons in a dataset obtained by serial block-face electron microscopy of a zebra finch brain. Using our method, we achieved a mean error-free neurite path length of 1.1 mm, and we observed only four mergers in a test set with a path length of 97 mm. The performance of flood-filling networks was an order of magnitude better than that of previous approaches applied to this dataset, although with substantially increased computational costs.

Anatomical identification of extracellularly recorded cells in large-scale multielectrode recordings.

This study combines for the first time two major approaches to understanding the function and structure of neural circuits: large-scale multielectrode recordings, and confocal imaging of labeled neurons. To achieve this end, we develop a novel approach to the central problem of anatomically identifying recorded cells, based on the electrical image: the spatiotemporal pattern of voltage deflections induced by spikes on a large-scale, high-density multielectrode array. Recordings were performed from identified ganglion cell types in the macaque retina. Anatomical images of cells in the same preparation were obtained using virally transfected fluorescent labeling or by immunolabeling after fixation. The electrical image was then used to locate recorded cell somas, axon initial segments, and axon trajectories, and these signatures were used to identify recorded cells. Comparison of anatomical and physiological measurements permitted visualization and physiological characterization of numerically dominant ganglion cell types with high efficiency in a single preparation.

A polyaxonal amacrine cell population in the primate retina.

Amacrine cells are the most diverse and least understood cell class in the retina. Polyaxonal amacrine cells (PACs) are a unique subset identified by multiple long axonal processes. To explore their functional properties, populations of PACs were identified by their distinctive radially propagating spikes in large-scale high-density multielectrode recordings of isolated macaque retina. One group of PACs exhibited stereotyped functional properties and receptive field mosaic organization similar to that of parasol ganglion cells. These PACs had receptive fields coincident with their dendritic fields, but much larger axonal fields, and slow radial spike propagation. They also exhibited ON-OFF light responses, transient response kinetics, sparse and coordinated firing during image transitions, receptive fields with antagonistic surrounds and fine spatial structure, nonlinear spatial summation, and strong homotypic neighbor electrical coupling. These findings reveal the functional organization and collective visual signaling by a distinctive, high-density amacrine cell population.

A cross-sectional study of optometrists' attitudes towards dry eye disease management in Hong Kong: A web-based survey in Hong Kong.

Purpose: This study aimed to explore the practices of optometrists in Hong Kong towards diagnosing and managing dry eye disease (DED). Methods: From September 2021 to March 2022, an online questionnaire was distributed to optometrists in Hong Kong through several professional associations. The questionnaire included questions about the importance and usefulness of various diagnostic tests, as well as inquiries about management strategies and recommended follow-up schedules for DED. Responses were compared between optometrists who were more or less proactive in continuing education to identify potential differences. Results: The analysis included 68 valid responses. Sixty-one of them were Part 1 optometrists that represents 5.5 % of registered Part 1 optometrists back in 2022. Assessment of clinical symptoms was the most commonly performed investigation (93 %) and considered the most important (75 %) procedure in DED assessments, followed by corneal staining and fluorescein tear break-up time. Traditional diagnostic tests were preferred over newer methods, such as osmolarity, which were not yet commonly used. Unpreserved lubricants (90 %) and lid hygiene (63 %) were the primary treatments recommended for mild DED. Optometrists who had more experience and frequent participation in continuing education were more confident in diagnosing and managing DED, and more likely to recommend omega-3 supplements for moderate DED. Conclusion: The diagnostic and management strategies of optometrists in Hong Kong were generally consistent with the recommendations of the Dry Eye Workshop II report. However, standardized DED questionnaires and newer diagnostic tools were not commonly used. Evidence-based optometric care for dry eye management should be encouraged in Hong Kong optometric practice.

Mapping of neuronal and glial primary cilia contactome and connectome in the human cerebral cortex.

Primary cilia act as antenna receivers of environmental signals and enable effective neuronal or glial responses. Disruption of their function is associated with circuit disorders. To understand the signals these cilia receive, we comprehensively mapped cilia's contacts within the human cortical connectome using serial-section EM reconstruction of a 1 mm3 cortical volume, spanning the entire cortical thickness. We mapped the "contactome" of cilia emerging from neurons and astrocytes in every cortical layer. Depending on the layer and cell type, cilia make distinct patterns of contact. Primary cilia display cell-type- and layer-specific variations in size, shape, and microtubule axoneme core, which may affect their signaling competencies. Neuronal cilia are intrinsic components of a subset of cortical synapses and thus a part of the connectome. This diversity in the structure, contactome, and connectome of primary cilia endows each neuron or glial cell with a unique barcode of access to the surrounding neural circuitry.

A petavoxel fragment of human cerebral cortex reconstructed at nanoscale resolution.

To fully understand how the human brain works, knowledge of its structure at high resolution is needed. Presented here is a computationally intensive reconstruction of the ultrastructure of a cubic millimeter of human temporal cortex that was surgically removed to gain access to an underlying epileptic focus. It contains about 57,000 cells, about 230 millimeters of blood vessels, and about 150 million synapses and comprises 1.4 petabytes. Our analysis showed that glia outnumber neurons 2:1, oligodendrocytes were the most common cell, deep layer excitatory neurons could be classified on the basis of dendritic orientation, and among thousands of weak connections to each neuron, there exist rare powerful axonal inputs of up to 50 synapses. Further studies using this resource may bring valuable insights into the mysteries of the human brain.

Gap-junctional coupling of mammalian rod photoreceptors and its effect on visual detection.

The presence of gap junctions between rods in mammalian retina suggests a role for rod-rod coupling in human vision. Rod coupling is known to reduce response variability, but because junctional conductances are not known, the downstream effects on visual performance are uncertain. Here we assessed rod coupling in guinea pig retina by measuring: (1) the variability in responses to dim flashes, (2) Neurobiotin tracer coupling, and (3) junctional conductances. Results were consolidated into an electrical network model and a model of human psychophysical detection. Guinea pig rods form tracer pools of 1 to ∼20 rods, with junctional conductances averaging ∼350 pS. We calculate that coupling will reduce human dark-adapted sensitivity ∼10% by impairing the noise filtering of the synapse between rods and rod bipolar cells. However, coupling also mitigates synaptic saturation and is thus calculated to improve sensitivity when stimuli are spatially restricted or are superimposed over background illumination.

Myopia Control Efficacy and Long-Term Safety of a Novel Orthokeratology Lens (MESOK Study)-A Randomized Controlled Clinical Trial Combining Clinical and Tear Proteomics Data.

Myopia control efficacy and long-term safety of the Breath-O-Correct orthokeratology (OK) lens was evaluated in a 2-year randomized, single vision (SV) spectacle lens-controlled, single-blind clinical trial combining clinical and tear proteomics data. A total of 71 children (43 OK, 9.8 ± 1.3 years; 28 SV, 9.5 ± 1.4 years) completed the 2-year study. Axial length (AL), cycloplegic refraction, clinical safety parameters (best-corrected visual acuity, central cornea thickness, corneal endothelial health, ocular surface disease index), and quantitative tear proteomics were evaluated by masked examiners. Mean 2-year-normalized AL elongations in the OK and SV groups differed significantly (p = 0.03) and were 0.37 ± 0.37 mm and 0.60 ± 0.41 mm, respectively. OK-mediated myopia control efficacy was 37.1%. No significant difference was found in clinical safety parameters of both groups (p > 0.10), except for a thinner central corneal thickness in the OK group (p = 0.01). Proteomics revealed modest OK lens-mediated effects on immune response proteins, including an increased abundance of haptoglobin at 6 and 12 months and a decreased abundance of two proteins (neutrophil defensin 3 and histone 4) at 6 months. The changes were further validated using a high-resolution multiple-reaction monitoring (MRMHR) mass spectrometry. In summary, the Breath-O-Correct OK lens significantly reduced AL elongation in schoolchildren without adverse clinical effects or subclinical inflammatory responses.

Probing Molecular Diversity and Ultrastructure of Brain Cells with Fluorescent Aptamers.

Detergent-free immunolabeling has been proven feasible for correlated light and electron microscopy, but its application is restricted by the availability of suitable affinity reagents. Here we introduce CAptVE, a method using slow off-rate modified aptamers for cell fluorescence labeling on ultrastructurally reconstructable electron micrographs. CAptVE provides labeling for a wide range of biomarkers, offering a pathway to integrate molecular analysis into recent approaches to delineate neural circuits via connectomics.

Multiplexed volumetric CLEM enabled by antibody derivatives provides new insights into the cytology of the mouse cerebellar cortex.

Mapping neuronal networks that underlie behavior has become a central focus in neuroscience. While serial section electron microscopy (ssEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide the molecular information that helps identify cell types or their functional properties. Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and ssEM on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (green fluorescent protein, glial fibrillary acidic protein, calbindin, parvalbumin, voltage-gated potassium channel subfamily A member 2, vesicular glutamate transporter 1, postsynaptic density protein 95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with spectral unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure with superimposition of the multiple fluorescence channels. Using this approach we could document a poorly described cell type in the cerebellum, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.

A large-scale volumetric correlated light and electron microscopy study localizes Alzheimer's disease-related molecules in the hippocampus.

Connectomics is a nascent neuroscience field to map and analyze neuronal networks. It provides a new way to investigate abnormalities in brain tissue, including in models of Alzheimer's disease (AD). This age-related disease is associated with alterations in amyloid-ß (Aß) and phosphorylated tau (pTau). These alterations correlate with AD's clinical manifestations, but causal links remain unclear. Therefore, studying these molecular alterations within the context of the local neuronal and glial milieu may provide insight into disease mechanisms. Volume electron microscopy (vEM) is an ideal tool for performing connectomics studies at the ultrastructural level, but localizing specific biomolecules within large-volume vEM data has been challenging. Here we report a volumetric correlated light and electron microscopy (vCLEM) approach using fluorescent nanobodies as immuno-probes to localize Alzheimer's disease-related molecules in a large vEM volume. Three molecules (pTau, Aß, and a marker for activated microglia (CD11b)) were labeled without the need for detergents by three nanobody probes in a sample of the hippocampus of the 3xTg Alzheimer's disease model mouse. Confocal microscopy followed by vEM imaging of the same sample allowed for registration of the location of the molecules within the volume. This dataset revealed several ultrastructural abnormalities regarding the localizations of Aß and pTau in novel locations. For example, two pTau-positive post-synaptic spine-like protrusions innervated by axon terminals were found projecting from the axon initial segment of a pyramidal cell. Three pyramidal neurons with intracellular Aß or pTau were 3D reconstructed. Automatic synapse detection, which is necessary for connectomics analysis, revealed the changes in density and volume of synapses at different distances from an Aß plaque. This vCLEM approach is useful to uncover molecular alterations within large-scale volume electron microscopy data, opening a new connectomics pathway to study Alzheimer's disease and other types of dementia.

Multiplexed volumetric CLEM enabled by antibody derivatives provides new insights into the cytology of the mouse cerebellar cortex.

Mapping neuronal networks that underlie behavior has become a central focus in neuroscience. While serial section electron microscopy (ssEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide the molecular information that helps identify cell types or their functional properties. Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and ssEM on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (green fluorescent protein, glial fibrillary acidic protein, calbindin, parvalbumin, voltage-gated potassium channel subfamily A member 2, vesicular glutamate transporter 1, postsynaptic density protein 95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with spectral unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure with superimposition of the multiple fluorescence channels. Using this approach we could document a poorly described cell type in the cerebellum, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.

Decomposition of retinal ganglion cell electrical images for cell type and functional inference.

Identifying neuronal cell types and their biophysical properties based on their extracellular electrical features is a major challenge for experimental neuroscience and the development of high-resolution brain-machine interfaces. One example is identification of retinal ganglion cell (RGC) types and their visual response properties, which is fundamental for developing future electronic implants that can restore vision. The electrical image (EI) of a RGC, or the mean spatio-temporal voltage footprint of its recorded spikes on a high-density electrode array, contains substantial information about its anatomical, morphological, and functional properties. However, the analysis of these properties is complex because of the high-dimensional nature of the EI. We present a novel optimization-based algorithm to decompose electrical image into a low-dimensional, biophysically-based representation: the temporally-shifted superposition of three learned basis waveforms corresponding to spike waveforms produced in the somatic, dendritic and axonal cellular compartments. Large-scale multi-electrode recordings from the macaque retina were used to test the effectiveness of the decomposition. The decomposition accurately localized the somatic and dendritic compartments of the cell. The imputed dendritic fields of RGCs correctly predicted the location and shape of their visual receptive fields. The inferred waveform amplitudes and shapes accurately identified the four major primate RGC types (ON and OFF midget and parasol cells), a substantial advance. Together, these findings may contribute to more accurate inference of RGC types and their original light responses in the degenerated retina, with possible implications for other electrical imaging applications.

Structured sampling of olfactory input by the fly mushroom body.

Associative memory formation and recall in the fruit fly Drosophila melanogaster is subserved by the mushroom body (MB). Upon arrival in the MB, sensory information undergoes a profound transformation from broadly tuned and stereotyped odorant responses in the olfactory projection neuron (PN) layer to narrowly tuned and nonstereotyped responses in the Kenyon cells (KCs). Theory and experiment suggest that this transformation is implemented by random connectivity between KCs and PNs. However, this hypothesis has been challenging to test, given the difficulty of mapping synaptic connections between large numbers of brain-spanning neurons. Here, we used a recent whole-brain electron microscopy volume of the adult fruit fly to map PN-to-KC connectivity at synaptic resolution. The PN-KC connectome revealed unexpected structure, with preponderantly food-responsive PN types converging at above-chance levels on downstream KCs. Axons of the overconvergent PN types tended to arborize near one another in the MB main calyx, making local KC dendrites more likely to receive input from those types. Overconvergent PN types preferentially co-arborize and connect with dendrites of αß and α'ß' KC subtypes. Computational simulation of the observed network showed degraded discrimination performance compared with a random network, except when all signal flowed through the overconvergent, primarily food-responsive PN types. Additional theory and experiment will be needed to fully characterize the impact of the observed non-random network structure on associative memory formation and recall.

Blue-yellow opponency in primate S cone photoreceptors.

The neural coding of human color vision begins in the retina. The outputs of long (L)-, middle (M)-, and short (S)-wavelength-sensitive cone photoreceptors combine antagonistically to produce "red-green" and "blue-yellow" spectrally opponent signals (Hering, 1878; Hurvich and Jameson, 1957). Spectral opponency is well established in primate retinal ganglion cells (Reid and Shapley, 1992; Dacey and Lee, 1994; Dacey et al., 1996), but the retinal circuitry creating the opponency remains uncertain. Here we find, from whole-cell recordings of photoreceptors in macaque monkey, that "blue-yellow" opponency is already present in the center-surround receptive fields of S cones. The inward current evoked by blue light derives from phototransduction within the outer segment of the S cone. The outward current evoked by yellow light is caused by feedback from horizontal cells that are driven by surrounding L and M cones. Stimulation of the surround modulates calcium conductance in the center S cone.

A connectome and analysis of the adult Drosophila central brain.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly's brain.

Animal brains of all sizes, from the smallest to the largest, work in broadly similar ways. Studying the brain of any one animal in depth can thus reveal the general principles behind the workings of all brains. The fruit fly Drosophila is a popular choice for such research. With about 100,000 neurons ­ compared to some 86 billion in humans ­ the fly brain is small enough to study at the level of individual cells. But it nevertheless supports a range of complex behaviors, including navigation, courtship and learning. Thanks to decades of research, scientists now have a good understanding of which parts of the fruit fly brain support particular behaviors. But exactly how they do this is often unclear. This is because previous studies showing the connections between cells only covered small areas of the brain. This is like trying to understand a novel when all you can see is a few isolated paragraphs. To solve this problem, Scheffer, Xu, Januszewski, Lu, Takemura, Hayworth, Huang, Shinomiya et al. prepared the first complete map of the entire central region of the fruit fly brain. The central brain consists of approximately 25,000 neurons and around 20 million connections. To prepare the map ­ or connectome ­ the brain was cut into very thin 8nm slices and photographed with an electron microscope. A three-dimensional map of the neurons and connections in the brain was then reconstructed from these images using machine learning algorithms. Finally, Scheffer et al. used the new connectome to obtain further insights into the circuits that support specific fruit fly behaviors. The central brain connectome is freely available online for anyone to access. When used in combination with existing methods, the map will make it easier to understand how the fly brain works, and how and why it can fail to work correctly. Many of these findings will likely apply to larger brains, including our own. In the long run, studying the fly connectome may therefore lead to a better understanding of the human brain and its disorders. Performing a similar analysis on the brain of a small mammal, by scaling up the methods here, will be a likely next step along this path.

Gap-junctional coupling and absolute sensitivity of photoreceptors in macaque retina.

We investigated gap-junctional coupling of rods and cones in macaque retina. Cone voltage responses evoked by light absorption in neighboring rods were briefer and smaller than responses recorded in the rods themselves. Rod detection thresholds, calculated from noise and response amplitude histograms, closely matched the threshold for an ideal detector limited by quantal fluctuations in the stimulus. Surprisingly, cone thresholds were only approximately two times higher. Amplitude fluctuations in cones could be explained by a Poisson distribution of photoisomerizations within a pool of seven or more coupled rods. Neurobiotin coupling between rods and cones was consistent with our electrical recordings, with approximately six rods labeled per injected cone. The spatial distribution of tracer-coupled rods matched the light-evoked cone receptive field. The gap junction inhibitor carbenoxolone abolished both electrical and tracer coupling. Amplitude fluctuations in most rods were accounted for by the expected rate of light absorption in their outer segments. The fluctuations in some rods, however, were consistent with a summation pool of up to six rods. When single rods were injected with Neurobiotin, up to 10 rods were labeled. Rod-rod and rod-cone electrical coupling is expected to extend the range of scotopic vision by circumventing saturation at the rod to rod-bipolar cell synapse; however, because coupling also renders the rod synapse less effective at separating out photon signals from dark noise, coupling is expected to elevate the absolute threshold of dark-adapted observers.

Identification of a Retinal Circuit for Recurrent Suppression Using Indirect Electrical Imaging.

Understanding the function of modulatory interneuron networks is a major challenge, because such networks typically operate over long spatial scales and involve many neurons of different types. Here, we use an indirect electrical imaging method to reveal the function of a spatially extended, recurrent retinal circuit composed of two cell types. This recurrent circuit produces peripheral response suppression of early visual signals in the primate magnocellular visual pathway. We identify a type of polyaxonal amacrine cell physiologically via its distinctive electrical signature, revealed by electrical coupling with ON parasol retinal ganglion cells recorded using a large-scale multi-electrode array. Coupling causes the amacrine cells to fire spikes that propagate radially over long distances, producing GABA-ergic inhibition of other ON parasol cells recorded near the amacrine cell axonal projections. We propose and test a model for the function of this amacrine cell type, in which the extra-classical receptive field of ON parasol cells is formed by reciprocal inhibition from other ON parasol cells in the periphery, via the electrically coupled amacrine cell network.

Mapping nonlinear receptive field structure in primate retina at single cone resolution.

The function of a neural circuit is shaped by the computations performed by its interneurons, which in many cases are not easily accessible to experimental investigation. Here, we elucidate the transformation of visual signals flowing from the input to the output of the primate retina, using a combination of large-scale multi-electrode recordings from an identified ganglion cell type, visual stimulation targeted at individual cone photoreceptors, and a hierarchical computational model. The results reveal nonlinear subunits in the circuity of OFF midget ganglion cells, which subserve high-resolution vision. The model explains light responses to a variety of stimuli more accurately than a linear model, including stimuli targeted to cones within and across subunits. The recovered model components are consistent with known anatomical organization of midget bipolar interneurons. These results reveal the spatial structure of linear and nonlinear encoding, at the resolution of single cells and at the scale of complete circuits.