The noncoding circular RNA circHomer1 regulates developmental experience-dependent plasticity in mouse visual cortex.

Circular RNAs (circRNAs) are noncoding RNAs abundant in brain tissue, and many are derived from activity-dependent, linear mRNAs encoding for synaptic proteins, suggesting that circRNAs may directly or indirectly play a role in regulating synaptic development, plasticity, and function. However, it is unclear if the circular forms of these RNAs are similarly regulated by activity and what role these circRNAs play in developmental plasticity. Here, we employed transcriptome-wide analysis comparing differential expression of both mRNAs and circRNAs in juvenile mouse primary visual cortex (V1) following monocular deprivation (MD), a model of developmental plasticity. Among the differentially expressed mRNAs and circRNAs following 3-day MD, the circular and the activity-dependent linear forms of the Homer1 gene, circHomer1 and Homer1a respectively, were of interest as their expression changed in opposite directions: circHomer1 expression increased while the expression of Homer1a decreased following MD. Knockdown of circHomer1 prevented the depression of closed-eye responses normally observed after 3-day MD. circHomer1-knockdown led to a reduction in average dendritic spine size prior to MD, but critically there was no further reduction after 3-day MD, consistent with impaired structural plasticity. circHomer1-knockdown also prevented the reduction of surface AMPA receptors after 3-day MD. Synapse-localized puncta of the AMPA receptor endocytic protein Arc increased in volume after MD but were smaller in circHomer1-knockdown neurons, suggesting that circHomer1 regulates plasticity through mechanisms of activity-dependent AMPA receptor endocytosis. Thus, activity-dependent circRNAs regulate developmental synaptic plasticity, and our findings highlight the essential role of circHomer1 in V1 plasticity induced by short-term MD.

Patch-walking: Coordinated multi-pipette patch clamp for efficiently finding synaptic connections.

Significant technical challenges exist when measuring synaptic connections between neurons in living brain tissue. The patch clamping technique, when used to probe for synaptic connections, is manually laborious and time-consuming. To improve its efficiency, we pursued another approach: instead of retracting all patch clamping electrodes after each recording attempt, we cleaned just one of them and reused it to obtain another recording while maintaining the others. With one new patch clamp recording attempt, many new connections can be probed. By placing one pipette in front of the others in this way, one can "walk" across the tissue, termed "patch-walking." We performed 136 patch clamp attempts for two pipettes, achieving 71 successful whole cell recordings (52.2%). Of these, we probed 29 pairs (i.e., 58 bidirectional probed connections) averaging 91 µm intersomatic distance, finding 3 connections. Patch-walking yields 80-92% more probed connections, for experiments with 10-100 cells than the traditional synaptic connection searching method.

Inefficient tissue immune response against MPXV in an immunocompromised mpox patient.

The recent outbreak of monkeypox virus (MPXV) was unprecedented in its size and distribution. Those living with uncontrolled HIV and low CD4 T cell counts might develop a fulminant clinical mpox course with increased mortality, secondary infections, and necrotizing lesions. Fatal cases display a high and widespread MPXV tissue burden. The underlying pathomechanisms are not fully understood. We report here the pathological findings of an MPXV-driven abscess in gastrocnemius muscle requiring surgery in an immunocompromised patient with severe mpox. Presence of virus particles and infectivity were confirmed by electron microscopy, expansion microscopy, and virus culture, respectively. MPXV tissue distribution by immunohistochemistry (IHC) showed a necrotic core with infection of different cell types. In contrast, at the lesion rim fibroblasts were mainly infected. Immune cells were almost absent in the necrotic core, but were abundant at the infection rim and predominantly macrophages. Further, we detected high amounts of alternatively activated GPNMB+-macrophages at the lesion border. Of note, macrophages only rarely colocalized with virus-infected cells. Insufficient clearance of infected cells and infection of lesion-associated fibroblasts sustained by the abundance of profibrotic macrophages might lead to the coalescing of lesions and the severe and persistent clinical mpox course observed in immunocompromised patients.

The neuron mixer and its impact on human brain dynamics.

A signal mixer facilitates rich computation, which has been the building block of modern telecommunication. This frequency mixing produces new signals at the sum and difference frequencies of input signals, enabling powerful operations such as heterodyning and multiplexing. Here, we report that a neuron is a signal mixer. We found through ex vivo and in vivo whole-cell measurements that neurons mix exogenous (controlled) and endogenous (spontaneous) subthreshold membrane potential oscillations, producing new oscillation frequencies, and that neural mixing originates in voltage-gated ion channels. Furthermore, we demonstrate that mixing is evident in human brain activity and is associated with cognitive functions. We found that the human electroencephalogram displays distinct clusters of local and inter-region mixing and that conversion of the salient posterior alpha-beta oscillations into gamma-band oscillations regulates visual attention. Signal mixing may enable individual neurons to sculpt the spectrum of neural circuit oscillations and utilize them for computational operations.

Pixel-wise programmability enables dynamic high-SNR cameras for high-speed microscopy.

High-speed wide-field fluorescence microscopy has the potential to capture biological processes with exceptional spatiotemporal resolution. However, conventional cameras suffer from low signal-to-noise ratio at high frame rates, limiting their ability to detect faint fluorescent events. Here, we introduce an image sensor where each pixel has individually programmable sampling speed and phase, so that pixels can be arranged to simultaneously sample at high speed with a high signal-to-noise ratio. In high-speed voltage imaging experiments, our image sensor significantly increases the output signal-to-noise ratio compared to a low-noise scientific CMOS camera (~2-3 folds). This signal-to-noise ratio gain enables the detection of weak neuronal action potentials and subthreshold activities missed by the standard scientific CMOS cameras. Our camera with flexible pixel exposure configurations offers versatile sampling strategies to improve signal quality in various experimental conditions.

QuATON: Quantization Aware Training of Optical Neurons.

Optical processors, built with "optical neurons", can efficiently perform high-dimensional linear operations at the speed of light. Thus they are a promising avenue to accelerate large-scale linear computations. With the current advances in micro-fabrication, such optical processors can now be 3D fabricated, but with a limited precision. This limitation translates to quantization of learnable parameters in optical neurons, and should be handled during the design of the optical processor in order to avoid a model mismatch. Specifically, optical neurons should be trained or designed within the physical-constraints at a predefined quantized precision level. To address this critical issues we propose a physics-informed quantization-aware training framework. Our approach accounts for physical constraints during the training process, leading to robust designs. We demonstrate that our approach can design state of the art optical processors using diffractive networks for multiple physics based tasks despite quantized learnable parameters. We thus lay the foundation upon which improved optical processors may be 3D fabricated in the future.

Post-transcriptional splicing can occur in a slow-moving zone around the gene.

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

Dense, Continuous Membrane Labeling and Expansion Microscopy Visualization of Ultrastructure in Tissues.

Lipid membranes are key to the nanoscale compartmentalization of biological systems, but fluorescent visualization of them in intact tissues, with nanoscale precision, is challenging to do with high labeling density. Here, we report ultrastructural membrane expansion microscopy (umExM), which combines a novel membrane label and optimized expansion microscopy protocol, to support dense labeling of membranes in tissues for nanoscale visualization. We validated the high signal-to-background ratio, and uniformity and continuity, of umExM membrane labeling in brain slices, which supported the imaging of membranes and proteins at a resolution of ~60 nm on a confocal microscope. We demonstrated the utility of umExM for the segmentation and tracing of neuronal processes, such as axons, in mouse brain tissue. Combining umExM with optical fluctuation imaging, or iterating the expansion process, yielded ~35 nm resolution imaging, pointing towards the potential for electron microscopy resolution visualization of brain membranes on ordinary light microscopes.

Multisensory gamma stimulation promotes glymphatic clearance of amyloid.

The glymphatic movement of fluid through the brain removes metabolic waste1-4. Noninvasive 40 Hz stimulation promotes 40 Hz neural activity in multiple brain regions and attenuates pathology in mouse models of Alzheimer's disease5-8. Here we show that multisensory gamma stimulation promotes the influx of cerebrospinal fluid and the efflux of interstitial fluid in the cortex of the 5XFAD mouse model of Alzheimer's disease. Influx of cerebrospinal fluid was associated with increased aquaporin-4 polarization along astrocytic endfeet and dilated meningeal lymphatic vessels. Inhibiting glymphatic clearance abolished the removal of amyloid by multisensory 40 Hz stimulation. Using chemogenetic manipulation and a genetically encoded sensor for neuropeptide signalling, we found that vasoactive intestinal peptide interneurons facilitate glymphatic clearance by regulating arterial pulsatility. Our findings establish novel mechanisms that recruit the glymphatic system to remove brain amyloid.

Fast light-field 3D microscopy with out-of-distribution detection and adaptation through conditional normalizing flows.

Real-time 3D fluorescence microscopy is crucial for the spatiotemporal analysis of live organisms, such as neural activity monitoring. The eXtended field-of-view light field microscope (XLFM), also known as Fourier light field microscope, is a straightforward, single snapshot solution to achieve this. The XLFM acquires spatial-angular information in a single camera exposure. In a subsequent step, a 3D volume can be algorithmically reconstructed, making it exceptionally well-suited for real-time 3D acquisition and potential analysis. Unfortunately, traditional reconstruction methods (like deconvolution) require lengthy processing times (0.0220 Hz), hampering the speed advantages of the XLFM. Neural network architectures can overcome the speed constraints but do not automatically provide a way to certify the realism of their reconstructions, which is essential in the biomedical realm. To address these shortcomings, this work proposes a novel architecture to perform fast 3D reconstructions of live immobilized zebrafish neural activity based on a conditional normalizing flow. It reconstructs volumes at 8 Hz spanning 512x512x96 voxels, and it can be trained in under two hours due to the small dataset requirements (50 image-volume pairs). Furthermore, normalizing flows provides a way to compute the exact likelihood of a sample. This allows us to certify whether the predicted output is in- or ood, and retrain the system when a novel sample is detected. We evaluate the proposed method on a cross-validation approach involving multiple in-distribution samples (genetically identical zebrafish) and various out-of-distribution ones.

Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding.

Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here, we present an ExM variant, decrowding expansion pathology (dExPath), that can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and, perhaps, diagnosis.

HiExM: high-throughput expansion microscopy enables scalable super-resolution imaging.

Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells at nanoscale resolution; however, current methods cannot be performed in multi-well cell culture plates which limits the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables consistent ~4.2x expansion within individual wells, across multiple wells, and between plates processed in parallel. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, in human cardiomyocytes (CMs) based on Hoechst signal intensity. We show a dose dependent effect on nuclear chromatin that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

Temporally multiplexed imaging of dynamic signaling networks in living cells.

Molecular signals interact in networks to mediate biological processes. To analyze these networks, it would be useful to image many signals at once, in the same living cell, using standard microscopes and genetically encoded fluorescent reporters. Here, we report temporally multiplexed imaging (TMI), which uses genetically encoded fluorescent proteins with different clocklike properties-such as reversibly photoswitchable fluorescent proteins with different switching kinetics-to represent different cellular signals. We linearly decompose a brief (few-second-long) trace of the fluorescence fluctuations, at each point in a cell, into a weighted sum of the traces exhibited by each fluorophore expressed in the cell. The weights then represent the signal amplitudes. We use TMI to analyze relationships between different kinase activities in individual cells, as well as between different cell-cycle signals, pointing toward broad utility throughout biology in the analysis of signal transduction cascades in living systems.

Optogenetic control of neural activity: The biophysics of microbial rhodopsins in neuroscience.

Optogenetics, the use of microbial rhodopsins to make the electrical activity of targeted neurons controllable by light, has swept through neuroscience, enabling thousands of scientists to study how specific neuron types contribute to behaviors and pathologies, and how they might serve as novel therapeutic targets. By activating a set of neurons, one can probe what functions they can initiate or sustain, and by silencing a set of neurons, one can probe the functions they are necessary for. We here review the biophysics of these molecules, asking why they became so useful in neuroscience for the study of brain circuitry. We review the history of the field, including early thinking, early experiments, applications of optogenetics, pre-optogenetics targeted neural control tools, and the history of discovering and characterizing microbial rhodopsins. We then review the biophysical attributes of rhodopsins that make them so useful to neuroscience - their classes and structure, their photocycles, their photocurrent magnitudes and kinetics, their action spectra, and their ion selectivity. Our hope is to convey to the reader how specific biophysical properties of these molecules made them especially useful to neuroscientists for a difficult problem - the control of high-speed electrical activity, with great precision and ease, in the brain.

Non-invasive temporal interference electrical stimulation of the human hippocampus.

Deep brain stimulation (DBS) via implanted electrodes is used worldwide to treat patients with severe neurological and psychiatric disorders. However, its invasiveness precludes widespread clinical use and deployment in research. Temporal interference (TI) is a strategy for non-invasive steerable DBS using multiple kHz-range electric fields with a difference frequency within the range of neural activity. Here we report the validation of the non-invasive DBS concept in humans. We used electric field modeling and measurements in a human cadaver to verify that the locus of the transcranial TI stimulation can be steerably focused in the hippocampus with minimal exposure to the overlying cortex. We then used functional magnetic resonance imaging and behavioral experiments to show that TI stimulation can focally modulate hippocampal activity and enhance the accuracy of episodic memories in healthy humans. Our results demonstrate targeted, non-invasive electrical stimulation of deep structures in the human brain.

Expansion microscopy using a single anchor molecule for high-yield multiplexed imaging of proteins and RNAs.

Expansion microscopy (ExM), by physically enlarging specimens in an isotropic fashion, enables nanoimaging on standard light microscopes. Key to existing ExM protocols is the equipping of different kinds of molecules, with different kinds of anchoring moieties, so they can all be pulled apart from each other by polymer swelling. Here we present a multifunctional anchor, an acrylate epoxide, that enables proteins and RNAs to be equipped with anchors in a single experimental step. This reagent simplifies ExM protocols and reduces cost (by 2-10-fold for a typical multiplexed ExM experiment) compared to previous strategies for equipping RNAs with anchors. We show that this united ExM (uniExM) protocol can be used to preserve and visualize RNA transcripts, proteins in biologically relevant ultrastructures, and sets of RNA transcripts in patient-derived xenograft (PDX) cancer tissues and may support the visualization of other kinds of biomolecular species as well. uniExM may find many uses in the simple, multimodal nanoscale analysis of cells and tissues.

Theta and gamma rhythmic coding through two spike output modes in the hippocampus during spatial navigation.

Hippocampal CA1 neurons generate single spikes and stereotyped bursts of spikes. However, it is unclear how individual neurons dynamically switch between these output modes and whether these two spiking outputs relay distinct information. We performed extracellular recordings in spatially navigating rats and cellular voltage imaging and optogenetics in awake mice. We found that spike bursts are preferentially linked to cellular and network theta rhythms (3-12 Hz) and encode an animal's position via theta phase precession, particularly as animals are entering a place field. In contrast, single spikes exhibit additional coupling to gamma rhythms (30-100 Hz), particularly as animals leave a place field. Biophysical modeling suggests that intracellular properties alone are sufficient to explain the observed input frequency-dependent spike coding. Thus, hippocampal neurons regulate the generation of bursts and single spikes according to frequency-specific network and intracellular dynamics, suggesting that these spiking modes perform distinct computations to support spatial behavior.

Fast light-field 3D microscopy with out-of-distribution detection and adaptation through Conditional Normalizing Flows.

Real-time 3D fluorescence microscopy is crucial for the spatiotemporal analysis of live organisms, such as neural activity monitoring. The eXtended field-of-view light field microscope (XLFM), also known as Fourier light field microscope, is a straightforward, single snapshot solution to achieve this. The XLFM acquires spatial-angular information in a single camera exposure. In a subsequent step, a 3D volume can be algorithmically reconstructed, making it exceptionally well-suited for real-time 3D acquisition and potential analysis. Unfortunately, traditional reconstruction methods (like deconvolution) require lengthy processing times (0.0220 Hz), hampering the speed advantages of the XLFM. Neural network architectures can overcome the speed constraints at the expense of lacking certainty metrics, which renders them untrustworthy for the biomedical realm. This work proposes a novel architecture to perform fast 3D reconstructions of live immobilized zebrafish neural activity based on a conditional normalizing flow. It reconstructs volumes at 8 Hz spanning 512 × 512 × 96 voxels, and it can be trained in under two hours due to the small dataset requirements (10 image-volume pairs). Furthermore, normalizing flows allow for exact Likelihood computation, enabling distribution monitoring, followed by out-of-distribution detection and retraining of the system when a novel sample is detected. We evaluate the proposed method on a cross-validation approach involving multiple in-distribution samples (genetically identical zebrafish) and various out-of-distribution ones.

Pixel-wise programmability enables dynamic high-SNR cameras for high-speed microscopy.

High-speed widefield fluorescence microscopy has the potential to capture biological processes with exceptional spatiotemporal resolution. However, conventional cameras suffer from low signal-to-noise ratio (SNR) at high frame rates, limiting their ability to detect faint fluorescent events. Here we introduce an image sensor where each pixel has individually programmable sampling speed and phase, so that pixels can be arranged to simultaneously sample at high speed with a high SNR. In high-speed voltage imaging experiments, our image sensor significantly increases the output SNR compared to a low-noise scientific CMOS camera (~2-3 folds). This SNR gain enables the detection of weak neuronal action potentials and subthreshold activities missed by the standard scientific CMOS cameras. Our proposed camera with flexible pixel exposure configurations offers versatile sampling strategies to improve signal quality in various experimental conditions.