Fractured columnar small-world functional network organization in volumes of L2/3 of mouse auditory cortex.

The sensory cortices of the brain exhibit large-scale functional topographic organization, such as the tonotopic organization of the primary auditory cortex (A1) according to sound frequency. However, at the level of individual neurons, layer 2/3 (L2/3) A1 appears functionally heterogeneous. To identify if there exists a higher-order functional organization of meso-scale neuronal networks within L2/3 that bridges order and disorder, we used in vivo two-photon calcium imaging of pyramidal neurons to identify networks in three-dimensional volumes of L2/3 A1 in awake mice. Using tonal stimuli, we found diverse receptive fields with measurable colocalization of similarly tuned neurons across depth but less so across L2/3 sublayers. These results indicate a fractured microcolumnar organization with a column radius of ∼50 µm, with a more random organization of the receptive field over larger radii. We further characterized the functional networks formed within L2/3 by analyzing the spatial distribution of signal correlations (SCs). Networks show evidence of Rentian scaling in physical space, suggesting effective spatial embedding of subnetworks. Indeed, functional networks have characteristics of small-world topology, implying that there are clusters of functionally similar neurons with sparse connections between differently tuned neurons. These results indicate that underlying the regularity of the tonotopic map on large scales in L2/3 is significant tuning diversity arranged in a hybrid organization with microcolumnar structures and efficient network topologies.

Time-dependent homeostatic mechanisms underlie brain-derived neurotrophic factor action on neural circuitry.

Plasticity and homeostatic mechanisms allow neural networks to maintain proper function while responding to physiological challenges. Despite previous work investigating morphological and synaptic effects of brain-derived neurotrophic factor (BDNF), the most prevalent growth factor in the central nervous system, how exposure to BDNF manifests at the network level remains unknown. Here we report that BDNF treatment affects rodent hippocampal network dynamics during development and recovery from glutamate-induced excitotoxicity in culture. Importantly, these effects are not obvious when traditional activity metrics are used, so we delve more deeply into network organization, functional analyses, and in silico simulations. We demonstrate that BDNF partially restores homeostasis by promoting recovery of weak and medium connections after injury. Imaging and computational analyses suggest these effects are caused by changes to inhibitory neurons and connections. From our in silico simulations, we find that BDNF remodels the network by indirectly strengthening weak excitatory synapses after injury. Ultimately, our findings may explain the difficulties encountered in preclinical and clinical trials with BDNF and also offer information for future trials to consider.

Filament displacement image analytics tool for use in investigating dynamics of dense microtubule networks.

The fate and motion of cells is influenced by a variety of physical characteristics of their microenvironments. Traditionally, mechanobiology focuses on external mechanical phenomena such as cell movement and environmental sensing. However, cells are inherently dynamic, where internal waves and internal oscillations are a hallmark of living cells observed under a microscope. We propose that these internal mechanical rhythms provide valuable information about cell health. Therefore, it is valuable to capture the rhythms inside cells and quantify how drugs or physical interventions affect a cell's internal dynamics. One of the key dynamical entities inside cells is the microtubule network. Typically, microtubule dynamics are measured by end-protein tracking. In contrast, this paper introduces an easy-to-implement approach to measure the lateral motion of the microtubule filaments embedded within dense networks with (at least) confocal resolution image sequences. Our tool couples the computer vision algorithm Optical Flow with an anisotropic, rotating Laplacian of Gaussian filtering to characterize the lateral motion of dense microtubule networks. We then showcase additional image analytics used to understand the effect of microtubule orientation and regional location on lateral motion. We argue that our tool and these additional metrics provide a fuller picture of the active forcing environment within cells.

Development of an accelerated cellular model for early changes in Alzheimer's disease.

Alzheimer's Disease (AD) is a leading cause of dementia characterized by amyloid plaques and neurofibrillary tangles, and its pathogenesis remains unclear. Current cellular models for AD often require several months to exhibit phenotypic features due to the lack of an aging environment in vitro. Lamin A is a key component of the nuclear lamina. Progerin, a truncated protein resulting from specific lamin A mutations, causes Hutchinson-Gilford Progeria Syndrome (HGPS), a disease that prematurely ages individuals. Studies have reported that lamin A expression is induced in the brains of AD patients, and overlapping cellular phenotypes have been observed between HGPS and AD cells. In this study, we investigated the effects of exogenous progerin expression on neural progenitor cells carrying familial AD mutations (FAD). Within three to four weeks of differentiation, these cells exhibited robust AD phenotypes, including increased tau phosphorylation, amyloid plaque accumulation, and an elevated Aß42 to Aß40 ratio. Additionally, progerin expression significantly increased AD cellular phenotypes such as cell death and cell cycle re-entry. Our results suggest that progerin expression could be used to create an accelerated model for AD development and drug screening.

Protocol for electrotaxis of large epithelial cell sheets.

Here, we present a protocol for electrotaxis of large epithelial cell sheets without compromising the integrity of cell epithelia in a high-throughput customized directed current electrotaxis chamber. We describe the fabrication and use of polydimethylsiloxane stencils to control the size and shape of human keratinocyte cell sheets. We detail cell tracking, cell sheet contour assay, and particle image velocimetry to reveal the spatial and temporal motility dynamics of cell sheets. This approach is applicable to other collective cell migration studies. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1.

Nanotopography modulates intracellular excitable systems through cytoskeleton actuation.

Cellular sensing of most environmental cues involves receptors that affect a signal-transduction excitable network (STEN), which is coupled to a cytoskeletal excitable network (CEN). We show that the mechanism of sensing of nanoridges is fundamentally different. CEN activity occurs preferentially on nanoridges, whereas STEN activity is constrained between nanoridges. In the absence of STEN, waves disappear, but long-lasting F-actin puncta persist along the ridges. When CEN is suppressed, wave propagation is no longer constrained by nanoridges. A computational model reproduces these experimental observations. Our findings indicate that nanotopography is sensed directly by CEN, whereas STEN is only indirectly affected due to a CEN-STEN feedback loop. These results explain why texture sensing is robust and acts cooperatively with multiple other guidance cues in complex, in vivo microenvironments.

NeuroWRAP: integrating, validating, and sharing neurodata analysis workflows.

Multiphoton calcium imaging is one of the most powerful tools in modern neuroscience. However, multiphoton data require significant pre-processing of images and post-processing of extracted signals. As a result, many algorithms and pipelines have been developed for the analysis of multiphoton data, particularly two-photon imaging data. Most current studies use one of several algorithms and pipelines that are published and publicly available, and add customized upstream and downstream analysis elements to fit the needs of individual researchers. The vast differences in algorithm choices, parameter settings, pipeline composition, and data sources combine to make collaboration difficult, and raise questions about the reproducibility and robustness of experimental results. We present our solution, called NeuroWRAP (www.neurowrap.org), which is a tool that wraps multiple published algorithms together, and enables integration of custom algorithms. It enables development of collaborative, shareable custom workflows and reproducible data analysis for multiphoton calcium imaging data enabling easy collaboration between researchers. NeuroWRAP implements an approach to evaluate the sensitivity and robustness of the configured pipelines. When this sensitivity analysis is applied to a crucial step of image analysis, cell segmentation, we find a substantial difference between two popular workflows, CaImAn and Suite2p. NeuroWRAP harnesses this difference by introducing consensus analysis, utilizing two workflows in conjunction to significantly increase the trustworthiness and robustness of cell segmentation results.

Enabled primarily controls filopodial morphology, not actin organization, in the TSM1 growth cone in Drosophila.

Ena/VASP proteins are processive actin polymerases that are required throughout animal phylogeny for many morphogenetic processes, including axon growth and guidance. Here we use in vivo live imaging of morphology and actin distribution to determine the role of Ena in promoting the growth of the TSM1 axon of the Drosophila wing. Altering Ena activity causes stalling and misrouting of TSM1. Our data show that Ena has a substantial impact on filopodial morphology in this growth cone but exerts only modest effects on actin distribution. This is in contrast to the main regulator of Ena, Abl tyrosine kinase, which was shown previously to have profound effects on actin and only mild effects on TSM1 growth cone morphology. We interpret these data as suggesting that the primary role of Ena in this axon may be to link actin to the morphogenetic processes of the plasma membrane, rather than to regulate actin organization itself. These data also suggest that a key role of Ena, acting downstream of Abl, may be to maintain consistent organization and reliable evolution of growth cone structure, even as Abl activity varies in response to guidance cues in the environment.

Elongate mineral particles (EMP) characteristics and mesothelioma: Summary and resolution for session I of the Monticello II conference.

The summary contains a consensus opinion regarding the current state of the science about the dimensions of Elongate Mineral Particles (EMPs) as a factor impacting their carcinogenicity.

Excitable systems: A new perspective on the cellular impact of elongate mineral particles.

We investigate how the geometry of elongate mineral particles (EMPs) in contact with cells influences esotaxis, a recently discovered mechanism of texture sensing. Esotaxis is based on cytoskeletal waves and oscillations that are nucleated, shaped, and steered by the texture of the surroundings. We find that all EMPs studied trigger an esotactic response in macrophages, and that this response dominates cytoskeletal activity in these immune cells. In contrast, epithelial cells show little to no esotactic response to the EMPs. These results are consistent with the distinct interactions of both cell types with ridged nanotopographies of dimensions comparable to those of asbestiform EMPs. Our findings raise the question of whether narrow, asbestiform EMPs may also dominate cytoskeletal activity in other types of immune cells that exhibit similar esotactic effects. These findings, together with prior studies of esotaxis, lead us to the hypothesis that asbestiform EMPs suppress the migration of immune cells and activate immune signaling, thereby outcompeting signals that would normally stimulate the immune system in nearby tissue.

Decoding Natural Astrocyte Rhythms: Dynamic Actin Waves Result from Environmental Sensing by Primary Rodent Astrocytes.

Astrocytes are key regulators of brain homeostasis, equilibrating ion, water, and neurotransmitter concentrations and maintaining essential conditions for proper cognitive function. Recently, it has been shown that the excitability of the actin cytoskeleton manifests in second-scale dynamic fluctuations and acts as a sensor of chemophysical environmental cues. However, it is not known whether the cytoskeleton is excitable in astrocytes and how the homeostatic function of astrocytes is linked to the dynamics of the cytoskeleton. Here it is shown that homeostatic regulation involves the excitable dynamics of actin in certain subcellular regions of astrocytes, especially near the cell boundary. The results further indicate that actin dynamics concentrate into "hotspot" regions that selectively respond to certain chemophysical stimuli, specifically the homeostatic challenges of ion or water concentration increases. Substrate topography makes the actin dynamics of astrocytes weaker. Super-resolution images demonstrate that surface topography is also associated with the predominant perpendicular alignment of actin filaments near the cell boundary, whereas flat substrates result in an actin cortex mainly parallel to the cell boundary. Additionally, coculture with neurons increases both the probability of actin dynamics and the strength of hotspots. The excitable systems character of actin thus makes astrocytes direct participants in neural cell network dynamics.

Finite-size correlation behavior near a critical point: A simple metric for monitoring the state of a neural network.

In this article, a correlation metric κ_{c} is proposed for the inference of the dynamical state of neuronal networks. κ_{C} is computed from the scaling of the correlation length with the size of the observation region, which shows qualitatively different behavior near and away from the critical point of a continuous phase transition. The implementation is first studied on a neuronal network model, where the results of this new metric coincide with those obtained from neuronal avalanche analysis, thus well characterizing the critical state of the network. The approach is further tested with brain optogenetic recordings in behaving mice from a publicly available database. Potential applications and limitations for its use with currently available optical imaging techniques are discussed.

Protocol for electrical modulation of ERK signaling dynamics in cell lines.

Here, we describe a protocol for modulating the dynamics of the extracellular signal-regulated kinases (ERK) pathway in a customized alternating current (AC) electric field stimulation chamber. We use an ERK translocation reporter that can accurately represent the intracellular ERK activity in real time without chemical agents or gene disruption. ERK activation is assessed by comparing the relative intensity of nuclear fluorescence to cytosolic fluorescence in live-cell conditions. The approach can be applied to other signaling pathways as well. For complete details on the use and execution of this protocol, please refer to Guo et al. (2021).

Propagation dynamics of electrotactic motility in large epithelial cell sheets.

Directional migration initiated at the wound edge leads epithelia to migrate in wound healing. How such coherent migration is achieved is not well understood. Here, we used electric fields to induce robust migration of sheets of human keratinocytes and developed an in silico model to characterize initiation and propagation of epithelial collective migration. Electric fields initiate an increase in migration directionality and speed at the leading edge. The increases propagate across the epithelial sheets, resulting in directional migration of cell sheets as coherent units. Both the experimental and in silico models demonstrated vector-like integration of the electric and default directional cues at free edge in space and time. The resultant collective migration is consistent in experiments and modeling, both qualitatively and quantitatively. The keratinocyte model thus faithfully reflects key features of epithelial migration as a coherent tissue in vivo, e.g. that leading cells lead, and that epithelium maintains cell-cell junction.

Experimentally Measuring Rolling and Sliding in Three-Dimensional Dense Granular Packings.

We experimentally measure a three-dimensional (3D) granular system's reversibility under cyclic compression. We image the grains using a refractive-index-matched fluid, then analyze the images using the artificial intelligence of variational autoencoders. These techniques allow us to track all the grains' translations and 3D rotations with accuracy sufficient to infer sliding and rolling displacements. Our observations reveal unique roles played by 3D rotational motions in granular flows. We find that rotations and contact-point motion dominate the dynamics in the bulk, far from the perturbation's source. Furthermore, we determine that 3D rotations are irreversible under cyclic compression. Consequently, contact-point sliding, which is dissipative, accumulates throughout the cycle. Using numerical simulations whose accuracy our experiment supports, we discover that much of the dissipation occurs in the bulk, where grains rotate more than they translate. Our observations suggest that the analysis of 3D rotations is needed for understanding granular materials' unique and powerful ability to absorb and dissipate energy.

Label-free cell tracking enables collective motion phenotyping in epithelial monolayers.

Collective cell migration is an umbrella term for a rich variety of cell behaviors, whose distinct character is important for biological function, notably for cancer metastasis. One essential feature of collective behavior is the motion of cells relative to their immediate neighbors. We introduce an AI-based pipeline to segment and track cell nuclei from phase-contrast images. Nuclei segmentation is based on a U-Net convolutional neural network trained on images with nucleus staining. Tracking, based on the Crocker-Grier algorithm, quantifies nuclei movement and allows for robust downstream analysis of collective motion. Because the AI algorithm required no new training data, our approach promises to be applicable to and yield new insights for vast libraries of existing collective motion images. In a systematic analysis of a cell line panel with oncogenic mutations, we find that the collective rearrangement metric, D2 min, which reflects non-affine motion, shows promise as an indicator of metastatic potential.

Actin Dynamics as a Multiscale Integrator of Cellular Guidance Cues.

Migrating cells must integrate multiple, competing external guidance cues. However, it is not well understood how cells prioritize among these cues. We investigate external cue integration by monitoring the response of wave-like, actin-polymerization dynamics, the driver of cell motility, to combinations of nanotopographies and electric fields in neutrophil-like cells. The electric fields provide a global guidance cue, and approximate conditions at wound sites in vivo. The nanotopographies have dimensions similar to those of collagen fibers, and act as a local esotactic guidance cue. We find that cells prioritize guidance cues, with electric fields dominating long-term motility by introducing a unidirectional bias in the locations at which actin waves nucleate. That bias competes successfully with the wave guidance provided by the bidirectional nanotopographies.

Cortical waves mediate the cellular response to electric fields.

Electrotaxis, the directional migration of cells in a constant electric field, is important in regeneration, development, and wound healing. Electrotaxis has a slower response and a smaller dynamic range than guidance by other cues, suggesting that the mechanism of electrotaxis shares both similarities and differences with chemical-gradient-sensing pathways. We examine a mechanism centered on the excitable system consisting of cortical waves of biochemical signals coupled to cytoskeletal reorganization, which has been implicated in random cell motility. We use electro-fused giant Dictyostelium discoideum cells to decouple waves from cell motion and employ nanotopographic surfaces to limit wave dimensions and lifetimes. We demonstrate that wave propagation in these cells is guided by electric fields. The wave area and lifetime gradually increase in the first 10 min after an electric field is turned on, leading to more abundant and wider protrusions in the cell region nearest the cathode. The wave directions display 'U-turn' behavior upon field reversal, and this switch occurs more quickly on nanotopography. Our results suggest that electric fields guide cells by controlling waves of signal transduction and cytoskeletal activity, which underlie cellular protrusions. Whereas surface receptor occupancy triggers both rapid activation and slower polarization of signaling pathways, electric fields appear to act primarily on polarization, explaining why cells respond to electric fields more slowly than to other guidance cues.

Electrically synchronizing and modulating the dynamics of ERK activation to regulate cell fate.

Intracellular signaling dynamics play fundamental roles in cell biology. Precise modulation of the amplitude, duration, and frequency of signaling activation will be a powerful approach to investigate molecular mechanisms as well as to engineer signaling to control cell behaviors. Here, we showed a practical approach to achieve precise amplitude modulation (AM), frequency modulation (FM), and duration modulation (DM) of MAP kinase activation. Alternating current (AC) electrical stimulation induced synchronized ERK activation. Amplitude and duration of ERK activation were controlled by varying stimulation strength and duration. ERK activation frequencies were arbitrarily modulated with trains of short AC applications with accurately defined intervals. Significantly, ERK dynamics coded by well-designed AC can rewire PC12 cell fate independent of growth factors. This technique can be used to synchronize and modulate ERK activation dynamics, thus would offer a practical way to control cell behaviors in vivo without the use of biochemical agents or genetic manipulation.

The social shape of sperm: using an integrative machine-learning approach to examine sperm ultrastructure and collective motility.

Sperm is one of the most morphologically diverse cell types in nature, yet they also exhibit remarkable behavioural variation, including the formation of collective groups of cells that swim together for motility or transport through the female reproductive tract. Here, we take advantage of natural variation in sperm traits observed across Peromyscus mice to test the hypothesis that the morphology of the sperm head influences their sperm aggregation behaviour. Using both manual and automated morphometric approaches to quantify their complex shapes, and then statistical modelling and machine learning to analyse their features, we show that the aspect ratio of the sperm head is the most distinguishing morphological trait and statistically associates with collective sperm movements obtained from in vitro observations. We then successfully use neural network analysis to predict the size of sperm aggregates from sperm head morphology and show that species with relatively wider sperm heads form larger aggregates, which is consistent with the theoretical prediction that an adhesive region around the equatorial region of the sperm head mediates these unique gametic interactions. Together these findings advance our understanding of how even subtle variation in sperm design can drive differences in sperm function and performance.