Biology subjects

Samuel, A. D. T.

Publications and source records attributed to Samuel, A. D. T..

15 recordsLinked to original sources

bioRxiv2025

The anticonvulsant and mood-stabilizing drug valproic acid attracts C. elegans and activates chemosensory neurons via a cGMP signaling pathway

Valproic acid (VPA) is a drug with both anticonvulsant and antimanic properties. It has been widely prescribed to treat epilepsy, bipolar disorder, and other neuropsychiatric conditions for decades, but prenatal exposure is linked to developmental anomalies, cognitive deficits, and an increased risk of autism. Remarkably, the molecular basis of VPA action in the brain is not fully understood. To determine VPAs effect on the nervous system without requiring systemic drug application that induce toxic effects, we exploited the well-characterized chemotaxis behavior of the nematode Caenorhabditis elegans. We found that C. elegans are attracted to VPA, and this behavior is missing in animals lacking the tax-4 ion channel and the tax-4-expressing AWC chemosensory neurons. To test the idea that VPA directly activates the AWC neurons, we performed calcium imaging studies in a line expressing GCaMP6s in all amphid chemosensory neurons in the head. We found that VPA evoked calcium transients consistently in the AWC neurons and variably in AWB and ASH. As cyclic nucleotide-gated channels, the active state of tax-4 increases upon the binding of cGMP. Within the worms chemosensory nervous system, receptor guanylate cyclases (rGCs) facilitate the synthesis of cGMP and serve as chemoreceptors for various chemical cues. By performing chemotaxis assays and calcium imaging experiments with rGC mutants against VPA, we found that odr-1 and gcy-28 are essential for mediating VPA attraction, as their absence disrupts both behavior and control AWC calcium transients. However, given their broad use in chemosensation, odr-1 and gcy-28 most likely act as downstream effectors rather than as direct targets. These findings compelled us to investigate whether chemoreceptors belonging to the G protein-coupled receptor (GPCR) family contribute to VPA sensing. We approached this hypothesis by conducting chemotaxis assays with G mutants and identified several (odr-3, egl-30, gpa-2;gpa-3) whose absence leads to a loss of VPA attraction. Thus, future studies should focus on elucidating the mechanisms by which GPCRs contribute to VPA sensing and cGMP signaling.

neuroscience↗

bioRxiv2025

Permeabilization with fenchone enhances cryopreservation of Drosophila embryos

The difficulty of cryopreservation has long been a limitation of Drosophila melanogaster as a genetic model organism. Here we report a statistically significant improvement in the efficiency of Drosophila cryopreservation by substituting limonene with the monoterpenoid fenchone in the embryo permeabilization step of a previously published method. We found that fenchone-permeabilized embryos exhibit greater uptake of cryoprotectant compared with those permeabilized by limonene, and a ~6-fold increase in the rate of egg-to-adult survival for wild-type flies. Using this improved protocol, we successfully cryopreserved and revived precious strains after 12 months of storage in liquid nitrogen. These results suggest that fenchone is a superior permeabilizing agent for fly embryo cryopreservation, expanding possibilities for the long-term maintenance of Drosophila and other insect species. Further refinement of this approach may enable cryopreservation to replace continuous culture as the method of choice for routine maintenance of fly stocks.

physiology↗

bioRxiv2025

The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

The bacterial flagellar motor drives bacterial swimming and chemotaxis by rotating helical flagellar filaments. When Escherichia coli navigates chemical gradients, the motor switches from counterclockwise (CCW) during forward swimming to clockwise (CW) during direction-changing tumbles. The motor responds indirectly to extracellular chemosensory input to membrane-bound chemoreceptors using an intervening intracellular signaling pathway. How the motor responds to its direct input signal - the diffusible messenger CheY-P - remains poorly understood. Steady-state motor measurements have been modeled as an allosteric switch between CCW/CW states that depend on mean CheY-P levels. Allosteric models have suggested that as many as 20 CheY-P molecules can be bound to the motor when it switches rotational direction. But steady-state models cannot predict the sensitivity of the motor to dynamic changes in CheY-P that essentially modulate chemotactic behavior. We present an optogenetic reagent that precisely controls the direct dynamical input signal to the motor. We designed a "caged" molecule, Opto-CheY, that is transiently activated by photon absorption. We find that activation and binding of 1-3 CheY-P molecules is sufficient to switch the motor from the CCW to CW state. The sensitivity of the motor to small changes in CheY-P occupancy helps resolve a long-standing paradox about the high sensitivity of the chemotactic response to external sensory input. Optogenetic biochemistry by light-activated uncaging of signal molecules is a new strategy to dissect information-processing in the living cell. Significance StatementMotile bacteria swim to better environments by modulating the rotation of the bacterial flagellar motor. How this motor responds to intracellular signaling activity is poorly understood. The physiologically-relevant response of the motor is to transient activation of intracellular signaling molecules on the sub-second time scale of bacterial decision-making. Here, we report the first optogenetic probe that targets in vivo the output module of the the chemotactic network. We demonstrate that the motor has high dynamical sensitivity to the binding of single intracellular signaling molecules. This solves a long-standing problem of high sensitivity and signal amplification in the bacterial chemotactic response.

biophysics↗

bioRxiv2025

Torque-generating units of the bacterial flagellar motor are rotary motors

E. coli swims using helical flagellar filaments driven at their base by a rotary motor. Torque-generating stator units drive the bacterial flagellar motor (BFM) by transmitting mechanical power to a cytoplasmic rotor, the C-ring. Each stator unit is a proton-conducting heteromer. A central dimer of two MotB proteins anchor to the cell wall. A surrounding pentamer of five MotA proteins transmit mechanical power to the C-ring. This asymmetrical 5:2 structure is consistent with rotation as the mechanism of torque generation. Here, we test the hypothesis that the MotA5MotB2 stator units are rotary motors themselves and interact with the rotor like intermeshed gearwheels, where rotation of the C-ring is directly coupled to MotA5 rotation around the MotB2. We used in vivo polarized photo-bleaching microscopy. When a subset of fluorescent domains inside a multimer is rapidly photo-bleached by a strong pulse of polarized light, the induced polarization-dependent fluorescence of unbleached domains becomes a reporter of angular orientation. We applied polarized photo-bleaching microscopy to tethered cells rotating by single flagellar motors. We probed fluorescently-labeled MotA pentamer and MotB dimer calibrated to motor rotation. The MotB dimer rotates at the same angular speed as the cell body, consistent with its anchor to the cell wall. The MotA pentamer rotates [~]6.2x faster than the flagellar motor, revealing the gear ratio between stator and rotor. Significance StatementBacteria swim by rotating rigid helical flagellar filaments. Here, we find that the torque-generating unit that drives flagellar rotation is itself a rotary motor. Each torque-generating unit is a heteromeric macromolecular machine - a pentamer of MotA subunits that surround a dimer of proton-conducting MotB subunits. Torque is generated as the MotA spins around MotB. The MotA pentamer interacts with rotor of the flagellar motor in a manner resembling intermeshing gearwheels. The bacterial flagellar motor is driven by the first set of enmeshed gearwheels that has been described in any living cell.

microbiology↗

bioRxiv2025

Laterality and interhemispheric integration in the larval Drosophila olfactory system

All animals with bilateral symmetry must integrate the sensory input from the left and right sides of their bodies to make coherent perceptual decisions. In the Drosophila larva, olfactory receptor neurons project largely ipsilaterally, providing a tractable system for asking where and how interhemispheric integration arises downstream. We combined volumetric calcium imaging with unilateral sensory perturbations, connectomic analysis, and optogenetic manipulations to trace the propagation of left-right olfactory information across successive layers of the olfactory system. This approach implicates the mushroom body (MB) as a key substrate for interhemispheric integration of odor representations. Kenyon cell (KC) odor responses were almost entirely ipsilateral, indicating minimal functional coupling between the two MBs at the input level. In contrast, modulatory neurons (MBINs) exhibited highly symmetric responses to unilateral stimulation, suggesting that reinforcement signals are broadly shared across hemispheres. Nevertheless, odor responses in some MB output neurons (MBONs), up to 5 synapses downstream from the sensory periphery, preserve information about stimulus laterality. Moreover, we show that asymmetric activation of these MBONs can modulate the animals turning behavior in a side-biased manner. Finally, we provide direct evidence that larvae can exploit instantaneous spatial comparisons for navigation in certain sensory contexts. These findings suggest that the deeply lateralized architecture of the larval olfactory system balances the need for interhemispheric integration with the advantages of parallel sensory processing.

neuroscience↗

bioRxiv2024

Efficient pheromone navigation via antagonistic detectors

Chemotaxis to a moving potential mate that emits a volatile sex pheromone poses a navigation challenge requiring rapid, precise responses to maximize reproductive success. Volatile chemicals form gradients that differ from soluble compounds, potentially making navigation based on comparisons between spatially separated sensors unreliable for small-bodied animals. Here we show that, rather than a simple spatial comparison, Caenorhabditis elegans males employ an antagonistic strategy, comparing inputs from sex-shared head (AWA) and male-specific tail (PHD) sensory neurons with distinct response properties. Despite sharing a receptor, SRD-1, these detectors play different roles: AWAs promote forward movement and acceleration, while PHDs induce reversals and deceleration. In rising pheromone gradients, AWA activity dominates; in falling gradients, AWA inactivates, allowing PHD to correct trajectories. AWAs are essential for mate-searching, while PHDs are crucial for complex tasks. A minimal computational model reproduces these behaviors and infers how head-tail signals are combined. Thus, a sexually dimorphic, antagonistic sensory system enables adaptive navigation in dynamic environments

neuroscience↗

bioRxiv2024

EM-Compressor: Electron Microscopy Image Compression in Connectomics with Variational Autoencoders

The ongoing pursuit to map detailed brain structures at high resolution using electron microscopy (EM) has led to advancements in imaging that enable the generation of connectomic volumes that have reached the petabyte scale and are soon expected to reach the exascale for whole mouse brain collections. To tackle the high costs of managing these large-scale datasets, we have developed a data compression approach employing Variational Autoencoders (VAEs) to significantly reduce data storage requirements. Due to their ability to capture the complex patterns of EM images, our VAE models notably decrease data size while carefully preserving important image features pertinent to connectomics-based image analysis. Through a comprehensive study using human EM volumes (H01 dataset), we demonstrate how our approach can reduce data to as little as 1/128th of the original size without significantly compromising the ability to subsequently segment the data, outperforming standard data size reduction methods. This performance suggests that this method can greatly alleviate requirements for data management for connectomics applications, and enable more efficient data access and sharing. Additionally, we developed a cloud-based application named EM-Compressor on top of this work to enable on-thefly interactive visualization: https://em-compressor-demonstration.s3.amazonaws.com/EM-Compressor+App.mp4.

neuroscience↗

bioRxiv2023

Evolutionarily-conserved behavioral plasticity enables context-dependent performance of mating behavior in C. elegans

Behavioral plasticity helps humans and animals to achieve their goals by adapting their behaviors to different environments. Although behavioral plasticity is ubiquitous, many innate species-specific behaviors, such as mating, are often assumed to be stereotyped and unaffected by plasticity or learning, especially in invertebrates. Here, we describe a novel case of behavioral plasticity in the nematode C. elegans - under a different set of naturalistic conditions the male uses a unique, previously undescribed set of behavioral steps for mating. Under standard lab conditions (agar plates with bacterial food), the male performs parallel mating, a largely two-dimensional behavioral strategy where his body and tail remain flat on the surface and slide alongside the partner s body from initial contact to copulation. But when placed in liquid medium, the male performs spiral mating, a distinctly three-dimensional behavioral strategy where he winds around the partner s body in a helical embrace. The performance of spiral mating does not require a long-term change in growing conditions but it does improve with experience. This experience-dependent improvement involves a critical period - a time window around the L4 to early adult stage, which coincides with the development of most male-specific neurons. We tested several wild isolates of C. elegans and other Caenorhabditis species and found that most were capable of parallel mating on surfaces and spiral mating in liquids. We suggest that two- and three-dimensional mating strategies in Caenorhabditis are plastic, conditionally expressed phenotypes conserved across the genus, and which can be genetically "fixed " in some species.

animal behavior and cognition↗

bioRxiv2023

mEMbrain: an interactive deep learning MATLAB tool for connectomic segmentation on commodity desktops

Connectomics is fundamental in propelling our understanding of the nervous systems organization, unearthing cells and wiring diagrams reconstructed from volume electron microscopy (EM) datasets. Such reconstructions, on the one hand, have benefited from ever more precise automatic segmentation methods, which leverage sophisticated deep learning architectures and advanced machine learning algorithms. On the other hand, the field of neuroscience at large, and of image processing in particular, has manifested a need for user-friendly and open source tools which enable the community to carry out advanced analyses. In line with this second vein, here we propose mEMbrain, an interactive MATLAB-based software which wraps algorithms and functions that enable labeling and segmentation of electron microscopy datasets in a user-friendly user interface compatible with Linux and Windows. Through its integration as an API to the volume annotation and segmentation tool VAST, mEMbrain encompasses functions for ground truth generation, image preprocessing, training of deep neural networks, and on-the-fly predictions for proofreading and evaluation. The final goals of our tool are to expedite manual labeling efforts and to harness MATLAB users with an array of semi-automatic approaches for instance segmentation. We tested our tool on a variety of datasets that span different species at various scales, regions of the nervous system and developmental stages. To further expedite research in connectomics, we provide an EM resource of ground truth annotation from 4 different animals and 5 datasets, amounting to around 180 hours of expert annotations, yielding more than 1.2 GB of annotated EM images. In addition, we provide a set of 4 pre-trained networks for said datasets. All tools are available from https://lichtman.rc.fas.harvard.edu/mEMbrain/. With our software, our hope is to provide a solution for lab-based neural reconstructions which does not require coding by the user, thus paving the way to affordable connectomics.

neuroscience↗

bioRxiv2022

Automated neuron tracking inside moving and deforming animals using deep learning and targeted augmentation

Advances in functional brain imaging now allow sustained rapid 3D visualization of large numbers of neurons inside behaving animals. To decode circuit activity, imaged neurons must be individually segmented and tracked. This is particularly challenging when the brain itself moves and deforms inside a flexible body. The field has lacked general methods for solving this problem effectively. To address this need, we developed a method based on a convolutional neural network (CNN) Awith specific enhancements which we apply to freely moving Caenorhabditis elegans. For a traditional CNN to track neurons across images of a brain with different postures, the CNN must be trained with ground truth (GT) annotations of similar postures. When these postures are diverse, an adequate number of GT annotations can be prohibitively large to generate manually. We introduce targeted augmentation, a method to automatically synthesize reliable annotations from a few manual annotations. Our method effectively learns the internal deformations of the brain. The learned deformations are used to synthesize annotations for new postures by deforming the manual annotations of similar postures in GT images. The technique is germane to 3D images, which are generally more difficult to analyze than 2D images. The synthetic annotations, which are added to diversify training datasets, drastically reduce manual annotation and proofreading. Our method is effective both when neurons are represented as individual points or as 3D volumes. We provide a GUI that incorporates targeted augmentation in an end-to-end pipeline, from manual GT annotation of a few images to final proofreading of all images. We apply the method to simultaneously measure activity in the second-layer interneurons in C. elegans: RIA, RIB, and RIM, including the RIA neurite. We find that these neurons show rich behaviors, including switching entrainment on and off dynamically when the animal is exposed to periodic odor pulses.

neuroscience↗

bioRxiv2020

Natural sensory context drives diverse brain-wide activity during C. elegans mating

Natural goal-directed behaviors often involve complex sequences of many stimulus-triggered components. Understanding how brain circuits organize such behaviors requires mapping the interactions between an animal, its environment, and its nervous system. Here, we use continuous brain-wide neuronal imaging to study the full performance of mating by the C. elegans male. We show that as each mating unfolds in its own sequence of component behaviors, the brain operates similarly between instances of each component, but distinctly between different components. When the full sensory and behavioral context is taken into account, unique roles emerge for each neuron. Functional correlations between neurons are not fixed, but change with behavioral dynamics. From the contribution of individual neurons to circuits, our study shows how diverse brain-wide dynamics emerge from the integration of sensory perception and motor actions within their natural context.

neuroscience↗

bioRxiv2020

Extracting neural signals from semi-immobilized animals with deformable non-negative matrix factorization

Extracting calcium traces from populations of neurons is a critical step in the study of the large-scale neural dynamics that govern behavior. Accurate activity extraction requires the correction of motion and movement-induced deformations as well as demixing of signals that may overlap spatially due to limitations in optical resolution. Traditionally, non-negative matrix factorization (NMF) methods have been successful in demixing and denoising cellular calcium activity in relatively motionless or pre-registered videos. However, standard NMF methods fail in animals undergoing significant non-rigid motion; similarly, standard image registration methods based on template matching can fail when large changes in activity lead to mismatches with the image template. To address these issues simultaneously, we introduce a deformable non-negative matrix factorization (dNMF) framework that jointly optimizes registration with signal demixing. On simulated data and real semi-immobilized C. elegans microscopy videos, dNMF outperforms traditional demixing methods that account for motion and demixing separately. Finally, following the extraction of neural traces from multiple imaging experiments, we develop a quantile regression time-series normalization technique to account for varying neural signal intensity baselines across different animals or different imaging setups. Open source code implementing this pipeline is available at https://github.com/amin-nejat/dNMF.

neuroscience↗

bioRxiv2020

Connectomes across development reveal principles of brain maturation in C. elegans

From birth to adulthood, an animals nervous system changes as its body grows and its behaviours mature. The form and extent of circuit remodelling across the connectome is unknown. We used serial-section electron microscopy to reconstruct the full brain of eight isogenic C. elegans individuals across postnatal stages to learn how it changes with age. The overall geometry of the brain is preserved from birth to adulthood. Upon this constant scaffold, substantial changes in chemical synaptic connectivity emerge. Comparing connectomes among individuals reveals substantial connectivity differences that make each brain partly unique. Comparing connectomes across maturation reveals consistent wiring changes between different neurons. These changes alter the strength of existing connections and create new connections. Collective changes in the network alter information processing. Over development, the central decision-making circuitry is maintained whereas sensory and motor pathways substantially remodel. With age, the brain progressively becomes more feedforward and discernibly modular. Developmental connectomics reveals principles that underlie brain maturation.

neuroscience↗

bioRxiv2017

Invariances in a combinatorial olfactory receptor code

Animals can identify an odorant type across a wide range of concentrations, as well as detect changes in concentration for individual odorant type. How olfactory representations are structured to support these functions remains poorly understood. Here, we studied how a full complement of ORNs in the Drosophila larva encodes a broad input space of odorant types and concentrations. We find that dose-response relationships across odorants and ORN types follow the Hill function with shared cooperativity but different activation thresholds. These activation thresholds are drawn from a power law statistical distribution. A fixed activation function and power law distribution of activation thresholds underlie invariances in the encoding of odorant identity and intensity. Moreover, we find similar temporal response filters of ORNs across odorant types and concentrations. Such uniformity in the temporal filter may allow identity invariant coding in fluctuating or turbulent odor environments. Common patterns in ligand-receptor binding and sensory transduction across olfactory receptors may give rise to these observed invariances in the olfactory combinatorial code. Invariant patterns in the activity responses of individual ORNs and the ORN ensemble may simplify decoding by downstream circuits.

neuroscience↗

bioRxiv2017

Integration of plasticity mechanisms within a single sensory neuron of C. elegans actuates a memory

Neural plasticity--the ability of a neuron to change its cellular properties in response to past experiences--underpins the nervous systems capacity to form memories and actuate behaviors. How different plasticity mechanisms act together in vivo and at a cellular level to transform sensory information into behavior is not well understood. Here we show that in the nematode C. elegans two plasticity mechanisms--sensory adaptation and presynaptic plasticity--act within a single cell to encode thermosensory information and actuate a temperature-preference memory. Sensory adaptation enables the primary thermosensory neuron, AFD, to adjust the temperature range of its sensitivity to the local environment, thereby optimizing its ability to detect temperature fluctuations associated with migration. Presynaptic plasticity transforms this thermosensory information into a behavioral preference by gating synaptic communication between sensory neuron AFD and its postsynaptic partner, AIY. The gating of synaptic communication is regulated at AFD presynaptic sites by the conserved kinase nPKC{varepsilon}. Bypassing or altering AFD presynaptic plasticity predictably changes the learned behavioral preferences without affecting sensory responses. Our findings indicate that two distinct and modular neuroplasticity mechanisms function together within a single sensory neuron to encode multiple components of information required to enact thermotactic behavior. The integration of these plasticity mechanisms result in a single-cell logic system that can both represent sensory stimuli and guide memory-based behavioral preference.

neuroscience↗