Biology subjects

Gilad, A.

Publications and source records attributed to Gilad, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Cortex-wide representational drift of different layers

Representational drift, the gradual evolution of neural population codes over days, has been widely documented at the level of single neurons. However, whether drift is organized across larger cortical populations and spatial scales remains unclear. Here, we examined cortex-wide population dynamics under tightly controlled sensory input and behavior. Using widefield calcium imaging, we longitudinally recorded excitatory activity in Layers 2/3 (L2/3) or Layer 5 (L5) across 25 cortical areas while mice performed a whisker-based texture discrimination task. Sensory-evoked activity was tracked over five consecutive days during stable task performance. Population activity patterns reorganized over days, across the cortex, in both layers. This reorganization followed distinct laminar motifs: notably L5 responses exhibited a widespread, monotonic decrease in activity across most cortical areas, whereas L2/3 responses showed spatially localized and heterogeneous changes that were strongest during the sensory period. These laminar differences extended beyond the stimulus period, with L5 exhibiting more prolonged temporal engagement than L2/3. Together, these findings indicate that representational drift can unfold as a process that is coordinated across cortex with distinct laminar profiles. Thus, drift may reflect a structured feature of cortex-wide circuit dynamics.

neuroscience↗

bioRxiv2026

Layer-specific wide-field calcium imaging of neocortical activity

The mammalian neocortex is highly organized in local microcircuits and through long-range projection patterns between different regions. Since various features of local and long-range connectivity are determined by the cortical layer in which the respective neurons reside, understanding the flow of cortical information within and across layers is essential. Wide-field calcium imaging enables mesoscale functional mapping of genetically identified neurons across cortical areas. However, it has been applied primarily to superficial cortical layers, and systematic comparisons of wide-field signals across cortical layers are scarce. Here, we apply wide-field calcium imaging to different cortical layers using transgenic mouse lines with selective expression of GCaMP6f in layers 2/3, 5, and 6. We address several challenges of layer-specific wide-field imaging and provide possible solutions. First, to improve the registration of functional data to standard atlases, we demonstrate the benefit of layer-specific registration maps that are warped on the basis of the depth-dependent surface projections of the labeled cell populations. These maps help to assign the imaged calcium signals to the specific regions from which they originate. Second, we measure the depth-dependent blurring of wide-field fluorescence signals induced by light scattering and reveal stronger blurring in deep vs. superficial layers, in line with previous theoretical predictions from simulations. We used measured point spread functions to deconvolve single-whisker-evoked calcium signals in the barrel cortex and demonstrate improved signal confinement to individual barrel columns across layers. Finally, we investigate cross-regional functional connectivity during awake resting state periods for distinct layers. We find that mesoscopic functional connectivity is largely conserved between the cortical layers, with subtle differences for key regions of the default mode network (retrosplenial cortex and medial prefrontal cortex). Our approaches facilitate the comprehensive characterization of layer-specific cortico-cortical interactions, expanding wide-field calcium imaging as a powerful tool to investigate the layered organization of distributed brain dynamics.

neuroscience↗

bioRxiv2026

Interhemispheric transfer of sensory and working memory information is dictated by behavioral strategy

The cerebral hemispheres process sensory information largely independently, yet coherent perception and behavior requires rapid interhemispheric communication. Which cortical area transfer information across hemispheres and what governs transfer location remains poorly understood. Here, we combined bilateral whisker-based texture-matching tasks with cortex-wide calcium imaging in mice to investigate the interhemispheric transfer of sensory and working memory information. Mice compared textures presented either simultaneously to both whisker pads or sequentially with a delay, thereby requiring the transfer of sensory or working memory content across hemispheres. Despite identical task demands, animals adopted distinct active or passive behavioral strategies that dictate the cortical location of interhemispheric transfer. Passive mice route both sensory and working memory information through the posterior lateral association cortex, whereas active mice rely primarily on bilateral barrel cortices. During delayed comparisons, active mice rapidly shift information to the contralateral barrel cortex, exhibit impatience, and display reduced performance, indicating that barrel cortices are suboptimal for transferring working memory. These findings identify behavioral strategy as a key determinant of interhemispheric communication and demonstrate a flexible, state-dependent routing of information across the cortex.

neuroscience↗

bioRxiv2025

Magneto-Photonic Gene Circuit for Minimally Invasive Control of Gene Expression in Mammalian Cells

Precise control of gene expression is one of the fundamental goals of synthetic biology. Whether the objective is to modify endogenous cellular function or induce the expression of molecules for diagnostic and therapeutic purposes, gene regulation remains a key aspect of biological systems. Over time, advances in protein engineering and molecular biology have led to the creation of gene circuits capable of inducing the expression of specific proteins in response to external stimulus such as light. These optogenetic, or light-activated circuits hold significant potential for gene therapy as a tool for regulating the expression of therapeutic genes within cells. However, the applications of optogenetic systems can be limited by the lack of efficient ways for light delivery inside cells or tissue. Our approach to address this challenge is to harness the power of bioluminescence to produce light directly inside cells using a luminescent enzyme. Combined with a photosensitive transcription factor, we report the development of a fully genetically encoded optogenetic circuit for control of gene expression. Furthermore, we utilized a magneto sensitive protein to engineer a split protein version of this luminescent enzyme, where its reconstitution is driven by a 50mT magnetic stimulus. Thus, resulting in a first-of-its-kind gene circuit activated by a combination of light and magnetic stimulus. We expect this work to advance the implementation of light-controlled systems without the need of external light sources, as well as serve as a basis for the development of future magneto-sensitive tools.

synthetic biology↗

bioRxiv2025

Brain-Wide Subnetworks within and between Naturally Socializing Typical and Autism Model Mice

Social interaction is inherently asymmetric, requiring coordinated activity between non-homologous brain regions across individuals. However, the brain-wide dynamics underlying such inter-brain coordination remain poorly understood. We used multi-fiber photometry to simultaneously record from 24 brain regions in pairs of freely interacting mice, including a model of autism. Social interactions evoked widespread, dynamic activity across brains, with inter-brain synchrony, especially between non-homologous areas, exceeding intra-brain synchrony, particularly in dominant mice. Network analysis revealed three subnetworks: (1) Emotional, intra-brain enhanced in subordinates; (2) Sensory, spanning both mice; (3) Decision/consolidation, linking dominant prefrontal cortex to subordinate hippocampus. These subnetworks encoded dominance, identity, and interaction roles, and followed a clear temporal sequence around social events. In an autism model, socially evoked activity was hyperactive displaying mostly within brain synchrony but lacked inter-brain synchrony. Our results uncover dynamic inter-brain circuits as a hallmark of social behavior and reveal their disruption in autism.

neuroscience↗

bioRxiv2025

Cortex-wide laminar dynamics diverge during learning

Learning to link sensory information to motor actions involves dynamic coordination across cortical layers and regions. However, the involvement of a particular layer in learning, especially from a cortex-wide perspective, is relatively unknown. Using wide-field calcium imaging in mice as they learn a whisker-based go/no-go task, we tracked activity in layers 2/3 or 5 (L2/3 or L5) across 25 cortical areas. A surprising initial effect of learning was that activity in L5 but not in L2/3 was globally suppressed at auditory cue onset. As the texture comes into touch, we found that L2/3 displayed learning-related enhancements in higher order association areas rostrolateral (RL) and secondary somatosensory (S2), whereas L5 in these areas oppositely decreased. During texture touch, the barrel cortex (BC) displayed similar learning-related enhancement in both layers. As sensory information is transformed into a motor action, there was a frontal/posterior divergence that emerges after learning, in which L5 was enhanced in the frontal cortex and L2/3 was suppressed in the posterior cortex. In general, learning related correlations were often stronger between distant cortical layers than within the same column, suggesting that learning drives laminar interactions that transcend traditional columnar organization. Together, these results reveal that learning orchestrates a dynamic interplay of activity across space, time and cortical layers. Our findings emphasize the critical role of laminar architecture in shaping cortical plasticity and support the view that layer-specific circuits are fundamental to sensorimotor learning.

neuroscience↗

bioRxiv2025

Using Manganese-Enhanced MRI to visualize Magnetogenetic-based Neuromodulation

PurposeInvestigation of the electromagnetic perceptive gene (EPG) protein and garnering evidence to suggest its use as a magnetogenetic tool for neuromodulation. Activation of EPG by electromagnetic field increases intracellular calcium levels, thus, we aimed to determine whether EPG influences the analogous manganese ion. This work yields potential for manganese-enhanced MRI (MEMRI) to be used to monitor EPG acting as a neuromodulator. MethodsHEK293FT cells expressing EPG were exposed to a MnCl2 solution and stimulated with a static or electromagnet. Excess MnCl2 was washed off the cells, followed by their collection and lysis. T1 map measurements of the lysates were obtained to gauge the presence of intracellular Mn2+. Several controls were employed to critically evaluate whether EPG can influence Mn2+ dynamics. ResultsLysate of cells expressing EPG showed significantly lower T1 values compared to cells without EPG that were exposed to the same MnCl2 solution and magnetic stimulus. ConclusionMagnetic activation of EPG increases the uptake of Mn2+ into the cell. By influencing ions pertinent to neuronal function, we demonstrate the potential of MEMRI to monitor EPG neuromodulatory activity.

biophysics↗

bioRxiv2025

'What' and 'where' brain-wide pathways are dominated by internal strategies

It is long thought that higher-order sensory processing is divided into two specialized cortical streams that encode in parallel either the identity of an object or its location (i.e., what and where streams). Here, using the mouse whisker system, we challenge this concept by demonstrating an existence of two alternating brain-wide (beyond cortex) subnetworks that are not primarily driven by external parameters, but rather by internal strategies. We combine simultaneous brain-wide neuronal recordings in mice trained to identify or locate a certain stimulus. We find that mice deploy either an active search or a passive sensation strategy during task performance. These strategies respectively drive two distinct and brain-wide subnetworks, frontal and posterior, regardless of the type of task performed. The posterior subnetwork encoded additional internal strategic parameters such as trial history, the first task of the day, and training history. A subgroup of trials that were not dominated by frontal cortex, contained meaningful task information in the posterior cortex and several thalamic areas. Integrated together, these two subnetworks may comprise normal cognitive function.

neuroscience↗

bioRxiv2025

Distinct roles of cortical layer 5 subtypes in associative learning

Adaptive behavior is critically dependent on associative learning, where environmental cues are linked with subsequent positive or negative outcomes. In mammals, primary neocortical sensory areas serve as pivotal nodes in this process, processing stimuli and distributing information to cortical and subcortical networks. Layer 5 (L5) of the cortex comprises two types of pyramidal projection neurons--intratelencephalic (IT) and extratelencephalic (ET) neurons--each with distinct downstream targets. Despite the crucial function of L5 as a main output node of the cortex, the specific contributions of these L5 neuronal subtypes to associative learning remain poorly understood. In the present study, by leveraging transgenic mouse lines, we distinguished IT and ET neurons in the primary somatosensory cortex and examined their roles in a whisker-based frequency-discrimination learning task. Longitudinal two-photon calcium imaging revealed distinct response characteristics between IT and ET neurons throughout learning. Interestingly, the activity of IT neurons hardly changed over the five days of learning, while the activity of ET neurons developed robustly. Furthermore, IT neurons appeared to show stimuli encoding from the beginning, whereas the ET neurons became increasingly responsive to stimuli associated with reward. Chemogenetic silencing of either IT or ET neurons both impaired learning, but in strikingly distinct ways, each associated with a different phase of learning. By modeling the response characteristics of IT and ET neurons using a reinforcement learning framework, we show that IT neurons primarily encode sensory stimuli, and their representations are critical for forming stimulus-reward associations. ET neurons instead represent the value of the stimulus, used for refining behavior. Thus, our results delineate the distinct roles of L5 IT and ET neurons, underscoring their integral and complementary contributions to associative learning.

neuroscience↗

bioRxiv2024

The Proton Resonance Enhancement for CEST imaging and Shift Exchange (PRECISE) family of RF pulse shapes for Chemical Exchange Saturation Transfer MRI

PurposeTo optimize a 100 msec pulse for producing CEST MRI contrast and evaluate in mice. MethodsA gradient ascent algorithm was employed to generate a family of 100 point, 100 msec pulses for use in CEST pulse trains ( PRECISE). Gradient ascent optimizations were performed for exchange rates (kca) = 500 s-1, 1,500 s-1, 2,500 s-1, 3,500 s-1 and 4,500 s-1 and offsets ({Delta}{omega}) = 9.6, 7.8, 4.2 and 2.0 ppm. 7 PRECISE pulse shapes were tested on an 11.7 T scanner using a phantom containing three representative CEST agents with peak saturation B1 = 4 T. The pulse producing the most contrast in phantoms was then evaluated for CEST MRI pH mapping of the kidneys in healthy mice after iopamidol administration. ResultsThe most promising pulse in terms of contrast performance across all three phantoms was the 9.6 ppm, 2500 s-1 optimized pulse with [~]2.7 x improvement over Gaussian and [~]1.3xs over Fermi pulses. This pulse also displayed a large improvement in contrast over the Gaussian pulse after administration of iopamidol in live mice. ConclusionA new 100 msec pulse was developed based on gradient ascent optimizations which produced better contrast compared to standard Gaussian and Fermi pulses in phantoms. This shape also showed a substantial improvement for CEST MRI pH mapping in live mice over the Gaussian shape and appears promising for a wide range of CEST applications.

bioengineering↗

bioRxiv2024

A conserved phenylalanine motif among Teleost fish provides insight for improving electromagnetic perception.

Magnetoreceptive biology as a field remains relatively obscure; compared to the breadth of species believed to sense magnetic fields, it remains under-studied. Here, we present grounds for the expansion of magnetoreception studies among Teleosts. We begin with the electromagnetic perceptive gene (EPG) from Kryptopterus vitreolus and expand to identify 72 Teleosts with homologous proteins containing a conserved three-phenylalanine (3F) motif. Phylogenetic analysis provides insight as to how EPG may have evolved over time, and indicates that certain clades may have experienced a loss of function driven by different fitness pressures. One potential factor is water type with freshwater fish significantly more likely to possess the functional motif version (FFF), and saltwater fish to have the non-functional variant (FXF). It was also revealed that when the 3F motif from the homolog of Brachyhypopomus gauderio (B.g.) is inserted into EPG - EPG(B.g.) - the response (as indicated by increased intracellular calcium) is faster. This indicates that EPG has the potential to be engineered to improve upon its response and increase its utility to be used as a controller for specific outcomes.

synthetic biology↗

bioRxiv2023

Magnetogenetic stimulation inside MRI induces spontaneous and evoked changes in neural circuits activity in rats

The ability to modulate specific neural circuits and simultaneously visualize and measure brain activity with MRI would greatly impact understanding brain function in health and disease. The combination of neurostimulation methods and MRI in animal models have already shown promise in elucidating fundamental mechanisms associated with brain activity. We developed an innovative magnetogenetics neurostimulation technology that can trigger neural activity through magnetic fields. Similar to other genetic-based neuromodulation methods, magnetogenetics offers cell-, area- and temporal- specific control of neural activity. However, the magnetogenetics protein (Electromagnetic Preceptive Gene (EPG)) are activated by non-invasive magnetic fields, providing a unique way to target neural circuits by the MRI gradients while simultaneously measure their effect on brain activity. EPG was expressed in rats visual cortex and the amplitude of low-frequency fluctuation (fALFF), resting-state functional connectivity (FC), and sensory activation was measured using a 7T MRI. The results demonstrate that EPG-expressing rats had significantly higher signal fluctuations in the visual areas and stronger FC in sensory areas consistent with known anatomical visuosensory and visuomotor connections. This new technology complements the existing neurostimulation toolbox and provides a mean to study brain function in a minimally-invasive way which was not possible previously.

neuroscience↗

bioRxiv2023

Chaperone Complexes From The Endoplasmic Reticulum (ER) And The Cytosol Inhibit wt-p53 By Activation The ER To Cytosol Signaling.

The Endoplasmic Reticulum (ER) is an essential sensing organelle responsible for the folding and secretion of almost one-third of eukaryotic cells total proteins. The ER contains numerous enzymes and chaperones which assist in oxidative protein folding and other posttranslational modifications. However, environmental, chemical, and genetic insults often lead to protein misfolding in the ER, accumulating misfolded proteins, altering homeostasis, and causing ER stress. Recently, we reported a novel ER surveillance mechanism by which proteins from the secretory pathway are refluxed to the cytosol to relieve the ER of its content during stress. In cancer cells, the refluxed proteins gain new pro-survival functions, thereby increasing cancer cell fitness. We termed this phenomenon ER to CYtosol Signaling (or "ERCY"). In yeast, ERCYS is regulated by HLJ1 (an ER-resident tail-anchored HSP40 cochaperone). Here, we found that in mammalian cells, HLJ1 has five putative orthologs possessing J-domains facing the cytosol. Among those, DNAJB12 and DNAJB14 appear to be the most significant. Mechanistically, we found that DNAJB12 and DNAJB14 bind the cytosolic HSC70 and its cochaperone - SGTA - through their cytosolically localized J-domains to facilitate ER-protein reflux to the cytosol. Moreover, we found that DNAJB12 is necessary and sufficient to drive this phenomenon to increase AGR2 reflux and inhibit wt-p53 during ER stress. Thus, we concluded that targeting the DNAJB12/14-HSC70/SGTA axis is a promising strategy to inhibit ERCYS and impair cancer cell fitness.

cell biology↗

bioRxiv2023

A Novel Protein for the Bioremediation of Gadolinium Waste

Several hundreds of tons of gadolinium-based contrast agents (GBCAs) are being dumped into the environment every year. Although macrocyclic GBCAs exhibit superior stability compared to their linear counterparts, we have found that the structural integrity of chelates are susceptible to ultraviolet light, regardless of configuration. In this study, we present a synthetic protein termed GLamouR that binds and reports gadolinium in an intensiometric manner. We then explore the extraction of gadolinium from GBCA-spiked artificial urine samples and investigate if the low picomolar concentrations reported in gadolinium-contaminated water sources pose a barrier for bioremediation. Based on promising results, we anticipate GLamouR can be used for detecting and mining REEs beyond gadolinium as well and hope to expand the biological toolbox for such applications.

bioengineering↗

bioRxiv2022

Proposed three-phenylalanine motif involved in magnetoreception signaling of an Actinopterygii protein expressed in mammalian cells

Studies at the cellular and molecular level of magnetoreception - sensing and responding to magnetic fields - is a relatively new research area. As it appears that different mechanisms of magnetoreception in animals evolved from different origins, many questions about the mechanisms remain left open. Here we present new information regarding the Electromagnetic Perceptive Gene (EPG) from Kryptopterus vitreolus that may serve as part of the foundation to understanding and applying magnetoreception. Using HaloTag coupled with fluorescent ligands and phosphatidylinositol specific phospholipase C (PI-PLC) we show that EPG is associated to the membrane via glycosylphosphatidylinositol (GPI) anchor. EPGs function of increasing intracellular calcium was also used to generate an assay using GCaMP6m to observe the function of EPG and to compare its function with homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues in order to function - stably swapping these residues using site directed mutagenesis resulted in a loss of function in EPG. This information not only expands upon our current understanding of magnetoreception but may provide a foundation and template to continue characterizing and discovering more within the field. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/519643v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@19a4f54org.highwire.dtl.DTLVardef@1ce17b2org.highwire.dtl.DTLVardef@175b97borg.highwire.dtl.DTLVardef@1e45294_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefEPG is a magnetoreceptive GPI anchored protein. Critical to its function is a three-phenylalanine motif which allows EPG to sense and respond to EMF. When expressed in mammalian cell, an increase in intracellular calcium is observed using GCaMP6m. This work represents progress towards understanding magnetoreception for use in future technologies. HighlightsO_LIEPG is associated to the cell membrane via glycosylphosphatidylinositol anchoring C_LIO_LIIn mammalian cells, EPG increases intracellular calcium upon EMF stimulation C_LIO_LIHomologs of EPG from the uPAR/Ly6 family show different responses to EMF C_LIO_LIA three-phenylalanine motif in EPG is critical to its magnetoreceptive ability C_LI

molecular biology↗

bioRxiv2022

History information emerges in the cortex during learning

We learn from our experience but the underlying neuronal mechanisms incorporating past information to facilitate learning is relatively unknown. Specifically, which cortical areas encode history-related information and how is this information modulated across learning? To study the relationship between history and learning, we continuously imaged cortex-wide calcium dynamics as mice learn to use their whiskers to discriminate between two different textures. We mainly focused on comparing the same trial type with different history information, i.e., a different preceding trial. We found history information in barrel cortex (BC) during stimulus presentation. Importantly, history information in BC emerged only as the mouse learned the task. Next, we also found learning-dependent history information in rostrolateral (RL) association cortex that emerges before stimulus presentation, preceding activity in BC. History information was also found in other cortical areas and was not related to differences in body movements. Interestingly, a binary classifier could discriminate history information at the single trial level just as well as current information both in BC and RL. These findings suggest that past experience emerges in the cortex around the time of learning, starting from higher-order association area RL and propagating down (i.e., top-down projection) to lower-order BC where it can be integrated with incoming sensory information. This integration between the past and present may facilitate learning.

neuroscience↗

bioRxiv2022

Protein Optimization Evolving Tool (POET) based on Genetic Programming

Proteins are used by scientists to serve a variety of purposes in clinical practice and laboratory research. To optimize proteins for greater function, a variety of techniques have been developed. For the development of reporter genes used in Magnetic Resonance Imaging (MRI) based on Chemical Exchange Saturation Transfer (CEST), these techniques have encountered a variety of challenges. Here we develop a mechanism of protein optimization using a computational approach known as "genetic programming". We developed an algorithm called Protein Optimization Evolving Tool (POET). Starting from a small library of literature values, use of this tool allowed us to develop proteins which produce four times more MRI contrast than what was previously state-of-the-art. Next, we used POET to evolve peptides that produced CEST-MRI contrast at large chemical shifts where no other known peptides have previously demonstrated contrast. This demonstrated the ability of POET to evolve new functions in proteins. Interestingly, many of the peptides produced using POET were dramatically different with respect to their sequence and chemical environment than existing CEST producing peptides, and challenge prior understandings of how those peptides function. This suggests that unlike existing algorithms for protein engineering that rely on divergent evolution, POET relies on convergent evolution.

synthetic biology↗

bioRxiv2021

A Dual-reporter Platform for Screening Tumor-targeted Extracellular Vesicles

Extracellular vesicle (EV)-mediated transfer of biomolecules plays an essential role in intercellular communication and may improve targeted drug delivery. In the past decade, various approaches to EV surface modification for targeting specific cells or tissues have been proposed, including genetic engineering of parental cells or postproduction EV engineering. However, due to technical limitations, targeting moieties of engineered EVs have not been thoroughly characterized. Here, we report the bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL-based dual-reporter platform for characterizing the cellular uptake of tumor homing peptide (THP)-engineered EVs, targeting PDL1, uPAR, or EGFR proteins expressed in MDA-MB-231 breast cancer cells, simultaneously by bioluminescence measurement and fluorescence microscopy. Bioluminescence analysis of cellular EV uptake revealed the highest binding efficiency of uPAR-targeted EVs, whereas PDL1-targeted EVs showed slower cellular uptake. EVs engineered with two known EGFR-binding peptides via lipid nanoprobes did not increase cellular uptake, indicating that designs of EGFR-binding peptide conjugation to the EV surface are critical for functional EV engineering. Fluorescence analysis of cellular EV uptake allowed us to track individual PalmReNL-EVs bearing THPs in recipient cells. These results demonstrate that the PalmReNL-based EV assay platform can be a foundation for high-throughput screening of tumor-targeted EVs.

bioengineering↗