Biology subjects

Levy, A. D.

Publications and source records attributed to Levy, A. D..

12 recordsLinked to original sources

bioRxiv2026

Embodied Dynamic Causal Modelling: Brain and Cardiac Effective Connectivity

Brain-body interactions underpin embodied inference and interoception, and are central to both general health and the pathophysiology of mental health disorders. These interactions are orchestrated across a hierarchy extending from the cortex, through the brainstem, to peripheral autonomic effectors. Existing methods either reduce this communication to scalar summaries or lack an invertible biophysical model, and so cannot resolve where in this extended architecture an observed effect originates. To address this, we introduce Embodied Dynamic Causal Modelling, an extension of the generative causal modelling framework that formalises hierarchical message passing across the full brain-body axis. We instantiate the framework for brain-heart interactions with the Brainstem Cardiac Network, a closed-loop neural mass model of brainstem autonomic circuitry and sinoatrial node dynamics. By incorporating five brainstem nuclei, a viscerosensory thalamic relay and the cardiac pacemaker, the network enables estimation of directed effective connectivity across the cortical-brainstem-cardiac axis. As an illustrative case study, representative of the experiments and extended physiological systems that Embodied DCM and the BCN can now interrogate, we applied the framework to simultaneously recorded electroencephalography and electrocardiography during an auditory Go/No-Go paradigm. A multimodal observation model permits joint inversion of electrophysiological and cardiac responses, so that neural and bodily measurements together constrain an account of central and peripheral dynamics. The case study shows how the framework can arbitrate between competing hypotheses about functional cytoarchitecture, distinguishing granular from agranular microcircuits within the viscero-sensorimotor hierarchy. It also shows how time-varying estimates of synaptic efficacy can be recovered alongside continuous latent neuronal dynamics, over extended windows spanning anticipation, target processing and motor inhibition. By linking neural and cardiac signals to a single generative architecture, this work moves the study of brain-heart interactions beyond correlation, providing a framework for testing theories of embodied inference and for identifying circuit-level disruptions in disorders of interoceptive and autonomic regulation.

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bioRxiv2026

Nanoarchitecture of the presynaptic cell adhesion molecule PTPσ in relation to prospective vesicle fusion sites and postsynaptic NMDA receptors

Protein organization at individual synapses tunes synaptic function. Presynaptic release-regulating proteins and postsynaptic receptors each concentrate into subsynaptic nanoclusters, and their precise alignment across the cleft into nanocolumns can maximize receptor activation following vesicle fusion. However, both the molecular organization defining presynaptic fusion sites and the mechanisms by which they are spatially coordinated with postsynaptic complexes remains unclear. Presynaptic transmembrane cell adhesion molecules may mediate both processes as they could simultaneously anchor cytosolic fusion machinery to the presynaptic membrane and bridge the cleft to coordinate PSD organization. The LAR-RPTP family member PTP{sigma} is an important possibility, as its trans-synaptic interactors that bind PSD-95 are associated with NMDAR dysfunction, suggesting PTP{sigma} may coordinate NMDAR positioning. However, PTP{sigma}'s subsynaptic organization has not been determined. Here, we investigate the cellular expression and subsynaptic nano-organization of endogenous PTP{sigma} in cultured rat hippocampal neurons. We find that PTP{sigma} is enriched at most excitatory synapses, but unexpectedly, PTP{sigma} nanoclusters do not colocalize with the vesicle priming protein Munc13-1. Instead, they adopt a stereotyped position adjacent to this machinery, without preferential association with sites in nanocolumns. PTP{sigma} nanoclusters also lie offset from postsynaptic NMDA receptors, mirroring their relationship to vesicle fusion sites, yet the presence of PTP{sigma} at a nanocolumn does not predict NMDAR abundance. Together, these findings suggest that PTP{sigma} does not directly coordinate release site - NMDAR alignment but instead occupies defined nanoscale positions adjacent to vesicle fusion sites, potentially helping to shape or stabilize the vesicle priming landscape of the active zone.

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bioRxiv2026

Btbd11 regulates glutamatergic synapse organization in GABAergic inhibitory interneurons

The mechanisms that underlie glutamatergic synapse organization and function in GABAergic inhibitory interneurons (INs) are not well described, despite evidence that impaired glutamatergic excitation of INs is implicated in psychiatric disorders such as schizophrenia and anxiety. Glutamatergic synapses received by INs have unique basal transmission properties and exhibit distinct synaptic plasticity compared to those received by excitatory neurons, likely due to cell-type specific differences in postsynaptic density (PSD) composition and maintenance mechanisms. In the present study, we show that the interneuron-specific protein Btbd11 regulates excitatory synapse transmission in hippocampal interneurons through promotion of phase separation and support of postsynaptic nanoarchitecture. Btbd11 forms a phase separated protein complex with Psd-95 and TARP{gamma}2 and impacts the stability of TARP{gamma}2 and GluA1 within glutamatergic IN synapses in an expression- and phase separation-dependent manner. Using super resolution imaging, we show that Btbd11 displays nanoscale clustering properties within IN synapses that correlate with Psd-95 nanostructure. Furthermore, genetic deletion of Btbd11 decreases PSD protein expression, reduces synapse size, and disrupts Psd-95 nanocluster organization. These effects manifest as a drastic reduction in glutamatergic synaptic transmission onto INs when Btbd11 is deleted. Together, these data provide insights into a novel cell type-specific synaptic regulatory mechanism in an understudied synapse population.

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bioRxiv2026

Time-Varying Dynamic Causal Modelling for Sequential Responses: Neural Mechanisms of Slow Cortical Potentials, Preparation, Planning and Beyond

Cognitive processes such as decision-making, working memory, and motor planning operate across a hierarchy of timescales, manifesting as rapid neural transients alongside slower physiological mechanisms like short-term plasticity. Conventional Dynamic Causal Modelling (DCM) limits our ability to study these dynamics by assuming stationary parameters, whilst recent time-varying approaches often rely on segmenting data into epochs. This segmentation artificially resets neural states between windows, fundamentally obscuring the continuous hysteresis essential to sequential processing. To address this limitation, we introduce DCM for Sequential Responses (DCM-SR), a generative framework that embeds parameter evolution directly within the first-level model whilst employing a continuous state-space formulation that removes the requirement for epoching. This approach generalises non-stationarity to all neural mass parameters, including synaptic gains and time constants, modelling them as piecewise smooth trajectories that evolve alongside continuous neural states. Consequently, the model explicitly captures two distinct forms of temporal memory: transient history dependence, where responses are shaped by the carryover effect of recent perturbations, and path dependence, where the systems trajectory through parameter space determines its responsiveness. The framework accommodates both exogenous, stimulus-locked transitions and endogenous, autonomous state changes, permitting inference on both external perturbations and internal drivers of network evolution. Simulations establish the models face validity, demonstrating robust parameter recovery and conservative model selection that accurately discriminates between genuine parameter evolution and spurious complexity. We applied the framework to empirical data from an auditory go/no-go task, modelling a full sequence of cognitive phases from initial cue processing and anticipation through to motor preparation and execution. This analysis established construct validity by resolving the biophysical generators of the contingent negative variation, attributing this slow potential to sustained thalamocortical drive and deep-layer hyperpolarisation rather than superficial-layer activity. Furthermore, the model captured trial-specific modulations of the hyperdirect pathway during motor inhibition, tracking the dynamic interplay between prefrontal executive control and basal ganglia gating. DCM-SR offers the first principled approach to decomposing compound signals such as slow cortical potentials into evolving synaptic mechanisms and continuous state trajectories, and provides a necessary bridge for investigating the biophysical implementation of extended cognitive phenomena including evidence accumulation and physiological hysteresis.

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bioRxiv2026

When the beat drops - The functional anatomy of cardiac-induced sensory attenuation of auditory processing

Sensory processing is continuously shaped by internal bodily states, yet the neural mechanisms underlying this interoceptive-exteroceptive integration remain poorly understood. Predictive processing theories propose that bodily states modulate perceptual inference through precision-weighting (the contextual adjustment of prediction error gain according to sensory reliability), but empirical validation with neurobiologically realistic models has been lacking. We address this gap by combining cardiac phase-locked magnetoencephalography with systematic dynamic causal modelling to test competing mechanistic hypotheses about systolic-induced sensory attenuation. Using an auditory oddball paradigm, we observed selective suppression of deviant responses during cardiac systole (200-250ms post-stimulus), affecting prediction errors from unexpected tones whilst sparing expected tones. To identify the underlying synaptic mechanisms, we implemented three methodological innovations: (1) systematic comparison across 20 architectures representing precision-weighting (via intrinsic gains and/or modulatory connections), sensory gating (via forward connections), and predictive suppression (via backward connections) hypotheses; (2) Bayesian model reduction testing all 256 parameter configurations within the winning architecture to handle distributed model evidence; and (3) sensitivity analysis quantifying both direct effects and second-order interactions across the cortical hierarchy. Model comparison decisively favoured precision-weighting implementations (>99.99% posterior probability), with Bayesian model averaging revealing distributed gain control: superficial pyramidal self-inhibition in primary auditory cortex (94%) and inferior frontal gyrus (100%), inhibitory interneuron modulation in superior temporal gyrus (99%), and top-down modulatory connections from superior temporal to primary auditory cortex (96%). Critically, sensitivity analysis demonstrated that intrinsic inhibitory mechanisms exerted order-of-magnitude larger effects than hierarchical modulatory connections, with superior temporal gyrus emerging as an integration nexus showing extensive parameter interactions. These findings provide the first empirical validation of precision-weighting mechanisms during cardiac-sensory integration, establishing that systolic attenuation operates primarily through coordinated local inhibitory gain control rather than hierarchical (expected) attentional modulation. This modelling framework bridges computational theories of interoceptive-exteroceptive integration with laminar-specific cortical mechanisms, offering a generalisable methodology for testing predictive coding hypotheses about embodied perception.

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bioRxiv2024

Dissecting the functional heterogeneity of glutamatergic synapses with high-throughput optical physiology

Fluorescent reporters for glutamate release and postsynaptic Ca2+ signaling are essential tools for quantifying synapse functional heterogeneity across neurons and circuits. However, leveraging these probes for neuroscience requires scalable experimental frameworks. Here, we devised a high-throughput approach to efficiently collect and analyze hundreds of optical recordings of glutaamate release activity at presynaptic boutons in cultured rat hippocampal neurons. Boutons exhibited remarkable functional heterogeneity and could be separated into multiple functional classes based on their iGluSnFR3 responses to single action potentials, paired stimuli, and synaptic parameters derived from mean-variance analysis. Finally, we developed a novel all-optical assay of pre- and postsynaptic glutamatergic synapse function. We deployed iGluSnFR3 with a red-shifted, postsynaptically-targeted Ca2+ sensor, enabling direct imaging and analysis of NMDA receptor-mediated synaptic transmission at large numbers of dendritic spines. This work enables direct observation of the flow of information at single synapses and should speed detailed investigations of synaptic functional heterogeneity.

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bioRxiv2024

Large donor CRISPR for whole-CDS replacement of cell adhesion molecule LRRTM2

The cell adhesion molecule LRRTM2 is crucial for synapse development and function. However, our understanding of its endogenous trafficking has been limited due to difficulties in manipulating its coding sequence (CDS) using standard genome editing techniques. We instead replaced the whole LRRTM2 CDS by adapting the recent CRISPR method Targeted Knock-in using Two Guides (TKIT), enabling complete control of LRRTM2. In primary rat hippocampal cultures, N-terminally tagged, endogenous LRRTM2 was found in 80% of synapses, and synaptic LRRTM2 content correlated with PSD-95 and AMPAR levels. LRRTM2 was also enriched with AMPARs outside synapses, demonstrating the sensitivity of this method to detect relevant new biology. Finally, we leveraged total genomic control to increase the synaptic levels of LRRTM2 via simultaneous mutation of its C-terminal domain, which did not correspondingly increase AMPAR enrichment. The coding region of thousands of genes span lengths suitable for whole-CDS replacement, suggesting this simple approach will enable straightforward structure-function analysis in diverse cellular systems.

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bioRxiv2024

Loss of postsynaptic NMDARs drives nanoscale reorganization of Munc13-1 and PSD-95

Nanoscale protein organization within the active zone (AZ) and post-synaptic density (PSD) influences synaptic transmission. Nanoclusters of presynaptic Munc13-1 are associated with readily releasable pool size and neurotransmitter vesicle priming, while postsynaptic PSD-95 nanoclusters coordinate glutamate receptors across from release sites to control their opening probability. Nanocluster number, size, and protein density vary between synapse types and with development and plasticity, supporting a wide range of functional states at the synapse. Whether or how the receptors themselves control this critical architecture remains unclear. One prominent PSD molecular complex is the NMDA receptor (NMDAR). NMDARs coordinate several modes of signaling within synapses, giving them the potential to influence synaptic organization through direct protein interactions or through signaling. We found that loss of NMDARs results in larger synapses that contain smaller, denser, and more numerous PSD-95 nanoclusters. Intriguingly, NMDAR loss also generates retrograde reorganization of the active zone, resulting in denser, more numerous Munc13-1 nanoclusters, more of which are aligned with PSD-95 nanoclusters. Together, these changes to synaptic nanostructure predict stronger AMPA receptor-mediated transmission in the absence of NMDARs. Notably, while prolonged antagonism of NMDAR activity increases Munc13-1 density within nanoclusters, it does not fully recapitulate these trans-synaptic effects. Thus, our results confirm that NMDARs play an important role in maintaining pre- and postsynaptic nanostructure and suggest that both decreased NMDAR expression and suppressed NMDAR activity may exert distinct effects on synaptic function, yet through unique architectural mechanisms. Significance StatementSynaptic transmission is shaped by the trans-synaptic coordination of molecular ensembles required for neurotransmitter release and receptor retention, but how receptors themselves influence this critical architecture remains unclear. Using state-of-the-art super-resolution microscopy, we report that loss of NMDA receptors from excitatory synapses alters both pre- and postsynaptic nano-organizational features. Notably, pharmacological antagonism of NMDA receptors also alters presynaptic features, but without fully mimicking effects of the knockout. This suggests that both NMDA receptor activity and presence at the synapse exert retrograde influence on active zone organization. Because numerous disease and activity states decrease expression or function of NMDA receptors, our results suggest that distinct nanostructural states contribute to the unique functional status of synapses in these disorders.

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bioRxiv2023

Trans-synaptic molecular context of NMDA receptor nanodomains

Tight coordination of the spatial relationships between protein complexes is required for cellular function. In neuronal synapses, many proteins responsible for neurotransmission organize into subsynaptic nanoclusters whose trans-cellular alignment modulates synaptic signal propagation. However, the spatial relationships between these proteins and NMDA receptors (NMDARs), which are required for learning and memory, remain undefined. Here, we mapped the relationship of key NMDAR subunits to reference proteins in the active zone and postsynaptic density using multiplexed super-resolution DNA-PAINT microscopy. GluN2A and GluN2B subunits formed nanoclusters with diverse configurations that, surprisingly, were not localized near presynaptic vesicle release sites marked by Munc13-1. Despite this, we found a subset of release sites was enriched with NMDARs, and modeling of glutamate release and receptor activation in measured synapses indicated this nanotopography promotes NMDAR activation. This subset of release sites was internally denser with Munc13-1, aligned with abundant PSD-95, and associated closely with specific NMDAR nanodomains. Further, NMDAR activation drove rapid reorganization of this release site/receptor relationship, suggesting a structural mechanism for tuning NMDAR-mediated synaptic transmission. This work reveals a new principle regulating NMDAR signaling and suggests that synaptic functional architecture depends on the assembly of and trans-cellular spatial relationships between multiprotein nanodomains.

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bioRxiv2023

Molecular definition of distinct active zone protein machineries for Ca2+ channel clustering and synaptic vesicle priming

Action potentials trigger neurotransmitter release with minimal delay. Active zones mediate this temporal precision by co-organizing primed vesicles with CaV2 Ca2+ channels. The presumed model is that scaffolding proteins directly tether primed vesicles to CaV2s. We find that CaV2 clustering and vesicle priming are executed by separate machineries. At hippocampal synapses, CaV2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins, but distinct interaction motifs independently execute these functions. In heterologous cells, Liprin- and RIM from co- assemblies that are separate from CaV2-organizing complexes upon co-transfection. At synapses, Liprin-1-4 knockout impairs vesicle priming, but not CaV2 clustering. The cell adhesion protein PTP{sigma} recruits Liprin-, RIM and Munc13 into priming complexes without co- clustering of CaV2s. We conclude that active zones consist of distinct complexes to organize CaV2s and vesicle priming, and Liprin- and PTP{sigma} specifically support priming site assembly.

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bioRxiv2023

Distinct SAP102 and PSD-95 nano-organization defines multiple types of synaptic scaffold protein domains at single synapses

The MAGUK family of scaffold proteins plays a central role in maintaining and modulating synaptic signaling, providing a framework to retain and position receptors, signaling molecules, and other synaptic components. Of these scaffold proteins, SAP102 and PSD-95 are essential for synaptic function at distinct developmental timepoints and perform overlapping as well as unique roles. While their similar structures allow for common binding partners, SAP102 is expressed earlier in synapse development and is required for synaptogenesis, whereas PSD-95 expression peaks later in development and is associated with synapse maturation. PSD-95 and other key synaptic proteins organize into subsynaptic nanodomains that have a significant impact on synaptic transmission, but the nanoscale organization of SAP102 is unknown. How SAP102 is organized within the synapse, and how it relates spatially to PSD-95 on a nanometer scale, could impact how SAP102 clusters synaptic proteins and underlie its ability to perform its unique functions. Here we used DNA-PAINT super-resolution microscopy to measure SAP102 nano-organization and its spatial relationship to PSD-95 at individual synapses. We found that like PSD-95, SAP102 accumulates in high-density subsynaptic nanoclusters. However, SAP102 nanoclusters were smaller and denser than PSD-95 nanoclusters across development. Additionally, only a subset of SAP102 nanoclusters co-organized with PSD-95, revealing that within individual synapses there are nanodomains that contain either one or both proteins. This organization into both shared and distinct subsynaptic nanodomains may underlie the ability of SAP102 and PSD-95 to perform both common and unique synaptic functions. Significance statementSAP102 and PSD-95 are two key members of the MAGUK family of synaptic scaffold proteins that are critical for synapse development, maintenance, and modification during plasticity. Because PSD-95 has a highly complex subsynaptic nanostructure that impacts synaptic function, we asked if SAP102 is similarly organized into nanoclusters at individual synapses and how it relates to PSD-95 within synapses. We found that SAP102 forms subsynaptic nanoclusters with unique properties, and that within individual synapses proteins both concentrate into overlapping nanodomains, as well as form MAGUK-specific nanodomains. This demonstrates that organization of synaptic proteins into nanoclusters is likely to be maintained within the family of MAGUK proteins and reveals potential mechanism for specializing functions within individual synapses based on scaffold protein nanodomains.

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bioRxiv2023

Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons

A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters whose precise alignment across the cleft in a trans-synaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses - those forming onto excitatory principal neurons (Ex[->]Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex[->]PV synapses). As in Ex[->]Ex synapses, we find that in Ex[->]PV synapses presynaptic Munc13-1 and postsynaptic PSD-95 both form nanoclusters that demonstrate alignment, underscoring synaptic nanostructure and the trans-synaptic nanocolumn as conserved organizational principles of excitatory synapses. Despite the general conservation of these features, we observed specific differences in the characteristics of pre-and postsynaptic Ex[->]PV nanostructure. Ex[->]PV synapses contained larger PSDs with fewer PSD-95 NCs when accounting for size than Ex[->]Ex synapses. Furthermore, the PSD-95 NCs were larger and denser. The identity of the postsynaptic cell also had a retrograde impact on Munc13-1 organization, as Ex[->]PV synapses hosted larger Munc13-1 puncta that contained less dense but larger and more numerous Munc13-1 NCs. Moreover, we measured the spatial variability of trans-synaptic alignment in these synapse types, revealing protein alignment in Ex[->]PV synapses over a distinct range of distances compared to Ex[->]Ex synapses. We conclude that while general principles of nanostructure and alignment are shared, cell-specific elements of nanodomain organization likely contribute to functional diversity of excitatory synapses. Understanding the rules of synapse nanodomain assembly, which themselves are cell-type specific, will be essential for illuminating brain network dynamics.

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