bioRxiv Subject Collection: Neuroscience's Journal

Voluntary movement initiation is associated with cardiac input in Libet's task.
The relationship between motor intention and initiation of voluntary movement remains a fundamental topic in neuroscience, originating from the B. Libet seminal framework introduced in 1983. Libet's paradigm significantly influenced discussions on intentionality, motor control, and free will. However, methodological critiques continue to challenge its interpretations, specifically the accuracy and validity of the 'urge to move' phenomenon. One understudied factor in this debate is the potential influence of interoceptive signals--particularly cardiac activity--in shaping the experience of motor intention and movement initiation. In our study, we addressed this gap by examining whether cardiac signals modulate participants' experience of the 'urge to move', using behavioural and electrophysiological measures in 34 healthy human participants performing Libet's task. Crucially, when participants were asked to report the perceived 'urge to move', their button press timings were predominantly aligned with the diastolic phase of the cardiac cycle, indicating cardiac modulation of motor intention perception. However, analysing heart evoked potential (HEP) amplitudes as a measure of cardiac input perception, we observed no differences in HEP amplitudes associated with changes in introspective demands during the task in both source and sensor spaces. Our results suggest that implicit perception of cardiac signals biases subjective experience of voluntary action initiation, independent from cortical interoceptive markers. These findings have implications for models of motor preparation, intentionality and the bodily basis of voluntary action, challenging conventional interpretations of motor intention and informing debates on volition and interoception.

Dual effect of alpha-synuclein disease variants on condensate formation
Alpha-synuclein is a pre-synaptic protein implicated in synucleinopathies like Parkinson's disease and Dementia with Lewy Bodies, where it accumulates in intracellular aggregates termed Lewy bodies and Lewy neurites. Recent studies have reported that alpha-synuclein undergoes phase separation to form biomolecular condensates both in vitro and in mammalian cells. Alpha-synuclein condensates are thought to contribute to disease through progressive aggregation. Here we show that specific PD-associated alpha-synuclein variants fail to form biomolecular condensates. We demonstrate that only two alpha-synuclein variants associated with familial disease, E46K and E83Q, enhance condensate formation in vitro and in cells. However, variants including A30G, G51D, and A53E fail to form or have reduced levels of condensate formation in cells. The same phenotypes are reflected in the budding yeast model showing differential inclusion formation. In iPSC-derived neurons, the propensity of alpha-synuclein variants to undergo VAMP2-mediated phase separation reflects the level of synaptic enrichment. We show that the intrinsic propensity of alpha-synuclein to form condensates and the ability to bind lipid membranes are important to mediate condensate formation in cells. Our results emphasize that alpha-synuclein pathology follows divergent pathways, with both increased and decreased condensate formation contributing to disease. This study establishes biomolecular condensates as a key intermediate in alpha-synuclein dysfunction, providing a novel foundation for translational research.

Ionic Mechanisms Underlying Bistability in Spinal Motoneurons: Insights from a Computational Model
Spinal motoneurons are the final output of spinal circuits that engage skeletal muscles to generate motor behaviors. Many motoneurons exhibit bistable behavior, alternating between a quiescent resting state and a self-sustained firing mode. This bistability is traditionally attributed to plateau potentials, which are driven by persistent inward currents. This intrinsic property is important for normal movement control, but can become dysregulated, causing motor control deficits, like spasticity. Here, we use a conductance-based single-compartment model to investigate the ionic conductances underlying the bistable behaviour of motoneurons. Our simulations demonstrate that the motoneuron bistability and how its emergence is regulated mainly depends on the interplay between several intrinsic ionic mechanisms. In particular, the calcium-activated nonspecific cation current (ICAN), which is amplified by ICaL and calcium-induced calcium release (CICR), primarily drives the plateau potential to sustain bistability. Additional modulation is provided by the persistent sodium current (INaP) and the calcium- dependent potassium current (IKCa). This study provides a mechanistic model of motoneuron bistability, offering insights into its disruption in pathological conditions and setting the stage for future research and therapeutic development.

Emergent brain-like representations in a goal-directed neural network model of visual search
Visual search, the act of locating a target among distractors, is a fundamental cognitive behavior and a core paradigm for studying visual attention. While its behavioral properties are well characterized in humans and non-human primates, the underlying neural mechanisms remain largely unspecified. To address this gap, we developed a biologically aligned neural network model trained to perform visual search directly from pixels in natural scenes. This model exhibits strong generalization to novel scenes and objects, produces human-like scanpaths, and replicates previously known behavioral biases in humans. By analyzing the internal representations of the model, we found that it naturally develops a retinocentric cue-similarity map and prospective fixation signals, features that closely resemble neural activity in the primate fronto-parietal network. Beyond reproducing known behavior and neural signatures, the model makes testable predictions about the geometry and dynamics of internal representations underpinning cue-driven prioritization, fixation preferences, their perspective memories, and prospective plans. These findings offer a computational framework for understanding visual search and a roadmap for future neurophysiological and behavioral studies.

Perirhinal cortex structure and function is dysregulated by corticosteroid treatment
Glucocorticoids play a crucial role in the stress response and in the regulation of circadian function, as well as cognitive, cardiovascular, metabolic and immunological processes. While synthetic glucocorticoids (sGC) are widely used in treating inflammatory disorders, their impact on cognitive functions, which include memory deficits and dysregulation of mood, remains less understood. Here, we demonstrate that chronic treatment with the sGC methylprednisolone (MPL) dysregulates the synaptic proteome and impairs cognitive function in the perirhinal cortex, a region critical for recognition memory and visual perception. Further, we show that synaptic structure and plasticity are altered by MPL treatment, highlighting the mechanisms through which sGCs disrupt mnemonic processing. These results may have broad implications for understanding the cognitive side effects of widely used sGC treatments

fMRI BOLD signals in the left angular gyrus and hippocampus are associated with memory precision
Prior functional magnetic resonance (fMRI) studies examining the neural correlates of retrieval success and precision have reported inconsistent results. Here, we examined the neural correlates of success and precision in a test of memory for spatial location. The present study extended prior findings by employing an experimental design that minimized temporal overlap between mnemonic and visuomotor processing. At study, participants viewed a series of object images, each placed at a randomly selected location on an imaginary circle. At test, studied images were intermixed with new images and presented to the participants. The requirement was to make a covert recognition memory judgment to each image and to attempt to recall its studied location, guessing if necessary. A cue signaling the requirement to make a location memory judgment was presented 4 seconds after image onset. Memory precision was quantified as the angular difference between the studied location and the location selected by the participant. In an analysis that combined the data from the present study and a closely similar prior study, we replicated prior reports that fMRI BOLD activity in the left angular gyrus (AG) and the hippocampus tracks memory precision on a trial-wise basis. Linear mixed effects modeling indicated that the activity in the two regions explained independent sources of variability in these judgments. In addition, multivoxel pattern similarity analysis revealed robust evidence for an item-level reinstatement effect (as indexed by encoding-retrieval overlap) in the left AG that was restricted to items associated with high precision judgments. These findings suggest that the hippocampus and the left AG play non-redundant roles in the retrieval and behavioral expression of high precision episodic memories.

CRISPR-mediated knockdown of oxytocin receptor in extended amygdala reduces stress-induced social avoidance and vigilance
Oxytocin receptors (OTR) within the extended amygdala and nucleus accumbens have been implicated in modulating social behaviors, particularly following stress. The effects of OTR could be mediated by modulating the activity of pre-synaptic axon terminals or via post-synaptic neurons or glia. Using a viral-mediated CRISPR/Cas9 gene editing system in California mice (Peromyscus californicus), we selectively knocked down OTR in the anteromedial bed nucleus of the stria terminalis (BNST) or the nucleus accumbens (NAc) to examine their roles modulating social approach and vigilance behaviors. Knockdown of OTR in the BNST attenuated stress-induced decreases of social approach and increases of social vigilance behaviors in adult female California mice, similar to prior pharmacological studies. These effects were more prominent in the large arena social interaction where mice could control proximity to a social target with a barrier (wire cage). In a small arena interaction test where focal mice freely interacted with target mice, effects of BNST OTR knockdown were muted. This suggests that within the BNST OTR are more important for modulating behavioral responses to more distal stimuli versus more proximal social contexts. In mice with OTR knockdown in the NAc, few behavioral changes were observed which is consistent with previous findings of the importance of presynaptic OTR, which were unaffected by our gene editing strategy, driving social approach behaviors in the NAc. Interestingly, BNST OTR knockdown increased exploratory behavior toward a non-social stimulus after stress, pointing to a potentially broader role for BNST OTR function. Our findings highlight the region- and context-specific functions of OTR in social behavior and the advantages of using a selective gene-editing tool to dissect the neural circuits that influence social and stress-related responses.

Robust Input Disentanglement Through Dendritic Calcium-Mediated Action Potentials
In daily life, living beings encounter a continuous stream of mixed information, which has to be disentangled by the brain to form proper representations. Using computational modeling, we demonstrate that the interplay between dendritic calcium-mediated action potentials (dCaAPs) with synaptic plasticity and rewiring can enable single neurons to successfully perform this complex task. Compared to other types of dendritic spikes, dCaAPs exhibit a high triggering threshold, large, but graded spike amplitude, with lower amplitudes for stronger synaptic inputs. We show that these properties enable neurons to successfully learn to represent discrete items from a continuous input stream by facilitating the clustering of synapses with temporally correlated presynaptic activities onto the same dendritic branch. In comparison to NMDA spikes, dendrites generating dCaAPs can form representations of individual items more efficiently, independent of the temporal order of their presentation during learning - whether randomly, sequentially, as part of a random stream of simultaneously shown input items, or even as items with shared properties. Thus, our results provide further evidence about the critical role of dCaAPs for the computational capabilities of single neurons.

Brain cholesterol metabolites cause significant neurodegeneration in human iPSC-derived neurons
Disrupted cholesterol metabolism is increasingly recognised as a contributing factor in neurodegeneration; however, the specific effects of key brain-derived cholesterol metabolites, 24S-hydroxycholesterol (24S-HC) and 27-hydroxycholesterol (27-HC), remain poorly understood. Using human iPSC-derived i3 cortical neurons, we demonstrate that both 24S-HC and 27-HC significantly impair neuronal calcium signalling by elevating resting calcium levels, reducing spike amplitude, and disrupting network synchrony. These functional deficits are accompanied by widespread organelle dysfunction. Both oxysterols induce mitochondrial fragmentation, decrease spare respiratory capacity, and impair lysosomal degradation. Notably, 27-HC uniquely triggers lysosomal swelling and membrane permeabilisation. Additional signs of cellular stress, including axonal swellings and elevated endoplasmic reticulum calcium levels, were also observed. Furthermore, both 24S-HC and 27-HC were found to directly interact with alpha-synuclein (aSyn), promoting its accumulation in cellular models. In contrast, cholesterol itself had minimal impact, highlighting the distinct toxicity of its hydroxylated metabolites. Together, these findings reveal a mechanistic link between oxysterol accumulation and neuronal dysfunction, supporting the hypothesis that elevated levels of 24S-HC and 27-HC, commonly observed in Parkinson's and Alzheimer's disease, may actively drive neurodegenerative processes. Targeting oxysterol metabolism may therefore represent a promising therapeutic avenue for intervention in neurodegenerative disorders.

Neural responses underlying ITD discrimination as a function of sensory reliability in the barn owl
Discrimination of sensory stimuli is fundamentally constrained by the information encoded in neuronal responses. In the barn owl, interaural time difference (ITD) serves as a primary cue for azimuthal sound localization and is represented topographically in the midbrain auditory space map in the external nucleus of the inferior colliculus (ICx). While prior studies have demonstrated a correspondence between spatial tuning and behavioral acuity, it remains unclear how changes in sensory reliability influence this relationship. Here, we examined how behavioral and neuronal ITD discrimination thresholds vary with binaural correlation (BC), which manipulates ITD cue reliability. Using the pupil dilation response (PDR) as a behavioral metric in head-fixed owls, we found that ITD just-noticeable-differences increased exponentially as BC decreased. In contrast, the widths of ICx ITD tuning curves increased more modestly, indicating that tuning resolution alone does not account for behavioral discrimination performance. By computing the Fisher information from ICx neuronal responses, we showed that the average neuronal discriminability predicts behavioral thresholds across BC levels. A habituation-based model incorporating BC-dependent changes in tuning width, firing rate, and response variability successfully accounted for both direction and ITD discrimination. These findings support a model in which perceptual acuity is governed by the combined influence of neuronal tuning and variability and provide a unified framework for understanding how midbrain auditory representations underlie adaptive spatial hearing.

Interdependency between oxytocin and dopamine in trust-based learning in mice
Oxytocin (OT) is a neuropeptide implicated in complex social behaviors including trust and attachment, yet the neural mechanisms underlying its effects remain unclear. OT is thought to modulate behavior by enhancing the salience of social cues and attenuating prediction error (PE) processing, the discrepancy between expected and actual outcomes that drives learning. Since both salience coding and PE processing involve dopamine (DA) neurons as well, the current study investigated the putative interdependence between OT and DA in social safety learning using the social transmission of food preference (STFP) paradigm. STFP is based on the observation that mice (observers) display neophobia toward novel food, but develop a preference for it after a conspecific demonstrator signals its safety. We interpreted STFP acquisition as a functional parallel to human trust-based learning and found that OT enhanced learning in a trust acquisition condition, but only when DA signaling was intact. In a trust violation condition, where demonstrated food was later paired with lithium chloride (LiCl)-induced food aversion, both OT and DA depletion blocked learning, resulting in retained preference for demonstrated food, but not when OT was administered under DA depletion. These findings reveal a functional interaction between the OT and DA systems to modulate social safety learning, which may have important implications for the potential of OT in treating disorders involving DA dysfunction.

Target configuration determines how and what we learn during sensorimotor adaptation
Motor adaptation--the process of correcting movement errors through feedback and practice--is a fundamental human capacity that keeps our actions well-calibrated amid changes in the environment and the body. However, how training context--specifically, the configuration of targets in the workspace--shapes how we learn and what we learn during motor adaptation remains unknown. To investigate this, we conducted two reaching experiments in which participants experienced a visuomotor gain perturbation, with feedback scaled to 1.3x (Exp 1) or 0.7x (Exp 2) the actual movement distance. In both experiments, participants were assigned to either the Extent Group, which trained with targets of varying amplitudes in a single direction, or the Angular Group, which trained with targets of equal amplitude in different directions. We found marked differences in how the two groups learned: the Angular Group learned more implicitly than the Extent Group, as evidenced by larger post-perturbation aftereffects when participants were instructed to forgo re-aiming strategies. Just as striking were the differences in what the two groups learned: the Angular Group learned a translation rule, which generalized to new directions but not amplitudes, while the Extent Group learned the imposed gain rule, which generalized to new amplitudes but not directions. Together, these findings underscore the importance of training context in determining how and what we learn.

Lowering the HTT1a transcript as an effective therapy for Huntingtons disease
Lowering the levels of HTT transcripts has been a major focus of therapeutic development for Huntingtons disease (HD), but which transcript should be lowered? HD is caused by a CAG repeat expansion in exon 1 of the HTT gene, and the rate of somatic expansion of this CAG repeat throughout life is now known to drive the age of onset and rate of disease progression. As the CAG repeat expands, the extent to which the HTT mRNA is alternatively processed to generate the HTT1a transcript and highly aggregation-prone and pathogenic HTT1a protein increases. Several HTT-lowering modalities have entered clinical trials that either target both HTT and HTT1a together, or full-length HTT alone. We have developed siRNAs that target the Htt1a mouse transcript (634/486) and used these, together with a potent Htt-targeting siRNA (10150) to compare the efficacy of lowering either full-length Htt or Htt1a. zQ175 and wild-type mice were treated with 10150 or 634/486 alongside control groups at 2 months of age with treatment to 6 or 10 months, or at 6 months with treatment to 10 months. The siRNA potency and durability were most effective in the hippocampus. Whilst both strategies showed benefits, despite the greater potency of 10150, targeting Htt1a was more effective at delaying HTT aggregation and transcriptional dysregulation than targeting full-length Htt. These data support HTT-lowering strategies that are designed to target the HTT1a transcript, either alone, or together with lowering full-length HTT.

Large-scale bidirectional arrayed genetic screens identify OXR1 and EMC4 as modifiers of α-synuclein aggregation
In Parkinson's disease and other synucleinopathies, -synuclein (-Syn) misfolds and forms Ser129-phosphorylated aggregates (pSyn129). The factors controlling this process are largely unknown. Here, we used arrayed CRISPR-mediated gene activation and ablation to discover new pSyn129 modulators. Using quadruple-guide RNAs (qgRNAs) and Cas9, or an inactive Cas9 version fused to a synthetic transactivator, we ablated 2304 and activated 2428 human genes related to mitochondrial, trafficking and motility function in HEK293 cells. After exposure of cells to -Syn fibrils, pSyn129 signals were recorded by high-throughput fluorescent microscopy and aggregates were identified by image analysis. We found that pSyn129 was increased by activating the mitochondrial protein OXR1, which decreased ATP levels and altered the mitochondrial membrane potential. Instead, pSyn129 was reduced by ablation of the endoplasmic reticulum (ER)-associated protein EMC4, which enhanced ER-driven autophagic flux and lysosomal clearance. OXR1 activation preferentially modulated cellular reactions to fibrils derived from multiple system atrophy (MSA) patients, whereas EMC4 ablation broadly reduced pSyn129 across diverse -Syn polymorphs. These findings were confirmed in human iPSC-derived cortical and dopaminergic neurons, where OXR1 preferentially promoted somatic aggregation and EMC4 reduced both somatic and neuritic aggregates. These results uncover previously unrecognized roles for OXR1 and EMC4 in -Syn aggregation, thereby broadening our mechanistic understanding of synucleinopathies.

Small tau aggregates exhibit disease-specific molecular profiles across tauopathies
Tauopathies are neurodegenerative diseases marked by pathological tau aggregation. While disease-specific folds of insoluble tau filaments have been established, it remains unclear whether the smaller, earlier species also differ across tauopathies. Here, we characterise these small tau aggregates from post-mortem brain of individuals with Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration, Pick's disease, and healthy controls. Using two complementary single-molecule assays, we confirm that small tau aggregates vary in abundance, morphology, and post-translational modifications. AD features specific long, fibrillar-shaped aggregates enriched in phospho-epitopes, while PSP aggregates are shorter, round, and selectively phosphorylated at serine-356, a site we identify as correlating with markers of inflammation and apoptosis. Aggregate properties co-vary with cellular stress signatures and align with disease-specific seeding profiles, suggesting distinct pathological mechanisms. These findings suggest that small tau aggregates are not a shared intermediate, but instead encode disease-specific mechanisms, with potential as both biomarkers and therapeutic targets.

Ultra-wide-field, deep, adaptive two-photon microscopy
Observing the activity patterns of large neural populations throughout the brain is essential for understanding neural functions. However, capturing neural interactions across widely distributed brain regions, including both superficial and deep cortical layers, remains challenging with existing microscopy technologies. Here, we introduce a state-of-the-art two-photon microscopy system capable of single-cell resolution imaging across an ultra-large field of view (FOV) exceeding 50 mm2, enabling deep in vivo imaging. To demonstrate its capabilities, we conducted a series of experiments under multiple imaging conditions, successfully visualizing brain structures and neuronal activities spanning a wide spatial range (> 7 mm) from superficial layers to depth reaching up to 900 um in the mouse brain. This versatile imaging platform overcomes traditional spatial constraints, providing a powerful tool for comprehensive exploration of neuronal circuitry over extensive spatial scales with cellular resolution.

Morphology and ultrastructure of pharyngeal sense organs of Drosophila larvae
This study provides a comprehensive ultrastructural analysis of the pharyngeal sensory system in Drosophila melanogaster larvae, focusing on the four major pharyngeal sense organs: the ventral pharyngeal sensilla (VPS), dorsal pharyngeal sensilla (DPS), dorsal pharyngeal organ (DPO), and posterior pharyngeal sensilla (PPS). Our analysis revealed 15 sensilla across these organs, comprising four mechanosensory, nine chemosensory, and two dual-function sensilla. We identified 35 Type I neurons (six mechanosensory and 29 chemosensory) and six Type II neurons with putative chemosensory functions. Additional sensory structures, including papilla sensilla and chordotonal organs in the cephalopharyngeal region, were characterized. This detailed mapping and classification of pharyngeal sensory structures completes their structural characterization and provides a foundation for future anatomical and functional studies of sensory perception in insects. This work represents a significant step towards a complete analysis of the larval sensory system, providing new opportunities for investigating how an organism processes sensory information to navigate and interact with its environment.

PRESYNAPTIC MUSCARINIC-NICOTINIC MODULATION of GABAergic INPUTS to NEURONS in the REM SLEEP (REM-S) EXECUTIVE AREA of the ROSTRAL PONS
A large body of evidence indicates a pivotal role of cholinergic neurons of brainstem tegmental nuclei in the control of neurons of the reticular nucleus pontis oralis (PnO) considered executive for rapid eye movement sleep (REM-S) onset and maintenance. More recent data suggest that the PnO is also under GABAergic control that gate REM-S and highlights the role of interactions between cholinergic and GABAergic processes as a key mechanism for REM-S control. Here we employed an in vitro model of REM-S motor suppression to investigate the modulation of GABAergic inputs to PnO neurons by cholinergic agents. We found that carbachol, a mixed muscarinic-nicotinic cholinergic agonist, provoked either depression or facilitation of single evoked monosynaptic GABAergic IPSCs. Both effects were presynaptic in origin and likely due to the activation of different presynaptic cholinergic receptors as depression was replicated by muscarine (presynaptic inhibition) and facilitation by nicotine (presynaptic facilitation). IPSCs evoked by presumed physiological patterns of stimulation (short trains of stimuli at 15 Hz) were also affected by cholinergic agonists. Muscarine caused presynaptic inhibition accompanied by frequency facilitation and nicotine promoted the opposite effect. Notably, both agonists reduced the total inhibitory charge transferred to postsynaptic neurons during the train. Our results shed light on a possible additional mechanism for a cholinergic-mediated relief of GABAergic inhibition of PnO neurons under presumed physiological conditions, as a local component of the mutual inhibitory interaction design between sleep controlling systems at REM-s brainstem executive areas.