bioRxiv Subject Collection: Neuroscience's Journal

PRMix: Primary Region Mix Augmentation and Benchmark Dataset for Precise Whole Mouse Brain Anatomical Delineation
The architecture of the mouse brain shares remarkable similarities with the human brain, making it an essential model for studying brain pathologies, synaptic diversity, and regional specialization. A key step in such studies involves registering molecular images to reference brain atlases, a process hindered by the difficulty of accurately delineating brain regions. Toward this, we have curated a collection of high-resolution, dual-fluorescence microscopy images, termed as dual-fluorescence mouse brain microscopy (DMBM) dataset, complemented by expert annotations of 118 subregions. This dataset provides unprecedented insights into the molecular and structural complexity of the mouse brain. However, its full potential for detailed whole-brain analysis is compromised by challenges such as boundary ambiguity and sample scarcity in existing automated segmentation methods, prompting the development of the primary region mix (PRMix) augmentation method. PRMix is specifically designed to expand these datasets, enhance the realism of synthetic data and minimize overlap between adjacent regions. Our approach, together with the curated dataset, achieves superior segmentation performance across the mouse brain compared with existing methods, setting a new benchmark in brain imaging research. Code and data are available at https://git-pages.ecdf.ed.ac.uk/dmbm-datasets-5c13cd/.

Hyperexcitability of female serotonin neurons underlies sex-specific anxiety responses
Mood disorders display robust sex differences in prevalence, symptom profile, and treatment outcomes, with women nearly twice as likely as men to be diagnosed. However, the neural substrates mediating sex-specific regulation of mood and related disorders remain incompletely understood. Here, we identify a neural circuit mechanism involving serotonergic (5-HT) projections from the median raphe region (MRR) to the ventral hippocampus (vHP) responsible for sex-specific regulation of anxiety-like behavior in mice. Using a multimodal approach combining electrophysiology, fiber photometry, and optogenetics, we show that 5-HT neurons targeting the vHP (5-HTvHP neurons) display heightened intrinsic excitability and delayed deactivation in female mice during exposure to aversive environments. Optogenetic activation of this pathway enhanced anxiety-like behavior, and decreased risk-assessment behavior in females, but not in males. Collectively, our findings establish 5-HTvHP neurons as a critical circuit for the regulation of anxiety and provide a mechanistic framework for understanding how serotonergic modulation contributes to sex-specific vulnerability to mood disorders.

Age-related decline of synaptic plasticity is regulated by neuro-androgen and neuro-estrogen in normal aging of hippocampus
We revealed a good relationship between age-dependent decrease in the hippocampal dendritic spine density and age-dependent decrease in hippocampal androgen and estrogen levels with normal aging of male rats. Approximately 25% decrease in the spine density was observed in hippocampal CA1 region by going from 3 month-old (3m; young adult) to 24 month-old (24m; aged). We found a significant age-induced decrease in hippocampal neuro-androgen levels by going from 3m to 24 m using mass-spectrometric analysis. The hippocampal levels of testosterone (T) and dihydrotestosterone (DHT) dramatically decreased from 17 nM T and 7 nM DHT at 3 m to 17/100 nM T and 7/15 nM DHT at 24 m. On the other hand, hippocampal estradiol (E2) was moderately decreased with aging, from 8 nM at 3 m to 2 nM at 24m. Comprehensive analysis of mRNAs of hippocampal steroidogenic enzymes and receptors showed an age-dependent decrease in their expression levels by approximately 50% (P450(17alpha)), 25% (17beta-hydroxysteroid dehydrogenase) and 0% (5-reductase and P450arom). Androgen receptor AR was moderately decreased but estrogen receptor ER was not decreased with aging. The 25% decrease in the spine density with aging may be due to a balance between considerably decreased T and DHT levels (spine decrease factor) and remained moderately high E2 level (spine increase factor) in the 24m hippocampus. Aged hippocampus still has moderate capacity of sex-steroid synthesis and their functions. Interestingly, DHT-supplementation and T-supplementation recovered the spine density at 24m.

NSF is required for diverse endocytic modes by promoting fusion and fission pore closure in secretory cells
The ATPase N-ethylmaleimide-sensitive factor (NSF), known for disassembling SNARE complexes, plays key roles in neurotransmitter release, neurotransmitter (AMPA, GABA, dopamine) receptor trafficking, and synaptic plasticity, and its dysfunction or mutation is linked to neurological disorders. These roles are largely attributed to SNARE-mediated exocytosis. Here, we reveal a previously unrecognized role for NSF: mediating diverse modes of endocytosis, including slow, fast, ultrafast, overshoot, and bulk, by driving closure of both fusion and fission pores. This function was consistently observed across large calyx nerve terminals, small hippocampal boutons, and chromaffin cells using capacitance recordings, synaptopHluorin imaging, electron microscopy, and multi-color pore-closure imaging. Results were robust across four NSF inhibitors, gene knockout, knockdown, and specific mutations. These findings establish NSF as a central regulator of membrane fission, kiss-and-run fusion, endocytosis, and exo-endocytosis coupling, offering new mechanistic insights into its diverse physiological and pathological roles in synaptic transmission, receptor trafficking, and neurological diseases.

Characterizing Post-Mortem Brain Molecular Taxonomy of Cognitive Resilience and Translating it to Living Humans
Here, we define cognitive resilience as slower or faster cognitive decline after we regress out the effects of common brain neuropathologies. Its understanding could provide important insights into the biology underlying cognitive health, enabling the development of more effective strategies to prevent cognitive decline and dementia. However, this requires the development of a practical method to quantify resilience and measure it in living individuals, as well as identifying heterogenous pathways associated with resilience in different individuals. Here, we approach this problem by using a data-driven framework to quantify and characterize molecular signatures underlying cognitive resilience. Using multimodal contrastive trajectory inference (mcTI) on bulk RNA sequencing and tandem mass tag (TMT) proteomic data from 898 post-mortem brain samples from the Religious Orders Study and the Rush Memory and Aging Project (ROSMAP), we derived individual-level molecular pseudotime values reflecting the molecular path from high to low resilience across individuals. Additionally, we identified two distinct molecular subtypes of resilience, each characterized by unique transcriptomic and proteomic signatures, and differing associations with several phenotypes. To translate our brain-derived pseudotime and subtypes to living individuals, we developed prediction models with paired genetics, ante-mortem blood omics, clinical, psychosocial, imaging and device data from the same individuals, demonstrating the potential to predict brain molecular resilience profiles in living persons. Our findings establish a framework for quantifying resilience based on multi-level molecular signatures, identify molecularly distinct resilience subtypes, and demonstrate the feasibility of translating brain-derived molecular profiles to living individuals, laying the groundwork for the development of targeted resilience-promoting interventions in cognitive aging.

Spinal Motor Neuron Pools May be Partly Driven by Impulsive Common Inputs
Spinal motor neurons serve as the link between the nervous system and muscles. As the final common pathway of the neuromuscular system, they receive inputs from both higher-level controllers and afferent pathways. It is often assumed that spinal motor neurons are primarily driven by continuous common inputs (cCI) within different frequency bands. Within this framework, the motor neuron pool behaves as a linear amplifier of the cCI. However, this framework overlooks the possibility that motor neurons could also be driven by impulsive common inputs (iCI), which can induce synchronization among them and disrupt the linear transmission of other synaptic inputs at the pool level. To test this hypothesis, computational simulations and experimental data from human subjects were used to characterize different aspects related to motor neuron spiking synchronization at the pool level. Our findings suggest that, indeed, iCI can account for relevant features observed in experimental data such as the presence of synchronization events at the pool level. We also observed that such impulsive inputs can affect the linearity in the transmission of cCI by the motor neuron pool. This study represents pioneering indirect evidence of the existence of iCI as inputs to motor neurons.

Human aged astrocytes induce neurotoxicity in response to inflammatory stimuli
Astrocytes play a critical role in neuroinflammation and the pathogenesis of neurodegenerative diseases. Here we found that human induced pluripotent stem cell (iPSC)-derived astrocytes responded differently to inflammatory triggers compared to rodent astrocytes, showing increased neurotoxicity when exposed to TNF- and IFN-{gamma}. Furthermore, astrocytes with senescent features showed even higher levels of neurotoxicity in the presence of TNF- and IFN-{gamma}, suggesting a potential link between aging and neurodegenerative diseases. It was also demonstrated that LPS-activated neuron/astrocyte/microglia tri-culture produced TNF-, leading to neurotoxicity in the tri-culture when IFN-{gamma} was present. Through compound screening, we identified Janus kinase inhibitors capable of preventing neurotoxicity in astrocytes induced by TNF- and IFN-{gamma}, demonstrating the potential use of neurotoxic astrocytes as a platform for drug screening. These results provide insight into the complex relationship between aging, inflammation, and neurodegenerative diseases, emphasizing the potential of targeting astrocytes as a novel therapeutic approach for addressing neurodegenerative diseases.

Perceptual Novelty in Tinnitus a Causative Factor for its Persistence. A Stimulus Novelty Based P300 Paradigm on Acute, Chronic, and Non-Tinnitus Controls
Our understanding of tinnitus pathophysiology may be greatly advanced by understanding how the condition evolves from its initial onset or acute stage to its chronic manifestation. Such a transition likely reflects dynamic neurophysiological changes within central auditory and non-auditory networks. Previous studies have highlighted that individuals with acute tinnitus tend to have increased activity in the regions of anterior cingulate cortex, inferior parietal lobe, and insula all of which are essential in constituting the salience network. We therefore aimed at tapping into the salience network of tinnitus through a novelty based P300 paradigm in individuals with Acute, Post Acute (six months follow up since acute tinnitus) Chronic, and Controls. Participants were presented with an auditory oddball paradigm comprising three deviant types: (1) novel environmental sounds, (2) low-frequency tonal deviants, and (3) high-frequency tonal deviants, embedded within a sequence of frequent standard tones. Our results indicate a significant drop in P300 amplitude during the Post Acute stage, highlighting the substantial influence of the anterior cingulate cortex/salience network in possible generation of tinnitus and inferior parietal lobe in the persistence of tinnitus.

Tyrosine kinase inhibitors affect sweet taste and dysregulate fate selection of specific taste cell subtypes via KIT inhibition.
Taste dysfunction, or dysgeusia, is a common side effect of many cancer drugs. Dysgeusia is often reported by patients treated with anti-angiogenic tyrosine kinase inhibitors (TKIs), which inhibit receptor tyrosine kinases (RTKs). However, the mechanisms by which TKIs cause dysgeusia are not understood, as the role of RTKs in adult taste homeostasis is unknown. Here, we find that treating adult mice with the TKI cabozantinib shifts the fate of differentiating functional taste cell subtypes within taste buds. Through behavioral assays, we find this cell fate shift leads to blunted responses to sweet tastant in cabozantinib-treated mice. Finally, we show that inducible knockout of the RTK KIT, which is inhibited by cabozantinib, largely phenocopies drug treatment. Our results establish KIT as a regulator of taste cell homeostasis and suggest that KIT inhibition may underlie TKI-induced dysgeusia in patients.

Non-neuronal, TGF-β- extracellular matrix restructuring promotes neurodegeneration in a PSP-Richardson syndrome model
Progressive supranuclear palsy-Richardson syndrome (PSP-RS) is a rapidly progressive tauopathy lacking effective therapies. Although tau aggregation is a defining feature, the initiating mechanisms remain elusive. Here we used patient-derived induced pluripotent stem cell midbrain organoids, integrating single-cell transcriptomics, bulk RNA profiling, and quantitative proteomics, to dissect early pathogenic events. We identified vascular leptomeningeal-like cells (VLMCs) as the first altered population, exhibiting TGF-{beta}-driven extracellular matrix (ECM) remodeling enriched in collagens, integrins, and TGFBI. The resulting pathological ECM increased stiffness, induced integrin clustering, and activated RhoA-ROCK-mediated cytoskeletal disorganization. These changes sustained PI3K-AKT and MAPK-ERK signaling, suppressed PP2A, hyperactivated mTOR, and impaired autophagy, culminating in tau hyperphosphorylation and mislocalization. Pharmacological inhibition of TGF{beta}, AKT, ERK, or mTORC1 restored autophagic flux, reduced tau burden, and rescued neuronal architecture. Our findings establish non-neuronal, matrix-producing niche cells as upstream drivers of tauopathy and reveal TGF-{beta}-mediated ECM restructuring as a mechanochemical trigger of neurodegeneration, opening multiple therapeutic avenues for PSP-RS and related tauopathies.

The Latency of a Domain-General Visual Surprise Signal is Attribute Dependent
Predictions concerning upcoming visual input play a key role in resolving percepts. Sometimes input is surprising, under which circumstances the brain must calibrate erroneous predictions so that perception is veridical. Despite the extensive literature investigating the nature of prediction error signalling, it is still unclear how this process interacts with the functionally segregated nature of the visual cortex, particularly within the temporal domain. Here, we recorded electroencephalography (EEG) from humans whilst they viewed static image trajectories containing a bound object that sequentially changed along different visual attribute dimensions (shape and colour). Crucially, the context of this change was designed to appear random (and unsurprising) or violate the established trajectory (and cause a surprise). Event-related potential analysis found no effects of surprise after controlling for cortical adaptation. However, multivariate pattern analyses found whole-brain neural representations of visual surprise that overlapped between attributes, albeit at distinct, attribute-specific latencies. These findings suggest that visual surprise results in whole-brain, generalised (i.e., attribute-agnostic) prediction error responses that conform to an attribute-dependent temporal hierarchy.

RatDISCO, a tissue clearing and immunolabelling protocol for large rat brains.
RatDISCO is a simple, cost-effective, and reproducible tissue-clearing protocol optimised for immunolabeling in adult rat brains. It enables robust detection of diverse neuronal subtypes, glial populations, and vasculature, overcoming key limitations in antibody penetration and optical transparency. It is compatible with virally labelled- and transgenic mice, as well as human-derived brain organoids, highlighting its versatility across species and models. Functionally, it enables the detection of activity-dependent markers after behaviour, verified in a rat model of neurodevelopmental disorders, the Fragile X syndrome. These findings highlight RatDISCO as a broadly applicable tool for investigating neuroanatomical and functional alterations in rat models of disorders.

Cortical GABAergic inhibition dynamics around hippocampal sharp-wave ripples
Cortical inhibition, mediated by GABA, is essential in balancing excitation and modulating neural processing, but it is unclear to what extent inhibitory dynamics are responsive to internally generated hippocampal activity, such as sharp-wave ripples (SWRs). Employing widefield imaging of extracellular GABA with iGABASnFR2 and hippocampal recordings, we characterized cortical inhibition during sleep and awake states and in the neighborhood of SWRs. Sleep and awake transitions drastically reorganized cortical levels of GABA, with increased inhibition in wakefulness and decreased upon entering NREM sleep. In the neighborhood of SWRs, inhibition was state- and region-specific: during NREM, medial cortices (e.g., retrosplenial) increased GABA before SWRs, whereas sensory areas in the lateral cortices decreased it; during wake, GABA increased in lateral cortices after SWRs. These findings reveal that cortical inhibition is not static and ubiquitous but instead is dynamically characterized by brain state, orchestrating the flow of hippocampal outputs to the rest of the cortex. Inhibitory modulation, as our data reveal, forms a mechanism allowing selective gating of memory-related activity during wakefulness and sleep.

Scratcher: An automated machine-vision tool for dissecting the neural basis of itch
Itch or pruritus invokes a specific reflexive and repetitive directed nocifensive behavioural response, known as scratching. Recent decades have revealed neural circuits that are involved in the sensory and affective-motivational aspects of itch-induced scratching. However, most of these studies relied on manual subjective methods of quantifying scratching in laboratory mice and rats. Recent advances in deep learning have opened avenues for the development of computational tools to analyze animal behaviour in a reliable and automated manner. Further, combined with optogenetic and chemogenetic strategies, these tools can accelerate our understanding of neural circuits underlying itch and scratching. To that end, we have developed Scratcher, a GUI-based computational tool based on a real-time object detection algorithm that allows semi-supervised automated analysis of scratching behaviour in mice in a computationally inexpensive manner. We recorded chloroquine-induced acute itch as it developed, and determined the consequence of nail-trimming on acute-itch induced scratching with Scratcher. To probe the neural mechanisms underlying itch, we combined Scratcher with genetic circuit dissection using the Fos-TRAP mouse line. By targeting itch-activated neurons in the lateral parabrachial nucleus (LPBN) - a key brainstem hub for pruritic signal transmission - we demonstrated that LPBN activity modulates itch-evoked scratching. Together, we present a novel, easy-to-use computational tool to dissect molecular, cellular, and circuit mechanisms of itch and scratching.

Functional muscle networks reveal the mechanistic effects of post-stroke rehabilitation on motor impairment and therapeutic responsiveness
Standardised assessment of post-stroke motor impairment and treatment responsiveness remains a major clinical challenge. In this study, we tackle this challenge by applying a novel muscle network analysis framework to stroke survivors undergoing intensive upper-limb motor training. Our approach revealed distinct patterns of redundant and synergistic muscle interactions, collectively reflecting the diverse biomechanical roles of flexor- and extensor-driven networks. From these patterns, we derived new biomarkers that stratified patients by impairment severity and therapeutic responsiveness, each associated with unique physiological signatures. Notably, we identified a shift from redundancy to synergy in muscle coordination as a hallmark of effective rehabilitation--a transformation supported by a more precise quantification of treatment outcomes. These findings offer an in-depth mechanistic account of post-stroke motor recovery and establish a robust, independent tool for evaluating rehabilitation efficacy.

Dpp6 Homozygous Knockout Mice Exhibit Increased Ethanol Conditioned Place Preference and Acute Ethanol-Induced Anxiolytic Behavior
The gene DPP6 has been associated with behavioral phenotypes of alcohol use disorder (AUD) in recent human genome wide association studies. To further assess the role of this gene in ethanol-related traits, we tested Dpp6 knockout (KO) mice for ethanol conditioned place preference (CPP), locomotor activity, and ethanol-induced anxiolysis. Male homozygous KO mice (HOM) showed greater preference for the ethanol-paired context compared to wild type littermates (WT) and heterozygous KO mice (HET), while female mice showed no genotypic difference. HOM of both sexes exhibited greater novelty-induced hyperactivity in the CPP apparatus than HET and WT mice in the first two minutes. In a separate experiment, HOM mice showed enhanced locomotor activity following a 1.5 g/kg ethanol injection; however, they also displayed greater locomotor activity during habituation, suggesting basal locomotor differences. Following 1.5 and 2 g/kg injections, HOM mice exhibited EtOH-induced anxiolysis in the first 5 minutes, while the HET and WT mice did not. Lastly, HOM mice displayed a significant sedative response compared to WT animals following a 2 g/kg injection of ethanol. Ultimately, these findings validate a role for Dpp6 in modulating ethanol's rewarding, anxiolytic, and sedative effects in a sex-dependent manner.

Regional patterns of neurodegeneration in a mouse model of proteinopathy
The aggregation of misfolded proteins is a hallmark of many neurodegenerative diseases, suggesting shared pathological mechanisms. However, the pathways by which protein misfolding in these proteinopathies lead to neuronal death remain unclear. Proteinopathies can be modelled in transgenic animals by expressing disease-causing mutations that promote protein aggregation, or in wild-type animals by injecting misfolded proteins (e.g. RML scrapie) that spread in a prion-like manner and recapitulate key neurodegenerative features, including gliosis, ER stress, and neuronal loss. Here, we map region-specific histopathological features of scrapie-induced neurodegeneration in the hippocampus, thalamus, cortex, and cerebellum during early (12 weeks post-inoculation) and late (20 weeks) stages of disease. Using a streamlined time-efficient protocol, we achieve reproducible paired sample collection and high-quality immunohistochemistry that is compatible with best practice in decontamination and containment. We found that among the tested markers of early pathology, thalamic astrocytic activation and spongiform degeneration were the most sensitive. By the late stage, there was widespread upregulation of IBA1+ microglia and GFAP+ astrocytes, accompanied by strong immunoreactivity of lysosomal marker LAMP1. LAMP1 expression in healthy brains was largely neuronal, but by 20 weeks it was significantly upregulated in astrocytes, suggesting their involvement in lysosomal pathology. The ER stress marker p-PERK was elevated in CA1/CA3 pyramidal neurons but minimal in the thalamus and cerebellum, where neuronal loss was most pronounced, suggesting region-specific mechanisms of degeneration. Overall, the thalamus and hippocampal CA1/CA3 areas exhibited the greatest pathological burden. Our shorter time-course, new pathological insights and safe handling protocols, and improved welfare, supports broader adoption of the RML scrapie model for resource-efficient studies of neurodegeneration and its prevention.

Distinct developmental changes in linear and nonlinear neural interactions across infancy and adulthood
The development of functional brain connectivity during early life depends on social experience and is best understood in the context of interactions with a caregiver. It is still an open question to what extent distinct modes of functional connectivity dominate in different stages of human brain development, and whether cognitive tasks can differentially modulate them. Using electroencephalography (EEG), we investigated the development of linear and nonlinear functional brain connectivity in infants and adults while they socially interacted. Using simultaneous EEG recordings in parent-infant dyads performing two different experiments (N = 160; 80 adults and 80 infants), we computed functional connectivity capturing distinct dynamics: a linear measure capturing phase synchronization (weighted phase lag index; WPLI), and a nonlinear measure capturing information sharing (weighted symbolic mutual information; WSMI). In both tasks, adults showed higher WSMI than infants, whereas infants showed higher WPLI than adults. Moreover, infant age predicted only task-related connectivity values computed with the nonlinear measure and not with its linear counterpart, suggesting that the information-theoretic measure was more sensitive to developmental changes in task-relevant neural processing. These findings suggest that over development, a shift in dominance from linear to nonlinear modes of brain communication may be essential for supporting emerging higher cognitive abilities, such as precursors to executive function (here, attention shifting) and social decision-making. Further, this work highlights the importance of using nonlinear measures in addition to traditional linear ones, which collectively permit a more robust capture of maturational changes.

Reduced TRPC3 conductance underlies altered SNr activity under dopamine depletion: predictions from data-driven network models
Sufficient loss of dopamine within the basal ganglia (BG) leads to neuronal activity changes, including altered firing rates and firing patterns, thought to underlie parkinsonian motor symptoms. Yet, within BG neuronal populations, baseline activity and responses to inputs are highly variable, complicating efforts to identify key factors associated with pathological changes. We introduce a novel approach to constructing a computational neuron population model that, when applied to the mouse substantia nigra pars reticulata (SNr), captures the firing heterogeneity observed across slice and in vivo recordings. This model reproduces the diversity of SNr neuron responses to stimulation of GABAergic input terminals, yielding new insights into the mechanisms underlying this variability. Moreover, our modeling pinpoints significant decreases in TRPC3 conductance in SNr dendrites as a key determinant of altered SNr activity in the dopamine depleted state, with important implications for efforts to restore functional SNr activity in this condition.

Temporal Deconvolution of Mesoscale Recordings
Mesoscale calcium imaging techniques, such as wide-field imaging, enable high temporal resolution recordings of extensive neuronal activity across one or more brain regions. However, since the recordings capture light emission generated by the fluorescence of the calcium indicator, the neural activity that drives the calcium changes is masked by the dynamics of the calcium indicator. In this study, we develop and evaluate new methods to deconvolve fluorescence traces into the underlying neuronal spiking rates driving them. Our new inference methods take into account both the noise in the recordings and the temporal dynamics of the calcium indicator response. Our first proposed method, termed 'Dynamical-Binning', estimates spiking rates that are constant over discrete time bins. The size of each time bin depends on the data and is determined dynamically. Our second method, 'Continuously-Varying,' estimates the spiking rate as a continuous function. This method aims at studies seeking to find slow rate fluctuations rather than identifying abrupt changes in the spiking rate. The third method, 'First-Differences', aims to give a quick estimate of the spiking rates, which is beneficial for exceptionally large datasets, typical of mesoscale recordings. Our fourth method, is a modified 'Weiner Filter.' It estimates spiking rates by efficiently removing noise with a fixed ratio compared to the signal. This approach is beneficial for datasets exhibiting large fluctuations in fluorescence magnitudes. We compare the accuracy of our methods against the existing 'Lucy-Richardson' image recovery algorithm in its adapted form to recover temporal dynamics. Our results demonstrate that all our proposed methods surpass the performance of 'Lucy-Richardson' on both synthetic and recording datasets, including concurrent recordings of fluorescence and spike counts from the exact origin by multichannel silicon probes. Furthermore, we illustrate that our findings are indifferent to the choice of removing hemodynamic signals. Lastly, we demonstrate that the reliance on calcium signals for advanced analytical approaches can lead to distorted results. For example, they can show correlations between the activity of different brain regions that are unrealistically high compared to the correlations of the underlying spiking rate between the same areas. This highlights the critical importance of temporal inference for further accurate and reliable analysis in understanding the complexities of brain activity.

Helix-to-Beta-Sheet Transition Drives Self-Assembly of Glutamate Transporter EAA1 Splice Peptides
Truncated isoforms play a critical role in understanding the structural and functional properties of membrane proteins, including glutamate transporters. Here, we molecularly characterize two helical truncated isoforms of the human glutamate transporter EAA1. Using an integrative multi-omics and computational approach, we show that these isoforms, particularly one derived from the N-terminus, do not adopt the canonical transporter fold. Instead, they self-assemble into stable, {beta}-sheet-enriched oligomers, a structure previously unobserved for this protein family. Furthermore, we identified a water-soluble truncated isoform (A0A7P0TAF5) of the membranous canonical EAA1, revealing that self-assembly is not confined to membranous isoforms of EAA1. This finding uncovers a previously unrecognized functional class of truncated isoforms capable of initiating assembly in the soluble state. Our 500ns molecular dynamics simulations further reveal that the N-terminal truncation alters the native conformational dynamics, promoting a transition into semi-helical {beta}-structures over time. In a model bilayer, {beta}-Sheet-driven octamerization of the helical EAA1 isoform A0A7P0Z4F7 induces localized upper leaflet membrane pitting during 250ns all-atom simulation. Helix to {beta}-sheet oligomer transitions is a known pathological hallmark of neurodegenerative disorders such as Alzheimer's disease. Our findings thus uncover a potential new mechanism for glutamate transporter involvement in neurodegeneration and identify the N-terminal domain as a promising therapeutic target. This work highlights how alternative splicing can generate isoforms with novel interaction patterns and distinct molecular conformations.

Open-source modular FPGA system for two-photon mesoscope enabling multi-layer, multi-depth neural activity recording and lifetime imaging
Large field-of-view (FOV) two-photon microscopy makes it possible to record a large number of neural activities from multiple brain regions simultaneously. However, the larger the field of view, the longer it takes to scan the entire FOV. To increase imaging speed, we have developed open-source software to digitize analogue signals from a photomultiplier tube using a field-programmable gate array (FPGA) at a rate of 3.2 GS/s. By combining this with a newly developed a circular delay-path module for a custom two-photon mesoscope (Diesel2p), we succeeded in simultaneous recording of >10,000 neurons from the entire bilateral dorsal cortex at up to four depths. We also demonstrated large FOV lifetime imaging using the same system. Our modular, open-source FPGA system can be readily integrated into any type of two-photon microscope and will accelerate the biomedical application of multi-scale two-photon imaging in a wide range of pathophysiological investigations.

Older Adults Show Altered Network Connectivity during Fairness Decisions with Similar and Dissimilar Partners
Objectives: The ability to navigate diverse social contexts, such as interacting with different individuals, is crucial across the lifespan and has implications for fraud susceptibility. However, the neural mechanisms supporting social decisions in older adults within varied social contexts remain largely unknown. This study investigated how age and partner similarity modulate neural activation and network connectivity during fairness-related decision making and whether individual differences in behavioral sensitivity to fairness norm violations correlate with neural activities. Methods: Younger (18-35) and older (65-80) adults underwent fMRI while playing an ultimatum game with ostensible partners of a similar or dissimilar age. We used regression to model choice behavior and whole-brain fMRI analyses to examine functional activation and connectivity of the Default Mode (DMN) and Executive Control (ECN) networks. Results: Behaviorally, choices did not differ by age, partner similarity, or their interactions. In contrast, neuroimaging revealed interactions between age, partner similarity, and individual sensitivity to norm violation. Younger adults exhibited positive DMN-anterior cingulate connectivity when interacting with a similar-aged partner, whereas older adults showed a negative connectivity. Furthermore, younger adults demonstrated a negative correlation between their sensitivity to norm violation and the partner-identity effect in ECN-medial prefrontal cortex connectivity, whereas older adults showed a positive correlation. Discussion: Our findings indicate a brain-behavior dissociation where different neural mechanisms support similar behavioral outcomes across age groups. These opposing patterns of neural responses suggest age-related functional reorganizations, which may represent compensatory strategies that enable older adults to preserve behaviors similar to that of their younger counterparts.

Dominant MLC-causing mutations alter hepaCAM subcellular localization and protein interactome in astrocytes of the developing mouse cortex
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy characterized by early-onset macrocephaly, white matter edema, seizures, and motor and cognitive decline. Missense mutations to hepatic and glial cell adhesion molecule (hepaCAM), also known as GlialCAM, are responsible for approximately twenty-five percent of MLC cases. HepaCAM is highly enriched in astrocytes and plays important roles in astrocyte territory establishment, gap junction coupling, branching organization, synaptic function, and development of the gliovascular unit. The molecular mechanisms through which MLC-causing missense mutations alter hepaCAM function in vivo and facilitate MLC pathogenesis during brain development remain largely unknown. Here, we used new viral tools and proximity-based proteomics to examine how three different dominant MLC-causing mutations impact hepaCAM subcellular localization and protein interactome in astrocytes of the developing mouse cortex. We found dramatic defects in hepaCAM distribution throughout the astrocyte, which were common to all mutants tested. We also observed significant changes in protein interactome between wild type and mutant hepaCAM, including decreased association with previously described hepaCAM-interacting proteins Connexin 43 and CLC-2. Moreover, we identified changes in association between hepaCAM and a number of previously undescribed potential hepaCAM-interaction partners, including the epilepsy-associated potassium channel KCNQ2. Collectively, our data provide new insights into hepaCAM function in astrocytes during brain development, reveal altered hepaCAM protein dynamics with MLC missense mutations, and provide a new resource to explore the molecular underpinnings of MLC pathogenesis.

A Practical Preprocessing Pipeline for Concurrent TMS-iEEG: Critical Steps and Methodological Considerations
Transcranial magnetic stimulation combined with intracranial EEG (TMS-iEEG) has emerged as a powerful approach for probing the causal organization and dynamics of the human brain. Despite its promise, the presence of TMS-induced artifacts poses significant challenges for accurately characterizing and interpreting evoked neural responses. In this study, we present a practical preprocessing pipeline for single pulse TMS-iEEG data, incorporating key steps of re-referencing, filtering, artifact interpolation, and detrending. Using both real and simulated data, we systematically evaluated the effects of each step and compared alternative methodological choices. Our results demonstrate that this pipeline effectively attenuated various types of artifacts and noise, yielding cleaner signals for the subsequent analysis of intracranial TMS-evoked potentials (iTEPs). Moreover, we showed that methodological choices can substantially influence iTEPs outcomes. In particular, referencing methods might strongly affect iTEP morphology and amplitude, underscoring the importance of tailoring the referencing strategy to specific signal characteristics and research objectives. For filtering, we recommend a segment-based strategy, i.e., applying filters to data segments excluding the artifact window, to minimize distortion from abrupt TMS-related transients. Overall, this work represents an important step toward establishing a general preprocessing framework for TMS-iEEG data. We hope it encourages broader adoption and methodological development in concurrent TMS-iEEG research, ultimately advancing our understanding of brain organization and TMS mechanisms.

Cerebrovascular Claudin-5 Isoform Expression Correlates with Worsened Stroke Outcomes Following Thromboembolic Stroke
Background and Purpose: Claudin-5 plays a crucial role in the maintenance of the blood-brain barrier (BBB) integrity through its role in endothelial tight junction formation. Alternative splicing of claudin-5 within the microvascular endothelium may modulate BBB structural and functional dynamics, potentially influencing neuronal damage and recovery following ischemic stroke. We hypothesized that ischemic stroke induces temporal changes in claudin-5 protein isoform expression that correlates with worsened neurological outcomes. Methods: Male Wistar rats underwent thromboembolic stroke. Claudin-5 isoform expression was assessed at 3, 6, and 24h post-stroke onset, with additional groups receiving recombinant tissue plasminogen activator (rt-PA) at 4 hours post-stroke. Brain edema, infarct volume, hemorrhage, and cerebral blood flow was evaluated using 9.4T MRI. Ipsilateral and contralateral cerebrovascular claudin-5 expression was quantified via western blotting while neurological function was assessed by 28-point neuroscore. In addition, RNA sequencing analysis was performed to identify novel splice variants. Results: A time-dependent increase in claudin-5 isoform 1 (35kDa) expression levels in the ipsilateral cerebrovasculature at 6 h was observed. Isoform 2 (25kDa) and fragment (10kDa) isoforms of claudin-5 remain unchanged. Treatment with rt-PA maintained the elevated levels of isoform 1 claudin-5 protein expression within the ipsilateral hemisphere. Increased claudin-5 isoform 1 expression within the ipsilateral hemisphere correlated with increased brain edema, hemorrhage, and worsened neurological function at 24h post-stroke onset. RNA sequencing revealed novel CLDN5 splice isoforms in post-stroke rat brain tissue which resemble structural similarity to known human CLDN5 isoforms. Conclusion: These findings demonstrate that ischemic stroke induces temporal, hemisphere-specific alterations in claudin-5 isoform expression that correlate with BBB dysfunction and poor neurological outcomes. The potential indication of novel alternative splice variants suggests that post-transcriptional regulation of claudin-5 represents a previously unrecognized mechanism contributing to endothelial tight junction dysfunction and stroke pathophysiology. These results highlight claudin-5 isoform expression as a potential therapeutic target for preserving BBB integrity following cerebral ischemia.

Distinct Electrophysiological Signatures Define Neuronal Subtypes in the Fasciola Cinereum
The fasciola cinereum (FC) is a small, conserved hippocampal subregion whose function has remained largely unexplored. Anatomically situated between dorsal CA1 and the third ventricle in rodents, the FC receives diverse cortical and subcortical inputs yet is often omitted from hippocampal circuit models. There remains a fundamental knowledge gap regarding the cell types and intrinsic properties of neurons in FC and whether they are distinct from neighboring hippocampal subregions. Here, we performed ex vivo whole-cell patch-clamp recordings in mouse hippocampal slices to characterize FC neurons. We found that FC cells are functionally distinct from neighboring CA1 pyramidal cells, exhibiting significantly reduced excitability, delayed spike initiation, and enhanced afterhyperpolarization (AHP) currents, consistent with strong potassium conductance. Notably, we identified two electrophysiologically distinguishable FC neuron excitatory cell subtypes, differing in excitability and potassium channel activity. Pharmacological analyses demonstrated that Kv2.1 and Kv7 potassium channels play a key role in shaping the intrinsic properties of FC neurons, underlying their reduced excitability. These findings suggest that the FC is a heterogeneous structure, molecularly and functionally specialized for gating excitability within the hippocampal circuit.

Semi-Automated Detection, Annotation, and Prognostic Assessment of Ictal Chirps in Intracranial EEG from Patients with Epilepsy
We analyzed the spectro-temporal characteristics of ictal chirp events in intracranial EEG (iEEG) recordings from 13 epilepsy patients, using a custom derivative dataset of 22,721 spectrograms that we generated from the Epilepsy-iEEG-Multicenter Dataset. Ictal chirps, transient frequency-modulated patterns, were semi-automatically annotated to assess their relationship with seizure onset zones (SOZs) and surgical outcomes. Preprocessing included notch filtering (60 Hz, 120 Hz) and bandpass filtering (1-60 Hz), followed by segmentation into 60-second windows. Spectrograms were generated via Short-Time Fourier Transform (STFT) with a Hann window (87.5% overlap) and converted to dB scale. Chirps were annotated by manually drawing bounding boxes, followed by automated ridge detection, model fitting, and feature extraction (start/end time-frequency, duration, direction, RMSE, R2). Spatial, Spectro-temporal, and clinical features were analyzed using heatmaps, hierarchical clustering, statistical tests (Mann-Whitney U), and outcome prediction models. Patient-channel mappings revealed clustering of chirp patterns among specific patient pairs, correlating with shared clinical profiles. Flow-based analysis demonstrated prognostic value: very high spectral durations in SOZ regions were associated with favorable surgical outcomes (80.43% success rate), whereas very high temporal durations in SOZ correlated with poorer outcomes (51.35% risk). Statistical comparisons showed significant differences between SOZ and non-SOZ chirps: SOZ chirps exhibited longer spectral durations (*p* < 0.001), shorter temporal durations (*p* = 0.006), and higher spectro-temporal ratios (*p* < 0.001). Distribution analyses further indicated that prolonged temporal chirps were more prevalent in non-SOZ regions.

Measuring Joint Pain Through Tibio-Femoral Flexion: technique validation and assessment of behavior in response to different analgesics in rats
Background: Assessing knee joint pain in experimental OA (EOA) models remains a significant challenge. Our study demonstrates that the use of an adapted electronic von Frey (aVF) device, featuring a modified tip, surpasses the standard von Frey (sVF) in detecting knee joint pain behavior and evaluating the efficacy of analgesic treatments in OA model induced by monoiodine acetate (MIA). Results: The sVF was able to induce a behavior profile in naive animals characterized by a hind paw flinching and withdrawal reflex. This behavioral response was affected by intraplantar lidocaine, which prone to the increase in mechanical thresholds related to sVF and validate it as pain related behavior. On the aVF method, the animals displayed no alterations at their mechanical thresholds in presence or absence of lidocaine, suggesting minimal stimulation of hind paw by the modified methodology. In animals where an osteoarthritic phenotype was induced by MIA, the aVF was able to detect a significant reduction on joint mechanical thresholds. The behavior linked to aVF was significantly affected by systemic delivery of morphine, confirming a nociceptive-like phenotype and suggesting a pain behavior predominantly triggered by joint flexion. The aVF was also able to detect an analgesic profile in MIA-OA rats treated with dexamethasone, LPS-RS, fucoidan, and morphine, indicating effectiveness in measure different the response profile triggered by analgesic drugs that affect joint pain perception. Conclusions: Our results suggest aVF as a more appropriate method to evaluate joint pain in rats.

Aging of the blood-brain barrier: Metabolic signatures of mouse brain endothelial cell senescence
The blood-brain barrier (BBB) serves as a critical boundary between the peripheral circulation and the brain. Brain endothelial cells (BECs), which form the inner lining of cerebral blood vessels, play a key role in maintaining this barrier. They express tight junction proteins that prevent molecules from leaking between cells into the brain and efflux transporters that actively remove substances from the brain. As we age, the BBB becomes leaky and dysfunctional, leading to cerebrovascular diseases and dementia. Here, we investigated how BECs become senescent, a major feature of aging, and change their metabolic profile. First, we demonstrate that both the chemotherapeutic doxorubicin, a known inducer of cellular senescence, as well as hydrogen peroxide, a mediator of oxidative stress, induce BEC senescence. This was indicated by positive senescence-associated {beta}-galactosidase staining (P = 0.002 and P = 0.001 for doxorubicin and H2O2, respectively) and increased P16 gene expression (both P = 0.004). Next, we analyzed the metabolic profile of the senescent cells using mass spectrometry. We identified 47 significantly altered metabolites (P < 0.05) in cells and 65 in the supernatant samples of doxorubicin-induced senescent cells, as well as, 53 significantly altered metabolites in cells and 82 in the supernatant samples of H2O2-induced senescent cells. There was an overlap of 10 and 44 metabolites between both treatments in the cell and supernatant samples, respectively. Of those, 9 cellular and 12 supernatant metabolites have already been reported in the literature to be changed during senescence in the brain or other organs. We also identified new metabolites and provide pathway analyses of the significantly altered metabolites. These analyses are the first to characterize BBB cell senescence at a metabolite level and provide the basis for the development of novel therapeutic interventions for age-related neurological disorders.

A Toolkit for In Vivo Mapping and Modulating Neurotransmission at Single-Cell Resolution
Understanding the organization and regulation of neurotransmission at the level of individual neurons and synapses requires tools that can track and manipulate transmitter-specific vesicles in vivo. Here, we present a suite of genetic tools in Caenorhabditis elegans to fluorescently label and conditionally ablate the vesicular transporters for glutamate, GABA, acetylcholine, and monoamines. Using a structure-guided approach informed by protein topology and evolutionary conservation, we engineered endogenously tagged versions for each transporter that maintain their physiological function while allowing for cell-specific, bright, and stable visualization. We also developed conditional knockout strains that enable targeted disruption of neurotransmitter synthesis or packaging in single neurons. We applied this toolkit to map co-expression of vesicular transporters across the C. elegans nervous system, revealing that over 10% of neurons exhibit co-transmission. Using the ADF sensory neuron as a case study, we demonstrate that serotonin and acetylcholine are trafficked in partially distinct vesicle pools. Our approach provides a powerful platform for mapping, monitoring, and manipulating neurotransmitter identity and use in vivo. The molecular strategies described here are likely applicable across species, offering a generalizable approach to dissect synaptic communication in vivo.