bioRxiv Subject Collection: Neuroscience's Journal

A connectomic resource for neural cataloguing and circuit dissection of the larval zebrafish brain
We present a correlated light and electron microscopy (CLEM) dataset from a 7-day-old larval zebrafish, integrating confocal imaging of genetically labeled excitatory (vglut2a) and inhibitory (gad1b) neurons with nanometer-resolution serial section EM. The dataset spans the brain and anterior spinal cord, capturing >180,000 segmented soma, >40,000 molecularly annotated neurons, and 30 million synapses, most of which were classified as excitatory, inhibitory, or modulatory. To characterize the directional flow of activity across the brain, we leverage the synaptic and cell body annotations to compute region-wise input and output drive indices at single cell resolution. We illustrate the dataset's utility by dissecting and validating circuits in three distinct systems: water flow direction encoding in the lateral line, recurrent excitation and contralateral inhibition in a hindbrain motion integrator, and functionally relevant targeted long-range projections from a tegmental excitatory nucleus, demonstrating that this resource enables rigorous hypothesis testing as well as exploratory-driven circuit analysis. The dataset is integrated into an open-access platform optimized to facilitate community reconstruction and discovery efforts throughout the larval zebrafish brain.

The role of amygdala calcitonin gene-related peptide receptors on the development of persistent bladder pain in mice.
Bladder pain significantly impacts millions worldwide, severely affecting their quality of life and posing a major clinical challenge. Understanding the mechanisms underlying persistent bladder pain is critical for developing better therapeutic strategies. In this study, we investigate the effects of cyclophosphamide (CYP)-induced persistent bladder sensitization to explore the lateralized contribution of amygdala calcitonin gene-related peptide receptors (CGRP-Rs) on pain-like changes in mice. We demonstrate that CYP induces hypersensitivity lasting up to 14 days post-injury (DPI) in the urinary bladder distention assay and up to 21 DPI when assessing abdominal mechanical sensitivity. Despite persistent pain-like changes, no significant bladder histological changes were observed. Based on previous findings that CGRP signaling from the parabrachial nucleus contributes to central amygdala (CeA) lateralization, we hypothesized that CGRP-Rs play a key role in driving visceral bladder pain-related hemispherical differences. We show that inhibiting CGRP-R activity with the antagonist CGRP8-37, in the right CeA attenuates bladder pain-like behavior, whereas left CeA inhibition sustains CYP-induced hypersensitivity. Electrophysiological recordings revealed increased firing frequency in CGRP-R positive cells in the right CeA 7 DPI. In vivo single photon calcium imaging demonstrated increased Ca transients in CGRP-R-positive cells in the right CeA, upon the presentation of a stimulus at 0 DPI, and overall at 2DPI, further confirming the pronociceptive role of CGRP-Rs in the right CeA. Taken together, these findings provide a crucial foundation for understanding pain-induced CeA lateralization and for further studies identifying how targeting CGRP signaling could provide bladder pain relief.

Non-inertial scan angle multiplier for expanded fields-of-view
In many laser scan engines, mechanical inertia imposes a tradeoff between mirror diameter (mass) and scan speed (cycle time) and/or scan angle amplitude. Ultimately, this limits the field-of-view and/or scan speed in multiphoton and confocal imaging, laser-based manufacturing, and other applications. To push parameters past this inertia limit, we present a non-inertial (stationary) add-on unit that doubles the scan angle amplitude while preserving both the beam size and cycle time, thus doubling the etendue (optical invariant) and overall system throughput. We demonstrate its use in two-photon calcium imaging of neural activity in living mice. We also adapt the approach to present a phase doubling unit that doubles the maximum range of phase modulators. Finally, we describe further variants including generalizing the approach to Nx scan angle multiplication (N = 2, 3, 4, ...) with diffraction limited performance across a wide scan angle range and over a broad wavelength bandwidth. These non-inertial scan angle multipliers (called Nisam2x, 3x, ...) can expand the capabilities of a range of technologies including imaging, adaptive optics, sensing, marking, and manufacturing.

Human placental stem cells induce a novel multiple myeloid cell-driven immunosuppressive program that ameliorates proinflammatory CNS pathology
Despite a growing interest in Amniotic Epithelial Cell (AEC)-based therapies, the immune responses triggered by AEC transplantation in vivo remain poorly characterized. In particular, how direct exposure to AECs within the central nervous system (CNS) shapes the local immune environment is currently unknown. Herein we describe a novel CNS-specific immunoregulatory pathway induced by intracisternal delivery of human AECs. Local immune responses induced by AECs in the brain led to recruitment of immunosuppressive Arginase 1+ (ARG1+) macrophages and a novel population of myeloid-derived suppressor cells with eosinophilic characteristics, which we term Eo-MDSCs. We further demonstrate that Eo-MDSCs produce Maresin 2 (MaR2), a specialized pro-resolving mediator (SPM) involved in the resolution of inflammation. In a mouse model of Multiple Sclerosis (MS), treatment of established disease with AECs induced immunological responses that resulted in reduced numbers of pathogenic macrophages and T helper (TH)17 cells, increased anti-inflammatory T cell subsets, and enhanced myelin phagocytosis, all of which led to functional recovery. These findings suggest that AEC therapy has the potential to target CNS-intrinsic inflammatory processes in MS, providing a strong rationale for translation into the clinic.

Integration of Motor Planning and Execution through Latent Structure Reorganization in the Posterior Parietal Cortex
The posterior parietal cortex (PPC) plays a central role in sensorimotor control, performing visuomotor transformations, supporting planning, and providing visual feedback. However, it is unknown how the neural populations in different PPC areas organize their activity during this process. It has been proposed that PPC activity reflects population-level dynamics rather than distinct subpopulations, raising the question of how the population flexibly reorganize between the two main phases of motor control, planning and execution. To address this question, we analyzed neural dynamics in three PPC areas (PE, PEc, V6A) in the context of a delayed reaching task, applying dimensionality reduction techniques. This approach allows identifying whether activity in each area is organized into independent or partially overlapping dynamics across task phases. We found evidence of area-specific population subspaces, distinct for movement planning and execution. Specifically, the analysis revealed that in PE, which is a predominantly somatomotor area, neural activity occupied nearly orthogonal subspaces between the two phases, suggesting independent dynamics for movement planning and execution. In contrast, in V6A and PEc, which are involved in visuomotor transformations, we identified both shared and exclusive subspaces, indicating a more flexible representation of motor information in these areas. Overall, our findings suggest that parietal circuits combine both separation and sharing of neural representation to support computations during the different movement stages, providing new insights into the role of the PPC in generating flexible motor behavior.

Cholesterol Inhibits HCN Channels through Dual Mechanisms in Neuropathic Pain
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate the excitability of dorsal root ganglion (DRG) neurons, particularly in the context of neuropathic pain. Cholesterol, a major component of lipid-ordered membrane domains (OMDs), has recently been identified as a critical modulator of HCN channel function. Using FLIM-FRET-based OMD probes and a fluorescent cholesterol sensor GRAM-W, we investigated the effects of cholesterol supplementation on nociceptor DRG neurons from a rat model of spared nerve injury (SNI). We developed a method to distinguish at least two phases of membrane remodeling during cholesterol enrichment: an initial phase marked by OMD expansion and increased accessible cholesterol, followed by a second phase with continued cholesterol accumulation without further OMD expansion. These cholesterol dynamics were further validated through changes in fluorescence anisotropy and homo-FRET measurements of GRAM-W. Temporal analysis of cholesterol enrichment revealed two mechanisms of HCN channel modulation: through expansion of OMDs and elevation of free cholesterol. In SNI DRG neurons with low cholesterol and small OMDs, both mechanisms are active, while in naive DRG neurons--characterized by high cholesterol and large OMDs--modulation occurs only via increased free cholesterol. These findings deepen our understanding of cholesterol's role in modulating ion channels and contributing to neuropathic pain.

Slow Wave Sleep Reduces CSF Concentrations of Beta-amyloid and Tau: A Randomized Crossover Study in Healthy Adults
Background: Slow-wave sleep has been proposed to facilitate the removal of proteins, implicated in neurodegeneration, from the brain. While mechanistic evidence from animal models is accumulating, direct human data on how slow-wave sleep shapes cerebrospinal fluid (CSF) proteostasis remain limited, constraining our understanding of physiological resilience to neurodegenerative disease. Methods: Twelve healthy adults (aged 20-40 years) underwent CSF sampling following three controlled sleep conditions in a randomized crossover design; (1) one night of sleep followed by afternoon CSF sampling, (2) one night of sleep followed by morning CSF sampling, and (3) one night of total sleep deprivation followed by morning CSF sampling. Sleep and wakefulness were verified using polysomnography and actigraphy, with >4-week washout periods between conditions. Measured CSF biomarkers included Alzheimer's disease-related proteins: beta-amyloid isoforms (1-38, 1-40, and 1-42), total and phosphorylated tau, glial fibrillary acidic protein (GFAP), and neurofilament light, as well as orexin, albumin (also measured in serum), and osmolality. Differences between conditions were assessed using Friedman tests with Dunn's post hoc correction. Results: CSF levels of beta-amyloid; and tau tended to be consistently lower after sleep compared with both afternoon sampling and post-sleep deprivation. Concurrently, CSF albumin levels increased after sleep, while neurofilament light and GFAP remained unchanged. Orexin levels rose markedly during sleep deprivation but showed no circadian variation. Conclusions: These findings support a model in which slow wave sleep enhances CSF turnover, reducing concentrations of specific proteins, including beta-amyloid; and tau. Understanding how sleep regulates the homeostasis of neurodegeneration-related proteins may inform strategies to mitigate disease progression.

Neurons, Muscles, and Venom: Identifying Drivers of Cephalopod Predation
Coleoid cephalopods (octopuses, squids, and cuttlefish) produce venoms in their posterior salivary glands. Despite venom's importance in cephalopod evolution and ecology, its regulation and secretion processes remain unresolved. Here, we performed multimodal histological profiling and live imaging to map the glandular architecture and innervation patterns across multiple coleoid species. Micro-computed tomography and tetrachrome alongside hematoxylin and eosin stains verified the proposed differentiation of gland tubular structures into (i) secretory tubules - for venom production - and (ii) striated tubules - potentially propelling venom towards the salivary duct. By tracing five neuronal markers, including nicotine acetylcholine receptors and synapsin, we observed dense neural networks intimately associated with localized muscular signals surrounding the tubules. These findings reveal a spatially distinct organization of venom production and release sites and strongly indicating that neuromuscular signals coordinate venom release into the salivary duct. Although the precise neural circuitry remains to be mapped, our results offer novel understanding of venom gland regulation in marine venomous invertebrates.

Integrate-and-fire neurons with potassium dynamics that capture switches in neuronal excitability class and firing regime
In conductance-based models, spiking-induced ion concentrations fluctuations can modify single neurons' excitability. What are the consequences in networks? To study this, simple models capturing ion concentration dynamics realistically are needed. We propose a method to derive a phenomenological model capturing the coupled extracellular potassium and voltage dynamics from a given class I conductance-based model. Rather than fitting voltage traces, we fit the bifurcation structure of the target model, thereby capturing parameter heterogeneity and rich dynamics. The resulting model extends the quadratic integrate-and-fire model, with extracellular potassium accumulation altering voltage dynamics by increasing the reset voltage. We apply our systematic reduction procedure to the Wang-Buzáski model. Its phenomenological version exhibits quantitatively comparable dynamics and replicates the reshaping of the phase-response curve associated with the transition from SNIC to HOM spikes at elevated potassium. To illustrate the derived model's applicability, we explore how changes in potassium concentration influence synchronization in networks.

iSleep: Continuous, binocular pupil tracking in sleep and reduced consciousness for physiological monitoring, predictions and interventions
Monitoring pupil dynamics is a key tool in understanding arousal. Pupil size can serve as a biomarker for the autonomic nervous system balance as well as for identifying brain states. While internal states can also be self-reported when awake, automated detection and non-invasive monitoring is crucial during sleep and reduced consciousness. Here, we introduce iSleep, an innovative pupil tracking and analysis framework for sleep in humans. It features comfortable, humidified eye-tracking goggles and a platform for integrated analysis and prediction capabilities. We show that iSleep allows safe and continuous access to binocular pupil size and ocular dynamics during sleep and anesthesia. iSleep reveals that pupillary fluctuations correlate tightly with brain activity, heartbeat, and breathing, and can reliably predict brain states. Pupil constrictions reflect parasympathetic drive and likely serve a protective function for deep sleep stability; while dilations indicate arousals. Unexpectedly, we observed a decoupling of binocular movements during periods of sleep, indicating alterations in reflexes which usually govern voluntary eye movements. Finally, iSleep was tested in surgery patients under general anesthesia, revealing dynamic pupil changes to noxious stimuli, suggesting the potential for nociception monitoring during surgeries. In summary, iSleep offers an easy-to-use, robust alternative to read out brain states during sleep and anesthesia, opening new avenues in monitoring, diagnostics, and treatments, previously obscured by closed eyelids.

Precision of reaches and proprioception in motor control and adaptation
How do precision of movement and proprioception influence motor control and adaptation? Several theories - such as the exploration-exploitation hypothesis - propose that variability plays a key role in motor performance and learning. However, empirical measures of motor and proprioceptive precision are often limited by small sample sizes, and proprioceptive estimates, especially those relying on efferent signals, are difficult to isolate and quantify. In this study, we leveraged a large dataset of 270 participants - including a subsample of older adults (ages 54-84) - to assess the precision of hand movements and proprioceptive estimates, and to examine whether these factors predict individual differences in motor learning and adaptation. We found that baseline reach variance did not predict learning or changes in hand localization. Although active hand localization (which includes efferent contributions) was slightly more precise - showing an 8.6% reduction in variance - this suggests that unseen hand estimates rely primarily on proprioception. Neither motor nor sensory precision varied with age. However, reach aftereffects were modestly associated with proprioceptive precision before training and proprioceptive recalibration after training. No other measure of learning or variance was reliably associated. These findings suggest that reach aftereffects may partly reflect changes in hand proprioception, but overall, we identified no predictors of adaptation to a rotated visual cursor.

Shared Neural Signatures of Socioeconomic Status, Scarcity, and Neighborhood Threat in Youth
Early life adversity is a known risk factor for psychopathology, yet the neurodevelopmental impacts of distinct types of adversity remain unclear. Using machine learning, we examined how adversity (physical and sexual abuse, neighborhood threat, scarcity, household dysfunction, prenatal substance exposure, parental psychopathology) and socioeconomic status are associated with cortical thickness in youth. We used data from the Adolescent Brain Cognitive Development Study at three time points: baseline (N=6,908, ages 9-10), two-year follow-up (N=5,808), and four-year follow-up (N=2,245). Cortical thickness was linked to socioeconomic status and neighborhood threat at all time points, and scarcity at baseline. The strongest negative associations were in medial temporal and occipital regions. Our analyses revealed neural effects of socioeconomic status, neighborhood threat, and scarcity, converging on regions involved in memory, visual processing, and higher-order cognition. These findings suggest consistent neural signatures linked to socioeconomic disadvantage, highlighting the importance of addressing inequality to promote neurodevelopmental health.

Drosophila Modeling of Insomnia-Associated Genes Reveals Diverse Underlying Sleep Phenotypes
Insomnia is a prevalent sleep disorder with highly heterogenous manifestations. While data-driven approaches to insomnia subtyping have revealed potential differences between proposed insomnia subtypes and their impacts on overall health, little is known about the genetic factors that underly and differentiate these potential insomnia subtypes. We employ a human-genetics-driven approach to Drosophila modeling to identify the range of sleep traits regulated by insomnia-associated genes. Modeling pan-neuronal loss of Drosophila orthologs of a set of insomnia genes reveals a broad range of sleep phenotypes. Through systematic characterization of traits related to sleep quantity, timing, and quality, we identify genetic factors that co-regulate aspects of the insomnia-associated phenotypic landscape. Out of the 75 insomnia-associated genes identified, only 1/3 had at least one Drosophila ortholog that regulated overall sleep quantity. In contrast, 1/3 of the insomnia-associated genes had at least one Drosophila ortholog that regulated either sleep timing or sleep quality, without impacting sleep quantity. Together, this work in Drosophila provides support for a genetic influence on the differences between insomnia subtypes.

DCPS modulates TDP-43 mediated neurodegeneration through P-body regulation
The proteinopathy of the RNA-binding protein TDP-43, characterized by nuclear clearance and cytoplasmic inclusion, is a hallmark of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD). Through CRISPR interference (CRISPRi) screening in human neurons, we identified the decapping enzyme scavenger (DCPS) as a novel genetic modifier of TDP-43 loss-of-function (LOF)-mediated neurotoxicity. Our findings reveal that TDP-43 LOF leads to aberrant mRNA degradation, via disrupting the properties and function of processing bodies (P-bodies). TDP-43 interacts with P-body component proteins, potentially influencing their dynamic equilibrium and assembly into ribonucleoprotein (RNP) granules. Reducing DCPS restores P-body integrity and RNA turnover, ultimately improving neuronal survival. Overall, this study highlights a novel role of TDP-43 in RNA processing through P-body regulation and identifies DCPS as a potential therapeutic target for TDP-43 proteinopathy-related neurodegenerative diseases.

Arousal-Driven Serial Dependence: Internal States Modulate Perceived Duration
Emotional experience shapes not only how we perceive the present but also how the past influences our judgments. In this study, we examined how emotional arousal and valence modulate serial dependence in time perception: a phenomenon in which prior experiences bias current estimates. Using a duration reproduction task, participants viewed affective images drawn from the International Affective Picture System (IAPS) to induce high-arousal (positive or negative) or low-arousal (neutral) emotional states. We found that participants consistently overestimated durations in high-arousal conditions compared to those in low-arousal conditions, regardless of valence. Crucially, serial dependence effects were significantly amplified during high-arousal states. This effect was strongest when high-arousal trials were preceded by similar high-arousal trials, indicating a state-dependent intensification of sequential bias. Emotional valence, by contrast, had no significant effect on either temporal distortion or serial dependence. These findings suggest that emotional arousal, rather than valence, plays a key role in shaping the temporal integration of past and present, offering new insight into how affective states shape temporal cognition and serial dependence.

Circadian Rhythms Time Seizure Severity in Drosophila
It is established that epilepsy patients can exhibit 24-hour rhythms in seizure severity and occurrence. While the pathways underlying seizure rhythmicity remain poorly understood, it seems likely that a contribution from the biological clock is involved. A better understanding of any such contribution may translate to better treatments. Here, the influence of the 24 h circadian rhythm on seizure activity in Drosophila melanogaster is investigated. Seizure-susceptible bang-sensitive julius seizure (jus) mutants were subjected to mechanically induced seizure at six different zeitgeber points. A clear sex-dependent phenotype was observed, with seizure severity showing a greater time-of-day effect in females, than males. The temporal pattern of seizure recovery time was bimodal, exhibiting both a morning and an evening peak. Rearing flies in constant light, which renders the molecular clock dysfunctional, abolished the seizure rhythm. Conversely, female seizure mutants reared in constant darkness, allowing free running of the circadian clock, continued to exhibit a bimodal rhythm of seizure severity. These findings support a role for the biological clock in seizure activity, at least in female Drosophila. Thus, this study validates Drosophila as a potential model for the identification of mechanisms modulating seizure rhythmicity, with the potential to aid future treatment of epilepsy.

GPR68, a proton-sensing GPCR, mediates acid-induced visceral nociception
Background & Aims: Localised acidification from immune cell infiltration and heightened glycolysis contributes to colitis pathology by activating acid-sensing receptors such as GPR68, a proton-sensing GPCR expressed on immune and stromal cells. Single-cell RNAseq analysis revealed GPR68 is also expressed in colonic sensory neurons, prompting us to investigate its role in acid-induced colonic nociception. Methods: Expression of GPR68 in colonic nociceptors and tissue from people with colitis was confirmed by in silico analysis of our RNAseq databases. Its contribution to disease activity was assessed using the acute dextran sulphate sodium (DSS) model of colitis. Acid-evoked sensory signalling was evaluated via colonic afferent recordings and Ca2+; imaging in DRG neurons from wild-type and GPR68-/- mice, supported by pharmacological studies using Ogerin (a GPR68 positive allosteric modulator) and Ogremorphin (a GPR68 antagonist). Results: RNAseq analysis showed GPR68 is robustly expressed in Trpv1+; colonic nociceptors and upregulated in tissue from people with inflammatory bowel disease, consistent with reduced disease activity in DSS-treated GPR68-/- mice. Genetic deletion of GPR68 abolished colonic afferent responses to acid, which were also attenuated by Ogremorphin and enhanced by Ogerin. In Ca2+;-free buffer, DRG neurons from GPR68-/- mice or those pre-treated with Ogremorphin showed significantly reduced acid-evoked intracellular Ca2+; responses. By contrast the colonic afferent and DRG Ca2+ response (in Ca2+-containing buffer) to capsaicin was comparable between tissue from wild-type and GPR68-/- mice highlighting the involvement of divergent proton-dependent cellular signalling cascades. Conclusions: These findings identify GPR68 as a key mediator of acid-induced colonic nociception and highlight its potential as a therapeutic target for the treatment of pain in colitis.

Induction of Human Pruriceptors from Pluripotent Stem Cells via Transcription Factors
Pruriception is crucial for defense against external stimuli but can lead to chronic pruritus, a debilitating condition affecting millions worldwide. Our understanding of the cellular and molecular mechanisms behind the sensation of itch has been hindered by the lack of functional human models. Here, we address this limitation by developing a protocol to generate induced pruriceptors (iPruriceptors) from human pluripotent stem cells (hPSCs). We compared two differentiation approaches: a direct method via forced expression of transcription factors (TFs) in hPSCs, and a 2-step process through expression of TFs in hPSC-derived neural crest cells (NCCs). The 2-step protocol proved superior in inducing a transcriptional program that closely resembles that of human pruriceptors. Our optimized protocol employs forced expression of NGN1 and ISL1 to drive differentiation from NCCs into pruriceptors, enhancing the expression of known pruritogen receptors such as IL31RA, which pairs with OSMR, and HRH1. The induction of this transcriptional program leads to functional maturation of iPruriceptors. Accordingly, iPruriceptors exhibit robust responses to itch stimuli and in vivo-like itch pharmacology such as treatment with ABT-317, a JAK1 inhibitor tool compound, similar to those targeting intensive pruritus in atopic dermatitis (AD). Importantly, iPruriceptors can be generated without viral vectors or safe-harbor gene editing, using a PiggyBac-based transfection method that simplifies scalability. Our protocol offers a robust platform for investigating itch biology, modeling chronic pruritus, and enabling high-throughput screening for therapeutic target discovery.

Dynamics of mesoscale brain network during decision-making learning revealed by chronic, large-scale single-unit recording
Associating unfamiliar stimuli with appropriate behavior through experience is crucial for survival. While task-relevant information has been found to be distributed across multiple brain regions, how regional nodes in this distributed network reorganize their functional interactions throughout learning remains to be elucidated. Here, we performed chronic, large-scale single-unit recording across 10 cortical and subcortical regions using ultra-flexible microelectrode arrays in mice performing a visual decision-making task and tracked mesoscale functional network dynamics throughout learning. Task learning reshaped interregional functional connectivity, leading to the emergence of a subnetwork involving visual and frontal regions during the acquisition of correct No-Go responses. This reorganization was accompanied by a more widespread representation of visual stimulus across regions, and a region's network rank strongly predicted its peak timing of visual information encoding. Together, our findings reveal that mesoscale networks undergo dynamic restructuring during learning, with functional connectivity ranks influencing the propagation of sensory information across the network.

Neural Mechanisms Underlying Approach and Avoidance Tendencies in Alcohol Use: An Electrophysiological Investigation
Background: A growing body of research highlights the differential role of approach and avoidance tendencies toward alcohol cues in the development and maintenance of harmful drinking behavior. Some individuals involved in alcohol consumption show an automatic approach towards alcohol-related stimuli, whereas others show avoidance, suggesting the need to understand the neurocognitive mechanisms underlying these automatic tendencies. Methods: The current study employed an Alcohol Approach-Avoidance Task (A-AAT) with electroencephalography (EEG) to investigate neural responses among individuals with alcohol approach and avoidance tendencies. Alcohol group participants were categorized into approach and avoidance subgroups based on their behavioral tendencies following A-AAT administration. Findings: Results revealed significant attenuation in P3 and FN400 amplitudes at frontal and parietal sites, respectively, in the alcohol-approach participants compared to both the alcohol-avoidance and non-alcohol participants. These findings suggest weakened controlled cue processing and impaired stimulus-response conflict resolution in individuals with stronger approach tendencies. Notably, right prefrontal activity exhibited prominent differences between the approach and avoidance groups, highlighting its potential role in regulating automatic alcohol-related responses. Implications: The identified ERP markers provide clinical utility for assessing alcohol approach tendencies and monitoring intervention progress. Findings further emphasize the importance of individually tailored targeted interventions aimed at reducing harmful alcohol consumption behavior by altering alcohol approach tendencies.

Characterization of neuronal ensembles in a model of dual reward conditioned place preference
Substance use disorder (SUD) is associated with maladaptive alterations in behavior. Drug-seeking behavior has been associated with neuronal ensembles, defined here as a small group of neurons exhibiting coordinated activity patterns, in the prelimbic prefrontal cortex (PL) and nucleus accumbens core (NAcore). Most SUD preclinical research focuses on the effects of single-reward exposure on the general population of neurons within the reward pathway, rather than on poly-reward exposure. Here, we seek to characterize and compare the ensembles linked to cocaine and chocolate using a within-subject approach. We used Ai14xFos2A-iCreER (c-Fos-TRAP2) transgenic mice to tag neuronal ensembles in a novel dual cocaine and chocolate conditioned place preference (CPP) model, where each chamber was associated with a different reward, either cocaine or chocolate. We found that, after successful dual conditioning and in the absence of the rewards, mice preferred cocaine over chocolate. Additionally, in mice exposed to both cocaine and chocolate, cortical and accumbal ensembles linked to each reward were comparable in size to reward ensembles in mice exposed only to one reward. However, reward-seeking ensembles were larger than ensembles tagged in homecage control mice across groups. We also found that ensemble size does not correlate with the level of reward seeking across single and dual CPP models. These results offer a new paradigm for studying drug-related neuronal ensembles in comparison to natural rewards in non-contingent behavioral models.

Response dynamics of discrete subiculum->retrosplenial cortex projections underlying trace fear conditioning
Associating events separated in time depends on the CA1, subiculum (SUB), and retrosplenial cortex (RSP). The degree to which their connectivity and underlying circuit mechanisms contribute to the association of such temporally discontiguous events is not known. Here we showed, using trace fear conditioning (TFC), wherein mice learn to associate tone and shock separated by a temporal trace, that molecularly distinct excitatory VGluT1+ and VGluT2+ SUB[->]RSP projections subserve the associative and temporal components of TFC. During trace memory formation, VGluT2+ SUB[->]RSP projections showed increased and decreased bulk calcium activity at tone and trace onset, respectively, an activity pattern that was reestablished during memory recall. Such pattern was not observed in CA subfields, suggesting that associative and temporal components of TFC are integrated at the SUB or SUB[->]RSP synapses before being presented to the RSP. Our findings establish a circuit mechanism for representing complex temporal information in episodic memory.

Sleep strengthens successor representations of learned sequences
Experiences reshape our internal representations of the world. However, the neural and cognitive dynamics of this process are largely unknown. Here, we investigated how sequence learning reorganizes neural representations and how sleep-dependent consolidation contributes to this transformation. Using high-density electroencephalography and multivariate decoding, we found that learning temporal sequences of visual information led to the incorporation of successor representations during a subsequent perceptual task, despite temporal information being task-irrelevant. Importantly, individuals with better sequence memory performance exhibited stronger successor incorporation during the perceptual task. Representational similarity analyses comparing neural patterns with different layers of a deep neural network revealed a learning-induced shift in representational format, from low-level visual features to higher-level abstract properties. Critically, both the strength and transformation of successor representations correlated with the proportion of slow-wave sleep during a post-learning nap. These findings support the idea that sequence learning induces lasting changes in visual representational geometry and that sleep strengthens these changes, providing mechanistic insights into how the brain updates internal models after exposure to environmental regularities.

Aβ-HMGB1 complex is a pathogenic molecule at the advanced stage of Alzheimer's disease
Multiple molecules including A{beta}, tau and other inflammatory molecules mediate Alzheimer's disease (AD) pathology. High mobility group box 1 (HMGB1), which is released from necrotic cells and binds to Toll-like receptors (TLRs) of surrounding neurons and microglia, also mediates AD pathology from the early stage. Paradoxically, HMGB1 concentration in cerebrospinal fluid (CSF) at the advanced stage of AD is not higher than that at the early stage. Here we show that A{beta}-HMGB1 complexes are generated in neurons undergoing secondary necrosis around A{beta} plaques in the AD brain at the advanced stage. A{beta}-HMGB1 complex triggers neurite degeneration and necrosis of human normal iPSC-derived neurons via binding to TLR4. Further, two anti-HMGB1 antibodies that inhibit its interaction with TLR4 successfully suppress the toxicity of A{beta}-HMGB1 complex to human iPSC-derived neurons, and recover cognitive impairment and A{beta}-HMGB1 complex-related brain pathology in AD model mice, while such therapeutic effects were not obvious with an anti-A{beta} antibody (lecanemab) approved for human AD patients. Enzyme-Linked Immuno Sorbent Assay (ELISA) revealed plasma level of A{beta}-HMGB1 complex was increased in a part of AD patients at the advanced stage. These findings for the molecular basis of toxicity to neurons suggest the significance of A{beta}-HMGB1 complex at the advanced stage of AD pathology, and might explain the discrepancy between A{beta} burdens and clinical symptoms of human AD patients treated with anti-A{beta} antibody.

Selective-plane Functional Ultrasound Neuroimaging
Functional ultrasound (fUS) is a sensitive neuroimaging technique that uses high frame rate ultrasound to monitor brain hemodynamics as a proxy for neural activity. Recent studies have demonstrated its potential for brain-machine interfaces (BMIs) in both primates and humans. However, current 2D fUS approaches are limited to a single brain slice, restricting the ability to decode widespread neural activity. While 3D fUS could overcome this, it demands high data throughput, increased computation, and higher temperature increases due to the requirement of a higher number of transmitted waves. To address this, we present selective-plane fUS, a method that leverages the wide field of view of row-column addressed (RCA) transducer arrays to capture activity in targeted brain regions without moving the probe. By electronically selecting imaging planes, this approach achieves higher spatio-temporal resolution with lower data and pulse repetition rate compared to 3D fUS, while preserving sensitivity to neurovascular signals. Our pipeline begins with a 3D functional activation scan to guide plane selection, followed by high frame rate focused wave (FW) imaging in coronal or sagittal slices. This method offers robust detection of visually evoked responses in rodents and reduces signal variability compared to 3D fUS. By imaging only functional regions of interest, selective-plane fUS cuts computational load by an order of magnitude, enables continuous 1000 Hz recordings, and reduces functional signal variability fivefold. We envision that this method will allow tailored continuous functional imaging of widespread neuronal activity in the human brain in a BMI context.

FiPhoPHA - A fiber photometry python package for post-hoc analysis
Fiber photometry is a neuroscience technique that can continuously monitor in vivo fluorescence to assess population neural activity or neuropeptide/transmitter release in freely behaving animals. Despite the widespread adoption of this technique, methods to statistically analyse data in an unbiased, objective, and easily adopted manner are lacking. Various pipelines for data analysis exist, but they are often system-specific, only for pre-processing data, and/or lack usability. Current post hoc statistical approaches involve inadvertently biased user-defined time-binned averages or area under the curve analysis. To date, no post-hoc user-friendly and assumption-free tool for a standardised unbiased analysis exists, yet such a tool would improve reproducibility and statistical reliability for all users. Hence, we have developed a user-friendly post hoc statistical analysis package in Python that is easily downloaded and applied to data from any fiber photometry system. This Fibre Photometry Post Hoc Analysis (FiPhoPHA) package incorporates a variety of tools, a downsampler, bootstrapped confidence intervals (CIs) for analyzing peri-event signals between groups and compared to baseline, and permutation tests for comparing peri-event signals across comparison periods. We also include the ability to quickly and efficiently sort the data into mean time bins, if desired. This provides an open-source, user-friendly python package for unbiased and standardised post-hoc statistical analysis to improve reproducibility using data from any fiber photometry system.

Overnight circuit remodelling drives juvenile alloparental care
Parental care is critical for the survival of altricial young and is mediated by neural circuits that are well characterised in adult rodents. Although adults can exhibit caregiving even before becoming parents, the developmental origins of this so-called alloparental behaviour remain unclear. Here, we show that alloparental behaviour in mice emerges abruptly between postnatal day (P)14 and 15, independently of prior social experience. This behavioural transition coincides with the onset of pup-specific activity in galanin-expressing medial preoptic area (MPOA-Gal) neurons, which are essential for parental behaviour in adulthood. Chemogenetic silencing of MPOA-Gal neurons abolishes caregiving in juveniles, suggesting that similar circuits control parenting across life stages. Viral trans-synaptic tracing and whole-cell recordings reveal extensive input remodelling of MPOA-Gal neurons between P14 and 15, marking a rapid transition from a highly connected, immature network to a sparser, adult-like circuit configuration. We identify microglia as key mediators of this process, as their ablation prevents both synaptic reorganisation and the emergence of alloparenting. Together, these findings uncover a previously unrecognised, microglia-dependent developmental switch that enables caregiving in juveniles through rapid circuit reconfiguration.

Ultrasound neuromodulation reveals distinct roles of the dorsal anterior cingulate cortex and anterior insula in Pavlovian biases
Pavlovian biases reflect the notorious influence of hard-wired, evolutionarily conserved cue-response tendencies on instrumental action selection: people show automatic action invigoration in face of potential rewards, but action suppression in face of potential punishments. The neural origin of these biases is unclear. Past evidence suggests dorsal anterior cingulate cortex (dACC) and anterior insula (aIns) as part of a reset network that rapidly responds to salient information and might contribute to these biases. We used transcranial ultrasonic stimulation (TUS) in twenty-nine healthy participants to interfere with neural activity in these regions and test their causal role in a within-subject, counter-balanced design across three sessions (sham, TUS-dACC, TUS-aIns). Computational modelling revealed a double dissociation, with distinct roles of both regions in learning from feedback: while TUS to the aIns changed people's tendency to overly take credit for rewards following action and to ignore punishments following inaction, TUS to dACC reduced participants' tendency to take cue valence as a reinforcer signal. Although the dACC and aIns are part of the same network and often co-activate during decision-making tasks, TUS interference reveals their distinct roles: the dACC selects feedback signals from the environment to inform subsequent choices while the aIns infers whether these signals were caused by one's own actions.

Hibernation improves neural performance during energy stress in regions across the central nervous system in the American bullfrog
Neuronal signaling requires high rates of ATP production via the oxidative metabolism of glucose. The American bullfrog is intriguing, as this species has typical brain energy requirements for an average vertebrate but modifies synaptic physiology and metabolism after hibernation to maintain function during hypoxia and ischemia. Given the importance of the respiratory system in restoring metabolic homeostasis during emergence from underwater hibernation, work to date has addressed this response in the brainstem respiratory network. Thus, metabolic plasticity has been interpreted as an adaptation used to restart respiratory motor behavior under hypoxic conditions during the transition from skin breathing to air breathing. It remains unclear whether these improvements are specific to the brainstem regions critical for breathing versus a global response within the central nervous system (CNS). To address this question, we recorded neural activity from the spinal cord, forebrain, and brainstem respiratory network in vitro. As expected, hypoxia disrupted the function of each network in control animals. After hibernation, each network improved its activity in hypoxia compared to controls. These results suggest that plasticity that improves neural function during energy stress following hibernation reflects a global response that may impact many behaviors controlled by the CNS and is not limited to regions involved in metabolic homeostasis.

Viral Infection Induces Alzheimer's Disease-Related Pathways and Senescence in iPSC-Derived Neuronal Models
INTRODUCTION: The Pathogen Infection Hypothesis proposes that beta-Amyloid functions as an antimicrobial peptide, with pathogen-induced aggregation potentially contributing to Alzheimer's disease (AD) pathology. METHODS: We used human iPSC-derived 2D neurons and 3D cerebral organoids from wild-type and familial AD (PSEN1/2 mutant) lines to model acute infections with HSV-1 and TBEV and beta-Amyloid aggregation. Transcriptomic and proteomic analyses were conducted to assess molecular responses. RESULTS: HSV-1, but not TBEV, induced robust beta-Amyloid clustering, which was, however, dependent on extracellular amyloid peptides. Transcriptomic profiling revealed widespread HSV-1-induced changes, including activation of neurodegeneration-related pathways. Proteomic profiling confirmed enrichment of neurodegeneration- and senescence-associated secretome signatures. PSEN1/2 mutations did not alter the acute infection response. Reanalysis of independent datasets confirmed our findings and revealed a limited protective effect of acyclovir. DISCUSSION: Results directly support the Pathogen Infection Hypothesis and suggest that preventing viral infections via vaccinations may represent a feasible approach to reducing AD risk.

Long-term Nrf2-driven microglial repopulation mitigates microgliosis, neuronal loss and cognitive deficits in tauopathy
A prominent pathological feature of tauopathies, including Alzheimer disease, is a chronic microglial reactivity, which contributes to neuroinflammation and disease progression. Microglia, the innate immune cells of the brain, can be pharmacologically eliminated by inhibiting the colony stimulating factor 1 receptor (e.g. with PLX5622), which is essential for their survival and proliferation. Upon inhibitor withdrawal, microglia rapidly repopulate, replenishing the central nervous system niche with naive cells. In recent years, microglia repopulation strategies have gained great interest as a means to reprogram dysfunctional microglia, while avoiding the detrimental effects of prolonged immune depletion. Despite promising short-term results, the long-term efficacy and pharmacological modulation of repopulated microglial remain poorly understood. Here, we investigate whether repopulated microglia after PLX5622 treatment sustains their beneficial effects over time in an AAV-hTauP301L induced model. Additionally, we assessed whether activating the cytoprotective nuclear factor erythroid 2 p45-related factor 2 (Nrf2) during microglial repopulation enhanced and prolonged the therapeutic outcomes. While microglial repopulation alone failed to maintain its neuroprotection in the long-term, when combined with an Nrf2 inducer, it improved cognitive deficits, reverted hippocampal neuronal loss and restored microglial phenotypes and mitochondrial energetics homeostasis, in our tauopathy-induced model. These results highlight the importance of shaping the fate of self-renewed microglia and propose Nrf2-mediated microglia repopulation as a potential pharmacological strategy for the treatment of tauopathies.

NiCLIP: Neuroimaging contrastive language-image pretraining model for predicting text from brain activation images
Predicting tasks or cognitive domains based on brain activation maps has remained an open question within the neuroscience community for many years. Meta-analytic functional decoding methods aim to tackle this issue by providing a quantitative estimation of behavioral profiles associated with specific brain regions. Existing methods face intrinsic challenges in neuroimaging meta-analysis, particularly in consolidating textual information from publications, as they rely on limited metrics that do not capture the semantic context of the text. The combination of large language models (LLMs) with advanced deep contrastive learning models (e.g., CLIP) for aligning text with images has revolutionized neuroimaging meta-analysis, potentially offering solutions to functional decoding challenges. In this work, we present NiCLIP, a contrastive language-image pretrained model that predicts cognitive tasks, concepts, and domains from brain activation patterns. We leveraged over 23,000 neuroscientific articles to train a CLIP model for text-to-brain association. We demonstrated that fine-tuned LLMs (e.g., BrainGPT models) outperform their base LLM counterparts. Our detailed evaluation of NiCLIP predictions revealed that performance is optimized when using full-text articles instead of abstracts, as well as a curated cognitive ontology with precise task-concept-domain mappings. Our results indicated that NiCLIP accurately predicts cognitive tasks from group-level activation maps provided by the Human Connectome Project across multiple domains (e.g., emotion, language, motor) and precisely characterizes the functional roles of specific brain regions, including the amygdala, hippocampus, and temporoparietal junction. However, NiCLIP showed limitations with noisy subject-level activation maps. NiCLIP represents a significant advancement in quantitative functional decoding for neuroimaging, offering researchers a powerful tool for hypothesis generation and scientific discovery.

Multiplexed Neuromodulatory-Type-Annotated EM-Reconstruction of Larval Zebrafish
The brain is complementarily assembled by the sensorimotor and neuromodulatory (NM) pathways. Mapping the neural connectome is essential for elucidating the synaptic organization principles of this bi-pathway architecture. However, most electron microscopy (EM) reconstructions provide limited information about cell types, in particular NM neurons. Here we present a synapse-level, multiplexed molecular annotated reconstruction of an intact larval zebrafish brain, comprising over 170 thousand cells and 25 million synapses. Noradrenergic, dopaminergic, serotonergic, hypocretinergic/orexinergic, and glycinergic neurons were identified using subcellular localization of APEX2, while glutamatergic and GABAergic neurons were inferred from the Zebrafish Mesoscopic Atlas. NM neurons with varying indegree scale-distinctly innervate various sensory-motor brain regions, exhibiting heterogeneity in synapse number and strength. As a critical NM system, individual locus coeruleus noradrenergic (LC-NE) neurons integrate extensive brain-wide inputs displaying modality-specific spatial organization. Motor-related and sensory-related inputs preferentially target proximal and distal dendrites of these neurons, respectively. While the majority of inputs are one-to-one, approximately 16% synapse onto multiple targets, forming one-to-many connections. Notably, these shared input patterns extend across different monoaminergic systems, serving as a structural basis for coordinated neuromodulation. Our results demonstrate the organization principles of the NM system's input architecture, particularly within the LC-NE system. This brain-wide, multiplexed molecular annotated microscale reconstruction of a vertebrate brain, combined with multi-modal mesoscopic datasets, offers a reliable resource for the precise identification of diverse neuronal types within EM connectomes, and provides critical reference for elucidating the synaptic architecture principles underlying sensorimotor and NM pathways in the vertebrate brain.

Close Packing of Cells in Vestibular Epithelia Supports Local Electrical Potentials that Reduce Latency of Action Potential Generation
In the vestibular system, upon transduction of head motion, ionic currents from type I sensory hair cells alter [K+] and electrical potentials in an extended synaptic cleft formed by a calyx terminal of the associated afferent neuron. During excitatory stimuli, these changes in turn modulate post-synaptic currents across the calyx inner face to depolarize the afferent and initiate action potentials. Within the tightly packed columnar vestibular sensory epithelium, electrical currents from the hair cell and calyx must also traverse non-synaptic extracellular spaces and generate local extracellular potentials before dispersing into the perilymph beneath the basement membrane. Here we show that such dynamic electrical potentials enhance action potential generation by reducing outward K+ currents on both the inner and outer faces of the calyx. This effect also influences adjacent calyces and may explain the abundance of calyx terminals in amniotes where there is a need for rapid recognition of changes in head orientation and acceleration.

RubyACRs Enable Red-Shifted Optogenetic Inhibition in Freely Behaving Drosophila
Optogenetic neuronal activators with red-shifted excitation spectra, such as Chrimson, have significantly advanced Drosophila neuroscience. However, until recently, available optogenetic inhibitors required shorter activation wavelengths, which do not penetrate tissue as effectively and are stronger visual stimuli to the animal, potentially confounding behavioral results. Here, we assess the efficacy of two newly identified anion-conducting channelrhodopsins with spectral sensitivities similar to Chrimson: A1ACR and HfACR (RubyACRs). Electrophysiology and functional imaging confirmed that RubyACRs effectively hyperpolarize neurons, with stronger and faster effects than the widely used inhibitor GtACR1. Activation of RubyACRs led to circuit-specific behavioral changes in three different neuronal groups. In glutamatergic motor neurons, activating RubyACRs suppressed adult locomotor activity. In PPL1-{gamma}1pedc dopaminergic neurons, pairing odors with RubyACR activation during learning produced odor responses consistent with synaptic silencing. Finally, activation of RubyACRs in the pIP10 neuron suppressed pulse song during courtship. Together, these results demonstrate that RubyACRs are effective and reliable tools for neuronal inhibition in Drosophila, expanding the optogenetic toolkit for circuit dissection in freely behaving animals.

A systemic clock brake: Period1 stabilizes the circadian network under environmental stress
Precise alignment between internal circadian clocks and environmental light cycles is essential for physiological homeostasis and survival. However, the molecular mechanisms that preserve this synchrony across central and peripheral tissues remain poorly defined. Here, we uncover an unexpected role for the core clock gene Period1 (Per1) as a systemic modulator of circadian stability, regulating light-induced re-entrainment across the brain and body. In Per1-deficient mice, we show that loss of Per1 accelerates clock realignment, influencing transcriptomic, metabolic, hormonal, and behavioral indicators of circadian realignment across multiple organ systems, including the suprachiasmatic nucleus (SCN) and peripheral tissues such as the liver, adipose tissue, and adrenal glands. Notably, this accelerated adaptation confers protection against jetlag-induced sleep disturbances, weight gain, and metabolic imbalance, underscoring a systemic role for Per1 in maintaining circadian network stability. Mechanistically, unbiased spatial transcriptomics identified reduced expression of the arginine vasopressin (AVP), a key neuropeptide mediating SCN intercellular coupling, as the driver of circadian network instability. Weakened SCN synchrony permits enhanced flexibility of peripheral oscillator responses, expediting whole-body adaptation to shifted light-dark schedules. These findings position Per1 as a critical regulator of circadian robustness, a buffer against light over-responsiveness, identifying a potential molecular target for mitigating circadian misalignment in contexts such as jetlag, shift work, and metabolic disease.

The Taco Setup: A Novel TMS-fMRI Setup for High Resolution Whole Brain Imaging
Simultaneous TMS-fMRI holds significant opportunities for advancing basic and translational neuroscience. However, current configurations face technical limitations, particularly the need to accommodate TMS hardware within the MRI environment. This often necessitates reducing the number of radio-frequency (RF) channels, compromising fMRI data quality and whole-brain coverage. Here, we introduce a novel 22-channel Taco TMS-fMRI configuration that re-purposes flexible RF coils, wrapping them around both the participant's head and the TMS coil to preserve whole brain signal reception. Guided by precision fMRI principles, we optimized acquisition protocols to achieve to achieve high temporal signal-to-noise ratio (tSNR) across the cortex. Data from three pilot participants demonstrate robust signal quality, including in regions proximal to the TMS coil. This setup offers a relatively simple and cost-effective approach to integrating precision fMRI into TMS-fMRI research.

Precision targeting of C3+ reactive astrocyte subpopulations with endogenous ADAR in an iPSC-derived model
Astrocytes play pivotal roles in maintaining neural architecture and function. However, their pronounced heterogeneity, especially in reactive states where distinct subtypes can adopt potentially opposing functions (e.g., neuroprotective vs. neuroinflammatory), complicates our understanding of their net contributions to neurological disorders. A critical challenge arises because these functionally distinct subpopulations often coexist, and the lack of precise tools to separately monitor or manipulate them has significantly hindered efforts to dissect their specific roles in disease progression. Here, we address this gap by developing and optimizing fluorescent RNA sensors mediated by endogenous adenosine deaminase acting on RNA (ADAR) for application in induced pluripotent stem cell (iPSC)-derived astrocytes. We employed a streamlined screening methodology to enhance sensor specificity and functionality for complement component 3 (C3), a key marker predominantly associated with neuroinflammatory astrocytes, thus enabling subtype-specific tracking and providing a crucial tool for distinguishing these cells within heterogeneous populations. By integrating the biological complexity of astrocytes with the technological precision of ADAR-mediated sensing, this study establishes a robust framework for investigating astrocyte dynamics.

An intracortical brain-machine interface based on macaque ventral premotor activity
The majority of brain-machine interface (BMI) studies have focused on decoding intended movements based on neural activity of primary motor (M1) and dorsal premotor cortex (PMd). The ventral premotor cortex (PMv), and more specifically area F5c, has been implicated in object grasping and action observation, and may represent an alternative for motor BMI control due to its phasic modulation during action observation. Using chronically implanted Utah arrays in F5c, PMd, and M1 in two male macaques, we compared the efficacy of controlling a motor BMI based on neural activity of each area. PMv decoding reached similar or even higher success rates than M1 and PMd in a 2D cursor control task, especially when controlling for the number of motion selective channels that were used by the decoder. We found similar results during a 2D robot avatar control task in a simulated 3D environment. At both the multi-unit and the population level, neural responses were highly similar during the training phase (passive observation of cursor movements) and the online decoding phase, and only a small subset of neurons modulated its selectivity for the direction of motion. Thus, ventral premotor area F5c may represent an alternative for online motor BMI control.

Modulation of SLP-2 expression protects against alpha-synuclein neuropathology by mitigating mitochondrial dysfunction
Parkinson's Disease (PD) is a progressive neurodegenerative disorder characterized by dopaminergic neuron loss and the accumulation of alpha-synuclein (aSyn)-rich aggregates known as Lewy bodies. Mitochondrial dysfunction is a key contributor to PD pathology, and mitochondrial defects are part of the pathogenic mechanisms induced by aSyn. Stomatin-Like protein 2 (SLP-2) is a mitochondrial scaffold protein that regulates mitochondrial integrity and function. Here, we investigated whether SLP-2 induction can counteract aSyn-induced mitochondrial dysfunction and neurodegeneration. We found that SLP-2 levels were reduced in human PD brains and an A53T aSyn mouse model. Mild overexpression of SLP-2 improved mitochondrial function, reduced oxidative stress, and prevented aSyn-mitochondria interactions in human iPSC-derived neurons. In vivo, SLP-2 overexpression protected dopaminergic neurons and motor function, while its depletion exacerbated degeneration and motor deficits in both mouse and Drosophila models. These findings suggest SLP-2 as a key regulator of mitochondrial resilience and a potential therapeutic target for PD and alpha-synucleinopathies.

Action observation responses in macaque frontal cortex
Neurons that are active during action execution and action observation (i.e. Action Observation/Execution Neurons, AOENs) are distributed across the brain in a network of parietal, motor, and prefrontal areas. In a previous study, we showed that most AOENs in ventral premotor area F5c, where they were discovered three decades ago, responded in a highly phasic way during the observation of a grasping action, did not require the perception of causality or a meaningful action, and even responded to static frames of the action videos. To assess whether these characteristics are shared with AOENs in other areas of the AOE network, we performed the first large-scale neural recordings during action execution and action observation in multiple frontal areas including dorsal premotor (PMd) area F2, primary motor (M1) cortex, ventral premotor area F5p, frontal eye field (FEF) and 45B. In all areas, AOENs displayed highly phasic responses during specific epochs of the action video and strong responses to simple movements of an object, similar to F5c. In addition, the population dynamics in PMv, PMd and M1 showed a shared representation between action execution and action observation, with an overlap that was as large as the overlap between action execution and passive viewing of simple translation movements. These results pose important constraints on the interpretation of action observation responses in frontal cortical areas.

Mapping of individual somatosensory representations - comparison of fMRI and TMS
Invasive brain mapping, functional magnetic resonance imaging (fMRI) and navigated transcranial magnetic stimulation (nTMS) results indicate that somatosensory representations vary between individuals. However, it is unknown how well somatosensory representations determined using nTMS and fMRI correspond. Here, we used single-pulse nTMS and fMRI in 17 right-handed subjects to determine the S1 representation of the tip of the right index finger stimulated mechanically with a Braille device. In an nTMS mapping experiment, the S1 site at which tactile sensation was blocked by nTMS, was considered the S1 representation site (S1HS) of the fingertip. The location of S1HS varied up to 36 mm between subjects. In the fMRI experiment, passive and oddball tactile tasks were employed. The location of the somatosensory peak fMRI activation varied up to 39 mm between subjects in the passive condition and up to 84 mm in the oddball condition. Within subjects, the mean distance between the S1HS and the peak fMRI activation was 10 + 2 mm (S.E.M.) in the passive condition and 14 + 3 mm in the oddball task. Taken together, our findings underscore the importance of multimodal approaches to brain mapping in future studies.

Dynamic respiration-neural coupling in substantia nigra across sleep and anesthesia
Respiration is increasingly recognized as a coordinator of neural activity across widespread brain regions and behavioral states. Even during sleep, respiration rhythms modulate sleep-related oscillations. While the basal ganglia are known to play roles in both sleep and respiratory regulation, their interaction with respiration rhythms remains poorly understood. Here, we examined respiration-neural couplings in the substantia nigra pars reticulata (SNr), a major output nucleus of the basal ganglia, and the primary motor cortex (M1) across multiple states in mice, including non-rapid eye movement (NREM) sleep, rapid eye movement (REM) sleep, quiet wakefulness, and anesthesia. Simultaneous recordings of local field potentials (LFPs) from M1 and SNr along with diaphragm muscle activities revealed state-dependent, region specific patterns of respiration-neural coupling. Coupling strength in both SNr and M1 was attenuated during NREM sleep compared to REM sleep and quiet wakefulness. However, under ketamine/xylazine anesthesia, coupling was markedly enhanced in the SNr, but not in M1, indicating region-specific sensitivity to arousal and anesthesia state. Notably, respiration-neural coupling was systematically related to delta sub-band power; coupling strength was reduced with increased slow delta (0.5-2 Hz) and decreased fast delta (2.5-4 Hz) powers. In addition, slow delta was associated with SNr-M1 synchronization, suggesting that inter-regional communication during deep sleep may suppress respiration locking. Together, these findings highlight dynamic, state-dependent modulation of respiration-neural couplings in cortico-basal ganglia circuits, underscoring its potential role in coordinating body-brain interactions during sleep and anesthesia.

Challenges in Replay Detection by TDLM in Post-Encoding Resting State
We investigated, using temporally delayed linear modelling (TDLM) and magnetoencephalography (MEG), whether items associated with an underlying graph structure are replayed during a post-learning resting state. In these same data, we have previously provided evidence for replay during on-line (non-rest) memory retrieval. Despite successful decoding of brain activity during a localizer task, and contrary to predictions, we did not detect evidence for replay during a post-learning resting state. To better understand this, we performed a hybrid simulation analysis in which we inserted synthetic replay events into a control resting state recorded prior to the actual experiment. This simulation revealed that replay detection using our current pipeline requires extremely high replay densities to reach significance (>1 replay sequence per second, with "replay" defined as a sequence of reactivations within a certain time lag). Furthermore, when scaling the number of replay events with a behavioural measure we were unable to experimentally induce a strong correlation between sequenceness and this measure. We infer that even if replay was present at plausible rates in our resting state dataset we would lack statistical power to detect it with TDLM. We discuss ways for optimizing the analysis approach and how to find boundary conditions under which TDLM can be expected to detect replay successfully. We conclude that solving these methodological constraints is likely to be crucial to optimise measuring replay non-invasively using MEG in humans.

Problem-solving without a cortex: inferior lobe drives goal-directed object manipulation in cichlid fish
Goal-directed object manipulation and problem-solving, which are necessary to evolve tool use behaviors, have long been linked to the expansion of the telencephalon in mammals and birds. Here, we show that goal-directed object manipulation in cichlid fish is driven by a non-telencephalic brain structure, the inferior lobe. Using manganese-enhanced MRI (MEMRI) on an ultra-high field 17.2 Tesla MRI system, we show that the inferior lobe is activated during a puzzle-box opening task. Furthermore, magnetic resonance (MR)-guided High Intensity Focused Ultrasound (HIFU) lesions profoundly impair fine motor coordination during this task without affecting general locomotion or motivation. These results reveal that cortex-like cognitive functions can arise from non-telencephalic brain structures in teleosts. With no homolog in tetrapods, the inferior lobe is a critical hub for flexible behavior in teleost fish. Our findings highlight the existence of alternative neural architectures for the emergence of complex cognition.

Directed cell-type recruitment during consolidation of a remote memory
Memories are consolidated into a distributed neocortical network for long-term storage. Long-term memory retrieval relies on cells that are active during learning and undergo necessary plasticity. However, remote memory retrieval activates a broader circuit, with learning-activated cells representing only a small subset. What are the rules and cell-types governing memory trace reorganization? We identified a class of prefrontal projection neurons that are gradually recruited to a memory trace through synaptic activity of learning-activated cells. This population, which projects to the temporal association area (TEa), progressively strengthens its encoding of memory-induced behaviors, mirroring increases in TEa activity. Notably, the prefrontal-TEa pathway is required for remote but not recent memory retrieval. Our findings reveal a cell type-specific mechanism underlying memory trace reorganization during consolidation.

Astrocytes control motor neuronal mitochondrial axonal transport deficits in C9ORF72 ALS.
Disrupted axonal transport and astrocyte dysfunction are implicated in amyotrophic lateral sclerosis (ALS). Here, we show these are intrinsically linked: human induced pluripotent stem cell-derived astrocytes carrying the C9ORF72 mutation disrupt axonal transport and mitochondrial function in motor neurons (MNs). Conversely, either isogenic gene-corrected astrocytes or selectively boosting C9ORF72-astrocyte mitochondrial bioenergetics rescue axonal transport deficits in C9ORF72-MNs. Thus, astrocytes have a dominant effect on axonal mitochondrial transport in the context of ALS.

Post-translational modifications distinguish amyloid-β isoform patterns extracted from vascular deposits and parenchymal plaques
Deposition of amyloid-beta (AB) aggregates is a core pathological hallmark of both cerebral amyloid angiopathy (CAA) and extracellular parenchymal plaques in Alzheimer disease (AD). While both disease processes share progressive, decades-long deposition of fibrillar AB peptide, they differ in isoform composition. We hypothesized that post-translational modifications (PTMs) on AB would also differ between CAA and parenchymal plaques. Using Lys-N enzymatic digestion followed by quantitative mass spectrometry, we profiled AB isoforms and N-terminus PTMs (aspartic acid isomerization and pyroglutamate formation) across CAA severity and compared them to parenchymal plaque AB in AD. Moderate to severe CAA primarily featured intact N-terminus (AB1-x) (~95%) with minimal N-truncated species (AB2-x, AB3pGlu-x, AB4-x), whereas parenchymal plaques displayed diverse N-terminus truncations and PTMs. Increasing CAA severity correlated with a shift from longer, hydrophobic C-terminal isoforms (AB41, AB42, AB43) to shorter, less hydrophobic C-terminal isoforms (AB37, AB38, AB39, AB40). Importantly, moderate and severe CAA displayed minimal isomerization of Asp-1 and Asp-7 residues, which correlated significantly (r > 0.9) with shorter C-terminal isoforms (AB37, AB38, AB39, AB40). These patterns suggest distinct AB aggregation mechanisms in CAA versus parenchymal plaques. We propose that the intact N-terminus found in CAA with limited Asp isomerization is due to its inclusion within the protofibril structure (less disordered and inaccessible to PTMs), unlike the parenchymal plaques, where the N-terminus is more disordered and accessible to PTMs. These biochemical differences may reflect distinct protofibril architectures with potential implications for biomarker development for early CAA detection and therapeutic targeting of vascular and parenchymal AB.

Supervised white matter bundle segmentation in glioma patients with transfer learning
In clinical settings, the virtual dissection of white matter tracts represents an informative source of information for monitoring neurological conditions or to support the planning of a treatment. The automation of this task using data-driven approaches, and in particular deep learning models, provides promising evidence of good accuracy when carried out on healthy individuals. However, the lack of large clinical datasets and the profound differences between healthy and clinical populations hinder the translation of these results to patients. Here, we investigated for the first time the effectiveness of transfer learning in adapting a deep learning architecture trained on a healthy population to glioma patients. Importantly, we provided the first thorough characterization of domain shift and its complexity, distinguishing systematic (i.e. measurement and pre-processing related) from tumor specific components. Our results suggest that (i) models trained on a large normative healthy population have a significant performance drop when the inference is carried out on patients; (ii) transfer learning can be an effective strategy to overcome the shortage of clinical data and to manage the systematic shift; (iii) fine tuning of the learning model cannot accommodate large white matter deformations induced by the tumor. The results were coherent across the five white matter bundles and three different input modalities tested, highlighting their robustness and generalizability. Our work provides valuable insights for advancing automated white matter segmentation in clinical populations and enhancing clinical transfer learning applications.

Semantics across the globe: A universal neurocognitive semantic structure adaptive to climate
Thousands of languages are used worldwide as the primary means of human thought communications. While both similarities and variations in word meaning (semantics) across different languages are well recognized, the underlying mechanisms remain enigmatic without a coherent theoretical model for semantic representation. Given that semantic representation is a product of the human brain, we address this issue through the lens of neurocognitive theories, with the consensus framework that semantics are derived from sensory experiences, with a set of dimensions being identified as biologically salient in neuroscientific studies. We operationalized word semantic representations with this set of specific dimensions, using computational models (53 languages' word embedding data; Study 1), human behavioral ratings (253 subjects, 8 languages; Study 2), and brain activity data (86 subjects, 45 languages; Study 3), and analyzed the similarity and variation patterns of concepts across different languages. These three approaches converge on the finding that, across diverse language samples, word semantic representations along the neurocognitive dimensional structures exhibit strong commonalities, with variations along this structure being significantly and uniquely explained by climate, beyond sociocultural-centered variables. These results present a universal, biologically constrained semantic structure that is adaptive to environmental inputs, reconciling the classical universality and relativity debate.

Structurally Constrained Functional Connectivity Reveals Efficient Visuomotor Decision-Making Mechanisms in Action Video Gamers
Long-term action video game (AVG) playing has been linked to improved response times (~190 ms) without accuracy tradeoffs in time-sensitive visuomotor decisions, but how it reshapes neural circuits is unclear. In this study, Cognitive Resource Reallocation (CRR) is introduced as a candidate mechanism for how sustained engagement with AVGs drives behaviorally relevant neuroplasticity through neuroplastic refinement. Using the AAL3 structural connectivity atlas, we apply structural constraints to functional connectivity (SC-FC) and directed functional connectivity (SC-dFC) in gamers and non-gamers. Our results provide strong support for the CRR hypothesis and demonstrate that the brain plausibly reallocates cognitive resources over time to optimize task-relevant networks in high-demand environments like AVGs, enhancing the integration of contextual information and refining motion processing, which may be a key mechanism in explaining more efficient visuomotor decision-making. These findings position action video games as powerful tools for studying experience-driven neuroplasticity, with implications for cognitive training, rehabilitation, and optimizing real-world visuomotor decisions.

Generation of knock-in Cre and FlpO mouse lines for precise targeting of striatal projection neurons and dopaminergic neurons
The basal ganglia and midbrain dopaminergic systems are critical for motor control, reward processing, and reinforcement learning, with dysfunction in these systems implicated in numerous neurodegenerative and neuropsychiatric disorders. To enable precise genetic targeting of key neuronal populations, we generated and characterized five knock-in mouse lines: Drd1-Cre, Adora2a-Cre, Drd1-FlpO, Adora2a-FlpO, and DAT-FlpO. These lines allow for Cre- or FlpO-mediated recombination in dopamine D1 receptor-expressing spiny projection neurons (SPNs), adenosine A2a receptor-expressing SPNs, and dopamine transporter (DAT)-expressing neurons in the midbrain. Histological analyses confirmed recombinase activity in expected brain regions, and whole-cell electrophysiological recordings validated the intrinsic excitability profiles of each neuronal subpopulation. These tools provide high specificity and reliability for studying basal ganglia circuitry and dopaminergic neurons. By enabling targeted manipulations, these openly available knock-in lines will advance research into the neural mechanisms underlying motor control, reward, and neuropsychiatric diseases.

Sex-dependent effects of maternal high-fat diet during lactation in adult THY-Tau22 mice offspring
The perinatal environment has been suggested to participate to the development of tauopathies and Alzheimers disease but the molecular and cellular mechanisms involved remain contradictory and under-investigated. Here, we evaluated the effects of a maternal high-fat diet (HFD) during lactation on the development of tauopathy in the THY-Tau22 mouse strain, a model of progressive tau pathology associated with cognitive decline.

During lactation, dams were fed either a chow diet (13.6% of fat) or a HFD (58% of fat). At weaning, offspring was fed a chow diet until sacrifice at 4 months of age (the onset of tau pathology) or 7 months of age (the onset of cognitive impairment).

During lactation, maternal HFD increased body weight gain in offspring. At 3 months of age, maternal HFD led to a mild glucose intolerance only in male offspring. Moreover, it impaired spatial memory in both male and female 6-month-old offspring, with males being more impacted. These cognitive deficits were associated with increased phosphorylation of hippocampal tau protein-observed at 4 months in males and at 7 months in females, highlighting a sex-specific temporal shift. Additionally, maternal HFD modified adult hippocampal neurogenesis (AHN), leading to an increase of mature neuronal cells number in females and of dendritic arborization length in males. Synaptic analysis further revealed that maternal HFD led to synaptic loss only in males. Finally, multi-omics approaches showed that maternal HFD has long-term consequences on both transcriptome, proteome and regulome, this effect being also sex-dependent with mitochondrial pathways, ribosomal activity, cilium and the extracellular matrix predominantly impacted in males, while gliogenesis, myelination and synaptic plasticity were primarily affected in females. Regulome analysis suggested that this sex-dependent phenotype was more related to a temporal shift rather than distinct sex-specific alterations. Collectively, our data suggest that maternal malnutrition accelerates the development of tauopathy in THY-Tau22 offspring, with sex-dependent effects, males being impacted earlier than females. These findings highlight the critical role of the perinatal environment as a key window of opportunity for interventions aimed at preventing the development of neurodegenerative diseases.

The vagus nerve promotes memory via septo-hippocampal acetylcholine: Implications for obesity-induced cognitive dysfunction
The vagus nerve relays critical metabolic information between the gastrointestinal (GI) tract and the brain. Recent findings highlight a role for vagus nerve-mediated gut-brain signaling in regulating higher-order cognitive processes, although the underlying mechanisms remain poorly understood. Here we demonstrate that nutrient consumption promotes hippocampal-dependent memory function via vagus nerve-mediated acetylcholine (ACh) release in the dorsal hippocampus (HPCd) from medial septum (MS) neurons. In vivo analyses reveal that HPCd ACh release is engaged during nutrient consumption, and that this response is abolished in animals that received MS cholinergic neuron ablation, subdiaphragmatic vagotomy (SDV), or early-life Western Diet (WD) maintenance. MS cholinergic neuron ablation, SDV, and early-life WD also impaired memory for meal location, suggesting that this gut-brain pathway functions to encode memories for eating events. Collectively, results identify a neurobiological mechanism whereby nutrient consumption promotes memory function, and reveal that disruption of this vagal-brain signaling system mediates WD-associated memory impairments.

Two Specialized Intrinsic Cardiac Neuron Types Safeguard Heart Homeostasis and Stress Resilience
The intrinsic cardiac nervous system (ICNS), often referred to as "the little brain on the heart", plays a central role in heart-brain communication and is increasingly recognized as both a contributor to cardiac disorders, including atrial fibrillation, heart failure, and sudden cardiac death, and a growing target for therapeutic intervention. Despite its clinical relevance, the molecular and functional organization of the ICNS remains poorly understood. Using single-cell transcriptomics, high-resolution imaging, and cell-specific genetic tools in mice, we identified two molecularly distinct intrinsic cardiac neuron (ICN) subtypes marked by Npy and Ddah1, each exhibiting unique anatomical innervation patterns. Npy ICNs function as parasympathetic postganglionic neurons essential for maintaining coronary perfusion and cardiac homeostasis during routine physiological activity. In contrast, Ddah1 ICNs are crucial for preserving cardiac electrical stability and preventing sudden death under extreme stress. These findings uncover specialized ICNS pathways that support cardiac homeostasis and resilience, providing a foundation for developing targeted neurocardiac therapies for autonomic dysfunction in human heart disease.

Increased Synapse Elimination by Inflammatory Cells Contributes to Long-lasting Post-Stroke Memory Dysfunction in Old Mice
Old patients are more likely to experience memory dysfunction than young patients after a stroke. It has been reported that brain astrocytes and microglia cause excessive removal of synapses at the acute and subacute stages of stroke, and inhibition of their phagocytosis improved neurobehavioral outcomes. We hypothesized that memory dysfunction in old subjects is associated with increased synapse removal by inflammatory cells. Ischemic stroke was induced in young (2-month-old) and old (15-18-month-old) mice. Memory functions were analyzed by the Y-maze test weekly for 8 weeks and the novel object recognition (NOR) test at 7 days before and 8 weeks post-stroke. We have also created a tibia fracture 6 hours before stroke injury in young mice, to test if the activation of 7-nicotinic acetylcholine receptor (nAchRs) reduces inflammatory cells and synapse elimination. Brains were collected 8 weeks after the induction of ischemic stroke. Transcriptome changes, neuronal injuries, neuroinflammation, synapse removal, and neurite outgrowth were analyzed. We found that old mice developed long-term memory dysfunction after ischemic stroke, which was not seen in young mice. Old mice showed larger infarct volume, higher neuroinflammation, and more synapses engulfed by microglia/macrophages and astrocytes in the peri-atrophic region and hippocampi than young mice. More synapse-engulfing astrocytes than microglia/macrophages were present in the peri-atrophic region and the ipsilateral hippocampi, suggesting that reactive astrocytes contributed more than activated microglia/macrophages in synapse removal. Activation of 7-nAchRs in mice subjected to tibia fracture 6 hours before ischemic injury reduced synapse removal by microglia/macrophages and astrocytes in the hippocampi. Our study indicated that an increase in synaptic elements by inflammatory cells contributes to the long-lasting memory deficit after stroke in old mice. Astrocytes may contribute more than microglia/macrophages in synapse removal. Inhibition of neuroinflammation by activating 7-nAchRs can reduce synapse loss and thus may improve post-stroke memory function.

A Reappraisal of the Role of the Mammillothalamic Tract in Memory Deficits Following Stroke in the Thalamus
The thalamus, traditionally viewed as a sensory relay station, is now recognized for its critical role in higher-order cognitive functions, including memory. While most research has focused on its distinct nuclei, the thalamus also contains crucial white matter tracts, such as the mammillothalamic tract (MTT), part of the Papez circuit. Despite its established role in memory, the MTT remains underappreciated in studies on the cognitive role of the thalamus, increasing the risk of misattributing memory functions to nearby thalamic nuclei. This study investigates the memory impact of thalamic strokes by considering disruption to the MTT.

We examined 40 patients with chronic ischemic thalamic lesions and 45 healthy controls using neuropsychological assessments, focusing on the Free and Cued Selective Reminding Test (FCSRT) and high-resolution structural neuroimaging. Advanced imaging techniques, such as symptom mapping and disconnectome analyses, were employed to analyze the relationships between lesion sites, tract disconnections and cognitive outcomes. Additionally, the expression of calbindin-rich matrix cells and parvalbumin-rich core cells was examined to assess how the connectivity property of thalamic cells, and its disruption, can relate to cognitive deficits following a thalamic stroke.

Patients with left-sided thalamic lesions, especially those involving the MTT, showed significant memory impairments. Symptom mapping identified a specific cluster of lesioned voxels in the left anterior-lateral thalamus, including the MTT, as being strongly associated with poorer memory performance. Disconnectome analysis confirmed that verbal memory deficits were associated with disruption of the MTT. Furthermore, a striking overlap was observed between the critical regions linked to memory deficits and calbindin-rich thalamic areas. However, this calbindin-rich region was also disrupted in patients without memory impairment, revealing that MTT disruption, and not lesions to this region, induced memory deficits in these patients. The identified FCSRT deficit cluster overlapped with brain regions consistently linked to memory processes following fMRI meta-analytic mapping of memory-related keywords.

These findings challenge the traditional focus on thalamic nuclei and connector hubs, indicating a need to reappraise the importance of the MTT in memory impairment after a thalamic stroke. This study advocates for a shift in thalamic research, emphasizing the need to investigate tract-specific contributions and disruptions of the MTT but also of other tracts such as the interthalamic adhesion or amygdalo-fugal pathway, rather than holding only to a "nuclei-centric" view of the thalamus.

Canagliflozin reprograms the aging hippocampus in genetically diverse UM-HET3 mice and attenuates Alzheimer's-like pathology
Aging is the strongest risk factor for cognitive decline and Alzheimers disease (AD), yet the mechanisms underlying brain aging and their modulation by pharmacological interventions remain poorly defined. The hippocampus, essential for learning and memory, is particularly vulnerable to metabolic stress and inflammation. Canagliflozin (Cana), an FDA-approved sodium-glucose co-transporter 2 inhibitor (SGLT2i) for type 2 diabetes, extends lifespan in male but not female mice, but its impact on brain aging is unknown. Here, we used a multi-omics strategy integrating transcriptomics, proteomics, and metabolomics to investigate how chronic Cana treatment reprograms brain aging in genetically diverse UM-HET3 mice. In males, Cana induced mitochondrial function, insulin and cGMP-PKG signaling, and suppressed neuroinflammatory networks across all molecular layers, resulting in improved hippocampal-dependent learning and memory. In females, transcriptional activation of neuroprotective pathways did not translate to protein or metabolite-level changes and failed to rescue cognition. In the 5xFAD AD model, Cana reduced amyloid plaque burden, microgliosis, and memory deficits in males only, despite comparable peripheral glucose improvements in both sexes. Our study reveals sex-specific remodeling of hippocampal aging by a clinically available SGLT2i, with implications for AD pathology and lifespan extension, and highlights Canas potential to combat brain aging and AD through sex-specific mechanisms.

Glutaminolysis Fuels Reactive Astrocytes, Exacerbating Amyloid Pathology in Alzheimer's Disease.
Amyloid-{beta} (A{beta}) plaques with progressively increasing reactive astrocytes characterize Alzhemer's disease (AD). Reactive astrocytes are regulated by cellular and molecular mechanisms that are known to progress A{beta} pathology. However, the metabolic adaptation and metabolites required to fuel these molecular changes in reactive astrocytes remain unknown. Using human AD samples, in vivo amyloid mouse models, and in vitro approaches, we demonstrate that reactive astrocytes utilize glutamine to fuel anaplerosis and meet their metabolic demands, thereby progressing amyloidosis. We show that reactive astrocytes increase Na+-coupled neutral amino acid transporters for glutamine uptake that are interdependent on Na+/K+ ATPase. Furthermore, increasing brain-glutamine levels with a high-glutamine diet exacerbated reactive astrocytes, increasing A{beta} burden in an amyloid mouse model. We demonstrate that glutamine undergoes glutaminolysis via glutaminase-2/glutamate dehydrogenase-1 enzymes to be incorporated into TCA metabolites for anaplerosis. Pharmacologically or genetically blocking glutaminolysis reduces reactive astrocytes and decreases A{beta} pathology in an amyloid mouse. Our findings reveal the first glutamine-dependent metabolic adaptation of reactive astrocytes affecting A{beta} pathology, which may be harnessed for AD therapeutic strategies.

Effects of non-invasive vagus nerve stimulation on pupil dilation are dependent on sensory matching
Transcutaneous auricular vagus nerve stimulation (taVNS) is a promising non-invasive method to modulate motivation, cognition, and affect. According to a recent meta-analysis, pulsed taVNS induces larger pupil dilation (vs. sham), but sensory aspects of the stimulation may contribute to these differences. Moreover, included studies were often small and stimulation was only applied to the left ear, so that questions about the robustness and generalization remain. Here, we investigated the effects of pulsed taVNS (1s, 20Hz, 400s pulse width) at the right ear on phasic pupil dilation using a randomized crossover design in 94 participants (47 women). We initially calibrated the stimulation amplitude to match the perceived sensation across conditions and tracked sensation over time. Contrary to our hypothesis, taVNS did not elicit greater pupil dilation versus sham (b = -0.42, 95% CI [-1.30; 0.46], p = .35). However, taVNS effects were larger if sham was perceived as less intense despite the initial matching (b = 3.39, 95% CI [0.62; 6.17], p = .017). Differences in intensity ratings were mainly associated with sham-induced pupil dilation (r = -.25, 95% CI [-.43; -.05], p = .013). To summarize, our results recapitulate the findings of other studies using fixed amplitudes by showing that right-sided pulsed taVNS only induced larger pupil dilation against sham if differences in sensation arose. These findings highlight the challenge of establishing a suitable sham condition since both taVNS and sham affect sensory nerves, potentially leading to confounding effects.

LSD Restores Synaptic Plasticity in VTA of Morphine-Treated Mice and Disrupts Morphine-Conditioned Place Preference
Psychedelics are emerging as a promising treatment option for a range of neuropsychiatric disorders, including substance use disorders. One potential mechanism underlying their therapeutic benefits may involve a reversal of maladaptive plasticity induced by drug exposure. Here, we identify physiological, behavioral, and epigenetic impacts of lysergic acid diethylamide (LSD) on morphine-treated male and female mice. Morphine was selected due to the high leverage capacity to address the opioid epidemic. A single treatment of LSD, or 4 microdoses of LSD, cause accelerated extinction of morphine-induced conditioned place preference. Whole-cell electrophysiology revealed that excitatory synaptic plasticity, which was eliminated in VTA GABA neurons following morphine exposure, was restored 24 hours after a single high dose of LSD. To explore the impact of LSD treatment on potential epigenetic changes, whole-brain DNA methylation analysis in morphine-treated mice that received either saline or LSD post-morphine treatment revealed significant differences in methylation profiles associated with LSD treatment. Collectively, these findings suggest that LSD may reverse or prevent morphine-induced changes in reward circuit plasticity and attenuate measures of morphine-preference.

Unlocking the Neuropeptidome using a Novel Endogenous Peptidomics Framework
Endogenous peptides have garnered increasing attention over the past decade driven by the development of advanced analytical methods. However, large-scale investigations of peptides as potential disease biomarkers or drug candidates are still hindered by their challenging biochemical properties and the scarcity of specialized analytical tools. Among these, neuropeptides are particularly challenging to study due to their low in vivo concentration, rapid turnover rate, and high structural variability. Data-independent acquisition (DIA) mass spectrometry (MS) has shown great ability in profiling low-abundance ions. Nevertheless, most available DIA analytical tools are designed for proteomics studies and are not suitable for endogenous peptides, as there is no set enzymatic cleavage for these peptides. Here, we introduce the novel EndoGenius platform, paired with DIA-NN, to achieve high-confidence neuropeptide identification using an updated spectral library for DIA MS analysis. By employing orthogonal offline fractionation, ion mobility instrumentation, and an optimized database searching algorithm specifically for neuropeptides, we have constructed the largest crustacean neuropeptide spectral library to date. With this library, in combination with neural networking technology, we report a 100-fold increase in the number of neuropeptides identified in all Cancer borealis tissues analyzed. We also cross-validated these findings with transcriptomics data to enhance identification confidence. This workflow presents a novel analytical framework for DIA peptidomics analysis, offering a robust approach to studying neuropeptides and other endogenous peptides.

Proximity labelling reveals the compartmental proteome of murine sensory neurons
Understanding the molecular architecture of peripheral sensory neurons is critical as we pursue novel drug targets against pain and neuropathy. Sensory neurons in the dorsal root ganglion (DRG) show extensive compartmentalization, thus understanding each compartment - from the peripheral to central terminals - is key to this effort. To systematically profile this spatial complexity, we generated a TurboIDfl/fl transgenic mouse line (ROSA26em1(TurboID)Bros), enabling targeted proximity labelling and deep proteomic profiling of DRG neuron compartments via AdvillinCre. Our data reveal distinct proteomic signatures across neuronal compartments that reflect specialized neuronal functions. We provide proteomic insights into previously inaccessible nerve terminals both in the periphery (innervating the skin) and in the spinal cord. Further, using a DRG explant model of chemotherapy-induced peripheral neuropathy (CIPN), we uncovered novel and discrete proteome changes, highlighting neuronal vulnerability. Together, our findings provide a unique proteome atlas of the sensory neuron proteome across anatomical domains and demonstrate the utility of proximity labelling proteomics for detecting compartment-specific molecular alterations in a disease model. This dataset serves as a valuable resource for mechanistic studies of sensory neuron function and pathology.

Preventing Microglial Reactivity Protects from Acute and Progressive Neuronal Dysfunction, Motor Impairments and Sedation following Alcohol Abuse
Alcohol abuse is the primary risk factor for alcohol use disorder (AUD), a leading cause of preventable morbidity and mortality, characterized by systemic inflammation, multi-organ damage, and neurological impairments. While direct effects of alcohol on brain function are well-established, the role of microglia in acute and chronic neurological dysfunction in AUD remains unclear. Using longitudinal in vivo imaging in mice during acute and repeated alcohol abuse, we found that microglia exhibit dynamic morphological responses that precede but parallel ethanol-induced sedation. Ethanol also induced microglia-dependent synapse elimination and reduced neuronal activity and density. Genetic disruption of microglial MyD88 reversed these ethanol-associated changes in microglial reactivity, neuronal structure and function, while protecting against alcohol-induced intoxication and motor impairments. These findings identify microglia as cellular drivers of acute and chronic brain dysfunction following alcohol abuse, and highlight MyD88 as a critical therapeutic target for the detrimental neurological consequences of AUD.

Effects of aging on upper body express visuomotor responses while reaching under varying postural demands
Humans can react remarkably quickly to novel or displaced visual stimuli when time is of the essence. Such movements are thought to be initiated by a subcortical fast visuomotor network, but it is unclear how this network declines with age. Past work in the upper limb has detailed delayed reaching corrections to jumped visual stimuli in the elderly, but the underlying mechanisms contributing to these changes of the fast visuomotor network are poorly understood. Conversely, work in the lower limb has reported delayed muscle recruitment during obstacle avoidance, but such findings may be confounded by age-related challenges in postural control. The output of the fast visuomotor network can be quantified by measuring express visuomotor responses (EVRs), which are the earliest and very short-latency bursts of muscle activity that follow visual target presentation. Here, we compare the prevalence, latency, and magnitude of EVRs in elderly (58-80 years old) and younger (18-25 years old) participants performing visually-guided reaches. We also investigated the impact of postural stability by having participants reach either while seated on a stable chair, or on a wobble stool. Both the elderly and younger cohorts expressed EVRs, but EVRs in the elderly were comparatively less frequent, and had longer latencies and smaller magnitude. Postural instability had no effects on these outcomes. Our results suggest age-related declines in the fast visuomotor network, potentially resulting from deterioration of underlying circuits and a prioritization of stability over speed. This study serves as an important standard for future research investigating clinical populations.

HighlightsO_LIDo aging and postural stability affect express visuomotor responses (EVRs)?
C_LIO_LIEVRs were recorded from the pectoralis muscle during rapid goal-directed reaches
C_LIO_LIEVRs were smaller and delayed, and movement was slower, in elderly participants
C_LIO_LIAge-related differences were independent of our postural stability manipulation
C_LIO_LIOur results suggest age-related declines of the fast visuomotor network
C_LI

Innervation of Retrodiscal Tissues in Patients with Temporomandibular Joint Disorder
Temporomandibular joint (TMJ) disorders (TMJDs) are a group of musculoskeletal conditions affecting the orofacial region and often associated with facial pain. Understanding the sensory innervation of TMJ structures, particularly the retrodiscal tissue, is essential for identifying pain mechanisms in TMJD because to study these mechanisms, we must first determine the sensory neuronal makeup of the TMJ. However, data on nerve types within TMJ tissues remain limited. This study examined the sensory and sympathetic nerve profiles in retrodiscal tissues from TMJD patients with osteoarthritis (OA), rheumatoid arthritis (RA), or condylar hyperplasia (CH) who underwent bilateral TMJ replacement (TMJR). Immunohistochemistry with specific nerve markers was used to visualize and quantify nerve subtypes. CH tissues had significantly lower densities of pgp9.5+ sensory fibers compared to RA and OA, which showed similar levels. Across all subtypes, the ratio of unmyelinated (pgp9.5+/NFH-) to myelinated (pgp9.5+/NFH+) fibers was approximately 70:30. Most sensory nerves were CGRP+ (peptidergic), while a smaller portion were CGRP- (non-peptidergic), some of which were parvalbumin-positive (PV+). Both myelinated and non-myelinated peptidergic as well as non-peptidergic fibers were present in the retrodiscal tissues. In addition to sensory innervation, all retrodiscal tissues contained tyrosine hydroxylase positive (TH+) sympathetic fibers, primarily innervating blood vessels (alpha-smooth muscle actin+). These vessels were also predominantly innervated by unmyelinated sensory fibers, with limited input from myelinated sensory nerves. In summary, all TMJD subtypes shared similar nerve compositions, but CH tissues exhibited reduced sensory nerve density, a potential explanation for the lower association with pain compared to OA and RA. For all TMJD subtypes, retrodiscal tissue vasculature was mainly innervated by sympathetic and unmyelinated sensory nerves. These findings enhance understanding of the neural basis of TMJD related pain.

Automated 3D Segmentation of Human Vagus Nerve Fascicles and Epineurium from Micro-Computed Tomography Images Using Anatomy-Aware Neural Networks
Objective. Precise segmentation and quantification of nerve morphology from imaging data are critical for designing effective and selective peripheral nerve stimulation (PNS) therapies. However, prior studies on nerve morphology segmentation suffer from important limitations in both accuracy and efficiency. This study introduces a deep learning approach for robust and automated 3D segmentation of human vagus nerve fascicles and epineurium from high-resolution micro-computed tomography (microCT) images. Methods. We developed a multi-class 3D U-Net to segment fascicles and epineurium that incorporates a novel anatomy-aware loss function to ensure that predictions respect nerve topology. We trained and tested the network using subject-level five-fold cross-validation with 100 microCT sub-volumes (11.4 [&mu]m isotropic resolution) from cervical and thoracic vagus nerves stained with phosphotungstic acid from five subjects. We benchmarked the 3D U-Net's performance against a 2D U-Net using both standard and anatomy-specific segmentation metrics. Results. Our 3D U-Net generated high-quality segmentations (average Dice similarity coefficient: 0.93). Compared to the 2D U-Net, our 3D U-Net yielded significantly better volumetric overlap, boundary delineation, and fascicle-level accuracy. The 3D approach reduced anatomical errors by 2.5-fold, provided more consistent inter-slice boundaries, and improved detection of fascicle splits/merges by nearly 6-fold. Significance. Our automated 3D segmentation pipeline provides anatomically accurate 3D maps of peripheral neural morphology from microCT data. The automation allows for high throughput, and the substantial improvement in segmentation quality and anatomical fidelity enhances the reliability of morphological analysis, vagal pathway mapping, and the implementation of realistic computational models. These advancements provide a foundation for understanding the functional organization of the vagus and other peripheral nerves and optimizing PNS therapies.

DeepFace: A High-Precision and Scalable Deep Learning Pipeline for Predicting Large-Scale Brain Activity from Facial Dynamics in Mice
We present DeepFace, a next-generation facial analysis pipeline that enhances orofacial tracking and cortical activity prediction in mice. Rather than replacing existing tools, DeepFace builds upon DeepLabCut and Facemap to address scalability bottlenecks and improve behavioral quantification. It offers high precision, keypoint customization, and robust performance across GCaMP6s, GCaMP6f, and jGCaMP8m lines. With scalable batch processing and high-performance computing compatibility, DeepFace enables high-throughput brain-behavior analysis in large-scale preclinical neuroscience.

Multi-Scale Parcellation of Dynamic Causal Models of the Brain
The hierarchical organization of the brain's distributed network has received growing interest from the neuroscientific community, largely because of its potential to enhance our understanding of the human cognition and behavior, in health and disease. While most multiscale connectivity analyses focus on structural and functional networks, characterizing the effective connectome across multiple scales has been somewhat overlooked - mostly for computational reasons. The difficulty of estimating large cyclic causal models on the one hand, and the scarcity of theoretical frameworks for systematically moving between scales on the other hand, have hindered progress in this direction. This technical note introduces a top-down multi-scale parcellation scheme for dynamic causal models, with application to neuroimaging data. The method is based on Bayesian model comparison, as a generalization to the well-known delta-BIC method. To facilitate the computations, recent developments in linear dynamic causal modeling (DCM) and Bayesian model reduction (BMR) are deployed. Specifically, a naive version of BMR is introduced, using which the parcellation scheme can be scaled up to hundreds or thousands of regions. Notably, the derivations reveal an analytical relationship between the model comparison score and the concept of cut size in graph theory. This duality puts the tools of graph theory at the service of model evidence optimization and significance testing. The proposed method was applied to simulated and empirical causal models, for face and construct validity. Notably, the large causal network (inferred from a neuroimaging dataset) showed scale-invariance in multiple measures. Future generalizations of this technique and its potential applications in systems and clinical neuroscience have been discussed.

Lycium barbarum polysaccharide alleviates neurobehavioral deficits in mice with ischemic cerebral injury
Objective: To investigate the effects of Lycium barbarum polysaccharides (LBP) on neurobehavioral impairments in mice with ischemic stroke and explore the underlying mechanisms. Methods: A middle cerebral artery occlusion (MCAO) model was established using the filament occlusion method to evaluate the therapeutic effects of LBP on pathological brain tissue damage after cerebral ischemia-reperfusion injury (I/R). Mice were randomly divided into three groups: sham surgery (Sham), I/R, and I/R + LBP. Behavioral tests, including the Y-maze test, rotarod test, and balance beam test, were systematically conducted to assess the impact of LBP on neurobehavioral impairments. Enzyme-linked immunosorbent assay (ELISA) was used to quantify peripheral blood levels of pro-inflammatory cytokines tumor necrosis factor- (TNF-) and interleukin-6 (IL-6), reflecting inflammatory status. Results: LBP significantly ameliorated neuroinflammation and oxidative stress in mice with cerebral I/R injury, demonstrating marked protection against I/R-induced neurofunctional damage. LBP notably improved motor and memory deficits caused by ischemic stroke. Compared to the I/R group, LBP improved neuroinflammation and oxidative stress levels post-I/R injury. Conclusion: This study demonstrates that LBP alleviates ischemic stroke-induced neurological damage by attenuating inflammatory responses.

Differential effect of low and high frequency transcranial magnetic stimulation on cortical excitability and myelination after neonatal hypoxia in mice
Premature infants are highly susceptible to intermittent hypoxic brain injury, which is associated with adverse motor, cognitive, and behavioral outcomes, including deficits in attention, hyperactivity, and learning. Previous animal studies have shown myelination deficits and increased glutamatergic synaptic strength in the sensory-motor cortex. The study investigates the feasibility, safety, and therapeutic potential of repetitive transcranial magnetic stimulation (rTMS) in ameliorating central hypomyelination, reducing excessive glutamatergic transmission in cortical neurons following neonatal intermittent hypoxia (IH), and improving behavioral outcomes. In the mouse model of neonatal intermittent hypoxia, low-frequency (LF-rTMS) stimulation at 1 Hz or high-frequency (HF-rTMS) at 10 Hz stimulation was applied for 5 days shortly after the IH injury. The rTMS regimen did not induce apoptosis or inflammation. HF-rTMS significantly ameliorated hypomyelination in the corticospinal tract, with a larger increase in MPF in the stimulated hemisphere and also in the contralateral hemisphere. LF-rTMS reduced locomotor hyperactivity in female IH mice and decreased elevated glutamatergic synaptic excitability in motor cortex slices. This study provides evidence that rTMS can modulate both myelination and synaptic excitability, leading to improved behavioral outcomes after neonatal hypoxic brain injury. These findings highlight the therapeutic potential of rTMS as an early intervention strategy for neurological sequelae of perinatal hypoxia.

High-throughput single-cell CRISPRi screens stratify neurodevelopmental functions of schizophrenia-associated genes
Schizophrenia is a complex neuropsychiatric disorder with strong genetic underpinnings, yet the molecular mechanisms linking genetic risk to disrupted brain development remain poorly understood. Transcription factors (TFs) and chromatin regulators (CRs) are increasingly implicated in neuropsychiatric disorders, where their dysregulation may disrupt neurodevelopmental programs. Despite this, systematic functional interrogation in human models has been limited. Here, we combine pooled CRISPR interference (CRISPRi) screens with high-throughput single-cell multiomic profiling in hiPSC-derived neural progenitors and neurons to functionally assess 65 schizophrenia-associated genes. Based on public datasets and literature review, we selected 55 TFs and CRs, along with ten additional risk genes whose loss-of-function has been linked to schizophrenia. Our single-cell CRISPRi readouts revealed that perturbations in TFs and CRs converge on disrupting neurodevelopmental timing. CRISPRi of several factors delayed neural differentiation, whereas others, such as the knockdown of MCRS1, drove precocious neural commitment. Validation screens combined with cell cycle and metabolic indicators confirmed the differentiation-restricting or -promoting roles of these TFs and CRs. Multimodal trajectory analysis uncovered discrete transcriptional and epigenomic states representing delayed and accelerated neurodevelopment, enriched for schizophrenia GWAS loci and disease-relevant pathways. Gene regulatory network (GRN) inference identified TCF4 and ZEB1 as critical mediators opposing the neural differentiation trajectory. Functional overexpression of these TFs followed by chromatin profiling demonstrated that TCF4 restrains, while ZEB1 promotes, neural differentiation in a stage-specific and competitive manner. Furthermore, we show that MCRS1 represses ZEB1 expression, positioning MCRS1 as a key brake on premature neurodevelopment. Together, our study establishes a scalable framework that integrates genetic perturbation, single-cell multiomics, and GRN modeling to functionally annotate disease-linked genes. We reveal convergent regulatory axes that underlie altered neurodevelopmental timing in schizophrenia, offering mechanistic insights into how chromatin misregulation contributes to disease pathogenesis.

Proteomic and metabolic profiling reveals APOE4-dependent shifts in whole brain, neuronal, and astrocytic mitochondrial function and glycolysis
Apolipoprotein E (APOE) genetic variation is the strongest genetic risk factor for late onset Alzheimers disease (LOAD). Studies on APOE genotype dependent changes have largely focused on amyloid beta aggregation, disease pathology, and lipid metabolism. Recently, there has been increased interest in the relationship between metabolic function and APOE genetic variation. In this study, we examined how APOE genotype can alter metabolism in the brains of young male and female APOE3 and APOE4 targeted replacement (TR) mice. In combination with this, we also examined cell type-specific differences using induced pluripotent stem cell (iPSC) derived astrocytes and neurons. We found sex and genotype dependent changes to metabolism in the brains of young APOE TR mice. Specifically, APOE4 mice show signs of metabolic stress and compensatory mechanisms in the brain. Using proteomics and stable isotope tracing metabolomics, we found that APOE4 iAstrocytes and iNeurons exhibit signs of inflammation, mitochondrial dysfunction, altered TCA cycle and malate-aspartate shuttle activity, and a metabolic shift toward glycolysis. Taken together, this data indicates APOE4 causes early changes to metabolism within the central nervous system. While this study establishes a relationship between APOE genotype and alterations in bioenergetics, additional studies are needed to investigate underlying mechanisms.

Elucidating the Contribution of OVLT Glutamatergic Neurons to Mineralocorticoid Hypertension in TASK-/- Mice
Background: Aldosterone overactivity intensifies central sodium sensitivity and sympathetic output, driving salt-sensitive hypertension, but specific mechanisms remain incompletely defined. Herein, we aimed to explore the role of organum vasculosum of the lamina terminalis glutamatergic neurons (OVLTGlut) and their hyperexcitability mechanisms in hyperaldosteronism-associated hypertension. Methods: Adult age matched male TASK-/- mice (primary aldosteronism model) and wild-type controls (TASK+/+) mice were used. Neuronal excitability was assessed via patch-clamp techniques. Arterial blood pressure (BP) monitored via telemetry or carotid catheterization. Chronic drug delivery used minipumps. RNA-seq/qPCR profiled gene expression, and intracerebroventricular hypertonic saline tested sodium sensitivity. Results: In TASK-/- mice, heightened OVLTGlut activity increased sympathetic outflow and hypertension, mitigated by OVLTGlut neuron ablation. Optogenetic activation of these neurons or their paraventricular nucleus (PVN) / rostral ventrolateral medulla (RVLM) projections acutely elevated BP, with ablation reducing BP selectively in TASK-/- mice. Aldosterone dependence of OVLTGlut-PVN/RVLM neuron hyperactivity was evident in both TASK-/- mice and TASK+/+ mice with chronic aldosterone infusion. Aldosterone chronic infusion enhanced central sodium pressor effects, that were nullified by OVLTGlut-PVN/RVLM neuron lesioning. RNA-seq indicated that aldosterone-induced ion channel expression spectrum changes, including potassium channels and the epithelial sodium channel, underlie the neuronal hyperexcitability. Conclusion: Overactivation of OVLTGlut neurons contributes to hypertension in TASK-/- mice through regulation of OVLTGlut-PVN/RVLM circuits. The hyperexcitability of these neurons, possibly due to aldosterone-induced changes in ion channel expression spectrum, contribute to hypertension by amplifying central sodium sensitivity.