bioRxiv Subject Collection: Neuroscience's Journal

Charting the Normal Development of Structural Brain Connectivity in Utero using Diffusion MRI
Understanding the structural connectivity of the human brain during fetal life is critical for uncovering the early foundations of neural function and vulnerability to developmental disorders. Diffusion-weighted MRI (dMRI) enables non-invasive mapping of white matter pathways and construction of the brain's structural connectome, but its application to the fetal brain has been limited by data scarcity and technical difficulties in analyzing fetal dMRI data. Here, we present the largest study to date of in utero brain connectivity, analyzing high-quality dMRI data from 198 fetuses between 22 and 37 gestational weeks, from the Developing Human Connectome Project. We employed advanced fetal-specific tools for brain segmentation, parcellation, and tractography. For connection weighting, we relied on the notion of fiber bundle capacity. We reconstructed individual structural connectomes and characterized the developmental trajectories. Graph-theoretical analysis revealed consistent increases in integration and segregation metrics over gestation, alongside stable small-world properties. Bootstrapping confirmed the robustness of nodal and edge-wise developmental patterns, and a sigmoid growth model identified a narrow time window (around 27.5-30.5 weeks) of rapid connectivity strengthening. Furthermore, we proposed a novel method for constructing age-specific connectome templates based on aggregation of individual subject connectomes. Our analysis shows that this approach is superior to spatial alignment and averaging of the data in image space, with the connectome templates preserving individual topology and supporting accurate age prediction. Together, our findings provide a reasonable normative map of fetal brain structural connectivity and establish a foundation for future studies of atypical development and early indicators of neurological risk.

Imaging progenitor cell differentiation during central nervous system remyelination using an MRI gene reporter
Demyelination, the loss of the myelin sheath from around otherwise intact axons, occurs in several diseases, most notably multiple sclerosis (MS). Demyelinated axons that are not remyelinated are vulnerable to irreversible degeneration and therefore therapies that enhance remyelination have been sought. However, there remains a paucity of suitable outcome measures to assess their efficacy. Magnetic Resonance Imaging (MRI) is a non-invasive imaging modality that is used both preclinically and clinically for the assessment of anatomy and tissue function. Here we describe an MRI technique for following the differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes during the spontaneous regenerative process of remyelination in vivo. OPCs were transduced in situ with a lentiviral vector expressing an organic anion transporting polypeptide (Oatp1a1) under the control of the differentiation-specific Myelin Basic Protein (MBP) promoter. Oatp1a1 mediates cell uptake of a gadolinium-based MRI contrast agent (Primovist), allowing detection of the cells in T1-weighted MR images. Uptake of the contrast agent is restricted to MBP-expressing cells, which is most highly expressed during myelin sheath formation, thereby allowing progenitor-mediated, and potentially oligodendrocyte-mediated, remyelination to be monitored non-invasively in vivo using MRI. These findings provide the foundation for the development of direct methods for assessing the efficacy of pro-remyelination therapies.

Linking brain structure to stress reactivity: Cingulate surface area predicts acute cortisol responses
Background: Altered stress responses are closely linked to mental disorders, but the role of brain structure in acute cortisol responses to psychosocial stress remains underexplored, particularly in healthy individuals. Previous studies, with predominantly small samples, primarily focused on selected limbic regions and functional measures. Thus, this study investigates associations between brain structure and cortisol responses to psychosocial stress, exploring if hypothalamic-pituitary-adrenal axis reactivity can be predicted from brain morphology. Methods: Our study included 291 subjects (157 females, 18-62 years) and consisted of two parts. First, a confirmatory analysis examined associations between specific cortical surface area, thickness, and subcortical volume with stress-induced cortisol increases using Permutation Analysis of Linear Models (PALM). Second, we conducted an exploratory whole-brain vertex-wise analysis, followed by out-of-sample prediction of cortisol increases from structural measures. Results: We found consistent negative associations between cingulate cortex (CC) sub-structures and acute cortisol increases. In PALM- and whole-brain analysis, a smaller surface area of the left rostral and caudal anterior cingulate cortex (cACC), posterior cingulate cortex, and right cACC were associated with higher cortisol stress responses, particularly in males. The left cACC surface area emerged as the most promising predictor in machine learning analyses. Additionally, other fronto-limbic structures were also associated with or predictive of acute cortisol reactivity. Conclusions: Our findings demonstrate that cortical and subcortical structural measures, particularly smaller surface areas of the CC, predict acute hormonal stress responses. Notably, the left cACC emerged as the most consistent predictor, underlining its potential as a biomarker for stress-related diseases.

Atypical low-frequency and high-frequency neural entrainment to rhythmic audiovisual speech in adults with dyslexia
Developmental dyslexia has been linked to atypical neural processing of the temporal dynamics of speech, but there has been disagreement concerning whether faster or slower dynamics are impaired. According to the Temporal Sampling (TS) theory, dyslexia arises from impaired entrainment of low-frequency neural oscillations - particularly in the delta (1-4 Hz) and theta (4-8 Hz) bands - to the rhythmic modulations of speech. This hypothesis was tested for adults with and without dyslexia using electroencephalography (EEG) during a rhythmic audiovisual speech paradigm, previously delivered to children. Participants viewed a "talking head" repeating the syllable "ba" at a 2-Hz rate. Measures were neural phase entrainment and band power in the delta, theta, beta (15-25 Hz), and low gamma (25-40 Hz) bands, and broadband event-related potentials (ERPs), focusing on the N1 and P2 components. Phase-amplitude coupling (PAC) and phase-phase coupling (PPC) were also assessed for delta-theta, delta-beta, theta-beta, delta-gamma, and theta-gamma interactions. Both groups exhibited significant delta- and theta-band phase entrainment; however, the two groups differed significantly in the preferred phase for the theta band. While the control group showed consistent beta- and low gamma-band phase entrainment, this was not observed for the dyslexic group. There was significantly greater delta-band power for the dyslexic group across the whole brain and in the right temporal region. Additionally, the P2 ERP component differed significantly between groups. The data are interpreted with respect to TS theory.

sAPPalpha inhibits neurite outgrowth in primary mouse neurons via GABA B Receptor subunit 1a
Neurite outgrowth is essential for neural circuit formation and is tightly regulated by secreted factors and their receptors. The secreted extracellular domain of the amyloid precursor protein (sAPPalpha) has been shown to modulate neurite outgrowth. Recently, the gamma amino butyric acid receptor type-B subunit 1a (GABABR1a) was identified as an sAPPalpha binding partner that mediates its effects on synaptic transmission. Here, we investigated whether this interaction also regulates neurite outgrowth. In primary hippocampal neurons, the GABABR agonist baclofen reduced axon length; whereas, its antagonist CGP54626 increased axon length in primary hippocampal neurons. Moreover, GABABR1a knockout increased axon length and abolished the effect of baclofen. Application of sAPPalpha reduced axon length, an effect that required the presence of both GABABR1a and the extension domain of sAPPalpha, which mediates its binding to GABABR1a. Similarly, the APP 17mer peptide, which is sufficient to bind GABABR1a and mimic the effects of sAPP on synaptic transmission, reduced axon outgrowth in wildtype but not in GABABR1a-deficient neurons. Together, these findings indicate that the 1a isoform contributes to GABABR-dependent suppression of neurite outgrowth and mediates the inhibitory effect of sAPPalpha on neurite outgrowth.

Distribution of metabotropic serotonergic receptors in GABAergic and glutamatergic neurons in the auditory midbrain
The neurotransmitter serotonin modulates a variety of behavioral and physiological responses in the brain. Serotonergic neurons from the dorsal raphe nuclei send a dense projection to the auditory system, including the inferior colliculus (IC), the midbrain hub of the central auditory system. In the IC, serotonin alters how neurons respond to complex sounds, and it has been implicated in the generation or perception of tinnitus. However, the distribution of serotonergic receptors and the identity of neurons that express serotonergic receptors in the IC remains unclear. Here, we hypothesized that IC GABAergic and glutamatergic neurons differentially express serotonergic receptors. To test this hypothesis, we performed in situ hybridization in IC brain slices of male and female mice using probes for Vgat (GABAergic neuron marker) and Vglut2 (glutamatergic neuron marker), along with probes for six subtypes of metabotropic serotonergic receptors: 5-HT1A and 5-HT1B (Htr1a and Htr1b, inhibitory, Gi/o G protein receptors), 5-HT2A, 5-HT2B and 5-HT2C (Htr2a, Htr2b, and Htr2c excitatory, Gq11 G protein receptors) and, 5-HT7 (excitatory, Gs G protein receptors). Our data show that glutamatergic IC neurons primarily express inhibitory serotonergic receptors. In contrast, a larger proportion of GABAergic neurons express excitatory serotonergic receptors. Our data suggest that serotonin exerts an inhibitory net effect on IC neuronal circuits. These findings contribute to our understanding of how serotonin signaling influences auditory processing. The differential expression of serotonergic receptors may help shape the balance of excitation and inhibition in the auditory midbrain, affecting sound processing.

Disease-specific tau polymorphs define unique protein interaction networks across proteinopathies
Tau aggregates exhibit distinct conformations across tauopathies, but their disease-specific interactors remain poorly understood. We demonstrate that disease-specific tau conformations determine unique interactors across Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB). Through interactome profiling of misfolded tau aggregates from PBS- and sarkosyl-soluble fractions, we identified 493 high-confidence proteins with disease specificity and no overlap across diseases. Machine learning classification achieved compelling discrimination between diseases, demonstrating molecular signatures underlying clinical heterogeneity. AD tau aggregates engaged cellular metabolism machinery and glutamate/GABA cycling components, showing 27-fold enrichment over other tauopathies. PSP tau displayed the most distinctive profile, with protein depletion and selective enrichment of proteasome components. DLB tau associated with neurogenesis modulators while depleting neuroinflammatory mediators. These interactors were validated through proximity ligation assays and correlated with distinct post-translational modifications. Critically, sarkosyl-soluble fractions revealed reduced interactome complexity across diseases, except PSP tau which maintained interactions with GPCR-ERK signaling and kinetochore proteins, suggesting unique aggregation mechanisms. Our findings establish that distinct tau strains dictate disease-specific interacting networks, providing molecular insights into tauopathy diversity.

Individuals born with congenital cataracts exhibit both persisting impairment and considerable recovery of white matter microstructure after sight restoration
Individuals born with dense bilateral cataracts, for whom sight was restored later in life (congenital cataract reversal individuals), provide a unique opportunity to explore the impact of early visual experience on the development of the human brain. Using diffusion MRI we assessed white matter integrity in a sample of 20 congenital cataract reversal individuals using along-the-tract analysis of major visual white matter tracts and non-visual control tracts. We additionally recruited three control groups: 8 permanently congenitally blind individuals, 11 individuals with reversed later-onset cataracts (developmental cataract reversal individuals), and 24 age- and sex-matched typically sighted controls. Diffusion tensor metrics exhibited significant group differences which were specific to visual tracts. Compared to normally sighted controls, congenitally blind and congenital cataract reversal individuals both showed impaired white matter integrity. However, differences were much more spatially extensive for permanently congenitally blind individuals, and a direct comparison revealed relatively higher tract integrity for congenital cataract reversal individuals, suggesting a high degree of recovery following sight restoration. The present results are compatible with the idea of both a sensitive period for white matter development and significant white matter plasticity later in life. We additionally speculate that life-long myelin plasticity remains higher than neuronal structural plasticity.

Continuous monitoring of cerebrovascular autoregulation using functional ultrasound imaging in the piglet brain.
Continuous real-time assessment of cerebral blood flow (CBF) and cerebrovascular autoregulation (CA) remains a major unmet clinical need in acute brain injury. Methods such as laser Doppler flowmetry (LDF), transcranial Doppler, or indirect indices lack accuracy and robustness. Functional ultrasound (fUS) is an emerging modality combining high spatiotemporal resolution, large field-of-view, and sensitivity to blood velocity and volume, making it a promising neuromonitoring tool. Piglets were equipped with arterial blood pressure (ABP), intracranial pressure (ICP), and LDF probes, plus cranial windows for fUS and red blood cell (RBC) flux imaging. CA was challenged by non-pharmacological ABP manipulation via intraaortic or intracaval balloon inflation. fUS hemodynamic parameters were compared with other modaliters across a CPP range of 10-150 mmHg. fUS provided continuous, stable intensity- and velocity-derived parameters across vessels types. CBF estimates correlated strongly with RBC flux and showed reproducibility comparable to LDF, with lower inter-animal variability. Autoregulation breakpoints were reliably identified by fUS, particularly the lower limit, while the upper limit was more variable. Parcellation confirmed robustness of fUS across brain regions. fUS images CBF and CA with higher stability and reproducibility than standard approaches, supporting its applicability for bedside neuromonitoring and clinical translation.

Variational autoencoder for interpretable seizure onset phases detection
In this study, we describe a deep learning framework for automated seizure annotation in stereo electroencephalography (SEEG) data of patients with focal epilepsy. We use a one-dimensional Variational Autoencoder (VAE) for feature extraction of single-channel temporal series and a linear classifier for segment classification. We trained the network using data from 37 patients containing manual annotations of ictal and Low-Voltage Fast Activity (LVFA) by clinicians. The 1D VAE encodes two-second SEEG segments into a low-dimensional representation in latent space and then classifies them as interictal, ictal, or LVFA segments. We used 5-fold cross-validation for training and validation. Our system classified ictal vs. interictal 2 second segments with an average recall of 0.88. For whole-channel seizure annotation, we combine the probabilities of all its segments and compute the Area Under Curve (AUC) of the exponentially smoothed probability signal, marking the onset of both ictal and LVFA, a high average recall of 0.86, with even higher performance (0.91) on channels identified by clinicians as belonging to the Seizure Onset Zone (SOZ). The markers were also temporally accurate, with a mean (median) time lag of 9.8 (5.0) seconds for the ictal onset and a mean (median) lag of 2.0 (0.8) seconds for LVFA. Additionally, latent space analysis suggests that multiple dimensions correlate with class-relevant SEEG features that the classifier pays attention to, most notably amplitude and spectral power, which provides an explainability component to the network. As a secondary objective, we obtain a seizure detection recall of 99% with a specificity of 95%. Our findings suggest that a VAE-based approach can produce a meaningful latent space from SEEG data and leverage it to detect seizures and fast onset patterns, potentially helping clinicians reduce the workload of SEEG review.

A subtype of ultrasonic vocalizations during highly palatable food consumption in rats identified by machine learning-assisted classification
Identifying behavioral and physiological responses to rewarding stimuli is essential for understanding positive emotional states in animals and for investigating their neural basis. Ultrasonic vocalizations (USVs) in rats are modulated by various behavioral contexts and are thought to represent affective-like states. However, their diversity during palatable food consumption remains underexplored. In this study, we investigated acoustic features of USVs in male rats consuming chocolate, a highly palatable food. Using a machine-learning-assisted logistic regression model trained on spectrogram features, we identified a distinct USV subtype -- the 40-kHz inverted-U type -- that was selectively emitted during chocolate consumption. The emission of this subtype was tightly time-locked to chocolate feeding behavior. Systemic administration of naloxone, an opioid receptor antagonist, significantly reduced the emission of the 40-kHz inverted-U USVs during chocolate intake. These findings suggest that these USVs may reflect internal states associated with palatable feeding and are under modulation by the endogenous opioid system. Moreover, our data demonstrate the utility of machine learning for high-throughput, objective classification of USV subtypes. This framework provides a promising approach for decoding emotion-related vocal expressions in rats and highlights the potential of specific USVs as behavioral readouts for studying the neurobiology of positive emotions.

A canonical gated neural circuit model for flexible perceptual decisions
Flexible perceptual decision-making requires rapid, context-dependent adjustments, yet its underlying neural circuit mechanisms remain unclear. Here, we reverse-engineer a canonical neural circuit model that integrates sensory evidence and selects actions via distributed neuronal encoding, guided by data from a task that dissociates perceptual choice from motor response. The model's nonlinear gating of action selective (AS) neurons replicates parietal cortical activity observed during task performance. Critically, recurrent excitation within the evidence integration (EI) population supports sensory evidence accumulation, working memory for sequential sampling, and reward rate optimisation. Moreover, the dynamics of EI and AS neurons respectively mirror parietal activity related to sensory evidence encoding and ramping-to-threshold firing in a separate task, suggesting that decision readout engages both populations. The model also explains decision interference in a two-stage decision task, capturing observed accuracy decrements while predicting slower decisions. Together, these findings propose a foundational circuit-level mechanism unifying perceptual, memory-based, and abstract decision-making.

A paradigm shift of SERPINA3N in neurobehavioral development and brain injury
Murine serine protease inhibitor clade A member 3N (SERPINA3N) and its human ortholog SERPINA3 are dysregulated in neurological disorders. SERPINA3N has been proposed as a protective factor, enhancing neurobehavioral development under homeostasis and mitigating neuronal/glial and vascular damage under neurological conditions. Here, we employed powerful, non-invasive genetic tools to revisit the concept of SERPINA3N in brain development and injury. Brain-specific SERPINA3N overexpression neither alters neuronal or glial development nor improves cognitive ability under homeostasis. In stark contrast, SERPINA3N drives a pro-inflammatory response to brain injury and exacerbates blood-brain barrier dysfunction. Its overexpression promotes neurodegeneration through apoptosis-mediated neuronal loss and disrupts oligodendroglial differentiation and myelination following neonatal brain injury. Collectively, our findings challenge the prevailing paradigm of SERPINA3N in neurodevelopment and injury, revealing that while SERPINA3N is dispensable for neurobehavioral development, it aggravates neural injury and vascular damage under pathological conditions.

Striatal spinophilin enhances D2R interaction with cytosolic proteins to mediate persistent D2R agonist-induced locomotor suppression.
Loss of dopamine neurons in Parkinson disease (PD) leads to motor deficits. Dopamine D2 receptor (D2R) agonists treat PD-associated motor deficits by acting on postsynaptic receptors located within the striatum that have been upregulated due dopamine loss. However, mechanisms that contribute to increased D2R activity in PD that enhance D2R function are poorly described. Spinophilin is a protein phosphatase 1 targeting protein that is expressed in postsynaptic dendritic spines and interacts with postsynaptic D2Rs. However, how spinophilin regulates D2R function is unknown. In the current study, we found that genetic knockout of spinophilin limited the suppression of locomotion caused by the D2R agonist, quinpirole. Mechanistically, we found that spinophilin is required for quinpirole-induced increases in the interaction of the D2R with intracellular proteins, suggesting spinophilin mediates agonist-induced D2R internalization. Therefore, our data support future studies targeting the spinophilin/D2R interaction to enhance the efficacy of current PD therapeutics.

Interareal and interlaminar differences in sound envelope encoding in core and parabelt auditory cortex
Amplitude-modulation (AM) plays an important role in the perception of complex sounds, and transformations in AM encoding may underlie aspects of complex sound perception. Previous studies have described a hierarchical progression across the auditory pathway, characterized by a decrease in the temporal precision of AM encoding. In human and nonhuman primates (NHP), the left hemisphere exhibits enhanced temporal encoding relative to the right hemisphere. The NHP model provides an opportunity to understand what circuit mechanisms generate these transformations in encoding by characterizing AM encoding in different intracortical circuits, and across the cortical hierarchy. To address this, here we report the encoding of AM signals as a function of cortical layer and hemisphere in NHP core and parabelt auditory cortex (AC). We recorded electrophysiological activity using linear array multielectrodes positioned across cortical layers while AM noise and click trains were presented to awake NHPs. Core AC typically encoded all AM frequencies (1.6-200 Hz) with high ( > 90%) classification accuracy, while sites in the parabelt encoded a subset of lower (~1.6-25 Hz) frequencies. Across both areas, the granular and infragranular layers displayed enhanced AM encoding relative to the supragranular layers. Both areas displayed enhanced AM encoding in the left hemisphere, restricted to the supragranular layers. These results represent the first analysis of AM encoding in the parabelt, indicating that significant temporal encoding of AM is still present in tertiary auditory cortex, and the layer-specific hemispheric differences suggest a potential supragranular layer origin of previously documented left-hemisphere dominance in temporal encoding.

Claustrum volume in human lifespan trajectory and effect of age, hemisphere, and sex
The human claustrum is a bilateral, thin, irregularly shaped gray matter structure located between the striatum and insula. While previous research demonstrated the effect of distinct medical conditions, such as prematurity, schizophrenia, and Alzheimer's disease, on claustrum function and structure, it is poorly understood how non-pathologic biological conditions effect the claustrum. This study aimed to investigate the effect of age, hemisphere, and sex on claustrum volume. We used T1-weighted 3 Tesla MRI scans of 3,474 healthy participants ranging from 1 to 80 years of age, deep learning-based automated claustrum segmentation, and a normative modelling approach to delineate lifespan trajectories of claustrum volumes for both hemispheres and sexes. Additionally, ordinary least squares regression analyses were applied to further characterize age, hemisphere, and sex effect. Lifespan analysis revealed a trajectory of rapid claustrum volume increase from infancy to adolescence (~ 1 - 15 years, annual growth 39.300 mm3/year), a plateau phase from early to middle adulthood (~ 15 - 40 years, annual change 0.153 mm3/year), and a subsequent decline from middle adulthood to old age (~ 40 - 80 years, annual decrease 10.325 mm3/year). The right claustrum was on average larger than the left one across all ages. Finally, overall, females had larger total intracranial volume-adjusted claustrum volumes than males across the lifespan. Results demonstrate a distinct effect of age, hemisphere, and sex on claustrum volume. Data provide a comprehensive framework for sex- and hemisphere-sensitive claustrum structure lifespan trajectories relevant for studying neurodevelopmental and neurodegenerative effects on the claustrum.

From Chewing to Chirping: The Misophonia Audiovisual Trigger Archive (MATA)
Misophonia is an emerging condition in which everyday sounds, such as chewing, sniffing, or tapping, evoke disproportionately intense emotional and physiological responses. Despite growing recognition of its clinical significance, progress in understanding misophonia has been hindered by the limited availability of standardized and ecologically valid stimulus sets. Here, we present a large, open-access archive of 1,300 five-second audiovisual clips spanning 12 empirically validated categories of misophonic triggers. This resource extends beyond orofacial movement-related sounds to include a diverse array of real-world triggers, and its audiovisual format enables systematic investigation of how visual context shapes responses to misophonic sounds. The archive lowers the barrier for laboratories to study misophonia, promotes reproducibility across sites, and offers applications ranging from crowdsourced assessments of population-level sensitivities to machine learning approaches for automated trigger detection. By providing the largest and most diverse audiovisual misophonia stimulus repository to date, this resource is designed to accelerate mechanistic, clinical, and translational research on misophonia and related sensory-emotional phenomena.

SFRP1 drives glycolytic activation in astrocytes during neuroinflammation
Astrocytes and microglia maintain brain homeostasis and respond to inflammation through functions coordinated by molecular mediators they produce. Growing evidence shows that cellular metabolism is key to how these cells adapt to challenges. However, little is known about what drives glial metabolic reprogramming or whether molecules involved in astrocyte microglia crosstalk also regulate this process. Here, we explored this question focusing on Secreted Frizzled-Related Protein 1 (SFRP1). SFRP1 is an astrocyte-derived factor induced by inflammatory cues and overexpressed in neurodegeneration, which fosters microglial response to inflammation through NFkappaB and HIF dependent programs. We combined mitochondrial morphometry (MitoTracker Red and MiNA analysis) with Seahorse extracellular flux assays (Mito Stress Test) to determine whether SFRP1 modulates glial bioenergetics in primary cultures of astrocytes and microglia from wild-type and Sfrp1-/- mice. We report that SFRP1 acts as a driver of astrocytic metabolic activation, preferentially enhancing glycolysis over mitochondrial respiration. This effect is most pronounced during inflammation, when oxidative phosphorylation is restricted and SFRP1 enhances glycolytic flexibility to sustain energy demands. By contrast, microglia showed the expected LPS-driven glycolytic shift with minimal dependence on SFRP1 under monoculture conditions. These findings position SFRP1 as a candidate regulator of astrocyte-centered metabolic tuning during neuroinflammation, with implications for disorders such as Alzheimer s disease, in which SFRP1 is elevated.

Age-related inflammatory changes and perineuronal net dynamics: implications for neurodegenerative disorders.
Background: Healthy aging alone can lead to cognitive decline, decreased brain size, protein aggregation, accumulation of senescent cells and neuroinflammation. Furthermore, age is the primary risk factor for several neurodegenerative disorders such as Parkinson's and Alzheimer's disease. Age-related neuroinflammation, as known as inflammaging, is thought to restrict brain plasticity. Perineuronal nets (PNNs), specialized extracellular matrix structures surrounding fast-spiking parvalbumin (PV) interneurons, regulate plasticity and protect neurons from oxidative stress. Given the known impact of inflammaging on neural circuits, this study examines age-associated changes in PNN homeostasis, glial activation, and neuroinflammation in two brain regions relevant to age-related neurodegenerative diseases. Methods: We analyzed young (4-month-old) and aged (22-month-old) C57BL/6J male mice for several behavioral phenotypes [hippocampal-dependent spatial learning using the Barnes maze; locomotion and anxiety-related behaviors using Open field and T-maze]. Using immunostaining, PNNs (Wisteria floribunda agglutinin and aggrecan), PV interneurons, and microglial activation (Iba1) were quantified in both the hippocampus and dorsal striatum. Glial morphology was examined using a battery of cell body, branching, and endpoint analyses. Quantitative RT-PCR was used to analyze changes in the gene expression of inflammatory and extracellular matrix markers. Results: Aged mice exhibited hippocampal-dependent memory deficits without alterations in locomotion or anxiety-related behavior. PNN counts increased in the aged hippocampus, particularly in CA2, with a higher proportion of WFA and aggrecan PNNs. In contrast, PNN homeostasis was maintained in the dorsal striatum. In general, Aged mice showed increases in microglial activation and a subset of inflammatory markers. We report brain region- and age- specific gene expression changes in complement, matrix metalloproteinases, and other inflammatory markers. Aged striatal microglia displayed an activated morphology with larger cell bodies and reduced branching, as well as increased expression of markers for microgliosis (Iba1, TREM2, CD68). Conclusions: These findings suggest that aging differentially affects neuroinflammation and PNN integrity across brain regions. The hippocampus exhibits PNN accumulation, neuroinflammation, and behavioral changes, whereas the striatum maintains PNN homeostasis concurrent with increased microglial activation. This work suggests that neuroinflammation contributes to age-related changes in PNNs and behavior underscoring the importance of region-specific therapeutic strategies targeting PNN regulation.

Design and Implementation of a Decision Making System for Controlling a Hand Exoskeleton Based on EEG/EMG Signals
This paper presents an approach of combining Electroencephalography (EEG) and Electromyography (EMG) signals to create a hybrid Brain Interface Computer (BCI) device for controlling a hand exoskeleton through classifying the flexion attempt and resting states of the subjects. We analyzed data of 51 healthy and patients with brain lesions that involved the motor cortex and different hand movement disorders. Their EMG and EEG activity were recorded while the subjects attempted to move their index finger. The signals are analyzed through deep neural network, restricted Boltzmann machine and LDA classifier methods to access a reliable accuracy. Further, to evaluate the proposed approach, we designed and implemented a robotic system for rehabilitation of the hand movement to show that it is able to derive assistive control, by detecting flexion movement from the signals as a command and send it to robot. The system is combined with control signals that determine the information of flexion according to measured EMG signals.

Building the blood-brain barrier: a scalable self-assembling 3D model of the brain microvasculature under unidirectional flow
The blood vessels of the central nervous (CNS) system form a tight, protective blood-brain barrier (BBB). This barrier is essential for healthy CNS function but also poses a hurdle in the treatment of increasingly common neurological disorders. Additionally, BBB dysfunction is a hallmark of many neurological diseases, further emphasizing a need for a better understanding of BBB function in health and disease. We present a human self-assembling 3D model of the BBB in a microfluidic cell culture platform that allows culture of 48 models in parallel on one tissue culture plate. Human brain microvascular endothelial cells, pericytes, and astrocytes form highly reproducible BBB vascular networks under unidirectional perfusion and remain viable for a minimum of 14 days. Immunostaining reveals close cell-cell interactions with pericytes and astrocyte end-feet in direct contact with the brain microvasculature. Compared to endothelial monocultures, co-culture with astrocytes or pericytes results in improved barrier function, lower vessel diameters, increased branching, and alignment of the vessels in the direction of fluid flow. These results were most pronounced in tri-cultures containing all three cell types. Unlike similar models previously reported, this brain microvasculature model allows for unidirectional perfusion without the need for pumps and syringes. Combined with its high-throughput nature, this feature renders the model suitable for studies of BBB function in health and disease, and assessment of potential BBB restorative therapies.

Sex Differences in Plasma Levels of Endocannabinoids and Related Lipids Before and After acute and repeated mTBI: an exploratory study for plasma biomarkers for mTBI
Mild traumatic brain injury (mTBI) is common diagnosis across all age groups and while most symptoms resolve within a few weeks; between 10 and 25 percent of mTBI patients suffer long-term problems. Known as post-concussion syndrome (PCS), symptoms include headache, a range of cognitive deficits, and depression. Currently, there are no established treatments for PCS and no clear predictive biometrics to determine which patients are at increased risk. Previous studies have identified some protein-derived plasma biomarkers for mTBI, however, the effects of mTBI on lipid signaling molecules and metabolites in blood is largely unknown. Endogenous lipids (endolipids) such as the endocannabinoids (eCBs) and their congeners are lipid signaling molecules that are associated with promoting neuroprotective responses after head trauma in animal models. Here, we examine the plasma lipidome using a rat model of acute and repeated mTBI that we previously demonstrated had a sex dependent change in neuroinflammation wherein females showed a higher degree of neurodegeneration after repeated head-injury than males. Key results of this exploratory lipidomics screen here demonstrates that acute head injury drives significantly more changes in plasma endolipids in males (32%) than females (8%), whereas, on the second day of head injury, only 11% change in males but 15% in females. Some key endolipids were modified in both males are precursors for resolving molecules and this was lacking in females. Given that females with repeated mTBI in this model demonstrated aspects of PCS, this could be an important component in evaluating clinical cases. Endolipids in the screen were measurable in plasma using only 100 microL, a volume necessary to be able to perform multiple blood draws on these rodent subjects. This threshold provides evidence that the levels of these endolipids could be readily measured throughout patient recovery. Therefore, this family of endolipids has the potential to provide data on the progression of the injury and could be another crucial aspect in predicting mTBI outcomes.

Temporal dynamics of neuroplasticity and neurodegeneration in the central auditory system following noise-induced hearing loss: A multimodal imaging and histological study
Noise-induced hearing loss (NIHL) is a sensorineural disorder that triggers profound neuroplastic and neurodegenerative consequences in the central auditory nervous system (CNS). This study examined how neuronal density, axonal integrity and glutamatergic and GABAergic neurotransmission are affected after acute overstimulation in the central inferior colliculus (CIC) and the ventral medial geniculate body of the thalamus (MGV). To achieve this, a correlative multimodal approach combining audiometric, magnetic resonance imaging (MRI) and histological biomarkers was performed. Adult mice were noise-exposed to broadband white noise (5-20 kHz) for 3 hours at either high (115 dB SPL) or moderate (90 dB SPL) intensity, while unexposed mice were used as controls. Separate cohorts of mice were investigated 1-, 7-, 56- and 84-days post-exposure using in vivo magnetic resonance imaging (MRI) techniques: Voxel-based morphometry (VBM) of gray matter density (GMD), diffusion MRI (dMRI) of microstructure and connectivity, and single-voxel proton magnetic resonance spectroscopy (1H-MRS) for glutamate and GABA quantification. Frequency-specific auditory brainstem responses (ABR) were recorded at 4, 8, 16 and 32 kHz before and after exposure to examine hearing threshold (HT) shifts. Following imaging, brains were processed for fluorescence immunohistochemistry (FIHC) targeting Neuronal Nuclear Protein (NeuN), 4',6-diamidino-2-fenilindo (DAPI), Neurofilament (SMI312), vesicular GABA transporter (VGAT), and vesicular glutamate transporters 1 (VGLUT1) and 2 (VGLUT2). HTs were significantly elevated 7d, 56d and 84d after 115 dB noise exposure, suggesting a NIHL phenotype. Neurofilament density significantly increased in the CIC and MGV 1d after 115 dB noise exposure, and returned to baseline at later time points. dMRI transient microstructural alterations were observed 7d after 90 dB noise exposure. Glutamate and GABA decreases 84d after 90 dB exposure were also detected. Moreover, correlation analysis between audiometric, histological and MRI datasets revealed scarce relationships between the investigated parameters. These findings advance our understanding of the CNS adaptations of NIHL and emphasizes the need for more sensitive and integrative approaches to identify robust biomarkers of central auditory dysfunction.

From ambiguous object to illusory faces: EEG decoding reveals a dynamic cascade of face pareidolia
Face pareidolia, perceiving faces in inanimate objects, provides a unique window into how the brain constructs perceptual meaning from ambiguous visual input. In this work, we investigated the neural dynamics of face pareidolia, testing the hypothesis that it emerges from a temporal cascade that integrates global (low spatial frequency, LSF) and local (high spatial frequency, HSF) visual information. With a pareidolia detection task, we combined event-related potentials (ERP), multivariate pattern analysis (MVPA), and representational similarity analysis (RSA) to map the evolving neural code for illusory face perception. Behaviorally, participants experienced pareidolia from both LSF and HSF stimuli, but the percept was strongest for broadband images containing both frequency bands. Electrophysiological results revealed a clear temporal progression. Early processing (<150 ms) was driven by low-level attributes, with distinct P100/N100 components and neural representations for LSF and HSF signals. A critical shift occurred around 145 ms, after which MVPA decoding could reliably distinguish between images perceived as faces and those that were not. Concurrently, RSA showed that the geometry of neural representations shifted to reflect participants' subjective face-likeness ratings, rather than the initial physical properties of the stimuli. This transition to a higher-order, subjective evaluation was marked by a later N250 component, whose amplitude indexed the cognitive load of integrating the visual cues. Together, these findings demonstrate that face pareidolia is a dynamic neural cascade, from early sensory encoding to higher-order perceptual evaluation, driven by the integration of global configuration and local features of visual stimuli. Our results illuminate how the brain transforms ambiguous input into meaningful social perception, offering insight into the temporal dynamics of visual awareness.

Neurophysiological Sensitivity to Envelope and Pulse Timing Interaural Time Differences in Cochlear Implanted Rats with Different Hearing Experiences
Cochlear implants (CIs) have successfully restored hearing in more than one million patients with severe to profound hearing loss worldwide. While CIs effectively restore speech perception in quiet environments, sound localization remains challenging for bilateral CI users, particularly their ability to utilize interaural time differences (ITDs). The majority of clinical CI processors use a coding strategy that encodes ITD information only in the envelope of electrical pulse trains rather than their pulse timing, which may contribute to the poorer spatial hearing perception of CI users. We recently demonstrated in a behavioral study on early deafened, bilaterally CI-implanted rats that pulse timing ITDs completely dominate ITD perception, while sensitivity to envelope ITDs is almost negligible in comparison. Building on this, we here investigated the neurophysiological sensitivity of the inferior colliculus (IC) to envelope and pulse timing ITDs at two different pulse rates (900 and 4500 pulses/s) and three different stimulation modulations (5, 20 and 100 Hz) in CI rats with different hearing experiences. Our results indicate that IC neurons exhibit far greater sensitivity to pulse timing ITD than envelope ITD independent of pulse rate, modulation rate or hearing experience. These findings suggest that to improve binaural hearing outcome in bilateral CI users, clinical stimulation strategies should provide informative pulse timing ITDs.

High-frequency, low-intensity pulsed electric field and N-acetylcysteine synergistically protect SH-SY5Y cells against hydrogen peroxide-induced cell damage in vitro
Oxidative stress plays an important role in the progression of neurodegenerative diseases (NDDs), and N-acetylcysteine (NAC) has gained attention as a potential agent due to its antioxidant capabilities. This study investigated the synergistic neuroprotective effects of combining NAC with non-contact high-frequency low-intensity pulsed electric field (H-LIPEF) stimulation on SH-SY5Y human neuronal cells subjected to hydrogen peroxide (H2O2)-induced oxidative damage. It was found that after SH-SY5Y cells were pretreated with NAC and exposed to H-LIPEF stimulation, the oxidative stress of cells was reduced in the subsequent treatment with H2O2. The results showed that the combined NAC and H-LIPEF treatment significantly improved cell viability and better preserved cellular morphology compared to either treatment alone. Additionally, this combination treatment more effectively reduced mitochondrial apoptosis. Mechanistic analyses revealed that the combination substantially decreased levels of superoxide and intracellular H2O2, which was associated with enhanced activation of the p-Akt/Nrf2/SOD2 signaling pathway. Furthermore, the treatment reduced the accumulation of 8-oxo-dG accumulation and elevated MTH1 expression, indicating a protective effect against oxidative DNA damage. These results suggest that H-LIPEF enhances the neuroprotective efficacy of low-dose NAC, highlighting the potential of this combination approach as a new therapeutic strategy for the treatment of NDDs.

Ectopic expression of two cone opsins in mouse RGCs results in opposite responses to light stimulation, likely due to differential G protein activation
Retinitis pigmentosa, a leading cause of inherited blindness, results in photoreceptor degeneration that current optogenetic approaches aim to address through microbial opsin expression in retinal ganglion cells (RGCs). While these microbial proteins restore light sensitivity, their clinical potential remains limited by low light sensitivity and immunogenicity risks. Recent efforts have focused on vertebrate opsins as safer alternatives, with mid-wavelength cone opsin (MW-opsin) demonstrating RGC depolarization via endogenous G-protein signaling. In this study, we reveal a surprising divergence in signaling outcomes of expressing short-wavelength mouse cone opsin (Opn1sw) in RGCs of blind mice. Using multielectrode array recordings and whole-cell patch clamping, we demonstrate that Opn1sw induces membrane hyperpolarization in RGCs - a stark contrast to MW-opsin's depolarizing effects. This unexpected inversion suggests differential engagement of intracellular signaling pathways, potentially stemming from distinct G-protein coupling preferences. Comparative analysis of native G-protein expression profiles in RGCs versus cone photoreceptors supports this hypothesis, revealing mismatches that may explain ectopic opsin behavior. Our findings challenge the assumption of conserved opsin signaling across spectral subtypes and cell types, highlighting critical gaps in understanding vertebrate opsin-G protein interactions in non-native cellular environments. This discovery points to the necessity of systematic characterization of opsin signaling networks in target retinal cells, a prerequisite for engineering optimized optogenetic tools that reliably produce desired electrophysiological outcomes. By establishing spectral subtype-dependent signaling divergence, our work redefines parameters for developing next-generation vision restoration therapies.

Multiomic profiling reveals pericyte and smooth muscle cell contributions to CADASIL pathology in cell-specific Notch3 mutant mice
Cerebral ischemic small vessel disease (SVD) is the leading cause of vascular dementia and a major contributor to stroke. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of familial SVD. CADASIL is caused by dominant missense mutations in Notch3, a receptor expressed in mural cells, including smooth muscle cells (SMCs) and pericytes. However, the cell-type specific contributions driving the CADASIL pathology remain unknown due to lack of animal models. Here, we generated two conditional knock-in mouse models carrying the CADASIL-causing Notch3R170C mutation selectively in SMC and brain pericytes. Both Notch3R170C models showed perivascular accumulation of the NOTCH3 extracellular domain, yet developed distinct neurovascular changes depending on the affected cell type. Pericyte-specific Notch3R170C mice displayed pronounced region-selective microglial activation and vascular changes, whereas SMC-specific Notch3R170C mice showed localized perivascular gliosis with minimal vascular remodeling. Proteomic profiling of isolated brain vessels revealed largely unique cell-specific responses. Pericytes Notch3R170C expression dysregulated metabolic pathways, whereas SMC Notch3R170C expression induced immune signaling related pathways. Integration with single-cell RNA-seq data revealed that many of the proteomic and phosphoproteomic shifts might also include brain endothelial cells, including metabolic changes in the presence of pericyte Notch3R170C and inflammatory signaling in the presence of SMC-Notch3R170C. Together, these findings define mural cell-specific mechanisms that contribute to the CADASIL-associated vascular pathology.