bioRxiv Subject Collection: Neuroscience's Journal

Ablation of VEGFA following a lumbar intervertebral disc injury attenuates intradiscal neurovascular features and prevents chronic low back pain symptoms
There are currently no therapies for the staggering disability and public health costs of chronic low back pain (LBP). Innervation of the degenerating intervertebral disc (IVD) is suspected to cause discogenic LBP, but the mechanisms that orchestrate the IVD's neo-innervation and subsequent symptoms of LBP remain unknown. We hypothesize that Vascular Growth Endothelial Factor-A (VEGFA) critically mediates the neurite invasion in the IVD and contributes to prolonged LBP. Initiating IVD degeneration through a mechanical injury, we evaluated the progression of neurovascular features into the IVD, as well as resultant LBP symptoms and locomotive performance at acute (3-weeks) and prolonged (12-weeks) time points following IVD injury. To determine the role of VEGFA, we used a mouse model with ubiquitous inducible recombination of the floxed VEGFA allele (UBC-CreERT2; VEGFAfl/fl). The ablation of VEGFA attenuated the neurite and vessel infiltration into the degenerating IVD, and the VEGFA-null animals exhibited alleviated mechanical allodynia and improved locomotive performance. To determine the effects of IVD-derived VEGFA on endothelial cells and neurons, we cultured HMEC-1 endothelial cells and SH-SY5Y neurons using conditioned media from VEGFA-silenced (siRNA) human primary IVD cells stimulated by IL1b. The endothelial cells and neurons exposed to the secretome of the VEGFA-silenced IVD cells exhibited reduced growth, suggesting that the inhibition of IVD-derived VEGFA may be sufficient to attenuate intradiscal neurovascular features. Together, we show that VEGFA orchestrates the growth of intradiscal vessels and neurites that cause low back pain and impaired function, and the inhibition of VEGFA can prevent prolonged low back pain.

Development of a network formation assay for developmental neurotoxicity hazard screening using 3D human iPSC derived BrainSpheres
Exposure of the developing brain to environmental neurotoxicants can result in permanent alterations in structure and/or function. To investigate the effects of chemical exposures on neurodevelopment, the human induced-pluripotent stem cell (iPSC)-derived neural BrainSphere model has been utilized due to its ability to form mature neuronal populations and exhibit spontaneous electrical activity. To model network formation for developmental neurotoxicity screening, developing BrainSpheres were plated on high-density microelectrode arrays (hdMEA) three weeks after beginning differentiation. Starting two days post-plating, BrainSpheres were treated three times per week with compounds known to disrupt in vitro network formation (i.e. assay positive controls; loperamide, dieldrin and deltamethrin), or with an assay negative control, glyphosate, expected to have no effect. For 29 days, BrainSphere activity was recorded to measure neural network activity, general activity, and features of action potential propagation. Concentration-dependent disruption in neural network formation was observed for positive controls at concentrations below cytotoxicity. Dieldrin, deltamethrin, and loperamide exposure disrupted several features of general activity, neural network formation, and action potential propagation. BrainSpheres on hdMEAs detected chemically induced perturbations in neural network formation and may represent a valuable complex in vitro model useful for developmental neurotoxicity screening.

Hippocampal reactivation of aversive experience enables safety learning and slow-breathing state for recovery from stress
Adaptive threat responses require both defensive behaviours to minimize danger and recovering from the induced physiological stress. However, the behavioural and neural basis of these recuperative strategies are still elusive. Using a novel two-location fear conditioning paradigm in mice, we have identified a slow-breathing immobility state of recovery that emerges when animals identify safe environments after threat avoidance. This immobile state was characterized by a 2-4 Hz breathing profile and replay of the aversive experience in the hippocampus. Suppressing hippocampal sharp-wave ripples (SWRs) inhibited the emergence of this recovery state, suggesting their role in learning safe locations. Anxiolysis with diazepam directly promoted the recovery state while suppressing SWRs, showing this treatment to be a double-edged sword that facilitates immediate relief but impairs long-term safety learning. These results demonstrate the importance of hippocampal replay for emotional resilience through its role in recovery.

Interindividual differences in predicting words versus sentence meaning:Explaining N400 amplitudes using large-scale neural network models
Prediction error, both at the level of sentence meaning and at the level of the next presented word, has been shown to successfully account for N400 amplitudes. Here we address the question of whether people differ in the representational level at which they implicitly predict upcoming language. To this end, we compute a measure of prediction error at the level of sentence meaning (magnitude of change in hidden layer activation, termed semantic update, in a neural network model of sentence comprehension, the Sentence Gestalt model) and a measure of prediction error at the level of the next presented word (surprisal from a next word prediction language model). When using both measures to predict N400 amplitudes during the reading of naturalistic texts, results showed that both measures significantly accounted for N400 amplitudes even when the other measure was controlled for. Most important for current purposes, both effects were significantly negatively correlated such that people with a reversed or weak surprisal effect showed the strongest influence of semantic update on N400 amplitudes, and random-effects model comparison showed that individuals differ in whether their N400 amplitudes are driven by semantic update only, by surprisal only, or by both, and that the most common model in the population was either semantic update or the combined model but clearly not the pure surprisal model. The current approach of combining large-scale models implementing different theoretical accounts with advanced model comparison techniques enables fine-grained investigations into the computational processes underlying N400 amplitudes, including interindividual differences.

High reelin expression can explain why the entorhinal cortex is a cradle for Alzheimer's disease
The entorhinal cortex (EC) plays a crucial role in memory functions. Long before the clinical symptoms of Alzheimer's disease (AD) emerge, it has already undergone significant degeneration, making it a primary site for the onset of the disease. The reasons for this remain elusive. It was recently shown that in layer II neurons of the anterolateral entorhinal cortex (alECLII neurons), which are especially prone to display a very early increase in intracellular amounts of amyloid-{beta} peptide (A{beta}) and hyperphosphorylated tau protein (p-tau), the large glycoprotein reelin binds to A{beta}, suggesting that reelin functions as a sink for intracellular A{beta}. The expression of reelin is extraordinarily high in alECLII neurons compared to most other cortical neurons. Here, we show by computational modeling that, in a senescent physiology predisposing to frequent inflammation-driven A{beta}42 production bursts, the intracellular amount of A{beta}42-reelin complexes can accumulate to extraordinarily high levels in alECLII neurons compared to the vast majority of cortical neurons. This explains experimental data showing that intracellular accumulations of A{beta}42 positive material ranged from 20 to 80% of the total cytoplasmic volume in EC neurons from patients with sporadic AD. We also show that this extreme intracellular aggregation can cause the accumulation of detrimental hyperphosphorylated tau fragments. Thus, when exposed to recurrent AD-promoting stress, the exceptionally high expression of reelin in alECLII neurons appears to be instrumental in their early demise relative to other cortical neurons.

Localization of Realistic Spatial Patches of Complex Source Activity in MEG
Accurate localization of neural sources in Magnetoencephalography (MEG) and Electroencephalography (EEG) is essential for advancing clinical and research applications in neuroscience. Traditional approaches like dipole fitting (e.g., MUSIC, RAP-MUSIC) are limited to discrete focal sources, while distributed source imaging methods (e.g., MNE, sLORETA) assume sources distributed across the cortical surface. These methods, however, often fail to capture sources with complex spatial extents, limiting their accuracy in realistic settings. To address these limitations, we introduce PATCH-AP, an enhanced version of the Alternating Projection (AP) method that effectively localizes both discrete and spatially extended sources. We evaluated PATCH-AP against leading source localization methods, including distributed source imaging techniques (MNE, sLORETA), traditional dipole fitting (AP), and recent extended source methods (Convexity-Champagne (CC), FLEX-AP). PATCH-AP consistently outperformed these methods in simulations, achieving lower Earth Mover's Distance (EMD) scores a metric indicating closer alignment with the true source distribution. In tests with real MEG data from a face perception task, PATCH-AP demonstrated high alignment with the fusiform face area, a region critical for face processing. These results highlight PATCH-AP's potential to enhance source localization accuracy, promising significant advancements in neuroscience research and clinical diagnostics.

BARTharm: MRI Harmonization Using Image Quality Metrics and Bayesian Non-parametric
Image derived phenotypes (IDPs) harmonization from Magnetic Resonance Imaging (MRI) data is essential for reducing scanner-induced, non-biological variability and enabling accurate multi-site analysis. Existing methods like ComBat, while widely used, rely on linear assumptions and explicit scanner IDs - limitations that reduce their effectiveness in real-world scenarios involving complex scanner effects, non-linear biological variation, or anonymized data. We introduce BARTharm, a novel harmonization framework that uses Image Quality Metrics (IQMs) instead of Scanner IDs and models scanner and biological effects separately using Bayesian Additive Regression Trees (BART), allowing for flexible, data-driven adjustment of IDPs. Through extensive simulation studies, we demonstrate that IQMs provide a more informative and flexible representation of scanner-related variation than categorical Scanner IDs, enabling more accurate removal of non-biological effects. Leveraging this and its ability to model complex relationships, BARTharm, consistently outperforms ComBat across a range of challenging scenarios, including model misspecification and confounded scanner-biological relationships. Applied to real-world datasets, BARTharm successfully removes scanner-induced bias while preserving meaningful biological signals, resulting in stronger, more reliable associations with clinical outcomes. Overall, we find that BARTharm is a robust, data-driven improvement over traditional harmonization approaches, particularly suited for modern, large-scale neuroimaging studies.

Ketogenic diet dampens excitatory neurotransmission by shrinking synaptic vesicle pools
The ketogenic diet (KD) is a common dietary intervention for treating seizures in intractable childhood epilepsies and has been proposed to improve disease outcome in neurodegenerative disorders. Despite its clinical applications, we know little about how this diet impacts brain circuitry and neuronal function to elicit its protective effects. Here, we examined the impact of the KD on hippocampal function through integrative analysis of gene expression, epigenetics and neurotransmission. We found that KD induces profound transcriptional reprogramming of the hippocampus, including dampened expression of numerous synaptic genes. Through proteomic analysis of histone variants and post-translational modifications, we uncovered significant changes in activating and repressive histone marks in the hippocampus of KD mice. To determine how transcriptional rewiring of the hippocampus under KD impacts neurotransmission, we performed electrophysiological recordings of neurotransmission and synaptic dynamics at excitatory CA3-CA1 synapses. We found that KD diminishes synaptic gain and dampens short-term plasticity at excitatory synapses, resulting in reduced integration of synaptic inputs at the circuit level. Combining electrophysiology and electron microscopy, we determined that effects of KD in excitatory synapses are caused by a reduction in size of the readily releasable pool of synaptic vesicles, as well as the total vesicle pool. Our findings show that the ketogenic diet triggers synaptic remodeling in the hippocampus, driven by broad transcriptional and epigenetic changes that reduce synaptic vesicle pools and short-term plasticity at excitatory synapses ultimately dampening excitatory synaptic gain and integration at the circuit level. These synaptic adaptations may represent a major mechanism underlying the anti-epileptic effects of this diet.

Different Multiword Verb Categories are Processed Differentially in the Brain: An Evidence from EEG Analysis and Decoding
The mental representation of multiword verb constructions is a central question in neurolinguistics: are they stored as single lexical units or completely compositional items? This study investigates the neurocognitive processing of two multiword verb types: phrasal verbs (e.g., look up) and prepositional verbs (e.g., decide on). Taking in consideration verb-to-infinitive constructions (e.g., want to go) as a control group. We analyse event-related potentials from eleven native English speakers who completed a listening task while EEG data were recorded. Grand-averaged waveforms and root-mean-square amplitudes were analysed across four-time windows. Therefore, statistical comparisons showed a significantly larger N400 amplitudes for prepositional verbs compared to phrasal verbs, while no significant differences were found between prepositional and to-infinitive constructions. Multivariate pattern analyses confirmed neural discriminability between phrasal and prepositional verbs, but not between prepositional and verb-to-infinitive structures. These results confirm that prepositional and verb-to-infinitive constructions are processed compositionally via valency-based integration, whereas phrasal verbs are stored as lexicalized units. The findings support a theoretical model in which multiword verb constructions differ in their degree of lexicalization, with measurable consequences for real-time neural processing.

Direct electrical stimulation of the human amygdala enhances recognition memory for objects but not scenes
Research from neuroscience studies using invasive neuroanatomy-inspired direct manipulations suggests the basolateral amygdala (BLA) mediates a generalized modulation of many different types of memory. In contrast, noninvasive, psychology-inspired indirect correlations suggest that specificity exists in how the BLA prioritizes experiences in memory. We used direct electrical stimulation of the BLA to investigate the specificity of the memory enhancement in the human brain. Patients undergoing intracranial monitoring via depth electrodes viewed object and scene images, half of which were followed by BLA stimulation. Stimulation enhanced long-term memory for object but not scene images. Furthermore, BLA stimulation elicited stronger evoked responses in the anterior vs. posterior medial temporal lobe (MTL), regions that preferentially process object and scene learning, respectively. These results suggest the BLA exerts an important influence over the specificity of what information is prioritized in memory, rather than a general enhancement of all memory, and provide insight into how BLA-MTL projections contribute to the dynamics of memory prioritization.

Mapping neurogenetic characteristics of psychopathological procrastination using normative modeling in a prospective twin cohort
Procrastination, affecting over 70% global population, is pervasively incurring negative outcomes in human society. This has long been studied as a bad daily habit, but it loses in delineating neurogenetic substrates underlying its psychopathological phenotyping. Using a prospective twin adolescent cohort, we demonstrate moderate heritability of this subclinical condition - PPS. Neuroimaging normative modeling analysis, further reveals that neurodevelopmental deviations in nucleus accumbens during adolescence, are predictive of PPS in adulthood, while such deviations-PPS mappings were highly genetically shared . Beyond to regional anomalies, PPS-specific whole-brain deviation patterns, notably in the default mode network, are neurobiologically enriched with changes in cortical manifolds (gradients) and neurotransmitter systems. Integrating these neuroimaging markers with transcriptomic atlas, we capture significant PPS-specific neurogenetic signatures associated with molecular transport system, neuroimmune responses, and neuroinflammation, particularly in serotonergic and dopaminergic pathways. These findings shed light on the multisystem neurogenetic architecture underlying PPS, providing evidence to theoretically conceptualize this psychopathological phenotype as a subclinical "brain disorder".

Neurodevelopmental Impact of Bipolar Disorder Genetic Risk on Cortical Thickness and Network Topology in Adolescents
Bipolar disorder (BD) is a highly heritable psychiatric condition characterized by recurrent mood episodes that commonly manifest during adolescence. Although polygenic risk scores (PRS) effectively quantify genetic susceptibility to BD, the neurobiological correlates of this genetic risk during adolescent brain development remain unclear. Using normative modeling and longitudinal neuroimaging data from the Adolescent Brain Cognitive Development (ABCD) cohort (N = 4519), we examined cortical thickness (CT) deviations and structural covariance network (SCN) alterations in adolescents stratified into high and low BD genetic risk groups based on PRS. Adolescents with high PRS for bipolar disorder exhibited greater cortical thickening in the inferior frontal gyrus and primary visual cortex, whereas those with low PRS demonstrated greater thickening in the posterior cingulate cortex and middle precentral gyrus. These patterns suggest PRS-related variations in cortical maturation, potentially reflecting distinct neurodevelopmental trajectories associated with genetic susceptibility to bipolar disorder. Furthermore, high PRS individuals displayed altered SCN topology, characterized by decreased local clustering and enhanced global network efficiency. Longitudinal data show that these abnormal regions exhibit atypical developmental trajectories, accompanied by a global reorganization of network topology. Additionally, high genetic risk was associated with lifestyle factors, especially correlated with increased positive expectancies toward substance use. Eventually, we found three potential BD risk genes during adolescence, including PLEKHA2, ZSCAN31 and ANK3, encoding the function of the postsynaptic membrane and synaptic membrane. These findings elucidate early neurodevelopmental deviations linked to genetic risk for BD, highlighting potential biomarkers for early identification and targeted interventions.

Molecular and functional dissection using CaMPARI-seq reveals the neuronal organization for dissociating optic flow-dependent behaviors
Optic flow processing is critical for the visual control of body and eye movements in many animals. Rotational and translational binocular optic flow patterns need to be clearly distinguished to induce different behavior outputs. However, the specific neuron types and their connectivity involved in this computation remain unclear. Here, we developed a method to link the functional labeling using a photoconvertible calcium indicator called CaMPARI2 and single-cell RNA-sequencing (CaMPARI-seq) to investigate the transcriptional profile of the pretectum, a center for processing optic flow in larval zebrafish. Using this technique, we identified a pretectal cluster expressing tcf7l2, which can be further classified into molecularly distinct subclusters. In vivo calcium imaging and cell ablation revealed that nkx1.2lb-positive pretectal neurons are commissural inhibitory neurons required for the optomotor response but not for the optokinetic response. Our genetic and functional dissection using CaMPARI-seq uncovered the neuronal organization essential for dissociating different optic flow-dependent behaviors.