bioRxiv Subject Collection: Neuroscience's Journal

Dissecting Medullary Raphe Neurons Regulating Multiple Thermogenic Pathways
Thermogenesis is critical for survival and health in mammals. Although the thermoregulatory systems in the preoptic area are well documented, the downstream processing of these central signals--particularly by medullary neurons involved in the control of shivering and sympathetic activation of brown adipose tissue (BAT)--remains incompletely understood. Here we show that vesicular glutamate transporter type 3 (vGluT3)-expressing neurons in the medullary raphe pallidus (RPa) become active immediately before a spontaneous increase in body temperature. These neurons remain inactive under experimentally induced hypometabolic conditions and are necessary for rapid recovery from hypothermia. Furthermore, they communicate with multiple brainstem systems involved in the integration of thermal cues. Notably, RPa-vGluT3 neurons can drive shivering via specific brainstem premotor neurons, in addition to regulating sympathetic outflows for BAT thermogenesis and heat-conserving piloerection. These data indicate that RPa-vGluT3 neurons function as medullary hubs, coordinating sympathetic and somatic motor outputs to increase body temperature.

Neural Subspaces Encode Sequential Working Memory, but Neural Sequences Do Not
The neural mechanisms of multiple-item working memory are not well understood. In the current study, we address two competing hypotheses about the neural basis of sequential working memory: neural subspaces versus neural sequences. Using broadband MEG data from human participants, we applied dimensionality reduction and multivariate decoding techniques to test whether sequential items are maintained during the retention period through the reactivation of individual items in sequence (neural sequences), or by organizing them into distinct low-dimensional subspaces (neural subspaces). Our results revealed behaviorally relevant, low-dimensional neural subspaces that organized memory representations during the retention period but not during stimulus encoding, supporting the neural subspaces hypothesis. In contrast, we found no evidence of sequential neural replay during the delay period, contrary to predictions from the neural sequences hypothesis. Together, our findings suggest that sequential working memory is maintained through structured geometric organization in low-dimensional representational space, rather than through the sequential reactivation of individual items.

A universal dynamic routing architecture governs the flow of human cortical activity
Behaviour requires distributed neural processing to be flexibly integrated and segregated, which in turn demands that information be dynamically routed across the brain. However, whether a unifying principle governs the routing of neural activity remains unknown. Here, we report that the flow of cortical activity is directed through canonical routing modes that are conserved across individuals, robust to variations in age, frequency band, brain state, and even the presence of neurodegeneration. These modes are constructed from the divergence and vorticity of the time-varying unit-phase vector field derived from electroencephalogram (EEG) recordings. We show that the canonical routing modes are flexibly combined to generate distinct, state- and function-dependent flow architectures that remain consistent across individuals. Disruption to the routing mode dynamics mechanistically links structural abnormalities and cognitive impairment in Alzheimer's disease. Together, these canonical routing modes provide a unified framework for describing, modelling, and predicting the dynamic integration and segregation of macroscopic brain activity underpinning behaviour.

Complementary regulation of memory flexibility and stabilization by dentate gyrus granule cells and mossy cells
Accurate memory formation requires hippocampal spatial representations to balance stability, for later recall, with flexibility, to incorporate new information. The dentate gyrus (DG) is essential to memory formation, but the distinct roles of its excitatory cell types, granule cells (GCs) and mossy cells (MCs), remain unclear. To evaluate how GC and MC activity affect hippocampal output, we recorded from CA1 using two-photon calcium imaging as head-fixed mice navigated familiar and novel virtual environments. DREADD-mediated MC inhibition disrupted initial map stabilization, decreasing spatial stability in novel, but not familiar, environments. In contrast, GC inhibition increased map stability in familiar, but not novel, environments by disrupting drift of spatial maps across distinct experiences (episodes) within an environment. These results reveal how distinct DG cell types support hippocampal memory formation in context-dependent ways; MCs promote stabilization of new spatial maps to support accurate memory recall, while GCs promote flexibility to update existing representations.

Cross-Species Evidence for Hippocampal CACNA1C as a Therapeutic Target for Alcohol Use Disorder
Context-induced relapse is a major barrier to recovery from alcohol use disorder (AUD). Identifying molecular targets involved in contextual memories associated with alcohol use may serve as novel pharmacotherapies. Our RNAseq profiling study of the hippocampus from rhesus monkeys with chronic alcohol use identified the voltage-gated calcium channel CACNA1C as a promising therapeutic target. However, data regarding CACNA1C expression in AUD and whether inhibition of CACNA1C can attenuate ethanol contextual memories remains limited. We tested the hypothesis that hippocampal CACNA1C expression is increased in human and nonhuman primates (NHPs) with chronic alcohol use. Further, we used a mouse conditioned place preference (CPP) paradigm to test the hypothesis that Nifedipine, a CACNA1C-selective L-type calcium channel antagonist, can attenuate ethanol-induced CPP. CACNA1C mRNA expression was increased in the hippocampus of subjects with AUD (p<0.03). Increased densities of CACNA1C neurons (p<0.01) and glia (p<0.02) were observed in rhesus monkeys with chronic alcohol use. Ethanol-treated mice spent more time in the ethanol-paired chamber compared to the vehicle animals (p<0.04), demonstrating ethanol-induced CPP. This effect was attenuated by Nifedipine, as time spent in the ethanol-paired chamber in the ethanol + Nifedipine group was not significantly different from the vehicle group. These findings demonstrate that chronic alcohol use increases CACNA1C expression in the hippocampus across species and that a CACNA1C subtype-selective antagonist reduces ethanol-induced CPP. Together, these results support CACNA1C as a promising therapeutic target for context-induced relapse in AUD.

Differential Effects of Inducible Cerebellar Granule Cell and Purkinje Cell Ablation on Motor Coordination and Motor Learning in Adult Mice
The cerebellum regulates motor coordination and motor learning through highly organized circuits composed mainly of granule cells (GCs) and Purkinje cells (PCs). To investigate their distinct roles, we generated two lines of inducible transgenic mice in which either GCs or PCs could be selectively ablated in adulthood by administration of the progesterone receptor antagonist RU-486. This system combined a Cre recombinase-progesterone receptor fusion, in which Cre activity is induced in an RU-486-dependent manner, with a Cre-dependent diphtheria toxin A expression to achieve cell-type-specific ablation. High-dose RU-486 induced nearly complete loss of either GCs or PCs and resulted in severe ataxia. When partial ablation was induced by low-dose RU-486, different phenotypes emerged. Mice retaining about 20% of PCs were still able to improve motor coordination in the rotarod test and maintained performance in the balance beam test comparable to that of controls. In contrast, mice with about 30% of GCs remaining showed marked deficits, failing to improve across rotarod trials and exhibiting reduced latency to fall in the balance beam test. These results suggest that while both GCs and PCs are indispensable for motor coordination, a sufficient number of GCs is required for both motor coordination and motor learning. This inducible ablation model highlights the differential contributions of cerebellar neurons and provides a valuable tool to dissect circuit-specific functions in the adult brain.

A cortical output channel for perceptual categorization
Perceptual categorization allows the brain to transform diverse sensory inputs into discrete representations that support flexible behavior. Auditory cortex (ACtx) has been implicated in this process, but the cell-type-specific circuits that implement category learning remain unknown. We trained head-fixed mice to categorize the temporal rate of amplitude-modulated noise while performing longitudinal two-photon imaging of layer (L)5 extratelencephalic (L5 ET) neurons alongside comparison populations of L2/3 and L5 intratelencephalic (L5 IT) neurons. With learning, L5 ET neurons underwent pronounced tuning modifications and developed robust, categorical responses, whereas L2/3 and L5 IT neurons did not. This categorical code was task engagement-dependent: it was present during behavior and absent during passive listening in the same neurons on the same day, indicating context-gated expression. Using a generalized linear model to dissociate stimulus- from choice-related signals, we confirmed that categorical selectivity in L5 ET neurons reflected sensory encoding rather than motor confounds. All three populations carried choice signals, but these were strongest in L5 ET neurons, suggesting a role in linking sensory categorization to action selection. These findings identify a projection-specific, deep-layer cortical output channel in which L5 ET neurons acquire categorical representations and selectively propagate behaviorally relevant signals to downstream targets.

WNK-Dependent Phosphorylation of Gephyrin Tunes GABAA Receptors at Inhibitory Synapses and Modulates Anxiety Behavior
The role of the chloride-sensitive kinase WNK1 and its effector SPAK in the brain remains poorly understood. Here, we identify a regulatory mechanism involving WNK signaling that directly controls the synaptic diffusion and clustering, as well as the membrane stability and endocytosis of inhibitory GABAA receptors (GABAARs). We show that activation of WNK signaling stabilizes GABAARs at inhibitory synapses, while inhibition enhances receptor internalization. This regulation depends on the phosphorylation state of two previously uncharacterized residues in the central linker region of the gephyrin scaffold protein. Modulating WNK activity alters neuronal activity and the kinetics of GABAergic currents. In vivo, expression of a phospho-mimetic form of gephyrin at WNK-targeted sites produces anxiolytic effects. By orchestrating the recruitment of GABAARs at inhibitory synapses, the WNK pathway emerges as a master regulator of GABAergic transmission and establishes chloride as a bona fide second messenger in inhibitory synaptic signaling.

Weakening of subcortical and strengthening of cortical visual pathways across early adolescence
Background: Mounting evidence suggests that amygdalar nuclei receive visual information via both a well-characterized cortical pathway through the inferior temporal cortex and a subcortical route through the superior colliculus and pulvinar. This subcortical pathway may facilitate rapid responses to salient visual stimuli and could explain phenomena such as blindsight. However, controversies remain about the organization of the subcortical pathway, its role in visual processing, and how the cortical and subcortical pathways mature across development. Methods: To address these questions we used longitudinal diffusion magnetic resonance imaging (dMRI) data from 4361 participants in the Adolescent Brain Cognitive Development (ABCD) study, reconstructing every major segment of the cortical and subcortical amygdala pathways. We tested the existence of the subcortical pathway against null tractography models, characterized cortical and subcortical pathways development across early adolescence, and investigated their association with visual processing speed. Results: We provide evidence for the existence of bilateral pulvinar-amygdala pathways against a null model (all p < 0.001, corrected). While cortical tracts involving the primary and extrastriate visual cortex and the inferior-temporal cortex strengthened with chronological age and over pubertal development, we demonstrate that subcortical pulvinar-amygdala connectivity decreased over pubertal development. Greater connectivity strength of the right pulvinar-amygdala tract was associated with faster responses on a visual task for both emotional face and place stimuli, a relationship also seen for cortical tracts. Conclusion: This study provides evidence for the existence of pulvinar to amygdala tracts in the largest sample of adolescent participants studied to date. Greater connectivity in both cortical and subcortical tracts were associated with faster reaction time on a visual task, but further work will be needed to investigate the specificity of this association in terms of both task and tract. In line with the hypothesized importance of the subcortical pathway in early development, we show that the developmental trajectories of cortical and subcortical pathways diverge and highlight the influence of pubertal development, with cortical pathways generally strengthening and subcortical pathways weakening across early adolescence.

Rate of functional network maturity and the role of environmental factors
The developmental period from childhood to adolescence is marked by significant changes to the functional properties of the brain that support various aspects of higher-level cognition. Environmental factors such as socioeconomic status and adversity can have an outsized influence on neurocognitive development. However, not all environmental factors have the same influence on cognitive and brain development. In the current study, we examined the differential influences of SES (i.e., parental education and neighbourhood safety) and adversity on the maturation rate of functional networks in children and adolescents. Using resting-state fMRI data, independent component analysis with dual regression was computed to identify six networks (Default Mode Network (DMN), Left Executive Control Network (ECN), Right ECN, Hippocampal (HPC), Salience, and Sensorimotor networks) of interest in children and adolescents aged 7 to 15 (N=216, acquired from the Healthy Brain Network). A neural maturity index was generated based on the degree of similarity between the spatial configuration of the six networks in each youth brain to that of an adult template (1243 total, with a mean age of 26; independently by sex). Regression analyses were used to determine the association between neural maturity, social-cognitive abilities and environmental factors such as parental education, neighbourhood safety and number of negative life events (adversity). We found one sensory (sensorimotor) and two association (default mode and executive control) networks matured faster than other networks. Only the rate of maturity of the DMN and HPC were associated with environmental factors. Maturity of the DMN was associated with less adversity and better social cognitive ability, whereas maturity of the HPC network was associated with younger participants with higher IQs. Moreover, these effects were stronger in females than males. Our results highlight the importance of examining the unique contributions of distinct dimensions of childhood environments on neurocognitive development.

The human factor: development and characterization of a scalable calcium imaging assay using human iPSC-derived neurons
Neuroscience drug discovery is challenged by the brain's structural and cell-type complexity, which is difficult to model in cellular systems compatible with high-throughput screening methods. Calcium oscillation assays, that harness neurons' intrinsic capability to develop functional neural networks in cell culture, are currently the closest cellular models with a relevant functional endpoint to model human neuronal circuitry in a dish. Here we further develop this useful assay towards scalable drug discovery applications. We show the importance of defined neuron-to-astrocyte ratios for optimal cellular distribution and surface adherence in HTS-compatible cell culture vessels and how the cell type ratios determine network firing patterns. We identify DAPT, a molecule previously shown to promote neuronal maturation and synapse formation, as a negative regulator of astrocyte viability. Addition of GABA-ergic inhibitory neurons increases the network spike frequency while reducing network spike amplitudes. We develop a pixel-based analysis for plate reader data to access local field activity in an automated and scalable calcium imaging environment. Using this method, we identify a desynchronization of excitatory neuronal activity by GABA neurons that echoes GABA action in vivo, and dysregulation thereof in pathological conditions.

HuD controls widespread RNA stability to drive neuronal activity-dependent responses
Neuronal activity shapes brain development and refines synaptic connectivity in part through dynamic changes in gene expression. While activity-regulated transcriptional programs have been extensively characterized, the holistic effects of neuronal activity on the full RNA life cycle remain relatively unexplored. Here, we show that neuronal activity influences multiple stages of RNA metabolism in vitro and in vivo. Among these, RNA stability emerges as a previously underappreciated regulator of gene expression, exerting a stronger influence than transcription on total RNA levels for ~10% of activity-dependent genes. We go on to profile 3'UTR mRNA motifs that are sufficient to modulate activity-dependent mRNA stability and employ machine learning to identify the neuronal-specific RNA-binding protein HuD as a key regulator of activity-dependent mRNA stabilization. We demonstrate that HuD shapes activity-dependent mRNA abundance of hundreds of transcripts in both soma and distal neuronal processes and that neuronal activity drives the reorganization of HuD-interacting proteins, thereby stabilizing HuD-bound mRNAs and directing them into translationally active granules. Finally, we find that many variants associated with autism spectrum disorder (ASD) and other neurodevelopmental disorders disrupt or promote aberrant activity-dependent changes in mRNA stability. These findings reveal mRNA stability as a widespread mechanism of stimulus-responsive gene regulation in neurons with direct implications for the understanding of neurodevelopmental disorders.

Compositional neural dynamics during reaching
The complex mechanics of the arm make the neural control of reaching inherently posture dependent. Because previous reaching studies confound reach direction with final posture, it remains unknown how neural population dynamics in the motor cortex account for arm posture. Here we address this gap with high-density neural recordings and a reaching task in which the same targets serve as start points on some trials and end points on others. We show that neural population dynamics in monkey primary motor cortex and dorsal premotor cortex exhibit a compositional structure with three components that enable posture-dependent control: first, a posture subspace containing fixed points visited whenever the arm is in a specific posture; second, rotational dynamics that transition between these fixed points, systematically organized so that similar rotations produce similar movements while continuously updating the posture representation; third, a condition-independent shift dimension that tracks trial progression across all movements. This compositional structure advances the population-level account of how motor cortical dynamics support skilled reaching.

Benefits of Music Training for Learning to Read: Evidence from Cortical Tracking of Speech in Children
Musical training has long been argued to boost early phonological and reading abilities. Cortical tracking of speech (CTS) has been proposed as a mechanism for this music-to-literacy transfer. In this study, we examined how musical training shapes CTS in young readers and whether it facilitates literacy benefits. In a sample of 57 children aged 5-9, musical training was linked to enhanced reading and phonological awareness (PA). EEG during story listening revealed that higher left-hemispheric and lower right-hemispheric CTS were also associated with higher reading scores. However, children with higher musicality exhibited stronger reading skills at lower levels of left-hemispheric CTS, suggesting more adult-like speech analysis. Critically, PA mediated the relationship between musicality and reading: greater musicality was associated with stronger PA, which in turn predicted higher reading performance, independent of demographic and cognitive factors. These findings indicate that musical training supports literacy by enhancing PA and shaping left-lateralized speech processing.

Spatial grouping modulates the link between individual alpha frequency and temporal integration windows in crowding
Previous research has linked endogenous alpha oscillations (~7-13 Hz) to temporal integration windows in visual perception, with higher individual alpha frequency (IAF) predicting improved temporal segregation. Here, we investigated whether alpha-rhythmic temporal integration is a factor in visual crowding and whether this relationship is mediated by spatial grouping mechanisms. 47 participants performed a Vernier discrimination task, in which we manipulated both the stimulus onset asynchrony (SOA) between flankers and targets, and the spatial configuration of the flankers. Specifically, flankers were arranged to either induce crowding or "uncrowding", through the manipulation of good-Gestalt properties. Our results show that crowding has a temporal integration period of around 170 ms but this varies substantially across individuals. Importantly, resting-state IAF predicted individual variance in temporal integration windows: individuals with faster endogenous alpha rhythms could begin to segregate targets from distractors at shorter SOAs. Crucially, this effect was specific for crowding-inducing flankers and disappeared when flankers led to uncrowding. These results suggest that top-down spatial grouping can overwrite the temporal integration constraint imposed by alpha oscillations, highlighting both the relevance of alpha for understanding limits on peripheral visual processing as well as the flexible and context-dependent role of alpha in temporal integration.

Sensory Compression as a Unifying Principle for Action Chunking and Time Coding in the Brain
The brain seamlessly transforms sensory information into precisely-timed movements, enabling us to type familiar words, play musical instruments, or perform complex motor routines with millisecond precision. This process often involves organizing actions into stereotyped "chunks''. Intriguingly, brain regions that are critical for action chunking, such as the dorsolateral striatum (DLS), also exhibit neural dynamics that encode the passage of time. How such brain regions support both task-specific motor habits and task-invariant internal timing, two seemingly distinct functions, remains a fundamental question. Here we show, using recurrent neural network models, that these two functions emerge from a single computational principle: sensory compression, the functional compression of high-dimensional sensory information into a low-dimensional representation. We find that a sensory bottleneck forces the network to develop stable internal dynamics that implicitly encode time, which in turn serve as a scaffold upon which the brain learns action chunks in response to predictable environmental regularities. This mechanism unifies task-invariant time coding and sensory-guided motor timing as two outcomes of the same process of sensory compression, providing a general principle for how the brain mirrors environmental regularities in both internal stable neural trajectories and external consistent motor habits.

Fast spiking interneurons autonomously generate fast gamma oscillations in the medial entorhinal cortex with excitation strength tuning ING-PING transitions
Gamma oscillations (40-140 Hz) play a fundamental role in neural coordination, facilitating communication and cognitive functions in the medial entorhinal cortex (mEC). While previous studies suggest that pyramidal-interneuron network gamma (PING) and interneuron network gamma (ING) mechanisms contribute to these oscillations, the precise role of inhibitory circuits remains unclear. Using optogenetic stimulation and whole-cell electrophysiology in acute mouse brain slices, we examined synaptic input and spike timing in neurons across layer II/III mEC. We found that fast-spiking interneurons exhibited robust gamma-frequency firing, while excitatory neurons engaged in gamma cycle skipping. Stellate and pyramidal cells received minimal recurrent excitation, whereas fast-spiking interneurons received strong excitatory input. Both excitatory neurons and fast-spiking interneurons received gamma frequency inhibition, emphasizing the role of recurrent inhibition in gamma rhythm generation. Notably, gamma activity persisted after AMPA/kainate receptor blockade, indicating that interneurons can sustain gamma oscillations independently through an ING mechanism. Selective activation of PV+ interneurons confirmed their ability to sustain fast gamma inhibition autonomously. To further assess the interplay of excitation and inhibition, we developed computational network models constrained by our experimental data. Simulations revealed that weak excitatory input to interneurons supports fast ING-dominated rhythms (~100-140 Hz), while strengthening excitatory drive induces a transition to slower PING-dominated oscillations (60-80 Hz). These findings highlight the dominant role of inhibitory circuits in sustaining gamma rhythms, demonstrate how excitation strength tunes the oscillatory regime, and refine models of entorhinal gamma oscillations critical for spatial memory processing.

Knockout of Perilipin 2 in Microglia Alters Lipid Droplet Accumulation and Response to Alzheimer's Disease Stimuli
Lipid droplets (LDs) are emerging as critical regulators of cellular metabolism and inflammation, with their accumulation in microglia linked to aging and neurodegeneration. Perilipin 2 (Plin2) is a ubiquitously expressed LD associated protein that stabilizes lipid stores, and in peripheral tissues, its upregulation promotes lipid retention, inflammation, and metabolic dysfunction. However, the role of Plin2 in brain-resident microglia remains undefined. Here, we used CRISPR engineered Plin2 knockout (KO) BV2 microglia to investigate the contribution of Plin2 to lipid accumulation, bioenergetics, and immune function. Compared to wild-type (WT) cells, Plin2 KO microglia exhibited markedly reduced LD burden under both basal and oleic acid loaded conditions. Functionally, this was associated with enhanced phagocytosis of zymosan particles, even after lipid loading, indicating improved clearance capacity in the absence of Plin2. Transcriptomic analyses revealed genotype specific responses to amyloid beta (AB), particularly in pathways related to mitochondrial metabolism. Seahorse assays confirmed that Plin2 KO cells exhibit a distinct bioenergetic profile, characterized by reduced basal respiration and glycolysis, yet preserved mitochondrial capacity, increased spare respiratory reserve, and a blunted glycolytic response to AB. Together, these findings identify Plin2 as a regulator of microglial lipid storage and metabolic state, with its loss alleviating lipid accumulation, improving phagocytic function, and altering AB induced metabolic reprogramming. Targeting Plin2 may therefore represent a potential strategy to modulate microglial metabolism and function in aging and neurodegeneration.

DRP1 induces neuroinflammation via transcriptional regulation of NF-ĸB
Neuroinflammation is a major pathogenic mechanism in neurodegenerative diseases. Understanding the regulation of neuroinflammation is critical to therapeutic development. We report here that dynamin related protein 1 (DRP1), well-recognized for its role in mitochondrial fission, is a transcription factor that regulates neuroinflammation. Using multiple inflammatory models, we provide evidence demonstrating that DRP1, when challenged with pro-inflammatory lipopolysaccharides, translocates from the cytosol to the nucleus, then binds to the promoter region of Rela (encoding NF-{kappa}B) to activate its gene products and other downstream inflammatory cytokines. Our data also demonstrate the significant role of the proinflammatory lipocalin 2 in the brain. In combination, this study highlights a previously unidentified function of DRP1 in mediating neuroinflammation via the NF-{kappa}B-lipocalin 2 axis. Through such mechanisms of DRP1, this study also provides potential therapeutic targets for neurodegenerative diseases and other conditions linked to inflammation.