bioRxiv Subject Collection: Neuroscience's Journal

Torsional Force by Helical Pericytes Regulates Blood Flow in Downstream Capillaries
This study investigates the contractile properties of downstream capillaries, which are traditionally regarded as passive conduits, and addresses ongoing debates surrounding blood flow regulation. It is widely accepted that these capillaries passively dilate in response to increased blood flow in upstream microvessels, and that the helical pericytes located on them lack contractile capability, largely due to the absence of detectable a-SMA expression. Challenging this prevailing view, we demonstrate that downstream capillary pericytes do express both a-SMA and Myosin11, as shown using in situ hybridization on whole-mount intact retinas -unlike prior studies that relied on dissociated cells. Furthermore, Forster resonance energy transfer (FRET) analysis reveals that a-SMA and Myosin11 are in sufficiently close proximity to permit actomyosin bridge cycling, a process essential for contraction. We also show that pericyte contraction can be inhibited by disrupting this molecular interaction. Distinct from the nodal constrictions caused by circular pericyte processes in upstream microvessels, we identify torsional contractions in the downstream capillaries in the retina of living mice using two-photon laser scanning microscopy (TPLSM), which regulates blood flow. These contractions provide direct evidence that downstream capillaries actively contribute to blood flow regulation. Notably, such contractions were overlooked in previous TPLSM studies that monitored only luminal diameter, unless the specialized analytical techniques we employed were applied. Our 3D modeling confirms that these torsional contractions correlate with the helical morphology of pericytes in downstream capillaries -a structure previously thought incapable of producing significant constrictive force. In conclusion, our findings provide direct evidence that downstream pericytes play an active role in regulating blood flow. They highlight a previously unrecognized mechanism -torsional contraction- that aligns with the helical structure of these pericytes and contributes to flow regulation in small-caliber capillaries located nearest to regions of high oxygen demand.

Early Diabetic Ca2+ Handling Impairments in the Rod Bipolar Pathway
Background: Previous work showed that electrically-evoked inhibition to Rod Bipolar Cells (RBC) is reduced in a mouse model of early diabetes. It is hypothesized that this is due to impaired Ca2+ handling in the presynaptic amacrine cell, either through increased Ca2+ buffering or decreased influx. To test this hypothesis and develop a mechanism for this effect, a model where direct optogenetic activation of inhibitory amacrine cells that expressed the light-activated channel ChR2 was used to isolate amacrine cell inputs to RBCs. Application of selective Ca2+ channel blockers could then assess potential locations of amacrine Ca2+ disruption. Using whole cell patch clamp electrophysiology, recordings were made from a 6 week diabetic population (DM) and vehicle injected non-DM animals. Results: Robust GABA C receptor inhibitory currents were recorded from RBCs after ChR2 stimulus that were significantly diminished by the application of nifedipine to block L-typeCa2+ channels in both DM and non-DM conditions. There were significant differences in the peak amplitude of these responses between DM and non- DM groups (p = 0.0146). However, in the non-DM group the decay tau of the response to the 50ms stimulus was significantly diminished by nifedipine ( p =0.0498, n = 5), but this was not seen in the DM group ( p = 0.9498, n=7). A 1s nifedipine-reduced response saw its decay tau increase in the DM group but not the non-DM. Ca2+ - induced Ca2+ release (CICR) blockade with ryanodine decreased responsivity equally between groups in the 1s stimulus but showed no significant kinetic changes. CICR blockade for a 50ms stimulus response showed significant kinetic changes in diabetes but otherwise reduced the response equally between DM and non-DM. Blockade of the mitochondrial Ca2+ uniporter (MCU) had little effect on the optogenetic response. Conclusion: This study presents evidence that diabetes alters amacrine cell output to the RBC unmasked through blockade of the L-type calcium channel, and the Endoplasmic Reticulum (ER). An apparent explanation for our results is that DM calcium buffering is dysregulated, leading to prolonged responses. The underlying mechanism for this alteration is complex and not yet clearly elucidated.

The emergence of intentional action in early human infancy
Intention lies at the core of human behavior, allowing flexible and goal-directed interaction with the environment. However, how it emerges in infancy remains poorly understood. Here we show that intentional action can emerge before overt reaching, revealing that 3-month-old infants display pupil dilation preceding self-initiated actions during interaction with a suspended mobile toy. This phasic pupil dilation first appeared after movement onset but gradually shifted to precede it through environmental interaction, reflecting the emergence of action preparation and prediction of action outcomes. This shift also implies that infants are capable of anticipating the timing of their forthcoming movements. Furthermore, the antecedent pupil dilation occurred only with efficient movements characterised by reduced muscle co-contractions. These findings suggest that infants learn to predict outcomes of their own actions and initiate particular movements at self-determined timings, marking the emergence of intentional action.

Dissociating mechanisms of heart-voice coupling
In a landmark paper in JASA, Orlikoff & Baken (1989a) demonstrated that the heartbeat echoes through acoustic features of the human voice. Heart-voice coupling questions how vocal control is functionally regulated under these perturbations. It has many potential engineering applications that extract heart rate from human voice and speech, with underutilized potential for cardiac monitoring from (wild) animal calls. However, laboratory phonology and bioacoustics seem to have forgotten about the phenomenon, such that the physiological underpinnings of heart-voice coupling have been left unexplained. In the current contribution, we show that vocal acoustics contain signatures of the heartbeat as originally reported by Orlikoff & Baken (1989a). More specifically, we analysed the steady-state vowel vocalizations of 17 participants and found a significant relationship between the vocal amplitude envelope and the cardiac activity (ECG). Furthermore, we conducted two empirical tests to dissociate between glottal (vocal fold vascularization) and subglottal (heart-lung interaction) mechanisms of heart-voice coupling. In addition to a stable heart-voice coupling, we also found a significant heart-expiration coupling during conditions of pure expiration. Since the vocal folds are abducted during expiration, this result demonstrates that subglottal mechanisms that modulate expiratory flow must be involved in heart-voice coupling. We argue that the heart rhythm needs to be considered as a weakly coupled pattern generator that possibly shaped the evolution of vocal and speech production.

Distinct functional roles across hippocampal subfields in anticipation to event boundaries during schema learning
Continuous experiences are constantly segmented into separate events in memory, which is accompanied by increased hippocampal activity at the boundaries between these events. Extracting knowledge across all these experiences about what type of events to expect in a certain environment leads to event schemas being represented in the brain. These event schemas help to predict future events. What are the differences across the various distinct hippocampal subfields in peaks of activity around event boundaries? And what is the influence of event schema learning on these hippocampal subfield peaks? Here, this was investigated using data from an fMRI experiment in which participants learned two (related) event schemas by exposure to many similar animated videos of wedding ceremonies in a novel fictional culture. Results revealed that the hippocampus (CA1, CA2/3, dentate gyrus and subiculum) showed peaks in activity before event boundaries. Interestingly, the CA1, CA2/3 and dentate gyrus pre-boundary peaks attenuated due to event schema learning. This provides evidence for these subfields signaling uncertainty about upcoming events in the seconds prior to event boundaries. These results give a more precise understanding about the temporal dynamics around event boundaries in the distinct hippocampal subfields, and how these hippocampal subfields' responses interact with schema learning.

Neuronal excitability is permanently altered by activity manipulation during an embryonic critical period in Drosophila
Neuronal intrinsic excitability provides the baseline that homeostatic mechanisms act to preserve, yet the processes that establish a baseline remain poorly defined. Developmental critical periods (CPs) are thought to play a central role, but the link between early activity and long-term intrinsic properties is not well characterised. To address this, we used the genetic tractability of the Drosophila larval locomotor circuit to manipulate individual neurons during an embryonic CP. Following optogenetic excitation or inhibition, during the CP, we assessed intrinsic excitability of the same neurons in third-instar larvae (i.e. ~5 days thereafter). We compared an excitatory premotor interneuron (A27h), an inhibitory premotor interneuron (A31k), and a motor neuron (aCC). Both interneurons exhibited anti-homeostatic responses: excitatory perturbation increased intrinsic excitability, while inhibitory perturbation decreased it, effects that persisted throughout larval development. In contrast, motor neurons showed no significant changes under the same conditions, revealing cell type-specific sensitivity to early activity. These findings build on the general principles of the functional relationships between CP activity and neuronal excitability and how intrinsic excitability is not passively set but actively shaped during these windows, with long-lasting, neuron-specific consequences. More broadly, our results highlight how developmental perturbations can alter the excitatory:inhibitory balance of mature neural circuits that may contribute to the aetiology of neurodevelopmental disorders.

MOSAIC: A scalable framework for fMRI dataset aggregation and modeling of human vision
Recent large-scale vision fMRI datasets have been invaluable resources to the vision neuroscience community for their deep sampling of individual subjects and diverse stimulus sets. However, practical limitations to the number of subjects, stimuli, and trials that can be collected prevent individual fMRI datasets from reaching the scale necessary for modern modeling approaches and robust conclusions. Here, we introduce MOSAIC (Meta-Organized Stimuli And fMRI Imaging data for Computational modeling), a fMRI dataset aggregation framework designed to leverage the richness of individual datasets for computationally intensive modeling and robust tests of generalization. MOSAIC is composed of eight large-scale vision fMRI datasets totaling 93 subjects, 430,007 fMRI-stimulus pairs, and 162,839 naturalistic and artificial stimuli. A shared fMRI preprocessing pipeline and a filtered test-train split minimizes dataset-specific confounds and test-set leakage when aggregating the datasets. Crucially, additional datasets can be integrated into MOSAIC post hoc, allowing MOSAIC to evolve according to the community's interests. We use MOSAIC to show that perceptually diverse stimulus sets consistently improve decoding accuracy and stability, carrying implications for future fMRI stimulus set design. We then jointly train brain-optimized encoding models across subjects and datasets to predict fMRI activity of all visual cortex and even the whole brain. In silico functional localizer experiments performed on these digital twin models can recover subject-specific category-selective cortical regions, thereby validating our approach. Together, MOSAIC provides a scalable and community-driven solution to build robust, larger-scale models of human vision.

Hypothesis Testing Governs an Efficiency-Flexibility Trade-off in Strategic Motor Learning
It remains unknown how people discover an effective movement strategy when the environment changes (e.g., when adapting to a new computer trackpad). We propose that strategic adaptation operates through hypothesis testing: learners generate candidate hypotheses, discard those inconsistent with feedback, and iteratively refine their actions through practice. A core prediction of this account is an efficiency-flexibility trade-off. In constrained environments, where few hypotheses are viable, learning slows as people eliminate competing hypotheses but supports broader generalization. In unconstrained environments, where many hypotheses are viable, learning accelerates as learners adopt one of many expedient hypotheses but yields poorer generalization. In two reaching experiments (N = 560), we varied the arrangement of target positions to manipulate how tightly the hypothesis space was constrained. As predicted, the constrained group learned more slowly but generalized more - an efficiency-flexibility trade-off that highlights hypothesis testing as a novel process governing human motor learning.

The Human Hippocampus Is Involved in Auditory Short-Term Memory Trace Formation
The human hippocampus has been claimed to play an important role in long-term memory, or episodic memory. It has long been argued whether the hippocampus does contribute to short-term memory. Here, we demonstrate human hippocampus has been involved in auditory short-term memory (ASTM) trace formation. We used a classic oddball paradigm and intracranial recordings across various human brain areas. High frequency activities evoked by deviant stimuli in subjects indicate the generation of ASTM trace. Auditory response latencies recorded from the hippocampus, insula, temporal lobe, parietal lobe, and frontal lobe showed the early processing of ASTM trace at a pre-attention stage. Moreover, Granger causality analyses showed ASTM trace is processed in hierarchical cortical areas and interactions among this memory process have been clarified. Specifically, bottom-up auditory signals processed by the insula, and auditory regions of the temporal lobe and the frontal lobe at an early stage, then auditory top-down information transmitted from the frontal lobe to the hippocampus, the inferior temporal lobe, as well as insula at a relatively late pre-attention stage of the auditory information processing. These results provide evidence that the human hippocampus contributes to the short-term memory of auditory perception at a pre-attention stage and suggests a challenge to entrenched beliefs in the classification/definition of memory systems.

Informational and Normative Influence on Conformity in Autism
This preregistered study examined whether adults with autism spectrum disorder (ASD) show reduced social conformity and whether any such reduction depends on the type of social influence. Social conformity, defined as the tendency to adjust judgments to align with those of others, is typically driven by normative (acceptance-seeking) and informational (accuracy-seeking) motives. Thirty adults with ASD and 30 matched neurotypical (NT) adults completed two tasks: a preference rating task indexing normative influence, and a dot-counting task indexing informational influence with monetary rewards. Contrary to our predictions, adults with ASD conformed as much as NT adults in the preference rating task but showed significantly reduced conformity in the dot-counting task. Exploratory analyses indicated that this reduction was driven by a distinct subgroup of nine adults with ASD who never revised their initial estimates despite informative social cues, resulting in poorer accuracy and lower rewards. When this subgroup was excluded, group differences in conformity were no longer evident. These findings suggest that, overall, adults with ASD are as susceptible as NT adults to normative influence but less responsive to informational influence, highlighting the importance of distinguishing between types of social influence and considering individual differences when characterizing social behavior in ASD.

DENDRO: Recovery and denoising of whole-tree dendritic voltagefrom 2D voltage movies
The ability to image voltage at high spatiotemporal resolution across an entire dendritic tree would represent a major advance in systems and circuit neuroscience. Recent advances in genetically encoded voltage indicators (GEVIs) have brought this possibility closer to reality. However, due to fundamental tradeoffs between imaging speed, resolution, SNR, and volume, this goal has remained out of reach. Here we develop a computational method that fuses 3D anatomical information with 2D voltage video data, yielding full time-varying 3D voltage estimates. Our method, termed DENDRO, comprises two steps. In step one, we use the anatomical data to build a microscope model which maps from voltages along the tree to observed fluorescence at the imaging plane. By exploiting local spatial smoothness of the voltage signal, we parameterize the voltage signal using a set of local basis functions, which reduces the dimensionality of the problem and allows us to approximately invert the microscope model. This step leverages spatial but not temporal smoothness of the underlying signal and yields noisy 3D estimates. In step two, we train a lightweight self-supervised neural network to perform spatiotemporal denoising of the inferred voltages. On simulated data, we find that DENDRO is able to recover voltages at high accuracy across an entire dendritic tree. On real voltage movies from hippocampal slices, DENDRO recovers known dendritic phenomena at single trial resolution and millisecond time-scales, and allows visualization of backpropagating action potentials in 3D.

Modeling Hierarchical Brain Dynamics Outperforms Hormonal Biomarkers in Predicting Menstrual Cycle Phases
Hormonal fluctuations across the menstrual cycle influence large-scale brain dynamics, yet the underlying neurobiological mechanisms remain poorly understood. In this study, 60 naturally cycling women were scanned using resting-state fMRI during the early follicular, pre-ovulatory, and mid-luteal phases. We then applied a thermodynamics-inspired framework to explore the functional hierarchical organization of whole-brain dynamics across these phases. First, we found that brain dynamics are significantly modulated by estradiol, progesterone, and age across multiple resting-state networks. Second, to elucidate underlying mechanisms, we estimated generative effective connectivity (GEC) matrices using whole-brain models and trained support vector machine classifiers to predict menstrual phases. These model-based biomarkers outperformed traditional functional connectivity and hormone measures in classifying menstrual cycle phases. These findings reveal that menstrual cycle-related changes modulate the hierarchical reorganization of brain dynamics, highlighting the potential of model-based approaches to advance women's brain health research.

Category learning disentangles representation of trial events in hippocampus CA1
The hippocampus (HC) is known to encode task-relevant variables, including latent ones, capturing and parsing critical information from sequences into episodes. This is thought to be the basis to form a cognitive map of possible abstract states beyond mere perceptual details, akin a state machine, with predictive value, contributing to an internal world model. However, its specific role in categorization tasks in general, where learning a category may depend on independent, unordered (non sequential) experienced examples, remains unclear. Here, we investigate CA1 population coding during a categorization paradigm in mice in combination with calcium imaging at different stages of category training with interleaved generalization tests. Our results reveal that hippocampal coding changes critically throughout the different stages of categorization training. Specifically, the disentangling of choice and outcome variables emerges as factorized, abstract, representations and fundamentally distinguishes simple discrimination from categorization training. Furthermore, this factorized geometry relates to improved behavioral performance. Our findings suggest that trial encoding on HC adapts in response to the category structure presented by representing critical events into the appropriate computational format to support generalization of category membership.

Elucidating an anterior cingulate circuit for self-initiated actions and rescue of Parkinsonian akinesia
Dopamine (DA) depletion is known to result in Parkinsonian symptoms such as the inability to initiate movements (akinesia). While Parkinsonian akinesia is traditionally associated with reduced DA signaling in the striatum, the contribution of cortical regions that also receive DA projections and project to the striatum remains unclear. Here, we identify a previously unexplored cortical circuit involving D1-like dopamine receptor-expressing neurons in the anterior cingulate cortex (ACC) that is critical for initiating goal-directed movements. We find that a selective activation of ACC-D1+ neurons can flexibly drive targeted movement and locomotion even in akinetic mice after dopamine depletion or receptor antagonism. These findings uncover a cortical mechanism for movement initiation and offer promising new therapeutic targets for treating Parkinsonian akinesia.

Biochemical Regulation of Brain Kynurenic Acid Synthesis and Inhibition in Rats is Sensitive to the Time of Day
Neurochemical imbalances, including elevations of the tryptophan metabolite kynurenic acid (KYNA), an endogenous antagonist of glutamatergic and cholinergic receptors, are linked to cognitive and sleep disturbances in psychiatric and neurocognitive disorders. Therapeutic strategies to reduce brain KYNA by inhibiting kynurenine aminotransferase II (KAT II) are under investigation. However, few studies consider time as a biological variable, despite recent evidence that the time of day can affect brain metabolism and drug effectiveness. Therefore, we explore the hypothesis that KYNA formation and synthesis inhibition change throughout the day. Using rats of both sexes, we measured basal KYNA levels and the effects of kynurenine (100 mg/kg, i.p.), to stimulate de novo KYNA, and/or PF-04859989 (KAT II inhibitor, 30 mg/kg, s.c.), at the beginning of light or dark phases. Microdialysis was used to assess extracellular KYNA in the dorsal hippocampus, and ex vivo assays evaluated KAT enzyme activity in separate animals. Additionally, we examined KYNA levels and the effects of PF-04859989 during acute sleep deprivation in male rats. PF-04859989 significantly decreased stimulated KYNA synthesis in both sexes and basal KYNA in males. In the dark phase, compared with the light phase, male rats treated with kynurenine and/or PF-04859989 showed higher KYNA levels and greater KYNA synthesis inhibition, respectively. In vitro, KAT II activity was similarly higher, and PF-04859989 was more effective, in the dark than in the light phase. Sleep deprivation increased extracellular KYNA levels, and PF-04859989 prevented this increase. Overall, our findings highlight the need to consider time-dependent factors when developing therapies impacting KYNA synthesis.

Reward Modality and Task Complexity jointly shape learning Dynamics in mice
Learning efficacy relies on balancing reward-driven motivation with the difficulty of reaching a goal. Disentangling these factors is essential for understanding learning dynamics. Spatial navigation is a standard paradigm for probing these processes in rodents, but variation in mazes, cues, and protocols makes it difficult to determine how reward properties, rather than task design, shape learning and exploration-exploitation strategies. To date, no studies have directly compared reward type and learning difficulty within a single behavioral context. Here, we used the Starmaze to examine how reward modality and task complexity jointly influence learning and the transition from exploration to exploitation. We created three versions of the same sequential egocentric task in which a fixed goal was rewarded with either a hidden escape platform (aquatic version), food pellets, or medial forebrain bundle stimulation. Task difficulty varied by increasing the number of required decision points from zero to three. Our results reveal distinct behavioral dynamics across reward modalities and provide a unified framework for studying reward-dependent learning in sequential navigation.

The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrPC Levels and Prion Replication
Prions are misfolded, self-propagating versions of the cellular prion protein (PrPC) that cause invariably fatal transmissible neurodegenerative diseases in humans and animals. Little is known about how prions replicate in the brain, including whether other proteins participate in prion replication in vivo. Several members of the protein disulfide isomerase family have been shown to reside in close spatial proximity to PrPC in cells and mice, implying that they could be involved in prion biogenesis. Here, we show that stable knock-down of the protein disulfide isomerase P4HB (also called PDIA1) in prion-susceptible CAD5 cells reduces PrPC levels and results in lower levels of protease-resistant PrP (PrPres), a marker of infectious prions, following infection with two different prion strains. Partial reduction of P4HB activity using the P4HB-selective inhibitor KSC-34 also decreases PrPC levels in uninfected CAD5 cells whereas treatment of prion-infected CAD5 cells with KSC-34 results in higher levels of PrPres. A proportion of P4HB reaches the cell surface where PrPC is located, and a secreted P4HB variant increases PrPres levels in cells. Collectively, these results suggest that P4HB facilitates prion replication in cells by stabilizing PrPC and potentially acting as a chaperone that directly modulates prion conversion. Thus, targeting P4HB during prion disease may have therapeutic benefit.

Directed cortico-limbic dialogue in the human brain
How can one trace the brain's orderly directed signals amid a tangle of nerve fibers? Because direct access to actual brain signaling is rare in humans, the precise wiring diagrams for cortico-limbic communication during sleep and wake remain essentially unmapped, hampering progress in neuroscience. Now, a unique neurosurgical window on the human brain allows for electrically mapping cortical connections at the hospital, but studies so far have relied on average signals, masking the dynamic nature of signal flow across brain regions. To causally estimate signaling dynamics, we repeatedly probed cortico-limbic networks with short-lived electrical pulses over days and assessed the variable fate of each transmitted signal on a single-trial basis. In the resulting openly available dataset, we characterized signaling probabilities and directionality across thousands local and long-range cortico-limbic connections over days. Challenging established views, we found that limbic structures send twice as many signals as they receive, in both wakefulness and sleep. Our findings provide a fundamental framework for causally interpreting signal flow in the brain and formulating therapeutic strategies for brain networks disorders.

Neonatal morphine and HIV synergy induce persistent neuroimmune and anxiety-related transcriptional states
Background: Early life opioid exposure can disrupt neurodevelopment and heighten vulnerability to anxiety and affective disorders, particularly in individuals with HIV. Methods: Using single-cell RNA sequencing (scRNA-seq), we profiled adolescent brains from wild-type and HIV1 transgenic (Tg26) mice exposed to morphine during postnatal days 2 to 7. Results: Morphine exposure in Tg26 mice resulted in a highly dysregulated microglial phenotype, characterized by the ectopic upregulation of genes encoding neuronpeptides (Avp, Hcrt, and Pmch), while simultaneously showing a reduction in both inflammatory and homeostatic markers (Map3k6, Lgals3, Ccl3). Microglia also showed enhanced expression of dynorphin (Pdyn) and {kappa}-opioid receptor (Oprk1) signaling modules implicated in dysphoria and stress-induced negative effects. Furthermore, transcriptomic mapping revealed cell-type-specific neuronal adaptations: cholinergic neurons upregulated genes linked to anxiety and arousal (Avp, Oxt), GABAergic neurons upregulated genes linked to condition and aversive behavior, whereas glutamatergic neurons enriched for transcripts associated with thigmotaxis and fear behaviors. Conclusions: Together, these findings demonstrate that brief neonatal morphine exposure in an HIV-inflamed milieu induces persistent, cell-type-specific neuroimmune and neurotransmitter reprogramming that engages the dynorphin KOR pathway and predisposes to anxiety- and aversion-related behaviors.

Sex differences define the molecular and cellular phenotypes of pain resolution in dorsal root ganglia
The dorsal root ganglion (DRG), a key site for the initiation and maintenance of neuropathic pain, was examined for sex-dependent phenotypes in sensory neurons, satellite glial cells (SGCs), and local macrophages following traumatic nerve injury and during natural pain resolution. Systematic analysis of 7,495 DRG immunofluorescence images and 62 transcriptomes revealed pronounced sex-specific, multicellular DRG phenotypes, especially during pain resolution. System parameters, including tissue size and neuron density also showed sex-dependent differences. Neuropathic pain resolved without tissue or sensory neuron loss. After injury, macrophages invaded the space between sensory neurons and satellite glial cells (SGCs); this was partially reversed during pain resolution, particularly in males. In females, immune-related gene expression and macrophage phenotypes persisted longer, while SGC activation and contact to sensory neurons was more persistent in males. During resolution, synaptic and excitability-related processes were pronounced in both sexes. However, while injury responses were largely shared between sexes, the resolution phase displayed distinctly sex-specific molecular and cellular signatures.

Dual-targeting snRNA gene therapy rescues STMN2 and UNC13A splicing in TDP-43 proteinopathies
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder caused by the selective deterioration of motor neurons in the central nervous system (CNS). A key driver of this pathogenesis is nuclear loss of ALS-associated protein TDP-43, leading to mis-splicing of TDP-43 targets including important neuronal genes STMN2 and UNC13A. Here, we have developed a gene therapy strategy for ALS and related TDP-43 proteinopathies, to correct mis-splicing of both STMN2 and UNC13A cryptic exons using small nuclear RNAs (snRNAs) encoded from a single vector. We identified promoter sequence elements to increase therapeutic snRNA expression by 10-fold, then further optimized the expression cassette with combinatorial snRNA targeting to rescue multiple cryptic splicing targets. The engineered snRNAs restored normal pre-mRNA processing of both STMN2 and UNC13A transcripts despite TDP-43 loss of function, rescuing stathmin-2 protein levels in iPSC derived motor neurons, restoring their axonal regeneration capacity to wild-type levels. In addition, adeno-associated virus (AAV) delivery of the snRNAs to the murine central nervous system in the constitutive cryptic splicing model Stmn2Hum{Delta}GU fully restored cortical Stmn2 pre-mRNA processing, highlighting the utility of snRNAs as a therapeutic modality in vivo. Together, this study demonstrates that snRNAs are a promising and versatile therapeutic strategy for the simultaneous correction of multiple aberrant transcripts affected by cryptic splicing in TDP-43 proteinopathies.

Human Mediodorsal Thalamus in Seizure Propagation
Background: How different thalamic sites are recruited during seizure propagation remains poorly understood. Simultaneous recordings from multiple thalamic sites in patients with focal seizures provide a rare opportunity to investigate the spatiotemporal pattern of thalamic involvement during human epilepsy. Objective: To characterize the recruitment patterns of mediodorsal (MD) thalamic subregion during seizures and their generalization to the contralateral hemisphere. Methods: We analyzed 119 seizures from 23 patients (12 male, age range: 20-57y) undergoing multisite thalamic recordings. In accordance with current clinical standards, we determined the spatial and temporal features of thalamic seizure activity by visually reviewing intracranial EEG recordings from different seizure types in each individual patient. Results: The procedure of multisite thalamic recordings had no complications. In total, we captured seizures originating from temporal lobes (63%), orbitofrontal (11%), frontotemporal (8%), occipital (8%), lateral frontal (4%), parietal (3%), and cingulate (2%) regions. Seizures were focal (76% in 21 patients), focal-to-bilateral tonic-clonic (FBTC, 9% in nine patients), or only electrographic (15% in six patients). Thalamic engagement was seen in 100% of patients occurring typically early during seizure evolution (83% within 15 seconds of seizure onset). Majority of FBTC seizures (73%) had faster thalamic recruitment, often within the first 5 seconds. The pulvinar (PLV) subregion was the most common first-activated thalamic site, particularly in temporal lobe seizures. Although the MD was involved in most seizures (88.2%), it was rarely the initial or sole thalamic structure engaged and more often followed anterior (ANT) and/or PLV sites. Contralateral propagation occurred in 66% of seizures and was strongly linked to MD involvement: the ipsilateral and contralateral MDs were engaged in about 95% of these cases. When ipsilateral MD engagement was absent, contralateral spread of seizures was uncommon. In majority of seizures (60%) that generalized to the contralateral hemisphere, the ipsilateral MD was involved before or simultaneously with the contralateral cortical sites. Importantly, seizures that first activated the MD originated mainly from the medial temporal lobes, whereas those spreading primarily to the contralateral cortex were mostly neocortical in onset. Conclusions: The thalamic MD subregion was often involved after the other thalamic sites, but the MD sites, along with the massa intermedia connecting the two thalami, were significantly involved when seizures spread to contralateral hemisphere. Our findings suggest that a single thalamic lead capturing both MD subregions may yield important clinical information about laterality, origin, and generalization of seizures.

Cognitive cartography of mammalian brains using meta-analysis of AI experts
The complexity of the brain is increasingly mirrored by the complexity of the neuroscientific literature, yet no individual mind can fully grasp the diversity of scales, methodologies and model organisms. Where human experts flag, the latest AI models excel: large language models can seamlessly integrate knowledge across scientific domains. Here we show how large language models can systematically and quantitatively synthesise literature-wide neuroscientific knowledge about the cognitive operations and dysfunctions associated with each brain region. Meta-analysis of AI experts reveals structurefunction mappings to which existing meta-analytic frameworks are blind, demonstrated by lesions and direct intracranial stimulation. It also unlocks the possibility of extending quantitative literature metaanalysis and decoding of brain maps to other model organisms beyond human. As proof of concept, we integrate LLM meta-analysis with species-specific transcriptomics in human, macaque, and mouse, to discover an evolutionarily conserved molecular circuit for cognition. Altogether, meta-analysis of AI experts can fundamentally catalyze neuroscientific discovery by overcoming the barrier of data aggregation from heterogeneous studies, finally bringing together a scattered literature to identify emergent patterns and latent insights across disparate subfields, modalities, and species.

GABA co-release guides the functional maturation of glycinergicsynapses in an auditory sound localization circuit.
In the mammalian brainstem and spinal cord, glycine is the primary inhibitory neurotransmitter. However, during development, many glycinergic neurons also co-release the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Although the acute effects of GABA co-release on immature synaptic transmission have been increasingly characterized, its role in synapse maturation and circuit formation remains poorly understood. Here, we investigated the developmental roles of GABA co-release at glycinergic synapses from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO), an auditory pathway essential for binaural integration and sound localization. During the first two postnatal weeks, MNTB-LSO synapses co-release GABA and undergo pronounced synaptic and circuit refinement. Using conditional knockout mice with severely diminished GABA co-release from MNTB neurons, we found that key aspects of circuit refinement, including synaptic silencing and strengthening, occurred normally. However, a disruption of GABA co-release resulted in significantly larger quantal amplitudes and a reduced readily releasable vesicle pool, impairing the high fidelity and temporal precision of synaptic transmission, which are essential for accurate binaural processing. These results reveal a critical developmental role for GABA co-release in shaping the functional synaptic architecture of glycinergic synapses involved in sound localization.

Significance StatementGlycinergic neurons in the brainstem and spinal cord often co-release GABA during development, but the role of this co-release in circuit refinement or synapse maturation remains poorly understood. This study found that disruption of developmental GABA co-release at the MNTB-LSO synapse, a key part of the sound localization circuit, did not affect topographic refinement by synapse elimination or strengthening. However, impaired GABA co-release prevented synapses from developing the characteristic features that enable the high-fidelity, temporally accurate transmission required for sound localization. This highlights a critical role for developmental GABA co-release in shaping the specialized functional architecture of glycinergic synapses critical for binaural processing and sound localization.

Model-based separation of visual and oculomotor signals in marmoset lateral intraparietal area
The lateral intraparietal area (LIP) integrates visual information with task demands to guide saccadic eye movements. Most prior studies have relied on imposed prolonged delays to separate sensory and motor activity, whereas natural behavior involves rapid sensory-motor transformations. To examine LIP function during more naturalistic oculomotor behavior, we recorded multi-unit activity from LIP in two marmosets performing a task requiring rapid selection of salient visual stimuli. Because sensory and motor signals temporally overlap in such fast tasks, we used a Generalized Linear Model (GLM) that separately captured visually driven and oculomotor-related components. The model explained substantial neural variance during correct and missed saccades, as well as spontaneous eye movements, and revealed heterogeneous sensory and motor encoding across recorded neural units. Our approach demonstrated that the origins of neural activity can be segregated without relying on imposed delays, and showed the presence of a strong post-saccadic motor signal in LIP.

Top-down control of sustained attention by the medial prefrontal cortex (mPFC)- locus coeruleus (LC) circuit during the rodent continuous performance task (rCPT)
The medial prefrontal cortex (mPFC) plays a pivotal role in attention by exerting top-down control to allocate cognitive resources toward behaviorally relevant stimuli based on learned context and expectations. mPFC neurons project to multiple cortical and subcortical regions, including the locus coeruleus (LC), the brain's primary source of norepinephrine (NE). The mPFC also receives inputs from the LC, which release NE to modulate mPFC neuronal activity and downstream cellular signaling. While enhanced functional connectivity between the mPFC and LC in mice during sustained attention tasks suggest an important role for the mPFC-LC circuit, functional evidence directly implicating this circuit in attention is lacking. Here, we investigated the role of the mPFC-LC circuit in attention by comparing selective chemogenetic manipulation of mPFC neurons that project to the LC (mPFC-LC projectors) to non-specific chemogenetic manipulation of mPFC neurons. Selective activation of mPFC-LC projectors in mice performing the rodent continuous performance test (rCPT), a translational sustained attention task, robustly improves attentional performance by enhancing discrimination while non-selective activation of mPFC neurons increases attentional performance by increasing responsiveness and impulsivity. Behavioral effects of mPFC-LC projector activation were mediated by recruitment of a microcircuit involving LC-NE neurons and glutamate and GABA peri-LC neurons. while effects of non-selective activation of mPFC neurons were mediated by engaging downstream targets such as the nucleus accumbens (NAc) as well as the LC/peri-LC region.

Advancing High-Resolution 7T Diffusion MRI: Evaluating Phase-Encoding Correction Strategies for Distortion Correction from Basic to Four-Way Acquisitions
Purpose: High-resolution 7T diffusion MRI (dMRI) is limited by image artifacts that compromise anatomical accuracy. The purpose of this study was to systematically evaluate phase-encoding (PE) acquisition and correction strategies to determine which methods best mitigate geometric distortions and improve data reproducibility. Methods: Five healthy adults were each scanned twice on a 7T MRI scanner (0.9 mm isotropic resolution), using a highly oversampled dMRI protocol with four PE directions (AP, PA, RL, LR). From this dataset, we created and processed eleven time-equivalent, 10-minute acquisitions, ranging from uncorrected single-PE data to comprehensive 4-way PE schemes. These strategies were quantitatively compared on their geometric alignment with T1-weighted images and on the scan-rescan reproducibility of DTI-derived metrics. Results: (1) All distortion-corrected schemes significantly improved geometric accuracy over uncorrected data; (2) Strategies correcting with a full set of reversed-PE (2-way) diffusion weighted images (DWIs) outperformed the common approach of using only a single reversed b=0 image; and (3) a 4-way PE acquisition consistently provided the highest image fidelity and reproducibility. The optimized acquisition enabled high-quality reconstruction of both long-range and fine-scale superficial white matter pathways. Conclusion: For high-resolution 7T dMRI, multi-PE acquisition is essential to achieve accurate geometry and stable microstructural estimates (i.e., less residual EPI distortion and better scan-rescan agreement). A 4-way PE scheme provides the most accurate and reproducible results for microstructural and connectivity modeling. Data statement: Data will be made available in BIDS format in openneuro sharing platform upon acceptance of the manuscript. To be updated with DOI.

Anterior thalamic nucleus inputs to the retrosplenial cortex are unperturbed in the hAPP-J20 mouse model of Alzheimer's disease
Alzheimers disease (AD) is a neurodegenerative condition characterised by progressive loss of memory and general decline in cognitive function. Although research has traditionally focused on the hippocampus and entorhinal cortex, other regions important for memory and spatial navigation, such as the anterior thalamic nuclei (ATN) and retrosplenial cortex (RSC), are also affected. The RSC is an important brain region for both memory and spatial navigation, and displays dysfunction in humans during prodromal phases of AD. The ATN provide a major input to RSC, with loss of this input to RSC causing a dramatic reduction in the expression of the immediate early gene product c-Fos. The ATN to RSC connection may thus present a new target for therapeutic intervention in AD.

Here, we set out to determine whether AD-like pathology perturbed projections from ATN to RSC using the hAPP-J20 amyloidopathy mouse model, using optogenetic activation of thalamic inputs combined with ex vivo patch clamp electrophysiology. We found that RSC displayed an age-dependent decline in basal c-Fos activity, with no additional effect of amyloid pathology on c-Fos expression. At all time points (3, 6 and 9 months) measured, we found no evidence of impairments in the synaptic strength or efficacy of the ATN projections to either granular or dysgranular subdivisions of RSC. We conclude that, in the hAPP-J20 mouse model, this pathway is unaffected by amyloid-{beta} overexpression.

Molecular dynamics of the pathogenic KCNQ2 variant G256W reveal mechanisms of channel dysfunction in epileptic encephalopathy
Brain voltage-gated potassium channels containing the subunit KCNQ2 are essential for regulating electrical signals contributing to sensation, learning, memory, and motor control. De novo KCNQ2 variants are among the more common Mendelian causes of early life epilepsy and neurodevelopmental impairment. Some patients with KCNQ2 variants are affected with KCNQ2 developmental and epileptic encephalopathy (KCNQ2 DEE) characterized by seizures and developmental delays. Children with KCNQ2 DEE exhibit a range of impairment patterns that appear to be correlated with specific consequences of the variant for protein function. Here, we used all-atom molecular dynamics to analyze a pathogenic missense variant KCNQ2 G256W, located in the pore turret. G256W subunit simulations showed migration of the hydrophobic W256 sidechain towards the lipid membrane. This movement affected turret structure and mobility prominently involving K255. We identified novel hydrogen bonding interactions in the wild type KCNQ2 turret region which formed a network that extended to the selectivity filter and identified N258, H260P, and K283 as key residues. Simulations comparing WT and G256W tetrameric channels exhibited more conformationally unstable ion selectivity filters for G256W. We analyzed how different stoichiometries of wild type and G256W subunits, as expected in heterozygous individuals, impacted dynamics and compared the G256W results to three additional variants of the turret-selectivity filter network. Our results provide additional support for an integral role of the KCNQ2 turret selectivity filter stability. The majority of severe KCNQ2 DEE variants are clustered near the selectivity filter in the pore domain. Our study provides insights that may be broadly applicable to this clinically important allele subgroup.

N-Linked Glycosylation as a Driver of Tau Pathology and Neuronal Transmission
N-Linked glycosylation of tau is implicated in Alzheimers disease, yet how it influences tau propagation and neuronal uptake remains unclear. Here, we use chemically defined N-glycoforms of 2N4R tau and the K18 repeat domain to map how glycan site and structure reprogram tau-polyanion binding, seeding, and entry into human neurons. Site-specific GlcNAc or high-mannose glycans at Asn359 or Asn410 remodel heparin and RNA binding kinetics in biolayer interferometry assays, revealing avidity-dominated, slow-off heparin interactions that are dampened by N-glycans and faster RNA interactions that can be potentiated by small GlcNAc modifications. Cross-seeding experiments show that N-glycosylation selectively tunes nucleation compatibility between tau proteoforms; GlcNAc at Asn410 in full-length tau and small glycans at Asn359 in K18 most strongly accelerate aggregation. In HEK293T FRET biosensor cells and iPSC-derived cortical neurons, N-glycans enhance or suppress seeding and pHrodo-reported uptake in a site- and glycan-dependent manner, with Asn410 glycosylation markedly potentiating cellular seeding. Pharmacological inhibition with atropine, dynasore, and cytochalasin D further indicates that N-glycans rebalance muscarinic, dynamin-dependent, and actin-dependent uptake routes. Together, these data establish N-linked glycosylation as a tunable regulator of tau trafficking and prion-like spread, coupling extracellular receptor usage to intracellular cofactor engagement, and reveals druggable nodes in uptake.

Early Diabetic Ca2+ Handling Impairments in the Rod Bipolar Pathway
BackgroundPrevious work showed that electrically-evoked inhibition to Rod Bipolar Cells (RBC) is reduced in a mouse model of early diabetes. It is hypothesized that this is due to impaired Ca2+ handling in the presynaptic amacrine cell, either through increased Ca2+ buffering or decreased influx. To test this hypothesis and develop a mechanism for this effect, a model where direct optogenetic activation of inhibitory amacrine cells that expressed the light-activated channel ChR2 was used to isolate amacrine cell inputs to RBCs. Application of selective Ca2+ channel blockers could then assess potential locations of amacrine Ca2+ disruption. Using whole cell patch clamp electrophysiology, recordings were made from a 6 week diabetic population (DM) and vehicle injected non-DM animals.

ResultsRobust GABAC receptor inhibitory currents were recorded from RBCs after ChR2 stimulus that were significantly diminished by the application of nifedipine to block L-type Ca2+ channels in both DM and non-DM conditions. There were significant differences in the peak amplitude of these responses between DM and non-DM groups (p = 0.0146). However, in the non-DM group the decay tau of the response to the 50ms stimulus was significantly diminished by nifedipine ({tau} p =0.0498, n = 5), but this was not seen in the DM group ({tau} p = 0.9498, n=7). A 1s nifedipine-reduced response saw its decay tau increase in the DM group but not the non-DM. Ca2+ - induced Ca2+ release (CICR) blockade with ryanodine decreased responsivity equally between groups in the 1s stimulus but showed no significant kinetic changes. CICR blockade for a 50ms stimulus response showed significant kinetic changes in diabetes but otherwise reduced the response equally between DM and non-DM. Blockade of the mitochondrial Ca2+ uniporter (MCU) had little effect on the optogenetic response.

ConclusionThis study presents evidence that diabetes alters amacrine cell output to the RBC unmasked through blockade of the L-type calcium channel, and the Endoplasmic Reticulum (ER). An apparent explanation for our results is that DM calcium buffering is dysregulated, leading to prolonged responses. The underlying mechanism for this alteration is complex and not yet clearly elucidated.

Signatures of remote planning in hippocampal replay
During brief, intermittent "replay" events, hippocampal activity can express navigational trajectories disconnected from both when and where they originally occurred. While replay biased toward immediate future goals has been observed, there is no evidence yet linking replay to planning beyond the next action. Here, we designed a sequential spatial working memory task which required rats to utilize information across multiple temporally separated actions. Remote replay events matched the animal's future navigational choices made after completing an intervening subtask. Critically, this occurred only when the replayed information was useful for reducing memory load, consistent with it being an active process. Our findings suggest these remote replay events are a neural correlate of episodic forethought, allowing animals to use memories to plan beyond their immediate surroundings.

Rapid robust high-fidelity 3D neuronal extraction from multi-view projections
Recent developments in imaging facilitate large-scale three-dimensional (3D) neuronal recording. While these huge amounts of data shed new light on population-level neural coding, it is much harder to extract neuronal calcium dynamics from 3D volumes than 2D images with the existence of noise and scattering. Here, we presented DeepWonder3D, a general end-to-end pipeline for rapid and robust 3D neuronal extraction with high fidelity. Instead of processing voxel by voxel, DeepWonder3D works on the multi-view projections of 3D imaging data obtained either digitally or optically through specific point spread functions (PSFs) for general applicability to diverse techniques, including point-scanning microscopy, light-field microscopy, and two-photon synthetic aperture microscopy. Integrating denoising, resolution registration, background removal, neuronal extraction, and multi-view fusion into a unified pipeline tailored for large-scale high-resolution datasets contaminated by noise and scattering, DeepWonder3D outperforms state-of-the-art methods in 3D localization accuracy with a 10-fold reduction in computational costs, validated by numerical simulations and a hybrid two-photon/light-field imaging system. With the RUSH3D mesoscope, DeepWonder3D achieves rapid high-fidelity 3D calcium extraction of tens of thousands of neurons across the mouse cortex within hours.

Awareness is necessary for predictive learning and prediction-based motor attenuation
Predictive coding theories posit that the brain continuously updates internal models of the world to anticipate sensory input. Attenuation of neural responses to predictable sensory stimuli is thought to aid in this process of prediction error detection and model updating. The topic of whether predictive processes such as sensory attenuation can occur in minimally conscious states has been hotly debated. The current study provides a novel test for the role of awareness in prediction-based attenuation using a transcranial magnetic stimulation (TMS) prediction task, whereby predictable stimulation is known to lead to attenuated motor system excitability. The experiment tested whether motor attenuation can occur without awareness using a dual task paradigm to mask a predictive cueing relationship. Participants simultaneously completed an n-back task and a TMS-counting task; critically, a visual cue (in the n-back task) consistently predicted the occurrence of a TMS pulse (in the counting task). Instruction conditions manipulated how much participants were informed of this relationship, and awareness was assessed in a post-experimental questionnaire. The results reveal that motor attenuation was present only in participants instructed about the relationship, or in those who became aware of the cue-TMS association. Implicit, predictable cue-TMS exposure was not sufficient to elicit motor attenuation in unaware participants. These findings show that awareness of the relationship is necessary for predictive learning and prediction-based attenuation in the motor system, challenging the assumption that predictive coding operates automatically.

A deformable attractor manifold organizes human resting-state brain dynamics
Intrinsic brain activity is often described as wandering within a continuous multivariate space, yet the organizing principles that constrain these dynamics remain unclear. Here, we show that spontaneous human brain activity during rest is structured by a deformable attractor manifold. Using large-scale fMRI datasets and a latent dynamical model, we find that cortical activity occupies two reproducible regimes: a low-coherence state with a unimodal latent distribution and a high-coherence state that exhibits bimodality, consistent with transient bistability across association networks. A compact two-parameter energy landscape explains these dynamics, revealing that transitions arise not from switching between discrete states, but from continuous deformation of the manifold that reshapes attractor geometry. Excursions into the bistable regime occur as rapid "jumps", whereas returns follow slow drifts along the manifold, reflecting network-specific timescales. Individuals with greater expression of the bistable regime show higher cognitive fluidity, and manifold parameters differentiate mild cognitive impairment from matched controls. These findings identify an organizing geometric and dynamical principle of resting activity, linking large-scale cortical coordination, cognitive variability, and vulnerability to pathology.

DNM2-CMT neuropathy stems from disrupted Schwann cell function and shows limited therapeutic reversibility
Dominant loss-of-function mutations in DNM2 cause Charcot-Marie-Tooth (CMT) neuropathy characterized by sensory and motor deficits associated with myelin and/or axonal abnormalities and muscle atrophy. Increasing DNM2 activity from embryogenesis has been reported to ameliorate neuromuscular phenotypes in the Dnm2K562E/+ CMT mouse; however, this model displays predominantly muscle pathology and limited nerve involvement, precluding rigorous evaluation of neuropathic mechanisms and potential therapies. Here, we performed comprehensive behavioral, electrophysiological, histological and molecular analyses to characterize the Dnm2K562E/SC- mouse, which combines systemic heterozygosity for the common K562E mutation together with Schwann cell (SC)-specific deletion of wild-type Dnm2. This model faithfully reproduces key clinical and pathological features of DNM2-CMT, including motor deficits, reduced general force and coordination, and severe sensory and motor conduction deficits associated with axonal loss, demyelination, and inflammation. Mechanistically, we delineate a coherent pathological sequence that explains the profound functional deficits. In particular, a downregulation of the transcription factor EGR2, a master regulator of myelin gene expression, and of the myelin protein MPZ correlates with demyelination. To evaluate the therapeutic potential of DNM2 supplementation, post-symptomatic intrathecal delivery of AAV9-DNM2 driven by the Schwann cell-specific MPZ promoter was performed at 4 weeks. Although DNM2 expression increased in peripheral nerves (~1.9-fold), no significant improvements were observed across behavioural, electrophysiological, structural, or molecular parameters. Together, these findings establish the Dnm2K562E/SC- mouse as a robust preclinical model, recapitulating key features of DNM2-CMT, and provides crucial insight into the biological and temporal constraints that must guide future therapeutic strategies for DNM2-CMT.

Selective impairment of spatial recognition memory and reduced frontal corticothalamic spine density following adolescent alcohol consumption
Heavy alcohol use is common during adolescence and is associated with increased risk of alcohol use disorder and prevalence of residual cognitive deficits, especially in behaviors associated with the latently developing prefrontal cortex (PFC). A major need for advancing our understanding of this relationship is replicable and accessible preclinical behavioral batteries that can be used to disassociate the effects of adolescent alcohol on select PFC circuits. Electrophysiological evidence implicates projections from the PFC to mediodorsal thalamus (PFC-MdT) as being uniquely impacted by adolescent intermittent alcohol consumption in mice. The present study aims to evaluate if voluntary consumption of alcohol during adolescence impacts anxiety or PFC-associated spatial and recognition memory and if they are associated with morphological changes to the PFC-MdT circuit. Our results indicate that compared to water-only controls, male and female mice that voluntarily consumed alcohol during adolescence demonstrate performance deficits in the object-in-place recognition task, without affecting the novel object recognition task, Y-maze alternation, anxiety or locomotion, an outcome consistent with PFC and MdT dysfunction. Morphological assessment of PFC neurons from male and female mice following behavioral tasks found a reduction in spine density in basal and apical, but not oblique dendrites of PFC-MdT neurons. Collectively, these results implicate the PFC to MdT circuit integrity as a potential locus of the effects of adolescent alcohol on spatial recognition memory.

Astrocyte senescence impairs synaptogenesis due to Thrombospondin-1 loss.
Cellular senescence is an irreversible state linked to aging that involves molecular and functional alterations. The mammalian hippocampus, a key brain region for learning and memory, is highly vulnerable to damage in age-related neurodegenerative diseases, yet the role of cellular senescence in hippocampal aging remains underexplored. Here, we report an early onset of senescence signatures in hippocampal astrocytes of the accelerated aging and frailty mouse model SAMP8. We examine how astrocyte senescence affects excitatory synapse formation, focusing on soluble signals released by astrocytes. Astrocytes isolated from SAMP8 brain and those differentiated from SAMP8 neural stem cells show senescence hallmarks (SA-{beta}-gal, p16INK4a, Lamin B1 loss), alongside a significant reduction in synaptogenic function. While astrocyte-conditioned medium (ACM) from control mice promotes excitatory synaptogenesis through thrombospondin-1 / 2{delta}-1 neuronal receptor signalling, ACM from senescent SAMP8 astrocytes lacks this capacity. Supplementing senescent ACM with thrombospondin-1 protein, or overexpressing thrombospondin-1 gene in senescent astrocytes, reinstates synaptogenesis. At the hippocampal level, thrombospondin-1 and synaptic puncta are reduced in SAMP8 mice. Our findings reveal that senescent astrocytes exhibit reduced synaptogenic capacity due to thrombospondin-1 loss, highlighting their contribution to synaptic dysfunction during aging. Preventing senescence in hippocampal astrocytes may thus restore astrocyte-mediated synaptogenesis in the aged brain.

An extended structure of the intracellular domain of the Torpedo nicotinic acetylcholine receptor and its proposed interactions with rapsyn
To gain insight into the interactions between rapsyn and the nAChR that induce clustering at the post-synaptic membrane, we refined a cryo-EM dataset using an intracellular domain focused strategy to obtain a 3.0 [A] map with the most extensive density yet for the intracellular domain of the Torpedo nAChR. The improved map allowed us to extend the structure beyond the MX -helix and prior to the MA -helix of the intracellular domain. The new structure defines a sharp N-terminal boundary of each MA -helix to place agrin-dependent phosphorylated tyrosines unambiguously within the flexible regions of the MX-MA loops. Two distinct conformations of the {delta} M4 -helix were also resolved, indicating that M4 conformational heterogeneity reflects intrinsic flexibility rather than a change in gating state. The new structural constraints defined for the MX-MA loop were then used to evaluate AlphaFold3-predicted full-length models of the nAChR, rapsyn, and various rapsyn-nAChR complexes, identifying a consistent, asymmetric 3:1 binding architecture where each rapsyn is always sandwiched between the MX-MA loops from two subunits and where each phospho-tyrosine is lodged in a cationic pocket formed by conserved residues implicated in congenital myasthenic syndromes. The defined architecture fits published cryo-ET maps of Torpedo post-synaptic membranes and explains how both phosphorylated tyrosines and myasthenic syndrome-causing rapsyn mutations modulate receptor clustering.

A paradoxical impact of alcohol on sleep-memory coupling
Sleep serves a fundamental role in memory consolidation, and yet it must adapt to the organism's physiological state. Acute ethanol consumption has a profound impact on animal physiology, but whether intoxication affects the role of sleep in memory consolidation remains unexplored. We demonstrate that acute ethanol exerts a paradoxical dual impact on sleep-memory coupling in Drosophila. Typically, satiated flies require sleep for memory consolidation, but starved flies that must forage for food switch to sleep-independent memory. Ethanol selectively impairs memory consolidation in satiated flies, whereas memories in starved flies remain intact despite intoxication. The observed impairment in satiated flies is due to a switch to sleep-independent memory, which then can't be supported because of ethanol-induced sedation. Mechanistically, the ethanol-induced switch to sleep-independent memory is driven by neuropeptide F-mediated modulation of dopamine signaling. These findings reveal that ethanol intoxication inverts the canonical function of sleep, wherein it becomes detrimental to memory consolidation.

Genetic rescue of disrupted synaptic protein interaction network dynamics following SYNGAP1 reactivation
Synaptic protein interaction networks (PINs) dynamically translate neural activity into biochemical signals that regulate synaptic structure and plasticity. Disruption of these coordinated networks is a common feature of autism spectrum disorder (ASD) risk genes, yet it remains unclear whether the molecular organization of a perturbed network can be restored after development. Here, we examined how post-developmental re-expression of the synaptic Ras GTPase-activating protein SynGAP1 affects network structure and signaling dynamics in a conditional SynGAP1 haploinsufficient mouse. Quantitative multiplex co-immunoprecipitation (QMI) across development revealed that SynGAP haploinsufficiency selectively reduced SynGAP-containing complexes without broadly disrupting NMDA-dependent network responses. Tamoxifen-inducible re-expression of SynGAP at postnatal day 21 fully restored both steady-state and activity-dependent interactions within the SynGAP module in hippocampus, and additionally normalized secondary alterations in Shank-Homer scaffolding complexes in somatosensory cortex. These data demonstrate that biochemical restoration of a disrupted synaptic network is achievable, even after early developmental windows have closed. Our findings suggest that while critical periods may constrain functional recovery, molecular network normalization remains possible through genetic reactivation of haploinsufficient synaptic regulators.

Real-life ear-EEG Recordings of Auditory Responses to Ambient Sounds
Despite being one of the most researched neural responses, auditory potentials have primarily been measured in controlled laboratory environments. However, there is a big interest in measuring auditory potentials in more naturalistic environments, with stimuli resembling, to a higher degree, natural sounds. The objective of this study is to investigate auditory evoked potentials elicited by naturally occurring sounds in real-life environments. The paper presents an integration of a portable hearing aid research platform and a portable ear-EEG platform. This setup was validated through recordings of an Auditory Steady State Response (ASSR), elicited by real-time amplitude modulation of the ambient sound at 40Hz. Recordings were conducted in real-life under three conditions: walking and sitting with open and closed eyes. In addition to ear-EEG, scalp EEG was recorded as a reference. For analysis, EEG signals were categorized into three groups: scalp channels, cross-ear channels, and within-ear channels. For each condition and channel group, the signal-to-noise (SNR) of the ASSR was calculated. The ASSR was statistically significant across all channel groups and conditions. A post-hoc analysis assessed the recording duration required to obtain a significant ASSR, revealing that 10 minutes were enough for within ear ear-EEG for all but two cases, whereas less than 7 minutes were sufficient for all but one case when using scalp-EEG.

Asymmetrical modulation of fear expression via GABAB receptors in the mouse medial habenula
The medial habenula (MHb) is implicated in regulating emotional responses to aversive events. Studies in zebrafish have identified a remarkable morphological left-right asymmetry in the dorsal habenula (zebrafish equivalent of mammalian MHb) to interpeduncular nucleus (IPN) pathway and its left-sided-specific role in modulating fear responses. However, there is little evidence for structural or functional lateralization in the mammalian MHb-IPN pathway. Here, we investigated the synaptic properties of left- and right-MHb afferents to the IPN and their roles in the expression of conditioned fear in mice. We found that each IPN neuron receives inputs from both left and right MHb, but the left MHb-originating synapses exhibit lower release probability and higher {gamma}-aminobutyric acid type B receptor (GABABR)-mediated potentiation compared to the right MHb-originating synapses. Interestingly, these asymmetrical properties persist in the inversus visceral mutant mice with normal laterality of the internal organs (situs solitus), but nearly disappear in those with reversed internal organ laterality (situs inversus). Behaviorally, chemogenetic inhibition of cholinergic neurons and conditional deletion of GABABR in the left, but not the right, MHb significantly attenuated cue-dependent fear recall. Our results demonstrate functional asymmetry of the MHb under partial influence of the nodal flow in mice, revealing a predominant role of GABABR-mediated signaling in the left MHb-IPN pathway in modulating fear memories. These findings suggest that lateralized MHb pathways could represent a fundamental principle in the neural regulation of emotion across species, but that they develop differently in zebrafish and mice.

Theta activity in the RSC anchors space to the body cardinal axes
Understanding human navigation in ecological, freely moving conditions requires uncovering how the brain anchors directional representations to the 's orientation. Using high-density mobile electroencephalography and immersive virtual reality during goal-directed whole-body rotations, we found that theta bursts reconstructed in the retrosplenial complex (RSC) encode both acceleration and alignment with the body's principal axes. Crucially, this body-axis-anchored neural signal emerged only during goal-directed rotations, and its strength correlated with individual navigation performance, suggesting an adaptive mechanism which provides a stable egocentric scaffold for orientation. These results provide compelling evidence for a self-motion-gated, body-centered reference frame that supports efficient navigation, and bridge the gap between static neuroimaging findings in humans and rodent research on RSC geometry codes. Overall, our findings advance an embodied, mechanistic account of human navigation, opening new avenues for investigating brain dynamics in naturalistic, movement-rich settings using non-invasive neural recordings.

Combining membrane potential and calcium imaging in brain slices using the voltage sensitive dye ElectroFluor630 and the calcium indicator Calbryte520
Wide-field imaging from brain slices stained with a voltage sensitive dye (VSD) and simultaneously loaded with a Ca2+ indicator allows investigating neuronal excitability and synaptic transmission at multi-cellular scale. So far, achieving this type of combined imaging has been limited by experimental constraints. We assessed the ability of the red-IR emitting VSD ElectroFluor630 (EF-630) to be combined with blue-excitable green-emitting Ca2+ indicators to record signals elicited by electrical stimulation in hippocampal slices. Transversal mouse hippocampal slices were stained with EF-630. Ca2+ indicators, either Fluo-4, Fluo-8, Cal520 or Calbryte520, were loaded using their AM-ester forms. Fluorescence, during stimulation of the CA3 region was imaged at 5 kHz from hippocampal areas of [~]750X250 square microns at 1 micron pixel resolution. After assessing all Ca2+ indicators, we selected Calbryte520 for achieving >30 minutes stable recordings in combination with EF-630. Action potentials and related Ca2+ transients were detected in the CA3 stimulated area whereas synaptic signals were observed in the CA1 region. On these signals, we tested the pharmacological blockade of either action potentials or glutamatergic synaptic potentials. We report novel optical measurements of both electrical and Ca2+ transients in brain slices, providing unique information on neuronal excitability and network activity.

Beyond Inheritance: De novo Fast Motion Computation in Primate Visual Cortex
Objects move through space and time, generating sequential visuotopic activations in all sighted animals leading to motion perception of velocity defined by direction and speed. Humans can effortlessly see motion with speeds ranging from 0.25 to 500deg/s. However, direction-selective neurons in the primary visual cortex (V1)-from which all subsequent processing is presumed to derive-only encode directionality at low speeds. To resolve this paradox, we recorded neuronal responses to moving dots, gratings, and movies across the LGN, V1, MT and MST of the macaque motion pathway. Regardless of cell type and motion stimuli, V1 neurons lost direction selectivity at ~29deg/s while MT and MST neurons maintained it up to ~82deg/s and ~183deg/s, respectively. A cascaded spatiotemporal integration model reveals that at each cortex direction-selective neurons can generate velocity selectivity de novo, by integrating sequential visuotopic activations from preceding areas, irrespective of speed and directionality. By computing velocity anew, the primate brain effectively uses the cortical hierarchy itself to 'shift gears' to efficiently encode slow and fast motion. Thus, five visual areas from the retina into the brain's processing hierarchy, external spatiotemporal information is being computed afresh, offering insights for motion processing in other species, modalities and machine vision.

NVUAtlas: A Comprehensive Single-Nucleus RNA-Seq Resource for the Human Neurovascular Unit in Alzheimer's Disease
Background: Neurovascular unit (NVU) dysfunction is being recognized as one of the earliest contributors to Alzheimer's disease (AD) pathogenesis. However, systematic investigation of NVU dysfunction is currently limited by lack of access to molecular level information due to underrepresentation of vascular and mural cells in standard single-nucleus RNA sequencing (snRNA-seq) datasets. Consequently, existing transcriptomic atlases lack the resolution necessary to capture the coordinated intercellular signaling and dysfunction across vascular components of NVU, including endothelial cells and pericytes. Methods: We constructed the Human NVU Atlas by integrating 11 publicly available snRNA-seq datasets, including vascular-enriched samples, from the human prefrontal cortex. This comprehensive dataset aggregates over 4.2 million nuclei from 748 donors, including AD patients and age-matched controls. We utilized a unified probabilistic pipeline based on deep generative models (SCVI) to perform batch-aware integration and employed an ensemble of supervised and deep-learning classifiers to rigorously re-annotate cell types. Differential expression and ligand-receptor interaction analyses were subsequently performed to identify cell-type-specific disruptions in males versus females. Results: The atlas successfully curated vascular populations from 11 studies to assemble the largest publicly available NVU cohort of endothelial cells (2.8%) and pericytes (1.9%) alongside astrocytes and neurons. Differential expression analysis revealed that while neurons predominantly exhibited gene downregulation in AD, vascular cells displayed a pattern of transcriptional hyperactivity with significant gene upregulation. We also identified pronounced sex-specific vulnerabilities; females exhibited distinct inflammatory signatures and downregulation of basement membrane collagen genes (e.g., COL4A1, COL4A2) in pericytes, whereas these changes were not observed in males. Moreover, cell-cell interaction analysis revealed a widespread loss of collagen-integrin signaling between pericytes and neurons, suggesting the involvement of extracellular matrix disruptions in NVU dysfunction observed in AD. Conclusion: The Human NVU Atlas provides a high-resolution, integrated transcriptomic framework for dissecting the cellular heterogeneity of the neurovascular unit. By uncovering sex-specific vascular mechanisms and disrupted intercellular communication, this resource highlights the critical role of vascular cells in AD progression and serves as a foundational reference for investigating cerebrovascular contribution to AD.

Ventromedial striatal dopamine dynamically integrates motivated action and reward proximity
Dopamine release in the ventromedial striatum (VMS) both invigorates actions and encodes reward-related information, yet how these functions are integrated remains under active debate. To investigate this further, we designed four different versions of a rat Go/No-go task, where we systematically manipulated response requirements, temporal task demands, and controllability of reward pursuit. Dopamine release increased reliably during action initiation (Go) but was delayed during action suppression (No-go), and was insensitive to augmented response demands or controllability. Following response completion, dopamine rose gradually until animals arrived at the reward location, irrespective of reward-delivery timing, prior action demands, or controllability. This proximity dopamine-signal was exaggerated after animals exhibited Pavlovian consummatory behavior during No-go trials, revealing a motivational signal component. Together, these findings indicate that in reward contexts, VMS-dopamine signals successively integrate the invigoration of action initiation with the continuous estimation of spatial - but not temporal - proximity to rewards.