bioRxiv Subject Collection: Neuroscience's Journal

GABAAR-PPT1 palmitoylation homeostasis controls synaptic transmission and circuitry oscillation in CLN1 disease
CLN1 disease, also called infantile neuronal ceroid lipofuscinosis, is a fatal neurodegenerative disease caused by mutations in the CLN1 gene encoding palmitoyl protein thioesterase 1 (PPT1). To identify depalmitoylation substrate of PPT1 is crucial to understand CLN1 disease. In this study, we found that PPT1 depalmitoylates GABAAR 1 subunit at Cystein-260, while binding to Cystein-165 and -179. Mutations of PPT1 or its GABAAR 1 subunit binding site result in enhanced inhibitory synaptic transmission, strengthened and oscillation but disrupted phase coupling in CA1 region and impaired learning and memory in 1- to 2-months-old PPT1-deficient and Gabra1em1 mice. Our study highlights the critical role of PPT1 in maintaining GABAAR palmitoylation homeostasis and reveals a previously unknown molecular pathway in PPT1 induced diseases.

ACSS2 upregulation enhances neuronal resilience to aging and tau-associated neurodegeneration
Epigenetic mechanisms, including histone acetylation, are pivotal for learning and memory, with a role in neuronal function in Alzheimer's disease and Related Dementia (ADRD). Acetyl-CoA synthetase 2 (ACSS2), an enzyme that generates acetyl-CoA, is central to histone acetylation and gene regulation, particularly in neurons, due to their unique metabolic demands and postmitotic state. ACSS2 can be recruited to the nucleus and chromatin, locally supplying acetyl-CoA to directly fuel histone acetyltransferase enzymes and key neuronal gene expression. This regulatory mechanism may be a promising target for therapeutic intervention in neurodegenerative diseases. Previously we showed that systemic ACSS2 deletion in mice, although largely normal in physiology, is greatly impaired in memory. Here we investigated whether increasing ACSS2 levels could protect neurons against disease and age-associated cognitive decline. Given the role of tau in ADRD, we used primary hippocampal neurons that mimic the sporadic development of tau pathology and the P301S transgenic mouse model for tau-induced memory decline. Our results show that ACSS2 upregulation mitigates tau-induced transcriptional alterations, enhances neuronal resilience against tau pathology, improves long-term potentiation, and ameliorates memory deficits. Expanding upon these findings, we reveal that increasing histone acetylation through ACSS2 upregulation improves age-associated memory decline. These findings indicate that increasing ACSS2 is highly effective in countering age- and tau-induced transcriptome changes, preserving elevated levels of synaptic genes, and safeguarding synaptic integrity. We thus highlight ACSS2 as a key player in the epigenetic regulation of cognitive aging and ADRD, providing a foundation for targeted therapeutics to enhance brain resilience and function.

Evidence for flexible motor costs in vertical arm movements: reduced gravity-related effort minimization under high accuracy constraints
The central nervous system (CNS) is thought to use motor strategies that minimize several criteria, such as end-point variability or effort, to plan optimal motor patterns. In the case of vertical arm movements, a large body of literature demonstrated that the brain uses a motor strategy that takes advantage of the mechanical effects of gravity to minimize muscle effort. Results from other studies suggested that the relative importance of each criterion may vary according to the task's constraints. For example, it could be hypothesized that reduced end-point variability driven by high accuracy demands is detrimental to effort minimization. The present study probes this specific hypothesis using the framework of gravity-related effort minimization. We asked twenty young healthy participants to perform vertical arm reaching movements towards targets whose size varied across conditions. We recorded the arm kinematics and electromyographic activities of the anterior deltoid to study two well-known motor signatures of the gravity-related optimization process; i.e., directional asymmetries on velocity profiles and negative epochs on phasic muscular activities. The results showed that both indices were reduced as target size decreased, demonstrating that the gravity-related optimization process was reduced under high accuracy constraints. This phenomenon is consistent with the use of a trade-off strategy between effort and end-point variability. More generally, it suggests that the CNS is able to appropriately modulate the relative importance of varied motor costs when facing varying task demands.

Sustained ON alpha retinal ganglion cells in the temporal retina exhibit task-specific regional adaptions in dendritic signal integration
Various retinal ganglion cells (RGCs) show regional adaptations, for instance, to increase visual acuity. However, for many RGC types, it is not known how they vary in their signal-processing properties across the retina. In the mouse retina, sustained ON alpha (sONalpha) RGCs were found to have differences in morphology and receptive field sizes along the naso-temporal axis, and temporal sONalpha RGCs are likely to play a role in visually guided hunting. Thus, we hypothesised that this cell type also exhibits regional adaptations on the level of dendritic signal processing and that these adaptations are advantageous for prey capture. Here, we measured dendritic signals from individual sONalpha RGCs at different locations in the ex-vivo whole-mount mouse retina using two-photon microscopy. We measured both postsynaptic Ca2+ signals at the dendrites of individual RGCs and presynaptic glutamate signals from bipolar cells (BCs). We found that temporal sONalpha RGC dendrites exhibit, in addition to the expected sustained-ON signals with only weak surrounds, signals with strong surround suppression, which were not present in nasal sONalpha RGCs. This difference was also present in the excitatory presynaptic inputs from BCs, suggesting a presynaptic origin. Finally, using population models in an encoder-decoder paradigm, we showed that these adaptations might be beneficial for detecting crickets in hunting behaviour.

Navigating Human Astrocyte Differentiation: Direct and Rapid one-step Differentiation of Induced Pluripotent Stem Cells to Functional Astrocytes Supporting Neuronal Network development
Astrocytes play a pivotal role in neuronal network development. Despite the well-known role of astrocytes in the pathophysiology of neurologic disorders, the utilization of human induced pluripotent stem cell (hiPSC)-derived astrocytes in neuronal networks remains limited. Here, we present a streamlined one-step protocol for the differentiation of hiPSCs directly into functional astrocytes without the need for ectopic gene expression or neural progenitor cell generation. We found that culturing hiPSCs directly in commercial astrocyte medium, was sufficient to differentiate hiPSCs into functional astrocytes within five weeks. Validation to varying extents across thirty hiPSC-lines demonstrated consistent astrocyte differentiation with minimal batch-to-batch variability. We confirmed astrocyte identity and functionality of the hiPSC-astrocyte monocultures by immunofluorescence, flowcytometry, RNA sequencing, glutamate uptake assays and calcium signaling recordings. Optimization of the protocol enabled co-culture of hiPSC-astrocytes with Ngn2 hiPSC-derived neurons (iNeurons), promoting neuronal differentiation and synapse formation. Lastly, we used single-cell electrophysiology and multi-electrode arrays to confirm robust neuronal network development in 5-week-old hiPSC-astrocyte and iNeuron co-cultures. This protocol offers a rapid and efficient method to establish all-human astrocyte-neuron co-cultures, facilitating the investigation of cell-type-specific contributions to disease pathogenesis. While validated across multiple hiPSC lines, we actively encourage researchers to test and provide feedback on this protocol to enhance its validation for future iterations.

Role of the NOS-cGMP pathways in time-dependent sensitization of behavioral responses in zebrafish
Nitric oxide (NO) is a molecule involved in plasticity across levels and systems. The role of NOergic pathways in time-dependent sensitization, a behavioral model of translational relevance to trauma and stress-related disorders, was assessed in adult zebrafish. In this model, adult zebrafish acutely exposed to a fear-inducing conspecific alarm substance (CAS) and left undisturbed for an incubation period show increased anxiety-like behavior 24 h after exposure. CAS increased forebrain glutamate immediately after stress and 30 min after stress, an effect that was accompanied by increased nitrite levels immediately after stress, 30 min after stress, 90 min after stress, and 24 h after stress. CAS also increased nitrite levels in the head kidney, where cortisol is produced in zebrafish. CAS-elicited nitrite responses in the forebrain 90 min (but not 30 min) after stress were prevented by a NOS-2 blocker. Blocking NOS-1 30 min after stress prevents TDS; blocking NOS-2 90 min after stress also prevents TDS, as does blocking calcium-activated potassium channels in this latter time window. TDS is also prevented by blocking guanylate cyclase activation in both time windows, and cGMP-dependent channel activation in the second time window. These results suggest that different NO-related pathways converge at different time windows of the incubation period to induce TDS.

Selective regulation of corticostriatal synapses by astrocytic phagocytosis
In the adult brain, neural circuit homeostasis depends on the constant turnover of synapses via astrocytic phagocytosis mechanisms. However, it remains unclear whether this process occurs in a circuit-specific manner. Here, we reveal that astrocytes target and reorganize excitatory synapses in the striatum. Using model mice lacking astrocytic phagocytosis receptors in the dorsal striatum, we found that astrocytes constantly remove corticostriatal synapses rather than thalamostriatal synapses. This preferential elimination suggests that astrocytes play a selective role in modulating corticostriatal plasticity and functions via phagocytosis mechanisms. Supporting this notion, corticostriatal long-term potentiation (LTP) and the early phase of motor sequence learning are dependent on astrocytic phagocytic receptors. Together, our findings demonstrate that astrocytes contribute to the connectivity and plasticity of the striatal circuit by preferentially engulfing a specific subset of excitatory synapses within brain regions innervated by multiple excitatory sources.

Origins of noise in both improving and degrading decision making
Unlike the predictions of normative choice theories, the real-world accuracy of human and animal decision-making depends on context. Observed impairments of choice performance by context can be explained by computations like divisive normalization. However, recent behavioral evidence suggests that context can sometimes enhance choice performance, a finding that challenges existing explanations. Here, we propose and test an extension of existing frameworks for context-dependent choice that incorporates a richer notion of noise. Model simulations show that noise arising before (early noise) and after (late noise) normalization predict opposing effects: early noise can cause contextual information to enhance choice performance while late noise can cause context to degrade choice performance. In experiments with human subjects, we confirm key elements of this model. Manipulating early and late noise - by inducing uncertainty in option values and controlling time pressure - produces dissociable positive and negative effects of context. Together, these findings suggest a new unifying mechanism for contextual modulation, highlighting the significant role of noise source in neural computation and behavior.

DIETARY REGULATION OF SILENT SYNAPSES IN THE DORSOLATERAL STRIATUM
Obesity results in circuit adaptations that closely resemble those induced by drugs of abuse. AMPA-lacking silent synapses are critical in circuit generation during early development, but largely disappear by adulthood. Drugs of abuse increase silent synapses during adulthood and may facilitate the reorganization of brain circuits around drug-related experience, facilitating addiction and relapse Whether obesity causes addiction-related synaptic circuit reorganization via alterations in silent synapse expression has not been examined. Using a dietary-induced obesity paradigm, we show that mice that chronically consumed high-fat diet (HFD) exhibit upregulated silent synapses in both direct and indirect pathway medium spiny neurons in the dorsolateral striatum. Both the onset of silent synapses and their re-silencing after HFD withdrawal occur on an extended time scale of weeks rather than days. Our data suggest that HFD-related silent synapses likely arise from AMPA receptor internalization rather than through de novo synaptogenesis of NR2B-containing NMDA receptors. These data demonstrate that chronic consumption of high-fat diet can alter mechanisms of circuit plasticity, likely facilitating neural reorganization analogous to that observed with drugs of abuse.

Transcranial direct current stimulation modulates primate brain dynamics across states of consciousness.
Background: Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation approach that has been reported to perturb task activity and to benefit patients with a variety of diseases. Nevertheless, the effects of tDCS on brain dynamics and transitions in brain patterns across states of consciousness remain poorly understood. Objective: Here, we investigated the modulatory effect of prefrontal cortex (PFC) tDCS on brain dynamics, both in the awake state and during anesthesia-induced loss of consciousness in non-human primates. Methods: We acquired functional magnetic resonance imaging (fMRI) data before, during, and after the application of high-density tDCS (HD-tDCS) utilizing a prefrontal-occipital montage with two electrodes. In the awake state, macaques received either anodal or cathodal PFC stimulation. Under anesthesia, macaques underwent two consecutive anodal PFC stimulations of increasing intensity. Dynamical functional connectivity was measured using fMRI, and the resulting connectivity matrices were clustered into distinct brain patterns. Results: We found that cathodal PFC HD-tDCS robustly disrupted the rich dynamic repertoire of brain patterns of the awake state. It increased the brain structure-function correlation, decreased Shannon entropy, and strongly favored Markov chain transition probabilities towards patterns closest to anatomy. Under anesthesia, 2 mA anodal PFC HD-tDCS significantly changed the distribution of brain patterns and reduced the structure-function correlation. Conclusion: Our findings offer compelling evidence that prefrontal tDCS induces a striking modification in the fMRI-based dynamic organization of the brain across varying states of consciousness. This contributes to an enhanced understanding of the neural mechanisms underlying tDCS neuromodulation.

A Set of FMRI Quality Control Tools in AFNI: Systematic, in-depth and interactive QC with afni_proc.py and more
Quality control (QC) assessment is a vital part of FMRI processing and analysis, and a typically under-discussed aspect of reproducibility. This includes checking datasets at their very earliest stages (acquisition and conversion) through their processing steps (e.g., alignment and motion correction) to regression modeling (correct stimuli, no collinearity, valid fits, enough degrees of freedom, etc.) for each subject. There are a wide variety of features to verify throughout any single subject processing pipeline, both quantitatively and qualitatively. We present several FMRI preprocessing QC features available in the AFNI toolbox, many of which are automatically generated by the pipeline-creation tool, afni_proc.py. These items include: a modular HTML document that covers full single subject processing from the raw data through statistical modeling; several review scripts in the results directory of processed data; and command line tools for identifying subjects with one or more quantitative properties across a group (such as triaging warnings, making exclusion criteria or creating informational tables). The HTML itself contains several buttons that efficiently facilitate interactive investigations into the data, when deeper checks are needed beyond the systematic images. The pages are linkable, so that users can evaluate individual items across group, for increased sensitivity to differences (e.g., in alignment or regression modeling images). Finally, the QC document contains rating buttons for each "QC block", as well as comment fields for each, to facilitate both saving and sharing the evaluations. This increases the specificity of QC, as well as its shareability, as these files can be shared with others and potentially uploaded into repositories, promoting transparency and open science. We describe the features and applications of these QC tools for FMRI.

Behavior and alpha-band oscillations reveal cross-modal interactions between chemosensory and auditory processing
The perceived location of a sound can be mislocated towards a spatially discordant but temporally synchronous visual stimulus. This is referred to as the spatial ventriloquist effect. However, whether chemosensory cues can similarly bias sound localization remains largely unaddressed. Hence, the present EEG study adopted a dynamic sound localization paradigm with concurrent bimodal odorant stimulation. Participants heard sequences of sounds varying in location. After each sound, participants made a two-alternative forced choice localization judgment (left vs. right). Critically, in a subset of occasions, but unbeknown to the participants, the sounds originated from a central location. Furthermore, in the first half of the sequence, sound presentation could be accompanied by a task-irrelevant, trigeminally potent odorant in the left, right, or both nostril(s). Auditory-only trials and birhinal stimulation served as controls. For central sounds in the second half of the sequence, the proportion of right-ward responses increased with right-nostril stimulation but decreased with left-nostril stimulation relative to the control conditions, showing an after-effect of odorant stimulation for ambiguous sound cues. This odorant-induced localization bias diminished with increasing spatial discernability of the sounds. On the contrary, alpha power lateralization, a correlate of auditory spatial attention, was most susceptible to odorant stimulation when the spatial disparity between the senses was largest, as reflected in diminished alpha lateralization for incongruent chemosensory-sound stimulation. No such effect was present in a multivariate decoding analysis of alpha power. We discuss the present findings in light of cross-modal interactions and a proposed common attentional control system between the senses.

Dynamic Gamma Modulation of Hippocampal Place Cells Predominates Development of Theta Sequences
The experience-dependent spatial cognitive process requires sequential organization of hippocampal neural activities by theta rhythm, which develops to represent highly compressed information for rapid learning. However, how the theta sequences were developed in a finer time scale within theta cycles remains unclear. In this study, we found that sweep-ahead structure of theta sequences developing with exploration was predominantly dependent on a relatively large proportion of FG-cells, i.e. a subset of place cells dominantly phase-locked to fast gamma rhythms. These ensembles integrated compressed spatial information entrained in a theta sequence by cells consistently firing at precessing slow gamma phases within the theta cycle. Accordingly, the sweep-ahead structure of FG-cell sequences was positively correlated with the intensity of slow gamma phase precession, in particular during early sequence development. These findings highlight the dynamic network-modulation by fast and slow gamma in the development of theta sequences which may further facilitate memory encoding and retrieval.

Neural Correlates of Different Randomization Tasks
In some cases, when we are making decisions, the available choices can appear to be equivalent. When this happens, our choices appear not to be constrained by external factors and instead we can believe to be selecting "randomly". Furthermore, randomness is sometimes even explicitly required by task conditions such as in random sequence generation (RSG) tasks. This is a challenging task that involves the coordination of multiple cognitive processes, which can include the inhibition of habitual choice patterns and monitoring of the running choice sequence. It has been shown that random choices are strongly influenced by the way they are instructed. This raises the question whether the brain mechanisms underlying random selection also differ between different task instructions. To assess this, we measured brain activity while participants were engaging in three different variations of a sequence generation task: Based on previous work, participants were instructed to either (1) "generate a random sequence of choices", (2) "simulate a fair coin toss", or (3) "choose freely". Our results reveal a consistent frontoparietal activation pattern that is shared across all tasks. Specifically, increased activity was observed in bilateral inferior and right middle frontal gyrus, left pre-supplementary motor area, bilateral inferior parietal lobules and portions of anterior insular cortex in both hemispheres. Activity in the mental coin toss condition was higher in right dorsolateral prefrontal cortex, left (pre-) supplementary motor area, a portion of right inferior frontal gyrus, bilateral superior parietal lobules and bilateral anterior insula. Additionally, our multivariate analysis revealed a distinct region in the right frontal pole to be predictive of the outcome of choices, but only when randomness was explicitly instructed. These results emphasize that different randomization tasks involve both shared and unique neural mechanisms. Thus, even seemingly similar randomization behavior can be produced by different neural pathways.

Sensory processing sensitivity is associated with neural synchrony and functional connectivity during threatening movies
Sensory processing sensitivity (SPS) is an evolutionarily conserved trait describing a person's sensitivity to subtle stimuli, their depth of processing, emotional reactivity, and susceptibility to being overwhelmed. SPS is considered a fundamental and evolutionarily conserved trait, yet its neural mechanisms remain insufficiently understood. Therefore, we investigated whether SPS relates to processing movies differently in the central executive (CEN), default mode (DMN), and salience (SN) networks. We obtained positive and negative dimension Sensory Processing Sensitivity Questionnaire (short-form) scores and (neutral and threat aural framing) movie-fMRI data from a population-based sample (Healthy Brain Study, N=238, age mean=34years). We performed a priori inter-subject representation similarity, activation, and inter-subject functional connectivity analyses to characterize SPS-dimension-related neural responses during movie-viewing. More similar negative dimension SPS score related to more neural synchrony in the CEN and SN during threat. Higher negative dimension SPS score related to reduced CEN-DMN functional connectivity during threat, an effect shared across between-network regions but most strongly driven by reduced connectivity between right dorsomedial prefrontal cortex and left lateral prefrontal cortex. Our findings suggest that highly sensitive individuals exhibit distinct CEN differences shaping environmental perception, process threat differently, and each SPSQ-SF dimension may involve unique neurological mechanisms.

Immediate glucose signaling transmitted via the vagus nerve in gut-brain neural communication
Sucrose consumption is influenced by certain gut-brain signaling mechanisms. Among these, one pathway involves neuropod cells, which form synaptic connections with the vagus nerve, leading to the immediate activation of central dopaminergic pathways. This study explored the role of the frontal cortex in its process. We found that the vagus nerve's immediate activation is mediated by the sodium-glucose cotransporter 1 (SGLT1) of neuropod cells after the intragastric glucose injection in mice. Also, we showed that the involvement of both astrocytes and neurons in the frontal cortex via D2 and D1 dopamine receptors, respectively, by in vivo Ca2+ imaging. Finally, we revealed that psychological stress, which induces a reduction in sucrose preference, significantly diminishes the activation levels of both the vagus nerve and the frontal cortex. These findings highlight the role of a comprehensive gut-brain network in modulating sucrose preference, involving neuropod cells, the vagus nerve, and the frontal cortex.

Violated predictions enhance the representational fidelity of visual features in perception
Predictive coding theories argue that recent experience establishes expectations that generate prediction errors when violated. In humans, brain imaging studies have revealed unique signatures of violated predictions in sensory cortex, but the perceptual consequences of these effects remain unknown. We had observers perform a dual-report task on the orientation of briefly presented gratings within predictable or random sequences, while we recorded their pupil size as an index of surprise. Observers first made a speeded response to categorize grating orientation (clockwise or counterclockwise from vertical), then reproduced the orientation without time pressure by rotating a bar. This allowed us to separately assess response speed and precision for the same stimuli. Critically, on half the trials, the target orientation deviated from the spatiotemporal structure established by the preceding gratings. Observers responded faster and more accurately to unpredicted gratings, and pupillometry provided physiological evidence of observers' surprise in response to these events. In a second experiment, we cued the spatial location and timing of the grating and found the same pattern of results, demonstrating that unpredicted orientation information is sufficient to produce faster and more precise responses, even when the location and timing of the relevant stimuli are fully expected. These findings reveal the perceptual consequences of prediction errors and indicate that unexpected events are prioritized by the visual system both in terms of processing speed and representational fidelity.

Multiple and subject-specific roles of uncertainty in reward-guided decision-making
Decision-making in noisy, changing, and partially observable environments entails a basic tradeoff between immediate reward and longer-term information gain, known as the exploration-exploitation dilemma. Computationally, an effective way to balance this tradeoff is by leveraging uncertainty to guide exploration. Yet, in humans, empirical findings are mixed, from suggesting uncertainty-seeking to indifference and avoidance. In a novel bandit task that better captures uncertainty-driven behavior, we find multiple roles for uncertainty in human choices. First, stable and psychologically meaningful individual differences in uncertainty preferences actually range from seeking to avoidance, which can manifest as null group-level effects. Second, uncertainty modulates the use of basic decision heuristics that imperfectly exploit immediate rewards: a repetition bias and win-stay-lose-shift heuristic. These heuristics interact with uncertainty, favoring heuristic choices under higher uncertainty. These results, highlighting the rich and varied structure of reward-based choice, are a step to understanding its functional basis and dysfunction in psychopathology.

Human chorionic gonadotropin decreases cerebral cystic encephalomalacia and parvalbumin interneuron degeneration in a pro-inflammatory model of mouse neonatal hypoxia-ischemia.
The pregnancy hormone, human chorionic gonadotropin (hCG) is an immunoregulatory and neurotrophic glycoprotein of potential clinical utility in the neonate at risk for cerebral injury. Despite its well-known role in its ability to modulate the innate immune response during pregnancy, hCG has not been demonstrated to affect the pro-degenerative actions of inflammation in neonatal hypoxia-ischemia (HI). Here we utilize a neonatal mouse model of mild HI combined with intraperitoneal administration of lipopolysaccharide (LPS) to evaluate the neuroprotective actions of hCG in the setting of endotoxin-mediated systemic inflammation. Intraperitoneal treatment of hCG shortly prior to LPS injection significantly decreased tissue loss and cystic degeneration in the hippocampal and cerebral cortex in the term-equivalent neonatal mouse exposed to mild HI. Noting that parvalbumin immunoreactive interneurons have been broadly implicated in neurodevelopmental disorders, it is notable that hCG significantly improved the injury-mediated reduction of these neurons in the cerebral cortex, striatum and hippocampus. The above findings were associated with a decrease in the amount of Iba1 immunoreactive microglia in most of these brain regions. These observations implicate hCG as an agent capable of improving the neurological morbidity associated with peripheral inflammation in the neonate affected by HI. Future preclinical studies should aim at demonstrating added neuroprotective benefit by hCG in the context of therapeutic hypothermia and further exploring the mechanisms responsible for this effect. This research is likely to advance the therapeutic role of gonadotropins as a treatment for neonates with neonatal brain injury.

Lineage-tracing reveals an expanded population of NPY neurons in the inferior colliculus
Growing evidence suggests that neuropeptide signaling shapes auditory computations. We previously showed that neuropeptide Y (NPY) is expressed in the inferior colliculus (IC) by a population of GABAergic stellate neurons and that NPY regulates the strength of local excitatory circuits in the IC. NPY neurons were initially characterized using the NPY-hrGFP reporter mouse, in which hrGFP expression indicates NPY expression at the time of assay, i.e., an expression-tracking approach. However, studies in other brain regions have shown that NPY expression can vary based on a range of factors, suggesting that the NPY-hrGFP mouse might miss NPY neurons not expressing NPY proximal to the experiment date. Here, we hypothesized that neurons with the ability to express NPY represent a larger population of IC GABAergic neurons than previously reported. To test this hypothesis, we used a lineage-tracing approach to irreversibly tag neurons that expressed NPY at any point prior to the experiment date. We then compared the physiological and anatomical features of neurons labeled with this lineage-tracing approach to our prior data set, revealing a larger population of NPY neurons than previously found. In addition, we used optogenetics to test the local connectivity of NPY neurons and found that NPY neurons routinely provide inhibitory synaptic input to other neurons in the ipsilateral IC. Together, our data expand the definition of NPY neurons in the IC, suggest that NPY expression might be dynamically regulated in the IC, and provide functional evidence that NPY neurons form local inhibitory circuits in the IC.

Synaptic function and sensory processing in ZDHHC9-associated neurodevelopmental disorder: a mechanistic account
Loss-of-function ZDHHC9 variants are associated with X-linked intellectual disability (XLID), rolandic epilepsy (RE) and developmental language difficulties. This study integrates human neurophysiological data with a computational model to identify a potential neural mechanism explaining ZDHHC9-associated differences in cortical function and cognition. Magnetoencephalography (MEG) data was collected during an auditory roving oddball paradigm from eight individuals with a ZDHHC9 loss-of-function variant (ZDHHC9 group) and seven age-matched individuals without neurological or neurodevelopmental difficulties (control group). Evoked responses to auditory stimulation were larger in amplitude and showed a later peak latency in the ZDHHC9 group but demonstrated normal stimulus-specific properties. Magnetic mismatch negativity (mMMN) amplitude was also increased in the ZDHHC9 group, reflected by stronger neural activation during deviant processing relative to the standard. A recurrent neural network (RNN) model was trained to mimic recapitulate group-level auditory evoked responses, and subsequently perturbed to test the hypothesised impact of ZDHHC9-driven synaptic dysfunction on neural dynamics. Results of model perturbations showed that reducing inhibition levels by weakening inhibitory weights recapitulates the observed group differences in evoked responses. Stronger reductions in inhibition levels resulted in increased peak amplitude and peak latency of RNN prediction relative to the pre-perturbation predictions. Control experiments in which excitatory connections were strengthened by the same levels did not result in consistently stable activity or AEF-like RNN predictions. Together, these results suggest that reduced inhibition is a plausible mechanism by which loss of ZDHHC9 function alters cortical dynamics during sensory processing.

Transcriptomic analysis of the juvenile to adult transition in the mouse corpus callosum
The corpus callosum, a major white matter tract in the brain, undergoes age-related functional changes. To extend our investigation of age-related gene expression dynamics in the mouse corpus callosum, we compared RNA-seq data from 2-week-old and 12-week-old wild-type C57BL/6J mice and identified the differentially expressed genes (e.g., Serpinb1a, Ndrg1, Dnmt3a, etc.) between these ages. Furthermore, by comparing these genes with the datasets from 20-week-old and 96-week-old mice, we identified novel sets of genes with age-dependent variations in the corpus callosum. These gene expression changes potentially affect key biological pathways and may be closely linked to age-related neurological disorders, including dementia and stroke. Therefore, our results provide an additional dataset to explore age-dependent gene expression dynamics in the corpus callosum.

α-Synuclein strain propagation is independent of cellular prion protein expression in transgenic mice
The cellular prion protein, PrPC, has been postulated to function as a receptor for -synuclein, potentially facilitating cell-to-cell spreading and/or toxicity of -synuclein aggregates in neurodegenerative disorders such as Parkinson's disease. To test this hypothesis, we compared the propagation behavior of two different -synuclein aggregate strains in M83 transgenic mice that either expressed or did not express PrPC. Following intracerebral inoculation with the S or NS strain, the presence of PrPC had minimal influence on -synuclein strain-specified attributes such as the kinetics of disease progression, the extent of cerebral -synuclein deposition, selective targeting of specific brain regions and cell types, the morphology of induced -synuclein deposits, and the structural fingerprints of protease-resistant -synuclein aggregates. Likewise, there were no appreciable differences in disease manifestation between PrPC-expressing and PrPC-lacking M83 mice following intraperitoneal inoculation of the S strain. Interestingly, intraperitoneal inoculation with the NS strain resulted in two distinct disease phenotypes, indicative of -synuclein strain evolution, but this was also independent of PrPC expression. Overall, these results suggest that PrPC plays at most a minor role in the propagation, neuroinvasion, and evolution of -synuclein strains. Thus, other putative receptors or cell-to-cell propagation mechanisms may play a larger role in the spread of -synuclein aggregates during disease.

The Neural Basis of Attentional Blink as a Selective Control Mechanism in Conscious Perception
Conscious perception of visual stimuli involves large-scale brain networks with multiple activation-deactivation dynamics. Previous works have shown that early detection networks may be switched off about 200ms to 300ms after presentation of a visual stimulus. We hypothesize that these deactivations represent a selective control mechanism of the brain to conserve resources for post-perceptual processing. To this end, we used attentional blink as a behavioral measure for this mechanism. We showed that attentional blink is more likely to occur when a previous visual stimulus was consciously perceived. Using high-resolution eye-tracking, we found prolonged decrease in pupil diameter and transient decrease in blink probability associated with attentional blink. Using scalp EEG data, we further showed that attentional blink is associated with more pronounced event-related potentials related to visual processing and report.

Human visual performance for identifying letters affected by physiologically-inspired scrambling
In human vision, the retinal input is transformed into internal representations through a series of stages. In earlier stages, the signals from a particular visual field locus are passed in parallel from one visual processing area to the next. The connections at each stage may therefore introduce "error", where incorrect or convergent projections result in a loss of spatial precision. Psychophysical and physiological studies have implicated spatial scrambling of this sort as a cause of the visual deficits in amblyopia. Several methods to measure scrambling (both in amblyopia and in healthy vision) have been developed in recent decades. In this work, we introduce a new approach. We consider two stages of visual processing where scrambling may occur: either at the input to or the output from the simple cell stage in V1. We refer to these as "subcortical" and "cortical" scrambling respectively. We investigated the impact of these two types of scrambling on a letter identification task. A physiologically-inspired decomposition and resynthesis algorithm was used to generate letter stimuli that simulate scrambling at each of these two stages. To establish a performance benchmark, we trained separate Convolutional Neural Networks (CNNs) to perform the task with each scrambling type. Comparing CNN performance against that of eight humans with normal healthy vision, we found humans exhibited greater resilience to subcortical scrambling compared to cortical scrambling. We further investigated performance by comparing confusion matrices. Compared to a simple template matching model, we found the human strategy to be more consistent with our CNNs. We conclude: i) the human resilience for subcortical scrambling suggests this may be the stage at which a greater degree of scrambling is introduced in the visual hierarchy, and ii) humans employ flexible strategies for identifying scrambled stimuli, more sophisticated than a simple template match to the expected target.

The laminar organization of cell types in macaque cortex and its relationship to neuronal oscillations
The canonical microcircuit (CMC) has been hypothesized to be the fundamental unit of information processing in cortex. Each CMC unit is thought to be an interconnected column of neurons with specific connections between excitatory and inhibitory neurons across layers. Recently, we identified a conserved spectrolaminar motif of oscillatory activity across the primate cortex that may be the physiological consequence of the CMC. The spectrolaminar motif consists of local field potential (LFP) gamma-band power (40-150 Hz) peaking in superficial layers 2 and 3 and alpha/beta-band power (8-30 Hz) peaking in deep layers 5 and 6. Here, we investigate whether specific conserved cell types may produce the spectrolaminar motif. We collected laminar histological and electrophysiological data in 11 distinct cortical areas spanning the visual hierarchy: V1, V2, V3, V4, TEO, MT, MST, LIP, 8A/FEF, PMD, and LPFC (area 46), and anatomical data in DP and 7A. We stained representative slices for the three main inhibitory subtypes, Parvalbumin (PV), Calbindin (CB), and Calretinin (CR) positive neurons, as well as pyramidal cells marked with Neurogranin (NRGN). We found a conserved laminar structure of PV, CB, CR, and pyramidal cells. We also found a consistent relationship between the laminar distribution of inhibitory subtypes with power in the local field potential. PV interneuron density positively correlated with gamma (40-150 Hz) power. CR and CB density negatively correlated with alpha (8-12 Hz) and beta (13-30 Hz) oscillations. The conserved, layer- specific pattern of inhibition and excitation across layers is therefore likely the anatomical substrate of the spectrolaminar motif.

Force Reserve Predicts Compensation in Reaching Movement with Induced Shoulder Strength Deficit
Following events such as fatigue or stroke, individuals often move their trunks forward during reaching, leveraging a broader muscle group even when only arm movement would suffice. In previous work, we showed the existence of a 'force reserve' - a phenomenon where individuals, when challenged with a heavy weight, adjusted their motor coordination to preserve approximately 40% of their shoulder's force. Here, we investigated if such reserve can predict hip, shoulder, and elbow movements and torques resulting from an induced shoulder strength deficit. We engaged 20 healthy participants in a reaching task with incrementally heavier dumbbells, analyzing arm and trunk movements via motion capture and joint torques through inverse dynamics. We simulated these movements using an optimal control model of a 3-degree-of-freedom upper body, contrasting three cost functions: traditional sum of squared torques, a force reserve function incorporating a nonlinear penalty, and a normalized torque function. Our results demonstrate a clear increase in trunk movement correlated with heavier dumbbell weights, with participants employing compensatory movements to maintain a shoulder force reserve of approximately 40% of maximum torque. Simulations showed that while traditional and reserve functions accurately predicted trunk compensation, only the reserve function effectively predicted joint torques under heavier weights. These findings suggest that compensatory movements are strategically employed to minimize shoulder effort and distribute load across multiple joints in response to weakness. We discuss the implications of the force reserve cost function in the context of optimal control of human movements and its relevance for understanding of compensatory movements post-stroke.

Epigenomic landscape of the human dorsal root ganglion: sex differences and transcriptional regulation of nociceptive genes
Gene expression is influenced by chromatin architecture via controlled access of regulatory factors to DNA. To better understand regulation of gene expression in the human dorsal root ganglion (hDRG) we used bulk and spatial transposase-accessible chromatin technology followed by sequencing (ATAC-seq). We detected a total of 3005 differentially accessible chromatin regions (DARs) between sexes using bulk ATAC-seq. DARs in female hDRG mapped mainly to the X chromosome. In males, DARs were found in autosomal genes. We also found differential transcription factor binding motifs within DARs. EGR1/3 and SP1/4 were abundant in females, and JUN, FOS and other AP-1 family members in males. With the aim of dissecting the open chromatin profile in hDRG neurons, we used spatial ATAC-seq. Consistent with our bulk ATAC-seq data, most of the DARs in female hDRG were located in X chromosome genes. Neuron cluster showed higher chromatin accessibility in GABAergic, glutamatergic, and interferon-related genes in females, and in Ca2+-signaling-related genes in males. Sex differences in open chromatin transcription factor binding sites in neuron-proximal barcodes were consistent with the bulk data, having EGR1 transcription factor activity in females and AP-1 family members in males. Accordingly, we showed higher expression of EGR1 in female hDRG compared to male with in-situ hybridization. Our findings point to epigenomic sex differences in the hDRG that likely underlie divergent transcriptional responses that determine mechanistic sex differences in pain.

Uncertainty in Thermosensory Expectations Enhances an Illusion of Pain
The human brain has a remarkable ability to learn and update its beliefs about the world. Here, we investigate how thermosensory learning shapes our subjective experience of temperature and the misperception of pain in response to harmless thermal stimuli. Through computational modeling, we demonstrate that the brain uses a probabilistic predictive coding scheme to update beliefs about temperature changes based on their uncertainty. We find that these expectations directly modulate the perception of pain in the thermal grill illusion. Quantitative microstructural brain imaging revealed that the myeloarchitecture and iron content of the somatosensory cortex, the posterior insula and the amygdala reflect inter-individual variability in computational parameters related to learning and the degree to which uncertainty modulates illusory pain perception. Our findings offer a new framework to explain how the brain infers pain from innocuous thermal inputs. Our model has important implications for understanding the etiology of thermosensory symptoms in chronic pain conditions.

Elevated pyramidal cell firing orchestrates arteriolar vasoconstriction through COX-2-derived prostaglandin E2 signaling
Neurovascular coupling (NVC), linking neuronal activity to cerebral blood flow (CBF), is essential for brain function and underpins functional brain imaging. Whereas mechanisms involved in vasodilation are well-documented, those controlling vasoconstriction remain overlooked. This study unravels the mechanisms by which pyramidal cells elicit arteriole vasoconstriction. Using patch-clamp recording, vascular and Ca2+ imaging in Emx1-Cre;Ai32 mouse cortical slices, we show that strong optogenetic activation of layer II/III pyramidal cells induces arteriole vasoconstriction, correlating with firing frequency and somatic Ca2+ increase. Ex vivo and in vivo pharmacological investigations indicate that this neurogenic vasoconstriction extends beyond glutamatergic transmission and predominantly recruits prostaglandin E2 (PGE2) through the cyclooxygenase-2 (COX-2) pathway, and activation of EP1 and EP3 receptors. Single-cell RT-PCR further identifies layer II/III pyramidal cells as a key source of COX-2-derived PGE2. Additionally, we present evidence that specific neuropeptide Y (NPY) interneurons acting on Y1 receptor, and astrocytes, through 20-hydroxyeicosatetraenoic acid (20-HETE) and COX-1-derived PGE2, contribute to this process. By revealing the mechanisms by which the activity of pyramidal cells leads to vasoconstriction, our findings shed light on the complex regulation of CBF.

Large-scale RNA-seq mining reveals ciclopirox triggers TDP-43 cryptic exons
Nuclear clearance and cytoplasmic aggregation of TDP-43 in neurons, initially identified in ALS-FTD, are hallmark pathological features observed across a spectrum of neurodegenerative diseases. We previously found that TDP-43 loss-of-function leads to the transcriptome-wide inclusion of deleterious cryptic exons in brains and biofluids post-mortem as well as during the presymptomatic stage of ALS-FTD, but upstream mechanisms that lead to TDP-43 dysregulation remain unclear. Here, we developed a web-based resource (SnapMine) to determine the levels of TDP-43 cryptic exon inclusion across hundreds of thousands of publicly available RNA sequencing datasets. We established cryptic exon inclusion across a variety of human cells and tissues to provide ground truth references for future studies on TDP-43 dysregulation. We then explored studies that were entirely unrelated to TDP-43 or neurodegeneration and found that ciclopirox olamine (CPX), an FDA-approved antifungal, can trigger the inclusion of TDP-43-associated cryptic exons in a variety of mouse and human primary cells. CPX induction of cryptic exon occurs via heavy metal toxicity and oxidative stress, suggesting that similar vulnerabilities could play a role in neurodegeneration. Our work demonstrates how diverse datasets can be linked through common biological features and underscores that public archives of sequencing data represent a vastly underutilized resource with tremendous potential for uncovering novel insights into complex biological mechanisms and diseases.

Low-Dimensional Representations of Visuomotor Coordination for Natural Behavior
Understanding how the eyes, head, and hands coordinate in natural contexts is a critical challenge in visuomotor coordination research, often limited by sedentary tasks in constrained settings. To address this gap, we conducted an experiment where participants proactively performed pick-and-place actions on a life-size shelf in a virtual environment and recorded concurrent gaze and body movements. Subjects exhibited intricate translation and rotation movements of the eyes, head, and hands during the task. We employed time-wise principal component analysis to study the relationship between the eye, head, and hand movements relative to the action onset. We reduced the overall dimensionality into 2D representations, capturing over 50% of the explained variance and up to 65% just in time of the actions. Our analysis revealed a synergistic coupling of the eye-head and eye-hand systems. While generally loosely coupled, they synchronized at the moment of action, with variations in coupling observed in horizontal and vertical planes, indicating distinct mechanisms for coordination in the brain. Crucially, the head and hand were tightly coupled throughout the observation period, suggesting a common neural code driving these effectors. Notably, the low-dimensional representations demonstrated maximum predictive accuracy ~200ms before the action onset, highlighting a just-in-time coordination of the three effectors. This study emphasizes the synergistic nature of visuomotor coordination in natural behaviors, providing insights into the dynamic interplay of eye, head, and hand movements during reach-to-grasp tasks.

The fMRI global signal and its association with the signal from cranial bone
The nature of the global signal, i.e. the average signal from sequential functional imaging scans of the brain or the cortex, is not well understood, but is thought to include vascular and neural components. Using resting state data, we report on the strong association between the global signal and the average signal from the part of the volume that includes the cranial bone and subdural vessels and venous collectors, separated from each other and the subdural space by multispectral segmentation procedures. While subdural vessels carried a signal with a phase delay relative to the cortex, the association with the cortical signal was strongest in the parts of the scan corresponding to the laminae of the cranial bone, reaching 80% shared variance in some individuals. These findings suggest that in resting state data vascular components may play a prominent role in the genesis of fluctuations of the global signal. Evidence from other studies on the existence of neural sources of the global signal suggests that it may reflect the action of multiple mechanisms (including cerebrovascular reactivity and autonomic control) concurrently acting to regulate global cerebral perfusion.

Hippocampal Signal Complexity and Rate-of-Change Predict Navigational Performance: Evidence from a Two-Week VR Training Program
The hippocampus is believed to be an important region for spatial navigation, helping to represent the environment and plan routes. Evidence from rodents has suggested that the hippocampus processes information in a graded manner along its long-axis, with anterior regions encoding coarse information and posterior regions encoding fine-grained information. Brunec et al. (2018) demonstrated similar patterns in humans in a navigation paradigm, showing that the anterior-posterior gradient in representational granularity and the rate of signal change exist in the human hippocampus. However, the stability of these signals and their relationship to navigational performance remain unclear. In this study, we conducted a two-week training program where participants learned to navigate through a novel city environment. We investigated inter-voxel similarity (IVS) and temporal auto-correlation hippocampal signals, measures of representational granularity and signal change, respectively. Specifically, we investigated how these signals were influenced by navigational ability (i.e., stronger vs. weaker spatial learners), training session, and navigational dynamics. Our results revealed that stronger learners exhibited a clear anterior-posterior distinction in IVS in the right hippocampus, while weaker learners showed less pronounced distinctions. Additionally, lower general IVS levels in the hippocampus were linked to better early learning. Successful navigation was characterized by faster signal change, particularly in the anterior hippocampus, whereas failed navigation lacked the anterior-posterior distinction in signal change. These findings suggest that signal complexity and signal change in the hippocampus are important factors for successful navigation, with IVS representing information organization and auto-correlation reflecting moment-to-moment updating. These findings support the idea that efficient organization of scales of representation in an environment may be necessary for efficient navigation itself. Understanding the dynamics of these neural signals provides insights into the mechanisms underlying navigational learning in humans.

Neurophysiology of effortful listening: Decoupling motivational modulation from task demands
In demanding listening situations, a listener's motivational state may affect their cognitive investment. Here, we aim to delineate how domain-specific sensory processing, domain-general neural alpha power, and pupil size as a proxy for cognitive investment encode influences of motivational state under demanding listening. Participants performed an auditory gap-detection task while pupil size and the magnetoencephalogram (MEG) were simultaneously recorded. Task demand and a listener's motivational state were orthogonally manipulated through changes in gap duration and monetary-reward prospect, respectively. Whereas task difficulty impaired performance, reward prospect enhanced it. Pupil size reliably indicated the modulatory impact of an individual's motivational state. At the neural level, the motivational state did not affect auditory sensory processing directly but impacted attentional post-processing of an auditory event as reflected in the late evoked-response field and alpha power change. Both pre-gap pupil dilation and higher parietal alpha power predicted better performance at the single-trial level. The current data support a framework wherein the motivational state acts as an attentional top-down neural means of post-processing the auditory input in challenging listening situations.

Circadian control of a sex-specific behaviour in Drosophila
An endogenous circadian clock controls many of the behavioral traits of Drosophila melanogaster. This clock relies on the activity of interconnected clusters of neurons that harbor the clock machinery. The hierarchy among clusters involved in the control of rest-activity cycles has been extensively studied. Sexually dimorphic behaviors, on the other hand, have received less attention. Even though egg-laying, a female characteristic behavior, has been shown to be rhythmic, it remains largely unexplored possibly due to metholodological constraints. The current study provides the first steps towards determining the neural substrates underlying the circadian control of egg-laying. We show that, whereas the lateral ventral neurons (LNvs) and the dorsal neurons (DNs) are dispensable, the lateral dorsal neurons (LNds) are necessary for rhythmic egg-laying. Systematically probing the Drosophila connectome for contacts between circadian clusters and oviposition-related neurons, we found no evidence of direct connections between LNvs or DNs and neurons recruited during oviposition. Conversely, we did find bidirectional connections between Cryptochrome (Cry) expressing LNd (Cry+ LNds) and oviposition related neurons. Taken together, these results reveal that cry positive LNd neurons have a leading role in the control of the egg-laying rhythm in Drosophila females.

Tonic dendritic GABA release by substantia nigra dopaminergic neurons
Recent studies have demonstrated the importance of extrastriatal dopamine release in the emergence of the network dysfunction underlying motor deficits in Parkinson's disease (PD). To better characterize the actions of dopamine on substantia nigra pars reticulata (SNr) GABAergic neurons, optogenetic and electrophysiological tools were used in ex vivo mouse brain slices to monitor synaptic transmission arising from globus pallidus externa (GPe) neurons. As predicted by previous work, activation of D2 dopamine receptors (D2Rs) suppressed GABA release evoked by stimulation of GPe axons. However, D2R activation also suppressed a tonic, GABA-A receptor mediated inhibition of SNr spiking. D2R-mediated inhibition of tonic GABA release led to a roughly 30% increase in SNr spiking rate. Chemogenetic inhibition of GPe terminals or excitation of astrocytes had no effect on tonic GABA release in the SNr. In contrast, chemogenetic inhibition of dopaminergic neurons or knocking down the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) blunted tonic GABAergic signaling. Lastly, in a progressive mouse model of PD targeting dopaminergic neurons, the tonic inhibition of SNr neurons by GABA release also was lost. Taken together, these observations suggest that dopamine and GABA are co-released by the dendrites of ALDH1A1-expressing dopaminergic neurons that course through the SNr. The co-release of these transmitters could serve to promote movement by making SNr neurons less responsive to phasic activity arising from the indirect pathway circuitry and by lowering basal spiking rates.

Nonuniform scaling of synaptic inhibition in the dorsolateral geniculate nucleus in a mouse model of glaucoma.
Elevated intraocular pressure (IOP) triggers glaucoma by damaging the output neurons of the retina called retinal ganglion cells (RGCs). This leads to the loss of RGC signaling to visual centers of the brain such as the dorsolateral geniculate nucleus (dLGN), which is critical for processing and relaying information to the cortex for conscious vision. In response to altered levels of activity or synaptic input, neurons can homeostatically modulate postsynaptic neurotransmitter receptor numbers, allowing them to scale their synaptic responses to stabilize spike output. While prior work has indicated unaltered glutamate receptor properties in the glaucomatous dLGN, it is unknown whether glaucoma impacts dLGN inhibition. Here, using DBA/2J mice, which develop elevated IOP beginning at 6-7 months of age, we tested whether the strength of inhibitory synapses on dLGN thalamocortical relay neurons is altered in response to the disease state. We found an enhancement of feed-forward disynaptic inhibition arising from local interneurons along with increased amplitude of quantal inhibitory synaptic currents. A combination of immunofluorescence staining for the GABAA-1 receptor subunit, peak-scaled nonstationary fluctuation analysis, and measures of homeostatic synaptic scaling indicated this was the result of an approximately 1.4-fold increase in GABA receptor number at post-synaptic inhibitory synapses, although several pieces of evidence strongly indicate a non-uniform scaling across inhibitory synapses within individual relay neurons. Together, these results indicate an increase in inhibitory synaptic strength in the glaucomatous dLGN, potentially pointing toward homeostatic compensation for disruptions in network and neuronal function triggered by increased IOP.

Resting-state fMRI reveals altered functional connectivity associated with resilience and susceptibility to chronic social defeat stress in mouse brain
Chronic stress is a causal antecedent condition for major depressive disorder and associates with altered patterns of neural connectivity. There are nevertheless important individual differences in susceptibility to chronic stress. How stress-induced alterations in functional connectivity amongst depression-related brain regions associates with resilience and susceptibility to chronic stress is largely unknown. We used resting-state functional magnetic resonance imaging (rs-fMRI) to examine functional connectivity between established depression-related regions in susceptible (SUS) and resilient (RES) adult mice following chronic social defeat stress (CSDS). Seed-seed FC analysis revealed that the ventral dentate gyrus (vDG) exhibited the greatest number of group differences in functional connectivity with targeted brain regions. SUS mice showed greater functional connectivity between the vDG and subcortical regions compared to both control (CON) or RES groups. Whole brain vDG seed-voxel analysis supported seed-seed findings in SUS mice and indicated significantly decreased connectivity between the vDG and anterior cingulate area compared to CON mice. Interestingly, RES mice exhibited enhanced connectivity between the vDG and anterior cingulate area compared to SUS mice. Moreover, RES mice showed greater connectivity between the infralimbic prefrontal cortex and the nucleus accumbens shell. These findings indicate unique differences in functional connectivity patterns in SUS and RES mice that could represent a neurobiological basis for vulnerability for stress-induced depression.

Optogenetics-integrated gut organ culture system connects enteric neurons dynamics and gut homeostasis
The enteric nervous system (ENS) senses microbiota-derived signals and orchestrates mucosal immunity and epithelial barrier functions, in health and disease. However, mechanistic dissections of intestinal neuro-immune-microbiota communications remain challenging and existing research methods limit experimental controllability and throughput. Here, we present a novel optogenetics-integrated gut organ culture system that enables real-time, whole-tissue stimulation of specific ENS lineages, allowing for detailed analysis of their functional impact. We demonstrate that optogenetic activation of enteric cholinergic neurons rapidly modulates intestinal physiology. Interestingly, distinct neuronal firing patterns differentially modulate neuro-immunological gene expression and epithelial barrier integrity. Furthermore, diverse enteric neuronal lineages exert distinct regulatory roles. While cholinergic activation promotes gene-sets associated with type-2 immunity, tachykininergic enteric neurons differentially control mucosal defense programs. Remarkably, luminal introduction of the immunomodulatory bacterium C. ramosum significantly remodeled cholinergic-induced neuro-immunological transcription. These findings suggest that complex combinatorial signals delivered by gut microbes and enteric neurons are locally integrated to fine-tune intestinal immunity and barrier defense. Collectively, we provide a powerful platform for systematic discovery and mechanistic exploration of functional neuroimmune connections, and their potential modulation by drugs, microbes, or metabolites.