bioRxiv Subject Collection: Neuroscience's Journal

The gut-brain vagal axis governs mesolimbic dopamine dynamics and reward events
Reward-related processes have traditionally been ascribed to neural circuits centered on the dopamine (DA) system. While exteroceptive stimuli, such as food and drugs of abuse, are well-established activators of DA-neuron activity, growing evidence indicates that interoceptive signals also play a critical role in modulating reward. Among these, the gut-brain vagal axis has emerged as a key pathway, yet its precise contribution to mesolimbic DA-dependent signaling, dynamics and behaviors remains poorly defined. Here, we combine complementary ex vivo and in vivo approaches across multiple scales to investigate how the gut-brain vagal axis regulates DA dynamics and reward-related behaviors. We show that gut-brain vagal tone is essential for gating mesolimbic DA system activity and functions, modulating DA-dependent molecular and cellular processes, and scaling both food- and drugs-induced reinforcement. These findings challenge the traditional brain-centric view of reward processing, supporting a more unified and integrated model in which gut-derived and vagus-mediated interoceptive signals are pivotal in intrinsically shaping motivation and reinforcement. By uncovering the influence of gut-brain vagal communication on mesolimbic DA functions, this work offers new insights into the neurobiological mechanisms underlying both adaptive and maladaptive reward processes, with broad implications for eating disorders and addiction.

Assessing analysis methods of brain synchrony in social interaction: Simulation-based comparison
Inter-brain synchrony is an essential measure for investigating social interactive behaviour via hyperscanning. While functional near-infrared spectroscopy is a unique modality for measuring this index in dynamic, real-world interactions, methods to adequately assess inter-brain relationships have not been firmly established, and the overall picture remains unclear. Consequently, in this article, we first briefly summarize analysis methods for examining social interaction by dividing them into static and dynamic measures. Among these, we focus on measures of synchrony and their assessment in correlating behaviours, conducting a simulation-based comparison and analysis. Specifically, we directly compared static and dynamic variants of wavelet transform coherence (WTC), Pearson's correlation coefficient (CC), and phase mutual information (pMI) using a real fNIRS dataset. Results showed a significant divergence between WTC and CC, while WTC and pMI exhibited similar patterns as static measures. Overall, WTC was suggested to better identify synchrony due to its non-linear, instantaneous, and robust nature. For the latter part, based on other simulation analyses, we propose a new method using generalized linear model (GLM) regression. Simulations with synthetic fNIRS data supported the effectiveness of our proposed method, which can capture the dynamic relationships between inter-brain synchrony and behaviour, even during free interaction.

Human- and Rodent-derived Extracellular Vesicles Mediate the Spread of Pathology in MSA-like Models
Multiple system atrophy (MSA) is characterized by the presence of protein-rich inclusions mainly within oligodendrocytes, comprised primarily by the neuronal protein alpha-synuclein and the oligodendroglial-specific phosphoprotein TPPP/p25alpha. Mature oligodendrocytes do not normally express detectable alpha-synuclein levels, suggesting that its oligodendroglial accumulation may arise from intercellular transfer, potentially via extracellular vesicles (EVs); however, the precise role of oligodendroglial-derived EVs in MSA progression remains relatively understudied. Herein, we characterized the cargo/features and pathogenic potential of EVs released by oligodendrocytes treated with human alpha-synuclein fibrils amplified from MSA or Parkinson's disease patient brains (or human recombinant alpha-synuclein fibrils) and EVs isolated from murine and human MSA (or respective control) brains. Our findings reveal that both oligodendroglial cell- and brain-derived EVs harbor pathological alpha-synuclein and TPPP/p25alpha conformations, similar to those accumulating in human MSA brains. These EVs are readily taken up by both neurons and oligodendrocytes, driving alpha-synuclein propagation in vitro. Importantly, inoculation of these MSA-like EVs in animal models induces robust pSer129-alpha-synuclein accumulation along the nigrostriatal axis, colocalizing with markers of mature oligodendrocytes and dopaminergic neurons. These findings underscore the pivotal role of oligodendroglial-derived EVs in pathology progression and neuronal-oligodendroglial communication, positioning them as promising targets for therapeutic strategies aimed at combating alpha-synucleinopathies.

Robust activity-dependent mitochondrial calcium dynamics at the AIS is dispensable for action potential generation
Mitochondria are diverse and multifaceted intracellular organelles regulating oxidative energy supply, lipid metabolism and calcium (Ca2+) signaling. In neurons the spatial sequestration of cytoplasmic Ca2+ by mitochondria plays a critical role in determining activity-dependent spine plasticity, shaping the presynaptic transmitter release characteristics and contributing to sustained action potential firing. Here, we tested the hypothesis that mitochondria at the axon initial segment (AIS) affect the microdomain cytoplasmic Ca2+ transients, thereby regulating Ca2+-dependent voltage-gated ion channels at the plasma membrane and initiation of action potentials. Using 3D electron microscopy (EM) reconstructions and virally injecting genetically encoded fluorescence indicators we visualized the ultrastructure and distribution of mitochondria selectively in thick-tufted layer 5 pyramidal neurons. We found that most mitochondria were stably clustered to the proximal AIS, while few were observed at distal sites. Simultaneous two-photon imaging of action potential-dependent cytoplasmic and mitochondrial Ca2+, combined with electrophysiological recordings showed the AIS mitochondria exhibit powerful activity-dependent cytosolic Ca2+ uptake. However, while intracellular application of the mitochondrial Ca2+ uniporter inhibitor Ru360 fully blocked mitochondrial Ca2+ import, it did not affect action potential input-output function, action potential dynamics nor the ability to produce high-frequency burst output. Together, the results indicate that AIS mitochondria are dispensable for temporal and rate encoding, suggesting that mt-Ca2+ buffering at the AIS may be involved in non-electrical roles, including AIS maintenance or axonal transport.

Data Heterogeneity Limits the Scaling Effect of Pretraining Neural Data Transformers
A key challenge in analyzing neuroscience datasets is the profound variability they exhibit across sessions, animals, and data modalities--i.e., heterogeneity. Several recent studies have demonstrated performance gains from pretraining neural foundation models on multi-session datasets, seemingly overcoming this challenge. However, these studies typically lack fine-grained data scaling analyses. It remains unclear how different sources of heterogeneity influence model performance as the amount of pretraining data increases, and whether all sessions contribute equally to downstream performance gains. In this work, we systematically investigate how data heterogeneity impacts the scaling behavior of neural data transformers (NDTs) in neural activity prediction. We found that explicit sources of heterogeneity, such as brain region mismatches among sessions, reduced scaling benefits of neuron- and region-level activity prediction performances. For tasks that do exhibit consistent scaling, we identified implicit data heterogeneity arising from cross-session variability. Through our proposed session-selection procedure, models pretrained on as few as five selected sessions outperformed those pretrained on the entire dataset of 84 sessions. Our findings challenge the direct applicability of traditional scaling laws to neural data and suggest that prior claims of multi-session scaling benefits may be premature. This work both highlights the importance of incremental data scaling analyses and suggests new avenues toward optimally selecting pretraining data when developing foundation models on large-scale neuroscience datasets.

Skeletonization of neuronal processes using Discrete Morse techniques from computational topology
To understand biological intelligence we need to map neuronal networks in vertebrate brains. Mapping mesoscale neural circuitry is done using injections of tracers that label groups of neurons whose axons project to different brain regions. Since many neurons are labeled, it is difficult to follow individual axons. Previous approaches have instead quantified the regional projections using the total label intensity within a region. However, such a quantification is not biologically meaningful. We propose a new approach better connected to the underlying neurons by skeletonizing labeled axon fragments and then estimating a volumetric length density. Our approach uses a combination of deep nets and the Discrete Morse (DM) technique from computational topology. This technique takes into account nonlocal connectivity information and therefore provides noise-robustness. We demonstrate the utility and scalability of the approach on whole-brain tracer injected data. We also define and illustrate an information theoretic measure that quantifies the additional information obtained, compared to the skeletonized tracer injection fragments, when individual axon morphologies are available. Our approach is the first application of the DM technique to computational neuroanatomy. It can help bridge between single-axon skeletons and tracer injections, two important data types in mapping neural networks in vertebrates.

Automated high-throughput patch clamp electrophysiology of hiPSC-derived neuronal models
The advent of human induced pluripotent stem cells (hiPSCs) and their differentiation into neurons and brain organoids has revolutionized our ability to model brain disorders in a human context. However, current technologies to assay the electrophysiological properties of human neurons in these models remain limited by throughput, as single-cell manual patch clamp is laborious and resource intensive. Here, we provide methods to perform automated high-throughput patch-clamp (APC) on hiPSC-derived neurons. We describe how to dissociate and perform voltage-clamp recordings on human neurons from three well-established protocols - 2D directed differentiation of cortical neurons, NGN2-induced neurons, and 3D cortical organoids - using the Nanion Syncropatch 384, a commercially available high-throughput APC system. Using this approach, we investigated the biophysical properties of voltage-gated sodium channels (VGSCs) and provide direct comparisons between manual and APC recordings across all three hiPSC-derived model systems. We demonstrate the capability of this automated system for pharmacological analysis of native human VGSC isoforms, which will enable compound screening approaches. Lastly, we provide methods to sort specific cellular populations within these hiPSC models using fluorescence-activated cell sorting (FACS) followed by APC. These methods and results provide a transformative and novel high-throughput technique for quantifying passive and active membrane properties in cell-type specific and/or genetically modified hiPSC-derived neurons.

Nanoporous Microelectrodes for Neural Electrophysiology Recordings in Organotypic Culture
Organotypic cultures, specifically brain slices, have been used in neuroscience studies for many years to prolong the lifetime of the biological tissue outside of the host organism. However, the cultures must be kept in a sterile environment, maintaining supply of gas/nutrients for tissue survival and physiological relevance. Electrophysiological recordings from cultured tissue are challenging as the conventional approaches implicate a compromise on biological stability or environmental integrity. In this article, a novel approach has been used to design and print nanoporous microelectrodes on culture wells enabling in situ recording of electrophysiological neural activities. Optimized ink formulations are developed for conductive nanocarbon microelectrodes, and furthermore, fluoropolymer (polytetrafluoroethylene-based AF2400) ink has been inkjet printed for the first time acting as an insulator layer for microelectrodes. To keep the biocompatible nanoporous structure of culture wells, the microelectrodes have been printed on the bottom of the culture cells and only small connector pads have been produced on top of the culture membrane. Neural activity has been recorded by such a microelectrode structure for rodent brain slices cultured for three weeks. Furthermore, aerosol jet printing has been used for printing of nanocarbon microelectrodes allowing to produce much smaller size features compared to the inkjet printing.

Frontoparietal functional dedifferentiation during naturalistic movie watching among older adults at risk of emotional vulnerability
Functional dedifferentiation, a hallmark of brain aging particularly evident within the frontoparietal network (FPN), has been extensively investigated in the context of cognitive decline, yet its implications for late-life mental health remain poorly understood. Leveraging naturalistic fMRI combined with gradient mapping techniques, the present study investigated FPN functional dedifferentiation-quantified by functional dispersion of FPN in the multidimension gradient manifold-during real-life emotional experiences and its link to affective outcomes. Here, we estimated functional dispersion during naturalistic movie watching in both younger (N=72, 34 female, 19-36 yrs) and older (N=68, 36 female, 65-82 yrs) adult groups with 7T MRI scanner and assessed their emotion regulation difficulties, anxiety, and depression symptoms as indicators of mental health status. The results demonstrated that greater FPN dispersion (i.e., more dissimilar connectivity) was linked to increased depressive symptoms in older adults and highlighted emotion regulation difficulties as a full mediator of this relationship. Moreover, FPN dispersion could distinguish emotionally resilient from vulnerable older individuals. These findings suggest that functional dedifferentiation of the FPN during ecologically valid emotional context constitutes a promising neural signature of affective vulnerability in older adults. By bridging age-related functional dedifferentiation to real-world emotional scenario, this work underscores the translational value of naturalistic paradigms in geriatric psychiatry and identifies potential intervention targets aimed at enhancing FPN specificity to promote mental health in aging population.

Magnetic Fields Influence Visual Responses in Mice
Many animals use the Earth's magnetic field for the purposes of orientation and navigation, although the sensory mechanisms remain unclear. It has been proposed that retinal responses to light may be modulated by magnetic fields. However, to date, there is no evidence for a retinal response to magnetic fields in mammals. Here we show that magnetic fields affect expression of the neuronal activity marker c-Fos in the mouse retina in a light dependent manner. These retinal responses to magnetic fields are abolished in mice lacking the candidate magnetoreceptor cryptochrome. To characterise the signalling pathways involved, we then used RNAseq and cell-type mapping. We also show that magnetic fields increase exploratory behaviour in a visually dependent task and lengthen the period of the retinal circadian clock. Together, our data provide the first evidence for a mammalian retinal response to magnetic fields at a cellular, molecular and functional level, which may influence vision.

AI-based decoding of long covid cognitive impairments in mice using automated behavioral system and comparative transcriptomic analysis
Long COVID (LC) following SARS-CoV-2 infection affects millions of individuals world-wide and manifests with a variety of symptoms including cognitive dysfunction also known as brain fog . This is characterized by difficulties in executive functions, planning, decision-making, working memory, impairments in complex attention, loss of ability to learn new skills and perform sophisticated brain tasks. No effective treatment options currently exist for LC-related cognitive dysfunction. Here, we use the IntelliCage, which is an automated tracking system of cognitive functions, following SARS-CoV-2 infection in mice, measuring the ability of each mouse within a group to perform tasks that mimic complex human behaviors, such as planning, decision-making, cognitive flexibility, and working memory. Artificial intelligence and machine learning analyses of the tracking data classified LC mice into distinct behavioral categories from non-infected control mice, permitting precise identification and quantification of complex cognitive dysfunction in a controlled, replicable manner. Importantly, we find that brains from LC mice with cognitive dysfunction exhibit transcriptomic alterations similar to those observed in humans suffering from LC-related cognitive impairments, including altered expression of genes involved in learning, executive functions, synaptic functions, neurotransmitters and memory. Together, our findings establish a validated murine model and an automated unbiased approach to study LC-related cognitive dysfunction for the first time, and providing a valuable tool for screening potential treatments and therapeutic interventions.

Fornix subdivisions and spatial learning: a diffusion MRI study
The fornix is the major white matter tract linking the hippocampal formation with distal brain sites. Human and animal lesion studies show that the connections comprising the fornix are vital for specific attributes of episodic and spatial memory. The fornix, however, interconnects the hippocampal formation with an array of subcortical and cortical sites and it is not known which specific connections support spatial-mnemonic function. To address this, making use of a partly previously published dataset (Hodgetts et al., 2020), we applied a novel deterministic tractography protocol to diffusion-weighted magnetic resonance imaging (dMRI) data from a group of healthy young adult humans who separately completed a desktop-based virtual reality analogue of the Morris water maze task. The tractography protocol enabled the two main parts of the fornix, delineated previously in axonal tracing studies in rodents and primates, to be reconstructed in vivo, namely the pre-commissural fornix (connecting the hippocampus to the medial prefrontal cortex and the basal forebrain) and the post-commissural fornix (connecting the hippocampus to the medial diencephalon). We found that inter-individual differences in pre-commissural- but not, surprisingly, post-commissural- fornix microstructure (indexed by free water corrected fractional anisotropy, FA) were significantly correlated with individual differences in spatial learning, indexed by reduction in search error as individuals learned to navigate to a hidden target location from multiple starting points. This study provides novel evidence that flexible and/or precise spatial learning involves a hippocampal-basal forebrain/prefrontal network underpinned in part by the pre-commissural fornix.

Temporal characteristics of hemodynamic responses during active and passive hand movements in schizophrenia spectrum disorder
In healthy individuals active compared to passive movements exhibit earlier neural processing, reflected by more positive contrast estimates of the first-order temporal derivative (TD) of the hemodynamic response function (HRF) in functional MRI (fMRI) analyses. This temporal advantage may play a critical role in self-other distinction. However, whether Schizophrenia Spectrum Disorder (SSD) is associated with deficits in sensory-motor predictive mechanisms that influence this earlier processing remains unknown. Patients with SSD (n = 20) and healthy control subjects (HC; n = 20) performed active and passive hand movements, while detected delays in video feedback of their own or another person's hand movement. 3T fMRI data was recorded during the task. To assess response dynamics, we applied the TD to examine timing and the second-order dispersion derivative (DD) to evaluate duration of the HRF. Compared to HC, patients with SSD exhibited delayed BOLD activation during active vs. passive movements in the right caudate nucleus, lobule VIII of right cerebellar hemisphere, left superior temporal gyrus, left postcentral gyrus, left thalamus, and left putamen/insula. For active movement with own hand feedback, HC showed earlier activation in the bilateral putamen and insula, whereas patients with SSD exhibited earlier activation in the left precentral gyrus, supplementary motor area, and postcentral gyrus. Delayed BOLD responses in patients with SSD, particularly in the right cerebellar lobule VIII, bilateral putamen and insula, suggest impaired predictive mechanisms affecting feedback monitoring. These delays may contribute to disturbances in the sense of agency and self-action awareness, potentially underpinning core symptoms of SSD.

Hierarchical recurrent temporal prediction as a model of the mammalian dorsal visual pathway
A major goal of neuroscience is to identify whether there are generalized principles that can explain the diverse structures and functions of the brain. The principle of temporal prediction provides one approach, arguing that the sensory brain is optimized to represent stimulus features that efficiently predict the immediate future input. Previous work has demonstrated that feedforward hierarchical temporal prediction models can capture the tuning properties of neurons along the visual pathway, and that recurrent temporal prediction models can explain local functional connectivity within primary visual cortex. However, the visual system is also characterized by extensive inter-areal feedback recurrency, which existing models lack. We aimed to better account for the dynamic features of neurons in the visual cortex by incorporating both local recurrency and inter-areal feedback connectivity into a hierarchical temporal prediction model. The resulting model captured tuning for pattern motion, surround suppression and elements of inter-areal functional connectivity in visual cortex. Moreover, compared with several alternative normative models, the hierarchical recurrent temporal prediction model provided the closest fit to these tuning properties and was best able to explain the emergence of neuronal response properties across the visual cortex. Accordingly, temporal prediction can account for information processing throughout the visual pathway.

The Ataxin-2 protein is required in Kenyon cells for RNP-granule assembly and appetitive long-term memory formation.
Ribonucleo-protein granules (mRNP granules) are thought to contribute to the control of neuronal mRNA translation required for consolidation of long-term memories. Consistent with this, the function of Ataxin-2 in mRNA granule assembly has been shown to be required for long-term olfactory habituation (LTH) in Drosophila, a form of non-associative memory. Knockdown of Ataxin-2 in either local interneurons (LNs) or projection neurons (PNs) of the insect antennal lobe disrupts LTH, leading to a model in which Ataxin-dependent translational control is required in both presynaptic and postsynaptic elements of the LN-PN synapse, whose potentiation has been causally linked to LTH. Here we use novel and established methods for cell-type specific perturbation to ask: (a) whether Ataxin-2 controls mRNA granule assembly in cell types beyond the few that have been examined; and (b) whether it functions not only in LTH, but also for long-term olfactory associative memory (LTM). We show that Ataxin-2 controls mRNP granule assembly in additional neuronal types, namely Kenyon Cells that encode associative memory, as well as more broadly in non-neuronal cells, e.g. in nurse cells in the egg chamber. Furthermore, selective knockdown of Atx2 in alpha/beta and alpha' / beta' KCs blocks appetitive long-term but not short-term associative memories. Taken together these observations support a hypothesis that Ataxin-2 dependent translational control is widely required across different mnemonic circuits for consolidation of respective forms of long-term memories.

Benchmarking cerebellar organoids to model autism spectrum disorder and human brain evolution
While cortical organoids have been used to model different facets of neurodevelopmental conditions and human brain evolution, cerebellar organoids have not yet featured so prominently in the same context, despite increasing evidence of this brain regions importance for cognition and behavior. Here, we provide a longitudinal characterization of cerebellar organoids benchmarked against human fetal data and identify at very early stages of development a significant number of dynamically expressed genes relevant for neurodevelopmental conditions such as autism and attention deficit hyperactivity disorders. Then, we model an ASD mutation impacting CHD8, showing both granule cell and oligodendrocyte lineages prominently affected, resulting in altered network activity in more mature organoids. Lastly, using CRISPR/Cas9 editing, we also model an evolution-relevant mutation in a regulatory region of the CADPS2 gene. We investigate the effect of such ancestral allele exclusively carried by archaic hominis, identifying a rerouting of the CADPS2-expression in rhombic lip cells, coupled with a different sensitivity to hypoxia which in turn lead to a differential timing of granule cell differentiation.

High-Resolution Spatial Profiling of Microglia Reveals Proximity Associated Immunometabolic Reprogramming in Alzheimers Disease
Single-cell RNA sequencing has demonstrated that the presence of parenchymal amyloid plaques and intracellular hyperphosphorylated tau pathology is associated with distinctive (and possibly disease-driving) microglial heterogeneity. However, our understanding of how proximity to these Alzheimers disease (AD) pathological hallmarks in situ relates to microglial gene expression remains obscure. Here, we utilized high-resolution spatial transcriptomics (ST) via the Xenium platform with a fully customized gene panel to elucidate disease-associated microglial subtypes in tandem with examining metabolic signatures across AD-relevant mouse models and well-characterized human postmortem tissue. Three mouse models were evaluated: PS19, APP/PS1, and 5xFAD. Analyzing anatomical features across entire hemisections, our approach resolved the distribution of five disease-associated microglial subtypes, while deciphering how proximity to cerebral amyloid plaques influenced transcriptional mediators governing metabolic pathways. We observed robust alterations in glycolytic and cholesterol/lipid processing pathways in plaque-associated microglia, consistent with a specific switch to glycolysis and lipid-fueled metabolism in the plaque niche. Extending our analysis to human postmortem dorsolateral prefrontal cortex (dlPFC), we identified conserved disease-reactive microglial states, i.e., similar proximity-dependent metabolic shifts around amyloid plaques. Further, integrating spatial transcriptomics with machine-learning approaches revealed novel anatomic domain-specific cellular gene expression profiling features, highlighting differential vulnerabilities of neuronal populations near specific microglial subtypes. Together, our findings provide one of the first comprehensive and high-resolution atlas of microglial immunometabolic states across species, anatomical regions, and AD pathological burden.

NETSseq Reveals Inflammatory and Aging Mechanisms in Distinct Cell Types Driving Cerebellar Decline in Ataxia Telangiectasia
Ataxia-telangiectasia (A-T) is a rare, autosomal recessive, multisystem disorder caused by mutations in the Ataxia-Telangiectasia Mutated (ATM) gene and is characterized by a devastating and progressive neurological pathology. The cellular and molecular changes driving the neurological abnormalities associated with A-T are not well understood. Here, we applied our proprietary Nuclear Enriched Transcript Sort sequencing (NETSseq) platform to investigate changes in cell type composition and gene expression in human cerebellar post-mortem tissue from A-T and control donors. We found dysregulation in neurotransmitter signaling in granule neurons, potentially underlying the impaired motor coordination in A-T. Astrocytes and microglia have evidence of accelerated aging, with astrocytes being characterized by neurotoxic signatures, while microglia showed activation of DNA damage response pathways. Compared to single-nuclei technologies, NETSseq provided a more robust detection of genes with low abundance, a higher cell type specific expression pattern, and significantly lower levels of cross-contamination. These findings highlight the importance of NETSseq as a resource for investigating mechanisms and biological processes associated with disease, providing high-sensitivity, cell-specific insights to advance targeted therapies for neurodegenerative diseases.

Differential effects of ischemia and inflammation on plasma-derived extracellular vesicle characteristics and function
Extracellular vesicles (EVs) have long been understood to be important mediators of cell-to-cell communication and may lead to the molecular aftermath and exacerbation of brain injuries such stroke. This study explored how the source of the EVs influenced their characteristics and the effect these differences had on naive brain tissue. EVs were isolated from animals post-stroke in the acute or chronic stages of recovery in animals with and without reperfusion, and from a model of systemic inflammation (intraperitoneal lipopolysaccharide). The data show that neither stroke nor inflammation significantly increase EV numbers compared to sham or naive animals. Post-stroke EVs exhibited a panel of different platelet and inflammatory markers, when compared to EVs derived from a model of inflammation, reflecting differences between stroke and systemic immune activation. When injected into the brain, both stroke-derived and inflammation-derived EVs induced expression of pro-inflammatory cytokine gene expression, suggesting a potential role in neuroinflammation. However, there was a lack of distinct glial and astrocyte reactivity in response to any EVs, despite robust changes in ICAM reactivity. The findings here underscore the complexity of EV roles in pathophysiology and highlight the need for improved EV isolation methods. With further longitudinal studies we may be able to more accurately determine how the context of the injury (reperfusion vs no reperfusion vs inflammation) might contribute to the EV populations and their function. Understanding more about EVs in different contexts will improve our ability to use EVs as biomarkers, but also our capacity to interfere with EV biology as a novel therapeutic approach.

EPIGENETIC REPROGRAMMING OF CELL IDENTITY IN THE RAT PRIMARY NEURON-GLIA CULTURES INVOLVES HISTONE SEROTONYLATION
Epigenetic rearrangements can create a favorable environment for the intrinsic plasticity of brain cells, leading to cellular reprogramming into virtually any cell type through the induction of cell-specific transcriptional programs. In this study, we assessed how chromatin remodeling induced by broad-spectrum HDAC inhibitors affects cellular differentiation trajectories in rat primary neuron-glia cultures using a combination of transcriptomics, qPCR and cytochemistry. We described epigenetic regulation of transcriptional programs controlled by master transcription factors and neurotrophins in the context of neuronal and glial differentiation and evaluated the expression of representative cell-specific markers. The results obtained suggest that HDAC inhibitors reduce proliferative potential of cultured cells and induce transcriptomic changes associated with cell differentiation and specialization. Particularly, we revealed a significant upregulation of genes typically expressed in neuromodulatory neurons, and downregulation of genes expressed in glia and inhibitory neurons. Transcriptional changes were accompanied by continuous elevation of histone serotonylation levels in both neurons and glia. We assume that early appearance of the enhanced histone serotonylation marks, and the persistence of these changes over many hours in distinct brain cells may indicate that chromatin remodeling induced by histone serotonylation contributes to the maintainance of a new transcriptional programs associated with cellular reprogramming.

Retinal nerve fibre layer thickness reflects characteristics of brain grey and white matter
The retina is a relatively accessible part of the central nervous system compared to the brain. Using high resolution optical imaging we investigated the relationship between retinal thickness, obtained with optical coherence tomography, and structural features of the brain obtained with magnetic resonance imaging. In a population-based sample of over 500 subjects, we hypothesized: (i) that there are structural associations between circumpapillary retinal nerve fibre layer thickness and brain grey matter density and white matter microstructural properties in visual information processing areas, and specifically contralateral associations for nasal retinal fibers, and (ii) that retinal findings reflect broader changes in brain grey and white matter related to cardiovascular risk factors. In support of the first hypothesis, we showed associations of circumpapillary retinal nerve fibre layer thickness with visual cortex grey matter density and with optic radiation fractional anisotropy. These correlations were stronger for the right eye, possibly reflecting right ocular dominancy. Regarding the second hypothesis, while we confirmed the broad impact of cardiovascular risk factors such as body mass index, diabetes, and hypertension on brain structure, we didn't find (adequate) significant partial correlations between circumpapillary retinal nerve fibre layer thickness and cardiovascular risk factors to support the hypothesis. As such, we couldn't confirm that circumpapillary retinal nerve fibre layer thickness is associated with the impact of cardiovascular risk factors on the brain structure. However, when the effects of cardiovascular risk factors were accounted for statistically, circumpapillary retinal nerve fibre layer thickness (particularly on the right side) was associated with fractional anisotropy of limbic system tracts, i.e., the fornix and stria terminalis including hippocampus and amygdala. To further explore the structural associations between eye and brain, in terms of a possible common underlying pathology related to cardiovascular risk factors and progressive neurodegenerative diseases on the central nervous system, longitudinal and interventional studies are necessary.

Alcohol use disorder is associated with increases in frontocentral phase-amplitude coupling strength during resting state
Compromised functional connectivity in the brain seen in fMRI and EEG coherence is a key feature of alcohol use disorder (AUD). Phase-amplitude coupling (PAC) represents another mechanism by which functional connectivity is achieved, and to date, the role played by PAC in understanding the development of AUD has yet to be investigated. In the current study, we compare PAC strengths between participants with severe AUD, defined as presenting with 6 or more symptoms (DSM-5), and unaffected controls. Associations between phase-amplitude coupling and AUD status were assessed using frontal EEG signals acquired during the resting state eyes closed condition as part of the Collaborative Study on the Genetics of Alcoholism (COGA). COGA is a national project following families affected with AUD and comparison families for over 35 years with data collection in multiple domains. COGA participants between 25 and 50 years old with severe AUD were compared to an age-matched group of unaffected controls (Women: N = 360; Men: N = 406). PAC calculations were made on EEG recordings from midfrontal channel FZ to generate high resolution comodulograms covering phase frequencies between 0.1-13 Hz and amplitude frequencies between 4-50 Hz. Comodulograms were generated for each 30 second EEG segment for a given visit. Average comodulogram for a visit was calculated for the first two minutes of EEG in the resting state condition for subsequent statistical analyses. PAC differences between AUD and unaffected controls were assessed at each frequency pair using Mann-Whitney test, and the resulting effect sizes and p-values were used to generate difference comodulograms and significance comodulograms, respectively. Results showed that severe AUD was associated with greater alpha-gamma PAC in both men and women compared to the unaffected groups. Men with AUD exhibited significant increases in PAC across large parts of theta-gamma. In women with AUD, increases in theta-gamma PAC were restricted to smaller domains, and accompanied significant decreases in neighboring theta-gamma subdomains. PAC strength in theta-alpha and alpha-beta frequency pair domains were also significantly greater in AUD for both men and women. The AUD-associated changes in PAC strength within the alpha-gamma, theta-beta, alpha-beta, and to a lesser degree in theta-gamma domains indicates some form of aberrant hyperconnectivity between networks within the medial prefrontal cortex during resting state of the brain.

Layer-specific spatiotemporal dynamics of feedforward and feedback in human visual object perception
Visual object perception is mediated by information flow between regions of the ventral visual stream along feedforward and feedback anatomical connections. However, feedforward and feedback signals during naturalistic vision are rapid and overlapping, complicating their identification and precise functional specification. Here we recorded human layer-specific fMRI responses to naturalistic object images in early visual cortex (EVC) and lateral occipital complex (LOC) to isolate feedforward and feedback information signals spatially by their cortical layer specific termination pattern. We combined these layer-specific fMRI responses with electroencephalography (EEG) responses for the same images to segregate feedforward and feedback signals in both time and space. Feedforward signals emerge early in the middle layers of EVC and LOC, followed by feedback signals in the superficial layer of both regions, and the deep layer of EVC. Comparing the identified dynamics in LOC to a visual deep neural network (DNN), revealed that early feedforward signals in LOC encode medium complexity features, whereas later feedback signals increase the representational format to high complexity features. Together this specifies the spatiotemporal dynamics and functional role of feedforward and feedback information flow mediating visual object perception.

Distinct Ca2+ signatures of leptomeningeal fibroblast subgroups in awake mouse brains in health and inflammation
The leptomeninges, composed of the arachnoid mater, and pia mater, contains distinct subgroups of fibroblasts that differ in location and transcriptomic profiles. These fibroblasts contribute to the blood cerebrospinal fluid barrier under physiological conditions, participate in fibrosis, and support blood brain barrier integrity during injury and disease. However, their Ca2+ signaling profiles and underlying mechanisms in health and disease remain poorly understood. In this study, we divided leptomeningeal fibroblasts into three subgroups based on their locations: arachnoid fibroblasts, pia mater fibroblasts and perivascular fibroblasts. We employed two-photon microscopy in awake transgenic mice expressing Ca2+; indicators in leptomeningeal fibroblasts to investigate spontaneous and behaviorally evoked Ca2+; transients across different fibroblast subgroups. We found that each subgroup exhibits a distinct Ca2+; activity profile, with pia mater fibroblasts showing the highest-amplitude Ca2+; transients. Moreover, these fibroblasts displayed unique responses to both whisker air-puff stimulation and locomotion. We further demonstrated, using a chronically implanted cannula beneath the cranial window, that locomotion-associated vasodilation is followed by TRPV4 channel-mediated fibroblast Ca2+; elevations. Finally, systemic inflammation induced by lipopolysaccharide (LPS) reduced spontaneous Ca2+; transients in pia mater fibroblasts, likely due to macrophage infiltration following the inflammatory response. For the first time, this study characterizes spontaneous and behaviorally evoked Ca2+; dynamics in distinct leptomeningeal fibroblast subgroups in awake animals, providing novel insights into the functional roles of leptomeningeal fibroblasts in the healthy and diseased brain.