bioRxiv Subject Collection: Neuroscience's Journal

An integrated anatomical, functional and evolutionary view of the Drosophila olfactory system
The Drosophila melanogaster olfactory system is one of the most intensively studied parts of the nervous system in any animal. Composed of ~60 independent olfactory neuron classes, with several associated hygrosensory and thermosensory pathways, it has been subject to diverse types of experimental analyses. However, synthesizing the available data is limited by the incompleteness and inconsistent nomenclature found in the literature. In this work, we first 'complete' the peripheral sensory map through the identification of a previously uncharacterized antennal sensory neuron population expressing Or46aB, and the definition of an exceptional 'hybrid' olfactory neuron class comprising functional Or and Ir receptors. Second, we survey developmental, anatomical, connectomic, functional and evolutionary studies to generate an integrated dataset of these sensory neuron pathways -- and associated visualizations -- creating an unprecedented comprehensive resource. Third, we illustrate the utility of the dataset to reveal relationships between different organizational properties of this sensory system, and the new questions these stimulate. These examples emphasize the power of this resource to promote further understanding of the construction, function and evolution of these neural circuits.

Experience-dependent reorganization of inhibitory neuron synaptic connectivity
Organisms continually tune their perceptual systems to the features they encounter in their environment. We have studied how ongoing experience reorganizes the synaptic connectivity of neurons in the olfactory (piriform) cortex of the mouse. We developed an approach to measure synaptic connectivity in vivo, training a deep convolutional network to reliably identify monosynaptic connections from the spike-time cross-correlograms of 4.4 million single-unit pairs. This revealed that excitatory piriform neurons with similar odor tuning are more likely to be connected. We asked whether experience enhances this like-to-like connectivity but found that it was unaffected by odor exposure. Experience did, however, alter the logic of interneuron connectivity. Following repeated encounters with a set of odorants, inhibitory neurons that responded differentially to these stimuli exhibited a high degree of both incoming and outgoing synaptic connections within the cortical network. This reorganization depended only on the odor tuning of the inhibitory interneuron and not on the tuning of its pre- or postsynaptic partners. A computational model of this reorganized connectivity predicts that it increases the dimensionality of the entire network's responses to familiar stimuli, thereby enhancing their discriminability. We confirmed that this network-level property is present in physiological measurements, which showed increased dimensionality and separability of the evoked responses to familiar versus novel odorants. Thus, a simple, non-Hebbian reorganization of interneuron connectivity may selectively enhance an organism's discrimination of the features of its environment.

Endothelial tPA-dependent recruitment of microglia to vessels protects the blood-brain barrier after stroke in mice
Thrombolysis with tissue-type plasminogen activator (tPA) remains the only pharmacological treatment for the acute phase of ischemic stroke. In this study, we hypothesize that endothelial tPA plays a key role in modulating the microglial response and maintaining blood brain barrier (BBB) integrity after stroke. Using a mouse model with conditional deletion for endothelial tPA (VeCadCre-tPAFlox) combined with a thrombotic stroke model and high-resolution imaging, we investigated the effects of endothelial tPA on vascular inflammation and microglia activation during the acute phase of stroke. Our results demonstrate that microglia-vessel contacts increase post-stroke. Notably, endothelial tPA deletion reduces vascular VCAM1 expression associated with decreased microglial activation and fewer microglia-vessel contacts. Following stroke, endothelial tPA deletion is associated with increased BBB permeability and heightened risk of haemorrhagic transformation. Collectively, these findings indicate that endothelial tPA mediates microglial recruitment to blood vessels, thereby exerting a protective effect on BBB integrity following ischemic stroke.

Isometric handgrip contraction increases tibialis anterior intrinsic motoneuron excitability in a dose-dependent manner
Persistent inward currents (PICs) contribution to motoneuron firing in the lower limb typically increase after a remote handgrip contraction, believed to result from diffuse increases of serotonergic input on the spinal cord. We investigated whether handgrip contraction intensity, duration, and/or impulse would affect tibialis anterior estimates of PICs. High-density electromyograms were recorded from the tibialis anterior of 21 participants (18-40 years) during 20% dorsiflexion before and after four handgrip conditions: i) 80%15s, 80% of their maximal handgrip strength sustained for 15s; ii) 40%15s, 40% sustained for 15s; iii) 40%30s, 40% sustained for 30s; and iv) Control (no handgrip). PICs contribution to motoneuron firing was estimated with the delta frequency ({Delta}F) using the paired motor unit analysis. The brace height, normalised as a percentage of a right triangle (%rTri), was used to quantify the effects of PICs on the non-linearity of firing patterns, representing the neuromodulatory drive (regulation of neural activity via neurotransmitter actions) onto the motoneurons. {Delta}F increased by 0.30 peaks per second (pps; 95%CI 0.11-0.49, d=0.37) after 40%30s and by 0.20 pps (0.04-0.36, d=0.24) after 80%15s but remained unchanged after 40%15s and Control. Similarly, brace height increased by 2.39 %rTri (0.55-4.23, d=0.25) after 40%30s and by 2.74 %rTri (1.14-4.34, d=0.28) after 80%15s; remaining unchanged after 40%15s and Control. The increase in PICs contribution to motoneuron firing induced by a remote handgrip contraction is impulse-dependent rather than intensity or duration. The parallel increases in {Delta}F and brace height suggest augmented neuromodulatory input onto the spinal cord.

A preparatory cranial potential for saccadic eye movements in macaque monkeys
Response preparation is accomplished by gradual accumulation in neural activity until a threshold is reached. In humans, such a preparatory signal, referred to as the lateralized readiness potential, can be observed in the EEG over sensorimotor cortical areas before execution of a voluntary movement. Although well-described for manual movements, less is known about preparatory EEG potentials for saccadic eye movements in humans and nonhuman primates. Hence, we describe a lateralized readiness potential over the frontolateral cortex in macaque monkeys. Homologous to humans, we observed lateralized electrical potentials ramping before the execution of both rewarded and non-rewarded contralateral saccades. This potential parallels the neural spiking of saccadic movement neurons in the frontal eye field, suggesting that it may offer a non-invasive correlate of intracortical spiking activity. However, unlike neural spiking in the frontal eye field, polarization in frontolateral channels did not distinguish between saccade generation and inhibition. These findings provide new insights into non-invasive electrophysiological signatures of saccadic preparation in nonhuman primates, highlighting the potential of EEG measures to bridge invasive neural recordings and non-invasive studies of eye movement control in humans.

The quantitative spatiotemporal relationship of whole brain activity of human brains revealed by fMRI
Human brain consists of many functional systems from the essential sensory, motor, attention and memory systems to higher order cognitive functions such as reasoning and language. Performing even a simple task may evoke multiple systems and cognitive functions, resulting in a whole brain activity across the entire brain. Despite the importance of studying task-evoked brain activated networks, investigating this whole brain activity may be crucial for understanding the neural bases of individual behavioral and clinical traits. BOLD-fMRI measures the four-dimensional (3 spatial and 1 temporal) neural activity across the entire brain at large-scale systems level. All local activities across the entire brain constitute the whole brain activity and each local activity is a part of that whole brain activity. Unlike a local activity that is characterized by its temporal neural activity, the whole brain activity is characterized by its spatial variation across the entire brain. We present a novel data-driven method to analyze the whole brain activity when performing tasks. The method enabled us to analyze the whole brain activity for each task trial and each individual subject with no requirement of a priori knowledge of task-evoked BOLD response. Our study revealed a quantitative spatiotemporal relationship of the whole brain activity with the local activities. The whole brain activity demonstrated a remarkable dynamic activity that varied from trial to trial when performing the same task repeatedly, showing the importance of analyzing the whole brain activity for investigating the neural bases of personal traits.

Human and Rodent Seizures Demonstrate a Dynamic Interplay with Spreading Depolarizations
Seizure termination has been linked to spreading depolarizations (SDs) in experimental epilepsy models, and SDs have recently been suggested to protect against seizures. The precise mechanism, however, remains unclear. Additionally, the co-occurrence of SDs with human seizures remains debated. In this study, we found that SDs are a prominent feature following ictal events in both human clinical recordings (n=20 seizures from 7 epilepsy patients) using direct-current amplifiers and in the 0 Mg2+ model of ictogenesis from rodent brain slices (n=17). Approximately one-third of rodent seizure-like events (SLEs) were associated with SDs, while all human seizures analyzed had associated infraslow shifts, a hallmark of SDs. SDs were more prominent in the lateral frontal, medial frontal, and lateral temporal lobes, as well as the insular cortex in human patients, but were observed in all recorded brain regions. In rodents, SDs clustered toward the end of ictal events, resulting in significantly shorter SLEs (SLE without SD: 32.60 {+/-} 5.31 s; SLE with SD: 16.03 {+/-} 4.45 s) and delayed onset of subsequent SLEs. These SDs also caused significant AC band silencing compared to SLEs without SDs. Interestingly, SLEs with SDs displayed significantly more low gamma activity during ictal events. Using ion-selective microelectrodes, we found no significant correlation between extracellular [K+] levels and SLEs ending in SDs, questioning the role of [K+]o in SD induction during seizures. We observed more heterogeneity in human seizures than in rodent SLEs, with some human seizures showing SD-associated termination and others demonstrating SDs in the middle of ictal events; such intra-seizure SDs were never observed in our rodent model. The human data, collected from patients with intractable epilepsy, demonstrate clear SD propagation during seizures and show that SDs appear and propagate, in multiple brain regions simultaneously with ictal events. Collectively, these results indicate that SDs are a hallmark of ictal activity and may contribute to seizure termination in both experimental and clinical settings. Furthermore, these findings provide unique insight into the neuronal dynamics that promote SD induction by showing that increased low gamma activity during SLEs is more predictive of SD induction than extracellular [K+] levels. We also add further support for the hypothesis that SDs are both anti-seizure and anti-ictogenic as they not only limit and delay subsequent ictal activity but also reduce the duration of SLEs. Taken together, these findings provide rationale for further exploration of SDs as a means to prematurely terminate life-threatening seizures.

In vivo Morphological Dynamics of Single Laser-Axotomized Corneal Nerve Fibers in Sarm1-Null Adult Mice
Axonal regeneration represents a pivotal aspect of the adult peripheral nervous system (PNS). When an injury occurs, peripheral axons are able to regenerate and reestablish connections with their original targets. Although several precision techniques for severing axons in various non-mammalian model organisms have allowed to study the dynamics of regeneration after injury, similar axonal injury models have yet to be fully developed in mammals. In this study, we introduced a novel experimental method for laser-induced axotomy of single corneal sensory subbasal nerve fibers. This method enables the in vivo monitoring and quantification of axonal degeneration and regeneration morphological dynamics in adult mice. Results revealed that the degeneration of the distal stump of the subbasal fiber was delayed in mice lacking the protein SARM1, which promotes degenerative process after injury. Meanwhile, the proximal stump maintained the regenerative dynamics of the subbasal fiber, but regeneration of its nerve terminals was impaired. The present study introduces a valuable model for the in vivo study of the morphological plasticity dynamics of peripheral axons after injury in genetically tractable adult mammals.

Plectin Regulates Focal Adhesion Dynamics and Cytoskeletal Organization in Mouse Astrocytes: Implications for Reactive Astrogliosis
Reactive astrogliosis, a hallmark of central nervous system pathologies, involves cellular responses, including morphological remodelling and upregulation of intermediate filaments such as vimentin. These changes are driven by cytoskeletal dynamics and are mediated by focal adhesions (FAs). Our study identifies plectin, a versatile cytoskeletal linker protein, as a critical modulator of FA-associated processes in mouse astrocytes. We demonstrate that plectin localizes to astrocyte FAs, where it regulates their number, maturation, turnover, and the mobility of FA components. Plectin also polarizes within FAs, depending on their maturation state, and controls the recruitment of key cytoskeletal elements particularly vimentin. In plectin-deficient astrocytes, the vimentin network exhibits impaired connectivity, accompanied by altered viscoelastic properties of the cells. In a model of reactive astrocytes FA number and size were elevated along with the expression of plectin, highlighting involvement of plectin in pathological conditions.

Persistent large-scale changes in alternative splicing in prefrontal cortical neuron types following psychedelic exposure
Psychedelics engage the serotonergic system as potent neuromodulators, increasing neuroplasticity in humans and rodents. Persistent changes in cognitive flexibility, emotional regulation, and social cognition are thought to underlie the therapeutic effects of psychedelics. However, the underlying molecular and cellular basis of psychedelic-induced plasticity remains unclear. Here, we identify persistent, cell type-specific alternative splicing changes in the mouse medial prefrontal cortex (mPFC) induced by a single dose of psychedelics. Combining deep RiboTag sequencing and bioinformatics, we find that a single dose of psychedelics modestly alters gene expression while dramatically shifting patterns of alternative splicing lasting at least a month. We connect our functional enrichment and alternative splicing analysis with changes in the extracellular matrix, synaptic physiology, and intrinsic physiology in parvalbumin interneurons days to a week after psychedelic treatment. Our dataset is an essential resource for understanding the persistent, cell type-specific effects of psychedelics on cortical cell types and functions.

VeCell: A Fiji Plugin to unveil the spatio-temporal dynamics of macroglial cell development and proximity to blood vessels
Cortical development results from the proliferation, differentiation, migration and maturation of many cell types. While neuronal development has been extensively studied, the mechanisms regulating the development and maturation of macroglial cells (oligodendrocytes and astrocytes) are still largely to be determined. Here we present VeCell, a Fiji plugin designed to analyse the development of macroglial cells. Using immunolabeling for two transcription factors, Sox9 (specific to macroglial progenitors and astrocytes) and Sox10 (specific to oligodendrocyte lineage), we determined their density, distribution within and across cortical layers and distance to the blood vessels from postnatal day (P) 5 and P60 in the somatosensory cortex. We found that Sox9+ cells are evenly distributed in the cortex with regular intercellular distances and comparable densities between the upper and lower parts of the cortex. In contrast, Sox10+ cells are predominantly concentrated in the lower cortical layers, and exhibited a more random distribution with variable distances between cells. Finally, while confirming the increased density and branching of the vascular network after P5, we also showed that macroglial cells are closer to blood vessels from P15 onward. In summary, VeCell provides a valuable tool for analyzing the development and spatial organization of macroglial cells. Our findings in the cortex reveal distinct patterns of distribution and proximity to the vasculature for macroglial cells during postnatal development. These insights contribute to a deeper understanding of glial maturation and highlight the dynamic interplay between macroglial cells and the vascular network, offering new avenues for future research into brain development and function.

Uncertainty during visual search: Insights from a computational model and behavioral experiment in natural stimuli
Visual search, driven by bottom-up and top-down processes, offers a unique framework for investigating decision-making. This study examines the awareness of the individuals to their own visual search by combining computational modeling with behavioral experiments. Fifty-seven participants performed a classical visual search task in which the goal was to find an object in a natural scene. Crucially, in some trials, the search was interrupted by clearing the screen before the gaze reached the target object. Participants had to report their best guess of the location of the target and the uncertainty on their response. We show that a modified version of the Entropy-Limit Minimization (ELM) model captures scanpaths and perceived target locations, while also revealing that uncertainty is influenced by scanpath length, the distance between the perceived and true target location, and the entropy of decision maps. These findings highlight the capacity of the model to reflect cognitive processes underlying response selection and uncertainty judgment.

Effects of age in the strategic control of recollected content as reflected by modulation of scene reinstatement
A previous study employing fMRI measures of retrieval-related cortical reinstatement reported that young, but not older, adults employ retrieval gating to attenuate aspects of an episodic memory that are irrelevant to the retrieval goal. We examined whether the weak memories of the older adults in that study rendered goal-irrelevant memories insufficiently intrusive to motivate retrieval gating. Young and older participants studied words superimposed on rural or urban scenes, or on pixelated backgrounds. To strengthen memory for background information, word-image pairs were studied twice, initially centrally, and then in one of three locations. During scanning, one retrieval test probed memory for the test words studied backgrounds and another test assessed memory for their location. Background memory performance was markedly higher than in the prior study. Retrieval gating was examined in two scene-selective regions of interest, the parahippocampal place area (PPA) and the medial place area (MPA). In the background task, robust retrieval-related scene reinstatement effects were identified in both age groups. These effects were attenuated (gated) in the location task in the young age group only, replicating the prior finding. The results did not differ when the two groups were sub-sampled to match strength of scene reinstatement when scene information was goal relevant. The findings indicate that older adults failure to gate goal-irrelevant scene information does not reflect age differences in memory strength and may instead reflect an age-related decline in top-down inhibitory control.

Medial entorhinal-hippocampal desynchronization parallels the emergence of memory impairment in a mouse model of Alzheimer's disease pathology
Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive impairments in episodic and spatial memory, as well as circuit and network-level dysfunction. While functional impairments in medial entorhinal cortex (MEC) and hippocampus (HPC) have been observed in patients and rodent models of AD, it remains unclear how communication between these regions breaks down in disease, and what specific physiological changes are associated with the onset of memory impairment. We used silicon probes to simultaneously record neural activity in MEC and hippocampus before or after the onset of spatial memory impairment in the 3xTg mouse model of AD pathology. We found that reduced hippocampal theta power, reduced MEC-CA1 theta coherence, and altered phase locking of MEC and hippocampal neurons all coincided with the emergence of spatial memory impairment in 3xTg mice. Together, these findings indicate that disrupted temporal coordination of neural activity in the MEC-hippocampal system parallels the emergence of memory impairment in a model of AD pathology.

Validation of Structure Tensor Analysis for Orientation Estimation in Brain Tissue Microscopy
Accurate localization of white matter pathways using diffusion MRI is critical to investigating brain connectivity, but the accuracy of current methods is not thoroughly understood. A fruitful approach to validating accuracy is to consider microscopy data that have been co-registered with MRI of post mortem samples. In this setting, structure tensor analysis is a standard approach to computing local orientations for validation. However, structure tensor analysis itself has not been well-validated and is subject to uncertainty in its angular resolution, and selectivity to specific spatial scales. In this work, we conducted a simulation study to investigate the accuracy of using structure tensors to estimate the orientations of fibers arranged in configurations with and without crossings. We examined a range of simulated conditions, with a focus on investigating the method's behavior on images with anisotropic resolution, which is particularly common in microscopy data acquisition. We also analyzed 2D and 3D optical microscopy data. Our results show that parameter choice in structure tensor analysis has relatively little effect on accuracy for estimating single orientations, although accuracy decreases with anisotropy. On the other hand, when estimating the orientations of crossing fibers, the choice of parameters becomes critical, and poor choices result in orientation estimates that are essentially random. This work provides a set of recommendations for researchers seeking to apply structure tensor analysis effectively in the study of axonal orientations in brain imaging data and quantifies the method's limitations, particularly in the case of anisotropic data.

Backward alpha oscillations shape perceptual bias under probabilistic cues
Predictive coding theory suggests that prior knowledge is crucial for optimizing human decision-making, with recent studies emphasizing the role of alpha-band oscillations in this process. Here, we employed a traveling waves approach to investigate how alpha oscillations integrate prior expectations during a perceptual decision-making task. Our findings demonstrated that expectation-based knowledge triggers the propagation of alpha traveling waves from frontal to occipital areas, with this increase associated with enhanced modulation of brain regions involved in stimulus processing and directly linked to prior-driven bias at the behavioral level. Moreover, participants who relied more on prior expectations exhibited stronger top-down signaling, whereas those who focused on sensory input showed a contrasting forward signaling pattern. These results highlight the role of alpha-band traveling waves in predictive mechanisms, suggesting that rhythmic interactions across brain regions facilitate this process and contribute to inter-individual differences in its implementation.

Calcineurin-mediated regulation of GAP-43 is essential for neurite and synapse formation and protects against α-synuclein-induced degeneration
Rise in calcium (Ca2+) and hyperactivation of Ca2+-dependent phosphatase calcineurin are two key determinants of -synuclein (-syn) pathobiology implicated in synucleinopathies such as Dementia with Lewy Bodies and Parkinson's Disease. Calcineurin activity can be inhibited with FK506, a Food and Drug Administration (FDA)-approved compound. Our previous work demonstrated a protective effect of sub-saturating doses of FK506 against -syn pathology in a rat model of -syn neurodegeneration. This neuroprotection was associated with the phosphorylation of GAP-43. In this study, we present evidence that phosphorylation of GAP-43 regulated by calcineurin is a critical determinant for neurite branching and synapse formation. Phosphorylation of GAP-43 promotes neurite branching and synapse formation whereas dephosphorylation prevents it. Therefore, our findings provide a novel way in which GAP-43 activity can be regulated by calcineurin and provide the mechanistic basis for neuroprotection for FK506 effects in neurons experiencing -syn proteotoxic stress.

Individualized Alpha-tACS for Modulating Pain Perception and Neural Oscillations: A Sham-Controlled Study in Healthy Participants
Pain encompasses sensory, affective, and cognitive dimensions, with neural oscillations increasingly recognized as key mechanisms in their integration. However, the underlying processes remain inadequately understood. Transcranial alternating current stimulation (tACS) offers a promising tool for modulating these oscillations, yet the widespread reliance on 'one-size-fits-all' tACS protocols with fixed frequencies has led to limited and contradictory findings on its efficacy in pain treatment. In this study, we employed individualized tACS at individual peak alpha frequency (IAF) over the primary motor cortex (M1) contralateral to the dominant arm of 38 healthy participants, in a within-subject, sham-controlled design, to investigate its effects on pain perception and neural oscillations. Sustained and periodic 0.2 Hz thermonociceptive stimuli were applied to the dominant forearm before and after tACS. We measured participants pain perception and heat pain thresholds (HPT) before and after tACS stimulation. Scalp electroencephalography (EEG) measurements were used to measure neural activity during thermonociceptive stimuli. To calculate IAF, we used a discriminative approach based on independent component analysis (ICA) to separate sensorimotor related IAF (SM-IAF). The results revealed an overall increase in pain perception and a decrease in HPT in both sham and active conditions, with no significant interactions between conditions. However, a trend toward reduced sensitization post-tACS was observed. Exploratory analyses indicated a significant tACS effect on HPT in women. Furthermore, a significant correlation was found between SM-IAF and HPT. These findings provide a novel perspective on advancing individualized neuromodulation approaches for pain and neurobiological disorders.

Preclinical Evaluation of P4B-2412 as Phosphodiesterase 4B Radioligand for Positron Emission Tomography Imaging
Phosphodiesterase 4B (PDE4B) plays a critical role in cAMP hydrolysis and is highly expressed in brain regions associated with neuroinflammation and central nervous system (CNS) disorders. Selective PDE4B radioligands hold significant potential for elucidating disease mechanisms, such as those in Parkinson's disease and schizophrenia, and enabling target occupancy measurements. In this study, we developed [18F]P4B-2412, a novel PDE4B-selective radioligand, and evaluated its utility for positron emission tomography imaging (PET). [18F]P4B-2412 was synthesized in high radiochemical yield (27.2%), excellent radiochemical purity (99%), and favorable molar activity (66.2 +/- 2.5 GBq/mol. In vitro autoradiography and dynamic PET imaging demonstrated high specificity for PDE4B in rodent brain regions, with blocking studies confirming negligible interaction with PDE4D. [18F]P4B-2412 also exhibited robust in vitro and in vivo metabolic stability. These results establish [18F]P4B-2412 as a promising PET imaging agent for visualizing PDE4B activity, offering a valuable tool for investigating neuroinflammation and advancing CNS drug development.

Thalamic involvement defines distinct slow-wave subtypes in NREM sleep
Slow waves (0.5-4 Hz) are a key feature of non-rapid-eye-movement (NREM) sleep, traditionally believed to arise from neocortical circuits. However, growing evidence suggests that subcortical structures, particularly the thalamus, may play a crucial role in initiating and synchronizing slow waves. We tested the hypothesis that slow waves may arise from distinct cortico-cortical and thalamo-cortical mechanisms using simultaneous EEG-fMRI in healthy adults. Spatial mapping based on thalamic fMRI responses revealed two slow-wave associated clusters. Cluster-1 C1, characterized by an early thalamic fMRI-signal increase, corresponded to large, efficiently synchronized waves associated with sleep spindles and with markers of higher arousal and autonomic activation. Cluster-2 C2, marked by an initial negative fMRI response, corresponded to smaller slow waves potentially resulting from cortico-cortical synchronization. These waves tended to more often occur during low-fragility phases of NREM sleep. These findings highlight distinct slow-wave subtypes with different thalamic involvement and, potentially, synchronization mechanisms.

Sequential visual stimuli increase high frequency power in the visual cortex
Today, 40 Hz flickering full-field visual stimulation is used to entrain neuronal oscillations for a variety of therapeutic purposes. We here propose spatially organized sequential visual flickering stimulation as a newer tool to entrain the visual system. We show that sequential visual flickering can evoke increased power in high frequencies (100 to 190 Hz) in the visual cortex of mice. Consequently, sequential sensory stimulation should be regarded as a putative new way leading to power increases in high frequency domains.

Technical and biological sources of noise confound multiplexed enhancer AAV screening
Cis-acting regulatory enhancer elements are valuable tools for gaining cell type-specific genetic access. Leveraging large chromatin accessibility atlases, putative enhancer sequences can be identified and deployed in adeno-associated virus (AAV) delivery platforms. However, a significant bottleneck in enhancer AAV discovery is charting their detailed expression patterns in vivo, a process that currently requires gold-standard one-by-one testing. Here we present a barcoded multiplex strategy for screening enhancer AAVs at cell type resolution using single cell RNA sequencing and taxonomy mapping. We executed a proof-of-concept study using a small pool of validated enhancer AAVs expressing in a variety of neuronal and non-neuronal cell types across the mouse brain. Unexpectedly, we encountered substantial technical and biological noise including chimeric packaging products, necessitating development of novel techniques to accurately deconvolve enhancer expression patterns. These results underscore the need for improved methods to mitigate noise and highlight the complexity of enhancer AAV biology in vivo.

Category-biased patches encircle core domain-general regions in the human lateral prefrontal cortex
The fine-grained functional organization of the human lateral prefrontal cortex (PFC) remains poorly understood. Previous fMRI studies delineated focal domain-general, or multiple-demand (MD), PFC areas that co-activate during diverse cognitively demanding tasks. While there is some evidence for category-selective (face and scene) patches, in human and non-human primate PFC, these have not been systematically assessed. Recent precision fMRI studies have also revealed sensory-biased PFC patches adjacent to MD regions. To investigate if this topographic arrangement extends to other domains, we analysed two independent fMRI datasets (n=449 and n=37) utilizing the high-resolution multimodal MRI approaches of the Human Connectome Project (HCP). Both datasets included cognitive control tasks and stimuli spanning different categories: faces, places, tools and body parts. Contrasting each stimulus category against the remaining ones revealed focal interdigitated patches of activity located adjacent to core MD regions. The results were robust, replicating across different executive tasks, experimental designs (block and event-related) and at the single subject level. Our results paint a refined view of the fine-grained functional organization of the PFC, revealing a recurring motif of interdigitated domain-specific and domain-general circuits. This organization offers new constraints for models of cognitive control, cortical specialization and development.

The medial prefrontal cortex encodes procedural rules as sequential neuronal activity dynamics
The prefrontal cortex plays a crucial role in procedural rule learning; however, the specific neuronal mechanism through which it represents rules is unknown. We hypothesized that sequential neuronal activities in the prefrontal cortex encode these rules. To investigate this, we recorded neuronal activities in the medial prefrontal cortex of mice during rule learning using Ca2+ imaging. We utilized a method based on convolutional negative matrix factorization, iSeq, to automatically detect temporal neuronal sequences in the recorded data. As rule learning advanced, these neuronal sequences began to encode critical information for rule execution. In mice that had mastered the rule, the dynamics of neuronal sequences could predict success and failure of reward acquisition. Furthermore, the composition of cell populations within the neuronal sequences was rearranged throughout the learning process. These findings suggest that as animals learn a rule, the medial prefrontal cortex continually updates its neuronal sequences to assign significance to behavioral actions crucial for reward acquisition.

Microglia regulate nucleus accumbens synaptic development and circuit function underlying threat avoidance behaviors
Microglia are the resident immune cells of the CNS and modulate synaptic connectivity and function in the developing brain1-3. While microglia have well-established roles in synapse pruning4,5, only a few studies have identified roles for microglia in synapse formation6-8. These studies focused on the cortex and primary sensory circuits during restricted developmental time periods, leaving substantial gaps in our understanding of the early developmental functions of microglia. Here we investigated how the absence of microglia impacts synaptic development in the nucleus accumbens (NAc), a region critical for emotional regulation and motivated behaviors and where dysfunction is implicated in psychiatric disorders that arise early in life9-11. Using a genetically modified mouse that lacks microglia (Csf1r{Delta}FIRE/{Delta}FIRE)12, we found blunted excitatory synapse formation in the NAc. This effect was most prominent during the second and third postnatal weeks, when we previously found microglia to be overproduced13, and was accompanied by an increase in presynaptic release probability and alterations in postsynaptic kinetics. Tissue-level NAc proteomics confirmed that microglial absence impacted numerous proteins involved in synapse structure, trans-synaptic signaling, and pre-synaptic function. However, microglial absence did not perturb levels of astrocyte-derived cues and adhesive proteins that promote synaptogenesis, suggesting that reduced synapse number may be caused by absence of a microglial-derived synaptogenic cue. Although observed electrophysiological synaptic changes were largely normalized by adulthood, we identified lasting effects of microglial absence on threat avoidance behavior, and these behavioral effects were directly associated with alterations of NAc neuronal activity. Together, these results indicate a critical role for microglia in regulating the synaptic landscape of the developing NAc and in establishing functional circuits underlying adult behavioral repertoires.

Impaired glymphatic clearance independently contributes to poor outcomes in Parkinsons disease
Background: Impaired glymphatic clearance may contribute to pathological accumulations in Parkinsons (PD), but how it interacts with other processes causing dementia and poor outcomes remains unclear. Objectives: Clarify how glymphatic clearance impacts cognition in PD and its interaction with established imaging markers. Methods: We used diffusion tensor image analysis along the perivascular space (DTI-ALPS) as an indirect marker of glymphatic clearance in 98 PD patients (31 PD-poor outcomes: dementia, mild cognitive impairment, frailty or death within 3-year follow-up; 67 PD-good outcomes) and 28 controls. We assessed DTI-ALPS relationship to cognition, white matter (fibre cross-section), cortical thickness, iron accumulation (quantitative susceptibility mapping (QSM)), and plasma markers (phosphorylated tau-181 (p-tau181 and neurofilament light (NFL)) cross-sectionally and longitudinally. Results: DTI-ALPS was lower in PD-poor outcomes compared to PD-good outcomes and controls (p=0.005) with further longitudinal reductions only in PD-poor outcomes (group*time interaction: beta=-0.013, p=0.021). Lower DTI-ALPS was associated with lower fibre cross-section in PD, at baseline and longitudinally but with different spatial distribution from white matter changes relating to PD cognition. There was no correlation between baseline DTI-ALPS and plasma ptau-181 (p=0.642), NFL (p=0.448) or baseline cortical thickness. Lower DTI-ALPS was associated with accelerated cortical thinning within left precentral gyrus and changes in brain iron distribution. Conclusions: PD patients who develop poor outcomes show impaired glymphatic clearance at baseline that worsened longitudinally. DTI-ALPS correlated with white matter integrity and brain iron accumulation. However, both showed different spatial distribution than that seen in PD dementia; suggesting impaired glymphatic clearance contributes to cognitive decline in a distinct manner.

EEG Responses to Exercise Intensity in Parkinson's Disease
1. Abstract 1.1. Background Exercise is increasingly recognized as a beneficial intervention for Parkinson's disease (PD), yet the optimal type and intensity of exercise remain unclear. This study investigated the relationship between exercise intensity and neural responses in PD patients, using electroencephalography (EEG) to explore potential neural markers for optimal exercise intensity. 1.2. Method EEG data were collected from 14 PD patients (5 females) and 8 healthy controls (HC) performing stationary pedaling exercises at 60 RPM with resistance adjusted to target heart rates of 30%, 40%, 50%, 60%, and 70% of maximum heart rate. Subjects pedaled for 3 minutes at each intensity level in a counterbalanced order. Canonical Time-series Characteristics (Catch-22) features and Multi-set Canonical Correlation Analysis (MCCA) were utilized to identify common profiles of EEG features at increasing exercise intensity across subjects. 1.3. Results We identified a statistically significant MCCA component demonstrating a monotonic relationship with pedaling intensity. The dominant feature in this component was Periodicity Wang (PW), reflecting the autocorrelation of neural dynamics. Analysis revealed a consistent trend across features: six features increased with intensity, indicating heightened rhythmic engagement and sustained neural activation, while three features decreased, suggesting reduced variability and enhanced predictability in neural responses. Notably, PD patients exhibited more rigid, consistent response patterns compared to healthy controls (HC), who showed greater flexibility and variability in their neural adaptation across intensities. 1.4. Conclusion This study highlights the feasibility of using EEG-derived features to track exercise intensity in PD patients, identifying specific neural markers correlating with varying intensity levels. PD subjects demonstrate less inter-subject variability in motor responses to increasing intensity. Our results suggest that EEG biomarkers can be used to assess differing brain involvement with the same exercise of increasing intensity, potentially useful for guiding targeted therapeutic strategies and maximizing the neurological benefits of exercise in PD.

Detection of alpha synuclein seeding activity in tear fluid in patients with Parkinson's disease
Detection of alpha-synuclein seeding activity in tear fluid (TF) might provide a promising non-invasive biomarker for Parkinson's disease (PD) diagnosis. In this study, we applied the alpha-synuclein seeding amplification assay (aSynSAA) to detect misfolded alpha-synuclein (aSyn) aggregation in TF from PD patients. The discovery cohort included 11 PD patients and 13 controls, and the validation cohort consisted of 9 PD patients and 11 controls without synucleinopathies. The aSynSAA yielded positive results in over 55% of PD patients. These findings were confirmed in a second cohort, including patients with prion diseases as a negative control for synuclein pathology. Our results demonstrate for the first time the ability of aSynSAA to distinguish between PD and control groups in TF, with PD showing the highest seeding activity compared to prion disease and control groups. Further comparisons between cerebrospinal fluid (CSF) and TF samples from the same individuals revealed consistent seeding results across both biofluids. These findings highlight the potential of tear fluid as a novel, accessible medium for detecting Lewy body-specific misfolded synuclein aggregation in PD, which could aid in early diagnosis and disease progression monitoring.

Stimulus-driven cerebrospinal fluid dynamics is impaired in glaucoma patients
Cerebrospinal fluid (CSF) dynamics, driven by sensory stimulation-induced neuronal activity, is crucial for maintaining homeostasis and clearing metabolic waste. However, it remains unclear whether such CSF flow is impaired in age-related neurodegenerative diseases of the visual system. This study addresses this gap by examining CSF flow during visual stimulation in glaucoma patients and healthy older adults using functional magnetic resonance imaging. The findings reveal that in glaucoma, CSF inflow becomes decoupled from visually evoked blood-oxygenation-level-dependent (BOLD) response. Furthermore, stimulus-locked CSF patterns, characterized by decreases following stimulus onset and increases after offset, diminish as glaucoma severity worsens. Mediation analysis suggests that this flattened CSF pattern is driven by a flatter BOLD slope, resulting in a shallower CSF trough and a reduced rebound. These findings unveil a novel pathophysiological mechanism underlying disrupted stimulation-driven CSF dynamics in glaucoma and highlight potential in vivo biomarkers for monitoring CSF in the glaucomatous brain.

An amygdala-cortical circuit for encoding generalized fear memories.
Generalized learning is a fundamental process observed across species, contexts, and sensory modalities that enables animals to use past experiences to adapt to changing conditions. Evidence suggests that the prefrontal cortex (PFC) extracts general features of an experience that can be used across multiple situations. The anterior cingulate cortex (ACC), a region of the PFC, is implicated in generalized fear responses to novel contexts. However, the ACCs role in encoding contextual information is poorly understood, especially under increased threat intensity that promotes generalization. Here, we show that synaptic plasticity within the ACC and signaling from amygdala inputs during fear learning are necessary for generalized fear responses to novel encountered contexts. The ACC did not encode specific fear to the training context, suggesting this region extracts general features of a threatening experience rather than specific contextual information. Together with our previous work, our results demonstrate that generalized learning about threatening contexts is encoded, in part, within an ascending amygdala-cortical circuit, whereas descending ACC projections to the amygdala drive generalized fear responses during exposure to novel contexts. Our results further demonstrate that schematic learning can occur in the PFC after single-trial learning, a process typically attributed to learning over many repeated learning episodes.

Classifying Mild Cognitive Impairment from Normal Cognition: fMRI Complexity Matches Tau PET Performance
Background: Tau protein accumulation is closely linked to synaptic and neuronal loss in Alzheimers disease (AD), resulting in progressive cognitive decline. Although tau-PET imaging is a direct biomarker of tau pathology, it is costly, carries radiation risks, and is not widely accessible. Resting-state functional MRI (rs-fMRI) complexity-an entropy-based measure of BOLD signal variation-has been proposed as a non-invasive surrogate biomarker of early neuronal dysfunction associated with tau pathology. Objectives: To determine whether fMRI-based brain complexity (sample entropy and multiscale entropy) can match or exceed tau-PET in classifying cognitively normal (CN) versus cognitively impaired (MCI/AD) individuals. And to investigate and compare the most influential network regions-of-interest (ROIs) for classification between fMRI complexity and tau-PET, thereby identifying key neuroanatomical correlates of AD-related changes. Design: A cross-sectional study employing 3D convolutional neural network (CNN) classification with five-fold cross-validation and leave-one-network-out analysis. Setting: Data from the Alzheimers Disease Neuroimaging Initiative (ADNI) database. Participants: One hundred forty-seven older adults (age 72.5 (SD: 7.5) years), including 95 CN, 45 MCI, and 7 AD. Measurements: We created whole-brain complexity maps from rs-fMRI and standardized uptake value ratio (SUVR) maps from tau-PET. Each modality was separately fed into CNN classifiers. Region-based analyses (leave-one-network-out) were performed to identify critical ROIs for classification. Results: fMRI complexity showed classification accuracy comparable to tau-PET yet surpassed it in F1-score (0.64 vs. 0.61) and area under the curve (AUC; 0.73 vs. 0.67). Salience and dorsal attention networks contributed most to fMRI-based classification, and a dorsal attention network contributed most to tau-PET-based classification. Conclusions: fMRI complexity performs similarly to tau-PET in detecting cognitive impairment related to AD and identifies partially distinct critical ROIs, suggesting an alternative, radiation-free imaging biomarker for earlier detection and broader clinical application.

Apolipoprotein E ε4 exacerbates microglia-mediated complement-dependent synapse loss caused by neuronal Tpk deficiency
Thiamine pyrophosphokinse-1 (TPK) is a key enzyme that converts thiamine to functional thiamine diphosphate (TDP). TPK insufficiency and hence TDP reduction in neurons induced by amyloid-{beta} deposition and diabetes, an independent risk factor of Alzheimer's disease (AD), recapitulate multi-pathophysiological features in the brain of mice, similar to those in human AD. Apolipoprotein E {varepsilon}4 allele (APOE4) is the most well-known genetic risk factor for AD. Clinical trials by boosting TDP using benfotiamine, a derivative of thiamine that significantly elevates TDP level in human encrythrocytes, have shown the inferior clinical efficacies in APOE4 carriers compared to non-APOE4 carriers. Clarifying the relationship between APOE4 and TPK expression and multi-pathophysiological characteristics of AD induced by Tpk deficiency in neurons is imperative. Here, we find that humanized APOE4 didn't directly affect Tpk expression of mice, but markedly aggravates behavior abnormalities of Tpk-cKO mice. Pathologically, the Tpk-cKO mice with humanized APOE4 knock-in (AE-cKO mice) exhibit more synapse loss than the mice with only humanized APOE4 knock-in and the Tpk-cKO mice. Transcriptomics and pathologic analysis identified that APOE4 promoted the overactivation of microglia and the transition of microglia to a disease-associated and phagocytosis state via a complement-mediated pathway. Further, the C3aR antagonist significantly repressed microglia phagocytosis and synaptic elimination of the AE-cKO mice. Our results demonstrate that APOE4 exacerbates behavior dysfunction of Tpk-cKO mice through microglia-mediated complement-dependent synaptic elimination. These findings provide important insights into the role of APOE4 in the pathogenesis of AD.

Loss of excitatory inputs and decreased tonic and evoked activity of locus coeruleus neurons in aged P301S mice
Tau pathology in the locus coeruleus (LC) is associated with several neurodegenerative conditions including Alzheimers disease and frontotemporal dementia. Phosphorylated tau accumulates in the LC and results in inflammation, synaptic loss, and eventually cell death in other brain regions as the disease progresses. Loss of LC neurons and noradrenergic innervation is thought to contribute to the symptoms of cognitive decline later in disease. While loss and degeneration of LC neurons has been well studied, less is known about changes in LC physiology at advanced stages of tau pathology that precedes neurodegeneration. In this study, we investigated the ex vivo electrophysiological properties of LC neurons in male and female mice from the P301S mouse model of tauopathy at 9 months of age, a time-point when significant tau accumulation, cell death, and cognitive impairments are observed. We found a reduction in excitatory inputs and changes in excitatory post-synaptic current kinetics in male and female P301S. There was also a decrease in spontaneous discharge of LC neurons and an increase in AP threshold in P301S mice of both sexes. Finally, we observed a decrease in excitability and increase in rheobase current in P301S mice. Despite the decrease in LC activity in slice, we did not identify differences in total tissue norepinephrine (NE) or NE metabolites in prefrontal cortex or hippocampus. Together these findings demonstrate reductions in the activity and excitability of LC neurons at late stages of tau accumulation. However, compensatory mechanisms may maintain normal NE levels in LC projection regions in vivo.

Reciprocal roles of crowding and serial dependence on visual perception
Visual perception arises from the interplay between current and prior sensory inputs. Two perceptual phenomena-serial dependence and visual crowding-result from the mandatory integration of retinal information across time and space, respectively. This study investigated, for the first time, their functional relationship for orientation and brightness discrimination tasks. Participants performed orientation and brightness discrimination tasks (blocked) on peripheral targets surrounded by distractors of varying color, orientation, and proximity. Both serial dependence (the influence of prior stimuli on current judgments) and visual crowding (impaired peripheral recognition caused by nearby distractors) were observed. Similar double dissociations in task and stimulus specificity emerged for both phenomena, suggesting functional links mediated by distinct processing mechanisms for brightness and orientation judgments. Additionally, the two effects interacted: crowding reduced serial dependence on subsequent trials, indicating that spatial information is prioritized over temporal information when spatial redundancy is high. Conversely, serial dependence increased crowding for orientation judgments, while for brightness judgments, serial dependence operated additively without affecting crowding. These interactions were spatially specific, occurring only when targets appeared in the same location across consecutive trials. Notably, a positive correlation between crowding and serial dependence was found exclusively for orientation judgments and only when targets appeared at the same location across trials. The reciprocal interaction and positive correlation between serial dependence and crowding observed for orientation judgments indicate that that these processes are intrinsically linked, possibly via a common neural mechanism. In contrast, for brightness judgments, the unilateral effect of crowding on serial dependence-along with the absence of correlation-indicates distinct neural mechanisms. Here, brightness crowding likely precedes serial dependence, reflecting a prioritization of spatial redundancy minimization over temporal stabilization. These findings suggest that the brain strategically integrates spatial and temporal information through distinct yet overlapping mechanisms, balancing spatial precision with temporal stability based on task demands and stimulus characteristics.

Repopulation of the brain with microglia-like cells following intraperitoneal bone marrow cell transfer in microglia-deficient mice.
Germ-line deletion of a conserved enhancer (the Fms intrinsic regulatory element, FIRE) in the mouse Csf1r locus causes congenital absence of microglia. Homozygous FIRE deletion on a C57BL/6J background leads to perinatal lethality and hydrocephalus (HC) in surviving pups. We developed a congenic C57BL/6J line with defined regions of non-C57BL/6J genomic DNA, increased postnatal viability and reduced incidence of HC. Both perinatal lethality and HC were eliminated in F2 mice following outcross of the congenic line to CBA/J or BALBc/J backgrounds. To assess the impacts of microglial deficiency in postnatal neurodevelopment we analyzed deep total RNA-seq data from multiple brain regions of wild-type and mutant mice. Aside from the loss of microglial-specific transcripts, we found no significant alterations in relative abundance of any cell-type or region-specific transcriptomic signature. Transcripts associated with endosome/lysosome function, which are enriched in microglia, were not affected, suggesting compensatory expression by other cell types. On the C57BL/6J x CBA/J F2 background, congenital absence of microglia did not affect motor activity, behavior or myelination up to 7 months of age but was associated with astrocytosis and calcification in the thalamus. In the congenic C57BL/6J Csf1r mutant mouse line, intraperitoneal transfer of wild-type bone marrow cells (BMT) at weaning led to complete repopulation of the brain with microglia-like cells without giving rise to monocytic intermediates. Our results suggest novel strategies for treatment of microglial deficiency.

Gaze dynamics prior to navigation support hierarchical planning
The task of planning future actions in the context of an uncertain world results in massive state spaces that preclude exhaustive search and other strategies explored in the domains of both human decision-making and computational agents. One plausible solution to this dimensionality explosion is to decompose the task into subgoals that match the information geometry of the task at hand. However, how individuals identify a productive hierarchy, and perceive and select subgoals suitable to planning, is not well understood. To investigate this topic, we designed a virtual-reality based behavioral experiment which collected eye movements during a pre-navigation planning phase. By capturing gaze dynamics correlated with the simulative processes used in planning, we were able to identify the spatiotemporal evolution of visual search under uncertainty. Our results highlight gaze dynamics indicative of a search process that exhibits hierarchical structure. These include a decreasing trend seen in gaze distance from origin and a broad to narrow shift (with reducing saccade distances and longer fixation durations) as plans are established. In line with prior work, critical tiles to which landscape connectivity is most sensitive were the strongest predictors of visual attention. We also find that deeper planning was correlated with success only on the most complex maps (e.g. those with a larger number of information-nodes, higher branching factor, and more forks, according to an info-graphical map analysis). This study highlights the role of embodied visual search during planning, and the skill-dependence of the specific subgoals and hierarchical decomposition used which unlocked successful performance.