bioRxiv Subject Collection: Neuroscience's Journal

A deep learning-based strategy for producing dense 3D segmentations from sparsely annotated 2D images
Producing dense 3D reconstructions from biological imaging data is a challenging instance segmentation task that requires significant ground-truth training data for effective and accurate deep learning-based models. Generating training data requires intense human effort to annotate each instance of an object across serial section images. Our focus is on the especially complicated brain neuropil, comprising an extensive interdigitation of dendritic, axonal, and glial processes visualized through serial section electron microscopy. We developed a novel deep learning-based method to generate dense 3D segmentations rapidly from sparse 2D annotations of a few objects on single sections. Models trained on the rapidly generated segmentations achieved similar accuracy as those trained on expert dense ground-truth annotations. Human time to generate annotations was reduced by three orders of magnitude and could be produced by non-expert annotators. This capability will democratize generation of training data for large image volumes needed to achieve brain circuits and measures of circuit strengths.

Network nature of ligand-receptor interactions underlies disease comorbidity in the brain
Neurodegenerative disorders have overlapping symptoms and have high comorbidity rates, but this is not reflected in overlaps of risk genes. We have investigated whether ligand-receptor interactions (LRIs) are a mechanism by which distinct genes associated with disease risk can impact overlapping outcomes. We found that LRIs are likely disrupted in neurological disease and that the ligand-receptor networks associated with neurological diseases have substantial overlaps. Specifically, 96.8% of LRIs associated with disease risk are interconnected in a single LR network. These ligands and receptors are enriched for roles in inflammatory pathways and highlight the role of glia in cross-disease risk. Disruption to this LR network due to disease-associated processes (e.g. differential transcript use, protein misfolding) is likely to contribute to disease progression and risk of comorbidity. Our findings have implications for drug development, as they highlight the potential benefits and risks of pursuing cross-disease drug targets.

Functional Hierarchy of the Human Neocortex from Cradle to Grave
Recent evidence indicates that the organization of the human neocortex is underpinned by smooth spatial gradients of functional connectivity (FC). These gradients provide crucial insight into the relationship between the brain's topographic organization and the texture of human cognition. However, no studies to date have charted how intrinsic FC gradient architecture develops across the entire human lifespan. In this work, we model developmental trajectories of the three primary gradients of FC using a large, high-quality, and temporally-dense functional MRI dataset spanning from birth to 100 years of age. The gradient axes, denoted as sensorimotor-association (SA), visual-somatosensory (VS), and modulation-representation (MR), encode crucial hierarchical organizing principles of the brain in development and aging. By tracking their evolution throughout the human lifespan, we provide the first ever comprehensive low-dimensional normative reference of global FC hierarchical architecture. We observe significant age-related changes in global network features, with global markers of hierarchical organization increasing from birth to early adulthood and decreasing thereafter. During infancy and early childhood, FC organization is shaped by primary sensory processing, dense short-range connectivity, and immature association and control hierarchies. Functional differentiation of transmodal systems supported by long-range coupling drives a convergence toward adult-like FC organization during late childhood, while adolescence and early adulthood are marked by the expansion and refinement of SA and MR hierarchies. While gradient topographies remain stable during late adulthood and aging, we observe decreases in global gradient measures of FC differentiation and complexity from 30 to 100 years. Examining cortical microstructure gradients alongside our functional gradients, we observed that structure-function gradient coupling undergoes differential lifespan trajectories across multiple gradient axes.

Dopamine D1 receptor expression in dlPFC inhibitory parvalbumin neurons may contribute to higher visuospatial distractibility in marmosets versus macaques
Marmosets and macaques are common non-human primate models of cognition, but evidence suggests that marmosets perform more poorly and appear more distractible during cognitive tasks. Prior experimental and theoretical work in macaques suggests that dopaminergic modulation and inhibitory parvalbumin (PV) neurons could contribute to distractibility during cognitive performance. Thus, we compared the two species using a visual fixation task with distractors, performed molecular and anatomical analyses in the dorsolateral prefrontal cortex (dlPFC), and linked functional microcircuitry with cognitive performance using computational modeling. We found that marmosets are indeed more distractible than macaques, and that marmoset dlPFC PV neurons contain higher levels of dopamine-1 receptor (D1R) transcripts and protein. The computational model suggested that higher D1R expression in marmoset dlPFC PV neurons may induce distractibility within the typical, mid D1R stimulation range. Our interdisciplinary study can inform species choice for translational studies of cognition, and clarify microcircuit mechanisms for distractor resistance.

Single-cell variations of circadian clock and immune gene expression in microglia and neurodegeneration
We investigated the diurnal rhythmicity of gene expression in microglia, the resident macrophages of the brain, in health and disease. Using RNA sequencing and single-cell analysis by RNAscope, we examined wild-type mice and the R6/2 transgenic mouse model of Huntington's disease (HD). Our findings suggest context-dependent rhythmic gene expression in microglia, exhibiting substantial variability between individual cells and brain regions over 24 hours. Notably, we observed loss of rhythmic gene expression of key clock genes in microglia from symptomatic but not presymptomatic R6/2 mice. Moreover, we identified de novo 24-hour rhythmic gene expression and altered diurnal patterns of immune-related genes associated with neurodegenerative diseases in microglia from symptomatic R6/2 mice. Our findings suggest circadian reprogramming of microglia in the context of neurodegeneration.

Vocal error monitoring in the primate auditory cortex
Sensory-motor control requires the integration and monitoring of sensory feedback resulting from our behaviors. This self-monitoring is thought to result from comparisons between predictions of expected sensory consequences of action and the feedback actually received, resulting in activity that encodes feedback error. Although similar mechanisms have been proposed during speech and vocal production, including sensitivity to experimentally-perturbed auditory feedback, evidence for a vocal error signal has been limited. Here, we recorded from the auditory cortex of vocalizing non-human primates, using real-time frequency shifts to introduce feedback errors of varying magnitude and direction. We found neural activity that scaled with the magnitude of feedback error in both directions, consistent with vocal error monitoring at both the individual unit and population levels. This feedback sensitivity was greater than that predicted based upon passive sensory responses and was more specific for units in the vocal frequency range. Similar patterns of sensitivity were seen in response to natural variations in produced vocal acoustics. These results provide evidence that the auditory cortex encodes the degree of vocal feedback error using both unit-level error calculations and changes in the population of neurons involved. These mechanisms may provide critical error information necessary for feedback-dependent vocal control.

Complex harmonics reveal low-dimensional manifolds of critical brain dynamics
The brain needs to perform time-critical computations to ensure survival. A potential solution lies in the non-local, distributed computation at the whole-brain level made possible by criticality and amplified by the rare long-range connections found in the brain's unique anatomical structure. This non-locality can be captured by the mathematical structure of Schroedinger's wave equation, which is at the heart of the novel CHARM (Complex Harmonics decomposition) framework that performs the necessary dimensional manifold reduction able to extract non-locality in critical spacetime brain dynamics. Using a large neuroimaging dataset of over 1,000 people, CHARM captured the critical, non-local/long-range nature of brain dynamics and the underlying mechanisms were established using a precise whole-brain model. Equally, CHARM revealed the significantly different critical dynamics of wakefulness and sleep. Overall, CHARM is a promising theoretical framework for capturing the low-dimensionality of the complex network dynamics observed in neuroscience and provides evidence that networks of brain regions rather than individual brain regions are the key computational engines of critical brain dynamics.

Oral vancomycin treatment alters serum levels of indole derivatives and secondary bile acids modulating the expression of mTOR pathway genes in astrocytes during EAE
Astrocytes play important roles in the central nervous system (CNS) during health and disease. Prior studies have shown that gut commensals derived indole derivatives as well as secondary bile acids modulate astrocyte function during the late stage of EAE (recovery phase). Here we show that administering vancomycin to mice starting during the early stage of EAE improved disease recovery, an effect that is mediated by the gut microbiota. We observed that 6 taxa within the Clostridia vadinBB60 genus were enriched in vancomycin treated mice compared to untreated EAE mice. Vancomycin-treated EAE mice also had elevated serum levels of the anti-inflammatory tryptophan derived metabolite, indole-3-lactic acid and decreased levels of deoxycholic acid, a pro-inflammatory secondary bile acid. RNA sequencing revealed altered expression of several genes belonging to the mammalian target of rapamycin (mTOR) pathway in astrocytes obtained during the late stage of EAE from vancomycin treated EAE mice. Furthermore, we observed a link between serum levels of indole derivatives and bile acids and expression of several genes belonging to the mTOR pathway. Interestingly, the mTOR signaling cascades have been implicated in several key biological processes including innate (e.g., astrocyte) immune responses as well as neuronal toxicity/degeneration. In addition, rapamycin, a specific inhibitor of mTOR, has been shown to inhibit the induction and progression of established EAE. Collectively, our findings suggest that the neuroprotective effect of vancomycin is at least partially mediated by indole derivatives and secondary bile acids modulating the expression of mTOR pathway genes in astrocytes.

Single cell landscape of sex differences in the progression of multiple sclerosis
One of the major challenges in addressing multiple sclerosis is to understand the progression trajectory of patients. The pathological process evolves from acute phases predominantly driven by inflammation transitioning to progressive profiles where neurodegeneration takes precedence. It remains unresolved why this course is highly heterogeneous among patients. Currently we know that sex variable plays a crucial role in its understanding. Females are 2-3 times more likely to suffer from multiple sclerosis while males' progression is faster with greater severity. We investigate the potential molecular mechanisms underlying these sex-differential clinical traits analysing transcriptomic data at single cell resolution. 48,919 central nervous system and 336,934 peripheral immune cells, covering the multiple sclerosis spectrum, enabled us to provide the comprehensive landscape of sex differences by cell type. This includes signatures in gene expression patterns, functional profiling, pathways activation and cell-cell communication networks for females, males and their sex-differential profiles. Complete results can be explored in the user-friendly interactive webtool https://bioinfo.cipf.es/cbl-atlas-ms/. Among these findings, we unveiled that female neurons may exhibit protective mechanisms against excitotoxicity, glial cells dysregulated widely stress response genes in a sex-differential manner, and female oligodendrocytes increase expression of axon-myelin contact genes suggesting strong potential for myelin recovery. In the inflammatory-predominant forms, female immune cells present an inflammatory core driven by the AP-1 transcription factor, while male adaptive immune cells exhibit higher mitochondrial impairment. Conversely, larger differences are reported in CD8+ T cells, with females displaying homeostasis recovery patterns and males exhibiting cytolytic profiles. We consider that the molecular description of sex differences in multiple sclerosis progression may be a valuable resource for prevention and diagnosis through biomarker research and the development of personalised therapeutic strategies.

The value of initiating a pursuit in temporal decision-making
Reward rate maximization is a prominent normative principle commonly held in behavioral ecology, neuroscience, economics, and artificial intelligence. Here, we identify and compare equations for evaluating the worth of initiating pursuits that an agent could implement to enable reward-rate maximization. We identify two fundamental temporal decision-making categories requiring the valuation of the initiation of a pursuit - forgo and choice decision-making - over which we generalize and analyze the optimal solution for how to evaluate a pursuit in order to maximize reward rate. From this reward rate maximizing formulation, we derive expressions for the subjective value of a pursuit, i.e. that pursuits equivalent immediate reward magnitude, and reveal that times cost is composed of an apportionment, in addition to, an opportunity cost. By re-expressing subjective value as a temporal discounting function, we show precisely how the temporal discounting function of a reward rate optimal agent is sensitive not just to the properties of a considered pursuit, but to the time spent and reward acquired outside of the pursuit for every instance spent within it. In doing so, we demonstrate how the apparent discounting function of a reward-rate optimizing agent depends on the temporal structure of the environment and is a combination of hyperbolic and linear components, whose contributions relate the apportionment and opportunity cost of time, respectively. We further then show how purported signs of suboptimal behavior (hyperbolic discounting, the "Magnitude" effect, the "Sign" effect) are in fact consistent with reward rate maximization. In clarifying what features are, and are not signs of optimal decision-making, we then analyze the impact of misestimation of identified reward rate maximizing parameters to best account for the pattern of errors actually observed in humans and animals. We find that errors in agents assessment of the apportionment of time inside versus outside a considered pursuit type is the likely driver of suboptimal temporal decision-making observed behaviorally, which we term the Malapportionment Hypothesis. By providing a generalized form for reward rate maximization, and by relating it to subjective value and temporal discounting, the true pattern of errors exhibited by humans and animals can now be more deeply understood, identified, and quantified, being key to deducing the learning algorithms and representational architectures actually used by humans and animals to evaluate the worth of pursuits.

The proneurogenic and microglial modulatory properties of botulinum toxin in the hippocampus of aging experimental mice
This study explored the neurogenic and microglial modulatory properties of botulinum toxin in the hippocampus of aging experimental mice. Therapeutic botulinum toxin (BoNT) treatment is widely practiced to reduce the excessive discharge of acetylcholine (ACh) in the management of aging and neurological deficits. While the production of new neurons in the adult brain contributes to cognitive functions, age-related diseases with excessive release of ACh and progressive neuroinflammation have been characterized by impaired hippocampal neurogenesis and memory loss. Therefore, we investigated the effect of BoNT on the regulation of hippocampal neurogenesis, focusing on doublecortin (DCX)-positive immature neurons in the hippocampus of aging experimental mice. We also assessed ionized calcium-binding adapter molecule 1 (Iba1)-positive microglia and the expression of cyclooxygenase (COX)-2, a key inflammatory response element, using reverse transcription polymerase chain reaction (RT-PCR). Results revealed a prominent increase in DCX-positive cells in the BoNT-treated animals compared to the control group. Additionally, the reduced number of microglia accompanied by decreased mRNA expression of COX-2 was evident in the BoNT-treated animals. These dual effects suggest that BoNT could be a promising therapeutic agent for mitigating age-related neuroregenerative decline and neuroinflammation responsible for cognitive impairments.

Age-related changes in Higuchi's fractal dimension in healthy human EEG are anti-correlated with changes in oscillatory power and 1/f slope
Non-linear dynamical methods such as Higuchi's fractal dimension (HFD) are often used to study the complexities of brain activity. In human electroencephalogram (EEG), while power in the gamma band (30-70 Hz) and the slope of the power spectral density (PSD) have been shown to reduce with healthy aging, there are conflicting findings regarding how HFD and other measures of complexity vary with aging. Further, the dependence of HFD on features obtained from PSD (such as gamma power and slope) has not been thoroughly probed. To address these issues, we computed time and frequency resolved HFD for EEG data collected from elderly population (N=217), aged between 50-88 years, for baseline (BL) eyes open state and during a fixation task in which visual grating stimuli that induce strong gamma oscillations were presented. During BL, HFD increased with age at frequencies upto 150 Hz, but surprisingly showed an opposite trend at higher frequencies. Interestingly, this change in HFD was opposite to the age-related change in PSD 1/f slope. Further, stimulus-related changes in HFD were anti-correlated with the changes in oscillatory power. However, age classification using HFD was slightly better than classification using spectral features (power and slope). Therefore, HFD could effectively integrate various spectral features as well as some non-linearities not captured using spectral analysis, which could enhance our understanding of brain dynamics underlying healthy aging.

Parvalbumin interneuron activity induces slow cerebrovascular fluctuations in awake mice
Neuronal regulation of cerebrovasculature underlies brain imaging techniques reliant on cerebral blood flow (CBF) changes. However, interpreting these signals requires understanding their neural correlates. Parvalbumin (PV) interneurons are crucial in network activity, but their impact on CBF is not fully understood. Optogenetic studies show that stimulating cortical PV interneurons induces diverse CBF responses, including rapid increases, decreases, and slower delayed increases. To clarify this relationship, we measured hemodynamic and neural responses to optogenetic stimulation of PV interneurons expressing Channelrhodopsin-2 during evoked and ongoing resting-state activity in the somatosensory cortex of awake mice. Two-photon microscopy (2P) Ca2+ imaging showed robust activation of PV-positive (PV+) cells and inhibition of PV-negative (PV-) cells. Prolonged PV+ cell stimulation led to a delayed, slow CBF increase, resembling a secondary peak in the CBF response to whisker stimulation. 2P vessel diameter measurements revealed that PV+ cell stimulation induced rapid arterial vasodilation in superficial layers and delayed vasodilation in deeper layers. Ongoing activity recordings indicated that both PV+ and PV- cell populations modulate arterial fluctuations at rest, with PV+ cells having a greater impact. These findings show that PV interneurons generate a complex depth-dependent vascular response, dominated by slow vascular changes in deeper layers.

Activation of the muscle-to-brain axis ameliorates neurocognitive deficits in an Alzheimer disease mouse model via enhancing neurotrophic and synaptic signaling
INTRODUCTION: Skeletal muscle regulates central nervous system (CNS) function and health, activating the muscle-to-brain axis through the secretion of skeletal muscle originating factors (myokines) with neuroprotective properties. However, the precise mechanisms underlying these benefits in the context of Alzheimer disease (AD) remain poorly understood. METHODS: To investigate muscle-to-brain axis signaling in response to amyloid {beta} (A{beta})-induced toxicity, we generated 5xFAD transgenic female mice with enhanced skeletal muscle function (5xFAD;cTFEB;HSACre) at prodromal (4-months old) and late (8-months old) symptomatic stages. RESULTS: Skeletal muscle TFEB overexpression reduced A{beta} plaque accumulation in the cortex and hippocampus at both ages and rescued behavioral neurocognitive deficits in 8-months-old 5xFAD mice. These changes were associated with transcriptional and protein remodeling of neurotrophic signaling and synaptic integrity, partially due to the CNS-targeting myokine prosaposin (PSAP). DISCUSSION: Our findings implicate the muscle-to-brain axis as a novel neuroprotective pathway against amyloid pathogenesis in AD.

Deciphering the molecular landscape of human peripheral nerves: implications for diabetic peripheral neuropathy
Diabetic peripheral neuropathy (DPN) is a prevalent complication of diabetes mellitus that is caused by metabolic toxicity to peripheral axons. We aimed to gain deep mechanistic insight into the disease process using bulk and spatial RNA sequencing on tibial and sural nerves recovered from lower leg amputations in a mostly diabetic population. First, our approach comparing mixed sensory and motor tibial and purely sensory sural nerves shows key pathway differences in affected nerves, with distinct immunological features observed in sural nerves. Second, spatial transcriptomics analysis of sural nerves reveals substantial shifts in endothelial and immune cell types associated with severe axonal loss. We also find clear evidence of neuronal gene transcript changes, like PRPH, in nerves with axonal loss suggesting perturbed RNA transport into distal sensory axons. This motivated further investigation into neuronal mRNA localization in peripheral nerve axons generating clear evidence of robust localization of mRNAs such as SCN9A and TRPV1 in human sensory axons. Our work gives new insight into the altered cellular and transcriptomic profiles in human nerves in DPN and highlights the importance of sensory axon mRNA transport as an unappreciated potential contributor to peripheral nerve degeneration.

Pre-training artificial neural networks with spontaneous retinal activity improves motion prediction in natural scenes
The ability to process visual stimuli rich with motion represents an essential skill for animal survival and is largely already present at the onset of vision. Although the exact mechanisms underlying its maturation remain elusive, spontaneous activity patterns in the retina, known as retinal waves, have been shown to contribute to this developmental process. Retinal waves exhibit complex spatio-temporal statistics and contribute to the establishment of circuit connectivity and function in the visual system, including the formation of retinotopic maps and the refinement of receptive fields in downstream areas such as the thalamus and visual cortex. Recent work in mice has shown that retinal waves have statistical features matching those of natural visual stimuli, such as optic flow, suggesting that they could prime the visual system for motion processing upon vision onset. Motivated by these findings, we examined whether artificial neural network (ANN) models trained on natural movies show improved performance if pre-trained with retinal waves. We employed the spatio-temporally complex task of next-frame prediction, in which the ANN was trained to predict the next frame based on preceding input frames of a movie. We found that pre-training ANNs with retinal waves enhances the processing of real-world visual stimuli and accelerates learning. Strikingly, even when matching the total training time by merely replacing initial training epochs on naturalistic stimuli with exposure to retinal waves, an ANN trained on retinal waves temporarily outperforms one trained solely on natural movies. Similar to observations made in biological systems, we also found that pre-training with spontaneous activity refines the receptive field of ANN neurons. Overall, our work sheds light on the functional role of spatio-temporally patterned spontaneous activity in the processing of motion in natural scenes, suggesting it acts as a training signal to prepare the developing visual system for adult visual processing.

SOD1 Enzymatic Activity in CSF from ALS patients with and without SOD1 mutations
Superoxide dismutase-1 (SOD1) mutations are a common cause of amyotrophic lateral sclerosis (ALS). Intrathecal gene therapy using the antisense-oligo-nucleotide drug tofersen to reduce SOD1 expression shows significant effects on disease progression and has recently been approved in the United States and the European Union. However, the discovery of children homozygous for four different inactivating SOD1 mutations developing the Infantile SOD1 Deficiency Syndrome (ISODDES) with injury to both upper and lower motor systems suggests that low SOD1 activities may be deleterious in humans. Monitoring SOD1 activity in cerebrospinal fluid (CSF) from tofersen-treated patients is advisable but difficult due to low levels and the presence of the isoenzyme SOD3. We here present a method to efficiently remove SOD3 from CSF using highly specific immobilized antibodies and subsequent measurement of the SOD activity as a sensitive assay for the SOD1 activity in CSF. We applied the method on CSF samples from ALS patients and controls and used paired erythrocyte samples for comparison. In ALS patients with wildtype SOD1, the SOD1 activity in CSF was the same as in controls, but patients with mutant SOD1 show lower activity in CSF, even patients with mutants previously reported to have full activity in erythrocytes. Activity variation is large among patients carrying the same SOD1 mutation and larger than seen in erythrocytes and in post-mortem central nervous system tissue. SOD1 in CSF shows a high specific activity, indicating that it is mainly native. Lastly, we identified a discrepancy between the SOD1 activity and protein level measured with ELISA in both CSF and erythrocytes. Since antibodies for SOD1 ELISA-quantification are raised against the native wildtype enzyme, the content of mutant SOD1s may be underestimated. Direct analysis of SOD1 enzymatic activity in CSF is therefore a more reliable way to monitor the effect of SOD1-lowering compounds.

Taking the perspective of an embodied avatar modulates the temporal dynamics of vicarious pain and pleasure: a combined Immersive Virtual Reality and EEG study
Observing negative and positive valence virtual stimuli can influence the onlookers' subjective and brain reactivity. However, information about the complex link between vicarious pain and pleasure, observer's perspective taking, and cerebral activity is scarce. To address this knowledge gap, we asked twenty-four, VR-immersed healthy participants to report about pleasant, painful, and neutral stimuli delivered to a virtual hand seen from either a first-person perspective (1PP) or third-person perspective (3PP) while undergoing time and time-frequency EEG recording. Participants experienced a stronger sense of ownership over a virtual hand when they viewed it from a 1PP compared to a 3PP. Furthermore, participants rated pain-inducing and pleasure-inducing stimuli as most unpleasant and pleasant, and as more intense. We observed distinct EEG patterns in early (N2, early posterior negativity- EPN) and late (late positive potential-LPP) event-related potentials, as well as in EEG power. The N2 and EPN components showed higher amplitudes for pain and pleasure stimuli compared to neutral stimuli particularly when seen from a 1PP. Conversely, the LPP component exhibited a smaller amplitude for pleasure stimuli compared to both pain and neutral stimuli. We also found that theta-band power increased and alpha power decreased for pain and pleasure stimuli viewed from a 1PP vs a 3PP perspective. Also, in the ultra-late time-window, we observed a decrease in theta, alpha, and beta-band power specifically associated with pleasure stimuli. Our study provides novel evidence on how perspective taking influences the temporal dynamics of vicarious sensations and on distinct electrocortical markers of observed pain and pleasure.

FreeSurfer version-shuffling can boost brain age predictions
The influence of FreeSurfer version-dependent variability in reconstructed cortical features on brain age predictions is average small when varying training and test splits from the same data. FreeSurfer version differences can lead to some variability in brain age dependent on the choice of algorithm and individual differences in brain morphometry, highlighting the advantage of repeated random train-test splitting. Shuffling of differently processed FreeSurfer data dependent on the FreeSurfer version increases performance and generalizability of the brain age prediction model.