bioRxiv Subject Collection: Neuroscience's Journal

Repetitive neonatal pain increases spinal cord DNA methylation of the μ-opioid receptor
Repetitive neonatal painful procedures experienced in the neonatal intensive care unit (NICU) are known to alter the development of the nociceptive system and have long-lasting consequences, notably lower post-operative -opioid receptor levels in the spinal cord. Given the influence of the NICU on the epigenome, the present study hypothesized that neonatal procedural pain alters the DNA methylation status of the opioid receptor mu 1 encoding gene (Mor-1) in the spinal cord and dorsal root ganglions (DRGs). To this end, the needle prick model of repetitive neonatal pain was used, and methylation of Mor-1 promotor was assessed in the spinal cord and the DRG using bisulfite pyrosequencing. Our findings demonstrated that neonatal procedural pain increased spinal cord Mor-1 promotor DNA methylation in the ipsilateral side as compared to the contralateral side, an effect that was not observed in the control animals, nor in the DRG. We also identified a behaviorally-associated CpG site following neonatal needle pricks. This study is the first to highlight a localized and noxious-stimuli-dependent effect of repetitive neonatal procedural pain on Mor-1 promotor methylation and emphasizes the need to explore the effects of repetitive neonatal procedural pain on the epigenome.

A Tale of Two Environments: Divisive Normalization and the (In)Flexibility of Choice
The Divisive Normalization (DN) function has been described as a 'canonical neural computation' in the brain that achieves efficient representations of sensory and choice stimuli. Recent theoretical work indicates that it efficiently encodes a specific class of Pareto-distributed stimuli. Does the brain shift to different encoding functions in other types of environments, or is there evidence for DN encoding in other types of environments? In this paper, using a within-subject choice experiment, we show evidence of the latter. Our subjects made decisions in two distinct choice environments with choice sets either drawn from a Pareto distribution or from a uniform distribution. Our results indicate that subjects' choices are better described by a divisive coding strategy in both environments. Moreover, subjects appeared to calibrate a DN function to match, as closely as possible, the actual statistical properties of each environment. These results suggest that the nervous system may be constrained to use divisive representations under all conditions.

Perception/action coupling in children with autism: insights from looking time and pupil dilation measurements
The objective of this study was to characterize, through indices extracted from eye-tracking measurements, the spontaneous distinction of videos of daily actions with a variable perception/action coupling, depending on whether, for the same action, the video was presented in the forward reading direction (strong coupling), or in the backward reading direction (weaker coupling). 17 pairs of videos of daily actions performed by adults were viewed by 36 typically developing children and 28 children with ASD aged 7-18 years. During the exposure phase, they watched two videos of the same action (forward and backward) presented successively, before looking at these two videos in competition, in a second visual preference phase. During the exposure phase, all participants paid similar general attention to each of the videos. We found greater pupillary dilation for backward than forward actions in both groups, but significantly less in the ASD group. In the visual preference phase, both groups showed significantly greater looking times for backward actions over forward ones, with no difference between groups. If TD children perceived the kinematics of the backward videos as violating their expectations given the strong perception/action coupling they had already built over that action, on the contrary, the lower increased in pupil dilation found in ASD children could reflect altered perception/action coupling. This study confirms the validity of looking time and pupil dilation as behavioral and physiological markers that could be used in a 10-mn eye-tracking test to explore perception/action coupling in childhood and in ASD.

Intra-individual variability in the effects of transcranial directcurrent stimulation on free choice saccade behaviour
Transcranial direct current stimulation (tDCS) is used as a tool to causally influence neural activity in humans non-invasively. Although most studies recruit a large number of participants in order to uncover population-level effects, growing evidence suggests that tDCS may be expected to induce different effects in different individuals, leading to large inter-individual variability and confounds in population-level testing. Alternatively, this variability may arise from intra-individual sources that are difficult to assess in standard designs. Here, we performed between 8 and 10 sessions of tDCS within individuals to understand how intra-individual variability impacts the inference of tDCS effects. We recruited 5 participants who underwent functional MRI to localize the human frontal eye field (FEF) homologue. An HD-tDCS montage was used to stimulate the target location based on individual MRI localizations, alternating the polarity between anodal or cathodal current over 8-10 repeated sessions during a 5 week period. Participants performed a free choice task before and after stimulation while recording EEG activity. We then developed a difference-in-difference method based on permutation testing to assess the likelihood of a causal effect of tDCS at different levels of abstraction: group-level, inter-individual, and intra-individual. At the group-level, we found evidence for an influence of tDCS on choice reaction times, which followed a reaction-time dependent change in alpha-band activity, and on how choices depended on recent history. However, individuals showed heterogeneous, and often contradictory, effects. We then analyzed the distribution of session permutations at the intra-individual level, and found a discrepancy between the inter-individual effects that survived significance testing and the intra-individual effects that correlated on a session-session basis. We argue that, while the observed variability may have arisen from a combination of inter and intra-individual differences relevant to tDCS-dependent mechanisms of action, it may be equally well explained by spurious effects arising from history-dependence between repeated measures that are typically assumed to be independent. In light of this, we assess the counterfactuals that must be evaluated in order to make data-driven inferences about the causal effects of tDCS on free choice behaviour and its neural correlates.

The role of protein shape in multiphasic separation within condensates
Liquid liquid phase separation (LLPS) of biological macromolecules leads to the formation of various membraneless organelles. LLPS can not only form homogenous condensates but also multilayered and multiphase condensates, which can mediate complex cellular functions. However, the factors that determine the topological features of multiphase condensates are not fully understood. Herein, we focused on Ca2+/calmodulin dependent protein kinase II (CaMKII), a major postsynaptic protein that undergoes various forms of LLPS with other postsynaptic proteins, and present a minimalistic computational model that reproduces these forms of LLPS, including a form of two phase condensates, phase in phase (PIP) organization. Analyses of this model revealed that the competitive binding of two types of client proteins is required for the PIP formation. The PIP only formed when CaMKII had high valency and a short linker length. Such CaMKII proteins exhibited a low surface tension, a modular structure, and slow diffusion. These properties are consistent with the functions required by CaMKII to store information at the synaptic level. Thus, the computational modeling reveals new structure function relationships for CaMKII as a synaptic memory unit.

Simultaneous GCaMP imaging and focal recording of tonic and phasic synapses: Probing short-term plasticity within a defined microenvironment
GCaMP fluorescence has been widely used to monitor intracellular Ca2+. However, the physiological significance of the GCaMP signal in presynaptic terminals remains to be further elucidated. We investigated how the dynamics of GCaMP signals correlates with the activity dependence of short-term plasticity in synaptic transmission. We devised a local manipulation protocol that minimizes interference from muscular contraction during simultaneous Ca2+ imaging and focal recording at the Drosophila larval neuromuscular junction (NMJ), where the tonic and phasic excitatory synapses can be compared side-by-side. By confining the local ionic microenvironment, this protocol enabled stable measurements across extended concentration ranges of Ca2+ or Sr2+ in saline. Compared to tonic synapses, phasic synapses displayed stronger GCaMP signals, along with faster facilitation and more severe depression. Upon repetitive stimulation (40 Hz), facilitation of transmission occurred during or immediately prior to the early rising phase (0.25 s) of the GCaMP signal, which could subsequently convert into a depression phase of transmission decline, most evident during a steeper and longer rise of GCaMP signals in higher Ca2+ saline. Typically, deepest depression occurred when GCaMP signals rose to a plateau. Phasic synapses with stronger GCaMP signal and deeper depression, more often exhibited lingering post-stimulation releases. In both tonic and phasic synapses, replacing Ca2+ with Sr2+ induced extreme asynchronous transmission coupled with post-stimulation lingering releases during the decay of GCaMP signals. Further applications of this focal recording-local manipulation protocol may help to probe additional mechanisms underlying synaptic transmission and plasticity.

Exploring the role of the rich club in network control of neurocognitive states
We present a systematic analysis of the rich club as a possible control center of brain dynamics. Contrary to research stressing control properties of the rich club, our results indicate that the rich club and high-degree regions were not involved in optimally controlling the human connectome for a set of data-driven and task-evoked patterns of activity. Indeed, low-degree peripheral regions were more suited towards this. Findings were stable across group-level and individual-level rich club definitions, various cognitively meaningful brain state transitions, and different parameter settings. A region's contribution to optimal control processes was associated with its affiliation with certain intrinsic connectivity networks and its position on the secondary, but not primary, cortical gradient. These results do not negate an integratory role of the rich club, but merely question its proposed role as a driver of control. Indeed, if it would inhabit such a role, we would have expected opposite results. Our findings fit with a position describing the rich club as a passive "data-highway" which, by means of its high connectivity, can be easily controlled by peripheral regions and thus facilitate relevant communication channels between them. We call for methodological expansions of the control theoretical toolbox allowing for elaborations on the temporal dynamics of control processes.

Motion correction with subspace-based self-navigation for combined angiography, perfusion and structural imaging
Motion artifacts are problematic in many MRI modalities. Self-navigating approaches are desirable, since no additional scan time or hardware is required. However, the generation of a navigator image, to estimate and correct motion, is difficult in cases where the tissue contrast is changing during the navigator acquisition window, such as in magnetization-prepared methods. Here we propose a subspace approach to reconstruct accurate navigators in the presence of time-varying tissue contrast and apply it to a combined angiography, perfusion and structural imaging method using a golden ratio 3D cones trajectory. This arterial spin labeling-based pulse sequence relies on subtraction of label and control images to isolate the relatively weak blood signal, making it particularly susceptible to motion corruption. An inversion pulse leads to time-varying tissue contrast across the readout train, but by reconstructing subspace coefficient maps directly, artifacts due to the varying contrast were alleviated. This resulted in high-quality navigator images that were subsequently registered to estimate and correct for motion. In addition, a split-update method was proposed to efficiently reconstruct from mismatched label/control k-space data with locally low rank regularization enforced on the difference image. The correction process was tested with numerical simulation and in vivo data from 8 healthy subjects with and without cued motion. In numerical simulation, the subspace-based navigator achieved an 84% reduction in RMSE of residual motion compared to without motion correction. In vivo, motion correction resulted in noise-like and background artifacts being greatly reduced and vessel sharpness being noticeably improved. Correlation of angiography, perfusion and structural images with motion-free reference images also increased by 159%, 53% and 12%, respectively, after motion correction. These results show that subspace-based navigators can effectively improve the motion robustness of MR imaging in contrast-varying acquisitions.

A sleep disturbance method by novel objects in the home cage to minimize stress
Background The increasing prevalence of low sleep quality is a significant issue, particularly among adolescents, necessitating a deeper understanding of its biological consequences. In sleep research, various protocols are used for sleep deprivation or disturbance, each presenting its own set of confounding factors crucial to consider. New Method We developed a standardized seven-day sleep disturbance (SD) protocol using daily four-hour exposures to novel objects to minimize rodent stress. Objects were selected and characterized for wake-promoting properties, and exposure timing was structured to reduce variability and enhance experimental reliability and reproducibility. Results During the four hours of SD, the mice were efficiently sleep-deprived on the first and seventh day of SD. Thus, the selected objects efficiently sleep restricted the mice. On the first day of SD, the protocol induced sleep deprivation effect when measured over 24h, but by the seventh day, the mice recovered the sleep loss. Thus, this method is a sub-chronic sleep disturbance and not sleep deprivation. Fecal corticosterone concentrations remained unchanged during the seven days of SD. Comparison with existing methods This approach reduced the risk of stress through voluntary rather than forced wakefulness. Previously, novel objects have been exchanged randomly during mouse sleep initiation causing protocol variability and very frequent disturbances. Our protocol minimizes this by introducing the novel object in a structured manner. Conclusion We effectively disturbed the sleep of the mice during seven days without inflicting substantial stress. We further demonstrate the value of validating the efficiency of an SD protocol with 24h recordings.

Spatiotemporal Decomposition of Whole-Brain Alpha Traveling Waves
Spontaneously emerging traveling waves are present within the spatiotemporal patterns of alpha-band EEG oscillations, but current analysis methods are limited in parsing the diversity of global wave structures and their correlation with brain functions. To address this limitation, we constructed a rigorous mathematical framework, Weakly Orthogonal Conjugate Contrast Analysis (WOCCA), which decomposes the whole-brain EEG alpha oscillations into directionally independent traveling waves. For the first time, we systematically characterized propagating components in alpha-band resting-state EEG as a combination of rotational, longitudinal, and horizontal traveling wave patterns. The intensity, directionality, and morphological characteristics of these wave patterns account for the differences between cognitive states during rest and consciousness levels under sedation. Moreover, our WOCCA decomposition encompassed the state transition dynamics captured by EEG Microstate Analysis, a conventional analysis framework for alpha waves. These results not only established a novel approach for identifying and analyzing traveling waves but also provided evidence for the relationship between wave directionality and cooperative interactions in brain network.

EEG Correlates of Cognitive Dynamics in Task Resumption after Interruptions: The Impact of Available Time and Flexibility
Interruptions are a common aspect of everyday working life, negatively affecting both task performance and long-term psychological well-being. However, research suggests that the effects of interruptions can be mitigated in several ways, such as the opportunity to anticipate the interruptions and preparation time. Here, we used a retrospective visual working memory task to investigate the effects of duration and flexible resumption after interruptions, with 28 participants (18-30 years old) attending the experiment. For this main task participants were required to remember the orientations of a set of colored bars and retrieve one at the end of the trial in response to a retro-cue. This task was sometimes interrupted with an arithmetic task that was presented before the retro-cue. The period after the interruptions and the retro-cue was either short (no additional time), long (additional 1000 ms), or self-determined. Interruptions affected the main task performance irrespective of duration condition, while response times were shorter with the flexible condition. EEG analysis showed that having more time before resuming the interrupted task enabled stronger beta suppression which in turn modulated task performance, helping participants to safely disengage from the interrupting task, and refocus their attention back more efficiently. Further, flexibility in the timing of resumption provided additional benefits as seen in stronger oscillatory alpha and beta suppression to the retro-cue, also being related to better task performance. These results demonstrate the important role of resumption time and individual flexibility in dealing with interruptions.

Progressive changes in functional connectivity between thalamic nuclei and cortical networks across learning
The thalamus is connected to cerebral cortex and subcortical regions, serving as a node within cognitive networks. It is a heterogeneous structure formed of functionally distinct nuclei with unique connectivity patterns. However, their contributions to cognitive functioning within networks is poorly understood. Recent animal research suggests that thalamic nuclei such as the mediodorsal nucleus play critical roles in goal-directed behaviour. Our aim was to investigate how functional integration of thalamic nuclei within cortical and subcortical networks changes whilst transitioning from more controlled goal-directed behaviour towards more automatic or habitual behaviour in humans. We analysed functional magnetic resonance imaging (fMRI) data from a stimulus-response learning study to investigate functional connectivity (FC) changes across learning between thalamic nuclei with cortical networks and subcortical structures in healthy subjects. We defined subcortical regions-of-interest (ROIs) individually in native space, segmenting the thalamus into 47 nuclei and segmenting 38 subregions within the basal ganglia and hippocampus. Additionally, we defined 12 cerebral cortex ROIs via maximum-probability network templates. Learning-related connectivity changes were examined via ROI-to-ROI functional network analysis. Our results showed that learning was associated with: 1) decreasing FC between the frontoparietal network and higher order thalamic nuclei; 2) increasing FC between the cingulo-opercular network and pulvinar nuclei, 3) decreasing FC between the default mode network (DMN) and right mediodorsal nuclei; 4) increasing FC between the DMN and left mediodorsal nuclei, and 5) increasing intrathalamic FC. Together, this suggests that several thalamic nuclei are involved in the learning-related transition from controlled to more automatic behaviour.

Investigating microscopic angioarchitecture in the human visual cortex in 3D with angioMASH tissue clearing and labelling
Non-invasive imaging techniques, such as ultra-high field fMRI, are intricately connected to the underlying vasculature and are approaching ever higher resolutions. For the analysis of fMRI signals over cortical depth at such high resolutions, microvascular differences might have to be taken into account. Therefore, a better understanding of the laminar distribution and interareal differences in the cortical vasculature is becoming more important. However, in comparison to cyto- and myeloarchitecture, the study of angioarchitecture has received far less attention and relatively few methods have been described to visualise the vascular network in the human brain. Here we present angioMASH, a method for double labelling angioarchitecture and cytoarchitecture in archival human brain tissue, based on the cytoarchitecture labelling and optical clearing of the recently published MASH protocol. The double labelling and optical clearing of thick human brain slices can be accomplished within 16 days. We use this method to acquire multi-resolution 3D datasets of combined cyto- and angioarchitecture in large human samples covering visual areas V1 and V2. We demonstrate for the first time, that classical angioarchitectonic features can be visualised in the human cortex and in 3D using tissue clearing and light-sheet microscopy. Lastly, we show differences in the vessel density and orientation over cortical depth within and between the two areas. Especially in V1, the vascular density is not homogeneous over cortical depth, but shows distinct layering. These layers are also determined by changes in the orientation of the blood vessels from a predominantly radial to a more tangential distribution. In V2, differences in vascular density are less pronounced, but orientation profiles follow a similar trend over cortical depth. We discuss potential consequences of these differences for the interpretation of non-invasive functional imaging modalities such as fMRI or fNIRS.

Natural Language Processing Applied to Spontaneous Recall of Famous Faces Reveals Memory Dysfunction in Temporal Lobe Epilepsy Patients
Objective and Background. Epilepsy patients rank memory problems as their most significant cognitive comorbidity. Current clinical assessments are laborious to administer and score and may not always detect subtle memory decline. The Famous Faces Task (FF) has robustly demonstrated that left temporal lobe epilepsy (LTLE) patients remember fewer names and biographical details compared to right TLE (RTLE) patients and healthy controls (HCs). We adapted the FF task to capture subjects' entire spontaneous spoken recall, then scored responses using manual and natural language processing (NLP) methods. We expected to replicate previous group level differences using spontaneous speech and semi-automated analysis. Methods. Seventy-three (N=73) adults (28 LTLE, 18 RTLE, and 27 HCs) were included in a case-control prospective study design. Twenty FF in politics, sports, and entertainment (active 2008-2017) were shown to subjects, who were asked if they could recognize and spontaneously recall as much biographical detail as possible. We created human-generated and automatically-generated keyword dictionaries for each celebrity, based on a randomly selected training set of half of the HC transcripts. To control for speech output, we measured the speech duration, total word count and content word count for the FF task and a Cookie Theft Control Task (CTT), in which subjects were merely asked to describe a visual scene. Subjects' responses to FF and CTT tasks were recorded, transcribed, and analyzed in a blinded manner with a combination of manual and automated NLP approaches. Results. Famous face recognition accuracy was similar between groups. LTLE patients recalled fewer biographical details compared to HCs and RTLEs using both the gold-standard human-generated dictionary (24%+/-12% vs. 31%+/-12% and 30%+/-12%, p=0.007) and the automated dictionary (24%+/-12% vs. 31%+/-12% and 32%+/-13%, p=0.007). There were no group level differences in speech duration, total word count, or content word count for either the FF and CTT to explain difference in recall performance. There was a positive, statistically significant relationship between MOCA score and FF recall performance as scored by the human-generated (rho = .327, p= .029) and automatically-generated dictionaries (rho = .422, p= .004) for TLE subjects, but not HCs, an effect that was driven by LTLE subjects. Discussion. LTLE patients remember fewer details of famous people than HCs or RTLE patients, as discovered by NLP analysis of spontaneous recall. Decreased biographical memory was not due to decreased speech output and correlated with lower MOCA scores. NLP analysis of spontaneous recall can detect memory dysfunction in clinical populations in a semi-automated, objective, and sensitive manner.

Using Dual-Coil TMS-EEG to Probe Bilateral Brain Mechanisms in Healthy Aging and Mild Cognitive Impairment
Background: A widespread observation in the cognitive neuroscience of aging is that older adults show a more bilateral pattern of task-related brain activation. These observations are based on inherently correlational approaches. The current study represents a targeted assessment of the role of bilaterality using repetitive transcranial magnetic stimulation (rTMS). Objective: We used a novel bilateral TMS-stimulation paradigm, applied to a group of healthy older adults (hOA) and older adults with mild cognitive impairment (MCI), with two aims: First, to elucidate the neurophysiological effects of bilateral neuromodulation, and second to provide insight into the neurophysiological basis of bilateral brain interactions. Methods: Electroencephalography (EEG) was recorded while participants received six forms of transcranial magnetic stimulation (TMS): unilateral and bilateral rTMS trains at an alpha (8 Hz) and beta (18 Hz) frequency, as well as two sham conditions (unilateral, bilateral) mimicking the sounds of TMS. Results: First, time-frequency analyses of oscillatory power induced by TMS revealed that unilateral beta rTMS elicited rhythmic entrainment of cortical oscillations at the same beta-band frequency. Second, both bilateral alpha and bilateral beta stimulation induced a widespread reduction of alpha power. Lastly, healthy older adults showed greater TMS-related reductions in alpha power in response to bilateral rTMS compared to the MCI cohort. Conclusion: Overall, these results demonstrate frequency-specific responses to bilateral rTMS in the aging brain, and provide support for inhibitory models of hemispheric interaction across multiple frequency bands.

A novel iPSC model of Bryant-Li-Bhoj neurodevelopmental syndrome demonstrates the role of histone H3.3 in neuronal differentiation and maturation
Background Bryant-Li-Bhoj neurodevelopmental syndrome (BLBS) is neurogenetic disorder caused by variants in H3-3A and H3-3B, the two genes that encode the histone H3.3 protein. Ninety-nine percent of individuals with BLBS show developmental delay/intellectual disability, but the mechanism by which variants in H3.3 result in these phenotypes is not yet understood. As a result, only palliative interventions are available to individuals living with BLBS. Methods Here, we investigate how one BLBS-causative variant, H3-3B p.Leu48Arg (L48R), affects neurodevelopment using an induced pluripotent stem cell (iPSC) model differentiated to 2D neural progenitor cells (NPCs), 2D forebrain neurons (FBNs), and 3D dorsal forebrain organoids (DFBOs). We employ a multi-omic approach in the 2D models to quantify the resulting changes in gene expression and chromatin accessibility. We used immunofluorescence (IF) staining to define the identities of cells in the 3D DFBOs. Results In the 2D systems, we found dysregulation of both gene expression and chromatin accessibility of genes important for neuronal fate, maturation, and function in H3.3 L48R compared to control. Our work in 3D organoids corroborates these findings, demonstrating altered proportions of radial glia and mature neuronal cells. Conclusions These data provide the first mechanistic insights into the pathogenesis of BLBS from a human-derived model of neurodevelopment, which suggest that the L48R increases H3-3B expression, resulting in the hyper-deposition of H3.3 into the nucleosome which underlies changes in gene expression and chromatin accessibility. Functionally, this causes dysregulation of cell adhesion, neurotransmission, and the balance between excitatory and inhibitory signaling. These results are a crucial step towards preclinical development and testing of targeted therapies for this and related disorders.

Impact of intragenic NRXN1 deletions on early cortical development
Background: Deletions in NRXN1 are strongly associated with neurodevelopmental and psychiatric conditions. While exonic deletions are well-studied, intragenic deletions, particularly in intron 5, are less understood and generally consider benign. Recent studies show exonic deletions impact isoform diversity during neurodevelopment, affecting neurogenesis and neuronal function. However, whether intragenic deletions impact isoform expression and neurodevelopment remains underexplored. Methods: We used hiPSCs from typically developing individuals (control) and those with NRXN1 intron 5 deletions to study neurodevelopment. HiPSCs were differentiated towards a cortical fate, with NRXN1 isoform expression, molecular differences, and neuronal morphology examined. Results: We observed distinct NRXN1 isoform expression dynamics during early neurodevelopment, with two expression peaks post-neuronal induction and NRXN1{beta} being most highly expressed. Both NRXN1 deletion and control lines showed similar acquisition of regional and cell fate identity, but significant differences in NRXN1 isoform expression were observed between deletion and control lines, and between deletion lines. RNA sequencing revealed genotype-dependent alterations, particularly in pathways related to synaptic function and neuronal morphology. Consistent with these findings, NRXN1 deletion lines exhibited altered dendrite outgrowth, with variations between deletion lines. Conclusions: Our results indicate a potential role for intron 5 in controlling NRXN1 isoform expression during neurodevelopment. Alterations in gene expression profiles, correlated with morphological changes, suggest a role for NRXN1 isoforms in shaping dendritic morphology. Molecular and cellular differences observed between lines with identical intronic deletions suggest that additional factors, such as genetic background or biological sex, may also play an important role in these phenotypes. Collectively, these findings indicate that NRXN1 intronic deletions are not benign, influencing isoform expression, cellular phenotypes, and neurodevelopment.

Transcriptomically-measured gene expression predicts physiological variation across single neurons in humans and mice
Single-cell transcriptomics measures the molecular landscape of individual neurons with unprecedented efficiency and scale. This insight has the potential to advance our understanding of the molecular basis of neuronal function, and to identify druggable targets for disease treatments. However, transcriptomics also suffers from greater measurement noise than traditional techniques (e.g., RT-PCR), which raises questions about its ability to offer insight into function at true single-cell resolution. We tested if transcriptomic data could yield insight into function of individual neurons in human and mouse neocortex by analyzing two datasets collected via Patch-Seq, a powerful technique for obtaining transcriptomic and physiology data from the same neuron. We found that computational models trained on single-cell transcriptomic data robustly predicted physiology of individual neurons. Critically, models trained on single cells outperformed those trained on cell type averages when predicting single-cell physiology. Thus, the standard approach of denoising single-cell transcriptomic data by averaging on cell types sacrifices functionally-relevant information. Our analysis also revealed novel relationships between gene expression and physiology, including a potential molecular substrate of human-mouse cross-species differences in the speed of single-neuron computation. Broadly, our findings highlight the promise of Patch-Seq for generating new insight into the molecular basis of neuronal function.

Dynamic modeling of EEG responses to natural speech reveals earlier processing of predictable words
In recent years, it has become clear that EEG indexes the comprehension of natural, narrative speech. One particularly compelling demonstration of this fact can be seen by regressing EEG responses to speech against measures of how individual words in that speech linguistically relate to their preceding context. This approach produces a so-called temporal response function that displays a centro-parietal negativity reminiscent of the classic N400 component of the event-related potential. One shortcoming of previous implementations of this approach is that they have typically assumed a linear, time-invariant relationship between the linguistic speech features and the EEG responses. In other words, the analysis typically assumes that the response has the same shape and timing for every word - and only varies (linearly) in terms of its amplitude. In the present work, we relax this assumption under the hypothesis that responses to individual words may be processed more rapidly when they are predictable. Specifically, we introduce a framework wherein the standard linear temporal response function can be modulated in terms of its amplitude, latency, and temporal scale based on the expectedness of the current and prior words. We use the proposed approach to model EEG recorded from a set of participants who listened to an audiobook narrated by a single talker, and a separate set of participants who attended to one of two concurrently presented audiobooks. We show that expected words are processed faster - evoking lower amplitude N400-like responses with earlier peaks - and that this effect is driven both by the word's own predictability and the predictability of the immediately preceding word. Additional analysis suggests that this finding is not simply explained based on how quickly words can be disambiguated from their phonetic neighbors. As such, our study demonstrates that the timing and amplitude of brain responses to words in natural speech depend on their predictability. By accounting for these effects, our framework also improves the accuracy with which neural responses to natural speech can be modeled.

SpinalTRAQ: A novel volumetric cervical spinal cord atlas identifies the corticospinal tract synaptic projectome in healthy and post-stroke mice
Descending corticospinal tract (CST) connections to the neurons of the cervical spinal cord are vital for performance of forelimb-specific fine motor skills. In rodents, CST axons are almost entirely crossed at the level of the medullary decussation. While specific contralateral axon projections have been well-characterized using anatomic and molecular approaches, the field currently lacks a cohesive imaging modality allowing rapid quantitative assessment of the entire, bilateral cervical cord projectome at the level of individual laminae and cervical levels. This is potentially important as the CST is known to undergo marked structural remodeling in development, aging, and disease. We developed SpinalTRAQ (Spinal cord Tomographic Registration and Automated Quantification), a novel volumetric cervical spinal cord atlas and machine learning-driven microscopy acquisition and analysis pipeline that uses serial two-photon tomography- images to generate unbiased, region-specific quantification of the fluorescent pixels of anterograde AAV-labeled CST pre-synaptic terminals. In adult mice, the CST synaptic projectome densely innervates the contralateral hemicord, particularly in laminae 5 and 7, with sparse, monosynaptic input to motoneurons in lamina 9. Motor pools supplying axial musculature in the upper cervical cord are bilaterally innervated. The remainder of the ipsilateral cord has sparse labeling in a distinct distribution compared to the contralateral side. Following a focal stroke of the motor cortex, there is a complete loss of descending corticospinal axons from the injured side. Consistent with prior reports of axon collateralization, the CST spinal projectome increases at four weeks post-stroke and continues to elevate by six weeks post stroke. At six weeks post-stroke, we observed striking synapse formation in the denervated hemicord from the uninjured CST in a homotopic distribution. Additionally, CST synaptic reinnervation increases in the denervated lamina 9 in nearly all motoneuron pools, exhibiting novel patterns of connectivity. Detailed level- and lamina-specific quantification of the bilateral cervical spinal cord synaptic projectome reveals previously undescribed patterns of CST connectivity in health and injury-related plasticity.

Decoding the elite soccer players psychological profile
Soccer is arguably the most widely followed sport worldwide, and many dream of becoming soccer players. However, only a few manage to achieve this dream, which has cast a significant spotlight on elite soccer players who possess exceptional skills to rise above the rest. Originally, such attention was focused on their great physical abilities. However, recently, it a new perspective has emerged, suggesting that being an elite soccer player require a deep understanding of the game, rapid information processing and decision-making. This growing attention has led to several studies suggesting higher executive functions in soccer players compared to the general population. Unfortunately, these studies often had small and non-elite samples, focusing mainly on executive functions alone without employing advanced machine learning techniques. In this study, we used artificial neural networks to comprehensively investigate the personality traits and cognitive abilities of a sample of 328 participants, including 204 elite soccer players from the top teams in Brazil and Sweden. Our findings indicate that elite soccer players demonstrate heightened planning and memory capacities, enhanced executive functions, especially cognitive flexibility, elevated levels of conscientiousness, extraversion, and openness to experience, coupled with reduced neuroticism and agreeableness. This research provides insights into the psychology of elite soccer players, holding significance for talent identification, development strategies in soccer and offering insights into the psychological profiles associated with success.

Perception/action coupling is modulated by the age-related motor skills of the agent performing the action
The perception/action coupling, underpinned by Mirror Neurons, allows the understanding of others' action goal and intention picked up from cues conveyed by the individual's kinematics and the context of the action. This mechanism can be modulated by familiarity with the observed action, by motor experience and motor expertise for that action, but could it be modulated by the age-related motor skills of the agent performing the action? We used an eye-tracking visual preference paradigm to study the modulation of perception/action coupling in 62 adults when viewing videos of daily actions presented in a forward reading direction or, for the same action, in a backward reading direction. Video actions were performed by young actors aged 4, 8, and 13 years and by adults. We found greater pupil dilation for all backward videos compared to forward videos. Interestingly, pupil dilation was greater for the forward videos with child actors than for those with adolescent/adult actors. An overall comparison of looking times during the preference phase did not reveal a preference for either video but testing for a context effect revealed a preference for backward videos when the last video observed in the exposure phase was forward. This study demonstrated the influence of the agent's age-related motor abilities on the observer's perception/action coupling and revealed that pupil dilation and the context effect could be relevant cues for exploring this coupling in a non-invasive, passive experimental setup that is particularly suitable for exploring perception/action coupling in very young children with a developmental disorder.

A theory of rapid behavioral inferences under the pressure of time
To survive, animals must be able quickly infer the state of their surroundings. For example, to successfully escape an approaching predator, prey must quickly estimate the direction of approach from incoming sensory stimuli. Such rapid inferences are particularly challenging because the animal has only a brief window of time to gather sensory stimuli, and yet the accuracy of inference is critical for survival. Due to evolutionary pressures, nervous systems have likely evolved effective computational strategies that enable accurate inferences under strong time limitations. Traditionally, the relationship between the speed and accuracy of inference has been described by the "speed-accuracy tradeoff" (SAT), which quantifies how the average performance of an ideal observer improves as the observer has more time to collect incoming stimuli. While this trial-averaged description can reasonably account for individual inferences made over long timescales, it does not capture individual inferences on short timescales, when trial-to-trial variability gives rise to diverse patterns of error dynamics. We show that an ideal observer can exploit this single-trial structure by adaptively tracking the dynamics of its belief about the state of the environment, which enables it make more rapid inferences and more reliably track its own error but also causes it to violate the SAT. We show that these features can be used to improve overall performance during rapid escape. The resulting behavior qualitatively reproduces features of escape behavior in the fruit fly Drosophila melanogaster, whose escapes have presumably been highly optimized by natural selection.

Photoperiodicity in Glucose Metabolism in the Human Brain
Photoperiodicity in the human brain function, which is a critical factor for social well-being, has been widely debated. In this study, 432 healthy males underwent fasting-state brain [18F]fluorodeoxyglucose positron emission tomography (PET) scanning twice: first at the baseline and then at the 5-year follow-up. We analyzed the effect of day length on brain glucose uptake separately for the baseline and follow-up studies and examined changes in glucose consumption as a function of the day length deviation for each participant between the repeated PET scans. Glucose uptake in the cuneus was consistently predicted by the day length on the day of scanning and by within-participant day length deviations. This longitudinal large-scale PET study provides a landmark evidence for photoperiodicity in glucose metabolism in the human brain. The cuneus may be an essential part of the visual cortex, translating environmental photoperiodic changes into temporal cues that influence cognitive function and social behavior.

On-Demand Seizures Facilitate Rapid Screening of Therapeutics for Epilepsy
Animal models of epilepsy are critical in drug development and therapeutic testing, but dominant methods for pharmaceutical evaluation face a tradeoff between higher throughput and etiological relevance. For example, in temporal lobe epilepsy, a type of epilepsy where seizures originate from limbic structures like the hippocampus, the main screening models are either based on acutely induced seizures in wild type, naive animals or spontaneous seizures in chronically epileptic animals. Both types have their disadvantages - the acute convulsant or kindling induced seizures do not account for the myriad neuropathological changes in the diseased, epileptic brains, and spontaneous behavioral seizures are sparse in the chronically epileptic models, making it time-intensive to sufficiently power experiments. In this study, we took a mechanistic approach to precipitate seizures "on demand" in chronically epileptic mice. We briefly synchronized principal cells in the CA1 region of the diseased hippocampus to reliably induce stereotyped on-demand behavioral seizures. These induced seizures resembled naturally occurring spontaneous seizures in the epileptic animals and could be stopped by commonly prescribed anti-seizure medications such as levetiracetam and diazepam. Furthermore, we showed that seizures induced in chronically epileptic animals differed from those in naive animals, highlighting the importance of evaluating therapeutics in the diseased circuit. Taken together, we envision our model to advance the speed at which both pharmacological and closed loop interventions for temporal lobe epilepsy are evaluated.

Alterations in large-scale resting-state network nodes following transcranial focused ultrasound of deep brain structures
Background: Low-intensity transcranial focused ultrasound (tFUS) is a brain stimulation approach that holds immense promise for the treatment of brain-based disorders. Several studies in humans have shown that tFUS can successfully modulate perfusion in focal sonication targets including the amygdala; however, limited research has explored how tFUS impacts the function of large-scale neural networks. Objective: The aim of the current study was to address this gap and examine changes in resting-state connectivity between large-scale network nodes using a randomized, double-blind, within-subject crossover study design. Methods: Healthy adults (n=18) completed two tFUS sessions, 14 days apart. Each session included tFUS of either the right amygdala or the left entorhinal cortex (ErC). The inclusion of two active targets allowed for within-subjects comparisons as a function of the locus of sonication. Resting-state functional magnetic resonance imaging was collected before and after each tFUS session. Results: tFUS altered resting-state functional connectivity (rsFC) within and between rs-network nodes. Specifically, pre-to-post sonication of the right amygdala modulated connectivity within nodes of the salience network (SAN) and between nodes of the SAN and the default-mode network (DMN) and fronto-parietal network (FRP). A decrease in SAN to FPN connectivity was specific to the amygdala target. Pre-to-post sonication of the left ErC was found to modulate connectivity between the dorsal attention network (DAN) and FPN and DMN. An increase in DAN to DMN connectivity was specific to the ErC target. Conclusion: These preliminary findings may suggest that tFUS induces neuroplastic changes beyond the immediate sonication target.

Neural correlates of age-related changes in social decisions from episodic memory
Older adults are frequent victims of financial scams. Previous behavioral research suggests that this may be due to systematic biases in how they make decisions about whom to trust: for instance, Lempert et al. (2022) found that relative to younger adults, older adults were more likely to base decisions about whether to re-engage with someone on how generous that person looked, rather than on their memory for how they had previously behaved. Here, we aimed to identify the neural correlates of these age-dependent changes in social decision-making in order to clarify the mechanism by which they emerge. Using functional Magnetic Resonance Imaging (fMRI), we measured neural activity while a total of 86 participants -- 45 younger and 41 older adults -- learned about how much of a $10 endowment an individual, represented by a picture of their face, was willing to share with them in a dictator game. After this encoding phase, participants then made decisions about whom they wanted to play another round of the dictator game with. In line with previous findings, we found that older adults did not reliably prefer to re-engage with people who had proven themselves to be generous. This bias was the result of several factors: (1) older adults had worse associative memory for how much each person had shared, possibly due to an age-dependent decrease in neural activity in the medial temporal lobe (MTL) during encoding, (2) older adults had a stronger tendency to re-engage with familiar over novel faces regardless of their past behavior, and (3) while activity in value-responsive brain regions tracked with how generous a face looked across the age range, older adults were less able to inhibit the influence of these irrelevant perceptual features when it was necessary to do so. In line with this behavioral effect, younger adults showed greater activation in the inferior frontal gyrus (IFG) during choices that required suppressing irrelevant perceptual features in favor of associative memory. Taken together, our findings highlight age-dependent changes in both the ability to encode relevant information and to adaptively deploy it in service of social decisions.

Sodium channel inhibitors alter the progress of tangle development in a mouse model of dementia
Sodium channel inhibitors have been reported to protect against a range of neuroinflammatory and neurodegenerative diseases. Here the effect of chronic administration of two Na+ channel inhibitors with different mechanisms of action, phenytoin and GS967 are tested in mouse models of different stages of Alzheimers disease. Subtle changes in the distribution of plaque sizes were observed in AppNLGF/NLGF mouse at 3 months of age, after being fed control or drug-supplemented chow from weaning onwards, with phenytoin treatment resulting in a significant increase in the frequency of the smallest plaques and a decrease in large plaques. The later pathology of neurofibrillary tangles was studied, in old age, by supplementing the food of transgenic mice with a P301L mutation in Tau. Chronic administration of Na+ inhibitors from 15 months of age resulted in a decrease in the density of MC1-positive neurofibrillary tangles, possibly due to effects on microglial Na+ channels. The density of microglial cells was strongly correlated with the density of neurofibrillary tangles but only in mice treated with the Na+ inhibitors.

Center-surround inhibition by expectation: a neuro-computational account
Expectation is beneficial for adaptive behavior through quickly deducing plausible interpretations of information. The profile and underlying neural computations of this process, however, remain unclear. When participants expected a grating with a specific orientation, we found a center-surround inhibition profile in orientation space, which was independent from attentional modulations by task-relevance. Using computational modeling, we showed that this center-surround inhibition could be reproduced by either a sharpening of tuning curves of expected orientation or a shift of tuning curves of unexpected orientations. Intriguingly, these two computations were further supported by orientation-adjustment and orientation-discrimination experiments. Finally, the ablation studies in convolutional neural networks revealed that predictive coding feedback played a critical role in the center-surround inhibition in expectation. Altogether, our study reveals for the first time that expectation results in both enhancement and suppression, optimizing plausible interpretations during perception by enhancing expected and attenuating similar but irrelevant and potentially interfering representations.

Acetate enhances spatial memory in females via sex- and brain region-specific epigenetic and transcriptional remodeling
Metabolic control of chromatin and gene expression is emerging as a key, but largely unexplored aspect of gene regulation. In the brain, metabolic-epigenetic interactions can influence critical neuronal functions. Here, we use a combination of behavioral, proteomic and genomic approaches to demonstrate that the intermediary metabolite acetate enhances memory in a brain region- and sex-specific manner. We show that acetate facilitates the formation of dorsal hippocampus-dependent spatial memories in female but not in male mice, while having no effect on cortex-dependent non-spatial memories in either sex. Acetate-enhanced spatial memory is driven by increased acetylation of histone variant H2A.Z, and upregulation of genes implicated in spatial learning in the dorsal hippocampus of female mice. In line with the sex-specific behavioral outcomes, the effect of acetate on dorsal hippocampal histone modifications and gene expression shows marked differences between the sexes during critical windows of memory formation (consolidation and recall). Overall, our findings elucidate a novel role for acetate, a ubiquitous and abundant metabolite, in regulating dorsal hippocampal chromatin, gene expression and learning, and outline acetate exposure as a promising new approach to enhance memory formation.

Assessing neuronal correlates of salience and their adaptability with naturalistic textures in macaque V1 and V2
Salience is critical to vision. It allows stimuli that are different from their surroundings to 'pop out', drawing our attention. Perceptual salience is postulated to be encoded via a saliency map, based on differences in neuronal responsivity to simple image features at different spatial locations. Simple image features such as luminance, orientation and color are known to affect saliency and many of these features are encoded in primary visual cortex (V1), which several influential theories propose instantiate a saliency map. However, the degree to which more complex image features can determine salience, and whether there are neural correlates of salience which are computed outside of V1, remains unclear. Here we use displays of naturalistic textures to test for neural correlates of salience-termed pop-out responses-in V1 and area V2 of anesthetized macaque monkeys. Sensitivity to higher-order texture statistics arises in V2, so pop-out responses for these displays, if they exist, would be expected to be computed after V1. We presented displays in which a target texture, presented within the neuronal receptive field, was surrounded by distractors. Distractors could differ from the target texture in either higher-order texture statistics only, or in both lower- and higher-order statistics. We found little evidence for pop-out signals in either V1 or V2, for either display type. However, brief periods of adaptation could induce pop-out responses in V2. This suggests that adaptation might define which features of the environment are most salient, even if those features would otherwise not evoke pop-out responses.

Neural tracking of auditory statistical regularities is reduced in adults with dyslexia
Listeners implicitly use statistical regularities to segment continuous sound input into meaningful units, e.g., transitional probabilities between syllables to segment a speech stream into separate words. Implicit learning of such statistical regularities in a novel stimulus stream is reflected in a synchronisation of neural responses to the sequential stimulus structure. The present study aimed to test the hypothesis that neural tracking of the statistical stimulus structure is reduced in individuals with dyslexia who have weaker reading and spelling skills, and possibly also weaker statistical learning abilities in general, compared to healthy controls. To this end, adults with and without dyslexia were presented with continuous streams of (non-speech) tones, which were arranged into triplets, such that transitional probabilities between single tones were high within triplets and low between triplets. We found that neural tracking of the triplet structure, i.e., phase coherence of EEG response at the triplet rate relative to the tone rate, was reduced in adults with dyslexia compared to the control group. Moreover, enhanced neural tracking of the statistical structure was associated with better spelling skills. These results suggest that individuals with dyslexia have a rather broad deficit in processing structure in sound instead of a merely phonological deficit.

Would you agree if N is three?On statistical inference for small N.
Non-human primate studies traditionally use two or three animals. We previously used standard statistics to argue for using either one animal, for an inference about that sample, or five or more animals, for a useful inference about the population. A recently proposed framework argued for testing three animals and accepting the outcome found in the majority as the outcome that is most representative for the population. The proposal tests this framework under various assumptions about the true probability of the representative outcome in the population, i.e. its typicality. On this basis, it argues that the framework is valid across a wide range of typicalities. Here, we show (1) that the error rate of the framework depends strongly on the typicality of the representative outcome, (2) that an acceptable error rate requires this typicality to be very high (87% for a single type of outlier), which actually renders empirical testing beyond a single animal obsolete, (3) that moving from one to three animals decreases error rates mainly for typicality values of 70-90%, and much less for both lower and higher values. Furthermore, we use conjunction analysis to demonstrate that two out of three animals with a given outcome only allow to infer a lower bound to typicality of 9%, which is of limited value. Thus, the use of two or three animals does not allow a useful inference about the population, and if this option is nevertheless chosen, the inferred lower bound of typicality should be reported.