bioRxiv Subject Collection: Neuroscience's Journal

Riemannian Geometry for the Classification of Brain States with fNIRS
Background: Functional near-infrared spectroscopy (fNIRS) has recently gained momentum as a reliable and accurate tool for assessing brain states. This increase in popularity is due to its robustness to movement, non-invasive nature, portability, and user-friendly application. However, compared to functional magnetic resonance imaging (fMRI), fNIRS is less sensitive to deeper brain activity and offers less coverage. Additionally, due to fewer advancements in method development, the performance of fNIRS-based brain-state classification still lags behind more prevalent methods like fMRI. Methods: We introduce a novel classification approach grounded in Riemannian geometry for the classification of kernel matrices, leveraging the temporal and spatial channel relationships and inherent duality of fNIRS signals-more specifically, oxygenated and deoxygenated hemoglobin. For the Riemannian geometry-based models, we compared different kernel matrix estimators and two classifiers: Riemannian Support Vector Classifier and Tangent Space Logistic Regression. These were benchmarked against four models employing traditional feature extraction methods. Our approach was tested in two brain-state classification scenarios based on the same fNIRS dataset: an 8-choice classification, which includes seven established plus an individually selected imagery task, and a 2-choice classification of all possible 28 2-task combinations. Results: The novel approach achieved a mean 8-choice classification accuracy of 65%, significantly surpassing the mean accuracy of 42% obtained with traditional methods. Additionally, the best-performing model achieved an average accuracy of 96% for 2-choice classification across all possible 28 task combinations, compared to 78% with traditional models. Conclusion: To our knowledge, we are the first to demonstrate that the proposed Riemannian geometry-based classification approach is both powerful and viable for fNIRS data, considerably increasing the accuracy in binary and multi-class classification of brain activation patterns.

Astrocyte ezrin defines resilience to stress-induced depressive behaviours in mice
Astrocyte atrophy is the main histopathological hallmark of major depressive disorder (MDD) in humans and in animal models of depression. Here we demonstrated that manipulating with ezrin expression specifically in astrocytes significantly increases the resilience of mice to chronic unpredictable mild stress (CUMS). Overexpression of ezrin in astrocytes from prefrontal cortex (PFC) rescued depressive-like behaviours induced by CUMS, whereas down-regulation of ezrin in astrocytes from PFC increased mice susceptibility to CUMS and promoted depressive-like behaviours. These behavioural changes correlated with astrocytic morphology. Astrocytes from PFC of mice sensitive to CUMS demonstrated significant atrophy; similar atrophy was found in astrocytes from animals with down-regulated ezrin expression. To the contrary morphology remains unchanged astrocytes in animals resistant to CUMS and in animals with astrocytic overexpression of ezrin. Morphological changes also correlated with ezrin immunoreactivity which was low in mice with depressive-like behaviours and high in mice resistant to stress. We conclude that Ezrin-dependent morphological remodelling of astrocytes defines the sensitivity of mice to stress: high ezrin expression renders them stress resilient, whereas low ezrin expression promotes depressive-like behaviour in response to chronic stress.

Generating human parvalbumin interneurons through 3D glia reprogramming
Parvalbumin (PV) interneurons are crucial for synaptic plasticity, and their damage or loss is linked to various neurological disorders. Yet, generating these cells of human source in vitro is challenging, limiting advancements in cell repair and disease modelling. We introduce a novel approach to derive human PV neurons through direct reprogramming of glial precursor cells (GPCs). Using ectopic expression of GABAergic neuronal genes, GPCs efficiently convert into GABAergic interneurons in 3D culture environment within weeks and achieve functional neuronal maturity. Single-nuclei RNA sequencing identified a distinct PV neuronal cluster with high maturity and characteristics of PV chandelier subclass that are equivalent to bona fide human interneurons. Trajectory analysis revealed a distinct glia-to-PV interneuron conversion pathway, involving several new transitory genes, with potential for functional importance for PV derivation. Our data introduces a new strategy for generating human PV interneurons, promising significant implications for future generation of patient-specific PV neurons both in vitro and in vivo.

Systemic injection of scopolamine increased the variability of temporal prediction in head-fixed mice.
Several theories propose a close relationship between interval timing and the temporal properties of memory. Systemic administration of scopolamine, a muscarinic acetylcholine receptor antagonist, is known to induce memory deficits and impair temporal prediction. However, existing studies on timing using free-moving animals are challenging to interpret due to the confounding effects of movement on interval timing. In this study, we examined the effects of scopolamine on timing behavior in mice using a head-fixed experimental setup. Mice were trained on a fixed-time schedule task with a peak procedure, where a 10% sucrose solution was delivered every 10 seconds through a spout placed within the licking distance of the mice. Following training, the mice exhibited anticipatory licking behavior in response to the timing of sucrose delivery, indicating that they could predict the reward timing. Systemic administration of scopolamine increased the variability of temporal prediction in a dose-dependent manner but did not affect the mean temporal prediction. Single-trial bout analysis revealed that scopolamine impaired the duration of licking bouts without affecting the total number of licks in peak trials, suggesting that the mice were unable to sustain licking at the spout. Additionally, we assessed the effects of scopolamine on spontaneous locomotor activity and excretion in a free-moving open-field task. Scopolamine injections increased locomotor activity and decreased fecal output. Taken together, these findings suggest that the increased variability in timing behavior induced by scopolamine may be attributed to changes in transitions between behavioral states.

Disrupted development of sensory systems and the cerebellum in a zebrafish ebf3a mutant
Mutations in the transcription factor EBF3 results in a neurodevelopmental disorder, and studies in animal models indicate that it has a critical role in neuronal differentiation. The molecular pathways and neuron types disrupted by its loss, however, have not been thoroughly investigated. Nor have the outcomes of these changes on behavior and brain activity. Here, we generated and characterized a zebrafish ebf3a loss-of-function mutant. We discovered morphological and neural phenotypes, including an overall smaller brain size, particularly in the hypothalamus, cerebellum, and hindbrain. Brain function was also compromised, with activity strongly increased in the cerebellum and abnormal behavior at baseline and in response to visual and acoustic stimuli. From RNA-sequencing of developing larvae, notable changes included significant downregulation of genes that mark olfactory sensory neurons, the lateral line, and cerebellar Purkinje neurons. This study sets the stage for determining which downstream pathways underlie the emergence of the observed phenotypes and establishes multiple strong phenotypes that could form the basis of a drug screen.

Progressive overfilling of readily releasable pool underlies short-term facilitation at recurrent excitatory synapses in layer 2/3 of the rat prefrontal cortex
Short-term facilitation of recurrent excitatory synapses within the cortical network has been proposed to support persistent activity during working memory tasks, yet the underlying mechanisms remain poorly understood. We characterized short-term plasticity at the local excitatory synapses in layer 2/3 of the rat medial prefrontal cortex and studied its presynaptic mechanisms. Low-frequency stimulation induced slowly developing facilitation, whereas high-frequency stimulation initially induced strong depression followed by rapid facilitation. This non-monotonic delayed facilitation after a brief depression resulted from a high vesicular fusion probability and slow activation of Ca2+-dependent vesicle replenishment, which led to the overfilling of release sites beyond their basal occupancy. Pharmacological and gene knockdown (KD) experiments revealed that the facilitation was mediated by phospholipase C/diacylglycerol signaling and synaptotagmin 7 (Syt7). Notably, Syt7 KD abolished facilitation and slowed the refilling rate of vesicles with high fusion probability. Furthermore, Syt7 deficiency in layer 2/3 pyramidal neurons impaired the acquisition of trace fear memory and reduced c-Fos activity. In conclusion, Ca2+- and Syt7-dependent overfilling of release sites mediates synaptic facilitation at L2/3 recurrent excitatory synapses and contributes to temporal associative learning.

Early Life Neuroimaging: The Generalizability of Cortical Area Parcellations Across Development
The cerebral cortex comprises discrete cortical areas that form during development. Accurate area parcellation in neuroimaging studies enhances statistical power and comparability across studies. The formation of cortical areas is influenced by intrinsic embryonic patterning as well as extrinsic inputs, particularly through postnatal exposure. Given the substantial changes in brain volume, microstructure, and functional connectivity during the first years of life, we hypothesized that cortical areas in 1-to-3-year-olds would exhibit major differences from those in neonates and progressively resemble adults as development progresses. Here, we parcellated the cerebral cortex into putative areas using local functional connectivity gradients in 92 toddlers at 2 years old. We demonstrated high reproducibility of these cortical regions across 1-to-3-year-olds in two independent datasets. The area boundaries in 1-to-3-year-olds were more similar to adults than neonates. While the age-specific group parcellation fitted better to the underlying functional connectivity in individuals during the first 3 years, adult area parcellations might still have some utility in developmental studies, especially in children older than 6 years. Additionally, we provided connectivity-based community assignments of the parcels, showing fragmented anterior and posterior components based on the strongest connectivity, yet alignment with adult systems when weaker connectivity was included.

Omission-responsive neurons encode negative prediction error and probability in the auditory cortex
Predictive coding posits the brain predicts incoming sensory information and signals prediction errors when actual input differs from expectations. Positive prediction errors occur when input exceeds predictions, while negative prediction errors arise when input falls short. Specific neurons are theorized to encode negative prediction errors, linked to responses to omitted expected inputs. However, the information encoded in omission responses remains unclear. We recorded single-unit activity in rat auditory cortex during an omission paradigm with varying tone probabilities. We identified neurons selectively responding to omissions, with responses increasing with evidence accumulation and correlating with tone predictability; key characteristics of negative prediction-error neurons. Interestingly, these neurons showed selective omission responses but broad tone responses, revealing an asymmetry in error signaling. We propose a circuit model with laterally interconnected prediction-error neurons reproducing this asymmetry. Our model demonstrates that lateral connections enhance precision and efficiency of prediction encoding, supported by the free energy principle

Eye Movements in Silent Visual Speech track Unheard Acoustic Signals and Relate to Hearing Experience
Behavioral and neuroscientific studies have shown that watching a speaker's lip movements aids speech comprehension. Intriguingly, even when videos of speakers are presented silently, various cortical regions track auditory features, such as the envelope. Recently, we demonstrated that eye movements track low-level acoustic information when attentively listening to speech. In this study we investigated whether ocular speech tracking occurs during visual speech and how it influences cortical silent speech tracking. Furthermore, we compared the data of hearing individuals with congenitally deaf individuals, and those with acquired deafness or hearing loss (DHH; Deaf or hard of hearing) to assess how auditory deprivation (early vs. late onset) affects neural and ocular speech tracking during silent lip-reading. Using magnetoencephalography (MEG), we examined ocular and neural speech tracking of 75 participants observing silent videos of a speaker played forward and backward. Our main finding is a clear ocular unheard speech tracking effect with a dominance <1 Hz, which was not present for the lip movements. Similarly, we observed a <=1.3 Hz effect of neural unheard speech tracking in temporal regions for hearing participants. Importantly, neural tracking was not directly linked to ocular tracking in this study. Strikingly, across different listening groups, deaf participants with auditory listening experience showed higher ocular speech tracking than hearing participants, while no ocular speech tracking effect was revealed for congenitally deaf participants in a very small sample. This study extends our previous work by demonstrating the involvement of eye movements in speech processing, even in the absence of acoustic input.

Effects Of Extrinsic Reward Based Skill Learning On Motor Plasticity
Human motor skill acquisition is improved by performance feedback and coupling such feedback with extrinsic reward (such as money) can enhance skill learning. However, the neurophysiology underlying such behavioral effect is unclear. To bridge this gap, we assessed the effects of reward on multiple forms of motor plasticity during skill learning. Sixty-five healthy participants divided in three groups performed a pinch-grip skill task with sensory feedback only, sensory and reinforcement feedback or both feedback coupled with an extrinsic monetary reward during skill training. To probe motor plasticity, we applied transcranial magnetic stimulation on the left primary motor cortex at rest before, during and after training in the three groups. We evaluated the amplitude and variability of corticospinal output, GABA-ergic short-intracortical inhibition and use-dependent plasticity before training and at two time points during and after training. At the behavioral level, monetary reward accelerated skill learning. In parallel, corticospinal output became less variable early on during training in the presence of extrinsic reward. Interestingly, this effect was particularly pronounced for participants who were more sensitive to reward, as evaluated in an independent questionnaire. Other measures of motor excitability remained comparable across groups. These findings highlight that a mechanism underlying the benefit of reward on motor skill learning is the fine tuning of early-training resting-state corticospinal variability.

Shox2 is necessary for normal thalamic spindle function
The cellular identity of thalamocortical neurons (TCNs), namely their firing properties, dictates brain-wide activity patterns, such as sleep spindles. Transcription factors are critical to the determination of cellular identity. Previously, we discovered that a subset of TCNs express the transcription factor, Shox2, and, in a global Shox2 KO, established that TCNs within the anterior nucleus of the thalamus rely on the expression of Shox2 to regulate key ion channels that are necessary to maintain their firing properties. From this, we hypothesized that Shox2 expression, through the regulation of firing properties of TCNs, is critical for the thalamocortical circuit to generate spindle oscillations. We exploited the somatosensory thalamocortical circuit to investigate this by creating a primary somatosensory thalamus (VB) Shox2 knockdown mouse model. We delivered Cre into the VB of P21 Shox2fl/fl mice using viral infection and compared in vitro, patch-clamp recordings from Shox2+ and Shox2 knockdown TCNs, finding that Shox2 expression is indeed critical to maintain burst and tonic firing properties of VB TCNs. Since Shox2 is important developmentally and firing from TCNs to cortex during development structures the circuit, we performed ultrasound-guided P3 injections at P3 to generate an early-stage, Shox2 VB knockdown, but found no changes in the layer four, barrel map (VB cortical target). Despite this, Shox2 knockdown mice exhibit reduced sleep-spindle EEG density. Further, key behaviors associated with spindles and proper VB thalamic function, memory consolidation and somatosensory perception, are significantly impaired. These results indicate that the impact on spindle function is likely due to cell autonomous changes to TCNs rather than circuit changes, confirming our hypothesis that Shox2 is necessary for normal thalamic spindle function and implicating a potential role for Shox2 in autism and schizophrenia pathologies.

Curriculum Learning: sequential acquisition of task complexity enhances neuronal discriminability
Curriculum Learning (CL) is a strategy where concepts with increasing complexity are acquired sequentially. Despite its widespread application, its putative neural mechanisms are poorly understood. In this study, using a multi-staged Go/No-Go auditory discrimination paradigm for mice, we show that CL improves and accelerates learning in a unidirectional way. By combining behavioral data with in vivo electrophysiological recordings of the auditory cortex, we demonstrate that neuronal discriminability is enhanced with sequential training. This improvement suggests that CL involves a convergence of common discriminable features in the auditory stimuli shared across tasks of different complexity. Our findings uncover specific properties of CL at the level of neuronal discriminability, distinguishing it from other forms of learning strategies.

Multi-epitope immunocapture of huntingtin reveals striatum-selective molecular signatures
Huntington's disease (HD) is a debilitating neurodegenerative disorder affecting an individual's cognitive and motor abilities. HD is caused by mutation in the huntingtin gene producing a toxic polyglutamine-expanded protein (mHTT) and leading to degeneration in the striatum and cortex. Yet, the molecular signatures that underlie tissue-specific vulnerabilities remain unclear. Here, we investigate this aspect by leveraging multi-epitope protein interaction assays, subcellular fractionation, thermal proteome profiling, and genetic modifier assays. Use of human cell, mouse, and fly models afforded capture of distinct subcellular pools of epitope-enriched and tissue-dependent interactions linked to dysregulated cellular pathways and disease relevance. We established an HTT association with nearly all subunits of the transcriptional regulatory Mediator complex (20/26), with preferential enrichment of MED15 in the tail domain. Using HD and KO models, we find HTT modulates the subcellular localization and assembly of Mediator. We demonstrated striatal enriched and functional interactions with regulators of calcium homeostasis and chromatin remodeling, whose disease relevance was supported by HD fly genetic modifiers assays. Altogether, we offer insights into tissue- and localization-dependent (m)HTT functions and pathobiology.

Spatiotemporal evidence accumulation through saccadic sampling for object recognition
Visual object recognition has been extensively studied under fixation conditions, but our natural viewing involves frequent saccadic eye movements that scan multiple local informative features within an object (e.g., eyes and mouth in a face image). Such visual exploration can facilitate object recognition, but mechanistic accounts of the contribution of saccades are yet to be established due to the presumed complexity of the interactions between the visual and oculomotor systems. Here, we present a framework for formulating object recognition as a process of accumulating evidence from local features through saccades to render a decision. This approach offers a simple model that quantitatively explains human face and object categorization behavior, even under conditions in which people freely make saccades to scan local features, departing from past studies that required controlled eye movements to examine trans-saccadic integration. Notably, our experimental results showed that active saccade commands (efference copy) did not substantially contribute to behavioral performance and that the patterns of saccades were largely independent of the ongoing decision-making processes. Therefore, we propose that object recognition with saccades can be approximated using a parsimonious decision-making model without assuming complex interactions between the visual and oculomotor systems.

Aversion encoding and behavioral state modulation of lateral habenula neurons
The lateral habenula (LHb) integrates aversive information to regulate motivated behaviors. Despite recent advances in identifying neuronal diversity at the molecular level, in vivo electrophysiological diversity of LHb neurons remains poorly understood. Understanding this diversity is essential for deciphering how information is processed in the LHb. To address this gap, we conducted in vivo electrophysiological recordings in mice and applied unsupervised clustering algorithm to analyze firing patterns. This analysis identified four distinct spontaneous firing patterns of LHb neurons, which were consistent across both anesthetized and awake states. To determine whether these firing patterns correlate with function, we recorded neuronal responses to foot shock stimulation in anesthetized mice and monitored spontaneous behavior in awake mice. We found that low-firing, bursting neurons were preferentially modulated by foot shocks in anesthetized mice and also tracked behavioral states in awake mice. Collectively, our findings indicate significant electrophysiological diversity among LHb neurons, which is associated with their modulation by aversive stimuli and behavioral state.

Bridging the gap between grey-matter microstructural and functional connectomes: A multi-modal MRI study in preterm and full-term infants
Functional networks characterised by synchronous neural activity across distributed brain regions have been observed to emerge early in neurodevelopment. However, how the maturation of regional specialization and functional organization relates to developmental changes in structure is poorly understood. The covariation of regional microstructural properties, or microstructural connectivity (MC), could partially reflect synchronized maturation across regions that relate to functional connectivity (FC) during the early neurodevelopmental period. In this study, we investigated the evolution of MC and FC postnatally across a set of cortical and subcortical regions, focusing on 45 preterm infants scanned longitudinally, and compared to 45 matched full-term neonates as part of the developing Human Connectome Project (dHCP). Our findings revealed a global reinforcement of both MC and FC with age, with connection-specific variability dependent on the connection maturational stage and confirmed the impact of prematurity at term-equivalent age. We also reported a significant relationship between MC and FC during the preterm period. This relationship seemed to decrease with development when directly comparing the two modalities. However, evaluation of overlaps between MC- and FC-derived networks suggested increasing relationships with age, potentially due to a shared network structure underlying changes of microstructural and functional properties. Thus, our study offers novel insights on the complex interplay of functional and microstructural development and highlights the potential utility of MC as a complementary descriptor for characterizing the brain network development and alterations due to perinatal insults such as premature birth.

Measuring the neurodevelopmental trajectory of excitatory-inhibitory balance via visual gamma oscillations
Disruption of the balance between excitatory and inhibitory neurotransmission (E-I balance) underlies theories of many neurodevelopmental disorders, however, its study is typically restricted to adults, animal models and the lab-bench. Neurophysiological oscillations in the gamma frequency band relate closely to E-I balance, and a new technology - OPM-MEG - offers the possibility to measure such signals across the lifespan. We used OPM-MEG to measure gamma oscillations induced by visual stimulation in >100 participants, aged 2-34 years. We demonstrate a significantly changing spectrum with age, with low amplitude broadband gamma oscillations in children and high amplitude band limited oscillations dominating in adults. We used a canonical cortical microcircuit to model these gamma signals, revealing significant age-related shifts in E-I balance in superficial pyramidal neurons in visual cortex. Our findings detail the first MEG metrics of gamma oscillations and their underlying generators from toddlerhood, providing a benchmark against which future studies can contextualise.

What if...? Computational anatomy in the cerebellar microzone, the middle layer of the cerebellar network
It is generally assumed that the representation of information in the brain is in high resolution and replicable with high fidelity, and that those features make it precise and characterise the inputs to sophisticated computations. Replicability is an important assumption of neural network learning models, including cerebellar models. We question those ideas. The molecular layer of the cerebellar cortex is divided functionally into strips called microzones populated by Purkinje cells and inhibitory interneurons. We propose instead: (1) The microzone computation is a passive effect, unaided, of the triad of cell morphologies, cerebellar network geometry and linear transmission. (2) Control of Purkinje cells is by a chain of rate codes, and not supplanted by learning. (3) Striped topography of input to a network can represent collectively coherent information about body shape and movement. This is capable, passed through the network computation, of driving coordinated motor sequences. (4) A learning algorithm and replicability are unnecessary to explain the evidence.

Learning to read transforms phonological into phonographic representations: Evidence from a Mismatch Negativity study
Learning to read changes the nature of speech representation. One possible change consists in transforming phonological representations into phonographic ones. However, evidence for such transformation remains surprisingly scarce. Here, we used a novel word learning paradigm to address this issue. During a learning phase, participants were exposed to unknown words in both spoken and written forms. Following this phase, the impact of spelling knowledge on spoken input perception was assessed at two time points through an unattended oddball paradigm, while the Mismatch Negativity component was measured by high density EEG. Immediately after the learning phase, no influence of spelling knowledge on the perception of the spoken input was found. Interestingly, one week later, this influence emerged, making similar sounding words with different spellings more distinct than similar sounding words that also share the same spelling. Our finding provides novel neurophysiological evidence of an integration of phonological and orthographic representations that occurs once newly acquired knowledge has been consolidated. These novel phonographic representations may characterize how known words are stored in literates' mental lexicon.

Neural Correlates of the Embodied Sense of Agency
The Sense of Agency (SoA) is the subjective experience that 'I am in control of my actions'. Recent accounts have distinguished two levels in the formation of SoA - early implicit sensorimotor processes (feeling of agency) and later explicit higher-level processes, incorporating one's thoughts and beliefs (judgment of agency). Even though SoA is fundamental to our interactions with the external world and the construct of the self, its underlying neural mechanism remains elusive. In the current pre-registered electroencephalography (EEG) study, we used time-frequency and Multivariate Pattern Analysis (MVPA) to investigate the electrophysiological characteristics associated with SoA. We used an established embodied virtual reality paradigm in which visual feedback of a movement is modulated to examine the effect of conflicts between the predicted and perceived sensory feedback. Participants moved their finger and were shown a virtual hand that either mimicked their movement or differed anatomically (different finger) or spatially (angular shift), then judged their SoA over the observed movement while their brain activity was recorded. In accordance with our pre-registered hypothesis, we found that a reduction of SoA is associated with decreased attenuation in the alpha frequency band. Increased power in the theta frequency band was also associated with SoA reduction. Importantly, we show that trials containing a sensorimotor alteration vs. trials containing no alteration can reliably be decoded with up to 68% accuracy starting around 200ms after the movement onset. Finally, cross-decoding analyses revealed similar neural patterns for reduced SoA in the anatomical and spatial conditions, starting around 500ms after the movement onset. Together, our results reveal a cortical signature of loss of SoA and provide neural evidence supporting the hypothesis of a two-level formation of SoA - an early domain-specific component, possibly the equivalent of the implicit feeling of agency, and a late domain-general component, possibly the equivalent of the explicit judgment of agency.

KIF5A regulates axonal repair and time-dependent axonal transport of SFPQ granules and mitochondria in human motor neurons
Mutations in the microtubule binding motor protein, kinesin family member 5A (KIF5A), cause the fatal motor neuron disease, Amyotrophic Lateral Sclerosis. While KIF5 family members transport a variety of cargos along axons, it is still unclear which cargos are affected by KIF5A mutations. We generated KIF5A null mutant human motor neurons to investigate the impact of KIF5A loss on the transport of various cargoes and its effect on motor neuron function at two different timepoints in vitro. The absence of KIF5A resulted in reduced neurite complexity in young motor neurons (DIV14) and significant defects in axonal regeneration capacity at all developmental stages. KIF5A loss did not affect neurofilament transport but resulted in decreased mitochondria motility and anterograde speed at DIV42. More prominently, KIF5A depletion strongly reduced anterograde transport of SFPQ-associated RNA granules in DIV42 motor neuron axons. We conclude that KIF5A most prominently functions in human motor neurons to promote axonal regrowth after injury as well as to anterogradely transport mitochondria and, to a larger extent, SFPQ-associated RNA granules in a time-dependent manner.

Variation in moment-to-moment brain state engagement changes across development and contributes to individual differences in executive function
Neural variability, or variation in brain signals, facilitates dynamic brain responses to ongoing demands. This flexibility is important during development from childhood to young adulthood, a period characterized by rapid changes in experience. However, little is known about how variability in the engagement of recurring brain states changes during development. Such investigations would require the continuous assessment of multiple brain states concurrently. Here, we leverage a new computational framework to study state engagement variability (SEV) during development. A consistent pattern of SEV changing with age was identified across cross-sectional and longitudinal datasets (N>3000). SEV developmental trajectories stabilize around mid-adolescence, with timing varying by sex and brain state. SEV successfully predicts executive function (EF) in youths from an independent dataset. Worse EF is further linked to alterations in SEV development. These converging findings suggest SEV changes over development, allowing individuals to flexibly recruit various brain states to meet evolving needs.

Potent and selective repression of SCN9A by engineered zinc finger repressors for the treatment of neuropathic pain
Peripheral neuropathies are estimated to affect several million patients in the US with no long-lasting therapy currently available. In humans, the Nav1.7 sodium channel, encoded by the SCN9A gene, is involved in a spectrum of inherited neuropathies, and has emerged as a promising target for analgesic drug development. The development of a selective Nav1.7 inhibitor has been challenging, in part due to structural similarities among other Nav channels. Here we present preclinical studies for the first genomic medicine approach using engineered zinc finger repressors (ZFRs) specifically targeting human/nonhuman primate (NHP) SCN9A. AAV-mediated delivery of ZFRs in human iPSC-derived neurons resulted in 90% reduction of SCN9A with no detectable off-target activity. To establish proof-of-concept, a ZFR targeting the mouse Scn9a was assessed in the SNI neuropathic pain mouse model, which resulted in up to 70% repression of Scn9a in mouse DRGs and was associated with reduction in pain hypersensitivity as measured by increased mechanical- and cold-induced pain thresholds. AAV-mediated intrathecal delivery of ZFR in NHPs demonstrated up to 60% repression of SCN9A in bulk DRG tissue and on single-cell levels in the nociceptors. The treatment was well tolerated in NHPs, and no dose-limiting findings were observed four weeks after a single intrathecal injection. Taken together, our results demonstrate that AAV-delivered ZFR targeting the SCN9A gene is promising and supports further development as a potential therapy for peripheral neuropathies.

Death in the taste bud: Morphological features of dying taste cells and engulfment by Type I cells
Taste buds comprise 50-100 epithelial derived cells, which are renewed throughout the life of an organism. Immature cells enter the bud at its base, maturing into one of three distinct cell types. How taste cells die and/or exit the bud, however, remains unclear. Here we present morphological data obtained through Serial Blockface Scanning Electron Microscopy of murine circumvallate taste buds, revealing several taste cells at the end of their life (4-6 per bud). Cells we identify as dying share certain morphological features typical of apoptosis: swollen endoplasmic reticulum, large lysosomes, degrading organelles, distended outer nuclear membranes, heterochromatin reorganization, cell shrinkage, and cell and/or nuclear fragmentation. Based on these features, we divide the cells into early and late stage dying cells. Most early stage dying cells have Type II cell morphologies, while a few display Type III cell features. Many dying cells maintain contacts with nerve fibers, but those fibers often appear detached from the main trunk of an afferent nerve fiber. Dying cells, like mature Type II and Type III taste cells, are surrounded by Type I taste cells, the glial-like cells of the bud. In many instances Type I cells appear to be engulfing their dying neighbors, suggesting a novel, phagocytic role for Type I cells. Surprisingly, virtually no Type I cells, which have the shortest residence time in taste buds, display features of apoptosis. The ultimate fate of Type I cells therefore remains enigmatic.

Neurotransmitters Contribute Structure-Function Coupling: Evidence from Grey Matter Volume (GMV) and Brain Entropy (BEN)
The emergence of human brain function from macroscopic anatomical structures and its relationship with the coupling of structure and function have long been pivotal questions in neuroscience. Neurotransmitter receptors are critical in signal transmission, regulating brain function, and potentially enhancing the coupling between brain structure and function. Recent research suggests that neurotransmitter systems facilitate this coupling between structural and functional networks. However, the mechanisms of how neurotransmitters adapt to local anatomical structures and drive functional emergence are not yet fully understood. This study explores these mechanisms using gray matter volume (GMV) and brain entropy (BEN). BEN reflects the irregularity, unpredictability, and complexity of brain activity, and functional MRI (fMRI)-based BEN has identified its distribution in the normal human brain. BEN is correlated to cognitive and task performance, and aberrant BEN patterns link to various brain disorders. Notably, BEN can indicate neuroplasticity through non-invasive brain stimulation, and pharmacological intervention. We analyzed structural imaging data, as well as 7T resting-state and movie-watching fMRI data from 176 participants in the Human Connectome Project (HCP), to calculate GMV, resting-state BEN (rsBEN), and movie-watching BEN (mvBEN). By integrating data from the publicly available neurotransmitter receptors database, we evaluated the role of neurotransmitters in the coupling between GMV and rsBEN, GMV and mvBEN, and rsBEN and mvBEN. Our findings reveal that neurotransmitters significantly contribute to structural and functional coupling and influence the transition from task-free to movie-watching conditions.

Brain-wide projections of mouse dopaminergic zona incerta neurons
The zona incerta (ZI) supports diverse behaviors including binge feeding, sleep/wake cycles, nociception, and hunting. This diversity of functions can be attributed to the heterogenous neurochemicals, cytoarchitecture, and efferent connections that characterize the ZI. The ZI is predominantly GABAergic, but we recently identified a subset of medial ZI GABA cells that co-express dopamine (DA), as marked by the enzyme tyrosine hydroxylase (TH). While the role of GABA within the ZI is well studied, little is understood about the function of ZI DA cells. To identify potential roles of ZI DA cells we mapped the efferent fiber projections from Th-cre ZI cells. We first validated a Th-cre;L10-Egfp mouse line and found that medial Egfp ZI cells were more likely to co-express TH-immunoreactivity (TH-ir). We thus delivered a cre-dependent virus into the medial ZI of Th-cre or Th-cre;L10-Egfp mice and selected two injection cases for full brain mapping. We selected the cases with the lowest (17%) and highest (53%) percentage of colocalization between TH-ir and virus transfected cells labelled with DsRed. Overall, DsRed-labelled fibers were observed throughout the brain and were most prominent within motor-related regions of the midbrain (MBmot), notably the periaqueductal grey area and superior colliculus. We also observed considerable DsRed-labelled fibers within the polymodal cortex associated regions of the thalamus (DORpm), including the paraventricular thalamic nucleus and nucleus of reunions. Overall, ZI DA cells displayed a similar connectivity profile to ZI GABA cells, suggesting that ZI DA cells may perform synergistic or opposing functions at the same target sites.