bioRxiv Subject Collection: Neuroscience's Journal

Nanophysiology Approach Revealed Diversity in Calcium Microdomains in Zebrafish Retinal Bipolar Ribbon Synapses
Rapid and high local calcium (Ca2+) signals are essential for triggering neurotransmitter release from presynaptic terminals. In specialized bipolar ribbon synapses in the retina, these local Ca2+ signals control multiple processes, including the priming, docking, and translocation of vesicles on the ribbon before exocytosis and the replenishment of release-ready vesicles to the fusion sites to sustain neurotransmission. However, our knowledge about Ca2+ signals along the axis of the ribbon active zone is limited. Here, we used fast confocal quantitative dual-color ratiometric line-scan imaging of a fluorescently labeled ribbon binding peptide and Ca2+ indicators to monitor the spatial and temporal aspects of Ca2+ transients of individual ribbon active zones in zebrafish retinal rod bipolar cells. We observed that a Ca2+ transient elicited a much greater fluorescence amplitude when the Ca2+ indicator was conjugated to a ribeye-binding peptide than when using a soluble Ca2+ indicator, and the estimated Ca2+ levels at the ribbon active zone exceeded 26 M in response to a 10-millisecond stimulus, as measured by a ribbon-bound low-affinity Ca2+ indicator. Our quantitative modeling of Ca2+ diffusion and buffering is consistent with this estimate and provides a detailed view of the spatiotemporal [Ca2+] dynamics near the ribbon. Importantly, our data demonstrates that the local Ca2+ levels may vary between ribbons of different rod bipolar cells and within the same cells. The variation in local Ca2+ signals is likely due to heterogeneity in local Ca2+ channels clustered at the ribbon active zone, as serial electron microscopy provides new information about the heterogeneity in ribbon size, shape, and area of the ribbon in contact with the plasma membrane.

Notch signaling blockade links transcriptome heterogeneity in quiescent neural stem cells with their reactivation routes and potential
In the vertebrate brain, neural stem cell (NSC) quiescence is necessary for stemness maintenance. Using single cell RNA sequencing (scRNAseq) in the zebrafish adult telencephalon, we identified different molecular clusters of quiescent NSCs, interpreted to sign different quiescence depths(1). Here, we show that these clusters, when challenged in vivo with an inhibitor of Notch signaling -a major quiescence promoting pathway-, unfold different behaviors. Notably, deeply quiescent NSCs with astrocytic features display a unique activation phenotype that combines the maintenance of astrocytic markers with the rapid upregulation of activation and neuronal commitment genes, reminiscent to murine periventricular astrocytes activating upon lesion. In contrast, an NSC cluster predicted to be in the deepest quiescence state resists Notch blockade, and we demonstrate that the transcription factor Nr2f1b mediates this resistance to activation in vivo. These results together link the molecular heterogeneity of quiescent NSCs with bona fide biological properties and their molecular regulators.

Differential destinations, dynamics, and functions of high- and low-order features in the feedback signal during object processing
Brain is a hierarchical information processing system, in which the feedback signals from high-level to low-level regions are critical. The feedback signals may convey complex high-order features (e.g., category, identity) and simple low-order features (e.g., orientation, spatial frequency) to sensory cortex to interact with the feedforward information, but how these types of feedback information are represented and how they differ in facilitating visual processing is unclear. The current study used the peripheral object discrimination task, 7T fMRI, and MEG to isolate feedback from feedforward signals in human early visual cortex. The results showed that feedback signals conveyed both low-order features natively encoded in early visual cortex and high-order features generated in high-level regions, but with different spatial and temporal properties. The high-order feedback information targeted both superficial and deep layers, whereas the low-order feedback information reached only deep layers in V1. In addition, MEG results revealed that the feedback information from occipito-temporal to early visual cortex emerged around 200 ms after stimulus onset, and only the representational strength of high-order feedback information was significantly correlated with behavioral performance. These results indicate that the complex and simple components of feedback information play different roles in predictive processing mechanisms to facilitate sensory processing.

Cognitive networks interactions through communication subspaces in large-scale models of the neocortex
The neocortex-wide neural activity is organized into distinct networks of areas engaged in different cognitive processes. To elucidate the underlying mechanism of flexible network reconfiguration, we developed connectivity-constrained macaque and human whole-cortex models. In our model, within-area connectivity consists of a mixture of symmetric, asymmetric, and random motifs that give rise to stable (attractor) or transient (sequential) heterogeneous dynamics. Assuming sparse low-rank plus random inter-areal connectivity, we show that our model captures key aspects of the cognitive networks' dynamics and interactions observed experimentally. In particular, the anti-correlation between the default mode network and the dorsal attention network. Communication between networks is shaped by the alignment of long-range communication subspaces with local connectivity motifs and is switchable in a bottom-up salience-dependent routing mechanism. Furthermore, the frontoparietal multiple-demand network displays a coexistence of stable and dynamic coding, suitable for top-down cognitive control. Our work provides a theoretical framework for understanding the dynamic routing in the cortical networks during cognition.

Viral vector-mediated SLC9A6 gene replacement reduces cerebellar dysfunction in the shaker rat model of Christianson syndrome
Christianson syndrome (CS) is an x-linked recessive neurodevelopmental and neurodegenerative condition characterized by severe intellectual disability, cerebellar degeneration, ataxia, and epilepsy. Mutations to the SLC9A6 gene are responsible for CS, and we recently demonstrated that a mutation to the rat Slc9a6 gene causes a similar phenotype in the spontaneous shaker rat model, which exhibits cerebellar degeneration with motor dysfunction. In previous work, we used the PhP.eB-L7-Slc9a6-GFP adeno-associated viral (AAV) vector to demonstrate that gene replacement in Purkinje cells reduced the shaker motor and molecular phenotype. Here, we carried out a 20-week longitudinal study evaluating the impact of Purkinje cell-specific gene replacement on ataxia and tremor. Taking advantage of the high homology between human SLC9A6 and rat Slc9a6, we tested a more clinically relevant construct, AAV9-CAG-hSLC9A6 AAV vector in the shaker rat. Administration of either of PhP.eB-L7-Slc9a6-GFP or AAV9-CAG-hSLC9A6 AAV vectors led to significant improvement in the molecular and motor phenotypes. The abundance of several disease-relevant cerebellar proteins was significantly correlated to motor performance. Finally, we found evidence that tremor and ataxia phenotypes in may arise from dissociable cerebellar mechanisms in the context of CS. These findings impact future SLC9A6-targeted gene therapy efforts for CS and strongly support gene replacement as a viable therapeutic strategy.

Brain functional connectivity, but not neuroanatomy, captures the interrelationship between sex and gender in preadolescents
Understanding sex differences in the adolescent brain is crucial, as these differences are linked to neurological and psychiatric conditions that vary between males and females. Predicting sex from adolescent brain data may offer valuable insights into how these variations shape neurodevelopment. Recently, attention has shifted toward exploring socially-identified gender, distinct from sex assigned at birth, recognizing its additional explanatory power. This study evaluates whether resting-state functional connectivity (rsFC) or cortical thickness more effectively predicts sex and sex/gender alignment (the congruence between sex and gender) and investigates their interrelationship in preadolescents. Using data from the Adolescent Brain Cognitive Development (ABCD) Study, we employed machine learning to predict both sex (assigned at birth) and sex/gender alignment from rsFC and cortical thickness. rsFC predicted sex with significantly higher accuracy (86%) than cortical thickness (75%) and combining both did not improve the rsFC model's accuracy. Brain regions most effective in predicting sex belonged to association (default mode, dorsal attention, and parietal memory) and visual (visual and medial visual) networks. The rsFC sex classifier trained on sex/gender aligned youth was significantly more effective in classifying unseen youth with sex/gender alignment than in classifying unseen youth with sex/gender unalignment. In females, the degree to which their brains' rsFC matched a sex profile (female or male), was positively associated with the degree of sex/gender alignment. Lastly, neither rsFC nor cortical thickness predicted sex/gender alignment. These findings highlight rsFC's predictive power in capturing the relationship between sex and gender and the complexity of the interplay between sex, gender, and the brain's functional connectivity and neuroanatomy.

Simultaneous mesoscopic measurement and manipulation of mouse cortical activity
Dynamics of activity across the cerebral cortex at the mesoscopic scale - coordinated fluctuations of local populations of neurons - are essential to perception and cognition and relevant to computations like sensorimotor integration and goal-directed task engagement. However, understanding direct causal links between population dynamics and behavior requires the ability to manipulate mesoscale activity and observe the effect of manipulation across multiple brain regions simultaneously. Here, we develop a novel system enabling simultaneous recording and manipulation of activity across the dorsal cortex of awake mice, compatible with large-scale electrophysiology from any region across the brain. Transgenic mice expressing the GCaMP calcium sensor are injected systemically with an adeno-associated virus driving expression of the ChrimsonR excitatory opsin. This strategy drives expression of the blue-excited calcium indicator, GCaMP, in excitatory neurons and red-excited Chrimson opsin in inhibitory neurons. We demonstrate widefield single-photon calcium imaging and simultaneous galvo-targeted laser stimulation over the entire dorsal cortical surface. The light channels of the imaging and the opsin do not interfere. We characterize the spatial and temporal resolution of the method, which is suitable for targeting specific cortical regions and specific time windows in behavioral tasks. The preparation is stable over many months and thus well-suited for long-term behavioral experiments. This technique allows for studying the effect of cortical perturbations on cortex-wide activity, on subcortical spiking activity, and on behavior, and for designing systems to control cortical activity in closed-loop.

DJ-1 deficiency in SH-SY5Y cells reveals dysregulated networks of genes and pathways involved in neuronal function and disease
Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting between 2 - 3% of the population aged 65 and older. Although the etiology of idiopathic PD is still to be elucidated, the study of heritable forms of the disease can provide new understanding into disease mechanisms. Recessively inherited loss of function mutations in the PARK7/DJ-1 gene has been found to be causative for familial, early-onset PD. Importantly, PARK7/DJ-1 related forms of familial PD replicate common disease phenotypes seen in idiopathic PD, including degeneration of substantia nigra dopaminergic neurons, and Parkinsonism. In this study, we evaluate the loss of function of PARK7/DJ-1 on a human neuronal cell line, SH-SY5Y. Following ablation of the PARK7/DJ-1 gene via CRISPR-Cas9, RNA sequencing and the DESEQ2 tool kit were utilized to filter differentially expressed genes between PARK7/DJ-1 knockouts and control SH-SY5Y cells. 5684 genes were identified to be significantly differentially expressed. 3 genes from each of the top 10 upregulated (ATOH8, LAYN, TLX2) and downregulated (CACNA1B, CPLX2, SV2C) gene lists were selected and confirmed via RT-PCR. Differentially expressed gene lists were run through the WebGestalt functional enrichment analysis toolkit to identify enriched gene ontology (GO) terms for biological processes, cellular components, molecular function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways respectively. Among the top 10 significantly enriched KEGG pathways for upregulated genes were those related to neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease (p-adj [≤] 0.05). Differentially expressed genes were run through the STRING database to predict protein-protein interactions (PPI). A highly significant PPI enrichment was observed (p < 1.0e-16). Our results indicate that loss of DJ-1 function in human neuronal cells leads to dysregulation of networks of connected genes and pathways that are implicated in neurodegenerative disease as well as neuronal function.

Consolidation of Sequential Planning
Thriving in changing environments requires the capacity to evaluate novel courses of action. This ability is hypothesized to depend on sequential planning via step-by-step simulations of the future, using cognitive maps or schemas of task contingencies. However, it is still unclear if, how and where in the brain such flexible planning is enacted. In parallel, it is thought that consolidation transforms memory representations over time to promote adaptive behaviour. Here, we hypothesize that consolidation strengthens cognitive maps of task contingencies used for simulation during sequential planning. To test this, we developed a novel behavioural task and a new multivariate method for analysis of magnetoencephalography (MEG) data. Using choice and reaction time data we dissociated flexible sequential planning from alternative non-sequential strategies, and identified this behaviour with robust neural markers of step-by-step simulation, localized to the anterior medial temporal lobe. Retesting a week later we showed that consolidation enhanced sequential planning and strengthened markers of sequential simulation in the prefrontal cortex, consistent with systems consolidation theory. By revealing that consolidation improves future simulations for flexible planning we open up a new frontier for the investigation of the functional interactions between memory and decision-making.

Mitochondrial NADK2-dependent NADPH controls Tau oligomer uptake in human neurons
Alterations in NADH and NADPH metabolism are associated with aging, cancer, and Alzheimer's Disease. Using 2P-FLIM imaging of the mitochondrial NAD(P)H in live human neurons and PS19 mouse brains, we show that tau oligomers (TauO) upregulate the mitochondrial de novo NADPH synthesis through NADK2. This process controls LRP1-mediated internalization of TauO, setting a vicious cycle for further TauO internalization. Thus, mitochondrial NADK2-dependent NADPH controls a key step in TauO toxicity.

London taxi drivers leverage regional boundaries to optimise route choices and improve their navigation skill across three decades
The world is defined by boundaries. They segment our experience of time and space, and in virtual environments have been shown to impact navigational choices. Here, we test the impact of boundaries on route choices in a real-world environment (London, UK) with a group of expert navigators: licensed London taxi drivers who are required to memorise the layout of over 26,000 streets to obtain their licence. After presenting photographs of a start location and a goal location, taxi drivers were asked to either accept or reject a third target street as forming part of the direct route or not. Performance increased across the adult life-span period in this group (age: 34 to 67). Taxi drivers were faster and more accurate when the target location formed part of a street network boundary (e.g. streets on the edge of the London neighbourhood Soho). Our results are consistent with taxi drivers exploiting the graph structure of the street network to plan routes, as well as consistent with the formation of hierarchical state representations to reduce the dimensionality of the planning problem. Taken together, we show that navigational skill can improve over decades of exposure and that experts exploit regional boundaries for optimal choices, providing a scaffolding over which to form action plans.

DJ-1 mediates regulation of metabolism and immune response in Parkinsons disease astrocytes and Glioblastoma cells
An inverse correlation for the expression of Parkinsons disease (PD)- and cancer-associated genes has been previously reported. Genes that are upregulated in cancer are frequently downregulated in PD and vice versa. PARK7, encoding DJ-1, was initially identified as an oncogene, but loss of DJ-1 causes early-onset PD. However, it remains elusive how differential DJ-1 levels contribute to opposite cell fates in cancer and PD. Here, we demonstrate specific effects of differential DJ-1 protein levels on the energy metabolism and cell growth in patient-derived cellular models of PD and glioblastoma (GBM) cell lines. Impaired energy metabolism was associated with an increased immune response upon IL-1{beta} stimulation and increased apoptosis and decreased cell growth in models of PD, whereas in GBM cells increased metabolic activity translated into a reduced immune response and increased cell growth. Furthermore, we found decreased glutathione (GSH) synthesis and therefore increased levels of reactive oxygen species (ROS) and oxidized glutathione (GSSG) in models of DJ-1 deficiency and decreased ROS levels in GBM cell lines. Thus, the mechanism by which DJ-1 modulates these phenotypes is the same in both diseases.

Acute and early stress axis modulation in joint disease permanently reduces pain and emotional comorbidities
Chronic secondary musculoskeletal pain affects 20-30% of the population and includes sensory disturbances and emotional comorbidities, such as anxiety and depression, which reduce quality of life. We monitored a pre-clinical model of persistent joint pain for 6 months and found that sensory and functional changes were immediate while anxio-depressive comorbidities appeared from 3 months onwards. Modulating the stress axis via genetic deletion or pharmacological blockade of the stress driver FKBP51 reduced both sensory and emotional symptoms, including injury-induced disruptions in sleep and activity patterns, and did not worsen joint damage. FKBP51 pharmacological blockade after the establishment of the hypersensitive state provided temporary relief while acute inhibition at disease onset, when sensory and affective responses were most severe, provided at least 6 months of pain relief and reduced or prevented late anxiety- and depressive-like behavior. RNA sequencing suggested that FKBP51 inhibition at disease onset downregulated the Naaa gene in the superficial dorsal horn, a key regulator of the transition to chronic pain, and reorganized spinal cilia. Our findings suggest that acute and early, but not late, FKBP51 targeting can offer permanent relief from chronic pain and its emotional effects, highlighting the importance for fast therapeutic intervention.

Habenula-ventral tegmental area functional coupling and risk-aversion in humans
Maladaptive responses to uncertainty, including excessive risk avoidance, are linked to a range of mental disorders. One expression of these is a pro-variance bias (PVB), wherein risk-seeking manifests in a preference for choosing options with higher variances/uncertainty. Here, using a magnitude learning task, we provide a behavioural and neural account of PVB in humans. We show that individual differences in PVB are captured by a computational model that includes asymmetric learning rates, allowing differential learning from positive prediction errors (PPEs) and negative prediction errors (NPEs). Using high-resolution 7T functional magnetic resonance imaging (fMRI), we identify distinct neural responses to PPEs and NPEs in value-sensitive regions including habenula (Hb), ventral tegmental area (VTA), nucleus accumbens (NAcc), and ventral medial prefrontal cortex (vmPFC). Prediction error signals in NAcc and vmPFC were boosted for high variance options. NPEs responses in NAcc were associated with a negative bias in learning rates linked to a stronger negative Hb-VTA functional coupling during NPE encoding. A mediation analysis revealed this coupling influenced NAcc responses to NPEs via an impact on learning rates. These findings implicate Hb-VTA coupling in the emergence of risk preferences during learning, with implications for psychopathology.

Astrocytes control UP state and slow oscillation periodicity in human cortical networks
Astrocytes are well known for homeostatic roles in energy maintenance, neurotransmitter recovery and immunoreactivity, however their contributing role in active modulation of neuronal activity remains controversial. Recent evidence highlights astrocytes as a potential signalling partner in ongoing high-order neuronal activity, and a modulator of baseline activity. Here we utilise an iPSC-derived in vitro cortical network model to describe a slow-wave oscillation and examine the contributions of astrocytes to development of this oscillatory activity, dependant upon generation of UP/DOWN state phenomena. To examine the role of astrocytes we invoke an acetylcholinergic oscillation in the neuronal population by addition of carbachol, which proved to be a robust mechanism for eliciting prolonged and synchronised network bursting. Pharmalogical interrogation determined oscillatory maintenance was dependent on both glutamatergic and purinergic signalling pathways. Upon further interrogation, we determined that astrocytic calcium signalling was essential to timing of the oscillatory signal. By utilising chemogenetic actuators we showed that whilst neurons are essential and sufficient for instigating the oscillatory signal, astrocytes played a key role in timing. This previously unreported mechanism may contribute to initial development of brain activity, and may underlie a basal activity present later during adulthood. In collaboration with recent results highlighting the role of active gliotransmission, this highlights astrocytes as an important research target for understanding brain activity alterations during development and disease.

Functional diversity in the output of the primate retina
The visual image transmitted by the retina to the brain has long been understood in terms of spatial filtering by the center-surround receptive fields of retinal ganglion cells (RGCs). Recently, this textbook view has been challenged by the stunning functional diversity and specificity observed in ~40 distinct RGC types in the mouse retina. However, it is unclear whether the ~20 morphologically and molecularly identified RGC types in primates exhibit similar functional diversity, or instead exhibit center-surround organization at different spatial scales. Here, we reveal striking and surprising functional diversity in macaque and human RGC types using large-scale multi-electrode recordings from isolated macaque and human retinas. In addition to the five well-known primate RGC types, 18-27 types were distinguished by their functional properties, likely revealing several previously unknown types. Surprisingly, many of these cell types exhibited striking non-classical receptive field structure, including irregular spatial and chromatic properties not previously reported in any species. Qualitatively similar results were observed in recordings from the human retina. The receptive fields of less-understood RGC types formed uniform mosaics covering visual space, confirming their classification, and the morphological counterparts of two types were established using single-cell recording. The striking receptive field diversity was paralleled by distinctive responses to natural movies and complexity of visual computation. These findings suggest that diverse RGC types, rather than merely filtering the scene at different spatial scales, instead play specialized roles in human vision.

Mitochondrial Stress Disassembles Nuclear Architecture through Proteolytic Activation of PKCδ and Lamin B1 Phosphorylation in Neuronal Cells: Implications for Pathogenesis of Age-related Neurodegenerative Diseases
Mitochondrial dysfunction and oxidative stress are hallmarks of pathophysiological processes in age-related neurodegenerative diseases including Parkinson's, Alzheimer's and Huntington's diseases. Neuronal cells are highly vulnerable to mitochondrial stress, however, the cellular and molecular mechanisms underlying the enhanced vulnerability are not well understood. Previously, we demonstrated that the novel PKC isoform PKC{delta} is highly expressed in dopamin(DA)ergic neurons and plays a key role in inducing apoptotic cell death during neurotoxic stress via caspase-3-mediated proteolytic activation. Herein, we further uncovered a key downstream molecular event of PKC{delta} signaling following mitochondrial dysfunction that governs neuronal cell death by dissembling nuclear architecture. Exposing N27 DAergic cell line to the mitochondrial complex-1 inhibitor tebufenpyrad induced PKC{delta}phosphorylation at the T505 activation loop accompanied by caspase-3-dependent proteolytic activation of the kinase. Subcellular analysis using high-resolution 3D confocal microscopy revealed that proteolytically activated cleaved PKC{delta} translocates to the nuclear compartment, colocalizing with Lamin B1. Electron microscopy also enabled the visualization of nuclear membrane damage triggered by subjecting the DAergic neuronal cells by Tebufenpyrad (Tebu) toxicity. In silico analyses identified that the threonine site on Lamin B1 (T575) is likely phosphorylated by PKC{delta} suggesting that Lamin B1 serves as a key downstream target of the kinase. Interestingly, N27 DAergic cells stably expressing the PKC{delta} proteolytic cleavage site-resistant mutant failed to induce nuclear damage, PKC{delta} activation, and Lamin B1 phosphorylation. Furthermore, CRISPR/Cas9-based stable knockdown of PKC{delta} greatly attenuated Tebu-induced Lamin B1 phosphorylation. Also, studies using Lamin B1T575G mutated at phosphorylation and PKC{delta}-{Delta}NLS-overexpressing N27 cells showed that PKC{delta} activation and translocation to the nuclear membrane are critically required for phosphorylating Lamin B1 at T575 to induce nuclear membrane damage during Tebu insult. Additionally, Tebu failed to induce Lamin B1 damage and Lamin B1 phosphorylation in organotypic midbrain slices cultured from PKC{delta}-/- mouse pups. More importantly, we observed higher PKC{delta} activation, Lamin B1 phosphorylation and Lamin B1 loss in nigral DAergic neurons from the postmortem brains of PD patients as compared to age-matched healthy control brains, thus providing translational relevance of our finding. Collectively, our data reveal that PKC{delta} functions as a Lamin B1 kinase to disassemble the nuclear membrane during the neuronal cell death process triggered by mitochondrial stress. This mechanistic insight may have important implications for the etiology of age-related neurodegenerative diseases resulting from mitochondrial dysfunction as well as for the development of novel treatment strategies.

From the fly connectome to exact ring attractor dynamics
A cognitive compass enabling spatial navigation requires neural representation of heading direction (HD), yet the neural circuit architecture enabling this representation remains unclear. While various network models have been proposed to explain HD systems, these models rely on simplified circuit architectures that are incompatible with empirical observations from connectomes. Here we construct a novel network model for the fruit fly HD system that satisfies both connectome-derived architectural constraints and the functional requirement of continuous heading representation. We characterize an ensemble of continuous attractor networks where compass neurons providing local mutual excitation are coupled to inhibitory neurons. We discover a new mechanism where continuous heading representation emerges from combining symmetric and anti-symmetric activity patterns. Our analysis reveals three distinct realizations of these networks that all match observed compass neuron activity but differ in their predictions for inhibitory neuron activation patterns. Further, we found that deviations from these realizations can be compensated by cell-type-specific rescaling of synaptic weights, which could be potentially achieved through neuromodulation. This framework can be extended to incorporate the complete fly central complex connectome and could reveal principles of neural circuits representing other continuous quantities, such as spatial location, across insects and vertebrates.

The most significant differences between male and female rats regarding psychostimulant self-administration behavior are unrelated to biological sex
Background: The goals of this study were to 1) validate the MISSING (Mapping Intrinsic Sex Similarities as an Integral quality of Normalized Groups) model for psychostimulant self-administration (SA), and 2) utilize it to explain the inconsistencies in the observation of sex differences in psychostimulant SA. Methods: We allowed male and female Long Evans rats (n = 40) to self-administer methamphetamine METH dose 0.1 mg/kg (male n = 9, female n = 18) and saline (male n = 3, female n = 10) via the intravenous route, FR1 schedule, 6 h per day, 5 days per week for 4 weeks. For the MISSING model, we identified behavioral clusters of males and females using normal mixtures clustering analysis of baseline intake, total intake and total intake normalized-to-baseline intake (NBI), followed by unpaired t-tests to compare clusters and Two-way ANOVA to determine if there were any SEX by cluster interactions. For the current model, we grouped our subjects according to biological sex and compared the above variables using unpaired t-tests. For both models, we employed Two-way repeated measures ANOVA and linear regression analysis to analyze SA time course. Results: For saline and METH SA, there were no sex differences when we compared males and females generally, with sex differences evident only when we compared sexes from distinct clusters. The current model could not explain the inconsistencies in the observability of sex differences in METH SA. Conclusions: We validated the MISSING model -it can explain the inconsistencies around sex differences in METH SA.

Speech in noise performance in adults with cochlear implants using a combined channel deactivation strategy with a variable number of dynamic focused channels
Objectives and Methods: Cochlear implant listeners show difficulties in understanding speech in noise. Channel interactions from activating overlapping neural populations reduce the signal accuracy necessary to interpret complex signals. Optimizing programming strategies based on focused detection thresholds to reduce channel interactions has led to improved performance. In the current study, two previously suggested methods, channel deactivation and focused dynamic tripolar stimulation, were combined to create three cochlear implant programs. Utilizing an automatic channel selection algorithm from focused detection threshold profiles, three programs were created with the same deactivated channels but varying proportions of channels employing focused stimulation, monopolar, dynamic focused and a mixed program. Thirteen ears in eleven adult cochlear implant listeners with Advanced Bionics HiRes90k devices were tested. Vowel identification and sentence perception in quiet and noise served as outcome measures, and the influences of listening experience, age, clinical consonant-nucleus-consonant performance, and perceptual thresholds on speech performance were assessed. Results: Across subjects, different degrees of focusing showed individual performance improvements for vowels and sentences over the monopolar program. However, only slight trends and no significant group improvements were observed. Focused listening benefits were shown for individuals with less cochlear implant experience, and clinically poor performers seem to benefit more from focusing than good performers. Conclusion: The current findings suggest that deactivating and focusing subsets of channels improves speech performance for some individuals, especially poor performers, a possible effect of reduced channel interactions. The findings also show that individual performance is largely variable, possibly due to listening experience, age, or the underlying detection threshold.

Bias-accounting meta-analyses overcome cerebellar neglect to refine the cerebellar behavioral topography
The cerebellum plays important roles in motor, cognitive, and emotional behaviors. Previous cerebellar coordinate-based meta-analyses and mappings have attributed different behaviors to cerebellar subareas, but an accurate behavioral topography is lacking. Here, we show overrepresentation of superior activation foci, which may be exacerbated by historical cerebellar neglect. Unequal foci distributions render the null hypothesis of standard activation likelihood estimation unsuitable. Our new method, cerebellum-specific activation-likelihood estimation (C-SALE), finds behavioral convergence beyond baseline activation rates. It does this by testing experimental foci versus null models sampled from a data-driven, biased probability distribution of finding foci at any cerebellar location. Cerebellar mappings were made across five BrainMap task domains and thirty-five subdomains, illustrating improved specificity of the new method. Twelve of forty (sub)domains reached convergence in specific cerebellar subregions, supporting dual motor representations and placing cognition in posterior-lateral regions. Repeated subsampling revealed that whereas action, language and working memory were relatively stable, other behaviors produced unstable meta-analytic maps. Lastly, meta-analytic connectivity modeling in the same debiased framework was used to reveal coactivation networks of cerebellar behavioral clusters. In sum, we created a new method for cerebellar meta-analysis that accounts for data biases and can be flexibly adapted to any part of the brain. Our findings provide a refined understanding of cerebellar involvement in human behaviors, highlighting regions for future investigation in both basic and clinical applications.

Electroconvulsive therapy generates a hidden wave after seizure
Electroconvulsive therapy (ECT) is a fast-acting, highly effective, and safe treatment for medication-resistant depression. Historically, the clinical benefits of ECT have been attributed to generating a controlled seizure; however, the underlying neurobiology is understudied and remains largely unresolved. Using optical neuroimaging to probe neural activity and hemodynamics in a mouse model of ECT, we demonstrated that a second brain event follows seizure: cortical spreading depolarization (CSD). We further found that ECT stimulation pulse parameters and electrode configuration directly shaped the wave dynamics of seizure and subsequent CSD. To translate these findings to human patients, we tested for the presence of hemodynamic signatures of post-ictal CSD using non-invasive diffuse optical monitoring of cerebral blood flow and oxygenation during routine ECT treatments. We found evidence that humans generate hyperemic waves after ECT seizure which are highly consistent with CSD. These results challenge a long-held assumption that seizure is the primary outcome of ECT and point to new opportunities for optimizing ECT stimulation parameters to precisely modulate brain activity and treatment outcomes.

Lateralized nodose ganglia gene expression implicates cholecystokinin receptors in interoceptive reward signaling
The vagus nerves are important carriers of sensory information from the viscera to the central nervous system. Emerging evidence suggests that sensory signaling through the right, but not the left, vagus nerve evokes striatal dopamine release and reinforces appetitive behaviors. However, the extent to which differential gene expression within vagal sensory neurons contributes to this asymmetric reward-related signaling remains unknown. Here, we use single-cell RNA sequencing to identify genes that are differentially expressed between the left and right nodose ganglia (NG) to identify candidate genes likely to contribute to vagus-mediated reward signaling. We find that a group of neurons expressing Chrna3 (nicotinic acetylcholine receptor subunit 3) and Cckar (cholecystokinin A receptor) is preferentially expressed in the right NG of both rats and mice. This result suggests that differential expression of gut-innervating nutrient sensors in NG neurons may contribute to asymmetric encoding of interoceptive rewards by the vagus nerves.

The presynaptic vesicle cluster transitions from a compact to loose organization during long-term potentiation
Functional and structural elements of synaptic plasticity are tightly coupled, as has been extensively shown for dendritic spines. Here, we interrogated structural features of presynaptic terminals in 3DEM reconstructions from CA1 hippocampal axons that had undergone control stimulation or theta-burst stimulation (TBS) to produce long-term potentiation (LTP). We reveal that after LTP induction, the synaptic vesicle (SV) cluster is less dense, and SVs are more dispersed. The distances between neighboring SVs are greater in less dense terminals and have more SV-associated volume. We characterized the changes to the SV cluster by measuring distances between neighboring SVs, distances to the active zone, and the dispersion of the SV cluster. Furthermore, we compared the distribution of SVs with randomized ones and provided evidence that SVs gained mobility after LTP induction. With a computational model, we can predict the increment of the diffusion coefficient of the SVs in the cluster. Moreover, using a machine learning approach, we identify presynaptic terminals that were potentiated after LTP induction. Lastly, we show that the local SV density is a volume-independent property under strong regulation. Altogether, these results provide evidence that the SV cluster is undergoing a transition during LTP.

Tcf4 Deficiency Causes Recurrent Seizures in Mice
Transcription factor 4 (TCF4) is a transcription factor that is critical for the normal development and function of the central nervous system. Haploinsufficiency of TCF4 causes Pitt-Hopkins Syndrome (PTHS), a lifelong neurodevelopmental disorder characterized by seizures and intellectual disability. To expand our currently limited understanding of TCF4 function and PTHS pathophysiology, we created a mouse model of PTHS with largely astrocyte-specific heterozygous knockout of Tcf4. These mice developed severe recurrent seizures and had decreased lifespans. In addition, we found that these mice had astrogliosis as well as increased neuronal activity in the cortex, hippocampus, amygdala, and hypothalamus. Furthermore, single nucleus RNA sequencing analysis revealed global changes in the gene expression profiles of excitatory neurons, inhibitory neurons, astrocytes, and oligodendrocytes of PTHS compared to wild-type mice. Overall, this is the first report of a PTHS mouse model with seizures, providing the field with a tool to investigate the mechanisms of PTHS development and progression and develop therapeutics for PTHS and its associated epilepsy.

Daily electric field treatment improves functional outcomes after thoracic contusion spinal cord injury in rats
Spinal cord injury (SCI) can result in permanent loss of sensory, motor, and autonomic functions, with limited therapeutic options to recover the loss. Low-frequency electric fields with changing polarity have shown promise in promoting axon regeneration and improving outcomes. However, the metal electrodes used previously were prone to corrosion, and their epidural placement limited the penetration of the electric field into the spinal cord. Here, we demonstrate that a thin-film implant with supercapacitive electrodes placed under the dura mater can safely and effectively deliver electric field treatment in rats with thoracic SCI. Subdural stimulation enhanced hind limb function and touch sensitivity compared to controls, without inducing a neuroinflammatory response in the spinal cord. While axon density around the lesion site remained unchanged after 12 weeks, in vivo monitoring and electrochemical testing of electrodes indicated that treatment was administered throughout the study. These results highlight the promise of electric field treatment as a viable therapeutic strategy for achieving long-term functional recovery in SCI.

Neural synchrony is "good enough" for speech comprehension
Recent evidence indicates that neural populations exhibit synchronous firing at phrase boundaries to facilitate the encoding of syntactic units during speech comprehension. However, good-enough processing accounts of speech comprehension suggest that detailed syntactic analysis may not always be necessary for successful interpretation, especially when listeners can deduce meaning from lexical-semantic contexts. In this brief report, we evaluate this notion and assess whether neural synchrony to syntactic boundaries is modulated by local lexical-semantic content. To this end, we reanalyzed an open-source EEG dataset, consisting of brain recordings obtained while participants passively listened to an audiobook. To determine neural synchrony to phrase boundaries, we computed mutual information (MI) between delta band EEG activity (< 3Hz) and hierarchically derived syntactic structures for each sentence in the audiobook. We then quantified local-lexical semantic contexts using semantic dissimilarity values that were derived from high-dimensional vectors of co-occurrence. We then regressed MI values for each sentence against the sentence's semantic dissimilarity values, using linear mixed-effects models. Results indicated that neural synchrony to phrase boundaries showed a positive linear relationship with semantic dissimilarity. We interpret this finding as evidence that listeners' reliance on syntactic information during speech comprehension is modulated by local lexical-semantic contexts, consistent with good-enough processing accounts.

Context matters: Integrative NMDA receptor dysfunction reveals effective seizure treatment in mice with a human patient GluN1 variant
Intractable epilepsy and cognitive deficits arise from missense variants in GRIN genes encoding subunits of the N-Methyl-D-Aspartate receptor (NMDAR). Here, we go beyond typical assessments of isolated receptors to explore the impact of a human GluN1 variant across multiple scales of native NMDAR signaling. We show that isolated and integrated NMDAR signaling are differentially affected in brain slices of transgenic mice with the heterozygous GluN1 Y647S patient variant. Loss-of-function NMDARs paradoxically prolong NMDAR-dependent dendritic integration, extending cortical network activity and increasing vulnerability for seizure-like events. We identify that loss-of-function NMDARs fail to engage canonical negative feedback via calcium-activated potassium channels that prevent NMDAR overdrive. Therefore, we test an unorthodox treatment to increase NMDAR Mg2+ block and show that oral treatment with magnesium-L-threonate significantly reduces seizure occurrence and severity in the mice. We reveal a case where higher-order functional context predicts effective treatment for seizures arising from NMDAR disruption.