iScience

Instrumental learning typically requires hundreds to thousands of trials in which subjects learn to link motor responses to sensory cues. In standard rodent protocols, response accuracy is reported only at trial end, preventing subjects from correcting erroneously initiated responses. We hypothesized that within-trial, closed-loop sensory feedback would accelerate instrumental learning by providing real-time information about response correctness. Head-fixed mice performed a two-alternative forced-choice task by rotating a choice wheel in response to sensory cues. Mice received either no feedback (n = 18), auditory feedback (n = 16), or audiovisual feedback (n = 4) coupled to wheel movements. Feedback-receiving mice required significantly fewer trials to reach 70 % accuracy criterion (median: 3186, 4918 and 7329 trials for multimodal, unimodal and no feedback, respectively; p = 0.0245) and showed higher accuracy when modifying choices (expert stage: 17 %, 11 % and 9 % accuracy in trials with modified choices for multimodal, unimodal and no feedback, respectively; p=1.04x10-). Only feedback mice displayed movement refinements across training (p = 8.02x10-, p = 4.07x10-{superscript 1} and p = 0.2602 for multimodal, unimodal and no feedback, respectively). In summary, closed-loop sensory feedback accelerated instrumental acquisition, demonstrating its value as routine training protocol. HIGHLIGHTSO_LIMice provided with feedback require fewer trials to reach expert stage in an instrumental learning task C_LIO_LIMice provided with feedback perform with higher accuracy in trials involving changes of mind C_LIO_LIMovement trajectories of mice provided with feedback undergo refinement as training advances C_LIO_LISensory feedback can be used as a training aid to accelerate instrumental learning C_LI

The neural system at the periphery is a frontline for embodied cognition, yet an acute, mild perturbation to dissect functional causality is hard to achieve. Peripheral neural processes and the central nervous system may work in concert to generate sophisticated representations regarding self and environments in the brain. This hypothesis, together with the underlying mechanisms, is particularly difficult to test for certain sensory inputs due to the lack of reversible manipulation techniques. Long postulated as a component for path integration, proprioception is one of such modalities. In this study, we developed a murine experimental system to manipulate proprioceptive inputs during memory-dependent localization task (which required precise operant-conditioned licks) in spatial virtual reality (VR). Through bioluminescent optogenetics (luminopsin) selectively expressed in the parvalbumin-positive neurons of the dorsal root ganglia in mice, proprioceptive processing was compromised directly from the periphery to bypass the bottleneck of specific central targeting, which results from the lack of anatomically or genetically dedicated proprioceptive circuits in the brain. In-vivo IVIS imaging and behavior suggested the effects of luminopsin last for roughly 20 minutes. While mice exhibited normal performance in tasks relying on gross motor skills, they showed subtle deficits in challenging spatial tasks that required integration of past movements. These observations support a task-specific role for proprioception, and demonstrate a potential of chemogenetics-like, rapidly reversible strategies for characterizing peripherally defined sensory contribution to spatial cognition. Future work will optimize this approach; for instance, to activate opsins by light with millisecond precision. To our knowledge, this is a first causal demonstration for acute participation of proprioception in path-integration biomechanics, enabling the first temporally defined method for mild perturbation of path-integration mechanisms.

Primates are distinguished by large brains relative to body size, with humans showing the greatest expansion. This increase in brain size evolved alongside advanced cognitive abilities as well as an elevated energetic demand. Importantly, allometric scaling alone does not explain this increased metabolic requirement, suggesting that other cellular mechanisms may be driving the unique energetic capacity of the human brain. Brain metabolism is critical for neurological function by providing the energy necessary for neuron firing. Much of metabolism in the brain is carried out by astrocytes: a type of glial cell that have long been viewed as passive support cells for neurons. More recent research has highlighted the unique roles of astrocytes in many critical neurological processes; however, it is less understood how astrocytes differ among species. To better characterize this, we developed a cross-species co-culture model of astrocytes and neurons from human or chimpanzee-derived iPSCs. This co-culture system allowed us to assess cell-type specific effects as well as species-specific differences in cellular interactions that may be driving overall differences in brain metabolism. We conducted single-cell RNA-sequencing as well as Seahorse XF Mitochondrial Stress tests and observed that human neural co-cultures are more metabolically active than chimpanzee neural co-cultures. Cross-species co-culture systems also highlight that astrocytes are driving major species differences in metabolism, whereas neurons are highly responsive to astrocytic activity. We conclude that both neurons and astrocytes have evolved differently across primates, and that metabolic interactions between these cell types are key contributors in primate brain evolution.

The circadian clock regulates diverse cellular processes, including intestinal epithelial cell (IEC) proliferation. However, mechanisms regulating clock-dependent IEC differentiation remain unknown. We performed a time course RNA-Seq using the mouse small intestine and identified NOTCH signaling, a key mechanism regulating IEC differentiation, as one of the pathways under the control of circadian rhythms. Using mouse enteroids, we discovered that a NOTCH reporter, Hes1-luciferase, exhibits both ultradian or circadian oscillations depending on the stemness of mouse enteroids. Furthermore, single-cell analysis of Hes1-mCherry revealed that the period of Hes1 oscillations varies widely, but circadian rhythms modulate the number of Hes1-mCherry+ cells in the population. Finally, we show that Paneth cell numbers fluctuate over the circadian cycle, suggesting that circadian clock-regulated Hes1 drives the circadian dynamics of IEC composition. Our study provides a deeper insight into circadian regulation of IEC differentiation, which will be critical for applications of chronotherapies for digestive diseases.

Drosophila connectomic datasets provide increasingly comprehensive maps of neuronal morphology and synaptic connectivity, offering an unprecedented opportunity to explore the structural organization of its neural circuits. This calls for designing automated tools to interact with connectomic datasets at scale for efficiently exploring structural features embedded in the vast amount of data. Yet the central challenge remains the understanding of the functional logic of neural circuits. In order to understand how elements of the functional logic may emerge from this structural organization, it is critical to (i) characterize the objects in the natural environment in which brain circuits operate, and (ii) formulate how brain circuits represent and process the defined objects in the natural environment. To develop and demonstrate a methodology for these requirements, we focus on the Drosophila looming-evoked escape pathway. We modeled the trajectory of looming objects that are on a collision course (direct-hits) or pass-by the fly (near-misses): their projected images on the retina can be characterized by the solid angle (angular size) and elevation. We then analyzed the pathway's morphology across the OpticLobe, Hemibrain, and FlyWire connectome datasets. By abstracting their sub-neuronal structure and retinotopic organization, we constructed an executable circuit model that maps each structural element to a processing block. We demonstrate that this model separates direct hits from near misses well before the angular size could tell them apart. To accelerate the connectomic analysis step, we developed a Python toolset with an agentic, code-free workspace interface called NeuroGraphBench (NGB). NGB provides four composable morphology-analysis primitives and an AI agent that composes them to interactively respond to natural-language queries aided by visualization on an interactive 3D canvas. Thus, NGB automates tedious and repetitive tasks to enable faster and scalable connectomic exploration, keeping human reasoning, instead of writing code, at the center of an open-ended research inquiry.

Axon outgrowth requires forces generated by the growth cone. A key model explaining this force generation is the adhesion-clutch mechanism, in which adhesion and clutch molecules convert backward movement of actin filaments into the force that drives axon outgrowth. However, this mechanism has not been validated in three-dimensional (3D) environments. Additionally, a recent study reported that inhibiting actin dynamics or the cell adhesion molecule integrin did not affect axon outgrowth in a 3D collagen gel, challenging the adhesion-clutch paradigm. Here, we show that the adhesion molecule N-cadherin and the clutch molecule shootin1a form a non-integrin adhesion-clutch in a 3D environment containing an appropriate adhesive substrate, N-cadherin. We detected forces produced by growth cones when N-cadherin was present. Furthermore, inhibition of N-cadherin, shootin1a or actin dynamics suppressed 3D axon outgrowth. Our findings demonstrate that the adhesion-clutch is critical machinery for 3D neural network formation under the regulation of specific adhesions.

Cold acclimation holds therapeutic potential for improving metabolic health. We previously demonstrated that repeated cold-induced shivering enhances insulin sensitivity in humans. However, the molecular pathways that underlie the skeletal muscle shivering response, and how these relate to beneficial physiological effects, remain poorly understood. In this study, we combined complementary bioinformatics approaches to allow in-depth analysis of the transcriptomic response of human skeletal muscle to repeated shivering. We identified a robust transcriptional signature and show a sex-specific component in the shivering skeletal muscle response, which seemed to diminish following cold adaptation. Our findings provide mechanistic insights into cold-induced muscle adaptations, shed light on potential interesting molecular targets for further investigation, and emphasize the importance of including both sexes in future cold acclimation studies.

Milk fat is produced by mammary epithelial cells (MEC) through a conserved mechanism shared among all fat-producing cells across biological kingdoms. Although highly conserved, different tissues and organisms produce distinctive fat compositions. Notably, ruminant milk fat is characterized by enrichment in short and medium chain fatty acids. We hypothesized that this unique profile is driven by MEC-specific metabolic characteristics related to their response to lipogenic substrates. To study this, we compared bovine MEC and udder-derived fibroblasts in terms of their lipogenic capacity and fatty acid composition when exposed to lipogenic building blocks. When exposed to acetate, MEC showed coordinated upregulation of acyl-CoA short-chain synthetase 1 (ACSS1) and diacylglycerol transferase (DGAT), while expression of acyl-CoA synthetase long-chain 1 (ACSL1) decreased. Medium chain fatty acids were also elevated in acetate-treated MEC and not in fibroblasts. The role of ACSS1 in the production of medium chain fatty acids in MEC was confirmed by knockdown experiments. Metabolomics analysis showed that in MEC acetate treatment triggered a broad metabolic response, primarily amino acids catabolism, energy and polar lipid metabolism. Collectively, these findings demonstrate effective utilization of acetate for de novo fatty acid synthesis in MEC with preferred tendency to produce medium chain fatty acids.

H2O2 released to the extracellular space (ECS) regulates diverse physiological processes, yet its concentrations and spatial distribution in tissues remain poorly defined. This uncertainty hampers mechanistic understanding of redox signaling. Here, we used reaction-diffusion modeling to estimate extracellular H2O2 concentrations and transport ranges in various scenarios. Idealized analytical models were combined with numerical models incorporating localized NADPH oxidase (NOX) clusters, ECS microstructure, membrane permeability, and the thioredoxin- and GSH-dependent clearance systems. Using maximal neutrophil and NOX superoxide/H2O2 release rates, we obtained upper bounds for extracellular H2O2. Adjacent to isolated average-sized, fully active NOX2 clusters H2O2 peaked at ~540 nM at adhesion cell-cell separations, and decreased radially over ~50-100 nm. At the receptor cell surface, peak concentration decreased inversely with intercellular separation, to <5 nM at 1 m separation. Radial decrease here, for this wide separation, was over ~2.5 m. Even the former maximal extracellular concentrations induce just a minimal, highly localized oxidation of the intracellular Prdx, Trx and GSH pools. In turn, maximally activated neutrophils carry ~2000 such NOX2 clusters, inducing 10s of M peak ECS H2O2 concentrations. These cause extensive Prdx and Trx oxidation near the exposed membranes. However, the GSH-dependent system still sustains a strong transmembrane gradient if the permeation barrier remains intact, and ECS H2O2 concentrations decay to sub-M within a few m of the source cell. Extracellular H2O2 concentrations scaled linearly with source flux in all the examined conditions. These results establish stringent constraints on autocrine, juxtacrine and next-cell paracrine H2O2 signaling.

The activity of the inferior olive (IO), conveyed to the cerebellar cortex via climbing fibers and manifesting as the complex spikes (CSs) in Purkinje neurons, is at the core of major theories of cerebellar function and motor learning. While the computational meaning of the CSs remains under intense debate, it is well accepted that changes in their occurrence and timing play a critical role in guiding cerebellar plasticity. However, it has not been examined whether the plastic processes leading to these changes in cerebellar CS activity are expressed in the IO, or whether they are entirely caused by changes in brain structures upstream of the climbing fibers, including the cerebellar nuclei. Here, we examine whether prolonged, kinematically challenging motor experience can lead to detectable changes in the IO network structure that could underlie shifts in cerebellar CS activity. As the IO neurons communicate exclusively via electrical synapses (gap junctions) residing on dendritic structures, we hypothesized that if long-term increases in electrical coupling related to the insertion of new gap junctions occur in response to novel motor experiences, morphological changes in the dendrites would also be expected. To investigate this, we quantitatively characterized the geometry of the IO neuropil revealed by immunohistochemical staining in adult male mice. We found that the neuropil structure varies across olivary subnuclei in naive animals, possibly underlying known differences in cerebellar complex spike co-activation patterns. Geometrical measures related to structural complexity also revealed that exposing the animals to a challenging high-speed treadmill running task requiring full-body coordination led to localized changes suggestive of increased network connectivity. In addition, the density of immunofluorescence puncta labeling Cx36 increased in the same regions, supporting the notion that exposure to contexts where novel motor skills need to be acquired may lead to changes in the clustering strength among IO neurons and thereby restructuring of the olivo-cerebellar micromodules. To our knowledge, this is the first report of experience-related plasticity within the IO, calling for renewed attention to the role of the IO in olivocerebellar function.

Variability is a prominent feature of animal behavior across species. Despite highly controlled experimental conditions, the same individual often responds differently to repeated identical stimulation. Part of this variability can be explained by the sequence of previous sensory stimuli and decision-making events - the trial history. Most studies about trial history are limited to animals that have a cortex, such as rodents or primates, observed as they perform learned cognitive tasks. It is currently unknown whether trial history can shape the behavior of animals lacking cortical structures during untrained, naturalistic behaviors. Here, we address this question in larval zebrafish, performing the optomotor response, an innate sensorimotor behavior in which animals turn in the direction of whole-field visual motion. We observed that a substantial proportion of variability can be explained by recent stimulation and decision-making events, with influences lasting for tens of minutes. Intriguingly, sensory and response histories bias the current response in opposite directions - repulsive and attractive, respectively - analogous to what has been previously reported in humans and rodents. An integrator model operating across multiple timescales explains a considerable fraction of response variability based on trial history alone. Our findings demonstrate that history dependency in animal behavior is not an exclusive feature of higher-order cortical computation after learning, but that it reflects a fundamental and evolutionarily shared property of vertebrate sensorimotor systems. SIGNIFICANCE STATEMENTBehavioral variability is often attributed to noise in sensory and neural processing. We show instead that much of this variability can arise predictably from an animals recent sensory and response history. In freely swimming larval zebrafish performing an innate visual behavior, previous stimuli and responses exert opposing, long-lasting influences: sensory history biases behavior away from the past, whereas response history promotes repetition. Such trial-to-trial effects are increasingly recognized in cognitive neuroscience but, to our knowledge, have not previously been demonstrated in a nonmammalian vertebrate. Their presence in zebrafish, a model system offering brain-wide neural access and powerful molecular tools, creates new opportunities to identify potentially conserved circuit mechanisms underlying persistent sensorimotor computations. HIGHLIGHTSO_LIVisual motion and response histories have opposing influences on behavior C_LIO_LIStimulus history negatively biases response direction over tens of seconds C_LIO_LIAnimals tend to repeat responses across trials, a positive bias that slowly increases over minutes C_LIO_LIA deterministic model applied to experimental sessions explains considerable fractions of response variability C_LI

Equids are considered unique in having encapsulated embryos that are actively moved between the uteri of mares during the first two and three weeks of pregnancy and do not form a placenta until about 40 days, the longest pre-placentation period known amongst Eutheria. Their embryos are likely supplied through the glycoprotein capsule with selected nutrients by uterocalin, a carrier protein secreted by maternal endometrial glands. Uterocalin binds polyunsaturated fatty acids and retinol (Vitamin A), concentrates in the capsule, and is internalised by the embryo. Here it is shown that the structures of equid uterocalin comprise an excess of metabolism-related essential amino acids, and structural specialisations potentially involved in stabilising the capsule matrix until emergence and engagement with a receptor on the trophoblast surface. The genomes of other perissodactyls (rhinos and tapirs) encode uterocalins with amino acid sequences, excesses of essential amino acids, and predicted structural properties, all closely similar to horse uterocalin. These characteristics are unique to the perissodactyl uterocalins and are not found in similar proteins of other mammal groups. Hence, rhinos and tapirs probably have equid-like reproductive specialisations retained since before the Cretaceous-Paleogene mass extinction and that uterocalin is key to both embryo nutrition and capsule formation.

The quiescent adult stem cells are activated to maintain tissue integrity. Mitochondria have emerged as critical regulators of stem/progenitor cells. Scattered literature suggests existence of a high-potency stemness state with characteristic mitochondrial structure-function properties that may prime stem cell activation. Precise tuning of mitochondrial structure by the mitochondrial fission protein, Drp1, can prime a stem cell state for neoplasticity in vitro. However, the in vivo significance and regulation of such a proposed primed stemness state remains elusive. We use quantitative genetic and cell biological approaches to demonstrate that light-dark cycling driven precise tuning of Drp1 primes the Drosophila ovarian germline stem cell (GSC) response to protein-rich dietary supplement towards stimulating egg production. This can be reversed by repressing mitochondrial fusion protein, Opa1, and is disrupted in the absence of functional Cry protein that maintains light-dark rhythm. Mechanistically, this is achieved by light or dark allowing protein-rich supplements to stimulate distinct characteristic recruitment and organization of Drp1 on mitochondria in GSC subpopulations. Screening of essential amino acids reveals the importance of interaction between Threonine supplementation, Cry, Drp1 and Opa1 in such priming of the GSCs and egg production. The priming of the ovarian follicle stem cells happens at a different tuned level of Drp1. Such Drp1-tuning driven priming of Drosophila ovarian stem cells reverses the inhibitory impact of diabetes-like condition on egg development in the presence of protein-rich supplement. Thus, our study revealing the paradigm of adult stem cell regulation by integration of light-dark and dietary cues via precise mitochondrial modulation, is significant in regenerative health and degenerative diseases.

Brain microRNA (miRNA) expression can shift in response to the social environment, translating external cues into regulatory action that can influence brain structure, neural activity, and behavior. miRNAs are key drivers of behavioral flexibility, regulating gene networks involved in neuroplasticity. We manipulated social rearing conditions of brilliant-thighed poison frog tadpoles (Allobates femoralis) to test whether isolation-linked behavioral changes are associated with altered miRNA expression in the midbrain and eyes, tissues linked by the retinotectal tract, a known connection for trafficked precursor miRNAs. We asked whether isolation alters visually guided behavior using a light/dark preference assay and found that isolated tadpoles spent less of the trial on the dark side than did group-housed tadpoles. In a social place preference assay, group-housed tadpoles preferred a zone near a conspecific over an object control when provided with multimodal sensory access, but not when access was restricted to vision alone. We next tested whether social rearing conditions influenced miRNA expression and found that isolated tadpoles exhibited upregulation of miR-15-P1c_3p*, the canonically non-dominant arm of an understudied miRNA, in the midbrain and eyes. We detected a shift in arm dosing for miR-124, a miRNA with conserved roles in neural differentiation and plasticity. Finally, social isolation during development was associated with downregulation of parathyroid hormone 2 (pth2), a peptide associated with social isolation and mechanosensation in other aquatic larvae. This work contributes to a growing body of literature implicating miRNAs in developmental neuroplasticity, presenting precedented and novel signatures of the transcriptional and regulatory response to isolation.

Methylglyoxal (MGO) is a metabolic byproduct of sugar metabolism involved in the formation of advanced glycation end products (AGEs) and its accumulation disrupts protein function, redox balance and cellular viability. Yet the metabolic rewiring required to divert MGO overflow and prevent these cytotoxic effects remains largely unknown. To address these gaps, we used flux sampling and flux balance analysis in genome scale metabolic model iJO1366 of E. coli. High glucose increased MGO flux from 0.16 mmol/gDCW/h to 1.87 mmol/gDCW/h in simulation. Large scale in silico screening of metabolic reactions identified that increasing threonine uptake, thereby increasing ethanol flux, reduced glucose induced increase in MGO flux from 1.87 mmol/gDCW/h to 0.405 mmol/gDCW/h. In silico inhibition of ethanol production (NAD+ regeneration) inhibited the MGO lowering potential of threonine. Threonine supplementation actively drives the acetaldehyde to ethanol flux to provide localized NAD+ relief, which subsequently enhances the flux of payoff phase in glycolysis (glyceraldehyde 3-phosphate), thereby efficiently draining the stagnated DHAP pool and shutting down the overflow towards MGO production. Unexpectedly, under reduced oxygen conditions, high glucose caused only a minimal increase in MGO flux, which was not reduced by threonine supplementation. Our study also decoded the mechanistic details behind this paradox. Though this model driven hypothesis requires further validation in vivo, these stoichiometric predictions have applications in metabolic engineering, for commercial MGO production. Furthermore, in vivo studies focused on bacterial stress, gut microbiome imbalances and AGE related diseases might benefit from these simulations

Motor systems controlling locomotion must generate repetitive rhythmic activity, while also still retaining the ability to generate a diverse range of outputs. How motor systems monitor, regulate, and promote diversity of their own outputs is not well understood. Here, we perform single-step, variable-order and hidden-state Markov modelling (HSMM) on spontaneous fictive locomotor activity in the isolated Drosophila larval nervous system to examine how a motor system balances constraint and promotion of diversity amongst competing motor programs. We show that spontaneous fictive activity is structured by interacting mechanisms operating at multiple levels of sequence organisation. Analysis of one-step transition rules revealed a bias in activity towards activity states underlying exploration that in turn, promote transition to diverse outputs. In contrast, higher-order Markov, N-gram, and HSMM analysis indicated a memory biased towards revisiting recently executed motor programs. These mechanisms together suggest that the Drosophila larval locomotor system maintains a dynamic repertoire of possible motor outputs by monitoring recent activity and biasing future transitions accordingly. In this sense, fictive rhythmogenesis reflects a diversity-generating process: the larval locomotor network does not simply repeat a fixed motor programme or randomly transition from one state to another, but rather continually regulates access to rhythmic states based on recent experience. Together, these findings suggest that fictive locomotor dynamics are consistent with adaptive winner-takes-all competition between central pattern generating (CPG) modules that balance constraint and promotion of motor program diversity.

The regulatory factors underlying posttranscriptional adaptation to spaceflight are incompletely understood. We identified NAT10, an RNA acetyltransferase, among RNA modification enzymes significantly upregulated in spaceflight-exposed mouse liver. Transcriptome-wide correlation analysis linked NAT10 expression to an RNA-metabolism module enriched for CAG repeat density in coding sequences. Actinomycin D chase assays showed that NAT10 knockdown destabilized CAG repeat-containing transcripts, with DDX17 serving as the primary validated target; DCP1A showed a directionally consistent pattern. Wild-type NAT10 re-expression fully rescued DDX17 mRNA stability, the acetyltransferase-dead G641E mutant rescued mRNA stability to a comparable extent, and the K290A helicase-domain mutant failed to rescue, consistent with a requirement for helicase domain integrity, whereas acetyltransferase activity was not strictly required under these experimental conditions. RNA immunoprecipitation further supported preferential NAT10 association with CAG repeat-containing mRNAs, with reduced association after single-site CAGCAG deletion in DDX17 and DCP1A reporters. Gene set enrichment analysis associated NAT10 upregulation in spaceflight liver with suppressed fatty acid beta-oxidation and induced cholesterol biosynthesis; NAT10 depletion in hepatocellular carcinoma cells selectively opposed the cholesterol-associated, but not the broader metabolic, component of this signature. These findings support a model linking NAT10 to CAG repeat-associated mRNA stabilization in the context of hepatic metabolic and posttranscriptional adaptation to spaceflight.

Electrical synapses are prevalent throughout nervous systems, including the mammalian brain. Several lines of evidence implicate these gap junction connections in several important roles in neural networks. Yet, progress has been hampered by a shortage of experimental tools to investigate their function with sufficient precision. In an effort to address this deficit, we here take advantage of a curious species-difference in electrical coupling in tuberoinfundibular dopamine (TIDA) neurons of the rodent hypothalamus. In rats, these cells exhibit strong electrical coupling, generating stereotyped, slow oscillations that are synchronized across the population. In contrast, mouse TIDA neurons lack gap junctions and display diverse, faster, and asynchronous oscillations. Motivated by this natural discrepancy, we designed a gain-of-function strategy to induce localized and robust expression of the main pore-forming protein of neuronal gap junctions, connexin-36 (Cx36), in mouse TIDA neurons by viral vectors. This perturbation yielded electrical coupling that was weaker than the rat TIDA system, but on par with many other brain populations connected by gap junctions. Cx36 overexpression resulted in significantly increased auto- and cross-correlation, as well as augmented functional connectivity, in agreement with the induction of electrical synapses in the population. Yet, these measures were modest in comparison with rat TIDA neurons, and Cx36 overexpression failed to alter serum levels of the hormone prolactin, which is controlled by this neuroendocrine system. These findings highlight the potential and limitations of experimentally inducing electrical synapses in a natively uncoupled system, and suggest directions for future improvements.

During development, endothelial cells (ECs) migrate into the brain and acquire blood-brain barrier (BBB) properties such as tight junctions, limited transcellular transport, and high electrical resistance. Although key signaling pathways that are active in vivo have been identified, factors critical in inducing brain EC differentiation in vitro remain unclear. Here, we describe conditions that promote brain EC-like gene expression in human pluripotent stem cell (hiPSC)-derived ECs. Activation of Wnt/{beta}-catenin signaling upregulates the brain EC marker GLUT1 (SLC2A1) while suppressing the peripheral EC marker PLVAP. Simultaneously, stimulation of STAT3 by CNTF together with TGF-{beta} inhibition increases CLDN5 expression. We further found that hiPSC-derived ECs secrete high levels of angiopoietin-2 (ANGPT2) and that razuprotafib (AKB-9778), a PTPRB (VE-PTP) inhibitor, inhibits ANGPT2 and improves monolayer integrity. These results suggest that combinatorial modulation of specific signaling pathways stimulates the differentiation of human brain ECs in vitro.

Understanding how the brain integrates internal bodily signals to shape behavior is essential for characterizing the neural basis of interoception. Here, we investigate whether the nucleus of the solitary tract (NTS), a brainstem hub for viscerosensory input, communicates with the locus coeruleus (LC), a noradrenergic nucleus involved in arousal and cognition, to influence anxiety-like behavior. We utilized viral-induced optogenetics combined with electrocardiography and diaphragmatic electromyography to monitor heart rate and respiratory frequency. Prior to behavioral testing we photoactivated transduced NTS neurons either at their somata, or at their axonal terminals in the LC. Anxiety-related behaviors were assessed using the elevated plus maze (EPM) and novelty-suppressed feeding test (NSFT). Stimulation of the NTS somata, or their terminals in the LC, increased aspects of anxiety-like behaviors in the EPM and NSFT, including freezing time during the EPM and latency to eat in the center of the NSFT. Importantly, only NTS somal activation suppressed post-NSFT feeding. This suggests that the anxiety-like behaviors observed from activating NTS projections to the LC are not secondary to activating feeding circuits. Additionally, activation of NTS terminals in the LC altered heart rate and its variability following food deprivation. These findings reveal that the NTS to LC circuit enhances anxiety-like behavior and modulates cardiorespiratory dynamics state dependently, thereby identifying a likely brainstem pathway through which viscerosensory signals influence interoception.