Glia at Transition Zones.

Neural cells are segregated into their distinct central nervous system (CNS) and peripheral nervous system (PNS) domains. However, at specialized regions of the nervous system known as transition zones (TZs), glial cells from both the CNS and PNS are uniquely present with other specialized TZ cells. Herein we review the current understanding of vertebrate TZ cells. The article discusses the distinct cells at vertebrate TZs with a focus on cells that are located on the peripheral side of the spinal cord TZs. In addition to the developmental origin and differentiation of these TZ cells, the functional importance and the role of TZ cells in disease are highlighted. This article also reviews the common and unique features of vertebrate TZs from zebrafish to mice. We propose challenges and open questions in the field that could lead to exciting insights in the field of glial biology.

Coxiella burnetii in domestic doe goats in the United States, 2019-2020.

Coxiella burnetii is a bacterial pathogen capable of causing serious disease in humans and abortions in goats. Infected goats can shed C. burnetii through urine, feces, and parturient byproducts, which can lead to infections in humans when the bacteria are inhaled. Goats are important C. burnetii reservoirs as evidenced by goat-related outbreaks across the world. To better understand the current landscape of C. burnetii infection in the domestic goat population, 4,121 vaginal swabs from 388 operations across the United States were analyzed for the presence of C. burnetii by IS1111 PCR as part of the United States Department of Agriculture, Animal Plant Health Inspection Service, Veterinary Services' National Animal Health Monitoring System Goats 2019 Study. In total, 1.5% (61/4121) of swabs representing 10.3% (40/388) (weighted estimate of 7.8, 95% CI 4.4-13.5) of operations were positive for C. burnetii DNA. The quantity of C. burnetii on positive swabs was low with an average Ct of 37.9. Factors associated with greater odds of testing positive included suspected Q fever in the herd in the previous 3 years, the presence of wild deer or elk on the operation, and the utilization of hormones for estrus synchronization. Factors associated with reduced odds of testing positive include the presence of kittens and treatment of herds with high tannin concentrate plants, diatomaceous earth, and tetrahydropyrimidines. In vitro analysis demonstrated an inhibitory effect of the tetrahydropyrimidine, pyrantel pamoate, on the growth of C. burnetii in axenic media as low as 1 µg per mL. The final multivariable logistic regression modeling identified the presence of wild predators on the operation or adjacent property (OR = 9.0, 95% CI 1.3-61.6, p value = 0.0248) as a risk factor for C. burnetii infection.

High-dose atorvastatin therapy progressively decreases skeletal muscle mitochondrial respiratory capacity in humans.

BACKGROUNDWhile the benefits of statin therapy on atherosclerotic cardiovascular disease are clear, patients often experience mild to moderate skeletal myopathic symptoms, the mechanism for which is unknown. This study investigated the potential effect of high-dose atorvastatin therapy on skeletal muscle mitochondrial function and whole-body aerobic capacity in humans.METHODSEight overweight (BMI, 31.9 ± 2.0) but otherwise healthy sedentary adults (4 females, 4 males) were studied before (day 0) and 14, 28, and 56 days after initiating atorvastatin (80 mg/d) therapy.RESULTSMaximal ADP-stimulated respiration, measured in permeabilized fiber bundles from muscle biopsies taken at each time point, declined gradually over the course of atorvastatin treatment, resulting in > 30% loss of skeletal muscle mitochondrial oxidative phosphorylation capacity by day 56. Indices of in vivo muscle oxidative capacity (via near-infrared spectroscopy) decreased by 23% to 45%. In whole muscle homogenates from day 0 biopsies, atorvastatin inhibited complex III activity at midmicromolar concentrations, whereas complex IV activity was inhibited at low nanomolar concentrations.CONCLUSIONThese findings demonstrate that high-dose atorvastatin treatment elicits a striking progressive decline in skeletal muscle mitochondrial respiratory capacity, highlighting the need for longer-term dose-response studies in different patient populations to thoroughly define the effect of statin therapy on skeletal muscle health.FUNDINGNIH R01 AR071263.

Assessment of a Novel Tool for the Clinical Grading of Nasal Septal Perforation.

BACKGROUND: Nasal septal perforations (NSP) can have a heterogeneous appearance on endoscopic examination with varying degrees of crusting, inflammatory change, and associated septal deviation. The clinical applicability of these findings as contributors to patient symptoms may be enhanced by use of a standardized assessment. METHODS: Video nasal endoscopy recordings were obtained from 40 patients with NSP. Five raters with varied levels of training ranging from a senior resident to an experienced septal perforation surgeon independently reviewed the videos for the following exam findings: crusting, scarring, granulation tissue, septal deviation, and edema. Scoring for each item was reported on a 3-point (0-2) scale, and each reviewer repeated scoring at a 14-day interval. Interrater and intrarater agreement were calculated using Fleiss kappa for each item and the total scores. Additionally, endoscopy scores were correlated with patient-reported NOSE-Perf symptom scores. RESULTS: Interrater agreement for the overall instrument was in the "fair-to-moderate" range with the following interrater agreement for each item: crusting (0.458-0.575), scarring (0.286-0.308), granulation (0.403-0.406), deviation (0.487-0.494), and edema (0.253-0.406). Intrarater agreement was generally "substantial" for individual items as well as the overall instrument (0.688). The NSP endoscopy scores were moderately correlated with NOSE-Perf scores (r = 0.44, p = 0.008). CONCLUSIONS: An endoscopic evaluation of NSP comprising five exam findings has acceptable interrater and intrarater reliability and correlates with patient-reported outcomes. NSP endoscopy may be applied to future clinical studies for characterization of NSP and assessment of treatment outcomes. LEVEL OF EVIDENCE: 3 Laryngoscope, 134:3049-3053, 2024.

Huntingtin Plays a Role in the Physiological Response to Ethanol in Drosophila.

BACKGROUND: Huntingtin (htt) protein is an essential regulator of nervous system function through its various neuroprotective and pro-survival functions, and loss of wild-type htt function is implicated in the etiology of Huntington's disease. While its pathological role is typically understood as a toxic gain-of-function, some neuronal phenotypes also result from htt loss. Therefore, it is important to understand possible roles for htt in other physiological circumstances. OBJECTIVE: To elucidate the role of htt in the context of ethanol exposure, we investigated how loss of htt impacts behavioral and physiological responses to ethanol in Drosophila. METHODS: We tested flies lacking htt for ethanol sensitivity and tolerance, preference for ethanol using capillary feeder assays, and recovery of mobility after intoxication. Levels of dopamine neurotransmitter and numbers of dopaminergic cells in brains lacking dhtt were also measured. RESULTS: We found that dhtt-null flies are both less sensitive and more tolerant to ethanol exposure in adulthood. Moreover, flies lacking dhtt are more averse to alcohol than controls, and they recover mobility faster following acute ethanol intoxication. We showed that dhtt mediates these effects at least in part through the dopaminergic system, as dhtt is required to maintain normal levels of dopamine in the brain and normal numbers of dopaminergic cells in the adult protocerebrum. CONCLUSIONS: Our results demonstrate that htt regulates the physiological response to ethanol and indicate a novel neuroprotective role for htt in the dopaminergic system, raising the possibility that it may be involved more generally in the response to toxic stimuli.

Piezo1-mediated spontaneous calcium transients in satellite glia impact dorsal root ganglia development.

Spontaneous Ca2+ transients of neural cells is a hallmark of the developing nervous system. It is widely accepted that chemical signals, like neurotransmitters, contribute to spontaneous Ca2+ transients in the nervous system. Here, we reveal an additional mechanism of spontaneous Ca2+ transients that is mechanosensitive in the peripheral nervous system (PNS) using intravital imaging of growing dorsal root ganglia (DRG) in zebrafish embryos. GCaMP6s imaging shows that developing DRG satellite glia contain distinct spontaneous Ca2+ transients, classified into simultaneous, isolated, and microdomains. Longitudinal analysis over days in development demonstrates that as DRG satellite glia become more synchronized, isolated Ca2+ transients remain constant. Using a chemical screen, we identify that Ca2+ transients in DRG glia are dependent on mechanical properties, which we confirmed using an experimental application of mechanical force. We find that isolated spontaneous Ca2+ transients of the glia during development is altered by manipulation of mechanosensitive protein Piezo1, which is expressed in the developing ganglia. In contrast, simultaneous Ca2+ transients of DRG satellite glia is not Piezo1-mediated, thus demonstrating that distinct mechanisms mediate subtypes of spontaneous Ca2+ transients. Activating Piezo1 eventually impacts the cell abundance of DRG cells and behaviors that are driven by DRG neurons. Together, our results reveal mechanistically distinct subtypes of Ca2+ transients in satellite glia and introduce mechanobiology as a critical component of spontaneous Ca2+ transients in the developing PNS.

A paradox promoted by microglia cannibalism shortens the lifespan of developmental microglia.

The overproduction of cells and subsequent production of debris is a universal principle of neurodevelopment. Here we show an additional feature of the developing nervous system that causes neural debris - promoted by the sacrificial nature of embryonic microglia that irreversibly become phagocytic after clearing other neural debris. Described as long-lived, microglia colonize the embryonic brain and persist into adulthood. Using transgenic zebrafish to investigate the microglia debris during brain construction, we identified that unlike other neural cell-types that die in developmental stages after they have expanded, necroptotic-dependent microglial debris is prevalent when microglia are expanding in the zebrafish brain. Time-lapse imaging of microglia demonstrates that this debris is cannibalized by other microglia. To investigate features that promote microglia death and cannibalism, we used time-lapse imaging and fate-mapping strategies to track the lifespan of individual developmental microglia. These approaches revealed that instead of embryonic microglia being long-lived cells that completely digest their phagocytic debris, once most developmental microglia in zebrafish become phagocytic they eventually die, including ones that are cannibalistic. These results establish a paradox -- which we tested by increasing neural debris and manipulating phagocytosis -- that once most microglia in the embryo become phagocytic, they die, create debris and then are cannibalized by other microglia, resulting in more phagocytic microglia that are destined to die.

Exploring STEM Teaching Assistants' Self-Efficacy and Its Relation to Approaches to Teaching.

Undergraduate and graduate teaching assistants (TAs) play large roles in introductory undergraduate education despite having little to no teaching experience or professional development (PD). Self-efficacy and teaching approach have each been studied as independent variables that impact teaching performance and student learning in the absence of practiced skill or developed knowledge. This study explored relationships between TAs' teaching approaches and teaching self-efficacy. Self-efficacy was measured using the Graduate Teaching Self-Efficacy Scale (GTA-TSES), and teaching approach was measured using the Approaches to Teaching Inventory (ATI). The following research questions guided the study: What is the relationship between TAs' approaches to teaching and their self-efficacy? How do approaches to teaching and self-efficacy interact to impact the model of TA self-efficacy? Both ATI subscales correlated strongly with the GTA-TSES learning environment subscale and weakly with the instructional strategy subscale. High self-efficacy TAs demonstrated more concern with impacting student learning, which may contribute to a more student-centered teaching approach. Results indicate that TAs with more confidence in their teaching ability may have a more student-centered approach than teacher-centered approach to teaching. Implications include enhancing TA PD with peer mentoring, constructive feedback, and reflection and incorporating learning concerns in the model of TA teacher efficacy.

Pulsatile Intravascular Lithotripsy: A Novel Mechanism for Peripheral Artery Calcium Fragmentation and Luminal Expansion.

OBJECTIVE: To assess the feasibility and treatment effect of pulsatile intravascular lithotripsy (PIVL) on calcified lesions in a cadaveric model of peripheral artery disease. BACKGROUND: PIVL represents a novel potential approach to intravascular lithotripsy for the treatment of vascular calcification. METHODS: In this preclinical device-feasibility study, technical success, calcium morphology and luminal expansion before and after PIVL treatment were evaluated in surgically isolated, perfused atherosclerotic lower-leg arteries and in perfused whole cadaveric lower legs. Analytical methods included micro-computed tomography (µCT), intravascular optical coherence tomography, digital subtraction angiography, and quantitative coronary analysis. RESULTS: Treatment delivery was successful in all whole-leg specimens (N = 6; mean age 74.2, 66 % female) and in the 8 excised vessels with diameter appropriate to the PIVL balloon (2 vessels exceeding diameter specifications were excluded). There were no vessel perforations. After PIVL, excised vessels showed extensive evidence of new, full-thickness fractures in lesions with calcium arc exceeding 152° and with calcium wall thickness between 0.24 mm and 1.42 mm. PIVL fractures were observed in intimal nodules, sheets, shingles, and medial plates. Vessels within whole-leg specimens also showed full-thickness fracturing and a mean of 1.9 ± 0.9 mm in acute luminal gain, 101.6 ± 99.5 % gain in total minimum cross-sectional area, and a 31.7 ± 13.4 % relative reduction in stenosis (P < 0.001 for all analyses). CONCLUSIONS: In a cadaveric model, PIVL treatment was technically feasible, fractured both circumferential and eccentric calcium lesions, and resulted in acute luminal gain. A clinical feasibility study of PIVL is currently enrolling.

Evolutionarily conserved concepts in glial cell biology.

The evolutionary conservation of glial cells has been appreciated since Ramon y Cajal and Del Rio Hortega first described the morphological features of cells in the nervous system. We now appreciate that glial cells have essential roles throughout life in most nervous systems. The field of glial cell biology has grown exponentially in the last ten years. This new wealth of knowledge has been aided by seminal findings in non-mammalian model systems. Ultimately, such concepts help us to understand glia in mammalian nervous systems. Rather than summarizing the field of glial biology, I will first briefly introduce glia in non-mammalian models systems. Then, highlight seminal findings across the glial field that utilized non-mammalian model systems to advance our understanding of the mammalian nervous system. Finally, I will call attention to some recent findings that introduce new questions about glial cell biology that will be investigated for years to come.

Improving fluorescence lifetime imaging microscopy phasor accuracy using convolutional neural networks.

Introduction: Although a powerful biological imaging technique, fluorescence lifetime imaging microscopy (FLIM) faces challenges such as a slow acquisition rate, a low signal-to-noise ratio (SNR), and high cost and complexity. To address the fundamental problem of low SNR in FLIM images, we demonstrate how to use pre-trained convolutional neural networks (CNNs) to reduce noise in FLIM measurements. Methods: Our approach uses pre-learned models that have been previously validated on large datasets with different distributions than the training datasets, such as sample structures, noise distributions, and microscopy modalities in fluorescence microscopy, to eliminate the need to train a neural network from scratch or to acquire a large training dataset to denoise FLIM data. In addition, we are using the pre-trained networks in the inference stage, where the computation time is in milliseconds and accuracy is better than traditional denoising methods. To separate different fluorophores in lifetime images, the denoised images are then run through an unsupervised machine learning technique named "K-means clustering". Results and Discussion: The results of the experiments carried out on in vivo mouse kidney tissue, Bovine pulmonary artery endothelial (BPAE) fixed cells that have been fluorescently labeled, and mouse kidney fixed samples that have been fluorescently labeled show that our demonstrated method can effectively remove noise from FLIM images and improve segmentation accuracy. Additionally, the performance of our method on out-of-distribution highly scattering in vivo plant samples shows that it can also improve SNR in challenging imaging conditions. Our proposed method provides a fast and accurate way to segment fluorescence lifetime images captured using any FLIM system. It is especially effective for separating fluorophores in noisy FLIM images, which is common in in vivo imaging where averaging is not applicable. Our approach significantly improves the identification of vital biologically relevant structures in biomedical imaging applications.

Members of the majority need to actively promote diversity, equity, inclusion, and belonging.

The responsibility for promoting diversity, equity, inclusion, and belonging (DEIB) too often falls on scientists from minority groups. Here, I provide a list of potential strategies that members of the majority can easily do to step up and get involved in DEIB.

Diabetes and Impaired Fracture Healing: A Narrative Review of Recent Literature.

PURPOSE OF THE REVIEW: Diabetes mellitus is a chronic metabolic disorder commonly encountered in orthopedic patients. Both type 1 and type 2 diabetes mellitus increase fracture risk and impair fracture healing. This review examines complex etiology of impaired fracture healing in diabetes. RECENT FINDINGS: Recent findings point to several mechanisms leading to orthopedic complications in diabetes. Hyperglycemia and chronic inflammation lead to increased formation of advanced glycation end products and generation of reactive oxygen species, which in turn contribute to the disruption in osteoblast and osteoclast balance leading to decreased bone formation and heightening the risk of nonunion or delayed union as well as impaired fracture healing. The mechanisms attributing to this imbalance is secondary to an increase in pro-inflammatory mediators leading to premature resorption of callus cartilage and impaired bone formation due to compromised osteoblast differentiation and their apoptosis. Other mechanisms include disruption in the bone's microenvironment supporting different stages of healing process including hematoma and callus formation, and their resolution during bone remodeling phase. Complications of diabetes including peripheral neuropathy and peripheral vascular disease also contribute to the impairment of fracture healing. Certain diabetic drugs may have adverse effects on fracture healing. The pathophysiology of impaired fracture healing in diabetic patients is complex. This review provides an update of the most recent findings on how key mediators of bone healing are affected in diabetes.

A Subset of Oligodendrocyte Lineage Cells Interact With the Developing Dorsal Root Entry Zone During Its Genesis.

Oligodendrocytes are the myelinating cell of the CNS and are critical for the functionality of the nervous system. In the packed CNS, we know distinct profiles of oligodendrocytes are present. Here, we used intravital imaging in zebrafish to identify a distinct oligodendrocyte lineage cell (OLC) that resides on the dorsal root ganglia sensory neurons in the spinal cord. Our profiling of OLC cellular dynamics revealed a distinct cell cluster that interacts with peripheral sensory neurons at the dorsal root entry zone (DREZ). With pharmacological, physical and genetic manipulations, we show that the entry of dorsal root ganglia pioneer axons across the DREZ is important to produce sensory located oligodendrocyte lineage cells. These oligodendrocyte lineage cells on peripherally derived sensory neurons display distinct processes that are stable and do not express mbpa. Upon their removal, sensory behavior related to the DRG neurons is abolished. Together, these data support the hypothesis that peripheral neurons at the DREZ can also impact oligodendrocyte development.

The embryonic zebrafish brain is seeded by a lymphatic-dependent population of mrc1+ microglia precursors.

Microglia are the resident macrophages of the CNS that serve critical roles in brain construction. Although human brains contain microglia by 4 weeks gestation, an understanding of the earliest microglia that seed the brain during its development remains unresolved. Using time-lapse imaging in zebrafish, we discovered a mrc1a+ microglia precursor population that seeds the brain before traditionally described microglia. These early microglia precursors are dependent on lymphatic vasculature that surrounds the brain and are independent of pu1+ yolk sac-derived microglia. Single-cell RNA-sequencing datasets reveal Mrc1+ microglia in the embryonic brains of mice and humans. We then show in zebrafish that these early mrc1a+ microglia precursors preferentially expand during pathophysiological states in development. Taken together, our results identify a critical role of lymphatics in the microglia precursors that seed the early embryonic brain.

Identification of astroglia-like cardiac nexus glia that are critical regulators of cardiac development and function.

Glial cells are essential for functionality of the nervous system. Growing evidence underscores the importance of astrocytes; however, analogous astroglia in peripheral organs are poorly understood. Using confocal time-lapse imaging, fate mapping, and mutant genesis in a zebrafish model, we identify a neural crest-derived glial cell, termed nexus glia, which utilizes Meteorin signaling via Jak/Stat3 to drive differentiation and regulate heart rate and rhythm. Nexus glia are labeled with gfap, glast, and glutamine synthetase, markers that typically denote astroglia cells. Further, analysis of single-cell sequencing datasets of human and murine hearts across ages reveals astrocyte-like cells, which we confirm through a multispecies approach. We show that cardiac nexus glia at the outflow tract are critical regulators of both the sympathetic and parasympathetic system. These data establish the crucial role of glia on cardiac homeostasis and provide a description of nexus glia in the PNS.

Compliant vascular models 3D printed with the Stratasys J750: a direct characterization of model distensibility using intravascular ultrasound.

PURPOSE: The purpose of this study is to evaluate biomechanical accuracy of 3D printed anatomical vessels using a material jetting printer (J750, Stratasys, Rehovot, Israel) by measuring distensibility via intravascular ultrasound. MATERIALS AND METHODS: The test samples are 3D printed tubes to simulate arterial vessels (aorta, carotid artery, and coronary artery). Each vessel type is defined by design geometry of the vessel inner diameter and wall thickness. Vessel inner diameters are aorta = 30mm, carotid = 7mm, and coronary = 3mm. Vessel wall thickness are aorta = 3mm, carotid = 1.5mm, and coronary = 1mm. Each vessel type was printed in 3 different material options. Material options are user-selected from the J750 printer software graphical user interface as blood vessel wall anatomy elements in 'compliant', 'slightly compliant', and 'rigid' options. Three replicates of each vessel type were printed in each of the three selected material options, for a total of 27 models. The vessels were connected to a flow loop system where pressure was monitored via a pressure wire and cross-sectional area was measured with intravascular ultrasound (IVUS). Distensibility was calculated by comparing the % difference in cross-sectional area vs. pulse pressure to clinical literature values. Target clinical ranges for normal and diseased population distensibility are 10.3-44 % for the aorta, 5.1-10.1 % for carotid artery, and 0.5-6 % for coronary artery. RESULTS: Aorta test vessels had the most clinically representative distensibility when printed in user-selected 'compliant' and 'slightly compliant' material. All aorta test vessels of 'compliant' material (n = 3) and 2 of 3 'slightly compliant' vessels evaluated were within target range. Carotid vessels were most clinically represented in distensibility when printed in 'compliant' and 'slightly compliant' material. For carotid test vessels, 2 of 3 'compliant' material samples and 1 of 3 'slightly compliant' material samples were within target range. Coronary arteries were most clinically represented in distensibility when printed in 'slightly compliant' and 'rigid' material. For coronary test vessels, 1 of 3 'slightly compliant' materials and 3 of 3 'rigid' material samples fell within target range. CONCLUSIONS: This study suggests that advancements in materials and 3D printing technology introduced with the J750 Digital Anatomy 3D Printer can enable anatomical models with clinically relevant distensibility.

Tetris in the Nervous System: What Principles of Neuronal Tiling Can Tell Us About How Glia Play the Game.

The precise organization and arrangement of neural cells is essential for nervous system functionality. Cellular tiling is an evolutionarily conserved phenomenon that organizes neural cells, ensuring non-redundant coverage of receptive fields in the nervous system. First recorded in the drawings of Ramon y Cajal more than a century ago, we now have extensive knowledge of the biochemical and molecular mechanisms that mediate tiling of neurons. The advent of live imaging techniques in both invertebrate and vertebrate model organisms has enhanced our understanding of these processes. Despite advancements in our understanding of neuronal tiling, we know relatively little about how glia, an essential non-neuronal component of the nervous system, tile and contribute to the overall spatial arrangement of the nervous system. Here, we discuss lessons learned from neurons and apply them to potential mechanisms that glial cells may use to tile, including cell diversity, contact-dependent repulsion, and chemical signaling. We also discuss open questions in the field of tiling and what new technologies need to be developed in order to better understand glial tiling.

Coxiella burnetii infections in mice: Immunological responses to contemporary genotypes found in the US.

Coxiella burnetii is an obligate intracellular bacterium that causes the human disease Q fever, which can manifest as an acute flu-like illness or a long-term chronic illness, such as endocarditis. Three genotypes (ST8, ST16, and ST20) of Coxiella burnetii are commonly found in the contemporary US and are associated with specific animal hosts. Although all three genotypes have been isolated from humans with Q fever, studies comparing virulence between C. burnetii sequence types have been rare. Here, groups of mice were infected via aerosol inoculation with isolates derived from cow's milk, environmental, animal, and human samples. Mice were monitored for weight loss and blood samples were takenweekly. Animals were euthanized at 2- and 12-weeks post-infection, and bacterial burden was determined for tissues by real-time PCR. The levels of anti-Coxiella antibodies and selected inflammatory cytokines were determined for serum samples. Weight loss and splenomegaly were observed in mice infected with ST20 and ST16 isolates but were absent in the mice infected with ST8 isolates. Bacterial concentrations in the tissues were lower in the ST8 isolates at 2 weeks post-infection relative to all other isolates. ST16 and ST20 isolates induced robust antibody and cytokine responses, while ST8 isolates produced significantly lower anti-C. burnetii titers early in the infection but saw increased titers in some animals several weeks post-infection. The data suggest that the ST8 isolates are less virulent in this mouse model, as they produce less robust antibody responses that are slow to develop, relative to the ST16 and ST20 isolates.

Pioneer Axons Utilize a Dcc Signaling-Mediated Invasion Brake to Precisely Complete Their Pathfinding Odyssey.

Axons navigate through the embryo to construct a functional nervous system. A missing part of the axon navigation puzzle is how a single axon traverses distinct anatomic choice points through its navigation. The dorsal root ganglia (DRG) neurons experience such choice points. First, they navigate to the dorsal root entry zone (DREZ), then halt navigation in the peripheral nervous system to invade the spinal cord, and then reinitiate navigation inside the CNS. Here, we used time-lapse super-resolution imaging in zebrafish DRG pioneer neurons to investigate how embryonic axons control their cytoskeleton to navigate to and invade at the correct anatomic position. We found that invadopodia components form in the growth cone even during filopodia-based navigation, but only stabilize when the axon is at the spinal cord entry location. Further, we show that intermediate levels of DCC and cAMP, as well as Rac1 activation, subsequently engage an axon invasion brake. Our results indicate that actin-based invadopodia components form in the growth cone and disruption of the invasion brake causes axon entry defects and results in failed behavioral responses, thereby demonstrating the importance of regulating distinct actin populations during navigational challenges.SIGNIFICANCE STATEMENT Correct spatiotemporal navigation of neuronal growth cones is dependent on extracellular navigational cues and growth cone dynamics. Here, we link dcc-mediated signaling to actin-based invadopodia and filopodia dynamics during pathfinding and entry into the spinal cord using an in vivo model of dorsal root ganglia (DRG) sensory axons. We reveal a molecularly-controlled brake on invadopodia stabilization until the sensory neuron growth cone is present at the dorsal root entry zone (DREZ), which is ultimately essential for growth cone entry into the spinal cord and behavioral response.