RESUMEN
To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we studied neuronal networks that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. We show that a specific class of leg sensory neurons synapses directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM more accessible and affordable to the scientific community.
Asunto(s)
Envejecimiento/fisiología , Drosophila melanogaster/ultraestructura , Microscopía Electrónica de Transmisión , Neuronas Motoras/ultraestructura , Células Receptoras Sensoriales/ultraestructura , Animales , Automatización , Conectoma , Extremidades/inervación , Nervios Periféricos/ultraestructura , Sinapsis/ultraestructuraRESUMEN
The posterior parietal cortex exhibits choice-selective activity during perceptual decision-making tasks1-10. However, it is not known how this selective activity arises from the underlying synaptic connectivity. Here we combined virtual-reality behaviour, two-photon calcium imaging, high-throughput electron microscopy and circuit modelling to analyse how synaptic connectivity between neurons in the posterior parietal cortex relates to their selective activity. We found that excitatory pyramidal neurons preferentially target inhibitory interneurons with the same selectivity. In turn, inhibitory interneurons preferentially target pyramidal neurons with opposite selectivity, forming an opponent inhibition motif. This motif was present even between neurons with activity peaks in different task epochs. We developed neural-circuit models of the computations performed by these motifs, and found that opponent inhibition between neural populations with opposite selectivity amplifies selective inputs, thereby improving the encoding of trial-type information. The models also predict that opponent inhibition between neurons with activity peaks in different task epochs contributes to creating choice-specific sequential activity. These results provide evidence for how synaptic connectivity in cortical circuits supports a learned decision-making task.
Asunto(s)
Toma de Decisiones , Vías Nerviosas , Lóbulo Parietal , Sinapsis , Calcio/análisis , Calcio/metabolismo , Toma de Decisiones/fisiología , Interneuronas/metabolismo , Interneuronas/ultraestructura , Aprendizaje/fisiología , Microscopía Electrónica , Inhibición Neural , Vías Nerviosas/fisiología , Vías Nerviosas/ultraestructura , Lóbulo Parietal/citología , Lóbulo Parietal/fisiología , Lóbulo Parietal/ultraestructura , Células Piramidales/metabolismo , Células Piramidales/ultraestructura , Sinapsis/metabolismo , Sinapsis/ultraestructura , Realidad Virtual , Modelos NeurológicosRESUMEN
A deep understanding of how the brain controls behaviour requires mapping neural circuits down to the muscles that they control. Here, we apply automated tools to segment neurons and identify synapses in an electron microscopy dataset of an adult female Drosophila melanogaster ventral nerve cord (VNC)1, which functions like the vertebrate spinal cord to sense and control the body. We find that the fly VNC contains roughly 45 million synapses and 14,600 neuronal cell bodies. To interpret the output of the connectome, we mapped the muscle targets of leg and wing motor neurons using genetic driver lines2 and X-ray holographic nanotomography3. With this motor neuron atlas, we identified neural circuits that coordinate leg and wing movements during take-off. We provide the reconstruction of VNC circuits, the motor neuron atlas and tools for programmatic and interactive access as resources to support experimental and theoretical studies of how the nervous system controls behaviour.
Asunto(s)
Conectoma , Drosophila melanogaster , Neuronas Motoras , Tejido Nervioso , Vías Nerviosas , Sinapsis , Animales , Femenino , Conjuntos de Datos como Asunto , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Drosophila melanogaster/ultraestructura , Extremidades/fisiología , Extremidades/inervación , Holografía , Microscopía Electrónica , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Neuronas Motoras/ultraestructura , Movimiento , Músculos/inervación , Músculos/fisiología , Tejido Nervioso/anatomía & histología , Tejido Nervioso/citología , Tejido Nervioso/fisiología , Tejido Nervioso/ultraestructura , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Vías Nerviosas/ultraestructura , Sinapsis/fisiología , Sinapsis/ultraestructura , Tomografía por Rayos X , Alas de Animales/inervación , Alas de Animales/fisiologíaRESUMEN
Animal movement is controlled by motor neurons (MNs), which project out of the central nervous system to activate muscles1. MN activity is coordinated by complex premotor networks that facilitate the contribution of individual muscles to many different behaviours2-6. Here we use connectomics7 to analyse the wiring logic of premotor circuits controlling the Drosophila leg and wing. We find that both premotor networks cluster into modules that link MNs innervating muscles with related functions. Within most leg motor modules, the synaptic weights of each premotor neuron are proportional to the size of their target MNs, establishing a circuit basis for hierarchical MN recruitment. By contrast, wing premotor networks lack proportional synaptic connectivity, which may enable more flexible recruitment of wing steering muscles. Through comparison of the architecture of distinct motor control systems within the same animal, we identify common principles of premotor network organization and specializations that reflect the unique biomechanical constraints and evolutionary origins of leg and wing motor control.
Asunto(s)
Conectoma , Drosophila melanogaster , Extremidades , Neuronas Motoras , Vías Nerviosas , Sinapsis , Alas de Animales , Animales , Femenino , Masculino , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Extremidades/inervación , Extremidades/fisiología , Neuronas Motoras/fisiología , Movimiento/fisiología , Músculos/inervación , Músculos/fisiología , Red Nerviosa/anatomía & histología , Red Nerviosa/citología , Red Nerviosa/fisiología , Vías Nerviosas/anatomía & histología , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Sinapsis/fisiología , Alas de Animales/inervación , Alas de Animales/fisiologíaRESUMEN
The cerebellum is thought to help detect and correct errors between intended and executed commands1,2 and is critical for social behaviours, cognition and emotion3-6. Computations for motor control must be performed quickly to correct errors in real time and should be sensitive to small differences between patterns for fine error correction while being resilient to noise7. Influential theories of cerebellar information processing have largely assumed random network connectivity, which increases the encoding capacity of the network's first layer8-13. However, maximizing encoding capacity reduces the resilience to noise7. To understand how neuronal circuits address this fundamental trade-off, we mapped the feedforward connectivity in the mouse cerebellar cortex using automated large-scale transmission electron microscopy and convolutional neural network-based image segmentation. We found that both the input and output layers of the circuit exhibit redundant and selective connectivity motifs, which contrast with prevailing models. Numerical simulations suggest that these redundant, non-random connectivity motifs increase the resilience to noise at a negligible cost to the overall encoding capacity. This work reveals how neuronal network structure can support a trade-off between encoding capacity and redundancy, unveiling principles of biological network architecture with implications for the design of artificial neural networks.
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Corteza Cerebelosa , Red Nerviosa , Vías Nerviosas , Neuronas , Animales , Ratones , Corteza Cerebelosa/citología , Corteza Cerebelosa/fisiología , Corteza Cerebelosa/ultraestructura , Redes Neurales de la Computación , Neuronas/citología , Neuronas/fisiología , Neuronas/ultraestructura , Red Nerviosa/citología , Red Nerviosa/fisiología , Red Nerviosa/ultraestructura , Microscopía Electrónica de TransmisiónRESUMEN
We present an auxiliary learning task for the problem of neuron segmentation in electron microscopy volumes. The auxiliary task consists of the prediction of local shape descriptors (LSDs), which we combine with conventional voxel-wise direct neighbor affinities for neuron boundary detection. The shape descriptors capture local statistics about the neuron to be segmented, such as diameter, elongation, and direction. On a study comparing several existing methods across various specimen, imaging techniques, and resolutions, auxiliary learning of LSDs consistently increases segmentation accuracy of affinity-based methods over a range of metrics. Furthermore, the addition of LSDs promotes affinity-based segmentation methods to be on par with the current state of the art for neuron segmentation (flood-filling networks), while being two orders of magnitudes more efficient-a critical requirement for the processing of future petabyte-sized datasets.
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Procesamiento de Imagen Asistido por Computador , Neuronas , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
We develop an automatic method for synaptic partner identification in insect brains and use it to predict synaptic partners in a whole-brain electron microscopy dataset of the fruit fly. The predictions can be used to infer a connectivity graph with high accuracy, thus allowing fast identification of neural pathways. To facilitate circuit reconstruction using our results, we develop CIRCUITMAP, a user interface add-on for the circuit annotation tool CATMAID.
Asunto(s)
Encéfalo/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Sinapsis/fisiología , Animales , Encéfalo/citología , Bases de Datos Factuales , Drosophila melanogaster , Microscopía Electrónica , Vías NerviosasRESUMEN
High-resolution serial-section electron microscopy (ssEM) makes it possible to investigate the dense meshwork of axons, dendrites, and synapses that form neuronal circuits. However, the imaging scale required to comprehensively reconstruct these structures is more than ten orders of magnitude smaller than the spatial extents occupied by networks of interconnected neurons, some of which span nearly the entire brain. Difficulties in generating and handling data for large volumes at nanoscale resolution have thus restricted vertebrate studies to fragments of circuits. These efforts were recently transformed by advances in computing, sample handling, and imaging techniques, but high-resolution examination of entire brains remains a challenge. Here, we present ssEM data for the complete brain of a larval zebrafish (Danio rerio) at 5.5 days post-fertilization. Our approach utilizes multiple rounds of targeted imaging at different scales to reduce acquisition time and data management requirements. The resulting dataset can be analysed to reconstruct neuronal processes, permitting us to survey all myelinated axons (the projectome). These reconstructions enable precise investigations of neuronal morphology, which reveal remarkable bilateral symmetry in myelinated reticulospinal and lateral line afferent axons. We further set the stage for whole-brain structure-function comparisons by co-registering functional reference atlases and in vivo two-photon fluorescence microscopy data from the same specimen. All obtained images and reconstructions are provided as an open-access resource.
Asunto(s)
Encéfalo/ultraestructura , Microscopía Electrónica , Pez Cebra , Anatomía Artística , Animales , Atlas como Asunto , Axones/metabolismo , Axones/ultraestructura , Encéfalo/anatomía & histología , Encéfalo/citología , Conjuntos de Datos como Asunto , Larva/anatomía & histología , Larva/citología , Larva/ultraestructura , Microscopía de Fluorescencia por Excitación Multifotónica , Publicación de Acceso Abierto , Pez Cebra/anatomía & histología , Pez Cebra/crecimiento & desarrolloRESUMEN
Circuits in the cerebral cortex consist of thousands of neurons connected by millions of synapses. A precise understanding of these local networks requires relating circuit activity with the underlying network structure. For pyramidal cells in superficial mouse visual cortex (V1), a consensus is emerging that neurons with similar visual response properties excite each other, but the anatomical basis of this recurrent synaptic network is unknown. Here we combined physiological imaging and large-scale electron microscopy to study an excitatory network in V1. We found that layer 2/3 neurons organized into subnetworks defined by anatomical connectivity, with more connections within than between groups. More specifically, we found that pyramidal neurons with similar orientation selectivity preferentially formed synapses with each other, despite the fact that axons and dendrites of all orientation selectivities pass near (<5 µm) each other with roughly equal probability. Therefore, we predict that mechanisms of functionally specific connectivity take place at the length scale of spines. Neurons with similar orientation tuning formed larger synapses, potentially enhancing the net effect of synaptic specificity. With the ability to study thousands of connections in a single circuit, functional connectomics is proving a powerful method to uncover the organizational logic of cortical networks.
Asunto(s)
Corteza Visual/anatomía & histología , Corteza Visual/fisiología , Vías Visuales/citología , Vías Visuales/fisiología , Animales , Axones/fisiología , Calcio/análisis , Dendritas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Fotones , Células Piramidales/citología , Células Piramidales/fisiología , Sinapsis/metabolismo , Corteza Visual/citología , Corteza Visual/ultraestructura , Vías Visuales/anatomía & histología , Vías Visuales/ultraestructuraRESUMEN
In the cerebral cortex, local circuits consist of tens of thousands of neurons, each of which makes thousands of synaptic connections. Perhaps the biggest impediment to understanding these networks is that we have no wiring diagrams of their interconnections. Even if we had a partial or complete wiring diagram, however, understanding the network would also require information about each neuron's function. Here we show that the relationship between structure and function can be studied in the cortex with a combination of in vivo physiology and network anatomy. We used two-photon calcium imaging to characterize a functional property--the preferred stimulus orientation--of a group of neurons in the mouse primary visual cortex. Large-scale electron microscopy of serial thin sections was then used to trace a portion of these neurons' local network. Consistent with a prediction from recent physiological experiments, inhibitory interneurons received convergent anatomical input from nearby excitatory neurons with a broad range of preferred orientations, although weak biases could not be rejected.
Asunto(s)
Red Nerviosa/anatomía & histología , Red Nerviosa/citología , Neuronas/fisiología , Corteza Visual/anatomía & histología , Corteza Visual/citología , Animales , Señalización del Calcio , Interneuronas/fisiología , Masculino , Ratones , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Microtomía , Red Nerviosa/fisiología , Red Nerviosa/ultraestructura , Inhibición Neural/fisiología , Neuronas/ultraestructura , Células Piramidales/fisiología , Células Piramidales/ultraestructura , Sinapsis/fisiología , Corteza Visual/fisiología , Corteza Visual/ultraestructuraRESUMEN
Primary cilia on granule cell neuron progenitors in the developing cerebellum detect sonic hedgehog to facilitate proliferation. Following differentiation, cerebellar granule cells become the most abundant neuronal cell type in the brain. While granule cell cilia are essential during early developmental stages, they become infrequent upon maturation. Here, we provide nanoscopic resolution of cilia in situ using large-scale electron microscopy volumes and immunostaining of mouse cerebella. In many granule cells, we found intracellular cilia, concealed from the external environment. Cilia were disassembled in differentiating granule cell neurons-in a process we call cilia deconstruction-distinct from premitotic cilia resorption in proliferating progenitors. In differentiating granule cells, cilia deconstruction involved unique disassembly intermediates, and, as maturation progressed, mother centriolar docking at the plasma membrane. Unlike ciliated neurons in other brain regions, our results show the deconstruction of concealed cilia in differentiating granule cells, which might prevent mitogenic hedgehog responsiveness. Ciliary deconstruction could be paradigmatic of cilia removal during differentiation in other tissues.
Asunto(s)
Diferenciación Celular , Cerebelo , Cilios , Proteínas Hedgehog , Neuronas , Cilios/metabolismo , Cilios/ultraestructura , Animales , Neuronas/metabolismo , Neuronas/citología , Neuronas/ultraestructura , Ratones , Cerebelo/metabolismo , Cerebelo/citología , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Neurogénesis , Centriolos/metabolismo , Centriolos/ultraestructura , Ratones Endogámicos C57BLRESUMEN
We demonstrate limited-tilt, serial section electron tomography (ET), which can non-destructively map brain circuits over large 3D volumes and reveal high-resolution, supramolecular details within subvolumes of interest. We show accelerated ET imaging of thick sections (>500 nm) with the capacity to resolve key features of neuronal circuits including chemical synapses, endocytic structures, and gap junctions. Furthermore, we systematically assessed how imaging parameters affect image quality and speed to enable connectomic-scale projects.
RESUMEN
Molecular layer interneurons (MLIs) account for approximately 80% of the inhibitory interneurons in the cerebellar cortex and are vital to cerebellar processing. MLIs are thought to primarily inhibit Purkinje cells (PCs) and suppress the plasticity of synapses onto PCs. MLIs also inhibit, and are electrically coupled to, other MLIs, but the functional significance of these connections is not known. Here, we find that two recently recognized MLI subtypes, MLI1 and MLI2, have a highly specialized connectivity that allows them to serve distinct functional roles. MLI1s primarily inhibit PCs, are electrically coupled to each other, fire synchronously with other MLI1s on the millisecond timescale in vivo, and synchronously pause PC firing. MLI2s are not electrically coupled, primarily inhibit MLI1s and disinhibit PCs, and are well suited to gating cerebellar-dependent behavior and learning. The synchronous firing of electrically coupled MLI1s and disinhibition provided by MLI2s require a major re-evaluation of cerebellar processing.
Asunto(s)
Interneuronas , Inhibición Neural , Células de Purkinje , Animales , Células de Purkinje/fisiología , Interneuronas/fisiología , Inhibición Neural/fisiología , Ratones , Cerebelo/citología , Cerebelo/fisiología , Ratones Transgénicos , Potenciales de Acción/fisiología , Ratones Endogámicos C57BL , Corteza Cerebelosa/fisiología , Corteza Cerebelosa/citologíaRESUMEN
Comprehensive, synapse-resolution imaging of the brain will be crucial for understanding neuronal computations and function. In connectomics, this has been the sole purview of volume electron microscopy (EM), which entails an excruciatingly difficult process because it requires cutting tissue into many thin, fragile slices that then need to be imaged, aligned, and reconstructed. Unlike EM, hard X-ray imaging is compatible with thick tissues, eliminating the need for thin sectioning, and delivering fast acquisition, intrinsic alignment, and isotropic resolution. Unfortunately, current state-of-the-art X-ray microscopy provides much lower resolution, to the extent that segmenting membranes is very challenging. We propose an uncertainty-aware 3D reconstruction model that translates X-ray images to EM-like images with enhanced membrane segmentation quality, showing its potential for developing simpler, faster, and more accurate X-ray based connectomics pipelines.
RESUMEN
Primary cilia on granule cell neuron progenitors in the developing cerebellum detect sonic hedgehog to facilitate proliferation. Following differentiation, cerebellar granule cells become the most abundant neuronal cell type in the brain. While essential during early developmental stages, the fate of granule cell cilia is unknown. Here, we provide nanoscopic resolution of ciliary dynamics in situ by studying developmental changes in granule cell cilia using large-scale electron microscopy volumes and immunostaining of mouse cerebella. We found that many granule cell primary cilia were intracellular and concealed from the external environment. Cilia were disassembed in differentiating granule cell neurons in a process we call cilia deconstruction that was distinct from pre-mitotic cilia resorption in proliferating progenitors. In differentiating granule cells, ciliary loss involved unique disassembly intermediates, and, as maturation progressed, mother centriolar docking at the plasma membrane. Cilia did not reform from the docked centrioles, rather, in adult mice granule cell neurons remained unciliated. Many neurons in other brain regions require cilia to regulate function and connectivity. In contrast, our results show that granule cell progenitors had concealed cilia that underwent deconstruction potentially to prevent mitogenic hedgehog responsiveness. The ciliary deconstruction mechanism we describe could be paradigmatic of cilia removal during differentiation in other tissues.
RESUMEN
The cerebellar cortex contributes to diverse behaviors by transforming mossy fiber inputs into predictions in the form of Purkinje cell (PC) outputs, and then refining those predictions1. Molecular layer interneurons (MLIs) account for approximately 80% of the inhibitory interneurons in the cerebellar cortex2, and are vital to cerebellar processing1,3. MLIs are thought to primarily inhibit PCs and suppress the plasticity of excitatory synapses onto PCs. MLIs also inhibit, and are electrically coupled to, other MLIs4-7, but the functional significance of these connections is not known1,3. Behavioral studies suggest that cerebellar-dependent learning is gated by disinhibition of PCs, but the source of such disinhibition has not been identified8. Here we find that two recently recognized MLI subtypes2, MLI1 and MLI2, have highly specialized connectivity that allows them to serve very different functional roles. MLI1s primarily inhibit PCs, are electrically coupled to each other, fire synchronously with other MLI1s on the millisecond time scale in vivo, and synchronously pause PC firing. MLI2s are not electrically coupled, they primarily inhibit MLI1s and disinhibit PCs, and are well suited to gating cerebellar-dependent learning8. These findings require a major reevaluation of processing within the cerebellum in which disinhibition, a powerful circuit motif present in the cerebral cortex and elsewhere9-17, greatly increases the computational power and flexibility of the cerebellum. They also suggest that millisecond time scale synchronous firing of electrically-coupled MLI1s helps regulate the output of the cerebellar cortex by synchronously pausing PC firing, which has been shown to evoke precisely-timed firing in PC targets18.
RESUMEN
Our ability to sense and move our bodies relies on proprioceptors, sensory neurons that detect mechanical forces within the body. Different subtypes of proprioceptors detect different kinematic features, such as joint position, movement, and vibration, but the mechanisms that underlie proprioceptor feature selectivity remain poorly understood. Using single-nucleus RNA sequencing (RNA-seq), we found that proprioceptor subtypes in the Drosophila leg lack differential expression of mechanosensitive ion channels. However, anatomical reconstruction of the proprioceptors and connected tendons revealed major biomechanical differences between subtypes. We built a model of the proprioceptors and tendons that identified a biomechanical mechanism for joint angle selectivity and predicted the existence of a topographic map of joint angle, which we confirmed using calcium imaging. Our findings suggest that biomechanical specialization is a key determinant of proprioceptor feature selectivity in Drosophila. More broadly, the discovery of proprioceptive maps reveals common organizational principles between proprioception and other topographically organized sensory systems.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Células Receptoras Sensoriales/fisiología , Propiocepción/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Canales Iónicos/metabolismoRESUMEN
Across mammalian skin, structurally complex and diverse mechanosensory end organs respond to mechanical stimuli and enable our perception of dynamic, light touch. How forces act on morphologically dissimilar mechanosensory end organs of the skin to gate the requisite mechanotransduction channel Piezo2 and excite mechanosensory neurons is not understood. Here, we report high-resolution reconstructions of the hair follicle lanceolate complex, Meissner corpuscle, and Pacinian corpuscle and the subcellular distribution of Piezo2 within them. Across all three end organs, Piezo2 is restricted to the sensory axon membrane, including axon protrusions that extend from the axon body. These protrusions, which are numerous and elaborate extensively within the end organs, tether the axon to resident non-neuronal cells via adherens junctions. These findings support a unified model for dynamic touch in which mechanical stimuli stretch hundreds to thousands of axon protrusions across an end organ, opening proximal, axonal Piezo2 channels and exciting the neuron.
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Mecanotransducción Celular , Células de Merkel , Animales , Células de Merkel/fisiología , Mecanotransducción Celular/fisiología , Imagenología Tridimensional , Canales Iónicos/metabolismo , Mecanorreceptores/fisiología , Mamíferos/metabolismoRESUMEN
Specialized mechanosensory end organs within mammalian skin-hair follicle-associated lanceolate complexes, Meissner corpuscles, and Pacinian corpuscles-enable our perception of light, dynamic touch 1 . In each of these end organs, fast-conducting mechanically sensitive neurons, called Aß low-threshold mechanoreceptors (Aß LTMRs), associate with resident glial cells, known as terminal Schwann cells (TSCs) or lamellar cells, to form complex axon ending structures. Lanceolate-forming and corpuscle-innervating Aß LTMRs share a low threshold for mechanical activation, a rapidly adapting (RA) response to force indentation, and high sensitivity to dynamic stimuli 1-6 . How mechanical stimuli lead to activation of the requisite mechanotransduction channel Piezo2 7-15 and Aß RA-LTMR excitation across the morphologically dissimilar mechanosensory end organ structures is not understood. Here, we report the precise subcellular distribution of Piezo2 and high-resolution, isotropic 3D reconstructions of all three end organs formed by Aß RA-LTMRs determined by large volume enhanced Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging. We found that within each end organ, Piezo2 is enriched along the sensory axon membrane and is minimally or not expressed in TSCs and lamellar cells. We also observed a large number of small cytoplasmic protrusions enriched along the Aß RA-LTMR axon terminals associated with hair follicles, Meissner corpuscles, and Pacinian corpuscles. These axon protrusions reside within close proximity to axonal Piezo2, occasionally contain the channel, and often form adherens junctions with nearby non-neuronal cells. Our findings support a unified model for Aß RA-LTMR activation in which axon protrusions anchor Aß RA-LTMR axon terminals to specialized end organ cells, enabling mechanical stimuli to stretch the axon in hundreds to thousands of sites across an individual end organ and leading to activation of proximal Piezo2 channels and excitation of the neuron.