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Here, we present a protocol for staining murine skin innervation by either a pan-axonal marker or a genetic tracer of sensory neuron subtypes using floating sections. We also describe steps for using a new MATLAB-based semiautomated routine that facilitates the quantification of innervation density. This protocol can also be applied to other organs, such as the mouse's spinal cord and tongue. For complete details on the use and execution of this protocol, please refer to Dey et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dorsal root ganglia (DRG) somatosensory neurons detect mechanical, thermal, and chemical stimuli acting on the body. Achieving a holistic view of how different DRG neuron subtypes relay neural signals from the periphery to the CNS has been challenging with existing tools. Here, we develop and curate a mouse genetic toolkit that allows for interrogating the properties and functions of distinct cutaneous targeting DRG neuron subtypes. These tools have enabled a broad morphological analysis, which revealed distinct cutaneous axon arborization areas and branching patterns of the transcriptionally distinct DRG neuron subtypes. Moreover, in vivo physiological analysis revealed that each subtype has a distinct threshold and range of responses to mechanical and/or thermal stimuli. These findings support a model in which morphologically and physiologically distinct cutaneous DRG sensory neuron subtypes tile mechanical and thermal stimulus space to collectively encode a wide range of natural stimuli., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We describe how to reconstruct and quantify multi-signal neuronal morphology, using the dendritic distributions of microtubules and F-actin in sensory neurons from fly larvae as examples. We then provide a detailed procedure to analyze channel-specific morphometrics from these enhanced reconstructions. To illustrate applications, we demonstrate how to run a cytoskeleton-constrained simulation of dendritic tree generation and explain its validation against experimental data. This protocol is applicable to any species, developmental stage, brain region, cell class, branching process, and signal type. For complete details on the use and execution of this protocol, please refer to Nanda et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

In this protocol, we provide step-by-step instructions for dissection and culture of primary murine dorsal root ganglia (DRG), which provide an opportunity to study the functional properties of peripheral sensory neurons in vitro . Further, we describe the analysis of neuropeptide release by ELISA as a possible downstream application. In addition, isolated DRGs can be used directly for immunofluorescence, flow cytometry, RNA sequencing or proteomic approaches, electrophysiology, and calcium imaging. For complete details on the use and execution of this protocol, please refer to Perner et al. (2020)., Competing Interests: C.L.S. is a paid consultant for Bayer and Merck., (© 2021 The Authors.)

In many organs, stem cell function depends on communication with their niche partners. Cranial sensory neurons develop in close proximity to blood vessels; however, whether vasculature is an integral component of their niches is yet unknown. Here, two separate roles for vasculature in cranial sensory neurogenesis in zebrafish are uncovered. The first involves precise spatiotemporal endothelial-neuroblast cytoneme contacts and Dll4-Notch signaling to restrain neuroblast proliferation. The second, instead, requires blood flow to trigger a transcriptional response that modifies neuroblast metabolic status and induces sensory neuron differentiation. In contrast, no role of sensory neurogenesis in vascular development is found, suggesting unidirectional signaling from vasculature to sensory neuroblasts. Altogether, we demonstrate that the cranial vasculature constitutes a niche component of the sensory ganglia that regulates the pace of their growth and differentiation dynamics., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Universitat Pompeu Fabra. Published by Elsevier Inc. All rights reserved.)

Vagal sensory neurons relay viscero- and somatosensory information from within the body and play a key role in maintaining physiological homeostasis. We recently characterized the diversity of vagal sensory neurons in the mouse using a single-cell transcriptomics approach. Here, we provide an in-depth protocol for the extraction of mouse vagal ganglia and the production of high-quality single-cell suspensions from this tissue. This effective protocol can also be applied for use with other peripheral and central neuron populations with few modifications. For complete details on the use and execution of this protocol, please refer to Kupari et al. (2019)., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Efficient and homogeneous in vitro generation of peripheral sensory neurons may provide a framework for novel drug screening platforms and disease models of touch and pain. We discover that, by overexpressing NGN2 and BRN3A, human pluripotent stem cells can be transcriptionally programmed to differentiate into a surprisingly uniform culture of cold- and mechano-sensing neurons. Although such a neuronal subtype is not found in mice, we identify molecular evidence for its existence in human sensory ganglia. Combining NGN2 and BRN3A programming with neural crest patterning, we produce two additional populations of sensory neurons, including a specialized touch receptor neuron subtype. Finally, we apply this system to model a rare inherited sensory disorder of touch and proprioception caused by inactivating mutations in PIEZO2. Together, these findings establish an approach to specify distinct sensory neuron subtypes in vitro, underscoring the utility of stem cell technology to capture human-specific features of physiology and disease., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Coordinated movement requires the integration of many sensory inputs including proprioception, the sense of relative body position and force associated with movement. Proprioceptive information is relayed to the cerebellum via spinocerebellar neurons, located in the spinal cord within a number of major neuronal columns or as various scattered populations. Despite the importance of proprioception to fluid movement, a molecular understanding of spinocerebellar relay interneurons is only beginning to be explored, with limited knowledge of molecular heterogeneity within and between columns. Using fluorescent reporter mice, neuronal tracing, and in situ hybridization, we identify widespread expression of Hox cluster genes within spinocerebellar neurons. We reveal a "Hox code" based on axial level and individual spinocerebellar column, which, at cervico-thoracic levels, is essential for subtype regionalization. Specifically, we show that Hoxc9 function is required in most, but not all, cells of the thoracic spinocerebellar column, Clarke's column, revealing heterogeneity reliant on Hox signatures., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Long-term sensitization of the gill withdrawal reflex in Aplysia requires heterosynaptic, modulatory input that is mediated in part by the growth of new synaptic connections between sensory neurons and their follower cells (intrinsic mediating circuit). Whether modulatory interneurons (the extrinsic modulatory circuit) also display learning-related structural synaptic plasticity remains unknown. To test this idea, we added a bona fide serotonergic modulatory neuron, the metacerebral cell (MCC), to sensory-motor neuron co-cultures and examined the modulating presynaptic varicosities of MCCs before and after repeated pulses of serotonin (5-HT) that induced long-term facilitation (LTF). We observed robust growth of new serotonergic varicosities that were positive for serotonin and capable of synaptic recycling. Our findings demonstrate that, in addition to structural changes in the intrinsic mediating circuit, there are also significant learning-related structural changes in the extrinsic modulating circuit, and these changes might provide a cellular mechanism for savings and for spread of memory., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Proprioceptive sensory axons in the spinal cord form selective connections with motor neuron partners, but the strategies that confer such selectivity remain uncertain. We show that muscle-specific sensory axons project to motor neurons along topographically organized angular trajectories and that motor pools exhibit diverse dendritic arbors. On the basis of spatial constraints on axo-dendritic interactions, we propose positional strategies that can account for sensory-motor connectivity and synaptic organization. These strategies rely on two patterning principles. First, the degree of axo-dendritic overlap reduces the number of potential post-synaptic partners. Second, a close correlation between the small angle of axo-dendritic approach and the formation of synaptic clusters imposes specificity of connections when sensory axons intersect multiple motor pools with overlapping dendritic arbors. Our study identifies positional strategies with prominent roles in the organization of spinal sensory-motor circuits., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Activation of Gα q -coupled receptors by inflammatory mediators inhibits cold-sensing TRPM8 channels, aggravating pain and inflammation. Both Gα q and the downstream hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP 2 ) inhibit TRPM8. Here, I demonstrate that direct Gα q gating is essential for both the basal cold sensitivity of TRPM8 and TRPM8 inhibition elicited by bradykinin in sensory neurons. The action of Gα q depends on binding to three arginine residues in the N terminus of TRPM8. Neutralization of these residues markedly increased sensitivity of the channel to agonist and membrane voltage and completely abolished TRPM8 inhibition by both Gα q and bradykinin while sparing the channel sensitivity to PIP 2 . Interestingly, the bradykinin receptor B2R also binds to TRPM8, rendering TRPM8 insensitive to PIP 2 depletion. Furthermore, TRPM8-Gα q binding impaired Gα q coupling and signaling to PLCβ-PIP 2 . The crosstalk in the TRPM8-Gα q -B2R complex thus determines Gα q gating rather than PIP 2 as a sole means of TRPM8 inhibition by bradykinin., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Coordinated motor behaviors depend on feedback communication between peripheral sensory systems and central circuits in the brain and spinal cord. Relay of muscle- and tendon-derived sensory information to the CNS is facilitated by functionally and anatomically diverse groups of spinocerebellar tract neurons (SCTNs), but the molecular logic by which SCTN diversity and connectivity is achieved is poorly understood. We used single-cell RNA sequencing and genetic manipulations to define the mechanisms governing the molecular profile and organization of SCTN subtypes. We found that SCTNs relaying proprioceptive sensory information from limb and axial muscles are generated through segmentally restricted actions of specific Hox genes. Loss of Hox function disrupts SCTN-subtype-specific transcriptional programs, leading to defects in the connections between proprioceptive sensory neurons, SCTNs, and the cerebellum. These results indicate that Hox-dependent genetic programs play essential roles in the assembly of neural circuits necessary for communication between the brain and spinal cord., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Thermosensation is critical for avoiding thermal extremes and regulating body temperature. While thermosensors activated by noxious temperatures respond to hot or cold, many innocuous thermosensors exhibit robust baseline activity and lack discrete temperature thresholds, suggesting they are not simply warm and cool detectors. Here, we investigate how the aristal Cold Cells encode innocuous temperatures in Drosophila. We find they are not cold sensors but cooling-activated and warming-inhibited phasic thermosensors that operate similarly at warm and cool temperatures; we propose renaming them "Cooling Cells." Unexpectedly, Cooling Cell thermosensing does not require the previously reported Brivido Transient Receptor Potential (TRP) channels. Instead, three Ionotropic Receptors (IRs), IR21a, IR25a, and IR93a, specify both the unique structure of Cooling Cell cilia endings and their thermosensitivity. Behaviorally, Cooling Cells promote both warm and cool avoidance. These findings reveal a morphogenetic role for IRs and demonstrate the central role of phasic thermosensing in innocuous thermosensation. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Obesity is associated with many complications, including type 2 diabetes and painful neuropathy. There is no cure or prevention for obesity-induced pain, and the neurobiology underlying the onset of the disease is still obscure. In this study, we observe that western diet (WD)-fed mice developed early allodynia with an increase of ER stress markers in the sensory neurons of the dorsal root ganglia (DRG). Using cell-specific approaches, we demonstrate that neuronal liver X receptor (LXR) activation delays ER stress and allodynia in WD-fed mice. Our findings suggest that lipid-binding nuclear receptors expressed in the sensory neurons of the DRG play a role in the onset of obesity-induced hypersensitivity. The LXR and lipid-sensor pathways represent a research avenue to identify targets to prevent debilitating complications affecting the peripheral nerve system in obesity., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dendrite morphogenesis is a highly regulated process that gives rise to stereotyped receptive fields, which are required for proper neuronal connectivity and function. Specific classes of neurons, including Drosophila class IV dendritic arborization (C4da) neurons, also feature complete space-filling growth of dendrites. In this system, we have identified the substrate-derived TGF-β ligand maverick (mav) as a developmental signal promoting space-filling growth through the neuronal Ret receptor. Both are necessary for radial spreading of C4da neuron dendrites, and Ret is required for neuronal uptake of Mav. Moreover, local changes in Mav levels result in directed dendritic growth toward regions with higher ligand availability. Our results suggest that Mav acts as a substrate-derived secreted signal promoting dendrite growth within not-yet-covered areas of the receptive field to ensure space-filling dendritic growth., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cellular replacement therapies for neurological conditions use human embryonic stem cell (hESC)- or induced pluripotent stem cell (hiPSC)-derived neurons to replace damaged or diseased populations of neurons. For the spinal cord, significant progress has been made generating the in-vitro-derived motor neurons required to restore coordinated movement. However, there is as yet no protocol to generate in-vitro-derived sensory interneurons (INs), which permit perception of the environment. Here, we report on the development of a directed differentiation protocol to derive sensory INs for both hESCs and hiPSCs. Two developmentally relevant factors, retinoic acid in combination with bone morphogenetic protein 4, can be used to generate three classes of sensory INs: the proprioceptive dI1s, the dI2s, and mechanosensory dI3s. Critical to this protocol is the competence state of the neural progenitors, which changes over time. This protocol will facilitate developing cellular replacement therapies to reestablish sensory connections in injured patients., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Disruptions in lipid homeostasis have been observed in many neurodevelopmental disorders that are associated with dendrite morphogenesis defects. However, the molecular mechanisms of how lipid homeostasis affects dendrite morphogenesis are unclear. We find that easily shocked (eas), which encodes a kinase with a critical role in phospholipid phosphatidylethanolamine (PE) synthesis, and two other enzymes in this synthesis pathway are required cell autonomously in sensory neurons for dendrite growth and stability. Furthermore, we show that the level of Sterol Regulatory Element-Binding Protein (SREBP) activity is important for dendrite development. SREBP activity increases in eas mutants, and decreasing the level of SREBP and its transcriptional targets in eas mutants largely suppresses the dendrite growth defects. Furthermore, reducing Ca 2+ influx in neurons of eas mutants ameliorates the dendrite morphogenesis defects. Our study uncovers a role for EAS kinase and reveals the in vivo function of phospholipid homeostasis in dendrite morphogenesis., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

In senses as diverse as vision, hearing, touch, and the electrosense, sensory neurons receive bottom-up input from the environment, as well as top-down input from feedback loops involving higher brain regions [1-4]. Through connectivity with local inhibitory interneurons, these feedback loops can exert both positive and negative control over fundamental aspects of neural coding, including bursting [5, 6] and synchronous population activity [7, 8]. Here we show that a prominent midbrain feedback loop synthesizes a neural code for motion reversal in the hindbrain electrosensory ON- and OFF-type pyramidal cells. This top-down mechanism generates an accurate bidirectional encoding of object position, despite the inability of the electrosensory afferents to generate a consistent bottom-up representation [9, 10]. The net positive activity of this midbrain feedback is additionally regulated through a hindbrain feedback loop, which reduces stimulus-induced bursting and also dampens the ON and OFF cell responses to interfering sensory input [11]. We demonstrate that synthesis of motion representations and cancellation of distracting signals are mediated simultaneously by feedback, satisfying an accepted definition of spatial attention [12]. The balance of excitatory and inhibitory feedback establishes a "focal" distance for optimized neural coding, whose connection to a classic motion-tracking behavior provides new insight into the computational roles of feedback and active dendrites in spatial localization [13, 14]., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In contrast to our knowledge of mechanisms governing circuit formation, our understanding of how neural circuits are maintained is limited. Here, we show that Dicer, an RNaseIII protein required for processing microRNAs (miRNAs), is essential for maintenance of the spinal monosynaptic stretch reflex circuit in which group Ia proprioceptive sensory neurons form direct connections with motor neurons. In postnatal mice lacking Dicer in proprioceptor sensory neurons, there are no obvious defects in specificity or formation of monosynaptic sensory-motor connections. However, these circuits degrade through synapse loss and retraction of proprioceptive axonal projections from the ventral spinal cord. Peripheral terminals are also impaired without retracting from muscle targets. Interestingly, despite these central and peripheral axonal defects, proprioceptive neurons survive in the absence of Dicer-processed miRNAs. These findings reveal that Dicer, through its production of mature miRNAs, plays a key role in the maintenance of monosynaptic sensory-motor circuits., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Sensory neurons are often tuned to particular stimulus features, but their responses to repeated presentation of the same stimulus can vary over subsequent trials. This presents a problem for understanding the functioning of the brain, because downstream neuronal populations ought to construct accurate stimulus representations, even upon singular exposure. To study how trial-by-trial fluctuations (i.e., noise) in activity influence cortical representations of sensory input, we performed chronic calcium imaging of GCaMP6-expressing populations in mouse V1. We observed that high-dimensional response correlations, i.e., dependencies in activation strength among multiple neurons, can be used to predict single-trial, single-neuron noise. These multidimensional correlations are structured such that variability in the response of single neurons is relatively harmless to population representations of visual stimuli. We propose that multidimensional coding may represent a canonical principle of cortical circuits, explaining why the apparent noisiness of neuronal responses is compatible with accurate neural representations of stimulus features., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sensory neurons are an animal's gateway to the world, and their receptive endings, the sites of sensory signal transduction, are often associated with glia. Although glia are known to promote sensory-neuron functions, the molecular bases of these interactions are poorly explored. Here, we describe a post-developmental glial role for the PROS-1/Prospero/PROX1 homeodomain protein in sensory-neuron function in C. elegans. Using glia expression profiling, we demonstrate that, unlike previously characterized cell fate roles, PROS-1 functions post-embryonically to control sense-organ glia-specific secretome expression. PROS-1 functions cell autonomously to regulate glial secretion and membrane structure, and non-cell autonomously to control the shape and function of the receptive endings of sensory neurons. Known glial genes controlling sensory-neuron function are PROS-1 targets, and we identify additional PROS-1-dependent genes required for neuron attributes. Drosophila Prospero and vertebrate PROX1 are expressed in post-mitotic sense-organ glia and astrocytes, suggesting conserved roles for this class of transcription factors., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Precise patterning of dendritic arbors is critical for the wiring and function of neural circuits. Dendrite-extracellular matrix (ECM) adhesion ensures that the dendrites of Drosophila dendritic arborization (da) sensory neurons are properly restricted in a 2D space, and thereby facilitates contact-mediated dendritic self-avoidance and tiling. However, the mechanisms regulating dendrite-ECM adhesion in vivo are poorly understood. Here, we show that mutations in the semaphorin ligand sema-2b lead to a dramatic increase in self-crossing of dendrites due to defects in dendrite-ECM adhesion, resulting in a failure to confine dendrites to a 2D plane. Furthermore, we find that Sema-2b is secreted from the epidermis and signals through the Plexin B receptor in neighboring neurons. Importantly, we find that Sema-2b/PlexB genetically and physically interacts with TORC2 complex, Tricornered (Trc) kinase, and integrins. These results reveal a novel role for semaphorins in dendrite patterning and illustrate how epidermal-derived cues regulate neural circuit assembly., (Copyright © 2016 Elsevier Inc. All rights reserved.)

While isolated motor actions can be correlated with activities of neuronal networks, an unresolved problem is how the brain assembles these activities into organized behaviors like action sequences. Using brain-wide calcium imaging in Caenorhabditis elegans, we show that a large proportion of neurons across the brain share information by engaging in coordinated, dynamical network activity. This brain state evolves on a cycle, each segment of which recruits the activities of different neuronal sub-populations and can be explicitly mapped, on a single trial basis, to the animals' major motor commands. This organization defines the assembly of motor commands into a string of run-and-turn action sequence cycles, including decisions between alternative behaviors. These dynamics serve as a robust scaffold for action selection in response to sensory input. This study shows that the coordination of neuronal activity patterns into global brain dynamics underlies the high-level organization of behavior., (Copyright © 2015 Elsevier Inc. All rights reserved.)

While neurogenic stem cells have been identified in rodent and human skin, their manipulation and further characterization are hampered by a lack of specific markers. Here, we perform genetic tracing of the progeny of boundary cap (BC) cells, a neural-crest-derived cell population localized at peripheral nerve entry/exit points. We show that BC derivatives migrate along peripheral nerves to reach the skin, where they give rise to terminal glia associated with dermal nerve endings. Dermal BC derivatives also include cells that self-renew in sphere culture and have broad in vitro differentiation potential. Upon transplantation into adult mouse dorsal root ganglia, skin BC derivatives efficiently differentiate into various types of mature sensory neurons. Together, this work establishes the embryonic origin, pathway of migration, and in vivo neurogenic potential of a major component of skin stem-like cells. It provides genetic tools to study and manipulate this population of high interest for medical applications., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Breathing is essential for survival and under precise neural control. The vagus nerve is a major conduit between lung and brain required for normal respiration. Here, we identify two populations of mouse vagus nerve afferents (P2ry1, Npy2r), each a few hundred neurons, that exert powerful and opposing effects on breathing. Genetically guided anatomical mapping revealed that these neurons densely innervate the lung and send long-range projections to different brainstem targets. Npy2r neurons are largely slow-conducting C fibers, while P2ry1 neurons are largely fast-conducting A fibers that contact pulmonary endocrine cells (neuroepithelial bodies). Optogenetic stimulation of P2ry1 neurons acutely silences respiration, trapping animals in exhalation, while stimulating Npy2r neurons causes rapid, shallow breathing. Activating P2ry1 neurons did not impact heart rate or gastric pressure, other autonomic functions under vagal control. Thus, the vagus nerve contains intermingled sensory neurons constituting genetically definable labeled lines with different anatomical connections and physiological roles., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Precise regulation of microtubule (MT) dynamics is increasingly recognized as a critical determinant of axon regeneration. In contrast to developing neurons, mature axons exhibit noncentrosomal microtubule nucleation. The factors regulating noncentrosomal MT architecture in axon regeneration remain poorly understood. We report that PTRN-1, the C. elegans member of the Patronin/Nezha/calmodulin- and spectrin-associated protein (CAMSAP) family of microtubule minus-end-binding proteins, is critical for efficient axon regeneration in vivo. ptrn-1-null mutants display generally normal developmental axon outgrowth but significantly impaired regenerative regrowth after laser axotomy. Unexpectedly, mature axons in ptrn-1 mutants display elevated numbers of dynamic axonal MTs before and after injury, suggesting that PTRN-1 inhibits MT dynamics. The CKK domain of PTRN-1 is necessary and sufficient for its functions in axon regeneration and MT dynamics and appears to stabilize MTs independent of minus-end localization. Whereas in developing neurons, PTRN-1 inhibits activity of the DLK-1 mitogen-activated protein kinase (MAPK) cascade, we find that, in regeneration, PTRN-1 and DLK-1 function together to promote axonal regrowth., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Planarians can regenerate any missing body part in a process requiring dividing cells called neoblasts. Historically, neoblasts have largely been considered a homogeneous stem cell population. Most studies, however, analyzed neoblasts at the population rather than the single-cell level, leaving the degree of heterogeneity in this population unresolved. We combined RNA sequencing of neoblasts from wounded planarians with expression screening and identified 33 transcription factors transcribed in specific differentiated cells and in small fractions of neoblasts during regeneration. Many neoblast subsets expressing distinct tissue-associated transcription factors were present, suggesting candidate specification into many lineages. Consistent with this possibility, klf, pax3/7, and FoxA were required for the differentiation of cintillo-expressing sensory neurons, dopamine-β-hydroxylase-expressing neurons, and the pharynx, respectively. Together, these results suggest that specification of cell fate for most-to-all regenerative lineages occurs within neoblasts, with regenerative cells of blastemas being generated from a highly heterogeneous collection of lineage-specified neoblasts., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell-derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.

Mating induces pronounced changes in female reproductive behavior, typically including a dramatic reduction in sexual receptivity. In Drosophila, postmating behavioral changes are triggered by sex peptide (SP), a male seminal fluid peptide that acts via a receptor (SPR) expressed in sensory neurons (SPSNs) of the female reproductive tract. Here, we identify second-order neurons that mediate the behavioral changes induced by SP. These SAG neurons receive synaptic input from SPSNs in the abdominal ganglion and project to the dorsal protocerebrum. Silencing SAG neurons renders virgin females unreceptive, whereas activating them increases the receptivity of females that have already mated. Physiological experiments demonstrate that SP downregulates the excitability of the SPSNs, and hence their input onto SAG neurons. These data thus provide a physiological correlate of mating status in the female central nervous system and a key entry point into the brain circuits that control sexual receptivity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Climbing fibers (CFs) are thought to contribute to cerebellar plasticity and learning by triggering a large influx of dendritic calcium in the postsynaptic Purkinje cell (PC) to signal the occurrence of an unexpected sensory event. However, CFs fire about once per second whether or not an event occurs, raising the question of how sensory-driven signals might be distinguished from a background of ongoing spontaneous activity. Here, we report that in PC dendrites of awake mice, CF-triggered calcium signals are enhanced when the trigger is a sensory event. In addition, we show that a large fraction of the total enhancement in each PC dendrite can be accounted for by an additional boost of calcium provided by sensory activation of a non-CF input. We suggest that sensory stimulation may modulate dendritic voltage and calcium concentration in PCs to increase the strength of plasticity signals during cerebellar learning., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stem cells (MSCs) are typically defined by their in vitro characteristics, and as a consequence the in vivo identity of MSCs and their niches are poorly understood. To address this issue, we used lineage tracing in a mouse incisor model and identified the neurovascular bundle (NVB) as an MSC niche. We found that NVB sensory nerves secrete Shh protein, which activates Gli1 expression in periarterial cells that contribute to all mesenchymal derivatives. These periarterial cells do not express classical MSC markers used to define MSCs in vitro. In contrast, NG2(+) pericytes represent an MSC subpopulation derived from Gli1+ cells; they express classical MSC markers and contribute little to homeostasis but are actively involved in injury repair. Likewise, incisor Gli1(+) cells, but not NG2(+) cells, exhibit typical MSC characteristics in vitro. Collectively, we demonstrate that MSCs originate from periarterial cells and are regulated by Shh secretion from an NVB., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In this issue of Neuron, Han et al. (2014) develop powerful methods to visualize phagocytosis of Drosophila peripheral sensory neuron dendrites. Remarkably, epidermal cells rather than professional phagocytes are the primary mediators of debris clearance, using both familiar and new molecular players., (Copyright © 2014 Elsevier Inc. All rights reserved.)

During developmental remodeling, neurites destined for pruning often degenerate on-site. Physical injury also induces degeneration of neurites distal to the injury site. Prompt clearance of degenerating neurites is important for maintaining tissue homeostasis and preventing inflammatory responses. Here we show that in both dendrite pruning and dendrite injury of Drosophila sensory neurons, epidermal cells rather than hemocytes are the primary phagocytes in clearing degenerating dendrites. Epidermal cells act via Draper-mediated recognition to facilitate dendrite degeneration and to engulf and degrade degenerating dendrites. Using multiple dendritic membrane markers to trace phagocytosis, we show that two members of the CD36 family, croquemort (crq) and debris buster (dsb), act at distinct stages of phagosome maturation for dendrite clearance. Our finding reveals the physiological importance of coordination between neurons and their surrounding epidermis, for both dendrite fragmentation and clearance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Inhibitory microcircuits are wired with a precision that underlies their complex regulatory roles in neural information processing. In the spinal cord, one specialized class of GABAergic interneurons (GABApre) mediates presynaptic inhibitory control of sensory-motor synapses. The synaptic targeting of these GABAergic neurons exhibits an absolute dependence on proprioceptive sensory terminals, yet the molecular underpinnings of this specialized axoaxonic organization remain unclear. Here, we show that sensory expression of an NB2 (Contactin5)/Caspr4 coreceptor complex, together with spinal interneuron expression of NrCAM/CHL1, directs the high-density accumulation of GABAergic boutons on sensory terminals. Moreover, genetic elimination of NB2 results in a disproportionate stripping of inhibitory boutons from high-density GABApre-sensory synapses, suggesting that the preterminal axons of GABApre neurons compete for access to individual sensory terminals. Our findings define a recognition complex that contributes to the assembly and organization of a specialized GABAergic microcircuit., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The branching of axons is a fundamental aspect of nervous system development and neuroplasticity. We report that branching of sensory axons in the presence of nerve growth factor (NGF) occurs at sites populated by stalled mitochondria. Translational machinery targets to presumptive branching sites, followed by recruitment of mitochondria to these sites. The mitochondria promote branching through ATP generation and the determination of localized hot spots of active axonal mRNA translation, which contribute to actin-dependent aspects of branching. In contrast, mitochondria do not have a role in the regulation of the microtubule cytoskeleton during NGF-induced branching. Collectively, these observations indicate that sensory axons exhibit multiple potential sites of translation, defined by presence of translational machinery, but active translation occurs following the stalling and respiration of mitochondria at these potential sites of translation. This study reveals a local role for axonal mitochondria in the regulation of the actin cytoskeleton and axonal mRNA translation underlying branching., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Mammalian pain-related sensory neurons are derived from TrkA lineage neurons located in the dorsal root ganglion. These neurons project to peripheral targets throughout the body, which can be divided into superficial and deep tissues. Here, we find that the transcription factor Runx1 is required for the development of many epidermis-projecting TrkA lineage neurons. Accordingly, knockout of Runx1 leads to the selective loss of sensory innervation to the epidermis, whereas deep tissue innervation and two types of deep tissue pain are unaffected. Within these cutaneous neurons, Runx1 suppresses a large molecular program normally associated with sensory neurons that innervate deep tissues, such as muscle and visceral organs. Ectopic expression of Runx1 in these deep sensory neurons causes a loss of this molecular program and marked deficits in deep tissue pain. Thus, this study provides insight into a genetic program controlling the segregation of cutaneous versus deep tissue pain pathways., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

In mammalian spinal cord, group Ia proprioceptive afferents form selective monosynaptic connections with a select group of motor pool targets. The extent to which sensory recognition of motor neurons contributes to the selectivity of sensory-motor connections remains unclear. We show here that proprioceptive sensory afferents that express PlexinD1 avoid forming monosynaptic connections with neurons in Sema3E(+) motor pools yet are able to form direct connections with neurons in Sema3E(off) motor pools. Anatomical and electrophysiological analysis of mice in which Sema3E-PlexinD1 signaling has been deregulated or inactivated genetically reveals that repellent signaling underlies aspects of the specificity of monosynaptic sensory-motor connectivity in these reflex arcs. A semaphorin-based system of motor neuron recognition and repulsion therefore contributes to the formation of specific sensory-motor connections in mammalian spinal cord., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Expression of the Down syndrome cell-adhesion molecule (Dscam) is increased in the brains of patients with several neurological disorders. Although Dscam is critically involved in many aspects of neuronal development, little is known about either the mechanism that regulates its expression or the functional consequences of dysregulated Dscam expression. Here, we show that Dscam expression levels serve as an instructive code for the size control of presynaptic arbor. Two convergent pathways, involving dual leucine zipper kinase (DLK) and fragile X mental retardation protein (FMRP), control Dscam expression through protein translation. Defects in this regulation of Dscam translation lead to exuberant presynaptic arbor growth in Drosophila somatosensory neurons. Our findings uncover a function of Dscam in presynaptic size control and provide insights into how dysregulated Dscam may contribute to the pathogenesis of neurological disorders., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Neurotrophins control the development and adult plasticity of the vertebrate nervous system. Failure to identify invertebrate neurotrophin orthologs, however, has precluded studies in invertebrate models, limiting our understanding of fundamental aspects of neurotrophin biology and function. We identified a neurotrophin (ApNT) and Trk receptor (ApTrk) in the mollusk Aplysia and found that they play a central role in learning-related synaptic plasticity. Blocking ApTrk signaling impairs long-term facilitation, whereas augmenting ApNT expression enhances it and induces the growth of new synaptic varicosities at the monosynaptic connection between sensory and motor neurons of the gill-withdrawal reflex. Unlike vertebrate neurotrophins, ApNT has multiple coding exons and exerts distinct synaptic effects through differentially processed and secreted splice isoforms. Our findings demonstrate the existence of bona fide neurotrophin signaling in invertebrates and reveal a posttranscriptional mechanism that regulates neurotrophin processing and the release of proneurotrophins and mature neurotrophins that differentially modulate synaptic plasticity., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

When an animal is reminded of a prior experience and shortly afterward treated with a protein synthesis inhibitor, the consolidated memory for the experience can be disrupted; by contrast, protein synthesis inhibition without prior reminding commonly does not disrupt long-term memory [1-3]. Such results imply that the reminding triggers reconsolidation of the memory. Here, we asked whether the behavioral and synaptic changes associated with the memory for long-term sensitization (LTS) of the siphon-withdrawal reflex in the marine snail Aplysia californica [4, 5] could undergo reconsolidation. In support of this idea, we found that when sensitized animals were given abbreviated reminder sensitization training 48-96 hr after the original sensitization training, followed by treatment with the protein synthesis inhibitor anisomycin, LTS was disrupted. We also found that long-term (≥ 24 hr) facilitation (LTF) [6], which can be induced in the monosynaptic connection between Aplysia sensory and motor neurons in dissociated cell culture by multiple spaced pulses of the endogenous facilitatory transmitter serotonin (5-HT) [7, 8], could be eliminated by treating the synapses with one reminder pulse of 5-HT, followed by anisomycin, at 48 hr after the original training. Our results provide a simple model system for understanding the synaptic basis of reconsolidation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Size homeostasis is fundamental in cell biology, but it is not clear how large cells such as neurons can assess their own size or length. We examined a role for molecular motors in intracellular length sensing.Computational simulations suggest that spatial information can be encoded by the frequency of an oscillating retrograde signal arising from a composite negative feedback loop between bidirectional motor-dependent signals. The model predicts that decreasing either or both anterograde or retrograde signals should increase cell length, and this prediction was confirmed upon application of siRNAs for specific kinesin and/or dynein heavy chains in adult sensory neurons. Heterozygous dynein heavy chain 1 mutant sensory neurons also exhibited increased lengths both in vitro and during embryonic development.Moreover, similar length increases were observed in mouse embryonic fibroblasts upon partial downregulation of dynein heavy chain 1.Thus, molecular motors critically influence cell length sensing and growth control., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

The proliferation/differentiation balance of stem and progenitor cell populations must respond to the physiological needs of the organism [1, 2]. Mechanisms underlying this plasticity are not well understood. The C. elegans germline provides a tractable system to study the influence of the environment on progenitor cells (stem cells and their proliferative progeny). Germline progenitors accumulate during larval stages to form an adult pool from which gametes are produced. Notch pathway signaling from the distal tip cell (DTC) niche to the germline maintains the progenitor pool [3-5], and the larval germline cell cycle is boosted by insulin/IGF-like receptor signaling [6]. Here we show that, independent of its role in the dauer decision, TGF-β regulates the balance of proliferation versus differentiation in the C. elegans germline in response to sensory cues that report population density and food abundance. Ciliated ASI sensory neurons are required for TGF-β-mediated expansion of the larval germline progenitor pool, and the TGF-β receptor pathway acts in the germline stem cell niche. TGF-β signaling thereby couples germline development to the quality of the environment, providing a novel cellular and molecular mechanism linking sensory experience of the environment to reproduction., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Dendrites achieve characteristic spacing patterns during development to ensure appropriate coverage of territories. Mechanisms of dendrite positioning via repulsive dendrite-dendrite interactions are beginning to be elucidated, but the control, and importance, of dendrite positioning relative to their substrate is poorly understood. We found that dendritic branches of Drosophila dendritic arborization sensory neurons can be positioned either at the basal surface of epidermal cells, or enclosed within epidermal invaginations. We show that integrins control dendrite positioning on or within the epidermis in a cell autonomous manner by promoting dendritic retention on the basal surface. Loss of integrin function in neurons resulted in excessive self-crossing and dendrite maintenance defects, the former indicating a role for substrate interactions in self-avoidance. In contrast to a contact-mediated mechanism, we find that integrins prevent crossings that are noncontacting between dendrites in different three-dimensional positions, revealing a requirement for combined dendrite-dendrite and dendrite-substrate interactions in self-avoidance., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Dendrites of the same neuron usually avoid each other. Some neurons also repel similar neurons through dendrite-dendrite interaction to tile the receptive field. Nonoverlapping coverage based on such contact-dependent repulsion requires dendrites to compete for limited space. Here we show that Drosophila class IV dendritic arborization (da) neurons, which tile the larval body wall, grow their dendrites mainly in a 2D space on the extracellular matrix (ECM) secreted by the epidermis. Removing neuronal integrins or blocking epidermal laminin production causes dendrites to grow into the epidermis, suggesting that integrin-laminin interaction attaches dendrites to the ECM. We further show that some of the previously identified tiling mutants fail to confine dendrites in a 2D plane. Expansion of these mutant dendrites in three dimensions results in overlap of dendritic fields. Moreover, overexpression of integrins in these mutant neurons effectively reduces dendritic crossing and restores tiling, revealing an additional mechanism for tiling., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Posttranslational modifications (PTMs) such as acetylation, detyrosination, and polyglutamylation have long been considered markers of stable microtubules and have recently been proposed to guide molecular motors to specific subcellular destinations. Microtubules can be deglutamylated by the cytosolic carboxypeptidase CCP1. Loss of CCP1 in mice causes cerebellar Purkinje cell degeneration. Cilia, which are conserved organelles that play important diverse roles in animal development and sensation, contain axonemes comprising microtubules that are especially prone to PTMs., Results: Here, we report that a CCP1 homolog, CCPP-1, regulates the ciliary localization of the kinesin-3 KLP-6 and the polycystin PKD-2 in male-specific sensory neurons in C. elegans. In male-specific CEM (cephalic sensilla, male) cilia, ccpp-1 also controls the velocity of the kinesin-2 OSM-3/KIF17 without affecting the transport of kinesin-II cargo. In the core ciliated nervous system of both males and hermaphrodites, loss of ccpp-1 causes progressive defects in amphid and phasmid sensory cilia, suggesting that CCPP-1 activity is required for ciliary maintenance but not ciliogenesis. Affected cilia exhibit defective B-tubules. Loss of TTLL-4, a polyglutamylating enzyme of the tubulin tyrosine ligase-like family, suppresses progressive ciliary defects in ccpp-1 mutants., Conclusions: Our studies suggest that CCPP-1 acts as a tubulin deglutamylase that regulates the localization and velocity of kinesin motors and the structural integrity of microtubules in sensory cilia of a multicellular, living animal. We propose that the neuronal degeneration caused by loss of CCP1 in mammals may represent a novel ciliopathy in which cilia are formed but not maintained, depriving the cell of cilia-based signal transduction., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Pruning that selectively removes unnecessary axons/dendrites is crucial for sculpting neural circuits during development. During Drosophila metamorphosis, dendritic arborization sensory neurons, ddaCs, selectively prune their larval dendrites in response to the steroid hormone ecdysone. However, it is unknown whether epigenetic factors are involved in dendrite pruning. Here, we analyzed 81 epigenetic factors, from which a Brahma (Brm)-containing chromatin remodeler and a histone acetyltransferase CREB-binding protein (CBP) were identified for their critical roles in initiating dendrite pruning. Brm and CBP specifically activate a key ecdysone response gene, sox14, but not EcR-B1. Furthermore, the HAT activity of CBP is important for sox14 expression and dendrite pruning. EcR-B1 associates with CBP in the presence of ecdysone, which is facilitated by Brm, resulting in local enrichment of an active chromatin mark H3K27Ac at the sox14 locus. Thus, specific intrinsic epigenetic factors cooperate with steroid hormones to activate selective transcriptional programs, thereby initiating neuronal remodeling., (Copyright © 2011 Elsevier Inc. All rights reserved.)

It is a long-standing question how developing motor and sensory neuron projections cooperatively form a common principal grid of peripheral nerve pathways relaying behavioral outputs and somatosensory inputs. Here, we explored this issue through targeted cell lineage and gene manipulation in mouse, combined with in vitro live axon imaging. In the absence of motor projections, dorsal (epaxial) and ventral (hypaxial) sensory projections form in a randomized manner, while removal of EphA3/4 receptor tyrosine kinases expressed by epaxial motor axons triggers selective failure to form epaxial sensory projections. EphA3/4 act non-cell-autonomously by inducing sensory axons to track along preformed epaxial motor projections. This involves cognate ephrin-A proteins on sensory axons but is independent from EphA3/4 signaling in motor axons proper. Assembly of peripheral nerve pathways thus involves motor axon subtype-specific signals that couple sensory projections to discrete motor pathways., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The transcription factor SOX2 is widely known to play a critical role in the central nervous system; however, its role in peripheral neurogenesis remains poorly understood. We recently developed an hESC-based model in which migratory cells undergo epithelial to mesenchymal transition (EMT) to acquire properties of neural crest (NC) cells. In this model, we found that migratory NC progenitors downregulate SOX2, but then start re-expressing SOX2 as they differentiate to form neurogenic dorsal root ganglion (DRG)-like clusters. SOX2 downregulation was sufficient to induce EMT and resulted in massive apoptosis when neuronal differentiation was induced. In vivo, downregulation of SOX2 in chick and mouse NC cells significantly reduced the numbers of neurons within DRG. We found that SOX2 binds directly to NGN1 and MASH1 promoters and is required for their expression. Our data suggest that SOX2 plays a key role for NGN1-dependent acquisition of neuronal fates in sensory ganglia., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Highly branched class IV multidendritic sensory neurons of the Drosophila larva function as polymodal nociceptors that are necessary for behavioral responses to noxious heat (>39 degrees C) or noxious mechanical (>30 mN) stimuli. However, the molecular mechanisms that allow these cells to detect both heat and force are unknown. Here, we report that the pickpocket (ppk) gene, which encodes a Degenerin/Epithelial Sodium Channel (DEG/ENaC) subunit, is required for mechanical nociception but not thermal nociception in these sensory cells. Larvae mutant for pickpocket show greatly reduced nociception behaviors in response to harsh mechanical stimuli. However, pickpocket mutants display normal behavioral responses to gentle touch. Tissue-specific knockdown of pickpocket in nociceptors phenocopies the mechanical nociception impairment without causing defects in thermal nociception behavior. Finally, optogenetically triggered nociception behavior is unaffected by pickpocket RNAi, which indicates that ppk is not generally required for the excitability of the nociceptors. Interestingly, DEG/ENaCs are known to play a critical role in detecting gentle touch stimuli in Caenorhabditis elegans and have also been implicated in some aspects of harsh touch sensation in mammals. Our results suggest that neurons that detect harsh touch in Drosophila utilize similar mechanosensory molecules.

Low-threshold mechanoreceptor neurons (LTMs) of the dorsal root ganglia (DRG) are essential for touch sensation. They form highly specialized terminations in the skin and display stereotyped projections in the spinal cord. Functionally defined LTMs depend on neurotrophin signaling for their postnatal survival and functioning, but how these neurons arise during development is unknown. Here, we show that specific types of LTMs can be identified shortly after DRG genesis by unique expression of the MafA transcription factor, the Ret receptor and coreceptor GFRalpha2, and find that their specification is Ngn2 dependent. In mice lacking Ret, these LTMs display early differentiation defects, as revealed by reduced MafA expression, and at later stages their central and peripheral projections are compromised. Moreover, in MafA mutants, a discrete subset of LTMs display altered expression of neurotrophic factor receptors. Our results provide evidence that genetic interactions involving Ret and MafA progressively promote the differentiation and diversification of LTMs., (2009 Elsevier Inc. All rights reserved.)