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Piezo proteins have been identified as mechanosensitive ion channels involved in mechanotransduction. Several ion channel dysfunctions may be associated with diseases (including deafness and pain); thus, studying them is critical to understand their role in mechanosensitive disorders and to establish new therapeutic strategies. The current study investigated for the first time the expression patterns of Piezo proteins in zebrafish octavolateralis mechanosensory organs. Piezo 1 and 2 were immunoreactive in the sensory epithelia of the lateral line system and the inner ear. Piezo 1 (28.7 ± 1.55 cells) and Piezo 2 (28.8 ± 3.31 cells) immunopositive neuromast cells were identified based on their ultrastructural features, and their overlapping immunoreactivity to the s100p specific marker (28.6 ± 1.62 cells), as sensory cells. These findings are in favor of Piezo proteins' potential role in sensory cell activation, while their expression on mantle cells reflects their implication in the maintenance and regeneration of the neuromast during cell turnover. In the inner ear, Piezo proteins' colocalization with BDNF introduces their potential implication in neuronal plasticity and regenerative events, typical of zebrafish mechanosensory epithelia. Assessing these proteins in zebrafish could open up new scenarios for the roles of these important ionic membrane channels, for example in treating impairments of sensory systems.

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In mammals, action potentials fired by rapidly adapting mechanosensitive afferents are known to reliably time lock to the cycles of a vibration. How and where along the ascending neuraxis is the peripheral afferent temporal code transformed into a rate code are currently not clear. Here, we probed the encoding of vibrotactile stimuli with electrophysiological recordings along major stages of the ascending somatosensory pathway in mice. We discovered the main transformation step was identified at the level of the thalamus, and parvalbumin-positive interneurons in thalamic reticular nucleus participate in sharpening frequency selectivity and in disrupting the precise spike timing. When frequency-specific microstimulation was applied within the brainstem, it generated frequency selectivity reminiscent of real vibration responses in the somatosensory cortex and could provide informative and robust signals for learning in behaving mice. Taken together, these findings could guide biomimetic stimulus strategies to activate specific nuclei along the ascending somatosensory pathway for neural prostheses.

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Behavioral plasticity allows animals to modulate their behavior based on experience and environmental conditions. Caenorhabditis elegans exhibits experience-dependent changes in its behavioral responses to various modalities of sensory cues, including odorants, salts, temperature, and mechanical stimulations. Most of these forms of behavioral plasticity, such as adaptation, habituation, associative learning, and imprinting, are shared with other animals. The C. elegans nervous system is considerably tractable for experimental studies-its function can be characterized and manipulated with molecular genetic methods, its activity can be visualized and analyzed with imaging approaches, and the connectivity of its relatively small number of neurons are well described. Therefore, C. elegans provides an opportunity to study molecular, neuronal, and circuit mechanisms underlying behavioral plasticity that are either conserved in other animals or unique to this species. These findings reveal insights into how the nervous system interacts with the environmental cues to generate behavioral changes with adaptive values.

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Pacinian corpuscle neurons are specialized low-threshold mechanoreceptors (LTMRs) that are tuned to detect high-frequency vibration (∼50-2,000 Hz); however, it is unclear how Pacinians and other LTMRs encode mechanical forces encountered during naturalistic behavior. Here, we developed methods to record LTMRs in awake, freely moving mice. We find that Pacinians, but not other LTMRs, encode subtle vibrations of surfaces encountered by the animal, including low-amplitude vibrations initiated over 2 m away. Strikingly, Pacinians are also highly active during a wide variety of natural behaviors, including walking, grooming, digging, and climbing. Pacinians in the hindlimb are sensitive enough to be activated by forelimb- or upper-body-dominant behaviors. Finally, we find that Pacinian LTMRs have diverse tuning and sensitivity. Our findings suggest a Pacinian population code for the representation of vibro-tactile features generated by self-initiated movements and low-amplitude environmental vibrations emanating from distant locations.

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During locomotion, most vertebrates-and invertebrates such as Drosophila melanogaster-are able to quickly adapt to terrain irregularities or avoid physical threats by integrating sensory information along with motor commands. Key to this adaptability are leg mechanosensory structures, which assist in motor coordination by transmitting external cues and proprioceptive information to motor centers in the central nervous system. Nevertheless, how different mechanosensory structures engage these locomotor centers remains poorly understood. Here, we tested the role of mechanosensory structures in movement initiation by optogenetically stimulating specific classes of leg sensory structures. We found that stimulation of leg mechanosensory bristles (MsBs) and the femoral chordotonal organ (ChO) is sufficient to initiate forward movement in immobile animals. While the stimulation of the ChO required brain centers to induce forward movement, unexpectedly, brief stimulation of leg MsBs triggered a fast response and sustained motor activity dependent only on the ventral nerve cord (VNC). Moreover, this leg-MsB-mediated movement lacked inter- and intra-leg coordination but preserved antagonistic muscle activity within joints. Finally, we show that leg-MsB activation mediates strong avoidance behavior away from the stimulus source, which is preserved even in the absence of a central brain. Overall, our data show that mechanosensory stimulation can elicit a fast motor response, independently of central brain commands, to evade potentially harmful stimuli. In addition, it sheds light on how specific sensory circuits modulate motor control, including initiation of movement, allowing a better understanding of how different levels of coordination are controlled by the VNC and central brain locomotor circuits.

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Mechanosensory neurons located across the body surface respond to tactile stimuli and elicit diverse behavioral responses, from relatively simple stimulus location-aimed movements to complex movement sequences. How mechanosensory neurons and their postsynaptic circuits influence such diverse behaviors remains unclear. We previously discovered that Drosophila perform a body location-prioritized grooming sequence when mechanosensory neurons at different locations on the head and body are simultaneously stimulated by dust (Hampel et al., 2017; Seeds et al., 2014). Here, we identify nearly all mechanosensory neurons on the Drosophila head that individually elicit aimed grooming of specific head locations, while collectively eliciting a whole head grooming sequence. Different tracing methods were used to reconstruct the projections of these neurons from different locations on the head to their distinct arborizations in the brain. This provides the first synaptic resolution somatotopic map of a head, and defines the parallel-projecting mechanosensory pathways that elicit head grooming.

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In electroreceptive jawed vertebrates, embryonic lateral line placodes give rise to electrosensory ampullary organs as well as mechanosensory neuromasts. Previous reports of shared gene expression suggest that conserved mechanisms underlie electroreceptor and mechanosensory hair cell development and that electroreceptors evolved as a transcriptionally related "sister cell type" to hair cells. We previously identified only one transcription factor gene, Neurod4, as ampullary organ-restricted in the developing lateral line system of a chondrostean ray-finned fish, the Mississippi paddlefish (Polyodon spathula). The other 16 transcription factor genes we previously validated in paddlefish were expressed in both ampullary organs and neuromasts. Here, we used our published lateral line organ-enriched gene-set (arising from differential bulk RNA-seq in late-larval paddlefish), together with a candidate gene approach, to identify 25 transcription factor genes expressed in the developing lateral line system of a more experimentally tractable chondrostean, the sterlet (Acipenser ruthenus, a small sturgeon), and/or that of paddlefish. Thirteen are expressed in both ampullary organs and neuromasts, consistent with conservation of molecular mechanisms. Seven are electrosensory-restricted on the head (Irx5, Irx3, Insm1, Sp5, Satb2, Mafa and Rorc), and five are the first-reported mechanosensory-restricted transcription factor genes (Foxg1, Sox8, Isl1, Hmx2 and Rorb). However, as previously reported, Sox8 is expressed in ampullary organs as well as neuromasts in a catshark (Scyliorhinus canicula), suggesting the existence of lineage-specific differences between cartilaginous and ray-finned fishes. Overall, our results support the hypothesis that ampullary organs and neuromasts develop via largely conserved transcriptional mechanisms, and identify multiple transcription factors potentially involved in the formation of electrosensory versus mechanosensory lateral line organs.

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The present work intends to provide a closer look at histamine in Drosophila. This choice is motivated firstly because Drosophila has proven over the years to be a very simple, but powerful, model organism abundantly assisting scientists in explaining not only normal functions, but also derangements that occur in higher organisms, not excluding humans. Secondly, because histamine has been demonstrated to be a pleiotropic master molecule in pharmacology and immunology, with increasingly recognized roles also in the nervous system. Indeed, it interacts with various neurotransmitters and controls functions such as learning, memory, circadian rhythm, satiety, energy balance, nociception, and motor circuits, not excluding several pathological conditions. In view of this, our review is focused on the knowledge that the use of Drosophila has added to the already vast histaminergic field. In particular, we have described histamine's actions on photoreceptors sustaining the visual system and synchronizing circadian rhythms, but also on temperature preference, courtship behavior, and mechanosensory transmission. In addition, we have highlighted the pathophysiological consequences of mutations on genes involved in histamine metabolism and signaling. By promoting critical discussion and further research, our aim is to emphasize and renew the importance of histaminergic research in biomedicine through the exploitation of Drosophila, hopefully extending the scientific debate to the academic, industry, and general public audiences.

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The chapter provides an overview of the applications of magnetic tweezers in living cells. It discusses the advantages and disadvantages of magnetic tweezers technology with a focus on individual magnetic tweezers configurations, such as electromagnetic tweezers. Solutions to the disadvantages identified are also outlined. The specific role of magnetic tweezers in the field of mechanobiology, such as mechanosensitivity, mechano-allostery and mechanotransduction are also emphasized. The specific usage of magnetic tweezers in mechanically probing cells via specific cell surface receptors, such as mechanosensitive channels is discussed and why mechanical probing has revealed the opening and closing of the channels. Finally, the future direction of magnetic tweezers is presented.

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The honeybee waggle dance has been widely studied as a communication system, yet we know little about how nestmates assimilate the information needed to navigate toward the signaled resource. They are required to detect the dancer's orientation relative to gravity and duration of the waggle phase and translate this into a flight vector with a direction relative to the sun1 and distance from the hive.2,3 Moreover, they appear capable of doing so from varied, dynamically changing positions around the dancer. Using high-speed, high-resolution video, we have uncovered a previously unremarked correlation between antennal position and the relative body axes of dancer and follower bees. Combined with new information about antennal inputs4,5 and spatial encoding in the insect central complex,6,7 we show how a neural circuit first proposed to underlie path integration could be adapted to decoding the dance and acquiring the signaled information as a flight vector that can be followed to the resource. This provides the first plausible account of how the bee brain could support the interpretation of its dance language.

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Mechanotransduction is the process by which a mechanical force, such as touch, is converted into an electrical signal. Transmembrane channel-like (TMC) proteins are an evolutionarily conserved family of membrane proteins whose function has been linked to a variety of mechanosensory processes, including hearing and balance sensation in vertebrates and locomotion in Drosophila. TMC1 and TMC2 are components of ion channel complexes, but the molecular features that tune these complexes to diverse mechanical stimuli are unknown. Caenorhabditis elegans express two TMC homologs, TMC-1 and TMC-2, both of which are the likely pore-forming subunits of mechanosensitive ion channels but differ in their expression pattern and functional role in the worm. Here, we present the single-particle cryo-electron microscopy structure of the native TMC-2 complex isolated from C. elegans. The complex is composed of two copies of the pore-forming TMC-2 subunit, the calcium and integrin binding protein CALM-1 and the transmembrane inner ear protein TMIE. Comparison of the TMC-2 complex to the recently published cryo-EM structure of the C. elegans TMC-1 complex highlights conserved protein-lipid interactions, as well as a π-helical structural motif in the pore-forming helices, that together suggest a mechanism for TMC-mediated mechanosensory transduction.

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Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples. Key features • Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET). • Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms. • The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.

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The torus semicircularis (TS) of teleosts is a key midbrain center of the lateral line and acoustic sensory systems. To characterize the TS in adult zebrafish, we studied their connections using the carbocyanine tracers applied to the TS and to other related nuclei and tracts. Two main TS nuclei, central and ventrolateral, were differentiable by their afferent connections. From central TS, (TSc) numerous toropetal cells were labeled bilaterally in several primary octaval nuclei (anterior, magnocellular, descending, and posterior octaval nuclei), in the secondary octaval nucleus, in the caudal octavolateralis nucleus, and in the perilemniscular region. In the midbrain, numerous toropetal cells were labeled in the contralateral TSc. In the diencephalon, toropetal cells labeled from the TSc were observed ipsilaterally in the medial prethalamic nucleus and the periventricular posterior tubercle nucleus. TSc toropetal neurons were also labeled bilaterally in the hypothalamic anterior tuberal nucleus (ATN) and ipsilaterally in the parvicellular preoptic nucleus but not in the telencephalon. Tracer application to the medial octavolateralis nucleus revealed contralateral projections to the ventrolateral TS (TSvl), whereas tracer application to the secondary octaval nucleus labeled fibers bilaterally in TSc and neurons in rostral TSc. The TSc sends ascending fibers to the ipsilateral lateral preglomerular region that, in turn, projects to the pallium. Application of DiI to the optic tectum labeled cells and fibers in the TSvl, whereas application of DiI to the ATN labeled cells and fibers in the TSc. These results reveal that the TSvl and TSc are mainly related with the mechanosensory lateral line and acoustic centers, respectively, and that they show different higher order connections.

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Insecticides have been widely used for the control of insect pests that have a significant impact on agriculture and human health. A better understanding of insecticide targets is needed for effective insecticide design and resistance management. Pymetrozine, afidopyropen and flonicamid are reported to target on proteins that located on insect chordotonal organs, resulting in the disruption of insect coordination and the inhibition of feeding. In this study, we systematically examined the susceptibility of six Drosophila melanogaster mutants (five transient receptor potential channels and one mechanoreceptor) to three commercially used insecticides, in order to identify the receptor subunits critical to the insect's response to insecticides. Our results showed that iav1, nan36aand wtrw1 mutants exhibited significantly reduced susceptibility to pymetrozine and afidopyropen, but not to flonicamid. The number of eggs produced by the three mutant females were significantly less than that of the w1118 strain. Meanwhile, the longevity of all male mutants and females of nan36a and wtrw1 mutants was significantly shorter than that of the w1118 strain as the control. However, we observed no gravitaxis defects in wtrw1 mutants and the anti-gravitaxis of wtrw1 mutants was abolished by pymetrozine. Behavioral assays using thermogenetic tools further confirmed the bioassay results and supported the idea that Nan as a TRPV subfamily member located in Drosophila chordotonal neurons, acting as a target of pymetrozine, which interferes with Drosophila and causes motor deficits with gravitaxis defects. Taken together, this study elucidates the interactions of pymetrozine and afidopyropen with TRPV channels, Nan and Iav, and TRPA channel, Wtrw. Our research provides another evidence that pymetrozine and afidopyropen might target on nan, iav and wtrw channels and provides insights into the development of sustainable pest management strategies.

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We describe a pericapillary organ in the rat forebrain and cerebellar cortex. It consists of a series of tripartite synapses with synaptic extensions enveloped by astrocytic endfeet that are linked to the capillary wall by synaptic extensions. Reciprocal specializations of the pericyte-capillary blood vessel (CBV) with such specialized synapses suggest a mechanoreceptor role. In Golgi-impregnated and 3D reconstructions of the cerebral cortex and thalamus, a series of TSs appear to be sequentially ordered in a common dendrite, paralleled by synaptic outgrowths termed golf club synaptic extensions (GCE) opposed to a longitudinal crest (LC) from the capillary basal lamina (BL). Our results show that, in the cerebellar cortex, afferent fibers and interneurons display microanatomical structures that strongly suggest an interaction with the capillary wall. Afferent mossy fiber (MF) rosettes and ascending granule cell axons and their dendrites define the pericapillary passage interactions that are entangled by endfeet. The presence of mRNA of the mechanosensitive channel Piezo1 in the MF rosettes, together with the surrounding end-feet and the capillary wall form mechanosensory units. The ubiquity of such units to modulate synaptic transmission is also supported by Piezo1 mRNA expressing pyramidal isocortical and thalamic neurons. This scenario suggests that ascending impulses to the cerebellar and cortical targets are presynaptically modulated by the reciprocal interaction with the mechanosensory pericapillary organ that ultimately modulates the vasomotor response.

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Sexual stimulation triggers changes in female physiology and behavior, including sexual satiety and preparing the uterus for pregnancy. Serotonin (5-HT) is an important regulator of reproductive physiology and sexual receptivity, but the relationship between sexual stimulation and 5-HT neural activity in females is poorly understood. Here, we investigated dorsal raphe 5-HT neural activity in female mice during sexual behavior. We found that 5-HT neural activity in mating females peaked specifically upon male ejaculation and remained elevated above baseline until disengagement. Artificial intravaginal mechanical stimulation was sufficient to elicit increased 5-HT neural activity but the delivery of ejaculatory fluids was not. Distal penis expansion ("penile cupping") at ejaculation and forceful expulsion of ejaculatory fluid each provided sufficient mechanical stimulation to elicit 5-HT neuron activation. Our study identifies a female ejaculation-specific signal in a major neuromodulatory system and shows that intravaginal mechanosensory stimulation is necessary and sufficient to drive this signal.

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Undulatory locomotion relies on the propagation of a wave of excitation in the spinal cord leading to consequential activation of segmental skeletal muscles along the body. Although this process relies on self-generated oscillations of motor circuits in the spinal cord, mechanosensory feedback is crucial to entrain the underlying oscillatory activity and thereby, to enhance movement power and speed. This effect is achieved through directional projections of mechanosensory neurons either sensing stretching or compression of the trunk along the rostrocaudal axis. Different mechanosensory feedback pathways act in concert to shorten and fasten the excitatory wave propagating along the body. While inhibitory mechanosensory cells feedback inhibition on excitatory premotor interneurons and motor neurons, excitatory mechanosensory cells feedforward excitation to premotor excitatory interneurons. Together, diverse mechanosensory cells coordinate the activity of skeletal muscles controlling the head and tail to optimize speed and stabilize balance during fast locomotion.

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The Reissner fiber (RF) is an acellular thread positioned in the midline of the central canal that aggregates thanks to the beating of numerous cilia from ependymal radial glial cells (ERGs) generating flow in the central canal of the spinal cord. RF together with cerebrospinal fluid (CSF)-contacting neurons (CSF-cNs) form an axial sensory system detecting curvature. How RF, CSF-cNs and the multitude of motile cilia from ERGs interact in vivo appears critical for maintenance of RF and sensory functions of CSF-cNs to keep a straight body axis, but is not well-understood. Using in vivo imaging in larval zebrafish, we show that RF is under tension and resonates dorsoventrally. Focal RF ablations trigger retraction and relaxation of the fiber's cut ends, with larger retraction speeds for rostral ablations. We built a mechanical model that estimates RF stress diffusion coefficient D at 5 mm2/s and reveals that tension builds up rostrally along the fiber. After RF ablation, spontaneous CSF-cN activity decreased and ciliary motility changed, suggesting physical interactions between RF and cilia projecting into the central canal. We observed that motile cilia were caudally-tilted and frequently interacted with RF. We propose that the numerous ependymal motile monocilia contribute to RF's heterogenous tension via weak interactions. Our work demonstrates that under tension, the Reissner fiber dynamically interacts with motile cilia generating CSF flow and spinal sensory neurons.

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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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Sexual stimulation triggers changes in female physiology and behavior, including sexual satiety and preparing the uterus for pregnancy. Serotonin is an important regulator of reproductive physiology and sexual receptivity, but the relationship between sexual stimulation and serotonin neural activity in females is poorly understood. Here, we investigated dorsal raphe serotonin neural activity in females during sexual behavior. We found that serotonin neural activity in mating females peaked specifically upon male ejaculation, and remained elevated above baseline until disengagement. Artificial intravaginal mechanical stimulation was sufficient to elicit increased 5-HT neural activity but the delivery of ejaculatory fluids was not. Distal penis erectile enlargement ("penile cupping") at ejaculation and forceful expulsion of ejaculatory fluid each provided sufficient mechanical stimulation to elicit serotonin neuron activation. Our study identifies a female ejaculation-specific signal in a major neuromodulatory system and shows that intravaginal mechanosensory stimulation is necessary and sufficient to drive this signal.