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Independent, bidirectional NIR-optogenetic manipulation of C. elegans motor behavior is demonstrated by synergistic integration of orthogonal emissive upconversion nanoparticles (OUCNPs) with a single dual-light responsive sensor, BiPOLES.

Behaviors consist of distinct states and state transitions. How a neural system continuously modulates each state, which underlies smooth state transitions, is not well understood.C. elegansspontaneously switches between two mutually exclusive motor states, forward and backward movements, a behavior long thought to reflect the reciprocal antagonism between interneurons that separately gate the forward and backward motor circuits. We report here that during spontaneous locomotion, the two motor states are not separately controlled, and these interneurons are neither functionally equivalent nor strictly reciprocally inhibitory. AVA, the premotor interneuron previously thought to exclusively gate the backward motor circuit, regulates both motor states with opposite polarities at two-time scales. AVA’s transient, fast activation inhibits the forward circuit while initiating the backward movement; in parallel, AVA maintains a slow, but constitutive excitation of the forward circuit to transit from reversals into maintained forward movement. This excitation results from AVA’s sustained depolarized membrane potential, which makes its chemical synapse tonically active. A tonically active master neuron breaks the symmetry between the forward and backward motor circuits. It offers an elegant circuit solution for smooth transitions when animals switch between two mutually exclusive motor states.HighlightsTwo mutually exclusive, forward and backward motor states, are inherently asymmetricThe forward and backward motor states are not separately controlledA master neuron underlies continuous modulation and transitions of two motor statesA tonically active premotor interneuron of the reversal circuit is the master neuron

Caenorhabditis elegans (C. elegans) is an ideal model organism for studying neuronal functions at the system level. This article develops a customized system for whole-body motor neuron calcium imaging of freely moving C. elegans without the coverslip pressed. Firstly, we proposed a fast centerline localization algorithm that could deal with most topology-variant cases costing only 6 ms for one frame, not only benefits for real-time localization but also for post-analysis. Secondly, we implemented a full-time two-axis synchronized motion strategy by adaptively adjusting the motion parameters of two motors in every short-term motion step (~50 ms). Following the above motion tracking configuration, the tracking performance of our system has been demonstrated to completely support the high spatiotemporal resolution calcium imaging on whole-body motor neurons of wild-type (N2) worms as well as two mutants (unc-2, unc-9), even the instantaneous speed of worm moving without coverslip pressed was extremely up to 400 μm/s.

Light-field microscopy has emerged as a technique of choice for high-speed volumetric imaging of fast biological processes. However, artifacts, nonuniform resolution and a slow reconstruction speed have limited its full capabilities for in toto extraction of dynamic spatiotemporal patterns in samples. Here, we combined a view-channel-depth (VCD) neural network with light-field microscopy to mitigate these limitations, yielding artifact-free three-dimensional image sequences with uniform spatial resolution and high-video-rate reconstruction throughput. We imaged neuronal activities across moving Caenorhabditis elegans and blood flow in a beating zebrafish heart at single-cell resolution with volumetric imaging rates up to 200 Hz. Reconstruction of light-field microscopy data with a deep-learning network achieves high reconstruction speed and reduces artifacts, as illustrated for moving C. elegans and beating zebrafish hearts.

Assembly of SNARE complexes that mediate neurotransmitter release requires opening of a ‘closed’ conformation of UNC-64/syntaxin. Rescue of unc-13/Munc13 mutant phenotypes by overexpressed open UNC-64/syntaxin suggested a specific function of UNC-13/Munc13 in opening UNC-64/ syntaxin. Here, we revisit the effects of open unc-64/syntaxin by generating knockin (KI) worms. The KI animals exhibit enhanced spontaneous and evoked exocytosis compared to WT animals. Unexpectedly, the open syntaxin KI partially suppresses exocytosis defects of various mutants, including snt-1/synaptotagmin, unc-2/P/Q/N-type Ca2+ channel alpha-subunit and unc-31/CAPS, in addition to unc-13/Munc13 and unc-10/RIM, and enhanced exocytosis in tom-1/Tomosyn mutants. However, open syntaxin aggravates the defects of unc-18/Munc18 mutants. Correspondingly, open syntaxin partially bypasses the requirement of Munc13 but not Munc18 for liposome fusion. Our results show that facilitating opening of syntaxin enhances exocytosis in a wide range of genetic backgrounds, and may provide a general means to enhance synaptic transmission in normal and disease states., Opening of the UNC-64/syntaxin closed conformation by UNC-13/Munc13 to form the neuronal SNARE complex is critical for neurotransmitter release. Here the authors show that facilitating the opening of syntaxin enhances exocytosis not only in unc-13 nulls as well as in diverse C. elegans mutants.

Background potassium (K+) currents, conducted by two-pore domain K+ (K2P) channels, are critical for the stabilization of the membrane potential. The effect of K2P channels on motor rhythm remains enigmatic. We show here that the K2P TWK-40 regulates the rhythmic defecation motor program (DMP) in Caenorhabditis elegans. Disrupting TWK-40 suppresses the expulsion defects of nlp-40 and aex-2 mutants. By contrast, a gain-of-function (gf) mutant of twk-40 significantly reduces the expulsion frequency per DMP cycle. In situ whole-cell patch clamping demonstrates that TWK-40 forms an outward current that hyperpolarize the resting membrane potential of DVB neuron. In addition, TWK-40 substantially contributes to the rhythmic activity of DVB. Specifically, DVB Ca2+ oscillations exhibit obvious defects in twk-40 mutants. Expression of TWK-40(gf) in DVB recapitulates the expulsion deficiency of the twk-40(gf) mutant, and inhibits DVB Ca2+ oscillations in both wild-type and twk-40(lf) animals. Moreover, DVB innervated enteric muscles also exhibit rhythmic Ca2+ defects. Taken together, these results demonstrate that TWK-40 is an essential inhibitor of DMP, thus revealing a cellular mechanism linking K2P channels with rhythmic motor activity.

Motor rhythm is initiated and sustained by oscillatory neuronal activity. We recently discovered that the A-class excitatory motor neurons (MNs) (A-MNs) function as intrinsic oscillators. They drive backward locomotion by generating rhythmic postsynaptic currents (rPSCs) in body wall muscles. Molecular underpinning of the rPSCs, however, is not fully elucidated. We report here that there are three types of the rPSC patterns, namely the phasic, tonic, and long-lasting, each with distinct kinetics and channel-dependence. The Na

Cardiovascular diseases are closely associated with dysfunction of vascular endothelial cells (VECs), which can be influenced by various intrinsic and extrinsic factors, including fibroblast growth factor 21 (FGF21), but the effects of serum FGF21 on VECs remain unclear. We performed a cross-sectional study nested within a prospective cohort to assess the range of physiological concentrations of fasting serum FGF21 in 212 healthy individuals. We also treated human umbilical VECs (HUVECs) with recombinant FGF21 at different concentrations. The effects of FGF21 treatment on glycolysis, nitric oxide release and reduction of intracellular reactive oxygen species were assessed. The cells were also collected for RNA transcriptomic sequencing to investigate the potential mechanisms induced by FGF21 treatment. In addition, the roles of SIRT1 in the regulation of FGF21 were evaluated by SIRT1 knockdown. The results showed that the serum FGF21 concentration in healthy individuals ranged from 15.70 to 499.96 pg/mL and was positively correlated with age and pulse wave velocity. FGF21 at 400 pg/mL was sufficient to enhance glycolysis, increase nitric oxide release and protect cells from H

Escape is an evolutionarily conserved and essential avoidance response. Considered to be innate, most studies on escape responses focused on hard-wired circuits. We report here that peptidergic signaling is an integral and necessary component of the Caenorhabditis elegans escape circuit. Combining genetic screening, electrophysiology and calcium imaging, we reveal that a neuropeptide NLP-18 and its cholecystokinin receptor CKR-1 enable the escape circuit to execute a full omega (Ω) turn, the last motor step where the animal robustly steers away from its original trajectory. We demonstrate in vivo and in vitro that CKR-1 is a Gαq protein coupled receptor for NLP-18. in vivo, NLP-18 is mainly secreted by the gustatory sensory neuron (ASI) to activate CKR-1 in the head motor neuron (SMD) and the turn-initiating interneuron (AIB). Removal of NLP-18, removal of CKR-1, or specific knockdown of CKR-1 in SMD or AIB neurons lead to shallower turns hence less robust escape steering. Consistently, elevation of head motor neuron (SMD)’s Ca2+ transients during escape steering is attenuated upon the removal of NLP-18 or CKR-1. in vitro, synthetic NLP-18 directly evokes CKR-1-dependent currents in oocytes and CKR-1-dependent Ca2+ transients in SMD. Thus, cholecystokinin signaling modulates an escape circuit to generate robust escape steering.

Photodynamic theranostics integrating synchronous photodynamic therapy and real-time monitoring of therapeutic efficacy has attracted great interest in clinical cancer management. Recently, a series of cationic molecules have been reported as self-reporting photosensitizers for unimolecular photodynamic theranostics. However, the design rationality and mechanisms for such multifunctional photosensitizers are not well understood and need further exploration. In this work, we have designed and synthesized three multicationic AIEgen photosensitizers with similar core structures, but which are end-capped with pyridinium groups (BPCI and TPCI) or quaternary ammonium groups (TPCB), respectively. The tetra-pyridinium-tethered TPCI exhibits superior efficiency in reactive oxygen species generation and dead cell identification compared to both bis-pyridinium-tethered BPCI and tetra-ammonium-tethered TPCB. The structure–activity relationship of all three AIEgens has been studied in detail, which provides great insights into designing the new generation of photosensitizers for photodynamic theranostics. Strikingly, all the multicationic AIEgens have large two-photon absorption cross-sections, and TPCI can effectively kill Caenorhabditis elegans upon white light irradiation and differentiate the dead nematodes from the living ones through the redistribution of its two-photon fluorescence, demonstrating great potential in two-photon fluorescence imaging-guided photodynamic theranostics.

GABAergic neurotransmission constitutes a major inhibitory signaling mechanism that plays crucial roles in central nervous system physiology and immune cell immunomodulation. However, its roles in innate immunity remain unclear. Here, we report that deficiency in the GABAergic neuromuscular junctions (NMJs) of Caenorhabditis elegans results in enhanced resistance to pathogens, whereas pathogen infection enhances the strength of GABAergic transmission. GABAergic synapses control innate immunity in a manner dependent on the FOXO/DAF-16 but not the p38/PMK-1 pathway. Our data reveal that the insulin-like peptide INS-31 level was dramatically decreased in the GABAergic NMJ GABA(A)R-deficient unc-49 mutant compared with wild-type animals. C. elegans with ins-31 knockdown or loss of function exhibited enhanced resistance to Pseudomonas aeruginosa PA14 exposure. INS-31 may act downstream of GABAergic NMJs and in body wall muscle to control intestinal innate immunity in a cell-nonautonomous manner. Our results reveal a signaling axis of synapse–muscular insulin–intestinal innate immunity in vivo.

GABAergic neurotransmission constitutes a major inhibitory signaling mechanism that plays crucial roles in central nervous system physiology and immune cell immunomodulation. However, its roles in innate immunity remain unclear. Here, we report that deficiency in the GABAergic neuromuscular junctions (NMJs) of Caenorhabditis elegans results in enhanced resistance to pathogens, whereas pathogen infection enhances the strength of GABAergic transmission. GABAergic synapses control innate immunity in a manner dependent on the FOXO/DAF-16 but not the p38/PMK-1 pathway. Our data reveal that the insulin-like peptide INS-31 level was dramatically decreased in the GABAergic NMJ GABAAR-deficient unc-49 mutant compared with wild-type animals. C. elegans with ins-31 knockdown or loss of function exhibited enhanced resistance to Pseudomonas aeruginosa PA14 exposure. INS-31 may act downstream of GABAergic NMJs and in body wall muscle to control intestinal innate immunity in a cell-nonautonomous manner. Our results reveal a signaling axis of synapse–muscular insulin–intestinal innate immunity in vivo.

The development of functional synapses in the nervous system is important for animal physiology and behaviors, and its disturbance has been linked with many neurodevelopmental disorders. The synaptic transmission efficacy can be modulated by the environment to accommodate external changes, which is crucial for animal reproduction and survival. However, the underlying plasticity of synaptic transmission remains poorly understood. Here we show that in Caenorhabditis elegans, the male environment increases the hermaphrodite cholinergic transmission at the neuromuscular junction (NMJ), which alters hermaphrodites’ locomotion velocity and mating efficiency. We identify that the male-specific pheromones mediate this synaptic transmission modulation effect in a developmental stage-dependent manner. Dissection of the sensory circuits reveals that the AWB chemosensory neurons sense those male pheromones and further transduce the information to NMJ using cGMP signaling. Exposure of hermaphrodites to the male pheromones specifically increases the accumulation of presynaptic CaV2 calcium channels and clustering of postsynaptic acetylcholine receptors at cholinergic synapses of NMJ, which potentiates cholinergic synaptic transmission. Thus, our study demonstrates a circuit mechanism for synaptic modulation and behavioral flexibility by sexual dimorphic pheromones.

SUMMARYThe development of functional synapses in the nervous system is important for animal physiology and behaviors. The synaptic transmission efficacy can be modulated by the environment to accommodate external changes, which is crucial for animal reproduction and survival. However, the underlying plasticity of synaptic transmission remains poorly understood. Here we show that in C. elegans, the male pheromone increases the hermaphrodite cholinergic transmission at the neuromuscular junction (NMJ), which alters hermaphrodites’ locomotion velocity and mating efficiency in a developmental stage-dependent manner. Dissection of the sensory circuits reveals that the AWB chemosensory neurons sense those male pheromones and further transduce the information to NMJ using cGMP signaling. Exposure of hermaphrodites to male pheromones specifically increases the accumulation of presynaptic CaV2 calcium channels and clustering of postsynaptic receptors at cholinergic synapses of NMJ, which potentiates cholinergic synaptic transmission. Thus, our study demonstrates a circuit mechanism for synaptic modulation by sexual dimorphic pheromones.

The action potential (AP) is the basic signaling unit in various crucial physiological processing, for instance, in neurotransmission, muscle contraction, and glandular secretion (Koch, 1990). The classic model animal, Caenorhabditis elegans (or C. elegans), with a simple and compact nervous system, conservatively employs the calcium-mediated all-or-none APs for odor response in AWA olfactory neurons (Liu et al., 2018), as well as for muscle contraction in either body wall muscles (Gao and Zhen, 2011; Liu et al., 2011) and pharyngeal muscles (Davis et al., 1999). Plateau potentials were also observed in ASE and RMD neurons (Goodman et al., 1998; Mellem et al., 2008; Lockery et al., 2009; Lockery and Goodman, 2009), though the underlying roles in specific behavior are still elusive. Either in neurons or in muscles, the action potential firing is dependent on the excitatory pre-synaptic vesicles release. The minimum number of the presynaptic vesicles to elicit a single action potential in C. elegans has not been reported before. Here, by the combination of optogenetics with in-vivo patch clamping technology, we demonstrated that at least approximately 37 excitatory acetylcholinergic vesicles are required for the initiation of an action potential at post-synaptic body wall muscles.

Light-sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high-speed and with low photo-toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub-voxel-resolving (SVR) light-sheet add-on microscopy (SLAM), is presented to enable fast, resolution-enhanced light-sheet fluorescence imaging from a conventional wide-field microscope. This method contains two components: a miniature add-on device to regular wide-field microscopes, which contains a horizontal laser light-sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off-axis scanning strategy and a SVR algorithm that utilizes sub-voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost-effective solution to convert the vast number of in-service microscopes for fast 3D live imaging with voxel-super-resolved capability.

We report a novel fusion of microfluidics and light-field microscopy, to achieve high-speed 4D (space + time) imaging of moving C. elegans on a chip. Our approach combines automatic chip-based worm loading / compartmentalization / flushing / reloading with instantaneous deep-learning light-field imaging of moving worm. Taken together, we realized intoto image-based screening of wild-type and uncoordinated-type worms at a volume rate of 33 Hz, with sustained observation of 1 minute per worm, and overall throughput of 42 worms per hour. With quickly yielding over 80000 image volumes that four-dimensionally visualize the dynamics of all the worms, we can quantitatively analyse their behaviours as well as the neural activities, and correlate the phenotypes with the neuron functions. The different types of worms can be readily identified as a result of the high-throughput activity mapping. Our approach shows great potential for various lab-on-a-chip biological studies, such as embryo sorting and cell growth assays.

Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1.SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.

SummaryAssembly of SNARE complexes that mediate neurotransmitter release requires opening of a ‘closed’ conformation of UNC-64/syntaxin. Rescue of unc-13/Munc13 phenotypes by overexpressed open UNC-64/syntaxin suggested a specific function of UNC-13/Munc13 in opening UNC-64/ syntaxin. Here, we revisit the effects of open unc-64/syntaxin by generating knockin (KI) worms. The KI animals exhibited enhanced spontaneous and evoked exocytosis compared to wild-type animals. Unexpectedly, the open syntaxin KI partially suppressed exocytosis defects of various mutants, including snt-1/synaptotagmin, unc-2/P/Q/N-type Ca2+ channel alpha-subunit, and unc-31/CAPS in addition to unc-13/Munc13 and unc-10/RIM, and enhanced exocytosis in tom-1/Tomosyn mutants. However, open syntaxin aggravated the defects of unc-18/Munc18 mutants. Correspondingly, open syntaxin partially bypasses the requirement of Munc13 but not Munc18 for liposome fusion. Our results show that facilitating opening of syntaxin enhances exocytosis in a wide range of genetic backgrounds, and may provide a general means to enhance synaptic transmission in normal and disease states.

Amytrophic lateral sclerosis (ALS) is a neurodegenerative disorder that has been associated with multiple genetic lesions, including mutations in the gene FUS (Fused in Sarcoma), an RNA/DNA-binding protein. Expression of the ALS-associated human FUS in C. elegans results in mislocalization and aggregation of FUS outside the nucleus, and leads to impaired neuromuscular behaviors. However, the mechanisms by which mutant FUS disrupts neuronal health and function remain partially understood. Here we investigated the impact of ALS-associated FUS on motor neuron health using correlative light and electron microscopy, electron tomography, and electrophysiology. Expression of ALS-associated FUS impairs synaptic vesicle docking at neuromuscular junctions, and leads to the emergence of a population of large and electron-dense filament-filled endosomes. Electrophysiological recording of neuromuscular transmission revealed reduced transmission from motor neurons to muscles. Together, these results suggest a potential direct or indirect role of human FUS in the organization of synaptic vesicles, and reduced transmission from motor neurons to muscles.Summary statementAn ALS-associated mutation in a trafficking protein disrupts the organization of the C. elegans neuromuscular junction.

The cerebral cortex develops from dorsal forebrain neuroepithelial progenitor cells. Following the initial expansion of the progenitor cell pool, these cells generate neurons of all the cortical layers and then astrocytes and oligodendrocytes. Yet, the regulatory pathways that control the expansion and maintenance of the progenitor cell pool are currently unknown. Here we define six basic pathway components that regulate proliferation of cortically specified human neuroepithelial stem cells (cNESCs) in vitro without the loss of cerebral cortex developmental potential. We show that activation of FGF and inhibition of BMP and ACTIVIN A signalling are required for long-term cNESC proliferation. We also demonstrate that cNESCs preserve dorsal telencephalon-specific potential when GSK3, AKT and nuclear CATENIN-β1 activity are low. Remarkably, regulation of these six pathway components supports the clonal expansion of cNESCs. Moreover, cNESCs differentiate into lower- and upper-layer cortical neurons in vitro and in vivo. The identification of mechanisms that drive the neuroepithelial stem cell self-renewal and differentiation and preserve this potential in vitro is key to developing regenerative and cell-based therapeutic approaches to treat neurological conditions.

The optogenetic neuron ablation approach enables noninvasive remote decoding of specific neuron function within a complex living organism in high spatiotemporal resolution. However, it suffers from shallow tissue penetration of visible light with low ablation efficiency. This study reports a upconversion nanoparticle (UCNP)-based multiplex proteins activation tool to ablate deep-tissue neurons for locomotion modulation. By optimizing the dopant contents and nanoarchitecure, over 300-fold enhancement of blue (450-470 nm) and red (590-610 nm) emissions from UCNPs is achieved upon 808 nm irradiation. Such emissions simultaneously activate mini singlet oxygen generator and Chrimson, leading to boosted near infrared (NIR) light-induced neuronal ablation efficiency due to the synergism between singlet oxygen generation and intracellular Ca2+ elevation. The loss of neurons severely inhibits reverse locomotion, revealing the instructive role of neurons in controlling motor activity. The deep penetrance NIR light makes the current system feasible for in vivo deep-tissue neuron elimination. The results not only provide a rapidly adoptable platform to efficient photoablate single- and multiple-cells, but also define the neural circuits underlying behavior, with potential for development of remote therapy in diseases.

Mutations in pre-synaptic voltage gated calcium channels can lead to familial hemiplegic migraine type 1 (FHM1). While mammalian studies indicate that the migraine brain is hyperexcitable due to enhanced excitation or reduced inhibition, the molecular and cellular mechanisms underlying this excitatory/inhibitory (E/I) imbalance are poorly understood. We identified a gain-of-function (gf) mutation in the Caenorhabditis elegans CaV2 channel α1 subunit, UNC-2, which leads to increased calcium currents. unc-2(zf35gf) mutants exhibit hyperactivity and seizure-like motor behaviors. Expression of the unc-2 gene with FHM1 substitutions R192Q and S218L leads to hyperactivity similar to that of unc-2(zf35gf) mutants. unc-2(zf35gf) mutants display increased cholinergic-and decreased GABAergic-transmission. Moreover, increased cholinergic transmission in unc-2(zf35gf) mutants leads to an increase of cholinergic synapses and a TAX-6/calcineurin dependent reduction of GABA synapses. Our studies reveal mechanisms through which CaV2 gain-of-function mutations disrupt excitation-inhibition balance in the nervous system. Fil: Huang, Yung Chi. University of Massachussets; Estados Unidos Fil: Pirri, Jennifer K.. University of Massachussets; Estados Unidos Fil: Rayes, Diego Hernán. University of Massachussets; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Gao, Shangbang. Mount Sinai Hospital; Estados Unidos Fil: Mulcahy, Ben. Mount Sinai Hospital; Estados Unidos Fil: Grant, Jeff. University of Massachussets; Estados Unidos Fil: Saheki, Yasunori. The Rockefeller University; Estados Unidos Fil: Francis, Michael M.. University of Massachussets; Estados Unidos Fil: Zhen, Mei. University of Toronto; Canadá. Mount Sinai Hospital; Estados Unidos Fil: Alkema, Mark J.. University of Massachussets; Estados Unidos

Light-sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high-speed and with low photo-toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub-voxel-resolving light-sheet add-on microscopy (SLAM), is presented to enable fast, resolution-enhanced light-sheet fluorescence imaging from a conventional wide-field microscope. This method contains two components: a miniature add-on device to regular wide-field microscopes, which contains a horizontal laser light-sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off-axis scanning strategy and a sub-voxel-resolving (SVR) algorithm that utilizes sub-voxel spatial shifts to reconstruct the image volume that results in a two-fold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost-effective solution to convert the vast number of in-service microscopes for fast 3D live volume imaging with voxel-super-resolved capability.

Near-infrared (NIR) light penetrates tissue deeply, but its application to motor behavior stimulation has been limited by the lack of known genetic NIR light-responsive sensors. We designed and synthesized a Yb³⁺/Er³⁺/Ca²⁺-based lanthanide-doped upconversion nanoparticle (UCNP) that effectively converts 808 nm NIR light to green light emission. This UCNP is compatible with Chrimson, a cation channel activated by green light; as such, it can be used in the optogenetic manipulation of the motor behaviors of Caenorhabditis elegans. We show that this UCNP effectively activates Chrimson-expressing, inhibitory GABAergic motor neurons, leading to reduced action potential firing in the body wall muscle and resulting in locomotion inhibition. The UCNP also activates the excitatory glutamatergic DVC interneuron, leading to potentiated muscle action potential bursts and active reversal locomotion. Moreover, this UCNP exhibits negligible toxicity in neural development, growth, and reproduction, and the NIR energy required to elicit these behavioral and physiological responses does not activate the animal's temperature response. This study shows that UCNP provides a useful integrated optogenetic toolset, which may have wide applications in other experimental systems. This work was supported by the National Natural Science Foundation of China (31871069, 31671052, 81703031 and 81672937), National Basic Research Program of China (2015CB931802), the National Science Foundation of Hubei Province (2018CFA039), Wuhan Morning Light Plan of Youth Science and Technology (2017050304010295), the Junior Thousand Talents Program of China, and funds from Huazhong University of Science and Technology (2017KFYXJJ162, Dengfeng Initiative, Global Talents Recruitment Program).

Mutations in pre-synaptic voltage gated calcium channels can lead to familial hemiplegic migraine type 1 (FHM1). While mammalian studies indicate that the migraine brain is hyperexcitable due to enhanced excitation or reduced inhibition, the molecular and cellular mechanisms underlying this excitatory/inhibitory (E/I) imbalance are poorly understood. We identified a gain-of-function (gf) mutation in the Caenorhabditis elegans CaV2 channel α1 subunit, UNC-2, which leads to increased calcium currents. unc-2(gf) mutants exhibit hyperactivity and seizure-like motor behaviors. Expression of the unc-2 gene with FHM1 substitutions R192Q and S218L leads to hyperactivity similar to that of unc-2(gf) mutants unc-2(gf) mutants display increased cholinergic- and decreased GABAergic-transmission. Moreover, we reveal that and increased cholinergic transmission in unc-2(gf) mutants leads to reduction of GABA synapses in a TAX-6/calcineurin dependent manner. Our studies provide mechanistic insight into how CaV2 gain-of-function mutations disrupt excitation-inhibition balance in the nervous system.

How neurons are capable of decoding stimulus intensity and translate this information into complex behavioral outputs is poorly defined. Here, we demonstrate that the C. elegans interneuron AIB regulates two types of behaviors: reversal initiation and feeding suppression in response to different concentrations of quinine. Low concentrations of quinine are decoded in AIB by a low-threshold, fast-inactivation glutamate receptor GLR-1 and translated into reversal initiation. In contrast, high concentrations of quinine are decoded by a high-threshold, slow-inactivation glutamate receptor GLR-5 in AIB. After activation, GLR-5 evokes sustained Ca2+ release from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores and triggers neuropeptide secretion, which in turn activates the downstream neuron RIM and inhibits feeding. Our results reveal that distinct signal patterns in a single interneuron AIB can encode differential behavioral outputs depending on the stimulus intensity, thus highlighting the importance of functional mapping of information propagation at the single-neuron level during connectome construction., Little is known about how stimuli of different intensities result in different behavioral outcomes in C. elegans. In this study, the authors demonstrate how distinct signal patterns, involving different glutamate receptors, in a single interneuron AIB can encode differential behavioral outputs depending on the stimulus intensity

Light-field microscopy has emerged as a technique of choice for high-speed volumetric imaging of fast biological processes. However, artefacts, non-uniform resolution, and a slow reconstruction speed have limited its full capabilities for in toto extraction of the dynamic spatiotemporal patterns in samples. Here, we combined a view-channel-depth (VCD) neural network with light-field microscopy to mitigate these limitations, yielding artefact-free three-dimensional image sequences with uniform spatial resolution and three-order-higher video-rate reconstruction throughput. We imaged neuronal activities across moving C. elegans and blood flow in a beating zebrafish heart at single-cell resolution with volume rates up to 200 Hz.

The intrinsic oscillatory activity of central pattern generators underlies motor rhythm. We review and discuss recent findings that address the origin of Caenorhabditis elegans motor rhythm. These studies propose that the A- and mid-body B-class excitatory motor neurons at the ventral cord function as non-bursting intrinsic oscillators to underlie body undulation during reversal and forward movements, respectively. Proprioception entrains their intrinsic activities, allows phase-coupling between members of the same class motor neurons, and thereby facilitates directional propagation of undulations. Distinct pools of premotor interneurons project along the ventral nerve cord to innervate all members of the A- and B-class motor neurons, modulating their oscillations, as well as promoting their bi-directional coupling. The two motor sub-circuits, which consist of oscillators and descending inputs with distinct properties, form the structural base of dynamic rhythmicity and flexible partition of the forward and backward motor states. These results contribute to a continuous effort to establish a mechanistic and dynamic model of the C. elegans sensorimotor system. C. elegans exhibits rich sensorimotor functions despite a small neuron number. These findings implicate a circuit-level functional compression. By integrating the role of rhythm generation and proprioception into motor neurons, and the role of descending regulation of oscillators into premotor interneurons, this numerically simple nervous system can achieve a circuit infrastructure analogous to that of anatomically complex systems. C. elegans has manifested itself as a compact model to search for general principles of sensorimotor behaviours. This article is part of a discussion meeting issue ‘Connectome to behaviour: modelling C. elegans at cellular resolution’.