Changes in neuronal activity create local and transient changes in energy demands at synapses. Here we discover a metabolic compartment that forms in vivo near synapses to meet local energy demands ...and support synaptic function in Caenorhabditis elegans neurons. Under conditions of energy stress, glycolytic enzymes redistribute from a diffuse localization in the cytoplasm to a punctate localization adjacent to synapses. Glycolytic enzymes colocalize, suggesting the ad hoc formation of a glycolysis compartment, or a “glycolytic metabolon,” that can maintain local levels of ATP. Local formation of the glycolytic metabolon is dependent on presynaptic scaffolding proteins, and disruption of the glycolytic metabolon blocks the synaptic vesicle cycle, impairs synaptic recovery, and affects locomotion. Our studies indicate that under energy stress conditions, energy demands in C. elegans synapses are met locally through the assembly of a glycolytic metabolon to sustain synaptic function and behavior.
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•A metabolic compartment forms in vivo near synapses to meet local energy demands•Under energy stress, glycolytic proteins redistribute to form clusters at synapses•The glycolytic metabolon is needed for the synaptic vesicle cycle•Disruption of glycolytic metabolon impairs synaptic recovery and affects locomotion
Changes in synaptic activity cause local changes in energy demands. Jang and Nelson et al. discover glycolytic microcompartments, or “glycolytic metabolons,” that form dynamically near presynaptic sites to meet local energy demands and support synaptic function.
We describe the construction and use of a compact dual-view inverted selective plane illumination microscope (diSPIM) for time-lapse volumetric (4D) imaging of living samples at subcellular ...resolution. Our protocol enables a biologist with some prior microscopy experience to assemble a diSPIM from commercially available parts, to align optics and test system performance, to prepare samples, and to control hardware and data processing with our software. Unlike existing light sheet microscopy protocols, our method does not require the sample to be embedded in agarose; instead, samples are prepared conventionally on glass coverslips. Tissue culture cells and Caenorhabditis elegans embryos are used as examples in this protocol; successful implementation of the protocol results in isotropic resolution and acquisition speeds up to several volumes per s on these samples. Assembling and verifying diSPIM performance takes ∼6 d, sample preparation and data acquisition take up to 5 d and postprocessing takes 3-8 h, depending on the size of the data.
Autophagy is a cellular degradation process important for neuronal development and survival. Neurons are highly polarized cells in which autophagosome biogenesis is spatially compartmentalized. The ...mechanisms and physiological importance of this spatial compartmentalization of autophagy in the neuronal development of living animals are not well understood. Here we determine that, in Caenorhabditis elegans neurons, autophagosomes form near synapses and are required for neurodevelopment. We first determine, through unbiased genetic screens and systematic genetic analyses, that autophagy is required cell autonomously for presynaptic assembly and for axon outgrowth dynamics in specific neurons. We observe autophagosome biogenesis in the axon near synapses, and this localization depends on the synaptic vesicle kinesin, KIF1A/UNC-104. KIF1A/UNC-104 coordinates localized autophagosome formation by regulating the transport of the integral membrane autophagy protein, ATG-9. Our findings indicate that autophagy is spatially regulated in neurons through the transport of ATG-9 by KIF1A/UNC-104 to regulate neurodevelopment.
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•The autophagy pathway is required for presynaptic assembly in vivo•The autophagy pathway acts cell autonomously and in specific neurons in development•Autophagosome biogenesis occurs in compartmentalized axonal regions near synapses•The synaptic vesicle kinesin UNC-104/KIF1A transports ATG-9 to presynaptic sites
Autophagy is a degradation process important for neurodevelopment. Stavoe, Hill, et al. uncover spatial regulation of autophagy in C. elegans neurons. They show that autophagosomes form near synapses and are required for presynaptic assembly and axon outgrowth dynamics. Local autophagosome biogenesis depends on kinesin KIF1A/UNC-104-mediated transport of autophagy protein ATG-9.
Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with ...improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.
Neuropil is a fundamental form of tissue organization within the brain
, in which densely packed neurons synaptically interconnect into precise circuit architecture
. However, the structural and ...developmental principles that govern this nanoscale precision remain largely unknown
. Here we use an iterative data coarse-graining algorithm termed 'diffusion condensation'
to identify nested circuit structures within the Caenorhabditis elegans neuropil, which is known as the nerve ring. We show that the nerve ring neuropil is largely organized into four strata that are composed of related behavioural circuits. The stratified architecture of the neuropil is a geometrical representation of the functional segregation of sensory information and motor outputs, with specific sensory organs and muscle quadrants mapping onto particular neuropil strata. We identify groups of neurons with unique morphologies that integrate information across strata and that create neural structures that cage the strata within the nerve ring. We use high resolution light-sheet microscopy
coupled with lineage-tracing and cell-tracking algorithms
to resolve the developmental sequence and reveal principles of cell position, migration and outgrowth that guide stratified neuropil organization. Our results uncover conserved structural design principles that underlie the architecture and function of the nerve ring neuropil, and reveal a temporal progression of outgrowth-based on pioneer neurons-that guides the hierarchical development of the layered neuropil. Our findings provide a systematic blueprint for using structural and developmental approaches to understand neuropil organization within the brain.
Mortality rate for septic shock, despite advancements in knowledge and treatment, remains high. Treatment includes administration of broad-spectrum antibiotics and stabilization of the mean arterial ...pressure (MAP) with intravenous fluid resuscitation. Fluid-refractory shock warrants vasopressor initiation. There is a paucity of evidence regarding the timing of vasopressor initiation and its effect on patient outcomes.
This retrospective, single-centered, cohort study included patients with septic shock from January 2017 to July 2017. Time from initial hypotension to vasopressor initiation was measured for each patient. The primary outcome was 30-day mortality.
Of 530 patients screened,119 patients were included. There were no differences in baseline patient characteristics. Thirty-day mortality was higher in patients who received vasopressors after 6 h (51.1% vs 25%, p < .01). Patients who received vasopressors within the first 6 h had more vasopressor-free hours at 72 h (34.5 h vs 13.1, p = .03) and shorter time to MAP of 65 mmHg (1.5 h vs 3.0, p < .01).
Vasopressor initiation after 6 h from shock recognition is associated with a significant increase in 30-day mortality. Vasopressor administration within 6 h was associated with shorter time to achievement of MAP goals and higher vasopressor-free hours within the first 72 h.
•Delays in vasopressors lead to longer times to shock resolution.•Early initiation does not result in increases in cumulative vasopressor doses.•Higher mortality seen when vasopressors started after 6 h from hypotension.
Brain circuits endow behavioral flexibility. Here, we study circuits encoding flexible chemotaxis in C. elegans, where the animal navigates up or down NaCl gradients (positive or negative chemotaxis) ...to reach the salt concentration of previous growth (the set point). The ASER sensory neuron mediates positive and negative chemotaxis by regulating the frequency and direction of reorientation movements in response to salt gradients. Both salt gradients and set point memory are encoded in ASER temporal activity patterns. Distinct temporal activity patterns in interneurons immediately downstream of ASER encode chemotactic movement decisions. Different interneuron combinations regulate positive versus negative chemotaxis. We conclude that sensorimotor pathways are segregated immediately after the primary sensory neuron in the chemotaxis circuit, and sensory representation is rapidly transformed to motor representation at the first interneuron layer. Our study reveals compact encoding of perception, memory, and locomotion in an experience-dependent navigational behavior in C. elegans.
•Compact encoding of perception, memory, and movement for chemotaxis•Temporal activity patterns of a sensory neuron encodes perception and memory•Flexible relays of interneuronal network•Rapid synaptic transformation of sensory representation to motor representation
Luo et al. explore a chemotaxis circuit in C. elegans and find that it encodes behavior and plasticity via complex activity patterns in single sensory neurons representing both perception and experience, transforming these patterns into representations of motor decisions at the first postsynaptic relays.
Autophagy is essential for cellular homeostasis and function. In neurons, autophagosome biogenesis is temporally and spatially regulated to occur near presynaptic sites, in part via the trafficking ...of autophagy transmembrane protein ATG-9. The molecules that regulate autophagy by sorting ATG-9 at synapses remain largely unknown. Here, we conduct forward genetic screens at single synapses of C. elegans neurons and identify a role for the long isoform of the active zone protein Clarinet (CLA-1L) in regulating sorting of autophagy protein ATG-9 at synapses, and presynaptic autophagy. We determine that disrupting CLA-1L results in abnormal accumulation of ATG-9 containing vesicles enriched with clathrin. The ATG-9 phenotype in cla-1(L) mutants is not observed for other synaptic vesicle proteins, suggesting distinct mechanisms that regulate sorting of ATG-9-containing vesicles and synaptic vesicles. Through genetic analyses, we uncover the adaptor protein complexes that genetically interact with CLA-1 in ATG-9 sorting. We also determine that CLA-1L extends from the active zone to the periactive zone and genetically interacts with periactive zone proteins in ATG-9 sorting. Our findings reveal novel roles for active zone proteins in the sorting of ATG-9 and in presynaptic autophagy.
Synaptic contacts are largely established during embryogenesis and are then maintained during growth. To identify molecules involved in this process, we conducted a forward genetic screen in ...C. elegans and identified cima-1. In cima-1 mutants, synaptic contacts are correctly established during embryogenesis, but ectopic synapses emerge during postdevelopmental growth. cima-1 encodes a solute carrier in the SLC17 family of transporters that includes sialin, a protein that when mutated in humans results in neurological disorders. cima-1 does not function in neurons but rather functions in the nearby epidermal cells to correctly position glia during postlarval growth. Our findings indicate that CIMA-1 antagonizes the FGF receptor (FGFR), and does so most likely by inhibiting FGFR’s role in epidermal-glia adhesion rather than signaling. Our data suggest that epidermal-glia crosstalk, in this case mediated by a transporter and the FGF receptor, is vital to preserve embryonically derived circuit architecture during postdevelopmental growth.
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•Synapse location during postdevelopmental growth depends on glia location•Epidermal-glia interaction positions glia and limits synapse distribution•Solute transporter, CIMA-1, can modulate epidermal-glia interaction via FGFR•Reducing adhesion is important to maintain correct neuroarchitecture during growth
Synaptic contacts established during embryogenesis are maintained during growth. CIMA-1 is a solute carrier transporter that regulates glial positioning and synaptic maintenance during postdevelopmental growth. This regulation occurs via CIMA-1 function in the epidermis rather than neurons, likely through inhibition of FGF receptor function.
In neurons, defects in autophagosome clearance have been associated with neurodegenerative disease. Yet, the mechanisms that coordinate trafficking and clearance of synaptic autophagosomes are poorly ...understood. Here, we use genetic screens and in vivo imaging in single neurons of C. elegans to identify mechanisms necessary for clearance of synaptic autophagosomes. We observed that autophagy at the synapse can be modulated in vivo by the state of neuronal activity, that autophagosomes undergo UNC-16/JIP3-mediated retrograde transport, and that autophagosomes containing synaptic material mature in the cell body. Through forward genetic screens, we then determined that autophagosome maturation in the cell body depends on the protease ATG-4.2, but not the related ATG-4.1, and that ATG-4.2 can cleave LGG-1/Atg8/GABARAP from membranes. Our studies revealed that ATG-4.2 is specifically necessary for the maturation and clearance of autophagosomes and that defects in transport and ATG-4.2-mediated maturation genetically interact to enhance abnormal accumulation of autophagosomes in neurons.
•Retrograde transport of autophagic vacuoles requires the motor adaptor UNC-16/JIP3•Protease ATG-4.2, but not related ATG-4.1, is required for autophagosome maturation•ATG-4.2 cleaves LGG-1/Atg8 to remove it from membranes and enable lysosomal fusion•Transport and maturation mechanisms interact to clear autophagosomes from synapses
Hill et al. use genetic screening and live imaging of autophagosomes in C. elegans to observe activity-dependent synaptic autophagy and discover that autophagosome trafficking and clearance are coordinated in neurons. They discover a unique role for protease isoform ATG-4.2 in cleaving LGG-1/Atg8/GABARAP from autophagosomes to promote their maturation and clearance.
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