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Deep-brain microscopy is strongly limited by the size of the imaging probe, both in terms of achievable resolution and potential trauma due to surgery. Here, we show that a segment of an ultra-thin multi-mode fiber (cannula) can replace the bulky microscope objective inside the brain. By creating a self-consistent deep neural network that is trained to reconstruct anthropocentric images from the raw signal transported by the cannula, we demonstrate a single-cell resolution (< 10μm), depth sectioning resolution of 40 μm, and field of view of 200 μm, all with green-fluorescent-protein labelled neurons imaged at depths as large as 1.4 mm from the brain surface. Since ground-truth images at these depths are challenging to obtain in vivo, we propose a novel ensemble method that averages the reconstructed images from disparate deep-neural-network architectures. Finally, we demonstrate dynamic imaging of moving GCaMp-labelled C. elegans worms. Our approach dramatically simplifies deep-brain microscopy.

Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50–100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits., Author summary Germlines have evolved specialized mechanisms to protect themselves from invasions by transposons and viruses, which create barriers to genome editing techniques. For example, transgenes are silenced in the germline of the nematode C. elegans, thereby creating a barrier to CRISPR editing by Cas9. To facilitate gene editing, we built a collection of C. elegans strains in which Cas9 is never silenced. CRISPR is significantly more efficient in these animals, decreasing the effort researchers need to expend to get edited animals. The strains are available in multiple genetic backgrounds, and contain accessory transgenes to simplify downstream genetics. Together, these strains enable efficient, low-cost genome editing in C. elegans.

Synaptic vesicles fuse with the plasma membrane to release neurotransmitter following an action potential, after which new vesicles must refill vacated release sites. How many vesicles can fuse at a single active zone, where they fuse within the active zone, and how quickly they are replaced with new vesicles is not well-established. To capture synaptic vesicle exocytosis at cultured mouse hippocampal synapses, we induced single action potentials by electrical field stimulation then subjected neurons to high-pressure freezing to examine their morphology by electron microscopy. During synchronous release, multiple vesicles can fuse at a single active zone; this multivesicular release is augmented by increasing the extracellular calcium concentration. Synchronous fusions are distributed throughout the active zone, whereas asynchronous fusions are biased toward the center of the active zone. Immediately after stimulation a large fraction of vesicles become undocked. Between 8 and 14 ms, new vesicles are recruited to the plasma membrane and fully replenish the docked pool, but docking of these vesicles is transient and they either undock or fuse within 100 ms. These results demonstrate that recruitment of synaptic vesicles to release sites is rapid and reversible.

To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated approximately 20 s after fusion by the assembly of clathrin scaffolds or in approximately 1 s by the reversal of fusion pores via 'kiss-and-run' endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy--'flash-and-freeze' electron microscopy. Docked vesicles fuse and collapse into the membrane within 30 ms of the stimulus. Compensatory endocytosis occurs within 50 to 100 ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover, it is 200-fold faster than clathrin-mediated endocytosis. It is likely that 'ultrafast endocytosis' is specialized to restore the surface area of the membrane rapidly.

A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immunocytochemical electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies and the inaccessibility of antigens. Here we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. We tagged proteins with the fluorescent proteins Citrine or tdEos and expressed them in Caenorhabditis elegans, fixed the worms and embedded them in plastic. We imaged the tagged proteins from ultrathin sections using stimulated emission depletion (STED) microscopy or photoactivated localization microscopy (PALM). Fluorescence correlated with organelles imaged in electron micrographs from the same sections. We used these methods to localize histones, a mitochondrial protein and a presynaptic dense projection protein in electron micrographs.

SUMMARY Muscle contraction is normally mediated by the release of neurotransmitters from motor neurons. Here we demonstrate that protons can act as a direct transmitter from intestinal cells to stimulate muscle contraction. During the C. elegans defecation motor program the posterior body muscles contract even in the absence of neuronal inputs or vesicular neurotransmission. In this study, we demonstrate that the space between the intestine and the muscle is acidified just prior to muscle contraction and that the release of caged protons is sufficient to induce muscle contraction. PBO-4 is a putative Na + /H + ion exchanger expressed on the basolateral membrane of the intestine, juxtaposed to the posterior body muscles. In pbo-4 mutants the extracellular space is not acidified and the muscles fail to contract. The pbo-5 and pbo-6 genes encode subunits of a ‘‘cysloop’’ proton-gated cation channel required for muscles to respond to acidification. In heterologous expression assays the PBO receptor is half-maximally activated at a pH of 6.8. The identification of the mechanisms for release and reception of proton signals establishes a highly unusual mechanism for intercellular communication.

Summary Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-basepair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure., Graphical Abstract, eTOC Paragraph A non-coding DNA sequence pattern can prevent epigenetic silencing of transgenes in germ cells, revealing a functional role for an abundant class of non-coding DNA and a possible mechanism by which cells may recognize and silence foreign DNA

Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an endonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans . Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, ≈20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions.

The normal distribution of crossover events on meiotic bivalents depends on homolog recognition, alignment, and interference. We developed a method for precisely locating all crossovers on Caenorhabditis elegans chromosomes and demonstrated that wild-type animals have essentially complete interference, with each bivalent receiving one and only one crossover. A physical break in one homolog has previously been shown to disrupt interference, suggesting that some aspect of bivalent structure is required for interference. We measured the distribution of crossovers in animals heterozygous for a large insertion to determine whether a break in sequence homology would have the same effect as a physical break. Insertions disrupt crossing over locally. However, every bivalent still experiences essentially one and only one crossover, suggesting that interference can act across a large gap in homology. Although insertions did not affect crossover number, they did have an effect on crossover distribution. Crossing over was consistently higher on the side of the chromosome bearing the homolog recognition region and lower on the other side of the chromosome. We suggest that nonhomologous sequences cause heterosynapsis, which disrupts crossovers along the distal chromosome, even when those regions contain sequences that could otherwise align. However, because crossovers are not completely eliminated distal to insertions, we propose that alignment can be reestablished after a megabase-scale gap in sequence homology.

Molting is required for progression between larval stages in the life cycle of nematodes. We have identified four mutant alleles of a Caenorhabditis elegans metalloprotease gene, nas-37, that cause incomplete ecdysis. At each molt the cuticle fails to open sufficiently at the anterior end and the partially shed cuticle is dragged behind the animal. The gene is expressed in hypodermal cells 4 hours before ecdysis during all larval stages. The NAS-37 protein accumulates in the anterior cuticle and is shed in the cuticle after ecdysis. This pattern of protein accumulation places NAS-37 in the right place and at the right time to degrade the cuticle to facilitate ecdysis. The nas-37 gene has orthologs in other nematode species,including parasitic nematodes, and they undergo a similar shedding process. For example, Haemonchus contortus molts by digesting a ring of cuticle at the tip of the nose. Incubating Haemonchus larvae in extracted exsheathing fluids causes a refractile ring of digested cuticle to form at the tip of the nose. When Haemonchus cuticles are incubated with purified NAS-37, a similar refractile ring forms. NAS-37 degradation of the Haemonchus cuticle suggests that the metalloproteases and the cuticle substrates involved in exsheathment of parasitic nematodes are conserved in free-living nematodes.

Transposons have been enormously useful for genetic analysis in both Drosophila and bacteria. Mutagenic insertions constitute molecular tags that are used to rapidly clone the mutated gene. Such techniques would be especially advantageous in the nematode Caenorhabditis elegans, as the entire sequence of the genome has been determined. Several different types of endogenous transposons are present in C. elegans, and these can be mobilized in mutator strains (reviewed in ref. 1). Unfortunately, use of these native transposons for regulated transposition in C. elegans is limited. First, all strains contain multiple copies of these transposons and thus new insertions do not provide unique tags. Second, mutator strains tend to activate the transposition of several classes of transposons, so that the type of transposon associated with a particular mutation is not known. Here we demonstrate that the Drosophila mariner element Mos1 can be mobilized in C. elegans. First, efficient mobilization of Mos1 is possible in somatic cells. Second, heritable insertions of the transposon can be generated in the germ line. Third, genes that have been mutated by insertion can be rapidly identified using inverse polymerase chain reaction. Fourth, these insertions can subsequently be remobilized to generate deletion and frameshift mutations by imperfect excision.

Ultrafast endocytosis can retrieve a single, large endocytic vesicle as fast as 50–100 ms after synaptic vesicle fusion. However, the fate of the large endocytic vesicles is not known. Here we demonstrate that these vesicles transition to a synaptic endosome about one second after stimulation. The endosome is resolved into coated vesicles after 3 s, which in turn become small-diameter synaptic vesicles 5–6 s after stimulation. We disrupted clathrin function using RNA interference (RNAi) and found that clathrin is not required for ultrafast endocytosis but is required to generate synaptic vesicles from the endosome. Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature. In the absence of ultrafast endocytosis, synaptic vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis. These results may explain discrepancies among published experiments concerning the role of clathrin in synaptic vesicle endocytosis. Ultrastructural analysis of synaptic vesicle recycling reveals that clathrin is not required for the initial rapid step of vesicle recycling by ultrafast endocytosis at the plasma membrane and instead clathrin acts later at an endosome to regenerate synaptic vesicles; however, when ultrafast endocytosis does not occur (for example, in experiments at room temperature rather than physiological temperature), clathrin-mediated endocytosis does happen at the plasma membrane. Since the discovery of synaptic vesicle recycling in the 1970s, clathrin has been thought to act at the plasma membrane to reconstitute synaptic vesicles about 20 seconds after stimulation of the axon terminal. Erik Jorgensen and colleagues have revisited these original experiments, but combining optogenetic stimulation with high-pressure freezing to rapidly fix the samples after stimulation. This changes the picture: the previously hypothesized mechanism operates at a non-physiological, 'room temperature' of 22°C, when ultra-fast endocytosis is disrupted. At a physiological temperature (34°C), large vesicles are instead retrieved from the plasma membrane within about 50 milliseconds, through clathrin-independent, ultra-fast endocytosis; these then fuse to form endosomes from which small synaptic vesicles are regenerated by clathrin scaffolds, about 5 seconds after stimulation.

Synaptic vesicles can be released at extremely high rates, which places an extraordinary demand on the recycling machinery. Previous ultrastructural studies of vesicle recycling were conducted in dissected preparations using an intense stimulation to maximize the probability of release. Here, a single light stimulus was applied to motor neurons in intact Caenorhabditis elegans nematodes expressing channelrhodopsin, and the animals rapidly frozen. We found that docked vesicles fuse along a broad active zone in response to a single stimulus, and are replenished with a time constant of about 2 s. Endocytosis occurs within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions. These studies suggest that synaptic vesicle endocytosis may occur on a millisecond time scale following a single physiological stimulus in the intact nervous system and is unlikely to conform to current models of endocytosis. DOI: http://dx.doi.org/10.7554/eLife.00723.001, eLife digest Neurons communicate with one another at junctions called synapses. When an electrical signal travels along a neuron and arrives at a synapse, vesicles filled with small neurotransmitter molecules fuse with the cell membrane and release the neurotransmitter. These chemicals rapidly bind to receptors on the downstream neuron that induce an electrical response in that cell. Vesicles can be consumed at prodigious rates, up to 500 a second, so the cell must recover the membrane rapidly and regenerate more vesicles filled with neurotransmitter. Experiments in the 1970s and 1980s suggested that when vesicles empty their contents into the synapse, they fuse completely with the membrane and are lost. To recover the membrane, the cell forms ‘pits’, by means of a coat protein called clathrin, which then bud off into the cell as new vesicles. It takes roughly 15–20 s for vesicles to be recycled in this way. By contrast, synapses with very high firing rates are thought to recycle vesicles through a faster process known as ‘kiss and run’, in which vesicles are not fully integrated into the membrane, but instead fuse transiently with it to form a reversible pore within about a second. However, these studies triggered vesicle release using conditions that are unlikely to occur naturally inside cells. Now, Watanabe et al. have used optogenetics to study vesicle recycling in response to single stimuli at the synapse between neurons and muscles in an intact living animal, the nematode C. elegans. The worms had been genetically modified to express a light-sensitive ion channel called channelrhodopsin in their motor neurons. Watanabe et al. used a single pulse of light to stimulate vesicle release, and then rapidly froze the worms before studying their synapses with electron microscopy. They found that vesicle recycling occurred at the edges of the synapse or at a specialized structure in the middle of the synapse. Vesicle recycling took less than 50 ms—much faster than anything previously observed. This ultrafast recycling is unlikely to occur via ‘kiss and run’ since recycling occurred at sites lateral to the sites of fusion and because the recycled vesicles were larger than the originals, implying that they had not simply re-formed after a brief fusion event. By using physiologically relevant stimuli in an intact animal, Watanabe et al. reveal that vesicles can be recycled at synapses much more rapidly than previously thought, suggesting that our current models of this process may need to be reassessed. DOI: http://dx.doi.org/10.7554/eLife.00723.002

UNC119 is widely expressed among vertebrates and invertebrates. Here we report that UNC119 recognized the acylated N-terminus of the rod photoreceptor transducin α-subunit (Tα) as well as C. elegans G proteins Odr-3 and Gpa-13. The crystal structure of human UNC119 at 1.95 Å resolution revealed an immunoglobulin-like β-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. Co-crystallization of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound TαGTP inhibiting its GTPase activity, thereby providing a stable UNC119-TαGTP complex that is capable of diffusing from the inner segment back to the outer segment following light-induced translocation. UNC119 deletion in both mouse and C. elegans lead to G protein mislocalization. These results establish UNC119 as a novel Gα-subunit cofactor that is essential for G-protein trafficking in sensory cilia.

Excision of a Mos1 transposon in the germline of Caenorhabditis elegans generates a double-strand break in the chromosome. We demonstrate that breaks are most prominently repaired by gene conversion from the homolog, but also rarely by nonhomologous end-joining. In some cases, gene conversion events are resolved by crossing over. Surprisingly, expression of the transposase using an intestine-specific promoter can induce repair, raising the possibility that activation of transposase expression in somatic cells can lead to transposition of Mos1 in the germline.

Single-nucleotide polymorphism (SNP) mapping is the easiest and most reliable way to map genes in Caenorhabditis elegans. SNPs are extremely dense and usually have no associated phenotype, making them ideal markers for mapping. SNP mapping has three steps. First, recombinant mutant animals are generated over a polymorphic strain (usually CB4856) using standard genetic techniques. Second, the genotype of these animals at SNP loci is determined using one of a variety of SNP detection technologies. Third, linkage between the mutant and one or more SNPs is used to position the mutant on the chromosome relative to the SNPs. This chapter presents a detailed procedure for generating recombinant animals, for assaying SNPs using restriction enzymes, and for analyzing mapping data.

Natural Caenorhabditis elegans isolates exhibit either social or solitary feeding on bacteria. We show here that social feeding is induced by nociceptive neurons that detect adverse or stressful conditions. Ablation of the nociceptive neurons ASH and ADL transforms social animals into solitary feeders. Social feeding is probably due to the sensation of noxious chemicals by ASH and ADL neurons; it requires the genes ocr-2 and osm-9, which encode TRP-related transduction channels, and odr-4 and odr-8, which are required to localize sensory chemoreceptors to cilia. Other sensory neurons may suppress social feeding, as social feeding in ocr-2 and odr-4 mutants is restored by mutations in osm-3, a gene required for the development of 26 ciliated sensory neurons. Our data suggest a model for regulation of social feeding by opposing sensory inputs: aversive inputs to nociceptive neurons promote social feeding, whereas antagonistic inputs from neurons that express osm-3 inhibit aggregation.

The nematode pharynx has a potassium channel with unusual properties, which allows the muscles to repolarize quickly and with the proper delay. Here, the Caenorhabditis elegans exp-2 gene is shown to encode this channel. EXP-2 is a Kv-type (voltage-activated) potassium channel that has inward-rectifying properties resembling those of the structurally dissimilar human ether-à-go-go –related gene (HERG) channel. Null and gain-of-function mutations affect pharyngeal muscle excitability in ways that are consistent with the electrophysiological behavior of the channel, and thereby demonstrate a direct link between the kinetics of this unusual channel and behavior.

Ivermectin is a widely used anthelmintic drug whose nematocidal mechanism is incompletely understood. We have used Caenorhabditis elegans as a model system to understand ivermectin9s effects. We found that the M3 neurons of the C.elegans pharynx form fast inhibitory glutamatergic neuromuscular synapses. avr‐15 , a gene that confers ivermectin sensitivity on worms, is necessary postsynaptically for a functional M3 synapse and for the hyperpolarizing effect of glutamate on pharyngeal muscle. avr‐15 encodes two alternatively spliced channel subunits that share ligand binding and transmembrane domains and are members of the family of glutamate‐gated chloride channel subunits. An avr‐15 ‐encoded subunit forms a homomeric channel that is ivermectin‐sensitive and glutamate‐gated. These results indicate that: (i) an ivermectin‐sensitive chloride channel mediates fast inhibitory glutamatergic neuromuscular transmission; and (ii) a nematocidal property of ivermectin derives from its activity as an agonist of glutamate‐gated chloride channels in essential excitable cells such as those of the pharynx.

The FLP enzyme catalyzes recombination between specific target sequences in DNA. Here we use FLP to temporally and spatially control gene expression in the nematode C. elegans. Transcription is blocked by the presence of an “off cassette” between the promoter and the coding region of the desired product. The “off cassette” is composed of a transcriptional terminator flanked by FLP recognition targets (FRT). This sequence can be excised by FLP recombinase to bring together the promoter and the coding region. We have introduced two fluorescent reporters into the system: a red reporter for promoter activity prior to FLP expression and a green reporter for expression of the gene of interest after FLP expression. The constructs are designed using the multisite Gateway system, so that promoters and coding regions can be quickly mixed and matched. We demonstrate that heat-shock-driven FLP recombinase adds temporal control on top of tissue specific expression provided by the transgene promoter. In addition, the temporal switch is permanent, rather than acute, as is usually the case for heat-shock driven transgenes. Finally, FLP expression can be driven by a tissue specific promoter to provide expression in a subset of cells that can only be addressed as the intersection of two available promoters. As a test of the system, we have driven the light chain of tetanus toxin, a protease that cleaves the synaptic vesicle protein synaptobrevin. We show that we can use this to inactivate synaptic transmission in all neurons or a subset of neurons in a FLP-dependent manner., Author Summary Genes turn on and off as a natural part of development. The nematode C. elegans has been an important model system for studying the roles of genes in animal development and physiology. However, worm researchers have had a limited toolkit for controlling gene activation. These drawbacks have been particularly restrictive when studying the function of a gene that has different roles in several cell types or at different times in development. Here we describe a way to turn any gene on at a specific time in specific cells. We provide a set of mix-and-match reagents that give researchers a way to quickly build new combinations of regulatory elements. These reagents will allow researchers to express a single gene in a wide array of temporal or spatial patterns, or to serially express many genes in a single cell. As a proof of principle, we made an artificial worm gene composed of a neurotoxin that would block neurotransmission. When we activated the gene in a small number of neurons in adult animals, these cells ceased to function. We anticipate that this new technique will find a wide variety of uses by the C. elegans community.

BackgroundInC. elegans, single nucleotide polymorphisms (SNPs) can function as silent genetic markers, with applications ranging from classical two- and three-factor mapping to measuring recombination across whole chromosomes.ResultsHere, we describe a set of 48 primer pairs that flank SNPs evenly spaced across theC. elegansgenome and that work under identical PCR conditions. Each SNP in this set alters aDraI site, enabling rapid and parallel scoring. We describe a procedure using these reagents to quickly and reliably map mutations. We show that these techniques correctly map a known gene,dpy-5. We then use these techniques to map mutations in an uncharacterized strain, and show that its behavioral phenotype can be simultaneously mapped to three loci.ConclusionTogether, the reagents and methods described represent a significant advance in the accurate, rapid and inexpensive mapping of genes inC. elegans.