Currently transgenes in C. elegans are generated by injecting DNA into the germline. The DNA assembles into a semi-stable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method called MosSCI (Mos1-mediated Single Copy Insertion) that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double strand break in non-coding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single copy insertions can be obtained in less than two weeks by injecting approximately twenty animals. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.

To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated ~20 s after fusion by the assembly of clathrin scaffolds or in ~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 with 50-100 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 rapidly restore the surface area of the membrane.

We describe an electrophysiological preparation of the neuromuscular junction of the nematode C. elegans, which adds to its considerable genetic and genomic resources. Mutant analysis, pharmacology and patch-clamp recording showed that the body wall muscles of wild-type animals expressed a GABA receptor and two acetylcholine receptors. The muscle GABA response was abolished in animals lacking the GABA receptor gene unc-49. One acetylcholine receptor was activated by the nematocide levamisole. This response was eliminated in mutants lacking either the unc-38 or unc-29 genes, which encode alpha and non-alpha acetylcholine receptor subunits, respectively. The second, previously undescribed, acetylcholine receptor was activated by nicotine, desensitized rapidly and was selectively blocked by dihydro-beta-erythroidine, thus explaining the residual motility of unc-38 and unc-29 mutants. By recording spontaneous endogenous currents and selectively eliminating each of these receptors, we demonstrated that all three receptor types function at neuromuscular synapses.