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. 2021 Nov 5;10:e67723. doi: 10.7554/eLife.67723

Figure 1. Two RIM neurotransmitters affect spontaneous locomotion.

(A) RIM synapses with interneurons, motor neurons, and muscle implicated in spontaneous foraging behavior (Cook et al., 2019; White et al., 1986). Colors of neurons indicate associated locomotor states based on neural manipulations and functional calcium imaging (Alkema et al., 2005; Gray et al., 2005; Kato et al., 2015; Li et al., 2014; Pokala et al., 2014; Steuer Costa et al., 2019; Wang et al., 2020; Zheng et al., 1999). (B) Off-food foraging assay. (C) Mean reversals per minute of wild-type animals in foraging assays. Vertical lines indicate standard error of the mean. Gray shaded boxes indicate local search (4–8 min off food) and global search (36–40 min off food) intervals analyzed in subsequent figures. n = 324. (D) RIM neurotransmitter mutants. RIM glu KO: RIM-specific knockout of the vesicular glutamate transporter EAT-4 (Figure 1—figure supplement 1). tdc-1, tyrosine decarboxylase mutant, which lacks tyramine in RIM and octopamine in RIC. (E) Ethograms of 50 randomly chosen tracks per genotype during minute 5–6 of local search. Color code: white, forward runs; black, reversals; yellow, omega turns coupled to a reversal; gray, pauses, shallow turns, and omega turns that were not preceded by a reversal.

Figure 1.

Figure 1—figure supplement 1. CRISPR-Cas9-generated alleles enable RIM-specific glutamate transporter knockout.

Figure 1—figure supplement 1.

(A) Schematic of the sources of glutamate and tyramine in the C. elegans nervous system. Adapted from Pereira et al., 2015. (B) Schematic of cell-specific glutamate transporter knockout genetic strategy. Using CRISPR-Cas9, an FRT site was inserted immediately before the start codon of eat-4 (VGLUT) and let-858 3′-UTR::FRT::mCherry immediately after the stop codon of eat-4. let-858 3′-UTR stops transcription so mCherry is not expressed. To knock out glutamate release in this edited strain, nuclear-localized flippase (nFLP) was expressed under a tdc-1 promoter. The intersection of tdc-1 and eat-4 expression is limited to RIM, leading to excision of the eat-4 ORF in RIM, confirmed by mCherry expression in the targeted cells. (C) Validation of CRISPR-Cas9 recombination. Top panel: differential interference contrast (DIC) (left), mCherry fluorescence (middle), and merged (right) images of animals with edited endogenous eat-4 (VGLUT) locus. In the edited strain, there is no mCherry expression, confirming that the let-858 3′-UTR stops transcription. Bottom: DIC (left), mCherry (middle), and merged (right) images for edited eat-4 strain following RIM-specific nFLP expression (tdc-1p::nFLP). mCherry is expressed only in RIM neurons, the intersection of eat-4 and tdc-1 expression. mCherry specificity for RIM was confirmed by screening 30 animals. Scale bar is 20 μM.