Lateral Facilitation between Primary Mechanosensory Neurons Controls Nose Touch Perception in C. elegans

Summary The nematode C. elegans senses head and nose touch using multiple classes of mechanoreceptor neurons that are electrically coupled through a network of gap junctions. Using in vivo neuroimaging, we have found that multidendritic nociceptors in the head respond to harsh touch throughout their receptive field but respond to gentle touch only at the tip of the nose. Whereas the harsh touch response depends solely on cell-autonomous mechanosensory channels, gentle nose touch responses require facilitation by additional nose touch mechanoreceptors, which couple electrically to the nociceptors in a hub-and-spoke gap junction network. Conversely, nociceptor activity indirectly facilitates activation of the nose touch neurons, demonstrating that information flow across the network is bidirectional. Thus, a simple gap-junction circuit acts as a coincidence detector that allows primary sensory neurons to integrate information from neighboring mechanoreceptors and generate somatosensory perception.


Generation of FLP/PVD cameleon line ljEX19
The egl-46 promoter region was obtained from plasmid TU#307 (Wu et al., 2001), a gift from the lab of Martin Chalfie. A 3 kb HinDIII/NotI fragment was fused to cameleon YC2.3 in the vector pPD95.75 (A. Fire). Transgenic lines were obtained by germline injection of a lin-15(n765) mutant strain with the egl-46::YC2.3 plasmid at a concentration of 50 ng/µl along with lin-15(+) genomic DNA (30 ng/µl) as a coinjection marker. Once a stable transgenic line was obtained, the lin-15(n765) allele was then removed by backcrossing to wild-type (N2) animals.

Transgenic rescue lines:
The egl-46 promoter was obtained from plasmid TU#307 (Wu et al., 2001), a kind gift from the Chalfie Lab. Using the primers GGCCTTCTGAAATCAAAACG and AGTTCACGCCAGATGCAAGATG we PCR amplified a 3 kb fragment which was cloned into a Gateway 3-way P4-P1R donor vector. The ocr-4 promoter we used was a 4.8kb fragment previsously described by Kindt et al (Kindt et al., 2007b). A 3 kb sra-6 promoter fragment cloned into a Gateway P4-P1R vector was obtained in the form of plasmid MGW1.2, a gift by E. Busch from the de Bono group. The 5' end of the promoter is defined by the sequnce 5'-CTTTTAGATATAATAAATCGAAATTG-3' while the 3' end is defined by the sequence 5'-GGCAAAATCTGAAATAATAAATATTAAATTC-3' adjacent of the start site of sra-6.
A trpa-1 3.6 kb cDNA fragment was amplified from a plasmid generated previously (Kindt et al., 2007b) using the primers 5'-ATGTCGAAGAAATCATTAGG-3' and 5'-TCAGTTATCTTTCTCCTCAAGT-3'. A 2.2 kb mec-10 cDNA was amplified using Qiagen OneStep RT-PCR Kit from an RNA library using the primers 5'-ATGAATCGAAACCCGCGAATG-3' and 5'-TCAATACTCATTTGCAGCATTTTC-3'. In order to generate rescue plasmids, we used the MultiSite Gateway Three Fragment Vector Construction Kit (Invitrogen) to fuse promoter, ORF and an unc-54 3'UTR. All fragments generated by PCR were sequenced after cloning, and any mutations that were detected were rectified using the site-directed mutagenesis kit: QuickChange Lightning Site-Directed Mutagenesis Kit, from Stratagene.
To generate arrays ljEx381 and ljEx382 we injected mec-10(tm1552) with egl-46::mec-10(cDNA) at 75 ng/µl and elt-2::RFP at 25 ng/µl. Two independent arrays were obtained and tested for rescue of the behavioral defect; one of these was subsequently crossed into the various calcium indicator strains that we used for imaging. To genotype the animals that came out of the crosses we used primers in the introns flanking the tm1552 deletion.
The unc-7(e5) allele was crossed with ljIs104[cat-1::YCD3] to image from RIH and with ljEx19 ]. To generate rescue lines for unc-7 we co-injected egl-46::unc-7(cDNA) and cat-1::unc-7(cDNA) transgenes at a final concentration of 50 ng/µl each along with 25 ng/µl elt-2::RFP. Two independent arrays were obtained, ljEx375 and ljEx376, and ljEx375 was crossed into the appropriate imaging lines. In order to genotype the animals that came out of the crosses we used primers in the introns flanking the e5 point mutation to generate a PCR fragment which was subsequently sequenced. The "promoter only" controls were injected as follows: cat-1 promoter was injected at 50 ng/µl with elt-2::RFP at 25 ng/µl, the egl-46 promoter at 50ng/µl with 25 ng/µl of elt-2::RFP and cat-1;elg-46 at a final concentration of 50 ng/µl with 25ng/µl elt-2::RFP.
All empty promoters were injected in an unc-7(e5) background.

A-D. Averaged calcium responses to gentle body touch in the ALMs. Animals
expressing cameleon in the ALM gentle body touch neurons were given a 1 second buzz stimulus on the anterior body as described (Suzuki et al., 2003). Each red trace represents the average percentage change in R/R 0 for the indicated genotype, where R is the fluorescence emission ratio at a given time point and R 0 is its initial value. 11 animals per genotype were imaged. E. Scatter plot of peak calcium respones for each stimulus. The red line indicates mean ratio change; the error bars indicate SEM. C) Figure S5: sra-6::osm-9 rescues ASH-mediated osmotic avoidance behavior. Wildtype,, and osm-9(ky10); sra-6::osm-9(genomic) animals were tested for escape behavior evoked by 1M glycerol as described (Hilliard et al., 2002). Each data point represents 50 worms assayed on three independent days. Error bars show S.E.M.
*** indicates a significant difference between mutant and rescued strains according to the chi-square test (p <0.001).  Figure S7. 9 animals were imaged for each control strain. B. Nose touch escape behavior "Empty" plasmids containing only the indicated promoters were injected into unc-7 animals at the same concentrations as the rescue constructs in Figure S7. 100 animals were assayed on 3 independent days. Error bars indicate SEM.