Rose, Jacqueline et al. (2015) International Worm Meeting "Exposure to Nicotine or the Putative GABA agonist Toluene Differentially Modulates Expression of GABA Signaling Elements and Alters Mobility in C. elegans. ."

Stankowicz, Nicole, Leonti, Amanda, Stafford, Parker, Rose, Jacqueline

[International Worm Meeting, 2015]

In mammalian studies of nicotine addiction, nicotinic acetylcholine receptor activation reportedly acts to disinhibit reward signaling by suppressing GABA release (D'Souza and Markou, 2011). As well, it has been recently reported that a subset of neurons in the rodent hippocampus co-release GABA and acetylcholine (Saunders et al, 2015). Thus, it is of interest to describe how nicotinic receptor activation and GABA signaling may interact in neurons. C. elegans is an ideal model as both GABA and acetylcholine receptors are co-expressed at the NMJ. Mobility resulting from NMJ signaling has been well-described as relying on alternating activation of cholinergic and GABA receptors to produce contraction and simultaneous relaxation of opposing body wall muscles, respectively. Thus, the C. elegans NMJ is a site where direct interaction between excitatory cholinergic and inhibitory GABAergic signaling likely occurs. Studies from other labs confirm that cholinergic NMJ signaling can be modulated, seen as altered surface expression of acr-16 nicotinic Ach receptors (Jensen et al., 2012). Previous studies from this lab have quantified changes in nicotinic Ach and GABA receptor transcripts in adult worms following exposure to nicotine during development (Rose et al., 2013). More recently, we have found in adult worms that application of nicotine (30 microM) for 24 hours significantly increases UNC-49::GFP GABA receptor expression (p<0.05) while SNB-1::GFP synaptobrevin expression in motor neurons appears relatively unchanged. Conversely, exposure to the inhalant drug toluene (a GABA agonist) results in increased SNB-1::GFP expression in motor neurons (p<0.05) with seemingly no change in UNC-49::GFP expression. This increase in presynaptic GABA elements was further supported using qRT-PCR where a significant increase in unc-47 (vesicular GABA transporter) transcripts was found (p<0.01). With regards to mobility, these differences in expression appear to correlate with decreased swim motion (measured as body bends) following application of either nicotine or toluene (p<0.05 compared to controls). Interestingly, application of nicotine following toluene exposure seems to rescue the toluene-induced decrease in mobility. This suggests that the effects of one drug may offset the effects of the other potentially by returning to balance the drug altered pre- and post-synaptic GABA signaling elements. Taken together, these results demonstrate a potential role for GABA in both nicotine and toluene addiction. Funding support from WWU Research and Sponsored Programs Project Development Award..

H Van Luenen et al. (1999) International C. elegans Meeting "World-wide worm SNPs: Dissecting the molecular evolution of the species using a high density SNP map"

R Plasterk, R Koch, H Van Luenen

[International C. elegans Meeting, 1999]

We would like to reconstruct the history of the C. elegans species at the genome level, therefore we sampled the genomes of four natural isolates (strain CB4857 isolated in Claremont, California, RC301 from Freiburg, Germany, TR403 from Madison, Wisconsin and AB1 from Adelaide, Australia) for single nucleotide polymorphisms (SNPs). Random genomic DNA fragments from the 4 strains were shotgun cloned and sequenced. There was no selection for transcribed or non-transcribed regions of the genome. In total we sequenced 1572 clones resulting in over 1 Mb of sequence information. The sequences are compared to the canonical Bristol N2 sequence to ask the question whether the clone maps to a unique sequence, and -if so- whether it contains polymorphisms. Once a SNP is identified we check other strains for the presence of the same polymorphism by PCR amplification and sequence analysis. In an initial experiment we found approximately one SNP per 3000 bp sequenced. The SNPs are randomly spread over the genome. Based on these observations we expect to find approximately 500 SNPs, one in every 200 kb. In the initial experiment we found, as expected, that several SNPs initially detected in one strain were also present in some but not all other strains. For example: a T in the Australian AB1, is a G at the same position in Bristol N2 in cosmid K10D2 at position 27946, and we found it to be like AB1 in the Californian CB4857 strain and the German RC301 strain, while the TR403 strain from Wisconsin resembles the Bristol N2 strain. Thus different patches of the genome have different ancestors. With our high density SNP map we will generate a genome map for each isolate which will show how each genome is patched together from a limited set of parental strains. The SNP's will be added to ACeDB, and can also be used as markers on the genetic map. They can be recognised by PCR followed by sequencing, but we also found that the SNPs we looked at could be visualised by SSCP analysis. We thank Jane Rogers and Amanda McMurray for their assistance in sequencing the clones.