Worm Breeder's Gazette 13(2): 70 (February 1, 1994)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We are interested in the molecular mechanisms that neurons use to control the release of neurotransmitter by synaptic vesicles. This problem has been made tractable in C. elegans by the ability to isolate synaptic transmission mutants based on resistance to inhibitors of cholinesterase, such as aldicarb (Alfonso and Rand, WBG 10(3):48). Resistance to aldicarb could result from pre- or postsynaptic defects. The 18 recessive, aldicarb resistant genes identified to date include at least 5 encoding known presynaptic proteins: snt-1 (synaptotagmin), unc-18 , unc-104 , cha-1 and unc-17 .We have been trying to determine if mutations in any of the other 13 genes result in presynaptic defects. A new mutation, md250 ,was mapped to the interval between the right ends of sDf22 and sDf21 on LG IV and failed to complement unc-26 .No other alleles of unc-26 had previously been isolated in the aldicarb screen although multiple alleles of each of the other aldicarb resistant mutants were found. This could be due to weaker resistance, and we have indeed found that unc-26 ( md250 )is less resistant to aldicarb than the five known presynaptic mutants.
unc-26 ,however, has other phenotypes more closely resembling presynaptic mutants. Behaviorally, it is often found in a kinked or coiled posture and is strongly pumping defective (both characteristic of cholinergic defects), and is also a shrinker (suggestive of a GABA transmission defect). In addition, evidence for a defect in neuronal function in unc-26 mutants has been obtained through analysis of postsynaptic currents induced by the M3 and MC neurons of the pharyngeal nervous system (Raizen and Avery, WBG, this issue). To determine if unc-26 mutants have a defect in cholinergic transmission, we quantitated acetylcholine (ACh) in asynchronous populations of unc-26 ( md250 )and found a 5.1 fold elevation (+/- 1.9) relative to N2 when normalized to mg protein. Since all or part of this difference could be due to the larger size of N2 relative to its nervous system, a separate experiment was done with synchronous young adults, using unc-26 ( e1196 ).In this case ACh levels were normalized to the number of animals, and e1196 was found to have 39.9% +/-12.2% more ACh/ worm than N2 .It is unclear however, from this analysis, whether the accumulated ACh was cytosolic or in synaptic vesicles. To determine if unc-26 mutants have a GABA transmission defect, we quantitated the expulsion failure rate in 8 unc-26 alleles, since the expulsion step of the defecation cycle is mediated by GABA (McIntire et al, Nature 364:334, 364:337). 3-10 synchronous young adults were examined for 12 cycles each. Two alleles, md250 and e1 O48,showed expulsion failure rates of 35% (120 cycles, n=10) and 24% (36 cycles, n=3) respectively, while the other 6 alleles tested had a much lower failure rate (0-8% over 36 cycles, n=3). The controls, N2 and unc-17 ( e245 )(cholinergic specific), had failure rates of 0.8% (120 cycles, n=10) and 0% (120 cycles, n=10) respectively. We have also examined the distribution of cholinergic synaptic vesicles in several unc-26 alleles and N2 using a monoclonal antibody to the synaptic vesicle ACh transporter, UNC-17 ,as a marker for synaptic vesicles (courtesy of Janet Duerr). By immunofluorescence staining we found no obvious differences in the intensity or distribution of synaptic vesicles. Since many subcellular differences could be missed by light microscopy, unc-26 mutants are currently being examined by electron microscopy (Erik Jorgensen, personal communication).
With the exception of the intragenic mapping of 11 unc-26 alleles along with their ranking in an allelic series by Baillie and colleagues (Mol. Gen. Genet. 221:459), analysis of the unc-26 gene has lagged until recently, when Yuan et al (Cell 75:641) mapped Bristol/Bergerac RFLPs relative to ced-3 and unc-26 To complement ongoing phenotypic analysis and further define the role of UNC-26 ,we are proceeding to clone the gene. We are currently probing Southern blots of unc-26 alleles with cosmids identified by the RFLP mapping as well as attempting transformation rescue. Spontaneous alleles and revertants from tranpositionally active strains are also available (courtesy of Bob Horvitz and Dave Baillie) and are being outcrossed to identify Tc1 insertions in the unc-26 gene.