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Method Article
Neurexins and neuroligins are membrane-neuron adhesion proteins which perform essential roles in synaptic differentiation and transmission. Neuroligin deficient mutants of C. elegans are defective in detecting osmotic strength, but when they also contain a mutation in the gene coding neurexin, they recover the wild type phenotype.
Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network function1. These proteins are found at the presynaptic and postsynaptic membranes 2. Studies in mice indicate that neurexins and neurologins have an essential role in synaptic transmission 1. Recent reports have shown that altered neuronal connections during the development of the human nervous system could constitute the basis of the etiology of numerous cases of autism spectrum disorders 3.
Caenorhabditis elegans could be used as an experimental tool to facilitate the study of the functioning of synaptic components, because of its simplicity for laboratory experimentation, and given that its nervous system and synaptic wiring has been fully characterized. In C. elegans nrx-1 and nlg-1 genes are orthologous to human NRXN1 and NLGN1 genes which encode alpha-neurexin-1 and neuroligin-1 proteins, respectively. In humans and nematodes, the organization of neurexins and neuroligins is similar in respect to functional domains.
The head of the nematode contains the amphid, a sensory organ of the nematode, which mediates responses to different stimuli, including osmotic strength. The amphid is made of 12 sensory bipolar neurons with ciliated dendrites and one presynaptic terminal axon 4. Two of these neurons, named ASHR and ASHL are particularly important in osmotic sensory function, detecting water-soluble repellents with high osmotic strength 5. The dendrites of these two neurons lengthen to the tip of the mouth and the axons extend to the nerve ring, where they make synaptic connections with other neurons determining the behavioral response 6.
To evaluate the implications of neurexin and neuroligin in high osmotic strength avoidance, we show the different response of C. elegans mutants defective in nrx-1 and nlg-1 genes, using a method based on a 4M fructose ring 7. The behavioral phenotypes were confirmed using specific RNAi clones 8. In C. elegans, the dsRNA required to trigger RNAi can be administered by feeding 9. The delivery of dsRNA through food induces the RNAi interference of the gene of interest thus allowing the identification of genetic components and network pathways.
1: Osmotic avoidance assay.
2: Generation of knockdown worms by RNAi feeding.
3: Strains used.
The C. elegans strains used in this work are shown in Table 1. Mutant strains were out-crossed against N2 wild-type at least four times to remove unwanted random mutations that could have been generated during the mutagenesis protocol.
OP50 E. coli strain was suppliied by Caenorhabditis Genetic Center, University of Minnesota, USA. E.coli HT115 (DE3) with the plasmid pL4440 carrying nrx-1 (JA:C29A12.5) and nlg-1 (JA:C40C9.5) gene fragments were provided by Dr. Peter Askjaer, Centro Andaluz de Biología del Desarrollo (CABD), CSIC Universidad Pablo Olavide, Sevilla, Spain.
4: Representative results.
Illustrative results are represented in Figures 1 and 2. Ten animals of each strain and a minimum of three replica experiments were carried out.
Figure 1. Experiments of osmotic avoidance behavior.
Control strains and nrx-1 (ok1649 and tm1961)V deficient mutantworms respond by reversing backwards when they encounter an osmotic barrier (4M fructose). nlg-1 (ok259 and tm474) X mutants fail to detect this barrier. Double mutants deficient in nlg-1 and nrx-1, strains CRR21 (ok1649 ; ok259)VX and CRR23 (tm1961; ok259)VX recovered the wild type phenotype. The * indicates significant differences (P ≤ 0.001) by t-student test, in the response of each strain to the Bristol N2 wild-type by t-student test
Figure 2. Experiments with knockdown worms by RNAi feeding.
E.coli HT115 (DE3) transformed with empty pL4440 vector or containing a fragment corresponding to the target genes nrx-1 or nlg-1 were used to fed different worm strains. The * indicates significant differences (P ≤ 0.001) by t-student test, in the response of the N2 strain fed with bacteria carrying the pL4440 vector with a fragment targeting the nlg-1 gene in comparison with the N2 strain fed with empty pL4440 vector.
Neurexins and neuroligins perform essential roles in synaptic transmission11 and differentiation of synaptic connections 12. Both molecules have been identified as candidate genes for autism 13,14.
In this video we show a simple method which allows us to study the effect of genes affecting the osmotic avoidance response in C. elegans. Neuroligin deficient mutants are defective in detecting osmotic strength; but mutants deficient in neurexin show a res...
We would like to thanks Dr Antonio Miranda-Vizuete for his valuable help. We also like to express our gratitude to Salma Boulayoune and Isabel Caballero for valuable technical assistance. This work was financed by a grant from the Junta de Andalucıa (BIO-272). This research has been carried out in agreement with current laws governing genetic experimentation in Europe.
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Table 1. C. elegans strains.
a. Caenorhabditis Genetic Center, University of Minnesota, USA. |
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