One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction

Abstract

We describe an electrophysiological preparation of the neuromuscular junction of the nematode C. elegans, which adds to its considerable genetic and genomic resources. Mutant analysis, pharmacology and patch-clamp recording showed that the body wall muscles of wild-type animals expressed a GABA receptor and two acetylcholine receptors. The muscle GABA response was abolished in animals lacking the GABA receptor gene unc-49. One acetylcholine receptor was activated by the nematocide levamisole. This response was eliminated in mutants lacking either the unc-38 or unc-29 genes, which encode alpha and non-alpha acetylcholine receptor subunits, respectively. The second, previously undescribed, acetylcholine receptor was activated by nicotine, desensitized rapidly and was selectively blocked by dihydro-β-erythroidine, thus explaining the residual motility of unc-38 and unc-29 mutants. By recording spontaneous endogenous currents and selectively eliminating each of these receptors, we demonstrated that all three receptor types function at neuromuscular synapses.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: C. elegans neuromuscular preparation.
Figure 2: Voltage-gated potassium and calcium currents are present in body wall muscles.
Figure 3: A functional GABA receptor is expressed in the muscle and encoded by the unc-49 gene.
Figure 4: The levamisole-sensitive current is abolished in unc-38(e264) and unc-29(e1072) mutants.
Figure 5: A nicotine-sensitive, rapidly desensitizing acetylcholine receptor is expressed in the muscle.
Figure 6: The nicotine-sensitive acetylcholine receptor is selectively blocked by the nicotinic antagonist dihydro-β-erythroidine (DHβE, 5 μM).
Figure 7: All three receptors are functionally active at the synapse.

References

  1. 1

    Chalfie, M. & Jorgensen, E. M. C. elegans neuroscience: genetics to genome. Trends Genet. 14, 506–512 (1998).

  2. 2

    Jorgensen, E. M. & Nonet, M. L. Neuromuscular junctions in the nematode C. elegans. Dev. Biol. 6, 207–220 (1995).

  3. 3

    Raizen, D. M. & Avery, L. Electrical activity and behavior in the pharynx of Caenorhabditis elegans. Neuron 12, 483–495 (1994).

  4. 4

    Nonet, M. L. et al. Caenorhabditis elegans rab-3 mutant synapses exhibit impaired function and are partially depleted of vesicles. J. Neurosci. 17, 8061–8073 (1997).

  5. 5

    Tempia, F., Bravin, M. & Strata, P. Postsynaptic currents and short-term synaptic plasticity in Purkinje cells grafted onto an uninjured adult cerebellar cortex. Eur. J. Neurosci. 8, 2690–2701 (1996).

  6. 6

    Eshhar, N., Petralia, R. S., Winters, C. A., Niedzielski, A. S. & Wenthold, R. J. The segregation and expression of glutamate receptor subunits in cultured hippocampal neurons. Neuroscience 57, 943–964 (1993).

  7. 7

    Nusser, Z., Sieghart, W. & Somogyi, P. Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. J. Neurosci. 18, 1693–1703 (1998).

  8. 8

    Sillar, K. T. & Roberts, A. Segregation of NMDA and non-NMDA receptors at separate synaptic contacts: evidence from spontaneous EPSPs in Xenopus embryo spinal neurons. Brain Res. 545, 24–32 (1991).

  9. 9

    Nimnual, A. S., Chang, N. S., Ross, A. F., Gelman, M. S. & Prives, J. M. Identification of phosphorylation sites on AChR delta-subunit associated with dispersal of AChR clusters on the surface of muscle cells. Biochemistry 37, 14823–14832 (1998).

  10. 10

    Nishizaki, T. & Sumikawa, K. Effects of PKC and PKA phosphorylation on desnsitization of nicotinic acetylcholine receptors. Brain Res. 812, 242–245 (1998).

  11. 11

    Paradiso, K. & Brehm, P. Long-term desensitization of nicotinic acteylcholine receptors is regulated via protein kinase A-mediated phosphorylation. J. Neurosci. 18, 9227– 9237 (1998).

  12. 12

    Liao, D., Hessler, N. A. & Malinow, R. Activation of postsynaptically silent synapses during pairing-induced LTP in CA1 region of hippocampal slice. Nature 375, 400–404 (1995).

  13. 13

    De Koninck, P. & Cooper, E. Differential regulation of neuronal nicotinic ACh receptor subunit genes in cultured neonatal rat sympathetic neurons: specific induction of alpha 7 by membrane depolarization through a Ca2+/calmodulin-dependent kinase pathway. J. Neurosci. 15, 7966–7978 (1995).

  14. 14

    McIntire, S. L., Jorgensen, E. & Horvitz, H. R. Genes required for GABA function in Caenorhabditis elegans. Nature 364, 334– 337 (1993).

  15. 15

    McIntire, S. L., Jorgensen, E., Kaplan, J. & Horvitz, H. R. The GABAergic nervous system of Caenorhabditis elegans. Nature 364, 337–341 (1993).

  16. 16

    Lewis, J. A., Wu, C. H., Levine, J. H. & Berg, H. Levamisole-resistant mutants of the nematode Caenorhabditis elegans appear to lack pharmacological acetylcholine receptors. Neuroscience 5, 967–989 (1980).

  17. 17

    Bamber, B. A., Beg, A. A., Twyman, R. E. & Jorgensen, E. M. The C. elegans unc-49 locus encodes multiple subunits of a heteromultimeric GABA receptor. J. Neurosci. 19, 5348– 5359 (1999).

  18. 18

    Lewis, J. A. et al. Cholinergic receptor mutants of the nematode Caenorhabditis elegans. J. Neurosci. 7, 3059– 3071 (1987).

  19. 19

    Lackner, M. R., Kornfeld, K., Miller, L. M., Horvitz, H. R. & Kim, S. K. A MAP kinase homolog, mpk-1, is involved in ras-mediated induction of vulval cell fates in Caenorhabditis elegans. Genes Devel. 8, 160– 173 (1994).

  20. 20

    Fleming, J. T. et al. Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits. J. Neurosci. 17, 5843–5857 (1997).

  21. 21

    Lewis, J. A., Wu, C. H., Berg, H. & Levine, J. H. The genetics of levamisole resistance in the nematode Caenorhabditis elegans. Genetics 95, 905–928 (1980).

  22. 22

    Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

  23. 23

    Robertson, S. J. & Martin, R. J. Levamisole-activated single-channel currents from muscle of the nematode parasite Ascaris suum. Br. J. Pharmacol. 108, 170– 178 (1993).

  24. 24

    Zoli, M., Lena, C., Picciotto, M. R. & Changeux, J. P. Identification of four classes of brain nicotinic receptors using β2 mutant mice. J. Neurosci. 18, 4461– 4472 (1998).

  25. 25

    Walrond, J. P. & Stretton, A. O. Excitatory and inhibitory activity in the dorsal musculature of the nematode Ascaris evoked by single dorsal excitatory motonerons. J. Neurosci. 5, 16–22 (1985).

  26. 26

    Moss, B. L. & Role, L. W. Enhanced ACh sensitivity is accompanied by changes in ACh receptor channel properties and segregation of ACh receptor subtypes on sympathetic neurons during innervation in vivo. J. Neurosci. 13, 13–28 (1993).

  27. 27

    Hartman, D. S. & Claudio, T. Coexpression of two distinct muscle acetylcholine receptor alpha-subunits during development. Nature 343, 372–375 (1990).

  28. 28

    Owens, J. L. & Kullberg, R. Three conductance classes of nicotinic acetylcholine receptors are expressed in developing amphibian skeletal muscle. J. Neurosci. 9, 2575–2580 (1989).

  29. 29

    Gardette, R., Listerud, M. D., Brussard, A. B. & Role, L. W. Developmental changes in transmitter sensitivity and synaptic transmission in embryonic chicken sympathetic neurons innervated in vitro. Dev. Biol. 147, 83–95 (1991).

  30. 30

    Hume, R. I. & Honig, M. G. Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons. J. Neurobiol. 22, 249– 262 (1991).

  31. 31

    Role, L. W. Diversity in primary structure and function of neuronal nicotinic acetylcholine receptor channels. Curr. Opin. Neurobiol. 2, 254–262 (1992).

  32. 32

    Byerly, L. & Masuda, M. O. Voltage-clamp analysis of the potassium current that produces a negative-going action potential in Ascaris muscle. J. Physiol. (Lond.) 288, 263–284 (1979).

Download references

Acknowledgements

The authors thank K. Broadie, T. Fergestad and D.E. Featherstone for technical assistance, L. Avery and A.V. Maricq for discussions, M. Hammarlund for providing the F21D12.3-GFP strain, K. Knobel for confocal images and K. Broadie and D.E. Featherstone for reading the manuscript. This work was supported by NIH grant RO3 MHS9820-01 (J.E.R.) and the Damon Runyon Fund (E.M.J.).

Author information

Correspondence to Janet E. Richmond.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Richmond, J., Jorgensen, E. One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nat Neurosci 2, 791–797 (1999). https://doi.org/10.1038/12160

Download citation

Further reading