Place: 4274 Chamberlin (refreshments will be served)
Speaker: Tony Stretton, UW Department of Zoology
Abstract: Numerically, nematodes have very simple nervous systems. The female parasitic nematode Ascaris suum has only 298 neurons, and the hermaphroditic free-living Caenorhabditis elegans has 302. A. suum is large (ca 35 cm), and has large neurons suitable for electrophysiological recording. We assembled a functional circuit from the morphological synapses, scored by electron microscopy, and the physiological properties of the neurons and their synapses. The predicted activity of this circuit matched that actually recorded from neurons in dissected preparations that were opened to allow microelectrode penetration. However, it differed dramatically from the activity recorded from these same neurons in semi-intact behaving preparations. Something was missing from the circuit description. We have now shown that there are numerous neuropeptides (at least 250) present in A. suum, and the ones we have sequenced have potent activity on individual neurons. We think that they were washed out of the dissected preparations, thus losing their modulatory activity on individual neurons. For peptide identification, initially peptides were purified by HPLC and sequenced by Edman degradation. Now we are using mass spectrometry, which has speeded up the discovery process more than one hundred-fold. In particular, we are now dissecting single identified neurons and subjecting them to MALDI-TOF MS and tandem MS for sequence determination. All neurons examined so far contain peptides. Most contain previously unknown peptides, and the unknown peptides often outnumber the known peptides. This is a powerful method of peptide discovery. It has the distinct advantage that it simultaneously solves the identity and the cellular expression of the peptide. It also has the advantage that it identifies the peptide actually expressed by a particular neuron, rather than relying on predictions from cDNA or genomic DNA sequences, and on reporter constructs for expression patterns. Neuropeptides are processed from precursor proteins, and the rules of the proteolytic cleavage are not yet robust enough for accurate prediction of processing.