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Journal of Experimental Biology, Vol 202, Issue 4 333-341, Copyright © 1999 by Company of Biologists

JOURNAL ARTICLES

Single-cell analyses of nitrergic neurons in simple nervous systems

LL Moroz, R Gillette and JV Sweedler
Department of Chemistry and Beckman Institute, Department of Molecular and Integrative Physiology, University of Illinois, Urbana, IL 61801, USA. moroz@whitney.ufl.edu

Understanding the role of the gaseous messenger nitric oxide (NO) in the nervous system is complicated by the heterogeneity of its nerve cells; analyses carried out at the single cell level are therefore important, if not critical. Some invertebrate preparations, most especially those from the gastropod molluscs, provide large, hardy and identified neurons that are useful both for the development of analytical methodologies and for cellular analyses of NO metabolism and its actions. Recent modifications of capillary electrophoresis (CE) allow the use of a small fraction of an individual neuron to perform direct, quantitative and simultaneous assays of the major metabolites of the NO-citrulline cycle and associated biochemical pathways. These chemical species include the products of NO oxidation (NO2-/NO3-), l-arginine, l-citrulline, l-ornithine, l-argininosuccinate, as well as selected NO synthase inhibitors and cofactors such as NADPH, biopterin, FMN and FAD. Diverse cotransmitters can also be identified in the same nitrergic neuron. The sensitivity of CE methods is in the femtomole to attomole range, depending on the species analysed and on the specific detector used. CE analysis can be combined with prior in vivo electrophysiological and pharmacological manipulations and measurements to yield multiple physiological and biochemical values from single cells. The methodologies and instrumentation developed and tested using the convenient molluscan cell model can be adapted to the smaller and more delicate neurons of other invertebrates and chordates.

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