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Symposium Reports |
1 Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK
(Received 20 December 2006;
accepted after revision 2 January 2007; first published online 4 January 2007)
Corresponding author J. Deuchars: Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK. Email: j.deuchars{at}leeds.ac.uk
Since nitric oxide was revealed as a gaseous transcellular signalling molecule there has been a huge volume of research dedicated to uncovering its physiological functions, as well as to how it may contribute to pathophysiological scenarios. A PubMed search on ‘nitric oxide’ retrieves nearly 80 000 hits (111 in Experimental Physiology alone!), whilst limiting the search with the addition of ‘brain’ reveals nearly 10 000 references (conducted 12.12.06). Despite these efforts an apparent puzzle remains in the CNS-NO influences both excitatory and inhibitory transmission, even within the same local area. Is it sensible that NO acts to cause release of multiple transmitters simultaneously? This scenario appears to occur in the nucleus tractus solitarii (NTS), an important brainstem nucleus where integration of central and sensory information takes place, ultimately to determine autonomic nervous output. In the NTS, NO has previously been found to enhance both excitatory and inhibitory synaptic transmission within baroreceptor reflex pathways.
Here, Wang et al. (2007) show that NO can differentially influence glutamate and GABA release depending on its concentration. A significant step is that the concentration of NO was carefully controlled. However, firstly, the authors had to establish the NO concentration, which was not straightforward because the sensitivity of their NO sensors was too low to accurately record likely physiological levels (around 1 nM). To determine NO levels, a solution saturated with NO gas (to 
2 mM) was diluted to measurable levels and the calculated NO content plotted against the measured concentrations. This plot could be fitted with a monoexponential curve, allowing extrapolation to lower, immeasurable levels. Similar measurements were applied to the NO resulting from application of an NO donor, diethylamine NONOate (DEA/NO). This ability to apply NO in known, low concentrations provided the key to the outcomes of the experiments.
Whole-cell patch clamping of NTS neurones in rat brainstem slices revealed that NO in low concentrations (1.1 nM aqueous NO) depolarized some neurones. Interestingly, the population of neurones depolarized by NO did not increase much when higher concentrations were used, suggesting that this effect might be limited to a specific subset of NTS neurones. In addition, local electrical stimulation evoked monosynaptic EPSPs which could be potentiated with a low concentration of NO (threshold 0.3 nM, apparent EC50 0.9 nM). A significantly higher concentration of NO was required for an effect in monosynaptic IPSPs (> 3 nM). The lowest concentration of the NO donor DEA/NO tested (0.1 µM, equivalent to 15 nM NO) provided a maximal increase in EPSP and also increased amplitude of monosynaptic IPSPs. Blocking soluble guanylate cyclase (sGC), the enzyme through which NO exerts its influences, prevented the NO actions, whilst direct pharmacological activation of sGC mimicked the effects of NO. To be certain that the effects on EPSPs and IPSPs resulted from an action on presynaptic release and not a change in sensitivity of the postsynaptic receptors, sGC was blocked in recorded neurones by inclusion of the sGC blocker within the patch pipette. This eliminated NO-induced depolarization in all recorded neurones, indicating that the block was effective. Importantly, potentiation of postsynaptic potentials was retained, indicating a presynaptic site of action.
These findings provide a critical indicator for experiments on NO, in that it is imperative to know its concentration. This factor has so far received little attention in previous experiments with NO donors. Consider that the lowest concentration of NO donor used here is below that commonly used in many experimental protocols, yet was sufficient to enhance both EPSPs and IPSPs! Since NO applied to the NTS may enhance or depress the baroreceptor reflex, a key factor in future will be to determine physiological NO concentrations in the NTS. This will probably depend on the source of the NO, given that both neuronal and endothelial nitric oxide synthase appear to play important roles in neuronal circuits influencing cardiovascular control (Paton et al. 2005), and so it will continue to be important to determine the circumstances under which each isoform contributes to NO-mediated signalling in the CNS.
References
Paton JFR, Deuchars J, Wang S & Kasparov S (2005). Nitroxergic modulation in the NTS: implications for cardiovascular function. In Advances in Vagal Afferent Neurobiology, ed. Undem B J & Weinreich D, pp. 209–246. CRC Press, Boca Raton, London, New York, Singapore.
Wang S, Paton JFR & Kasparov S (2007). Differential sensitivity of excitatory and inhibitory synaptic transmission to modulation by nitric oxide in rat nucleus tractus solitarii. Exp Physiol 92, 371–382.
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