moiety onto other thiols, or to concentrations of exogenous NO that produce nitrosating species on reaction with oxygen (e.g. N2O3), or by unphysiologically exposing cells to a Ca2+ ionophore (reviewed by Hogg, 2002), but there are no unambiguous instances of neural function being regulated physiologically through this modiﬁcation. S-nitrosation of synaptic NMDA receptors was once advertised as a negative feedback mechanism (Lipton et al., 2002) but, on experi- mental testing, was found to be an artifact (Hopper et al., 2004). Working with NO is undoubtedly difﬁcult and experiments are susceptible to various artifacts arising from unintended chemical and biological reactions (reviewed in Keynes & Garthwaite, 2004). Even seemingly innocuous ingredients, such as Hepes buffer, and conduct- ing experiments using tissue culture media under normal laboratory lighting conditions, can cause problems (Beckman & Koppenol, 1996; Keynes et al., 2003). In vivo, S-nitrosation may occur mainly in pathological states where the changed redox environment may facilitate the appearance of species capable of converting thiols to nitrosothiols (see review by Zhang & Hogg, 2005). Another speculative physiological target for NO is mitochondrial cytochrome c oxidase (Erusalimsky & Moncada, 2007), which reduces O2 to water. NO can compete with O2 for binding to this complex and thereby inhibit respiration but higher concentrations are needed than to activate receptors, the EC50 value at physiological O2 concentrations (20–30 lm) being 120 nm (Bellamy et al., 2002). In an in vivo investigation, cytochrome c oxidase in brain was unaffected by inhibition of NO production either before or after a period of ischaemia (De Visscher et al., 2002) and, in in vitro studies on brain slices, the prevailing NO concentrations appeared to remain in the low nanomolar range or below (too low to affect mitochondrial respiration) despite intense NMDA receptor activation (Bellamy et al., 2002; Keynes et al., 2004), transient simulated ischaemia (Grifﬁths et al., 2002), or the expression of active iNOS in microglia (Duport & Garthwaite, 2005). Only by artiﬁcially increasing the numbers of activated microglia in hippocampal slice cultures could NO receptors be seen to be saturated, suggesting ambient concentrations in excess of 10 nm (Duport & Garthwaite, 2005). Hence, mitochondrial inhibition by NO in the brain may only become relevant under certain pathological conditions, for example when iNOS is expressed in active inﬂammatory plaques in multiple sclerosis (reviewed in Smith & Lassmann, 2002). NO inactivation It is often stated that NO does not need a specialised inactivation mechanism because it is disposed of by virtue of its natural reactivity. However, at the low nanomolar concentrations and below that are likely to be physiological, NO is remarkably unreactive. Reaction with O2, for example, is obvious at micromolar NO concentrations but negligible at low nanomolar concentrations (Ford et al., 1993). Reaction with superoxide ions, giving peroxynitrite, is extremely rapid (Nauser & Koppenol, 2002) but, over the presumed physiological NO concentration range, superoxide dismutase is greatly in excess of NO so that superoxide ions are removed too quickly to allow reaction with NO (Beckman & Koppenol, 1996). Low NO concentrations will, however, react avidly with lipid peroxyl radicals in a beneﬁcial process that stops lipid peroxidation (O’Donnell et al., 1997; Keynes et al., 2005). In physiological conditions, one pathway for NO degradation will be through reaction with haemoglobin in circulating erythrocytes, forming nitrate and methaemoglobin (Liu et al., 1998). Calculations based on the geometry of the microcirculation suggest that this pathway would impose on NO a tissue half-life of 1 s (J. Wood and J. Garthwaite, unpublished result). Neural nitric oxide transmission 2787 A much more active mechanism for NO consumption exists in brain tissue itself. Reminiscent of the effect of transporters on the potency of exogenous glutamate (Garthwaite, 1985), it was found that almost 1000-fold higher NO concentrations were required to saturate NO receptors in incubated slices of cerebellum than in dispersed cells (where diffusional barriers are lacking), implying rapid consumption of NO as it diffuses into the slices (Hall & Garthwaite, 2006). At a gross level, consumption appeared to be uniform across the slice, suggesting that the mechanism is present in all cerebellar cell layers. Moreover, NO consumption could not be detected in blood platelets and leukocytes (Keynes et al., 2005), and appears to be very weak in the intact aorta (Liu et al., 2008), pointing to a specialised mechanism and, parenthetically, also explaining why exogenous NO is so much less potent at activating receptors in cerebellar tissue than in blood vessels (Southam & Garthwaite, 1991). From a diffusion-inactivation model, the mechanism in the cerebellar slices conformed to a Michaelis–Menten-type reaction having a maximum velocity of 1–2 lm ⁄ s and a Michaelis constant of 10 nm. From these values, it is predicted that inactivation would impose a very short half-life (< 10 ms) on NO in concentrations up to 10 nm (Hall & Garthwaite, 2006). This process would have little effect on single sources of NO, where diffusional dispersion would dominate, but would impinge strongly where there are multiple sources within a tissue volume, such as when a plexus of nerve ﬁbres are active (Philippides et al., 2005) or when NO is generated in the microvascular network. The mechanism could not be explained by any known method of NO consumption and it remains to be identiﬁed. It is worth noting, however, that its activity may vary from one brain region to another: in slices containing the nucleus of the solitary tract (Wang et al., 2007), exogenous NO affected neurotransmission at concentrations ( 1 nm) that would be expected to be active without any tissue NO consumption. Because of the steep NO concentration gradients across the slice thickness imposed by NO consumption (Hall & Garthwaite, 2006), the location of the cell under study relative to the slice surface would also dictate its sensitivity to externally applied NO. Neurotransmission by NO: general features Depending on the circuit, NO may be produced pre- or postsynap- tically. In many brain areas, the prototypic coupling with postsynaptic NMDA receptors appears to apply. In others, NO may derive from presynaptic axon terminals, much as it does in peripheral nitrergic nerves. In these nerves, the stimulus for NO synthesis is usually the action potential-dependent opening of presynaptic N-type and ⁄ or other Ca2+ channels, with the resulting NO effecting smooth muscle relaxation (see review by Toda & Herman, 2005). Clues to the shapes of the synaptic NO signals come from experiments where it has been possible to record downstream electrophysiological responses at the single-cell level. The ﬁrst example (Fig. 1A) shows recordings from neurones in the pond snail (Lymnaea stagnalis), speciﬁcally at synapses between a nitrergic neuron and its partner (Park et al., 1998). Bursts of presynaptic activity resulted in slow NO-mediated excitatory post- synaptic potentials (EPSPs) that could cause postsynaptic spiking whereas single presynaptic action potentials normally had no observable effect. When ampliﬁed by increasing the Ca2+ and Mg2+ concentrations in the bathing medium, however, unitary NO-mediated EPSPs could be visualised. They consisted of slow depolarizations taking off after a ﬁxed delay of 200 ms, peaking after 0.5 s and decaying back to baseline after 1 s. When viewed in this way NO neurotransmission appears quite familiar, being evocative of the ª The Author (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 2783–2802