(Pawlik et al., 1981). Furthermore, the three-dimensional geometry of the capillary circulation would be just as well suited for delivering NO globally to the tissue as it is for delivering O2 (Fig. 3A and B). The ﬁrst evidence for endothelial NO inﬂuencing parenchymal cells was from the liver where it potentiated Ca2+ signalling in hepatocytes (Patel et al., 1999). In the CNS, electrophysiological recordings from the isolated optic nerve showed that there was an endogenous source of NO that persistently depolarized the axons by activating HCN channels; the source was tracked down to being eNOS in the capillary endothelial cells (Garthwaite et al., 2006) (Fig. 3C). In hippocampal slices also, eNOS was the isoform responsible for the tonic, low level of NO (estimated to be 0.1 nm) that, along with the burst of NO associated with synaptic NMDA receptor activation, contributed to LTP (Bon & Garthwaite, 2003; Hopper & Garthwaite, 2006). The concerted roles of nNOS and eNOS helps understand the earlier observations on hippocampal LTP in mice lacking nNOSa and eNOS (Son et al., 1996) although the data on nNOSa knockouts may be complicated by activity from residual nNOS splice variants (see above). As eNOS is mostly, if not exclusively, found in endothelial cells, effects of knocking out eNOS on brain function may be interpreted as the consequence of eliminating a signal from endothelial cells to neuronal or glial cells. eNOS-knockout mice suffer disrupted synaptic plasticity not only in the hippocampus (Wilson et al., 1999; see also Kantor et al., 1996) but also in the cerebral cortex and striatum (Haul et al., 1999; Doreulee et al., 2003). In the solitary tract nucleus, eNOS regulates autonomic function and angiotensin II may inﬂuence the baroreceptor reﬂex here by releasing NO from endothelial cells (reviewed by Paton et al., 2007). Mice lacking eNOS exhibit greatly reduced NMDA- evoked GABA release in the cerebral cortex, hippocampus and striatum, as measured using microdialysis in vivo (Kano et al., 1998), reduced aggression (Demas et al., 1999), accelerated turnover of serotonin in the frontal cortex and of dopamine in the ventral striatum (Frisch et al., 2000), and decreased neurogenesis (Reif et al., 2004; Chen et al., 2005). Endothelial eNOS offers a pathway for many different blood-borne agents, including hormones, to inﬂuence brain function. In some speciﬁc regions, such as the hypothalamic median eminence, eNOS may regulate the output of hormones by effecting structural adapta- tions (De Seranno et al., 2004). Bringing eNOS into the picture also has repercussions for experimental design and interpretation because endothelial cells could be an active source of NO following application of many different experimental agents, bearing, as they can do, a long list of receptors (for acetylcholine, ADP, bradykinin, serotonin, histamine, etc) that are coupled to eNOS activation. Concluding remarks This review has attempted to encapsulate the substantial progress made in recent years towards understanding the cellular and molecular mechanisms through which NO acts in the mammalian CNS, mechanisms that must ultimately explain the multifarious behavioural effects of NO evident at the whole-animal level. The broad conclusion to be drawn is that NO functions in the mammalian CNS more or less like a conventional neurotransmitter, in much the way it does in animals that evolved hundreds of millions of years ago. The main distinguishing features are the ways that it is produced and its ability to spread extremely rapidly through cell membranes away from its point of synthesis, properties that allow it to operate economically (dispensing with the need for complex storage and release devices) and in a much more versatile fashion than can be achieved with a transmitter acting only extracellularly while, at the same time, achieving a similar degree of synapse speciﬁcity. Adding to the economy is the need for so few NO molecules to do the job because Neural nitric oxide transmission 2795 the sensitivity of the NO detectors is so high: were our hypothetical NO-generating synapse (Fig. 2) going at full blast for a second, it would use up only 1000 l-arginine molecules, or roughly a quarter of the number of amino acid neurotransmitter molecules released from a single synaptic vesicle. Most often, NO is seen to operate in concert with other transmitters rather than in a stand-alone mode; yet, by eliciting subtle alterations in the functioning of ion channels or other proteins, it can elicit effects whose consequences can be profound and sometimes very long- lasting. One of the next steps must be to establish an anatomically coherent picture of the workings of the signalling pathway at individual synapses because, unlike with conventional transmitters, there are no rules to follow and so each synapse must be treated on its own merit. A distinct shortcoming has been the lack of methods capable of measuring physiologically meaningful NO signals in real time and in subcellular dimensions. The recent development of genetically encoded ﬂuorescent sensors for monitoring NO and cGMP with the necessary speciﬁcity, sensitivity and dynamics (Sato et al., 2005; Russwurm et al., 2007; Nausch et al., 2008) are likely to herald progress on this issue, as well as on the subcellular regulation of nNOS and NO receptors. Further downstream, untangling the molecular mechanisms of short-term and long-term alterations occur- ring as a result of cGMP elevation, particularly when PKG activation is involved, will be challenging. Finally, there appears to be another, hitherto unsuspected, source of NO that may impinge importantly on CNS function, namely the capillary endothelial cells. The meaning of this line of communication is speculative but it is likely to convey a different type of message from that generated at synapses because of its global and more persistent (but low-level) nature, one that may translate peripheral signals, such as from hormones, into alterations in CNS function, or ‘prime’ neuronal responsiveness to synaptically released NO or other transmitters. Acknowledgements I am ﬂattered to have been awarded the 2006 Federation of European Neurosciences EJN prize, for which this article was written, and am indebted to the many talented people whom I have been fortunate to have in my laboratory doing the research on NO signalling, the most long-standing (and long- suffering) being Giti Garthwaite. There are too many others to list individually but I would particularly like to acknowledge the more recent contributions of Tomas Bellamy, Victoria Wykes, Elaine Mo and Brijesh Roy to the study of NO receptors, of Christelle Bon and Rachel Hopper to the study of NO in LTP, and of Charmaine Grifﬁths, Robert Keynes and Catherine Hall to the study of NO inactivation in the brain. Barrie Lancaster also contributed immeasurable to our electrophysiology. I would also like to acknowledge the long-term research support from the Wellcome Trust and the Sir Jules Thorn Charitable Trust, as well as funding from the Biotechnology and Biological Sciences Research Council (UK) and the Medical Research Council (UK). Abbreviations CaMK, Ca2+ ⁄ calmodulin-dependent protein kinase; CNG channel, cyclic nucleotide-gated ion channel; CNS, central nervous system; eNOS, endothelial NOS; EPSP, excitatory postsynaptic potential; HCN channel, hyperpolariza- tion-activated, cyclic nucleotide-modulated ion channel; Hsp, heat-shock protein; iNOS, inducible NOS; IPSC ⁄ P, inhibitory postsynaptic cur- rent ⁄ potential; LTD, long-term depression; LTP, long-term potentiation; nNOS, neuronal NOS; NO, nitric oxide; NOS, NO synthase; PDE, phosphodiesterase; PKG, cGMP-dependent protein kinase. References Abudara, V., Alvarez, A.F., Chase, M.H. & Morales, F.R. (2002) Nitric oxide as an anterograde neurotransmitter in the trigeminal motor pool. J. Neuro- physiol., 88, 497–506. ª The Author (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 2783–2802