added at 5–10 nm, elicited an enduring increase in the frequency of spontaneous miniature EPSPs in cultured hippocampal neurones (O’Dell et al., 1991). Subsequent work extended this observation to provide a convincing case for NO serving as a retrograde messenger at these synapses in response to brief tetanic stimulation of the presynaptic neurone, and that it acts through cGMP and PKG (Arancio et al., 1995, 2001), leading to the rapid (within a minute) formation of new clusters of presynaptic proteins, coordinating with the slightly later appearance of new postsynaptic GluR1 clusters (Wang et al., 2005). A protein well known in the cardiovascular ﬁeld as a PKG substrate, vasodilator-stimulated phosphoprotein (VASP), together with RhoA, a member of the Rho GTPase family, apparently contributed to the clustering on both sides of the synapse, suggesting a cytoskeletal involvement, with the conversion of the clusters into functional units, perhaps under the control of CaMKII activity (Ninan & Arancio, 2004; Wang et al., 2005). The results are consistent with the functioning of VASP in ﬁlopodial dynamics (Dwivedy et al., 2007) and with evidence that NO mediates NMDA receptor-dependent growth of presynaptic protrusions and the remodelling of presynaptic varicosities in hippocampal slice cultures (Nikonenko et al., 2003). Good evidence from other synapses also implicates a presynaptic site of action of NO although, in most cases, the mechanisms remain to be explored (Wu et al., 1997; Grassi & Pettorossi, 2000; Volgushev et al., 2000; Hardingham & Fox, 2006; Sjostrom et al., 2007). In rat rostral ventral medulla neurones, endogenous NO was found to operate though cGMP, PKG and presynaptic N-type Ca2+ channels to potentiate glutamate release for 10–20 min (Huang et al., 2003). In the cerebellar parallel ﬁbre–Purkinje cell synapse, NO was required for a presynaptic form of LTP which, curiously, depended on cAMP but not cGMP (Jacoby et al., 2001) and which reﬂected an augmentation of action potential-evoked presynaptic Ca2+currents (Qiu & Knopfel, 2007). LTP at the cerebellar mossy ﬁbre–granule cell synapse, however, engaged the NO–cGMP pathway to increase the presynaptic terminal excitability (Maffei et al., 2003). NO–cGMP-dependent potentiation of inhibitory, presumed glycinergic (Wu & Dun, 1996) and GABAergic (Nugent et al., 2007), synapses through the presynaptic route have also been reported. Finally, NO may also contribute to LTD by suppressing presynaptic excitatory transmitter release cGMP-dependently, as has been observed in the hippocampus (Zhang et al., 2006b) and the neuromuscular junction (Wang et al., 1995; Etherington & Everett, 2004). A role for NO in plastic changes of other neurotransmitter systems is also likely. For example, it has been found that PKG-dependent phosphorylation of a threonine residue on the serotonin transporter enhanced serotonin uptake (Ramamoorthy et al., 2007), an effect that may contribute to the lowering of extracellular serotonin brought about by the NO–cGMP pathway observed in the hypothalamus (Kaehler et al., 1999), and to obsessive–compulsive disorder in humans (Zhang et al., 2007a). Alternatively, nNOS may physically interact with the serotonin transporter in such a way as to reduce its cell surface expression, which should increase extracellular serotonin levels (Chanrion et al., 2007). Plasticity of intrinsic excitability Plasticity can also be expressed outside the synapse, through alterations in intrinsic neuronal excitability (reviewed in Daoudal & Debanne, 2003) and here too NO may be involved. In the cerebellum, Purkinje cell ﬁring became moderately higher, but in a sustained manner, in response to NO–cGMP (Smith & Otis, 2003). A more pronounced effect was observed in Aplysia sensory neurones which, Neural nitric oxide transmission 2793 much like in mammals, become hyperexcitable (decreased threshold for action potential generation, and increased depolarization-induced and spontaneous ﬁring) following injury to their axons (Sung et al., 2004). Underlying the hyperexcitability was an upregulation of nNOS, stimulation of which, apparently at the level of the axon, led to local activation of PKG which then was transported to the neuronal cell body. Here it phosphorylated mitogen-activated protein kinase which then entered the nucleus to initiate transcriptional activity. As well as providing a mechanism for enduring hyperexcitability, the results give an example of how NO acting remotely on neuronal processes can engage the gene expression machinery in the cell body. Extending the results to mammals, it was found that compression injury to rat dorsal root ganglion neurones, or simply dissociating the neurones, resulted in them adopting a hyperexcitable state that was maintained by cAMP and cGMP acting through their respective kinases (Song et al., 2006; Zheng et al., 2007). Furthermore, in hypoglossal motoneurones, while brief NO exposure normally generated only a small cGMP-dependent depolarization, upregulation of nNOS as a result of nerve injury, or prolonged (4 h) exposure to exogenous NO, led to sustained hyperexcitability through PKG-mediated inhibition of resting K+ currents, particularly those produced by the pH-sensitive, two- pore-domain TASK-like channels (Gonzalez-Forero et al., 2007), channels that are widely distributed in the CNS (reviewed by Bayliss et al., 2003). The gradual onset of the NO-mediated suppression of the channels suggests a mechanism operating through trafﬁcking or gene ⁄ protein expression (Gonzalez-Forero et al., 2007). Although these last two examples are of speciﬁc relevance to the development of heightened pain sensitivity and other responses to injury, they may also exemplify changes enacted under normal conditions to contribute, along with synaptic changes, to the development of activity-dependent alterations in excitability that link cellular plasticity to learning and memory formation (Daoudal & Debanne, 2003). Roles of individual NO receptors The development of mice lacking speciﬁc NO receptor subunits (Friebe et al., 2007) offers new opportunities for understanding the roles of each receptor isoform in neuroplasticity and other phenomena. Fascinatingly, it has so far been shown that knocking out either the a1b1 or a2b1 virtually abolished NMDA receptor-dependent LTP in the visual cortex, with rescue being effected by an exogenous cGMP derivative in both cases (Haghikia et al., 2007). Why both isoforms are essential remains a mystery. Perhaps one (a2b1) transduces the NO signal associated with NMDA receptor activity at synapses and the other (a1b1) transduces the signal from endothelial cells (see below). The precise distribution of the isoforms in the cortex remains to be determined but, in the hippocampus, they were found in different cells, a1b1protein appearing to be exclusive to a population of GABAergic interneurones, whereas the mRNA for a2b1 was conﬁned to pyramidal neurones (Szabadits et al., 2007). Other lines of communication involving NO Glia as NO targets The highest concentration of cGMP in the cerebellum is in astrocytes (de Vente et al., 1990; Southam et al., 1992; Southam & Garthwaite, 1993), because these cells have an extremely low phosphodiesterase activity, some 6000- to 9000-fold less than in platelets (Garthwaite, 2005), which allows cGMP to accumulate to near millimolar concentrations on persistent activation of NO receptors (Bellamy & ª The Author (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 2783–2802