channels appear to be located in the transition zone just before the terminal itself and to regulate terminal excitability in such a way as to suppress aberrant action potential ﬁring and associated neurotrans- mitter release (Dodson et al., 2003; Ishikawa et al., 2003). Possibly, therefore, cGMP-dependent phosphorylation directly or indirectly inhibits the channels, resulting in enhanced GABA release. Interest- ingly, Kv3 channels, which affect transmitter release by shaping the presynaptic action potential, have been found, when expressed in CHO cells, to be suppressed by the NO–cGMP pathway, not through direct phosphorylation by PKG but through the intermediary of a phosphatase, which probably removes a phosphate group from the channel protein that normally promotes activity (Moreno et al., 2001). Should this mechanism occur in nerve terminals, spike broadening, leading to increased transmitter release, would be anticipated (Ishik- awa et al., 2003). While these investigations focus on K+ channel inhibition as a mechanism of increasing presynaptic transmitter release, K+ channel activation may paradoxically improve transmitter release as well. Speciﬁcally, stimulation of Ca2+-activated K+ channels by cGMP- dependent phosphorylation enhanced presynaptic spiking by increas- ing the spike afterhyperpolarization, allowing more Na+ channels to recover from inactivation which, by facilitating spike conduction, increased Ca2+inﬂux in response to trains of stimuli (Klyachko et al., 2001). One shortcoming in many studies of synaptic NO signalling is ignorance of exactly where the NO comes from and where it acts. Considerations based on individual synapses (Fig. 2) would predict that the sources and targets would need to be close together for the signalling pathway to work, although summation of signals extrasy- naptically would be plausible if groups of synapses were simulta- neously active (Philippides et al., 2005; Hall & Garthwaite, 2006). According with this idea, high resolution studies of hippocampal CA1 pyramidal neurones showed that, in a population of synapses, NO receptors were located in the active zone in excitatory nerve terminals whereas nNOS was concentrated in juxtaposed dendritic spines just below the plasma membrane, an arrangement well suited to a retrograde messenger role for NO at these synapses (Burette et al., 2002). Unexpectedly, in the same neurones an additional line of communication has materialised from nNOS also being found in the postsynaptic densities of GABAergic terminals (on somata, dendrites and axon initial segments) with the NO receptors being presynaptic (Szabadits et al., 2007). The NO receptor at GABAergic synapses was, interestingly, the a1b1 isoform whereas the a2b1 receptor, which associates with pre- and ⁄ or postsynaptic scaffold proteins (see above), appeared to service the excitatory synapses. This arrangement immediately suggests a new type of retrograde NO signalling wherein local rises in cytosolic Ca2+ as a result of back-propagating action potentials or of local synaptic input activates postsynaptic nNOS and the NO then signals to abutting inhibitory nerve terminals to inﬂuence GABA release (Szabadits et al., 2007). Supporting this line of communication being functional, when studied in the presence of a cholinergic agonist, depolarization (for 1 s) of the postsynaptic neurone caused a transient ( 20 s) suppression of GABAergic inhibitory postsynaptic currents (IPSCs), an effect mediated through NO receptors and cGMP (Makara et al., 2007), with endocannabi- noids also being intimately involved (see below). In these experiments, spontaneous quantal GABAergic IPSCs (recorded in the presence of tetrodotoxin) were unaffected by NOS or NO receptor blockade (Makara et al., 2007), indicating that cGMP suppressed action potential-dependent GABA release ultimately, perhaps, by inhibiting presynaptic voltage-gated Ca2+ channels. In this respect there is a large literature dealing with NO–cGMP and Ca2+ Neural nitric oxide transmission 2791 homeostasis in cells of relevance to both its pre- and postsynaptic actions (reviewed in Garthwaite & Boulton, 1995; Clementi, 1998; Ahern et al., 2002; Grassi et al., 2004) but no general rules: in some cells Ca2+ currents are inhibited, in others they are enhanced; sometimes Ca2+ release from internal stores is reduced, elsewhere it is increased. Regarding the latter, recordings in the nucleus of the solitary tract found that, via cGMP, low concentrations of exogenous NO reversibly potentiated both glutamatergic EPSPs and GABAergic IPSPs, apparently through a presynaptic mechanism (Wang et al., 2007). In the case of the IPSPs, NO–cGMP seemed to act by evoking Ca2+ release from presynaptic ryanodine-sensitive stores through the intervention of cyclic ADP ribose (Wang et al., 2006b), whose generation can be stimulated by PKG phosphorylating the synthes- ising enzyme (Willmott et al., 1996). Elsewhere, NO may enhance spontaneous neurotransmitter release by the action of cGMP on presynaptic CNG channels, leading to a raised intraterminal Ca2+ concentration (Savchenko et al., 1997; Murphy & Isaacson, 2003). A major NO–PKG transduction pathway in smooth muscle and platelets is through the IP3 receptor-associated protein IRAG which, when phosphorylated by PKG1b, inhibits Ca2+ release from IP3-sensitive stores (reviewed in Hofmann et al., 2006). However, IRAG appears to have a minor presence in the brain, the mRNA being prominent only in thalamic relay nuclei (Geiselhoringer et al., 2004) where, perhaps signiﬁcantly, PKGII is also concentrated (El-Husseini et al., 1999; de Vente et al., 2001). Interaction with other signalling pathways Apart from these serial signalling cascades inﬂuencing primary synaptic neurotransmission, the interplay between NO–cGMP and other neuromodulators introduces another layer of regulation. This is presaged by ﬁndings in the peripheral nervous system at junctions between nitrergic nerves and effector organs, where a multitude of interactions with cholinergic, adrenergic, purinergic and peptidergic nerves, often presynaptic, has been described (reviewed in Toda & Okamura, 2003; Toda & Herman, 2005). Similarly, based on in vivo sampling of the extracellular ﬂuid, myriad interactions with other transmitters in the brain are to be expected (reviewed in Prast & Philippu, 2001), although details of what may be happening at the level of the synapse are sparse. In the pond snail, serotonin transmission was markedly (up to 80%) dependent on endogenous NO acting postsynaptically through cGMP and PKG, perhaps bringing about phosphorylation of the serotonin receptor (Straub et al., 2007). There is also long-standing evidence for an interaction between NO, cGMP and acetylcholine in the brain that remains poorly understood (reviewed by de Vente, 2004). One intriguing link with acetylcholine may involve the endocannabinoids. These are fatty acid derivatives that act in synapses somewhat like NO, in that they are generated enzymatically in response to a rise in Ca2+, usually postsynaptically, and then act retrogradely on presynaptic CB1 receptors, typically to depress neurotransmitter release (reviewed in Hashimotodani et al., 2007). In the vertebrate (lizard) neuromuscular junction, endocanna- binoid release underlay the transient suppression of acetylcholine release produced by activation of muscarinic (M3) receptors but, in order for the pathway to be effective, NO–cGMP and associated PKG were needed (Newman et al., 2007). NO on its own did not affect acetylcholine release (Graves et al., 2004), so there seems to be a step occurring in the presynaptic nerve terminal where endocannabinoid and NO signalling cascades meet. Somewhat similarly, the depression of GABAergic IPSCs brought about by depolarizing hippocampal neurones in the presence of a cholinergic agonist depended on both ª The Author (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 2783–2802