2792 J. Garthwaite NO and endocannabinoids, the two of them apparently being produced postsynaptically and acting presynaptically (Makara et al., 2007). Unlike in the lizard neuromuscular junction, cannabinoids could depress transmission on their own but, in the presence of the cholinergic agonist, the NO–cGMP and cannabinoid pathways converged, apparently at the presynaptic location (where CB1 receptors and NO-evoked cGMP accumulation were located) to elicit the transient depression of GABAergic transmission. Further interplay between endocannabinoids and NO can be found in relation to long- lasting alterations in synaptic efﬁcacy (see below and Sergeeva et al., 2007; Kyriakatos & El Manira, 2007). NO and neuroplasticity There has been much interest in an involvement of NO in long-term changes in synaptic strength, initially because its diffusible nature and link with NMDA receptors made it an appealing candidate for a retrograde trans-synaptic messenger, relaying information about NMDA receptor activity to the presynaptic terminal to coordinate alterations in transmitter release (Garthwaite et al., 1988). Despite some confusing results early on, it is now established that the NO– cGMP pathway plays a role in long-term potentiation (LTP) or long- term depression (LTD) at many synapses throughout the CNS, and even at the neuromuscular junction. As the topic has been reviewed many times before, both generally (Garthwaite & Boulton, 1995; Holscher, 1997; Prast & Philippu, 2001; Susswein et al., 2004) and with respect to speciﬁc brain regions (Daniel et al., 1998; Hawkins et al., 1998; Centonze et al., 1999; Grassi & Pettorossi, 2001; Hartell, 2002), the emphasis here will be on mechanisms and, as with the acute synaptic actions of NO (above), these can be pre- and ⁄ or postsyn- aptically located, or they can involve more general alterations in neuronal excitability. Postsynaptic plasticity One of the most developed instances of NO participating in plasticity is with LTD in the cerebellum, which has long been considered part of motor learning behaviour and which occurs when a powerful excitatory input to Purkinje cells from the climbing ﬁbres is repeatedly active just after another, from parallel ﬁbres. The climbing ﬁbre input is viewed as an error signal which dampens input from parallel ﬁbres that are inappropriately active just beforehand (reviewed by Ito, 2001). According to the current model, NO is produced either in parallel ﬁbres themselves (Shibuki & Kimura, 1997) or via NMDA receptor activity in interneurones simultaneously receiving parallel ﬁbre excitation (Shin & Linden, 2005) and acts postsynaptically at parallel ﬁbre synapses to raise cGMP and thence activate PKG. Purkinje cells are enriched in the PKG substrate, known as G-substrate, that, on phosphorylation, functions as a phosphatase inhibitor (Endo et al., 1999). This, together with ongoing protein kinase C activity, leads to persistent phosphorylation of AMPA receptors at a particular serine residue, which disrupts AMPA receptor clusters and favours receptor endocytosis (Launey et al., 2004; Steinberg et al., 2006). Consistent with the mechanism being behaviourally signiﬁcant, knocking down PKG1a speciﬁcally in Purkinje cells impaired LTD and introduced a deﬁcit in a motor learning behaviour (adaptation of the vestibulo-ocular reﬂex) although their general motor performance was normal (Feil et al., 2003). Endocannabinoids ﬁgure in cerebellar LTD as well and it appears that NO functions downstream of this pathway because endocannabinoid-induced LTD could be blocked by NOS inhibition, suggesting that endocannabinoids may somehow promote the NO synthesis needed for LTD (Safo & Regehr, 2005). A long-term potentiation of NO release from the parallel ﬁbres following tetanic stimulation has been described (Kimura et al., 1998) but this depended on cAMP, whose levels are usually reduced by endocannabinoids (reviewed in Hashimotodani et al., 2007). Even so, there are precedents for endocannabinoid CB1 receptors being coupled to increased NO synthesis (Poblete et al., 2005; Romano & Lograno, 2006). Long-term potentiation in the hippocampus also involves changes in postsynaptic AMPA receptor density, but in the opposite direction (reviewed in Collingridge et al., 2004) and NO–cGMP may play a part in this process. A key AMPA receptor subunit required for NMDA receptor-dependent LTP is the GluR1 subunit, whose insertion into the synapse depends on complex interactions with synaptic scaffold proteins and protein phosphorylation. In dissociated hippocampal cultures brief application of glutamate elicited an enduring potentia- tion of spontaneous glutamatergic excitatory postsynaptic currents (EPSCs) comprising a rapid enhancement in frequency, originating presynaptically, and an increase in amplitude, associated with increased numbers of postsynaptic protein clusters containing GluR1 (Antonova et al., 2001). A surprising ﬁnding was that the increase in GluR1 clusters required PKG and could be replicated by exposure to 8-Br-cGMP (Wang et al., 2005). In a comprehensive series of experiments, Serulle et al. (2007) have now discovered a key participant to be PKGII. Binding of cGMP led to the formation of a complex between PKGII and GluR1 and the phosphorylation of GluR1 on a serine residue (serine-845) that facilitates its delivery to extrasynaptic sites, priming insertion into the synapse. Previously, serine-845 phosphorylation had been linked with cAMP but Serulle et al. (2007) showed in cultured hippocampal neurones that it also happens in response to exogenous NO, and to NMDA receptor activity in a NOS- and PKG-dependent manner. Increased cell surface GluR1 expression correlated with changes in synaptic transmission that shared the same NO–cGMP–PKG-dependent properties. The receptor clustering seen in these experiments is reminiscent of that occurring in the developing neuromuscular junction, where agrin secreted from active motor nerve terminals induces, through the NO– cGMP–PKG pathway, the formation of postsynaptic acetylcholine receptor clusters (reviewed in Godfrey & Schwarte, 2003). However, Serulle et al. (2007) were able show that blocking PKGII selectively using a dominant negative fragment also reduced LTP in adult mouse hippocampal slices, indicating that the mechanism may not be conﬁned to the relatively immature circuitry of the tissue culture model. These two examples of NO–cGMP effecting opposite changes in postsynaptic AMPA receptor density at two different synapses may help explain the participation of NO in LTP and LTD elsewhere. However, other mechanisms must also at be work. For hippocampal LTP to persist beyond the ﬁrst hour or so, RNA and protein synthesis are needed. In slice preparations, it has been shown that NO–cGMP–PKG resulted in the phosphorylation of the transcription factor CREB (cAMP response element binding protein) in the cell bodies of postsynaptic neurones by a mechanism involving Ca2+ release from ryanodine-sensitive stores (Lu et al., 1999; Lu & Hawkins, 2002), implicating cyclic ADP ribose as an intermediary. CREB regulates the expression of many different genes. There are also several other ways by which cGMP can directly and indirectly regulate gene expression in cells. In all, > 60 RNA species have so far been shown to be increased or decreased by cGMP in various cell types (reviewed in Pilz & Broderick, 2005). Presynaptic plasticity The ﬁrst direct evidence for NO persistently augmenting neurotrans- mitter release came from recordings showing that exogenous NO, ª The Author (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 2783–2802