2784 J. Garthwaite Table 1. Key papers leading to the identiﬁcation of NO as a brain transmitter Reference Ferrendelli et al. (1974, 1976) Arnold et al. (1977) and Miki et al. (1977) Deguchi & Yoshioka (1982) Garthwaite (1985) Garthwaite & Garthwaite (1987) Palmer et al. (1987) and Ignarro et al. (1987) Garthwaite et al. (1988) Main ﬁndings Glutamate elicits Ca2+-dependent elevation of cGMP in brain slices; speculated on an intervening transmitter NO activates soluble guanylyl cyclase in brain homogenates l-arginine identiﬁed as the endogenous activator of soluble guanylyl cyclase in brain extracts; activation similar to that induced by NO-releasing agents cGMP response to glutamate in dispersed brain cells mediated exclusively through NMDA receptors cGMP response to NMDA involves cell-cell communication; missing transmitter presumed unstable and could be substituted by exogenous NO (from sodium nitroprusside) Endothelium-derived relaxing factor (EDRF) in blood vessels identiﬁed as NO Brain transmitter identiﬁed as EDRF ⁄ NO; released Ca2+-dependently on NMDA receptor stimulation 2004). iNOS is usually linked with pathological situations and will not be considered here. nNOS The ﬁrst NO synthase to be puriﬁed and cloned (Bredt & Snyder, 1990; Bredt et al., 1991b) and the most abundant isoform generally found in the central and peripheral nervous systems is nNOSa, which is activated by Ca2+ complexed with calmodulin and has a wide but uneven distribution in the mammalian brain, resembling in extent that of a major neuro- transmitter (Bredt et al., 1991a; Vincent & Kimura, 1992). The physical association of nNOSa and the NMDA receptor subunit NR2B with postsynaptic density protein-95 (PSD-95) through their specialised PDZ domains (Brenman et al., 1996) helps explain the preferential link between NMDA receptors and NO production (Garthwaite, 1985; Garthwaite et al., 1988). Other splice variants also exist, namely nNOSb and nNOSc, both of which lack the amino terminal PDZ domain (Brenman et al., 1996). nNOSc has little or no enzymatic activity but nNOSb is active and is upregulated in the striatum and cortex in mice lacking the nNOSa isoform (Eliasson et al., 1997; Langnaese et al., 2007), which probably accounts for the relatively mild phenotype of such animals compared to ones lacking the b and c variants as well (Gyurko et al., 2002). In addition to regulation by Ca2+ ⁄ calmodulin, nNOS possesses several putative sites for phosphorylation. Phosphorylation by cAMP- and cGMP-dependent protein kinases, by protein kinase C and by Ca2+ ⁄ calmodulin-dependent protein kinase (CaMK)II were reported for the puriﬁed enzyme early on, but the effects on activity were generally quite modest and sometimes contradictory (Nakane et al., 1991; Bredt et al., 1992; Dinerman et al., 1994b). A resurgence of interest in this type of post-translational regulation has come from research carried out on cells. CaMKII, a co-resident with NMDA receptors and nNOS at synapses (Kennedy, 2000), was found to phosphorylate the enzyme on serine-847 and inhibit NO formation by 50%, probably by affecting Ca2+ ⁄ calmodulin binding (Hayashi et al., 1999; Komeima et al., 2000). When studied in cultured hippocampal neurones, glutamate had a dual effect on nNOS phosphorylation on serine-847, increasing it at low concentrations (5 lm) and decreasing it at the high concentrations more usually associated with excitotoxicity (100 lm or more). Both were mediated by NMDA receptors, with the phosphorylation blocked by CaMKII inhibition and dephosphorylation by concentrations of okadaic acid active on protein phosphatase 1 (Rameau et al., 2004). nNOS phosphorylated on serine-847 was found to exist in rat brain (Hayashi et al., 1999), indicating it to be a physiologically relevant modiﬁcation. In the cultured neurones, phosphorylated nNOS was concentrated in dendritic spines but the phosphorylation process was slow, taking 15 min to be detectable (Rameau et al., 2004), suggesting that CaMKII is likely to be not a dynamic regulator of nNOS activity but more a longer-term gain controller. The protein kinase Akt (also known as protein kinase B) phos- phorylated nNOS in cultured cortical neurones on serine-1412 (Rameau et al., 2007). This site is equivalent to a key one in eNOS (see below) and its phosphorylation was evoked by 5 min exposure to low glutamate concentrations (5–30 lm) or by briefer periods (1 min) at higher concentrations, after which dephosphorylation became overwhelming. Serine-1412 phosphorylation was NMDA receptor- dependent and led to a rapid enhancement of NOS activity (by how much remains unclear), with dephosphorylation being dependent on AMPA receptors and L-type Ca2+channels. Blockers of either of these activities also enhanced serine-1412 phosphorylation in the absence of glutamate, suggesting that Ca2+inﬂux through L-type channels in response to AMPA receptor-mediated depolarization tonically stimu- lates phosphatases that regulate NMDA receptor-associated nNOS activity. The presence of serine-1412-phosphorylated nNOS in a rat brain lysate (Rameau et al., 2007) indicates that the modiﬁcation occurs in vivo and it may produce a stimulatory effect (as with eNOS; see below) by increasing the sensitivity of nNOS to Ca2+ ⁄ calmodulin (Adak et al., 2001; Rameau et al., 2007). Phosphorylation of nNOS in neuroblastoma cells incubated with a phosphatase inhibitor occurred on threonine-1296 resulting in an 50% reduction in activity although a mutation mimicking threonine-1296 phosphorylation showed a stronger effect (Song et al., 2005). CaMKI may inhibit nNOS activity though serine-741 (Song et al., 2004). No evidence yet exists for phosphorylation of either of these sites in vivo. In addition to phosphorylation, nNOS activity or its location may be inﬂuenced by interactions with a number of proteins (Rodriguez- Crespo et al., 1998; Billecke et al., 2002; Jaffrey et al., 2002; Dreyer et al., 2004) but additional work is needed to clarify their functional signiﬁcance. Recent evidence indicates that nNOS can bind to the serotonin transporter in the plasma membrane such that serotonin uptake then couples to NO formation (Chanrion et al., 2007), a hitherto unique ‘metabotropic’ transporter activity (Garthwaite, 2007). These new ﬁndings suggest that the regulation of nNOS activity in neurones is more complex than previously thought and suggest intriguing options for cross-talk with other signalling pathways. Phosphorylation may transpire to be at least as important for nNOS as it is for eNOS. eNOS Based on immunocytochemical staining, this isoform was originally claimed to be present in neurones of the hippocampus (Dinerman et al., 1994a; O’Dell et al., 1994) but later studies inferred that the staining was artifactual (Demas et al., 1999; Blackshaw et al., 2003). Its presence or otherwise in astrocytes remains unsettled, some ﬁnding by immunocytochemistry or in situ hybridisation that only endothelial cells in the brain were labelled (Seidel et al., 1997; Stanarius et al., 1997; Topel et al., 1998; Demas et al., 1999; Blackshaw et al., 2003) ª The Author (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 2783–2802