2788 J. Garthwaite Fig. 1. NO-mediated synaptic transmission. (A) In a synaptically coupled pair of Lymnea stagnalis neurones, single presynaptic action potentials produce constant- latency, one-to-one unitary EPSPs mediated through NO; ﬁve sweeps are superimposed. Modiﬁed from Park et al. (1998) with permission. (B) Slow inhibitory post- junction potentials in mouse colonic circular muscles induced by afferent nerve stimulation (electrical ﬁeld stimulation, single pulses, 0.3 ms duration) in the absence and presence of a NOS inhibitor (100 lm l-nitroarginine), showing the slow hyperpolarizing component to be NO-mediated. Note that inhibition of NOS also depolarizes the muscle by 14 mV as a result of the removal of a tonic hyperpolarizing NO source. Modiﬁed from Hwang et al. (2008) with permission. slower synaptic transmission occurring through metabotropic GABA or glutamate receptors (Dutar & Nicoll, 1988; Batchelor & Garthwa- ite, 1997). Moreover, the much more sustained response of the neurones seen after exogenous NO application (Park et al., 1998) suggests that the falling phase of the EPSP is not caused by tachyphylaxis (loss of responsiveness) but probably reﬂects the earlier dissipation of the NO signal. Hence, the unitary synaptic NO signal is likely to be quite brief. The second example (Fig. 1B) shows synaptic NO having the opposite effect (hyperpolarization) when released from nitrergic nerves innervating the colon (Hwang et al., 2008). In this case, a single shock to the nerves elicited a biphasic inhibitory potential, an initial rapid phase caused by release of a purine and then a second phase that is the result of NO release. The time-course of the nitrergic potential is a little slower than that of the snail neurone EPSP, peaking after 3 s and then falling to baseline after 8 s. Although there are no equivalent data dealing with postsynaptically- generated NO, we can begin to envisage what happens. A typical excitatory synapse in the brain would have 50 NMDA receptors dispersed over a 400-nm-diameter postsynaptic density (Kennedy, 2000). Taking the extreme situation of each being associated with an nNOS molecule and the nNOS molecules all being maximally active, generating 20 NO molecules each per second, we can see the resulting NO concentration proﬁle that would be set up purely as a result of diffusion of the molecule away from the sources (Fig. 2A). At steady state (with continuous NMDA receptor activation) we expect to ﬁnd only 1 nm NO just the other side of the synaptic cleft, reducing to 0.25 nm at the periphery of the nerve terminal. A high degree of synapse speciﬁcity is implied, although closely neighbouring struc- tures would also be penetrated by low NO concentrations. When synaptic NMDA receptors are activated by a pulse of glutamate, the resulting local rise in intracellular Ca2+ follows a time- course resembling that of the current passing through the membrane, rising to peak in 50 ms and then declining to baseline within 500 ms (Sabatini et al., 2002). The active Ca2+ ⁄ calmodulin required for nNOS activity has four bound Ca2+. On removal of Ca2+, unbinding of the ﬁrst two Ca2+ is rapid, causing arrest of NOS activity with a half-time of < 70 ms. Unbinding of the remaining two Ca2+ is at least 10-fold slower and dissociation of calmodulin from nNOS slower still, with an estimated half-time of 7 s (Persechini et al., 1996). A scenario for efﬁcacious NO generation, therefore, would be in a recently activated synapse where calmodulin with two Ca2+ bound is already associated with nNOS. Synaptic NMDA receptor activation should then result in almost contemporaneous binding of the ﬁnal two Ca2+ and subsequent NO production. The resulting NO pulses predicted at different distances from the source are illustrated in Fig. 2B. There would be no perceptible delay in the peak of the pulse arriving at different parts of the synapse although, of course, the amplitude falls with distance and does not quite achieve that seen with a continuous NO output. By placing NO receptors at different distances we can gauge how effective they would be at transducing the signals (Fig. 2C, upper panel). Assuming the empirical kinetic scheme drawn up previously (Garthwaite, 2005) and a receptor density the same as is found in platelets (Mo et al., 2004), an NO pulse arriving mid-way through the nerve terminal, peaking at 0.3 nm, would evoke 0.4 lm cGMP, an impressive 1000-fold ampliﬁcation taking place within 1 s. Hence, the receptors are beautifully tuned to capture and translate even brief, low-amplitude NO pulses. The simulation ignores hydrolysis of cGMP by phosphodiesterases but, at these concentrations, much of the cGMP would probably bind to signalling proteins in the vicinity which would protect it from degradation (Kotera et al., 2003). The submicromolar afﬁnity of cGMP-dependent protein kinases for cGMP (Wall et al., 2003) provides a mechanism for such an NO pulse being biologically signiﬁcant and it is notable that the time-courses of the simulated responses fall reasonably well in line with those of the potential changes recorded electrophysiologically (Fig. 1), bearing in mind the steps downstream from cGMP that will help shape the physiological response. It is probably unrealistic to imagine all synaptic NMDA receptors being momentarily active together but the simulation in Fig. 2C (lower panel) shows that if there were repeated stimuli leading to a 2-s period of continuous NOS activity, cGMP may accumulate into the low micromolar range (a 10 000-fold ampliﬁcation over the NO concen- tration), meaning that many fewer NMDA receptors would need to be active to generate a biological response. The temporal summation of the cGMP concentration in this scenario helps explain why NO- mediated transmission is generally seen to operate most effectively following short periods of higher frequency activity. That endogenous NO should be acting at synapses in concentrations of only 1 nm, which is a tenth of the concentration needed to activate cellular receptors by 50% (see above), may seem strange. ª The Author (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 2783–2802