2794 J. Garthwaite Garthwaite, 2001a). Immunocytochemical studies have also detected astrocytic cGMP staining in response to NO in other brain regions (de Vente et al., 1998). Astrocyte processes frequently enwrap synapses and so could be within range of synaptically generated NO. The correlation of cerebellar cGMP levels in vivo (largely reﬂecting glial cGMP) with motor activity (Wood, 1991) also suggests a link with synaptic function. The slow kinetics of cGMP degradation in cerebellar astrocytes could mean that cGMP provides a time-averaged readout of ongoing synaptic activity. Alternatively, it could allow cGMP to diffuse intracellularly to targets distant from the site of synthesis. One report found that, in the cerebellum, parallel ﬁbre stimulation led to a NO-mediated increase in Ca2+ in the Bergmann glia (Matyash et al., 2001) but this was not replicated in other laboratories (Beierlein & Regehr, 2006; Piet & Jahr, 2007). Never- theless, in forebrain cultures, brief (100 ms) puffs of NO elicited a rise in glial Ca2+ and the propagation of intercellular Ca2+ waves, due to cGMP–PKG promoting release from ryanodine-sensitive stores (Willmott et al., 2000). In cultured astrocyte-like cells (tanycytes) from the median eminence of the hypothalamus, where neurosecretory terminals are normally found close to blood vessels, co-culture with endothelial cells from the same region caused remodelling of the actin cytoskeleton through NO–cGMP and evidence was obtained in the intact tissue that endogenous NO helped position the nerve terminals next to the capillaries to facilitate delivery of secreted hormone, and hence regulate reproductive function (De Seranno et al., 2004). In other astrocytes also, NO–cGMP (through PKG) regulated the expression of glial ﬁbrillary acidic protein, the principal intermediate ﬁlament of astrocytes (Brahmachari et al., 2006), and altered cytoskeletal dynamics (Boran & Garcia, 2007). Neurovascular communication Cerebral blood ﬂow is closely linked with neuronal activity. Larger blood vessels are supplied with nitrergic nerves derived mainly from the pterygopalatine ganglion, activity in which results in NO release, vasodilatation and increased blood ﬂow (reviewed by Toda & Okamura, 2003). The ﬁrst evidence linking local synaptic activity to NO-mediated vascular relaxation came with the demonstration that the NMDA-induced dilatation of cerebral pial arterioles in vivo can be blocked by NOS inhibitors (Faraci & Breese, 1993; Faraci & Brian, 1995). NMDA application in hippocampal slices also resulted in NO- dependent vasodilatation of microvessels, adding support to the existence of a local mechanism (Lovick et al., 1999). However, this response to NMDA, and that occurring in vivo (Faraci & Breese, 1993), was nulliﬁed by tetrodotoxin, implying that the NO causing the vasodilatation is not produced directly by NMDA receptor stimulation but through a secondary, action potential-dependent mechanism. In the cerebellum and cortex, this may involve ﬁring in speciﬁc classes of nNOS-containing GABAergic interneurone that innervate the micro- vasculature (Yang et al., 2000; Cauli et al., 2004; Rancillac et al., 2006), with NO working alongside several other local constrictor and dilator molecules, including peptides and prostanoids (reviewed by Iadecola, 2004). Vasculoneuronal communication The role of endothelial eNOS in relaxing vascular smooth muscle is ﬁrmly established, but most of the eNOS in the brain lies in the capillary circulation which, by deﬁnition, is devoid of smooth muscle layers (although some modiﬁed smooth muscle cells, or pericytes, are present) raising the possibility that capillary eNOS serves another function. Fig. 3. Signalling from capillaries to axons in optic nerve. (A) Whole-mount preparation of 10-day old rat optic nerve immunostained for eNOS. The dotted line depicts the shape of the nerve, including its cut end (ellipse). The image is taken from Garthwaite et al. (2006) with permission. (B) Confocal micrograph of optic nerve co-stained for neuroﬁlament-68 (green, identifying axons) and eNOS (red) in the optic nerve (also whole-mount preparation). The picture was kindly provided by Dr G. Garthwaite. (C) Cartoon of the proposed mechanism whereby NO generation from eNOS in the capillary circulation persistently depolarizes axons by raising the levels of cGMP which then acts on HCN channels (from Bartus et al., 2007 with permission). Scale bar (in A), 200 lm (A), 100 lm (B). Blood vessels are usually far from the mind of the synaptically orientated neuroscientist, yet any region of the brain parenchyma is, at most, only a typical cell diameter (25 lm) away from a capillary endothelial cell ª The Author (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 2783–2802