Our findings support the hypothesis that glial cells mediate neurovascular coupling in the CNS. In accordance with this hypothesis, our data show the following. First, selective stimulation of glial cells results in large, short-latency vasodilations and vasoconstrictions. These glial-evoked vasomotor responses are a result of direct glia-to-vessel signaling and do not involve neurons as intermediaries. Second, light-evoked and glial-evoked vasomotor responses are mediated by the same arachidonic acid metabolites. Both light- and glial-evoked dilation is mediated by epoxygenase production of EETs, whereas light- and glial-evoked constriction is mediated by ω-hydroxylase production of 20-HETE. Third, light-evoked vasodilations and vasoconstrictions are blocked when neuron-to-glia signaling is interrupted. Together, these results indicate that light-evoked vasomotor responses are mediated, in large part, by signaling through glial cells.
The results reveal that glia can evoke both vasodilations and vasoconstrictions mediated by EET and 20-HETE production, respectively. As shown previously, glial cells can synthesize both arachidonic acid metabolites (Amruthesh et al., 1993
; Alkayed et al., 1996
; Harder et al., 1998
). Glial production of EETs and 20-HETE could be initiated by increases in glial Ca2+
, which would activate Ca2+
-dependent phospholipase A2 and result in arachidonic acid production. Light-evoked neuronal activity has been shown previously to increase glial Ca2+
in the retina (Newman, 2005
). However, our results do not exclude a downstream site of EET or 20-HETE production. For instance, synthesis of these arachidonic acid metabolites could occur in vascular smooth muscle cells after release of other signaling molecules from activated glial cells. Moreover, unlike COX metabolites, EETs and 20-HETE can also be stored in membranes for later release, adding to the complexity of this pathway. Release of these metabolites from the membrane could account for the residual vasomotor responses seen in the presence of epoxygenase and ω
We observed no correlation between the amplitude, duration, or spatial extent of glial Ca2+ increases after glial stimulation and the type of vasomotor response observed. Indeed, no such correlation would be expected if the nature of the vasomotor response (dilation or constriction) is determined by NO levels, as we argue in the following paragraphs. We should note that our results do not exclude the possibility of glial-evoked vasomotor responses that are generated by a Ca2+ -independent mechanism.
Our findings suggest that NO levels may regulate whether neuronal activity results in vasodilation or constriction in the retina. NO has multiple effects on vasomotor responses. NO can induce vasodilation by stimulating guanylyl cyclase, leading to the production of cGMP and activation of K+
channels in vascular smooth muscle. NO can also induce vasodilation by inhibiting ω
-hydroxylase, resulting in a decrease in the vasoconstricting metabolite 20-HETE (Alonso-Galicia et al., 1998
; Sun et al., 2000
). Both of these mechanisms contribute to vasodilation in the brain (Alonso-Galicia et al., 1999
). However, NO could also favor vasoconstriction. NO could inhibit cytochrome P450 epoxygenase, the enzyme that metabolizes arachidonic acid to vasodilating EETs (Udosen et al., 2003
). By reducing EET production, NO would promote vasoconstriction.
Although NO has both vasodilating and vasoconstricting effects, it is generally considered to be a vasodilating agent. However, our results suggest that in the retina, NO promotes vasoconstriction. Activity-evoked vasodilations were generally observed in the presence of NOS inhibitors and NO scavengers, whereas activity-evoked vasoconstrictions were seen in the presence of NO donors. Arterioles that initially dilated in response to light in nominally zero NO conditions constricted as NO levels were raised. These findings suggest that EET production by epoxygenase is more sensitive to NO inhibition than is 20-HETE production by ω-hydroxylase. High NO levels would inhibit epoxygenase production of vasodilatory EETs, resulting in activity-dependent vasoconstriction mediated by 20-HETE production. Low NO levels would release epoxygenase from inhibition, resulting in greater activity-dependent vasodilation. Comparative studies to determine the relative sensitivity of epoxygenase and ω-hydroxylase to NO inhibition have not been conducted.
The net effect of NO on vasomotor responses is most likely complex and may depend on the precise site of its synthesis, which would determine which cells it acts on. In the proximal retina, nNOS is expressed in processes of amacrine cells lying close to capillaries and larger vessels (Roufail et al., 1995
). In addition, Müller cells and astrocytes both express eNOS (Haverkamp et al., 1999
). Precise control of NO synthesis within retinal neurons and glial cells could determine whether glial activation results in vasodilation or constriction and may control the spatial distribution of these responses. Further studies are needed to determine the relationship between the cellular sources of NO, regulation of its synthesis, and its effects on neurovascular coupling.
Our results are in agreement with previous studies demonstrating the involvement of glial cells in mediating neurovascular coupling. Zonta et al. (2003)
found that in the hippocampal slice, glial stimulation results in arteriole dilation mediated by production of COX metabolites. Recently, Takano et al. (2006)
reported that in somatosensory cortex in vivo
, glial stimulation elicited vasodilation via production of COX-1 metabolites. In contrast, we found that in the retina, EET production, but not prostaglandin production, mediates vasodilation. This discrepancy could be a result of differences in the neurovascular coupling mechanisms operating in different CNS regions.
Mulligan and MacVicar (2004)
found that in hippocampal slices, glial stimulation results in vasoconstriction mediated by 20-HETE production, in agreement with our own findings. However, they did not observe glial-evoked vasodilations. The lack of a vasodilation response could be caused by high levels of NO in their preparation, leading to inhibition of EET production, because they report that glial-evoked vasodilations were observed after NOS inhibition by L-NAME.
Filosa et al. (2004)
reported that in cortical slices, evoked Ca2+
increases in astrocytes result in a decrease in Ca2+
oscillations in adjacent vascular smooth muscle cells and to changes in vasomotor activity, demonstrating functional glia to vascular signaling. Cauli et al. (2004)
report that stimulation of interneurons in cortical slices can induce either vasodilation or vasoconstriction, in agreement with our own findings that both types of vasomotor responses can be evoked by neuronal activity.
Our finding that light stimulation and glial stimulation evoke both vasodilation and vasoconstriction suggests that regulation of blood flow in the CNS may also involve both vasomotor responses. For instance, neuronal activity in a localized CNS region could evoke vasodilation within that region and vasoconstriction in more distant vessels, resulting in an increased redistribution of blood flow to the vasodilating region. It is noteworthy that fMRI studies of activity-dependent hemodynamic changes indicate that a center-surround-like regulation of blood flow may occur in vivo
(Harel et al., 2002
; Shmuel et al., 2002
; Devor et al., 2005
). Active vasoconstriction may contribute to such an effect. It is difficult to predict what the spatial distribution of glial-evoked vasodilations and vasoconstrictions will be in vivo
. The distribution will depend on a number of factors that could not be reproduced in our experiments, including the spatial distribution of neuronal activation, the resting level of NO, and the spatial distribution of NO production.
Regulation of blood flow in response to neuronal activity is a complex process and is undoubtedly mediated by several mechanisms. Our results suggest that glial cells are involved in generating activity-dependent vasomotor responses and can induce both vasodilation and vasoconstriction. NO, which plays a central role in neurovascular coupling, may exert its effect, in part, by modulating glial cell regulation of these vasomotor responses.