Active neurons increase their energy supply by dilating nearby arterioles and capillaries to increase blood flow, but the mechanisms underlying neurovascular coupling are debated. In this paper, the authors show that different calcium-dependent signaling pathways regulate blood flow at the level of capillary pericytes and arteriole smooth muscle. Active neurons increase their energy supply by dilating nearby arterioles and capillaries. This neurovascular coupling underlies blood oxygen level–dependent functional imaging signals, but its mechanism is controversial. Canonically, neurons release glutamate to activate metabotropic glutamate receptor 5 (mGluR5) on astrocytes, evoking Ca2+ release from internal stores, activating phospholipase A2 and generating vasodilatory arachidonic acid derivatives. However, adult astrocytes lack mGluR5, and knockout of the inositol 1,4,5-trisphosphate receptors that release Ca2+ from stores does not affect neurovascular coupling. We now show that buffering astrocyte Ca2+ inhibits neuronally evoked capillary dilation, that astrocyte [Ca2+]i is raised not by release from stores but by entry through ATP-gated channels, and that Ca2+ generates arachidonic acid via phospholipase D2 and diacylglycerol lipase rather than phospholipase A2. In contrast, dilation of arterioles depends on NMDA receptor activation and Ca2+-dependent NO generation by interneurons. These results reveal that different signaling cascades regulate cerebral blood flow at the capillary and arteriole levels.

Fast excitatory synapses are generally thought to act as private communication channels between presynaptic and postsynaptic neurons. Some recent findings, however, suggest that glutamate may diffuse out of the synaptic cleft and bind to several subtypes of receptors, either in the perisynaptic membrane or at neighboring synapses. It is not known whether activation of these receptors can occur in response to the release of a single vesicle of glutamate. Here we estimate the spatiotemporal profile of glutamate in the extrasynaptic space after vesicle exocytosis, guided by detailed ultrastructural measurements of the CA1 neuropil in the adult rat. We argue that the vicinity of the synapse can be treated as an isotropic porous medium, in which diffusion is determined by the extracellular volume fraction and the tortuosity factor, and develop novel stereological methods to estimate these parameters. We also estimate the spatial separation between synapses, to ask whether glutamate released at one synapse can activate NMDA and other high-affinity receptors at a neighboring synapse. Kinetic simulations of extrasynaptic glutamate uptake show that transporters rapidly reduce the free concentration of transmitter. Exocytosis of a single vesicle is, however, sufficient to bind to high-affinity receptors situated in the immediate perisynaptic space. The distance separating a typical synapse from its nearest neighbor is ∼465 nm. Whether glutamate can reach a sufficient concentration to activate NMDA receptors at this distance depends critically on the diffusion coefficient in the extracellular space. If diffusion is much slower than in free aqueous solution, NMDA receptors could mediate crosstalk between neighboring synapses.

Extrasynaptic actions of glutamate are limited by high-affinity transporters expressed by perisynaptic astroglial processes (PAPs): this helps maintain point-to-point transmission in excitatory circuits. Memory formation in the brain is associated with synaptic remodeling, but how this affects PAPs and therefore extrasynaptic glutamate actions is poorly understood. Here, we used advanced imaging methods, in situ and in vivo, to find that a classical synaptic memory mechanism, long-term potentiation (LTP), triggers withdrawal of PAPs from potentiated synapses. Optical glutamate sensors combined with patch-clamp and 3D molecular localization reveal that LTP induction thus prompts spatial retreat of astroglial glutamate transporters, boosting glutamate spillover and NMDA-receptor-mediated inter-synaptic cross-talk. The LTP-triggered PAP withdrawal involves NKCC1 transporters and the actin-controlling protein cofilin but does not depend on major Ca2+-dependent cascades in astrocytes. We have therefore uncovered a mechanism by which a memory trace at one synapse could alter signal handling by multiple neighboring connections.