Increases in brain blood flow, evoked by neuronal activity, power neural computation and form the basis of BOLD (blood-oxygen-level-dependent) functional imaging. Whether blood flow is controlled solely by arteriole smooth muscle, or also by capillary pericytes, is controversial. We demonstrate that neuronal activity and the neurotransmitter glutamate evoke the release of messengers that dilate capillaries by actively relaxing pericytes. Dilation is mediated by prostaglandin E2, but requires nitric oxide release to suppress vasoconstricting 20-HETE synthesis. In vivo, when sensory input increases blood flow, capillaries dilate before arterioles and are estimated to produce 84% of the blood flow increase. In pathology, ischaemia evokes capillary constriction by pericytes. We show that this is followed by pericyte death in rigor, which may irreversibly constrict capillaries and damage the blood-brain barrier. Thus, pericytes are major regulators of cerebral blood flow and initiators of functional imaging signals. Prevention of pericyte constriction and death may reduce the long-lasting blood flow decrease that damages neurons after stroke.

BOLD fMRI (blood oxygenation level dependent functional magnetic resonance imaging) is increasingly used to detect developmental changes of human brain function that are hypothesized to underlie the maturation of cognitive processes. BOLD signals depend on neuronal activity increasing cerebral blood flow, and are reduced by neural oxygen consumption. Thus, developmental changes of BOLD signals may not reflect altered information processing if there are concomitant changes in neurovascular coupling (the mechanism by which neuronal activity increases blood flow) or neural energy use (and hence oxygen consumption). We review how BOLD signals are generated, and explain the signalling pathways which convert neuronal activity into increased blood flow. We then summarize in broad terms the developmental changes that the brain's neural circuitry undergoes during growth from childhood through adolescence to adulthood, and present the changes in neurovascular coupling mechanisms and energy use which occur over the same period. This information provides a framework for assessing whether the BOLD changes observed during human development reflect altered cognitive processing or changes in neurovascular coupling and energy use.

The cerebral circulation is highly specialized, both structurally and functionally, and it provides a fine-tuned supply of oxygen and nutrients to active regions of the brain. Our understanding of blood flow regulation by cerebral arterioles has evolved rapidly. Recent work has opened new avenues in microvascular research; for example, it has been demonstrated that contractile pericytes found on capillary walls induce capillary diameter changes in response to neurotransmitters, suggesting that pericytes could have a role in neurovascular coupling. This concept is at odds with traditional models of brain blood flow regulation, which assume that only arterioles control cerebral blood flow. The investigation of mechanisms underlying neurovascular coupling at the capillary level requires a range of approaches, which involve unique technical challenges. Here we provide detailed protocols for the successful physiological and immunohistochemical study of pericytes and capillaries in brain slices and isolated retinae, allowing investigators to probe the role of capillaries in neurovascular coupling. This protocol can be completed within 6-8 h; however, immunohistochemical experiments may take 3-6 d.

Astrocytes are important regulators of excitatory synaptic networks. However, astrocytes regulation of inhibitory synaptic systems remains ill defined. This is particularly relevant since GABAergic interneurons regulate the activity of excitatory cells and shape network function. To address this issue, we combined optogenetics and pharmacological approaches, two-photon confocal imaging and whole-cell recordings to specifically activate hippocampal somatostatin or paravalbumin-expressing interneurons (SOM-INs or PV-INs), while monitoring inhibitory synaptic currents in pyramidal cells and Ca2+ responses in astrocytes. We found that astrocytes detect SOM-IN synaptic activity via GABABR and GAT-3-dependent Ca2+ signaling mechanisms, the latter triggering the release of ATP. In turn, ATP is converted into adenosine, activating A1Rs and upregulating SOM-IN synaptic inhibition of pyramidal cells, but not PV-IN inhibition. Our findings uncover functional interactions between a specific subpopulation of interneurons, astrocytes and pyramidal cells, involved in positive feedback autoregulation of dendritic inhibition of pyramidal cells.

Nitric oxide (NO) functions as a diffusible transmitter in most tissues of the body and exerts its effects by binding to receptors harboring a guanylyl cyclase transduction domain, resulting in cGMP accumulation in target cells. Despite its widespread importance, very little is known about how this signaling pathway operates at physiological NO concentrations and in real time. To address these deficiencies, we have exploited the properties of a novel cGMP biosensor, named δ-FlincG, expressed in cells containing varying mixtures of NO-activated guanylyl cyclase and cGMPhydrolyzing phosphodiesterase activity. Responsiveness to NO, signifying a physiologically relevant rise in cGMP to 30 nM or more, was seen at concentrations as low as 1 pM, making cells by far the most sensitive NO detectors yet encountered. Even cells coexpressing phosphodiesterase-5, a cGMP-activated isoform found in many NO target cells, responded to NO in concentrations as low as 10 pM. The dynamics of NO capture and signal transduction was revealed by administering timed puffs of NO from a local pipette. A puff lasting only 100 ms, giving a calculated peak intracellular NO concentration of 23 pM, was detectable. The results could be encapsulated in a quantitative model of cellular NO-cGMP signaling, which recapitulates the NO responsiveness reported previously from crude cGMP measurements on native cells, and which explains how NO is able to exert physiological effects at extremely low concentrations, when only a tiny proportion of its receptors would be occupied.is an evolutionarily ancient transmitter of fundamental importance to the physiology of the mammalian cardiovascular, nervous, and other systems (1, 2). NO signal transduction takes place through the simplest one-component type of receptor, comprising an NO binding site (a prosthetic heme) coupled to a guanylyl cyclase (GC) transduction domain (3, 4). The elevation in cellular cGMP that follows NO binding is curtailed by one or more phosphodiesterase (PDE) enzymes that convert it to GMP. There have been countless descriptions of changes in the levels of cGMP in association with NOmediated transmission in different cells and tissues. It has become increasingly evident, however, that the levels of cGMP measurable by traditional methods, such as radioimmunoassay, may not be relevant to physiological NO signaling. For example, smooth muscle relaxation (5) and cGMP-dependent phosphorylation events in platelets (6) are seen at NO concentrations below those giving increases in cGMP measurable with such methods; NO can still relax vascular smooth muscle despite deletion of 94% of the NO-activated GC (7); and engagement of 2% or less of the available GC activity is sufficient to stimulate cGMPdependent phosphorylation in platelets (8). These findings indicate that cells possess a very large receptor excess and that new approaches are required to understand the elementary properties of NO capture and signal amplification by cells under physiological conditions. A related gap in knowledge is in...