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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 Ca 2+ responses in astrocytes. We found that astrocytes detect SOM-IN synaptic activity via GABA B R and GAT-3-dependent Ca 2+ signaling mechanisms, the latter triggering the release of ATP. In turn, ATP is converted into adenosine, activating A 1 Rs 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. Astrocytes have been shown to regulate glutamatergic transmission in the brain. Here, the authors show that astrocytes also detect and modulate GABAergic transmission from somatostatin but not parvalbumin-positive interneurons, thus regulating dendritic inhibition via a feedback loop.

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.

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 cGMP-hydrolyzing 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.

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 E 2 , 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. Neuronal activity relaxes pericytes, leading to capillary dilation and increased blood flow, before arterioles dilate, suggesting that pericytes initiate blood-oxygen-level-dependent (BOLD) functional imaging signals; pericytes constrict and die in rigor in ischaemia, which will cause a long-lasting blood flow decrease after stroke, and damage the blood–brain barrier. Blood flow response to neural activity Cerebral blood flow dynamics have long been linked to neural activity, and form the basis of BOLD (blood-oxygen-level-dependent) functional imaging. But how such blood flow changes are mediated has remained controversial. Here, David Attwell and colleagues reveal how neuronal activity can hyperpolarize pericytes, leading to their relaxation and capillary dilation. Capillary dilation is responsible for 84% of the blood increase linked to neural activity, so irreversible capillary closure due to pericyte death during ischaemia can injure the blood–brain barrier and exacerbate injury. Pericyte death under pathological conditions can be reduced if glutamate receptor signalling is inhibited. This work suggests that pericytes are major regulators of cerebral blood flow and may initiate BOLD imaging signals.

Nat. Protoc. 9, 323–336 (2014); doi: 10.1038/nprot.2014.019; published online 16 January 2014; corrected after print 2 May 2014 In the version of this article initially published, the authors omitted the following from the acknowledgments: “We thank A. Nishiyama and D. Dietrich for providing NG2-DsRed mice”.