Catherine N. Hall

Capillary pericytes regulate cerebral blood flow in health and disease

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.

What is the real physiological NO concentration in vivo

Clarity about the nitric oxide (NO) concentrations existing physiologically is essential for developing a quantitative understanding of NO signalling, for performing experiments with NO that emulate reality, and for knowing whether or not NO concentrations become abnormal in disease states. A decade ago, a value of about 1 μM seemed reasonable based on early electrode measurements and a provisional estimate of the potency of NO for its guanylyl cyclase-coupled receptors, which mediate physiological NO signal transduction. Since then, numerous efforts to measure NO concentrations directly using electrodes in cells and tissues have yielded an irreconcilably large spread of values. In compensation, data from several alternative approaches have now converged to provide a more coherent picture. These approaches include the quantitative analysis of NO-activated guanylyl cyclase, computer modelling based on the type, activity and amount of NO synthase enzyme contained in cells, the use of novel biosensors to monitor NO release from single endothelial cells and neurones, and the use of guanylyl cyclase as an endogenous NO biosensor in tissue subjected to a variety of challenges. All these independent lines of evidence suggest the physiological NO concentration range to be 100 pM (or below) up to ∼5 nM, orders of magnitude lower than was once thought.

Pericyte-mediated regulation of capillary diameter: a component of neurovascular coupling in health and disease

Because regional blood flow increases in association with the increased metabolic demand generated by localized increases in neural activity, functional imaging researchers often assume that changes in blood flow are an accurate read-out of changes in underlying neural activity. An understanding of the mechanisms that link changes in neural activity to changes in blood flow is crucial for assessing the validity of this assumption, and for understanding the processes that can go wrong during disease states such as ischaemic stroke. Many studies have investigated the mechanisms of neurovascular regulation in arterioles but other evidence suggests that blood flow regulation can also occur in capillaries, because of the presence of contractile cells, pericytes, on the capillary wall. Here we review the evidence that pericytes can modulate capillary diameter in response to neuronal activity and assess the likely importance of neurovascular regulation at the capillary level for functional imaging experiments. We also discuss evidence suggesting that pericytes are particularly sensitive to damage during pathological insults such as ischaemia, Alzheimers disease and diabetic retinopathy, and consider the potential impact that pericyte dysfunction might have on the development of therapeutic interventions and on the interpretation of functional imaging data in these disorders.

Oxidative Phosphorylation, Not Glycolysis, Powers Presynaptic and Postsynaptic Mechanisms Underlying Brain Information Processing

Neural activity has been suggested to initially trigger ATP production by glycolysis, rather than oxidative phosphorylation, for three reasons: glycolytic enzymes are associated with ion pumps; neurons may increase their energy supply by activating glycolysis in astrocytes to generate lactate; and activity increases glucose uptake more than O2 uptake. In rat hippocampal slices, neuronal activity rapidly decreased the levels of extracellular O2 and intracellular NADH (reduced nicotinamide adenine dinucleotide), even with lactate dehydrogenase blocked to prevent lactate generation, or with only 20% superfused O2 to mimic physiological O2 levels. Pharmacological analysis revealed an energy budget in which 11% of O2 use was on presynaptic action potentials, 17% was on presynaptic Ca2+ entry and transmitter release, 46% was on postsynaptic glutamate receptors, and 26% was on postsynaptic action potentials, in approximate accord with theoretical brain energy budgets. Thus, the major mechanisms mediating brain information processing are all initially powered by oxidative phosphorylation, and an astrocyte–neuron lactate shuttle is not needed for this to occur.

What is a pericyte

Pericytes, spatially isolated contractile cells on capillaries, have been reported to control cerebral blood flow physiologically, and to limit blood flow after ischaemia by constricting capillaries and then dying. Paradoxically, a recent paper dismisses the idea of pericytes controlling cerebral blood flow, despite confirming earlier data showing a role for pericytes. We show that these discrepancies are apparent rather than real, and depend on the new paper defining pericytes differently from previous reports. An objective definition of different sub-classes of pericyte along the capillary bed is needed to develop novel therapeutic approaches for stroke and disorders caused by pericyte malfunction.

Inactivation of nitric oxide by rat cerebellar slices

Nitric oxide (NO) functions as an intercellular messenger throughout the brain. For this role to be performed efficiently, there must be a mechanism for neutralizing NO, but whether an active biological process exists, or whether NO is lost mainly through diffusion is unclear. To investigate this issue, rat cerebellar slices were exposed to constant levels of NO and the cGMP generated within the slice used as an indicator of NO concentrations therein. NO was about 1000‐fold less potent in slices (EC50, 1 μm) than in separated cells from the same tissue (EC50, 1.6 nm), consistent with access of NO to the slice interior being greatly hindered by inactivation. Supporting this interpretation, immunohistochemical analysis indicated a marked concentration gradient of cGMP across the thickness of slices exposed to subsaturating NO concentrations, signifying a marked NO gradient. Several known NO‐degrading processes, including reaction with lipid peroxyl radicals, erythrocytes and superoxide ions, were eliminated as contributing factors, indicating a novel mechanism. A diffusion–inactivation model was used to estimate the kinetics of NO consumption by the slices. The best fits to experimental data indicated a Michaelis‐Menten‐type reaction having a Vmax of 1–2 μm s−1 and a Km of around 10 nm. The rates predict that inactivation would impose a very short half‐life (<10 ms) on NO in physiological concentrations (up to 10 nm) and that it would play an important role in shaping the NO concentration profiles when it is synthesized by multiple nearby sites.

Assessing the physiological concentration and targets of nitric oxide in brain tissue

Low nanomolar concentrations of nitric oxide activate guanylyl cyclase to produce cGMP, which has diverse physiological effects. Higher concentrations inhibit mitochondrial respiration at cytochrome c oxidase and this has been proposed to be important physiologically, increasing oxygen permeation into tissue (by reducing the oxygen use of cells near blood vessels), activating AMP kinase, and regulating the relationship between cerebral blood flow and oxygen use. It is unclear, however, whether nitric oxide can accumulate physiologically to concentrations at which inhibition of respiration occurs. In rat cerebellar slices, we activated nitric oxide production from each isoform of nitric oxide synthase. Only activation of inducible nitric oxide synthase, which is expressed pathologically, caused any significant inhibition of respiration. Modelling oxygen and nitric oxide concentrations predicted that, in vivo, physiological nitric oxide levels are too low to affect respiration. Even pathologically, the nitric oxide concentration may only rise to 2.5 nm, producing a 1.5% inhibition of respiration. Thus, under physiological conditions, nitric oxide signals do not inhibit respiration but are well‐tuned to the dynamic range of guanylyl cyclase activation.

Imaging pericytes and capillary diameter in brain slices and isolated retinae

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.

Cytochrome P450 oxidoreductase participates in nitric oxide consumption by rat brain

In low nanomolar concentrations, NO (nitric oxide) functions as a transmitter in brain and other tissues, whereas near-micromolar NO concentrations are associated with toxicity and cell death. Control of the NO concentration, therefore, is critical for proper brain function, but, although its synthesis pathway is well-characterized, the major route of breakdown of NO in brain is unclear. Previous observations indicate that brain cells actively consume NO at a high rate. The mechanism of this consumption was pursued in the present study. NO consumption by a preparation of central glial cells was abolished by cell lysis and recovered by addition of NADPH. NADPH-dependent consumption of NO localized to cell membranes and was inhibited by proteinase K, indicating the involvement of a membrane-bound protein. Purification of this activity yielded CYPOR (cytochrome P450 oxidoreductase). Antibodies against CYPOR inhibited NO consumption by brain membranes and the amount of CYPOR in several cell types correlated with their rate of NO consumption. NO was also consumed by purified CYPOR but this activity was found to depend on the presence of the vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), included in the buffer as a precaution against inadvertent NO consumption by lipid peroxidation. In contrast, NO consumption by brain membranes was independent of Trolox. Hence, it appears that, during the purification process, CYPOR becomes separated from a partner needed for NO consumption. Cytochrome P450 inhibitors inhibited NO consumption by brain membranes, making these proteins likely candidates.

Non-signalling energy use in the developing rat brain

Energy use in the brain constrains its information processing power, but only about half the brains energy consumption is directly related to information processing. Evidence for which non-signalling processes consume the rest of the brains energy has been scarce. For the first time, we investigated the energy use of the brains main non-signalling tasks with a single method. After blocking each non-signalling process, we measured oxygen level changes in juvenile rat brain slices with an oxygen-sensing microelectrode and calculated changes in oxygen consumption throughout the slice using a modified diffusion equation. We found that the turnover of the actin and microtubule cytoskeleton, followed by lipid synthesis, are significant energy drains, contributing 25%, 22% and 18%, respectively, to the rate of oxygen consumption. In contrast, protein synthesis is energetically inexpensive. We assess how these estimates of energy expenditure relate to brain energy use in vivo, and how they might differ in the mature brain.