Nicola B. Hamilton

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.

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.

Regulation of Oligodendrocyte Development and Myelination by Glucose and Lactate

In the gray matter of the brain, astrocytes have been suggested to export lactate (derived from glucose or glycogen) to neurons to power their mitochondria. In the white matter, lactate can support axon function in conditions of energy deprivation, but it is not known whether lactate acts by preserving energy levels in axons or in oligodendrocytes, the myelinating processes of which are damaged rapidly in low energy conditions. Studies of cultured cells suggest that oligodendrocytes are the cell type in the brain that consumes lactate at the highest rate, in part to produce membrane lipids presumably for myelin. Here, we use pH imaging to show that oligodendrocytes in the white matter of the rat cerebellum and corpus callosum take up lactate via monocarboxylate transporters (MCTs), which we identify as MCT1 by confocal immunofluorescence and electron microscopy. Using cultured slices of developing cerebral cortex from mice in which oligodendrocyte lineage cells express GFP (green fluorescent protein) under the control of the Sox10 promoter, we show that a low glucose concentration reduces the number of oligodendrocyte lineage cells and myelination. Myelination is rescued when exogenous l-lactate is supplied. Thus, lactate can support oligodendrocyte development and myelination. In CNS diseases involving energy deprivation at times of myelination or remyelination, such as periventricular leukomalacia leading to cerebral palsy, stroke, and secondary ischemia after spinal cord injury, lactate transporters in oligodendrocytes may play an important role in minimizing the inhibition of myelination that occurs.

Mechanisms of ATP‐ and glutamate‐mediated calcium signaling in white matter astrocytes

Neurotransmitters released at synapses mediate Ca2+ signaling in astrocytes in CNS grey matter. Here, we show that ATP and glutamate evoke these Ca2+ signals in white matter astrocytes of the mouse optic nerve, a tract that contains neither neuronal cell bodies nor synapses. We further demonstrate that action potentials along white matter axons trigger the release of ATP and the intercellular propagation of astroglial Ca2+ signals. These mechanisms were studied in astrocytes in intact optic nerves isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the human glial fibrillary acidic protein promoter (GFAP) by Fura‐2 ratiometric Ca2+ imaging. ATP evoked astroglial Ca2+ signals predominantly via metabotropic P2Y1 and ionotropic P2X7 purinoceptors. Glutamate acted on both AMPA‐ and NMDA‐type receptors, as well as on group I mGlu receptors to induce an increase in astroglial [Ca2+]i. The direct Ca2+ signal evoked by glutamate was small, and the main action of glutamate was to trigger the release of the “gliotransmitter” ATP by a mechanism involving P2X7 receptors; propagation of the glutamate‐mediated Ca2+ signal was significantly reduced in P2X7 knock‐out mice. Furthermore, axonal action potentials and mechanical stimulation of astrocytes both induced the release of ATP, to propagate Ca2+ signals in astrocytes and neighboring EGFP‐negative glia. Our data provide a model of multiphase axon–glial signaling in the optic nerve as follows: action potentials trigger axonal release of ATP, which evokes further release of ATP from astrocytes, and this acts by amplifying the initiating signal and by transmitting an intercellular Ca2+ wave to neighboring glia.

Properties and Fate of Oligodendrocyte Progenitor Cells in the Corpus Callosum, Motor Cortex, and Piriform Cortex of the Mouse

Oligodendrocyte progenitor cells (OPCs) in the postnatal mouse corpus callosum (CC) and motor cortex (Ctx) reportedly generate only oligodendrocytes (OLs), whereas those in the piriform cortex may also generate neurons. OPCs have also been subdivided based on their expression of voltage-gated ion channels, ability to respond to neuronal activity, and proliferative state. To determine whether OPCs in the piriform cortex have inherently different physiological properties from those in the CC and Ctx, we studied acute brain slices from postnatal transgenic mice in which GFP expression identifies OL lineage cells. We whole-cell patch clamped GFP-expressing (GFP+) cells within the CC, Ctx, and anterior piriform cortex (aPC) and used prelabeling with 5-ethynyl-2′-deoxyuridine (EdU) to assess cell proliferation. After recording, slices were immunolabeled and OPCs were defined by strong expression of NG2. NG2+ OPCs in the white and gray matter proliferated and coexpressed PDGFRα and voltage-gated Na+ channels (INa). Approximately 70% of OPCs were capable of generating regenerative depolarizations. In addition to OLIG2+ NG2+ INa+ OPCs and OLIG2+ NG2neg INaneg OLs, we identified cells with low levels of NG2 limited to the soma or the base of some processes. These cells had a significantly reduced INa and a reduced ability to incorporate EdU when compared with OPCs and probably correspond to early differentiating OLs. By combining EdU labeling and lineage tracing using Pdgfrα–CreERT2 : R26R–YFP transgenic mice, we double labeled OPCs and traced their fate in the postnatal brain. These OPCs generated OLs but did not generate neurons in the aPC or elsewhere at any time that we examined.

Axons and astrocytes release ATP and glutamate to evoke calcium signals in NG2-glia

NG2‐glia are an abundant population of cells in the adult CNS that make up a novel glial cell type. Here, we have examined calcium signals in NG2‐glia identified by expression of the fluorescent protein DsRed under the control of the NG2 promoter in the white matter of the mouse optic nerve. We focused on mice aged postnatal day (P)12–16, after the main period of oligodendrocyte generation. Using fluo‐4 and fura‐2 calcium imaging in isolated intact nerves, we show that glutamate and ATP evoke Ca2+ signals in NG2‐glia in situ, acting on AMPA‐type glutamate receptors and P2Y1 and P2X7 purine receptors; NMDA evoked a weak Ca2+ signal in a small proportion of NG2‐glia. We show that axonal action potentials and mechanical stimulation of astrocytes effect the release of glutamate and ATP to act on NG2‐glia; ATP alone evokes robust Ca2+ signals, whereas glutamate did not unless AMPA receptor desensitization was blocked with cyclothiazide. We identify the precise contacts that NG2‐glia form with axons at nodes of Ranvier, and the intricate bipartite sheaths formed between the processes of NG2‐glia and astrocytes. In addition, we provide evidence that NG2‐glia express synaptophysin, indicating they have mechanisms for transmitting as well as receiving signals. This study places NG2‐glia within a neuron‐glial network, and identifies roles for glutamate and ATP in communication with astrocytes as well as axons.

Glutamatergic signaling in the brain's white matter.

Glutamatergic signaling has been exceptionally well characterized in the brains gray matter, where it underlies fast information processing, learning and memory, and also generates the neuronal damage that occurs in pathological conditions such as stroke. The role of glutamatergic signaling in the white matter, an area until recently thought to be devoid of synapses, is less well understood. Here we review what is known, and highlight what is not known, of glutamatergic signaling in the white matter. We focus on how glutamate is released, the location and properties of the receptors it acts on, the interacting molecules that may regulate trafficking or signaling of the receptors, the possible functional roles of glutamate in the white matter, and its pathological effects including the possibility of treating white matter disorders with glutamate receptor blockers.

Proton-gated Ca2+-permeable TRP channels damage myelin in conditions mimicking ischaemia

The myelin sheaths wrapped around axons by oligodendrocytes are crucial for brain function. In ischaemia myelin is damaged in a Ca2+-dependent manner, abolishing action potential propagation. This has been attributed to glutamate release activating Ca2+-permeable N-methyl-d-aspartate (NMDA) receptors. Surprisingly, we now show that NMDA does not raise the intracellular Ca2+ concentration ([Ca2+]i) in mature oligodendrocytes and that, although ischaemia evokes a glutamate-triggered membrane current, this is generated by a rise of extracellular [K+] and decrease of membrane K+ conductance. Nevertheless, ischaemia raises oligodendrocyte [Ca2+]i, [Mg2+]i and [H+]i, and buffering intracellular pH reduces the [Ca2+]i and [Mg2+]i increases, showing that these are evoked by the rise of [H+]i. The H+-gated [Ca2+]i elevation is mediated by channels with characteristics of TRPA1, being inhibited by ruthenium red, isopentenyl pyrophosphate, HC-030031, A967079 or TRPA1 knockout. TRPA1 block reduces myelin damage in ischaemia. These data suggest that TRPA1-containing ion channels could be a therapeutic target in white matter ischaemia.

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.

The effect of N-acetyl-aspartyl-glutamate and N-acetyl-aspartate on white matter oligodendrocytes.

Elevations of the levels of N-acetyl-aspartyl-glutamate (NAAG) and N-acetyl-aspartate (NAA) are associated with myelin loss in the leucodystrophies Canavans disease and Pelizaeus-Merzbacher-like disease. NAAG and NAA can activate and antagonize neuronal N-methyl-D-aspartate (NMDA) receptors, and also act on group II metabotropic glutamate receptors. Oligodendrocytes and their precursors have recently been shown to express NMDA receptors, and activation of these receptors in ischaemia leads to the death of oligodendrocyte precursors and the loss of myelin. This raises the possibility that the failure to develop myelin, or demyelination, occurring in the leucodystrophies could reflect an action of NAAG or NAA on oligodendrocyte NMDA receptors. However, since the putative subunit composition of NMDA receptors on oligodendrocytes differs from that of neuronal NMDA receptors, the effects of NAAG and NAA on them are unknown. We show that NAAG, but not NAA, evokes an inward membrane current in cerebellar white matter oligodendrocytes, which is reduced by NMDA receptor block (but not by block of metabotropic glutamate receptors). The size of the current evoked by NAAG, relative to that evoked by NMDA, was much smaller in oligodendrocytes than in neurons, and NAAG induced a rise in [Ca2+]i in neurons but not in oligodendrocytes. These differences in the effect of NAAG on oligodendrocytes and neurons may reflect the aforementioned difference in receptor subunit composition. In addition, as a major part of the response in oligodendrocytes was blocked by tetrodotoxin (TTX), much of the NAAG-evoked current in oligodendrocytes is a secondary consequence of activating neuronal NMDA receptors. Six hours exposure to 1 mM NAAG did not lead to the death of cells in the white matter. We conclude that an action of NAAG on oligodendrocyte NMDA receptors is unlikely to be a major contributor to white matter damage in the leucodystrophies.