Brian A. MacVicar

Glial and neuronal control of brain blood flow

Blood flow in the brain is regulated by neurons and astrocytes. Knowledge of how these cells control blood flow is crucial for understanding how neural computation is powered, for interpreting functional imaging scans of brains, and for developing treatments for neurological disorders. It is now recognized that neurotransmitter-mediated signalling has a key role in regulating cerebral blood flow, that much of this control is mediated by astrocytes, that oxygen modulates blood flow regulation, and that blood flow may be controlled by capillaries as well as by arterioles. These conceptual shifts in our understanding of cerebral blood flow control have important implications for the development of new therapeutic approaches.

Calcium transients in astrocyte endfeet cause cerebrovascular constrictions

Cerebral blood flow (CBF) is coupled to neuronal activity and is imaged in vivo to map brain activation. CBF is also modified by afferent projection fibres that release vasoactive neurotransmitters in the perivascular region, principally on the astrocyte endfeet that outline cerebral blood vessels. However, the role of astrocytes in the regulation of cerebrovascular tone remains uncertain. Here we determine the impact of intracellular Ca2+ concentrations ([Ca2+]i) in astrocytes on the diameter of small arterioles by using two-photon Ca2+ uncaging to increase [Ca2+]i. Vascular constrictions occurred when Ca2+ waves evoked by uncaging propagated into the astrocyte endfeet and caused large increases in [Ca2+]i. The vasoactive neurotransmitter noradrenaline increased [Ca2+]i in the astrocyte endfeet, the peak of which preceded the onset of arteriole constriction. Depressing increases in astrocyte [Ca2+]i with BAPTA inhibited the vascular constrictions in noradrenaline. We find that constrictions induced in the cerebrovasculature by increased [Ca2+]i in astrocyte endfeet are generated through the phospholipase A2–arachidonic acid pathway and 20-hydroxyeicosatetraenoic acid production. Vasoconstriction by astrocytes is a previously unknown mechanism for the regulation of CBF.

Brain metabolism dictates the polarity of astrocyte control over arterioles

Calcium signalling in astrocytes couples changes in neural activity to alterations in cerebral blood flow by eliciting vasoconstriction or vasodilation of arterioles. However, the mechanism for how these opposite astrocyte influences provide appropriate changes in vessel tone within an environment that has dynamic metabolic requirements remains unclear. Here we show that the ability of astrocytes to induce vasodilations over vasoconstrictions relies on the metabolic state of the rat brain tissue. When oxygen availability is lowered and astrocyte calcium concentration is elevated, astrocyte glycolysis and lactate release are maximized. External lactate attenuates transporter-mediated uptake from the extracellular space of prostaglandin E2, leading to accumulation and subsequent vasodilation. In conditions of low oxygen concentration extracellular adenosine also increases, which blocks astrocyte-mediated constriction, facilitating dilation. These data reveal the role of metabolic substrates in regulating brain blood flow and provide a mechanism for differential astrocyte control over cerebrovascular diameter during different states of brain activation.

Imaging cell volume changes and neuronal excitation in the hippocampal slice

Brain cell swelling is a consequence of seizure, ischemia or excitotoxicity. Changes in light reflectance from cortical surface are now used to monitor brain activity but these intrinsic signals are poorly understood. The objectives of this study were first, to show that changes in light transmittance were correlated with cell volume and second, to image increases in light transmittance as they related to neuronal activation. Transverse hippocampal slices from the rat were used for the study. Brief exposure (4-6 min) to hypo-osmotic artificial cerebrospinal fluid (-40 mOsm) elevated light transmittance consistently and reversibly in most regions of the slice and particularly in CA1 dendritic regions. Neither zero-Ca2+ artificial cerebrospinal fluid nor tetrodotoxin altered the transmittance increase and its subsequent reversal, suggesting that it was dependent on osmolality but independent of synaptic transmission and neuronal firing. The amplitude of the CA1 population spike evoked from Schaffer collaterals increased concomitantly with the hypo-osmotic increase in light transmittance, providing evidence that the extracellular tissue resistance increased. Hyper-osmotic artificial cerebrospinal fluid (+40 mOsm) containing impermeant mannitol consistently lowered light transmittance and the amplitude of the population spike. Glycerol (+40 mOsm), which is cell permeant, did not have an affect. Taken together these observations indicate that osmotic challenge alters light transmittance by inducing changes in cell volume. Transmittance increases induced by hypo-osmotic artificial cerebrospinal fluid or 10 microM kainate were small in the CA1 cell body region compared to dendritic regions. Similarly, orthodromic stimulation of axons terminating in stratum oriens or in stratum radiatum evoked transmittance increases only in their respective postsynaptic areas. In contrast, the cell body region and its adjacent proximal-apical dendrites (both sites of action potential initiation) could display dramatic increases in light transmittance upon brief exposure to 20 mM K+. The response, which may represent neuronal damage, was blocked in tetrodotoxin. Antidromic stimulation evoked a weak response in these same proximal areas. We conclude that activity-dependent increases in light transmittance across brain slices primarily reveal glial and neuronal swelling associated with excitatory synaptic input and action potential discharge. The signal can be imaged in real time to reveal neuronal activation, not only among hippocampal areas, but among neuronal regions. Cell swelling is a known consequence of excessive neuronal discharge. Therefore, the imaging of changes in light transmittance across brain slices should prove useful in monitoring epileptiform and excitotoxic states.

Activation of Pannexin-1 Hemichannels Augments Aberrant Bursting in the Hippocampus

Pannexin-1 (Px1) is expressed at postsynaptic sites in pyramidal neurons, suggesting that these hemichannels contribute to dendritic signals associated with synaptic function. We found that, in pyramidal neurons, N-methyl-d-aspartate receptor (NMDAR) activation induced a secondary prolonged current and dye flux that were blocked with a specific inhibitory peptide against Px1 hemichannels; knockdown of Px1 by RNA interference blocked the current in cultured neurons. Enhancing endogenous NMDAR activation in brain slices by removing external magnesium ions (Mg2+) triggered epileptiform activity, which had decreased spike amplitude and prolonged interburst interval during application of the Px1 hemichannel blocking peptide. We conclude that Px1 hemichannel opening is triggered by NMDAR stimulation and can contribute to epileptiform seizure activity.

Astrocyte control of the cerebrovasculature

The control of cerebral vessel diameter is of fundamental importance in maintaining healthy brain function because it is critical to match cerebral blood flow (CBF) to the metabolic demand of active neurons. Recent studies have shown that astrocytes are critical players in the regulation of cerebral blood vessel diameter and that there are several molecular pathways through which astrocytes can elicit these changes. Increased intracellular Ca2+ in astrocytes has demonstrated a dichotomy in vasomotor responses by causing the constriction as well as the dilation of neighboring blood vessels. The production of arachidonic acid (AA) in astrocytes by Ca2+ sensitive phospholipase A2 (PLA2) has been shown to be common to both constriction and dilation mechanisms. Constriction results from the conversion of AA to 20‐hydroxyeicosatetraenoic acid (20‐HETE) and dilation from the production of prostaglandin E2 (PGE2) or epoxyeicosatrienoic acid (EET) and the level of nitric oxide (NO) appears to dictate which of these two pathways is recruited. In addition the activation of Ca2+ activated K+ channels in astrocyte endfeet and the efflux of K+ has also been suggested to modify vascular tone by hyperpolarization and relaxation of smooth muscle cells (SMCs). The wide range of putative pathways indicates that more work is needed to clarify the contributions of astrocytes to vascular dynamics under different cellular conditions. Nonetheless it is clear that astrocytes are important albeit complicated regulators of CBF.

Pannexin channels are not gap junction hemichannels

Pannexins, a class of membrane channels, bear significant sequence homology with the invertebrate gap junction proteins, innexins, and more distant similarities in their membrane topologies and pharmacological sensitivities with the gap junction proteins, connexins. However, the functional role for the pannexin oligomers or pannexons, is different from connexin oligomers, the connexons. Many pannexin publications have used the term “hemichannels” to describe pannexin oligomers while others use the term “channels” instead. This has led to confusion within the literature about the function of pannexins that promotes the idea that pannexons serve as gap junction hemichannels and thus, have an assembly and functional state as gap junctional intercellular channels. Here, we present the case that unlike the connexin gap junction intercellular channels, so far, pannexin oligomers have repeatedly been shown to be channels that are functional in single membranes, but not as intercellular channels in appositional membranes. Hence, they should be referred to as channels and not hemichannels. Thus, we advocate that in the absence of firm evidence that pannexins form gap junctions, the use of the term “hemichannel” be discontinued within the pannexin literature.

Non-junction functions of pannexin-1 channels.

Pannexins are large-pore ion channels with broad expression in the central nervous system (CNS). The channels function by releasing large signaling molecules, such ATP and arachidonic acid derivatives, from neurons and possibly astrocytes. They might also contribute to novel forms of non-synaptic communication in the CNS, thereby affecting synaptic function, astrocytic Ca(2+) wave propagation and possibly regulation of vascular tone in the brain. Panx1 activation in various in vitro pathological conditions implicates these channels in ischemic, excitotoxic and ATP-dependent cell death, whereas Panx coupling with purinergic receptors triggers the inflammasome. Novel functions for the pannexin channels are likely to be discovered as current understanding of how they are regulated in physiological and pathological situations improves.

Tumor-Suppressive Effects of Pannexin 1 in C6 Glioma Cells

Mammalian gap junction proteins, connexins, have long been implicated in tumor suppression. Recently, a novel family of proteins named pannexins has been identified as the mammalian counterpart of the invertebrate gap junction proteins, innexins. To date, pannexin 1 (Panx1) and pannexin 2 (Panx2) mRNAs are reported to be expressed in the brain. Most neoplastic cells, including rat C6 gliomas, exhibit reduced connexin expression, aberrant gap junctional intercellular communication (GJIC), and an increased proliferation rate. When gap junctions are up-regulated by transfecting C6 cells with connexin43, GJIC is restored and the proliferation is reduced. In this study, we examined the tumor-suppressive effects of Panx1 expression in C6 cells. Reverse transcription-PCR analysis revealed that C6 cells do not express any of the pannexin transcripts, whereas its nontumorigenic counterpart, rat primary astrocytes, exhibited mRNAs for all three pannexins. On generation of stable C6 transfectants with tagged Panx1 [myc or enhanced green fluorescent protein (EGFP)], a localization of Panx1 expression to the Golgi apparatus and plasma membrane was observed. In addition, Panx1 transfectants exhibited a flattened morphology, which differs greatly from the spindle-shaped control cells (EGFP only). Moreover, Panx1 expression increased gap junctional coupling as shown by the passage of sulforhodamine 101. Finally, we showed that stable expression of Panx1 in C6 cells significantly reduced cell proliferation in monolayers, cell motility, anchorage-independent growth, and in vivo tumor growth in athymic nude mice. Altogether, we conclude that the loss of pannexin expression may participate in the development of C6 gliomas, whereas restoration of Panx1 plays a tumor-suppressive role.

Local neuronal circuitry underlying cholinergic rhythmical slow activity in CA3 area of rat hippocampal slices.

1. Intracellular and extracellular recordings were obtained from the CA3 area of rat hippocampal slices to study cellular and synaptic mechanisms underlying rhythmic slow activity (RSA). In all impaled CA3 pyramidal neurones, continuous applications of carbachol, a non‐hydrolysable cholinergic agonist, induced first a brief non‐rhythmic excitation and then periodic bursts of RSA which could persist for several hours. Each burst of RSA consisted of 4‐10 Hz oscillatory depolarizations which had a rise time much slower than conventional EPSPs recorded in the same cell. 2. The carbachol‐induced RSA was blocked by atropine; therefore the cholinergic stimulation involved muscarinic receptors. 3. Analyses of simultaneous recordings from pairs of neurones, or a neurone and a glial cell, or a neurone and the extracellular field, indicated that carbachol‐induced RSA was synchronous in a large population of CA3 pyramidal neurones. 4. Complete removal of the dentate gyrus and CA1 region did not block carbachol‐induced RSA in CA3, but applications of tetrodotoxin or inorganic Ca2+ channel blockers (Cd2+, Co2+ or Mn2+) abolished carbachol‐induced RSA. This suggested that the RSA involved propagation of action potentials through a local synaptic network in the CA3 area. 5. Carbachol‐induced RSA was reversibly blocked by a broad‐spectrum excitatory amino acid antagonist (kynurenic acid), but not by two selective N‐methyl‐D‐aspartate (NMDA) antagonists (DL‐2‐amino‐7‐phosphonoheptanoic acid or DL‐2‐amino‐5‐phosphonovaleric acid), a GABAA antagonist (bicuculline), or a GABAB antagonist (phaclofen), suggesting that carbachol‐induced RSA involved primarily non‐NMDA excitatory amino acid, but not GABAergic, synapses. 6. Raising extracellular [Ca2+] beyond 7 mM, which should significantly weaken the polysynaptic recurrent excitation among CA3 pyramidal neurones, abolished carbachol‐induced RSA. This suggests that the recurrent excitation among CA3 pyramidal neurones is necessary for carbachol‐induced RSA in the CA3 area. However, our experiments cannot clarify whether the recurrent excitation, alone, is sufficient for carbachol‐induced RSA.