Grant R. J. Gordon

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.

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.

Metabolic Communication between Astrocytes and Neurons via Bicarbonate-Responsive Soluble Adenylyl Cyclase

Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO₃⁻) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response to HCO₃⁻ entry via the electrogenic NaHCO₃ cotransporter (NBC). Activated sAC increases intracellular cAMP levels, causing glycogen breakdown, enhanced glycolysis, and the release of lactate into the extracellular space, which is subsequently taken up by neurons for use as an energy substrate. This process is recruited over a broad physiological range of [K⁺](ext) and also during aglycemic episodes, helping to maintain synaptic function. These data reveal a molecular pathway in astrocytes that is responsible for brain metabolic coupling to neurons.

Transient Swelling, Acidification, and Mitochondrial Depolarization Occurs in Neurons but not Astrocytes during Spreading Depression

Cortical spreading depression (SD) is a propagating wave of neuronal and glial depolarization that manifests in several brain disorders. However, the relative contribution of neurons and astrocytes to SD genesis has remained controversial. This is in part due to a lack of utilizing sophisticated experimental methodologies simultaneously to quantify multiple cellular parameters. To address this, we used simultaneous two-photon imaging, intrinsic optical imaging, and electrophysiological recordings to ascertain the changes in cellular processes that are fundamental to both cell types including cell volume, pH, and metabolism during SD propagation. We found that SD was correlated in neurons with robust yet transient increased volume, intracellular acidification, and mitochondrial depolarization. Our data indicated that a propagating large conductance during SD generated neuronal depolarization, which led to both calcium influx triggering metabolic changes and H(+) entry. Notably, astrocytes did not exhibit changes in cell volume, pH, or mitochondrial membrane potentials associated with SD, but they did show alterations induced by changing external [K(+)]. This suggests that astrocytes are not the primary contributor to SD propagation but are instead activated passively by extracellular potassium accumulation. These data support the hypothesis that neurons are the crucial cell type contributing to the pathophysiological responses of SD.

Glutathione Restores the Mechanism of Synaptic Plasticity in Aged Mice to That of the Adult

Glutathione (GSH), the major endogenous antioxidant produced by cells, can modulate the activity of N-methyl-D-aspartate receptors (NMDARs) through its reducing functions. During aging, an increase in oxidative stress leads to decreased levels of GSH in the brain. Concurrently, aging is characterized by calcium dysregulation, thought to underlie impairments in hippocampal NMDAR-dependent long-term potentiation (LTP), a form of synaptic plasticity thought to represent a cellular model for memory. Here we show that orally supplementing aged mice with N-acetylcysteine, a precursor for the formation of glutathione, reverses the L-type calcium channel-dependent LTP seen in aged animals to NMDAR-dependent LTP. In addition, introducing glutathione in the intrapipette solution during whole-cell recordings restores LTP obtained in whole-cell conditions in the aged hippocampus. We conclude that aging leads to a reduced redox potential in hippocampal neurons, triggering impairments in LTP.

Bidirectional control of arteriole diameter by astrocytes

Astrocytes are the most numerous cells in the CNS. It is a defining feature of brain anatomy that every astrocyte has at least one contact with the vasculature, termed an endfoot. Collectively, all endfeet completely circumscribe all vessels in the brain. This unique anatomical feature has profound functional significance, as astrocyte endfeet have been discovered to release diffusible messengers that communicate directly with underlying smooth muscle cells to change arterial diameter and thereby regulate cerebral blood flow. A growing body of data now demonstrates that astrocytes serve as a bridge, relaying information on the level of neural activity to blood vessels in order to co‐ordinate oxygen and glucose delivery with the energy demands of the tissue. In particular, astrocytes respond to elevations in extracellular glutamate as a consequence of synaptic transmission through the activation of group 1 metabotropic glutamate receptors. These Gq‐coupled receptors elevate intracellular calcium via IP3 signalling, which activates phospholipase A2 and generates arachidonic acid. Arachidonic acid acts as a signalling molecule or is converted to several lipid derivates, including prostaglandin E2 and epoxyeicosatrienoic acids. Each of these lipids acts on vascular smooth muscle cells via different mechanisms to affect vessel diameter. Arachidonic acid initiates the production of 20‐hydroxyeicosatetraenoic acid to cause vasoconstriction, whereas prostaglandin E2 and epoxyeicosatrienoic acids cause vasodilatation. Factors that determine whether constrictor or dilatory pathways predominate involve nitric oxide and brain metabolic elements, such as oxygen, lactate and adenosine. Thus, astrocytes are thought to be capable of bidirectional control of arterial diameter, and the type of influence depends on the state of brain activity.

Noradrenaline Triggers Multivesicular Release at Glutamatergic Synapses in the Hypothalamus

The origin of large-amplitude miniature EPSCs (mEPSCs) at central synapses remains to be firmly established. Here, we show that at excitatory synapses onto magnocellular neurosecretory cells in the hypothalamus, noradrenaline induces a rapid and robust increase in mEPSC amplitude that requires α1-adrenoceptor activation but is impervious to postsynaptic manipulations that block the putative insertion of AMPA receptors. In response to noradrenaline, mEPSCs exhibit a putative multimodal amplitude histogram distribution that is not attibutable to random temporal summation, the unveiling of a quiescent synapse, or the release of large vesicles. Large-amplitude mEPSCs are sensitive to a high dose of ryanodine and are associated with an enhanced glutamate cleft concentration. Together, these data are consistent with the hypothesis that large-amplitude mEPSCs result from the synchronous release of multiple vesicles via rapid presynaptic calcium expulsion from intracellular stores.

A practical guide to the synthesis and use of membrane-permeant acetoxymethyl esters of caged inositol polyphosphates.

This protocol describes a method for efficient chemical synthesis of an analog of inositol-1,4,5-trisphosphate (IP3) hexakis acetoxymethyl ester having an ortho-nitroveratryl photochemical caging group on the 6-hydroxyl position. The six esters render the probe membrane permeant, such that it can be loaded into intact living cells in vitro or in vivo. Inside cells, the caged IP3 is inert until activated by two-photon excitation at 720 nm. The photoliberated signaling molecule can mobilize release of Ca2+ from intracellular stores on the endoplasmic reticulum. When co-loaded with the fluorescent Ca2+ indicator rhod-2, one laser can be used for stimulating and monitoring intracellular Ca2+ signaling with single-cell resolution. This protocol has chemistry and biology sections; the former describes the organic synthesis of the caged IP3, which requires 12 d, and the latter an application to a day-long study of astrocyte-regulated neuronal function in living brain slices acutely isolated from rats. As Ca2+ is the single most important intracellular second messenger and the IP3-Ca2+ signaling cascade is used by many cells to produce increases in Ca2+ concentration, this method should be widely applicable for the study of a variety of physiological processes in intact biological systems.

Astrocyte control of blood flow

Astrocytes have recently been shown to be essential participants in the control of cerebral blood flow (CBF) through their prominent control of cerebral vessel diameter. Although the unique close relationship of astrocytes with cerebral blood vessels has long been recognized it is only within the last few years that evidence has shown how astrocytes might translate information to the vasculature on the activity level and energy demands of neurons. These findings suggest that astrocytes are key players in the system for the delivery and clearance of molecules important to brain function.

Bidirectional Control of Blood Flow by Astrocytes: A Role for Tissue Oxygen and Other Metabolic Factors

Altering cerebral blood flow through the control of cerebral vessel diameter is critical so that the delivery of molecules important for proper brain functioning is matched to the activity level of neurons. Although the close relationship of brain glia known as astrocytes with cerebral blood vessels has long been recognized, it is only recently that these cells have been demonstrated to translate information on the activity level and energy demands of neurons to the vasculature. In particular, astrocytes respond to elevations in extracellular glutamate as a consequence of synaptic transmission through the activation of group 1 metabotropic glutamate receptors. These Gq-protein coupled receptors elevate intracellular calcium via IP3 signaling. A close examination of astrocyte endfeet calcium signals has been shown to cause either vasoconstriction or vasodilation. Common to both vasomotor responses is the generation of arachidonic acid in astrocytes by calcium sensitive phospholipase A2. Vasoconstriction ensues from the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid, while vasodilation ensues from the production of epoxyeicosatrienoic acids or prostaglandins. Factors that determine whether constrictor or dilatory pathways predominate include brain oxygen, lactate, adenosine as well as nitric oxide. Changing the oxygen level itself leads to many downstream changes that facilitate the switch from vasoconstriction at high oxygen to vasodilation at low oxygen. These findings highlight the importance of astrocytes as sensors of neural activity and metabolism to coordinate the delivery of essential nutrients via the blood to the working cells.