Protocol for the longitudinal study of neuroinflammation and reactive astrocytes in Lcn2CreERT2 mice.

During brain disease, astrocytes can reprogram into a reactive state that alters many of their functions. Here, we present a protocol for studying neuroinflammation and reactive astrogliosis in mice using lipopolysaccharide (LPS) from E. coli.

Astrocyte regulation of extracellular space parameters across the sleep-wake cycle.

Multiple subfields of neuroscience research are beginning to incorporate astrocytes into current frameworks of understanding overall brain physiology, neuronal circuitry, and disease etiology that underlie sleep and sleep-related disorders. Astrocytes have emerged as a dynamic regulator of neuronal activity through control of extracellular space (ECS) volume and composition, both of which can vary dramatically during different levels of sleep and arousal. Astrocytes are also an attractive target of sleep research due to their prominent role in the glymphatic system, a method by which toxic metabolites generated during wakefulness are cleared away.

A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes.

Astrocytes are vital support cells that ensure proper brain function. In brain disease, astrocytes reprogram into a reactive state that alters many of their cellular roles. A long-standing question in the field is whether downregulation of reactive astrocyte (RA) markers during resolution of inflammation is because these astrocytes revert back to a non-reactive state or die and are replaced.

Structure-function relationships of the LRRC8 subunits and subdomains of the volume-regulated anion channel (VRAC).

Volume Regulated Anion Channels (VRAC) are critical contributors to cell volume homeostasis and are expressed ubiquitously in all vertebrate cells. VRAC sense increases in cell volume, and act to return cells to baseline volume in a process known as regulatory volume decrease (RVD) through the efflux of anions and organic osmolytes. This review will highlight seminal studies that elucidated the role of VRAC in RVD, their characteristics as a function of subunit specificity, and their clinical relevance in physiology and pathology.

Contributions of Astrocyte and Neuronal Volume to CA1 Neuron Excitability Changes in Elevated Extracellular Potassium.

Rapid increases in cell volume reduce the size of the extracellular space (ECS) and are associated with elevated brain tissue excitability. We recently demonstrated that astrocytes, but not neurons, rapidly swell in elevated extracellular potassium ([K] ) up to 26 mM. However, effects of acute astrocyte volume fluctuations on neuronal excitability in [K] have been difficult to evaluate due to direct effects on neuronal membrane potential and generation of action potentials.

Honey, I shrunk the extracellular space: Measurements and mechanisms of astrocyte swelling.

Astrocyte volume fluctuation is a physiological phenomenon tied closely to the activation of neural circuits. Identification of underlying mechanisms has been challenging due in part to use of a wide range of experimental approaches that vary between research groups. Here, we first review the many methods that have been used to measure astrocyte volume changes directly or indirectly.

Astrocyte-Selective Volume Increase in Elevated Extracellular Potassium Conditions Is Mediated by the Na/K ATPase and Occurs Independently of Aquaporin 4.

Astrocytes and neurons have been shown to swell across a variety of different conditions, including increases in extracellular potassium concentration (^[K]). The mechanisms involved in the coupling of K influx to water movement into cells leading to cell swelling are not well understood and remain controversial. Here, we set out to determine the effects of ^[K] on rapid volume responses of hippocampal CA1 pyramidal neurons and stratum radiatum astrocytes using real-time confocal volume imaging.

Multiple Lines of Evidence Indicate That Gliotransmission Does Not Occur under Physiological Conditions.

A major controversy persists within the field of glial biology concerning whether or not, under physiological conditions, neuronal activity leads to Ca-dependent release of neurotransmitters from astrocytes, a phenomenon known as gliotransmission. Our perspective is that, while we and others can apply techniques to cause gliotransmission, there is considerable evidence gathered using astrocyte-specific and more physiological approaches which suggests that gliotransmission is a pharmacological phenomenon rather than a physiological process. Approaches providing evidence against gliotransmission include stimulation of Gq-GPCRs expressed only in astrocytes, as well as removal of the primary proposed source of astrocyte Ca responsible for gliotransmission.

Hippocampal and Cortical Pyramidal Neurons Swell in Parallel with Astrocytes during Acute Hypoosmolar Stress.

Normal nervous system function is critically dependent on the balance of water and ions in the extracellular space (ECS). Pathological reduction in brain interstitial osmolarity results in osmotically-driven flux of water into cells, causing cellular edema which reduces the ECS and increases neuronal excitability and risk of seizures. Astrocytes are widely considered to be particularly susceptible to cellular edema due to selective expression of the water channel aquaporin-4 (AQP4).

Turning down the volume: Astrocyte volume change in the generation and termination of epileptic seizures.

Approximately 1% of the global population suffers from epilepsy, a class of disorders characterized by recurrent and unpredictable seizures. Of these cases roughly one-third are refractory to current antiepileptic drugs, which typically target neuronal excitability directly. The events leading to seizure generation and epileptogenesis remain largely unknown, hindering development of new treatments.

GLT-1-Dependent Disruption of CNS Glutamate Homeostasis and Neuronal Function by the Protozoan Parasite Toxoplasma gondii.

The immune privileged nature of the CNS can make it vulnerable to chronic and latent infections. Little is known about the effects of lifelong brain infections, and thus inflammation, on the neurological health of the host. Toxoplasma gondii is a parasite that can infect any mammalian nucleated cell with average worldwide seroprevalence rates of 30%.

Osmotic Edema Rapidly Increases Neuronal Excitability Through Activation of NMDA Receptor-Dependent Slow Inward Currents in Juvenile and Adult Hippocampus.

Cellular edema (cell swelling) is a principal component of numerous brain disorders including ischemia, cortical spreading depression, hyponatremia, and epilepsy. Cellular edema increases seizure-like activity in vitro and in vivo, largely through nonsynaptic mechanisms attributable to reduction of the extracellular space. However, the types of excitability changes occurring in individual neurons during the acute phase of cell volume increase remain unclear.

Recent molecular approaches to understanding astrocyte function in vivo.

Astrocytes are a predominant glial cell type in the nervous systems, and are becoming recognized as important mediators of normal brain function as well as neurodevelopmental, neurological, and neurodegenerative brain diseases. Although numerous potential mechanisms have been proposed to explain the role of astrocytes in the normal and diseased brain, research into the physiological relevance of these mechanisms in vivo is just beginning. In this review, we will summarize recent developments in innovative and powerful molecular approaches, including knockout mouse models, transgenic mouse models, and astrocyte-targeted gene transfer/expression, which have led to advances in understanding astrocyte biology in vivo that were heretofore inaccessible to experimentation.

Astrocyte calcium microdomains are inhibited by bafilomycin A1 and cannot be replicated by low-level Schaffer collateral stimulation in situ.

Astrocyte Gq GPCR and IP3 receptor-dependent Ca(2+) elevations occur spontaneously in situ and in vivo. These events vary considerably in size, often remaining confined to small territories of astrocyte processes called "microdomains" and sometimes propagating over longer distances that can include the soma. It has remained unclear whether these events are driven by constitutive (basal) GPCR signaling activity, neuronal action potential-dependent or quantal vesicular release, or some combination of these mechanisms.

Astrocytic group I mGluR-dependent potentiation of astrocytic glutamate and potassium uptake.

One of the most important functions of astrocytes is removal of glutamate released during synaptic transmission. Surprisingly, the mechanisms by which astrocyte glutamate uptake is acutely modulated remain to be clarified. Astrocytes express metabotropic glutamate receptors (mGluRs) and other G protein-coupled receptors (GPCRs), which are activated during neuronal activity.

Glial cell changes in epilepsy: overview of the clinical problem and therapeutic opportunities.

It is estimated that one in 26 people will develop epilepsy in their lifetime, amounting to almost 12 million people in the United States alone (Hesdorffer et al., 2011). Epilepsy is a group of conditions characterized by sporadic occurrence of seizures and unconsciousness.

Calcium Signaling and Gliotransmission in Normal vs. Reactive Astrocytes.

A prominent area of neuroscience research over the past 20 years has been the acute modulation of neuronal synaptic activity by Ca(2+)-dependent release of the transmitters ATP, D-serine, and glutamate (called gliotransmitters) by astrocytes. Although the physiological relevance of this mechanism is under debate, emerging evidence suggests that there are critical factors in addition to Ca(2+) that are required for gliotransmitters to be released from astrocytes. Interestingly, these factors include activated microglia and the proinflammatory cytokine Tumor Necrosis Factor α (TNFα), chemotactic cytokine Stromal cell-Derived Factor-1α (SDF-1α), and inflammatory mediator prostaglandin E2 (PGE(2)).

Cofilin under control of β-arrestin-2 in NMDA-dependent dendritic spine plasticity, long-term depression (LTD), and learning.

Dendritic spines are dynamic, actin-rich structures that form the postsynaptic sites of most excitatory synapses in the brain. The F-actin severing protein cofilin has been implicated in the remodeling of dendritic spines and synapses under normal and pathological conditions, by yet unknown mechanisms. Here we report that β-arrestin-2 plays an important role in NMDA-induced remodeling of dendritic spines and synapses via translocation of active cofilin to dendritic spines.

Hippocampal short- and long-term plasticity are not modulated by astrocyte Ca2+ signaling.

The concept that astrocytes release neuroactive molecules (gliotransmitters) to affect synaptic transmission has been a paradigm shift in neuroscience research over the past decade. This concept suggests that astrocytes, together with pre- and postsynaptic neuronal elements, make up a functional synapse. Astrocyte release of gliotransmitters (for example, glutamate and adenosine triphosphate) is generally accepted to be a Ca2+-dependent process.

Sorting out astrocyte physiology from pharmacology.

A number of exciting findings have been made in astrocytes during the past 15 years that have led many researchers to redefine how the brain works. Astrocytes are now widely regarded as cells that propagate Ca(2+) over long distances in response to stimulation, and, similar to neurons, release transmitters (called gliotransmitters) in a Ca(2+)-dependent manner to modulate a host of important brain functions. Although these discoveries have been very exciting, it is essential to place them in the proper context of the approaches used to obtain them to determine their relevance to brain physiology.

What is the role of astrocyte calcium in neurophysiology?

Astrocytes comprise approximately half of the volume of the adult mammalian brain and are the primary neuronal structural and trophic supportive elements. Astrocytes are organized into distinct nonoverlapping domains and extend elaborate and dense fine processes that interact intimately with synapses and cerebrovasculature. The recognition in the mid 1990s that astrocytes undergo elevations in intracellular calcium concentration following activation of G protein-coupled receptors by synaptically released neurotransmitters demonstrated not only that astrocytes display a form of excitability but also that astrocytes may be active participants in brain information processing.

Loss of IP3 receptor-dependent Ca2+ increases in hippocampal astrocytes does not affect baseline CA1 pyramidal neuron synaptic activity.

Astrocytes in the hippocampus release calcium (Ca(2+)) from intracellular stores intrinsically and in response to activation of G(q)-linked G-protein-coupled receptors (GPCRs) through the binding of inositol 1,4,5-trisphosphate (IP(3)) to its receptor (IP(3)R). Astrocyte Ca(2+) has been deemed necessary and sufficient to trigger the release of gliotransmitters, such as ATP and glutamate, from astrocytes to modulate neuronal activity. Several lines of evidence suggest that IP(3)R type 2 (IP(3)R2) is the primary IP(3)R expressed by astrocytes.