Fushun Wang

Uniquely hominid features of adult human astrocytes.

Defining the microanatomic differences between the human brain and that of other mammals is key to understanding its unique computational power. Although much effort has been devoted to comparative studies of neurons, astrocytes have received far less attention. We report here that protoplasmic astrocytes in human neocortex are 2.6-fold larger in diameter and extend 10-fold more GFAP (glial fibrillary acidic protein)-positive primary processes than their rodent counterparts. In cortical slices prepared from acutely resected surgical tissue, protoplasmic astrocytes propagate Ca2+ waves with a speed of 36 μm/s, approximately fourfold faster than rodent. Human astrocytes also transiently increase cystosolic Ca2+ in response to glutamatergic and purinergic receptor agonists. The human neocortex also harbors several anatomically defined subclasses of astrocytes not represented in rodents. These include a population of astrocytes that reside in layers 5–6 and extend long fibers characterized by regularly spaced varicosities. Another specialized type of astrocyte, the interlaminar astrocyte, abundantly populates the superficial cortical layers and extends long processes without varicosities to cortical layers 3 and 4. Human fibrous astrocytes resemble their rodent counterpart but are larger in diameter. Thus, human cortical astrocytes are both larger, and structurally both more complex and more diverse, than those of rodents. On this basis, we posit that this astrocytic complexity has permitted the increased functional competence of the adult human brain.

Adenosine A1 receptors mediate local anti-nociceptive effects of acupuncture

Acupuncture is an invasive procedure commonly used to relieve pain. Acupuncture is practiced worldwide, despite difficulties in reconciling its principles with evidence-based medicine. We found that adenosine, a neuromodulator with anti-nociceptive properties, was released during acupuncture in mice and that its anti-nociceptive actions required adenosine A1 receptor expression. Direct injection of an adenosine A1 receptor agonist replicated the analgesic effect of acupuncture. Inhibition of enzymes involved in adenosine degradation potentiated the acupuncture-elicited increase in adenosine, as well as its anti-nociceptive effect. These observations indicate that adenosine mediates the effects of acupuncture and that interfering with adenosine metabolism may prolong the clinical benefit of acupuncture.

Forebrain Engraftment by Human Glial Progenitor Cells Enhances Synaptic Plasticity and Learning in Adult Mice

Human astrocytes are larger and more complex than those of infraprimate mammals, suggesting that their role in neural processing has expanded with evolution. To assess the cell-autonomous and species-selective properties of human glia, we engrafted human glial progenitor cells (GPCs) into neonatal immunodeficient mice. Upon maturation, the recipient brains exhibited large numbers and high proportions of both human glial progenitors and astrocytes. The engrafted human glia were gap-junction-coupled to host astroglia, yet retained the size and pleomorphism of hominid astroglia, and propagated Ca2+ signals 3-fold faster than their hosts. Long-term potentiation (LTP) was sharply enhanced in the human glial chimeric mice, as was their learning, as assessed by Barnes maze navigation, object-location memory, and both contextual and tone fear conditioning. Mice allografted with murine GPCs showed no enhancement of either LTP or learning. These findings indicate that human glia differentially enhance both activity-dependent plasticity and learning in mice.

Critical role of aquaporin-4 (AQP4) in astrocytic Ca2+ signaling events elicited by cerebral edema

Aquaporin-4 (AQP4) is a primary influx route for water during brain edema formation. Here, we provide evidence that brain swelling triggers Ca2+ signaling in astrocytes and that deletion of the Aqp4 gene markedly interferes with these events. Using in vivo two-photon imaging, we show that hypoosmotic stress (20% reduction in osmolarity) initiates astrocytic Ca2+ spikes and that deletion of Aqp4 reduces these signals. The Ca2+ signals are partly dependent on activation of P2 purinergic receptors, which was judged from the effects of appropriate antagonists applied to cortical slices. Supporting the involvement of purinergic signaling, osmotic stress was found to induce ATP release from cultured astrocytes in an AQP4-dependent manner. Our results suggest that AQP4 not only serves as an influx route for water but also is critical for initiating downstream signaling events that may affect and potentially exacerbate the pathological outcome in clinical conditions associated with brain edema.

Astrocytes Modulate Neural Network Activity by Ca2+-Dependent Uptake of Extracellular K+

Astrocytes modulate synaptic activity by changing the local concentration of extracellular K+. Increasing Synaptic Fidelity Although astrocytes have long been implicated in maintenance of extracellular K+ homeostasis and in the mediation of propagated Ca2+ signals, the relationship between these two properties has been unclear. Here, Wang et al. show that increases in astrocyte cytosolic Ca2+ secondary to activation of G protein–coupled receptors (GPCRs) lead to increased Na+ influx through the Na+/Ca2+ exchanger, which, in turn, stimulates astrocyte Na+,K+-ATPase activity and K+ uptake and consequently decreases the local extracellular K+ concentration. Decreased K+ increased neuronal hyperpolarization, with a consequent suppression of basal—but not evoked—synaptic activity, and enhanced synaptic fidelity. The authors thus conclude that Ca2+-dependent activation of the astrocyte Na+,K+-ATPase through GPCR-activated signaling pathways enables astrocytes to dynamically regulate the extracellular K+ concentration and thereby modulate neuronal function. Astrocytes are electrically nonexcitable cells that display increases in cytosolic calcium ion (Ca2+) in response to various neurotransmitters and neuromodulators. However, the physiological role of astrocytic Ca2+ signaling remains controversial. We show here that astrocytic Ca2+ signaling ex vivo and in vivo stimulated the Na+,K+-ATPase (Na+- and K+-dependent adenosine triphosphatase), leading to a transient decrease in the extracellular potassium ion (K+) concentration. This in turn led to neuronal hyperpolarization and suppressed baseline excitatory synaptic activity, detected as a reduced frequency of excitatory postsynaptic currents. Synaptic failures decreased in parallel, leading to an increase in synaptic fidelity. The net result was that astrocytes, through active uptake of K+, improved the signal-to-noise ratio of synaptic transmission. Active control of the extracellular K+ concentration thus provides astrocytes with a simple yet powerful mechanism to rapidly modulate network activity.

α1-Adrenergic receptors mediate coordinated Ca2+ signaling of cortical astrocytes in awake, behaving mice.

Astrocyte Ca2+ signals in awake behaving mice are widespread, coordinated and differ fundamentally from the locally restricted Ca2+ transients observed ex vivo and in anesthetized animals. Here we show that the synchronized release of norepinephrine (NE) from locus coeruleus (LC) projections throughout the cerebral cortex mediate long-ranging Ca2+ signals by activation of astrocytic α1-adrenergic receptors. When LC output was triggered by either physiological sensory (whisker) stimulation or an air-puff startle response, astrocytes responded with fast Ca2+ transients that encompassed the entire imaged field (positioned over either frontal or parietal cortex). The application of adrenergic inhibitors, including α1-adrenergic antagonist prazosin, potently suppressed both evoked, as well as the frequently observed spontaneous astroglial Ca2+ signals. The LC-specific neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), which reduced cortical NE content by >90%, prevented nearly all astrocytic Ca2+ signals in awake mice. The observations indicate that in adult, unanesthetized mice, astrocytes do not respond directly to glutamatergic signaling evoked by sensory stimulation. Instead astrocytes appear to be the primary target for NE, with astrocytic Ca2+ signaling being triggered by the α1-adrenergic receptor. In turn, astrocytes may coordinate the broad effects of neuromodulators on neuronal activity.

Ammonia triggers neuronal disinhibition and seizures by impairing astrocyte potassium buffering

Ammonia is a ubiquitous waste product of protein metabolism that can accumulate in numerous metabolic disorders, causing neurological dysfunction ranging from cognitive impairment to tremor, ataxia, seizures, coma and death. The brain is especially vulnerable to ammonia as it readily crosses the blood-brain barrier in its gaseous form, NH3, and rapidly saturates its principal removal pathway located in astrocytes. Thus, we wanted to determine how astrocytes contribute to the initial deterioration of neurological functions characteristic of hyperammonemia in vivo. Using a combination of two-photon imaging and electrophysiology in awake head-restrained mice, we show that ammonia rapidly compromises astrocyte potassium buffering, increasing extracellular potassium concentration and overactivating the Na+-K+-2Cl− cotransporter isoform 1 (NKCC1) in neurons. The consequent depolarization of the neuronal GABA reversal potential (EGABA) selectively impairs cortical inhibitory networks. Genetic deletion of NKCC1 or inhibition of it with the clinically used diuretic bumetanide potently suppresses ammonia-induced neurological dysfunction. We did not observe astrocyte swelling or brain edema in the acute phase, calling into question current concepts regarding the neurotoxic effects of ammonia. Instead, our findings identify failure of potassium buffering in astrocytes as a crucial mechanism in ammonia neurotoxicity and demonstrate the therapeutic potential of blocking this pathway by inhibiting NKCC1.

Extracellular Ca²⁺ acts as a mediator of communication from neurons to glia.

Local decreases in extracellular calcium trigger signaling events in astrocytes that activate inhibitory neurotransmission. Calcium Signals Outside the Cell Excitatory glutamatergic signaling is accompanied by calcium influx into neurons through voltage-dependent calcium channels and ionotropic glutamate receptors. The crucial role of calcium as an intracellular second messenger makes it easy to overlook the consequences of a localized decrease in extracellular calcium. Here, Torres et al. explored the functional consequences of a localized decrease in extracellular calcium in hippocampal slices. They found that a localized decrease in extracellular calcium—elicited either directly through activation of a photosensitive calcium buffer or secondary to glutamate-stimulated calcium influx in neurons—evoked astrocytic release of ATP, as did high-frequency stimulation, which elicited a comparable decrease in extracellular calcium. Moreover, decreased extracellular calcium increased the activity of inhibitory interneurons bearing purinergic receptors, suggesting that, under conditions of high-intensity glutamatergic signaling, a local decline in extracellular calcium could, by stimulating astrocyte calcium signals and ATP release, activate a compensatory increase in synaptic inhibition. Defining the pathways through which neurons and astrocytes communicate may contribute to the elucidation of higher central nervous system functions. We investigated the possibility that decreases in extracellular calcium ion concentration ([Ca2+]e) that occur during synaptic transmission might mediate signaling from neurons to glia. Using noninvasive photolysis of the photolabile Ca2+ buffer diazo-2 {N-[2-[2-[2-[bis(carboxymethyl)amino]-5-(diazoacetyl)phenoxy]ethoxy]-4-methylphenyl]-N-(carboxymethyl)-, tetrapotassium salt} to reduce [Ca2+]e or caged glutamate to simulate glutamatergic transmission, we found that a local decline in extracellular Ca2+ triggered astrocytic adenosine triphosphate (ATP) release and astrocytic Ca2+ signaling. In turn, activation of purinergic P2Y1 receptors on a subset of inhibitory interneurons initiated the generation of action potentials by these interneurons, thereby enhancing synaptic inhibition. Thus, astrocytic ATP release evoked by an activity-associated decrease in [Ca2+]e may provide a negative feedback mechanism that potentiates inhibitory transmission in response to local hyperexcitability.

Anti-Cancer Activities of Tea Epigallocatechin-3-Gallate in Breast Cancer Patients under Radiotherapy

The purpose of this study was to test the hypothesis that administration of epigallocatechin-3-gallate (EGCG), a polyphenol present in abundance in widely consumed tea, inhibits cell proliferation, invasion, and angiogenesis in breast cancer patients. EGCG in 400 mg capsules was orally administered three times daily to breast cancer patients undergoing treatment with radiotherapy. Parameters related to cell proliferation, invasion, and angiogenesis were analyzed while blood samples were collected at different time points to determine efficacy of the EGCG treatment. Compared to patients who received radiotherapy alone, those given radiotherapy plus EGCG for an extended time period (two to eight weeks) showed significantly lower serum levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and reduced activation of metalloproteinase-9 and metalloproteinase-2 (MMP9/MMP2). Addition of sera obtained from patients treated with combination of radiotherapy and EGCG feeding for 2-8 weeks to in vitro cultures of highly-metastatic human MDA-MB-231 breast cancer cells resulted in the following significant changes: (1) suppression of cell proliferation and invasion; (2) arrest of cell cycles at the G0/G1 phase; (3) reduction of activation of MMP9/MMP2, expressions of Bcl-2/Bax, c-Met receptor, NF-κB, and the phosphorylation of Akt. MDA-MB-231 cells exposed to 5-10 μM EGCG also showed significant augmentation of the apoptosis inducing effects of γ-radiation, concomitant with reduced NF-κB protein level and AKT phosphorylation. These results provide hitherto unreported evidence that EGCG potentiated efficacy of radiotherapy in breast cancer patients, and raise the possibility that this tea polyphenol has potential to be a therapeutic adjuvant against human metastatic breast cancer.

Rapid manifestation of reactive astrogliosis in acute hippocampal brain slices

A flurry of studies over the past decade has shown that astrocytes play a more active role in neural function than previously recognized. Hippocampal slices prepared from young rodent pups have served as a popular model for studying the pathways by which astrocytes participate in synaptic transmission. It is, however, not known how well astrocytes tolerate traumatic injury and hypoxia, which are unavoidable when preparing acute slices. We here showed that astrocytes exhibit striking changes in expression of several receptors and structural proteins, including re‐expression of the developmental marker nestin within 90 min following preparation of live vibratome slices. Moreover, immunoelectron microscopy showed a 2.7‐fold loss of astrocytic processes in acute hippocampal slices prepared from glial fibrillary acidic protein‐green fluorescent protein reporter mice. A sharp decrease in the number of mitochondria was also noted in acute slices, concurrently with an increase in mitochondrial size. Glycogen content decreased 3‐fold upon slice preparation and did not recover despite stable recordings of field excitatory postsynaptic current. Analysis of Ca2+ signaling showed that astrocytic responses to purine receptor and mGluR5 agonists differed in slice versus in vivo. These observations suggest that the functional properties and the fine structure of astrocytes in slices may be reflective of early stages of reactive gliosis and should be confirmed in vivo when possible. GLIA 2013;62:78–95