Individual Astrocytes Research Articles

Disentangling astroglial physiology with a realistic cell model in silico

Electrically non-excitable astroglia take up neurotransmitters, buffer extracellular K+ and generate Ca2+ signals that release molecular regulators of neural circuitry. The underlying machinery remains enigmatic, mainly because the sponge-like astrocyte morphology has been difficult to access experimentally or explore theoretically. Here, we systematically incorporate multi-scale, tri-dimensional astroglial architecture into a realistic multi-compartmental cell model, which we constrain by empirical tests and integrate into the NEURON computational biophysical environment. This approach is implemented as a flexible astrocyte-model builder ASTRO. As a proof-of-concept, we explore an in silico astrocyte to evaluate basic cell physiology features inaccessible experimentally. Our simulations suggest that currents generated by glutamate transporters or K+ channels have negligible distant effects on membrane voltage and that individual astrocytes can successfully handle extracellular K+ hotspots. We show how intracellular Ca2+ buffers affect Ca2+ waves and why the classical Ca2+ sparks-and-puffs mechanism is theoretically compatible with common readouts of astroglial Ca2+ imaging.

Dynamic Neuron-Glia Interactions in an Oscillatory Network Controlling Behavioral Plasticity in the Weakly Electric Fish, Apteronotus leptorhynchus

The involvement of glial cells in the regulation of physiological functions is being increasingly recognized, yet their role in plasticity of neural oscillators has remained largely elusive. An excellent model system to address the latter function is the pacemaker nucleus of the weakly electric fish, Apteronotus leptorhynchus. This brainstem oscillator drives the fish's electric organ discharge in a one-to-one fashion, with median frequencies of 880 Hz in males and 740 Hz in females. Morphometric analysis of the pacemaker nucleus has shown that astrocytes outnumber mature neurons seven-fold, and oscillator neurons even 200-fold. A similar dominance of astrocytes occurs among the adult-born cells that differentiate into glia and neurons. The astrocytes form a dense meshwork of cells interconnected by gap junctions. The degree of association of astrocytic fibers with the neural oscillator cells, and the gap-junction coupling between individual astrocytes, exhibit a sexual dimorphism, which parallels the sexual dimorphisms in the output frequency of the pacemaker nucleus, and ultimately in the electric organ discharge of the fish. It is hypothesized that the dynamics in astroglial structure mediate differences in the capacity to buffer potassium, which increases during the generation of action potentials. These differences, in turn, affect the excitability of the neural oscillator cells, and thus the output frequency of the pacemaker nucleus. Comparison of the pacemaker nucleus with other brain oscillators suggests that modulation of the output activity is one of the chief functions of the interaction of glia with the neural oscillator cells.

The role of astrocytes in amyloid production and Alzheimer's disease.

Alzheimer's disease (AD) is marked by the presence of extracellular amyloid beta (Aβ) plaques, intracellular neurofibrillary tangles (NFTs) and gliosis, activated glial cells, in the brain. It is thought that Aβ plaques trigger NFT formation, neuronal cell death, neuroinflammation and gliosis and, ultimately, cognitive impairment. There are increased numbers of reactive astrocytes in AD, which surround amyloid plaques and secrete proinflammatory factors and can phagocytize and break down Aβ. It was thought that neuronal cells were the major source of Aβ. However, mounting evidence suggests that astrocytes may play an additional role in AD by secreting significant quantities of Aβ and contributing to overall amyloid burden in the brain. Astrocytes are the most numerous cell type in the brain, and therefore even minor quantities of amyloid secretion from individual astrocytes could prove to be substantial when taken across the whole brain. Reactive astrocytes have increased levels of the three necessary components for Aβ production: amyloid precursor protein, β-secretase (BACE1) and γ-secretase. The identification of environmental factors, such as neuroinflammation, that promote astrocytic Aβ production, could redefine how we think about developing therapeutics for AD.

Astrocyte-Mediated Neuronal Synchronization Properties Revealed by False Gliotransmitter Release.

Astrocytes spontaneously release glutamate (Glut) as a gliotransmitter (GT), resulting in the generation of extrasynaptic NMDAR-mediated slow inward currents (SICs) in neighboring neurons, which can increase local neuronal excitability. However, there is a deficit in our knowledge of the factors that control spontaneous astrocyte GT release and the extent of its influence. We found that, in rat brain slices, increasing the supply of the physiological transmitter Glut increased the frequency and signaling charge of SICs over an extended period. This phenomenon was replicated by exogenous preexposure to the amino acid D-aspartate (D-Asp). Using D-Asp as a “false” GT, we determined the extent of local neuron excitation by GT release in ventrobasal thalamus, CA1 hippocampus, and somatosensory cortex. By analyzing synchronized neuronal NMDAR-mediated excitation, we found that the properties of the excitation were conserved in different brain areas. In the three areas, astrocyte-derived GT release synchronized groups of neurons at distances of >;200 μm. Individual neurons participated in more than one synchronized population, indicating that individual neurons can be excited by more than one astrocyte and that individual astrocytes may determine a neuron's synchronized network. The results confirm that astrocytes can act as excitatory nodes that can influence neurons over a significant range in a number of brain regions. Our findings further suggest that chronic elevation of ambient Glut levels can lead to increased GT Glut release, which may be relevant in some pathological states.SIGNIFICANCE STATEMENT Astrocytes spontaneously release glutamate (Glut) and other gliotransmitters (GTs) that can modify neuronal activity. Exposing brain slices to Glut and D-aspartate (D-Asp) before recording resulted in an increase in frequency of GT-mediated astrocyte–neuron signaling. Using D-Asp, it was possible to investigate the effects of specific GT release at neuronal NMDARs. Calcium imaging showed synchronized activity in groups of neurons in cortex, hippocampus, and thalamus. The size of these populations was similar in all areas and some neurons were involved in more than one synchronous group. The findings show that GT release is supply dependent and that the properties of the signaling and activated networks are largely conserved between different brain areas.

Astrocyte heterogeneity across the brain and spinal cord occurs developmentally, in adulthood and in response to demyelination.

Astrocytes have emerged as essential regulators of function and response to injury in the brain and spinal cord, yet very little is known about regional differences that exist. Here we compare the expression of key astroglial markers (glial fibrillary acidic protein (GFAP) and Aldehyde Dehydrogenase-1 Family Member L1 (ALDH1L1)) across these disparate poles of the neuraxis, tracking their expression developmentally and in the context of demyelination. In addition, we document changes in the astrocyte regulatory cytokine interleukin 6 (IL-6), and its signaling partner signal transducer and activator of transcription 3 (STAT3), in vivo and in vitro. Results demonstrate that GFAP expression is higher in the developing and adult spinal cord relative to brain. Comparisons between GFAP and ALDH1L1 expression suggest elevations in spinal cord GFAP during the early postnatal period reflect an accelerated appearance of astrocytes, while elevations in adulthood reflect higher expression by individual astrocytes. Notably, increases in spinal cord compared to whole brain GFAP were paralleled by higher levels of IL-6 and STAT3. Equivalent elevations in GFAP, GFAP/ALDH1L1 ratios, and in IL-6, were observed in primary astrocyte cultures derived from spinal cord compared to cortex. Also, higher levels of GFAP were observed in the spinal cord compared to the brain after focal demyelinating injury. Altogether, these studies point to key differences in astrocyte abundance and the expression of GFAP and IL-6 across the brain and spinal cord that are positioned to influence regional specialization developmentally and responses occurring in the context of injury and disease.

VEGF treatment during status epilepticus attenuates long-term seizure-associated alterations in astrocyte morphology.

Vascular endothelial growth factor (VEGF) treatment during pilocarpine-induced status epilepticus (SE) causes sustained preservation of behavioral function in rats in the absence of enduring neuroprotection (Nicoletti et al., 2010), suggesting the possibility that other cells or mechanisms could be involved in the beneficial effects of VEGF during SE. Astrocytes have been reported to contribute to epileptiform discharges in the hippocampus (Tian et al., 2005; Kang et al., 1998) and to express VEGF receptors (Krum & Rosenstein, 2002). We report here that VEGF treatment significantly alters multiple astrocyte parameters. This study investigated astrocyte morphology one month after SE in animals treated with VEGF or inactivated VEGF control protein during SE. Individual GFAP-immunostained astrocytes from CA1 and dentate gyrus hilus were traced and morphologically quantified, and both somatic and process structures were analyzed. VEGF treatment during SE significantly prevented post-SE increases in number of branch intersections, process length, and node count. Furthermore, analysis of distance to nearest neighboring astrocytes revealed that VEGF treatment significantly increased inter-astrocyte distance. Overall, VEGF treatment during SE did not significantly alter the shape of the astrocytes, but did prevent SE-induced changes in branching complexity, size, and spatial patterning. Because astrocyte morphology may be related to astrocyte function, it is possible that VEGF's enduring effects on astrocyte morphology may impact the functioning of the post-seizure hippocampus.

Astrocyte Sodium Signalling and Panglial Spread of Sodium Signals in Brain White Matter.

In brain grey matter, excitatory synaptic transmission activates glutamate uptake into astrocytes, inducing sodium signals which propagate into neighboring astrocytes through gap junctions. These sodium signals have been suggested to serve an important role in neuro-metabolic coupling. So far, it is unknown if astrocytes in white matter-that is in brain regions devoid of synapses-are also able to undergo such intra- and intercellular sodium signalling. In the present study, we have addressed this question by performing quantitative sodium imaging in acute tissue slices of mouse corpus callosum. Focal application of glutamate induced sodium transients in SR101-positive astrocytes. These were largely unaltered in the presence of ionotropic glutamate receptors blockers, but strongly dampened upon pharmacological inhibition of glutamate uptake. Sodium signals induced in individual astrocytes readily spread into neighboring SR101-positive cells with peak amplitudes decaying monoexponentially with distance from the stimulated cell. In addition, spread of sodium was largely unaltered during pharmacological inhibition of purinergic and glutamate receptors, indicating gap junction-mediated, passive diffusion of sodium between astrocytes. Using cell-type-specific, transgenic reporter mice, we found that sodium signals also propagated, albeit less effectively, from astrocytes to neighboring oligodendrocytes and NG2 cells. Again, panglial spread was unaltered with purinergic and glutamate receptors blocked. Taken together, our results demonstrate that activation of sodium-dependent glutamate transporters induces sodium signals in white matter astrocytes, which spread within the astrocyte syncytium. In addition, we found a panglial passage of sodium signals from astrocytes to NG2 cells and oligodendrocytes, indicating functional coupling between these macroglial cells in white matter.

Astrocytes in the Optic Nerve Head of Glaucomatous Mice Display a Characteristic Reactive Phenotype.

PurposeOptic nerve head astrocytes, a subtype of white-matter astrocytes, become reactive early in the course of glaucoma. It was shown recently that in the DBA/2J mouse model of inherited glaucoma optic nerve astrocytes extend new longitudinal processes into the axon bundles before ganglion cell loss becomes apparent. The present study aims at testing whether this behavior of astrocytes is typical of early glaucomatous damage.MethodsMice expressing green fluorescent protein in individual astrocytes were used to evaluate the early response of astrocytes in the glial lamina of the optic nerve head after increasing the IOP using the microbead occlusion method. Tissue sections from the glial lamina were imaged consecutively by confocal and electron microscopy.ResultsConfocal and electron microscope images show that astrocytes close to the myelination transition zone in the hypertensive nerve heads extend new processes that follow the longitudinal axis of the optic nerve and invade axon bundles in the nerve head. Ultrastructurally, the longitudinal processes were largely devoid of subcellular organelles except for degenerating mitochondria.ConclusionsThe longitudinal processes are a common feature of glaucomatous optic nerve astrocytes, whereas they are not observed after traumatic nerve injury. Thus, astrocytes appear to fine-tune their responses to the nature and/or timing of the injury to the neurons that they surround.

Monitoring Ca2+ elevations in individual astrocytes upon local release of amyloid beta in acute brain slices

The pathogenesis of Alzheimer’s disease (AD) is thought to involve acute neurotoxic effects exerted by oligomeric forms of amyloid-β 1-42 (Aβ). Application of Aβ oligomers in physiological concentrations have been shown to transiently elevate internal Ca2+ in cultured astroglia. While the cellular machinery involved has been extensively explored, to what degree this important signalling cascade occurs in organised brain tissue has remained unclear. Here we adapted two-photon excitation microscopy and calibrated time-resolved imaging (FLIM), coupled with patch-clamp electrophysiology, to monitor Ca2+ concentration ([Ca2+]) inside individual astrocytes and principal neurons in acute brain slices. Inside the slice tissue local micro-ejection of Aβ in sub-micromolar concentrations triggered prominent [Ca2+] elevations in an adjacent astrocyte translated as an approximately two-fold increase (averaged over ∼5min) in basal [Ca2+]. This elevation did not spread to neighbouring cells and appeared comparable in amplitude with commonly documented spontaneous [Ca2+] rises in astroglia. Principal nerve cells (pyramidal neurons) also showed Ca2+ sensitivity, albeit to a lesser degree. These observations shed light on the extent and dynamics of the acute physiological effects of Aβ on brain cells in situ, in the context of AD.

The correlation between expression profiles measured in single cells and in traditional bulk samples.

Reverse transcription quantitative PCR (RT-qPCR) is already an established tool for mRNA detection and quantification. Since recently, this technique has been successfully employed for gene expression analyses, and also in individual cells (single cell RT-qPCR). Although the advantages of single cell measurements have been proven several times, a study correlating the expression measured on single cells, and in bulk samples consisting of a large number of cells, has been missing. Here, we collected a large data set to explore the relation between gene expression measured in single cells and in bulk samples, reflected by qPCR Cq values. We measured the expression of 95 genes in 12 bulk samples, each containing thousands of astrocytes, and also in 693 individual astrocytes. Combining the data, we described the relation between Cq values measured in bulk samples with either the percentage of the single cells that express the given genes, or the average expression of the genes across the single cells. We show that data obtained with single cell RT-qPCR are fully consistent with measurements in bulk samples. Our results further provide a base for quality control in single cell expression profiling, and bring new insights into the biological process of cellular expression.

Mechanisms of CO2/H+ Sensitivity of Astrocytes.

Brainstem astrocytes detect physiological changes in pH, activate neurons of the neighboring respiratory network, and contribute to the development of adaptive respiratory responses to the increases in the level of blood and brain PCO2/[H+]. The mechanisms underlying astroglial pH sensitivity remained unknown and here we show that in brainstem astrocytes acidification activates Na+/HCO3- cotransport, which brings Na+ inside the cell. Raising [Na+]i activates the Na+/Ca2+ exchanger to operate in a reverse mode leading to Ca2+ entry. This identifies a plausible mechanism of functional CO2/H+ sensitivity of brainstem astrocytes, which play an important role in homeostatic regulation of brain pH and control of breathing.

Astrocyte membrane properties are altered in a rat model of developmental cortical malformation but single-cell astrocytic glutamate uptake is robust

Developmental cortical malformations (DCMs) are linked with severe epilepsy and are caused by both genetic and environmental insults. DCMs include several neurological diseases, such as focal cortical dysplasia, polymicrogyria, schizencephaly, and others. Human studies have implicated astrocyte reactivity and dysfunction in the pathophysiology of DCMs, but their specific role is unknown. As astrocytes powerfully regulate glutamate neurotransmission, and glutamate levels are known to be increased in human epileptic foci, understanding the role of astrocytes in the pathological sequelae of DCMs is extremely important. Additionally, recent studies examining astrocyte glutamate uptake in DCMs have reported conflicting results, adding confusion to the field. In this study we utilized the freeze lesion (FL) model of DCM, which is known to induce reactive astrocytosis and cause significant changes in astrocyte morphology, proliferation, and distribution. Using whole-cell patch clamp recording from astrocytes, we recorded both UV-uncaging and synaptically evoked glutamate transporter currents (TCs), widely accepted assays of functional glutamate transport by astrocytes. With this approach, we set out to test the hypothesis that astrocyte membrane properties and glutamate transport were disrupted in this model of DCM. Though we found that the developmental maturation of astrocyte membrane resistance was disrupted by FL, glutamate uptake by individual astrocytes was robust throughout FL development. Interestingly, using an immunolabeling approach, we observed spatial and developmental differences in excitatory amino acid transporter (EAAT) expression in FL cortex. Spatially specific differences in EAAT2 (GLT-1) and EAAT1 (GLAST) expression suggest that the relative contribution of each EAAT to astrocytic glutamate uptake may be altered in FL cortex. Lastly, we carefully analyzed the amplitudes and onset times of both synaptically- and UV uncaging-evoked TCs. We found that in the FL cortex, synaptically-evoked, but not UV uncaging-evoked TCs, were larger in amplitude. Additionally, we found that the amount of electrical stimulation required to evoke a synaptic TC was significantly reduced in the FL cortex. Both of these findings are consistent with increased excitatory input to the FL cortex, but not with changes in how individual astrocytes remove glutamate. Taken together, our results demonstrate that the maturation of astrocyte membrane resistance, local distribution of glutamate transporters, and glutamatergic input to the cortex are altered in the FL model, but that single-cell astrocytic glutamate uptake is robust.

CAMP-Dependent Calcium Oscillations of Astrocytes: An Implication for Pathology.

Astrocytes in various brain regions exhibit spontaneous intracellular calcium elevations both in vitro and in vivo; however, neither the temporal pattern underlying this activity nor its function has been fully evaluated. Here, we utilized a long-term optical imaging technique to analyze the calcium activity of more than 4000 astrocytes in acute hippocampal slices as well as in the neocortex and hippocampus of head-restrained mice. Although astrocytic calcium activity was largely sparse and irregular, we observed a subset of cells in which the fluctuating calcium oscillations repeated at a regular interval of ∼30 s. These intermittent oscillations i) depended on type 2 inositol 1,4,5-trisphosphate receptors; ii) consisted of a complex reverberatory interaction between the soma and processes of individual astrocytes; iii) did not synchronize with those of other astrocytes; iv) did not require neuronal firing; v) were modulated through cAMP-protein kinase A signaling; vi) were facilitated under pathological conditions, such as energy deprivation and epileptiform hyperexcitation; and vii) were associated with enhanced hypertrophy in astrocytic processes, an early hallmark of reactive gliosis, which is observed in ischemia and epilepsy. Therefore, calcium oscillations appear to be associated with a pathological state in astrocytes.

Astrocytes Modulate Local Field Potential Rhythm?

Astrocytes Modulate Local Field Potential Rhythm?

Reciprocal Regulation of Mitochondrial Dynamics and Calcium Signaling in Astrocyte Processes.

In neurons, the movement and positioning of mitochondria at sites of elevated activity are important for matching local energy and Ca(2+) buffering capacity. Previously, we demonstrated that mitochondria are immobilized in astrocytes in response to neuronal activity and glutamate uptake. Here, we demonstrate a mechanism by which mitochondria are immobilized in astrocytes subsequent to increases in intracellular [Ca(2+)] and provide evidence that mitochondria contribute to the compartmentalization of spontaneous Ca(2+) signals in astrocyte processes. Immobilization of mitochondria at sites of glutamate uptake in astrocyte processes provides a mechanism to coordinate increases in activity with increases in mitochondrial metabolism.

Gap junction coupling confers isopotentiality on astrocyte syncytium.

Astrocytes are extensively coupled through gap junctions into a syncytium. However, the basic role of this major brain network remains largely unknown. Using electrophysiological and computational modeling methods, we demonstrate that the membrane potential (VM) of an individual astrocyte in a hippocampal syncytium, but not in a single, freshly isolated cell preparation, can be well-maintained at quasi-physiological levels when recorded with reduced or K(+) free pipette solutions that alter the K(+) equilibrium potential to non-physiological voltages. We show that an astrocyte's associated syncytium provides powerful electrical coupling, together with ionic coupling at a lesser extent, that equalizes the astrocyte's VM to levels comparable to its neighbors. Functionally, this minimizes VM depolarization attributable to elevated levels of local extracellular K(+) and thereby maintains a sustained driving force for highly efficient K(+) uptake. Thus, gap junction coupling functions to achieve isopotentiality in astrocytic networks, whereby a constant extracellular environment can be powerfully maintained for crucial functions of neural circuits.

Heterogeneity of Notch signaling in astrocytes and the effects of GFAP and vimentin deficiency.

Astrocytes have multiple roles in the CNS including control of adult neurogenesis. We recently showed that astrocyte inhibition of neurogenesis through Notch signaling depends on the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Here, we used real-time quantitative PCR to analyze gene expression in individual mouse astrocytes in primary cultures and in GFAP(POS) or Aldh1L1(POS) astrocytes freshly isolated from uninjured, contralesional and lesioned hippocampus 4 days after entorhinal cortex lesion. To determine the Notch signaling competence of individual astrocytes, we measured the mRNA levels of Notch ligands and Notch1 receptor. We found that whereas most cultured and freshly isolated astrocytes were competent to receive Notch signals, only a minority of astrocytes were competent to send Notch signals. Injury increased the fraction of astrocyte subpopulation unable to send and receive Notch signals, thus resembling primary astrocytes in vitro. Astrocytes deficient of GFAP and vimentin showed decreased Notch signal sending competence and altered expression of Notch signaling pathway-related genes Dlk2, Notch1, and Sox2. Furthermore, we identified astrocyte subpopulations based on their mRNA and protein expression of nestin and HB-EGF. This study improves our understanding of astrocyte heterogeneity, and points to astrocyte cytoplasmic intermediate filaments as targets for neural cell replacement strategies.

Diversity of astrocyte functions and phenotypes in neural circuits.

Astrocytes tile the entire CNS. They are vital for neural circuit function, but have traditionally been viewed as simple, homogenous cells that serve the same essential supportive roles everywhere. Here, we summarize breakthroughs that instead indicate that astrocytes represent a population of complex and functionally diverse cells. Physiological diversity of astrocytes is apparent between different brain circuits and microcircuits, and individual astrocytes display diverse signaling in subcellular compartments. With respect to injury and disease, astrocytes undergo diverse phenotypic changes that may be protective or causative with regard to pathology in a context-dependent manner. These new insights herald the concept that astrocytes represent a diverse population of genetically tractable cells that mediate neural circuit-specific roles in health and disease.

Isolation of intact astrocytes from the optic nerve head of adult mice

The astrocytes of the optic nerve head are a specialized subtype of white matter astrocytes that form the direct cellular environment of the unmyelinated ganglion cell axons. Due to their potential involvement in glaucoma, these astrocytes have become a target of research. Due to the heterogeneity of the optic nerve tissue, which also contains other cell types, in some cases it may be desirable to conduct gene expression studies on small numbers of well-characterized astrocytes or even individual cells. Here, we describe a simple method to isolate individual astrocytes. This method permits obtaining astrocytes with intact morphology from the adult mouse optic nerve and reduces contamination of the isolated astrocytes by other cell types. Individual astrocytes can be recognized by their morphology and collected under microscopic control. The whole procedure can be completed in 2–3 h. We also discuss downstream applications like multiplex single-cell PCR and quantitative PCR (qPCR).

Role for astroglial α1-adrenoreceptors in gliotransmission and control of synaptic plasticity in the neocortex.

Communication between neuronal and glial cells is thought to be very important for many brain functions. Acting via release of gliotransmitters, astrocytes can modulate synaptic strength. The mechanisms underlying gliotransmission remain uncertain with exocytosis being the most intriguing and debated pathway. We demonstrate that astroglial α1-adrenoreceptors are very sensitive to noradrenaline (NA) and make a significant contribution to intracellular Ca2+-signaling in layer 2/3 neocortical astrocytes. We also show that astroglial α1-adrenoreceptors are prone to desensitization upon prolonged exposure to NA. We show that within neocortical slices, α-1adrenoreceptors can activate vesicular release of ATP and D-serine from cortical astrocytes which initiate a burst of ATP receptor-mediated currents in adjacent pyramidal neurons. These purinergic currents can be inhibited by intracellular perfusion of astrocytes with Tetanus Toxin light chain, verifying their origin via astroglial exocytosis. We show that α1 adrenoreceptor-activated release of gliotransmitters is important for the induction of synaptic plasticity in the neocortex:long-term potentiation (LTP) of neocortical excitatory synaptic potentials can be abolished by the selective α1-adrenoreceptor antagonist terazosin. We show that weak sub-threshold theta-burst stimulation (TBS) can induce LTP when astrocytes are additionally activated by 1 μM NA. This facilitation is dependent on the activation of neuronal ATP receptors and is abolished in neocortical slices from dn-SNARE mice which have impaired glial exocytosis. Importantly, facilitation of LTP by NA can be significantly reduced by perfusion of individual astrocytes with Tetanus Toxin. Our results strongly support the physiological importance of astroglial adrenergic signaling and exocytosis of gliotransmitters for modulation of synaptic transmission and plasticity.