Adenosine signalling to astrocytes coordinates brain metabolism and function.

Brain computation performed by billions of nerve cells relies on a sufficient and uninterrupted nutrient and oxygen supply1,2. Astrocytes, the ubiquitous glial neighbours of neurons, govern brain glucose uptake and metabolism3,4, but the exact mechanisms of metabolic coupling between neurons and astrocytes that ensure on-demand support of neuronal energy needs are not fully understood5,6. Here we show, using experimental in vitro and in vivo animal models, that neuronal activity-dependent metabolic activation of astrocytes is mediated by neuromodulator adenosine acting on astrocytic A2B receptors. Stimulation of A2B receptors recruits the canonical cyclic adenosine 3',5'-monophosphate-protein kinase A signalling pathway, leading to rapid activation of astrocyte glucose metabolism and the release of lactate, which supplements the extracellular pool of readily available energy substrates. Experimental mouse models involving conditional deletion of the gene encoding A2B receptors in astrocytes showed that adenosine-mediated metabolic signalling is essential for maintaining synaptic function, especially under conditions of high energy demand or reduced energy supply. Knockdown of A2B receptor expression in astrocytes led to a major reprogramming of brain energy metabolism, prevented synaptic plasticity in the hippocampus, severely impaired recognition memory and disrupted sleep. These data identify the adenosine A2B receptor as an astrocytic sensor of neuronal activity and show that cAMP signalling in astrocytes tunes brain energy metabolism to support its fundamental functions such as sleep and memory.

K+ efflux through postsynaptic NMDA receptors suppresses local astrocytic glutamate uptake.

Glutamatergic transmission prompts K+ efflux through postsynaptic NMDA receptors. The ensuing hotspot of extracellular K+ elevation depolarizes presynaptic terminal, boosting glutamate release, but whether this also affects glutamate uptake in local astroglia has remained an intriguing question. Here, we find that the pharmacological blockade, or conditional knockout, of postsynaptic NMDA receptors suppresses use-dependent increase in the amplitude and duration of the astrocytic glutamate transporter current (IGluT ), whereas blocking astrocytic K+ channels prevents the duration increase only. Glutamate spot-uncaging reveals that astrocyte depolarization, rather than extracellular K+ rises per se, is required to reduce the amplitude and duration of IGluT . Biophysical simulations confirm that local transient elevations of extracellular K+ can inhibit local glutamate uptake in fine astrocytic processes. Optical glutamate sensor imaging and a two-pathway test relate postsynaptic K+ efflux to enhanced extrasynaptic glutamate signaling. Thus, repetitive glutamatergic transmission triggers a feedback loop in which postsynaptic K+ efflux can transiently facilitate presynaptic release while reducing local glutamate uptake.

Maturation and phenotype of pathophysiological neuronal excitability of human cells in tau-related dementia.

Frontotemporal dementia and parkinsonism (FTDP-17) caused by the 10+16 splice-site mutation in the gene encoding microtubule-associated protein tau (MAPT) provides an established platform to model tau-related dementia in vitro Neurons derived from human induced pluripotent stem cells (iPSCs) have been shown to recapitulate the neurodevelopmental profile of tau pathology during in vitro corticogenesis, as in the adult human brain. However, the neurophysiological phenotype of these cells has remained unknown, leaving unanswered questions regarding the functional relevance and the gnostic power of this disease model. In this study, we used electrophysiology to explore the membrane properties and intrinsic excitability of the generated neurons and found that human cells mature by ∼150 days of neurogenesis to become compatible with matured cortical neurons. In earlier FTDP-17, however, neurons exhibited a depolarized resting membrane potential associated with increased resistance and reduced voltage-gated Na+- and K+-channel-mediated conductance. Expression of the Nav1.6 protein was reduced in FTDP-17. These effects led to reduced cell capability of induced firing and changed the action potential waveform in FTDP-17. The revealed neuropathology might thus contribute to the clinicopathological profile of the disease. This sheds new light on the significance of human in vitro models of dementia.

A genetically encoded fluorescent sensor for in vivo imaging of GABA.

Current techniques for monitoring GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in vertebrates, cannot follow transients in intact neural circuits. To develop a GABA sensor, we applied the design principles used to create the fluorescent glutamate receptor iGluSnFR. We used a protein derived from a previously unsequenced Pseudomonas fluorescens strain and performed structure-guided mutagenesis and library screening to obtain intensity-based GABA sensing fluorescence reporter (iGABASnFR) variants. iGABASnFR is genetically encoded, detects GABA release evoked by electric stimulation of afferent fibers in acute brain slices and produces readily detectable fluorescence increases in vivo in mice and zebrafish. We applied iGABASnFR to track mitochondrial GABA content and its modulation by an anticonvulsant, swimming-evoked, GABA-mediated transmission in zebrafish cerebellum, GABA release events during interictal spikes and seizures in awake mice, and found that GABA-mediated tone decreases during isoflurane anesthesia.

Electrodiffusion phenomena in neuroscience: a neglected companion.

The emerging technological revolution in genetically encoded molecular sensors and super-resolution imaging provides neuroscientists with a pass to the real-time nano-world. On this small scale, however, classical principles of electrophysiology do not always apply. This is in large part because the nanoscopic heterogeneities in ionic concentrations and the local electric fields associated with individual ions and their movement can no longer be ignored. Here, we review basic principles of molecular electrodiffusion in the cellular environment of organized brain tissue. We argue that accurate interpretation of physiological observations on the nanoscale requires a better understanding of the underlying electrodiffusion phenomena.

Mitochondrial ROS control neuronal excitability and cell fate in frontotemporal dementia.

INTRODUCTION: The second most common form of early-onset dementia-frontotemporal dementia (FTD)-is often characterized by the aggregation of the microtubule-associated protein tau. Here we studied the mechanism of tau-induced neuronal dysfunction in neurons with the FTD-related 10+16 MAPT mutation. METHODS: Live imaging, electrophysiology, and redox proteomics were used in 10+16 induced pluripotent stem cell-derived neurons and a model of tau spreading in primary cultures. RESULTS: Overproduction of mitochondrial reactive oxygen species (ROS) in 10+16 neurons alters the trafficking of specific glutamate receptor subunits via redox regulation. Increased surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors containing GluA1 and NR2B subunits leads to impaired glutamatergic signaling, calcium overload, and excitotoxicity. Mitochondrial antioxidants restore the altered response and prevent neuronal death. Importantly, extracellular 4R tau induces the same pathological response in healthy neurons, thus proposing a mechanism for disease propagation. DISCUSSION: These results demonstrate mitochondrial ROS modulate glutamatergic signaling in FTD, and suggest a new therapeutic strategy.

Conductance of porous media depends on external electric fields.

In obstacle-filled media, such as extracellular or intracellular lumen of brain tissue, effective ion-diffusion permeability is a key determinant of electrogenic reactions. Although this diffusion permeability is thought to depend entirely on structural features of the medium, such as porosity and tortuosity, brain tissue shows prominent nonohmic properties, the origins of which remain poorly understood. Here, we explore Monte Carlo simulations of ion diffusion in a space filled with overlapping spheres to predict that diffusion permeability of such media decreases with stronger external electric fields. This dependence increases with lower medium porosity while decreasing with radial (two-dimensional or three-dimensional) compared with homogenous (one-dimensional) fields. We test our predictions empirically in an electrolyte chamber filled with microscopic glass spheres and find good correspondence with our predictions. A theoretical insight relates this phenomenon to a disproportionately increased dwell time of diffusing ions at potential barriers (or traps) representing geometric obstacles when the field strength increases. The dependence of medium ion-diffusion permeability on electric field could be important for understanding conductivity properties of porous materials, in particular for the accurate interpretation of electric activity recordings in brain tissue.

Fluorescence lifetime imaging reveals regulation of presynaptic Ca2+ by glutamate uptake and mGluRs, but not somatic voltage in cortical neurons.

Brain function relies on vesicular release of neurotransmitters at chemical synapses. The release probability depends on action potential-evoked presynaptic Ca2+ entry, but also on the resting Ca2+ level. Whether these basic aspects of presynaptic calcium homeostasis show any consistent trend along the axonal path, and how they are controlled by local network activity, remains poorly understood. Here, we take advantage of the recently advanced FLIM-based method to monitor presynaptic Ca2+ with nanomolar sensitivity. We find that, in cortical pyramidal neurons, action potential-evoked calcium entry (range 10-300 nM), but not the resting Ca2+ level (range 10-100 nM), tends to increase with higher order of axonal branches. Blocking astroglial glutamate uptake reduces evoked Ca2+ entry but has little effect on resting Ca2+ whereas both appear boosted by the constitutive activation of group 1/2 metabotropic glutamate receptors. We find no consistent effect of transient somatic depolarization or hyperpolarization on presynaptic Ca2+ entry or its basal level. The results unveil some key aspects of presynaptic machinery in cortical circuits, shedding light on basic principles of synaptic connectivity in the brain.

Imaging tripartite synapses using super-resolution microscopy.

Astroglia are vital facilitators of brain development, homeostasis, and metabolic support. In addition, they are also essential to the formation and regulation of synaptic circuits. Due to the extraordinary complex, nanoscopic morphology of astrocytes, the underlying cellular mechanisms have been poorly understood. In particular, fine astrocytic processes that can be found in the vicinity of synapses have been difficult to study using traditional imaging techniques. Here, we describe a 3D three-colour super-resolution microscopy approach to unravel the nanostructure of tripartite synapses. The method is based on the SMLM technique direct stochastic optical reconstruction microscopy (dSTORM) which uses conventional fluorophore-labelled antibodies. This approach enables reconstructing the nanoscale localisation of individual astrocytic glutamate transporter (GLT-1) molecules surrounding presynaptic (bassoon) and postsynaptic (Homer1) protein localisations in fixed mouse brain sections. However, the technique is readily adaptable to other types of targets and tissues.

Extracellular GABA waves regulate coincidence detection in excitatory circuits.

KEY POINTS: Rapid changes in neuronal network activity trigger widespread waves of extracellular GABA in hippocampal neuropil. Elevations of extracellular GABA narrow the coincidence detection window for excitatory inputs to CA1 pyramidal cells. GABA transporters control the effect of extracellular GABA on coincidence detection. Small changes in the kinetics of dendritic excitatory currents amplify when reaching the soma. ABSTRACT: Coincidence detection of excitatory inputs by principal neurons underpins the rules of signal integration and Hebbian plasticity in the brain. In the hippocampal circuitry, detection fidelity is thought to depend on the GABAergic synaptic input through a feedforward inhibitory circuit also involving the hyperpolarisation-activated Ih current. However, afferent connections often bypass feedforward circuitry, suggesting that a different GABAergic mechanism might control coincidence detection in such cases. To test whether fluctuations in the extracellular GABA concentration [GABA] could play a regulatory role here, we use a GABA 'sniffer' patch in acute hippocampal slices of the rat and document strong dependence of [GABA] on network activity. We find that blocking GABAergic signalling strongly widens the coincidence detection window of direct excitatory inputs to pyramidal cells whereas increasing [GABA] through GABA uptake blockade shortens it. The underlying mechanism involves membrane-shunting tonic GABAA receptor current; it does not have to rely on Ih but depends strongly on the neuronal GABA transporter GAT-1. We use dendrite-soma dual patch-clamp recordings to show that the strong effect of membrane shunting on coincidence detection relies on nonlinear amplification of changes in the decay of dendritic synaptic currents when they reach the soma. Our results suggest that, by dynamically regulating extracellular GABA, brain network activity can optimise signal integration rules in local excitatory circuits.

Disentangling calcium-driven astrocyte physiology.

Astrocytes seem to rely on relatively sluggish and spatially blurred Ca(2+) waves to communicate with fast and point-precise neural circuits. This apparent discrepancy could, however, reflect our current inability to understand the microscopic mechanisms involved. Difficulties in detecting and interpreting astrocyte Ca(2+) signals may have led to some prominent controversies in the field. Here, we argue that a deeper understanding of astrocyte physiology requires a qualitative leap in our experimental and analytical strategies.

Unveiling the Extracellular Space of the Brain: From Super-resolved Microstructure to In Vivo Function.

The extracellular space occupies approximately one-fifth of brain volume, molding a spider web of gaps filled with interstitial fluid and extracellular matrix where neurons and glial cells perform in concert. Yet, very little is known about the spatial organization and dynamics of the extracellular space, let alone its influence on brain function, owing to a lack of appropriate techniques (and a traditional bias toward the inside of cells, not the spaces in between). At the same time, it is clear that understanding fundamental brain functions, such as synaptic transmission, memory, sleep, and recovery from disease, calls for more focused research on the extracellular space of the brain. This review article highlights several key research areas, covering recent methodological and conceptual progress that illuminates this understudied, yet critically important, brain compartment, providing insights into the opportunities and challenges of this nascent field.

ARACHNE: A neural-neuroglial network builder with remotely controlled parallel computing.

Creating and running realistic models of neural networks has hitherto been a task for computing professionals rather than experimental neuroscientists. This is mainly because such networks usually engage substantial computational resources, the handling of which requires specific programing skills. Here we put forward a newly developed simulation environment ARACHNE: it enables an investigator to build and explore cellular networks of arbitrary biophysical and architectural complexity using the logic of NEURON and a simple interface on a local computer or a mobile device. The interface can control, through the internet, an optimized computational kernel installed on a remote computer cluster. ARACHNE can combine neuronal (wired) and astroglial (extracellular volume-transmission driven) network types and adopt realistic cell models from the NEURON library. The program and documentation (current version) are available at GitHub repository https://github.com/LeonidSavtchenko/Arachne under the MIT License (MIT).

Dopamine elevates and lowers astroglial Ca2+ through distinct pathways depending on local synaptic circuitry.

Whilst astrocytes in culture invariably respond to dopamine with cytosolic Ca2+ rises, the dopamine sensitivity of astroglia in situ and its physiological roles remain unknown. To minimize effects of experimental manipulations on astroglial physiology, here we monitored Ca2+ in cells connected via gap junctions to astrocytes loaded whole-cell with cytosolic indicators in area CA1 of acute hippocampal slices. Aiming at high sensitivity of [Ca2+ ] measurements, we also employed life-time imaging of the Ca2+ indicator Oregon Green BAPTA-1. We found that dopamine triggered a dose-dependent, bidirectional Ca2+ response in stratum radiatum astroglia, a jagged elevation accompanied and followed by below-baseline decreases. The elevation depended on D1/D2 receptors and engaged intracellular Ca2+ storage and removal whereas the dopamine-induced [Ca2+ ] decrease involved D2 receptors only and was sensitive to Ca2+ channel blockade. In contrast, the stratum lacunosum moleculare astroglia generated higher-threshold dopamine-induced Ca2+ responses which did not depend on dopamine receptors and were uncoupled from the prominent inhibitory action of dopamine on local perforant path synapses. Our findings thus suggest that a single neurotransmitter-dopamine-could either elevate or decrease astrocyte [Ca2+ ] depending on the receptors involved, that such actions are specific to the regional neural circuitry and that they may be causally uncoupled from dopamine actions on local synapses. The results also indicate that [Ca2+ ] elevations commonly detected in astroglia can represent the variety of distinct mechanisms acting on the microscopic scale. GLIA 2017;65:447-459.

Probing nano-organization of astroglia with multi-color super-resolution microscopy.

Astroglia are essential for brain development, homeostasis, and metabolic support. They also contribute actively to the formation and regulation of synaptic circuits, by successfully handling, integrating, and propagating physiological signals of neural networks. The latter occurs mainly by engaging a versatile mechanism of internal Ca2+ fluctuations and regenerative waves prompting targeted release of signaling molecules into the extracellular space. Astroglia also show substantial structural plasticity associated with age- and use-dependent changes in neural circuitry. However, the underlying cellular mechanisms are poorly understood, mainly because of the extraordinary complex morphology of astroglial compartments on the nanoscopic scale. This complexity largely prevents direct experimental access to astroglial processes, most of which are beyond the diffraction limit of optical microscopy. Here we employed super-resolution microscopy (direct stochastic optical reconstruction microscopy; dSTORM), to visualize astroglial organization on the nanoscale, in culture and in thin brain slices, as an initial step to understand the structural basis of astrocytic nano-physiology. We were able to follow nanoscopic morphology of GFAP-enriched astrocytes, which adapt a flattened shape in culture and a sponge-like structure in situ, with GFAP fibers of varied diameters. We also visualized nanoscopic astrocytic processes using the ubiquitous cytosolic astrocyte marker proteins S100ß and glutamine synthetase. Finally, we overexpressed and imaged membrane-targeted pHluorin and lymphocyte-specific protein tyrosine kinase (N-terminal domain) -green fluorescent protein (lck-GFP), to better understand the molecular cascades underlying some common astroglia-targeted fluorescence imaging techniques. The results provide novel, albeit initial, insights into the cellular organization of astroglia on the nanoscale, paving the way for function-specific studies. © 2017 Wiley Periodicals, Inc.

Tonic GABAA conductance bidirectionally controls interneuron firing pattern and synchronization in the CA3 hippocampal network.

The spiking output of interneurons is key for rhythm generation in the brain. However, what controls interneuronal firing remains incompletely understood. Here we combine dynamic clamp experiments with neural network simulations to understand how tonic GABAA conductance regulates the firing pattern of CA3 interneurons. In baseline conditions, tonic GABAA depolarizes these cells, thus exerting an excitatory action while also reducing the excitatory postsynaptic potential (EPSP) amplitude through shunting. As a result, the emergence of weak tonic GABAA conductance transforms the interneuron firing pattern driven by individual EPSPs into a more regular spiking mode determined by the cell intrinsic properties. The increased regularity of spiking parallels stronger synchronization of the local network. With further increases in tonic GABAA conductance the shunting inhibition starts to dominate over excitatory actions and thus moderates interneuronal firing. The remaining spikes tend to follow the timing of suprathreshold EPSPs and thus become less regular again. The latter parallels a weakening in network synchronization. Thus, our observations suggest that tonic GABAA conductance can bidirectionally control brain rhythms through changes in the excitability of interneurons and in the temporal structure of their firing patterns.

Long-term potentiation depends on release of D-serine from astrocytes.

Long-term potentiation (LTP) of synaptic transmission provides an experimental model for studying mechanisms of memory. The classical form of LTP relies on N-methyl-D-aspartate receptors (NMDARs), and it has been shown that astroglia can regulate their activation through Ca(2+)-dependent release of the NMDAR co-agonist D-serine. Release of D-serine from glia enables LTP in cultures and explains a correlation between glial coverage of synapses and LTP in the supraoptic nucleus. However, increases in Ca(2+) concentration in astroglia can also release other signalling molecules, most prominently glutamate, ATP and tumour necrosis factor-alpha, whereas neurons themselves can synthesize and supply D-serine. Furthermore, loading an astrocyte with exogenous Ca(2+) buffers does not suppress LTP in hippocampal area CA1 (refs 14-16), and the physiological relevance of experiments in cultures or strong exogenous stimuli applied to astrocytes has been questioned. The involvement of glia in LTP induction therefore remains controversial. Here we show that clamping internal Ca(2+) in individual CA1 astrocytes blocks LTP induction at nearby excitatory synapses by decreasing the occupancy of the NMDAR co-agonist sites. This LTP blockade can be reversed by exogenous D-serine or glycine, whereas depletion of D-serine or disruption of exocytosis in an individual astrocyte blocks local LTP. We therefore demonstrate that Ca(2+)-dependent release of D-serine from an astrocyte controls NMDAR-dependent plasticity in many thousands of excitatory synapses nearby.

Cannabinoid- and lysophosphatidylinositol-sensitive receptor GPR55 boosts neurotransmitter release at central synapses.

G protein-coupled receptor (GPR) 55 is sensitive to certain cannabinoids, it is expressed in the brain and, in cell cultures, it triggers mobilization of intracellular Ca(2+). However, the adaptive neurobiological significance of GPR55 remains unknown. Here, we use acute hippocampal slices and combine two-photon excitation Ca(2+) imaging in presynaptic axonal boutons with optical quantal analysis in postsynaptic dendritic spines to find that GPR55 activation transiently increases release probability at individual CA3-CA1 synapses. The underlying mechanism involves Ca(2+) release from presynaptic Ca(2+) stores, whereas postsynaptic stores (activated by spot-uncaging of inositol 1,4,5-trisphosphate) remain unaffected by GPR55 agonists. These effects are abolished by genetic deletion of GPR55 or by the GPR55 antagonist cannabidiol, a constituent of Cannabis sativa. GPR55 shows colocalization with synaptic vesicle protein vesicular glutamate transporter 1 in stratum radiatum. Short-term potentiation of CA3-CA1 transmission after a short train of stimuli reveals a presynaptic, Ca(2+) store-dependent component sensitive to cannabidiol. The underlying cascade involves synthesis of phospholipids, likely in the presynaptic cell, but not the endocannabinoids 2-arachidonoylglycerol or anandamide. Our results thus unveil a signaling role for GPR55 in synaptic circuits of the brain.