Publications by authors named "Dmitri A Rusakov"

109 Publications

Conductance of porous media depends on external electric fields.

Biophys J 2021 Feb 18. Epub 2021 Feb 18.

UCL Queen Square Institute of Neurology, University College London, London, United Kingdom. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bpj.2021.02.012DOI Listing
February 2021

Release probability increases towards distal dendrites boosting high-frequency signal transfer in the rodent hippocampus.

Elife 2021 Jan 13;10. Epub 2021 Jan 13.

Queen Square UCL Institute of Neurology, University College London, London, United Kingdom.

Dendritic integration of synaptic inputs involves their increased electrotonic attenuation at distal dendrites, which can be counterbalanced by the increased synaptic receptor density. However, during network activity, the influence of individual synapses depends on their release fidelity, the dendritic distribution of which remains poorly understood. Here, we employed classical optical quantal analyses and a genetically encoded optical glutamate sensor in acute hippocampal slices of rats and mice to monitor glutamate release at CA3-CA1 synapses. We find that their release probability increases with greater distances from the soma. Similar-fidelity synapses tend to group together, whereas release probability shows no trends regarding the branch ends. Simulations with a realistic CA1 pyramidal cell hosting stochastic synapses suggest that the observed trends boost signal transfer fidelity, particularly at higher input frequencies. Because high-frequency bursting has been associated with learning, the release probability pattern we have found may play a key role in memory trace formation.
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http://dx.doi.org/10.7554/eLife.62588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837677PMC
January 2021

Biodegradable Microcapsules Loaded with Nerve Growth Factor Enable Neurite Guidance and Synapse Formation.

Pharmaceutics 2020 Dec 25;13(1). Epub 2020 Dec 25.

Department of Clinical and Experimental Epilepsy, UCL Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK.

Neurological disorders and traumas often involve loss of specific neuronal connections, which would require intervention with high spatial precision. We have previously demonstrated the biocompatibility and therapeutic potential of the layer-by-layer (LbL)-fabricated microcapsules aimed at the localized delivery of specific channel blockers to peripheral nerves. Here, we explore the potential of LbL-microcapsules to enable site-specific, directional action of neurotrophins to stimulate neuronal morphogenesis and synaptic circuit formation. We find that nanoengineered biodegradable microcapsules loaded with nerve growth factor (NGF) can guide the morphological development of hippocampal neurons in vitro. The presence of NGF-loaded microcapsules or their clusters increases the neurite outgrowth rate while boosting neurite branching. Microcapsule clusters appear to guide the trajectory of developing individual axons leading to the formation of functional synapses. Our observations highlight the potential of NGF-loaded, biodegradable LbL-microcapsules to help guide axonal development and possibly circuit regeneration in neuropathology.
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http://dx.doi.org/10.3390/pharmaceutics13010025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823884PMC
December 2020

Astrocytes regulate brain extracellular pH via a neuronal activity-dependent bicarbonate shuttle.

Nat Commun 2020 10 8;11(1):5073. Epub 2020 Oct 8.

Centre for Cardiovascular and Metabolic Neuroscience, Neuroscience, Physiology and Pharmacology, University College London, London, UK.

Brain cells continuously produce and release protons into the extracellular space, with the rate of acid production corresponding to the levels of neuronal activity and metabolism. Efficient buffering and removal of excess H is essential for brain function, not least because all the electrogenic and biochemical machinery of synaptic transmission is highly sensitive to changes in pH. Here, we describe an astroglial mechanism that contributes to the protection of the brain milieu from acidification. In vivo and in vitro experiments conducted in rodent models show that at least one third of all astrocytes release bicarbonate to buffer extracellular H loads associated with increases in neuronal activity. The underlying signalling mechanism involves activity-dependent release of ATP triggering bicarbonate secretion by astrocytes via activation of metabotropic P2Y receptors, recruitment of phospholipase C, release of Ca from the internal stores, and facilitated outward HCO transport by the electrogenic sodium bicarbonate cotransporter 1, NBCe1. These results show that astrocytes maintain local brain extracellular pH homeostasis via a neuronal activity-dependent release of bicarbonate. The data provide evidence of another important metabolic housekeeping function of these glial cells.
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http://dx.doi.org/10.1038/s41467-020-18756-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545092PMC
October 2020

LTP Induction Boosts Glutamate Spillover by Driving Withdrawal of Perisynaptic Astroglia.

Neuron 2020 12 24;108(5):919-936.e11. Epub 2020 Sep 24.

UCL Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK. Electronic address:

Extrasynaptic actions of glutamate are limited by high-affinity transporters expressed by perisynaptic astroglial processes (PAPs): this helps maintain point-to-point transmission in excitatory circuits. Memory formation in the brain is associated with synaptic remodeling, but how this affects PAPs and therefore extrasynaptic glutamate actions is poorly understood. Here, we used advanced imaging methods, in situ and in vivo, to find that a classical synaptic memory mechanism, long-term potentiation (LTP), triggers withdrawal of PAPs from potentiated synapses. Optical glutamate sensors combined with patch-clamp and 3D molecular localization reveal that LTP induction thus prompts spatial retreat of astroglial glutamate transporters, boosting glutamate spillover and NMDA-receptor-mediated inter-synaptic cross-talk. The LTP-triggered PAP withdrawal involves NKCC1 transporters and the actin-controlling protein cofilin but does not depend on major Ca-dependent cascades in astrocytes. We have therefore uncovered a mechanism by which a memory trace at one synapse could alter signal handling by multiple neighboring connections.
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http://dx.doi.org/10.1016/j.neuron.2020.08.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736499PMC
December 2020

Extracellular GABA waves regulate coincidence detection in excitatory circuits.

J Physiol 2020 09 4;598(18):4047-4062. Epub 2020 Aug 4.

UCL Queen Square Institute of Neurology, University College London, Queen Square, London, WC1N 3BG, UK.

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 I 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 GABA receptor current; it does not have to rely on I 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.
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http://dx.doi.org/10.1113/JP279744DOI Listing
September 2020

Publisher Correction: Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles.

Nat Neurosci 2020 Sep;23(9):1176

Department of Neuroscience, Physiology & Pharmacology, University College London, London, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41593-020-0680-0DOI Listing
September 2020

Polylactic Acid-Based Patterned Matrixes for Site-Specific Delivery of Neuropeptides On-Demand: Functional NGF Effects on Human Neuronal Cells.

Front Bioeng Biotechnol 2020 12;8:497. Epub 2020 Jun 12.

Center for Neurobiology and Brain Restoration, Skolkovo Institute of Science and Technology, Moscow, Russia.

The patterned microchamber arrays based on biocompatible polymers are a versatile cargo delivery system for drug storage and site-/time-specific drug release on demand. However, functional evidence of their action on nerve cells, in particular their potential for enabling patterned neuronal morphogenesis, remains unclear. Recently, we have established that the polylactic acid (PLA)-based microchamber arrays are biocompatible with human cells of neuronal phenotype and provide safe loading for hydrophilic substances of low molecular weight, with successive site-specific cargo release on-demand to trigger local cell responses. Here, we load the nerve growth factor (NGF) inside microchambers and grow N2A cells on the surface of patterned microchamber arrays. We find that the neurite outgrowth in local N2A cells can be preferentially directed towards opened microchambers (upon-specific NGF release). These observations suggest the PLA-microchambers can be an efficient drug delivery system for the site-specific delivery of neuropeptides on-demand, potentially suitable for the migratory or axonal guidance of human nerve cells.
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http://dx.doi.org/10.3389/fbioe.2020.00497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304324PMC
June 2020

Noisy Synaptic Conductance: Bug or a Feature?

Trends Neurosci 2020 06 21;43(6):363-372. Epub 2020 Apr 21.

Gatsby Computational Neuroscience Unit, University College London, 25 Howland Street, London, W1T 4JG, UK. Electronic address:

More often than not, action potentials fail to trigger neurotransmitter release. And even when neurotransmitter is released, the resulting change in synaptic conductance is highly variable. Given the energetic cost of generating and propagating action potentials, and the importance of information transmission across synapses, this seems both wasteful and inefficient. However, synaptic noise arising from variable transmission can improve, in certain restricted conditions, information transmission. Under broader conditions, it can improve information transmission per release, a quantity that is relevant given the energetic constraints on computing in the brain. Here we discuss the role, both positive and negative, synaptic noise plays in information transmission and computation in the brain.
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http://dx.doi.org/10.1016/j.tins.2020.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902755PMC
June 2020

Academia Europaea Position Paper on Translational Medicine: The Cycle Model for Translating Scientific Results into Community Benefits.

J Clin Med 2020 May 19;9(5). Epub 2020 May 19.

Institute for Translational Medicine, Medical School, University of Pécs, 7624 Pécs, Hungary.

Introduction: Translational science has gained prominence in medicine, but there is still much work to be done before scientific results are used optimally and incorporated into everyday health practice. As the main focus is still on generating new scientific data with financial resources primarily available for that purpose, other activities that are necessary in the transition from research to community benefit are considered less needy. The European Statistical Office of the European Commission has recently reported that 1.7 million people under 75 years of age died in Europe in 2016, with around 1.2 million of those deaths being avoidable through effective primary prevention and public health intervention. Therefore, Academia Europaea, one of the five Pan-European networks that form SAPEA (Science Advice for Policy by European Academies), a key element of the European Commission's Scientific Advice Mechanism (SAM), has launched a project to develop a model to facilitate and accelerate the utilisation of scientific knowledge for public and community benefit.

Methods: During the process, leaders in the field, including prominent basic and clinical researchers, editors-in-chief of high-impact journals publishing translational research articles, translational medicine (TM) centre leaders, media representatives, academics and university leaders, developed the TM cycle, a new model that we believe could significantly advance the development of TM.

Results: This model focuses equally on the acquisition of new scientific results healthcare, understandable and digestible summation of results, and their communication to all participants. We have also renewed the definition in TM, identified challenges and recommended solutions.

Conclusion: The authors, including senior officers of Academia Europaea, produced this document to serve as a basis for revising thinking on TM with the end result of enabling more efficient and cost-effective healthcare.
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http://dx.doi.org/10.3390/jcm9051532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290380PMC
May 2020

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

J Neurochem 2021 01 20;156(1):48-58. Epub 2020 Jun 20.

Queen Square Institute of Neurology, University College London, London, UK.

Brain function relies on vesicular release of neurotransmitters at chemical synapses. The release probability depends on action potential-evoked presynaptic Ca entry, but also on the resting Ca 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 Ca with nanomolar sensitivity. We find that, in cortical pyramidal neurons, action potential-evoked calcium entry (range 10-300 nM), but not the resting Ca level (range 10-100 nM), tends to increase with higher order of axonal branches. Blocking astroglial glutamate uptake reduces evoked Ca entry but has little effect on resting Ca 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 Ca 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.
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http://dx.doi.org/10.1111/jnc.15094DOI Listing
January 2021

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

J Cell Sci 2020 05 27;133(10). Epub 2020 May 27.

Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, WC1N 3BG, UK

Frontotemporal dementia and parkinsonism (FTDP-17) caused by the 10+16 splice-site mutation in the gene encoding microtubule-associated protein tau () provides an established platform to model tau-related dementia Neurons derived from human induced pluripotent stem cells (iPSCs) have been shown to recapitulate the neurodevelopmental profile of tau pathology during 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 Na1.6 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 models of dementia.
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http://dx.doi.org/10.1242/jcs.241687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272359PMC
May 2020

Correction to: Optical monitoring of glutamate release at multiple synapses in situ detects changes following LTP induction.

Mol Brain 2020 Mar 25;13(1):48. Epub 2020 Mar 25.

Queen Square Institute of Neurology, University College London, Queen Square, London, WC1N 3BG, UK.

In the original publication of this article [1], text has been introduced erroneously to Figs. 4a and 5d due to a typesetting mistake.
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http://dx.doi.org/10.1186/s13041-020-00590-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093947PMC
March 2020

Optical monitoring of glutamate release at multiple synapses in situ detects changes following LTP induction.

Mol Brain 2020 03 13;13(1):39. Epub 2020 Mar 13.

Queen Square Institute of Neurology, University College London, Queen Square, London, WC1N 3BG, UK.

Information processing and memory formation in the brain relies on release of the main excitatory neurotransmitter glutamate from presynaptic axonal specialisations. The classical Hebbian paradigm of synaptic memory, long-term potentiation (LTP) of transmission, has been widely associated with an increase in the postsynaptic receptor current. Whether and to what degree LTP induction also enhances presynaptic glutamate release has been the subject of debate. Here, we took advantage of the recently developed genetically encoded optical sensors of glutamate (iGluSnFR) to monitor its release at CA3-CA1 synapses in acute hippocampal slices, before and after the induction of LTP. We attempted to trace release events at multiple synapses simultaneously, by using two-photon excitation imaging in fast frame-scanning mode. We thus detected a significant increase in the average iGluSnFR signal during potentiation, which lasted for up to 90 min. This increase may reflect an increased amount of released glutamate or, alternatively, reduced glutamate binding to high-affinity glutamate transporters that compete with iGluSnFR.
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http://dx.doi.org/10.1186/s13041-020-00572-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071671PMC
March 2020

Local Resting Ca Controls the Scale of Astroglial Ca Signals.

Cell Rep 2020 03;30(10):3466-3477.e4

Institute of Neurology, University College London, London, UK; Institute of Cellular Neurosciences, Medical Faculty, University of Bonn, Bonn, Germany; German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany. Electronic address:

Astroglia regulate neurovascular coupling while engaging in signal exchange with neurons. The underlying cellular machinery is thought to rely on astrocytic Ca signals, but what controls their amplitude and waveform is poorly understood. Here, we employ time-resolved two-photon excitation fluorescence imaging in acute hippocampal slices and in cortex in vivo to find that resting [Ca] predicts the scale (amplitude) and the maximum (peak) of astroglial Ca elevations. We bidirectionally manipulate resting [Ca] by uncaging intracellular Ca or Ca buffers and use ratiometric imaging of a genetically encoded Ca indicator to establish that alterations in resting [Ca] change co-directionally the peak level and anti-directionally the amplitude of local Ca transients. This relationship holds for spontaneous and for induced (for instance by locomotion) Ca signals. Our findings uncover a basic generic rule of Ca signal formation in astrocytes, thus also associating the resting Ca level with the physiological "excitability" state of astroglia.
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http://dx.doi.org/10.1016/j.celrep.2020.02.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068654PMC
March 2020

Serotonin 5-HT receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin.

Commun Biol 2020 02 14;3(1):76. Epub 2020 Feb 14.

Cellular Neurophysiology, Hannover Medical School, Carl-Neuberg Str. 1, 30625, Hannover, Germany.

Activity-dependent remodeling of excitatory connections underpins memory formation in the brain. Serotonin receptors are known to contribute to such remodeling, yet the underlying molecular machinery remains poorly understood. Here, we employ high-resolution time-lapse FRET imaging in neuroblastoma cells and neuronal dendrites to establish that activation of serotonin receptor 5-HT (5-HTR) rapidly triggers spatially-restricted RhoA activity and G13-mediated phosphorylation of cofilin, thus locally boosting the filamentous actin fraction. In neuroblastoma cells, this leads to cell rounding and neurite retraction. In hippocampal neurons in situ, 5-HTR-mediated RhoA activation triggers maturation of dendritic spines. This is paralleled by RhoA-dependent, transient alterations in cell excitability, as reflected by increased spontaneous synaptic activity, apparent shunting of evoked synaptic responses, and enhanced long-term potentiation of excitatory transmission. The 5-HTR/G13/RhoA signaling thus emerges as a previously unrecognized molecular pathway underpinning use-dependent functional remodeling of excitatory synaptic connections.
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http://dx.doi.org/10.1038/s42003-020-0791-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021812PMC
February 2020

Author Correction: Disentangling astroglial physiology with a realistic cell model in silico.

Nat Commun 2019 Nov 4;10(1):5062. Epub 2019 Nov 4.

UCL Institute of Neurology, University College London, London, WC1N 3BG, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-019-12712-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828709PMC
November 2019

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

Nat Methods 2019 08 15;16(8):763-770. Epub 2019 Jul 15.

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.

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.
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http://dx.doi.org/10.1038/s41592-019-0471-2DOI Listing
August 2019

Glutamate Imaging Reveals Multiple Sites of Stochastic Release in the CA3 Giant Mossy Fiber Boutons.

Front Cell Neurosci 2019 4;13:243. Epub 2019 Jun 4.

UCL Queen Square Institute of Neurology, University College London, London, United Kingdom.

One of the most studied central synapses which have provided fundamental insights into cellular mechanisms of neural connectivity is the "giant" excitatory connection between hippocampal mossy fibers (MFs) and CA3 pyramidal cells. Its large presynaptic bouton features multiple release sites and is densely packed with thousands of synaptic vesicles, to sustain a highly facilitating "detonator" transmission. However, whether glutamate release sites at this synapse act independently, in a stochastic manner, or rather synchronously, remains poorly understood. This knowledge is critical for a better understanding of mechanisms underpinning presynaptic plasticity and postsynaptic signal integration rules. Here, we use the optical glutamate sensor SF-iGluSnFR and the intracellular Ca indicator Cal-590 to monitor spike-evoked glutamate release and presynaptic calcium entry in MF boutons. Multiplexed imaging reveals that distinct sites in individual MF giant boutons release glutamate in a probabilistic fashion, also showing use-dependent short-term facilitation. The present approach provides novel insights into the basic mechanisms of neurotransmitter release at excitatory synapses.
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http://dx.doi.org/10.3389/fncel.2019.00243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558140PMC
June 2019

Imaging tripartite synapses using super-resolution microscopy.

Methods 2020 03 31;174:81-90. Epub 2019 May 31.

UCL Queen Square Institute of Neurology, University College London, London, United Kingdom. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ymeth.2019.05.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144327PMC
March 2020

Multiplex imaging relates quantal glutamate release to presynaptic Ca homeostasis at multiple synapses in situ.

Nat Commun 2019 03 29;10(1):1414. Epub 2019 Mar 29.

UCL Queen Square Institute of Neurology, University College London, Queen Square, London, WC1N 3BG, UK.

Information processing by brain circuits depends on Ca-dependent, stochastic release of the excitatory neurotransmitter glutamate. Whilst optical glutamate sensors have enabled detection of synaptic discharges, understanding presynaptic machinery requires simultaneous readout of glutamate release and nanomolar presynaptic Ca in situ. Here, we find that the fluorescence lifetime of the red-shifted Ca indicator Cal-590 is Ca-sensitive in the nanomolar range, and employ it in combination with green glutamate sensors to relate quantal neurotransmission to presynaptic Ca kinetics. Multiplexed imaging of individual and multiple synapses in identified axonal circuits reveals that glutamate release efficacy, but not its short-term plasticity, varies with time-dependent fluctuations in presynaptic resting Ca or spike-evoked Ca entry. Within individual presynaptic boutons, we find no nanoscopic co-localisation of evoked presynaptic Ca entry with the prevalent glutamate release site, suggesting loose coupling between the two. The approach enables a better understanding of release machinery at central synapses.
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http://dx.doi.org/10.1038/s41467-019-09216-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441074PMC
March 2019

Polymer microchamber arrays for geometry-controlled drug release: a functional study in human cells of neuronal phenotype.

Biomater Sci 2019 May;7(6):2358-2371

Department of Clinical and Experimental Epilepsy, UCL Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK.

Polyelectrolyte multilayer (PEM) microchambers can provide a versatile cargo delivery system enabling rapid, site-specific drug release on demand. However, experimental evidence for their potential benefits in live human cells is scarce. Equally, practical applications often require substance delivery that is geometrically constrained and highly localized. Here, we establish human-cell biocompatibility and on-demand cargo release properties of the PEM or polylactic acid (PLA)-based microchamber arrays fabricated on a patterned film base. We grow human N2A cells (a neuroblastoma cell line widely used for studies of neurotoxicity) on the surface of the patterned microchamber arrays loaded with either a fluorescent indicator or the ubiquitous excitatory neurotransmitter glutamate. The differentiating human N2A cells show no detrimental effects on viability when growing on either PEM@PLA or PLA-based arrays for up to ten days in vitro. Firstly, we use two-photon (2P) excitation with femtosecond laser pulses to open individual microchambers in a controlled way while monitoring release and diffusion of the fluorescent cargo (rhodamine or FITC fluorescent dye). Secondly, we document the increases in intracellular Ca2+ in local N2A cells in response to the laser-triggered glutamate release from individual microchambers. The functional cell response is site-specific and reproducible on demand and could be replicated by applying glutamate to the cells using a pressurised micropipette. Time-resolved fluorescence imaging confirms the physiological range of the glutamate-evoked intracellular Ca2+ dynamics in the differentiating N2A cells. Our data indicate that the nano-engineering design of the fabricated PEM or PLA-based patterned microchamber arrays could provide a biologically safe and efficient tool for targeted, geometrically constrained drug delivery.
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http://dx.doi.org/10.1039/c8bm01499jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873774PMC
May 2019

Extreme statistics may govern avalanche-type biological reactions: Comment on "Redundancy principle and the role of extreme statistics in molecular and cellular biology" by Z. Schuss, K. Basnayake, D. Holcman.

Phys Life Rev 2019 03 20;28:85-87. Epub 2019 Feb 20.

Queen Square UCL Institute of Neurology, University College London, Queen Square, London WC1N 3BG, United Kingdom.

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http://dx.doi.org/10.1016/j.plrev.2019.02.001DOI Listing
March 2019

A Method to Visualize the Nanoscopic Morphology of Astrocytes In Vitro and In Situ.

Methods Mol Biol 2019 ;1938:69-84

UCL Queen Square Institute of Neurology, University College London, Queen Square, London, UK.

In recent years it has become apparent that astroglia are not only essential players in brain development, homeostasis, and metabolic support but are also important for the formation and regulation of synaptic circuits. Fine astrocytic processes that can be found in the vicinity of synapses undergo considerable structural plasticity associated with age- and use-dependent changes in neural circuitries. However, due to the extraordinary complex, essentially nanoscopic morphology of astroglia, the underlying cellular mechanisms remain poorly understood.Here we detail a super-resolution microscopy approach, based on the single-molecule localisation microscopy (SMLM) technique direct stochastic optical reconstruction microscopy (dSTORM) to visualize astroglial morphology on the nanoscale. This approach enables visualization of key morphological changes that occur in nanoscopic astrocyte processes, whose characteristic size falls below the diffraction limit of conventional optical microscopy.
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http://dx.doi.org/10.1007/978-1-4939-9068-9_5DOI Listing
July 2019

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

J Neurosci 2018 10;38(44):9355-9363

Institut Interdisciplinaire des Neurosciences, Université de Bordeaux, 33077 Bordeaux, France, and

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.
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http://dx.doi.org/10.1523/JNEUROSCI.1664-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706003PMC
October 2018

Differential Nanoscale Topography and Functional Role of GluN2-NMDA Receptor Subtypes at Glutamatergic Synapses.

Neuron 2018 10 27;100(1):106-119.e7. Epub 2018 Sep 27.

Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, 33000 Bordeaux, France; CNRS, IINS UMR 5297, Bordeaux, France. Electronic address:

NMDA receptors (NMDARs) play key roles in the use-dependent adaptation of glutamatergic synapses underpinning memory formation. In the forebrain, these plastic processes involve the varied contributions of GluN2A- and GluN2B-containing NMDARs that have different signaling properties. Although the molecular machinery of synaptic NMDAR trafficking has been under scrutiny, the postsynaptic spatial organization of these two receptor subtypes has remained elusive. Here, we used super-resolution imaging of NMDARs in rat hippocampal synapses to unveil the nanoscale topography of native GluN2A- and GluN2B-NMDARs. Both subtypes were found to be organized in separate nanodomains that vary over the course of development. Furthermore, GluN2A- and GluN2B-NMDAR nanoscale organizations relied on distinct regulatory mechanisms. Strikingly, the selective rearrangement of GluN2A- and GluN2B-NMDARs, with no overall change in NMDAR current amplitude, allowed bi-directional tuning of synaptic LTP. Thus, GluN2A- and GluN2B-NMDAR nanoscale organizations are differentially regulated and seem to involve distinct signaling complexes during synaptic adaptation.
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http://dx.doi.org/10.1016/j.neuron.2018.09.012DOI Listing
October 2018

Disentangling astroglial physiology with a realistic cell model in silico.

Nat Commun 2018 09 3;9(1):3554. Epub 2018 Sep 3.

UCL Institute of Neurology, University College London, London, WC1N 3BG, UK.

Electrically non-excitable astroglia take up neurotransmitters, buffer extracellular K and generate Ca 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 Ca buffers affect Ca waves and why the classical Ca sparks-and-puffs mechanism is theoretically compatible with common readouts of astroglial Ca imaging.
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http://dx.doi.org/10.1038/s41467-018-05896-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120909PMC
September 2018

An optical sensor to monitor dynamics of extracellular glycine.

Authors:
Dmitri A Rusakov

Nat Chem Biol 2018 09;14(9):835-836

UCL Queen Square Institute of Neurology, University College London, Queen Square, London, UK.

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http://dx.doi.org/10.1038/s41589-018-0123-3DOI Listing
September 2018

Astrocytic Atrophy Following Parallels Reduced Ca Activity and Impaired Synaptic Plasticity in the Rat Hippocampus.

Front Mol Neurosci 2018 26;11:215. Epub 2018 Jun 26.

UNN Institute of Neuroscience, N. I. Lobachevsky State University of Nizhny Novgorod, University of Nizhny Novgorod, Nizhny Novgorod, Russia.

Epilepsy is a group of neurological disorders commonly associated with the neuronal malfunction leading to generation of seizures. Recent reports point to a possible contribution of astrocytes into this pathology. We used the lithium-pilocarpine model of (SE) in rats to monitor changes in astrocytes. Experiments were performed in acute hippocampal slices 2-4 weeks after SE induction. Nissl staining revealed significant neurodegeneration in the pyramidal cell layers of hippocampal CA1, CA3 areas, and the hilus, but not in the granular cell layer of the dentate gyrus. A significant increase in the density of astrocytes stained with an astrocyte-specific marker, sulforhodamine 101, was observed in CA1 . Astrocytes in this area were also whole-cell loaded with a morphological tracer, Alexa Fluor 594, for two-photon excitation imaging. Sholl analyses showed no changes in the size of the astrocytic domain or in the number of primary astrocytic branches, but a significant reduction in the number of distal branches that are resolved with diffraction-limited light microscopy (and are thought to contain Ca stores, such as mitochondria and endoplasmic reticulum). The atrophy of astrocytic branches correlated with the reduced size, but not overall frequency of Ca events. The volume tissue fraction of nanoscopic (beyond the diffraction limit) astrocytic leaflets showed no difference between control and SE animals. The results of spatial entropy-complexity spectrum analysis were also consistent with changes in ratio of astrocytic branches vs. leaflets. In addition, we observed uncoupling of astrocytes through the gap-junctions, which was suggested as a mechanism for reduced K buffering. However, no significant difference in time-course of synaptically induced K currents in patch-clamped astrocytes argued against possible alterations in K clearance by astrocytes. The magnitude of long-term-potentiation (LTP) was reduced after SE. Exogenous -serine, a co-agonist of NMDA receptors, has rescued the initial phase of LTP. This suggests that the reduced Ca-dependent release of -serine by astrocytes impairs initiation of synaptic plasticity. However, it does not explain the failure of LTP maintenance which may be responsible for cognitive decline associated with epilepsy.
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http://dx.doi.org/10.3389/fnmol.2018.00215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028739PMC
June 2018

Multiplexed calcium imaging of single-synapse activity and astroglial responses in the intact brain.

Neurosci Lett 2019 01 19;689:26-32. Epub 2018 Jun 19.

UCL Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK. Electronic address:

All-optical registration of neuronal and astrocytic activities within the intact mammalian brain has improved significantly with recent advances in optical sensors and biophotonics. However, relating single-synapse release events and local astroglial responses to sensory stimuli in an intact animal has not hitherto been feasible. Here, we present a multiplexed multiphoton excitation imaging approach for assessing the relationship between presynaptic Ca entry at thalamocortical axonal boutons and perisynaptic astrocytic Ca elevations, induced by whisker stimulation in the barrel cortex of C57BL/6 mice. We find that, unexpectedly, Ca elevations in the perisynaptic astrocytic regions consistently precede local presynaptic Ca signals during spontaneous brain activity associated with anaesthesia. The methods described here can be adapted to a variety of optical sensors and are compatible with experimental designs that might necessitate repeated sampling of single synapses over a longitudinal behavioural paradigm.
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http://dx.doi.org/10.1016/j.neulet.2018.06.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335263PMC
January 2019