Biology subjects

Rusakov, D. A.

Publications and source records attributed to Rusakov, D. A..

4 recordsLinked to original sources

bioRxiv2018

Astroglia withdraw from potentiated synapses boosting inter-synaptic cross-talk

Extrasynaptic actions of glutamate are limited by high-affinity transporters on perisynaptic astroglial processes (PAPs), which helps to maintain point-to-point transmission in excitatory circuits. Memory formation in the brain is associated with synaptic remodelling, but how this remodelling 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 localisation reveal that LTP induction thus prompts a spatial retreat of glial 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 Ca2+-dependent cascades in astrocytes. We have therefore uncovered a mechanism by which synaptic potentiation could alter signal handling by multiple nearby connections.

neuroscience↗

bioRxiv2018

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

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 Ca2+ entry at thalamocortical axonal boutons and perisynaptic astrocytic Ca2+ elevations, induced by whisker stimulation in the barrel cortex of C57BL/6 mice. We find that, unexpectedly, Ca2+ elevations in the perisynaptic astrocytic regions consistently precede local presynaptic Ca2+ 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.\n\nHighlightsO_LIWe applied multiplexed multiphoton imaging to optically assess activity of individual synapses and the surrounding astroglia in the intact brain\nC_LIO_LIPerisynaptic astrocytic regions display localiased, context-dependent Ca2+ elevations in vivo\nC_LIO_LISuch elevations may precede spontaneous Ca2+ entry at presynaptic boutons\nC_LIO_LIThis method paves the way for optical investigation of glutamate release probability in vivo\nC_LI

neuroscience↗

bioRxiv2018

Multiplex imaging of quantal glutamate release and presynaptic Ca2+ at multiple synapses in situ

Information processing by brain circuits depends on Ca2+-dependent, stochastic release of the excitatory neurotransmitter glutamate. Recently developed optical sensors have enabled detection of evoked and spontaneous release at common glutamatergic synapses. However, monitoring synaptic release probability, its use-dependent changes, and its underpinning presynaptic machinery in situ requires concurrent, intensity-independent readout of presynaptic Ca2+ and glutamate release. Here, we find that the red-shifted Ca2+ indicator Cal-590 shows Ca2+-sensitive fluorescence lifetime, and employ it in combination with the novel green glutamate sensor SF-iGluSnFR variant to document quantal release of glutamate together with presynaptic Ca2+ concentration, in multiple synapses in an identified neural circuit. At the level of individual presynaptic boutons, we use multi-exposure and stochastic reconstruction procedures to reveal nanoscopic co-localisation of presynaptic Ca2+ entry and glutamate release, a fundamental unknown in modern neurobiology. This approach opens a new horizon in the quest to understand release machinery of central synapses.

neuroscience↗

bioRxiv2018

A genetically encoded fluorescent sensor for in vivo imaging of GABA

Current techniques for monitoring GABA, the primary inhibitory neurotransmitter in vertebrates, cannot follow ephemeral transients in intact neural circuits. We applied the design principles used to create iGluSnFR, a fluorescent reporter of synaptic glutamate, to develop a GABA sensor using a protein derived from a previously unsequenced Pseudomonas fluorescens strain. Structure-guided mutagenesis and library screening led to a usable iGABASnFR ({Delta}F/Fmax ~ 2.5, Kd ~ 9 M, good specificity, adequate kinetics). iGABASnFR is genetically encoded, detects single action potential-evoked GABA release events in culture, and produces readily detectable fluorescence increases in vivo in mice and zebrafish. iGABASnFR enabled tracking of: (1) mitochondrial GABA content and its modulation by an anticonvulsant; (2) swimming-evoked GABAergic transmission in zebrafish cerebellum; (3) GABA release events during inter-ictal spikes and seizures in awake mice; and (4) GABAergic tone decreases during isoflurane anesthesia. iGABASnFR will permit high spatiotemporal resolution of GABA signaling in intact preparations.

neuroscience↗