Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue

Tønnesen, Jan; Inavalli, V. V. G. Krishna; Nägerl, U. Valentin

doi:10.1016/j.cell.2018.02.007

Cited by 245 publications

(310 citation statements)

References 53 publications

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“…For example, it enables the study of neuronal plasticity in deeper layers of the brain cortex, the dynamic nanoscale organization of complex structures such as glomeruli in kidney or the structural reorganization of chromatin during cell differentiation in tissue. Furthermore, it could be combined with the SUSHI labeling technique [42] which, by labeling the extracellular space, enables the investigation of cellular relationships and morphology in living tissue. Our technology can extend the benefits of this labelling technique deep inside the living mouse brain.…”

Section: Discussionmentioning

confidence: 99%

3D super-resolution deep-tissue imaging in living mice

Velasco

Zhang

Antonello

et al. 2019

Preprint

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Stimulated emission depletion (STED) microscopy enables the three-dimensional (3D) visualization of dynamic nanoscale structures in living cells, offering unique insights into their organization. However, 3D-STED imaging deep inside biological tissue is obstructed by optical aberrations and light scattering.We present a STED system that overcomes these challenges. Through the combination of 2-photon excitation, adaptive optics, far-red emitting organic dyes, and a long-working distance water-immersion objective lens, our system achieves aberration-corrected 3D super-resolution imaging, which we demonstrate 164 µm deep in fixed mouse brain tissue and 76 µm deep in the brain of a living mouse.

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Section: Discussionmentioning

confidence: 99%

3D super-resolution deep-tissue imaging in living mice

Velasco

Zhang

Antonello

et al. 2019

Preprint

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“…STED microscopy enables monitoring nanoscopic cellular compartments in live preparations, far beyond the optical diffraction limit (Tonnesen et al, 2018;Tonnesen et al, 2014). We therefore turned to two-colour STED imaging, combined with electrophysiology, in organotypic hippocampal slices (Panatier et al, 2014) (Methods, Supplementary Figure 2A; we used acute slices in all other methods).…”

Section: Sted Imaging Reveals Decreased Pap Presence Near Spines Uponmentioning

confidence: 99%

LTP induction boosts glutamate spillover by driving withdrawal of astroglia

Henneberger

Bard

Panatier

et al. 2018

Preprint

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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 LTPtriggered PAP withdrawal involves NKCC1 transporters and the actin-controlling protein cofilin but does not depend on major Ca 2+ -dependent cascades in astrocytes. We have therefore uncovered a mechanism by which synaptic potentiation could alter signal handling by multiple nearby connections.

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“…6a-c). Clearly, such accuracy in the z direction appears more problematic: it will require specific optics that would enable registration of optical aberration (such as astigmatism) against the z coordinate 40 or otherwise a specifically modified PSF shape 41 .…”

Section: Nanoscopic Localisation Of Presynaptic Glutamate Release Andmentioning

confidence: 99%

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

Jensen

Zheng

Cole

et al. 2018

Preprint

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Information processing by brain circuits depends on Ca 2+-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 Ca 2+ and glutamate release. Here, we find that the red-shifted Ca 2+indicator Cal-590 shows Ca 2+-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 Ca 2+ concentration, in multiple synapses in an identified neural circuit. At the level of individual presynaptic boutons, we use multiexposure and stochastic reconstruction procedures to reveal nanoscopic co-localisation of presynaptic Ca 2+ 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. Stochastic, Ca2+ -dependent release of the excitatory neurotransmitter glutamate by individual synapses is what underpins information handling and storage by neural networks. However, in many central circuits glutamate release occurs with a low probability and a high degree of heterogeneity among synapses 1, 2 . Therefore, methods to probe presynaptic function in an intact brain aim to reliably detect presynaptic action potentials, record the presynaptic Ca 2+ dynamics, and register release of individual glutamate quanta with high temporal resolution and broad dynamic range. The optical quantal analysis method went some way toward this goal, by providing quantification of release probability at individual synapses in brain slices 3,4 . In parallel, advances in the imaging techniques suited to monitor membrane retrieval at presynaptic terminals have enabled detection of synaptic vesicle exocytosis in cultured neurons 5 . Recently developed optical glutamate sensors 6,7 have drastically expanded the sensitivity and the dynamic range of glutamate discharge detection in organised brain tissue 8 . However, such methods on their own cannot relate neurotransmitter release to presynaptic Ca 2+ dynamics, which . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/336891 doi: bioRxiv preprint first posted online Jun. 2, 2018; 2 is the key to understanding presynaptic release machinery, as demonstrated in elegant studies of giant synapses permitting direct electrophysiological probing 9-11 . In parallel, we have developed a glutamate sensor variant SF-iGluSnFR.A184S whose kinetic properties permit reliable registration of individual quanta of released neurotransmitter at multiple gl...

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Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue

Cited by 245 publications

References 53 publications

3D super-resolution deep-tissue imaging in living mice

3D super-resolution deep-tissue imaging in living mice

LTP induction boosts glutamate spillover by driving withdrawal of astroglia

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

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Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue

Cited by 245 publications

References 53 publications

3D super-resolution deep-tissue imaging in living mice

3D super-resolution deep-tissue imaging in living mice

LTP induction boosts glutamate spillover by driving withdrawal of astroglia

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

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