Two-photon multiplane imaging of neural circuits (Conference Presentation)

To capture the emergent properties of neural circuits, high-speed volumetric imaging of neural activity at cellular resolution is desirable. But while conventional two-photon calcium imaging is a powerful tool to study population activity in vivo, it is restrained to two-dimensional planes. Expanding it to 3D while maintaining high spatiotemporal resolution appears necessary. Here, we developed a two-photon microscope with dual-color laser excitation that can image neural activity in a 3D volume. We imaged the neuronal activity of primary visual cortex from awake mice, spanning from L2 to L5 with 10 planes, at a rate of 10 vol/sec, and demonstrated volumetric imaging of L1 long-range PFC projections and L2/3 somatas. Using this method, we map visually-evoked neuronal ensembles in 3D, finding a lack of columnar structure in orientation responses and revealing functional correlations between cortical layers which differ from trial to trial and are missed in sequential imaging. We also reveal functional interactions between presynaptic L1 axons and postsynaptic L2/3 neurons. Volumetric two-photon imaging appears an ideal method for functional connectomics of neural circuits.

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All-optical interrogation of neural circuits in behaving mice

10.1101/2021.06.29.450430 ◽

2021 ◽

Author(s):

Lloyd E. Russell ◽

Henry W.P. Dalgleish ◽

Rebecca Nutbrown ◽

Oliver Gauld ◽

Dustin Herrmann ◽

...

Keyword(s):

Neural Circuits ◽

Neural Circuit ◽

Barrel Cortex ◽

Brain Area ◽

Imaging Data ◽

Temporal Precision ◽

Two Photon ◽

All Optical ◽

The Impact

Recent advances combining two-photon calcium imaging and two-photon optogenetics with digital holography now allow us to read and write neural activity in vivo at cellular resolution with millisecond temporal precision. Such 'all-optical' techniques enable experimenters to probe the impact of functionally defined neurons on neural circuit function and behavioural output with new levels of precision. This protocol describes the experimental strategy and workflow for successful completion of typical all-optical interrogation experiments in awake, behaving head-fixed mice. We describe modular procedures for the setup and calibration of an all-optical system, the preparation of an indicator and opsin-expressing and task-performing animal, the characterization of functional and photostimulation responses and the design and implementation of an all-optical experiment. We discuss optimizations for efficiently selecting and targeting neuronal ensembles for photostimulation sequences, as well as generating photostimulation response maps from the imaging data that can be used to examine the impact of photostimulation on the local circuit. We demonstrate the utility of this strategy using all-optical experiments in three different brain areas - barrel cortex, visual cortex and hippocampus - using different experimental setups. This approach can in principle be adapted to any brain area for all-optical interrogation experiments to probe functional connectivity in neural circuits and for investigating the relationship between neural circuit activity and behaviour.

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Motor Learning Drives Dynamic Patterns of Intermittent Myelination on Behaviorally-Activated Axons

10.1101/2021.10.13.464319 ◽

2021 ◽

Author(s):

Clara M. Bacmeister ◽

Rongchen Huang ◽

Michael A. Thornton ◽

Lauren Conant ◽

Anthony R. Chavez ◽

...

Keyword(s):

Motor Learning ◽

Computational Modeling ◽

Learning And Memory ◽

Neuronal Activity ◽

Neural Circuits ◽

Cortical Layers ◽

Nodes Of Ranvier ◽

Two Photon ◽

Myelin Sheaths

Myelin plasticity occurs when newly-formed and pre-existing oligodendrocytes remodel existing myelination. Recent studies show these processes occur in response to changes in neuronal activity and are required for learning and memory. However, the link between behaviorally-relevant neuronal activity and circuit-specific changes in myelination remains unknown. Using longitudinal, in vivo two-photon imaging and targeted labeling of behaviorally-activated neurons, we explore how the pattern of intermittent myelination is altered on individual cortical axons during learning of a dexterous reach task. We show that learning-induced plasticity is targeted to behaviorally-activated axons and occurs in a staged response across cortical layers. During learning, myelin sheaths retract, lengthening nodes of Ranvier. Following learning, addition of new sheaths increases the number of continuous stretches of myelination. Computational modeling suggests these changes initially slow and subsequently increase conduction speed. Thus, behaviorally-activated, circuit-specific changes to myelination may fundamentally alter how information is transferred in neural circuits during learning.

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Two-photon photostimulation and imaging of neural circuits

Nature Methods ◽

10.1038/nmeth1105 ◽

2007 ◽

Vol 4 (11) ◽

pp. 943-950 ◽

Cited By ~ 165

Author(s):

Volodymyr Nikolenko ◽

Kira E Poskanzer ◽

Rafael Yuste

Keyword(s):

Neural Circuits ◽

Two Photon

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Multiplexed temporally focused light shaping through a GRIN lens for precise in-depth optogenetic photostimulation

10.1101/515908 ◽

2019 ◽

Author(s):

Nicolò Accanto ◽

I-Wen Chen ◽

Emiliano Ronzitti ◽

Clément Molinier ◽

Christophe Tourain ◽

...

Keyword(s):

Neural Circuits ◽

Three Dimensions ◽

Optical Methods ◽

Optical Investigation ◽

Two Photon ◽

Grin Lens ◽

All Optical ◽

Living Tissues ◽

And Control

AbstractIn the past 10 years, the use of light has become irreplaceable for the optogenetic study and control of neurons and neural circuits. Optical techniques are however limited by scattering and can only see through a depth of few hundreds µm in living tissues. GRIN lens based micro-endoscopes represent a powerful solution to reach deeper regions. In this work we demonstrate that cutting edge optical methods for the precise photostimulation of multiple neurons in three dimensions can be performed through a GRIN lens. By spatio-temporally shaping a laser beam in the two-photon regime we project several tens of targets, spatially confined to the size of a single cell, in a volume of 150×150×400 μm3. We then apply such concept to the optogenetic stimulation of multiple neurons simultaneously in vivo in mice. Our work paves the way for an all-optical investigation of neural circuits at previously unattainable depths.

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