Wide field-of-view, multi-region two-photon imaging of neuronal activity in the mammalian brain

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Abstract

Two-photon calcium imaging provides an optical readout of neuronal activity in populations of neurons with subcellular resolution. However, conventional two-photon imaging systems are limited in their field of view to ~1 mm ², precluding the visualization of multiple cortical areas simultaneously. Here, we demonstrate a two-photon microscope with an expanded field of view (>9.5 mm ²) for rapidly reconfigurable simultaneous scanning of widely separated populations of neurons. We custom designed and assembled an optimized scan engine, objective, and two independently positionable, temporally multiplexed excitation pathways. We used this new microscope to measure activity correlations between two cortical visual areas in mice during visual processing.

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Most cited references 26

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Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance.

Linda Madisen, Aleena Garner, Daisuke Shimaoka … (2015)

An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA-dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity.

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Imaging large-scale neural activity with cellular resolution in awake, mobile mice.

Daniel A Dombeck, Anton N Khabbaz, Forrest Collman … (2007)

We report a technique for two-photon fluorescence imaging with cellular resolution in awake, behaving mice with minimal motion artifact. The apparatus combines an upright, table-mounted two-photon microscope with a spherical treadmill consisting of a large, air-supported Styrofoam ball. Mice, with implanted cranial windows, are head restrained under the objective while their limbs rest on the ball's upper surface. Following adaptation to head restraint, mice maneuver on the spherical treadmill as their heads remain motionless. Image sequences demonstrate that running-associated brain motion is limited to approximately 2-5 microm. In addition, motion is predominantly in the focal plane, with little out-of-plane motion, making the application of a custom-designed Hidden-Markov-Model-based motion correction algorithm useful for postprocessing. Behaviorally correlated calcium transients from large neuronal and astrocytic populations were routinely measured, with an estimated motion-induced false positive error rate of <5%.

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Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex.

Kenichi Ohki, Sooyoung Chung, Yeang Ch'ng … (2005)

Neurons in the cerebral cortex are organized into anatomical columns, with ensembles of cells arranged from the surface to the white matter. Within a column, neurons often share functional properties, such as selectivity for stimulus orientation; columns with distinct properties, such as different preferred orientations, tile the cortical surface in orderly patterns. This functional architecture was discovered with the relatively sparse sampling of microelectrode recordings. Optical imaging of membrane voltage or metabolic activity elucidated the overall geometry of functional maps, but is averaged over many cells (resolution >100 microm). Consequently, the purity of functional domains and the precision of the borders between them could not be resolved. Here, we labelled thousands of neurons of the visual cortex with a calcium-sensitive indicator in vivo. We then imaged the activity of neuronal populations at single-cell resolution with two-photon microscopy up to a depth of 400 microm. In rat primary visual cortex, neurons had robust orientation selectivity but there was no discernible local structure; neighbouring neurons often responded to different orientations. In area 18 of cat visual cortex, functional maps were organized at a fine scale. Neurons with opposite preferences for stimulus direction were segregated with extraordinary spatial precision in three dimensions, with columnar borders one to two cells wide. These results indicate that cortical maps can be built with single-cell precision.

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Author and article information

Journal

Journal ID (nlm-journal-id): 9604648

Journal ID (pubmed-jr-id): 20305

Journal ID (nlm-ta): Nat Biotechnol

Journal ID (iso-abbrev): Nat. Biotechnol.

Title: Nature biotechnology

ISSN (Print): 1087-0156

ISSN (Electronic): 1546-1696

Publication date Nihms-submitted: 14 May 2016

Publication date (Electronic): 27 June 2016

Publication date (Print): August 2016

Publication date PMC-release: 27 December 2016

Volume: 34

Issue: 8

Pages: 857-862

Affiliations

[1 ]Neuroscience Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599

[2 ]Carolina Institute for Developmental Disabilities, University of North Carolina School of Medicine, Chapel Hill, NC 27599

[3 ]Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599

[4 ]Neurobiology Curriculum, University of North Carolina School of Medicine, Chapel Hill, NC 27599

[5 ]Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599

[6 ]Department of Electrical and Computer Engineering, North Carolina State University, University of North Carolina School of Medicine, Chapel Hill, NC 27599

Author notes

Correspondence should be addressed to: slab@ 123456unc.edu (S.L.S.)

Article

Manuscript ID: NIHMS785666

DOI: 10.1038/nbt.3594

PMC ID: 4980167

PubMed ID: 27347754

SO-VID: 8b0fc272-d6bc-4e36-ba10-122c1f1fea4c

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Wide field-of-view, multi-region two-photon imaging of neuronal activity in the mammalian brain

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Genome Engineering using CRISPR

Most cited references 26

Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance.

Imaging large-scale neural activity with cellular resolution in awake, mobile mice.

Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex.

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