Fast 3D Imaging of Spine, Dendritic, and Neuronal Assemblies in Behaving Animals

Szalay, Gergely; Judák, Linda; Katona, Gergely; Ócsai, Katalin; Juhász, Gábor; Veress, Máté; Szadai, Zoltán; Fehér, András; Tompa, Tamás; Chiovini, Balázs; Maák, Pál; Rózsa, Balázs

doi:10.1016/j.neuron.2016.10.002

Cited by 108 publications

(106 citation statements)

References 61 publications

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“…Point-scanning based mesoscopes have achieved pixel rates of ~2×10 7 /s over 0.6 × 0.6 mm FOVs but the requirement to translate the beam long distances limits pixel rates over large FOVs (4.4 × 4.2 mm) to 5.6×10 6 /s. Acousto-optical steering allows fast 2P random-access imaging 38 For precisely targeted single-cell stimulation, 2P optics are essential, but for wide-area optogenetic stimulation, 1P optics are preferable, as follows: 2P optogenetic stimulation requires timeaverage optical powers of 20 -80 mW/cell, [2][3][4] . Maximal safe steady-state 2P optical power into intact brain tissue is ~200 mW 39 , limiting simultaneous 2P stimulation to at most a few tens of neurons at a time.…”

Section: Discussionmentioning

confidence: 99%

Wide-area all-optical neurophysiology in acute brain slices

Farhi

Parot

Grama

et al. 2018

Preprint

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Optical tools for simultaneous perturbation and measurement of neural activity open the possibility of mapping neural function over wide areas of brain tissue. However, spectral overlap of actuators and reporters presents a challenge for their simultaneous use, and optical scattering and out-of-focus fluorescence in tissue degrade resolution. To minimize optical crosstalk, we combined an optimized variant (eTsChR) of the most blue-shifted channelrhodopsin reported to-date with a nuclear-localized red-shifted Ca 2+ indicator, H2B-jRGECO1a. To perform wide-area optically sectioned imaging in tissue, we designed a structured illumination technique that uses Hadamard matrices to encode spatial information. By combining these molecular and optical approaches we made wide-area maps, spanning cortex and striatum, of the effects of antiepileptic drugs on neural excitability and on the effects of AMPA and NMDA receptor blockers on functional connectivity. Together, these tools provide a powerful capability for wide-area mapping of neuronal excitability and functional connectivity in acute brain slices. ResultsA spectrally orthogonal Ca 2+ sensor and channelrhodopsin for 1-photon AON AON requires a spectrally orthogonal optogenetic actuator and activity reporter ( Fig. 1a). Examination of channelrhodopsin action spectra and Ca 2+ reporter excitation spectra suggested that the best approach for 1-photon AON was to use a blue-shifted channelrhodopsin and a red-shifted genetically encoded Ca 2+ indicator (RGECI). We thus set out to identify protein pairs suitable for this purpose.We began by comparing the single action potential responses of RGECIs in cultured neurons. jRGECO1a was the most sensitive (∆F/F = 54 ± 10%, n = ~120 neurons), followed by R-CaMP2 and jRCaMP1a, consistent with previous reports (Supplementary Fig. 1a-b, Supplementary Table 1) 15 . R-CaMP2 had the fastest kinetics (τon = 26 ± 10 ms, τoff = 270 ± 20 ms, n = ~120 neurons), followed by jRGECO1a (τon = 47 ± 1 ms, τoff = 440 ± 40 ms, n = ~120 neurons) and jRCAMP1a ( Supplementary Fig. 1ab, Supplementary Table 1). In HEK293T cells, under basal Ca 2+ conditions, jRGECO1a had the longest photobleaching time constant (τbleach = 81 ± 5 s, I561 = 44 W/cm 2 , n = 9 cells), followed by R-CaMP2 and jRCaMP1a ( Supplementary Table 1).Under typical imaging conditions (I561 = 0.1 W/cm 2 ), photobleaching of jRGECO1a was thus < 10% during 1 hr of continuous imaging. While photobleaching is often a concern for 1P imaging, these results established that this effect was minor for wide-area imaging of jRGECO1a. We selected jRGECO1a for its superior sensitivity and photostability.mApple-based fluorescent sensors, including jRGECO1a, are known to undergo photoswitching under blue light illumination 14,16 . We thus sought a blue-shifted channelrhodopsin that could drive spikes in jRGECO1a-expressing neurons at blue intensities low enough to avoid optical crosstalk. TsChR is the most blue-shifted published ChR (Fig. 1a), but was initially reported to produce only ~40% as much ph...

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Section: Discussionmentioning

confidence: 99%

Wide-area all-optical neurophysiology in acute brain slices

Farhi

Parot

Grama

et al. 2018

Preprint

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“…Because their settling times are on the order of a few milliseconds, these technologies are not fast enough to capture relevant in vivo depth-imaging with high z-resolution, limiting their use to in neural physiological imaging only a few defined z planes. Other systems use a high-speed axial scanner such as an acoustic-optical scanner [15]or an ultrasound-driven lens [16], but they can suffer from focusing aberrations. Another solution to imaging in depth is the technique of implanting microprisms in the brain to image orthogonal to the skull surface while still using a traditional xy scanner [17,18].…”

Section: Introductionmentioning

confidence: 99%

Deformable mirror-based two-photon microscopy for axial mammalian brain imaging

Peinado¹,

Bendek

Yokoyama³

et al. 2019

Preprint

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This work presents the design and implementation of an enhanced version of a traditional two-photon (2P) microscope with the addition of high-speed axial scanning for live mammalian brain imaging. Our implementation utilizes a deformable mirror (DM) that can rapidly apply different defocus shapes to manipulate the laser beam divergence and consequently control the axial position of the beam focus in the sample. We provide a mathematical model describing the DM curvature, then experimentally characterize the radius of curvature as well as the Zernike terms of the DM surface for a given set of defocuses. A description of the optical setup of the 2P microscope is detailed. We conduct a thorough calibration of the system, determining the point spread function, the total scanning range, the axial step size, and the intensity curvature as a function of depth. Finally, the instrument is used for imaging different neurobiological samples, including fixed brain slices and in vivo mouse cerebral cortex.

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“…The major advancement in measuring Ca 2+ signals was the invention and the application of two-photon microscopy in the nervous system (Denk et al, 1990; Yuste and Denk, 1995). Over many years, particularly with the help of the continuous development of Ca 2+ indicators, two-photon Ca 2+ imaging has become widely used for detecting neural activities on multiple scales ranging from networks to single synapses in both anesthetized and behaving animals (Stosiek et al, 2003; Chen et al, 2011, 2013; Nadella et al, 2016; Szalay et al, 2016). Another commonly used approach for in vivo brain Ca 2+ imaging is based on the use of charged coupled detector/complementary metal-oxide-semiconductor-based cameras, which are particularly useful for recording large-field Ca 2+ dynamics in the superficial cortical layers (Berger et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Locomotion-Related Population Cortical Ca2+ Transients in Freely Behaving Mice

Zhang

Yao

et al. 2017

Front. Neural Circuits

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Locomotion involves complex neural activity throughout different cortical and subcortical networks. The primary motor cortex (M1) receives a variety of projections from different brain regions and is responsible for executing movements. The primary visual cortex (V1) receives external visual stimuli and plays an important role in guiding locomotion. Understanding how exactly the M1 and the V1 are involved in locomotion requires recording the neural activities in these areas in freely moving animals. Here, we used an optical fiber-based method for the real-time monitoring of neuronal population activities in freely moving mice. We combined the bulk loading of a synthetic Ca2+ indicator and the optical fiber-based Ca2+ recordings of neuronal activities. An optical fiber 200 μm in diameter can detect the coherent activity of a subpopulation of neurons. In layer 5 of the M1 and V1, we showed that population Ca2+ transients reliably occurred preceding the impending locomotion. Interestingly, the M1 Ca2+ transients started ~100 ms earlier than that in V1. Furthermore, the population Ca2+ transients were robustly correlated with head movements. Thus, our work provides a simple but efficient approach for monitoring the cortical Ca2+ activity of a local cluster of neurons during locomotion in freely moving animals.

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Fast 3D Imaging of Spine, Dendritic, and Neuronal Assemblies in Behaving Animals

Cited by 108 publications

References 61 publications

Wide-area all-optical neurophysiology in acute brain slices

Wide-area all-optical neurophysiology in acute brain slices

Deformable mirror-based two-photon microscopy for axial mammalian brain imaging

Locomotion-Related Population Cortical Ca2+ Transients in Freely Behaving Mice

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