scholarly journals Three-dimensional Two-Photon Optogenetics and Imaging of Neural Circuits in vivo

2017 ◽  
Author(s):  
Weijian Yang ◽  
Luis Carrillo-Reid ◽  
Yuki Bando ◽  
Darcy S. Peterka ◽  
Rafael Yuste
Keyword(s):  
Visual Cortex ◽  
Neural Activity ◽  
Calcium Imaging ◽  
Pyramidal Neurons ◽  
Neural Circuits ◽  
Three Dimensional ◽  
Two Photon ◽  
Cellular Resolution ◽  
Photon Excitation

We demonstrate a holographic system for simultaneous three-dimensional (3D) two-photon stimulation and imaging of neural activity in the mouse neocortex in vivo with cellular resolution. Dual two-photon excitation paths are implemented with independent 3D targeting for calcium imaging and precision optogenetics. We validate the usefulness of the microscope by photoactivating local pools of interneurons in awake mice visual cortex in 3D, which suppress the nearby pyramidal neurons’ response to visual stimuli.

scholarly journals Dual-color volumetric imaging of neural activity of cortical columns

2018 ◽  
Author(s):  
Shuting Han ◽  
Weijian Yang ◽  
Rafael Yuste
Keyword(s):  
Neural Activity ◽  
High Speed ◽  
Neural Circuits ◽  
Emergent Properties ◽  
Spatiotemporal Resolution ◽  
Population Activity ◽  
Volumetric Imaging ◽  
Two Photon ◽  
Dual Color

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.

scholarly journals All-optical imaging and patterned stimulation with a one-photon endoscope

2021 ◽  
Author(s):  
Jinyong Zhang ◽  
Ryan N Hughes ◽  
Namsoo Kim ◽  
Isabella P Fallon ◽  
Konstantin I bakhurin ◽  
...  
Keyword(s):  
Neural Activity ◽  
Calcium Imaging ◽  
Neural Circuit ◽  
Integrated System ◽  
Striatal Neurons ◽  
Indirect Pathway ◽  
Cellular Resolution ◽  
All Optical ◽  
Freely Moving

While in vivo calcium imaging makes it possible to record activity in defined neuronal populations with cellular resolution, optogenetics allows selective manipulation of neural activity. Recently, these two tools have been combined to stimulate and record neural activity at the same time, but current approaches often rely on two-photon microscopes that are difficult to use in freely moving animals. To address these limitations, we have developed a new integrated system combining a one-photon endoscope and a digital micromirror device for simultaneous calcium imaging and precise optogenetic photo-stimulation with near cellular resolution (Miniscope with All-optical Patterned Stimulation and Imaging, MAPSI). Using this highly portable system in freely moving mice, we were able to image striatal neurons from either the direct pathway or the indirect pathway while simultaneously activating any neuron of choice in the field of view, or to synthesize arbitrary spatiotemporal patterns of photo-stimulation. We could also select neurons based on their relationship with behavior and recreate the behavior by mimicking the natural neural activity with photo-stimulation. MAPSI thus provides a powerful tool for interrogation of neural circuit function in freely moving animals.

scholarly journals Long-term Transverse Imaging of the Hippocampus with Glass Microperiscopes

2021 ◽  
Author(s):  
William T Redman ◽  
Nora S Wolcott ◽  
Luca Montelisciani ◽  
Gabriel Luna ◽  
Tyler D Marks ◽  
...  
Keyword(s):  
Cognitive Processes ◽  
Calcium Imaging ◽  
Pyramidal Neurons ◽  
Spatial Information ◽  
Anatomical Distribution ◽  
Two Photon ◽  
Information Finding ◽  
Apical Dendrites

The hippocampus consists of a stereotyped neuronal circuit repeated along the septal-temporal axis. This transverse circuit contains distinct subfields with stereotyped connectivity that support crucial cognitive processes, including episodic and spatial memory. However, comprehensive measurements across the transverse hippocampal circuit in vivo are intractable with existing techniques. Here, we developed an approach for two-photon imaging of the transverse hippocampal plane in awake mice via implanted glass microperiscopes, allowing optical access to the major hippocampal subfields and to the dendritic arbor of pyramidal neurons. Using this approach, we tracked dendritic morphological dynamics on CA1 apical dendrites and characterized spine turnover. We then used calcium imaging to quantify the prevalence of place and speed cells across subfields. Finally, we measured the anatomical distribution of spatial information, finding a non-uniform distribution of spatial selectivity along the DG-to-CA1 axis. This approach extends the existing toolbox for structural and functional measurements of hippocampal circuitry.

Three-Dimensional Mapping of Unitary Synaptic Connections by Two-Photon Macro Photolysis of Caged Glutamate

2008 ◽  
Vol 99 (3) ◽  
pp. 1535-1544 ◽  
Author(s):  
Masanori Matsuzaki ◽  
Graham C. R. Ellis-Davies ◽  
Haruo Kasai
Keyword(s):  
Visual Cortex ◽  
Single Cell ◽  
Pyramidal Neurons ◽  
Three Dimensional ◽  
Core Region ◽  
Synaptic Connectivity ◽  
Synaptic Connections ◽  
Two Photon ◽  
Layer 2 ◽  
Caged Glutamate

To understand the precise microarchitecture of the cortical circuitry, it is crucial to know the distribution of synaptic connections and their synaptic strengths at the level of a single cell, rather than a group of cells. Here, we describe a new application of two-photon photolysis of caged glutamate that enabled us to induce an action potential in only a small number (about five) of pyramidal neurons by increasing the volume of two-photon excitation by reducing the effective numerical aperture of the objective. We performed whole cell patch-clamp recordings from layer 2/3 pyramidal neurons in the rat visual cortex and stimulated many neurons in a large three-dimensional space (∼600 × 600 × 100 μm) including neurons in layers 2/3 and 4 using this new technique. We mapped the density and amplitude of unitary excitatory postsynaptic currents and found that the basic microarchitecture of excitatory synaptic connections consists of two regions: a columnar, dense core region with a radius of 150 μm and an outer, sparse region. The dense core region includes the majority of strong synaptic connections in layer 2/3. Our results reveal the columnar organization of synaptic connectivity in the rat visual cortex, where functional columns have not been clearly demonstrated. Thus this technique will be a uniquely powerful tool for quantifying synaptic connectivity and manipulating neural activity at the single-cell level.

scholarly journals Navigating the translational roadblock: Towards highly specific and effective all-optical interrogations of neural circuits

2020 ◽  
Author(s):  
Ting Fu ◽  
Isabelle Arnoux ◽  
Jan Döring ◽  
Hirofumi Watari ◽  
Ignas Stasevicius ◽  
...  
Keyword(s):  
Visual Cortex ◽  
Calcium Imaging ◽  
Cortical Neurons ◽  
Activity Patterns ◽  
Detection Algorithm ◽  
Two Photon ◽  
All Optical ◽  
Laser Sources ◽  
Mouse Visual Cortex

AbstractTwo-photon (2-P) all-optical approaches combine in vivo 2-P calcium imaging and 2-P optogenetic modulations and have the potential to build a framework for network-based therapies, e.g. for rebalancing maladaptive activity patterns in preclinical models of neurological disorders. Here, our goal was to tailor these approaches for this purpose: Firstly, we combined in vivo juxtacellular recordings and GCaMP6f-based 2-P calcium imaging in layer II/III of mouse visual cortex to tune our detection algorithm towards a 100 % specific identification of AP-related calcium transients. False-positive-free detection was achieved at a sensitivity of approximately 73 %. To further increase specificity, secondly, we minimized photostimulation artifacts as a potential source for false-positives by using extended-wavelength-spectrum laser sources for optogenetic stimulation of the excitatory opsin C1V1. We achieved artifact-free all-optical experiments performing photostimulations at 1100 nm or higher and simultaneous calcium imaging at 920 nm in mouse visual cortex in vivo. Thirdly, we determined the spectral range for maximizing efficacy of optogenetic control by performing 2-P photostimulations of individual neurons with wavelengths up to 1300 nm. The rate of evoked transients in GCaMP6f/C1V1-co-expressing cortical neurons peaked already at 1100 nm. By refining spike detection and defining 1100 nm as the optimal wavelength for artifact-free and effective stimulations of C1V1 in GCaMP-based all-optical interrogations, we increased the translational value of these approaches, e.g. for the use in preclinical applications of network-based therapies.One Sentence SummaryWe maximize translational relevance of 2-P all-optical physiology by increasing specificity, minimizing artifacts and optimizing stimulation efficacy.

scholarly journals Two-photon calcium imaging of the medial prefrontal cortex and hippocampus without cortical invasion

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Masashi Kondo ◽  
Kenta Kobayashi ◽  
Masamichi Ohkura ◽  
Junichi Nakai ◽  
Masanori Matsuzaki
Keyword(s):  
Prefrontal Cortex ◽  
Medial Prefrontal Cortex ◽  
Neural Activity ◽  
Calcium Imaging ◽  
Cortical Surface ◽  
Intact Mouse ◽  
Two Photon ◽  
Ca1 Region ◽  
Hippocampal Ca1 Region

In vivo two-photon calcium imaging currently allows us to observe the activity of multiple neurons up to ~900 µm below the cortical surface without cortical invasion. However, many important brain areas are located deeper than this. Here, we used an 1100 nm laser that underfilled the back aperture of the objective together with red genetically encoded calcium indicators to establish two-photon calcium imaging of the intact mouse brain and detect neural activity up to 1200 μm from the cortical surface. This imaging was obtained from the medial prefrontal cortex (the prelimbic area) and the hippocampal CA1 region. We found that neural activity before water delivery repeated at a constant interval was higher in the prelimbic area than in layer 2/3 of the secondary motor area. Reducing the invasiveness of imaging is an important strategy to reveal the intact brain processes active in cognition and memory.

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