Multiphoton Imaging of Neuronal Activity in vivo in Behavioral Paradigms

Transcription

1 Multiphoton Imaging of Neuronal Activity in vivo in Behavioral Paradigms

2 2 Outline Surgical preparation Experimental apparatus Synchronization of behavioral trials with imaging Off axis imaging

3 3 Multiphoton in vivo imaging in awake behaving animals Multiphoton imaging capable of imaging at over 1 mm depths with cellular (and sub-cellular resolution) Deeper imaging with multiphoton microscopes can be achieved with GRIN lenses and cannulas Multiphoton imaging with resonant scanners provide temporal resolution to identify neurons that have fired, allowing observation of firing patterns of groups of neurons Specific brain regions can be observed with multiphoton microscopy in awake behaving animals Surgical procedures for implanting clear observation windows Procedures for implanting cranial restraint, keeps head still while allowing limbs to move Peripherals for conducting conditioning trials during microscope observation Allows reading the brain during controlled behavior

4 4 Surgical Preparation Cranial Window Cranial window necessary in order to reduce scatter and aberations Thinned skull Cranial window Cover larger areas - up to several microns Window can be removed for treatments, e.g. viral infection Yang et al, SFN Goldey et al doi: /nprot

5 5 Experimental apparatus Microscope Multiphoton microscope Raster scanning galvos will need to be run with ROI to get faster frame rates Spiral scanning can achieve faster scanning Resonant scanner 30 frames per second full 512x512 frame Faster speeds, 100s of frames per second, with ROIs

6 6 Peripherals Stimuli Sound Whiskers Olfaction Single lights Patterns on LCD screens Virtual reality screens Response Licking Locomotion Linear treadmills Spherical treadmills

7 7 Synchronization Behavioral trials typically being generated on separate computer Computer running behavioral trials typically masters experiment Many times each trial of a behavioral trial series is of a fixed length, and inter-trial intervals are fixed. Requires imaging system to be able to cue up repetitions of acquisitions of a defined length, with each acquisition triggered by the behavioral computer.

8 8 Synchronization In other cases each trial of a behavioral trial series can be of a variable length Different trial lengths set up a priori Trial length varies during experiment based on animal response or state Requires that image acquisition for each series matches in duration to behavioral trial Behavioral control software needs to provide start and stop trigger Imaging software needs to be able to respond to start and stop triggers

9 An example of a an experiment utilizing multiphoton imaging in an awake animal during behavioral trials 1/21/2015 Bruker Confidential 9 Distinct roles of visual, parietal, and frontal motor cortices in a memoryguided sensorimotor decision Michael J Goard, Gerald Pho, Mriganka Sur Picower Institute for Learning and Memory Massachusetts Institute of Technology

10 10 Distinct roles of visual, parietal, and frontal motor cortices in a memory-guided sensorimotor decision V1, primary visual cortex; PPC, posterior parietal cortex; fmc, frontal motor cortex

11 11 Experimental Setup Bruker Ultima Intravital with moving base, resonant scanner and piezo focus LCD display

12 12 Protocol Imaging calcium with GCaMP6 Behavioral task with visual stimulation and lick response

13 Behavioral training 1/21/2015 Bruker Confidential 13

14 Example movie of neurons firing during behavioral trial 1/21/2015 Bruker Confidential 14

15 Single cells have reliable trial-evoked responses

16 Imaging on multiple focal planes 1/21/

17 Imaging with Prism Perpendicular to Optical Axis PV+ interneurons labeled with a red fluorophor (TD-tomato). The green channel shows GCaMP6 activity. Goldey et al doi: /nprot Michael Goard MIT 1/21/2015 Bruker Confidential 17

18 Imaging Deeper Structures P. Kaifosh, M. Lovett-Barron, G. Turi, T. Reardon, A. Losonczy, Nature Neuroscience 16, (2013 1/21/

19 Behavioral apparatus used for providing stimuli and locomotion measurement while imaging hippocampal neurons P. Kaifosh, M. Lovett-Barron, G. Turi, T. Reardon, A. Losonczy, Nature Neuroscience 16, (2013 1/21/2015 Bruker Confidential 19

20 Calcium signals visualized with GCaMP in hippocampal pyramidal neurons during behavioral training paradigm in awake mouse 1/21/2015 Bruker Confidential 20

21 21 Off Axis Imaging Imaging off the standard vertical microscope axis Access to areas if interest Reduced scatter when imaging perpendicular to cortex surface Brainmaps.org

22 22 Motorized Orbital Nose Piece Motors on objective tilt and nose piece rotation Software reads motor position and calculates how to move microscope X,Y,Z motors together to move objective along optical axis of objective for focusing and in X,Y perpendicular to objective optical axis for sample navigation Collection optics in ONP coupled via fiber optic to detectors, increases collection efficiency while allowing freedom of motion

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37 Copyright Bruker Corporation. All rights reserved.