Optical Neuroimaging: Investigating Plasticity Multi-Modal Neuroimaging Program Presenter: Santresda Johnson B.A., M.S. Neuropsychology, Howard University Optical Imaging Mentor: Dr. Justin Crowley Ph.D. Biology Department, Carnegie Mellon University

Santresda Johnson B.A., M.S. Santresda Johnson B.A., M.S. Neuropsychology Doctoral Candidate, at Howard University Neuropsychology Doctoral Candidate, at Howard University Research interests: Research interests: Plasticity of Brain Plasticity of Brain Abnormal Behavior Abnormal Behavior Mood Disorders Mood Disorders

Objectives in Optical Neuroimaging Lab To learn and use different optical imaging techniques to investigate plasticity To learn and use different optical imaging techniques to investigate plasticity Techniques learned and used: Techniques learned and used: Intrinsic signal optical imaging (to assess function) Intrinsic signal optical imaging (to assess function) Two photon microscopy (to assess structure) Two photon microscopy (to assess structure) Fluorescence microscopy (to explore protein localization) Fluorescence microscopy (to explore protein localization)

Intrinsic Signal Overview The setup: A craniotomy was preformed and images were collected from the exposed visual cortex of the ferret. The visual cortex was illuminated with light of 700nm wavelength (to observe photon scatter). The images were acquired with a CCD camera while the ferret was visually stimulated with square wave gratings of different orientations. The images were digitized and processed by a computer which regulated the experiment. Multiple iterations of each stimulus were averaged to improve the signal to noise ratio in functional maps.

Experimental Design Physiological imaging (intrinsic signal) for ocular dominance and orientation preference maps Lid Suture for 5-6 days Repeat physiological imaging Immunohistochemistry

Imaging Paradigm Blockwise experimental design Blockwise experimental design 5 data frames per stimulus (8 seconds) 5 data frames per stimulus (8 seconds) 5 stimuli per block (blank, two stimuli to left eye, two stimuli to right eye) 5 stimuli per block (blank, two stimuli to left eye, two stimuli to right eye) 16 blocks per experiment 16 blocks per experiment 2 experiments needed to cover 4 stimulus orientations for each eye 2 experiments needed to cover 4 stimulus orientations for each eye Visual Stimuli Visual Stimuli Block 1 Block 2

Difference Image Equations Orientation Preference  [(RE-0°) + (LE-0°)] – [(Reye-90°) + (LE-90°)] = Horizontal vs. Vertical difference image Horizontal vs. Vertical difference image  [(RE-45 0 ) + (LE-45 0 )] – [(RE ) + (LE )] = 45 0 vs difference image 45 0 vs difference image Ocular Dominance  (RE: )- (LE: )

Analysis Objective Compare pre and post deprivation maps Compare pre and post deprivation maps IS the Ocular Dominance Map different between the experiment days? IS the Ocular Dominance Map different between the experiment days? IS the Orientation Map similar between days (control)? IS the Orientation Map similar between days (control)? HYPOTHESIS: We expect the OD map to be different and the Orientation Map be similar. HYPOTHESIS: We expect the OD map to be different and the Orientation Map be similar.

Ocular Dominance Plasticity Results Pre deprivation Pre deprivation The area in RED is larger on Day5 The area in RED is larger on Day5 The area in BLUE is smaller on Day5 The area in BLUE is smaller on Day5 Post deprivation Post deprivation The Right Eye has been sutured for 5 days The Right Eye has been sutured for 5 days The Left Eye results indicate the Left eye is more dominant in the visual cortex (V1). The Left Eye results indicate the Left eye is more dominant in the visual cortex (V1).

Orientation Maps Pre Deprivation Pre Deprivation Post Deprivation

Two Photon Overview Two photons hit the fluorophore at the same moment (each has ~1/2 the energy of the single photon excitation wavelength) Two photons hit the fluorophore at the same moment (each has ~1/2 the energy of the single photon excitation wavelength) nm excitation wavelength (infrared light) nm excitation wavelength (infrared light) One photon is emitted from the eGFP fluorophore One photon is emitted from the eGFP fluorophore eGFP= Enhanced Green Fluorescent Protein eGFP= Enhanced Green Fluorescent Protein eGFP is expressed in a subset of neurons in the GFP-O transgenic mouse line eGFP is expressed in a subset of neurons in the GFP-O transgenic mouse line Advantages: -Allows imaging at deeper depths due to better tissue penetration of IR light -images are sharply focused because two photon event only occurs at focal plane -Less tissue damage and bleaching Two photon anatomical imaging enables detection of structural correlates of plasticity (dendrites and axons) in live animals

Project Objectives: Objectives: -learn to perform thinned bone surgery in mouse -learn to use two photon microscopy for structural imaging of neurites -learn different techniques to correct for physiology artifact (i.e. EKG, respiration) Example experiment in neural plasticity: Two photon technique can be used to image OD deprivation effects at the level of the neurite or to identify neuronal structure in OD columns.

Two Photon Imaging Low magnification image of pyramidal cell apical dendrites Low magnification image of pyramidal cell apical dendrites High magnification image of dendrites High magnification image of dendrites

Conclusions Optical imaging of intrinsic signal is a good tool to measure physiological change in ocular dominance columns during the critical period. Optical imaging of intrinsic signal is a good tool to measure physiological change in ocular dominance columns during the critical period. Monocular deprivation results in less of the visual cortex responding to the deprived eye Monocular deprivation results in less of the visual cortex responding to the deprived eye In vivo two-photon microscopy enables time lapse imaging at the scale of neurites and would facilitate studies of anatomical correlates of neural plasticity In vivo two-photon microscopy enables time lapse imaging at the scale of neurites and would facilitate studies of anatomical correlates of neural plasticity Respiratory and cardiac artifacts can be corrected for using triggering software that synchronizes microscope scanning with animal physiology. Respiratory and cardiac artifacts can be corrected for using triggering software that synchronizes microscope scanning with animal physiology.

Acknowledgements Krishnan Padmanabhan Krishnan Padmanabhan Corey Flynn Corey Flynn Nicole Marthaler Nicole Marthaler David Whitney David Whitney Danielle Fisher Danielle Fisher Dr. Alberto Vasquez Dr. Alberto Vasquez Dr. Justin Crowley Dr. Justin Crowley Dr. SEONG-GI KIM Dr. SEONG-GI KIM Dr. William Eddy Dr. William Eddy Tomika Cohen Tomika Cohen Rebecca Clark Rebecca Clark