Optical Optical Neuroimaging: Neuroimaging: Investigating Investigating

PlasticityPlasticityMulti-Modal Neuroimaging ProgramMulti-Modal Neuroimaging Program

Presenter: Santresda Johnson B.A., Presenter: Santresda Johnson B.A., M.S.M.S.

Neuropsychology, Howard UniversityNeuropsychology, Howard UniversityOptical Imaging Mentor: Dr. Justin Optical Imaging Mentor: Dr. Justin

Crowley Ph.D.Crowley Ph.D.Biology Department, Carnegie Mellon Biology Department, Carnegie Mellon

University University

Santresda Johnson B.A., Santresda Johnson B.A., M.S.M.S.

Neuropsychology Neuropsychology Doctoral Doctoral Candidate, at Candidate, at Howard UniversityHoward University

Research interests:Research interests: Plasticity of BrainPlasticity of Brain Abnormal BehaviorAbnormal Behavior Mood Disorders Mood Disorders

Objectives in Optical Objectives in Optical Neuroimaging LabNeuroimaging Lab

To learn and use different optical To learn and use different optical imaging techniques to investigate imaging techniques to investigate plasticityplasticity Techniques learned and used:Techniques learned and used:

Intrinsic signal optical imaging (to assess Intrinsic signal optical imaging (to assess function)function)

Two photon microscopy (to assess Two photon microscopy (to assess structure)structure)

Fluorescence microscopy (to explore Fluorescence microscopy (to explore protein localization) protein localization)

Intrinsic Signal OverviewIntrinsic Signal Overview

The setup: The setup: A craniotomy was preformed and A craniotomy was preformed and images images were were collected from the collected from the exposed exposed visual visual cortex of the cortex of the ferret.ferret. The The visual visual cortex cortex waswas illuminated with light of illuminated with light of 700700nm wavelengthnm wavelength (to observe photon scatter) (to observe photon scatter) . The images . The images werewere acquired with acquired with a a CCD CCD camera camera while while the the ferretferret was visually was visually stimulated withstimulated with square wave gratings of different orientations.square wave gratings of different orientations. The The images wereimages were digitized digitized and processed and processed by a computer by a computer which regulated thewhich regulated the experiment. experiment. Multiple iterations of each stimulus were averaged Multiple iterations of each stimulus were averaged to improve the signal to noise ratio in functional maps. to improve the signal to noise ratio in functional maps.

Experimental DesignExperimental DesignPhysiological Physiological

imaging (intrinsic imaging (intrinsic signal) for ocular signal) for ocular dominance and dominance and orientation orientation preference mapspreference maps

Lid Suture Lid Suture for 5-6 for 5-6 daysdays

Repeat Repeat physiologicphysiological imagingal imaging

ImmunohistocheImmunohistochemistrymistry

Imaging ParadigmImaging Paradigm Blockwise Blockwise

experimental designexperimental design 5 data frames per 5 data frames per

stimulus (8 seconds)stimulus (8 seconds) 5 stimuli per block 5 stimuli per block

(blank, two stimuli to (blank, two stimuli to left eye, two stimuli to left eye, two stimuli to right eye)right eye)

16 blocks per 16 blocks per experimentexperiment

2 experiments needed to 2 experiments needed to cover 4 stimulus cover 4 stimulus orientations for each eyeorientations for each eye

Visual StimuliVisual Stimuli

Block 1 Block 2

Difference Image Difference Image Equations Equations

Orientation PreferenceOrientation Preference [(RE-0°) + (LE-0°)] – [(Reye-90°) + (LE-90°)][(RE-0°) + (LE-0°)] – [(Reye-90°) + (LE-90°)]

== Horizontal vs. Vertical difference imageHorizontal vs. Vertical difference image

[(RE-45[(RE-4500) + (LE-45) + (LE-4500)] – [(RE-135)] – [(RE-13500) + (LE-135) + (LE-13500)])]==

454500 vs. 135 vs. 13500 difference image difference image

Ocular DominanceOcular Dominance (RE: 0(RE: 000 + 90 + 9000 +45 +4500 + 135 + 13500 )- (LE: 0 )- (LE: 000 + 90 + 9000 +45 +4500

+ 135+ 13500 ) )

Analysis ObjectiveAnalysis Objective

Compare pre and post deprivation Compare pre and post deprivation maps maps IS the Ocular Dominance Map different IS the Ocular Dominance Map different

between the experiment days? between the experiment days? IS the Orientation Map similar between IS the Orientation Map similar between

days (control)?days (control)?

HYPOTHESIS: We expect the OD HYPOTHESIS: We expect the OD map to be different and the map to be different and the Orientation Map be similar.Orientation Map be similar.

Ocular Dominance Ocular Dominance Plasticity Results Plasticity Results

Pre deprivationPre deprivation

The area in RED is larger The area in RED is larger on Day5on Day5

The area in BLUE is The area in BLUE is smaller on Day5smaller on Day5

Post deprivationPost deprivation

The Right Eye has been sutured The Right Eye has been sutured for 5 daysfor 5 days

The Left Eye results indicate the The Left Eye results indicate the Left eye is more dominant in the Left eye is more dominant in the visual cortex (V1).visual cortex (V1).

Orientation MapsOrientation Maps Pre DeprivationPre Deprivation Post DeprivationPost Deprivation

Two Photon OverviewTwo Photon Overview

Two photons hit the Two photons hit the fluorophore at the same fluorophore at the same moment (each has ~1/2 moment (each has ~1/2 the energy of the single the energy of the single photon excitation photon excitation wavelength) wavelength) 860-890 nm excitation 860-890 nm excitation

wavelength (infrared wavelength (infrared light)light)

One photon is emitted One photon is emitted from the eGFP fluorophorefrom the eGFP fluorophore eGFP= Enhanced eGFP= Enhanced

Green Fluorescent Green Fluorescent Protein Protein

eGFP is expressed in a eGFP is expressed in a subset of neurons in subset of neurons in the GFP-O transgenic the GFP-O transgenic mouse linemouse line

Advantages:Advantages:

-Allows imaging at deeper -Allows imaging at deeper depths due to better depths due to better tissue penetration of IR tissue penetration of IR lightlight

-images are sharply -images are sharply focused because two focused because two photon event only photon event only occurs at focal planeoccurs at focal plane

-Less tissue damage and -Less tissue damage and bleachingbleaching

Two photon anatomical imaging enables detection of structural correlates of plasticity (dendrites and axons) in live animals

Project Project Objectives:Objectives:

-learn to perform thinned bone surgery in mouse-learn to perform thinned bone surgery in mouse-learn to use two photon microscopy for -learn to use two photon microscopy for structural imaging of neuritesstructural imaging of neurites-learn different techniques to correct for -learn different techniques to correct for physiology artifact (i.e. EKG, respiration)physiology artifact (i.e. EKG, respiration)

Example experiment in neural plasticity: Two Example experiment in neural plasticity: Two photon technique can be used to image OD photon technique can be used to image OD deprivation effects at the level of the neurite or deprivation effects at the level of the neurite or to identify neuronal structure in OD columns. to identify neuronal structure in OD columns.

Two Photon ImagingTwo Photon Imaging

Low magnification Low magnification image of pyramidal image of pyramidal cell apical cell apical dendritesdendrites

High magnification High magnification image of dendritesimage of dendrites

ConclusionsConclusions Optical imaging of intrinsic signal is a good Optical imaging of intrinsic signal is a good

tool to measure physiological change in ocular tool to measure physiological change in ocular dominance columns during the critical period.dominance columns during the critical period.

Monocular deprivation results in less of the Monocular deprivation results in less of the visual cortex responding to the deprived eyevisual cortex responding to the deprived eye

In vivoIn vivo two-photon microscopy enables time two-photon microscopy enables time lapse imaging at the scale of neurites and lapse imaging at the scale of neurites and would facilitate studies of anatomical would facilitate studies of anatomical correlates of neural plasticitycorrelates of neural plasticity

Respiratory and cardiac artifacts can be Respiratory and cardiac artifacts can be corrected for using triggering software that corrected for using triggering software that synchronizes microscope scanning with animal synchronizes microscope scanning with animal physiology. physiology.

AcknowledgementsAcknowledgements Krishnan PadmanabhanKrishnan Padmanabhan Corey FlynnCorey Flynn Nicole MarthalerNicole Marthaler David WhitneyDavid Whitney Danielle FisherDanielle Fisher Dr. Alberto VasquezDr. Alberto Vasquez Dr. Justin CrowleyDr. Justin Crowley Dr. SEONG-GI KIM Dr. SEONG-GI KIM Dr. William EddyDr. William Eddy Tomika CohenTomika Cohen Rebecca ClarkRebecca Clark