4D Functional Imaging in Freely Moving Animals

4D Functional Imaging in Freely Moving Animals

Randall L. Barbour SUNY Downstate Medical Center

OSA Biomedical Optics Meeting

Fort Lauderdale, FL,

March 20, 2005

09/07/2005 R.L. Barbour

Cell Free Preparation

Cell Culture

Organotypic Culture

Perfused Organ

Anesthetized Animal

Restrained Animal

Freely Moving Animal

Degree ofControl

Higher

Lower

PhenomenologicalComplexity

Maximal

Minimal

Levels of Analysis in Biological Investigation

09/07/2005 R.L. Barbour

Why Freely Moving Animals?

• Only preparation capable of expressing the full behavioral repertoire of a species.– Aggression– Mating– Fear– Perceptual – Locomotor– Manipulative

• Current imaging tools require investigation on restrained/anesthetized animals.– PET/SPECT– MR-fMRI– MEG

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Why Optical Methods?

• Inexpensive, compact instrumentation

• High intrinsic sensitivity

• Deep tissue penetration

• Fast data collection

• Easily overlaid on other sensing technologies

• Opportunity for dynamic studies

09/07/2005 R.L. Barbour

Objectives of current study

1. Determine feasibility of continuous functional imaging in freely moving animals while simultaneously recording behavioral, neural and hemodynamic responses.

2. Identify the temporal and spatial dependence of the vascular response as gated to EEG (theta) rhythms.

09/07/2005 R.L. Barbour

Detector channels

Power supplies

Laser controllers

Source fiber terminal

Optical switch

FRONT

BACK

Lasers / optics

Timing

Photo of 9s x 32d imager

09/07/2005 R.L. Barbour

Schematic of System Setup

DYNOT compact system

Laptop computer

Opticaltether

Arena w/ animal

Head stage w/Tracking LED

Electro-physiology

recording system

Environmentalchamber

synchronization

Electricaltether

Computer

Videocam

Computerw/ frame grabber

Figure 12. Schematic of Optical Imaging-EEG-Behavior Monitoring System.

09/07/2005 R.L. Barbour

Dual mode optical-EEG measuring head

Optical array: 4 source x 16 detector

Dual wavelength: 760, 830 nm

Framing rate: 17 Hz

EEG: 12, 0.1mm diameter electrodesOptical Fibers

1.8 mm dia.

Tracking LED’s

Electrode leads

Connecting Clips

Male part

Female part

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Dual mode optical-EEG measuring head

Optical fiber extension element

EEG Electrodes

Grounding wires

Male Part

Female Part

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Rat Brain Anatomy with Optical-EEG Overlay

Transmitting/receiving Fiber

Left Cortical Hemisphere

Right Cortical Hemisphere

Hippocampus

Cerebellum

Olfactory bulbs

Receiving Fiber

EEG Electrodes

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Rat with attached helmet and tether

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Movie of freely moving rat with attached tether

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Hippocampal EEG Rhythms

Theta

Am

plitu

deA

mpl

itude

Time

Time

Large Irregular Activity

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Data Analysis-Integration

Tim

e

Theta

Non-Theta

Theta

Non-Theta

Optical Image Time Series

EEG Time Series

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FEM Mesh for Rat Brain Model

S-D Geometry(3D View) FEM Mesh

(3D View)

7-compartment model of rat head anatomy obtained from CT scan. 2488 FEM nodes. From Bluestone et al. 2004.

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Approach• Capture simultaneous: EEG, behavior and dual

wavelength tomographic time-series.

• Compute volumetric images

• Determine temporal/spatial dependence of Hb on EEG/behavior states.

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• Time dependence of spatially integrated findings.

• Spatial dependence of temporally integrated findings.

RESULTS

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Exp. 1: EEG-Gated Hb Spatial Mean Time Series

Red – Non-Theta Green – Theta (animal moving)

Hboxy

Hbdeoxy

Hbtot

HbO2 Sat

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Exp 1: Time Averaged-Whole Brain EEG-Gated Hemoglobin Response

Hemoglobin

State

EEG

classification

Mean

(M)

Standard deviation

(M)

Number of time frames

t-statistic

(df)

p-value

Hboxy

Non-Theta -6.18e-9 1.46e-8 7976

-25.27

(935.92)

2.39e-104

Theta 1.06e-8 1.86e-8 828

Hbdeoxy

Non-Theta 1.93e-9 9.38e-9 7976

16.80

(1056.43)

2.57e-56

Theta -3.25e-9 8.34e-9 828

Hbtot

Non-Theta -4.25e-9 1.55e-8 7976

-15.98

(929.08)

5.93e-51

Theta 7.37e-9 2.03e-8 828

HbO2 Sat

Non-Theta 0.68787 0.00029 7976

-29.37

(1026.08)

4.29e-138

Theta 0.68817 0.00028 828

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Stationarity of EEG-Gated Hb Response

0 20 40 60 80

10-5

100

P-V

alu

e

Window Size: 1 min; Moving time steps: 30 sec

0 20 40 60 80-1

0

1

2x 10

-8

0 20 40 60 80-1

0

1

2x 10

-8

0 20 40 60 80-1

0

1

2x 10

-8

0 20 40 60 80

0.688

Sliding Window

HboxyHbredHbtotHbsatP-value

HbOxy

HbDeoxy

HbTotal

HbSat

.. ……

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Time Lag of Hb Response

0 10 20 30 40 50 60 70 80 900

1

2x 10

-8

Hbo

xy

Temporal and spatial averaged EEG gated Hb levels

0 10 20 30 40 50 60 70 80 90-1

-0.5

0x 10

-8

Hbr

ed

0 10 20 30 40 50 60 70 80 900

0.5

1x 10

-8

Hbt

ot

0 10 20 30 40 50 60 70 80 900.6879

0.688

0.6881

0.6882

Hbs

at

Percentage of initial response-period removed

Non-ThetaTheta

Figure 8. Hb response as a function of removal of fraction of initial period.

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Spatially Integrated findings of vascular response to theta rhythm

–Increased Hboxy

–Decreased Hbdeoxy

–Increase Hbtot

–Increased HbO2Sat

–i.e., BOLD effect

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EEG-Gated Hb Response

Rat 1 Session 1 (Sec 1 - 4) Rat 1 Session 2 (Sec 1 - 4)

Rat 2 Session 1 (Sec 1 - 4) Rat 2 Session 2 (Sec 1 - 4)

BA

C D

Hb

Oxy

Hb

Deo

xyH

bT

ot

Hb

Sat

Hb

Oxy

Hb

Deo

xyH

bT

ot

Hb

Sat

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Time Dependence of Gated Response

Hb

Sat

HbT

otH

bDeo

xyH

bOxy

Four sessions combined (0-1 sec) Four Sessions Combined (Sec 1 - 4)

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Spatial dependence

• Spatial response is reproducible across trials.

• Positive, negative and mixed BOLD effects are mainly spatially distinct.

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Autoregulatory dependent hemoglobin states

Hemoglobin State

State1

State2

State3

State 4

State 5

State 6

Hboxy

- - - + + +

Hbdeoxy

- + + + - -

Hbtot

- - + + + -

Balanced Uncomp. oxygen debt

Comp. oxygen debt

Balanced Uncomp. oxygen excess

Comp. oxygen excess

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Hboxy+

Hbdeoxy+

Hbtot+

Spatial Mean Time Series for Autoregulatory State 4 (Balanced)

Pixel No

2000 4000 6000 8000 10000 12000 14000 16000 180000

500

1000

1500

2000

2500

2000 4000 6000 8000 10000 12000 14000 16000 18000

2

4

6

8

10

12

14

x 10-7

2000 4000 6000 8000 10000 12000 14000 16000 18000

2

4

6

8

10

12

14

x 10-7

2000 4000 6000 8000 10000 12000 14000 16000 18000

2

4

6

8

10

12

14

x 10-7

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2000 4000 6000 8000 10000 12000 14000 16000 180000

500

1000

1500

2000

2000 4000 6000 8000 10000 12000 14000 16000 18000

0

2

4

6

x 10-7

2000 4000 6000 8000 10000 12000 14000 16000 18000

0

2

4

6

x 10-7

2000 4000 6000 8000 10000 12000 14000 16000 18000

0

2

4

6

x 10-7

Time Point

Hboxy+

Hbdeoxy-

Hbtot+

Spatial Mean Time Series for Autoregulatory State 5 (Uncompensated oxygen excess)

Pixel No

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Hboxy+

Hbdeoxy-

Hbtot-

Spatial Mean Time Series for Autoregulatory State 6 (Compensated oxygen excess)

Pixel No

2000 4000 6000 8000 10000 12000 14000 16000 180000

500

1000

1500

2000

2000 4000 6000 8000 10000 12000 14000 16000 18000

-4

-2

0

2

x 10-7

2000 4000 6000 8000 10000 12000 14000 16000 18000

-4

-2

0

2

x 10-7

2000 4000 6000 8000 10000 12000 14000 16000 18000

-4

-2

0

2

x 10-7

Time Point

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Nose

12

3

4 5

6

Spatial dependence of autoregulatory response

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Temporal Averaged Gated Maps of Hb States

condition I II III IV V VI ∑

Nodes (Theta) 188 1451 382 147 161 159 2488

Nodes (Non-theta)

159 110 53 801 1270 95 2488

Theta

Images

Non-

theta

Images

Diff. Images Blue: Non-theta

Red: Theta

09/07/2005 R.L. Barbour

P-values for Theta vs. Non-theta for Autoregulatory dependent

hemoglobin states

Hemoglobin State

State1

State2

State3

State 4

State 5

State 6

Hboxy <10-90 0 0 <10-90 0 0

Hbdeoxy <10-18 0 0 <10-90 0 0

Hbtot <10-58 0 0 <10-90 0 0Balanced Uncomp.

oxygen debtComp.

oxygen debtBalanced Uncomp.

oxygen excess

Comp. oxygen excess

09/07/2005 R.L. Barbour

Time-integrated Hb states: Theta

12

3

4 5

6

Composite

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Time-integrated Hb states: Non-Theta

12

3

45

6

Composite

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Conclusions

• Real-time recording of hemodynamic, EEG and behavorial responses is technically feasible in freely moving animals.

• Hemodynamic response to theta rhythms are reproducible and spatially distinct.

• Method provides for assessment of temporal-spatial dynamics of autoregulatory response to neural activation.

09/07/2005 R.L. Barbour

Future Considerations

• Imaging under defined behavioral paradigms to ascertain localizability of EEG dependent hemodynamic responses.

• Influence of pharmacoactive agents on measured responses.

• Technological improvements: >S-D pairs, wavelengths, etc.

• Development of human compatible system.