1. 4D Functional Imaging in Freely Moving Animals Randall L. Barbour SUNY Downstate Medical Center OSA Biomedical Optics Meeting Fort Lauderdale, FL, March 20, 2005

2. 09/07/2005R.L. Barbour Cell Free Preparation Cell Culture Organotypic Culture Perfused Organ Anesthetized Animal Restrained Animal Freely Moving Animal Degree of Control Higher Lower Phenomenological Complexity Maximal Minimal Levels of Analysis in Biological Investigation

3. 09/07/2005R.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

4. 09/07/2005R.L. Barbour 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

5. 09/07/2005R.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.

6. 09/07/2005R.L. Barbour Detector channels Power supplies Laser controllers Source fiber terminal Optical switch FRONT BACK Lasers / optics Timing Photo of 9s x 32d imager

7. 09/07/2005R.L. Barbour Schematic of System Setup DYNOT compact system Laptop computer Optical tether Arena w/ animal Head stage w/ Tracking LED Electro- physiology recording system Environmental chamber synchronization Electrical tether Computer Video cam Computer w/ frame grabber Figure 12. Schematic of Optical Imaging-EEG-Behavior Monitoring System.

8. 09/07/2005R.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 electrodes Optical Fibers 1.8 mm dia. Tracking LED’s Electrode leads Connecting Clips Male part Female part

9. 09/07/2005R.L. Barbour Dual mode optical-EEG measuring head Optical fiber extension element EEG Electrodes Grounding wires Male Part Female Part

10. 09/07/2005R.L. Barbour Rat Brain Anatomy with Optical-EEG Overlay Transmitting/receiving Fiber Left Cortical Hemisphere Right Cortical Hemisphere Hippocampus Cerebellum Olfactory bulbs Receiving Fiber EEG Electrodes

11. 09/07/2005R.L. Barbour Rat with attached helmet and tether

12. 09/07/2005R.L. Barbour Movie of freely moving rat with attached tether

13. 09/07/2005R.L. Barbour Hippocampal EEG Rhythms Theta Amplitude Time Large Irregular Activity

14. 09/07/2005R.L. Barbour Data Analysis-Integration Time Theta Non-Theta Theta Non-Theta Optical Image Time Series EEG Time Series

15. 09/07/2005R.L. Barbour 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.

16. 09/07/2005R.L. Barbour Approach Capture simultaneous: EEG, behavior and dual wavelength tomographic time-series. Compute volumetric images Determine temporal/spatial dependence of Hb on EEG/behavior states.

17. 09/07/2005R.L. Barbour Time dependence of spatially integrated findings. Spatial dependence of temporally integrated findings. RESULTS

18. 09/07/2005R.L. Barbour Exp. 1: EEG-Gated Hb Spatial Mean Time Series Red – Non-Theta Green – Theta (animal moving) Hb oxy Hb deoxy Hb tot HbO 2 Sat

19. 09/07/2005R.L. Barbour 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 Hb oxy Non-Theta-6.18e-91.46e-87976 -25.27 (935.92) 2.39e-104 Theta1.06e-81.86e-8828 Hb deoxy Non-Theta1.93e-99.38e-97976 16.80 (1056.43) 2.57e-56 Theta-3.25e-98.34e-9828 Hb tot Non-Theta-4.25e-91.55e-87976 -15.98 (929.08) 5.93e-51 Theta7.37e-92.03e-8828 HbO 2 Sat Non-Theta0.687870.000297976 -29.37 (1026.08) 4.29e-138 Theta0.688170.00028828

20. 09/07/2005R.L. Barbour Stationarity of EEG-Gated Hb Response P-value Hb Oxy Hb Deoxy Hb Total Hb Sat.. ……

21. 09/07/2005R.L. Barbour Time Lag of Hb Response Figure 8. Hb response as a function of removal of fraction of initial period.

22. 09/07/2005R.L. Barbour Spatially Integrated findings of vascular response to theta rhythm – Increased Hb oxy – Decreased Hb deoxy – Increase Hb tot – Increased HbO 2 Sat – i.e., BOLD effect

23. 09/07/2005R.L. Barbour 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) B A C D HbOxy HbDeoxy HbTot HbSat HbOxy HbDeoxy HbTot HbSat

24. 09/07/2005R.L. Barbour Time Dependence of Gated Response Hb Sat HbTot HbDeoxy HbOxy Four sessions combined (0-1 sec) Four Sessions Combined (Sec 1 - 4)

25. 09/07/2005R.L. Barbour Spatial dependence Spatial response is reproducible across trials. Positive, negative and mixed BOLD effects are mainly spatially distinct.

26. 09/07/2005R.L. Barbour Autoregulatory dependent hemoglobin states Hemoglobin State State 1 State 2 State 3 State 4 State 5 State 6 Hb oxy ---+++ Hb deoxy -+++-- Hb tot --+++- BalancedUncomp. oxygen debt Comp. oxygen debt BalancedUncomp. oxygen excess Comp. oxygen excess

27. 09/07/2005R.L. Barbour Hb oxy+ Hb deoxy+ Hb tot+ Spatial Mean Time Series for Autoregulatory State 4 (Balanced) Pixel No

28. 09/07/2005R.L. Barbour Hb oxy+ Hb deoxy- Hb tot+ Spatial Mean Time Series for Autoregulatory State 5 (Uncompensated oxygen excess) Pixel No

29. 09/07/2005R.L. Barbour Hb oxy+ Hb deoxy- Hb tot- Spatial Mean Time Series for Autoregulatory State 6 (Compensated oxygen excess) Pixel No

30. 09/07/2005R.L. Barbour Nose 1 2 3 4 5 6 Spatial dependence of autoregulatory response

31. 09/07/2005R.L. Barbour Temporal Averaged Gated Maps of Hb States conditionIIIIIIIVVVI∑ Nodes (Theta)18814513821471611592488 Nodes (Non- theta) 159110538011270952488 Theta Images Non- theta Images Diff. Images Blue: Non- theta Red: Theta

32. 09/07/2005R.L. Barbour P-values for Theta vs. Non-theta for Autoregulatory dependent hemoglobin states Hemoglobin State State 1 State 2 State 3 State 4 State 5 State 6 Hb oxy <10 -90 00 00 Hb deoxy <10 -18 00 <10 -90 00 Hb tot <10 -58 00 <10 -90 00 BalancedUncomp. oxygen debt Comp. oxygen debt BalancedUncomp. oxygen excess Comp. oxygen excess

33. 09/07/2005R.L. Barbour Time-integrated Hb states: Theta 1 2 3 4 5 6 Composite

34. 09/07/2005R.L. Barbour Time-integrated Hb states: Non-Theta 1 2 3 4 5 6 Composite

35. 09/07/2005R.L. Barbour 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.

36. 09/07/2005R.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.