What can we measure: An experimentalist’s perspective

1. What can we measure:An experimentalist’s perspective Mathematical Neuroscience conference Theoden Netoff Assistant Professor of Biomedical Engineering

2. Brain Signals that can be recorded • Electrical • Chemical • Magnetic • Response to magnetic pulses • Changes in blood flow • Metabolic changes in cells • Glucose consumption • Oxidative state of NADP • Scattering properties • X-ray absorption • How Sound waves pass through the brain • Gene expression

3. Multiple scales neurophysiology Cortex Networks Mossy fiber tract sprouting Changes in stim response Pathalogical EEG Cells Dendriticmorpohology Ion Channels Point mutations Upregulation Modulation Ramon y Cajal homepage.mac.com/dtrapp/eChem.f/labB3.html

4. Pros: Directly relates brain area to behavior Cons: Mostly case studies Cannot be repeated Lesions: Phineas Gage • What is the signal: • Functional loss following an injury • Wallarian degeneration used to track pathways neuraldump.com/2007/09/patient-hm.html Harlow 1868 3500 BC, Iron age Trepanation Natural History Museum, Lausanne

5. The canonical neuron • Soma • Dendrites: Inputs • Axon: Outputs

6. Pros: Very high fidelity recording Neuron can be identified morphologically by staining Neuron can be stimulated Cons: Limited number of cells that can be recorded Cell type cannot be definitively determined physiologically Patch clamp recording: • What is the signal: • Glass tube filled with salt solution applied to neuron to make low noise junction with neuron

7. Electrode-Cell circuit Axon Guide

8. Patch clamp recording Single channel recording Sakmann and Neher, 1984 Hamill et al., 1981 http://butler.cc.tut.fi/~malmivuo/bem/bembook/04/04.htm

9. Sodium and potassium channels Hille, 1992 http://butler.cc.tut.fi/~malmivuo/bem/bembook/04/04.htm

10. Whole cell recording The “Sag” current Hasselmo et al, J. Neurophysiol 2000

11. Intracellular and local field potentials Netoff and Schiff, 2002

12. Pros: Multiple cells can be recorded at once Can be done in vivo Cons: Cell that is being recorded cannot be positively identified Local field recordings: • What is the signal: • Electrode placed into the brain to record electric fields www.berkelab.org/Techniques.html

13. Tetrode Neural recordings Slide courtesy of Adam Johnson and David Redish

14. Place cells O’Keefe, 1978 Kneirim et al., 1995 Slide courtesy of Adam Johnson and David Redish

15. EEG signal source:Dendritic currents • Apical dendrite acts as a dipole • Dendritic currents, not action potentials, summate best • Electrical signal is strongly attenuated at the scalp. • Inhibitory neuron populations do not produce good dipoles http://www.biosemi.com/pin_electrode.htm

16. EEG Signal confounds correlation and activity • Populations of cells that are synchronous and have dendrites lined up produce strong signals

17. Sensitivity of neuron depends on orientation to electric field • Rushton, 1927: -Excitability roughly proportional to the cosine of the ange between current and nerve. Current perpendicular to the nerve cannot stimulate an action potential.

18. Degree of asymmetry of neuron determines strength of response to electric field and the field it produces Chan and Nicholson, 1986

19. Pros: High spatial resolution Non-invasive, but does require X-rays Cons: Poor constrast between tisses Limited to anatomical scans Computed Tomograpy :3d X-ray reconstruction of the head • What is the signal: • X-rays are projected through the head. The signal is rotated around the head and a 3D reconstruction is formed with the aid of a computer. www.csmc.edu/6158.html

20. Pros: High spatial resolution Non-invasive Good white/Gray matter contrast Cons: Water signals do not provide good temporal signals, but other signals can be measured Expensive to run Magnetic Resonance Imaging :Interaction with tissue and magnetic field • What is the signal: • Hydrogen atoms are pumped to high energy state with Electromagnetic wave. • Turn off EMF wave and measure time of relaxation to low energy state • Strength of signal varies with water density. Gray matter has higher water density than white matter • Frequency is different as field strength falls off allowing for depth imaging http://en.wikipedia.org/wiki/Image:MRI_head_saggital.jpg

21. Pros: Medium spatial resolution with depth Non-invasive Flexibility in signals that can be measured Can be laid directly over an anatomical scan Cons: Slow temporal scale Assumes relation of activity to blood flow, but what causes increase in metabolism is not a unique set Expensive to run Functional MRI:MRI of signals that change over time • What is the signal: • Changes in MR signal with time. • Depending on the RF pulse frequency, different molecules can be imaged besides hydrogen. • BOLD- hemoglobin changes from paramagnetic to diamagnetic when oxygenated http://www.cem.msu.edu/~reusch/VirtTxtJml/Spectrpy/nmr/nmr1.htm en.wikipedia.org/wiki/Positron_emission_tomography MRI Imaging signals: Hemoglobin Choline Creatine Lactate N-acetlyaspartate

22. Pros: Medium spatial resolution with depth Non-invasive Flexibility in signals that can be measured Cons: Only slow metabolic activity can be imaged Exposure to radiation Very Expensive to run Positron Emission Tomography:Sequestration of radioactive isotopes in tissue • What is the signal: • Radioactive isotope of a molecule is developed using an accelerator • A neurotransmitter or glucose are made with the isotope and injected into the subject • The brain sequesters the radioactive molecules into active spots where it makes hot spots of gamma • Using a scintillator material gamma radiation is converted to light and imaged. en.wikipedia.org/wiki/Positron_emission_tomography PET molecules: Fluorodeoxyglucose 2-Deoxy glucose Water Ammonia Dopamine Seretonin

23. Pros: Non-invasive Source localization is done to estimate depth from signal Provides signals that cannot be measured in any other way Cons: Low spatial resolution with respect to EEG Low signal to noise with respect to EEG Magnetoencephalography:Magnetic signals from the brain • What is the signal: • Magnetic fields generated by electrical activity of the brain • SQUIDs detect magnetic signal en.wikipedia.org/wiki/Magnetoencephalography www.acoustics.org/press/153rd/hertrich.html

24. Pros: Does not require dye High spatial resolution Cons: Signal not well understood Requires exposing the brain Signal is slow and on the order of a metabolic signal Cannot be used to resolve single cells Intrinsic Optical Imaging:Reflectance of the brain • What is the signal: • Infrared light is used to illuminate the tissue • CCD camera detects changes in brightness of tissue over time • Caused by changes extracellular space volume (scattered light) or by changes in blood flow to the area (absorbed light) Cerne and Haglund Neuroscience Letters(2002)

25. Pros: Does not require dye High spatial resolution Cons: Signals are of metabolic processes, not limited to neuronal activity Slow, on order of metabolic demand Intrinsic Optical Imaging:Flavoprotein imaging • What is the signal: • Excitation light is used to illuminate brain tissue • Autofluorescentflavoproteins (such as NADH) change fluorescence with oxidative state. This is a direct measurement of the metabolic process Reinert et al, J Neurosci Research, 2007

26. Pros: Non-invasive recording Useful on children Useful in MRI where electrode leads can be difficult to deal with Cons: Very low resolution spatially and low signal to noise ratio Signal, not very well understood Non-invasive optical imaging:Optical reflectance and scattering • What is the stimulus: • Bright long wavelength light is shined onto the surface of the scalp penetrating to the brain • Photo sensors detect reflected and scattered light • Signal changes caused by bloodflow or swelling of cells increasing reflectance http://rabi.nmr.mgh.harvard.edu/DOT/research/probe-gallery.htm

27. Pros: Signal is very fast Direct measure of neuronal activity Cons: Voltage dyes stain all membranes, neurons are only a small fraction To keep SNR high light must be very bright Cannot be used to image large numbers with single cell resolution Dyes can be toxic Voltage dye imaging:Directly imaging membrane voltage • What is the signal: • Membranes are stained with a fluorescent protein that indicates voltage change Zochowski, et al.

28. Pros: Intracellular measurement of activity Bath or intracellular application Very bright changes Cons: Slow Indirect measure of activity Calcium dye imaging :Directly imaging membrane voltage • What is the signal: • Cells stained with fluorescent dyes that indicate changes in intracellular calcium concentration. Ikegaya et al, Neuroscience Research, 2005 http://probes.invtrogen.com

29. Calcium dyes:Bath applied AM dyes Badea, et al. J. Neurobiol, 5;48(3):215-27 (2001)

30. Multiple cell recordings using Calcium dyes • Imaging population over time and parsing image up results in time series from multiple neurons Cossart et al, Cell Calcium (2005)

31. DIC Str. Rad Str. Pyr Str. Ori Resting Fluorescence 100 μm 4-AP induced Seizure activity in CA1

32. Calcium imaging of seizures in the hippocampus

33. Pros: Measure many different signals: Gasses:O2 pH Salts: K+, Na+, Ca++ Cons: Slow Low spatial resolution Interference across ion species Ion sensitive electrodes What is the signal: Ion selective exchange across membrane creates voltage. Membranes can be made of glass, crystal, resin or polymer. Enzyme electrodes Potassium concentrations after stimulation Gorji et al, Epilepsia, 2006

34. Stimulating the brain • Patch electrode: • can be used for recording and stimulating • Extracellular electrodes: • Playing back measured signal is like playing a tape recording of planes taking off at the airport to make planes take off. Walter Freeman

35. Deep brain stimultion http://www.sctimst.ac.in/hospital/neurology/movementdisorder.htm mcrais.googlepages.com/implants.htm en.wikipedia.org/wiki/Image:Parkinson_surgery.jpg

36. Pros: Non-invasive stimulation Used for treating depression Cons: Not localized Mechanism? Inducing Seizures! Electroconvulsive therapy:Electric fields and the brain • What is the stimulus: • Current is applied to the head to induce seizures http://www.humanillnesses.com/Behavioral-Health-Br-Fe/Electroconvulsive-Therapy.html

37. Pros: Non-invasive stimulation Short duration Some localization of field Used in place of electroconvulsive therapy Cons: Localization is poor (compared with an implanted electrode) Effects not well understood Transcranial magnetic stimulation:Magnetic fields and the brain • What is the stimulus: • Short pulse of magnetic field induces small current flows in the brain. www.princeton.edu/~napl/methods_h.htm

38. Stimulating neurons: Caged compounds Rothman et al, Epilepsy Research 74(2):201-209 (2007) Caged glutamate stimulation Caged GABA Jin et al, J. Neuroscience 26(18):4891-4900 (2006)

39. Closing the loop • Dynamic clamp • Brain Machine/Computer Interface • Neurotransmitter uncaging • Photosensitive ion channels

40. Pros: Flexible system for complex protocols Simulation of ion channels Simulation of synapses Simulation of neurons to make “hybrid” networks Cons: Limited number of channels Dynamic clamp:Real time computer interfaced with a neuron • What is the stimulus: • Voltage measurement can be used to calculate current to inject Iapp Vm

41. T Phase Response Curve

42.   Phase Response Curve

43. Phase Response Curve

44. Phase Response Curve Perturbation from period Stimulus time (since last spike)

45. Pros: Single cell resolution of 100s of cells Millisecond time scale Cons: Requires genetic alteration of cells Optical stimulation:Real time computer interfaced with a neuron What is the stimulus: Channel rhodposin-2 a light sensitive cation channel activated by yellow light Boyden et al, Nature Neuroscience (2005)

46. Inhibiting neurons • Light driven chloride pump • Genetically spliced to cell type specific promoter • Shining light hyperpolarizes the cell with msec time constants • Single action potentials can be inhibited. Han and Boyden, PLoS ONE, 2007

47. Summary Churchland and Sejnowski, 1988