Methods in brain research

1. Structurea. Morphologyb. Pathways

2. Function

1. Sacrificing the animal / or post-mortem studies

2. Replace the blood with saline (physiologic water)

3. Fixation – injection of formaline

Dissection

Grey matterWhite matter

frontal section horizontal section

ventricles

Thalamus

Nissl stain

Staining of cell bodies

Histology

Staining of axon fibers

Hard to see individual cells

Golgi stain

Individual cells

Microscopy

1. Light (up to x1500 magnification)

2. Electron Microscopy (EM; magnification of ~108 in tissue slices)1. Transmission EM (high resolution 2d information)2. Scanning EM – lower resolution but provides 3d information

3. Confocal microscopy – Uses laser and fluorescent dyes. Higher resolution than light microscopy Thicker slices (3d) in living tissue

Neural pathways

Anterograde labeling – chemicals that enter the cells (through dendrites), and travel along the axons to the terminal buttons

1. Injection of PHA-L

2. Substance is transported throughout the cell

3. Animal is sacrificed and brain is perfused

4. Histology – PHA-L is made visible through use of immunocytochemical methods

Autoradiography – using radioactive markers for labeling

Retrograde labeling – chemicals that enter the terminal buttons, and travel through the axons to the cell bodies

Auditory pathway

Tracer in the thalamus

Imaging techniques to study structure

Computerized Tomography (CT)

Magnetic Resonance Imaging (MRI)

Computed Tomography

Computed TomographyX ray

Computed TomographyX ray

Brain damage

StrokeHemorrhage

Computed Tomography X ray

Advantages of CT

• Accessible (and cheap)

• Efficient in detecting

• Stroke•

Hemorrhages• Tumors

• Uses X-Ray• No separation between white/grey

matter• Can only take horizontal sections

Drawbacks of CT

Good spatial resolution (~1mm)Separation between grey/white matter

Magnetic Resonance Imaging

Very strong magnetic field.Earth is ~0.5 Gauss10,000 Gauss is 1 TeslaTypical scanner today has a magnetic field of 3 Tesla

Magnetic resonance angiography (MRA)

Measuring proton density (from water molecules) with MRI

Advantages:

Non-invasive (and can be used in the living brain)

High spatial resolution (~1mm)

Safe (Does not damage tissue)

Diffusion Tensor Imaging (DTI)

Can NOT tell from _X_ to_Y_ - but gives the general direction of the fibers

Tools to study brain function

1. Manipulating brain function (e.g. by lesions) and inferring a region’s role in behavior/cognition

2. Manipulating behavior and measuring changes in brain activity

Naturally occurring lesions (stroke) or accidents (e.g. gun wounds etc.)

Broca

Phineas Gage’s lesion reconstructed

(H. Damasio and R. Frank, 1992)

Experimental ablation (removal of brain tissue)

1. Suction of brain tissue

2. Destruction of brain tissue Electrically (general)Chemically (more specific)for example – use of kianic

acid to over-excite cells until they die

3. Temporary lesions (cooling)

Recording of electrical activity / electrical stimulation of brain tissue

Spike trains

Free recall demo for hippocampal re-activation

Gelbard-Sagiv, Mukamel, Harel, Malach & Fried. Science 2008

Quiroga, Mukamel, Isham, Malach & Fried. PNAS 2008

Positron emission tomography (PET) – Enables tracking metabolic processes in various brain

regionsInvolves injection of a radio-active tracerEnables scanning in multiple planes

1. Seeing words2. Listening to words3. Saying words4. Generating verbs

PET-Positron Emission tomography

1 2

3 4

Functional Magnetic Resonance Imaging

What does the BOLD fMRI signal measure?

• Hemoglobin has two states: Oxygenated (diamagnetic), andDe-Oxygenated (paramagnetic)

• The BOLD fMRI signal is sensitive to the ratio between Oxy and De-oxy hemoglobin in a manner that an increase in Oxygenated blood results in an increase in the fMRI signal.

Following neural activation:

Consumption of oxygen

Overcompensation with fresh oxygenated blood

Time

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fMRI signal

Advantages of fMRI over PET

• Higher temporal resolution (seconds vs min)• No radioactive radiation• cheaper