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TECHNIQUES ~ TOOLS OF THE TRADE

TECHNIQUES ~ TOOLS OF THE TRADE. TECHNIQUES. NEUROANATOMICAL TECHNIQUES MANIPULATIONS OF THE BRAIN RECORD NEURAL ACTIVITY MEASURE NEUROCHMICALS VISUALIZE THE HUMAN BRAIN BEHAVIORAL PARADIGMS. NEUROANATOMCIAL TECHNIQUES. HISTOLOGICAL PROCEDURES TRACING NEURONAL CONNECTIONS

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TECHNIQUES ~ TOOLS OF THE TRADE

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  1. TECHNIQUES ~ TOOLS OF THE TRADE

  2. TECHNIQUES • NEUROANATOMICAL TECHNIQUES • MANIPULATIONS OF THE BRAIN • RECORD NEURAL ACTIVITY • MEASURE NEUROCHMICALS • VISUALIZE THE HUMAN BRAIN • BEHAVIORAL PARADIGMS

  3. NEUROANATOMCIAL TECHNIQUES • HISTOLOGICAL PROCEDURES • TRACING NEURONAL CONNECTIONS • LOCALIZATION OF NEUROCHEMICALS

  4. HISTOLOGICAL PROCEDURES • FIXATION OF THE BRAIN • soak in formalin ~ embalming solution • hardens tissue • blocks tissue decomposition • CUT THIN SECTIONS OF TISSUE • freeze tissue to -25°C or embed in paraffin • microtome slicing ~ 20-70 m • STAIN BRAIN TISSUE • dyes absorbed by cell bodies or myelin

  5. TRACING NEURONAL CONNECTIONS • ANTEROGRADE LABELING METHODS • AMINO ACID autoradiography • phaseolus vulgaris leukoagglutinin (PHA-L) ~ a plant protein, lectin. • horseradish peroxidase (HRP) • RETROGRADE LABELING METHODS • horseradish peroxidase* • fluorescent dyes: FAST BLUE, NUCLEAR YELLOW AND FLUOROGOLD

  6. LOCALIZATION OF NEUROCHEMICALS • IMMUNOHISTOCHEMISTRY • LOCATE the neurochemicals directly • LOCATE the enzymes which synthesize the neurochemicals

  7. MEASURING NEUROCHEMICALS • MICRODIALYSIS • MICRODISSECTIONS • HIGH PRECISION LIQUID CHROMATOGRAPHY (HPLC)

  8. MANIPULATIONS OF THE BRAIN • LESIONS • ELECTRICAL STIMULATION • CHEMICAL STIMULATION • MICROIONOTOPHORESIS • CANNULATION

  9. MANIPULATIONS OF THE BRAIN • LESIONS • ASPIRATIONS • KNIFE CUTS • RADIO-FREQUENCY, ELECTROLYTIC • NEUROTOXINS • IBOTENIC ACID • NMDA • 6-OHDA • SAPORIN • REVERSIBLE LESIONS • CRYOGENIC BLOCKADE • LIDOCAINE

  10. RECORDING ELECTRICAL ACTIVITYOF THE BRAIN • MICROELCTRODES • MACROELECTRODES • ELECTRO-ENCEPHLOGRAPH (EEG)

  11. ELECTROPHYSIOLOGY ~ STEP BY STEP • INSERT ELECTRODES (or attached EEG electrodes) • AMPLIFY ELECTRICAL SIGNAL • DISPLAY AND STORE CHANGES ON OUTPUT DEVICE • OSCILLOSCOPE • POLYGRAPH • VCR

  12. STEREOTAXIC SURGERGY • STEREOTAXIC APPARATUS • STEREOTAXIC BRAIN ATLAS ~ “brain map” • dorsal/ventral • medial/lateral • anterior/posterior • BREGMA ~ reference point

  13. VISUALIZING THE HUMAN BRAIN • X-RAY CCMPUTED TOMOGRAPHY (a.k.a., COMPUTERIZED AXIAL TOMOGRAPHY OR CAT scan) • POSITRON EMISSION TOMOGRAPHY (PET scan) • MAGNETIC RESONANCE IMAGING (MRI) • FUNCTIONAL MRI (fMRI)

  14. CAT SCANS... • Computer combines a series of contrast X-Rays taken from circling around head to create a CT scan of one 2-D horizontal section of the brain. • 1 regular X-RAY would not work.

  15. POSITRON EMISSION TOMOGRAPHY step by step... • Inject radioactive substance into blood • radioactively labeled 2-deoxy-d-glucose (2-DG) ~ [“false glucose”] is absorbed by “active” neurons and accumulates in cells since it can’t be broken down like normal glucose. • OR • radioactively labeled H2O (hydrogen with O15) carried by  blood flow to “active” neurons.

  16. PET scans ... • Inject radioactive substance • Radioactive substance emits positrons when its atoms disintegrate • Positrons interact with electrons and produce photons of light • Detectors measure the photons • Functional but NO SPATIAL resolution • ? = Baseline state - STATE of INTEREST

  17. MAGNETIC RESONANCE IMAGING (MRI) • High resolution images constructed from measurements of waves that H-atoms emit when activated by radio-frequency waves in a magnetic field. • Higher the density of Hydrogen atoms, the higher the density of tissue.

  18. FUNCTIONAL MAGNETIC RESONANCE IMAGING (fMRI) • Measures changes in blood flow, similar to PET except: • no radioactive substance needs to be injected • combines structural and functional images • spatial resolution is better • monitors changes in neural activity in real time, don’t need to a wait a minute to complete scan, like with PET

  19. NEUROPSYCHOLOGICAL TESTING • WECHSLER ADULT INTELLIGENCE SCALE (WAIS) ~ general abilities • DIGIT SPAN ~ short term memory/attention • PARAGRAPH RECALL ~ STM • BLOCK DESIGN ~ visuospatial ability • WISCONSIN CARD SORT TEST ~ STM/rules • REY-OSTERRIETH FIGURE TEST

  20. ANIMAL BEHAVIOR PARADIGMS • OPEN FIELD TEST • COLONY INTRUDER PARADIGM • ELEVATED PLUS MAZE • SEXUAL BEHAVIOR • PAVLOVIAN CONDITIONING • OPERANT CONDITIONING • SPATIAL MEMORY

  21. Neural Grafting

  22. Anders Bjorkland SEPTUM - Acetylcholine containing neurons Implant embryonic septal neurons here Cut axons Acetylcholine terminal boutons HIPPOCAMPUS Embryonic septal neurons survive and form synapses with hippocampal neurons

  23. Parkinson’s DiseaseSymptoms • Bilateral Rigidity • Bradykinesia Slowness of movement • Resting Tremor • Speech Impairment • Dementia

  24. PARKINSON’S DISEASE SUBSTANTIA NIGRA Dopamine - containing soma Degenerates in Parkinson’s Disease Dopamine terminal boutons GLOBUS PALLIDUS PUTAMEN Neostriatum

  25. L-DOPA (taken orally) Converted to DOPAMINE

  26. Pre - Surgery Neostriatal neurons [18F]fluorodopa uptake - terminal boutons Substantia nigra neurons

  27. Post - Surgery Neostriatal neurons Increased Fluorodopa uptake in fetal substantia nigra neurons Fetal substantia nigra neurons Substantia nigra neurons

  28. Surgical treatment - Parkinson’s Disease • Pallidotomy • Thalamotomy • Deep Brain Stimulation

  29. Implications of Grafting Technique for Reversal of Aging- related Disorders

  30. Gage et al. Aged rats are deficient in spatial memory Morris Water Maze Rat day 1 Submerged platform “milky water” Rat day 2

  31. SEPTUM Fornix lesion FORNIX Spatial Memory Impairment HIPPOCAMPUS

  32. Experimental Groups 1. Young Rats (2-3 mos) - Escape in 17.9 secs 2. Old Unimpaired rats (9-11 mos) - Escape similar to young rats 3. Old Impaired Rats (9-11 mos) - Escape > 38.5 secs - Two subgroups Old Impaired Treated -septal tissue into hippocampus Old Impaired Control - sham surgery, no implant

  33. Gage et al. Motor Coordination in Old Rats 1.Aged rats (21-23 months) 2. Round Bridge Deficit 3. Neostriatal Dopamine Deficiency?? 4. Implant fetal substantia nigra cells into neostriatum 5. Implant fetal septal cells into neostriatum 6. Test 11-14 weeks after implantation

  34. Implications of Grafting Technique for Recovery from Brain Damage

  35. Ridley et al. 1. Marmoset 2. Wisconsin General Testing Apparatus (WGTA) 3. Visuospatial Discrimination Task

  36. Trial 1 Bread Choose left Reward Correct Choice

  37. Trial 2 Choose Right Reward Correct Criterian = 27 Correct out of 30 trials

  38. Ridley’s Design 1. Three unoperated groups 2. Train on two Visuospatial tasks 3. Section fornix in two groups 4. Train all three groups on a third task 5. Implant septal tissue into hippocampus in one of the two fornix-lesioned groups 6. Train on a fourth task

  39. Fisher et al. 1. Harvest skin Fibroblasts from rat 2. Infect with retrovirus containing DNA for rat tyrosine hydroxylase Tyrosine occurs in substantia nigra L-dopa Dopamine Tyrosine hydroxylase(TH) Dopa-decarboxylase (DDC)

  40. DDC in neostriatum can convert fibroblast L-dopa to dopamine Neostriatal neuron L-dopa released here Lose DDC here TH - fibroblast Substantia nigra neurons

  41. 3. Grow in culture medium 4. Assay for release of L-dopa Experiment 1. lesion substantia nigra 2. Measure rotational behavior in three groups of rats a. control- no grafts b. graft with TH fibroblasts c. graft with beta-galactoside fibroblasts

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