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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

TECHNIQUES ~ TOOLS OF THE TRADE


Techniques
TECHNIQUES

  • NEUROANATOMICAL TECHNIQUES

  • MANIPULATIONS OF THE BRAIN

  • RECORD NEURAL ACTIVITY

  • MEASURE NEUROCHMICALS

  • VISUALIZE THE HUMAN BRAIN

  • BEHAVIORAL PARADIGMS


Neuroanatomcial techniques
NEUROANATOMCIAL TECHNIQUES

  • HISTOLOGICAL PROCEDURES

  • TRACING NEURONAL CONNECTIONS

  • LOCALIZATION OF NEUROCHEMICALS


Histological procedures
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


Tracing neuronal connections
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


Localization of neurochemicals
LOCALIZATION OF NEUROCHEMICALS

  • IMMUNOHISTOCHEMISTRY

    • LOCATE the neurochemicals directly

    • LOCATE the enzymes which synthesize the neurochemicals


Measuring neurochemicals
MEASURING NEUROCHEMICALS

  • MICRODIALYSIS

  • MICRODISSECTIONS

  • HIGH PRECISION LIQUID CHROMATOGRAPHY (HPLC)


Manipulations of the brain
MANIPULATIONS OF THE BRAIN

  • LESIONS

  • ELECTRICAL STIMULATION

  • CHEMICAL STIMULATION

    • MICROIONOTOPHORESIS

    • CANNULATION


Manipulations of the brain1
MANIPULATIONS OF THE BRAIN

  • LESIONS

    • ASPIRATIONS

    • KNIFE CUTS

    • RADIO-FREQUENCY, ELECTROLYTIC

    • NEUROTOXINS

      • IBOTENIC ACID

      • NMDA

      • 6-OHDA

      • SAPORIN

  • REVERSIBLE LESIONS

  • CRYOGENIC BLOCKADE

  • LIDOCAINE


  • Recording electrical activityof the brain
    RECORDING ELECTRICAL ACTIVITYOF THE BRAIN

    • MICROELCTRODES

    • MACROELECTRODES

    • ELECTRO-ENCEPHLOGRAPH (EEG)


    Electrophysiology step by step
    ELECTROPHYSIOLOGY ~ STEP BY STEP

    • INSERT ELECTRODES (or attached EEG electrodes)

    • AMPLIFY ELECTRICAL SIGNAL

    • DISPLAY AND STORE CHANGES ON OUTPUT DEVICE

      • OSCILLOSCOPE

      • POLYGRAPH

      • VCR


    Stereotaxic surgergy
    STEREOTAXIC SURGERGY

    • STEREOTAXIC APPARATUS

    • STEREOTAXIC BRAIN ATLAS ~ “brain map”

      • dorsal/ventral

      • medial/lateral

      • anterior/posterior

    • BREGMA ~ reference point


    Visualizing the human brain
    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)


    Cat scans
    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.


    Positron emission tomography step by step
    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.


    Pet scans
    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


    Magnetic resonance imaging mri
    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.


    Functional magnetic resonance imaging fmri
    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


    Neuropsychological testing
    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


    Animal behavior paradigms
    ANIMAL BEHAVIOR PARADIGMS

    • OPEN FIELD TEST

    • COLONY INTRUDER PARADIGM

    • ELEVATED PLUS MAZE

    • SEXUAL BEHAVIOR

    • PAVLOVIAN CONDITIONING

    • OPERANT CONDITIONING

    • SPATIAL MEMORY



    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


    Parkinson s disease symptoms
    Parkinson’s DiseaseSymptoms

    • Bilateral Rigidity

    • Bradykinesia

      Slowness of movement

    • Resting Tremor

    • Speech Impairment

    • Dementia


    PARKINSON’S DISEASE

    SUBSTANTIA NIGRA

    Dopamine -

    containing

    soma

    Degenerates in

    Parkinson’s Disease

    Dopamine

    terminal boutons

    GLOBUS PALLIDUS

    PUTAMEN

    Neostriatum


    L-DOPA (taken orally)

    Converted to

    DOPAMINE


    Pre - Surgery

    Neostriatal

    neurons

    [18F]fluorodopa

    uptake - terminal

    boutons

    Substantia nigra neurons


    Post - Surgery

    Neostriatal

    neurons

    Increased Fluorodopa

    uptake in fetal substantia nigra neurons

    Fetal substantia nigra

    neurons

    Substantia nigra neurons


    Surgical treatment parkinson s disease
    Surgical treatment - Parkinson’s Disease

    • Pallidotomy

    • Thalamotomy

    • Deep Brain Stimulation



    Gage et al. related Disorders

    Aged rats are deficient in spatial memory

    Morris Water Maze

    Rat

    day 1

    Submerged

    platform

    “milky water”

    Rat day 2


    SEPTUM related Disorders

    Fornix lesion

    FORNIX

    Spatial

    Memory Impairment

    HIPPOCAMPUS


    Experimental Groups related Disorders

    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


    Gage et al. related Disorders

    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



    Ridley et al. Damage

    1. Marmoset

    2. Wisconsin General Testing Apparatus (WGTA)

    3. Visuospatial Discrimination Task


    Trial 1 Damage

    Bread

    Choose left

    Reward

    Correct Choice


    Trial 2 Damage

    Choose Right

    Reward

    Correct

    Criterian = 27 Correct out of 30 trials


    Ridley’s Design Damage

    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


    Fisher et al. Damage

    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)


    DDC in neostriatum Damage

    can convert fibroblast

    L-dopa to dopamine

    Neostriatal neuron

    L-dopa

    released here

    Lose DDC here

    TH - fibroblast

    Substantia nigra neurons


    3. Grow in culture medium Damage

    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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