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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

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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


    Techniques tools of the trade

    Neural Grafting


    Techniques tools of the trade

    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


    Techniques tools of the trade

    PARKINSON’S DISEASE

    SUBSTANTIA NIGRA

    Dopamine -

    containing

    soma

    Degenerates in

    Parkinson’s Disease

    Dopamine

    terminal boutons

    GLOBUS PALLIDUS

    PUTAMEN

    Neostriatum


    Techniques tools of the trade

    L-DOPA (taken orally)

    Converted to

    DOPAMINE


    Techniques tools of the trade

    Pre - Surgery

    Neostriatal

    neurons

    [18F]fluorodopa

    uptake - terminal

    boutons

    Substantia nigra neurons


    Techniques tools of the trade

    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


    Implications of grafting technique for reversal of aging related disorders

    Implications of Grafting Technique for Reversal of Aging- related Disorders


    Techniques tools of the trade

    Gage et al.

    Aged rats are deficient in spatial memory

    Morris Water Maze

    Rat

    day 1

    Submerged

    platform

    “milky water”

    Rat day 2


    Techniques tools of the trade

    SEPTUM

    Fornix lesion

    FORNIX

    Spatial

    Memory Impairment

    HIPPOCAMPUS


    Techniques tools of the trade

    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


    Techniques tools of the trade

    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


    Implications of grafting technique for recovery from brain damage

    Implications of Grafting Technique for Recovery from Brain Damage


    Techniques tools of the trade

    Ridley et al.

    1. Marmoset

    2. Wisconsin General Testing Apparatus (WGTA)

    3. Visuospatial Discrimination Task


    Techniques tools of the trade

    Trial 1

    Bread

    Choose left

    Reward

    Correct Choice


    Techniques tools of the trade

    Trial 2

    Choose Right

    Reward

    Correct

    Criterian = 27 Correct out of 30 trials


    Techniques tools of the trade

    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


    Techniques tools of the trade

    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)


    Techniques tools of the trade

    DDC in neostriatum

    can convert fibroblast

    L-dopa to dopamine

    Neostriatal neuron

    L-dopa

    released here

    Lose DDC here

    TH - fibroblast

    Substantia nigra neurons


    Techniques tools of the trade

    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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