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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
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
slide22

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
slide24

PARKINSON’S DISEASE

SUBSTANTIA NIGRA

Dopamine -

containing

soma

Degenerates in

Parkinson’s Disease

Dopamine

terminal boutons

GLOBUS PALLIDUS

PUTAMEN

Neostriatum

slide25

L-DOPA (taken orally)

Converted to

DOPAMINE

slide26

Pre - Surgery

Neostriatal

neurons

[18F]fluorodopa

uptake - terminal

boutons

Substantia nigra neurons

slide27

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
slide30

Gage et al.

Aged rats are deficient in spatial memory

Morris Water Maze

Rat

day 1

Submerged

platform

“milky water”

Rat day 2

slide31

SEPTUM

Fornix lesion

FORNIX

Spatial

Memory Impairment

HIPPOCAMPUS

slide32

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

slide33

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

slide35

Ridley et al.

1. Marmoset

2. Wisconsin General Testing Apparatus (WGTA)

3. Visuospatial Discrimination Task

slide36

Trial 1

Bread

Choose left

Reward

Correct Choice

slide37

Trial 2

Choose Right

Reward

Correct

Criterian = 27 Correct out of 30 trials

slide38

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

slide39

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)

slide40

DDC in neostriatum

can convert fibroblast

L-dopa to dopamine

Neostriatal neuron

L-dopa

released here

Lose DDC here

TH - fibroblast

Substantia nigra neurons

slide41

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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