Transcript TECHNIQUES2001

TECHNIQUES ~ TOOLS OF
THE TRADE
TECHNIQUES
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NEUROANATOMICAL TECHNIQUES
MANIPULATIONS OF THE BRAIN
RECORD NEURAL ACTIVITY
MEASURE NEUROCHMICALS
VISUALIZE THE HUMAN BRAIN
BEHAVIORAL PARADIGMS
NEUROANATOMCIAL
TECHNIQUES
• HISTOLOGICAL
PROCEDURES
• TRACING
NEURONAL
CONNECTIONS
• LOCALIZATION OF
NEUROCHEMICALS
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
• 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
• IMMUNOHISTOCHEMISTRY
– LOCATE the neurochemicals directly
– LOCATE the enzymes which synthesize the
neurochemicals
MEASURING
NEUROCHEMICALS
• MICRODIALYSIS
• MICRODISSECTIONS
• HIGH PRECISION LIQUID
CHROMATOGRAPHY (HPLC)
MANIPULATIONS OF THE
BRAIN
• LESIONS
• ELECTRICAL STIMULATION
• CHEMICAL STIMULATION
» MICROIONOTOPHORESIS
» CANNULATION
MANIPULATIONS OF THE
BRAIN
• LESIONS
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ASPIRATIONS
KNIFE CUTS
RADIO-FREQUENCY, ELECTROLYTIC
NEUROTOXINS
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IBOTENIC ACID
NMDA
6-OHDA
SAPORIN
– REVERSIBLE LESIONS
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CRYOGENIC BLOCKADE
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LIDOCAINE
RECORDING ELECTRICAL
ACTIVITYOF THE BRAIN
• MICROELCTRODES
• MACROELECTRODES
• ELECTROENCEPHLOGRAPH
(EEG)
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 APPARATUS
• STEREOTAXIC BRAIN ATLAS ~ “brain
map”
– dorsal/ventral
– medial/lateral
– anterior/posterior
• BREGMA ~ reference point
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...
• Computer combines a series of contrast XRays 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...
• Inject radioactive substance into blood
– radioactively labeled 2-deoxy-d-glucose (2DG) ~ [“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 ...
 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)
• 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)
• Measures changes in blood flow, similar to
PET except:
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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
• 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
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OPEN FIELD TEST
COLONY INTRUDER PARADIGM
ELEVATED PLUS MAZE
SEXUAL BEHAVIOR
PAVLOVIAN CONDITIONING
OPERANT CONDITIONING
SPATIAL MEMORY
Neural Grafting
Anders Bjorkland
Acetylcholine containing
neurons
SEPTUM
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
• Bilateral Rigidity
• Bradykinesia
Slowness of movement
• Resting Tremor
• Speech Impairment
• Dementia
PARKINSON’S DISEASE
SUBSTANTIA NIGRA
Degenerates in
Parkinson’s Disease
Dopamine containing
soma
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
• Pallidotomy
• Thalamotomy
• Deep Brain Stimulation
Implications of Grafting Technique
for Reversal of Aging- related
Disorders
Gage et al.
Aged rats are deficient in spatial memory
Morris Water Maze
Rat
day 1
“milky water”
Submerged
platform
Rat day 2
SEPTUM
Fornix lesion
FORNIX
Spatial Memory Impairment
HIPPOCAMPUS
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
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
Ridley et al.
1. Marmoset
2. Wisconsin General Testing Apparatus (WGTA)
3. Visuospatial Discrimination Task
Trial 1
Bread
Choose left
Reward
Correct Choice
Trial 2
Choose Right
Reward
Correct
Criterian = 27 Correct out of 30 trials
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
Fisher et al.
1. Harvest skin Fibroblasts from rat
2. Infect with retrovirus containing DNA for
rat tyrosine hydroxylase
Tyrosine
occurs in
substantia
nigra
Tyrosine hydroxylase(TH)
L-dopa
Dopa-decarboxylase
(DDC)
Dopamine
Neostriatal neuron
Lose DDC here
DDC in neostriatum
can convert fibroblast
L-dopa to dopamine
L-dopa
released here
TH - fibroblast
Substantia nigra neurons
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