IMAGING CELLULAR MECHANISMS OF MIGRAINE

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IMAGING CELLULAR MECHANISMS OF MIGRAINE
Andrew Charles, M.D. Professor Director, Headache
Research and Treatment Program Department of
Neurology UCLA School of Medicine
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Bilateral Spreading Cerebral Hypoperfusion during
Spontaneous Migraine HeadacheRoger P. Woods,
Marco Iacoboni, and John C. MazziottaNEJM
3311689-92, 1994
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Oleson, et al. 1981
Woods et al., 1994
Hadjikhani et al., 2001
Cao et al., 1999
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CELLULAR IMAGING MODALITIES FOR INVESTIGATION OF
MIGRAINE

• Fluorescence imaging
• Cells are loaded or labeled with indicators that
  change their fluorescence intensity in response
  to changes in cellular characteristics (calcium,
  other ions, membrane potential, cellular
  messengers)
• Optical intrinsic signal imaging
• Brain tissue is directly illuminated and light
  that is either reflected or transmitted is
  recorded. Recording at different wavelengths
  emphasizes different components of signal (blood
  volume, hemoglobin oxygenation, change in
  extracellular space, ? mitochondrial swelling,
  etc.)

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POTENTIAL CELLULAR SUBSTRATES FOR PROPAGATED
CORTICAL PHENOMENA IN MIGRAINE

• NEURONS
• GLIAL CELLS
• ASTROCYTES, OLIGODENDROCYTES, MICROGLIA
• VASCULAR CELLS
• ENDOTHELIAL CELLS, PERICYTES, SMOOTH MUSCLE CELLS

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FLUORESCENCE IMAGING OF INTRACELLULAR
Ca2 Cortical Neuronal Culture
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ASTROCYTES

• OUTNUMBER NEURONS BY UP TO 5x
• RATIO OF NEURONS TO ASTROCYTES DIFFERENT IN
  DIFFERENT BRAIN REGIONS (E.G. GREATEST IN
  OCCIPTIAL CORTEX)
• CLOSE RELATIONSHIP WITH SYNAPSE AND WITH
  VASCULATURE
• RECEPTORS FOR A VARIETY OF NEUROTRANSMITTER
  RECEPTORS
• CAPABLE OF EXTENSIVE INTERCELLULAR SIGNALING

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Intercellular Calcium Wave Mixed Glial Culture
Real Time
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Neurotransmitters
Mechanical Stimulation
Low Extracellular Ca
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Ca
2
PLC
Ca
2
V
D
V
D
D
V
D
V
m
m
m
m
IP
IP
IP
3
3
PIP
3
2
IP
Ca
2
IP
3
3
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
?
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
A MODEL FOR GLIAL CALCIUM WAVES
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AN EXTRACELLULAR MESSENGER
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GLIAL CALCIUM WAVES ARE MEDIATED BY RELEASE OF ATP

• LUCIFERASE ASSAY DEMONSTRATES RELEASE OF ATP
• PURINERGIC RECEPTOR ANTAGONISTS BLOCK CALCIUM
  WAVES
• APYRASE (ATP DEGRADING ENZYME) BLOCKS CALCIUM
  WAVES
• DETECTOR CELLS EXPRESSING PURINERGIC RECEPTORS
  RESPOND TO GLIAL CALCIUM WAVES

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GLIAL CALCIUM WAVES

• ASSOCIATED WITH ACTIVE RELEASE OF
• ATP
• GLUTAMATE
• K
• EICOSANOIDS
• LACTATE
• MODULATE
• NEURONAL ACTIVITY VIA TRANSMITTER RELEASE (Araque
  et al., 1999 Fellin et al., 2004)
• NEURONAL/VASCULAR METABOLISM VIA LACTATE RELEASE
  (Paemeleire, 2000 Bernadelli et al., 2004)
• VASCULAR TONE VIA ARACHIDONIC ACID, EICOSANOID
  RELEASE, AND ATP (?) RELEASE (Zonta et al., 2003
  Filosa et al., 2004 Mulligan et al., 2004)

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.
.
Endothelial Cell
Endothelial Cell
Meningeal
Cell
Meningeal
Cell
Neurons
Neurons
Glutamate
ATP Adenosine
ATP Adenosine
Neurotransmitters
Neurotransmitters
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Ca
Ca
K
-
-
ATP
ATP
Mechanical stimulus
Mechanical stimulus
Mg
2
Mg

DAG
PLC
Glutamate
PLC
ATP
Glutamate
ATP
PLC
K
PLC


PIP
PIP
IP
IP
2
2
2
2
Ca
Ca
3
3
IP
IP
PIP
PIP
3
IP
3
IP
2
2
3
3
Connexin Hemichannel
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
2
2
Ca
Ca
Ca
2
Ca
2
?
?
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
Ca
2
2
2
2
2
Ca
Ca
Ca
Ca
2
2
Ca
Ca
Ca
2
Ca
2
Ca
2
Ca
2
ASTROCYTE
ASTROCYTE
ASTROCYTE
ASTROCYTE
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Glial Ca2 Waves in Hippocampal Slice
Culture Time Lapse Sequence of 5 Minute Recording
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DO GLIAL WAVES PLAY A ROLE IN MIGRAINE?

• SIMILAR TEMPORAL AND SPATIAL CHARACTERISTICS AS
  MIGRAINE EVENTS
• ASSOCIATED RELEASE OF ATP, GLUTAMATE, AND OTHER
  NEURO- AND VASO-ACTIVE SUBSTANCES
• ASSOCIATED CHANGES IN EXTRACELLULAR IONIC
  COMPOSITION
• INTIMATE SPATIAL RELATIONSHIPS WITH SYNAPSE AND
  VASCULATURE
• COULD EXPLAIN DRAMATIC PROPAGATION WITH
  RELATIVELY MILD NEUROLOGICAL SYMPTOMS

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ATP RELEASED BY GLIAL CELLS IS RAPIDLY METABOLIZED
TO ADENOSINE, WHICH INHIBITS NEURONS
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ADENOSINE CAUSES RAPID AND REVERSIBLE
INHIBTION OF NEURONAL ACTIVITY
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ADENOSINE INHIBITS SPONTANEOUS ACTIVITY IN
CORTICAL NEURONS
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ADENOSINE AS A NEURO-MODULATOR AND VASO-MODULATOR

• MULTIPLE RECEPTOR SUBTYPES WITH CELL-SPECIFIC
  ACTIONS
• PRIMARILY INHIBITORY EFFECT ON NEURONS
• MAY ACTIVATE GLIAL CELLS
• DILATES PIAL BLOOD VESSELS
• CAFFEINE IS AN ADENOSINE RECEPTOR ANTAGONIST

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HYPOTHESIS
GLIAL CALCIUM WAVES MEDIATE PROPAGATED CORTICAL
PHENOMENA IN MIGRAINE VIA RELEASE OF ATP WHICH IS
METABOLIZED TO ADENOSINE
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GLIAL CALCIUM WAVES AND SPREADING DEPRESSION

• GLIAL CALCIUM WAVES ARE OBSERVED IN CONJUNCTION
  WITH ELECTROPHYSIOLOGICAL SPREADING DEPRESSION IN
  BRAIN SLICES (Basarsky et al., 1998 Kunkler et
  al., 1998 Peters et al., 2003)
• GLIAL CALCIUM WAVES CAN BE DISSOCIATED FROM
  SPREADING DEPRESSION IN THESE PREPARATIONS
• RELATED BUT DISTINCT PHENOMENA WITH INDEPENDENT
  MECHANISMS OF PROPAGATION

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Luis Beltran-Parrazal
K.C. Brennan
Calcium imaging Electrophysiology
OIS Imaging and Electrophysiology Cortical
spreading depression
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OPTICAL INTRINSIC SIGNAL SPREADING DEPRESSION
EVOKED BY KCl PULSE RAT CORTEX (Compressed 5 min
recording)
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Regions of interest
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Arteriolar dilatation precedes parenchymal onset
of CSD
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OIS IMAGING OF CSD

• PROPAGATED WAVE OF CHANGE IN REFLECTANCE WITH AT
  LEAST 4 PHASES
• IMAGING AT DIFFERENT WAVELENGTHS EMPHASIZES
  DIFFERENT CELLULAR/VASCULAR PHENOMENA
• MULTIPHASIC ARTERIAL RESPONSE THAT MAY PRECEDE
  WAVE OF OPTICAL SIGNAL

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HYPOTHESIS
GLIAL CALCIUM WAVES MEDIATE PROPAGATED CORTICAL
PHENOMENA IN MIGRAINE VIA RELEASE OF ATP,
GLUTAMATE, AND K
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Glial Calcium Waves In Hippocampal Slice
Recurrent CSD Evoked By KCL Crystal
SIMILAR TEMPORAL AND SPATIAL PATTERNS OF GLIAL
CALCIUM WAVES AND OIS SIGNAL OF CSD
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CONNEXIN CHANNEL BLOCKER FLUFENAMIC ACID INHIBITS
GLIAL Ca2 WAVES AND ATP RELEASE
IN 50 mM FLUFENAMIC ACID
CONTROL
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FLUFENAMIC ACID REVERSIBLY INHIBITS SPREADING
DEPRESSION
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Control
CGS 15943 10 mM
AN ADENOSINE RECEPTOR ANTAGONIST (CGS15943)
INHIBITS THE VASCULAR RESPONSE TO CSD
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CGS 15943 attenuates vascular reactivity during
CSD
250 µm
250 µm
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RELATED PHENOMENA

• GLIAL CALCIUM WAVES
• SPREADING DEPRESSION
• SPREADING HYPER/OLIGEMIA OF MIGRAINE SEEN ON PET,
  fMRI
• CLINICAL MANIFESTATIONS OF MIGRAINE AURA

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NEURONAL ACTIVATION
GLIAL WAVES?
GLUTAMATE RELEASE
ATP RELEASE (?)
INCREASED K
SPREADING DEPRESSION
SPREADING HYPER/ OLIGEMIA
BRAINSTEM ACTIVATION
PEPTIDERELEASE
VESTIBULAR SYMPTOMS
PAIN
AURA
Nausea
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CONCLUSIONS

• MICROSCOPIC IMAGING REVEALS MULTIPLE POTENTIAL
  CELLULAR COMPONENTS OF MIGRAINE
• GLIAL CELLS MAY PLAY AN IMPORTANT ROLE
• INDIVIDUAL MIGRAINE PHENOMENA MAY INVOLVE
  DISTINCT CELLULAR SIGNALING PATHWAYS WITH
  DISTINCT THRESHOLDS

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ACKNOWLEDGEMENTS

• CHARLES LAB
• Marni Harris-White
• Luis Beltran-Parrazal
• K.C. Brennan
• UCLA LABORATORY OF NEUROIMAGING
• Arthur Toga
• Michael Guiou
• MICHAEL SANDERSON - University of Massachussetts