Ian Parker Department of Neurobiology

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Ian ParkerDepartment of Neurobiology Behavior,
UC Irvine
Imaging Calcium Signals in Neurons and Surrogate
Neurons From Single Channels to Alzheimers
Disease

• UCR Jan 20, 2004

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Why study intracellular Ca2 signaling?

• Its important!

Serves numerous functions in virtually all
cells. A life and death signal
Berridge et al., 1998
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2. Its simple!
3. We can see it!
Fluorescent calcium indicators let us visualize
intracellular Ca2 signals with mm and ms
resolution

• Unlike other second messengers Ca2 is an
  element. All a cell can do is move it from one
  place to another.

Information is encoded by the kinetics of a Ca2
signal, and the spatial localization of the Ca2
ions.
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On the importance of looking
You can observe a lot just by watching.
Yogi Berra Former Yankee Catcher andGreat
American Sage
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What am I going to be talking about?(you get the
taster menu)
1. Imaging single Ca2 channels total internal
reflection microscopy 2. Shaping of
intracellular Ca2 signals by buffers and Ca2
-binding proteins confocal microscopy caged
IP3 3. Ca2 signals in cortical neurons
involvement in Alzheimers disease 2-photon
imaging
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• 1. Imaging single Ca2 channels

Angelo Demuro
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Motivations to develop functional single-channel
Ca2 imaging
1. To provide a complimentary technique to
electrophysiological patch-clamp
recording. Patch-clamping has limitations
including - lack of spatial information regarding
channel location inability to obtain
simultaneous, independent recordings from
multiple channels need for physical access of
pipette possible disruption of channel function
during seal formation. 2. To image the
spatial and temporal distribution of
cytosolic Ca2 around an open Ca2 channel.
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Cloud of Ca2 in cytosol around the mouth of
an open Ca2 channel
Very low (50 nM) resting Ca2 in cytosol
High (6 mM) extracellular Ca2
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How much Ca2 goes through a single channel?Can
we detect it?

• Say single channel Ca2 current i 0.1 pA
  channel open duration 20 ms
• Then charge transfer 10-15 Coulombs
• So amount of Ca2 that moves 10-20 moles (10 z
  mol) or 6000 ions
• Assume 10 of Ca2 ions bind to fluorescent
  indicator
• So 600 dye/Ca2 molecules
• Each dye molecule can be excited to emit 103
  photons ms-1 (lt10 saturation)
• So 6 x 105 photons ms-1
• Even given poor (a few ) collection efficiency
  of microscope, this is plenty to work with.

Note the amplification provided by optical
recording Electrophysiological recording - 1
Ca2 ion 2 electron charges Optical recording
- 1 Ca2 ion gt100 photons
ms-1
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Ca2 signals are large and fast near the
channelmouth, but small and slow only 1 mm
away. So, to get a faithful record of channel
gating, we need to record local, near-membrane
signal.
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Total Internal Reflection Fluorescence
Microscopy(TIRFM)The evanescent wave formed by
refraction at a glass/water interface extends
only a few hundred nm into the aqueous phase.
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Cultured cells expressing GFP-tagged membrane
protein imaged by conventional epifluorescence
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The same cells viewed by TIRFM
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TIRFM imaging of single-channel Ca2 signals
Ca2 entry through voltage-gated N-type channels
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Local Ca2 signals (sparklets) resulting from
stochastic, voltage-gated openings of single Ca2
channels
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Optical single-channel records
Independent, simultaneous measurements from 40
channels
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Local Ca2 signals (sparklets) arise from Ca2
influx through individual N-type channels
1. Sparklets absent in control oocytes not
expressing N- type channels.
4. Voltage dependence of fluorescence matches
that expected for influx of extra- cellular Ca 2
2. Magnitude of fluorescence signals consistent
with single channel current 0.1 pA.
5. Sparklets are blocked by 2 mM Cd2
3. Stochastic, pulsatile nature of sparklets
consistent with channel openings
Control Cd2
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Optical images can provide kinetic data about
channel gating analogous to patch-clamp
measurements
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AND, single channel imaging can give information
that patch-clamping cannot.E.g. - Variability
in density and properties of channels
Membrane patches 200 mm apart in the same
oocyte showing very different channel densities
Variation in opening frequency among channels.
Colors show mean sparklets per depolarizing
pulse
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Simultaneous recordings show variation in opening
probability among channels within the same
membrane region
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Restricted mobility of channels in membrane
Diffusion coefficient for free motility of a
protein in membrane D 3 mm2 s-1 So mean
displacement after 270s would be 55 mm sqrt
(4 D 270)
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Conclusions optical single channel recording
vs. patch-clamp
Pro Less invasive Simultaneous, independent
monitor of many channels Spatial mapping of
channels Adaptable for high-throughput
screening? Con No absolute measure of current
(flux) magnitude Less good time
resolution Restricted to Ca2-permeable channels
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2. Buffer kinetics shape the spatio-temporal
patterns of IP3-evoked Ca2 signals in Xenopus
oocytes
Sheila Dargan

• Sheila Dargan

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Different cells (esp. neurons) express a wide
range of different Ca2 buffering proteins with
varying kinetics (ON and OFF rates for Ca2
binding)

• Why??

Most previous studies have considered buffer
actions on a fixed slug of Ca2 entering the
cytosol (e.g. Ca2 influx through voltage-gated
channels)
But, situation will be more complex for Ca2
signals generated by flux through channels that
are themselves Ca2 sensitive (e.g. IP3R and
RyR). We studied the influence of buffer
kinetics on IP3-mediated Ca2 signaling in
Xenopus oocytes by microinjecting two exogenous
buffers (EGTA and BAPTA) with similar affinities
but very different kinetics, and compared two
CaBP with fast (calretinin) and slow binding
kinetics (parvalbumin)
apparent affinity (nM)
ON rate (mM-1 s-1) OFF rate (s-1) EGTA
(slow) 150 3 10 0.5 1.5 BAPTA
(fast) 160 100 1000 16 - 160
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IP3 receptors are clustered on ER, so Ca2
interactions can take place on 2 different
distance and time scales
Local (tens of nm) scale between IP3R to
generate Ca2 puffs
Longer range (a few mm) interactions between
clusters to propagate Ca2 waves
Buffers with fast binding kinetics may disrupt
receptor-receptor interactions, while slow
buffers primarily affect cluster-cluster
interactions
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Video-Rate Confocal Microscope
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Experimental methods

• Fast (ms), high-resolution (ca. 250 nm) imaging
  in Xenopus oocytes of intracellular calcium
  signals using fluorescent indicator dyes and
  confocal microscopy
• Precise control of intracellular IP3 by
  photorelease from caged IP3.

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Local and global Ca2 signals co-exist under
physiological conditions
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Slow buffers shorten the duration of IP3-evoked
Ca2 transients, whereas fast buffers prolong
them.
Linescan images
distance
time
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Slow buffers dissociate global IP3-evoked Ca2
responses (waves) into spatially localized events
(puffs).
Fast buffers do not
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Ca2 buffers with differing kinetics
differentially modulate the amplitude of
IP3-evoked Ca2 signals.
slow buffers
fast buffers
Normalized concentration of 500mM Ca2 binding
sites per Ca2 buffer / CaBP
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A qualitative model to explain how the kinetics
of mobile buffers shape IP3-evoked Ca2 signals
Diffusion of Ca2 ions is normally slowed by
immobile buffers in the cytosol
Slow buffers cannot bind Ca2 ions diffusing
short distances between IP3R within a cluster, so
have little effect on CICR underlying puffs. But,
they bind Ca2 ions diffusing between clusters,
and shuttle them away, functionally uncoupling
clusters.
Fast buffers bind Ca2 ions diffusing within a
cluster to inhibit puffs and facilitate
inter-cluster communication to generate diffuse
Ca2 signals.
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Conclusions
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Two-photon imaging in brain slices Involvement
of disrupted Ca2 signaling in Alzheimers disease
Grace Stutzmann
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What is 2-photon microscopy?

1. Near simultaneous absorption of the energy of two
   infrared photons results in excitation of a
   fluorochrome that would normally be excited by a
   single photon of twice the energy.
2. The probability of excitation depends on the
   square of the infrared intensity and decreases
   rapidly with distance from the focal volume.

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And what can it do for me?

• Tissue imaging depth in hundreds of microns
• Excitation of fluorophore only in focal plane
• Reduced phototoxicity and photobleaching
• -Submicron resolution at video rate acquisition
  speeds

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Video-rate 2-photon microscope
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3-D reconstruction of 2-photon images of
DSred-labelled neuron
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Studying IP3-mediated signaling in mouse cortical
pyramidal neurons using Whole-cell patch clamp
fast 2-photon Ca2 imaging
caged IP3


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Spikes
7ms flash
30ms flash
soma dendrites
soma dendrites
dendrites soma
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IP3- and spike-evoked Ca2 signals
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IP3-evoked Ca2 signals activate a K conductance
that inhibits spike firing
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Ca2 hypothesis of AD (Khachaturian, 1989)
cellular mechanisms, which maintain the
homeostasis of cytosol Ca2 concentration, play a
key role in brain aging and that sustained
changes in Ca2i homeostasis provide the final
common pathway for age-associated brain changes
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Alzheimers disease is categorized in two forms
Famililal (1) Mutations in presenilin and APP
genes (autosomal dominant) early-onset
(30s!) Genetic testing Same phenotype as
sporadic
Sporadic (99) No known etiology late-onset
(over 65 yrs.) Post-mortem diagnosis Beta amyloid
plaques, tangles
Signal transduction Transcription Ionic
conductance Electrical excitability Apoptosis Etc.
..
Gq-coupled receptor
5-HT Ach Glu
extracellular
intracellular
IP3
Ca2

IP3 receptor
Presenilin
Ca2
Ca2
Ca2
Ca2
ER
Ca2
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PS1M146V knock-in mouse
Guo et al., 1996
-No overt mutant phenotype -Increased
susceptibility to oxidative stress and
apoptosis -Fibroblasts show enhanced Ca2
response to bradykinin -Lower threshold/enhanced
hippocampal LTP (field potentials)
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PS1 mutation specifically enhances
IP3-mediated Ca 2 signals in cortical neurons
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Conclusions
1). Alzheimers disease-causing mutations in PS1
enhance IP3-evoked Ca2 responses in cortical
neurons. 2). The PS-mediated enhancement of
Ca2 signals appears specific to intracellular
stores. 3). Ca2 liberation from
IP3sensitive stores suppresses membrane
excitability by activating outward membrane
currents.
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The End!
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Kinetic resolutionWith TIR presently limited by
speed of camera (33 ms per frame). But with fast
linescan confocal microscopy can resolve rise
times of 4 ms, and post-process records to
partially correct for distortions
Kinetic resolution of channel openings and
closings Correction for Baseline Ca2
accumulation High frequency rolloff
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Comparison of effects of buffers on Ca2 signals
arising from Ca2 entry through voltage-gated
channels, and IP3-evoked Ca2 liberation
Decay of IP3 -evoked Ca2 signals is slow (in
control conditions) because of lingering Ca2
liberation, not because Ca2 clearance from
cytosol is slow. EGTA speeds decay by inhibiting
this prolonged Ca2 liberation, not by slowly
complexing Ca2 already released into the
cytosol.
Ca2 entry through voltage-gated channels
IP3-evoked Ca 2 liberation
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Voltage-dependence of Ca2 influx and channel
gating
Biphasic voltage-dependence of total Ca2 influx
reflects both
Single-channel Ca2 flux decreasing at more
positive voltages due to decreased electrical
driving force
Increase in N-type channel open probability with
increasingly positive voltage