VII. Calcium signaling pathways (Uconjoint 532, Storm, Nov 22, 2013).
One of the most important signaling molecules in all eukaryotic cells is intracellular Ca2+. Ca2+controls
secretion, metabolism, muscle contraction cell shape, growth, proliferation, cell survival, synaptic plasticity in the
CNS and transcription. Like cAMP, increases in intracellular are usually transient and highly localized.
Cross Talk
Slide, cAMP and calcium crosstalk: There is extensive signal transduction cross-talk between calcium and
other signaling pathways including the Erk MAP kinase pathway and cAMP in most animal cells.
In animal cells changes in intracellular cAMP almost always affect intracellular free calcium ( eg PKA regulation
of Voltage-Sensitive calcium channels and cAMP stimulation of CNG). Conversely, changes in intracellular
calcium normally affect intracellular cAMP by stimulation or inhibition of adenylyl cyclases and
phosphodiesterases.
Mechanisms for increasing intracellular free Ca2+:
Slide, intracellular vs extracellular free caclium: Intracellular free Ca2+ in unstimulated cells is usually about
100 nM. In fact, prolonged increases in intracellular Ca2+ are toxic to most cells. This low Ca2+ is maintained
by various membrane associated Ca2+ pumps that pump Ca2+ outside of the cell, into the endoplasmic
reticulum or into mitochondria. Extracellular Ca2+ concentration is typically several mM. When cells are
activated, intracellular Ca2+ can increase from 0.1 µM to 10 µM or even to levels are high as several hundred
µM in subcellular compartments. Like cAMP, Ca2+ increases are transient and prolonged increases in Ca2+
can be toxic for animal cells.
Slide, local free calcium: The calcium signals within a cell can be quite local. Free calcium generated around
calcium channels is quickly bound to various calcium binding proteins leading to steep calcium transients. Local
concentration can be extremely high reaching localized transients as high as 400 µM.
Slide, control of intracellular free calcium:
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Mechanisms for increasing Ca2+ inside the cell include:
1. Calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate-type
receptors
2. N-methyl-D-aspartate (NMDA) glutamate-type receptors
3. Voltage-gated calcium channels (VGCC)
4. Nicotinic acetylcholine receptors (nAChR)
5. transient receptor potential type C (TRPC) channels.
6. Calcium release from internal stores mediated by inositol trisphosphate receptors (IP3R) and
ryanodine receptors (RyR). Ca2+ is a major physiological ligand that triggers opening of RyRs, but
there are modulatory proteins and small molecules in the cytoplasm and endoplasmic reticulum
lumen that stimulate RyRs.
7. Inositol trisphosphate can be generated by metabotropic glutamate receptors (mGluR) and other
receptors that are coupled to PLC.
Calcium efflux is mediated by:
1. The plasma membrane calcium ATPase (PMCA)
2. The sodium-calcium exchanger (NCX)
3.
The sarco-/endoplasmic reticulum calcium ATPase (SERCA). Also the mitochondria are important
for neuronal calcium homeostasis
Receptors Coupled to Phospholipase C and Generation of IP3
Slide, receptors, PLC and IP3: There are a number of membrane receptors including metabotropic-1
glutamate receptors, alpha-1 adrenergic receptors, calcitonin receptors, H-I histamine receptors as well as M1,
M3, and M5 muscarinic receptors that are coupled to the enzyme phospholipase C through Gq-alpha. PLC
hydrolyzes the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol
1,4,5-trisphosphate (IP3). DAG remains bound to the membrane, and IP3 is released into the cytosol. IP3
diffuses through the cytosol to bind to IP3 receptors coupled to calcium channels in the endoplasmic reticulum
(ER). This causes the cytosolic concentration of calcium to increase, causing a cascade of intracellular changes
and activity. In addition, calcium and DAG together work to activate protein kinase C.
Slide, structures of PIP2, IP3 and DAG:
For those of you interested in the chemical structures involved…here are the structures of PIP2, IP3 and DAG.
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Slide, model slide IP3 PKC:
The free calcium released from the ER can activate a number of downstream effectors including activation of
isozymes of PKC in combination with the DAG released from PIP2.
Slide, ER pump, calmodulin
Ca2+ is pumped back into the ER by a calcium ATPase and the free calcium liberated through IP3
receptors can activate a number of enzymes and protein systems through the calcium binding
protein, calmodulin.
Also IP3 receptors are regulated by PKA, PKC, and calmodulin kinase II (CaMK-II). In addition,
high calcium can inhibit IP3 receptors.
Calmodulin
Slide, CaM properties:
There are a number of Ca2+ binding proteins that mediate the effects of intracellular
Ca2+increases. One of the most important Ca2+ binding proteins is CaM which is found in all animal cells but
particularly abundant in the central nervous system.
Slide, calcium to CaM increased affinity for most proteins:
CaM binds four Ca2+ ions. This enhances its affinity for its target proteins. The binding of CaM to its target
proteins changes their activities. In most cases, binding of CaM to enzymes stimulates their activities
Slide, Neuromodulin is an exception:
There is one calmodulin binding protein which is different”
1. Neuromodulin is an abundant calmodulin binding protein that is localized in growth cones and synapses of
growing neurons.
2. Neuromodulin binds calmodulin in the absence of calcium and localizes calmodulin within growth cones and
synapses.
3. Neuromodulin releases calmodulin when calcium goes up and when it is phosphorylated within its IQ domain
by PKC, the site for calmodulin binding
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Slide, Neuromodulin model:
It is hypothesized that neuromodulin binds and localizes calmodulin at specific sites in the cell and release free
calmodulin locally when calcium increases and PKC is activated.
Slide, calmodulin hydrophobic domain:
When Ca2+ binds to CaM it undergoes a conformational change which exposes a hydrophobic domain. This
hydrophobic is a major component of the CaM/ protein interface There are a number of hydrophobic drugs, e.g.
the phenothiazines, that antagonize CaM-regulated systems by binding to CaM in the presence of Ca2+,
thereby blocking interactions with CaM binding proteins.
Slide, fluorescent dyes and calmodulin:
This was originally detected using a series of hydrophobic fluorescent dyes that only bind to CaM when Ca2+ is
bound to it. Several classes of fluorescent probes capable of sensing exposure of hydrophobic binding sites on
proteins were found to bind to CaM, and these interactions were greatly enhanced by Ca2+. In the presence of
Ca2+, the fluorescence intensity of 9-anthroylcholine (9AC) was increased 24-fold by CaM, with a shift in the
fluorescence emission maximum from 514 to 486 nm. The fluorescence intensity of 8-anilino-1naphthalenesulfonate (Ans) was enhanced 27-fold with an emission maximum shift from 540 to 488 nm in the
presence of CaM and Ca2+. Similar results were obtained with the uncharged fluorescent ligand, N-phyenyl-1naphthylamine. This hydrophobic domain on calmodulin is its interface with CaM-binding proteins.
Slide, structure of calmodulin:
Calmodulin has four EF-hand calcium binding sites that are separated by a flexible domain between
EF1/EF2 and EF3/EF4.
Slide, binding of calmodulin to a target proteins:
It is hypothesized that when calcium binds to the four calcium binding sites on calmodulin that it’s
hydrophobic domain wraps around the calmodulin domain of the target protein.
Slide, calmodulin binding proteins:
There are a large number of CaM regulated proteins including ACs, PDEs, protein kinases, transport systems,
and components of the cytoskelton.
a. Enzymes
Cyclic nucleotide phosphodiesterases (e.g. the PDE1 family)
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Adenylyl cyclases (AC1 and AC8)
ATP-dependent Ca2+ pumps (discovered by Frank Vincenzi at the UW)
myosin light chain kinase
CaM kinase I, II and IV
phosphorylase kinase
calcineurin (a protein phosphatase)
NO synthase
b. Non enzymatic proteins
RasGRF1, a CaM-activated GEF
Tubulin
Troponin I
Spectrin, fodrin, caldesmon, calspectin, cytosynalin
MAP-2 and Tau
neuromodulin and neurogranin
Ca2+ channels
Slide, calcium binding to calmodulin:
On initial inspection, CaM does not seem to have the Ca2+ binding properties that would explain the Ca2+
activation curves exhibited by CaM-regulated enzymes.
1. The affinity of CaM for Ca2+ is about 10 to 15 µM and the window of free Ca2+ during signaling is at much
lower levels of Ca2+.
2. The Ca2+ sensitivities of the CaM regulated enzymes vary over a large range. How could one Ca2+ binding
protein mediate Ca2+ stimulation over such a wide range of Ca2+ concentrations.
3. The Ca2+ activation curves of some of the enzymes are positively cooperative. Binding of Ca2+to CaM does
not show strong positive cooperativity.
When the energetics of Ca2+/CaM/protein complexes was examined a little more closely
we find that CaM is ideally designed to mediate Ca2+ regulation of a large number of different proteins because
of heterotropic positive cooperativity. This arises from multiple Ca2+ binding sites on CaM and heterologous
interactions between CaM and its target proteins.
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Because binding of Ca2+ enhances the affinity of CaM for its target proteins, it follows from the laws of
thermodynamics that CaM binding proteins must enhance the affinity of CaM for Ca2+. This is illustrated by
looking at Ca2+ binding to CaM in the absence and presence of a target protein (e.g. tropnin I). Since the
affinity of CaM for Ca2+ is enhanced by binding to the target protein, this means that Ca2+ activation curves for
the CaM/protein complexes are at much lower Ca2+ than Ca2+ binding to CaM alone.
Slide, energy coupling: This increase in Ca2+ affinity is due to energy coupling between the two proteins
which is different for each of the calmodulin/ protein complexes. The energy coupling is due to the specificity of
each CaM/ protein interface and is dependent upon the binding of four Ca2+ ions to the CaM/proteins
complexes
Slide, Ca2+ binding curves for several CaM-binding proteins:
Consequently, if you examine the Ca2+binding curves to CaM in the presence of a number of different CaM
binding proteins, you see different Ca2+ dependency, even though you use the same Ca2+ binding protein.
(Slide: Positive cooperativity) The other interesting feature of CaM regulated systems is that Ca2+stimulation
of enzymes through CaM is strongly positively cooperative, even though Ca2+ binding to CaM itself shows low
cooperativity.
As Ca2+ starts binding to CaM and fills one or two of the four this enhances the affinity of CaM for the
target enzyme, which leads to complex formation. The binding of subsequent Ca2+ to this complex is with
higher affinity and you thereby generate positive heterotropic cooperativity. ie steep activation curves.
Thus the unique properties of CaM allow:
1. Ca2+ activation of enzymes over the physiological Ca2+ range (0.1 to 10 µM).
2. Different Ca2+ activation curves for each enzyme, even though the same Ca2+ binding protein is used
throughout.
3. Positive cooperativity allowing enzymes and proteins to be activated over narrow Ca2+ ranges and finely
regulated.
Methods for Monitoring Intracellular Free Calcium
Slide, summary of calcium indicators:
Calcium Indicators.
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1. Bioluminescent protein. Binding of calcium ions to aequorin leads to the oxidation of the prosthetic
group coelenterazine (C, left side) to coelenteramide, (C, right side). Colenteramide relaxes to the
ground state while emitting a photon of 470 nm. Aequorin has three calcium binding sites.
Advantages:
a. This indicator has a high signal to noise ratio.
b. Aequorin can monitor calcium changes from 10-7 to 10-3
c. Does not require a external illumination
Disadvantages
a. Each molecule performs only one emission cycle and the
recharging process is slow
b. Has to be loaded to cells with a micro-pipette—aequorin can’t penetrate the cell
membrane. Solution is cloning and expression of the protein but you still have to
provide exogenous coelenterazine.
2.Chemical calcium indicator. Fura-2 is excitable by ultraviolet light (e.g., 350/380 nm) and its
emission peak is between 505 and 520 nm. The binding of calcium ions by fura-2 leads to changes in
the emitted fluorescence. Ratio method is useful because it doesn’t depend on the amount of Fura-2
loaded.
3. FRET-based genetically encoded calcium indicator (GECI). After binding of calcium ions two
fluorescent proteins, ECFP (donor) and Venus (acceptor), approach. This enables Förster resonance
energy transfer (FRET) and thus, the blue fluorescence of 480 nm decreases, whereas the fluorescence
of 530 nm increases. The donar and acceptor are coupled by calmodulin and a CaM binding peptide
M13. Calcium binding to CaM results in binding of M13 with a decrease in the distance between the
donar and acceptor.
4. Single-fluorophore genetically encoded calcium indicator (GECI). After binding of calcium to
GCaMP conformational intramolecular changes lead to an increase in the emitted fluorescence of
515 nm. It is a genetically engineered construct that has calmodulin and M13 attached to GFP.
When they interact you get enhanced GFP fluorescence. It is relatively easy to express GCaMP in
animal cells using AAV.
Slide, ratio Fura 2: Fluorescent Ca2+indicators can be of two types: The key advantage of ratiometric dyes
is that the measured ratio is independent of the amount of dye present but proportional to ion concentration.
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Under suitable conditions, fluorescence variations due to factors such as uneven cell thickness, unequal dye
distribution, dye leakage or photobleaching are normalized using the ratio approach. Fura-2 is excited at
340 nm and 380 nm of light, and the ratio of the emissions at those wavelengths is directly correlated to the
amount of intracellular calcium. Regardless of the presence of calcium, Fura-2 emits at 510 nm of light. The use
of the ratio automatically cancels out confounding variables, such as variable dye concentration and cell
thickness, making Fura-2 one of the most appreciated tools to quantify calcium levels. Despite this major
advantage of ratiometric dyes, they suffer from two significant drawbacks, both related to their absorption in the
UV range of the spectrum:
1. The first problem is that with UV excitation many cellular components (as well as glass, plastics, etc)
fluoresce and thus excitation in this range creates high non-specific fluorescence background.
2. The second problem is that UV excitation is not convenient to laser- or LED-based instrumentation.
As a result of these problems, a major effort has been devoted in recent years to the development of
calcium indicator dyes with excitation at longer wavelengths. This has resulted in the commercial
availability of several new dyes such as Calcium Green (Ex/Em at 488/535 nm), Calcium Orange
(545/575 nm), and Calcium Crimson (570/610 nm). Unfortunately, none of the available longwavelength dyes exhibit ratiometric behavior and therefore are not suited to quantitative analysis
based on traditional intensity-based detection methods.
Slide, Dye-Loading Approaches:
(A) Single-cell loading by sharp electode impalement (left panel), whole-cell patch-clamp configuration
(middle panel), and single-cell electroporation (right panel). Note that these approaches can be used for
loading of chemical and genetically encoded calcium indicators.
(B) “Acute” network loading. Many neurons are labeled simultaneously by acetoxymethyl ester (AM) loading
(left panel), by loading with dextran-conjugated dye (middle panel), and by bulk electroporation (left panel).
Loading Acetoxymethyl (AM) esters: the protection of carboxylic groups as AM esters makes the
dye neutral, so it can cross the cell membrane. Once inside the cell, esterases will cleave AM
groups. This process gives place to charged compounds that are entrapped inside the cell.
Complete hydrolysis of the AM esters is very important to avoid artifacts. If the experiment begins
before all AM ester dye is converted to free dye, total concentration increases during the
experiment and gives place to false fluorescence variations.
A drawback of AM esters is that they can accumulate inside intracellular compartments, making
indicator insensitive to calcium cytosolic levels. To avoid this behaviour, loading/de-esterification
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temperature can be decreased, which usually implies longer incubation periods.
Loading of dextran conjugates and salt form: these compounds are impermeable to cell
membranes, so they are less prone to accumulate in intracellular compartments. As a drawback,
they have to be loaded using reversible methods to make permeable large populations or
procedures such as microinjection and electroporation (cells can be damaged during these
processes). An important advantage of dextran conjugates is that dextran moiety not only avoids
compartmentalization, but also prevents conjugation of the indicator to proteins, membranes, etc.
This prevents indicator from being sequestered and provides enhanced resistance to leakage.
(C) Expression of Genetically Encoded Calcium Indicators (GECI) by viral transduction (left panel), in utero
electroporation (middle panel), and generation of transgenic mouse lines (right panel).
Common imaging devices:
Common Imaging Devices.
(A and B) Wide-field microcopy using a photodiode array (A) or a charged coupled device (CCD)-based
(B) detection unit. In both cases the light source can be a mercury or xenon lamp. Excitation and emission light
is separated by a dichroic mirror. A dichroic filter, thin-film filter, or interference filter is a very accurate color filter
used to selectively pass light of a small range of colors while reflecting other colors.
(C) Confocal microscopy using a continuous wave (CW) laser as light source. The excitation spot is
steered across the specimen by a scanner. The emission light is descanned and reaches the photomultiplier
tube (PMT) after passing a pinhole which is blocking out-of-focus fluorescence. Excitation and emission light is
separated by a dichroic mirror.
(D) Two-photon microscopy using a pulsed near-IR laser suitable for two-photon microscopy. Twophoton excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a
very high depth…..up to about one millimeter. Being a special variant of the multiphoton fluorescence
microscope, it uses red-shifted excitation light which can also excite fluorescent dyes. However, for each
excitation, two photons of infrared light are absorbed. Using infrared light minimizes scattering in the tissue. Due
to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased
penetration depth for these microscopes. Two-photon excitation can be a superior alternative to confocal
microscopy due to its deeper tissue penetration, efficient light detection, and reduced phototoxicity. The
excitation spot is steered across the specimen by a scanner. The emitted fluorescence is detected by a
photomultiplier tube (PMT). Two-photon excitation can be a superior alternative to confocal microscopy due to
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its deeper tissue penetration, efficient light detection, and reduced phototoxicity. However, the animal is
anesthesized and immobilized.
(E and F) Imaging devices used for calcium imaging in non-head-fixed behaving animals. (E)
Endoscopic approaches. (F) Portable head-mounted microscopes.
An example of Fura 2 imaging showing cAMP/Ca2+ interactions
In most cases we expect that increases in cAMP will stimulate increases in intracellular free calcium because
IP3 receptors are activated by PKA and various calcium channels are generally stimulated by PKA or gated by
cAMP. We wondered what would you expect for a cell expressing a calcium inhibited AC. So the calcium
inhibited adenylyl cyclase AC3 which is inhibited by CaM kinase II was expressed in 293 cells that also express
glucagon and beta-adrenergic receptors…both of which can couple to stimulation of AC3.
Slide, glucagon stimulates a slow calcium oscillation in AC3-expressing cells:
HEK-293 cells expressing the rat glucagon receptor (293-G), or the glucagon receptor with I-AC (I-AC-G) or IIIAC (III-AC-G) were treated with 100 nM glucagon and Ca2+ imaged using Fura-2. Representative traces from
individual cells are presented. Note that the time interval between peak calclium is 2 to 3 minutes…a slow
oscillation.
Slide, Calcium responses in the population of cells are heterogenous:
Typical examples of Ca2+ responses in III-AC-G cells treated with 100 nM glucagon are presented. A, 7% of the
cells showed only one Ca2+ spike; B, 8% showed a spike plateau; C, 85% showed Ca2+ oscillations.
Representative traces from individual cells are presented.
Side, Isoproterenol stimulation of Ca2+ oscillations in HEK-293 cells expressing III-AC: IP is a betaadrenrgic agonist that stimulates AC3. HEK-293 cells were treated with 10 μM isoproterenol and Ca2+ imaged
using Fura-2. Representative traces from individual cells are presented.
Slide, Forskolin stimulation of Ca2+ oscillations in HEK-293 cells expressing III-AC. Forskolin is a general
activator of adenylyl cyclases. It also stimulated Ca2+ oscillations.
Slide, Inhibitors of cAMP-dependent protein kinase block hormone-stimulated Ca2+ oscillations in IIIAC-G cells.
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H-89 and Rp-cAMP are PKA inhibitors. This data suggested that the oscillation depends on PKA activity.
Slide, Effect of dibutyryl cAMP and (Sp)-cAMP on intracellular free Ca2+ in HEK-293 cells.
Simply increasing cAMP and PKA activity doesn’t stimulate oscillations.
Slide, Ryanodine does not affect glucagon-stimulated Ca2+ oscillations in III-AC-G cells.
This indicates that ryanodine receptors in the ER are not responsible for the transients in intracellular calcium.
Slide, Extracellular Ca2+ is not required for hormone-stimulated Ca2+ oscillations in III-ACG cells:
When extracellular calcium is limited the oscillation still occurs but you see a progressive run down
in the signal suggesting that an internal calcium pool depends ultimately on external calcium. If
external calcium is not required for the oscillation than the most likely source would be ER calcium.
Thapsigargin is an inhibitor of the intracellular sarcoenodplasmic reticulum Ca21 ATPases and
pretreatment with the drug depletes ER calcium. This blocks oscillations. Since ryanodine
receptors aren’t involved this strongly implicates IP3 receptors in the ER.
Hormone-stimulated Ca2+ oscillations in III-AC-G cells are blocked by the CaM kinase
inhibitor KN-62. CaK kinase II inihibitors, including KN-62 blocked the oscillation. Since CaM
kinase II phosphorylates and inhibits AC3, you should be able to put all of these pieces together
and explain the oscillation.
Mechanism for hormone-stimulated Ca2+ oscillations in III-AC-G cells.
It is hypothesized that stimulation of III-AC-G by hormones or forskolin leads to activation of PKA,
stimulation of IP receptors, and increases in intracellular Ca2+. As intracellular Ca2+ increases, IIIAC activity is inhibited and cAMP levels are decreased by cAMP phosphodiesterases. When cAMP
drops below a threshold level, Ca2+ is resequestered and the cycle is repeated as long as
activators of III-AC are present. R, adenylyl cyclase stimulatory receptor; III-AC, type III adenylyl
cyclase; CaM, calmodulin; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; PLC,
phospholipase C; DAG, diacylglycerol; IP, inositol 1,4,5-trisphosphate; IPR, IP receptor/channel;
CaMK II/IV, CaM kinase type II or IV; PDE, cAMP phosphodiesterase.
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fiber-optic confocal endomicroscopy
Slide, Cellvio FEE system I am going to show you some FEE data obtained using the Cellvizio
NeuropakTM FEE system to continually monitor the activity of neurons in neurons of the mouse
hippocampus. These data were collected by Xuanmau Chen and Carlos Sindreau in my lab in
collaboration with Dr Larry Zweifel who has a Cellvio FEE system. These systems are very
expensive…about $250,000 to $300,000 and the probes cost $12,000. The expense is a major
drawback in using FEE. Endomicroscopic confocal imaging with cellular resolution is appropriate
for in vivo imaging of the brain in behaving mice. This system uses a permanent implant in the
mouse brain, attached to a flexible fiber optic bundle….to constantly monitor GFP fluorescence
real time.
Slide, the FEE system. In this system, a bundle of fiber optics encased in the laser microprobe
conveys excitation light to the tissue (488 nm laser beam) and collects the emitted fluorescence.
Light conveyed and collected by the same fiber reaches the photodetector. The laser probe has a
beveled tip of 350 um diameter that permits imaging large numbers of neurons at a time,
particularly in the densely compact cell body layers of the hippocampus (i.e. 200 neurons per field
of view). The temporal resolution is 85 ms per frame, which is above the 10 Hz acquisition rate
necessary for functional imaging, and suitable for detection of single calcium spikes with GCaMP3
given a half decay time of fluorescence of 0.4 s. This allows studies of temporally correlated activity
among neurons in local networks. The lateral and axial optical resolutions are ~3 um and ~10 um,
allowing clear detection of single neurons. The cannula implant only weights 0.29 g (~1% of adult
mouse body weight).
Slide, Mouse with FEE: Once the implant is secured the mouse can move freely about the cage
and the activity of individual neurons can be monitored real time. The implant is about the size of a
quarter.
Slide , the AAV construct. Before training, an FEE implant frame for the laser probe is attached to
the skull of the mice and the AAV1-CBA-GCaMP3 expression vector is administered by sterotaxic
injection into the hippocampus. GCaMP3 is a reporter of free calcium levels and hence a reporter
activated neurons, in vivo. The CBA promoter confers neuron specific expression. Two to three
weeks after virus administration, the laser probe is inserted at the sterotaxic coordinates used to
administer AAV1-DIOGCaMP3 and the mice are trained for context, continually monitoring the
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activity of individual neurons when mice are trained for context and during retrieval in freely moving
mice.
virus doesn’t affect normal expression of spatial of contextual memory: When expressing
GCaMP3 in any tissue you want to insure that the reporter isn’t perturbing normal calcium and
calmodulin signaling in mice. So…mice that received the AAV1-CBA-GCaMP3 were tested for two
forms of hippocampus memory that depends on calcium signaling and calmodulin regulated
enzymes. Spatial learning and memory using the Morris water task and contextual fear memory.
Both were normal in mice receiving the AAV1 construct.
Slide, extensive expression throughout area CA1: One of the problems with viral epression
experiments is the extent and range of the expression. AAV1 gives widespread expression in area
CA1 when injected into CA1.
Slide, stability of the recordings: Since the mouse is moving around you might worry about
movement of the recording unit and recording artifacts. This is an image of the hippocampus
showing stability of 11 neurons followed for 8 hrs while the mouse was moving around its home
cage,
kanic Acid Stimulation of Calcium transients in the hippocampus
Slide, Following one neuron during training and retrieval of contextual memory:
This is data following one neuron in the hippocampus when a mouse was trained for context by
shocking in the training context followed by return to context without shock (retrieval)
Slide, AC3-/- mice show dampened response: AC3-/- exhibit a depressed phenotype which
we think is due to lowered basal neuronal activity. This data compares the WT and AC3-/- when
they are trained for context. Note the lower activity in AC3-/- mice.
Advantages:
1. This approach overcomes the limitations of current optical and electrical techniques
that measure real-time neuronal activity in the intact brain, whether it be limited imaging
depth, the need of a tabletop microscope, the occurrence of motion artifacts, the lack of
cell type specificity, the poor temporal or cellular resolution, the low sampling efficiency,
or the physical limitations imposed on the animal.
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2. The use of a genetically-encoded calcium indicator allows chronic imaging experiments
necessary for the study of long-term processes.
3. Genetic expression of the calcium indicator allows cell specific expression using cellspecific promoters
4. One can monitor an unrestrained animal during various behavioral tests.
Disadvantages of FEE;
1. Expensive system
2. Resolution…only see cell bodies not axons and dendrites
3. Only monitoring one mouse at a time.
4. Only getting one signal at a time…would like to be able to monitor two or more
fluorescent signals simultaneously.
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