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be mosaic, not mapping directly to the known patterns of a CamKII expression.
Neocortical expression could also be imaged through the thinned skull in anaesthe-
tized mice. Two photon fluoresence recovery after photobleaching suggested that as
much as half the fluorescence signal was immobile and this together with punctuate
staining patterns suggested that this immobile sensor fraction might be due to
interaction between the M13 and CaM moieties of the sensors and their normal
cellular targets. Cellular and synaptic stimulation of pyramidal neurones in
cortical slices using sharp and patch microelectroded gave 5-10% increases
in 535 nm fluorescence using wide field imaging and around 20-100% for cam-
garoo-2 and
30% for inverse pericam using two photon imaging. In the retina, a
ganglion cell subset was strongly labeled in YC3.1-expressing mice, but no light-
evoked responses were detected. In camgaroo-2 expressing lines, bleaching occurred
in the retina too quickly for measurements to be made. In one inverse pericam-
expressing mouse, 7 of 12 ganglion cells tested showed a transient decrease in
fluorescence attributable to a calcium increase in response to light. Sensors were
imaged in the olfactory bulb in vivo using wide fieldmicroscopy. Camgaroo-2 expres-
sing mice showed a 1-3% increase in response to odors, while inverse pericam gave
8% decrease. Each distinct odor evoked a unique pattern of activity, similar odors
evoking similar patterns.
This thoughtful study established four main facts: around half of the transgeni-
cally expressed cameleon family sensor was immobile; this reduced sensitivity and
made quantitation of the calcium signals impossible; nonetheless, it was possible to
observe patterns of neuronal activity; YC3.12 was not an e
ective transgenic
sensor. The study also reports unpublished experiments in which transgenic mice
expressing YC3.0 under the control of a b -actin promoter gave only 1-2% ratio
changes during wide filed imaging in cerebellar slices. The high proportion of
immobile sensor in transgenic animals remains for the moment inexplicable—it
was not seen in the stably transfected fibroblast lines.
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4. GCaMP
G-CaMP ( Nakai et al. , 2001 ) was expressed in mice under the control of a
smooth muscle myosin heavy chain promoter and was expressed in vascular and
nonvascular smooth muscle ( Ji et al. , 2004 ). The signatures of inotropic (ion
channel) and metabotropic (InsP 3 -mediated) postsynaptic signaling could be dis-
tinguished in single excised smooth muscle cells.
In a set of experiments strikingly parallel to those with YC2.1 ( Diegelmann
et al. , 2002; Fiala et al. , 2002 ), but using two photon imaging, G-CaMP was
expressed in a subset of projection neurones in Drosophila antennal
lobe to
demonstrate that di
erent odorants activated specific patterns of glomeruli
( Wang et al. , 2003 ). Individual glomeruli are di
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erentially sensitive to a given
odorant and more are recruited as the odorant concentration is increased.
Increases of fluorescence of up to 50% (at 525 nm) were measured in responsive
glomeruli.
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