Biology Reference
In-Depth Information
Transgenic expression of GCaMP2 has been achieved in mouse heart (
Tallini
et al.
, 2006
). The TET system was used: the GCAMP2 sequence was placed
downstream of a weakened
a
myosin heavy chain promoter (
a
MHC) and seven
tetO enhancer sequences to permit suppression of gene expression using doxycy-
cline. These mice were crossed with others with a hemizygous
a
MHC-tetracycline
transactivator allele. The doubly transgenic mice expressed GCaMP2 only in the
heart. Doxycycline suppression of the transgene was essential, as mice constitu-
tively expressing GCaMP2 from birth showed markedly enlarged hearts, a pheno-
type comparable to that seen in mice overexpressing calmodulin. This phenotype
was avoided entirely by administering doxycycline in utero and until 13-15 weeks
postpartum. Subsequent removal of doxycycline for up to 6 weeks led to no
detectable cardiomegaly. Robust GCaMP signals were present 4 weeks after
doxycycline removal.
Striking wide field fluorescence images of cardiac calcium transients in whole
mouse heart beating at up to 300 beats/min were obtained with anaesthetized,
ventilated open-chested mice, the first to be recorded under wholly physiological
conditions with the heart under normal load. As expected sympathetic stimulation
with isoproterenol markedly increased the calcium signal and also increased end
diastolic calcium concentration. Signal-to-noise ratios were good and it was possi-
ble to record very clean signals from a single pixel of the 100
100 pixel imaging
array (tens of microns). Using a photodiode array in isolated perfused heart,
signals from a membrane potential sensitive dye and from GCaMP2 were acquired
simultaneously. Association and dissociation kinetics of calcium were rapid (
t¼
14
and 75 ms, respectively) and unaltered
in vivo
. Comparison with a fast calcium dye
Rhod2 nonetheless showed that the rise and decay times of the GCaMP2 signal in
beating heart was around 45% slower, but with a three times greater dynamic
range. Calcium sparks could not be observed in isolated ventricular myocytes
expressing GCaMP2. GCaMP2 imaging in open-chested embros from embryonic
day 10 allowed the analysis of the development of the atrio-ventricular node
conduction pathway.
GCaMP2 fused to synaptotagmin localizes to synaptic boutons. It reports the
location of synapses in zebrafish
in vivo
and shows a linear response over a wide
range of action potential frequencies (
Dreosti
et al.
, 2009
). It can report spiking
frequencies in optic tectum; it also reports activity in the graded synapses of retinal
bipolar cells. GCaMP2 has also been used to map functional connections in the
C.
elegans
nervous system (
Guo
et al.
, 2009
). Connections can be mapped grossly, but
the sensor's signals are too weak to distinguish direct from indirect connections.
5. TN-L15, TN-XL, and TN-XXL
A cerulean version of TN-L15, cerTN-L15, was used to create a transgenic mouse
line that expressed the sensor widely in brain, especially in the neocortex and
hippocampus (
Heim
et al.
, 2007
). Calcium changes resulting from two to three
action potentials could be resolved and calcium responses in spiny dendrites of
