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pyramidal cells could be detected after pu
ng on glutamate, an excitatory neuro-
transmitter (
Garaschuk
et al.
, 2007; Heim
et al.
, 2007
). TN-XL was expressed using
the UAS/Gal4 tissue-specific expression system in
Drosophila
neuromuscular
junction (
Mank
et al.
, 2006
). Its rapid o
Y
V
rate for calciummade it significantly better
at tracking calcium changes than its counterparts. TN-XXL showed improved
sensitivity and long term-stability in sensing calcium signals from fly neurones; in
mice, tuning curves for orientation-specific neurones in visual cortex could be
monitored repeatedly over timescales of days or weeks (
Mank
et al.
, 2008
).
6. Comparison of the Performance of Genetically Encoded Calcium Sensors
Though progress in the field has been periodically reviewed (
Barth, 2007;
Garaschuk
et al.
, 2007; Griesbeck, 2004; Mank and Griesbeck, 2008
), few studies
have systematically compared the performance of di
erent genetically encoded
calcium sensors, except to demonstrate the superiority of a novel sensor over its
predecessors. I have discussed above (Section III.B.3) the systematic comparisons
of camgaroo-1 and-2 when expressed in
Drosophila
mushroom bodies (
Yu
et al.
,
2003
) and of inverse pericam, camgaroo-2 and YC3.1 when expressed in mouse
brain (
Hasan
et al.
, 2004
).
The performance of GCaMP, inverse pericam, and camgaroo-2 was compared
with that of the low molecular mass synthetic indicators X-Rhod-5F and Fluo4-
FF in apical dendrites of pyramidal cells in hippocampal brain slices from 6- to 7-
day-old rats transfected using a biolistic approach and maintained at room
temperature (
Pologruto
et al.
, 2004
). Images were obtained using two-photon
microscopy. Action potentials were triggered using current injection into the cell
body. Under these conditions, X-Rhod-5F and Fluo4-FF could detect calcium
changes (signal twice that of noise) in the dendrite due to voltage-dependent
calcium channel activation after single action potentials while with the same
criterion GCaMP required five action potentials, camgaroo-2, 33 action potentials,
and inverse pericam over 20. For comparison, the dynamic ranges (
DF
/
F
) for the
three sensors under these conditions
in vitro
was1.8,
V
0.25, so the
sensitivity of camgaroo-2 was poor despite its larger dynamic range. Power spec-
trum analysis was used to analyze the fluorescence response during action potential
trains at 20 Hz. Most of the power in the frequency analysis of X-Rhod-5F and
Fluo4-FF fluorescence was at the fundamental frequency, 20 Hz, indicating that
the fluorescence signal tracked each action potential. For the genetically encoded
sensors, no clear peak was observed at 20 Hz, indicating that the sensors were too
slow to track individual action potentials at this stimulation frequency.
It was possible to measure calcium activation curves
in situ
for the three sensors
and thus their apparent dissociation constants by simultaneously measuring
calcium concentration using a calibrated X-Rhod-5F signal and the fluorescence
signal from the sensor at various levels of stimulation. For inverse pericam (
K
0
d
0.9
m
M) and camgaroo-2 (
K
0
d
8
m
M), these were comparable to those previously
reported
in vitro
; however, GCaMP showed a
K
0
d
(1.7
m
M) almost an order of
2, and
