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magnitude greater than that previously reported
in vitro
(
Nakai
et al.
, 2001
).
Because the calcium concentration profile of dendritic action potentials is well
characterized (
Pologruto
et al.
, 2004
), there seems little doubt that the calcium
dissociation characteristics of GCaMP vary markedly
in vitro
and
in vivo
. FRAP
studies in dendrites showed that the all three sensors were mobile, with mobilities
comparable to GFP itself. This result is quite firmly at odds with that reported in
mouse brain (
Hasan
et al.
, 2004
) and discussed above (Section III.B.3). It may be
that dendrites, being relatively free of organelles, mirror better the behavior of the
sensors in cytoplasm than cell bodies; it should be noted that punctuate staining
was reported in mouse brain (
Hasan
et al.
,2004
). It should also be borne in mind
that though the observations on mouse brain slices were carried out at room
temperature, as were these experiments in rat brain slices, in the mouse study,
the sensors had been expressed at body temperature, whereas the biolistically
transfected rat brain slices were maintained throughout at room temperature.
These data, as the authors point out (
Pologruto
et al.
, 2004
), demonstrate that
the genetically encoded sensors are better-suited to measuring summated neuronal
responses after multiple stimuli, not single action potentials, consistent with their
reported use to monitor patterns of neuronal activity (
Fiala
et al.
, 2002; Hasan
et al.
, 2004; Wang
et al.
, 2003
); as it happens, these three studies all described
odorant-specific patterns of neuronal signaling.
As an addendum to the study, Svoboda's group also provided
in vitro
solution
X-ray scattering evidence that showed that the calcium-dependent fluorescent
signal of GCaMP, as theorized, depends on a coupled structural change in which
calcium binding to CaM is closely linked to binding of CaM to M13; in contrast,
the calcium-dependent fluorescence signal in camgaroo-2 is solely due to binding
to CaM, the M13 peptide paradoxically playing no part in the sensor response
(
Pologruto
et al.
, 2004
).
A second comparative study was undertaken at the
Drosophila
larva neuromus-
cular junction (
Rei
V
et al.
, 2002
). The responses of 10 sensors from the three families to 40 and 80 Hz
stimulation of the synaptic bouton were compared. Camgaroos-1 and-2 and flash
pericam did not sense calcium changes in the bouton. YC2.0, 2.3, 3.3, TN-L15,
inverse pericam, and GCaMP1.3 and 1.6 all showed adequate responses (around
5% on average at 40 Hz and 10-15% at 80 Hz) to pulse train stimuli, but none
exhibited dynamic ranges anywhere near comparable to those measured
in vitro
(
Rei
V
et al.
, 2005
), using an approach previously reported (
Rei
et al.
, 2005
). None was comparable in performance in this system when
compared to the later developed TN-XL (
Mank
et al.
, 2006
). In an echo of the
work in rat brain slices, the performance of YC3.3, TN-L15, GCaMP1.6,
GCaMP2, YC2.60, YC3.60, cameleon D3, and TN-XL were compared one with
another and calibrated against a low molecular mass indicator, Oregon-Green-
BAPTA-1 (
Hendel
et al.
, 2008
). The latter four sensors were around twofold more
responsive than their earlier counterparts. None of the sensors were seen to detect
single action potentials, though YC3.60 and cameleon D3 could detect two action
potentials in succession. None showed the fast temporal response of the low
V
