Biology Reference
In-Depth Information
molecular mass indicator. A theoretical framework in which to consider the pros
and cons of calcium sensors in recording neuronal activity has been adumbrated
(
Hires
et al.
, 2008
).
GCaMP1.6 and GCaMP2 were compared in pyramidal cells dendrites in
mammalian brain slices transfected ballistically or by electroporation (
Mao
et al.
, 2008
) under conditions that allowed comparison with first generation
sensors (
Pologruto
et al.
, 2004
). Their performance was not significantly better
than GCaMP, even when localized using membrane and cytoskeletal targeting
chimeras (
Mao
et al.
, 2008
). GCaMP3, however, showed substantial gains in
sensitivity and discrimination (
Tian
et al.
,2009
): overall, the signal-to-noise
ratio was much improved and responses in dendrites to single action potentials
could be reliably detected. Direct comparison with TN-XXL and cameleon D3
showed that, although brighter, the two FRET sensors gave smaller fluorescence
changes and less favorable signal-to-noise ratios. GCaMP3 was also more photo-
stable. After either adenoviral transfection or in utero electroporation, calcium
responses in pyramidal neurones could be observed in awake, behaving mice
(
Tian
et al.
, 2009
). Parallel electrical recordings showed that detectable calcium
responses were associated with three or more action potentials. Calcium responses
were also readily observed in the glomeruli of
Drosophila
antennal lobe and in
sensory neurones of
C. elegans
, altogether a methodological tour de force (
Tian
et al.
, 2009
).
IV. Use of Genetically Encoded Calcium Sensors
For single cell applications, wide-field fluorescence imaging, spinning disk, or
confocal microscopy are appropriate methods. Dual excitation laser scanning
confocal imging is achievable (
Shimozono
et al.
, 2002
). For whole animal applica-
tions, particularly in intact brain or brain slices two photon microscopy is recom-
mended, as it reduces tissue damage and o
V
ers improved imaging within tissue (see
Chapter 9;
Fan
et al.
, 1999
).
Expression of sensors in cells and tissues, as we have seen, can be achieved by
transfection and transgenesis. One advantage of transgenic approaches is that
expression can be confined to a specific tissue or cell type, an advantage even if it
is excised for imaging. Random expression in a subset of cells can more simply be
achieved by using biolistic transfection of excised tissue.
Ratiometric sensors (in this context the FRET-based sensors, ratiometric peri-
cam and DRIP) o
er the advantage that the quantitative signal is in theory
independent of variations in sensor distribution and concentration within cells
(
Silver
et al.
, 1992
). This allows reliable calibration of the signal in terms of calcium
concentration (see chapter 1). Nonratiometric sensors (e.g., GCaMP3) are ade-
quate for determining changes in calcium concentration, for example, when mea-
suring overall spatial and temporal patterns of calcium signaling. Even in these
circumstances, caution should be exercised in case the responses are nonlinear,
V
