Biomedical Engineering Reference
In-Depth Information
cytotoxicity is required on a case-by-case basis. On the other hand, higher
expression levels make the detection of fl uorescence signals easier. Although a
possible perturbation of endogenous Ca
2+
signaling by the Ca
2+
-buffering proper-
ties of GECIs has not been detected in
Drosophila
so far (Diegelmann et al.
2002
;
Jayaraman and Laurent
2007
; Reiff et al.
2005
), we recommend independent tests
whether the functionality of the system you study is not affected by the expression
of GECIs, e.g., through behavioral experiments. A couple of methods can be used
to modify the level of expression. First, the use of homozygous versus heterozy-
gous fl ies for both GAL4 and UAS transgenes alters the expression level. Two (or
more) copies of a transgene typically result in higher levels of expression. Second,
fl ies with different insertion sites, or number of UAS sites, can be used to vary the
level of expression. More than one line for a single GECI is often available, such as
UAS-Cameleon 2.1 (Fiala et al.
2002
) and UAS-GCaMP3 (Tian et al.
2009
), kept
at the Bloomington
Drosophila
Stock Center. Third, the shift in temperature alters
the activity of GAL4 in
Drosophila
(Duffy
2002
). In fl ies maintained at 16 °C,
GAL4 shows a minimal ability to activate transcription. As the temperature at
which fl ies are raised is increased, the activity of GAL4 increases. 29 °C provides
a balance between maximal GAL4 activity and minimal effects on fertility and
viability of fl ies. This means that by altering the temperature, a wide range of
expression levels the GECI can be achieved.
7.5
Examples of Applications
Here we exemplify how Ca
2+
imaging in the brain or a sensory organ can be accom-
plished (Fig.
7.2
). We monitored the neural responses in olfactory sensory neurons
innervating the antennal lobe (AL), the primary olfactory center in the fl y brain, and
mechanosensory neurons in the Johnston's organ (JO), the auditory organ of the fl y
(Kamikouchi et al.
2010
). The fl y line UAS-Cameleon 2.1 (Diegelmann et al.
2002
)
was crossed to cell type-specifi c GAL4 lines, each of which expresses GAL4 in
olfactory receptor neurons (Fig.
7.2a
) or in the JO neurons (Fig.
7.2b
), leading to
restricted Cameleon 2.1 expression in the progeny. The fl ies were anesthetized on
ice and then affi xed to an imaging platform with a drop of glue. Images in the eYFP
and eCFP channels were recorded simultaneously several seconds before, during,
and after the stimuli as described previously (Fiala and Spall
2003
). A time point is
chosen before the onset of the stimulus to determine the baseline value for the inten-
sities of the eYFP and the eCFP fl uorescence (
F
0
) and the eYFP/eCFP ratio (
R
0
).
The change in fl uorescence relative to the baseline is calculated as
F
/
F
0
for eYFP
and eCFP, respectively. Likewise, the change in the ratio is calculated as
Δ
R
/
R
0
.
This ratio change is indicative of alterations in the intracellular calcium concentra-
tion. It is clearly visible that in the case of olfactory receptor neurons, an odor stimu-
lus causes an increase in intracellular Ca
2+
, whereas in the case of JO neurons, sound
is an adequate stimulus.
Δ
