Biomedical Engineering Reference
In-Depth Information
1.4.2
An Example of Calcium Imaging with GECI
We can selectively monitor the neuronal Ca
2+
change by cell specifi cally expressing
GECIs using cell-specifi c promoters. In the many cases of
C. elegans
neurons
,
Ca
2+
change is thought to be reliably refl ecting neural activity because
C. elegans
has no
voltage-gated sodium channel. Figure
1.3c
shows response of thermosensory neu-
ron AFD to thermal stimulus. In this case, 23 °C cultivated animal responds at just
before 23 °C with increase of temperature. YFP/CFP value is calculated with
defi ned neuronal region in the fl uorescence images, and average intensity among
the region is used for YFP/CFP. Slight differences of the value calculation protocol
cause slight change of the results but seldom produce qualitatively different results.
However, imaging with GECIs sometimes contains artifact caused by several rea-
sons such as photobleach, the animal movement, and microscopic focal plane
change; therefore, experimenters should be careful for these possible artifacts when
acquiring image data. Controls always should be accompanied with imaging experi-
ments; in the case of FRET-based GECIs, accepter bleaching or inspection of ratio-
metric intensity change between two fl uorescence proteins is useful to validate
FRET occurrences.
1.5
Simultaneous Monitoring for Behavior
and Neural Activity
Combination of individual tracking and calcium imaging enables to monitor simul-
taneous monitoring for stimulus, neural activity, and behavior (Ben Arous et al.
2010
; Clark et al.
2007
; Piggott et al.
2011
). To conduct such simultaneous monitor-
ing, fully or almost computerized system is necessary. Figure
1.4a
shows an exam-
ple of the simultaneous monitoring for thermotaxis. The system consists of thermal
gradient, tracking, and Ca
2+
imaging component. Each component is coordinately
controlled with computers. The system records trail of freely moving animal on
thermal gradient with time stamps (Fig.
1.4b
), thereby enabling the calculation of
the time course of temperature stimulus for freely moving animal (Fig.
1.4c
). From
time-lapse fl uorescence images of GECIs, time course of Ca
2+
concentration change
can be obtained as time course of YFP/CFP (Fig.
1.4d
). Note that Ca
2+
imaging for
moving object tends to include several kinds of artifacts, which essentially origi-
nated from the movement. We should thus particularly be careful for the artifact in
the case of Ca
2+
imaging for moving objects. Integration of Ca
2+
imaging data and
migrating trail comprehensively depict the relationship between the monitoring
neural activity and the corresponding behavior. Figure
1.4e
shows the correlation
between high activity of AFD thermosensory neuron and turning, or straight migra-
tion and low activity of AFD. Acquirement and detailed analysis of these quantita-
tive data are powerful for dissecting dynamic phenomena. Moreover, these data can
be reconciled with mathematical modeling and thus profi table to understand the
mechanisms for regulation of dynamic systems such as animal behavior.
