Biomedical Engineering Reference
In-Depth Information
Recently, electroporation techniques have been used to achieve bulk loading of
Ca
2+
indicators (Nagayama et al.
2007
; Hovis et al.
2010
; Bonnot et al.
2005
).
Following microinjection of the potassium salt or the dextran-conjugated fl uores-
cent dye into the brain, an electric fi eld is imposed across a population of cells to
transiently introduce membrane pores through which the Ca
2+
indicators can enter.
This method is capable of loading the Ca
2+
indicators into brain tissue that is inac-
cessible to the AM ester.
In summary, there are a variety of approaches for loading the Ca
2+
indicators into
the target cell or cells to be studied. The most effective method will depend upon the
nature of the preparation under study, the specifi c questions to be addressed, and
any technological limitations imposed by the recording method to be used.
5.3.2
Preparation for In Vivo Imaging
In vivo Ca
2+
imaging of nerve cells in awake behaving animals is ideal for neu-
roethogical research but presents a host of technical diffi culties beyond those asso-
ciated with dye loading. Problems related to stabilization of the target tissue and
accessibility of that tissue to a high-numerical-aperture microscope objective
require careful consideration. To illustrate some of these considerations, we intro-
duce three different types of preparations using the cricket nervous system for in
vivo Ca
2+
imaging.
The fi rst type of preparation is an isolated preparation, in which an entire gan-
glion and connected sensory organs are removed from the animal's body. For our
studies of the neuronal processing underlying the detection and analysis of air-
current direction and dynamics in the cricket cercal sensory system, we dissect the
terminal abdominal ganglion away from the abdomen, along with the pair of air-
current-sensitive organs called cerci that send sensory input into the ganglion. The
preparation is mounted in a glass chamber made of a 0.25-mm thick cover glass
(Ogawa et al.
2006
,
2008
). This recording chamber is mounted on the stage of an
inverted microscope confi gured for fl uorescent imaging. We can observe clear
images of dye-loaded neurons in this preparation, maintaining the neural circuits
and the sensory organs in conditions that are similar to their natural in vivo confi gu-
ration. By removing the standard condenser tube and transmitted light source from
the microscope, which normally obstruct access to sample from above, this setup
provides excellent accessibility of the preparation to electrodes and perfusion appa-
ratus and has the advantage that the objective lens (placed below the stage) does not
disturb the normal airfl ow stimulus across the sensory organs.
The second type of preparation is a “head-fi xed” preparation, in which the ner-
vous system is left within a restrained animal's body. The head-fi xed preparation
enables in vivo imaging experiments to be carried out in intact animals that contain
a complete, intact central nervous system with fully functional sensory capabilities.
Pioneer works of the in vivo Ca
2+
imaging in the head-fi xed preparation were per-
formed in fl y visual system, demonstrating dendritic Ca
2+
accumulation evoked by
