Biomedical Engineering Reference
In-Depth Information
visual motion stimuli (Borst and Egelhaaf
1992
; Single and Borst
1998
). At present,
two-photon imaging experiments in head-fi xed mice have successfully monitored
cortical Ca
2+
responses to whisker, odor, auditory, and visual stimuli (Sato et al.
2007
; Wachowiak et al.
2004
; Bandyopadhyay et al.
2010
; Rothschild et al.
2010
;
Smith and Häusser
2010
). A critical requirement for this preparation is the immobi-
lization of the nervous tissue with respect to the objective lens. We have carried out
optical recordings using awake crickets that were fi xed on a silicone elastomer plat-
form by insect pins, with their brain ganglion held up and immobilized using a
stainless steel spoon. In a preliminary study using this preparation, we have opti-
cally recorded the odor-evoked Ca
2+
signals in the calyx of the mushroom body in a
conditioned cricket (Fig.
5.3
; Ogawa and Oka
2008
).
The most ideal preparation for neuroethological studies is a tethered preparation,
which is designed for in vivo imaging of animals performing an actual behavior,
like locomotion. This type of preparation is more diffi cult with respect to stabiliza-
tion of the nervous tissue: it is important to only immobilize the nervous tissue to be
studied under the microscope, without encumbering the animal's behavior. One
novel imaging method has recently been developed to achieve this goal, through
which a microendoscope made of narrow optical fi ber bundles is penetrated into the
brain of a freely moving mouse enabling dendritic Ca
2+
imaging (Adelsberger et al.
2005
; Murayama et al.
2007
). Here we summarize a tethered preparation for in vivo
imaging of the cricket during locomotion on a spherical treadmill. A U-shaped steel
plate was slipped into the cervical segment between the head and thorax, and the
cuticular epicranium was fi xed to that plate with wax. One end of the plate was con-
nected to a stainless steel rod, controlled via a micromanipulator, for precise posi-
tioning of the head. This head-tethered cricket was placed so that its legs were
positioned on a treadmill in their natural arrangement, and fl uorescent images of the
brain ganglion were monitored under the microscope during the cricket's walking
behavior. Using similar head-fi xed preparations in mice and
Drosophila
, two-
photon Ca
2+
imaging has been performed while the animals were walking (Dombeck
et al.
2007
; Seelig et al.
2010
). To eliminate optical noise resulting from vibration
of the brain during the animals' movements, additional procedures were required
involving a surgical fi xture or fi lling with a silicone adhesive (Kwik-Sil, WPI).
5.3.3
Imaging Devices and Data Analysis
Various types of fl uorescent microscopes have been developed over the last few
decades, and most confi gurations have now been used to carry out Ca
2+
imaging in
neuroscience research. Likewise, multiple kinds of imaging devices have been
developed and are available for imaging experiments. In general, Ca
2+
imaging
requires cameras with very high sensitivity. Current implementations typically use
an electron-multiplying CCD (EM-CCD) or a complementary metal oxide semicon-
ductor (CMOS) sensor mounted to a conventional fl uorescent microscope. Recent
advances in microscope and imaging technologies are providing progressively
