Biomedical Engineering Reference
In-Depth Information
propagation induced by electrical tetanic stimulation in the earthworm giant axon
using Calcium Green-1 (Ogawa et al.
1994
; Ogawa and Oka
1996
). Furthermore, by
using voltage-dependent fl uorescent dyes (RH and JPW series dyes, see recent
review, Chemla and Chavane
2010
) and voltage-sensitive fl uorescent proteins
(VSFP series proteins, see Mutoh et al.
2011
), direct measurement of membrane
potential as an indicator of neuronal activity has been developed. Optical recording
using voltage-sensitive dyes and proteins enables us to study the spatial pattern of
stimuli-induced depolarization and hyperpolarization at a high time resolution.
However, the location of individual neurons activated by the mechanical stimuli and
their connectivity remains unknown. Lucifer yellow and sulforhodamine are often
used as markers of neuronal activity (Wilcox and Franceschini
1984
; Keifer et al.
1992
; Kimura et al.
1998
). On the other hand, the styryl dye
N
-(3- triethyl-
ammoniumpropyl)-4-(4-(dibutylamino)styryl)-pyridinium dibromide (FM1- 43 ,
Fig.
6.5a
is useful for visualizing active synapses (Betz and Bewick
1992
; Betz et al.
1992
). When the internalization of membrane vesicles occurs in the presence of
FM1-43, the dye is trapped inside the endocytosed vesicles. Then, when the dye is
removed from the external medium, the FM1-43-labeled vesicles can be detected
fl uorescently (Betz and Bewick
1992
; Betz et al.
1992
). The fl uorescence intensity
decreases when the dye-loaded vesicles are exocytosed again and release the dye to
the outside medium (Betz and Bewick
1992
; Betz et al.
1992
; Klingauf et al.
1998
).
We applied FM1-43 staining to the VNC of the earthworm and confi rmed the neu-
ronal activity-dependent staining of the VNC with FM1-43 (Shimizu et al.
1999
).
The same technique was also applied to intact neural preparations from
Caenorhabditis elegans
, lamprey, and rat (Kay et al.
1999
). We, therefore, applied
activity-dependent labeling with FM1-43 to the nervous system of the earthworm to
determine the location of neural activity during fi ctive locomotion.
We investigated fl uorescent staining of the VNC with FM1-43 and observed the
fl uorescent spots as a function of the concentration of octopamine (Mizutani et al.
2003
). The intensity of each fl uorescent spot increased with increasing octopamine
concentration. An analysis of the fl uorescence intensity of the spots relative to octopa-
mine concentration showed that two response patterns were present (Fig.
6.5b
). One
group of spots responded at low concentrations of octopamine (10
−7
M) and was satu-
rated at 10
−5
to 10
−4
M (high-affi nity group; Fig.
6.5b
, plotted as diamonds). The other
group of spots responded from 10
−5
M and was saturated at 10
−4
to 10
−3
M (low-affi nity
group; plotted as triangles), in a manner similar to the sensitivity of the motor pattern
response to exposure to octopamine (plotted as circles). The octopamine concentrations
for the half-maximal responses in the high- and low-affi nity groups were 1.0 × 10
−6
M
and 2.0 × 10
−5
M, respectively, while that of the motor pattern was 1.0 × 10
−6
M.
The voltage-sensitive dye ww-781 can be applied after monitoring FM1-43
uptake to visualize the location of neurons and presynaptic regions. Because
ww-781 is lipophilic and stains neuronal cell membranes, we inverted the acquired
fl uorescence images to visualize neuronal cell bodies and then superimposed the
FM1-43 fl uorescent images over the ww-781 inverted fl uorescent images (Fig.
6.5c
).
FM1-43 spots from the group with a high affi nity to octopamine were located at
10-15
m from the edge of the VNC, between the fi rst and second/third lateral
nerves (Fig.
6.5c
, yellow area), while those from the low-affi nity group were located
μ
