Biomedical Engineering Reference
In-Depth Information
However, the neuronal function of the earthworm and its relationship to behavior
are still mostly unknown.
Therefore, we have investigated the relationship between the nervous system and
behavior of the earthworm,
Eisenia fetida
, for the last 20 years using conventional
electrophysiology and optical imaging techniques for Ca, nitric monoxide (NO),
immunohistochemistry, and activity-dependent presynaptic imaging. In this chap-
ter, we describe the strategies we have used to understand the behavior of this ani-
mal with several powerful imaging techniques.
6.2
Optical Imaging of the Nervous System Using Specifi c
Fluorescent Dyes
Figure
6.2
contains a summary of several imaging techniques that are used to
investigate neuronal networks and their functions. Electron microscopy is the basic
tool for investigating synaptic connections in the nervous system (Fig.
6.2a
). This
fi gure reveals the huge axons of the MGF and LGFs in the ventral nerve cord (VNC)
and the complicated structure of the neuropile. Because electron microscopy is the
only method that can be used to identify the location of synapses and to understand,
the reconstruction of the synaptic connections is a laborious task. Recently, the
development of low-pressure scanning electron microscopy and sequential
large-fi eld sectioning instruments has enabled the high-throughput reconstruction
of the nervous system, and this approach has been named as “connemics” or the
“connectome” (Lichtman and Denk
2011
).
For neuroethological investigations, knowledge of the innervations and projec-
tions between neurons is crucial. One approach to visualize the nervous system is
the application of several kinds of fl uorescent dyes to the target neurons. Figure
6.2b
illustrates the application of two types of lipophilic fl uorescent dyes (DiI and DiA)
to the cut ends of the left and right segmental nerves. This double-staining technique
can visualize projection neurons to each and also both sides of the muscles and body
wall. The cell bodies of these projection neurons are clearly visualized in vivo, and
the combination of this technique with intracellular recording enables the investiga-
tion of the neural network and its function simultaneously. Furthermore, by backfi ll-
ing with fl uorescent Ca indicators from a specifi c segmental nerve, we can investigate
neuronal activity and also functional synaptic connections that are related to spe-
cifi c earthworm behavior.
Without staining, we can visualize neurons and several fi ne axons in the nervous
system. The nervous system of the earthworm is opaque, and the insertion of an
intracellular electrode or application of a patch electrode is diffi cult without staining.
However, by using differential interference contrast (DIC) microscope with infrared
illumination, we can easily apply electrophysiological techniques to the nervous
system without staining (Fig.
6.2c
). After the electrophysiological experiment is
complete, microinjection of a fl uorescent dye from the microelectrode enables the
visualization of the structure of the target neuron in detail.
