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HB9 -/-
Myotomal muscle cell
+21 hours
* * * *
a -Btx
100 m m
FIGURE 8.15 Accumulation of AChRs in the central region of
uninnervated muscle. Diaphragm muscle from wild-type (A and B)
or HB9 mutant (C and D) embryos were stained with Texas red-a-
bungarotoxin (aBGT) and antibodies to neurofilament and synap-
tophysin (anti-NF/Syn). At embryonic day 18.5, there is dense
innervation at the center of the wild-type muscle but no motor inner-
vation in the HB9 mutants. (A few sensory and/or autonomic axons
can be seen at the edge of HB9 muscle in panel D.) The insets show
individual AChR clusters at high magnification in wild-type (A) and
mutant (C) embryos. The bar is 100 mm for the low magnification
images. (Adapted from Yang et al., 2001)
Muscle cell
+39 hours
* * *
Nerve process
* * * *
they are associated with few AChR clusters. In con-
trast, neurites from spinal cord neurons, which pre-
sumably include motor neurons, are associated with
AChR clusters at 70% of contact sites (Cohen and
Weldon, 1980; Kidokoro et al., 1980). When spinal
motor neurons are selectively prelabeled in vivo and
then dissociated in the presence of myocytes, their
ability to induce AChR clusters can be compared to the
unlabeled spinal interneurons (Role et al., 1985). Motor
neurons and ciliary ganglion cells, both of which
secrete ACh, are able to induce AChR clusters on all
contacted myocytes, whereas the interneurons do not
induce significant AChR clustering.
The studies on NMJ suggest that appropriate nerve
terminals (i.e., cholinergic) support the induction of
AChR clusters. Does the neurotransmitter receptor
have to be activated in order to cluster? When myocytes
and spinal cord are cultured in the presence of an AChR
antagonist, D-tubocurarine, they develop an identical
number of functional contacts compared to control cul-
tures (Cohen, 1972). To directly assess AChR clustering
during the blockade of neurotransmission, fluores-
cently labeled a-Btx was first applied to visualize the
AChRs that were present on muscle cell membrane, and
excess unlabeled a-Btx was then added to prevent visu-
alization of newly inserted AChRs (Anderson and
Spinal cord neurons
Sensory neurons
Sympathetic neurons
% Btx stained contacts
FIGURE 8.16 ACh receptor clustering on muscle fibers is
induced by contact with spinal neurites. A. A culture of spinal
neurons and muscle cells was labeled with a fluorescent a-Btx (red)
at 21 and 39 hours after plating. Soon after the spinal neurite grew
across the muscle surface, fluorescent a-Btx appeared at the contact
site, indicating that AChR aggregation is induced. B. The ability to
induce AChR clusters is neuron-specific. When muscle cells were cul-
tured with spinal neurons, DRG neurons, and sympathetic neurons,
only the spinal neurons induced significant a-Btx labeling. (Adapted
from Anderson and Cohen, 1977; Cohen and Weldon, 1980)
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