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A
A
B
C
Place in
FM4-64
Depolarize
(FM uptake)
Loaded
Depolarize
(FM release)
Unloaded
Obtaining a patch
“Sniffer” in bath
“Sniffer” at growth cone
AChR
Muscle
cell
Growth
cone
ACh
B
“Sniffer”
t = 0
353 s
outside-out
patch
4 pA
10 s
Intensity:
FIGURE 8.6 Spontaneous release of neurotransmitter from
growth cones. A. A biological sensor for ACh (a “sniffer”) was
created by excising a patch of membrane from a muscle cell with a
recording pipet. The membrane contained AChRs that were facing
outward. B. Recording of ACh-evoked currents (downward deflec-
tions) when the “sniffer” pipet was distant from the growth cone,
and C. when it was within a few microns of the growth cone. The
increased activity indicates that the growth cone was releasing ACh.
(From Young and Poo, 1983)
Low
High
C
FM4-64
Synaptophysin
receptors (see BOX: Biophysics: Nuts and Bolts of
Functional Maturation). These electrodes are brought
within a few microns of a growth cone before it has
contacted a myocyte. If the growth releases ACh, then
the ACh receptor-coupled channels open, and current
flows across the electrode.
The release of transmitter is probably a general
property of all growth cones. For example, a different
neurotransmitter (GABA) is released from growth
cones of mammalian CNS neurons (Gao and van den
Pol, 2000). Growth cones are also able to release trans-
mitter in response to electrical stimulation of their cell
bodies. Therefore, some of the presynaptic neuro-
transmission machinery is present even before synap-
togenesis occurs, albeit in an immature form. It is not
yet known whether growth cones release transmitter
via the fusion of synaptic vesicles, as do mature
synapses. To demonstrate that vesicular release can
occur from growth cone filopodia (see Chapter 5), an
optical technique was employed. As shown in Figure
8.7A, neurons are incubated in the presence of a fluo-
rescent dye (FM4-64) that does not cross the cell mem-
brane. However, when the neuron is depolarized, the
dye enters vesicles that have fused transiently with the
membrane. Vesicles that are loaded with fluorescent
dye in this manner will then release the dye when they
next fuse with the membrane; that is, when the mem-
FIGURE 8.7 Apresynaptic vesicular release mechanism is
present in growth cone filopodia. A. A schematic of the FM dye tech-
nique. The dye is taken up into vesicles as they fuse with the mem-
brane. When the terminal is depolarized again, the vesicles fuse with
the membrane again and the terminal is unloaded of dye. B. Release
of FM4-64 from growth cone filopodia in response to depolarization
(yellow arrows). C. Co-localization of FM4-64 (red) and the synap-
tic vesicle protein, synaptophysin (green), in growth cone filopodia.
(Panels B and C from Sabo and McAllister, 2003)
brane is next depolarized. In fact, the growth cone
filopodia of cortex neurons can incorporate and release
dye in response to depolarization, suggesting that a
vesicular mechanism is present (Figure 8.7B). Several
synaptic vesicle proteins are co-localized within
growth cone filopodia (Figure 8.7C), raising the possi-
bility that neurotransmitter release occurs at the
moment of contact (Sabo and McAllister, 2003).
In some systems, particularly in the peripheral
nervous system or the NMJ, the timing of axon
ingrowth and synaptic transmission has been followed
with great precision in vivo. In the rat superior cervi-
cal ganglion, axons first enter the target between
embryonic (E) days 12 and 13, and afferent-evoked
postsynaptic potential are recorded by E13. Similarly,
 
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