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do not provide the stop signal. However, when
pontine nuclei were cultured on a bed of glia along
with dissociated granule cells, the outgrowth of neu-
rites was depressed. By closely examining individual
neurites it was found that the decreased growth was
due to contact with granule cells (Figure 8.13A). Neu-
rites that did not come upon a granule cell during their
outgrowth continued to grow for a normal distance.
Moreover, when granule cells were suspended above
the pontine explants, the neurites grew at a normal
rate. Thus, growth cones can be terminated at the
appropriate target by a contact-dependent mechanism.
The dialogue between pre- and postsynaptic cells
begins as soon as the growth cone filopodia makes a
contact. Calcium levels suddenly increase in the
growth cone (Dai and Peng, 1993; Zoran et al., 1993).
This was determined for both frog and snail motor
neurons that were grown in dissociated tissue culture
and filled with a Ca 2+ -sensitive indicator dye. When a
muscle cell is manipulated into contact with a growth
cone, the Ca 2+ increases locally within seconds (Figure
8.9). This response exhibits some target-specificity. The
Ca 2+ rise only occurs when appropriate postsynaptic
cells are manipulated into contact with the motor
neuron. This mechanism is similar to that observed
when growth cones collapse as they contact specific
pathfinding cues, which is often accompanied by an
elevation of intracellular Ca 2+ (see Chapter 5). It is not
yet clear how calcium levels increase, but one possi-
bility is that calcium is released from internal stores. In
the rat central nervous system, IP 3 receptors, which
transduce calcium release from endoplasmic reticu-
lum, are upregulated during the period of intense
synaptogenesis (Dent et al., 1996).
What is the evidence that a contact-evoked rise in
Ca 2+ provides a signal for growth cone differentiation?
Intracellular Ca 2+ can be manipulated in growth cones
by exposing them to an ionophore such as A23187, a
molecule that spontaneously inserts into a neuron
membrane, allowing Ca 2+ to pass freely into the cell
(Mattson and Kater, 1987). As calcium rises, growth
cones are often found to slow down and to assume a
rounded appearance. The effect of increased calcium
can even be detected on growth cones that have been
isolated from their cell body, indicating that calcium
acts locally. When the Ca 2+ concentration within the
growth cone is adjusted to differerent levels by setting
extracellular Ca 2+ concentration, cultured chick DRG
growth cones became stationary in all but a limited
range of Ca 2+ concentrations, from 200 to 300 nM
(Lankford and Letourneau, 1991).
The formation of new synapses may be regulated by
the presence of astrocytes. When retinal ganglian cells
are isolated and grown in a defined medium, they
FIGURE 8.9 Contact with target increases free calcium in the
growth cone. Dissociated neurons were filled with a Ca 2+ -sensitive
dye (top), and the growth cone was imaged while either a muscle
cell or a neuron was brought into contact (middle). Intracellular free
calcium increased only during contact with the muscle (red). The
muscle-evoked rise in Ca 2+ did not occur when the cells were bathed
in a Ca 2+ -free medium (bottom), indicating the involvement of
calcium channels. (Adapted from Dai and Peng, 1993)
display little synaptic activity. However, the addition
of astrocytes from their target region leads to a dra-
matic increase in the number and strength of synaptic
contacts (Ullian et al., 2001).The glial cells need not be
in contact with retinal neurons to elicit this response,
suggesting that they release a soluble factor. A search
for the synapse-inducing activity led to the discovery
that an essential membrane constituent, cholesterol,
plays an important role in synapse-formation. Appar-
ently, developing neurons manufacture only enough
cholesterol to survive and grow dendrites, but depend
on the delivery of additional cholesterol from astro-
cytes to produce synapses (Mauch et al., 2001). There
are likely to be many other glial signals; a large protein,
thrombospondin, can partly explain the ability of
astrocytes to induce synapse formation.
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