Biomedical Engineering Reference
In-Depth Information
a
Phase contrast
α-Tubulin
Matrigel
PDL
PDMS
b
Matrigel
PDL
PDMS
30 µm
FIGURE 6.49
Integration.of.biochemical.and.microtopographical.cues.by.the.growth.cone..(From.
Li,.N..and.Folch,.A.,.“Integration.of.topographical.and.biochemical.cues.by.axons.during.growth.on.
microfabricated.3-D.substrates,”.
Exp. Cell Res.
.311,.307,.2005..Figure.contributed.by.Nianzhen Li.)
if the steps did not exist. On the other extreme, neurons always turned at steps deeper than 22
μm. In the middle, approximately 50% of axons turned when the step was about 11-μm-deep,
possibly relecting the size of the growth cone in culture (see
Figure 6.48c
, inset). he reason
for this behavior is probably that the growth cone was “feeling” the substrate before deciding
the growth direction. Indeed, for the 11 μm step, the incidence of turning was a strong func-
tion of the angle of approach: 100% of the axons that arrived at the step very tangentially (0-15
degrees) continued on the plateau whereas approximately 90% of the axons that arrived at the
step orthogonally to it (76.90 degrees) ended up crossing it.
How about studying the reaction of the growth cone to topographical cues while conlicting
biochemical cues are also present? he author's laboratory added Matrigel (a good neural growth
substrate) to PDMS steps to observe the growth preferences of mouse cortical neurons (
Figure
6.49
). On lat
poly-D-lysine (PDL)
-coated PDMS substrates, the neurons preferred to grow on
the PDL-coated substrates (
Figure 6.49a
), suggesting that neurons prefer PDL compared with
Matrigel. However, on 22-μm-deep PDMS steps, the neurons that started growing on PDL-coated
substrates preferred to grow into Matrigel (
Figure 6.49b
); if they had made any efort to “feel”
the step, they would have turned, as they did for substrates that are not covered with Matrigel
(see
Figure 6.48
). It is clear that these neurons have a preference to grow straight if the substrate
is permissible, and that in doing so they integrate both biochemical and topographical cues.
6.5.1.3 Axon Guidance by Insoluble (Surface) Gradients
Studies in the last two decades have revealed that axons in the developing embryo are guided by
extracellular signals to grow along speciic paths toward their inal synaptic targets. Axon guid-
ance factors can be categorized according to their efect (attractive or repulsive) or according to
their acting range (contact or long range), thus yielding four categories of axon guidance mecha-
nisms. his is a simpliication because diferent axons can respond to a given signal diferently
and even the same axon's response may change with time and space (e.g., with developmental
clock, previous exposures to other signals, coincidence with other competing signals, etc.). he
growth cone senses contact guidance factors through cell-adhesion receptors (integrins,
cad-
herins, NCAM, L1
, etc.) that enable it to move by modulating its adhesion to the ECM and
to other cells. he growth cone also senses gradients of difusible long-range factors through
specialized receptors that are unique for each guidance factor and integrates all the stimuli
to produce growth and motility. Yet reproducing these interactions in vitro has been diicult
because neurons are not always are as responsive in culture as they are in the embryo and the
