Biomedical Engineering Reference
In-Depth Information
micropatterned discrete gradient assay to probe axon guidance responses, at least for retinal
axons.
FOR YOUR EYES ONLY
The retina has a
flat
anatomy
that (as brilliantly shown by modern luorescence
microscopy) is “painted” with
gradients
of axon guidance molecules. herefore, retina
gradients are ideally suited to mimicking by micropatterning and microluidics tech-
niques. Moreover, the retina, unlike the cortex and the hippocampus (the focus of most
axon guidance research eforts to date), is a good source of highly homogeneous cell popu-
lations, the retinal ganglion cells (RGCs). RGCs respond to Netrin-1 gradients. During the
development of the anterior visual pathway, the axon trajectories are simple, multiple rele-
vant guidance molecules have already been identiied (many tested with explants in vitro),
and a common cause of blindness (optic nerve hypoplasia) is associated with defects in
this process—which makes for great grant proposal opportunities. It is surprising that
until very recently, Friedrich Bonhoefer and his proliic academic ofspring (Drescher,
Bastmeyer, etc.) have been the only major group exploiting this rich mine with microlu-
idic tools. You might wonder how you didn't see this opportunity right before your eyes …
To further probe the role of bound protein gradients, a team led by Mu-ming Poo from the
University of California at Berkeley has investigated the responses of hippocampal neurons to
bound gradients of Netrin-1 and brain-derived neurotrophic factor (BDNF). heir novel hydro-
gel-based gradient generator is extremely easy to use and inexpensive, which should make it easy
to disseminate to other biological laboratories. he gradient generator consists of an agarose
stamp whose microchannels are illed with the protein solution of interest (
Figure 6.53a
). What
Protein solution
Agarose stamp
NH
+
NH
+
NH
+
NH
+
Surface-bounded
protein gradient
O
O
a
b
PLL-coated epoxy coverslip
10 min
18 mm
4 mm
Protein
Hippocampal neurons
350 µm
c
d
Netrin gradient
Tuj-1 neuron marker
40 hours
20 µm
smi-312 (axon marker)
Tuj-1
smi-312
FIGURE 6.53
Axon.guidance.by.surface-bound.gradients.created.with.an.agarose.stamp..(From.Junyu.
Mai,.Lee.Fok,.Hongfeng.Gao,.Xiang.Zhang,.and.Mu-ming.Poo,.“Axon.initiation.and.growth.cone.turning.
on.bound.protein.gradients,”.
J. Neurosci.
.29,.7450-7458,.2009..Figure.contributed.by.Mu-ming.Poo.)
