Biomedical Engineering Reference
In-Depth Information
Using the hook method, the microtubule polarity of neurites has been measured in excised nerves
(Burton and Paige, 1981, Heidemann et al., 1981) and cultured neurons (Baas et al., 1988, 1989). These
studies showed that early in neuronal development, all neurites have uniform polarity microtubules.
However, as the neurites mature and their identities as axons and dendrites are established, the micro-
tubule organization also evolves. The axons contain uniform polarity, plus-end-distal microtubules,
whereas dendrites contain mixed-polarity microtubules with equal numbers of plus- and minus-end-
distal microtubules. The distinct difference between axons and dendrites is maintained in the mature
neuronal cultures. There are exceptions to this
in vitro
developmental rule, for example, when the axon
is injured (Baas et al., 1987).
Can the
in vitro
microtubule organization principles be extrapolated to understand neurites
in vivo
?
SHG intensity is sensitive to microtubule polarity, providing a tool for measuring in native brain tissues
that are too thick for the hook method. An early study showed that in acute hippocampal brain slices
of young rats, postnatal 14- to 20-day-old, axons generated SHG signal, agreeing with earlier observa-
tions that axons contain uniform polarity microtubules (Dombeck et al., 2003). In a follow-up study
in mice over a wide range of ages, it was found that mature apical dendrites of pyramidal neurons, the
principal excitatory cell in the hippocampus and in the neocortex, also generate SHG (Figure 7.6). This
result implies that, in addition to axons, certain classes of dendrites can also contain polarized micro-
tubule arrays (Kwan et al., 2008). Recently, a separate study that tracked the movement of fluorescently
labeled microtubule plus-end-tracking proteins in
Drosophila
neurons found that their proximal den-
drites contain polarized, minus-end-distal microtubules (Stone et al., 2008). Taken together, these new
results demonstrate that uniform polarity, plus-end-distal microtubules in axons may be a universal
rule, whereas microtubule organization in dendrites can differ for proximal versus distal compartments
and for
in vivo
versus
in vitro
conditions.
Mapping the distribution of polarized microtubule ensembles using SHG imaging is applicable to
native brain tissues so it can be combined with electrophysiology and molecular manipulations. For
example, the dependence of long-term potentiation on microtubule-based axonal transport was investi-
gated in the acute brain slice, where SHG intensity was used to monitor the pharmacological destruction
of the microtubule network (Barnes et al., 2010). Another application area is the study of neurodegen-
erative disease. One hypothesis for the pathological mechanism in Alzheimer's disease is the disruption
of axonal transport. SHG imaging has been applied to study the structure of polarized microtubules
near senile plaques in brain slices from Alzheimer's disease mouse models (Kwan et al., 2009) and to
investigate how overexpression of tau, a protein genetically linked to the disease, can affect the SHG
intensity (Stoothoff et al., 2008). It is expected that SHG imaging will be a useful tool for assessing the
structural and functional integrity of microtubule networks within neurons.
7.3 Application 2: endogenous time Stamp and Marker for
cell Division for Whole-embryo Developmental Studies
A central problem in developmental biology is embryogenesis. From the beginning of a single cell, the
fertilized egg goes through successive series of cell divisions. The number of cells grows exponentially
until the organism takes shape. To unravel this process, it is essential to track cell differentiation and
movement with high spatiotemporal specificity. An ideal tool should be able to observe subcellular
details at a temporal resolution finer than the duration of each phase of cell division, over a time span
that covers the entire embryogenesis. Moreover, because later cell division cycles involve a large number
of cells, an automated process of segmentation and annotation is a necessity.
The multiple forms of nonlinear optical signals, including SHG, two-photon-excited fluorescence, and
third-harmonic generation, could excite different endogenous molecules to illuminate various aspects of
the tissue (Zipfel et al., 2003). An early study used a Cr:forsterite laser source in the ~1200-1350 nm range
to observe mitosis inside live zebra fish embryos (Chu et al., 2003). Long excitation wavelengths reduced
photodamage and enabled time-lapse recording for over 20 h (Chen et al., 2006). For the development
