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lines or the same transgenic lines observed variable effects on all phenotypes, in
particular with degeneration of DA neurons and climbing deficits. For example, a
couple of groups have reported no quantifiable defects in locomotion upon
expression of WT or mutant
,
2005). This may not be entirely surprising given that behavioral assays are well
known to be sensitive to subtle environmental fluctuations. Coupled with find-
ings that DA neuron dysfunction results in a hyperexcitable or startle phenotype
upon vigorous mechanical disturbance of flies raises the possibility that over-
vigorous handling of flies may impact the outcome of a climbing assay (Friggi-
Grelin
-synuclein (Auluck
et al.
, 2002; Pesah
et al.
, 2003). Furthermore, as described above, recent work has shown that
apparently homogeneous fly populations consist of subpopulations with varying
“high” or “low” locomotor activity (Lima and Miesenbock, 2005). While still
unresolved, such technical influences still deserve consideration.
Moreover, several groups have reported significantly less pronounced
DA neuron degeneration, seeing only a partial (
et al.
50%) decrease (Auluck and
Bonini, 2002; Auluck
et al.
, 2002; Bayersdorfer
et al.
, 2010). Again, reports vary as
to the differential effects of WT and mutated
-synuclein; for example, Auluck
et al.
concur with no difference observed between WT and A30P or A53T
-
synuclein (Auluck and Bonini, 2002; Auluck
et al.
(2008) describe only significant effects with A30P. Perhaps, most concerning is a
report describing total lack of effect for all phenotypes with multiple lines of WT,
A30P, or A53T
et al.
, 2002), while Botella
, 2005). Although singular in their formal
publication, these observations are somewhat corroborated by the
-synuclein (Pesah
et al.
very
modest
effects reported by Du
(2010) and others (T. Riemensperger and S. Birman,
personal communication). Consequently, other investigators have taken to sig-
nificantly increase the expression level of
et al.
-synuclein by expressing multiple
copies of a modified translation-efficient transgene, and still effecting only modest
loss of DA neurons (this time in the PPL1 cluster; Trinh
, 2008).
While these conflicts are not easily reconciled, several possible expla-
nations can be offered. The construction of equivalent lines is essentially identi-
cal; however, there may exist subtle differences, such as genetic background or
insertion site effects on expression levels. These aspects are hard to control with
traditional methodologies, and may have a profound effect on the toxicity of an
exogenous gene, but may be circumvented with the use of recent advances in
transgenesis such as site-specific genomic integration (Groth
et al.
et al.
, 2004). How-
ever, a recent study by Auluck
et al.
(2005) strongly suggests that the conflicting
results of DA neuron analysis in
-synuclein transgenic flies are likely explained
by differences in the methodology used to analyze DA neurons. While many of
the studies that report profound neuronal loss in
-synuclein-expressing flies
utilized paraffin-embedded sectioning and light microscopy techniques to visua-
lize TH-positive neurons (Auluck and Bonini, 2002; Auluck
et al.
, 2002; Chen
and Feany, 2005; Chen
et al.
, 2009; Feany and Bender, 2000), more recent work
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