Sykes 1 Paul A. Sykes

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Variation in serotonergic and dopaminergic neuronal survival in the adult Drosophila CNS.
Paul A. Sykes1,2, Heather S. Norman2 and Barry G. Condron2,*
1
Medical Scientist Training Program, University of Virginia, Charlottesville, VA 22903
2
Department of Biology, University of Virginia, Charlottesville, VA 22903
*
Corresponding author:
Barry G. Condron
University of Virginia
Department of Biology
Gilmer Hall 071
Box 400328
Charlottesville, VA 22903
Phone (434)243-6794
FAX (434)243-5315
[email protected]
Running title: SEROTONERGIC AND DOPAMINERGIC SURVIVAL
Keywords: neuronal death; metamorphosis; aging.
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Summary
Loss of serotonergic and dopaminergic neurons may have serious implications for normal
brain function. Drosophila models of neurodegenerative diseases utilize the short life-span and
simple anatomy of the fly to characterize the molecular and genetic processes characteristic to
each dysfunctional state. In fly embryonic and larval ventral nerve cords, serotonergic and
dopaminergic neurons are positioned in a stereotypic pattern which is reorganized during
metamorphosis. In this study, we examine the adult pattern of serotonergic and dopaminergic
neurons within the adult fly ventral nerve cord. We find that the number of cells lost following
metamorphosis is highly variable. Changes in cell number due to age is therefore likely to be
highly masked by developmental variation. The source of this variation is likely to be apoptosisbased cell loss during pupal development.
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Dopaminergic and serotonergic neurons are involved in numerous human disease states,
including Parkinson’s disease, depression and anxiety. These neurons are subject to age-related
changes that may have pathological consequences: in the rat, serotonergic fibers develop
aberrant branching patterns, and the entire density of serotonergic fibers in the brain is decreased
with age (Nishimura et al., 1998). Similarly, the incidence of Parkinson’s disease in humans
increases with age, presumably as dopaminergic neurons are lost to neurodegenerative processes
(Dauer and Przedborski, 2003). Model systems, such as the fruit fly, provide an opportunity to
dissect the molecular and genetic pathways that contribute to proper function of an organism, as
well as those pathways that dysfunction in human disease states. Several fly models of human
neurodegenerative diseases have been described, including Parkinson’s disease, Alzheimer’s
disease and polyglutamine disease (Chan and Bonini, 2000). Recent studies have specifically
targeted fly dopaminergic neurons to model the influence of genes implicated in Parkinson’s
Disease, such as α-synuclein and Parkin (Feany and Bender, 2000; Yang et al., 2003).
Developmentally, the major reorganization of the fly brain during metamorphosis typically
spares most cell bodies (White and Kankel, 1978), but may drastically modify the connectivity of
the neurons (Technau and Heisenberg, 1982). Furthermore, aging and experience modify nerve
fiber density within the fly brain as adult flies mature (Technau 1984). However, in the young
adult fly, specific cell clusters of catecholaminergic neurons show variation between individuals
upon eclosion (Budnik and White, 1988). Following pupation, a subset of about 300 fly neurons
undergo programmed cell death in response to changes in ecdysone levels coincident with
eclosion (Robinow et al., 1993). Previous studies in other invertebrate systems have
demonstrated asymmetry in cell distribution resulting from competitive interactions between
neighboring neurons (Blair et al., 1990; Martindale and Shankland, 1990), and neuronal
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degeneration via programmed cell death pathways has been observed prior to the time of
eclosion and persisting into early adulthood (Truman, 1983). In order to further characterize the
suitability of the fly serotonergic and dopaminergic systems for longitudinal studies involving
cell death and dysfunction, here we measure the variability of neuronal loss following
metamorphosis and as aging occurs in adult flies.
Results
Anatomy and Identification of Adult Serotonergic and Dopaminergic Neurons
Ventral nerve cords (VNCs) from UAS-CD8-GFP;ddc-GAL4 adult flies (wild-type) were
removed from aged fly populations and processed for serotonin and GFP immunohistochemistry.
These flies utilize the UAS-GAL4 system (Brand and Perrimon, 1993) to express membranelocalized CD8-GFP (Lee and Luo, 1999) in all cells that also express dopa decarboxylase (ddc)
(Li et al., 2000). In the fly VNC, the serotonergic and dopaminergic neurons are the principle cell
types that are ddc-positive (Lundell and Hirsh, 1994); these neurons can be distinguished by
anatomy and immunohistochemical detection of serotonin (Budnik and White, 1988; Valles and
White, 1988). Cells from these VNCs that express GFP (i.e., ddc-positive) but do not stain for
serotonin are most likely to be dopaminergic (Lundell and Hirsh, 1994; Friggi-Grelin et al.,
2003). A regular pattern of up to seven serotonergic neurons can be identified in the prothorax
and mesothorax of the adult Drosophila VNC (Figure 1A). Dopaminergic neurons are less easily
characterized into fixed positions within the VNC (Figure 1B), but can be identified on the basis
of GFP-staining within the cell soma (Figure 1C-F). Below each confocal image is a schematic
showing the scored serotonin and dopamine neurons. Antibody access to the tissue was verified
by immunostaining for GFP (rhodamine-conjugated probe), and all native GFP fluorescence was
co-localized (data not shown). As adult flies age, serotonergic and dopaminergic neurons
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maintain their approximate position within the tissue, and can be identified between specimens.
The abdominal region was not examined due to its highly compressed anatomy; in addition,
fluorescence from labeled serotonergic fibers in the abdomen obscures some of the fluorescence
from the cell bodies, preventing adequate visualization of all of the cell soma in this region.
Normal Variation of Serotonergic and Dopaminergic Neurons in the Thoracic VNC
The number of serotonergic and dopaminergic neurons present at any given age is
variable. This variation does not seem to be strain-specific, as other wild-type strains show early
variation in catecholaminergic cell number in the thoracic VNC (Budnik and White, 1988). No
difference in variation was seen between newly eclosed adults and other 1 day-old adults (data
not shown); these VNCs were grouped for analysis. A representative composite of the thoracic
variation is shown in Figure 2. Older flies occasionally had more serotonergic and dopaminergic
neurons in the thoracic region of the VNC than some younger flies (compare Figure 2, Day 17
with Day 1C and Day 5). With respect to serotonergic neurons, no wild-type VNC had more than
seven neurons, and the anatomical localization was constant, indicating that cell loss is the cause
of variation and new cells are not being added during this time. The maximum number of
dopaminergic neurons identified in the prothorax and mesothorax was sixteen. The variation of
the cell number was measured for adult flies aged 1-22 days (Figure 3). On average, there were
three serotonergic neurons in the thorax for any given age examined (Figure 3A), and six
dopaminergic neurons (Figure 3B). There is no statistical change in the number of serotonergic
or dopaminergic neurons over the first three weeks of the fly lifespan (i.e., the slope of the trend
lines in Figure 3 do not statistically deviate from zero). However, the number of serotonergic and
dopaminergic neurons widely vary between age-matched VNCs, even among young flies,
suggesting that longitudinal aging is not a major contributing factor in the observed cell loss.
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Apoptosis May Contribute to Serotonergic and Dopaminergic Neuronal Variability
During pupation, the larval central nervous system of the fly is reorganized into the adult
pattern (Valles and White, 1988); in this time, a number of neurons are removed through
apoptosis-mediated cell death (Draizen et al., 1999). In order to examine if apoptosis plays a role
in neuronal variation within adult VNCs, expression of the anti-apoptosis baculovirus protein
p35 (Hay et al., 1994) was specifically expressed in the serotonergic and dopaminergic neurons
(Li et al., 2000) using the UAS-GAL4 system (Brand and Perrimon, 1993).
The p35 protein has been shown to prevent apoptosis-regulated cell death as well as death
due to X-irradiation (Hay et al., 1994). Three representative VNCs from 1-day old UAS-CD8GFP/UAS-p35;ddc-GAL4/+ flies are shown in Figure 4A, with corresponding schematic of
scored neurons shown below each confocal image. Note the additional serotonergic neurons in
the anterior region of the thorax. The p35-expressing adult flies did not live more than one day
following eclosion. When wild-type adults eclose following pupation, the number of
serotonergic and dopaminergic neurons within the thoracic region is highly variable (Figure 4B,
WT); however, expression of the apoptosis inhibitor p35 results in a statistically significant
increase in the number of both serotonergic and dopaminergic neurons after metamorphosis
(Figure 4B, p35).
Discussion
Changes in the number of serotonergic and dopaminergic neurons due to aging are less
significant than the individual variation within any given time point. This variation is apparent at
the earliest stages following metamorphosis, consistent with previous descriptions (Budnik and
White, 1988). The lifespan of the fly ranges from 30-60 days, such that the examination of VNCs
from the first 3 weeks of adult life incorporates a large portion of the aging spectrum. Given the
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variation observed under normal conditions, the application of the fly as a model system for
neurodegenerative diseases may be complicated when studying serotonergic and dopaminergic
neurons.
Much of the intrinsic variation in neuronal distribution in the adult VNC may be due to
cell loss during metamorphosis. Apoptosis may be one way in which these cells are removed
during this transformation. Another possibility is that these cells may have lost marker staining
and/or gene expression such that we can no longer detect these cells using our methods (i.e., a
functional deletion of the cell). In either case, these developmental changes represent significant
variability in the anatomical pattern of serotonergic and dopaminergic neurons. In other systems,
local neuronal competition has been shown to influence cell identity and survival (Martindale
and Shankland, 1990). It was not possible to examine the longitudinal effects of the apoptosisinhibitor p35 on serotonergic and dopaminergic neurons, as these adult flies all died within one
day of eclosion. Possible explanations for this short lifespan include complications from
improper central neuronal innervation, or may be due to peripheral effects of ddc-expressing
cells; outside of the central nervous system, much of the gut neuronal innervation is ddc+, and
many pigment-producing cells utilize ddc (Budnik et al., 1989).
There may be as many as sixteen serotonin-negative, ddc+ neurons in the thorax of adult
VNCs. The dichotomy of ddc-positive cells into serotonergic and dopaminergic populations is
well-established (Lundell and Hirsh, 1994; Friggi-Grelin et al., 2003), suggesting that all ddc+,
serotonin-negative neurons are by default, dopaminergic. However, the initial characterization of
catecholamine-containing neurons within the thorax found only eight potential dopaminergic
neurons based on glyoxylic acid histofluorescence, and this pattern was variable in both newly
eclosed and other 1-day old adults (Budnik and White, 1988). Here we use the functional
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expression of ddc for cell identification of dopaminergic neurons, which does not rely on the
less-efficient histofluorescence method. In contrast, serotonin fixation conditions produce less
background than dopamine detection, and allow for improved immunohistochemical detection
(Lundell and Hirsh, 1994). In our study, we validate that the variation in dopaminergic neurons
and show these differences are not due to histochemical artifact; in addition, we quantify the
variation in cell number over time for both serotonergic and dopaminergic neurons.
In summary, the distribution of serotonergic and dopaminergic cells within the adult
VNC is highly variable, resulting in part due to reorganization of the central nervous system
during metamorphosis. Initial results suggest that apoptosis may be one mechanism involved in
producing this variation. Aging may contribute to cell loss through neurodegenerative pathways
in these cells, but the individual variation in wild-type flies is sufficient to interfere with direct
analysis.
Materials and Methods
The following fly stocks were obtained from Bloomington stock center
(http://flystocks.bio.indiana.edu/): UAS-mCD8-GFP (Lee and Luo 1999); ddc-GAL4 (Li et al.,
2000); and UAS-p35 (Zhou et al., 1997).
Newly eclosed adult flies were collected daily from a large colony and housed in clean
vials containing standard fly food. These vials were changed weekly to maintain the health of the
flies, and to prevent contamination of the sample from developing offspring. Adult flies were
lightly anesthetized with CO2 for sorting prior to VNC removal. Nerve cords were rapidly
dissected from awake flies in fresh Schneider’s insect media and immediately fixed in 4%
paraformaldehyde for at least one hour, followed by 1X PBS and 1X PBT washes. VNCs were
processed for serotonin and GFP immunohistochemistry: primary antibodies anti-serotonin
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(1:100, rabbit polyclonal, ImmunoStar) and anti-GFP (1:300, 3E6 mouse monoclonal, Molecular
Probes) were added to VNCs in 300 µL 1X PBT overnight at 4°C, followed by 1X PBT washes.
The VNCs were probed with secondary antibodies (1:150, fluorescein-conjugated goat-antimouse and rhodamine-conjugated goat-anti-rabbit, Jackson Laboratories) in 300 µL 1X PBT,
overnight at 4°C. Immunostained VNCs were cleared in 90% glycerol/2.5% DABCO (Sigma)
overnight at 25°C, mounted to microscope slides and stored at –20°C prior to imaging.
Adult VNCs were imaged with a Nikon eclipse E800 microscope (10X lens, NA=0.45),
Hamamatsu ORCA-ER camera, and a Perkin-Elmer spinning disc confocal unit. Optical sections
were taken 0.48 µm apart, with 1x1 binning. Exposure times varied from 150-500 milliseconds,
depending on the intensity of the fluorescence signal. The microscope and camera were
calibrated using a micron scale (Sigma) and the Focal Check Fluorescent Microspheres Kit
(6µm, Molecular Probes, F-24633). Confocal images were leveled in Adobe Photoshop and then
imported into Volocity 2.0 for 3-dimensional (3D) rendering. For 3D rendered images, black
levels were adjusted to the upper limit of background and brightness increased such that the
signal from the cell bodies appeared equivalently intense.
Statistical comparisons were made using Tukey-Kramer Multiple Comparisons Test and
ANOVA analyses Graphpad InStat 3.0 for Macintosh. These tests evaluate whether the means of
three or more independent variables differ. Graphpad Prism 4.0 for Macintosh was used for
graphical representations, and mean±s.d. are shown in all figures.
Acknowledgments
This work was supported by a Keck Scholars Award and NINDS R29 37322 to BGC and
by the University of Virginia Medical Scientist Training Program to PAS.
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References
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Budnik V, White K (1988) Catecholamine-containing neurons in Drosophila melanogaster:
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Budnik V, Wu CF, White K (1989) Altered branching of serotonin-containing neurons in
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Hay BA, Wolff T, Rubin GM (1994) Expression of baculovirus P35 prevents cell death in
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Lee T, Luo L (1999) Mosaic analysis with a repressible cell marker for studies of gene function
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neuropeptide expression by identified neurons of the leech. Devel Biol 139:210-226.
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aging in the rat serotonergic system. Acta Neuropathol 96:581-595.
Robinow S, Talbot WS, Hogness DS, Truman JW (1993) Programmed cell death in the
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Technau GM (1984) Fiber number in the mushroom bodies of adult Drosophila melanogaster
depends on age, sex and experience. J Neurogen 1:113-126.Valles AM, White K (1988)
Serotonin-containing neurons in Drosophila melanogaster: development and distribution. J
Comp Neurol 268:414-428.
Truman JW (1983) Programmed cell death in the nervous system of an adult insect. J Comp
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White K, Kankel DR (1978) Patterns of cell division and cell movement in the formation of the
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Figure Legends
Figure 1: Serotonergic and dopaminergic neurons in adult fly ventral nerve cords (VNCs).
(A-B) General anatomy of the adult fly VNC, (A) stained for serotonin immunohistochemistry
and (B) ddc-GAL4 expression of CD8-GFP. (A) There are up to seven serotonergic neurons in
the thoracic VNC (prothorax, PT, mesothorax, MS, and metathorax, MT), while the abdominal
cord (AB) has a less regular distribution of cells. The upper thorax (PT and MS), anterior to the
asterisk (“*”), were examined for all future analyses. (B) The number of ddc+ neurons in the
thoracic VNC may be as high as 22, including both serotonergic and dopaminergic neurons (CF) Representative thoracic VNCs from UAS-CD8-GFP;ddc-GAL4 adult flies, aged up to 3
weeks, were stained for serotonin immunohistochemisty (red); ddc-positive serotonergic and
dopaminergic cells are labeled by GFP expression (green). A schematic diagram of scored
neurons is shown below each confocal image. Serotonergic neurons are co-labeled with red and
green, while dopaminergic neurons are labeled in green only. Dopaminergic neurons have an
irregular pattern within the VNC. Scale bar is 100 µm.
Figure 2: Variation in serotonergic and dopaminergic neuronal survival in the upper
thorax (prothorax and mesothorax) of the adult fly VNC.
The upper thorax region of the adult VNC (see Fig. 1A, “*”) is shown for flies aged 1-20 days.
The number of neurons present within this region is highly variable for each time point (compare
Day 1A, 1B and 1C), and loss of these cells is not necessarily dependent upon age (compare Day
1 with older time points). The general pattern of serotonergic neurons can be identified in each
sample. Scale bar is 100 µm.
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Figure 3: Longitudinal study of serotonergic and dopaminergic neuronal survival.
The total number of (A) serotonergic and (B) dopaminergic neurons for the upper thorax (PT and
MS) were manually identified from confocal reconstructions for VNCs from adult flies aged 122 days post-eclosion. Each point is shown as mean±s.d.; n=38 for day 1, and n=1-8 for days 222 (average n=4). The linear trend line (solid line) is shown with 95% confidence intervals
(hatched lines). The best-fit values for the trend line are (A) y=0.0192x+2.998, r2=0.0151 and
(B) y=0.1281x+6.114, r2=0.0869.
Figure 4: Effects of an apoptosis inhibitor, p35, on serotonergic and dopaminergic
neuronal survival following pupation.
(A) Representative adult fly thoracic VNCs from UAS-CD8-GFP/UAS-p35;ddc-GAL4/+ are
shown. These VNCs were stained for serotonin immunoreactivity (red); ddc-driven GFP
expression (green) appears in both serotonergic and dopaminergic neurons. Additional
serotonergic cells are seen in the prothorax. A schematic diagram of scored neurons is shown
below each confocal image. (B) Serotonergic and dopaminergic neurons the prothorax and
mesothorax were manually counted for 1-day old adult UAS-CD8-GFP;ddc-GAL4 (wild-type,
WT) and UAS-CD8-GFP/UAS-p35;ddc-GAL4/+ flies (mean±s.d., n=38 and 11, respectively).
Scale bar is 100 µm. *=p<0.05, ***=p<0.001.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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