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Endocrinology 149(9):4632– 4637
Copyright © 2008 by The Endocrine Society
doi: 10.1210/en.2008-0448
Sexual Differentiation of Vasopressin Innervation of the
Brain: Cell Death Versus Phenotypic Differentiation
Geert J. de Vries, Michelle Jardon, Mohammed Reza, Greta J. Rosen, Eleanor Immerman,
and Nancy G. Forger
Center for Neuroendocrine Studies and Department of Psychology, University of Massachusetts, Amherst,
Massachusetts 01003
In most vertebrates studied, males have more vasopressin
(VP) cells in the bed nucleus of the stria terminalis, or homologous vasotocin cells in nonmammalian species, than females.
Previous research excluded differential cell birth and migration
as likely mechanisms underlying this difference, leaving just
differential cell death and phenotypic differentiation of existing
cells. To differentiate between these remaining possibilities, we
compared VP cell number in wild-type mice vs. mice overexpressing the anti-cell death factor, Bcl-2. All animals were gonadectomized in adulthood and given testosterone capsules.
Three weeks later, brains were processed for in situ hybridization to identify VP cells. Bcl-2 overexpression increased VP cell
number in both sexes but did not reduce the sex difference. We
repeated this experiment in mice with a null mutation of the
D
URING DEVELOPMENT, sex differences in gonadal
hormone levels cause a multitude of sex differences in
neurotransmitter systems, which presumably contribute to
differences in neural function and behavior (1–5). In principle, two fundamentally different sets of processes could
cause these differences: those that determine the absolute
number of cells capable of expressing a specific neurotransmitter (such as the birth, death, or migration of cells), or
processes that act on preexisting cells to alter their phenotype
(4, 6, 7). It has been surprisingly difficult to disentangle these
two possibilities, in part because gonadal hormones often
trigger sexual differentiation before the neurons of interest
assume their final phenotype.
A case in point is the sexually dimorphic vasopressin (VP)
innervation of the brain. This innervation shows one of the
most consistently found neural sex differences among vertebrates (8), with males having more VP neurons in the bed
nucleus of the stria terminalis (BNST) and medial amygdaloid nucleus and denser projections from these areas than do
females across many mammalian species (8). Nonmammalian vertebrates show similar sex differences in homologous
vasotocin projections (8 –10). This sex difference has been
well studied in rats, where exposure to gonadal steroids
during perinatal life determines the number of VP cells found
in adults (11, 12). Differential cell birth and migration are
pro-cell death gene, Bax, and obtained similar results; cell number was increased in Baxⴚ/ⴚ mice of both sexes, but males had
about 40% more VP cells, regardless of Bax gene status. Taken
together, cell death is unlikely to account for the sex difference
in VP cell number, leaving differentiation of cell phenotype as
the most likely underlying mechanism. We also used immunocytochemistry to examine VP projections in Bcl-2-overexpressing mice. As expected, males showed denser VP-immunoreactive
fibers than females in the lateral septum, a projection area of the
bed nucleus of the stria terminalis. However, even though Bcl-2
overexpression increased VP cell number, it did not affect fiber
density. Thus, a compensatory mechanism may control total
septal innervation regardless of the number of contributing
cells. (Endocrinology 149: 4632– 4637, 2008)
very unlikely to contribute to the sex differences in VP expression, because VP cells are born on embryonic d 12 and
13 (13, 14) at least a week before gonadal hormones trigger
their sexual differentiation (11, 12). This leaves differential
cell death or phenotypic differentiation as the two most likely
causes.
Mice may be especially useful for differentiating between
these two possibilities, because there are several genetically
engineered strains in which cell death is altered (15). One
would predict that mice overexpressing cell death-reducing
factors would show no, or reduced, sexual differentiation if
differentiation depended on cell death. The same would be
true for mice lacking proteins required for neuronal cell
death. Here, we compare VP expression in wild-type mice vs.
two genetically altered strains: those that overexpress the
antiapoptotic factor Bcl-2, specifically in neurons (16), or
mice with a null mutation in the gene encoding the cell death
factor Bax (17). In both mutants, neuronal cell death is markedly reduced throughout the brain, and sex differences in cell
number are abolished in several brain areas (18, 19). We
report that these mutations do indeed increase the total number of cells that produce VP. Critically, however, the sex
difference in cell number remains intact.
Materials and Methods
Animals
First Published Online May 22, 2008
Abbreviations: Bcl-2-OE, Bcl-2-overexpressing; BNST, bed nucleus of
the stria terminalis; VP, vasopressin.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
Wild-type and transgenic mice overexpressing human Bcl-2 under
the control of the neuron-specific enolase promoter [Bcl-2-overexpressing (Bcl-2-OE) mice] (16) were generated by mating Bcl-2-OE males with
B6D2F1 females (The Jackson Laboratory, Bar Harbor, ME). The line was
subsequently backcrossed to B6D2F1 for at least 10 generations. In these
mice, the human Bcl-2 transgene is expressed in neurons from embry-
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de Vries et al. • Sexual Differentiation of VP Expression
onic d 13 through adulthood at levels that far exceed expression of the
endogenous mouse Bcl-2 (16). Wild-type (Bax⫹/⫹) and Bax knockout
(Bax⫺/⫺) mice were generated by mating mice heterozygous for the Bax
gene deletion (Bax⫹/⫺; The Jackson Laboratory). The knockout was
originally on a mixed C57BL/6 ⫻ 129 background (17) but subsequently
was backcrossed to C57BL/6 for at least eight generations. Offspring
were genotyped by PCR amplification of tail DNA using published
primer sequences (20, 21). Mice were housed in groups of three to four
in a 12-h light, 12-h dark cycle at 24 C. Food and water were available
ad libitum.
Gonadectomy and hormone treatments
Sexual differentiation of VP cell numbers is defined by the observation that the number of VP-expressing cells is higher in males, even when
males and females are treated with equivalent levels of testosterone in
adulthood (22, 23). Thus, we gonadectomized all mice as adults (4 –9
months old) under ketamine-xylazine anesthesia and implanted them
with 5-mm SILASTIC brand capsules (1.57 mm inner diameter, 3.18 mm
outer diameter; Dow Corning, Midland, MI) filled with crystalline testosterone (Sigma Chemical Co., St. Louis, MO). Three weeks later, mice
were asphyxiated with carbon dioxide and rapidly decapitated. Brains
were removed and cut midsagittally. One side was processed for in situ
hybridization (all groups), and the other was used for immunostaining
(Bcl-2-OE mice and their wild-type littermates only). All procedures
conformed to National Institutes of Health guidelines and were in accordance with a protocol approved by the University of Massachusetts,
Amherst, Institutional Animal Care and Use Committee.
In situ hybridization
Brain tissue was quickly frozen in dry ice-cooled 2-methylbutane and
stored at ⫺80 C until sectioning. Tissue was cut in 25-␮m coronal
sections, which were thaw-mounted onto Superfrost Plus slides (Fisher
Scientific, Pittsburgh, PA) and stored at ⫺80 C until used. Slides were
postfixed in 4% paraformaldehyde for 5 min and rinsed in 0.1 m PBS (pH
7.4) for 2 min at 4 C. The in situ hybridization closely followed a procedure used previously (23), except that each slide was exposed to about
106 dpm of a mixture of two oligonucleotide probes complementary
to the mouse VP mRNA bases 383– 426 and 430 – 477, which code
mainly for the glycopeptide region at the carboxyl end of the VP
precursor peptide. The probes were labeled at the 3⬘ end with
[35S]dATP (NEN Life Science Products, Boston, MA) using terminal
deoxynucleotidyl transferase (Life Technologies Inc., Gaithersburg,
MD). After the hybridization procedure, slides were dipped in Kodak
NTB two-track emulsion under a safelight and stored desiccated in
light-tight boxes at 4 C. Four weeks later, the slides were developed,
lightly counterstained with methyl green, and coverslipped with
Cytoseal (Fisher Scientific).
Cell counts and silver grain density
All analyses were performed on slides coded to conceal the sex and
genotype of the animal. VP mRNA-labeled cells were examined in every
third section through the BNST of each mouse. Labeled cells were
identified under dark-field illumination (Fig. 1) and counted only if
examination under bright-field illumination confirmed a methyl greenstained nucleus underneath the silver grains.
FIG. 1. In situ hybridization for VP mRNA in a Bcl-2-OE
male. A and B, Dark- and bright-field views, respectively,
of a section through the BNST. Counts of VP cells were
made under dark-field microscopy. Arrows point to the
same cells in A and B. A magnocellular VP neuron, easily
distinguishable from the parvocellular BNST neurons of
interest, can also be seen in the upper right corner. St, Stria
terminalis. C, Higher-magnification view of the boxed area
in B showing sliver grains over individual VP-positive neurons. A standard circle, as shown here, was centered over
each cell for automated grain counting. Scale bars, 100 ␮m
(A and B) and 50 ␮m (C).
Endocrinology, September 2008, 149(9):4632– 4637
4633
For estimating the average number of silver grains over labeled cells,
the section containing the greatest number of VP mRNA-expressing cells
was identified for each subject. Images of all labeled cells in that section
(approximately 50 cells per animal) were obtained with a ⫻40 objective
and analyzed using the Image version 1.44 program developed by Dr.
Rasbaud (National Institutes of Health, Bethesda, MD). The light intensity and camera settings were kept constant to standardize measurements. Grains were counted by computerized gray-level thresholding
(24). A standardized circle (⬃20 ␮m in diameter) was centered over each
cell, and the number of pixels covered by grains was determined (Fig.
1). This number was converted into number of grains by dividing the
total number of pixels per cell by the average number of pixels representing one silver grain (this last number was determined by analyzing
32– 66 randomly chosen grains in three subjects of each experimental
group).
Immunocytochemistry
VP fibers in the lateral septum were examined in Bcl-2-OE mice. Brain
hemispheres were submerged in 5% acrolein in phosphate buffer (0.1 m,
pH 7.6) for 2 h. Fixed brains were transferred to 30% sucrose in phosphate buffer and stored at ⫺20 C until sectioning. Thirty-micrometerthick sections were cut with a freezing microtome and immunostained
for VP using anti-VP (ICN Immunobiologicals, Irvine, CA), diluted
1:16,000 in Tris-Triton, as described previously (11). VP antibodies were
visualized using the ABC Elite kit (Vector Laboratories, Burlingame, CA)
followed by incubation in a 0.05% 3,3⬘-diaminobenzidine solution,
0.0005% glucose oxidase, 0.15% ␤-d-glucose, and 0.03% nickel ammonium sulfate in Tris-NaCl for 20 min. After rinsing in Tris buffer, sections
were mounted on glass slides, air dried, and coverslipped.
The density of VP-immunopositive fibers in the lateral septum was
analyzed in two consecutive sections at the level containing the highest
fiber density. Computerized gray-level thresholding (24) was performed
using the National Institutes of Health Image program, as described
previously (11). Light intensity and camera settings were kept constant
across the sections to standardize measurements. The density was expressed as the area covered by VP-immunoreactive fibers in a 0.5- ⫻
0.3-mm sampling area immediately bordering the lateral ventricle midway between the dorsal and ventral extent of the ventricular wall.
Statistical analyses
Differences between groups were analyzed separately for each mutant using two-way ANOVA with sex and genotype as between-subjects
variables. Differences were considered significant if P ⬍ 0.05.
Results
VP cell number and mRNA expression
Male rats have more VP cells in the BNST cells than do
females, even when both sexes are treated with identical
hormone capsules in adulthood (23). In this study, we confirm that mice show a similar sex difference, albeit of smaller
magnitude than is seen in rats. In the first experiment with
Bcl-2-OE mice and their wild-type littermates, males had
about 25% more VP cells than did females [F(1, 35) ⫽ 14.570;
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Endocrinology, September 2008, 149(9):4632– 4637
FIG. 2. Number of VP neurons in the BNST of Bcl-2 OE mice and
their wild-type littermates. Bcl-2 overexpression increased the number of VP cells, but did not reduce the sex difference in cell number.
Lines over bars indicate main effects of sex and genotype. There was
no sex-by-genotype interaction.
P ⬍ 0.001]. We also observed a main effect of genotype, such
that Bcl-2-OE mice had more VP cells than did wild-type
mice [F(1,35) ⫽ 11.7; P ⬍ 0.002; Fig. 2]. The increase was
observed in both sexes, and no sex-by-genotype interaction
on VP cell number was seen [F(1,35) ⫽ 0.02; P ⬎ 0.8].
Bax⫺/⫺ mice and their wild-type (Bax⫹/⫹) controls displayed the same pattern. Males had 44% more VP cells than
did females irrespective of genotype [F(1,19) ⫽ 13.92; P ⬍
0.0015; Fig. 3]. As was seen with Bcl-2-OE mice, we also found
that Bax⫺/⫺ animals had about 30% more VP cells than did
wild-type mice [F(1,19) ⫽ 7.18; P ⬍ 0.015; Fig. 3]. There was
de Vries et al. • Sexual Differentiation of VP Expression
FIG. 4. The number of silver grains per cell after in situ hybridization
for VP mRNA in the BNST of Bcl-2-OE and wild-type mice. There
were no significant differences between groups.
no sex-by-genotype interaction [F(1,19) ⫽ 0.72; P ⬎ 0.7], confirming that the magnitude of the sex difference was not
affected by Bax gene status. Thus, based on both strains of
mice, there is a significant sex difference in VP cell number,
and VP cell number is subject to Bax- and Bcl-2-dependent
cell death. Importantly, however, the sex difference in VP cell
number is not affected in the cell death mutants.
We also quantified the number of silver grains over each
positive cell in Bcl-2-OE and WT mice (Fig. 1). We found no
effect of genotype on average number of silver grains per cell
[F(1,35) ⫽ 0.32; P ⬎ 0.5]. Thus, Bcl-2 overexpression does not
affect VP mRNA levels in positive cells. There was also no
effect of sex on this measure and no sex-by-genotype interaction (Fig. 4). Thus, males have more VP cells than do
females, but mRNA expression per cell did not differ significantly between sexes. This differs from what has been
found in rats, where both cell number and mRNA content per
cell are greater in males (23). If anything, the number of silver
grains per cell was slightly (but nonsignificantly) higher in
female mice (Fig. 4).
VP innervation of the septum
FIG. 3. Number of VP neurons in the BNST of Bax knockout mice
(Bax⫺/⫺) and their wild-type siblings. Bax gene deletion increased the
number of VP cells but did not reduce the sex difference in VP cell
number. Lines over bars indicate main effects of sex and genotype.
There was no sex-by-genotype interaction.
The greater number of VP cells in Bcl-2-OE mice, together
with evidence that expression per cell did not differ by genotype, suggested that Bcl-2-OE mice might have a denser
innervation of target sites by VP-immunoreactive nerve terminals. We assessed this by performing immunocytochemistry for VP in the lateral septum, a major projection site of
BNST VP neurons (8). The sex difference in VP cell number
was matched by an equally large sex difference (male more
than female) in the density of VP-immunoreactive fibers in
the lateral septum [F(1,34) ⫽ 8.47; P ⬍ 0.007; Figs. 5 and 6]. This
was true for both wild-type and Bcl-2-OE mice [i.e. there was
no interaction between sex and genotype; F(1,34) ⫽ 0.09; P ⬎
0.7]. Surprisingly, however, despite the larger number of
de Vries et al. • Sexual Differentiation of VP Expression
Endocrinology, September 2008, 149(9):4632– 4637
4635
Discussion
FIG. 5. VP immunoreactivity in the lateral septum of Bcl-2 OE and
wild-type mice. A, Wild-type male; B, wild-type female; C, Bcl-2-OE
male; D, Bcl-2-OE female. Medial is to the left in all views. VP
immunoreactivity was greater in males than in females and was
not influenced by genotype. ac, Anterior commissure; CP, caudate
putamen; fi, fimbria; LS, lateral septum; V, lateral ventricle. Scale
bar, 200 ␮m.
VP-expressing cells in Bcl-2-OE mice, VP innervation of the
septum was not distinguishable from wild-type controls
[F(1,34) ⫽ 0.08; P ⬎ 0.7; Figs. 5 and 6].
FIG. 6. Quantification of VP immunoreactivity in the lateral septum
of Bcl-2-OE mice and their wild-type siblings. Fiber density (expressed as labeled pixels per 1.5 ⫻ 105 ␮m2) was greater in males than
in females (P ⬍ 0.007). There was no effect of genotype and no sexby-genotype interaction on this measure.
The sex difference in VP innervation of the forebrain has
received considerable attention in recent years and has been
linked to sex differences in the modulation of autonomic
functions, learning and memory, and social or reproductive
behaviors in several species (2, 8, 10, 25, 26). Despite the
intense interest in this neuropeptide, the cellular basis for the
sex difference has not been established. Previous work tested
whether differential neurogenesis contributes to sexual differentiation of VP expression. Birth dating shows that VP
neurons are among the very earliest cells generated in the
region, with well over 80% born on embryonic d 12 and 13
and virtually none born after embryonic d 16 in either sex (13,
14). Gonadal hormones, however, act during the first 2 wk
postnatally to determine the number of BNST cells that will
express VP in adulthood (11, 12). Similarly, cell migration
into the BNST appears complete prenatally (27), before gonadal hormones determine the final number of VP cells, and
there is no evidence for differential placement of VP cells in
males and females. This leaves the differentiation of cell
phenotype or differential cell death as possible mechanisms
underlying the sex difference in VP number.
Prior evidence in rats favors the differentiation of cell
phenotype. Essentially all VP cells in the BNST coexpress the
neuropeptide galanin, but not all galanin cells coexpress VP
(28). Because the total number of galanin cells does not differ
between males and females, it was hypothesized that, during
development, higher levels of testosterone act on existing
galaninergic cells to increase the percentage that will coexpress VP (29). In support, VP and galanin neurons in the
BNST and medial amygdaloid nucleus of rats show the same
unusual birth profile, with both types of neuron born days
earlier than most surrounding cells (14), consistent with the
idea that these neurons belong to a single pool.
The hypothesis that testosterone directs pluripotent cells
to become vasopressinergic was based on circumstantial evidence, however, and is difficult to test directly. Gonadal
steroid hormones determine VP cell number soon after birth
(11, 12), yet the vast majority of presumptive VP neurons do
not begin expressing VP until days (in males) to weeks (in
females) later (30). Thus, one cannot identify the cells of
interest during the time the sex difference in their number is
determined. It has also been difficult to rule out a role for cell
death. In mice, the number of galanin cells in the BNST may
itself be sexually dimorphic (31), which is problematic for a
model that supposes that testosterone acts on a sexually
undifferentiated pool of galaninergic precursor cells to produce the sex difference in VP cell number. In rats, it remained
possible that testosterone increases the death of galanin-only
cells while decreasing the death of cells coexpressing galanin
and VP. The current observations, however, rule out cell
death as a likely factor.
We find that sex differences in VP cell number persist in
Bcl-2-OE and Bax⫺/⫺ mice. We previously used these same
mutants to examine the cellular basis of sex differences in
several other neural systems (18, 19, 32). In each case, and in
contrast to results presented here, sex differences in overall
cell number were eliminated by Bax deletion, or significantly
reduced by Bcl-2 overexpression, indicating that differential
4636
Endocrinology, September 2008, 149(9):4632– 4637
cell death in males and females was responsible for the differences. Here we report that although Bax deletion and Bcl-2
overexpression increase the number of VP cells, neither mutation reduces the sex difference.
Because there are multiple paths to cell death (33), these
observations alone would not completely rule out the possibility that cell death contributes to the sex difference in VP
cell number, but that in this case death is Bax and Bcl-2
independent. Several considerations make this possibility
rather unlikely, however. The Bcl-2 family proteins have
emerged as crucial regulators of survival in the developing
nervous system (15, 16, 34), and Bax in particular appears to
be required for most cell death in the developing nervous
system (21). VP cell number in this study was increased in
Bax⫺/⫺ mice, as is total cell number in the BNST of Bax
knockouts (19). Even more important, apoptosis is essentially
eliminated in the BNST of perinatal Bax⫺/⫺ mice of both
sexes (35). That is, there is no evidence that in Bax⫺/⫺ mice,
cells in the BNST find another, Bax-independent, road to
death. Nonetheless, preventing cell death by Bax deletion
does not have any impact on the sex difference in the number
of BNST cells that express VP.
Bcl-2 overexpression also increased total VP cell number
without affecting the sex difference in VP cells. A previous
report suggests that a Bcl-2 null mutation reduced VP synthesis in magnocellular neurons of the paraventricular and
supraoptic nuclei in the hypothalamus (36). If so, then overexpression of Bcl-2 might have increased cell counts simply
by increasing VP expression in the BNST, thereby enhancing
the detectability of individual VP neurons. We can rule out
this interpretation, however, because we did not find a difference in VP mRNA levels between wild-type and Bcl-2-OE
mice. Thus, the increased number of VP cells in Bcl-2-OE
mice is not due to changes in peptide expression and, therefore, detectability of cells.
As argued above, our present and previous studies rule
out differential cell birth, migration, and death as likely
causes for sexual differentiation of vasopressin innervation,
leaving differentiation of neuronal phenotype as the only
remaining plausible cause. Numerous other neurotransmitters and neuropeptides (e.g. dopamine, neurotensin, substance P, and enkephalin) show sex differences in cell number (reviewed in Ref. 1), and for none of these has the cellular
mechanism of sexual differentiation been established. The
search for these mechanisms may be amenable to the same
strategy used here. For example, we previously examined
dopaminergic neurons in the anteroventral periventricular
nucleus, which are much more numerous in females than in
males (37). Although neither Bax deletion nor Bcl-2 overexpression eliminated this sex difference (18, 19), the interpretation of this finding is muddied by the fact that the total
number of dopaminergic neurons in anteroventral periventricular nucleus also was unaffected in the cell death mutants. Thus, the mechanism regulating this sex difference
remains unresolved.
Because Bcl-2-OE mice had an increased number of VP
cells in the BNST, and mRNA expression per cell did not
differ, one might expect denser VP innervation of BNST
target sites. Interestingly, our data contradict this; the density
of VP fibers in the lateral septum did not differ between
de Vries et al. • Sexual Differentiation of VP Expression
Bcl-2-OE and wild-type mice. One possible explanation for
this result is that the extra VP neurons in the mutants do not
form functional connections with the septum. For example,
motoneurons rescued from developmental cell death by Bax
gene deletion may not project to target muscles (38). However, these supernumerary motoneurons are also severely
atrophic and fail to express cell-specific markers, which does
not appear to be the case for the VP cells examined here.
Alternatively, a mechanism independent of VP cell number
may regulate the density of VP innervation of the septum.
Signals from target cells influence axon branching and the
density of afferent innervation in many neural systems (39 –
41). Thus, the septum might limit the total input from the
BNST. If so, then increasing the number of BNST cells would
not be expected to increase the density of VP projections;
instead, axon branching and/or synapse number per cell
would be reduced. Our differentiation of cell phenotype
hypothesis predicts that the magnitude of the sex difference
in VP innervation is unlikely to be affected, because this
hypothesis posits that the number of cells projecting to targets such as the septum does not differ in males and females;
rather, it is the percentage of those cells expressing VP that
is differentiated. The fact that the sex difference in VP innervation was preserved in Bcl-2-OE mice is one more piece
of evidence in support of this hypothesis.
The next hurdle will be to identify the molecular mechanisms underlying differentiation of VP expression. The permanent effect of testosterone on the potential of cells to
express VP suggests that epigenetic mechanisms (e.g. methylation of the gene promoter region or modifications of its
associated histones) are likely to be involved (42). A more
complete understanding of the sexual differentiation of VP
innervation may provide clues to the origin of behavioral
disorders such as depression, autism, and schizophrenia (43–
47). Interestingly, each of these disorders shows sex differences in occurrence (48 –50) and has been linked to variability
in VP neurotransmission, e.g. elevated VP levels in cerebrospinal fluid or polymorphisms in the VP receptor gene (43,
51–56). Mechanisms that contribute to differences in VP innervation may therefore explain some of the variation between the sexes found in these disorders.
Acknowledgments
We thank Lynn Bengston for assistance with the figures.
Received April 1, 2008. Accepted May 15, 2008.
Address all correspondence and requests for reprints to: Geert J. de
Vries, Center for Neuroendocrine Studies, University of Massachusetts,
Amherst, Massachusetts 01003. E-mail: [email protected].
The research was funded by National Institutes of Health Grants KO2
MH01497 and RO1 MH47538 (G.J.d.V.) and KO2 MH072825 and RO1
MH068482 (N.G.F.).
Disclosure Statement: The authors have nothing to disclose.
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