The challenge hypothesis: behavioral
ecology to neurogenomics
John C. Wingfield
Journal of Ornithology
ISSN 2193-7192
J Ornithol
DOI 10.1007/s10336-012-0857-8
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DOI 10.1007/s10336-012-0857-8
REVIEW
The challenge hypothesis: behavioral ecology to neurogenomics
John C. Wingfield
Received: 8 July 2011 / Revised: 14 April 2012 / Accepted: 24 April 2012
Ó Dt. Ornithologen-Gesellschaft e.V. 2012
Abstract Male song sparrows (Melospiza melodia) are
territorial year-round. However, neuroendocrine responses
to simulated territorial intrusions (STI) differ between
breeding (spring) and non-breeding seasons (autumn). In
spring, exposure to STI leads to increases in plasma levels
of luteinizing hormone and testosterone (consistent with
the challenge hypothesis), but not in autumn. This suggests
that there are fundamental differences in the mechanisms
driving neuroendocrine responses to STI between seasons
despite apparently identical behavioral responses. Recent
studies have also shown that areas of the telencephalon and
diencephalon involved with singing behavior and aggression express the enzymes necessary to synthesize sex
steroids de novo from cholesterol. Of these, aromatase (that
regulates the conversion of testosterone to estradiol) and
3b-hydroxysteroid dehydrogenase (that regulates the
synthesis of biologically active steroids) are regulated
seasonally, whereas receptors for sex steroids such as
androgen receptor and estrogen receptor alpha and beta are
not. Functional analyses indicate specific genes that may be
involved in the mechanisms of differential neuroendocrine
responses to aggressive interactions in different life-history
stages. Microarrays were used to test the hypothesis that
gene expression profiles in the hypothalamus after territorial aggression differ between the seasons. Over 150 genes
were differentially expressed between spring and autumn
in the control birds and 59 genes were significantly affected
by STI in autumn, but only 14 genes in spring. Real-time
Communicated by Cristina Miyaki.
J. C. Wingfield (&)
Department of Neurobiology, Physiology and Behavior,
University of California, Davis, CA 95616, USA
e-mail: [email protected]
PCR was performed for validation, and it indicated that
STI drives differential genomic responses in the hypothalamus in the breeding versus non-breeding seasons. The
results suggest major underlying seasonal effects in the
hypothalamus that determine the differential response upon
social interaction.
Keywords Challenge hypothesis Sex steroids Aggression Gene expression Territoriality
Introduction
The Earth has always been a changeable planet where
organisms often must deal with rapid shifts in both physical
and social environments. With global changes in climate,
as well as different social interactions due to changed
population dynamics, an overarching question is, how do
organisms organize their life cycles, time components of
those cycles, and synchronize them with other individuals?
Clearly they use environmental signals from the physical
environment and the social environment to do this
(Wingfield 2008a, b). Such social interactions include
male–male interactions over territories and mates that in
turn are both dependent upon and regulate reproductive
hormone secretions. The evolution of traits associated with
hormone–behavior interactions is also receiving considerable attention in the face of global change, shifting ranges
of organisms, and the resultant social interactions in novel
environments (Wingfield 2008a; Duckworth 2009; Hau and
Wingfeld 2011).
The focus here will be on male–male interactions as
they establish and defend territories in seasonal contexts
and also attract mates. Aggressive behavior expressed in
these contexts is a complex suite of behaviors that can be
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both offensive and defensive (e.g., Wingfield et al. 2005).
Quantifying aggression in territorial contexts is a complex
task because of the varying postures and vocalizations
expressed and their intensity; see Fig. 1 for examples in the
Song Sparrow, Melospiza melodia (from Wacker et al.
2008). Although a detailed analysis of these traits is possible in captive conditions where video allows the quantification of even the most subtle differences in postures and
intensity, such detailed quantification in the field is
impossible because birds are moving in and out of vegetative cover and are not visible for the whole study session.
Thus, to quantify aggression in field conditions, we use
easily quantified behavioral traits such as latency to
respond to a simulated territorial intrusion (STI), where a
live decoy male Song Sparrow in a cage is placed in the
territory of a free-living male, and tape-recorded songs are
then played through a speaker placed alongside. When the
focal male responds, it is possible to record the total
number of songs given during a 10 min observation period,
the closest approach to the decoy, the time spent within
5 m of the decoy, and the total number of flights around the
decoy (e.g., Wingfield 1985; Wingfield and Hahn 1994). In
general, the behavioral results are similar in the laboratory
and field, but differences can be detected, especially when
investigating the effects of hormone manipulations on
suites of behavioral traits that are not easy to record in field
conditions (i.e., they can differ depending upon the detailed
information collected). Therefore, laboratory experiments
have the potential to reveal much more specific effects of
hormones and their metabolites (Wacker et al. 2008),
whereas field experiments assess environmental and social
effects more accurately but with more general behavioral
data.
Fig. 1 Behavioral traits studied in the laboratory allow a completely
different assessment of aggression from field studies. Different body
postures used in both offensive and defensive territorial aggression
can be scored, as can their intensity. This aggression can be directed
specifically at another individual, often at close range (\20 cm), or
indirectly in response to the presence of another individual meters
away. From Wacker et al. (2008), courtesy of Elsevier Press.
Drawings by Todd Sperry
Endocrine and paracrine control of aggression in birds
Agonistic behaviors are expressed in all phases of an
individual’s life, and it is not surprising that a suite of
hormonal and central nervous system paracrine secretions
have been implicated in the control of aggressive behavior
(e.g., Adkins-Regan 2005; Wingfield et al. 2005). In birds,
as in other vertebrates, central control involves complex
interactions of paracrine actions of arginine vasotocin
(AVT), vasoactive intestinal peptide (VIP), and serotonin
in relation to sociality and social networks in the CNS
(Adkins-Regan 2005; Goodson and Evans 2004; Goodson
2005; Goodson et al. 2006, 2009). Hormonal activation by
testosterone (T) or estradiol (E2) after conversion from
testosterone in target cells is also important, especially in
species that show marked seasonal changes in aggression,
such as those that are territorial only during the breeding
season (Wingfield et al. 2005; Mukai et al. 2009). In other
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contexts, prolactin regulates parental aggression (Buntin
1996) and corticosterone can increase competition for food
(Kitaysky et al. 2003). Hormonal and paracrine control of
agonistic behavior may also include deactivation (inhibition) by glucocorticoids of territorial aggression and sexual
aggression (e.g., Wingfield and Silverin 1986) and by the
activation of submissive behavior (Leshner 1978, 1981).
When expression of agonistic behavior occurs in different seasons of the year, and usually in very different
contexts such as breeding and non-breeding, an important
question arises: is control of, for example, seasonal
expression of territorial aggression regulated by the same
mechanisms, or by a completely different means? Given
the well-known role of T in the regulation of aggression in
relation to reproduction, it has been questioned whether
control over similar aggression in the non-breeding season
is also regulated by T (e.g., Wingfield and Hahn 1994;
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Wingfield and Soma 2002; Wingfield et al. 2001, 2005).
However, before we go on to discuss the regulation of
similar behavioral suites at different times of the year and
contexts, it is important to first reflect on the biological
actions of T.
Control of territorial aggression in seasonal contexts:
what does testosterone do?
There is very extensive evidence that T levels in blood are
higher during the breeding season than during the nonbreeding season. Furthermore, in many socially monogamous songbirds, plasma T concentrations peak early in
spring, when males arrive in breeding territories and attract
mates. There is a marked decline in T to a breeding
baseline during the parental phase when both males and
females may feed young (Fig. 2; Wingfield et al. 1990). In
species that are territorial virtually year-round, it is clear
that T levels in blood generally remain below the detectability threshold of the assay (e.g., Wingfield and Hahn
1994; Fig. 2). In the rare cases in which T levels do
increase in autumn, the non-breeding season, then these
peaks are involved in pair formation and may actually
represent the beginning of the breeding season (with a
Fig. 2 Plasma testosterone levels in free-living male Song Sparrows
in relation to territorial behavior. Challenges with simulated territorial
intrusions (STI) indicate that males are territorial throughout the
breeding season (February–August) but not during the molt in late
summer. By early autumn (October) they become territorial again,
and remain so (albeit with some decline in aggressive responses to
STI) during the worst weather of winter. Note that although
testosterone levels in blood are high and variable in the breeding
season, they are below the detectability threshold of the assay in molt,
autumn and winter. From Wingfield and Hahn (1994), courtesy of
Academic Press (Elsevier)
hiatus during most of winter, Wingfield et al. 1997). In the
northwestern subspecies of Song Sparrow, M.m. morphna,
males remain in territories during the post-breeding prebasic molt, but show greatly blunted aggressive responses
to STI. This is concomitant with a decrease in plasma T to
undetectable levels (Fig. 2). In autumn, after the molt is
complete, these males become once again very responsive
to STI in terms of aggressive responses, but plasma T
levels remain undetectable despite levels of territoriality
similar to those seen in spring (Wingfield and Hahn 1994;
Fig. 2). Therefore, what regulates this autumnal territorial
aggression becomes a key question.
Next, it is important to discuss how T secretion is
controlled (Fig. 3) and what its biological actions are.
Environmental information transduced through the CNS
regulates the release of gonadotropin-releasing hormone
(GnRH-1), which stimulates the release of two pituitary
gonadotropins: luteinizing hormone (LH) and folliclestimulating hormone (FSH). These gonadotropins circulate
in the peripheral blood to the gonads, where they regulate
sexual maturation and the production of sex steroids,
Fig. 3 Control mechanisms for territorial aggression in birds in
spring. There are three major components to the control mechanisms.
First, the regulation of hormone secretion from the hypothalamopituitary-gonad (HPG) axis (shown here). This figure summarizes
how sensory information, in this case social, is transduced through
neurotransmitter [e.g., glutamate (NMDA)] and neuroendocrine
secretions such as gonadotropin-releasing hormone (GnRH) and
gonadotropin-inhibiting hormone (GnIH) into the release of the
gonadotropins luteinizing hormone (LH) and follicle-stimulating
hormone (FSH—not shown) from the anterior pituitary into the
blood. LH circulates to the gonad, where it acts on cells that express
steroidogenic enzymes to stimulate the secretion of the sex steroid
hormone testosterone (T), which is in turn released into the blood.
Among the many actions of T are effects on territorial aggression
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Fig. 4 Control mechanisms for territorial aggression in birds in
spring. There are three major components to the control mechanisms.
First, the regulation of hormone secretion from the hypothalamopituitary-gonad (HPG) axis (Fig. 3), then the transport of hormones
such as testosterone in the blood (lines in red shown here). In birds, T
circulates while bound weakly to CBG, before entering target neurons
involved in the expression of territorial aggression (color figure
online)
particularly T in males (Fig. 3). There is now strong evidence that LH release in birds can be inhibited by the
action of another neuropeptide, gonadotropin-inhibitory
hormone (GnIH; e.g., Tsutsui et al. 2009). There is
extensive evidence that T affects spermatogenesis, muscle
hypertrophy, accessory organs such as the cloacal protuberance, some secondary sex characters (Wingfield and
Farner 1993; Owens and Short 1995), provides negative
feedback for GnRH and LH (Fig. 3; Wingfield et al. 2001),
and activates sexual behavior and aggression in reproductive contexts.
Recent evidence also suggests that T circulates in avian
blood while bound with medium affinity to corticosteroidbinding globulin (Fig. 4; Swett and Breuner 2008), and
thus a second level of regulation at the level of hormone
transport to its target cell is possible. Once T arrives at the
target cells (e.g., neurons in areas of the brain associated
with territorial aggression), it enters the cell passively and
can then have four fates (Fig. 5). First it can bind directly
to the androgen receptor (AR), a member of the type 1
genomic receptor family, which become gene transcription
factors once they are bound to T. Second, T can be converted to E2 by the enzyme aromatase. E2 can then bind to
either estrogen receptor alpha (Era) or estrogen receptor
beta (Erb), both of which are genomic receptors that
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regulate gene transcription, but different genes from those
regulated by AR. Third, T can be converted to 5a-dehydrotestosterone (5a-DHT), which also binds to AR and
cannot be aromatized, thus enhancing the AR gene
transcription pathway. Fourth, T can be converted to
5b-dihydrotestosterone, which binds to no known receptors
and also cannot be aromatized, indicating a deactivation
shunt. A complex system of co-repressors and co-activators
of genomic steroid receptor action are also known (Fig. 5;
Shibata et al. 1997; Edwards 2000). The products of gene
expression may then act in concert with neuropeptides such
as AVT, VIP, and others as mentioned above and in Fig. 6.
It is now clear that in regions of the brain associated
with the expression of agonistic behavior, neurons express
all of the enzymes necessary to synthesize T de novo from
cholesterol, or from the uptake of a circulating precursor
such as dehydroepiandrosterone (DHEA), androstenedione,
or even progesterone (Schlinger 1994; Schlinger and
Brenowitz 2002; Fig. 7). It is thought that during the
breeding season, the source of T acting on the brain comes
from gonadal sources and transport in the blood stream, but
local production of sex steroids in brain regions may still
be important (Wingfield et al. 2001; Fig. 7). It should also
be noted that brain nuclei associated with the songs used in
territorial aggression may also change rapidly, within a day
or perhaps less (Thompson et al. 2007; Thompson and
Brenowitz 2009).
In the non-breeding season, the gonads regress in seasonally breeding songbirds, and circulating levels of
gonadotropins and T become undetectable in the assay
systems employed (Fig. 2). From this perspective, an
increase in T secretion in Song Sparrows in autumn would
be inappropriate, because reproductive traits would be
expressed at the wrong season, and effects of T that
potentially promote reproductive function would reduce
survival during the autumn and winter periods (Wingfield
et al. 2001). Even during the breeding season, T levels do
not remain at their peak for long, and there are rapid
increases and declines in blood concentrations. What regulates these surges in T?
The challenge hypothesis
Behavioral endocrinology has many examples of ambiguous relationships of circulating hormones to expression
of patterns of behavior (Adkins-Regan 2005; Ball and
Balthazart 2008). A typical example in the breeding season
is that circulating levels of T in many vertebrates correlate
with aggression only during periods of social instability
(Wingfield 1984a, b; Wingfield et al. 1990). Male–male
interactions over status and access to sexually receptive
females tend to increase T secretion. However, expression
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Fig. 5 Control mechanisms for territorial aggression in birds in
spring. There are three major components to the control mechanisms.
First, the regulation of hormone secretion from the hypothalamopituitary-gonad (HPG) axis (left part of the figure), the transport of
hormones such as testosterone (T) in the blood (lines in red), and the
mechanisms associated with the action of the hormone in the target
cell, in this case a neuron in the brain (shown here). This three-part
control system for hormone secretion, transport, and effects on target
organs is an important concept because it profers many points of
potential regulatory mechanisms. Once T has entered a target neuron,
it has four potential fates. First, it can bind directly to the androgen
receptor (AR), a type 1 genomic receptor, which become gene
transcription factors once they are bound to T. Second, T can be
converted to estradiol (E2) by the enzyme aromatase. E2 can then
bind to either estrogen receptor alpha (Era) or estrogen receptor beta
(Erb), both of which are genomic receptors that regulate gene
transcription, but different genes from those regulated by AR. Third,
T can be converted to 5a-dihydrotestosterone (5a-DHT), which also
binds to AR and cannot be aromatized, thus enhancing the AR gene
transcription pathway. Fourth, T can be converted to 5b-dihydrotestosterone, which binds to no known receptors and also cannot be
aromatized, indicating a deactivation shunt. A complex system of corepressors and co-activators of genomic steroid receptor action are
also known (color figure online)
of parental behavior in males requires that T levels decline
to the reproduction baseline. Other potential costs of T at
high concentrations represent a classic trade-off, with high
T promoting male reproductive function but at the cost of
reduced survival and reproductive success (Dufty 1989;
Wingfield et al. 1990, 2001). An extensive meta-analysis
within the class Aves (Hirschenhauser et al. 2003) and of
over 150 vertebrates ranging from fish to mammals (Hirschenhauser and Oliveira 2006) suggests that this trade-off,
especially the relationship with parental care, persists
across a broad spectrum of taxa.
Is it possible that although baseline levels of T are
undetectable in the non-breeding season, they increase
during periods of social instability and when the individual
is challenged for a winter territory? STIs in the breeding
season lead to increased plasma levels of LH and T in
responding males (Wingfield 1985). However, although
males responded to STI in autumn with similar behavior to
that seen in spring, there were no increases in LH or T at
this time (Wingfield and Hahn 1994). In spring, males that
obtained a territory also had increased plasma levels of T
compared with males in an area where territories had
already been established (Wingfield 1994). In contrast in
autumn, males that established a new territory had undetectable levels of LH and T (Wingfield 1994). Autumn
males also did not respond to estrogen-implanted females
that were sexually receptive (Wingfield and Monk 1994).
Normally males exposed to sexually responsive females in
the breeding season show marked increases in T secretion
(e.g., Adkins-Regan 2005; Wingfield 2006). Moreover, the
castration of territorial male Song Sparrows in autumn had
no effect on territorial aggression in response to STI
compared with sham-operated controls (Wingfield 1994).
Together, these findings strongly suggest that sex steroids
of gonadal origin are not required for autumnal
territoriality.
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Fig. 6 Control mechanisms for territorial aggression in birds in
spring. There are three major components to the control mechanisms.
First, the regulation of hormone secretion from the hypothalamopituitary-gonad (HPG) axis (left part of the figure), the transport of
hormones such as testosterone (T) in the blood (lines in red), and the
mechanisms associated with action of the hormone in the target cell, in
this case a neuron in the brain (central part of the figure). The net result
is the regulation of territorial aggression by neural networks (right
hand part of the figure). This three-part system of control of hormone
secretion, transport, and effects on target organs is an important
concept because it profers many points of potential regulatory
mechanisms. The secretion component on the left summarizes how
sensory information, in this case social, is transduced through
neurotransmitter [e.g. glutamate (NMDA)] and neuroendocrine secretions such as gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibiting hormone (GnIH) into the release of the gonadotropins
luteinizing hormone (LH) and follicle-stimulating hormone (FSH—
not shown) from the anterior pituitary into the blood. LH circulates to
the gonad, where it acts on cells that express steroidogenic enzymes to
stimulate the secretion of the sex steroid hormone T, which is in turn
released into the blood. Local actions of T in the testis include the
regulation of spermatogenesis, but it is also released into the blood
in many avian species when breeding. Among the many actions of T
are effects on territorial aggression and negative feedback on
neuroendocrine and pituitary secretions (red lines). In birds, T
circulates while bound weakly to CBG, before entering target neurons
involved in the expression of territorial aggression (center of figure).
Once T has entered a target neuron, it has four potential fates. First, it
can bind directly to the androgen receptor (AR), a type 1 genomic
receptor, which become gene transcription factors once they are bound
to T. Second, T can be converted to estradiol (E2) by the enzyme
aromatase. E2 can then bind to either estrogen receptor alpha (Era) or
estrogen receptor beta (Erb), both of which are genomic receptors that
regulate gene transcription, but different genes from those regulated by
AR. Third, T can be converted to 5a-dihydrotestosterone (5a-DHT),
which also binds to AR and cannot be aromatized, thus enhancing
the AR gene transcription pathway. Fourth, T can be converted to
5b-dihydrotestosterone, which binds to no known receptors and also
cannot be aromatized, indicating a deactivation shunt. A complex
system of co-repressors and co-activators of genomic steroid receptor
action are also known. The end result is regulatory action on neural
networks that regulate the expression of territorial aggression. Several
neurotransmitters and neuromodulators such as arginine vasotocin,
vasoactive intestinal peptide (VIP), and serotonin are also involved at
this level. Evidence suggests that the basic secretory, transport, and
action mechanisms are conserved across vertebrates (color figure
online)
Testosterone and territorial aggression
in the non-breeding season: problems
(Wingfield and Monk 1992). However, in autumn there
were some single females as well as single males holding a
territory. In some cases, there were female–female pairs,
male–male pairs, and up to five males and females apparently responding to STI with territorial aggression. Other
Song Sparrows were ‘‘floaters,’’ apparently not associated
with a specific territory. Furthermore, of the 30 % or so of
the territorial pairs that were male–female in autumn, very
few actually mated and bred together the next spring
(Wingfield and Monk 1992). Clearly, autumnal territoriality
in northwestern Song Sparrows does not appear to be
Given the previous results, is territorial behavior in autumn
the same as that in the breeding season in spring? Is the
context of spring territoriality the same as that in autumn?
Over five years of observations of individually color-banded and free-living Song Sparrows of known sex in western
Washington State (USA) showed that, as predicted, 70 % of
the territorial pairs in spring were male–female. The other
approximately 30 % were single males without a mate
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Fig. 7 Control mechanisms for territorial aggression in birds in
autumn. Because this is the non-breeding season for most avian
species, the hypothalamo-pituitary-gonad (HPG) axis is essentially
shut down, as represented on the left hand side of the figure in faded
text (compared to Fig. 6, where the HPG axis is active). There is
evidence for some very low level testosterone (T) secretion that
maintains negative feedback (blue lines), but territorial aggression,
where it occurs in the non-breeding season, can be expressed even in
the absence of the gonads. However, all of the T response machinery
in target cells such as neurons involved in the regulation of territorial
aggression may still be fully functional (central part of the figure). In
this case, specific neurons appear to be able to express all of the
enzymes (CYP/HSD) needed to synthesize T and estradiol (E2) de
novo from cholesterol. There is also evidence that circulating
precursors to sex steroids such as dehydroepiandrosterone (DHEA),
androstenedione, and even progesterone may be taken up from the
blood and converted within neurons to biologically active sex steroids
such as T and E2. These then have varying fates, and may bind to
genomic receptors as described in Fig. 5. Neurotransmitters and
neuromodulators in neuron networks involved in territorial aggression
are probably also involved, as shown in Fig. 6. In this way, the major
pathways regulating territorial aggression in the breeding and nonbreeding seasons may have similar or identical bases, but different
points of the regulatory pathways are shut down or upregulated. Thus,
similar behavioral patterns may be regulated by a conserved pathway.
However, similar outcomes of behavioral regulation can be achieved
by regulating diverse combinations of the components (color figure
online)
associated solely with breeding territories. Indeed, in early
autumn, many birds moved from their breeding territory by
distances of a few meters up to several 100 m to establish a
winter territory. In western Washington State, Song Sparrows in autumn appear to form ‘‘alliances’’ with one or
more birds that were mostly not breeding pairs, as in spring.
Thus there appear to be two periods of territory formation,
one in early spring prior to breeding, and a second after the
molt in early autumn that is used mostly for the purposes of
wintering.
reproductive function, contributing to reduced reproductive
success (Ketterson et al. 1996; Wingfield et al. 2001;
Wingfield and Soma 2002). Experimental manipulation of
T increases activity and energetic costs (Wikelski et al.
1999), may have oncogenic effects, increases injury and
mortality (Dufty 1989), interferes with pair bonds, disrupts
parental care in some species (Hegner and Wingfield 1987;
Silverin 1980), and suppresses immune function and
recovery from oxidative damage (Ketterson et al. 1992,
1996; Owen-Ashley et al. 2006, 2008; Martin et al. 2008).
Furthermore, T secretion outside the breeding season
would be inappropriate, as it would induce morphological
and physiological effects that are normally conducive with
breeding at the wrong time, in addition to the costs associated with those effects. Nonetheless, territorial aggression in non-breeding individuals that is essentially identical
to that expressed in the breeding season is widespread
in many organisms (Wingfield et al. 1997, 2001, 2005).
Avoiding the costs of testosterone
Androgens such as T regulate male reproductive function
and if expressed outside the breeding season could
be unnecessarily costly. Moreover, there is considerable
evidence that androgens have negative effects on male
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Control of territorial aggression outside the breeding season becomes an important question, and if T and its
metabolites 5a-dihydrotestosterone (DHT) and estradiol
are involved, how do organisms avoid potential costs?
Some hypotheses have been proposed (Wingfield et al.
2001; Wingfield and Soma 2002; see also Schlinger 1994;
Remage-Healey et al. 2010):
1.
2.
3.
Testosterone hypersensitivity hypothesis. This predicts
that the brain is more sensitive to circulating sex steroid
hormones in autumn. Low plasma steroid levels from
nongonadal sources could support autumnal aggression. Increased hormone receptors or enzymes to
enhance sex steroid hormone actions in the brain could
be important mechanism. In a free-living tropical
suboscine, the Spotted Antbird, Hylophylax naevioides,
in Panama, mRNA for estrogen receptor (ERa) in the
preoptic area and androgen receptor (AR) in the
nucleus taeniae were higher in males during the nonbreeding season when plasma levels of T were lowest
(Canoine et al. 2007). Both of these brain regions are
involved in territorial behavior. These data are consistent with the ‘‘increased sensitivity’’ hypothesis, but it
is an unlikely hypothesis in other species such as the
Song Sparrow based on behavioral and neurobiological
studies (see below; Wacker et al. 2010).
Neurosteroid hypothesis. This predicts that the brain
synthesizes steroids de novo from cholesterol (Fig. 7),
so there should be high sex steroid levels in brain but
not in plasma. An important follow-up question is
then: how is enzyme expression in brain regulated
(Schlinger 1994; Schlinger and Brenowitz 2002; Soma
et al. 1999a, b; Soma et al. 2000a, b; Soma and
Wingfield 2001; Pradhan et al. 2010; Remage-Healey
et al. 2010; Figs. 6, 7)?
Circulating precursor hypothesis. A corollary of the
neurosteroid hypothesis predicts that a steroid precursor such as dehydroepiandrosterone (DHEA) is
secreted into the blood from, for example, the adrenal
cortex, and circulates to the brain or other target
tissues, where it is taken up by specific cells and
converted to biologically active steroids such as T,
DHT, and E2 (Soma and Wingfield 2001; Soma et al.
2002; Wingfield and Soma 2002; Remage-Healey
et al. 2010; Fig. 7).
These hypotheses are not mutually exclusive, and different ones or combinations of them may be prevalent in
different species, suggesting that control of territoriality
may have evolved numerous times in different contexts and
life-history stages (e.g., Wingfield and Soma 2002; AdkinsRegan 2005; Hau and Wingfeld 2011). We now turn our
focus to control of territoriality in the Song Sparrow, where
we compare breeding and non-breeding life-history stages.
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Testosterone and territorial aggression
in the non-breeding season: regulation
of neural steroids in Song Sparrows
Evidence from several vertebrate taxa clearly indicate that
enzymes in neurons convert T to E2, metabolize circulating
precursors such as DHEA (resulting in both T and E2), or
even synthesize sex steroids de novo from cholesterol
(Schlinger 1994; Schlinger and Brenowitz 2002; Soma
et al. 2002; Wingfield and Soma 2002; Remage-Healey
et al. 2010; Figs. 6, 7). The production of sex steroid
hormones locally in brain regions associated with the
expression of territorial aggression could be a powerful
way to avoid high circulating levels, such as those from
gonadal sources, which would then activate a morphology
and physiology typical of reproductive function and also
influence the expression of sexual behavior at the wrong
time (Wingfield and Soma 2002). For local neural production of sex steroids to be effective, those tissues should
also express androgen receptors (AR) that bind T and or
DHT, and E2 receptors (ERa or ERb types). These in turn
influence the transcription of different genes, thus determining patterns of behavior without affecting other regions
of the brain or peripheral tissues that in spring would be
responding to widespread sex hormone secretion from
gonadal tissue broadcast through the blood circulation
(Fig. 3). Local neural expression of specific steroidmetabolizing enzymes, the ability to take up circulating
steroid hormone precursors that are otherwise biologically
inactive and convert them to active steroids, plus the
potential regulation of AR and ER types to affect sensitivity to those locally produced steroids, provide unprecedented flexibility in how a neuron responds to a hormone
signal! AR appears to be particularly important for prebreeding territorial behavior, but less so later in the
breeding season (Sperry et al. 2010), whereas ERs may be
more important in the non-breeding season (Soma et al.
1999b). More work is needed to tease apart potential differences in neuron responses to steroid hormones in different seasons.
Whereas the hypothalamo-pituitary-gonad axis is activated in the breeding season, resulting in a cascade of
gonadotropin-releasing hormone expression, pituitary
release of the gonadotropins LH and follicle-stimulating
hormone (FSH), and thus gonadal development, and the
secretion of sex steroid hormones which then regulate
essentially all aspects of territorial aggression in reproductive contexts, this axis appears to be completely inactive in the non-breeding season (e.g., Dawson et al. 2001;
Wingfield and Silverin 2002, 2009; Bentley et al. 2007;
Fig. 7). How, then, is autumn territoriality regulated, and
more importantly, what aspect of the local neural steroidmetabolizing system is controlled? Ways in which this
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J Ornithol
could be achieved include changing enzyme expression
and activities, regulating the circulating levels of steroid
hormone precursors such as DHEA, and the differential
expression of steroid hormone receptors such as AR and
ERs in local brain regions. Again, each possibility is not
mutually exclusive, and combinations of changes are
possible.
Key enzymes whose expression and activity in local
brain regions could be regulated include 3b-hydroxysteroid
dehydrogenase (3b-HSD), which is essential for biological
activity; aromatase that is responsible for the conversion of
testosterone to estradiol thus favoring ERa and ERb
actions: 5a-reductase mediating the conversion of testosterone to 5a-DHT that cannot be aromatized thus favoring
AR actions; and 5b-reductase that is a de-activation shunt
converting testosterone to 5b-DHT that apparently does not
bind to any known genomic receptor (Schlinger 1994;
Schlinger and Brenowitz 2002; Wingfield and Soma 2002;
Figs. 5, 6). There is growing evidence that the expression
of some of these enzymes in local regions of the brain
occurs seasonally (e.g., Schlinger and Brenowitz 2002;
Soma 2006; Remage-Healey et al. 2010; Wacker et al.
2010), including in the Song Sparrow males that activate
territorial aggression in the autumn (Soma and Wingfield
2003; Pradhan et al. 2010). The enzymes 3b-HSD, aromatase, and others do appear to be regulated—but how?
In spring, but not in autumn, STI results in increases in
LH and T. However, male Song Sparrows captured after
STI in autumn had increased 3b-HSD activity in the central
medial and caudal telencephalon compared with controls
(Pradhan et al. 2010). Thus, it appears that although circulating biologically active sex steroids appear to be absent
in the autumn, the brain not only makes its own sex steroids by activating enzymes in specific brain regions, but
this enzymatic activity is regulated by social stimuli.
Mechanisms of this regulation remain to be determined.
Other ways in which costs of circulating T in the nonbreeding season could be avoided involve the regulation of
steroid hormone precursors such as DHEA. The most
probable source of DHEA is the adrenal cortical cells
(Schlinger et al. 2008). Circulating DHEA levels are
modulated with season: they are low during molt, when
territorial aggression is also at a nadir, and high during
breeding and again in autumn when territorial aggression is
elevated (Soma and Wingfield 2001; Soma et al. 2002).
Furthermore, implanting DHEA in autumn enhanced
singing behavior and produced morphological effects that
increased song control regions, HVC (Soma et al. 2002).
Thus, an increase in plasma levels of DHEA from molt to
autumn could result in more substrate for conversion to T
and/or E2, thereby activating autumnal territoriality
(Fig. 7). However, it is unclear how seasonal changes in
DHEA release are regulated, but it apparently does not
involve the hypothalamo-pituitary-adrenal axis or the HPG
axis (Soma et al. 2002).
As discussed earlier, yet another way to adjust control
mechanisms for autumn territorial behavior is to regulate
the expression of AR and ERs. In male Spotted Antbirds,
ERa and AR are upregulated in local brain regions during
the non-breeding season (Canoine et al. 2007) consistent
with the T hypersensitivity hypothesis. Note that this does
not appear to be the case in male Song Sparrows in autumn
(Wacker et al. 2010), where AR is lower than in the
breeding season and ERa and ERb do not appear to change.
These disparate results suggest a ‘‘diversity of mechanisms’’ by which autumn territoriality and its regulation
evolved. Such diversity and the potential trade-offs may be
widespread in endocrine and neuroendocrine control systems (Hau and Wingfeld 2011).
Challenge hypothesis revisited
Returning to the original hypothesis, social instability as
males compete for territories and mates results in increases
in LH and T, especially in those species in which males
show extensive parental care (Wingfield et al. 1990; Hirschenhauser et al. 2003; Lynn et al. 2005). Nonetheless,
because the HPG axis appears to be inactive in the nonbreeding season, STI fails to raise LH or T levels in male
Song Sparrows in autumn despite a very similar behavioral
response (Wingfield and Hahn 1994). However, STI in
male Song Sparrows in autumn does result in an increase in
3b-HSD activity in telencephalic areas associated with
territorial aggression. This activity required the presence of
co-factors such as NAD? (Pradhan et al. 2010). Such a
remarkable finding suggests that the challenge hypothesis
is still valid in the non-breeding season, but instead of
activating the classic HPG axis and peripheral levels of T,
local enzyme activity in the brain is activated to produce
sex steroids locally, thus avoiding peripheral effects and
potential costs (Pradhan et al. 2010; Fig. 7). Such mechanisms may enable the social modulation of sex steroid
production locally in many other contexts and species, and
further research is needed to explore these possibilities.
The neural and neuroendocrine mechanisms of this
reflex, the social modulation of T, in spring (when breeding) remain a mystery. Because the same behavioral
interactions in autumn do not result in an increase in LH
and T, then the hypothalamic activity of autumn birds will
be an important control for hypothalamic activity in spring
in response to STIs. We collected hypothalami from freeliving male Song Sparrows exposed to STI and from
control males in spring and autumn, and subjected the RNA
extract to microarray analysis (Mukai et al. 2009). The
cDNA microarray contained about 18,000 genes from the
123
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J Ornithol
Zebra Finch brain when a pooled Zebra Finch sample was
used as a reference. Although several hundred genes were
either upregulated or downregulated in relation to treatments, ingenuity pathway analysis revealed certain candidates that may be involved in the neuroendocrine pathway
leading to LH release in spring (Mukai et al. 2009).
Validation of the microarray results by real-time PCR
showed that the expression of glycoprotein hormones alpha
chain precursor (CGA) and transthyretin (TTR) were
upregulated in spring. Results were calculated in terms of
relative amounts of RNA using b-actin as an internal
control, and expressed as the mean fold-change to group
with the lowest expression value. Both CGA and TTR have
been shown to be important mediators of photoperiod
effects on GnRH and LH release in Japanese Quail,
Coturnix japonica (e.g., Yoshimura 2004; Watanabe et al.
2007). It is now possible that these genes may also be
involved in mediating social effects on GnRH as well.
Further work on transcriptomics will be illuminating.
Conclusions and future research
Testosterone and its metabolites DHT and E2 regulate
several behavioral traits associated with territorial aggression. The context of aggression in autumn Song Sparrows
is different from that in spring. Testosterone and E2 appear
to be involved in autumnal aggression, although circulating
levels are not important. The mechanisms by which the T
message gets to the brain vary with season, with neural
steroid production predominating in the non-breeding
season (Figs. 3, 4, 5, 6, 7). The neural and neuroendocrine
pathways that mediate the social modulation of T in spring
(breeding) do not appear to be functional in autumn. Brain
enzyme activity in autumn is socially modulated, and may
avoid the costs of high circulating blood sex steroids in the
non-breeding season.
Territorial aggression is a complex suite of traits. It is
not surprising, then, that when behavior is studied in
increasing detail, even more potentially complex regulatory pathways emerge (Wacker et al. 2008). In laboratory
studies that performed a detailed analysis of aggressive
behavioral traits in response to STI, it was demonstrated
that the aromatase inhibitor fadrozole did not inhibit nonbreeding season aggression in every aspect (as shown in
Fig. 1), compared with much broader observations of the
number of songs, closest approach, etc., made in the field
(Wacker et al. 2008). These data suggest that more detailed
analysis may reveal some traits regulated by ER pathways
and others regulated by AR pathways (see also Sperry et al.
2010). Furthermore, the possible role of potential neuromodulatory peptides such as VIP can also be determined
now (Wacker et al. 2008). Future studies will be
123
particularly interesting, as they can take advantage of the
rich diversity of patterns of territoriality in birds and begin
to tease apart intriguing mechanisms, as well as the
diversity of these mechanisms, by which different components of an otherwise highly conserved hormone system
may be adjusted in different contexts (Hau and Wingfeld
2011).
Behavioral ecology, evolutionary biology, and mechanistic approaches at the neural and endocrine levels mesh
together well. This diversity of mechanisms illustrates how
organisms manage state transitions in their life cycles in
ways that we could not have imagined. The future use of
genomic, proteomic and bioinformatics approaches will
allow us to integrate behavioral ecology, evolutionary
biology, and cell/molecular mechanisms (organism–environment interactions), and will allow even more powerful
analyses.
Acknowledgments JCW is grateful to the National Science Foundation for a series of grants that have supported much of the research
and ideas discussed in this manuscript. The most current grant is IOS0750540. He also acknowledges support from the Russell F. Stark
Professorship from the University of Washington and the Endowed
Chair in Physiology, University of California, Davis. The author is
also grateful to the following people who worked on this project over
the past 30 years: Lee B. Astheimer, Gregory F. Ball, Eliot Brenowitz, D. Shallin Busch, William Buttemer, David Clayton, Alfred
M. Dufty Jr., Wolfgang Goymann, Thomas P. Hahn, Michaela Hau,
Robert Hegner, Kathleen Hunt, Meta Landys, Sharon Lynn, Simone
Meddle, Ignacio Moore, Michael C. Moore. Motoko Mukai, Devleena
S. Pradhan, Marilyn Ramenofsky, Kiran Soma, Barney Schlinger,
Todd Sperry, Douglas Wacker, Gang Wang, and Martin Wikelski.
Their contributions were invaluable.
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