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11
To Flock or Fight:
Neurochemical Signatures of
Divergent Life Histories in Sparrows
JAMES L. GOODSON,* LEAH C. WILSON,
AND SARA E. SCHROCK
Many bird species exhibit dramatic seasonal switches between territorial-
ity and flocking, but whereas neuroendocrine mechanisms of territorial
aggression have been extensively studied, those of seasonal flocking are
unknown. We collected brains in spring and winter from male field spar-
rows (Spizella pusilla), which seasonally flock, and male song sparrows
(Melospiza melodia), which are territorial year-round in much of their
range. Spring collections were preceded by field-based assessments of
aggression. Tissue series were immunofluorescently multilabeled for
vasotocin, mesotocin (MT), corticotropin-releasing hormone (CRH), vaso-
active intestinal polypeptide, tyrosine hydroxylase, and aromatase, and
labeling densities were measured in many socially relevant brain areas.
Extensive seasonal differences are shared by both species. Many mea-
sures correlate significantly with both individual and species differences
in aggression, likely reflecting evolved mechanisms that differentiate
the less aggressive field sparrow from the more aggressive song spar-
row. Winter-specific species differences include a substantial increase of
MT and CRH immunoreactivity in the dorsal lateral septum (LS) and
medial amygdala of field sparrows, but not song sparrows. These spe-
cies differences likely relate to flocking rather than the suppression of
winter aggression in field sparrows, because similar winter differences
were found for two other emberizids that are not territorial in winter—
Department of Biology, Indiana University, Bloomington IN 47405. *To whom correspon-
dence should be addressed: E-mail: jlgoodso@indiana.edu.
193
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194 / James L. Goodson et al.
dark-eyed juncos (Junco hyemalis), which seasonally flock, and eastern
towhees (Pipilo erythropthalmus), which do not flock. MT signaling in
the dorsal LS is also associated with year-round species differences in
grouping in estrildid finches, suggesting that common mechanisms are
targeted during the evolution of different life histories.
A
t the termination of the breeding season, many bird species leave
their exclusive territories and join flocks that range from small
parties to thousands of individuals. This dramatic seasonal shift
in behavioral phenotype undoubtedly has profound fitness implications,
but to our knowledge, no studies have addressed the neural or endocrine
mechanisms that promote seasonal flocking. In contrast, mechanistic stud-
ies of avian territorial aggression are relatively extensive and have inar-
guably revolutionized the field of behavioral endocrinology (Wingfield,
2005; Soma, 2006). However, few of these studies explore the brain mecha-
nisms of territoriality (Soma, 2006; Maney and Goodson, 2011). Using
four emberizid songbird species that have evolved divergent life-history
strategies, we here examine seasonal variation and evolutionary diversity
in six neurochemical systems and demonstrate links of those systems to
both winter flocking and territorial aggression.
On the basis of the immediate early gene responses of (i) male rodents
to resident–intruder encounters, and (ii) male song sparrows (Melospiza
melodia) to simulated territorial intrusion (playback of song and presenta-
tion of a caged male decoy), it seems that the neural substrates of territorial
aggression are extensively comparable in birds and mammals. Thus, in
both taxa significant activation is observed in the medial bed nucleus of
the stria terminalis (BSTm), lateral septum (LS), paraventricular nucleus
of the hypothalamus (PVN), anterior hypothalamus (AH), lateral portion
of the ventromedial hypothalamus (VMH), and midbrain central gray
[Kollack-Walker et al. (1997), Maney and Ball (2003), Goodson and Evans
(2004), Goodson et al. (2005); also see Kingsbury et al. (2011)]. For the year-
round territorial song sparrow, immediate early gene results are largely
comparable in winter and summer (Goodson and Evans, 2004; Goodson et
al., 2005), although microarray data suggest that hypothalamic responses
to simulated intrusion are very different in winter and summer, perhaps
reflecting the fact that luteinizing hormone is released during territorial
challenges only in the breeding season (Mukai et al., 2009). Conversely,
neurons that produce steroidogenic enzymes such as aromatase (ARO)
may show greater activity in winter, given that territoriality in song spar-
rows shifts from reliance on gonadal steroids during the breeding season
to nongonadal hormone production during the fall and winter (Wingfield,
2005; Soma, 2006).
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Seasonal Sociality in Sparrows / 195
Remarkably, neural mechanisms that influence group-size decisions
have received very little attention, although recent studies have begun
to address this topic using five estrildid finch species that exhibit rela-
tively stable group sizes year-round. These studies show that multiple
neurochemical systems have evolved in relation to grouping behavior,
particularly within the LS and associated subnuclei of the posterior sep-
tum. Receptor densities for vasotocin (VT; homolog of the mammalian
nonapeptide vasopressin), mesotocin (MT; homolog of the mammalian
nonapeptide oxytocin), corticotropin-releasing hormone (CRH), and vaso-
active intestinal polypeptide (VIP) all exhibit patterns of parallel and
divergent evolution that closely track species-typical group size (Goodson
et al., 2006, 2009b). Furthermore, VT neurons in the BSTm that project to
the LS are sensitive to social valence and exhibit differential Fos responses
in territorial and flocking species (Goodson and Wang, 2006). Antisense
knockdown of VT production in those cells potently reduces gregarious-
ness in the highly social zebra finch (Taeniopygia guttata) (Kelly et al., 2011),
and antagonism of V1a-like and oxytocic receptors in the septum likewise
reduces preferred group sizes (Goodson et al., 2009b; Kelly et al., 2011).
The relative distribution of nonapeptide receptors across LS subnuclei
may also be relevant to species differences in grouping, because flocking
species have proportionally higher receptor binding in the dorsal (pallial)
LS, whereas territorial species exhibit proportionally more binding in the
subpallial LS (Goodson et al., 2006, 2009b). Consistent with these findings,
septal VT infusions reduce territorial aggression in emberizid sparrows
and estrildid finches (Goodson, 1998a,b). Finally, dopamine circuits are
likely also relevant to grouping behavior, as gregarious finch species
exhibit significantly more tyrosine hydroxylase-immunoreactive (TH-ir)
neurons in the caudal ventral tegmental area (VTA) than do territorial
species (Goodson et al., 2009a). The activity of these neurons is tightly
coupled to courtship behavior, and perhaps to other aspects of affiliation
as well (Goodson et al., 2009a).
These prior studies of avian sociality have focused exclusively on
species that exhibit stable, year-round variation in species-typical group
sizes (Goodson and Kingsbury, 2011). We hypothesize that the same neu-
rochemical systems have evolved to mediate seasonal transitions between
territoriality and flocking, but this remains to be determined. As a first
approach to this hypothesis, we here quantify the neurochemical inner-
vation of numerous brain areas in emberizid species that (i) alternate
between gregarious and territorial phenotypes (field sparrow, Spizella
pusilla, and dark-eyed junco, Junco hyemalis) (Carey et al., 1994; Nolan et
al., 2002), (ii) are territorial year-round in much of their range (song spar-
row) (Arcese et al., 2002), or (iii) switch from breeding territoriality to loose
distributions in fall and winter, without flocking (eastern towhee, Pipilo
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erythropthalmus) (Greenlaw, 1996). The four clades giving rise to these spe-
cies diverged at approximately the same time, relatively early in emberizid
phylogeny (Carson and Spicer, 2003). Our focus is on males, given that
breeding territoriality is typically most intense in males. Complete data
sets from spring and winter birds are reported for song and field sparrows,
including correlations with spring aggression. Winter differences that may
reflect flocking in field sparrows were further explored in comparisons of
winter juncos and towhees. Given that winter differences in neurochem-
istry between field and song sparrows potentially reflect differences in
either winter aggression or winter flocking, the junco-towhee comparison
is particularly useful. Specifically, we hypothesize that if winter differ-
ences between field and song sparrows reflect flocking, then juncos and
towhees should exhibit a comparable winter difference. If winter differ-
ences between field and song sparrows reflect a lack of aggression in field
sparrows, then juncos and towhees should not differ, because neither is
territorial in winter.
We hypothesized that flocking-related changes in neurochemistry
would be evidenced in one of two ways. Most obvious would be a winter
increase in field sparrows (which flock in winter) that is not exhibited by
song sparrows (which are territorial year-round). Alternatively, given that
neurochemical circuits that promote winter flocking may also be involved
in other affiliation behaviors that are expressed in the breeding season,
such as pair bonding and caring for young, we hypothesized that field
sparrows may maintain some neuroendocrine systems year-round that
show a winter collapse in song sparrows. Both patterns are observed and
are strongly supported by follow-up comparisons of juncos and towhees.
Finally, all of the substances examined here are made in multiple
cell groups in the brain and may be relevant to a wide variety of behav-
iors, including both flocking and territoriality, dependent upon the brain
area. For instance, whereas VT neurons in the BSTm respond primarily
to affiliation-related social stimuli, those in the PVN are responsive to
a diversity of stressors (Goodson and Kingsbury, 2011). TH cell groups
likewise show great variation in response profiles (Charlier et al., 2005;
Bharati and Goodson, 2006; Goodson et al., 2009a). We therefore do not
combine analyses across all brain areas for each neurochemical, given that
each neurochemical is not a unitary “system.”
RESULTS
General Approach
Tissue from field and song sparrows (n = 6 males per species and
season; 24 total) was immunofluorescently multilabeled for VT, VIP, and
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Seasonal Sociality in Sparrows / 197
TH (series 1), and MT, CRH, and ARO (series 2). We were not uniformly
satisfied with the quality of TH labeling in series 1, and therefore labeled
a third series for TH using an antibody that yielded robust labeling in all
subjects (Methods; a third series was not available for two spring subjects,
one field and one song sparrow, because of earlier processing errors). We
followed up on significant winter differences by labeling a single series
of junco and towhee tissue for MT, CRH, and TH; and labeled a limited
amount of tissue from a second junco–towhee series for VT and VIP. Note
that for logistical purposes related to antibody lineups, most antigens were
labeled using different fluorophores in the field–song and junco–towhee
datasets, and thus labeling densities can only be compared within each
species pair, not across.
Optical densities (ODs) of immunolabeling were measured in the
medial preoptic nucleus, several hypothalamic areas (PVN, AH, and lateral
and medial divisions of the VMH); anterior and posterior medial amygdala
(MeA); BSTm; lateral BST; central gray; nucleus intercollicularis; rostral
and caudal VTA; and nucleus accumbens. In addition, we quantified label-
ing in subnuclei of the septal complex that are differentiated on the basis of
chemoarchitecture, peptide receptor distributions, and/or transcriptional
responses to social stimuli (Goodson and Evans, 2004; Goodson et al.,
2004, 2006, 2009b; Leung et al., 2011). These are the nucleus of the pallial
commissure; caudocentral septum (CcS); rostral LS subdivision (LSr); and
both pallial and subpallial portions of the caudal LS subdivision, which are
denoted here as LSc.d and subpallial LSc (includes both ventral and ven-
trolateral subnuclei). The LSc.d and subpallial LSc were analyzed at rostral
and caudal levels. In addition to OD, we conducted counts of TH-ir) cells in
the VTA (A10 cell group), central gray (A11), dorsolateral tuberomammil-
lary area (external mammillary nucleus; A12), and subparaventricular area
(A14). VIP-ir cells were counted in the tuberal hypothalamus, and CRH,
VT, and MT cells were counted in the PVN. Alpha values after Benjamini-
Hochberg corrections for the false discovery rate (Benjamini and Hochberg,
1995) are reported in the figure captions and tables for the field and song
sparrows, for which we collected full datasets (Methods). Results of Spe-
cies × Season ANOVAs and within-species regressions with aggression are
reported in the SI Appendix of Goodson et al. (2012b).
Neurochemical Signatures of Seasonal Flocking
As described in the Introduction, we hypothesized that flocking-
related changes in neurochemistry would take the form of either (i) a
winter increase in flocking field sparrows that is not exhibited by song
sparrows, or (ii) the maintenance of some neuroendocrine systems year-
round in field sparrows that show a winter collapse in song sparrows.
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The first pattern is observed for both MT-ir and CRH-ir fiber densi-
ties in the anterior and posterior MeA (“nucleus taeniae”), and the rostral
LSc.d [SI Appendix, Tables S1 and S2, of Goodson et al. (2012b)]. CRH is
additionally increased in the LSr. The LS innervation consists of extremely
fine-caliber processes that arborize most extensively in the pallial LS. In
winter field sparrows, MT-ir processes form numerous light pericellular
baskets. Similarly fine processes are observed in the MeA.
ANOVA results for the LSc.d are shown in Fig. 11.1A and B. Impor-
tantly, both MT-ir and CRH-ir fiber densities in the rostral LSc.d and LSr
correlate negatively with multiple measures of aggression (Fig. 11.1C–F),
and thus the increased densities in winter field sparrows may serve to
suppress aggression rather than promote flocking. To address this issue,
we quantified MT and CRH immunolabeling in wintering dark-eyed jun-
cos, which flock, and eastern towhees, which loosely distribute in winter
and do not flock. This comparison reveals significantly higher MT-ir and
CRH-ir fiber densities in the rostral LSc.d of juncos relative to towhees
(Fig. 11.1G and H) but no differences in CRH OD in the LSr (P = 0.07).
A parallel set of results is obtained for MT and CRH OD in the anterior
MeA (field > song; junco > towhee; Fig. 11.2), but juncos and towhees do
not differ in the posterior MeA (MT, P = 0.28; CRH, P = 0.71). Notably,
colocalization of CRH and MT in PVN neurons is significantly greater in
winter field sparrows than song sparrows (Fig. 11.3A), and winter juncos
likewise tend to show more colocalization than towhees (P < 0.06; Fig.
11.3B). Double-labeling does not correlate with measures of aggression
(all P > 0.10).
The second pattern described above, in which field sparrows maintain
circuitry year-round that collapses during winter in song sparrows, is
observed for VT-ir cell number in the PVN; and VIP OD in the PVN, AH,
rostral subpallial LSc, CcS, and BSTm (in some cases field sparrows main-
tain relatively more but show a slight decline from spring). As shown in
Fig. 11.4A and B, the field–song difference in VT neurons is matched by a
similar difference between winter juncos and towhees, indicating a rela-
tionship to flocking. However, with the exception of VIP OD in the BSTm,
the Species × Season effects for VIP are complex, with species differences
in both winter and spring, but in different directions. That is, spring VIP
OD measures in the PVN, AH, and septal areas are actually higher in song
than in field sparrows. Furthermore, as described in the following section,
AH and CcS measures correlate positively with spring aggression, which
we did not anticipate for variables that promote flocking. Despite these
complexities, we conducted follow-up comparisons in juncos and towhees,
and although no differences are observed for VIP OD in the AH (P = 0.14)
or CcS (P = 0.85; areas where VIP immunolabeling correlates positively
with aggression), juncos do show greater VIP OD in the PVN and BSTm,
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FIGURE 11.1 OD (in arbitrary units) of (A) MT-ir fibers and (B) CRH-ir fibers in the LSc.d of field and song sparrows collected in
spring and winter, showing increased innervation density in winter field sparrows. (C–F) MT-ir and CRH-ir fiber densities correlate
negatively with song sparrow aggression (SS PC1) in both the LSc.d (C and D) and LSr (E and F), suggesting that the increased in-
nervation in winter field sparrows may suppress aggression rather than promote flocking. (G and H) However, comparisons of two
species that are not territorial in winter show that MT-ir and CRH-ir fiber densities are greater in the flocking species (dark-eyed
junco) than in the nonflocking species (eastern towhee). Data are shown as means ± SEM. *Significant after Benjamini-Hochberg
199
corrections (sparrows).
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FIGURE 11.2 OD (in arbitrary units) of (A) MT-ir fibers and (B) CRH-ir fibers
in the anterior MeA of field and song sparrows collected in spring and winter,
showing increased innervation density in winter field sparrows. (C and D) MT-ir
and CRH-ir fiber densities are greater in the flocking dark-eyed junco than in the
nonflocking eastern towhee. Data are shown as means ± SEM. *Significant after
Benjamini-Hochberg corrections (sparrows).
following the pattern of higher fiber density in winter field sparrows rela-
tive to song sparrows. Relevant data are shown in Fig. 11.4C–F.
In addition to the patterns described above, one other finding initially
suggested a possible relationship to flocking. This is a main effect of Spe-
cies for TH immunolabeling in the rostral and caudal VTA, where field
sparrows exhibit significantly higher TH-ir cell numbers and OD year-
round relative to song sparrows [SI Appendix, Table S3, of Goodson et
al. (2012b)]. Cell numbers also correlate negatively with aggression (next
section). However, comparable differences are not exhibited by winter
juncos and towhees, suggesting that the year-round difference between
field and song sparrows reflects their year-round differences in aggression,
as presented below.
Finally, no winter differences are exhibited for VT OD in the BSTm
(as would be predicted from estrildids), although VT-ir fiber density in
spring is significantly higher in field sparrows than in song sparrows [SI
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Seasonal Sociality in Sparrows / 201
FIGURE 11.3 (A) Number of PVN neurons double-labeled for MT and CRH in
field and song sparrows. Because of a lack of variance in winter song sparrows,
winter data were analyzed using Mann-Whitney tests. (B) A similar trend is ob-
served for winter juncos and towhees.
Appendix, Fig. S1, of Goodson et al. (2012b)]. Again, as described in the
next section, this is associated with species differences in aggression.
Neurochemical Signatures of Species-Specific Territorial Behavior
Before collections in the breeding season, we took three measures of
territorial behavior during 3 min of song playback: latency to respond (by
song, fly-by, or flyover), flights (defined as close fly-bys and flyovers), and
songs. We then erected a mist net, began another round of playback, and
took a second measure of response latency. Many measures of neurochem-
istry correlate significantly with these behavioral measures on a within-
species level (next section). However, relevant to our focus on divergent
life histories, we were particularly interested in determining whether
measures of neurochemistry predicted species differences in aggression,
given that that field sparrows are substantially less aggressive during the
breeding season than are song sparrows.
To quantify the species differences in aggression, we conducted a
principal component (PC) analysis of the four behavioral measures, com-
bining data for both species (P = 0.0029). This yields a single component
(PC1) that strongly loads all four measures (Fig. 11.5) and explains 68% of
the behavioral variance. A t test of PC scores confirms that song sparrows
are more aggressive than field sparrows during the breeding season (Fig.
11.5), and more striking, PC scores for the two species are nonoverlapping.
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FIGURE 11.4 (A–F) Left panels show VT-ir cell number in the PVN, VIP-ir OD
(in arbitrary units) in the PVN, and VIP-ir OD in the BSTm of field and song
sparrows. Right panels show corresponding data for juncos and towhees. Data
are shown as means ± SEM. *Significant after Benjamini-Hochberg corrections
(sparrows).
Thus, neurochemical measures that correlate with PC1 are strong candi-
dates as mechanisms underlying evolutionary divergence in territoriality
(although experience of aggression may also be a factor; see Discussion).
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Seasonal Sociality in Sparrows / 203
FIGURE 11.5 PC loadings from a combined analysis of field and song sparrow
aggression (Left) and a comparison of PC scores by species (Right). PC1 explains
68% of the variance and yields non-overlapping values for field and song spar-
rows. Data are shown as means ± SEM.
Note that because of the strong loadings of latency measures, the
direction of PC1 values is counterintuitive (i.e., higher PC scores reflect
lower aggression). The PC1 score for one of the field sparrows was 2.8
standard deviations above the mean and thus this subject was excluded
from the regressions.
Regression analyses reveal significant negative correlations with PC1
(and thus positive correlations with aggression) for VIP OD in the AH and
CcS; ARO OD in the posterior MeA (with a strong trend in the anterior
MeA, as well); CRH OD in the posterior MeA and nucleus accumbens; and
MT OD in the caudal subpallial LSc. In contrast, regression analyses reveal
positive correlations with PC1 (and thus negative correlations with aggres-
sion) for VIP OD in the medial and lateral VMH; VT OD in the BSTm,
central gray, and nucleus intercollicularis; CRH OD in the CcS; and TH OD
in the medial preoptic nucleus, AH, LSr, and nucleus intercollicularis. In
addition, TH-ir cell numbers in the rostral VTA, tuberomammillary hypo-
thalamus, and subparaventricular area correlate positively with PC1. Ten
of the strongest correlations are shown in Fig. 11.6. Note that significance
is not obtained solely on the basis of large species differences, because data
points within each species tend to follow the overall slope.
Individual Differences in Aggression
As just described, many neurochemical measures correlate with both
individual and species differences in aggression. However, neurochemi-
cal variables may relate to individual differences within a given spe-
cies without also relating to differences in aggression across species. We
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204
FIGURE 11.6 (A–J) Regressions of neurochemical measures (OD, A–H; cell counts, I and J) and an index of aggression (PC1; Fig.
11.5) in field and song sparrows (closed and open circles, respectively). See x-axes for neurochemical variable and brain area. *Sig-
nificant after Benjamini-Hochberg corrections (sparrows). CG, central gray; ICo, nucleus intercollicularis; SPa, subparaventricular
area.
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Seasonal Sociality in Sparrows / 205
therefore conducted behavioral PC analyses for field and song sparrows
independently. However, whereas a significant matrix is obtained for
song sparrows (P = 0.0318), this is not the case for field sparrows (P =
0.60), likely because the field sparrows displayed few flights and songs,
and little variation in those measures. Thus, we conducted regressions for
field sparrows based on the average of their two latency measures, and
for song sparrows based on a single-species PC (SS PC1), that explains
64% of the variance and exhibits strong loadings for flights (−0.913) and
both latencies (0.901 and 0.928, respectively), but a weak loading for songs
(−0.234). Results of these analyses are reported in the SI Appendix, Tables
S7–S12, of Goodson et al. (2012b).
DISCUSSION
Although neuroendocrine mechanisms of seasonal territoriality have
been extensively described (Wingfield, 2005; Soma, 2006; Maney and
Goodson, 2011), those of seasonal flocking have not, and brain mecha-
nisms that evolve in relation to species differences in the intensity of ter-
ritorial aggression are likewise unknown. We now show that in emberizid
songbirds, several neurochemical variables reflect seasonal shifts from
territoriality to flocking, whereas numerous other variables correlate with
both individual and species differences in territorial aggression. Given
that the relevant neurochemical systems may be influenced by social
interactions (e.g., via altered hormone levels), we must be cautious in our
interpretations, because neurochemical variation may be the product of
species differences in behavior rather than the drivers of it. However, as
expounded upon in the following sections, other relevant findings suggest
that many of the species differences are indeed products of evolution and
mechanistic drivers of behavioral variation. Finally, our results reveal a
remarkable degree of seasonal, neurochemical plasticity within socially
relevant brain areas that is far more extensive than previously appreciated.
Neurochemical Profiles of Seasonal Flockers
Estrildid finches that are gregarious year-round exhibit nonapeptide
binding sites in the rostral LSc.d (pallial LS) at much higher densities than
do territorial estrildids (Goodson et al., 2006, 2009b). The relevance of these
binding sites to flocking is supported by the demonstrations that intraven-
tricular and intraseptal infusions of nonapeptide receptor antagonists (V1a
and oxytocin receptor antagonists) reduce preferences for larger groups in
the highly gregarious zebra finch (Goodson et al., 2009b; Kelly et al., 2011),
as does antisense knockdown of VT-ir neurons in the BSTm (Kelly et al.,
2011)—neurons that seem to provide the majority of VT-ir innervation to
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the LS (De Vries and Buijs, 1983; De Vries and Panzica, 2006). Conversely,
preferences for larger groups are facilitated by intraventricular infusions
of MT (Goodson et al., 2009b). The present findings are strongly consistent
with those in estrildids: field sparrows show a significant increase in MT-ir
fiber density in the LSc.d during winter, when they form flocks, whereas
the year-round territorial song sparrow does not. Flocking dark-eyed jun-
cos likewise show a higher MT-ir fiber density in the LSc.d during winter
than do nonflocking, nonterritorial eastern towhees. This pattern of MT
results is replicated in the anterior MeA, and a very similar pattern of
CRH innervation is observed in both the rostral LSc.d and anterior MeA.
Social affiliation in rodents is also linked to nonapeptide signaling in
the LS. For instance, nonapeptide receptor densities in the LS increase in
response to communal rearing (Curley et al., 2009), promote pair bonding
(Liu Y et al., 2001), and correlate positively with both social investigation
(Ophir et al., 2009) and maternal behaviors [and in the pallial LS specifi-
cally (Curley et al., 2012)]. Although the specific significance of peptide
action in the pallial LS remains to be directly demonstrated, recent findings
in mice demonstrate that the pallial LS plays an important role in link-
ing contextual stimulus information to the activation of the mesolimbic
dopamine system, which influences incentive motivational processes and
reward (Luo et al., 2011). The functional properties of the anterior MeA
are relatively less clear. In mammals, the posterior subnuclei have been
far more extensively studied, although Newman (1999) has suggested that
the anterior MeA exerts broad effects on social arousal. Homology of MeA
subnuclei in birds and mammals remains to be demonstrated.
The finding that CRH innervation paralleled the MT innervation was
unexpected, but is consistent with the fact that these two peptides are
produced in many of the same neurons in the PVN and that colocaliza-
tion is greater in winter flockers (Fig. 11.3). CRH is generally linked to
anxiety-like processes and stress (Lovejoy and Balment, 1999), which may
be the connection to flocking, given that thermoregulatory and foraging
challenges lead to facultative grouping in many vertebrate species (Davies,
1976; Gilbert et al., 2010). Thus, we might hypothesize that winter flock-
ers are in some sense hyperresponsive to the challenges of winter. This
hypothesis also fits well with the observation that flocking birds exhibit
significantly greater numbers of VT-ir PVN neurons in the winter than do
nonflocking birds. Given that VT-ir fiber density collapses during winter
in almost every brain area that we examined, it seems likely that these
“extra” PVN neurons in flocking species project to the anterior pituitary,
where VT acts as a secretagogue for adrenocorticopin hormone (Goodson
and Bass, 2001) and thereby contribute to a higher glucocorticoid tone.
Finally, we observed complex patterns of VIP-ir fiber densities,
some of which correlate positively with aggression (next section). How-
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Seasonal Sociality in Sparrows / 207
ever, winter flocking (and not aggression) is associated with higher den-
sities of VIP-ir fibers in the PVN and BSTm. Similarly, gregarious finch
species exhibit higher densities of VIP binding sites in the BSTm than do
territorial species (Goodson et al., 2006), providing additional evidence
that VIP signaling in the BSTm promotes grouping.
Species Differences in Territorial Aggression
As shown here, field sparrows are significantly less aggressive than
are song sparrows. Thus, the present dataset allows us to identify neu-
rochemical mechanisms that may have evolved in relation to territorial
behavior, because we are able to correlate measures of neurochemis-
try with aggressive behavior across both individuals and species. As
a caveat to this approach, we observed widespread winter decreases
in immunolabeling, suggesting the likelihood of positive relationships
between gonadal hormones and labeling density. Thus, because male–
male interactions typically elevate levels of testosterone (Wingfield, 2005),
we must consider that any positive correlations between neurochemistry
and behavior may be the product of male–male interactions and not the
cause of it. For instance, ARO gene expression correlates positively with
both aggression and plasma T in juncos (Rosvall et al., 2012). Nonetheless,
most of the strongest relationships described here for neurochemistry and
aggression are negative.
For instance, VT-ir fiber density in the BSTm collapses in winter, yet
we also see that it correlates negatively with individual and species dif-
ferences in aggression. This observation is consistent with the findings
that (i) gregarious estrildids exhibit relatively more VT-ir neurons in the
BSTm than do territorial species (Goodson and Wang, 2006), (ii) those
neurons respond selectively to affiliation-related stimuli (Goodson and
Wang, 2006), and (iii) infusions of VT into the septum (a major recipient
of BSTm VT projections) reduce overt territorial aggression in both field
sparrows and territorial finches (Goodson, 1998a,b).
Similarly, VIP immunolabeling correlates negatively with sparrow
aggression in the lateral VMH and tuberal hypothalamus, but also posi-
tively in the AH and caudal septum. These results are strongly consistent
with a variety of findings in territorial finches. For instance, intraseptal
VIP infusions facilitate offensive aggression (Goodson, 1998b), whereas
antisense knockdown of VIP production in the AH virtually abolishes it
(Goodson et al., 2012a) (note that VIP-ir cells in the AH are only detect-
able after colchicine pretreatment and were thus not examined here).
VIP-ir cell numbers in the AH of control finches correlate positively with
aggression, but consistent with our present findings, VIP-ir cell numbers
relate negatively to aggression in the tuberal hypothalamus [SI Appendix
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208 / James L. Goodson et al.
in Goodson et al. (2012a)]. These finch data were obtained from birds in
nonbreeding condition, suggesting that the positive relationship between
AH VIP and aggression is not dependent upon gonadal steroids. Hence,
VIP circuitries in the AH-CcS and mediobasal hypothalamus, which bear
positive and negative relationships to aggression, respectively, are likely
both relevant to behavioral evolution in sparrows.
We observed many other correlations across species that cannot be as
readily interpreted because of a lack of direct functional data, but those
findings nonetheless provide the basis for many hypothesis-driven experi-
ments on the evolution of aggression.
Widespread Seasonal Plasticity
Although the present study was designed to focus on aggression and
flocking, the analyses in field and song sparrows reveal a remarkable
and unanticipated amount of seasonal plasticity, including all six neuro-
chemical systems and 21 brain areas that we examined. Most remarkable
are CRH and VIP. Seasonal plasticity has been shown for VIP within the
septum and infundibulum (Kosonsiriluk et al., 2008; Wacker et al., 2008),
but to our knowledge no such plasticity has been shown for the CRH
innervation of the brain. However, we observed significant seasonal varia-
tion in 13 of the sampling areas for CRH, and 11 of the sampling areas for
VIP. Seasonal plasticity for both peptides is exhibited in the MeA, BST,
septal complex, medial preoptic nucleus, hypothalamic nuclei, and mid-
brain. Even in the case of VT, for which extensive seasonal and hormone-
mediated plasticity is already known (as with VP in mammals) (Goodson
and Bass, 2001; De Vries and Panzica, 2006), the extent of seasonal remod-
eling came as a surprise. Interestingly, the most extensive plasticity known
for mammals comes from jerboas (Jaculus orientalis) that were collected in
the field (Lakhdar-Ghazal et al., 1995), as were the animals in the present
study, suggesting that exposure to a full range of seasonal cues is neces-
sary to reveal the natural extent of seasonal plasticity.
CONCLUSIONS
We here hypothesized that flocking-related changes in neurochemistry
take the form of either (i) a winter increase in flockers that is not exhibited
by nonflocking species, or (ii) the maintenance of some neuroendocrine
systems year-round in flockers that show a winter collapse in nonflockers.
The first pattern is exhibited in the MT and CRH innervation of the pal-
lial LS and anterior MeA, and in the colocalization of MT and CRH in the
PVN. The second pattern is observed for VT-ir cell numbers in the PVN,
and VIP innervation of the PVN and BSTm. A much larger number of neu-
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Seasonal Sociality in Sparrows / 209
rochemical variables seem to evolve in relation to territorial aggression,
and all neurochemicals and brain areas examined here exhibit remarkable
seasonal plasticity.
METHODS
Animals
Spring field and song sparrows were caught April thru May 2009
in the vicinity of Bloomington, IN. Wintering sparrows were caught in
the vicinity of Bloomington, IN, and in Davidson County, TN, between
December 2008 and February 2009. Juncos and towhees were collected
in the vicinity of Bloomington, IN, in January 2010. Collections were
made under applicable state and federal permits, and all procedures were
in accordance with guidelines established by the National Institutes of
Health for the ethical treatment of animals.
Tissue Processing and Image Analysis
Subjects were euthanized within 30 min of capture. Perfusions, tissue
processing, and immunofluorescent labeling followed standard protocols
(Goodson et al., 2004, 2009a; Kabelik et al., 2010). All Alexa Fluor (A.F.)
conjugates were purchased from Invitrogen. Secondaries were raised in
donkey. Sparrow series 1 was labeled using sheep anti-TH (Novus Biologi-
cals), guinea pig anti-VP (Bachem), and rabbit anti-VIP (Bachem), with A.F.
488, biotin followed with streptavidin-A.F. 594, and A.F. 680 secondaries,
respectively. Sparrow series 2 was labeled using custom sheep anti-ARO,
rabbit anti-MT (VA10; a kind gift of H. Gainer, National Institute of Neu-
rological Disorders and Stroke, Bethesda, MD), and guinea pig anti-CRH
(Bachem), using A.F. 488, 594, and 680 secondaries, respectively. Sparrow
series 3 was labeled using mouse anti-TH (Immunostar) and A.F. 594 sec-
ondary. The specificity of all antibodies has been addressed [Goodson et
al. (2004), Kabelik et al. (2010); see company datasheets for TH]. Each pro-
cessing run contained a mixture of species and seasons. Junco and towhee
series 1 was labeled using rabbit anti-MT, mouse anti-TH, and guinea pig
anti-CRH, with A.F. 488, 594, and 680 secondaries, respectively. Additional
junco and towhee tissue was labeled using guinea pig anti-VP and rabbit
anti-VIP, with A.F. 594 and 680 secondaries, respectively.
Although some larger areas with robust labeling were captured at 5×,
most photomicrographs were obtained at 10× using a Zeiss AxioImager
microscope outfitted with a Z-drive and optical dissector (Apotome; Carl
Zeiss). OD of label and background was measured in Adobe Photoshop
CS5 (Adobe Systems, Seattle, WA) from monochrome images, and back-
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210 / James L. Goodson et al.
ground values were subtracted for statistical analysis. Cell counts were
conducted as previously described (Goodson and Wang, 2006; Goodson
et al., 2009a). All cells were counted in each relevant section for smaller
cell groups and are represented as number of cells per section/gram body
weight. TH-ir cells in the VTA were counted within a standardized box
and are represented as number of cells per 100 μm2.
Statistics
All ANOVAs, regressions, and PC analyses described in the Results
were conducted using Statview 5.0 for Macintosh. Given the large number
of analyses, some concern arises with regard to type I error, although all
brain areas and neurochemicals examined here are known a priori to be
relevant to social behavior (although not in all possible combinations).
Corrections for multiple comparisons in such instances are usually too
conservative and not appropriate (Rothman, 1990), and we therefore do not
emphasize them in our interpretations. However, they may still provide a
useful metric for evaluation; thus each of our data tables and figure pan-
els provides information on significance relative to Benjamini-Hochberg
corrections for the false discovery rate (Benjamini and Hochberg, 1995).
Corrections were applied to each set of ANOVAs (e.g., for VT measures
across all brain areas) and to each corresponding set of regressions. Again,
though not emphasized in the Results, the robustness of our findings is
notable; for example, 73 of 78 ANOVAs that yield P values < 0.05 were sig-
nificant following corrections. Note that although the Benjamini-Hochberg
correction initially applies a Bonferroni criterion, it adjusts α in a stepwise
manner for remaining tests as long as P values continue to be significant
at each step.
ACKNOWLEDGMENTS
We thank Francisco Ayala, John Avise, and Georg Striedter for invit-
ing this contribution; Jacob Callis, Brian Gress, Alexis Howard, Aubrey
Kelly, Melissa Knisley, and Brittany Welsh for assistance with immunocy-
tochemistry and/or cell counts; Ellen Ketterson, Dawn O’Neal, and Ryan
Kiley for assistance with collections; and Drew King and Meredith West
for property access; Harold Gainer for the donation of antiserum. Support
for this study was provided by Indiana University.
