To flock or fight: Neurochemical signatures of
divergent life histories in sparrows
James L. Goodson1, Leah C. Wilson, and Sara E. Schrock
Department of Biology, Indiana University, Bloomington, IN 47405
Edited by John C. Avise, University of California, Irvine, CA, and approved April 20, 2012 (received for review February 27, 2012)
Many bird species exhibit dramatic seasonal switches between
territoriality and flocking, but whereas neuroendocrine mecha-
nisms of territorial aggression have been extensively studied,
those of seasonal flocking are unknown. We collected brains in
spring and winter from male field sparrows (Spizella pusilla),
which seasonally flock, and male song sparrows (Melospiza mel-
odia), 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), vasoactive 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 measures correlate significantly
with both individual and species differences in aggression, likely
reflecting evolved mechanisms that differentiate the less aggres-
sive field sparrow from the more aggressive song sparrow. Win-
ter-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
species differences likely relate to flocking rather than the sup-
pression of winter aggression in field sparrows, because similar
winter differences were found for two other emberizids that are
not territorial in winter—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.
from small parties to thousands of individuals. This dramatic
seasonal shift in behavioral phenotype undoubtedly has pro-
found fitness implications, but to our knowledge no studies have
addressed the neural or endocrine mechanisms that promote
seasonal flocking. In contrast, mechanistic studies of avian ter-
ritorial aggression are relatively extensive and have inarguably
revolutionized the field of behavioral endocrinology (1, 2).
However, few of these studies explore the brain mechanisms of
territoriality (1, 3). Using four emberizid songbird species that
have evolved divergent life-history strategies, we here examine
seasonal variation and evolutionary diversity in six neurochemi-
cal 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 presentation 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),
t the termination of the breeding season, many bird species
leave their exclusive territories and join flocks that range
and midbrain central gray (4–7; also see ref. 8). For the year-round
territorial song sparrow, immediate early gene results are largely
comparable in winter and summer (4, 5), although microar-
ray data suggest that hypothalamic responses to simulated in-
trusion are very different in winter and summer, perhaps
reflecting the fact that luteinizing hormone is released during
territorial challenges only in the breeding season (9). Conversely,
neurons that produce steroidogenic enzymes such as aromatase
(ARO) may show greater activity in winter, given that territori-
ality in song sparrows shifts from reliance on gonadal steroids
during the breeding season to nongonadal hormone production
during the fall and winter (1, 2).
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 relatively 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 septum. Receptor
densities for vasotocin (VT; homolog of the mammalian non-
apeptide vasopressin), mesotocin (MT; homolog of the mam-
malian nonapeptide oxytocin), corticotropin-releasing hormone
(CRH), and vasoactive intestinal polypeptide (VIP) all exhibit
patterns of parallel and divergent evolution that closely track
species-typical group size (10, 11). 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 (12). Antisense knockdown of VT production in those
cells potently reduces gregariousness in the highly social zebra
finch (Taeniopygia guttata) (13), and antagonism of V1a-like and
oxytocic receptors in the septum likewise reduces preferred
group sizes (11, 13). The relative distribution of nonapeptide
receptors across LS subnuclei may also be relevant to species
differences in grouping, because flocking species have propor-
tionally higher receptor binding in the dorsal (pallial) LS,
whereas territorial species exhibit proportionally more binding in
the subpallial LS (10, 11). Consistent with these findings, septal
VT infusions reduce territorial aggression in emberizid sparrows
and estrildid finches (14, 15). Finally, dopamine circuits are likely
also relevant to grouping behavior, because gregarious finch
species exhibit significantly more tyrosine hydroxylase-immuno-
reactive (TH-ir) neurons in the caudal ventral tegmental area
(VTA) than do territorial species (16). The activity of these
This paper results from the Arthur M. Sackler Colloquium of the National Academy of
Sciences, “In the Light of Evolution VI: Brain and Behavior,” held January 19–21, 2012, at
the Arnold and Mabel Beckman Center of the National Academies of Sciences and Engi-
neering in Irvine, CA. The complete program and audio files of most presentations are
available on the NAS Web site at www.nasonline.org/evolution_vi.
Author contributions: J.L.G. designed research; J.L.G., L.C.W., and S.E.S. performed
research; J.L.G. analyzed data; and J.L.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
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| suppl. 1
neurons is tightly coupled to courtship behavior, and perhaps to
other aspects of affiliation as well (16).
These prior studies of avian sociality have focused exclusively
on species that exhibit stable, year-round variation in species-
typical group sizes (17). We hypothesize that the same neuro-
chemical systems have evolved to mediate seasonal transitions
between territoriality and flocking, but this remains to be de-
termined. As a first approach to this hypothesis, we here quantify
the neurochemical innervation 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) (18, 19), (ii) are territorial year-
round in much of their range (song sparrow) (20), or (iii) switch
from breeding territoriality to loose distributions in fall and
winter, without flocking (Eastern towhee, Pipilo erythropthalmus)
(21). The four clades giving rise to these species diverged at
approximately the same time, relatively early in emberizid phy-
logeny (22). Our focus is on males, given that breeding territo-
riality is typically most intense in males. Complete datasets 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 neurochemistry 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 differences
between field and song sparrows reflect flocking, then juncos and
towhees should exhibit a comparable winter difference. If winter
differences between field and song sparrows reflect a lack of
nolabeling in the caudal septum and posterior hypothalamus. Asterisk corresponds to asterisk in D. (B) MT and CRH immunolabeling in the PVN and AH. Note
the extensive colabeling in the lateral subpopulation of neurons (arrows). (C) ARO and CRH immunolabeling in the BSTm and adjacent structures. Asterisk
corresponds to asterisk in E. (Scale bars, 100 μm in A, C, and D; 50 μm in B and E.) Arc, arcuate nucleus; CHCS, cortico-habenular and cortico-septal tract
(fimbria); CoS, commissural septal nucleus; ExM, external mammillary nucleus; Inf, infundibulum; LSc, caudal division of the lateral septum (.d, dorsal; .v,
ventral; .vl, ventrolateral); MM, medial mammillary area; MS, medial septum; nPC, nucleus of the pallial commissure; PH, posterior hypothalamic area; v, lateral
ventricle; vaf, ventral amygdalofugal tract.
Representative immunolabeling in field sparrows. B is from a winter field sparrow; all others are from spring subjects. (A) TH, VIP, and VT immu-
| www.pnas.org/cgi/doi/10.1073/pnas.1203394109Goodson et al.
aggression in field sparrows, then juncos and towhees should not
differ, because neither is territorial in winter.
We hypothesized that flocking-related changes in neuro-
chemistry 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 terri-
torial year-round). Alternatively, given that neurochemical cir-
cuits 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 pat-
terns 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 behaviors, including both flocking and territoriality,
dependent upon the brain area. For instance, whereas VT neu-
rons in the BSTm respond primarily to affiliation-related social
stimuli, those in the PVN are responsive to a diversity of stres-
sors (17). TH cell groups likewise show great variation in re-
sponse profiles (16, 23, 24). We therefore do not combine
analyses across all brain areas for each neurochemical, given that
each neurochemical is not a unitary “system.”
General Approach. Tissue from field and song sparrows (n = 6
males per species and season; 24 total) was immunofluorescently
multilabeled for VT, VIP, and TH (series 1), and MT, CRH, and
ARO (series 2). Representative photomicrographs are shown in
Fig. 1. 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 la-
beling 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 line-ups, most antigens were la-
beled using different fluorophores in the field–song and junco–
towhee datasets, and thus labeling densities can only be com-
pared within each species pair, not across.
sparrow. The approximate pallial–subpallial boundary is indicated by the
dashed line, highlighting the greater arborization within the pallial LS (LSc.d).
(Scale bar, 50 μm.) v, lateral ventricle.
Fine-caliber MT-ir and CRH-ir fibers in the LSc.d of a winter field
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 innervation 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 corrections (sparrows).
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
Goodson et al.PNAS
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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 nu-
cleus accumbens. In addition, we quantified labeling in subnuclei
of the septal complex that are differentiated on the basis of
chemoarchitecture, peptide receptor distributions, and/or tran-
scriptional responses to social stimuli (4, 10, 11, 25, 26). These
are the nucleus of the pallial commissure; caudocentral septum
(CcS); rostral LS subdivision (LSr); and both pallial and sub-
pallial portions of the caudal LS subdivision, which are denoted
here as LSc.d and subpallial LSc (includes both ventral and
ventrolateral 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 tuberomammillary area (exter-
nal 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 dis-
covery rate (27) are reported in the figure captions and tables for
the field and song sparrows, for which we collected full datasets
(Methods). Results of Species × Season ANOVAs and within-
species regressions with aggression are reported in SI Appendix,
Tables S1–S6 (ANOVAs) and S7–S12 (regressions) (corre-
sponding to MT, CRH, TH, VT, VIP, and ARO, respectively,
for ANOVAs and regressions).
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 in-
crease 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.
The first pattern is observed for both MT-ir and CRH-ir fiber
densities in the anterior and posterior MeA (“nucleus taeniae”)
and the rostral LSc.d (SI Appendix, Tables S1 and S2). CRH is
additionally increased in the LSr. As shown in Fig. 2, the LS
innervation consists of extremely fine-caliber processes that ar-
borize 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. 3 A and B.
Importantly, both MT-ir and CRH-ir fiber densities in the rostral
LSc.d and LSr correlate negatively with multiple measures of
aggression (Fig. 3 C–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 juncos, 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. 3 G 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 > to-
whee; Fig. 4), but juncos and towhees do not differ in the pos-
terior 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. 5A),
and winter juncos likewise tend to show more colocalization than
towhees (P < 0.06; Fig. 5B). 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 maintain relatively more but show
a slight decline from spring). Fig. 6A shows representative VT
and VIP immunolabeling in the PVN and AH, and as shown in
Fig. 6 B and C, the field–song difference in VT neurons is
matched by a similar difference between winter juncos and
towhees, indicating a relationship 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
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).
OD (in arbitrary units) of (A) MT-ir fibers and (B) CRH-ir fibers in the
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
observed for winter juncos and towhees.
(A) Number of PVN neurons double-labeled for MT and CRH in field
| www.pnas.org/cgi/doi/10.1073/pnas.1203394109 Goodson et al.
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, following the pattern of higher fiber density
in winter field sparrows relative to song sparrows. Relevant data
are shown in Fig. 6 D–G.
In addition to the patterns described above, one other finding
initially suggested a possible relationship to flocking. This is
a main effect of Species 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). 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
Finally, no winter differences are exhibited for VT OD in the
sparrows (SI Appendix, Fig. S1). 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 meas-
ures of territorial behavior during 3 min of song playback: la-
tency 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 neurochemistry 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 con-
ducted a principle component (PC) analysis of the four behav-
ioral measures, combining data for both species (P = 0.0029).
This yields a single component (PC1) that strongly loads all four
measures (Fig. 7) and explains 68% of the behavioral variance. A
t test of PC scores confirms that song sparrows are more ag-
gressive than field sparrows during the breeding season (Fig. 7),
and more striking, PC scores for the two species are non-
overlapping. Thus, neurochemical measures that correlate with
PC1 are strong candidates as mechanisms underlying evolu-
tionary divergence in territoriality (although experience of ag-
gression may also be a factor; Discussion).
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 SDs 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
AH of a winter field sparrow. (B, D, and F) 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. (C, E, and G) Corresponding data for juncos and towhees.
Data are shown as means ± SEM. *Significant after Benjamini-Hochberg
(A) Representative immunolabeling for VT and VIP in the PVN and
aggression (Left) and a comparison of PC scores by species (Right). PC1
explains 68% of the variance and yields nonoverlapping values for field and
song sparrows. Data are shown as means ± SEM.
PC loadings from a combined analysis of field and song sparrow
Goodson et al.PNAS
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MeA and nucleus accumbens; and MT OD in the caudal sub-
pallial LSc. In contrast, regression analyses reveal positive corre-
lations with PC1 (and thus negative correlations with aggression)
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 hypothalamus, and subparaventricular
area correlate positively with PC1. All regression results are shown
in SI Appendix, Table S13, and 10 of the strongest correlations are
shown in Fig. 8. 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 neu-
rochemical measures correlate with both individual and species
differences in aggression. However, neurochemical variables may
relate to individual differences within a given species without
also relating to differences in aggression across species. We
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 dis-
played few flights and songs, and little variation in those meas-
ures. 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 SI
Appendix, Tables S7–S12.
Although neuroendocrine mechanisms of seasonal territoriality
have been extensively described (1–3), those of seasonal flocking
have not, and brain mechanisms that evolve in relation to species
differences in the intensity of territorial 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 influ-
enced 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
aregregarious year-roundexhibit nonapeptide binding sitesin the
rostral LSc.d (pallial LS) at much higher densities than do terri-
torial estrildids (10, 11). The relevance of these binding sites to
flocking is supported by the demonstrations that intraventricular
and intraseptal infusions of nonapeptide receptor antagonists
(V1aand oxytocin receptor antagonists) reduce preferences for
larger groups in the highly gregarious zebra finch (11, 13), as does
antisense knockdown of VT-ir neurons in the BSTm (13)—neu-
rons that seem to provide the majority of VT-ir innervation to the
LS (28, 29). Conversely, preferences for larger groups are facili-
tated by intraventricular infusions of MT (11). The present find-
ings 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 juncos
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 theanterior 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 sig-
naling in the LS. For instance, nonapeptide receptor densities in
the LS increase in response to communal rearing (30), promote
pair bonding (31), and correlate positively with both social in-
vestigation (32) and maternal behaviors [and in the pallial LS
specifically (33)]. Although the specific significance of peptide
open circles, respectively). See x-axes for neurochemical variable and brain area. *Significant after Benjamini-Hochberg corrections (sparrows). CG, central
gray; ICo, nucleus intercollicularis; SPa, subparaventricular area.
(A–J) Regressions of neurochemical measures (OD, A–H; cell counts, I–J) and an index of aggression (PC1; Fig. 7) in field and song sparrows (closed and
| www.pnas.org/cgi/doi/10.1073/pnas.1203394109 Goodson et al.
action in the pallial LS remains to be directly demonstrated,
recent findings in mice demonstrate that the pallial LS plays an
important role in linking contextual stimulus information to the
activation of the mesolimbic dopamine system, which influences
incentive motivational processes and reward (34). The functional
properties of the anterior MeA are relatively less clear. In
mammals, the posterior subnuclei have been far more extensively
studied, although Newman (35) 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 in-
nervation was unexpected but is consistent with the fact that
these two peptides are produced in many of the same neurons in
the PVN (Fig. 1B) and that colocalization is greater in winter
flockers (Fig. 5). CRH is generally linked to anxiety-like pro-
cesses and stress (36), which may be the connection to flocking,
given that thermoregulatory and foraging challenges lead to
facultative grouping in many vertebrate species (37, 38). Thus,
we might hypothesize that winter flockers are in some sense
hyperresponsive to the challenges of winter. This hypothesis also
fits well with the observation that flocking birds exhibit signifi-
cantly 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 adrenocorticopic hormone (39) and thereby contribute to
a higher glucocorticoid tone.
Finally, we observed complex patterns of VIP-ir fiber densi-
ties, some of which correlate positively with aggression (next
section). However, winter flocking (and not aggression) is asso-
ciated with higher densities 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 (10),
providing additional evidence that VIP signaling in the BSTm
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 neurochemical
mechanisms that may have evolved in relation to territorial be-
havior, because we are able to correlate measures of neuro-
chemistry 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 (2), 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 (40). 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 differences in aggression. This observation is con-
sistent with the findings that (i) gregarious estrildids exhibit
relatively more VT-ir neurons in the BSTm than do territorial
species (12), (ii) those neurons respond selectively to affiliation-
related stimuli (12), 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 (14, 15).
Similarly, VIP immunolabeling correlates negatively with
but also positively in the AHand caudal septum. These results are
strongly consistent with a variety of findings in territorial finches.
For instance, intraseptal VIP infusions facilitate offensive ag-
gression (14), whereas antisense knockdown of VIP production in
the AH virtually abolishes it (note that VIP-ir cells in the AH are
only detectable 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 pres-
ent findings, VIP-ir cell numbers relate negatively to aggression in
the tuberal hypothalamus (SI Appendix, Fig. S2). These finch data
were obtained from birds in nonbreeding condition, suggesting
that the positive relationship between AH VIP and aggression is
AH-CcS and mediobasal hypothalamus, which bear positive and
negative relationships to aggression, respectively, are likely both
relevant to behavioral evolution in sparrows.
be as readily interpreted, because of a lack of direct functional
data, but those findings nonetheless provide the basis for many
hypothesis-driven experiments 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
brain areas that we examined. Most remarkable are CRH and VIP.
Seasonal plasticity has been shown for VIP within the septum and
infundibulum (41, 42), but to our knowledge no such plasticity has
been shown for the CRH innervation of the brain. However, we
observed significant seasonal variation 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 midbrain.
Even in the case of VT, for which extensive seasonal and
hormone-mediated plasticity is already known (as with VP in
mammals) (29, 39), the extent of seasonal remodeling came as
a surprise. Interestingly, the most extensive plasticity known for
mammals comes from jerboas (Jaculus orientalis) that were col-
lected in the field (43), as were the animals in the present study,
suggesting that exposure to a full range of seasonal cues is ne-
cessary to reveal the natural extent of seasonal plasticity.
We here hypothesized that flocking-related changes in neuro-
chemistry take the form of either (i) a winter increase in flockers
that is not exhibited by nonflocking species, or (ii) the mainte-
nance 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 pallial 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 neurochemical variables seem to evolve in re-
lation to territorial aggression, and all neurochemicals and brain
areas examined here exhibit remarkable seasonal plasticity.
Animals. Spring field and song sparrows were caught April through 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 killed within 30 min of
capture. Perfusions, tissue processing, and immunofluorescent labeling fol-
lowed standard protocols (16, 25, 44). 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 Biologicals) guinea pig anti-
VP (Bachem), and rabbit anti-VIP (Bachem), with A.F. 488, biotin followed
Goodson et al.PNAS
| June 26, 2012
| vol. 109
| suppl. 1
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 Neurological 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 secondary. The specificity of all
antibodies has been addressed [(25, 44); see company datasheets for TH].
Each processing 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) from monochrome images, and background values were
subtracted for statistical analysis. Cell counts were conducted as previously
described (12, 16). 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 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 (45), and we therefore do not emphasize them in our
interpretations. However, they may still provide a useful metric for evalua-
tion, thus each of our data tables and figure panels provides information on
significance relative to Benjamini-Hochberg corrections for the false dis-
covery rate (27). 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, although not emphasized in Results, the robustness of
our findings is notable; for example, 73 of 78 ANOVAs that yield P values
<0.05 were significant after 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 inviting this contribution; Jacob Callis, Brian Gress, Alexis
Howard, Aubrey Kelly, Melissa Knisley, and Brittany Welsh for assistance
with immunocytochemistry and/or cell counts; Ellen Ketterson, Dawn
O’Neal, and Ryan Kiley for assistance with collections; Drew King and Mer-
edith West for property access; and Harold Gainer for the donation of anti-
serum. Support for this study was provided by Indiana University.
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| www.pnas.org/cgi/doi/10.1073/pnas.1203394109Goodson et al.