Supplemental methods and results
All animal procedures were approved by the Autoridad Nacional del Ambiente del
República de Panamá (permit # SEX/A-87-06). We placed wild-caught reproductively
active frogs (n = 9-11 in each stimulus condition for each sex) in sound isolation
chambers for two hours to decrease egr-1 levels to baseline prior to 30 minutes
acoustic treatment or 30 additional minutes of sound isolation (silence group). Acoustic
stimuli comprised a single exemplar of a natural call of either P. petersi or P.
pustulosus, each selected to have acoustic properties close to the mean measures of all
calls sampled within the population. These same stimuli were used to demonstrate sex
differences in behavioral responses in this species (Bernal et al. 2007). The stimuli were
broadcast at 82 dB SPL (re. 20 P) in the center of the chamber at 1 call/second,
alternating from two speakers (SME-AFS, Saul Mineroff Electronics, Elmont, NY) on
either side of the chamber. Males of this species only vocalize from pools of water, and
as no standing water was provided to them, no males vocalized in response to the
stimuli. Females in this species do not vocalize. In this experimental chamber, therefore,
the behavioral responses of both males and females were limited to locomotive activity,
which we recorded under infrared illumination using a PC-6EX-2 IR video camera
(Supercircuits, Liberty Hill, TX) connected to a ZR60 miniDV digital camcorder (Canon,
Lake Success, NY). We manually scored frog behavior to measure the time spent in
motion for each animal (Table S1). We found that the sexes differed in the time each
spent in motion in each stimulus condition (two-factor ANOVA, sex by stimulus
interaction F2,57 = 6.605, p = 0.003).
Following acoustic treatment, we rapidly decapitated animals and froze their
heads in liquid nitrogen. We sectioned brains and performed radioactive in situ
hybridization to localize egr-1 mRNA as described previously (Hoke et al. 2004, 2005,
2007). We systematically spaced high magnification photomicrographs throughout the
superior olivary nucleus (n = 6 images, Fig. S1; Hoke et al. 2005) and four divisions of
the torus semicircularis (n = 6-12 images per division, Fig S1; Hoke et al. 2004). We
selected sections for analysis based on anatomical landmarks. The cell density in the
superior olivary nucleus is fairly low, so we selected three images among the four or five
with higher density in the ventral hindbrain. Anatomical appearance of the torus
semicircularis changes rapidly over its extent (typically spanning 5-6 sections, of which
we measured 4). We selected our first section based on the rostralmost appearance of
the laminar, principal, and midline regions. Our next section included the appearance of
a ventral cell-sparse region between the principal nucleus and the tegmentum. Our third
section was marked by the disappearance of the midline nucleus with continued good
distinction of laminar and principal nuclei. Our final section comprised the most rostral
section in which a distinct laminar nucleus was no longer visible, and we sampled only
the principal nucleus within this section. Within each of these sections, three
photomicrographs were spaced by 100 (ventral, midline) or 250 (laminar, principal)
microns along the long axis of the toral region (dorsal to ventral for midline, lateral to
medial for others) for each toral division present in the section. Due to errors with tissue
processing or difficulties in sectioning angle, we excluded individual frogs in which
reliable measures could not be estimated in the auditory regions, leaving sample sizes
listed in Table S1 for each group.
We used custom-made automated image processing methods (Adobe
Photoshop) to separate color photomicrographs into portions covered by cells (stained
with cresyl violet and thus purple) and portions covered by silver grains (brown-black in
our images). The image processing involved manually selecting the color range of the
cresyl-violet stained cell bodies, clearing all pixels outside that area covered by cells,
and using the thresholding function to contrast area containing cells in black and cellfree areas in white. The color range of the black silver grains was then selected on a
second copy of the original photomicrograph after clearing all pixels outside the area
covered by cells, then converted to a black and white image using thresholding. We
then calculated silver grain density for each photomicrograph, that is, the fraction of the
area covered by cells that contained silver grains based on area measurements of the
two images representing the cells and grains (Image J). As background levels adjacent
to tissue were uniformly low, we did not correct for background silver grain density. We
averaged the measurements for each brain region for each individual, and logtransformed the averages to achieve normal distributions (Shapiro Wilks p > 0.05).
We tested for sex differences using ANCOVA with log-transformed average silver
grain densities for a single brain region as the dependent measure (SPSS 11). We
tested for the main effects of sex and stimulus, the interaction of stimulus and sex, and
included two covariations: time in motion and a global covariate representing overall
activation throughout the brain (Hoke et al. 2007). Because the average egr-1 levels
throughout the brain regions we analyzed was correlated with the motor output
produced by the individual, we normalized our global covariate so as to avoid statistical
confounds (based on Desjardins et al. 2001). To calculate this global covariate, we first
normalized silver grain densities measured in 36 brain regions (including the auditory
regions in this study) by dividing each measure by the mean of all individuals in that
brain region. We then averaged the 36 normalized measures for each individual to give
an estimate of relative overall egr-1 levels. These global activity levels were influenced
by acoustic treatment and locomotive activity, so we calculated residuals from this
activity measure not explained by acoustic stimulus and behavioral response. These
residuals, representing whole-brain activity unrelated to stimulus or motor output, were
used as the global activity covariate in the ANCOVA analyses. The global activity levels
were significant covariates (p  0.001) in all auditory regions except the superior olivary
nucleus (p = 0.1). The movement covariate was significant or nearly significant (p <
0.06; Table 1) in all auditory regions except the superior olivary nucleus (p = 0.46).
Removing either or both of these covariates from the superior olivary nucleus ANCOVA
had only minor effects on the tests for sex, stimulus, or the sex* stimulus interactions,
and did not influence our conclusions, thus we present here the full ANCOVA models
for all brain regions (Table 1). Similarly, removing the time in motion covariate (p = 0.06)
from the ANCOVA model for the laminar nucleus did not alter our conclusions.
For the laminar nucleus, we performed post-hoc analyses to compare pair-wise
comparisons between stimulus pairs in each sex (sex*stimulus interaction) using
uncorrected t-tests of the estimated marginal means (Fisher’s Least Significant
Difference procedure).
References:
Bernal, X. E., Rand, A. S. & Ryan, M. J. 2007 Sexual differences in receiver
permissiveness to advertisement calls in túngara frogs, Physalaemus pustulosus. Anim.
Behav. 73, 955-964.
Desjardins, A. E., Kiehl, K. A., & Liddle, P. F. 2001 Removal of confounding effects of
global signal in functional MRI analyses. Neuroimage 13, 751-758.
Hoke, K. L., Burmeister, S. S., Fernald, R. D., Rand, A. S., Ryan, M. J. & Wilczynski, W.
2004 Functional mapping of the auditory midbrain during mate call reception. J.
Neurosci. 24, 11264-11272.
Hoke, K. L., Ryan, M. J. & Wilczynski, W. 2005 Social cues shift functional connectivity
in the hypothalamus. Proc. Natl. Acad. Sci. U.S.A. 102, 10712-10717.
Hoke, K. L., Ryan, M. J. & Wilczynski, W. 2007 Integration of sensory and motor
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Table S1: Sample sizes and average proportion of time in movement for males
and females in each treatment.
Acoustic Treatment
Females
Males
Time in
Sample
Time in
Sample
motion
Size
motion
Size
Silence
0.24
9
0.31
9
P. petersi
0.39
8
0.27
9
P. pustulosus
0.57
11
0.10
5
Notes: One male in silence condition lacked usable sections of the superior olivary
nucleus, hence sample size is reduced for that brain region. The time in motion
averages above include only those males in which auditory system activity was
measured. Inclusion of additional animals (those in which brainstem sections were
missing or too angled for accurate identification of location) altered average proportions
of time in motion by at most 0.01.
Figure S1:
We distinguish among three models for the neural basis of sex differences in stimulus
selectivity. Black type indicates the presence of a sex difference whereas gray type
signifies similar responses in males and females. Black arrows indicate neural pathways
that differ in males and females, and gray arrows indicate similar functional relationships
in the sexes. The first model posits differential sensory sensitivity, in which sex
differences in behavior arise due to sex-specific selectivity in the auditory periphery. The
sex differences in selectivity would be apparent at all stages of the auditory and motor
pathways. In the sensorimotor gateway model, the lower auditory system functions
similarly in males and females, with sex difference emerging at a key site gating which
sensory information activates motor control targets. The third model proposes
differential motor responsiveness underlies sex differences: auditory processing is
similar in males and females, but the influence of auditory regions on motor control
regions differs. The lack of sex difference in the superior olivary nucleus and presence
in the laminar nucleus is consistent with the sensorimotor gateway model, with the
laminar nucleus as the critical site establishing behavioral selectivity for stimuli.