Hormones & Behavior. 41(2):156-69
Natural Breeding Conditions and Artificial Increases in
Testosterone Have Opposite Effects on the Brains of Adult
Male Songbirds: A Meta-analysis
Tom V. Smulders
This document has been modified for the purposes of IT skills courses run by Rebecca
McCready and Sue Vecsey (LTMS), Newcastle University, UK, with permission from the author.
Abstract
A meta-analysis of the literature shows that in adult male songbirds, brain mass,
telencephalon volume and n. rotundus (a thalamic visual nucleus) volume increase from the
nonbreeding season (low testosterone) to the breeding season (higher testosterone). These
effects can at least partially be mimicked by photoperiod manipulations in captivity. In
contrast, an artificial testosterone (T) titer increase by chronic implants yields the opposite
results: telencephalon, n. rotundus, and n. pretectalis volumes are lower in T-treated animals
than in controls. These results suggest that artificial testosterone manipulations do not
necessarily mimic the effects of natural variations in hormone levels and that results from
experiments using T implants to mimic natural hormonal effects should be interpreted with
caution.
Key Words: song system; avian hippocampus; brain mass; androgens; corticosteroids;
telencephalon; n. rotundus; n. pretectalis; n. spiriformis medialis; oscines.
Introduction
Much research has been directed toward understanding seasonal behaviors and the associated
seasonal changes in the underlying neuroanatomy in birds. This research includes such
behaviors as foodhoarding, nest-searching in brood parasites, and singing. Nuclei of the song
system, which control learning and production of song in oscine songbirds, often are larger
during the breeding season than during the nonbreeding season. This variation is partly due to
the rise in testosterone during the breeding season, but other photoperiodically regulated
factors (e.g., melatonin and possibly thyroid hormone; recently reviewed by Tramontin and
Brenowitz, 2000) also contribute. The hippocampal formation in birds also changes
seasonally, and this seems to be related to a seasonal need for better spatial memory. In
food-hoarding birds, it is larger during the hoarding season than during the rest of the year. In
brood-parasitic cowbirds, the hippocampal formation is larger during the breeding season, but
only in the sex(es) that actively looks for host nests. In this system, the underlying
mechanisms have not yet been studied in detail.
When investigating changes in neuroanatomy, especially brain region volumes, it is usually
necessary to also measure control areas: regions of the brain that are not expected to vary as
a result of the experimental manipulation or condition. Typical control measures are the
volume of the entire telencephalon, or some variant such as total brain mass or
telencephalon width, as well as the volumes of small, easily delineated nontelencephalic
nuclei. Sometimes significant differences have also been found in these control regions. Most
often, these differences are assumed to be “nonspecific background variation” and are
controlled for statistically when investigating the differences in the areas under study.
In this paper, I investigate the changes in the most commonly used control regions in songbird
neurobiology using the quantitative review techniques of meta-analysis. One of the common
problems with meta-analyses is the “publication bias:” it is difficult to find negative results in
the literature because it is difficult to publish negative results. This is not a problem in the
current investigation, however, since the regions under investigation were not the primary
targets of the original papers. Therefore, any consistent and significant patterns across
studies would be especially meaningful. The regions that are included in this analysis are
whole brain mass and the volumes of the entire telencephalon (Tel), nucleus rotundus (Rt) (a
thalamic visual nucleus), nucleus pretectalis (Pt) (a thalamic visual nucleus), and nucleus
spiriformis medialis (SpM) (a thalamic sensorimotor nucleus) [Ref 3]. Whereas several parts of
the Tel contain androgen receptors, no androgen receptors have been described in any of the
Hormones & Behavior. 41(2):156-69
thalamic nuclei under investigation in this paper (Balthazart, Foidart, Wilson, and Ball, 1992;
Nastiuk and Clayton, 1995).
Methods
Data Analysis
I used Stouffer’s method for combining hypothesis tests. First, I tabulated the P values
associated with the relevant comparisons and the directions of all differences, whether they
were statistically significant or not. Then, all two-tailed P values were transformed to onetailed P values in the most commonly occurring direction of the difference for that structure.
These P values were then transformed to the corresponding Z scores using the inverse
function of a Gaussian distribution. Stouffer’s Zc was then calculated as
where zi represents the individual z value for each study included in the analysis, and N is the
total number of studies included. The one-tailed P value (Pc) resulting from this combined Z
score is then used as an indicator of the significance of the effect across all the tests of the
same hypothesis. Effects are considered significant if Pc , 0.05. In addition, I calculated a
common metric for the effect size (d) for each study (whether significant or not) as the
difference between two groups as measured in units of standard deviations (SD).
Results
Seasonal Changes in Wild-Caught Birds
Table 1 represents all the studies from which I obtained data on seasonal changes in male
songbirds. Birds collected during the breeding season have brains that are on average 1.05 (6
0.42 (SEM)) SD heavier (i.e., brain mass is larger) than those of birds collected in the
nonbreeding season (Zc 5 23.547, Pc 5 0.0002). Black-capped chickadees are seasonal food
hoarders, which have a larger hippocampal formation (HF) and septum in the fall. These
structures (and possibly other associated structures) make up a large part of the brain and
this would definitely influence total brain size.
Table 1. Summary of All the Studies Investigating Seasonal Changes under Natural Conditions.
Species
Direction
Effect size
(d)
Two-tailed
P
One-tailed
P
Z
score
Brain mass
Black capped chickadee
S>F
0.060
0.918
0.459
0.103
Bunting (Emberiza rutila)
S>F
0.120
0.076
0.381
0.303
Dark-eyed junco
S>F
0.820
0.248
0.124
1.155
Red-winged blackbird
S>F
6.500
0.170
0.085
1.372
Nuttall's white-crowned
sparrow
S>F
2.130
0.005
0.003
2.807
Dark-eyed junco
S>F
2.526
0.003
0.002
2.948
F>S
-0.780
0.152
0.924
1.433
Telencephalon
Dark-eyed junco
Hormones & Behavior. 41(2):156-69
Black capped chickadee
F>S
-0.900
0.164
0.918
1.392
Screaming cowbird
F>S
-0.480
0.492
0.754
0.687
Nuttall's white-crowned
sparrow
S>F
0.420
0.400
0.200
0.842
Dark-eyed junco
S>F
0.650
0.357
0.179
0.921
Rufous-sided towhee
S>F
0.880
0.260
0.130
1.126
Shiny cowbird
S>F
1.440
0.017
0.009
2.387
Discussion
Seasonal Changes in Brain Mass
In birds too, it is likely that at least part of the increase in brain mass during the breeding
season is the result of increased water content. Evidence for this comes from the fact that
most studies from which we collected data used perfusion-fixed brains, which were then cut
on a freezing microtome or on a cryostat. For this procedure, brains are typically
cryoprotected by immersion in a high concentration of sucrose (typically 20–30%). Such a high
osmolarity solution will, in addition to adding sucrose to the tissue (which cryoprotects it),
equalize the total osmolarity inside and outside the tissue. If the tissue osmolarity is lower in
the breeding season (i.e., if there is more water relative to solutes in the brain tissue), the
brains should lose more water (i.e., more weight) when immersed in sucrose if collected
during the breeding season than during the nonbreeding season. This is exactly what we found
in two independent data sets: one from black-capped chickadees and one from dark-eyed
juncos. In both cases, during the breeding season, immersion in sucrose resulted in a
significant decrease in brain weight, while during the nonbreeding season, the change was
much smaller (Figure 1).
Figure 1. Change in brain mass as a consequence of cryoprotection. The percentage (6SEM)
change in brain mass before and after immersion in a 30% sucrose solution for several days is
plotted against the time of year when the brains were collected and processed. (A) Brains
from adult black-capped chickadees (male and female) from upstate New York, captured at
five different times across the year.
Hormones & Behavior. 41(2):156-69
Figure 2. Change in brain mass in castrated adult male dark-eyed juncos with changing
photoperiodic condition.
Conclusions
A quantitative review of the literature shows that there are overall changes taking place in
the brains of adult male songbirds across the year and with artificial testosterone treatment.
Under natural conditions, brains tend to be larger during the breeding season than during the
nonbreeding season. Artificial elevations of T levels, however, seem to decrease the volumes
of these same brain areas. These patterns now require careful direct experimental testing,
both to verify the patterns (using experimental designs with high statistical power) and to
elucidate the possible underlying mechanisms. These results lead to three important
cautionary insights for researchers in the field of behavioral neuroendocrinology. First of all,
artificial hormone treatments are not necessarily a good mimic of natural hormone changes,
however well we think we know the system. Second, care should be taken when interpreting
the outcomes of behavioral experiments in which birds were implanted with exogenous
testosterone, since the behavioral implications of changes in nontargeted brain regions are
not known. Finally, these brain regions should not be used as statistical controls or
“standards” when investigating the effects of testosterone on other brain nuclei, such as the
song system, since they may distort the actual data.
References