Hormones & Behavior (2002) 41(2):156-69
Chapter 1 Natural Breeding Conditions and Artificial Increases
in Testosterone Have Opposite Effects on the Brains of Adult
Male Songbirds: A Meta-analysis
T 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.
1.1 Abstract
A meta-analysis of the literature shows that in adult male songbirds:
1. brain mass,
2. telencephalon volume and
3. 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 Ttreated 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:
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song system;
avian hippocampus;
brain mass;
androgens;
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Hormones & Behavior (2002) 41(2):156-69

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corticosteroids;
telencephalon;
n. rotundus;
n. pretectalis;
n. spiriformis medialis;
oscines.
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Hormones & Behavior (2002) 41(2):156-69
Table of Contents
Chapter 1
Natural Breeding Conditions and Artificial Increases in Testosterone Have
Opposite Effects on the Brains of Adult Male Songbirds: A Meta-analysis ..................................... i
1.1 Abstract ................................................................................................................................................... i
1.2 Introduction ......................................................................................................................................... 1
1.3 Methods ................................................................................................................................................. 2
1.3.1 Data Analysis ............................................................................................................................. 2
1.4 Results .................................................................................................................................................... 3
1.4.1 Seasonal Changes in Wild-Caught Birds ......................................................................... 3
1.5 Discussion ............................................................................................................................................. 3
1.5.1 Seasonal Changes in Brain Mass ........................................................................................ 3
1.6 Conclusions .......................................................................................................................................... 4
1.7 References ............................................................................................................................................ 5
List of Figures
List of Tables
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1.2 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 [Ref 1]. This varitation 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 [Ref 2]. 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
varitation” and are controlled for statistically when investigating the differences in the
areas under study.
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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 thalamic nuclei under investigation
in this paper (Balthazart, Foidart, Wilson, and Ball, 1992; Nastiuk and Clayton, 1995).
1.3 Methods
1.3.1 Data Analysis
I used Stouffer’s method for combining hypothesis tests [Ref 4]. 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 one-tailed 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
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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).
1.4 Results
1.4.1 Seasonal Changes in Wild-Caught Birds
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”.
. Summary of All the Studies Investigating Seasonal Changes under Natural Conditions
1.5 Discussion
1.5.1 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
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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 ().
. 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 [Web Ref]. (A) Brains from adult black-capped chickadees (male and
female) from upstate New York, captured at five different times across the year.
. Change in brain mass in castrated adult male dark-eyed juncos with changing
photoperiodic condition.
1.6 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
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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.
Appendix
1.7 References
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