modulation of monoamine neurotransmitters in fighting fish betta splendens exposed to waterborne...

Modulation of monoamine neurotransmitters in fighting fishBetta splendens exposed to waterborne phytoestrogens

Ethan D. Clotfelter • Meredith M. McNitt •

Russ E. Carpenter • Cliff H. Summers

Received: 25 July 2009 / Accepted: 29 November 2009 / Published online: 11 December 2009

� Springer Science+Business Media B.V. 2009

Abstract Endogenous estrogens are known to affect

the activity of monoamine neurotransmitters in verte-

brate animals, but the effects of exogenous estrogens

on neurotransmitters are relatively poorly understood.

We exposed sexually mature male fighting fish Betta

splendens to environmentally relevant and pharmaco-

logical doses of three phytoestrogens that are potential

endocrine disruptors in wild fish populations: geni-

stein, equol, and b-sitosterol. We also exposed fish to

two doses of the endogenous estrogen 17b-estradiol,

which we selected as a positive control because

phytoestrogens are putative estrogen mimics. Our

results were variable, but the effects were generally

modest. Genistein increased dopamine levels in the

forebrains of B. splendens at both environmentally

relevant and pharmacological doses. The environ-

mentally relevant dose of equol increased dopamine

levels in B. splendens forebrains, and the pharmaco-

logical dose decreased norepinephrine (forebrain),

dopamine (hindbrain), and serotonin (forebrain) lev-

els. The environmentally relevant dose of b-sitosterol

decreased norepinephrine and dopamine in the fore-

brain and hindbrain, respectively. Our results suggest

that sources of environmental phytoestrogens, such as

runoff or effluent from agricultural fields, wood pulp

mills, and sewage treatment plants, have the potential

to modulate neurotransmitter activity in free-living

fishes in a way that could interfere with normal

behavioral processes.

Keywords Serotonin � Dopamine �Norepinephrine � Genistein � Equol � b-Sitosterol

Introduction

Gonadal estrogens have a variety of pre- and post-

synaptic actions on monoamine neurotransmitters such

as serotonin, dopamine, and norepinephrine (Etgen

2002). Consequences of these actions in mammals

include variation in monoaminergic activity between the

sexes, across the estrous cycle (Crowley et al. 1978;

Becker 1999; Bethea et al. 2002), and perimenopausal

depression and post-partum mood disorders in humans

(McEwen and Alves 1999; Amin et al. 2005). One

molecular mechanism by which this occurs is the

binding of estrogens to estrogen receptor (ER) types aand b in brain regions such as the dorsal raphe, median

E. D. Clotfelter (&) � M. M. McNitt

Department of Biology, Amherst College,

Amherst, MA 01002, USA

e-mail: [email protected]

R. E. Carpenter � C. H. Summers

Department of Biology, University of South Dakota,

Vermillion, SD 57069, USA

C. H. Summers

Neuroscience Group, Division of Basic Biomedical

Sciences, Sanford School of Medicine, University of

South Dakota, Vermillion, SD 57069, USA

123

Fish Physiol Biochem (2010) 36:933–943

DOI 10.1007/s10695-009-9370-2

raphe, medial preoptic, and paraventricular nuclei,

which are major sites of monoaminergic neurons in

the vertebrate brain (Crowley et al. 1978; Lu et al. 2001;

Sheng et al. 2004). The binding of ligands to these

intracellular receptors results in the transcription of

genes whose products regulate neurotransmission (e.g.,

synthesis and degradation enzymes). Steroid hormones,

particularly 17b-estradiol, have rapid, non-genomic

effects via interactions with other receptor types (McE-

wen and Alves 1999; Maggi et al. 2004; Cornil et al.

2006).

Environmental estrogens also have potential to

affect the production and metabolism of monoamine

neurotransmitters. Hundreds of chemicals are known

to disrupt the endocrine system of vertebrates, and

many of these compounds bind to estrogen receptors

ERa or ERb, thus mimicking endogenous estrogens

(Keith 1997; Brody and Rudel 2003). The reproduc-

tive consequences of exposure to endocrine disrupt-

ing chemicals are well documented, and include

effects on sex determination, secondary sexual char-

acters, oogenesis, spermatogenesis, and the onset of

sexual maturation (Colborn et al. 1993; Toppari et al.

1996; Crisp et al. 1998). Some of these reproductive

impairments may be mediated through alteration of

serotonin, dopamine, or norepinephrine activity in the

brain (Khan and Thomas 2001). Many industrial

contaminants such as bisphenol A (Miyatake et al.

2006), naphthalene (Gesto et al. 2006), octylphenol

(Christian and Gillies 1999), nonylphenol (Ferguson

et al. 2002), polychlorinated biphenyls (Seegal et al.

1986; Khan and Thomas 2001), and heavy metals

(Tsai et al. 1995; Zhou et al. 1999; Sloman et al.

2005) affect monoamine neurotransmission. Further-

more, neurotransmitter activity is disrupted in ani-

mals exposed to municipal sewage effluent, which

contains synthetic estrogens such as 17a-ethinylest-

radiol from oral contraceptives (Gagne and Blaise

2003; Gagne et al. 2007).

Not all endocrine disrupting chemicals are anthro-

pogenic. More than 300 species of plants contain

estrogenic isoflavones, coumestans, or lignans (Wyn-

ne-Edwards 2001; Dixon 2004). Many of these

compounds occur in plants as biologically inactive

glycosides (e.g., genistin, daidzin), but are converted

to active aglycones (e.g., genistein, daidzein) in the

presence of gut microbes. Of these groups, isoflav-

ones such as genistein have been the most intensively

studied, particularly in the context of human health

(Adlercreutz 1995; Setchell and Cassidy 1999; Dixon

2004). Isoflavones have numerous estrogenic and

anti-estrogenic effects in animals via ERa and ERb

(Patisaul et al. 2001; Whitten and Patisaul 2001;

Lephart et al. 2002; Whitten et al. 2002). Phytoster-

ols, a structurally distinct class of phytoestrogen

present in the cell membranes of all plants, also have

estrogenic effects in vertebrates (MacLatchy and Van

Der Kraak 1995; Mellanen et al. 1996; MacLatchy

et al. 1997; Tremblay and Van der Kraak 1998).

Phytoestrogens can be metabolized and excreted by

animals and thus are distinct from many lipid-soluble

endocrine disruptors such as polychlorinated biphe-

nyls, but limited toxicokinetic data suggests some

storage in tissues (Janning et al. 2000).

Little is known about how phytoestrogens affect

brain neurochemistry. For example, dopamine release

is increased by the isoflavone genistein in both in vitro

and in vivo rodent studies, though differences exist

between baseline and amphetamine-stimulated dopa-

mine levels (Bare et al. 1995; Ferguson et al. 2002).

Several flavonoid constituents of licorice (Fabaceae)

have been found to inhibit serotonin reuptake (Ofir

et al. 2003). Previous work in our laboratory has shown

that exposure to environmentally relevant concentra-

tions of genistein (an isoflavone), equol (an isoflavone

metabolite), and b-sitosterol (a phytosterol) sup-

presses mirror-induced agonistic behavior in the fight-

ing fish Betta splendens (Clotfelter and Rodriguez

2006), a behavior known to be mediated by serotonin

(Clotfelter et al. 2007). Mirror-induced agonistic

behavior in rainbow trout (Oncorhynchus mykiss) fry

is affected by exposure to paper mill effluent, which

contains a mixture of phytoestrogens (Johnsson et al.

2003). Furthermore, phytoestrogen exposure is known

to cause reproductive abnormalities in several fishes,

such as gonadal intersex, production of the egg-yolk

protein vitellogenin in males, reduced sperm motility

and concentration in males, delayed oocyte maturation

in females, and altered levels of steroid hormones in

both sexes (Lehtinen et al. 1999; Bennetau-Pelissero

et al. 2001; Kiparissis et al. 2003; Sepulveda et al.

2003). Thus, in light of these behavioral and repro-

ductive pathologies, the goal of the current study was

to determine whether phytoestrogen exposure affects

the synthesis or metabolism of monoamine neuro-

transmitters in the brains of Betta splendens (family

Osphronemidae). To this end, we exposed fish to

environmentally relevant and pharmacological

934 Fish Physiol Biochem (2010) 36:933–943

123

concentrations of genistein, equol, and b-sitosterol,

and measured gross brain levels of serotonin (5-HT),

the serotonin metabolite 5-hydroxyindoleacetic acid

(5-HIAA), dopamine (DA), the dopamine metabolite

3,4-dihydroxyphenylacetic acid (DOPAC), and nor-

epinephrine (NE).

Materials and methods

Phytoestrogen exposure and fish maintenance

Sexually mature male B. splendens (1.64 ± 0.03 g)

were obtained from a commercial supplier and accli-

mated for a week in the laboratory prior to assignment

to treatment groups. We exposed fish to the following

phytoestrogens, which were dissolved in ethanol:

genistein (Sigma–Aldrich, G6649), equol (Apin,

N04392), and b-sitosterol (Sigma–Aldrich, S1270).

Two doses were selected for each compound, one to

represent an environmentally relevant dose based on

data collected in situ (reviewed in Clotfelter and

Rodriguez 2006), and one a pharmacological dose that

was 2–3 orders of magnitude greater. We also had two

positive control groups, which were exposed to one

of two different doses of the endogenous estrogen

17b-estradiol (Sigma–Aldrich, E8875) dissolved in

ethanol. We selected 17b-estradiol as a positive

control because phytoestrogens are putative estrogen

mimics; the doses were selected based on studies of

17b-estradiol’s synthetic analog 17a-ethinylestradiol

and its relative potency in teleost fishes (Balch et al.

2004; Bell 2004; Scholz et al. 2004). The relative

binding affinity of genistein to estrogen receptor, for

example, is 0.01 that of 17b-estradiol (Santell et al.

1997), thus our positive control exposure concentra-

tions were two orders of magnitude lower than our

phytoestrogen concentrations. Most importantly, sim-

ilar doses of 17b-estradiol as we used here are known

to alter agonistic behavior in male B. splendens

(Clotfelter and Rodriguez 2006). Our negative control

group was exposed only to the ethanol vehicle.

Fish were kept in 1-l glass beakers filled with

reconstituted reverse-osmosis water, dosed with the

appropriate compound, for 28 days. This exposure

duration was selected because it is commonly used in

endocrine disruption studies in fishes (Baatrup and

Junge 2001; Bjerselius et al. 2001; Scholz et al.

2004). Twenty-five percent of the water in each

beaker was replaced thrice weekly and re-dosed

accordingly. Stock solutions of our compounds were

replaced weekly. Fish were fed *5% of their body

mass in chironomid larvae (negligible phytoestrogen

content) each day and were maintained at 27�C on a

12:12 L:D cycle, conditions typical of their native

Thailand.

Sample sizes varied slightly due to incidental

mortality, and are as follows: negative control (N =

13 fish), 100 ng/l 17b-estradiol (N = 12), 10 lg/l

17b-estradiol (N = 12), 1 lg/l genistein (N = 11),

1000 lg/l genistein (N = 11), 10 lg/l equol (N =

13), 1000 lg/l equol (N = 9), 10 lg/l b-sitosterol

(N = 12), and 1000 lg/l b-sitosterol (N = 12).

Although these are nominal concentrations, water

samples analyzed by high performance liquid chroma-

tography (HPLC) from the 1 lg/l genistein group

revealed that actual concentrations (4.03 ± 1.54 lg/l;

N = 5) were reflective of nominal concentrations, and

were well within the environmentally relevant range

for this compound (Kiparissis et al. 2001).

Analysis of neurotransmitters and their

metabolites

After the 28-day exposure period, we killed fish,

removed their brains rapidly (\3 min), and separated

the telencephalon and diencephalon (hereafter fore-

brain) from the mesencephalon, cerebellum, and

medulla (hereafter, collectively referred to as hind-

brain). We analyzed forebrains and hindbrains sep-

arately because previous studies in other species have

demonstrated differences in monoamine concentra-

tions between these gross regions (Tsai et al. 1995;

Øverli et al. 1999, 2001; Gesto et al. 2006, 2008).

These tissues were weighed (±0.1 mg) and stored at

-80�C until analysis. We used high performance

liquid chromatography with electrochemical detec-

tion (HPLC-ED) to measure NE, DA, DOPAC, 5-HT,

and 5-HIAA in forebrains and hindbrains (Winberg

et al. 1996; Summers et al. 1998, 2003). Briefly, the

samples were sonicated in 10 w/v 25 mM sodium

acetate buffer (pH 5) containing 3,4-dihydroxyben-

zylamine (internal standard), centrifuged twice at

15,0009g for 2 min and filtered (0.45 lm). The

supernatant was removed and 45 ll was injected into

a chromatographic system (Waters Associates, Inc.)

and analyzed electrochemically with an LC-4B

potentiostat (Bioanalytical Systems, Inc.). We used

Fish Physiol Biochem (2010) 36:933–943 935

123

5-HIAA:5-HT and DOPAC:DA as indices of seroto-

nergic and dopaminergic activity, respectively (Shan-

non et al. 1986).

Statistical analysis

Data were checked for normality prior to analysis.

Ratio data were not normally distributed and were

subjected to an inverse transformation prior to

analysis. Table 1 presents the non-transformed ratios.

We used one-way analysis of variance (ANOVA)

with Dunnett’s post hoc tests (SPSS v. 15.0) to

compare treatment groups to negative control groups.

Differences were considered statistically significant if

P \ 0.05.

Results

Of the 112 fish whose brains were removed, seven

forebrain and 13 hindbrain samples were lost during

HPLC. For other samples, concentrations of some

neurotransmitters could not be resolved, resulting in

different sample sizes for each analysis.

Norepinephrine

Generally, higher concentrations of norepinephrine

(NE) were measured in B. splendens forebrains than

in hindbrains (Fig. 1). A comparison of forebrains

and hindbrains in negative control fish yielded a

significant difference in NE content (t11 = 3.82,

P = 0.003). We found that phytoestrogens and 17b-

estradiol generally decreased NE levels in the fore-

brain (Fig. 1), but in only two treatment groups were

these differences statistically significant: the

1000 lg/l dose of equol (F2,32 = 7.42, P = 0.002)

and the 10 lg/l dose of b-sitosterol (F2,34 = 8.69,

P = 0.001). Differences in hindbrain levels of nor-

epinephrine between fish exposed to phytoestrogens

and negative control fish were non-significant

(P C 0.14 for all).

Dopamine and DOPAC

Dopamine (DA) levels in the forebrain of B. splen-

dens were lower than those in the hindbrain (Fig. 2)

among negative control fish. The forebrain levels

were 22.67 pg/mg and the hindbrain levels were

47.98 pg/mg (t11 = -4.57, P = 0.001). The positive

control 17b-estradiol did not significantly affect

DA levels in B. splendens brains. In the fore-

brain, genistein (F2,32 = 5.83, P = 0.007) and equol

(F2,32 = 3.98, P = 0.029), including environmen-

tally relevant doses of both compounds, increased

DA levels (Fig. 2). By contrast, the pharmacological

dose of equol (F2,31 = 3.53, P = 0.042) and the envi-

ronmentally relevant dose of b-sitosterol (F2,28 =

5.33, P = 0.011) decreased DA in the hindbrain

(Fig. 2).

DOPAC, the principal metabolite of dopamine, was

not affected by any of the phytoestrogen or 17b-

estradiol treatments (Table 1). Similarly, dopaminer-

gic activity (DOPAC:DA) was unaffected by exposure

to phytoestrogens or 17b-estradiol (Table 1). Among

negative control fish, dopaminergic activity was sig-

nificantly higher in forebrains than in hindbrains

(t10 = -5.86, P \ 0.001).

Serotonin and 5-HIAA

Serotonin (5-HT) levels were higher in B. splendens

forebrains than in their hindbrains (Fig. 3). Among

negative control fish, this difference was highly

statistically significant (t11 = 5.76, P \ 0.001). The

only treatment group in which 5-HT levels were

affected by phytoestrogen exposure was equol

(F2,32 = 7.32, P = 0.002), where the pharmacologi-

cal dose caused a significant decline in forebrain 5-HT

(Fig. 3).

The principal metabolite of 5-HT, 5-HIAA, was

also significantly reduced in the forebrains of

B. splendens exposed to the pharmacological dose

of equol (F2,32 = 6.90, P = 0.003; Table 1). None of

the other treatment groups showed a significant

difference in 5-HIAA (Table 1) or in serotonergic

activity (Table 1). Serotonergic activity in negative

control fish was higher in B. splendens forebrains

than in hindbrains (t10 = -2.23, P = 0.048).

Discussion

We found that environmentally relevant doses of all

three phytoestrogens tested, genistein, equol, and

b-sitosterol, had the potential to modulate neurotrans-

mitter concentration in the brains of sexually mature

male Betta splendens relative to negative control fish.


123

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123

The lower doses of genistein and equol both increased

dopamine levels in B. splendens forebrains, while

b-sitosterol decreased norepinephrine and dopamine

levels in forebrains and hindbrains, respectively. At

pharmacological doses, both genistein and equol had

significant effects on monoamines, with equol being

0

50

100

150

200

250

300

350

400

450

500

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hri

ne

(NE

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pg

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HindbrainsForebrains

Fig. 1 Concentrations

of the monoamine

neurotransmitter

norepinephrine (NE) in the

brains of male Bettasplendens exposed to

17b-estradiol (positive

control) and various

phytoestogens for 28 days.

ANOVA with Dunnett’s

post hoc tests revealed that

the following treatment

groups differed significantly

in forebrain levels of NE

from the negative control

treatment group (indicated

by asterisks): 1000 lg/l

equol (P = 0.002) and

10 lg/l b-sitosterol

(P = 0.001)

0

10

20

30

40

50

60

70

Do

pam

ine

(DA

) co

nce

ntr

atio

n (

pg

/mg

tis

sue)

* * **

Forebrains Hindbrains

*

Fig. 2 Concentrations of the monoamine neurotransmitter

dopamine (DA) in the brains of male Betta splendens exposed

to 17b-estradiol (positive control) and various phytoestogens

for 28 days. ANOVA with Dunnett’s post hoc tests revealed

that the following treatment groups differed significantly in

forebrain levels of DA from the negative control treatment

group (indicated by asterisks): 1 lg/l genistein (P = 0.12),

1000 lg/l genistein (P = 0.13), and 10 lg/l equol (P = 0.048).

Furthermore, hindbrain levels of DA were decreased in the

1000-lg/l equol (P = 0.027) and the 10-lg/l b-sitosterol

(P = 0.015) groups


123

the most potent; high doses of equol reduced levels of

all three neurotransmitters.

The gross differences we found between forebrains

and hindbrains in monoamine concentrations among

our negative control fish were fairly consistent with

previously published research. We found higher

serotonin content in forebrains than in hindbrains

(caudal midbrains plus hindbrains), as has been

reported for rainbow trout and tilapia Oreochromis

mossambicus (Saligaut et al. 1990; Tsai et al. 1995;

Øverli et al. 2001). Differences in forebrain versus

hindbrain serotonergic activity, the ratio of metabo-

lite 5-HIAA to 5-HT, varies among studies in trout

(Øverli et al. 1999, 2001; Gesto et al. 2006, 2008);

we found slightly elevated serotonergic activity in

B. splendens forebrains. With respect to the cate-

cholamines, NE was higher in B. splendens forebrains

than in hindbrains, as is true of trout (Gesto et al.

2006). Higher DA content in the hindbrains of

B. splendens compared with forebrains is inconsistent

with the data for trout (Øverli et al. 2001; Jonsson

et al. 2003), where DA contents of both the

telencephalon and the hypothalamus (both included

in our forebrain samples) are consistently higher than

in brainstems. Dopaminergic activity is also higher in

trout forebrains than in hindbrains (Jonsson et al.

2003; Gesto et al. 2006, 2008), which is what we

found for B. splendens.

There are few data available documenting the effects

of phytoestrogens on neurochemistry of fishes, but our

findings find mixed support from rodent and cell culture

studies. The increase in forebrain DA that we observed

in B. splendens following exposure to genistein may be

related to the neuroprotective effects isoflavones impart

on mammalian dopaminergic neurons (Wang et al.

2005; Kyuhou 2008). Furthermore, our observation that

b-sitosterol reduced hindbrain dopamine content is

consistent with previous research showing toxicity of

b-sitosterol to dopaminergic neurons in rats (Kim et al.

2008). In bovine adrenal medullary cells, however,

treatment with the phytoestrogens daidzein (an isoflav-

one) and resveratrol (a polyphenol) caused dose-depen-

dent effects on total 14C-catecholamine (epinephrine,

norepinephrine, and dopamine) secretion (Yanagihara

et al. 2008). Low concentrations of phytoestrogens

stimulated catecholamine secretion, whereas high

0

50

100

150

200

250

300

350

Ser

oto

nin

(5-

HT

) co

nce

ntr

atio

n (

pg

/mg

tis

sue)

*

Forebrains Hindbrains

Fig. 3 Concentrations of the monoamine neurotransmitter

serotonin (5-HT) in the brains of male Betta splendens exposed

to 17b-estradiol (positive control) and various phytoestogens

for 28 days. ANOVA with Dunnett’s post hoc tests revealed

that only the 1000-lg/l equol treatment group (P = 0.009)

differed significantly in forebrain 5-HT levels from the

negative control treatment group (indicated by asterisk)


123

concentrations were inhibitory. This pattern is consistent

with our observation that dopamine levels were higher in

fish exposed to low doses of equol compared to those

exposed to high doses, but was not consistent with our

results on genistein, which was stimulating at both low

and high doses. This non-monotonic effect of genistein

merits further study, as such a phenomenon has already

been documented in rodents (Wisniewski et al. 2005),

and it has clearly important consequences for contam-

inant regulation. Similar non-monotonic dose–response

curves have been found for endogenous estrogens

(Welshons et al. 2003).

We generally found few effects of phytoestrogens

on the concentrations of serotonin or its metabolite

5-HIAA in the brains of B. splendens. A study on rats

showed that genistein treatment does not affect

serotonin content of the ventromedial nucleus of the

hypothalamus (Patisaul et al. 2008), but genistein and

other phytoestrogens are known to affect serotonin

re-uptake (Ofir et al. 2003). Furthermore, mammalian

5-HT neurons are ERb-rich (Bethea et al. 2002),

suggesting that genistein should be capable of

significant modulation of serotonergic activity (Whit-

ten and Patisaul 2001; Patisaul et al. 2002). Whether

this is true in fishes remains largely unknown. The

only significant effects we found for genistein were

similar for the environmentally relevant and the

pharmacological doses, which confirm that genistein

can exert significant neurobehavioral effects even at

low doses (Whitten et al. 2002).

Our positive control, 17b-estradiol, had no effect

on B. splendens neurochemistry. This finding is

somewhat surprising given that this potent endoge-

nous estrogen modulates monoamine activity in

mammals (Etgen 2002). Teleost fishes have two

estrogen receptor types that are homologous to

mammalian ERa and ERb (Socorro et al. 2000),

which are distributed throughout the telencephalon,

diencephalon, and hypothalamus (Kah et al. 1997).

Fishes also have a third receptor type, ERc, which is

derived from ERb and binds 17b-estradiol with high

affinity (Hawkins et al. 2000). The three ER types

have unique distributions in fish brains (Hawkins

et al. 2000), suggesting that steroid actions on

monoamines in fishes will be difficult to predict from

mammalian models and merit further investigation.

The lack of any effect of our positive control

17b-estradiol also suggests that phytoestrogen effects

on neurotransmitters in B. splendens may occur

independent of estrogen receptors. Equol has been

shown to bind dihydrotestosterone (Lund et al. 2004),

for example, so interference with androgens is a

viable alternative hypothesis. Another possibility is

that the sequestration of 17b-estradiol from water,

perhaps mediated by sex hormone-binding globulin

activity (Miguel-Queralt and Hammond 2008), dif-

fers from that of phytoestrogens, thus reducing the

effect of the endogenous estrogen on brain

neurochemistry.

The low dose we used for each phytoestrogen was

environmentally relevant based on available data on

sewage treatment plant effluent, wood pulp mill

effluent, and agricultural soils (reviewed in Clotfelter

and Rodriguez 2006). Thus, our findings suggest that

phytoestrogen exposure has the potential to exert

significant behavioral effects on free-living fish

populations, as has been documented in mummichogs

(Fundulus heteroclitus) in areas contaminated by

heavy metals (Smith et al. 1995; Smith and Weis

1997; Zhou et al. 1999). The neurochemical data we

present here, however, cannot be directly compared

to a previous behavioral study, in which Clotfelter

and Rodriguez (2006) found that mirror-induced

aggression was reduced in fighting fish exposed to 1

and 1000 lg/l genistein and 1000 lg/l equol. Future

work should focus on integrating behavioral and

neurochemical endpoints in the study of endocrine

disrupting chemicals such as phytoestrogens, with a

particular emphasis on establishing behavioral assays

for fishes that mimic natural conditions (Smith et al.

1995), which may not necessarily be true of mirror-

induced aggression (Oliveira et al. 2005). Multiple

exposure periods should also be used, as the

relationship between monoamines and agonistic

behavior is dynamic and may not be captured

adequately by a single exposure period (Summers

et al. 2005a, b).

Acknowledgments We thank Lexi Brown, Maureen Manning,

Kathy Nieves-Puigdoller, and Karina Zaveri for assistance in the

laboratory. Monica Giusti and Dante Vargas at Ohio State

University kindly performed the HPLC to validate our nominal

genistein concentrations. Cynthia Bethea, Lexi Brown, Heather

Patisaul, Brigitte Todd, and three anonymous reviewers provided

helpful comments on earlier versions of this paper. Funding for this

research was provided by the Webster Fund of the Department of

Biology at Amherst College, the H. Axel Schupf ‘57 Fund for

Intellectual Life at Amherst College, National Science Foundation

grant IOS-0725186 to E.D.C. and National Institutes of Health

COBRE grant P20 RR15567 to C.H.S.


123

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