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Impulse: The Premier Journal for Undergraduate Publications in the Neurosciences


2009

Effects of melatonin and ethanol on the heart rate of Daphnia magna


Bonnie Kaas1,2, Krithika Krishnarao2, Elizabeth Marion1, Larissa Stuckey1, and Rebecca
Kohn3
1

Department of Neuroscience, 2Department of Biochemistry and Molecular Biology, 3Department of Biology,


Ursinus College, Collegeville, Pennsylvania, 19426

Melatonin, an endogenous hormone that may regulate circadian rhythms by modulating


cholinergic activity, is increasing in popular use as a natural treatment for sleep disorders.
However, the effects of melatonin on the human heart are not well characterized, and the
consequences of combining alcohol with melatonin are unknown. The myogenic heart of the
water flea Daphnia magna (D. magna) is regulated by inhibitory cholinergic neurons that
modulate cardiac function, including heart rate. D. magna is a useful model organism for
cardiovascular function, due to its physical transparency and susceptibility to cardioactive drugs
known to affect the human heart. In this study, the effects of immersion in 10 mg/L melatonin
and 5% ethanol on the heart rate of D. magna were quantified. Two-hour exposure to melatonin
caused a significant decrease in heart rate, from 228 2 bpm to 167 8 bpm. Six-minute
immersion in ethanol also significantly depressed the heart rate to 176 10 bpm. Pretreatment
with melatonin prior to the addition of ethanol resulted in a greater decrease in heart rate (89 7
bpm) than ethanol or melatonin alone. These findings indicate that melatonin and alcohol may
combine to cause a greater depressive effect on cardiac function.
Abbreviations: ACh (acetylcholine), AChR (acetylcholine receptor), bpm (beats per minute), D.
magna (Daphnia magna)
Keywords: cardiovascular function, alcohol, acetylcholine, natural sleep aids, cholinergic
regulation

Introduction
Melatonin is a neuroendocrine hormone
secreted by the pineal gland. In humans, its
primary role is that of a circadian pacemaker,
and exogenous melatonin has a welldocumented soporific effect when taken during
the daytime or early evening (Hughes and Badia,
1997; Pires et al., 2001; Zhdanova et al., 1995).
Oral administration of melatonin has been
successfully used to treat various sleep-cycle
disturbances in children with chronic sleep
disorders by decreasing sleep onset latency (Jan
and ODonnell, 1996; Smits et al., 2003).
Melatonin may exert these effects by modulating
levels of the neurotransmitter acetylcholine

(ACh), or by altering the function of


acetylcholine receptors (AChR) (Paredes et al.,
1999; Brusco et al., 1998; Markus et al., 1996;
Zago and Markus, 1999). The heart of the water
flea, D. magna, is regulated by cholinergic
neurons, and may be useful as a model for the
effect of melatonin on cardiovascular function
(Stein et al., 1966).
D. magna are freshwater cladocerans
that exhibit a short life span, rapid maturation,
and reproduction (Teschner, 1995; Anderson
and Jenkins, 1942). Their physical transparency
allows for simple observation of internal
structures, including the heart (Teschner, 1995;
Pirow et al., 2004). In addition, they are highly

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Effects of Melatonin and Ethanol on the Heart Rate of Daphnia magna
2009

sensitive to environmental changes, which cause


them to adapt morphologically, physiologically,
and behaviorally (Gard et al., 2009; Ikenaka et
al., 2006). Water fleas are unusual among
crustaceans in that they possess myogenic hearts
(Yamagishi et al., 2000; McMahon, 2001).
Unlike neurogenic hearts, in which the cardiac
ganglion acts as the pacemaker to initiate
contraction, myogenic heart rhythms are
initiated in the cardiac muscular tissue and are
independent of neural, metabolic, or hormonal
stimulation (Cooke, 2002; Davis and Hill,
1999). Inhibition by extra-cardiac cholinergic
nerves allows for neuronal modulation in
addition to the myogenic activity (Stein et al.,
1966; Bekker and Krijgsman, 1951; Baylor,
1942). The heart of D. magna may therefore
resemble the vertebrate myogenic heart more
closely than higher orders of Crustacea (Baylor,
1942).
D. magna possess a globular myogenic
heart inhibited following administration of ACh,
which may act on conduction mechanisms in the
cardiovasculature (Spicer, 2001; Ellenbogen and
Obreshkove, 1949; Prosser, 1940; Stein et al.,
1966). Most of the heart is only a single celllayer thick, which may explain why it is easily
influenced by the application of drugs and other
substances (Stein et al., 1966). D. magna have
been shown to respond to a number of
cardioactive drugs that are known to affect
human heart function (Villegas-Navarro et al.,
2003; Postmes et al., 1987). Testing the effects
of these drugs is simplified in D. magna as the
fleas are responsive to pharmacological agents
added to the water in which they swim
(Campbell et al., 2004). The introduction of
these pharmacological agents to water fleas may
induce activity directly on the cardiac muscle
(Bekker and Krijgsman, 1951).
The ingestion of ethanol has been shown
to have a number of cardiovascular effects in
human beings. However, neither ingestion nor
intra-arterial infusion of ethanol has been shown

to affect human heart rate (Ahmed et al., 1973;


Tawakol et al., 2004). While melatonin is
generally considered a safe supplement, with
only rare and mild reported side effects, the
result of combining oral melatonin with alcohol
is unknown. The FDA strongly advises against
the use of alcohol in conjunction with
prescription or over-the-counter sleep aids
(FDA, 2007).
However, individuals with
insomnia commonly self-medicate with alcohol
(Kaneita et al., 2007; Johnson et al., 1998).
Melatonin has been shown to decrease heart rate
in humans and rats (Gilbert et al., 1999; Chuang
et al., 1993), but the combined effect of
melatonin and ethanol on heart functions has not
yet been studied.
Because melatonin is known to exert a
soporific effect, possibly through the modulation
of cholinergic transmission, we predict that
melatonin will slow the heart rate of D. magna
by increasing ACh activity in the inhibitory
regulatory neurons. We predict that ethanol will
increase the heart rate of D. magna by inhibiting
ACh release. Melatonin and ethanol combined
should exert antagonistic effects on the
cholinergic system, resulting in a minimal
change in the heart rate.

Materials and Methods


D. magna (WARDS Natural Science,
Rochester, NY) were maintained in jars of
spring water under a 130-Watt incandescent
bulb. Individual animals were transferred in a
drop of water to a depression slide coated in
petroleum jelly. The heart rate was counted
after a 30-second acclimation period using a
Nikon SMZ645 dissecting microscope with a
NI-150 High Intensity Illuminator (Nikon Inc.,
Melville, NY). For the water controls, multiple
heart rate readings were taken per flea, starting
30 s after immobilization and continuing for 2min intervals until a total of five counts had been

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Impulse: The Premier Journal for Undergraduate Publications in the Neurosciences
2009

completed. The animal was then discarded. A


total of 70 control heart rate readings from 14
individual fleas were collected in spring water.
For every other condition, only one average
heart rate was obtained per flea.
Effect of Melatonin on Heart Rate
Powdered melatonin (Sigma Aldrich, St.
Louis, MO) was dissolved in deionized water to
make a 10 mg/L solution, corresponding to the
maximum solubility of the melatonin powder.
This solution was stored at 4C and protected
from light due to its photosensitivity. The
average heart rate following 0-, 15-, 30-, 120-,
and 180-min exposure to melatonin was
determined.
Individual
animals
were
immobilized on depression slides in a drop of
water. The water was then withdrawn and
replaced with room-temperature melatonin
solution. Heart rate was assessed after 0, 15,
and 30 minutes had elapsed. For the 120- and
180-min time points, individuals were allowed
to swim freely in room-temperature melatonin
solution in the dark before the heart rate was
scored using the procedure described above.
This permitted examination of heart rate at
greater time intervals while avoiding any longterm negative effects of immobilization in
petroleum jelly.
The maximum effect of melatonin on
heart rate was quantified using a 120-min
exposure time (based on time lapse trial results,
Figure 1). Individual D. magna were allowed to
swim freely in room-temperature melatonin
solution for two hours in the dark before the
fleas were immobilized and the heart rate scored
using the procedure described above. As an
additional control, twenty individuals were kept
in spring water and protected from light for two
hours before counting the heart rate in order to
rule out any confounding effect of dark
conditions on heart rate.

Effect of Ethanol on Heart Rate


To test the effect of ethanol on heart
rate, fleas were immobilized as previously
described, and an initial heart rate was counted
in water. All water was withdrawn from the
slide and quickly replaced with 100 L of 5%
ethanol (EM Science, Gibbstown, NJ), which
was the lowest concentration found to elicit a
strong physiological response in preliminary
testing (data not shown). The heart rate was
counted in two minute intervals for a total of ten
minutes to determine when the maximal
response would occur. Additional trials were
then performed using a six-minute exposure
time to quantify the maximum effect of ethanol
on heart rate.

Combined Effect of Alcohol and Melatonin


on Heart Rate
Individual D. magna were treated for
two hours with melatonin solution as described
previously.
After immobilization on a
petroleum jelly-coated slide, the melatonin
solution was withdrawn and replaced with a 5%
ethanol/10 mg/L melatonin solution. Heart rate
was then assessed after six minutes of exposure
to the ethanol/melatonin solution.

Statistical Analysis
Results were analyzed with either oneway ANOVA followed by Tukey HSD post-hoc
comparison (family error rate = 5%) or
Dunnetts method for comparison with control
or a Students t-test with an alpha level of 0.05
using Minitab15.1.0.0 for Windows (LEAD
Technologies Inc., Haddonfield, NJ). All error
bars represent the standard error of the mean.

Results
A significant decrease in heart rate was
observed in response to 10 mg/L melatonin
solution only at the 120-min time point (Figure

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Effects of Melatonin and Ethanol on the Heart Rate of Daphnia magna
2009

1, Dunnetts Test, T= -4.578, p=0.0001).


Animals kept in dark conditions for two hours
did not exhibit a decrease in heart rate (244 7
bpm) in comparison with controls (228 2 bpm,
one-tailed t-test, t(23) = 2.14, p = 0.978).

Figure 2.Observed heart rate of D. magna following


exposure to 5% ethanol for various lengths of time.
A significant decrease is noted at 0 min (Dunnetts
Test, =0.05) and the heart rate does not change
significantly between 2 and 10 min (one-way
ANOVA, =0.05) (N=20 for each time point).

Figure 1. Observed average heart rate of D. magna


following exposure to melatonin for select times.
* indicates significant difference compared to initial
time point (Dunnetts Test, =0.05) (N=10 for each
time point).

D. magna exhibited a significant


decrease in heart rate immediately following the
addition of 5% ethanol, and the heart rate
remained significantly depressed over the 10min period (Dunnetts Test, =0.05). The heart
rate did not change significantly between two
and ten minutes (one-way ANOVA, F=0.08,
p=0.989). For logistical reasons, a six-minute
exposure time was used to assess the maximum
effect of ethanol on heart rate (reported in Figure
3), although the data indicate that any time
between two and ten minutes could have been
used.

The maximum depressive effects of 10


mg/L melatonin and 5% ethanol on the heart rate
were assessed using 120- and 6-min exposure
times, respectively. Treatment with ethanol
(176 10 bpm, T= -5.51, p<0.00005),
melatonin (167 8 bpm, T= -6.75, p<0.00005),
and ethanol and melatonin together (89 7 bpm,
T= -14.60, p<0.00005) were all found to
decrease the heart rate significantly compared to
controls (Dunnetts Test). Treatment means
were compared using Tukey HSD post hoc
comparisons. Ethanol and melatonin were found
to cause identical decreases in heart rate
compared to animals in spring water (T= 0.939,
p= 0.7838). Six-min ethanol exposure following
two-hour immersion in melatonin, however,
resulted in a greater decrease in heart rate than
either ethanol (T= -8.591, p<0.00005) or
melatonin (T= 7.977, p<0.00005) treatment
alone (Figure 3).

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2009

Figure 3. Observed average heart rate of D. magna in


water (control), following a two-hour exposure to
melatonin, and following an addition of 5% ethanol,
with and without two-hour pretreatment with
melatonin. (N=14, 54, 53, 50, respectively, * indicates
significant difference at the 0.05 level, determined by
Tukey post hoc comparison). All three treatments were
found to decrease heart rate compared to the control
(one-way ANOVA, p<0.001).

Discussion
Immersing D. magna in melatonin for
two hours caused a significant decrease in heart
rate afterward. This depressive effect on heart
rate has been demonstrated previously in human
beings (Gilbert et al., 1999). Melatonin likely
exerts its effects on the water flea heart by
altering ACh levels.Application of ACh inhibits
the heart rate of D. magna, and melatonin has
been shown to increase ACh secretion in the rat
brain (Baylor, 1942; Paredes et al., 1999; Brusco
et al., 1998). It is therefore likely that the
observed decrease in the D. magna heart rate
upon melatonin administration occurs due to
modulation of the activity of the inhibitory
cholinergic neurons that regulate the heart, most
likely through an increase in ACh release.
Alternatively, melatonin may affect the heart by
altering the binding properties of neuronal
nicotinic AChR (Markus et al., 1996; Zago and
Markus, 1999). Melatonin receptors have also
been identified in the vasculature, so it is
possible that melatonin has a direct effect on the
cardiovascular system that does not involve

modulation of cholinergic activity (Ekmekcioglu


et al., 2003).
Immersion in 5% ethanol was shown to
decrease the heart rate significantly in D. magna
after a six-minute exposure time (Figure 2).
Similarly, ethanol has been previously shown to
slow the heart rate of the invertebrate Ciona
intestinalis (Pope and Rowley, 2002).
The
mechanism by which this occurs is uncertain.
Ethanol has been shown to have a wide range of
effects on physiological function. Ethanol is
known to elicit a biphasic response, with
stimulatory effects produced at lower doses and
inhibitory effects at higher doses (Earleywine
and Martin, 1992). The high concentration used
in this study would be expected to result in an
inhibitory effect. Previously, ethanol has been
shown to inhibit the release of neurotransmitters,
including ACh, in rats (Carmichael and Israel,
1975; Erickson and Graham, 1973). Application
of ethanol, which would inhibit ACh release and
therefore decrease cholinergic transmission in
these inhibitory neurons, would thus be expected
to increase the heart rate of D. magna. It is
possible that the effect observed in D. magna is
not due to changes in the release of ACh, but
rather in receptor sensitivity to ACh. Ethanol
has also been shown to alter the properties of
neuronal nicotinic AChR, potentiating AChinduced currents (Cardoso et al., 1999; Aistrup
et al., 1999; Wu et al., 1994). Thus, ethanol may
slow the heart rate by acting on AChRs located
either on the heart itself or in the chain of
regulatory neurons.
Ethanol may also act
through an entirely different mechanism to cause
direct effects on the cardiac muscle of D.
magna. Ethanol has been shown to impair
myocardial contractility in the human heart
(Ahmed et al., 1973), and to decrease heart rate
in the isolated rat heart (Pagala et al., 1994).
The application of ethanol following a
two-hour exposure to melatonin was shown to
cause a greater decrease in heart rate than either
ethanol or melatonin alone. If melatonin causes

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Effects of Melatonin and Ethanol on the Heart Rate of Daphnia magna
2009

an increase in cholinergic activity, and ethanol


inhibits the release of ACh, these two drugs
would be expected to have antagonistic effects.
Due to their opposing effects on the cholinergic
system, these results suggest that ethanol and
melatonin act through different mechanisms to
influence the activity of the D. magna heart.
The high concentration of ethanol may have
severely impaired the myogenic cells of the
heart, and pre-treatment with melatonin may
have altered the activity of the cholinergic
neurons such that their normal regulatory
activity was hindered, causing a more severe
depression of the heart rate. According to this
mechanism, melatonin may make it more
difficult for the regulatory cholinergic system to
respond appropriately to stress on the heart (such
as the administration of an acute dose of
alcohol). Alternatively, the combined effect of
alcohol and melatonin could be the result of
different actions on the cholinergic system.
Melatonin may increase the release of ACh,
while ethanol may alter the sensitivity of
AChRs. According to this mechanism, ethanol
would enhance the response of the D. magna
heart to ACh, which is elevated by melatonin, to
cause the enhanced depression of heart rate. A
possible limitation to this study involved the use
of petroleum jelly to immobilize the fleas so that
heart rate could be counted. Although the heart
rate was scored in this manner for all conditions,
it is possible that this technique resulted in
additional stress for the animals, which may
have impacted their response to the drugs. More
studies are needed to elucidate the mechanisms
by which these two drugs influence the heart and
nervous system of D. magna.
These results suggest that combining
ethanol and melatonin may cause a potentially
unsafe decrease in heart rate. This is of concern
due to the tendency of those with sleep disorders
to self-medicate with alcohol. Melatonin is
increasing in popular use as a natural alternative
to prescription sleep aids, and due to the lack of

strong warning labels, individuals may be less


cautious about concomitant alcohol use. More
studies are needed to investigate the effect of
these two drugs in combination on the
mammalian heart. If these additive effects prove
to be significant in higher organisms, it may be
prudent to include an alcohol warning on
melatonin supplements similar to that included
with prescription sleep aids.

Corresponding Author
Bonnie Kaas
Ursinus College
2712 Flintridge Court
Myersville, MD 21773
bokaas@gmail.com

Acknowledgements
We thank Kenton Woodard for his assistance
and guidance in experiment design and
execution. We thank Roger Coleman for his
assistance with the statistical analysis.

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