1. Introduction
The heart of Procamarus clarkii, or crayfish, is neurogenic in that the basic
heartbeat and rhythm are determined by the neural output of the cardiac ganglion (Harper
The Effect of Nicotine on the Neurogenic Heart of
Procambarus Clarkii
Abigail Tirrell, Casey Nash, Nick Bielanksi, John Jacobs
University of Notre Dame
R-AM (Matt Leming)
Abstract
Nicotine dependency is a widespread problem due to the prevalence of consumer tobacco
products. An understanding of the neurological effects of nicotine is necessary to intelligently
and effectually combat this widespread health problem. This report studies the direct effect of
nicotine on neuron firing at various nicotine concentrations in order to establish an
understanding this compound’s mechanism of action in the brain. This study was carried out
by applying nicotine solutions of various concentrations directly to the neurogenic heart of a
vivisected Louisiana crayfish (Procamarus clarkii) and measuring the change in heart rate.
The basic heartbeat and rhythm of a neurogenic heart is determined by the neural output of
the cardiac ganglion, which can be influenced by neurotransmitters such as nicotine, a
stimulant. The presence of nicotine, regardless of concentration, was found to have no
significant effect on the heart rate of crayfish. This suggests that nicotine has no direct
correlation with neuronal activity, measured by the heart rates of P. clarkii. Thus, indirect
effects of nicotine may be attributable to neurotransmitters whose release is triggered by the
activity of nicotine as an acetylcholine receptor agonist, but this report shows no direct
relationship between nicotine and neuronal activity.

2. and Reiber, 2004). The cardiac ganglia of various crustaceans, including crayfish, are
influenced by several neurotransmitters. The resting heart rate and contractile force are
set by this neural input. Hence, the beats of neurogenic hearts are modulated by
neurotransmitters. Several experiments have been performed testing the effects of various
neurotransmitters on the cardiac ganglion. For example, cardioexcitatory peptides
increase the heart’s rate and contractile force (Listerman et al., 1999). It is also known
that the neuropeptide proctolin has excitatory effects on the isolated lobster cardiac
ganglion (Miller and Sullivan, 2004).
The widespread dependency associated with nicotine use through tobacco
products prompts the study of the neurological effects of this drug. Nicotine is a stimulant
and a nicotinic acetylcholine receptor (nAChR) agonist in the brain. In humans, it has
been found to increase heart rate and blood pressure due to the stimulation of sympathetic
neurotransmission. This occurs through the activation of nicotine acetylcholine receptors
localized on the cardiac ganglia, resulting in the influx of sodium and calcium ions due to
increased cation permeability of nAChRs (Fucile 2004). The resulting depolarization of
the sympathetic nerve stimulates calcium influx through voltage dependent calcium
channels, which sensitizes cardiac sympathetic nerves to the norepinephrine-releasing
effect of nicotine, shown in a study on the guinea pig heart (Haas and Kubler, 1997).
Applying nicotine to crayfish hearts will indicate the relationship between nicotine, an
acetylcholine receptor agonist, and neural cells in humans since crayfish hearts are
neurogenic.
Nicotine concentration in cigarettes has varied over time and by brand of
cigarette. For this reason, it is important to understand how the effect of nicotine on

3. neuronal activity varies with nicotine concentration. The Louisiana crayfish (Procamarus
clarkii) has a neurogenic heart structure so crayfish heart activity is a suitable analog for
measuring neuronal activity. In this study, the measured heart activity of crayfish that
have had nicotine applied directly to their heart ganglion after vivisection will reveal the
effect of nicotine concentration on brain activity in humans. It is hypothesized that an
increased concentration of nicotine, a stimulant, will increase the neurogenic heart
activity in a crayfish.
Materials and Methods
Pure liquid nicotine was obtained and four different aqueous solutions were made
of varied concentration. Solutions of 0.01 M, 0.001 M, 0.0001 M, and 0.00001 M were
prepared.
Crayfish were held on ice for 25 minutes to anesthetize them and limit mobility
during the vivisection procedure. With scissors, a shallow cut was made just above the
claws on the underside of the crayfish to sever the thoracic ganglion. This prevented
sensation in the crayfish and stopped cardiac regulatory nerve activity. Next, the claws
and walking legs were removed and discarded. The abdomen (crayfish tail) was cut and
removed.
A shallow cut along the entire cervical groove was made; upward pressure was
maintained to prevent internal organ damage. The rostral end of the exoskeleton was
pulled forward to remove head, stomach, gut, hepatopancreas and gonads. The carapace
was held with the dorsal side up to prevent digestive enzymes from harming the heart.

4. The remaining piece of crayfish was placed in a beaker of saline solution for a minute to
make sure no blood clots formed.
The number of heartbeats was counted over a 20 second period for four control
organisms, and heartbeats per minute were calculated. This procedure was then repeated
for each specific nicotine solution concentration. A milliliter of nicotine solution was
administered drop wise directly to the still-beating heart; the heartbeats were counted
directly after application of the solution. Statistical tests, including an Analysis of
Variance test and a Tukey test, were then run to determine if there was a significant
correlation between heart rate and nicotine concentration.
Results

5. Fig. 1. Data and descriptive statistics of the heart rates of P. clarkii after nicotine
administered. The P. clarkii specimen were vivisected and nicotine of
concentrations 0 M, 0.01 M, 0.001 M, 0.0001 M, and 0.00001 M were administered to
the neurogenic hearts, and heart rate was counted and recorded for 4 trials per nicotine
concentration. A control of 0 M solution nicotine was used. The above data was
obtained with the highest average heart rate occurring after the administration of the
0.0001 M nicotine solution, and the lowest average heart rate was produced by the
0.01 M nicotine solution. The data also shows the standard deviations and sample
errors of the average heart rates in each sample.
Fig. 2. Graph of the means and standard deviations of the average heart rates of
P. clarkii administered varying concentrations of nicotine. The hearts of P. Clarkii
were administered nicotine concentrations of 0 M, 0.01 M, 0.001 M, 0.0001 M, and
0.00001 M and the heart rates were measured and averaged. This graph shows the
average heart rates of the crayfish samples at each concentration with error bars that
show the standard deviations of the means. After statistical analysis, the means were
given a p-value of 0.0198, indicating a statistical difference between at least two of the
sample means. The bars of the heart rates at each concentration do not indicate a clear
correlation between nicotine solution concentration and average heart rate.

6. To determine if nicotine, a stimulant, increases neurogenic heart activity in P.
clarkii, four concentrations of nicotine were administered to the vivisected crayfish, and
Fig. 3. ANOVA statistical analysis of data of P, Clarkii heart rates
affected by nicotine. An Analysis of Variance (ANOVA) comparison
test was used to compare the multiple sample means and variability
between the varying concentrations of nicotine applied to the hearts of
P. clarkii. Since P=0.0198 and F>0 (F=4.07), the ANOVA test
indicates that there is a significant difference between at least two of the
mean heart rates.
Fig. 4. Results of a Tukey HSD Test on the
variance of the average mean heart rates
of P. Clarkii. A Tukey HSD test was run
using the results of the ANOVA summary
test to locate the significant difference in at
least two sample mean heart rates. The
results of the test show that there is a
significant difference between the M2 (mean
2, 0.01 M nicotine) and M4 (mean 4, 0.0001
M nicotine) since P<0.05. The differences
between the remaining means were not
statistically significant.

7. the heart rates were measured. The sample size for each concentration was 4; a control of
0.00 M nicotine solution was used along with concentrations of 0.01 M, 0.001 M, 0.0001
M, and 0.00001 M nicotine solutions. Averaging the measured sample heart rates for
each trial of a different concentration of nicotine showed that the crayfish administered a
0.0001 M solution of nicotine had the highest average heart rates, while crayfish
administered a 0.01 M solution of nicotine had the lowest average heart rates. Standard
deviation and standard errors were also calculated, and showed that the heart rates due to
0.001 M solutions had the greatest variance, while heart rates affected by 0.01 M
solutions had the least variance (Figure 1). From this data, a bar graph was constructed
that shows the crayfish sample average heart rates and the standard deviations of the
means, displayed through the use of error bars (Figure 2). The error bars of the graphed
data indicate that the averages of 0.01 M and 0.0001 M solutions differ significantly,
since their error bars do not overlap. Overall, the averages in the graph do not indicate a
clear correlation between nicotine solution concentration and average heart rate of P.
clarkii.
From the calculated averages and variance, an Analysis of Variance (ANOVA)
comparison test was used to compare the means and standard deviations between the
varying concentrations of nicotine applied to the neurogenic hearts of crayfish. This one-
way ANOVA was used to compare the means of several samples and compare the
variability between samples to the variability within samples. Since the calculated P-
value is less than 0.05 (P=0.01981) and F is greater than 0, which means that the
variation among sample means is more than expected by chance, it is implied that at least
two means are significantly different (Figure 3). This P-value, being below the

8. significance level (α), was determined from the F ratio and the degrees of freedom, as
shown in Figure 3, and indicates that there is only a 5% chance or less that the means are
the same; thus the null hypothesis that the mean heart rates are the same is rejected.
To determine which two or more means differed significantly, Tukey’s HSD
statistical test was used to locate the difference. HSD, the absolute difference between
any two sample means required for significance at different significant levels, was
determined (HSD[.05]=21.14). Comparing this HSD value to the differences between the
mean heart rates led determined that the difference between heart rate due to 0.01 M
nicotine and heart rate due to 0.0001 M nicotine was statistically significant, and P<0.05
(Figure 4). This difference has a low probability (less than 5%) of being due to chance at
α=0.05. The differences between the remaining average heart rates were not statistically
significant at α=0.05. Thus, there was a statistical difference between the heart rates that
resulted from 0.01 M and 0.0001 M solutions of nicotine, but no significant difference
between any other concentrations; therefore, there was no significant correlation or trend
in heart rate versus the decreasing concentration of nicotine solution applied to the hearts
of P. clarkii.
Discussion
This study provides no evidence of a positive relationship between nicotine
concentration and the heart activity of P. clarkii. A significant difference in heart rate was
measured between solutions at 0.01 M nicotine and 0.0001 M nicotine, but higher heart
activity was measured at the lower concentration, which refutes the hypothesis. Heart
activity did not differ significantly for any other pairs of nicotine concentration (Figure

9. 4). At the concentrations measured, the presence of nicotine was not shown to have any
significant effect on heart activity. Low sample size and uncontrolled variation in the
fitness of the crayfish measured can account for the unevenness of the heart rates
measured.
In the context of this study, it is reasonable to suspect that nicotine has no direct
effect on neuronal activity. Much of the neurological research on nicotine focuses on its
role as a nAChR agonist (Pomerleau & Rosecrans 1989, Balfour & Fagerstrom 1996).
These studies report that nicotine activity is characterized by imitation of acetylcholine.
This imitation is significant because the body does not naturally regulate nicotine levels
as it does acetylcholine (Sherman 2007). High sustained levels of nicotine trigger the
release of acetylcholine, epinephrine, norepinephrine, dopamine, ß-endorphin, serotonin,
and arginine vasopressin. It is through the activity of these neurotransmitters that nicotine
is “active” (Pomerleau & Rosecrans 1989). These findings suggest that nicotine does not
need to affect neuron firing directly in order to show efficacy. Rather, neurotransmitters
whose release was triggered by nicotine may be the cause of nicotine’s effects.
Further study must be carried out to elucidate the full neurological effect of
nicotine. Increased sample size of this experiment could continue to show no significant
difference in crayfish heart activity in the presence of nicotine. To discern how nicotine
affects neuron firing, study on neurotransmitters released as a result of the presence of
nicotine (as mentioned above) is necessary. If neurotransmitters whose release is
triggered by nicotine are shown to have an effect on crayfish heart activity, then the
indirect but important role of nicotine on neurological activity could be inferred. The
manifest effect of these substances on P. clarkii heart activity would represent a more

10. complete description of the effects of nicotine, as this study suggests that nicotine has no
direct effects.
Literature Cited
Balfour DJK, Fagerstrom KO. 1996. Pharmacology of nicotine and its theraputic use
in smoking cessation and neurodegenerative disorders. Pharmacology &
Therapeutics. 71: 51-81.
Fucile S. 2004. Ca2+ permeability of nicotinic acetylcholine receptors. Cell Calcium.
35: 1-8.
Haass M, Kubler W. 1997. Nicotine and sympathetic neurotransmission.
Cardiovascular Drugs Therapy. 10: 657-665.
Harper S, Reiber C. 2004. Physiological Development of the Embryonic and Larval
Crayfish Heart. The Biological Bulletin. 206: 78-86.
Listerman L, Deskins J, Bradacs H, Cooper R. 1999. Heart rate within male crayfish:
social interactions and effects of 5-HT. Comparative Biochemistry and
Physiology Part A: Molecular & Integrative Physiology. 125: 251-263.
Miller M, Sullivan R. 2004. Some effects of proctolin on the cardiac ganglion of the
maine lobster, Homarus americanus (Milne Edwards). Journal of Neurobiology.
12: 629-639.
Pomerleau OF, Rosecrans J. 1989. Neuroregulatory effects of nicotine.
Psychoneuroendocrinology. 14: 407-423.
Sherman C. 2007. Impacts of Drugs on Neurotransmission.
<http://http://www.drugabuse.gov/news-events/nida-notes/2007/10/impacts-
drugs-neurotransmission>. Accessed 2014 April 10.