Post on 22-Jan-2018
Assessing Crayfish Heart Rate Responses After Nicotinic Receptor Stimulation by Nicotine and Lobeline
Lukas Isenhart, Brittany Files, Cody Costley, Niariah Fields Birmingham-Southern College
Department of Biology
12 May, 2016
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Abstract
Crayfish were administered 0.05mL dosages of 3μM nicotine [(S)-3-[1-Methylpyrrolidin-2-yl]pyridine] and lobeline [2-((2R,6S)-6-((S)-2-Hydroxy-2-phenylethyl)-1-
methylpiperidin-2-yl)-1-phenylethanone] to determine effects on heart rate as a method for comparing cardiac stress. This would suggest lobeline’s possible use as a nicotine replacement therapy (NLT) in humans. Data proved insignificant for experimental groups
of three crayfish per drug. Further study is needed to determine the viability of lobeline as an effective nicotinic-cholinergic competitive agonist for nicotine in nicotinic receptors,
which would prove beneficial to cardiovascular medicine and its treatment of nicotine and other drug addictions.
1 Introduction
The nonscientific community often directly associates nicotine with cancer and
other negative physiological effects because it is the main psychoactive and addictive
ingredient in cigar and cigarette smoke, hookah and e-cigarette vapor, snuff, and other
various tobacco products. As a stimulant, high doses of nicotine can prove lethal, but the
nicotine concentration necessary to reach this isn’t possible with commonly available
tobacco products like cigarettes or snuff (Brandon, et al. 2015). Use of nicotine during
pregnancy is associated with a fetus’s future respiratory dysfunction, obesity,
hypertension, and neurobehavioral defects as well as increased spontaneous abortions,
premature delivery rates, and decreased final trimester birth weights (Schraufnagel, et
al.). Nicotine has demonstrated an in-vitro association to cancer, but nicotine’s
carcinogenic effects have yet to be demonstrated in-vivo, suggesting that common
nicotine ingestion methods have little effect on future cancer development (Hass and
Kuebler 1997). Nicotine use in pharmacological treatments for developed adults is
relatively risk-free to overall health: it can be said that the additional chemical
compounds found in nicotine products – such as carbon monoxide and the resulting gases
of combusted pesticides, herbicides, and fungicides, among others – pose a higher risk to
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users. However, nicotine poses a threat to long-term cardiovascular health in adults by
increasing heart rate, myocardial contractility, and blood pressure, which inadvertently
lead to increased likelihood of heart attacks, angina, and strokes (Haass and Kuebler
1997).
Nicotine is an alkaloid commonly found in cultivated Nicotiana tabacum, which,
when ingested, leads to reward-motivated addictive behavior as a result of dopamine
release inflection and increased concentrations of extracellular dopamine near brain
reward systems (Goutier, et al. 2016). Nicotine interacts with cholinergic receptors,
including multiple nicotinic acetylcholine receptors that are found throughout the human
body, but their interaction within the brain and central nervous system suggests nicotine’s
importance as an agonist. Due to nicotine’s agonist characteristics on nicotinic receptors,
it is possible to employ chemicals of similar structure for use as competitive nicotine
blockers. This led to research into chemicals like lobeline, which has a high binding
affinity to the nicotinic cholinergic receptors and was considered useful for treating
nicotine addiction (Stead and Hughes 2012).
Lobeline is an alkaloid found in Lobelia inflata, and, like nicotine, modulates
dopamine release and increases extracellular dopamine concentrations in reward centers
of the brain via its agonistic relationship with nicotinic receptors (Buchhalter, et al.
2008). Because of this, lobeline was used as a smoking cessation aid and nicotine
replacement therapy (NRT) until 1993, when the FDA found that lobeline lacked
sufficient efficacy in antismoking treatments (Stead and Hughes 2012). This is largely
due to improper use of controls in multiple studies, but also because the effective dose of
lobeline is nearly toxic to humans. Despite this, lobeline still has applications in
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respiratory health and there has been a revival of studies that delve into its use as an
addiction treatment for nicotine, amphetamines, alchohol, and cocaine because of its
effects on dopamine reuptake.
Because lobeline has potential to be used as an NRT, it is important to analyze its
efficacy in model organisms to suggest a reason for its further application to human
physiology. In rat models, lobeline has a significant impact on quelling
methamphetamine- induced behavior by limiting dopamine reuptake and by increasing
dopamine release from presynaptic storage vesicles (Dwoskin, Linda, and Crooks 2002).
Also in rats, lobeline displays similar cognitive effects to nicotine in terms of memory
retention (Decker, Majchrzak, and Arnerić 1993). Nicotine performs similarly to lobeline,
particularly in crayfish where nicotine significantly increased chemosensitivity (He,
Tucket, and English 1999).
Little is known about nicotine and lobeline’s effect on the heart rates of crayfish,
and therefore this study is important to the scientific community and possibly to the
cardiovascular realm of human medicine in providing a more effective method of
pharmacologic treatment for nicotine addiction. This study treated crayfish with lobeline
and nicotine solutions and used initial and resulting heart rate in beats per minute (bpm)
as a method for comparing the vascular health of the crayfish. Crayfish were used for
their simplistic heart anatomy as well was for ease of electrode implantation to measure
changes in heart rate. Because cardiovascular stress can lead to multiple long-term
conditions in smokers, it is essential to use model organisms like crayfish to compare the
cardiac stresses associated with nicotine or lobeline use. It was hypothesized that nicotine
and lobeline would have equivalent effects on heart rate due to the similarity of their
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nicotinic-antagonist properties (H0: bpmNicotine = bpmLobeline), but statistically significant
variation (Ha: bpmNicotine =/= bpmLobeline) could suggest that one drug is better suited to
addiction therapies by minimizing cardiac stress.
2 Materials and Methods
2.1 Initial Crayfish Preparation
Seven crayfish were removed from tanks of similar ion concentrations before being
wiped dry. The crayfish were then inverted and sexed by identifying longer swimmerettes
that extended upward above the back legs. Males possessed these swimmerrettes while
females did not. After sexing, the claws were glued for safety, covered with pieces of
aquarium tubing, and labeled. Crayfish were housed
in large, oxygenated beakers then covered to prevent
their escape and to minimize aggression and stress.
2.2 Preparation of Lobeline and Nicotine Doses
Using Ringer’s solution, 3 μM solutions of both
lobeline and nicotine were created and refrigerated
during storage. The solutions were gently swirled
before use.
2.3 Preparation of Impedance Converter Electrodes
The following methods follow Andrew T. Gannon’s procedure for electrode implantation
(Gannon, 2005). Two insulated copper wires, each 30cm long, were stripped at their ends
to reveal 1cm of exposed copper. A thicker gauge wire was then used as a guide to pull
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the electrode wiring through an aquarium tube so that both ends extend 6cm from the
tubing. These wires were then taped to the tubing to prevent dislocation. Two dental dam
squares, each 1cm x 1cm, were then
pierced with a syringe needle and
threaded onto each wire at one end by
inserting the wire into the needle point.
One end of each wire was then attached
to the impedance converter, which was
set to an AC Short time balance,
calibrated at 0.5Ω, and balanced in preparation for electrode implantation. Repeat for
every crayfish.
2.4 Surgical Insertion of Electrodes
Using a wooden block with four attached
metal rings, the crayfish were restrained
using rubber bands while paying
attention to not harm the eyestalk,
antennae, antennules, and rostrum. For
best restraint tightly cover the cranial
cephalothorax, chelipeds, and telson. A
dremel tool with an abrading stone
attachment was then used to pierce the
cephalothorax of the crayfish without
piercing the epidermis. While drilling, the exposed tissue will change from white to light
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red when the epidermis is reached. Holes were drilled in the left and right carapace,
laterally adjacent to the heart. A third hole was then similarly drilled caudal to the heart at
the carapace nearest the abdomen. Taking great care both to avoid harming the heart and
prevent the loss of hemolymph by piercing the pericardium or gonads, the wires were
inserted lateral to the heart. A steady reading for heart rate by the impedance converter
suggests that this is done correctly. Rebalance the impedance converter as needed. Using
minimal superglue, the wires were glued in place and covered with dental dam squares,
adding another drop of superglue to the wire above the dental dam for added security.
The caudal opening was closed by gluing the circumference of the hole and by applying a
square of dental dam.
2.5 Dosage Data Collection
Given the stressful nature of the implantation, an aggressive crayfish heart rate was
minimized by allowing the crayfish three hours of dark, covered rest before beginning the
doses. The crayfish were restrained as previously mentioned and given an additional five
minutes of covered rest to reach a submissive resting heart rate. Due to similar
favorability for nicotinic active sites, nicotine and lobeline experienced similar dosages,
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each requiring 3μM solutions and 0.05mL doses per three-minute period. These doses
were applied using a needle syringe inserted through the dental dam of the caudal
carapace. The doses accumulated over a 15-minute period to reach a total dosage of
0.25mL. This dose schedule applied for six dosing crayfish (N1, N2, N3, L1, L2, L3),
while a seventh crayfish (Test) was initially used for a dose-intensity analysis of nicotine
on heart rate, which was compared to outside data to determine the lethality of this assay.
Heart rate was analyzed using Loggerpro software and an impedance converter.
Rebalance the impedance converter as needed. The seven crayfish were subsequently
euthanized by applying a 0.3mL dosage of water saturated with potassium chloride (KCl)
through the caudal carapace to cause cardiac arrest. Using Microsoft Excel software,
lobeline and nicotine analyses were graphed along a polynomial trend line and a two-
tailed heteroscedastic test was performed, using a P-value of 0.05 to differentiate between
accepting the null or alternate hypotheses.
3 Data
Table 1. Individual and average weight of nicotine, lobeline, test, and total experimental
groups.
Identification Sex Weight (g)
Test M 33.0
N1 M 30.8
N2 F 39.3
N3 M 39.2
Nic. Average 36.4
L1 M 33.6
L2 M 41.3
L3 M 40.8
Lob. Average 38.6
Total Average 36.9
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Lobeline and nicotine doses did not account for the weights of individual crayfish,
instead relying on equivalent dosages across test subjects (Table 1.). Most crayfish
weighed over 33.0g, suggesting developmental maturity (Table 1.). There is no
significant correlation between crayfish sizes and their sex, as only one female crayfish
was tested (Table 1.).
Table 2. Total applied dosages for nicotine and lobeline experimental groups.
Time
(m)
Nicotine Dosage
(3 μM)
Lobeline Dosage
(3 μM)
0 0.00 mL 0.00 mL
3 0.05 mL 0.05 mL
6 0.10 mL 0.10 mL
9 0.15 mL 0.15 mL
12 0.20 mL 0.20 mL
15 0.25 mL 0.25 mL
Table 3. Individual heart rate responses by dosage for nicotine and lobeline crayfish groups. (N=7).
Heart Rate (bpm)
Time (m) Test N1 N2 N3 L1 L2 L3
0 73 61 67 72 52 63 56
3 204 87 79 84 114 90 82
6 x 96 96 81 120 96 85
9 x 96 99 95 114 95 106
12 x 99 92 94 102 101 99
15 x 109 95 99 108 103 101
Doses of 0.05mL of 3μM nicotine and lobeline solutions resulted from the analysis of a
test crayfish to determine the immediate effects of a 0.25mL dose of 3μM nicotine (Table
1., Table 2.). The test crayfish experienced a potentially harmful heightened heart rate
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response and abdomen hyperactivity, but heart rate normalized after 15 minutes (Table
3.).
Figure 1. Average heart rate responses at three-minute intervals due to 0.05 mL nicotine doses. Error bars denote standard deviation. (N=3).
The polynomial regression had a high coefficient of determination for nicotine (0.9593)
and displayed a relatively low peak for average heart rate at 101bpm + 7.2 (Fig. 1.)
y = -0.1798x2 + 4.7183x + 68.393R² = 0.9593
50
60
70
80
90
100
110
120
0 5 10 15 20
He
art
Ra
te (
bp
m)
Time (m)
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Figure 2. Average heart rate responses at three-minute intervals due to 0.05 mL lobeline doses. Error bars denote standard deviation. (N=3).
Compared to that of nicotine, the polynomial regression for lobeline had a lower
coefficient of determination (0.87813) and displayed a higher peak for average heart rate
at 104bpm + 3.6 (Fig. 1.) A T-test performed between the data sets resulted in a P-value
of 0.6108, supporting the null hypothesis.
4 Discussion
Given a P-value over 0.05, the null hypothesis was supported, suggesting that
lobeline-stimulated heart rate had no statistical significance in comparison to nicotine-
stimulated heart rate. Despite lobeline’s higher average heart rate peak at 104 bpm + 3.6,
lobeline did not have a greater effect on heart rate than nicotine (Fig. 2.). This suggests
that nicotine and lobeline can be used interchangeably in drug treatment therapies when
not considering other cardiological effects attributed to each drug. This said, crayfish
visibly experienced greater stress in lobeline trials, especially in regard to abdomen
shaking and quivering at higher dosages. In the test subject, where dosages were used to
determine lethality, larger upfront dosages appeared to have a much higher resulting heart
y = -0.421x2 + 8.7526x + 62.807R² = 0.8781
50
60
70
80
90
100
110
120
0 5 10 15 20
He
art
Ra
te (
bp
m)
Time (m)
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rate than in the 15-minute procedure. This study does not effectively demonstrate that one
drug is preferential for administration to crayfish and thus cannot suggest any new
association for nicotine and lobeline in terms of heart rate responses. If two identical
people were given equal doses of nicotine and lobeline they would be expected to have
the exact same responsive heart rate.
Obvious issues need to be fixed within this study, including a necessary increase
in sample size and a sample set of same-sex crayfish that are similar in size. More tests
are needed to verify these results; perhaps nicotine and lobeline have different cardiac
responses that weren’t observed within the confines of such a small data set. Additional
measures also need to be addressed as far as the handling and preparation of crayfish for
this procedure, as crayfish had a relatively high die-off rate as a result of the lateral
placement of the electrode wires, which may have pierced the pericardium in some
crayfish during implantation. These crayfish may have been too small to support the
procedure. A larger gauge electrode wire is also recommended, as the crayfish easily
pulled out their wires, leading to repeated procedures that caused unnecessary stress to
the crayfish.
5 Conclusion
When administered directly to the heart of crayfish, lobeline and nicotine did not have a
statistically significant variation in output heart rate at various dosages, suggesting no
new applicable knowledge to the realm of NRTs for people. Future trials are needed to
further support this claim.
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6 Acknowledgments
We would like to thank the Department of Biology at Birmingham-Southern College for
providing materials and laboratory space as well as Dr. Andrew T. Gannon for assistance
with his crayfish electrode implantation technique.
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