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Theses and Dissertations
8-2015
The Role of Zinc in the Regulation of Ultradian
Rhythms in Paramecium Tetraurelia
Brianne J. Seitz
Indiana University of Pennsylvania
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THE ROLE OF ZINC IN THE REGULATION OF ULTRADIAN RHYTHMS
IN PARAMECIUM TETRAURELIA
A Thesis
Submitted to the School of Graduate Studies and Research
in Partial Fulfillment of the
Requirements for the Degree
Master of Science
Brianne J. Seitz
Indiana University of Pennsylvania
August 2015
© 2015 Brianne J. Seitz
All Rights Reserved
ii
Indiana University of Pennsylvania
School of Graduate Studies and Research
Department of Biology
We hereby approve the thesis of
Brianne J. Seitz
Candidate for the degree of Master of Science
6/25/15
_____________________________________________________________________________________________________________
6/25/15
_____________________________________________________________________________________________________________
6/25/15
_____________________________________________________________________________________________________________
Signature on File
Robert Hinrichsen, Ph.D.
Associate Professor of Biology, Advisor
________________________________________________________________________________________________________________________________________________________________________________________________________________________
Signature on File
Robert Major, Ph.D.
Assistant Professor of Biology
____________________________________________________________________________________________________________________________________________________________________________________________________________________________
Signature on File
Cuong Diep, Ph.D.
Assistant Professor of Biology
___________________________________________________________________________________________________________________________________________________________________________________________________________________________
ACCEPTED
Signature on File
Randy L. Martin, Ph.D.
Dean
School of Graduate Studies and Research
___________________________________________________________________________________________________________________________________________________________________________________________________________
__________________________________________________________________________________________________________________
iii
Title: The Role of Zinc in the Regulation of Ultradian Rhythms in Paramecium tetraurelia
Author: Brianne J. Seitz
Thesis Chair: Dr. Robert Hinrichsen
Thesis Committee Members: Dr. Robert Major
Dr. Cuong Diep
Paramecium tetraurelia exhibit spontaneous behavioral responses, which operate under
an ultradian rhythm with a periodicity of 45-60 minutes 3,4. There was evidence that zinc plays a
role in the control of this rhythm. The goal of this study was to determine how zinc
concentrations affect the ultradian rhythm. Zinc was added to the cells’ media to increase the
concentration, and TPEN, a zinc chelator, was added to the media to lower the zinc
concentration. Results showed that increasing zinc has no effect on the ultradian rhythm, but
decreasing zinc eliminated the rhythm. When zinc and TPEN were added simultaneously, the
zinc overcame TPEN’s effects. Finally, the TPEN’s effect was not long-term, as removal from
TPEN allowed the cell to return to its normal ultradian rhythm. These results indicate that zinc is
necessary for the maintenance of the ultradian rhythm of spontaneous behavioral responses in
Paramecium tetraurelia, a novel finding.
iv
ACKNOWLEDGEMENTS
First, I would like to thank my family, especially my parents, Paul and Mary Seitz, who
have always pushed me to do my best, who never allowed me to give up on my goals, and who
believe that I can accomplish anything. I also want to thank my siblings, Adam, Laura, and
Meghan Seitz, who will always be my best friends and who keep me smiling and laughing every
time they are around. You all inspire me to do the very best I can.
Next, I would like to thank my thesis committee, Dr. Hinrichsen, Dr. Major, and Dr.
Diep, for sharing their extensive knowledge and enthusiasm, not only about my research but also
about all topics in Biology. A special thank you to Dr. Hinrichsen, who gave me great guidance
on this project, who was accommodating to my schedule, and who was always available to help.
Finally, I would like to thank my fiancé, Zachary, for being there for me throughout this
whole process. You have always encouraged me when I became discouraged, listened intently
when I needed to talk, and celebrated my accomplishments with much enthusiasm. I love you.
v
TABLE OF CONTENTS
Chapter
I
Page
INTRODUCTION .............................................................................................................. 1
Statement of the Problem .................................................................................................... 1
II
LITERATURE REVIEW ................................................................................................... 3
Paramecium Biology Overview .......................................................................................... 3
Cilia ..................................................................................................................................... 4
Swimming ........................................................................................................................... 5
Action Potentials ................................................................................................................. 5
Ion Channels and Currents .................................................................................................. 6
Circadian Rhythms.............................................................................................................. 7
Ultradian Rhythms .............................................................................................................. 8
Infradian Rhythms .............................................................................................................. 9
Neurobiology of Zinc .......................................................................................................... 9
III
MATERIALS AND METHODS ...................................................................................... 12
Cells and Growth Conditions ............................................................................................ 12
Outside Data...................................................................................................................... 12
Measuring Zinc Toxicity................................................................................................... 12
Membrane Depolarization ................................................................................................ 13
Recording Behavioral Responses ..................................................................................... 13
Adding Zinc to the Media ................................................................................................. 14
Adding TPEN to the Media .............................................................................................. 15
Timing of TPEN’s Effects ................................................................................................ 15
TPEN + Zinc ..................................................................................................................... 15
TPEN + Calcium ............................................................................................................... 16
Reversibility of TPEN....................................................................................................... 16
Measuring the Periodicity of the Ultradian Clock ............................................................ 17
Statistical Tests ................................................................................................................. 18
IV
RESULTS ......................................................................................................................... 19
Control Data ...................................................................................................................... 19
Measuring Zinc Toxicity................................................................................................... 19
Membrane Depolarization ................................................................................................ 20
TPEN Toxicity Data ......................................................................................................... 21
Effects of Added Zinc on the Ultradian Clock ................................................................. 22
vi
Chapter
Page
Effects of TPEN on the Ultradian Clock .......................................................................... 23
Timing of TPEN’s Effects ................................................................................................ 27
Cancelling Effects of Zinc on TPEN ................................................................................ 28
Adding Calcium Does Not Change TPEN’s Effects ........................................................ 29
Reversibility of TPEN....................................................................................................... 31
V
DISCUSSION ................................................................................................................... 33
REFERENCES ............................................................................................................................. 38
APPENDICES...............................................................................................................................41
Appendix A - 0.1 mM Zinc Graphs .................................................................................. 41
Appendix B - 20 µM TPEN Graphs ................................................................................. 43
Appendix C - 10 µM TPEN Graphs ................................................................................. 46
Appendix D - 5 µM TPEN Graphs ................................................................................... 48
Appendix E - TPEN Timing Graphs ................................................................................. 50
Appendix F - TPEN + Zinc Graphs .................................................................................. 52
Appendix G - TPEN + Calcium Graphs ........................................................................... 55
Appendix H - TPEN Reversal Graphs .............................................................................. 58
vii
LIST OF TABLES
Table
Page
1
Cell Viability in Zinc Acetate ............................................................................................ 20
2
Cell Viability in Media Containing Different Concentrations of KCl ............................... 21
3
TPEN Toxicity Data .......................................................................................................... 22
viii
LIST OF FIGURES
Figure
Page
1
Slide preparation for video recording ............................................................................... 14
2
A sine curve that shows a classic biological rhythm ........................................................ 17
3
Control data graph............................................................................................................. 19
4
0.1 mM zinc acetate average graph................................................................................... 23
5
20 µM TPEN average graph ............................................................................................. 25
6
10 µM TPEN average graph ............................................................................................. 26
7
5 µM TPEN average graph ............................................................................................... 27
8
TPEN timing average graph.............................................................................................. 28
9
20 µM TPEN + 0.1 mM zinc acetate average graph ........................................................ 29
10
20 µM TPEN + 0.01 mM calcium average graph............................................................. 30
11
TPEN reversibility average graph ..................................................................................... 32
12
Hypothesis that explains the biochemical pathway that produces an ultradian rhythm
in Paramecium .................................................................................................................. 36
13
Hypothesis that predicts how zinc depletion eliminates an ultradian rhythm .................. 36
ix
CHAPTER I
INTRODUCTION
Statement of the Problem
Paramecium tetraurelia is a ciliated protozoan that has been widely used to perform
behavioral studies. Paramecium is used as a study organism due to its large size, the ability to be
genetically manipulated, and the presence of numerous behavioral mutants 1. In particular,
paramecium is an ideal organism for the study of the genetic control of biological rhythms. A
paramecium exhibits backwards swimming behavior, an indication of an action potential
occurring, and this behavior is known to operate under particular biological rhythms.
The circadian rhythm is the most recognized type of biological rhythm. A biological
process is said to be under the influence of a circadian rhythm when the period of the rhythm is
about 24 hours. A related biological rhythm is the ultradian rhythm, which has a period of
rhythm that is less than 24 hours. Ultradian clocks seem to be involved in spontaneous
oscillations within individual cells. The overall significance of ultradian rhythms is unknown, but
it is believed that biochemical and physiological processes of a cell must have temporal
organization in order to overcome fluctuations in its environment. Therefore, ultradian rhythms
may have been the evolutionary forerunner to the circadian rhythm. In Paramecium tetraurelia,
Kippert 2 noted a 70 minute ultradian oscillation in swimming speed and time of cell division.
Evidence suggested that both of these processes are controlled by the same ultradian clock.
Finally, Hinrichsen 3,4 reported that spontaneous behavioral responses showed the characteristics
of an ultradian rhythm, occurring with a periodicity of 45-60 minutes. These spontaneous
behavioral responses were also found to operate under a circadian rhythm, showing that two
different biological processes affect one process and leading to questions about the molecular
interactions between the two biological rhythms.
The role of calcium has been widely studied in paramecium, and it is known to play a
large role in the spontaneous behavioral responses in paramecium 5. Like calcium, zinc is a
signal ion, but the roles of zinc have not been studied as much as those of calcium. In humans,
zinc moves through gated membrane channels and modulates protein function by binding to and
detaching from zinc-dependent proteins. In addition, like calcium, free zinc is toxic when it is
found in excess in bodily tissues. In humans, the biological function of zinc appears to be its
ability to modulate the excitability of the brain by affecting glutamate and GABA receptors 6.
Zinc has not been highly studied in paramecium, but because of its similarities to calcium and
ability to control the excitability of the human brain, it is possible that zinc may play a role in the
control of the ultradian rhythm of paramecium’s spontaneous behavioral responses.
2
CHAPTER II
LITERATURE REVIEW
Paramecium Biology Overview
Paramecium is a ciliated, unicellular protozoan that lives in fresh water aquatic habitats 7.
The taxonomic classification of the genus Paramecium is as follows 8:
Kingdom:
Protista
Subkingdom: Protozoa
Phylum:
Ciliophora
Class:
Oligohymenophorea
Subclass:
Hymenostomatia
Order:
Hymenostomatida
Suborder:
Peniculina
Family:
Parameciidae
Genus:
Paramecium
All species in the genus Paramecium share certain characteristics. First, all have cilia that is used
for swimming in aquatic environments and also for feeding. In order for a paramecium to swim,
the cilia beat synchronously in a direction to propel the organism. Likewise, the cilia beat in
synchrony to move food along the oral groove. The overall behavior of a paramecium is a result
of a variety of swimming activities displayed by the organism. Because of this, a paramecium is
used as a model to study excitable cells 9. When swimming forward, a paramecium’s cilia beat
3
toward the back of the cell. The cilia can rotate 180° and allow the cell to move in a backwards
direction. This backwards swimming motion is representative of an action potential occurring
within the cell 1. These action potentials can be stimulated by touch, heat, ions, and certain
organic compounds. An action potential results in an influx of calcium into the cell, causing the
cilia to rotate and beat in the opposite direction, leading to a backwards swimming movement 10.
Therefore, by counting the number of backwards swimming movements, one is also counting the
number of action potentials occurring.
Cilia
As stated, a prominent feature of a paramecium is cilia located on the outside of the cell.
Cilia are hair-like projections that are used for movement, and they are located all around the
outside of the paramecium. The cilia are arranged in longitudinal rows and beat in one direction
to propel the paramecium. During swimming, the cilia undergo a two-part movement. First, a
power stroke occurs that propels the paramecium forward. A recovery stroke follows in which
the cilia return to the starting position so that a power stroke may take place again 11. The power
stroke involves rigid, rod-like cilia that beat together in order to propel the paramecium in the
direction opposite to that of the ciliary beat. During the recovery stroke, the cilia are more
flexible and bent so that the paramecium is not propelled backwards through the media 12.
Though the cilia may appear to beat in synchrony, they actually move at slightly different
rates. They are said to beat metachronally 13. In reality when studying a single cilium, it can be
noted that that particular cilium beats slightly slower than the cilium in front of it and slightly
faster than the cilium behind it. This causes the cilia to move in a wave-like fashion when the
paramecium swims.
4
Swimming
As stated, a paramecium uses its cilia to move through a medium. It swims towards
favorable conditions and away from unfavorable ones. For example, if a paramecium detects a
food source in its environment, it will swim toward that food in order to feed. On the other hand,
if a paramecium detects a harmful substance in the environment, like a toxin, it will swim
backwards away from that toxin so that the paramecium can avoid it. Additional swimming
behavior occurs when faced with a harmful situation. Jennings 12 described it as an avoidance
reaction. After swimming into an unfavorable area, the paramecium will back away, turn to a
side, and swim forward again. If the harmful situation is still present, then the process of backing
up and approaching a new area is repeated until the paramecium is able to swim away from the
unfavorable area 14. Finally, paramecia have been known to swim at varying speeds depending
on the conditions in their environments. Numerous factors can affect swimming speed including
pH, temperature, and light. The speed of swimming in paramecia can range from 800µm/sec to 3
mm/sec 15.
Action Potentials
Action potentials in paramecia often occur in response to some external stimuli, such as
touch, heat, ions, and some organic compounds. When an action potential is generated, an influx
of calcium into the cell occurs. This influx of calcium leads to the rotation of the cilia so that
they may beat in a backwards direction 10. Therefore, as described earlier, by counting the
number of backwards swimming movements, one can also determine the number of action
potentials occurring.
Changes in the concentration of intracellular Ca2+ is regulated by the cell membrane. This
change in intracellular Ca2+ controls ciliary movement in paramecia 11. Normally, the
5
concentration of free intracellular Ca2+ is lower than the concentration of free extracellular Ca2+.
When depolarization of the cell membrane occurs, the membrane allows an influx of Ca2+ into
the cell, and the concentration of free intracellular Ca2+ increases. Again, this leads to a rotation
of the cilia allowing it to beat in a backwards direction so that the paramecium may swim
backwards. The backwards swimming motion will continue for the duration of the action
potential. When the cell membrane repolarizes, the paramecium will begin forward swimming
again.
Ion Channels and Currents
Several ion channels play a role in membrane excitability of paramecia. As discussed in
the section on action potentials, Ca2+ ion channels allow free Ca2+ to enter the cell when
depolarization occurs, leading to an action potential and backwards swimming. In addition, there
are other ion channels activated during an action potential. First, voltage-dependent K+ channels
open leading to K+ leaving the cell and leading to repolarization of the membrane 11. When
strong depolarization occurs, three ion currents can be generated. First, a Ca2+-dependent Na+
current may occur and will prolong the action potential when Na+ is present, as the Na+ will keep
the membrane depolarized. Next, a Ca2+ -dependent K+ current will lead to repolarization. Last, a
Ca2+-dependent Mg2+ current may occur, but the function of this current is unknown 16. In
addition, hyperpolarization of the membrane can generate two other currents. A voltagedependent K+ current and a Ca2+-dependent K+ current can be generated that are thought to be
involved in prolonging an action potential 16. Action potentials in paramecium occur according to
particular biological clocks, which will be discussed later. The involvement of these ions in
membrane excitability leads to the question of what other ions may be involved, including zinc,
and how they relate to the occurrence of action potentials under certain biological rhythms.
6
Circadian Rhythms
“[A] circadian rhythm is one in which the period of the rhythm is approximately 24
hours” 5. There are three criteria that a biological process must meet in order to be considered
under the influence of a circadian clock 17. First, in constant temperature and constant light or
dark conditions, there must be a persistent rhythm of about 24 hours. Second, there must be
temperature compensation. The periodicity of the rhythm must be the same at all physiological
temperatures. Finally, the internal circadian rhythm of about 24 hours must be entrainable to
external cues like light-dark cycles, temperature cycles, or other stimuli.
An example of a circadian rhythm in Paramecium tetraurelia involves sensitivity to
cadmium. Cadmium is lethal to Paramecium tetraurelia 18. Moreover, the sensitivity to cadmium
operates on a circadian clock. Paramecium tetraurelia are much more sensitive to cadmium in
the subjective morning that in the subjective late afternoon and evening. This rhythm displayed
all three criteria to be considered a circadian rhythm: temperature compensation, endogenous
rhythm in constant conditions, and resetting of the clock by pulses of light.
Paramecium tetraurelia also display a circadian rhythm in relation to the maximum
number of behavioral responses per minute 19. The maximum number of behavioral responses, in
other words action potentials, per minute changes over a 24-hour period. There is regular
oscillation in the frequency of responses per minute that exhibits an approximately 60 minute
periodicity. During the night, the maximum number of responses increases compared to the day.
This behavioral response pattern also operates on an ultradian clock, which will be discussed
next.
7
Ultradian Rhythms
An ultradian rhythm has a period of much less than 24 hours and also displays
temperature compensation 20. Ultradian rhythms seem to be involved in spontaneous oscillations
within individual cells. They are also regarded as the repetition of physiological events over a
24-hour period 20. The significance of ultradian rhythms has not yet been explained. However, it
is thought that in order for a cell to overcome random fluctuations within its internal and external
environments, that cell’s biochemical and physiological processes must have temporal
organization 21. In addition, Klevecz and Li 22 proposed that ultradian rhythms may have been
evolutionary forerunners to the longer circadian rhythms. Lloyd 23 also suggested that ultradian
rhythms could play a role in signaling, optimizing responsiveness, and spatial and temporal
organization. In most cases it is likely that independent ultradian rhythms regulate numerous
biochemical pathways.
In addition to the circadian rhythm of action potentials in Paramecium tetraurelia, those
action potentials also operate under an ultradian clock. Hinrichsen 4 was the first to report the
observation of an ultradian rhythm in the frequency of action potentials in Paramecium
tetraurelia. The spontaneous behavioral responses that occur due to the action potentials showed
a periodicity of 45-60 minutes. The periodicity remained constant over a range of temperatures,
meaning it displayed temperature compensation. This ultradian rhythm occurs within the
circadian rhythm. Over a 24 hour period, there is a regular oscillation in the frequency of
spontaneous behavioral responses per minute, with a periodicity of about 60 minutes. In addition,
the maximum number of behavioral responses increases during the night. Therefore, in the case
of Paramecium tetraurelia, both a circadian clock and ultradian clock affect the rhythm of
spontaneous behavioral responses.
8
Infradian Rhythms
Infradian rhythms occur with a periodicity of greater than 24 hours 17. One common
example of an infradian rhythm is the female menstrual period, which occurs on an approximate
28 day period 24. Infradian rhythms may be influenced by neurochemical rhythms or by
environmental cues. There are some seasonal rhythms that will still occur under constant
conditions and that display temperature compensation, indicating and endogenous clock 25. The
molecular mechanisms that control infradian rhythms are not well-studied. However, it has been
demonstrated that infradian rhythms do not rely on an active circadian clock 26.
Neurobiology of Zinc
Zinc is a metallic chemical element with an atomic number of 30. Recently, the role of
zinc in neurobiology has been under investigation. Zinc is a micronutrient, a component of
proteins, and an ionic signal. Zn2+ is stored in and released from presynaptic vesicles in neurons
that can be found in the cerebral cortex of mammalian brains 6. These neurons also release
glutamate, a neurotransmitter; therefore, these neurons have been termed “gluzinergic” 27,28. As
an ionic signal, zinc appears to modulate the overall excitability of the brain by affecting
glutamate and possibly γ-aminobutyric acid (GABA) receptors and may affect synaptic
plasticity 29. Zinc moves from presynaptic terminals into postsynaptic terminals through zincpermeable, gated channels 6. The zinc-permeable channels can be opened by both glutamate and
depolarization of the presynaptic neuron, which means the highest amount of zinc translocation
would occur during intense neuronal activity 30,31.
Because zinc is an ion, not a molecule, no synthetic or metabolic enzymes are available
that can inhibit or stimulate zinc release 6. There are amino acid receptors for zinc that have an
excitatory effect on the brain. First, NR2A, a subunit of the glutamate receptor NMDA, has a
9
high-affinity binding site for zinc. This site is normally partially occupied by zinc, depressing the
NMDA channel current and keeping the extracellular pZn between 8 and 9 32. When a zinc
chelator is introduced and extracellular pZn goes above 10, excitability of the brain tissue is
increased and the threshold for seizure induction is lowered 32,33. Additionally, zinc can cause a
delayed increase in NMDA receptor sensitivity to agonists 34. This indicates that, under normal
circumstances, the presence of zinc decreases brain excitability.
In addition to excitatory amino acid receptors, zinc also has the potential to bind to
inhibitory amino acid receptors. GABAA receptors are zinc-sensitive 35. It is believed that zinc
plays a vital role in brain function since there are neurons along the spinal cord that release
GABA along with zinc 36. In some studies, the growth of zinc-releasing axons in abnormal
locations can result in the ectopic release of zinc, which reduces GABAA receptor-mediated
inhibition and enhances seizure susceptibility, meaning zinc acts to inhibit the effects of
GABAA thereby increasing brain excitability37.
Because zinc acts on both excitatory and inhibitory receptors, zinc would be expected to
make the forebrain neurons more excitable, less excitable, or have no net effect 6. However, this
is not necessarily true. When zinc chelators are administered, epileptiform brain activity, lowered
threshold for seizure induction, or increased excitatory postsynaptic potentials (EPSPs) or
excitatory postsynaptic currents (EPCPs) at NMDA receptor synapses may all occur. This
indicates that the dominant action of Zn2+ on the brain is to reduce excitability and act as an
anticonvulsant. On the other hand, intracranial administration of zinc salts is proconvulsive 38.
Finally, zinc may play an important role in synaptic plasticity. In particular, “glutamateand zinc-releasing synapses might have a special role in the synaptic plasticity that underlies
learning and memory” 39,40. Plasticity of brains in mammalian young is commonly accompanied
10
by changes in innervation by zinc-containing neurons 6. Studies testing the role of glutamate- and
zinc-releasing synapses in experiential plasticity have yielded mixed results. Therefore, further
work on this particular subject is required.
Overall, little is known about what roles zinc plays in the human body as well as in other
organisms. In the future, it is important to understand not only what zinc does but also how it
affects different biochemical pathways as a signaling molecule. With new technologies and
techniques being developed, it will be possible to further study the neurobiology of zinc.
11
CHAPTER III
MATERIALS AND METHODS
Cells and Growth Conditions
All experiments were performed with Paramecium tetraurelia strain 51s (wild type). The
cells were grown in a wheat grass medium that was inoculated with Klebsiella aerogenes as a
food source (Hinrichsen et al., 2012). Unless otherwise noted, the cells were grown at 26C in a
14 hour light, 10 hour dark regiment. In experiments where the cells were grown in zinc acetate or
KCl, aliquots from stock solutions of 1 M zinc acetate and 1 M KCl were added directly to the
wheat grass media prior to the addition of the cells, and then treated the same as cells grown in
regular growth medium.
Outside Data
Control data and TPEN toxicity data were obtained from Hasawa’s 41 thesis research. The
control data was generated by video recording a single Paramecium for 3 hours in media
containing only wheat grass. Control data was used to perform t tests in order to compare the
periodicity (τ) of control data to the τ of the data generated from the following experiments.
TPEN toxicity data was obtained by putting a few Paramecium in media containing various
concentrations of TPEN, including 50 µM, 40 µM, 30 µM, 20 µM, 10 µM, 5 µM, and 2.5 µM.
The TPEN toxicity data was used to determine the maximum concentration of TPEN to be used
in the media.
Measuring Zinc Toxicity
The toxicity of zinc acetate was measured by placing individual cells into depression
wells that contained various concentrations of zinc in the wheat grass medium. The goal was to
determine the maximum concentration of zinc that would support P. tetraurelia. The
12
concentrations of zinc tested were 100 mM, 10 mM, 1 mM, 0.1 mM, and 0.01 mM. In addition, a
well with wheat grass only was used as a control. 100 µl of the medium of varying
concentrations was placed in each depression well along with 3-5 cells. The cells were placed at
26 C and were observed immediately, at 15 minutes, 30 minutes, 1 hour, and 24 hours. Cells
were scored as dead if there was no cellular movement over a five minute period. This process
was done twice.
Membrane Depolarization
KCl was added to the zinc acetate and wheat grass medium to depolarize the cells’
membranes. Depolarizing the membranes could cause zinc to have a different effect on the
ultradian rhythm since action potentials should occur more often when the membrane is more
depolarized. 40 µl, 20 µl, 10 µl, and 5 µl of KCl were added to 1.0 µl of 1.0 mM zinc and
varying amounts of wheat grass to obtain the needed concentrations. The concentrations tested
were 40 mM, 20 mM, 10 mM, and 5 mM KCl. Like the zinc toxicity process, 100 µl of each
solution was put into a depression well, and 3-5 cells were then added. The cells were observed
immediately, at 15 minutes, 30 minutes, 1 hour, and 24 hours to determine the toxicity of each
concentration of KCl. Cells were scored as dead if there was no cellular movement over a five
minute period. This process was done three times.
Recording Behavioral Responses
Avoiding reactions in Paramecium are classified as brief (less than 1 second) bouts of
backward swimming that cells frequently display in growth medium. In order to measure the
number of avoiding reactions for an individual cell over time, a single Paramecium was isolated
into a small drop of medium on a microscope slide. The drop was covered with a glass coverslip
that was positioned such that the cell was free to swim in the medium. The coverslip was
13
bordered with petroleum jelly to prevent evaporation of the drop (Fig. 1). The slide was
positioned under a dissecting microscope that was mounted with a video camera, and the cell
movements were recorded onto a video recorder over a several hour period. The videotape was
analyzed by counting the number of bouts of brief backward swimming episodes (avoiding
reactions) that were executed during a 1 minute time span; this was repeated every 5-10 minutes
over a 3 hour period of time.
Glass slide
Filter paper
Drop of media containing
Paramecium
Glass cover slip
Figure 1 Slide preparation for video recording
Adding Zinc to the Media
For the first video recording experiment, 50 µl of the 0.1 mM zinc acetate media from the
zinc toxicity experiment was put into a depression well. A Pasteur pipette was used to add 2-3
drops of cells to the well. The cells were allowed to sit for 15 minutes. Then, one cell was
isolated on the microscope slide and prepared for video recording as described. Three recordings
were taken.
14
Adding TPEN to the Media
The second condition recorded involved adding cells to varying concentrations of TPEN.
The TPEN media was made by adding 50 mg of TPEN to 10 µl ETOH to get 11.8 mM TPEN. 1
µl of this TPEN was then added to 700 µl wheat grass to get 20 µM TPEN media. A high
concentration of TPEN should have a more significant impact on the ultradian clock than lower
concentrations, so two more concentrations of TPEN were also tested. 10 µM TPEN was made
by adding 1 µl of the 11.8 mM TPEN to 1.4 ml wheat grass. Finally, 5 µM TPEN was made with
1 µl of the 11.8 mM TPEN added to 2.8 ml wheat grass. To set up for the video recordings, a
Pasteur pipette was again used to add 2-3 drops of cells to a depression well with 50 µl of one of
the concentrations of TPEN. The cells were allowed to sit for 15 minutes before one cell was
isolated on a microscope slide, and the recordings were taken. Four trials were done with the 20
µM TPEN media, while two trials each were done for the 10 µM and 5 µM TPEN media.
Timing of TPEN’s Effects
TPEN did not affect the Paramecium immediately after coming in contact with the cells.
Therefore, a timing experiment was necessary to determine how long TPEN takes to take effect.
For this process, 2-3 drops of cells were added to 50 µl of the 20 µM TPEN using a Pasteur
pipette. One cell was immediately isolated on a microscope slide for the video recording, and the
recording was started within minutes of the cells being added to the TPEN. Four recordings were
taken, and those recordings were analyzed to determine how long it took for TPEN to affect the
ultradian rhythm of the Paramecium.
TPEN + Zinc
Since TPEN and zinc are believed to have opposing effects on the ultradian rhythm of the
Paramecium, if both are added to the same depression well then their effects should cancel each
15
other out. To test this, equal parts of 20 µM TPEN and 0.1 mM zinc acetate media were added to
one depression well. For this experiment, 20 µl of each were used. After both were added to the
well, a Pasteur pipette was used to add 2-3 drops of cells to the well, and the cells were allowed
to sit for 15 minutes. One cell was isolated on a microscope slide for the video recording. This
test was run twice.
TPEN + Calcium
Calcium has a lower binding affinity for TPEN than zinc does. Calcium was added to the
TPEN solution in order to determine if it could cancel out the effects of TPEN on the ultradian
rhythm and to compare its effects on TPEN to the effects that added zinc has on TPEN. 1 mM
calcium was diluted with wheat grass to achieve 0.1 mM and a 0.01 mM calcium media. Three
concentrations of calcium were added to 20 µM TPEN; the concentrations of calcium tested
were 1.0 mM, 0.1 mM, and 0.01 mM. For each concentration, equal parts calcium and TPEN
were added to a depression well. In this experiment, 20 µl of each were used. After the calcium
and TPEN were mixed, a Pasteur pipette was used to add 2-3 drops of cells. The cells were
allowed to sit for 15 minutes in the solution. One cell was then isolated on a microscope slide for
the video recording. The 1.0 mM and 0.1 mM calcium media were not recorded, which will be
discussed later. The 0.01 mM calcium media was recorded four times.
Reversibility of TPEN
The effects of TPEN on the ultradian rhythm should be reversible when the Paramecium
are removed from a TPEN solution. In order to test this, 2-3 drops of cells from a Pasteur pipette
were put into 50 µl of 20 µM TPEN in a depression well. The cells were allowed to sit for 45
minutes so that the effects of TPEN could occur. After 45 minutes, some cells were transferred to
a wheat grass medium. Following the transfer, one cell was isolated on a microscope slide for
16
video recording. Four recordings were done, and the recordings were analyzed to determine if
the ultradian rhythm of the Paramecium would return to normal after being removed from TPEN
and how long it took for that reversal to take effect.
Measuring the Periodicity of the Ultradian Clock
For the determination of the periodicity of the ultradian rhythm, a single period (tau) was
defined as the interval from one maximum peak of a cycle to the maximum peak of the next
cycle. The periodicity was measured directly from the recorded graphs of the behavioral data.
The time between peaks of maximal activity was designated tau () (Fig. 2). The calculated tau
from several experiments was then averaged to provide the final value for the periodicity of the
ultradian clock. Graphs were also generated for each experiment using Microsoft Excel. An
average graph was then generated by aligning the graphs for each trial and calculating the
average number of avoidance reactions for each time point. Those averages were then plotted on
a graph to show the average periodicity for each experiment. All trials were done for 180
minutes; however, some average graphs will show less than 180 minutes due to losing some time
points when aligning the graphs from multiple trials. In addition, the standard deviation for each
time point was calculated using Microsoft Excel and shown on the graph as error bars.
τ
Figure 2 A sine curve that shows a classic biological rhythm. Tau (τ) represents the periodicity of
that rhythm
17
Statistical Tests
Three statistical tests were run: standard deviation, F tests, and t tests. Standard deviation
was calculated in Microsoft Excel and was shown on the averaged graphs as error bars for each
experiment. F tests were run on the data to determine if the variances of two datasets were equal
or unequal. The variances of all datasets were equal. The t tests were also calculated using
Microsoft Excel. Each t test was run as a two-tailed, two-sample test with equal variances. Five t
tests were run. The first compared the control data to the data generated when 0.1 mM zinc was
added. The second compared the control data to the 10 µM TPEN data. The third compared
control data to the 5 µM TPEN data. The fourth t test compared the control data to the 20 µM
TPEN + 0.1 mM zinc data. Last, the 10 µM TPEN data was compared to the 5 µM TPEN data.
The p value was generated for each comparison, with a value of <0.05 considered significant.
18
CHAPTER IV
RESULTS
Control Data
Control data obtained from Hasawa 41 was graphed using Microsoft Excel. These
Paramecium were recorded swimming in media that contained only wheat grass. A single trial
was run. The periodicity (τ) ranged from 45-55 minutes, and the mean τ for the control was
51.67 minutes (Fig. 3).
Figure 3 Control data graph. The number of avoidance reactions (AR’s) a single cell performed
in media containing only wheat grass. The cell was recorded for 180 minutes, and the number of
AR’s were counted for 1 minute every 5 minutes. The average periodicity (τ) was 51.67 minutes
Measuring Zinc Toxicity
In order to ascertain the maximal amount of zinc that could be utilized in the study, it was
necessary to do a dosage curve for survival of Paramecium in zinc. Two trials were run to
determine the maximum concentration of zinc that could maintain life of the Paramecium over a
19
24 hour period. For both trials, 100 mM and 10 mM zinc acetate supported no life past the 15
minute mark. The remaining concentrations, 1 mM, 0.1 mM, 0.01 mM, and 0 mM (all wheat
grass), all supported life up to the 1 hour mark. At 24 hours, 0.1 mM and 0.01 mM zinc acetate
supported living Paramecium (Table 1). Therefore, 0.1 mM zinc acetate was the maximum
concentration that supported life over the 24 hour period.
Table 1 Cell viability in zinc acetate at different time points
Zinc
Concentration
100 mM
10 mM
1 mM
0.1 mM
0.01 mM
0 mM
Time points
0 minutes
15 minutes
30 minutes
1 hour
24 hours
+
-
-
-
-
+
-
-
-
-
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+: cells alive, -: cells dead
Membrane Depolarization
There is evidence in the literature that suggests that depolarizing the membrane increases
the toxicity of zinc 42. This was tested in Paramecium by the addition of KCl, which depolarizes
the membrane. When KCl was added to the 1.0 mM zinc acetate and wheat grass media, the
toxicity of the media increased. 40 mM, 20 mM, 10 mM, and 5 mM KCl all supported life of the
20
Paramecium immediately after the addition of the cells to the media. In the first trial, none of the
concentrations of KCl supported life at the 15 minute time point. In the second and third trials,
none of the concentrations of KCl supported life at the 30 minute time point (Table 2). In the
zinc toxicity experiment, 1.0 mM zinc acetate supported Paramecium for at least one hour but
not up to 24 hours. Therefore, the addition of KCl to the 1.0 mM zinc acetate and wheat grass
media led to increased toxicity of the media. As a result, when KCl was present in the media, life
of the Paramecium could not be supported over a 24 hour period, regardless of KCl
concentration.
Table 2 Cell viability in media containing different concentrations of KCl
KCl
Concentration
40 mM
20 mM
10 mM
5 mM
Time Points
0 minutes
15 minutes
30 minutes
1 hour
24 hours
+
-
-
-
-
+
-
-
-
-
+
-/+
-
-
-
+
-/+
-
-
-
+: cells alive, -: cells dead
TPEN Toxicity Data
TPEN toxicity data was obtained from Hasawa’s thesis paper 41. The goal was to
determine the maximum concentration of TPEN that could support Paramecium tetraurelia life
over a 24 hour period. The maximum concentration of TPEN that supported life at 24 hours was
21
20 µM (Table 3). Therefore, this concentration was the maximum used in determining how
TPEN affects the ultradian rhythm in Paramecium tetraurelia.
Table 3 TPEN toxicity data from Hasawa’s thesis 41
TPEN concentration
Time Paramecium survived (hours)
50 µM
1
40 µM
2.5
30 µM
7.5
20 µM
> 24
10 µM
> 24
5 µM
> 24
2.5 µM
> 24
Effects of Added Zinc on the Ultradian Clock
In humans, zinc is known to affect the excitability of the brain, but its mechanisms are
not understood. Changes in calcium concentrations has been studied in Paramecium and is
known to change the periodicity of the ultradian rhythm. Since zinc is a signaling ion like
calcium, it is plausible that changing the concentrations of zinc may also affect the periodicity of
the ultradian rhythm. First, zinc was added to the cells’ media. For each of the three trials, a
single cell was recorded in 0.1 mM zinc acetate and analyzed over a three hour period. The
periodicity of the ultradian rhythm for each trial was determined by analyzing a graph that shows
22
the maximum peaks for each cycle. Each trial yielded three cycles with a periodicity that ranged
from 40-65 minutes. Appendix A contains the graphs from the three individual trials.
Paramecium tetraurelia isolated in 0.1 mM zinc acetate media experienced an ultradian rhythm
with an average periodicity of 53.33 minutes (Fig. 4). This was not significantly different than
the control (p=0.722). Therefore, the addition of zinc had no effect on the ultradian rhythm.
Average # of AR's for 0.1 mM Zn++
18
16
14
# of AR's
12
10
8
6
4
2
0
0
10
20
30
40
50
60
70
80
90
100 110 120 130 140 150 160 170 180
Time (min)
Figure 4 0.1 mM zinc acetate average graph. The average number of avoidance reactions (AR’s)
a single cell performed in media containing 0.1 mM zinc acetate. Three trials were performed,
and the average number of AR’s were graphed. The cells were recorded for 180 minutes each,
and the number of AR’s were counted for 1 minute every 5 minutes. The average periodicity (τ)
was 53.33 minutes
Effects of TPEN on the Ultradian Clock
After determining that zinc had no effect on Paramecium’s ultradian clock, it was
necessary to determine how removing zinc affects the periodicity of spontaneous behavioral
23
responses. In order to decrease zinc concentrations, TPEN, a zinc chelator, was added to the
media. For the 20 µM TPEN, four cells were analyzed over the three hour video recordings. The
graphs from the four individual recordings can be found in Appendix B. The 20 µM TPEN
essentially eliminated the ultradian rhythm of the Paramecium’s spontaneous behavioral
responses. There were no clear periods present in the graphs that were generated from analyzing
the video recordings (Fig. 5), so no t test was run. For the 10 µM TPEN media, two cells were
recorded. The first recording yielded five cycles over a three hour period, while the second
recording showed four cycles. Appendix C shows the graphs from the two trials. The periodicity
of the cycles ranged from 25-45 minutes. The average periodicity for the ultradian clock affected
by 10 µM TPEN was 36.11 minutes (Fig. 6). This was significantly different than the control
periodicity (p=0.006). Therefore, 10 µM TPEN still had some effect on the ultradian clock of the
spontaneous behavioral responses, though not as dramatic as 20 µM TPEN. Last, two cells were
recorded in the 5 µM TPEN media. In each trial, two cycles were present in the three hour
recorded period. Appendix D contains the graphs from both trials. The length of these cycles
ranged from 40-65 minutes. The average periodicity was 53.75 minutes (Fig. 7). In this case, the
ultradian rhythm was within the range that Paramecium exhibit under normal conditions
(p=0.782), so the 5 µM TPEN had no effect. When comparing the 10 µM TPEN periodicity to
the 5 µM TPEN periodicity, there was a significant difference (p=0.005). The ultradian rhythm
of Paramecium in 5 µM TPEN remained within the range normally exhibited, while the
periodicity of the rhythm in 10 µM was significantly shorter than in normal conditions.
24
Average # of AR's for 20 µM TPEN
18
16
14
# of AR's
12
10
8
6
4
2
0
0
10
20
30
40
50
60
70
80
90
100 110 120 130 140 150 160 170 180
Time (min)
Figure 5 20 µM TPEN average graph. The average number of avoidance reactions (AR’s) a
single cell performed in media containing 20 µM TPEN. Four trials were performed, and the
average number of AR’s were graphed. The cells were recorded for 180 minutes each, and the
number of AR’s were counted for 1 minute every 5 minutes. There was no clear periodicity (τ)
present
25
Average # of AR's for 10 µM TPEN
18
16
14
# of AR's
12
10
8
6
4
2
0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
Time (min)
Figure 6 10 µM TPEN average graph. The average number of avoidance reactions (AR’s) a
single cell performed in media containing 10 µM TPEN. Two trials were performed, and the
average number of AR’s were graphed. The cells were recorded for 180 minutes each, and the
number of AR’s were counted for 1 minute every 5 minutes. Only 160 minutes are shown after
aligning multiple graphs. The average periodicity (τ) was 36.11 minutes
26
Average # of AR's for 5 µM TPEN
20
18
16
# of AR's
14
12
10
8
6
4
2
0
0
10
20
30
40
50
60
70
80
90
100 110 120 130 140 150 160 170 180
Time (min)
Figure 7 5 µM TPEN average graph. The average number of avoidance reactions (AR’s) a single
cell performed in media containing 5 µM TPEN. Two trials were performed, and the average
number of AR’s were graphed. The cells were recorded for 180 minutes each, and the number of
AR’s were counted for 1 minute every 5 minutes. The average periodicity (τ) was 53.75 minutes
Timing of TPEN’s Effects
While analyzing the cells that were allowed to sit in 20 µM TPEN before recording, it
was noted that there was a delay before the TPEN took effect. Cells were immediately recorded
after being added to TPEN in order to determine the amount of time TPEN took to change the
ultradian clock. The cells were tested in 20 µM, since that concentration eliminated the ultradian
rhythm. Four trials were done in the 20 µM TPEN, and the time it took for TPEN to affect the
cells ranged from 85-110 minutes. Each of the graphs from the four trials can be found in
Appendix E. The average time for 20 µM TPEN’s effects to begin was 95 minutes (Fig. 8).
27
TPEN Timing Average
20
18
16
# of AR's
14
12
10
8
6
4
2
0
0
10
20
30
40
50
60
70
80
90
100 110 120 130 140 150 160 170 180
Timing (min.)
Figure 8 TPEN timing average graph. The average time 20 µM TPEN took to eliminate the
ultradian rhythm. Three trials were performed, and the average number of AR’s were graphed.
The cells were recorded for 180 minutes each, and the number of AR’s were counted for 1
minute every 5 minutes. The average time TPEN took to take effect was 95 minutes
Cancelling Effects of Zinc on TPEN
TPEN is a zinc chelator and works by binding free zinc. If there is more zinc present in
the media than the TPEN can bind, then TPEN’s effects should not be observed. This was tested
by adding TPEN and excess zinc to the media. When equal parts 0.1 mM zinc acetate and 20 µM
TPEN were added together, the effects of TPEN were not pronounced. Four trials were run, and
each of the four cells underwent three cycles in the three hour recorded period. Appendix F
contains the graphs for each of the four trials. The period lengths for each cycle ranged from 3560 minutes. The average periodicity for the ultradian rhythm exhibited under these conditions
was 47.08 minutes (Fig. 9). This is within the range that Paramecium tetraurelia exhibit under
28
normal conditions (p=0.406). This indicates that zinc can overcome the effect of TPEN in
Paramecium tetraurelia.
Average # of AR's for TPEN + Zn
20
18
16
# of AR's
14
12
10
8
6
4
2
0
0
10
20
30
40
50
60
70
80
90
100 110 120 130 140 150 160 170 180
Time (min)
Figure 9 20 µM TPEN + 0.1 mM zinc acetate average graph. The average number of avoidance
reactions (AR’s) a single cell performed in media containing 20 µM TPEN + 0.1 mM zinc
acetate. Four trials were performed, and the average number of AR’s were graphed. The cells
were recorded for 180 minutes each, and the number of AR’s were counted for 1 minute every 5
minutes. The average periodicity (τ) was 47.08 minutes
Adding Calcium Does Not Change TPEN’s Effects
Like zinc, calcium can be bind to TPEN; however, TPEN’s affinity for calcium is much
lower than its affinity for zinc. If enough calcium is added to the media with TPEN, it may be
able to reverse TPEN’s effects. Calcium was added to the media containing 20 µM TPEN in
order to determine whether calcium can effectively cancel TPEN’s effects on the ultradian clock.
Three concentrations of calcium were added to the 20 µM TPEN media: 1.0 mM, 0.1 mM, and
29
0.01 mM. When cells were added to the media that contained 1.0 mM calcium, the cells
immediately died. Similarly, when cells were added to the media that contained 0.1 mM calcium,
the cells all died. Because of this, the cells in 1.0 mM and 0.1 mM calcium were not recorded.
Cells that were added to the media containing 0.01 mM calcium were able to survive.
Four cells from this media were recorded, and those four graphs can be found in Appendix G.
Unlike zinc, calcium did not change how TPEN affected the cells (Fig. 10). The graphs
generated by this data looked similar to the graphs from cells put in 20 µM TPEN alone, and no t
test was run since there was no clear periodicity present. Therefore, calcium does not cancel the
effects that TPEN has on the ultradian rhythm of Paramecium tetraurelia.
Average # of AR's for TPEN + Ca
20
18
16
# of AR's
14
12
10
8
6
4
2
0
0
10
20
30
40
50
60
70
80
90
100 110 120 130 140 150 160 170 180
Time (min)
Figure 10 20 µM TPEN + 0.01 mM calcium average graph. The average number of avoidance
reactions (AR’s) a single cell performed in media containing 20 µM TPEN + 0.01 mM calcium.
Four trials were performed, and the average number of AR’s were graphed. The cells were
recorded for 180 minutes each, and the number of AR’s were counted for 1 minute every 5
minutes. No clear periodicity (τ) is present
30
Reversibility of TPEN
An ultradian rhythm should return to normal once any outside factors are removed from
the cells’ environment. In this case, adding cells to TPEN eliminated the ultradian clock of
Paramecium tetraurelia. If those cells are removed from the media containing TPEN, the cells’
ultradian clock should return to its normal periodicity. Cells were placed in TPEN and then taken
out to determine if removal from TPEN would reverse the effects TPEN had on the ultradian
rhythm. In each of the four trials, 20 µM TPEN initially caused the ultradian rhythm to be
eradicated, but after removal from TPEN, the cells’ ultradian clock reverted its normal pattern.
After removal from the 20 µM TPEN, the effects of the TPEN lasted for some time. The amount
of time the cells remained in the altered rhythm for the four trials was 60, 70, 65, and 75 minutes
for trials 1-4. Those four graphs are shown in Appendix H. The average time the cells took to
revert back to their normal ultradian rhythm was 67.5 minutes (Fig. 11). In summary, removal of
cells from 20 µM TPEN led to a reversal of the TPEN’s effects but only after some time
removed.
31
Average TPEN Reversal
18
16
14
# of AR's
12
10
8
6
4
2
0
0
10
20
30
40
50
60
70
80
90
100 110 120 130 140 150 160 170 180
Time (min)
Figure 11 TPEN reversibility average graph. The average time cells took to return to their normal
ultradian rhythm after being removed from 20 µM TPEN. Four trials were performed, and the
average number of AR’s were graphed. The cells were recorded for 180 minutes each, and the
number of AR’s were counted for 1 minute every 5 minutes. The average time it took for
TPEN’s effects to be reversed was 67.5 minutes
32
CHAPTER V
DISCUSSION
This study is one of the first to look at zinc in Paramecium tetraurelia, specifically how
varying levels of zinc affect the ultradian rhythm of Paramecium’s spontaneous behavioral
responses. Several novel findings were noted. The effects of both increasing and decreasing zinc
concentrations were investigated. Before trials were done on the effects on the ultradian rhythm,
the toxicology of zinc was tested. The highest concentration of zinc that supported life of
Paramecium over a 24 hour period was 0.1 mM. When the concentration of zinc was raised to 1
mM and higher, the zinc was toxic to the cells. A previous study suggested that depolarizing the
membrane increases toxicology of zinc 42. In this study, different concentrations of KCl were
added to the media containing zinc in order to depolarize the membrane. This caused the zinc to
be more toxic for all KCl concentrations, supporting previous evidence in the literature.
After the toxicity of zinc was determined, tests were run to test its effects on
Paramecium’s ultradian rhythm. First, additional zinc was added to the media. Zinc by itself
does not affect the ultradian rhythm, as shown in Figures 3 and 4. Zinc concentrations were then
decreased by adding TPEN, a zinc chelator, to the media. TPEN causes an elimination of the
ultradian rhythm. However, TPEN does not work immediately on the ultradian rhythm. Its effect
takes approximately 88 minutes before being seen. TPEN’s effect is also reversible by removing
the cells from the TPEN and the effect can be overcome with the addition of zinc. In contrast,
TPEN’s effect is not influenced by the addition of calcium. Therefore, TPEN is zinc-specific.
Again, this study confirmed the findings of Colvin, Davis, Nipper, and Carter 42, who
suggested that depolarization of the membrane increases zinc toxicity. This outcome indicates
that zinc goes through a voltage-dependent ion channel. As the membrane becomes increasingly
33
depolarized, the channels are more likely to be in an open state, allowing ions, like zinc, to move
through more easily. The identity of the ion channel in Paramecium is not yet known, but there
is evidence in other systems of calcium channels being involved. To test this, the zinc toxicity
test could be run with the addition of calcium. Since the calcium channels are more permeable to
calcium than zinc, the addition of calcium should allow the cells to live in higher concentrations
of zinc. The zinc would be less toxic because it would not be able to enter the cell as readily.
Another way to test if calcium channels are responsible for allowing the zinc to enter the cell is
to use pawn mutants, which have no calcium channels. If zinc is entering through the calcium
channels, then these mutants should be able to live in higher concentrations of zinc since it
would not be able to enter the cells as easily. In summary, depolarization of the membrane
appears to increase toxicity of zinc in Paramecium tetraurelia, most likely due to the opening of
voltage-gated calcium channels.
A novel finding of this study was the determination that zinc is required for maintaining
the ultradian rhythm of spontaneous behavioral responses in Paramecium tetraurelia. It is known
that zinc plays a role in the transcriptional events of the circadian clock 43 but was unknown for
the ultradian rhythm. When zinc was depleted with the addition of TPEN, the ultradian rhythm
was eliminated, showing that the presence of zinc is necessary to have the ultradian rhythm. The
effect is not a long-term cellular damage issue, as the effect is reversible within approximately
67.5 minutes of being removed from TPEN. In addition, the fact that the TPEN effect can be
reversed by the addition of zinc, but not calcium (for which TPEN has a lower affinity), indicates
that zinc is the actual ion required.
A major question that remains to be determined is what zinc is doing within the cell in
order to contribute to the control of the ultradian rhythm. A novel hypothesis has been put
34
forward that involves reactive oxygen species (ROS) in the control of the ultradian rhythm in
Paramecium. Mitochondrial activity has an ultradian rhythm, which results in an oscillation in
the concentration of ROS within cells. Therefore, the ultradian clock could be a result of periodic
changes in the cell’s redox state, since the mitochondria release redox active compounds into the
cell during respiration 44. Hasawa 41 did a study on Paramecium tetraurelia in which she added
oligomycin to the cells’ media; oligomycin is a mitochondrial inhibitor that increases the amount
of ROS in cells. When oligomycin was added to the media, the ultradian rhythm in the
Paramecium was eliminated, similar to that seen when zinc is removed. Furthermore, ROS
concentrations are affected by several enzymes, notably superoxide dismutase (SOD). SOD is an
antioxidant that lowers the concentration of ROS in cells. Zinc has been shown to be necessary
for SOD to function in other systems. Therefore, it can be postulated that when zinc is depleted
in a system, SOD is inhibited, and there is a buildup of ROS in the cell. Similar to the addition of
oligomycin, an increase in ROS due to depletion of zinc would lead to the elimination of the
ultradian rhythm. Figure 12 demonstrates the hypothesis explaining the biochemical pathway
that generates the ultradian rhythm in Paramecium 5. Figure 13 shows zinc and SOD’s
hypothesized roles in the regulation of the same biochemical pathway.
35
Figure 12 Hypothesis that explains the biochemical pathway that produces an ultradian rhythm in
Paramecium 5. The release of ROS from the mitochondria generates an ultradian rhythm for the
amount of free calcium in a cell. The change in calcium concentration alters the membrane
potential of the cell, which leads to changes in swimming speed and number of spontaneous
behavioral responses
Cell with Zinc Depletion
↓ Zinc
↓ SOD
↑ ROS
function
Figure 13 Hypothesis that predicts how zinc depletion eliminates an ultradian rhythm. A picture
demonstrating how zinc affects superoxide dismutase (SOD) concentrations, which in turn
changes reactive oxygen species (ROS) concentrations. Zinc is needed for SOD to function
properly. When SOD does not function, ROS concentrations are constantly up. When there is no
oscillation in the concentration of ROS, no ultradian rhythm is exhibited
36
In conclusion, zinc is toxic to Paramecium tetraurelia at a concentration of 1 mM and
higher. Depolarization of the membrane increases zinc’s toxicity to the cells. TPEN reduces the
concentration of zinc, and this depletion of zinc leads to an elimination of the ultradian rhythm.
This effect by TPEN is reversible upon removal of the cells from the TPEN, and the addition of
zinc to the media overcomes TPEN’s effect. Unlike zinc, calcium is unable to overcome TPEN’s
effect. Finally, zinc may contribute to the control of the ultradian rhythm through its effect on
superoxide dismutase (SOD), which normally functions to eliminate reactive oxygen species
(ROS) from the cell. An increase in the concentration of ROS has been known to disrupt the
ultradian rhythm in some systems. SOD requires zinc to function, and the depletion of zinc
prevents SOD from removing ROS from the cell leading to the elimination of the ultradian
rhythm.
37
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40
Appendix A
0.1 mM Zinc Graphs
# 0f AR's
0.1 mM Zn++ #1
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure A.1 The first trial of a cell in 0.1 mM zinc. The number of avoidance reactions (AR’s) a
single cell performed in media containing 0.1 mM zinc. The cell was recorded for 180 minutes,
and the number of AR’s were counted for 1 minute every 5 minutes
41
# 0f AR's
0.1 mM Zn++ #2
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure A.2 The second trial of a cell in 0.1 mM zinc. The number of avoidance reactions (AR’s)
a single cell performed in media containing 0.1 mM zinc. The cell was recorded for 180 minutes,
and the number of AR’s were counted for 1 minute every 5 minutes
# 0f AR's
0.1 mM Zn++ #3
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure A.3 The third trial of a cell in 0.1 mM zinc. The number of avoidance reactions (AR’s) a
single cell performed in media containing 0.1 mM zinc. The cell was recorded for 180 minutes,
and the number of AR’s were counted for 1 minute every 5 minutes
42
Appendix B
20 µM TPEN Graphs
# of AR's
20 uM TPEN #1
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure B.1 The first trial of a cell in 20 µM TPEN. The number of avoidance reactions (AR’s) a
single cell performed in media containing 20 µM TPEN. The cell was recorded for 180 minutes,
and the number of AR’s were counted for 1 minute every 5 minutes
43
# of AR's
20 uM TPEN #2
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure B.2 The second trial of a cell in 20 µM TPEN. The number of avoidance reactions (AR’s)
a single cell performed in media containing 20 µM TPEN. The cell was recorded for 180
minutes, and the number of AR’s were counted for 1 minute every 5 minutes
# of AR's
20 uM TPEN #3
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure B.3 The third trial of a cell in 20 µM TPEN. The number of avoidance reactions (AR’s) a
single cell performed in media containing 20 µM TPEN. The cell was recorded for 180 minutes,
and the number of AR’s were counted for 1 minute every 5 minutes
44
# of AR's
20 uM TPEN #4
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure B.4 The fourth trial of a cell in 20 µM TPEN. The number of avoidance reactions (AR’s)
a single cell performed in media containing 20 µM TPEN. The cell was recorded for 180
minutes, and the number of AR’s were counted for 1 minute every 5 minutes
45
Appendix C
10 µM TPEN Graphs
# of AR's
10 uM TPEN #1
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure C.1 The first trial of a cell in 10 µM TPEN. The number of avoidance reactions (AR’s) a
single cell performed in media containing 10 µM TPEN. The cell was recorded for 180 minutes,
and the number of AR’s were counted for 1 minute every 5 minutes
46
# of AR's
10 uM TPEN #2
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure C.2 The second trial of a cell in 10 µM TPEN. The number of avoidance reactions (AR’s)
a single cell performed in media containing 10 µM TPEN. The cell was recorded for 180
minutes, and the number of AR’s were counted for 1 minute every 5 minutes
47
Appendix D
5 µM TPEN Graphs
# of AR's
5 uM TPEN #1
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure D.1 The first trial of a cell in 5 µM TPEN. The number of avoidance reactions (AR’s) a
single cell performed in media containing 5 µM TPEN. The cell was recorded for 180 minutes,
and the number of AR’s were counted for 1 minute every 5 minutes
48
# of AR's
5 uM TPEN #2
18
16
14
12
10
8
6
4
2
0
5 15 25 35 45 55 65 75 85 95 105115125135145155165175
Time (min)
Figure D.2 The second trial of a cell in 5 µM TPEN. The number of avoidance reactions (AR’s)
a single cell performed in media containing 5 µM TPEN. The cell was recorded for 180 minutes,
and the number of AR’s were counted for 1 minute every 5 minutes
49
Appendix E
TPEN Timing Graphs
# of AR's
TPEN Timing # 1
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure E.1 The first trial timing a cell in 20 µM TPEN to determine how long TPEN takes to
affect the cell. The number of avoidance reactions (AR’s) a single cell performed in media
containing 20 µM TPEN. The cell was recorded for 180 minutes, and the number of AR’s were
counted for 1 minute every 5 minutes
50
# of AR's
TPEN Timing # 2
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure E.2 The second trial timing a cell in 20 µM TPEN to determine how long TPEN takes to
affect the cell. The number of avoidance reactions (AR’s) a single cell performed in media
containing 20 µM TPEN. The cell was recorded for 180 minutes, and the number of AR’s were
counted for 1 minute every 5 minutes
# of AR's
TPEN Timing # 3
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure E.3 The third trial timing a cell in 20 µM TPEN to determine how long TPEN takes to
affect the cell. The number of avoidance reactions (AR’s) a single cell performed in media
containing 20 µM TPEN. The cell was recorded for 180 minutes, and the number of AR’s were
counted for 1 minute every 5 minutes
51
Appendix F
TPEN + Zinc Graphs
# of AR's
TPEN + Zn #1
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure F.1 The first trial of a cell in 20 µM TPEN + 0.1 mM zinc. The number of avoidance
reactions (AR’s) a single cell performed in media containing 20 µM TPEN + 0.1 mM zinc. The
cell was recorded for 180 minutes, and the number of AR’s were counted for 1 minute every 5
minutes
52
# of AR's
TPEN + Zn #2
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure F.2 The second trial of a cell in 20 µM TPEN + 0.1 mM zinc. The number of avoidance
reactions (AR’s) a single cell performed in media containing 20 µM TPEN + 0.1 mM zinc. The
cell was recorded for 180 minutes, and the number of AR’s were counted for 1 minute every 5
minutes
18
16
14
12
10
8
6
4
2
0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
# of AR's
TPEN + Zn #3
Time (min)
Figure F.3 The third trial of a cell in 20 µM TPEN + 0.1 mM zinc. The number of avoidance
reactions (AR’s) a single cell performed in media containing 20 µM TPEN + 0.1 mM zinc. The
cell was recorded for 180 minutes, and the number of AR’s were counted for 1 minute every 5
minutes
53
# of AR's
TPEN + Zn #4
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure F.4 The fourth trial of a cell in 20 µM TPEN + 0.1 mM zinc. The number of avoidance
reactions (AR’s) a single cell performed in media containing 20 µM TPEN + 0.1 mM zinc. The
cell was recorded for 180 minutes, and the number of AR’s were counted for 1 minute every 5
minutes
54
Appendix G
TPEN + Calcium Graphs
# of AR's
TPEN + Ca #1
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure G.1 The first trial of a cell in 20 µM TPEN + 0.01 mM calcium. The number of avoidance
reactions (AR’s) a single cell performed in media containing 20 µM TPEN + 0.01 mM calcium.
The cell was recorded for 180 minutes, and the number of AR’s were counted for 1 minute every
5 minutes
55
18
16
14
12
10
8
6
4
2
0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
# of AR's
TPEN + Ca #2
Time (min)
Figure G.2 The second trial of a cell in 20 µM TPEN + 0.01 mM calcium. The number of
avoidance reactions (AR’s) a single cell performed in media containing 20 µM TPEN + 0.01
mM calcium. The cell was recorded for 180 minutes, and the number of AR’s were counted for 1
minute every 5 minutes
# of AR's
TPEN + Ca #3
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure G.3 The third trial of a cell in 20 µM TPEN + 0.01 mM calcium. The number of
avoidance reactions (AR’s) a single cell performed in media containing 20 µM TPEN + 0.01
mM calcium. The cell was recorded for 180 minutes, and the number of AR’s were counted for 1
minute every 5 minutes
56
# of AR's
TPEN + Ca #4
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure G.4 The fourth trial of a cell in 20 µM TPEN + 0.01 mM calcium. The number of
avoidance reactions (AR’s) a single cell performed in media containing 20 µM TPEN + 0.01
mM calcium. The cell was recorded for 180 minutes, and the number of AR’s were counted for 1
minute every 5 minutes
57
Appendix H
TPEN Reversal Graphs
# of AR's
TPEN Reversal # 1
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure H.1 The first trial timing a cell in wheat grass media to determine how long TPEN’s
effects take to wear off once the cell is removed from 20 µM TPEN. The number of avoidance
reactions (AR’s) a single cell performed in media containing wheat grass. The cell was recorded
for 180 minutes, and the number of AR’s were counted for 1 minute every 5 minutes
58
# of AR's
TPEN Reversal # 2
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure H.2 The second trial timing a cell in wheat grass media to determine how long TPEN’s
effects take to wear off once the cell is removed from 20 µM TPEN. The number of avoidance
reactions (AR’s) a single cell performed in media containing wheat grass. The cell was recorded
for 180 minutes, and the number of AR’s were counted for 1 minute every 5 minutes
# of AR's
TPEN Reversal # 3
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure H.3 The third trial timing a cell in wheat grass media to determine how long TPEN’s
effects take to wear off once the cell is removed from 20 µM TPEN. The number of avoidance
reactions (AR’s) a single cell performed in media containing wheat grass. The cell was recorded
for 180 minutes, and the number of AR’s were counted for 1 minute every 5 minutes
59
# of AR's
TPEN Reversal # 4
18
16
14
12
10
8
6
4
2
0
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180
Time (min)
Figure H.4 The fourth trial timing a cell in wheat grass media to determine how long TPEN’s
effects take to wear off once the cell is removed from 20 µM TPEN. The number of avoidance
reactions (AR’s) a single cell performed in media containing wheat grass. The cell was recorded
for 180 minutes, and the number of AR’s were counted for 1 minute every 5 minutes
60