This study seeks to analyze the molecular basis of circadian rhythms and behavioral modulation by hormones in the nudibranch Melibe leonina. The goals are to localize circadian clock neurons in the central nervous system using immunohistochemistry, stain for the neurotransmitters conopressin and melatonin, and assess behavioral responses to injections of these hormones through video recordings. Understanding the relationship between clock neurons, hormones, and behavior can provide insights into circadian rhythm regulation applicable to fields like medicine.

1. UNIVERSITY OF NEW HAMPSHIRE
COLLEGE OF LIFE SCIENCES AND AGRICULTURE
Localization of circadian clock neurons and analysis of behavioral
modulation by conopressin and melatonin in the Nudibranch Melibe leonina
Submitted by: Allison Nash
Sophomore – BMS:MLS major
9344 Granite Square Station
Durham, NH 03824
amh336@wildcats.unh.edu
Faculty Sponsor: Winsor Watson
179 Rudman Hall
(603) 862-1629
Date: February 15, 2015

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1. Abstract
Most cellular processes are regulated by circadian clock mechanisms responsible for
coordinating the metabolic and physiological activities of an organism. The Nudibranch Melibe
leonina has served as a model organism for neurobiological research of circadian rhythms and
the underlying neuronal circuitry patterns of expression and locomotion (Newcomb et al. 2014).
This project seeks to analyze the molecular basis of behavioral modulation by the circadian clock
in Melibe leonina. The first goal of the study will be to localize the clock neurons in the central
nervous system (CNS) of the Nudibranch. Sections of the brain will be stained with specific
antibodies using immunohistochemistry, and then mapped using fluorescent microscopy. The
second goal of the study will utilize the same immunohistochemistry techniques to stain for the
neurotransmitters conopressin and melatonin in the brain. The third goal of the study will be to
assess the behavioral activities of the Melibe in response to injections with each of these
hormones. Behavior will be monitored using time-lapse video recordings over 3 to 4 consecutive
24-hour light:dark (LD) cycles. The cumulative goals of the study should indicate the relation
between clock neuron functionality and the hormonal regulation of subsequent activity in the
Nudibranch.
2. Project History and Definition
Gastropods are an extremely diverse taxonomic class that includes snails, slugs, limpets
and sea hares (Holthius, 1995). Historically, the gastropod sea slug Aplysia californica has
served as a viable model organism for neurobiological research conducted by neuropsychiatrist
Eric Kandel. Kandel studied the molecular basis of memory storage in neurons by testing the
monosynaptic gill-withdrawal reflex in Aplysia (Kandel, 2006). Subsequent studies of Aplysia

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have been conducted to determine the role of the hormone conopressin. Conopressin is of
sequence homology with the hormone oxytocin, a neuromodulator in the brain, and has been
found to induce currents in molluscan neurons that function in sexual reproduction (Soest, 1998).
The study with Aplysia indicated that the vasopressin-like peptide conopressin is involved in the
regulation of the gill-withdrawal reflex at the synaptic level, that is, between the sensory and
motor neuron (Martinez-Padron, 1992).
Additional studies with the marine zooplankton Platynereis dumerilli have been conducted
to determine the role of melatonin in behavioral modulation. Melatonin is widely known to
regulate the sleep-cycle in humans, as well as other vertebrates, and has been used to treat people
that suffer from sleep disorders. This “hormone of darkness,” is secreted at night in synchronicity
with an organisms LD cycle, thereby regulating many biological functions (Tosches, 2014). The
study of zooplankton has indicated that when melatonin is released at night, it induces the firing
of neurons innervating locomotive cells, thus establishing a nocturnal swimming pattern in the
organism (Tosches, 2014).
Following the studies with conopressin and melatonin regulation in these marine
invertebrates, this research will attempt to determine how the circadian clock in the Nudibranch
Melibe leonina regulates the expression of these hormones. Melibe are soft-bodied marine slugs
that also exhibit nocturnal patterns of swimming, which have been investigated through
behavioral analysis (Watson et al. 2001). Much of behavior is regulated by hormonal secretions,
which are in turn modulated by circadian rhythms according to LD cycles for each organism.
These clock mechanisms regulate the metabolic and physiological activities of both terrestrial
and marine organisms based on 24-hour rhythms and the approximate 12-hour ebb and flow of
the tides, respectively (Wilcockson, Zhang, 2008). Behavioral analysis studies have shed light on

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the functional effect these clocks have on the locomotion of particular organisms. However, far
less is known regarding the cellular and physiological basis of these oscillators in the modulation
of behavioral expression (Newcomb et al. 2014).
As part of this study, circadian clock neurons and hormone neurotransmitters in the CNS of
Melibe leonina will be located with specialized antibodies custom made using Melibe-specific
clock protein sequences (Watson et al. 2001). The localization of clock neurons will provide
insight into any correlation between the location and corresponding function(s) of these
endogenous clock mechanisms in the regulation of hormonal secretions. Injecting the Melibe
with each of the two hormones and observing their behavior through time-lapse videos will
determine the effect these hormones have on behavior. The combination of neurobiology and
behavioral analysis will allow for a better visual of how clock neurons modulate hormonal output
and subsequent behavioral expression in the Nudibranch.
3. Approach/Methodology
Immunohistochemistry
The CNS of the Melibe leonina will be isolated through careful dissection, then fixed
and frozen. Sections will be cut using a cryostat and transferred to glass slides. Melibe-
specific clock protein sequences were used to create custom cryptochrome (CRY)
antibodies for the localization of clock neurons in the CNS of Melibe leonina. The brain
sections will be washed and blocked using a buffer solution. Then they will be diluted with
the rabbit primary antibody, specifically made to attach to the desired clock-N protein. The
slides will be washed again and diluted with the goat anti-rabbit secondary antibody, which
has a fluorescent tag attached, before being washed again and mounted for later

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examination using microscopy. The same procedure will be used to localize conopressin
and melatonin neurotransmitters in the brain with antibodies specific to each of these
hormones.
Fluorescent Microscopy
The prepared slides will be observed under a fluorescent microscope to identify the
clock neurons, which should fluoresce as bright bundles of dots under the microscope. The
staining patterns of the neurotransmitters will indicate their relative outputs and relation to
clock neurons. Images of the bundles of clock neurons and neurotransmitters will be
captured through the camera in the microscope. Slides that may not have stained well will
not be included in the final picture of the Melibe brain, but all viable sections will be
accounted for. The location of the clock neurons will be drawn on images of the brain and
presented as on a full layout of the Melibe brain to identify the particular regions of the
brain they are most present in. All pictorial representations of the clock neurons will be
compared to those completed previously, or by other lab members to ensure the most
accurate layout will be produced.
Pharmacology
The Melibe will be held in a 24-hour LD cycle for 3-4 days to assess normal behavior
using time-lapse video documentation. During the day, two Melibe will then be injected
with approximately 0.5mL of 0.1M melatonin, diluted with 0.1M sodium chloride (NaCl),
and another two will be injected with 0.1M NaCl to serve as the controls. A second set will
follow with injections of conopressin in the same amount and concentration. The behavior
of the Melibe will continue to be recorded for another 2 days to document any changes in
locomotion. Since melatonin is responsible for activating nocturnal swimming patterns in

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Melibe, it will be expected that injecting them during the day will off-set their circadian
rhythm, causing early patterns of swimming during the day.
4. Significance/Meaning/Applications
Circadian rhythms in most organisms, including humans, are responsible for maintaining
homeostasis through regulatory feedback loops, which control physiological functioning
(Wilcockson, Zhang, 2008). Regulatory behavior occurs at both the cellular and supracellular
level, and thus constitutes a fundamental aspect of animal physiology (Goldbeter, 2014). This
study takes on a traditional reductionist approach in tracing the origin of circadian regulatory
mechanisms to the intracellular level. Once the cellular basis for the functioning of clock neurons
in behavioral modulation is understood, such knowledge may be applied to broader scientific
fields such as medicine. For example, “epidemiological studies [have] demonstrated that
myocardial infarction, stroke, and sudden cardiac death, have a 24-hour daily pattern” (Ivanov et
al. 2007). Thus, determining how clock genes modulate physiological and locomotive behavior
may aid in the development of therapeutic medicine for the treatment of autonomic dysfunction
in the human body. The study conclusions are therefore applicable to further lines of research
focused on determining how disruptions of circadian rhythms may inhibit the proper
physiological functioning of organisms.
5. Personal Outcome
I am currently switching majors to Neuroscience and Behavior; thus, conducting this
research on circadian rhythms will be an invaluable experience in terms of its relation to my area
of interest. As a premed student and aspiring Neuropathologist, the medicinal application of the

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study intrigues me greatly, and working with pharmacological substances will aid in my
understanding of how they work in different organisms. Also, the experience will provide me
with a sense of how research may be conducted in medical laboratories focused on studying
diseases of the nervous system. The hands-on aspect of the study through the use of dissections
with surgical tools is also a great tool for improving dexterity, which can aid in the preparation of
slides and analysis of tissue samples.
Being awarded a SURF would provide me with the opportunity to expand my knowledge
beyond the general classroom setting, in addition to gaining direct experience with the many
facets of research, something I plan to continue in association with earning a medical degree.
Additionally, as a member of the Honors Program, I will be required to complete an Honors
Thesis my senior year. Conducting this research would provide me with grounds to base by
thesis off of in the future.
6. Location
The study will take place in Dr. Winsor Watson’s Laboratory, located in Rudman Hall on
the University of New Hampshire campus in Durham, NH. Melibe leonina specimens will be
collected off the Pacific coast by Dr. Watson’s associates, and transported to the campus
laboratory to be held in aquarium tanks.
7. My Role/Preparation/Experience
Since the start of winter break following the 2014 fall semester, I have been working in Dr.
Watson’s laboratory learning the techniques and skills necessary for conducting research in
this field of study. I participated in the dissection of the brain and ventral chord of the Atlantic

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horseshoe crab, Limulus polyphemus. I isolated and sectioned the Limulus brain using a
cryostat, and mounted the sections on slides. I then stained the slides for clock proteins using
immunohistochemistry techniques, and located the clock neurons in the protocerebrum
through fluorescent microscopy. The clock neurons were then mapped using drawing revisions
I made of the Limulus brain. Additionally, this semester I injected two Melibe with the desired
concentration of melatonin and began video recordings of their behavior. Pending
observations of this test, I will continue to research the effects these hormones have on the
behavioral patterns of the Melibe.
Localization of clock neurons and neurotransmitters in the Melibe brain will be an
extension of this research, and my experience with immunohistochemistry, fluorescent
staining/microscopy, and pharmacology has provided me with the necessary skills to proceed
with this study. As I continue research throughout the spring semester of 2015, I will be
working in loose collaboration with other undergraduate and graduate students, and extending
this research over the summer will allow for us to further our data compilation.
8. Timetable
The following table details the chronological stages of the research experiment to be
completed the summer of 2015. Some of these stages have already been started in lab to assess
the feasibility of the project and allow for repeatable tests to ensure the validity of the results.
The completion of the study will help to strengthen my application to medical school and aid
in the attainment of a career path oriented toward research in the medical field

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Experimental Stage Project Goals
1 Set up LD cycle experiment with 4
Melibe at a time. Record time-
lapse videos for 3-4 days
Assess normal behavior of the
Melibe to determine how they
express a daily rhythm
2 Inject 2 Melibe with melatonin and
2 with NaCl. Record time-lapse
videos for 2 days (repeat with
additional Melibe)
Determine how the hormone
influences the swimming behavior
and circadian rhythm of the
Melibe
3 Inject 2 Melibe with conopressin
and two with NaCl. Record time-
lapse videos for 2 days (repeat
with additional Melibe)
Determine how the hormone
influences the behavior and
circadian rhythm of the Melibe
4 Dissect and section the CNS of
the Melibe and stain for clock
neurons using the specified
antibodies
Locate the clock neurons using
fluorescent microscopy and
compare images to those
previously taken
5 Use the sections of the brain to
stain for conopressin and
melatonin neurotransmitters in the
brain
Localize the neurotransmitters in
relation to clock neurons in the
brain
6 Compile data from time-lapse
recording and images of the
Melibe brain stained for clock
neurons and hormone
neurotransmitters
Determine whether there is a
relation between the location of
clock neurons and hormonal
neurotransmitters in the brain that
influence patterns of locomotion in
the Melibe

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Appendices
Appendix 1 – Bibliography
Ivanov, P., Hu, K., Hilton, M., Shea, S., & Stanley, H. (2007). Endogenous circadian rhythm
in human motor activity uncoupled from circadian influences on cardiac dynamics. Proceedings
of the National Academy of Sciences, 104(52), 20702-20707.
Kandel, E. (2006). In Search of Memory: The emergence of a new science of mind (1st ed.).
New York: W.W. Norton &. Co.
Martinez-Padron, M., Edstrom, J., Wickham, M., & Lukowiak, K. (1992). Modulation of
Aplysia californica siphon sensory neurons by conopressin G. Journal of Experimental Biology,
2(4), 79-105.
Newcomb, J., Watson, W., Kirouac, L., Bixby, K., Lee, C., Malanga, S., & Raubach, M.
(2014). Circadian rhythm of locomotion in the nudibranch mollusc Melibe leonina. Frontiers in
Behavioral Neuroscience, 1(3), 263-273.
Soest, V., & Kits, K. (1998). Conopressin affects excitability, firing, and action potential shape
through stimulation of transient and persistent inward currents in mulluscan neurons. Journal of
Neurophysiology, 79(4), 19-32.
Tosches, M., Bucher, D., Vopalensky, P., & Arendt, D. (2014). Melatonin signaling controls
circadian swimming behavior in marine zooplankton. Developmental Biolody, 46-57.
Watson, W., Lawrence, K., & Newcomb, J. (2001). Neuroethology of Melibe leonina
swimming behavior. Integrative and Comparative Biology, 41(4), 1026-1035.
Wilcockson, D., & Zhang, L. (2008). Circatidal clocks. Current Biology, 18(17), R753-R755.