Grant

Proposal: Effect of 5HT Receptor

Concentration on Anxiety in Rodent Brain
Ionotropic Vs Metabotropic Receptors



Prepared for:













Prepared by:

Lynda Sharrett-Field, Ph.D.

Department of Psychology
University of Kentucky

Seth Jones, Ph.D.

Department of Biology
University of Kentucky

Alec Bradley, Neuroscience B.S.

University of Kentucky

December 9, 2016

5HT Receptor Effect on Anxiety

Bradley 2

Table of Contents
ABSTRACT .............................................................................................................................. 3
Background ............................................................................................................................ 4
Preliminary Studies ................................................................................................................ 5
Behavioral Data ................................................................................................................................ 6
Immunohistochemical Staining of Hippocampus .............................................................................. 7

Proposal ................................................................................................................................. 8
Research Design .................................................................................................................... 10
References ............................................................................................................................ 13

5HT Receptor Effect on Anxiety

Bradley 3

ABSTRACT
In my grant proposal, I have divided the different 5HT receptors into two categories: ionotropic
receptors vs metabotropic receptors. My goal is to test whether there is a difference in the
concentration of ionotropic receptors vs metabotropic receptors in the major areas of the
serotonin pathways in a mouse brain, and whether or not this difference leads to an alteration
of anxiety behavior. Recent literature has challenged effectiveness of the treatment of SSRIs on
anxiety and has suggested that a 5ht antagonist could provide more effective anxiolytic
properties. I am planning to antagonize the 5HT1a (metabotropic) and 5HT3 (ionotropic)
receptors in raphe nuclei, amygdala, and medial prefrontal cortex of 129 Sv mice. The
implications of this study could aid in the search for an effective pharmaceutical or other
treatment shown to be effective in alleviating the symptoms of anxiety.

5HT Receptor Effect on Anxiety

Bradley 4

Background
The purpose of this grant proposal is to request money to study anxiety in a
translational model of mice, focusing on the 5HT serotonergic receptor in the mouse brain.
Because rodents share similar brain structures to humans and can react and interact with their
environment, rodents serve as good test subjects in human translational experiment models.
Using a rodent model will allow us to analyze brain-behavior relationships to obtain an
understanding of the biochemical mechanisms of anxiety in the rodent brain, which can be
used to provide important information as to how anxiety may operate in the human brain.
Anxiety is the most common mental illness in the United States affecting over 40 million
adults, which is roughly 18% of the US population (ADAA, 2016). Per year, the United States
healthcare system spends $42 billion on anxiety alone, which is one third of the total mental
health cost to the US healthcare system (ADAA, 2016). In order to relieve Americans affected
by anxiety and alleviate this immense financial burden on our healthcare system, we must
invest in research that works to identify the mechanisms associated with anxiety. Once this is
accomplished, we can work to find a cure to the increasing threat of anxiety.
One study that has looked closely at the anxiety mechanism and how the serotonergic
pathways play a role in anxiety in mice shows that increased 5ht neurotransmission in the
raphe nucleus, amygdala, and prefrontal cortex enhances anxiety (Graeff et al., 1997).
Increasing serotonin levels in your brain can relieve depression, but causes anxiety. Recent
literature has challenged effectiveness of the treatment of SSRIs on anxiety and has suggested
that a 5ht antagonist could provide more effective anxiolytic properties (Mercola, 2015).

5HT Receptor Effect on Anxiety

Bradley 5

There are numerous types of 5HT receptors found throughout the mammalian brain
that can be subdivided into two different categories. These two categories are ionotropic
receptors and metabotropic receptors. I am interested to see if the difference between
ionotropic and metabotropic transmitter reception has any effect on anxiety as a whole, and if
so what the implications of that could mean in terms of finding a pharmaceutical that could
alleviate the symptoms of anxiety. The two specific types of 5HT receptors I will be testing for
are 5ht1a (metabotropic) receptors and 5ht3 (ionotropic) receptors in the raphe nuclei, the
amygdala, and the medial prefrontal cortex parts of the brain.

Preliminary Studies
In our prior experiments searching for a link between levels of acetylcholine and anxiety,
we used two different strains of mice. The first was a strain labeled 129 Sv, which were
genetically inbred to show high signs of anxiety. The second was a strain labeled C57, which
was genetically inbred to express low levels of anxiety. We used three separate methods of
collecting data including: (1) a behavioral study where we quantified anxiety-like behaviors, (2)
an analysis of the levels of acetylcholine (AcH) in the hippocampus and amygdala using
colorimetry, and (3) an immunohistochemical stain of AcH receptors in the hippocampus in
coordination with a cresyl violet cell body stain. We used all three of these methods to provide
our study with a higher validity. Our hypothesis leading into this study was that higher
acetylcholine levels in the hippocampus will lead to higher anxiety. From the data we collected,
we were able to see that the data supported our hypothesis. The hippocampus and amygdala

5HT Receptor Effect on Anxiety

Bradley 6

are primary targets for fear conditioning and acetylcholine activity. Our data collected from the
open field behavioral test consistently showed in terms of quantifiable anxiety behavior, that
the 129 strain demonstrated significantly higher anxious behavior than the C57 strain. The
behavioral data coming from the open field maze can be seen below in the form of multiple bar
graphs, and the fluorescent antibody stain is pictured directly below the behavioral data.

Behavioral Data

Time in Center

Grooming

(non-anxious behavior)

(non-anxious behavior)

20

10

0
d1

d2
c57

d1

129

c57

Total Crossing

Defecation

(anxious behavior)

200

0
day 1

day 2
c57

d1

129

d2
c57

129

(non-anxious behavior)

d2

129

5HT Receptor Effect on Anxiety

Bradley 7

Immunohistochemical Staining of Hippocampus
Antibody Stain of Hippocampus of 129
Primary antibody: Rabbit anti-NeuN (1:100; conjugated with FitC-green), goat anti-CHAT (1:100)
Secondary antibody: Donkey anti-goat Rhodamine (red stain predominant in hippocampus)

In this image, the red staining in the hippocampus is a result of the rhodamine, and the green
staining in the corpus callosum is the result of the FITC.

5HT Receptor Effect on Anxiety

Bradley 8

DAPI Stain of Hippocampus of 129

In this image, the blue staining in the hippocampus is a result of the DAPI.

Proposal
In order to study anxiety, I will measure the difference in receptor expression between
5HT1a and 5HT3 receptors in the raphe nuclei, amygdala, and medial prefrontal cortex of the
129 Sv brain. I will then examine whether the expression difference of these receptors causes
higher or lower levels of anxiety in the brain of the 129 mouse. Because antagonists of the 5HT
receptor are hypothesized to decrease overall levels of anxiety, I will use an antagonist for both
5HT receptors (Charney & Leger, 2010).
I will perform the study by observing the baseline behavior of the 129 Sv mice, which
have been genetically inbred to express high levels of anxiety. I will have the 129s preform an
open field test and elevated plus test as controls (Garner, 2009).

5HT Receptor Effect on Anxiety

Bradley 9

One important consideration to take into account when analyzing the behavior of a
mouse and how it translates to a human in the elevated plus maze and the open field
exploration test is the fact that mice show a preference towards dark places, whereas humans
show a preference for well-lit places. This is because mice are small prey animals have a very
good sense of smell and sense of sound, whereas humans have a better sense of vision than
smell or sound. The anxiety level in these two behavioral tests will be quantified by how many
times the mouse travels into the light as oppose to how many times it travels into darker places
or stays next to a wall, as oppose to traveling in the exposed center of the field. In the open
field test, moving from one square to the next will be quantified as all four paws being in a
single square. This method of quantification for where the mouse is will be the same for the
elevated plus test.
Once the baseline test is complete, I will antagonize the metabotropic receptor of 1/3 of
the 129 Sv mice targeting the previously outlined serotonergic pathway (raphe nuclei,
amygdala, and medial prefrontal cortex) via oral administration of a 5HT1a antagonist. I will
antagonize the ionotropic receptor of 1/3 of the 129 Sv mice targeting the outlined
serotonergic pathway via oral administration of a 5HT3 antagonist, and keep 1/3 of the 129 Sv
mice as a control. I will administer these drugs simultaneously to control for temporal
confounds. There are five major questions I would like to have the answer to after
administering the 5HT antagonist. (1) Is there an effect on anxiety? (2) Which antagonist has
greater efficacy (metabotropic or ionotropic)? (3) Do both antagonists have the same or
statistically similar efficacy? (4) Could ionotropic and metabotropic receptor antagonists be

5HT Receptor Effect on Anxiety

Bradley 10

used in conjunction with each other? And (5) is there a difference in temporal efficacy of the
antagonist between ionotropic and metabotropic receptors?
To answer these questions, I will re administer the behavioral tests run at the start of
the experiment; compare the data collected after the administration of the 5HT antagonist to
the baseline data; and then guillotine the mice and use immunohistochemistry to identify the
5HT1a and 5HT3 receptor density in the raphe nuclei, amygdala, and medial prefrontal cortex in
the brain of the 129 Sv. I will use a base cresyl violet stain to identify the soma of neurons in the
raphe nuclei, amygdala, and medial prefrontal cortex. I will then use immunohistochemical
fluorescent staining so that I can identify the neuronal synapses of the 5HT receptor sites. I will
use a rabbit anti-neuN conjugated with FITC and goat anti-CHAT as the primary antibody, and
Donkey anti-goat Rhodamine as the secondary antibody (Jones, Sharrett-Field, 2016). I will use
these antibodies because I have had experience in using them before, and because they have
stained well in the past. Given that one of the antagonists worked in reducing the symptoms of
anxiety in these mice, the additional microscopy should provide me with further details as to
how and why these antagonists reduced the effects of anxiety.

Research Design
In this experiment, the independent variable will be the oral administration of the
5HT1a antagonist and the oral administration of the 5HT3 antagonist. The dependent variable
will be the symptoms of anxiety displayed by the mice, such as defecation, grooming, crossing,
and time spent in the center for the open field test. Grooming, crossing, and time spent in the

5HT Receptor Effect on Anxiety

Bradley 11

center are all anxiolytic signs; defecation is an anxiogenic symptom. I hypothesize that the
antagonist of the 5HT1a and 5HT3 receptors will both have an anxiolytic effect on the 129 Sv
mice, and I further hypothesize that the 5HT3 antagonist will have a more immediate effect in
terms of relieving the symptoms of anxiety. I propose this because the 5HT3 receptor is an
ionotropic receptor, whereas the 5HT1a receptor is a ligand gated receptor. Ligand gated
receptors take longer to react because the agonist or antagonist causes a chain of events to
occur before the ion channel is opened, whereas an ionotropic receptor is directly acted upon
by an agonist or antagonist.
The required materials I am requesting grant money for include: The rabbit anti-neuN
conjugated with FITC and goat anti-CHAT primary antibody; The Donkey anti-goat Rhodamine
secondary antibody; and the 129 Sv genetically inbred mice expressing anxiety. The data I
expect to collect is that one or both of the 5HT antagonists will have an anxiolytic effect on the
129 Sv mice. I expect the largest anxiolytic effect to be identified in the amygdala, because
blocking the emotional stress response with a 5HT antagonist in this part of the brain should
relieve most, if not all of the effects of anxiety.
Looking forward to finding an effective anxiolytic drug in humans, finding a 5HT receptor
that responds to an antagonist and relieves anxiety symptoms in a mouse could lead to a major
breakthrough in terms of finding a 5HT antagonist that relieved anxiety in humans. Although
rodent models do not always prove to translate perfectly into human treatments, this study
would be helpful in initiating a step to identify one of the main causes of anxiety in humans; a
step to push back against the $42 billion that has been spent this year and, will be spent next
year, and every year after that until a cure is found. The biggest limitations involved with this

5HT Receptor Effect on Anxiety

Bradley 12

study include the fact that we are studying anxiety in mice, rather than humans. There are
significant differences in our neural environment, which is why we havent found an effective
cure for anxiety yet. We cannot ask the mice whether they are anxious or not, rather we must
put them through a series of tests that imply they are anxious. One final limitation is the fact
that we cannot know for certain whether this experiment will work until we perform it.

5HT Receptor Effect on Anxiety

Bradley 13

References
ADAA.org. (2016). Retrieved December 09, 2016, from https://www.adaa.org/splashify/1
Charnay, Y., & Leger, L. (2010). Brain serotonergic circuitries. Retrieved December 09, 2016,
from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3181988/
Garner, M., et al., Research in anxiety disorders: From the bench to the bedside, Eur.
Neuropsychopharmacol. (2009), doi:10.1016/j.euroneuro.2009.01.011
Graeff, F. (1997, November 7). Dual role of 5-HT in defense and anxiety. Retrieved December
09, 2016, from http://www.sciencedirect.com/science/article/pii/S0149763496000590
Mercola, D. (2015, July 2). Social Anxiety Disorder Linked to High Serotonin Levels. Retrieved
December 09, 2016, from
http://articles.mercola.com/sites/articles/archive/2015/07/02/social-anxietydisorder.aspx