EFFECT OF OLFACTORY STIMULI ON ANIMAL MODELS OF ANXIETY
Megha Andrews, Madlyn Kates, Christine Lu, Katherine Miao, Archana Raghunath, Angeli
Sharma, Grace Shen, Michael Tai, Mary Tresvalles, Elena Wei, Destiny West, Audrey Zhou
Advisor: Dr. Graham Cousens
Assistant: Runi Patel
ABSTRACT
In this study, the effect of predator pheromones and odorant stimuli on phasic fear and
immediate or sustained response was examined in the corticomedial amygdala of rats. To
identify which stimuli would produce anxiety-like symptoms, behavioral studies such as an
Acoustic Startle Response (ASR) experiment and an Open Maze experiment were conducted.
The Acoustic Startle Response, when conducted in conjunction with an exposure to predator
pheromones, served as a measure of phasic response to sudden stimuli. The Open Maze test,
based on thigmotaxis and aversion to open spaces, is used as an assay in anxiety-related
behavior. To see if neurons in the amygdala are responsive to sensory stimuli, an
Electrophysiology experiment was conducted. This measured electrical activity in the neurons of
the corticomedial amygdala, their firing patterns in relation to odorant triggers and subsequent
anxiety responses. Understanding the circuitry of the amygdaloidal complex and how sensory
stimuli are processed can provide insight into predator-odor induced anxiety responses, and aid
in further research on anxiety disorders in humans.
INTRODUCTION
Anxiety has provided humans with a means to react to potential threats and act
accordingly to ensure survival. However, anxiety as an evolutionary advantage has evolved into
pervasive disorders that negatively impact both physical and mental health. Human beings
exhibit anxiety disorders which range from phobias to panic disorders. Characterized by feelings
of dread over future events, anxiety disorders are commonly associated with general fear and
uneasiness, as well as chronic symptoms of heart and respiratory disease. Anxiety disorders and
its multiple variants have become one of the most prevalent forms of psychological illness. Thus,
a heightened scientific interest in its anatomical causes and neural mechanisms has followed suit.
However, as human experimentation of anxiety and fear necessitates stress and displeasure, a
standardized animal model is implemented as a practicable alternative.
Although many researchers believe that anxiety and fear are discrete emotions, there is
still behavioral similarity that allows scientists to look to phasic and sustained fear in animal
models to understand the role fear and anxiety play within the corticomedial amygdala and other
neural pathways. The phasic fear is a sudden response to specific stimuli that dissipates quickly
once the threat is eliminated, akin to specific phobias, while a sustained fear which is caused by
apprehension to a less specific and unpredictable threat, akin to anxiety disorders (3).
Anatomically, the processing of phasic and sustained fear is located in discrete areas of the
amygdala with evidence that the corticomedial nuclei is involved with sustained anxiety-like
responses. The use of pheromones on rat models examines the phasic fear and sustained fear
which cause anxiety responses and behavior.
[4-1]
Olfaction perception is processed within a plethora of neural pathways. The olfactory
portion of a rat's brain is known to have five subsections: the main olfactory system (MOS), the
accessory olfactory system (AOS), the trigeminal system, the Grueneberg ganglion, and the
septal organ of Masera (8). Pheromones are detected using the vomeronasal organ (VNO) (8).
Species may evince signs of fear and stress such as freezing or running due to activation of the
sympathetic nervous system from detecting predator odors. However, would a pheromone from a
natural predator cause a Sprague-Dawley domesticated rat, which has never been exposed to the
odor, to become aversive due to innate anxiety responses within the corticomedial amygdala? If
a prey species is exposed to the pheromone for the first time, the reluctance to stay in the same
vicinity as a pheromone may be seen due to innate behaviors passed on through breeding.
Although there has already been research indicating the role of the entorhinal cortex of a
rat brain in processing olfactory stimuli and pheromones, it is believed that the amygdala in the
temporal lobes also play a seminal part in response to pheromones (10). There are three subregions of the amygdala: the basolateral (BLA), central, and corticomedial amygdala (CMA).
While all three regions aid in olfactory perception, only the CMA receives input from both the
MOS and the AOS. Consequently, it is likely that the corticomedial amygdala is not only able to
receive signals generated by pheromones, but also regular, nonvolatile odors.
The objective of this study is to examine the effect of olfactory stimulation on animal
models of anxiety. Three experiments were conducted to evaluate potential contributions of the
corticomedial amygdala to processing olfactory information relevant to a predator odor model of
anxiety. Sustained fear response, tested with the Acoustic Startle Test, and aversion, tested with
the Open Maze, were measured after exposure to predator odors, while electrophysiology
quantified the neural activity when exposed to pheromones. These tests were then repeated with
the control variables of neutral odorants and a non-predator pheromone.
Open Maze
Open Maze Behavioral Response
To determine if predator pheromones would stimulate aversive behaviors, the rats were
placed within a tub and introduced to predator pheromones, cat urine and fox urine, which were
placed in specified zones on gauze. The rats explored the tub and the aversion due to the
exposure of pheromones was analyzed. In nature, due to sympathetic nervous system activation,
prey species elicit signs of fear and stress such as aversion or running, the fight or flight
response, as an innate survival mechanism. The prediction was that domesticated rats which
come in contact with a predator pheromone would become aversive to the area where the odor is
present even if exposed to that pheromone for the first time. Rats have innate behaviors which
must be taken into account within this study. Within an open maze, rats may tend to avoid open
spaces and stay around the perimeter of the maze, a phenomenon known as thigmotaxis (4). The
thigmotaxis that rats exhibit is due to innate stress and anxiety behavior, which in nature enables
avoidance of predator species to maintain survival. This aversive behavior may lead to less
exploration of the maze and the pheromones. In order to avoid this behavior affecting the study,
the rats were placed in the middle of the maze in order to begin exploration and given
consequent trials to acclimate to the maze.
[4-2]
Acoustic Startle Response
In order to determine the effects of the odorants on the subjects, the rats were placed in startle
chambers and observed for their acoustic startle response. When exposed to sound pulses of
approximately 85 decibels (dBA) or higher, the rats startle in response due to an inherent reflex.
In nature, such responses to sudden, loud stimuli may protect them from the attack of predators.
Modulation of Acoustic Startle Response
While the aforementioned pontine reticular nucleus is pivotal in generating motor output in the
acoustic startle response, it also receives input from other nuclei that can alter the intensity of the
response due to factors such as habituation, prepulse inhibition (PPI), and fear (5). Since it is
known that fear can enhance the startle response, studying the activity of rats in the presence of
pheromones known to elicit fear is a potential way of understanding whether the corticomedial
amygdala has a role in processing fear and the related emotion, anxiety.
The Effect of Neophobia on Acoustic Startle Response
One factor that had to be taken into account in the startle chamber portion of the study was
neophobia, which is defined as the fear of new experiences. In order to avoid the nebulous
situation of whether an enhanced startle response was caused by the pheromones or the fact that
the rodents were enclosed in a box they have never been in before, the rats were given a brief
acclimation period of one to five minutes in the chamber before any sound pulses and/or
pheromones were released.
Electrophysiology Channels and Variables
Unit Recordings of Odor Specific Neural Ensembles
Single-unit electric recordings of individual or ensembles of neurons provide valuable
insight into the function of specific brain regions in processing external information and signals.
The ability to monitor single-neuron activity allows for high spatial and temporal resolution in
the assessment of interneuron communication and flow. Such recordings enable easy
measurement of action potentials or enhanced neural activity via extracellular spike discharge
without damage to the cell. Subsequently, receptive field analysis is performed in which a
stimulus configuration activates neuron recording into an electrophysiology diagram.
In vitro single-unit extracellular recording of the anesthetized rat involving a Tungsten
microelectrode was performed to measure the electrical activity of cells in the corticomedial
amygdaloidal complex- thought to orchestrate a behavioral and physiological response to
predator stimuli. Recording of CMA neuronal activity in the rat when exposed to both volatile
odors and pheromones evinces the olfactory pathways that elicit anxiety or fear. Given that this
recording technique allows for the detection of neighboring neurons around the electrode tip,
neurons in the CMA may even generate a synchronous response or experience similar neuronal
tuning. This may provide additional insight into the interactions between neurons in processing
predator odors followed by an anxiety-like response.
Measure of Respiratory Rate using Nasal Thermocouple
[4-3]
Respiration is crucial to the olfactory perception of the rats, as the action potentials of the
neurons in the corticomedial amygdala of the brain fire in conjunction with every breath.
Entrained cells are usually found deep in the amygdala, and cells that are entrained with the sniff
cycle will fire together and regulate their activity. A nasal thermocouple was used to measure
sniffing behavior and respiration rate, and after being implanted into one nostril of the rats, was
able to record breathing patterns and their relation to neural activity. By measuring the
temperature differences between inspired and expired air as well as fluctuations of pressure in
the nasal cavity, the thermocouple was able to match the respiration rate with the action
potentials of the firing neurons.
Odorants and Triggers
In order to better understand the pathways of anxiety cortical processing, particularly in
relation to olfactory detection leading to predator odor fear, an array of pheromones and
chemical stimuli was presented to the rats. The 4 monomolecular odorants administered were as
follows: limonene, heptanone, isoamyl acetate, propyl butyrate, as well as the one pheromone,
fox urine- containing 2,3,4 dihydro 2,5 trimethyl azoline (TMT) - which possesses predatorial
properties that may elicit changes in neuronal activity. In line with prior research (8), TMT, a
remarkably volatile compound found in red fox urine, has been shown to generate drastic
behavioral and physiological effects in both rats and mice. Exposure to this predator odor in rats
induces an increase in c-fos expression, an indirect marker of heightened neural firing, in several
layers of the main olfactory bulb as well as increased vigilance via freezing behavior and
elevated EEG and neck muscle EMG recordings. In accordance with previous studies, in which
the rat was exposed to a wide range of emotional triggers, the 4 volatile stimuli were utilized to
elicit neutral and positive responses. As the electrical recordings are focused on the waveforms
engendered by the introduction of fox urine, using negative odors provide an articulate
comparison to the enhanced effect of fox urine, while a positive odor would cause a paucity of
activity in the brain regions associated with olfaction-induced fear. Volatile odors were chosen as
control stimuli as they elicit a presumably neutral rat response and do not indicate possible
threat.
One of such areas of the olfactory system that modulates predator odor conditioned fear
is the corticomedial amygdala (CMA). The CMA serves as the transmission center of direct and
indirect input from the main and accessory olfactory systems and relays the information to the
hypothalamic nuclei where hormone secretion is modulated to produce a defensive response.
With a key role in facilitating innate emotional behaviors, unit electrical recordings of this region
would shed considerable light on the neural circuitry that accompanies unconditioned pheromone
- evoked anxiety.
MATERIALS AND METHODS
Subjects
The subjects of the Startle Response study were eight male Sprague-Dawley rats. Each
subject was housed in a clear, plastic cage within a climate-controlled room and was kept on a
12-hour light cycle. Each subject was put through the same number of baseline tests and startle
tests, but four of the subjects were put through one extra startle test session. However, the data
[4-4]
from that session is not included in this study due to technical errors (see experimental error
section).
The subjects of the Open Maze study were six male Sprague-Dawley rats. Prior to the
experiment each subject was handled three times. Each time, they were handled by a different
person and were held for three minutes each before being returned to their cages.
The subjects of the Electrophysiology study were two male Sprague-Dawley rats. Each
subject experienced the same craniotomy and electrophysiology recording procedures on
different days of the week; however, the electrode was inserted in different coordinates in each
of the rats’ brains as described in the procedure.
Open Maze Experiment
Apparatus
The Maze test was conducted in an empty tub, which was put directly under a camera
that detected the rat's’ movement and location and convey data and a video onto the Any-Maze
software. The tub was divided into 12 sections in order to collect more accurate and precise data.
A small circle divided the inner side of the tub from the edge and six sections dividing the tub
into wedges. Within each wedge, an X was placed on which either an odorless gauze or gauze
with the odor was placed. The ANY-Maze computer software program was used to record and
analyze data. The ANY-Maze software consists of a camera that records and tracks the
movement of the rat in the tub. Each section was programmed as a zone in the Any-Maze
software. Each pheromone position was programmed as a point in the software. By dividing the
tub in such a manner, data could be collected on how long the rat spent in each section and
compared with the amount of time the rat spent in the section which contained the pheromone.
Within the tub, the amount of time the rat spent in each section and also the average distance
from the point was measured.
Variables and Procedure
Four different odors and their impact on the behavior of rats were tested. In this
experiment, cat urine was used as the weak predator pheromone and TMT from fox urine was
used as the strong predator pheromone. A positive pheromone, the urine of sexually active
female rats, was used as a counterbalance for the predator pheromones. The neutral smell of
propyl butyrate was used to compare both the positive and negative pheromones with. In order to
better facilitate diffusion of the odor, 5-6 drops of the odor were placed on cotton gauze. The rest
of the five cotton gauzes had 5-6 drops of water on them. Gauzes were taped down on the Xs in
the tub. Baseline trials were run in order to get the rats acquainted with the tub and the gauzes,
with each gauze containing drops of water instead of odorants. The data from the baseline testing
was used to gain a better understanding of the way the rats interacted with their environment.
All the rats were kept outside of the testing room with the door closed prior to each new
odor. Changing the location of the odor each trial blocks the possibility that the rats avoided or
gravitated towards a certain location despite the odor that is placed there. The lights inside the
room were turned off to ensure that an unbalanced distribution of light did not affect the location
of the rat. There was one red light in the room to allow for the camera to detect the rat's
[4-5]
movement. Five to six drops of cat urine was placed on one gauze, drops of water were put on
the other five gauzes, and the gauzes were taped on the Xs in the tub. The first subject was
brought into the room, held for 30 seconds, and then once it was placed in the center of the tub,
the three minute test on the Any-Maze program began and the software measured the previously
set parameters. Once the three minutes was complete, the rat was taken out of the tub and put
back in its cage. The tub was cleaned with ethanol solution and the process continued until all the
rats had finished the first trial. Once all the rats went through the first trial, the location of the
odor was moved, more drops of the odor was added to the gauze with odor, and the process was
repeated. When all three trials for this odor was completed, the process was repeated for the
propyl butyrate, fox urine, and female rat urine.
Acoustic Startle Response
Apparatus
The four apparatuses used in the Acoustic Startle Response test were Med Associates
sound attenuating enclosures. Each chamber had a platform for the subjects to step on, as well as
a load cell that recorded the force with which the subjects exerted on the platform when startled,
a red light, a speaker, an automatic fan, and an amplifier. For the entirety of the testing, the
speakers were emitting white noise at 65 decibels (dBA). An olfactometer was used to disperse
odorants into the apparatuses. These odorants were fox urine (which contained 2,4,5 dihydro 2,5
trimethylthiazoline (TMT)), cat urine, female rat urine, and propyl butyrate (PB).
Procedure
Three days prior to beginning the study, all subjects were handled by humans for 3
minutes a day to minimize any stress that may be caused by handling. Before starting the
Acoustic Startle Test sessions, each subject was put through three baseline tests in which their
individual startle responses to varied sound pulses were measured. Subjects N50-N53 (Group A)
were tested first and received a 2 minute acclimation period inside the chambers before the
session began. During the session, sound pulses of 75, 85, 95, and 105 dBA were emitted in a
pseudo-random sequence (the sequence was kept constant for each subject). The pulses had a
rising time of 15 milliseconds and were played in 15 second intervals. Each baseline session was
10 minutes long. Approximately 5 minutes later, subjects N54-N59 (Group B) were tested using
the same procedures as Group A. The chambers were cleaned with ethanol between every
session.
After the Baseline Test, vials containing 1 mL of each odorant were equipped with tubing
that connected the vials to the olfactometer and the chambers. Group A was the first group to be
tested. All subjects were placed in the four chambers in numerical order. Once placed in the
chambers, the subjects were given a 5 minute acclimation period. Afterward, 30 acoustic pulses
with an intensity of 95 dBA and rising time of 15 milliseconds were released in 15 second
intervals. Following those 30 pulses (on the 31st pulse), the olfactometer released odorants into
their respective chambers. During this portion of the test, the fan was running automatically to
filter the odorants outside of the chamber (this took approx. 30 seconds). Afterward, odorants
were released every fourth pulse (35th pulse, 39th pulse, 43rd pulse, etc.). Upon completing the
session, Group A’s subjects were taken out of the chambers, placed into their respective cages,
[4-6]
and given 20 minutes rest. Group B was then placed into the chambers in and were then
administered the test in the same fashion as Group A. Note that in between Sessions 2 and 3, a
fourth baseline test was administered to both groups. The order of the odors presented across the
sessions was counterbalanced to remove any possible confound of repeated testing.
Craniotomy and Electrophysiology Recording
Apparatus
A stereotax apparatus was used to perform the electrophysiology recording. It contained
ear and incisor bars to fix the rat’s head in place and dials that could be used to precisely move
and lower an electrode in an x-y-z-coordinate system. A 12 mega ohm tungsten recording
electrode from A-M system (Plano, Texas) was used. The electrode was connected to an A-M
Systems model 1800 microelectrode amplifier; the signal was amplified 10,000 times and it was
filtered between 500 Hz and 3000 Hz. The amplifier had 2 channels; 1 channel was used to
detect neuronal activity and the second channel received input from the thermocouple to record
respiration. The output of the amplifier was sent in parallel to the oscilloscope and a National
Instruments digital acquisition PCI card, which allowed the signal to be digitized at 20 kHz.
Procedure
The craniotomy was performed on two rats over two days, following the same procedure
each day. The rats were anesthetized with 3 grams per kilogram of urethane and isoflurane was
used as a supplemental anesthetic. Ear bars and an incisor bar were used to fix the rat’s head in
place in the sterotax and the top of the head was shaved. A scalpel was used to make an incision
along the back of the head and the skin was clipped to the side to expose the skull. The
connective tissue covering the skull was scraped aside. A window was drilled in the right parietal
bone and a hole for the electrode was drilled in the left parietal bone.
Electrophysiology recording was done over two days and the same procedure was
followed on both days. First, the rat was positioned in the stereotax and grounded by connecting
its tail to the faraday cage with wire in order to reduce electrical noise. A thermocouple was
connected to observe and record the respiration of the rat. Differential recording of the brain was
conducted by connecting 2 electrodes to the brain. A curved reference electrode was used to
record extraneous electrical noise while the straight unit electrode was used to record electrical
signals from neurons and noise. The unit electrode was positioned at bregma, the point where the
frontal and parietal bones of the skull come together, and the coordinates of this point were
measured. The target was then found and the electrode was lowered to the brain surface. On the
first day of recording, the target was 3 mm posterior and 2 mm lateral to bregma. For the second
day of recording, the target was changed to 3 mm posterior and 3.4 mm lateral to bregma in
order to reach the amygdala. After the oscilloscope settings were set to 1 ms per division and 50
V per division, the electrode was lowered into the brain. On the first day, the electrode was only
lowered until the multiple cells were found at 5.96 mm below the surface of the brain. On the
second day of the recording, the electrode was lowered to 8.45 mm below the surface until
neurons were observed. On the second day, researchers could hear breathing related neural
activity, which is typically only observed in ventral sites that receive olfactory input; this was not
[4-7]
noticed in initial recordings. This would suggest that on the second day, the electrode was in a
region that receives olfactory input.
Odor tubes were connected to present odors to the rat. Six odors were presented to the rat
on the first day, following the sequence: limonene, heptanol, isoamyl acetate, heptanone, propyl
butyrate, and cat urine. On the second day, the cat urine was replaced by fox urine, but the rest of
the odors presented were same and the sequence also remained the same. Odors were presented
for 2 seconds with a 1 minute odor onset latency and each odor was presented 10 times. Once the
recording was complete, the rat was decapitated and the rongeurs were used to remove the brain
from the skull. The brain was placed in a 10% formalin and 30% sucrose solution for a week to
preserve and harden it. After a week, the brain was sliced and sectioned with a vibratome and
mounted onto microscope slides. The slides were stained with neutral red nissl staining and
observed under a light microscope.
RESULTS
Open Maze
The objective of the Open Maze experiment was to measure any aversion the rats may
have to the various odors. This was done by placing the rats in an empty pool that acted as an
open environment for the rats to freely explore. The movements of the rats were recorded
through a camera placed over the maze that then analyzed the pattern of rat behavior through the
ANY-Maze program. At the conclusion of the experiment, the amount of time each rat spent in
each zone of the maze, the rats’ average speeds in each zone, and the average distance the rats
spent away from the points of odor placement were measured and recorded. The data was then
analyzed with two-tail T-tests that calculated the certainty of significant difference (t), the
degrees of freedom (df), and the probability of getting the specific t-value for the given degree of
freedom (p) in a (t, df, p) format. In order to analyze the data recorded for the experiment, two
sets of inferential statistical analyses were performed. One two-tail T-test analysis was
performed to compare the behavioral patterns of the rats in the zones containing the odors,
indicated as “target,” and those zones opposite of them. As can be seen in Table I, only the pvalues comparing the target and opposite zones for all three types of the data recorded were less
than 0.05. This suggests, with a 95% confidence, that there was a statistically significant
difference in the time, speed, and distance away from points of odor placement the rats spent in
each zone. The p-value for female rat urine the values was greater than 0.05, indicating that there
was no real statistical significant difference between the time spent in the target and opposite
zones.
Table I: Two-tail T-test results comparing odorants to control trial
Odorants
T
DF P
Cat urine vs. propyl butyrate
3.0031 5
Time in zone
[4-8]
.0300
Fox urine vs. propyl butyrate 1.280
5
.2775
Rat urine vs. propyl butyrate
1.8673 5
.1208
Cat urine vs. propyl butyrate
8.9576 5
.0003
Fox urine vs. propyl butyrate 3.2546 5
.0226
Rat urine vs. propyl butyrate
2.7240 5
.0416
Cat urine vs. propyl butyrate
3.8940 5
.0115
Fox urine vs. propyl butyrate 4.3691 5
.0072
Rat urine vs. propyl butyrate
.0115
Speed in zone
Table 1: The table displays the (t,
df, p) values calculated from the
two-tail T-Test analysis for
comparing the rats’ behaviors in
the target and opposite zones for
each odor. The general trend of
the table indicates that mainly
the rat behavior exhibited in the
cat urine and fox urine trial
showed a significant difference
between their target and opposite
zones.
Distance from point
3.8880 5
The other type of inferential statistical analysis was used to analyze the data comparing
the three odors: Cat Urine, Fox Urine, Female Rat Urine against the control: Propyl Butyrate. In
general it was found that there was little difference in the time the rats spent in target zones in the
odor trials than those in the control trial. For example, as shown in Table II , only the trial with
cat urine has p-values less than 0.05, suggesting that it is the only odor that can be said with a
95% confidence to have shown a statistically significant increase in the time the rats spent
exploring the cat odor zones compared to the propyl butyrate zones. Both the fox urine and rat
urine trials showed no statistically significant difference from the control trial, indicating that any
observed differences between time the rats spent in the fox and rat odor trials compared to the
controlled trial were the result of chance rather than an innate aversion or attraction to the
pheromones.
When analyzing the Average Speed of the Rats in the Target Zones containing the odors
compared to the Target Zones containing the control scent, it can be seen that there was a
statistically significant difference between the speed the rats spent in the odors compared to the
control. As shown in Table II, the p-values for cat urine, fox urine, and rat urine were 0.0002,
0.0149, 0.0269, respectively. All of these p- values are less than 0.05 which indicates at least a
95% confidence that the difference in rats’ speeds in the zones containing the odors compared to
Propyl Butyrate is statistically significant, and not a result of chance.
Finally when analyzing the data for the Average Distance the Rats kept away from the
points in the target zones in the odor trials compared to those in the control trial, it was found
that there was generally a statistically significant difference in the distance the rats stayed away
from the points in the odor trials compared to the points in the control trial. For example, the p[4-9]
values for Cat Urine and Fox Urine compared to Propyl Butyrate were 0.0418 and 0.0049,
respectively. T-test results for both odors suggest with at least a 95% confidence that the rats on
average stayed closer to the target points in the odor trials than those in the control trial.
Table II: Two-tail T-test results comparing odorants to control trial
Odorants
T
DF P
Cat urine vs. propyl butyrate
3.0031 5
Time in zone
Fox urine vs. propyl butyrate 1.280
.0300
5
.2775
Rat urine vs. propyl butyrate
1.8673 5
.1208
Cat urine vs. propyl butyrate
8.9576 5
.0003
Fox urine vs. propyl butyrate 3.2546 5
.0226
Rat urine vs. propyl butyrate
2.7240 5
.0416
Cat urine vs. propyl butyrate
3.8940 5
.0115
Fox urine vs. propyl butyrate 4.3691 5
.0072
Rat urine vs. propyl butyrate
.0115
Speed in zone
Table II: The table
displays the (t, df, p)
values calculated from
the two-tail T-Test
analysis for comparing
the rats’ behaviors in the
target zones from each
odor trial: Cat Urine,
Fox Urine, and Female
Rat Urine to a control
trial: Propyl Butyrate. In
general, mainly the rat
behaviors in the Cat and
Fox Urine target zones
showed a statistically
significant difference
from the rat behaviors
exhibited in the target
zones of the control trial.
Distance from point
3.8880 5
1a)
1b)
1c)
Figure 1: The data from the open maze tests with error bars representing 10% standard error.
Comparison of the average time the rats spent in the target zone (zone with odorant) and the zone
[4-10]
opposite of it expressed in seconds for each odorant, comparison of the average speed in the target zone
and the zone opposite of it expressed in meters per second for each odorant, and comparison of the
average distance the rats traveled in the target point and the point opposite of it expressed in meters for
each odorant.
Acoustic Startle Chamber
To monitor behavioral response in response to anxiety-inducing odors, rats were held in
startle chambers to measure and record its startles. The timing and amount of force was recorded
using the Startle Reflex program, which depicted the movements in waveforms. These
waveforms varied in amplitude and wavelength depending on the variations of the startle. If the
rat pushed off the bars of the chamber with greater force, the program would record a larger
amplitude. Along the same lines, if the rat startled quicker, the wavelength would be shorter. The
results are then entered into an ANOVA calculator for an analysis of variance using inferential
statistics.
Table III: Inferential Statistics and Analysis of Variance for Startle Test Results
F
P
Baseline
10.08
<0.001
Habituation
17.82
<0.001
Cat urine
0.15
0.929
Fox urine
0.07
0.975
Female rat urine
0.09
0.965
Propyl butyrate
0.02
0.996
Foremost, the data collected from the baseline sessions conducted at the commencement
of the experiment is represented in Figure 2a. One-Way ANOVA was used as a method of
inferential statistics in order to analyze variability, and sphericity was assumed that all groups
had equal variability. The results, seen in Table III, gives us above a 99% confidence that there is
a significant main effect of pulse intensity and that a greater sound pulse intensity produces a
greater startle reflex in the rats. Thus, reasonable confidence can be taken in the reliability of the
rats and equipment. Additionally, the baseline trials give evidence that habituation takes place as
well. As the mice are repeatedly exposed to the sound pulse, they appear to startle less as they
become accustomed to the noise. In Figure 2a, at 95 db, the rats appear to startle relatively more
[4-11]
during the first baseline session; as the sessions progress, the startle response decreases.
Inferential statistics indicates that this observation is significantly significant, as shown in Table
III. This indicates that there is more variance than expected by chance.
2a)
2b)
2c)
2d)
2e)
2f)
Figure 2: Startle amplitude of Olfactory Startle Tests Expressed in Arbitrary Units of Force.
[4-12]
The startle amplitude during the five baseline sessions, the thirty pre-odor baseline trials of the cat urine
session, and the olfactory sessions of cat urine, fox urine, female rat urine, and propyl butyrate
respectively. Included are error bars that represent the ±SEM.
Data is recorded for each rat, which underwent four sessions of different odorants. Each
rat was to be exposed to each odorant in a different order to counterbalance any lingering effects
of an odor. Despite this intention, a glitch occurred in which half the rats received exposure to
one odorant without proper startle measurement recording such that the data began recording 400
milliseconds after the pulse was emitted, effectively missing the rat’s startle reaction in the data
collected. Nevertheless, each rat was exposed to each odorant and the session was redone to
successfully record the startle peak value. In each session, the first thirty trials are baseline trials
with the sound pulse given when no odorant is present. These trial results are averaged together
in groups of ten, an example of which is shown in Figure 2b.
The last forty trials of every session were in the presence of an odorant. By representing
the data using graphs, one is able to see the average effects of various odors on the startle of the
rats. The graph in Figure 2c represents the average startles of the eight rats in the presence of cat
urine. Each bar is made up of interspersed trials, representing every fourth sound pulse trial. The
first bar is the average of the peak value of the startle at the moment the odor is released,
represented as the percentage of the average baseline value taken before each session. The
following bars represent the average of the startles 15, 30, and 45 seconds after the odor release.
The startles in the presence of cat urine odorant is higher than during the baselines without the
odorant present; each bar is above the average baseline peak value. However; there is not much
variance shown for the startles during the time between the odor releases. As shown in Table III,
there is only 1% confidence. Based on the data represented in Figure 2d, fox urine appears to
elicit the greatest behavioral response in a temporary, transient fashion. At the moment the
odorant is released, the rat startle is dramatically greater than the startles seen during the
baselines and in the period after the odor is released. Table III shows that these results are also
inconclusive. Female rat urine, represented in Figure 2e, elicited a higher response than baseline
though not much variance in the startle response. Analysis of variance in Table III shows that
there is little confidence in effect of female rat urine. Propyl butyrate appears have the least
variance in startle, as shown in Figure 2f. The startle in the presence of the odorant is greater
than baseline, but there is little variability between the trials. Inferential statistics in Table III
gives little confidence in the significance of any variance, showing any variance is likely due to
chance.
Electrophysiology Recording
Information from the electrodes were processed amplified, then displayed on a monitor
(Fig. 2). The waveforms displayed are the difference of the input from a reference electrode and
the signals picked up by the test electrode. This reduces electrical noise in the data, allowing for
the clear comparison of the morphologies between different electrical spikes of potential nearby
action potentials. The recording of the data began after the electrode had reached a region where
the amplitude of firing neurons reached around 2-3 times the ambient electrical noise. One cell
could easily be observed to consistently fire with a similar amplitude and frequency throughout
the experiment, regardless of the presence of an odorant.
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After the recording of electrical input was complete, the data was analyzed by the
program Offline Sorter. Every electrical signal that had an amplitude that exceeded a specified
threshold was recorded as a point on a graph with the peak-valley value (an indicator of
amplitude) on the x-axis and the time of the experiment on the y-axis (Fig. 3). The points were
then sorted into two groups, two clumps of points most likely to have originated from the firing
of a neuron near the inserted electrode. They were labeled cell 1a (region on the left) and cell 1b
(region on the right). At the beginning of the recording, the difference in amplitude between the
two cells is evident, but the amplitude of cell 1b decreases over the course of the experiment.
About ⅗ through the experiment, the electrode began to record either less frequent or weaker
action potentials, but began again to detect greater numbers of higher amplitude action potentials
after around ⅘ of the recording had progressed.
Figure 3: Electrical information displayed as
multiple cells fire action potentials with
amplitudes exceeding the preset threshold.
Figure 4: Graph of the electrical spikes that exceed
a set threshold with peak-valley values on the x-axis
and time on the right, separated into two separate
units.
Figure 5: Histogram (periform raster) of
the number of action potentials at
specific times relative to the release of 5
neutral odors (heptanol, isoamyl acetate,
propyl butyrate, heptanone, limonene)
and one experimental odor (fox urine) at
time 0 seconds to time 2 seconds
(experimental cell 1b).
The data was then processed by the Neuroexplorer software. It first generated periform
rasters, histograms of the frequency of action potentials at certain times relative to the
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presentation of an odorant from time 0 seconds to 2 seconds (Fig. 5). The first five odorants were
presented as neutral control odors to the rat while the sixth, the fox urine, was the pheromone
targeted by the experiment. The data was expected to match the results gathered from a different
experiment conducted on a rat brain exposed to the same 5 neutral control odors as well as
octadiene (Fig. 5). These cells demonstrated consistent and immediate spikes of activity during
the odorant presentation, especially during the presentation of the heptanol, isoamyl acetate and
octadiene.. It is clear that the firing of the experimental cells are far more random and less
clustered around the two-second odor presentation window than predicted across all odorants.
The bars over the histograms, marking the firing of neurons at specific times during each trial,
also demonstrate the decrease in amplitude seen over time in Figure 2.
Figure 6: Histogram (periform raster) of the
number of action potentials at specific times
relative to the release of 6 neutral odors
(heptanol, isoamyl acetate, propyl butyrate,
heptanone, limonene, and octadiene) at time 0
seconds to time 2 seconds (model cell 1c).
Figure 7: Cross correlogram of the two
experimental neurons detected by the inserted
electrode displaying the frequency of firing of cell
1b at a specific time relative to the firing of cell 1a
at time 0 seconds.
Figure 8: Cross correlogram of two of the model neurons
displaying the frequency of firing of cell 1c (model) at a
specific time relative to the firing of cell 1a (model) at time 0
seconds.
A cross correlogram was also generated by the program to indicate whether the proximity
of the neurons was reflected in connected activity. This would be demonstrated if a neuron
consistently fires at a specific time before or after another cell fires. A cross correlogram was
generated for the two cells identified by the experimental electrode data (Fig. 6). The time of
firing of cell 1b when compared to the firing of cell 1a (at time 0) seems random when compared
to the near synchronization seen in the two cells recorded in a separate experiment (Fig. 7).
Their spatial association is reflected in the close temporal relationship in their activity.
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DISCUSSION
This experiment focused on the relationship between predator pheromones and the
behavioral responses and responses on the corticomedial amygdala of rats to these odorant
stimuli. The three parts to the experiment analyzed anxiety in rats in different ways. The open
maze experiment found that while rats do show apparent recognition of odors from rat and fox
urine, there is not conclusive evidence to show that rats exhibit an innate fear or aversion
response to these odorants. The acoustic startle experiment found that the rats exhibited a phasic
fear response to the fox urine odorant, but had no definitive conclusions for any of the odorants
presented due to a lack of statistically significant difference in startle reaction for all of the
odorants. The data from the electrophysiology experiment did not support the hypothesis that
neurons would respond differently to the presentation of a predator odor.
Open Maze
Several predictions were made on the reactions the rats would have to the various
odorants. The first odorant tested was cat urine. Researchers predicted that the rats would show
at least a slight aversion to the cat urine due to an innate fear within the rats that would cause
aversion to the pheromones present in the odorant. However, the data (Fig. 1a) shows results that
contrasted with the expected results. The inferential statistical analysis indicates that there was a
significant difference in the amount of time the rat spent in the target zone and the zone opposite,
but unlike expected, it spent a greater amount of time in the opposite zone than the target zone.
These findings do not support that rats have an innate aversion to cat urine. This does, however,
indicate that the rats recognized the cat urine odor as something more than a regular odor such as
propyl butyrate (Fig. 1a). The researchers observed that the rat spent a great amount of time
smelling the odorant, as indicated by the results. Although the design of the maze does not allow
for a confident conclusion to be drawn with these results that there is an aversion, it can be noted
that the rats do in fact have an innate recognition to cat urine. Since none of these rats had
previously been exposed to a cat, they may not have immediately recognized that the odor
belonged to a predator and may not have sensed any apparent danger. Furthermore, fox urine
was also tested, and it was predicted that the fox urine would also cause an aversion as strong as
or even stronger than the cat urine. Similar to with cat urine, the rats spent a significantly higher
amount of time in the zone with the odorant than the opposite zone. Once again, this is peculiar
because previous studies have shown that rats have an aversion to the pheromones released from
fox urine (8). Conclusive evidence that the fox urine caused anxiety cannot be drawn from the
maze. The neutral propyl butyrate did not show a significant difference in time spent in target
and opposite zones, but both the cat urine and fox urine did, indicating recognition of the odors.
The results suggest that an open environment may not be ideal for testing anxiety levels in rats
because innate recognition of the odorants, although it may have caused some anxiety, was all
that could be directly measured rather than an innate fear.
The third odorant that was tested was female rat urine. Since all the rats used in the
experiment were male rats, it was expected that the rats would spend the greatest amount of time
in the zone with the female rat urine. It was found that for most of the rats (Fig. 1a), the rats did
spend more time in the zone with the female rat urine than the opposite zone. The results were
not statistically significant, but if more rats and more trials were used, the trend shows there
might have been a statistical significance. The excitement of the rats may account for the lack of
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statistically significant evidence even though the rats seemed sexually aroused. These qualitative
measures indicate that there may have been more than simply recognition of the odorant and that
there was some sexual excitement.
Error Analysis
The study was designed to test the effect olfactory senses have on innate fears in rats and
the behavioral reactions rats have to pheromones from various animals. A factor of concern
when evaluating the results is the potential effects of habituation in the rats in which repeated
exposure to the environment lessens the response to the stimulus. After repeated exposure to the
scents, realization that there was no apparent danger present may have affected the rats’
behaviors. Habituation to the maze itself is an important factor that may have affected rat
behavior. The experimental design did not fully accommodate for changes in rat behavior that
may have been due primarily to repeated exposure to the maze environment. This confounding
variable was not accounted for since baseline testing was not done each day of experimentation
due to time constraints. Daily measurement of a baseline would have blocked the effects of
habituation to the maze environment and limited the amount of variables in the experiment. In
future studies, varying the order of the odorants for each rat would also be beneficial as another
precaution in order to block the effects of increased exposure to the maze environment.
Acoustic Startle Chamber
The first portion of testing in the startle response experiment measured a baseline
reaction. In these sessions, two of which occurred on the first day of testing and one at the start
of each day of testing with odors, no odor was introduced and the pulse sequence was a pseudorandom sequence of pulses ranging from 75 to 105 decibels in magnitude. Researchers expected
that as the baseline testing progressed, the rats would demonstrate habituation i.e., the lessening
of response to a stimulus as the subject becomes familiar with, and learns the harmlessness of,
said stimulus. As predicted, a decrease in startle reaction was identified in the progression of the
baseline sessions seen by the statistically significant difference between the first and fifth (last)
baseline sessions, shown in figure 2a. This data indicates that the rats grew familiar with the
chamber such that their neophobia (fear of new things) subsided and that the equipment is acting
how it should in order to have confidence for the later results.
Aside from the separate sessions for baseline testing, each session in which an odorant
was presented commenced with a 30 baseline trials of 95 decibels 15 seconds apart. Researchers
expected to observe a decrease in startle magnitude, as evidence of additional habituation,
however, the results showed quite the opposite. Figure 2b shows that as the baseline trials
progress, the average magnitude actually increases. While unexpected, this can be easily
explained in terms of the study. As 3 out of the 4 sessions with odorant rats experienced
succeeded other sessions (and the rats were given the scents in all different orders) it is quite
likely that the rats had some recollection of the previous sessions, which may have included
odorants like fox urine and cat urine with their predator pheromones, and vague memory that the
odorants were only introduced after a while. Thus, the increasing startle response results from
anxiety from the rats anticipating the odorants’, which they might expect to be a negative
stimulus like the predatory pheromones, introduction coming nearer and nearer.
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In the actual experimental portion of this test, i.e. the sessions in which odorant was
presented, researchers had several predictions about what the data would show. Concerning the
cat urine, researchers predicted that the rats may not show full-blown anxiety-induced behaviors
as a result of presentation of the odorant. The data shown in Figure 2c demonstrated that while
there was an increase in the startle response as compared to the baseline, this was not a
significant increase. This finding indicates little confidence that such a reaction is from innate
fear and instead points to a heightened response as a result of neophobia or some other
confounding variable. The next odor researchers used was female rat urine which was intended
to be a positive stimulus to the rats (as the urine of the females would contain female sex
pheromones)and as such, it was predicted that rats would not show a significantly elevated startle
response to the odor. The data, presented in figure 2e, showed that there was an elevation in
startle response from the baseline trials, though, this difference was not statistically significant.
Once again this indicates that there is little confidence that the increased response is a result of
anxiety from exposure to the female rat pheromones. The observed heightened response can be
mostly likely explained by the fact that the rats are sexually inexperienced, such that this
exposure would have been their first encounter of such sex pheromones so that the rats were
likely quite sexually aroused, possibly resulting in a hypersensitivity to startle. Otherwise the
heightened response could be the result of neophobia, similar to the reaction to cat urine.
The third odorant presented was the propyl butyrate, or the pineapple oil, which acted as
a control odor in the experiment as it was the only odorant presented which contained no
pheromones. Because it was a control, researchers expected no enhanced startle response to the
odorant. The data, summarized in figure 2f, shows that the rats actually had the greatest overall
increase in startle response relative to its baseline (even though it technically was not statistically
significant) which was quite unexpected. It seems plausible that the elevated response was the
result of irritability from the rats having been kept in the small chamber for an extended period
of time and for more sessions than they would customarily experience.
Finally, researchers predicted that the rats would show an elevated, and likely sustained,
startle response in the presence of fox urine. The data shown in figure 2d however confirms most
of this prediction as it shows an increased response to the sound pulse when the odorant was
presented, but the increased response is only apparent at the sound pulse right when the odorant
is presented. Data such as this suggests that the rats definitely experienced innate anxiety to the
pheromones present in the fox urine, but that they experienced more of a phasic fear response,
characteristic of phobias, than the sustained anxiety, characteristic of generalized anxiety
disorder, which researchers expected to observe. Despite the apparent trends however, the
inferential analysis determined this test to be inconclusive, preventing the establishment of
definitive conclusion. Even still, the fox urine, as implied by the data, is the only odorant which
shows an anxiety response and hence the only odorant viable for use in further research on
anxiety.
Error Analysis
This experiment was designed to examine rat’s innate anxiety (or lack thereof) to
different odorants as measured by their tendency to startle (a common anxiety response) upon
hearing a 95 decibel sound pulse. Although researchers made extensive efforts to avoid
confounding variables in the data collection, technology errors and timing related inconsistencies
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have inadvertently done so. A major error in the testing was based around coding problem which
caused a delay in data collection such that the recording to miss the actual startle response and
showed data that falsely indicated little to no reaction to any of the odorants presented. While the
data was collected a second time at a later date, this error caused what should have been a first
exposure to odors, therefore definitely observing innate fear, to become a second exposure,
which introduces new confounds such as habituation or conditioned fear from previous
experience of being startled.
Electrophysiology Recording
One cell was recorded from the first rat. It was not located in the corticomedial amygdala
so it was not surprising that no relationship was found between odor presentation and frequency
of action potentials. Two cells were recorded from the second rat; it was expected that they were
located in the medial nucleus of the amygdala because background neural activity related to
breathing was heard. However, when the action potentials were compared with the breathing
data from the thermocouple in Figure 9, there was no clear relationship between the timing of
respiration and the timing of the firing. This indicates that although the electrode was recording
from the correct region of the brain, the cells that were recorded did not process olfactory input.
Action potentials from the two different cells were divided into separate units during data
analysis. However, neither cell showed activity that suggested a relationship between odor onset
and the frequency of action potentials. Experimental cell 1b in Figure 5 did not show a clear
increase in firing in response to any of the odors. Therefore, it can be concluded that the recorded
cells were not responding to odors. However, it is still possible that they were located in the
medial nucleus of the amygdala, because only 10-15% of cells in this area of the brain are
typically responsive to odors.
It was expected that during the recordings, the cells would increase firing during the 2
seconds of odor presentation. Sample data that was recorded prior to the current experiment has
been included in Figure 6 as an example of the expected cell responses. The cells in this figure
show a distinct increase in firing with the presentation of odors. It was hoped that the cells
recorded during the experiment would show the largest increase in firing in response to the
presentation of fox and cat urine. Such a response in the corticomedial amygdala would indicate
that this region processes anxiety related odors and pheromones.
Future Implications
This study was an investigation into the effect of olfactory stimulation on phasic and
sustained fear in the neurons of the corticomedial amygdala. Future researchers may want to
work to examine the effects of stronger predator pheromones, such as TMT in fox urine. The
experiment conducted involving fox urine showed the greatest potential for larger response in the
amygdala. As the neuronal circuitry of a rat and human are strikingly similar, the application of
rats in such studies would provide scientists with a more lucid understanding of the role discrete
areas of the brain play in anxiety disorders and behaviors.
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