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Transcript
BEHAVIORAL AND NEURONAL RESPONSES TO ODORANT STIMULI
Sevan Asadurian, Brittany Bistis, Daniel Kim, Reshma Modi, Taylor Morrison, Archana
Mupparapu, Sima Parekh, Chitra Parikh, Nikita Shah, Michelle Shui, Ming Wu, Alice Zhang
Advisor: Dr. Graham Cousens
Lab Assistant: Michael Clancy
ABSTRACT
The primary purpose of this research was to contribute to ongoing behavioral and
electrophysiological research examining the role of the olfactory tubercle (OT) in odor object
representation in rodents. Three experiments were conducted. First, procedures for establishing a
successive-cue go/no-go odor discrimination task were evaluated. By the end of the experiment,
the rats were able to discriminate between the positive and negative odors and respond to each
odor accordingly. Second, the effect of natural pheromones was assessed on the acoustic startle
response (ASR), an index of fear and anxiety. Although the ASR was sensitive to pulse intensity
and showed long-term habituation as predicted, no significant change in startle response was
observed in response to predator and rat odors. Finally, acute single-unit and local field potential
(LFP) electrophysiological signals were monitored in anesthetized rats in order to observe how
odors were processed in the OT and the hippocampus, which receives less prominent olfactory
input. In contrast to previous recordings conducted in the lab, none of the cells recorded showed
clear patterned responses to any of the odors presented, suggesting that they were not tuned.
However, a more clear relationship between the peak of the LFP signal and the firing of an
action potential was discovered. This suggests that cells in the brain are firing in groups. In sum,
these findings offer further insight into the olfactory system in rodents and in humans.
INTRODUCTION
As research in neurobiology is continuing to gain momentum, current studies are
focusing on understanding brain circuitry and related processes of learning and memory. More
specifically, experimentation on olfactory processing is necessary to provide data for ongoing
studies on the olfactory tubercle, the region in the brain essential for processing chemical signals
and initiating both behavioral and emotional responses. The results obtained from this research
may lead to greater insight on the functions and overall process of olfactory learning.
The continued study of olfactory systems has allowed researchers to perform clinical
studies to better treat complications such as debilitating diseases and human addictions.
Olfactory function tests can serve as early biomarkers of neurodegenerative diseases, including
Alzheimer’s disease, Parkinson’s disease, schizophrenia, and multiple sclerosis (1). The study of
olfactory function thus allows the exploration of diseases through a non-invasive method. In
addition, the involvement of the olfactory tubercle in motivation and reward has been employed
to study drug addiction through operant conditioning and other associative learning processes. In
one study, it was found that rats will “self-administer”, or press a lever, to directly infuse
cocaine, nicotine, and other addictive drugs directly into the olfactory tubercle (2). This suggests
[3-1]
that the olfactory tubercle may play a critical role in drug addiction, and as a result, drug
addiction has become a primary motivation for the current research on olfactory learning and the
olfactory tubercle. Understanding how the neurons in this region communicate is vital to
understanding how the olfactory tubercle may contribute to drug dependence in individuals.
Olfaction begins with the sensory neurons located in the olfactory epithelium. The axons
of those sensory neurons send impulses to the olfactory bulb, located on the interior surface of
the frontal lobe. After this first relay of information, output neurons send signals directly from
the olfactory bulb to the olfactory cortex, including the piriform/olfactory cortex, anterior
olfactory nucleus, entorhinal cortex, and the olfactory tubercle (OT). The OT, located near the
olfactory bulbs on the anterior portion of the brain, links to the olfactory system. It is also
involved with the brain’s reward circuitry; it is a component of a deep pathway in the brain that
regulates behavior through the release of dopamine (3). Furthermore, it is not necessary for the
neuronal messages to pass through the thalamus between the olfactory bulb or the primary
olfactory cortex and the orbitofrontal cortex.
The piriform cortex and the olfactory bulb send direct projections to the lateral entorhinal
cortex through the lateral olfactory tract. The lateral olfactory tract has been observed to project
into the hippocampus and contain a monosynaptic connection to the amygdala. The
interconnectedness of the olfactory cortices and the limbic system thus hint toward strong
linkage between olfaction, learning, memory, and emotion.
In order to continue current research on the role of the olfactory tubercle and other related
regions of the brain involved in odor processing, learning, and memory, three separate studies
were performed to increase a comprehension of such topics. The first study sought to establish
suitable training procedures for a successive-cue, go/no-go odor discrimination task that will be
used to examine functions of the OT during active odor processing. The second study
investigated the effects of predator and conspecific pheromones on the acoustic startle response
(ASR), a measure of fear and anxiety, which could lead to a better understanding of the role of
the OT in emotional processing. Finally, the third study directly measured the neural activity of
OT neurons in response to neutral odors.
PROJECT 1: SUCCESSIVE-CUE, GO/NO-GO ODOR DISCRIMINATION
Background
An important aspect of olfaction is the ability to detect and discriminate between
different odors. Odors can have informational value; they can be used to predict the availability
or absence of reward or punishment. The objective of the operant chamber experiment was to
condition the rats’ odor learning for a future electrophysical experiment in the lab this summer.
The rats were placed in empty operant chambers and conditioned to discriminate between the
positive odorant, heptanone, and the negative odorant, amyl acetate. When the rats were exposed
to the positive odorant, they were conditioned to move to the water cup to receive their reward of
sugar solution, but when the negative odorant was released, they were conditioned not to move
to the cup, as there was no reward. The experiment tracked the progression of the rats’ odor
[3-2]
discrimination as they realized not to poke their heads in the water cup. This go/no-go successive
cue olfactory discrimination task has been exercised during numerous occasions in previous
experiments including those conducted by Otto & Eichenbaum (4) and Cousens et al. (5). It has
proven to be an effective and efficient system that properly conditions the rats to discriminate
odors and prepares them for testing.
These conditioning procedures may be utilized to induce emotional olfactory learning,
thus allowing an experimental approach in a future laboratory environment. While the third
experiment included in this paper tested the neurobiology of an anesthetized rat through
electrodes, this experiment tested similar stimulants among conscious rats. The testing procedure
allowed the rats to develop a learned behavioral preference for specific odors and conditioned
rats for future experiments in which the local field potential (LFP) in these rats’ olfactory
systems will be tested. Specifically LFP in the rats’ main olfactory bulb and the olfactory
tubercle will be examined to test the coherence of the LFP signal in these two regions during
odor processing.
Materials and Methods
Subjects
A total of 21 Sprague-Dawley rats bred in the laboratories of Drew University were used
in the three experiments. Go/no-go odor discrimination results were obtained from 12 juvenile
male rats, while the acoustic startle project used eight adult female rats as its subjects. In the
third experiment, an adult female rat was used to record action potentials and neuronal activity in
its brain. The female rats were pair-housed while the male rats were individually housed. All
three procedures were in accord with Public Health Service guidelines and were approved by the
Drew University Institutional Animal Care and Use Committee.
Apparatus
The individual sessions were conducted inside three operant conditioning chambers (Med
Associates, St. Albans, VT) . Each chamber was equipped with a pump fitted with a syringe on
the right interior that delivered 0.1 mL of a 10% sucrose solution and a tube that dispensed the
odorant later in the experiment. Located on the left interior of the chamber was a nose poke
portal and a light above it to attract attention. All the data was monitored and recorded through
the MED-PC program. Odors utilized during the experiment were heptanone and amyl acetate
diluted to 350 ppm in odorless mineral oil.
[3-3]
Procedures
Prior to conditioning, the rats’ water supply was mildly restricted in order to ensure they
would value the sucrose water as a reward. The subjects were first given a 12-hour, nose poke
response-shaping session which terminated after a maximum of 100 responses. Next, the male
rats performed in a series of baseline trials. These baseline trials ran for an extended period of 45
minutes to allow them to adapt to the chamber and the task at hand. At the start of the
conditioning phase, the rats were given the sucrose solution in both the head entry cup, where the
solution was dispensed, and in the nose poke portal. This prompted the rats to associate these
markers with a produced outcome. The subjects ran three preliminary trials without exposure to
the odorants. Then, upon learning how to correctly enter the nose poke portal of the training
apparatus, they were given the sucrose solution as a reward. After completion of the three
baseline sessions, the experimental trials began. These trials ran for 30 minutes. During these
trials the rats were exposed to the positive scent - heptanone. Each rat underwent the trial in sets
of three. After all the rats had completed one day’s worth of the task, they were given 15 minutes
of unlimited access to water. These recorded trials differed from the baseline trials because they
employed the use of the first scent after each nose poke. After the completion of the heptanone
trial, the negative odorant was introduced - amyl acetate. When the amyl acetate was released, if
the rats poked their noses, a four second period of darkness ensued as punishment. In these trials,
after each nose poke, one of the odorants, was randomly selected by the coded software to be
released. Six seconds were given to register a head entry after a nose poke before the next trial
began. In these trials there were four possible outcomes, a hit (nose poke for heptanone and head
entry), miss (nose poke during heptanone and no head entry) , withhold (nose poke during amyl
acetate and withhold from head entry), and false positive (nose poke during amyl acetate and
head entry). The percent correct would be measured as the number of hits and correct withholds
divided by the number of total nose pokes.
Results
The percent correct was recorded and compared among each session: 53%, 71%, 75%,
82%, and 87% as displayed in Figure 2. Although an increase in percent correct was observed
throughout each session as seen in Figure 1, the strength of the results centered around the
increase of correct withholds, which signified that the rats effectively learned through this mode
of conditioning.
[3-4]
Figure 1: The tracking of the progress of the rats’ average number of misses, hits, correct
withholds, and false positives throughout all five sessions. The rats effectively learned through
their conditioning not to move to the water cup when amyl acetate was released but to retrieve
their sugar solution reward once heptanone was released.
Figure 2: Tracking of the progress of the rats’ average percent of correctness throughout all five
sessions.
The overall percent correct increase across the five odor discrimination session is
revealed by a oneway repeated-measures ANOVA, given by the statistic /F/_4,40 = 19.143
(/p/<0.001). The ANOVA F statistic is a test statistic that compares the variability within each
session to the variability among different sessions, and its value is the quotient of the mean
square between groups divided by the mean square within groups. The large quantity for the
ANOVA F statistic (19.143) is primarily due to the great increase in number of correct
withholds; this number more than doubled from the first session to the last one. The average
number of correct withholds increased throughout almost each session: 20, 30, 29, 44, and 45.
[3-5]
While not significant, the results signal a positive progression in the rats’ learning. In addition,
an interesting bias to note is that the rats consistently made a large number of nose pokes, but
despite this, they gradually learned not to respond as much when the negative odorant was
released. Similar ANOVA tests could be run on these measures to see how significant each
change was across the session.
Discussion
It was hypothesized that the male rats would be able to discriminate between the odors
heptanone and amyl acetate. This hypothesis was correct in predicting that the rats would
associate the presence of smell with a reward. As stated in Figure 2, the performance increased
after each consecutive session. As displayed in Figure 1, both the misses and false positive
responses decreased, while correct withhold and hits increased. The data presented in Figure 2
also shows that the number of misses per rat per session were low and had a slight decreasing
trend. The data describing the amount of hits was particularly interesting because of its slow,
steady increase. Despite the increase, however, the amount of hits had less significant change
over the sessions than the other measures. This denotes that the rats, through operant
conditioning, achieved the overarching goal of learning. The rats initial hit responses were larger
than any other data set collected, while the misses were initially lower than the rest of the data.
Both of these results are due to the preliminary baseline training that made the rats aware of the
risks associated with incorrectly performing the task and the reward associated upon successful
completion of the task. The rats’ ability to learn is clearly demonstrated through association of
odors with behavioral tasks.
Although the project was successful, there still remains room for improvement. While
handling the rats, many were not handled over an extended period of time. This made the rats
less docile and more susceptible to anxiety when exposed to human touch. In addition, three of
the subjects were exposed to lights during the trials which potentially provided a distraction for
the rats in completing the task and compromised their light cycle. Furthermore, when analyzing
the data it was evident that subject number 12 lacked data entries. During the crucial pre-lab
operant conditioning, it was apparent that the procedure was flawed due to an absence of the 10
percent sucrose solution. As a result, the subject continued to make nose entries and received no
reward. Therefore, it could not discriminate between the positive and negative odorants. The lack
of entries from this subject proved to be an outlier in the data and as a result, led to the removal
of this subject from the experiment. In addition, when the negative odorant was initially
introduced, it became apparent that there was not enough fluid for a full experiment worth of
trials. Therefore, the negative odorant, limonene, was switched to amyl acetate. Finally, towards
the end of the experiment the subjects were not properly water restricted, which delayed the set
times for experimenting and altered the scheduled trials.
[3-6]
PROJECT 2: PHEROMONE MODULATION OF ACOUSTIC STARTLE
Background
The Acoustic Startle Response (ASR) process is a means of assessing the behavior of
organisms to auditory stimuli. The behavior of rats is measured by a stabilimeter chamber that
measures a whole-body flinch elicited by acoustic input (6). Typically, a brief sound is emitted
within the subject’s chamber as the acoustic stimulus. The subject’s flinch response to the
stimulus is then measured as a waveform of the downward force on a platform within the
chamber. The maximum peak values of these waveforms were obtained, allowing for
comparisons between the rats’ reactions. It was expected that the intensity of a subject’s startle
response would be correlated with the level of the pulse released. Thus, a louder acoustic input
would be expected to elicit a more severe response from a subject.
Pheromones are chemical signals important for intraspecies communication. Essential
information transferred by these signals include sexual attraction, the presence of danger, and the
location of another individual. These chemicals are necessary for individuals of a species to
maintain constant communication amongst each other in order to ensure survival and
reproduction. Furthermore, the strength of the olfactory messages emitted by subjects remains
constant when animals are birthed or housed in laboratories rather than in their natural
environments (7). Pheromones are released partially via the urine of animals. In both a natural
environment and a laboratory setting, animals of the same species may detect pheromones this
way. Previous studies have shown that an increase of stress pheromones, natural alarm odors
emitted by rats, can increase anxiety in other rats that are in the presence of them (8).
Furthermore, although it has been observed that trimethylthiazoline emitted by foxes may not
affect the response of rat subjects in acoustic startle (9), ongoing work in our lab has shown that
rats will spend more time investigating a fluid cup with fox urine than one with neutral odors.
This procedure sought to explore the significance of the olfactory tubercle in emotional
learning and association by investigating whether the presence of specific odorants would
increase or decrease a rat’s startle response. Specifically, three odorants - male rat urine, female
rat urine, and fox urine - were utilized to emit pheromones specific to each variant. It was
believed that each odorant would elicit a different response from the rat subjects due to the innate
behavioral responses to the presence of male rats, other female rats, or predators.
Materials and Methods
Apparatus
The ASR was assessed using startle chambers (Med Associates, St. Albans, VT). Inside
each chamber was a cage for the rat to remain in during testing. A red light, fan, and speaker
were also present in the chamber. The platform of each cage was equipped with a load cell which
recorded the force with which the rats pushed off when startled. The interior of the chamber was
[3-7]
lined with a sound-insulating foam to isolate the sounds inside the chamber and prevent exterior
noise from entering the chamber.
Procedure
Eight adult female rats were used as subjects in the experiment. The subjects were
handled by the researchers for approximately five minutes a day for three days in order to reduce
startle response to human contact, a confounding variable. Three sessions of baseline testing
were then performed. Each subject was housed alone in a startle chamber with a constant 65 dB
white noise, as studies have shown that housing multiple rats in the same cage may alter
behavior (7). After a five minute acclimation period, during which no pulses were emitted, the
75, 85, 95, and 105 dB pulses began in a pseudo-random sequence, with thirty seconds between
pulses. Each subject underwent 40 pulses (10 of each decibel level). Startle responses were
recorded.
The urine-based testing was then performed using a within-groups design across four
days, consisting of eight sessions in total. Before each session, the startle chambers were cleaned
using isopropyl alcohol, and litter pans were rinsed thoroughly in order to ensure the subjects
were not exposed to other rats’ odors. Approximately 15 minutes before each session, the odors
were placed in the chambers to ensure proper permeation. About 7 mL of each liquid - water
(control), female urine, male urine, fox urine (trimethylthiazoline) - were kept in open bottles, in
front of the rats in their respective chambers. Once the rats were placed in the chambers, the
doors were closed, the lights were switched off, and an automated program was started to emit
pulses in a pseudo-random sequence and record the startle response.
Results
As the trials progressed, the rats’ responses decreased in magnitude. Before any odors
were introduced, three baseline tests were run to observe their initial responses and allow the rats
to adjust to the startle chambers. A T-Test calculator was used to calculate a t7-value of 2.744,
with a p-value of less than 0.05 for the changes between the first two baseline tests. Therefore,
between the first two baseline tests, it can be said that, with 95% confidence, there was a
statistically significant decrease in the rats’ responses. This intentional habituation served to
normalize the startle response before the odorant trials. In the third baseline test, the startle
chambers malfunctioned and did not provide the auditory pulses, rendering that data invalid.
However, following the third baseline test, this problem was corrected for all following tests.
There was also a marked difference in the rats’ responses depending on the intensity of
the auditory pulses. Using oneway repeated measures ANOVA, an F statistic was calculated,
given by F3, 21 = 2.214 (p < 0.001) for the variations between the rats’ responses to different
sound pulses in the first baseline. This suggests, with 99.9% confidence, that there was a
statistically significant increase in the rats’ responses as pulse intensity also increased.
[3-8]
Figure 3: The above graph, Startle Response (Force) vs. Volume (dB), displays how the rats
reacted to the auditory pulses with the various odors present in the startle chamber. Each colored
line represents the rats’ performances with the control and each odorant.
Finally, the effects of the pheromones on the rats were determined by comparing the rats’
reactions to the pulses while open containers of the respective odorants were placed in the startle
chambers. As seen in Figure 3, there was a slight suppression in the rats’ responses with the
odors present compared to the control. However, the p-values for the odor groups were much
greater than 0.05, implying that there were no statistically significant differences in the rats’
responses with the pheromones compared to the control. Therefore, it was concluded that the
pheromones did not significantly affect the startle response.
Discussion
There was no significant increase in startle response for subjects exposed to female and
male rat odors. It should be noted that this study did not analyze the impact of stress-induced or
alarm pheromones. Previous experimentation has focused on stress-induced pheromones, in
which rodents were exposed to pheromones of rodents that were stressed or in a state of panic. In
the study done by Inagaki et al., an increase in acoustic startle response was noted when the
rodents were exposed to the stress-induced pheromones (8). In this study, however, the rat urine
was extracted from rats that were relaxed, which could account for the lack of increased startle
response.
This study attempted to analyze the effects of predator pheromones by presenting fox
odor through trimethylthiazoline (TMT). No significant increase in startle response was observed
for rats exposed to the TMT compared to those in the control. This can be explained by several
factors. As indicated by a study done by Wallace KJ et al., an increased startle response would
not be predicted, but instead a freezing response would be expected. In each of four experiments,
the rats exposed to TMT elicited a significant freezing response when compared to those not
exposed to TMT (10). In contrast, a study done by Endres T et al. shows an increased startle
response when the rats were exposed to TMT (11). However, all of the previous conducted
experiments used male subjects, while this study used female subjects. The discrepancies in
[3-9]
results may be explained by discrepancies in behavior between male and female subjects.
Regardless, the results of this study indicated that the fox urine did not increase startle response
in rats.
Although all rats were handled prior to baseline testing, each rat may not have been
handled for an equal amount of time. This may account for startle response discrepancies
between rats observing the same decibel level pulses. During the third baseline session, pulses
may not have been administered, as startle responses were not observed. If so, the rats may not
have had sufficient exposure to the pulses before the odors were introduced, and therefore, would
demonstrate higher startle responses than anticipated. One technical issue arose in the analysis of
startle response peaks. In some instances, instead of the highest peak value, a lower peak value
was recorded, which falsely displayed lower startle responses than were actually observed. In an
attempt to reduce the impact of this error, peak values were averaged among sessions.
PROJECT 3: SINGLE-UNIT AND LOCAL FIELD POTENTIAL RECORDING
Background
The 2010 study Smelling sounds: olfactory sensory convergence in the olfactory tubercle
showed that neurons in the tubercle selectively responded to odors and that the tubercle may be
involved in cross-modal modulation and direct multimodal convergence. The 2015 study Impact
of olfactory fear conditioning on olfactory tuning in cortical and amygdaloid nuclei provided
data that neurons in the olfactory tubercle are odor selective and also demonstrated how firing
rates of single cells fluctuated with respiration. Furthermore, cells clustered in similar locations
appeared to be similarly tuned, indicating “functional local circuit organization” (12). Since the
olfactory tubercle receives direct input from the major olfactory bulb, significant activity in the
layer I of the olfactory tubercle would be anticipated (3). Therefore, a high frequency of LFP and
a high frequency of individual action potentials would be anticipated. The hippocampus is
disynaptically connected to the major olfactory bulb; the main olfactory bulb projects to the
lateral entorhinal cortex which then projects to the hippocampus. Since this route is less direct
than that in the olfactory tubercle, less odor representation would be expected.
The purpose of the present work is to add to the dataset collected in the 2015 study. To
determine how and with what frequency individual action potentials were firing, in vivo
extracellular single unit recordings were conducted. The frequencies of the neural activity in the
olfactory tubercle rat vary in response to different odorants being administered. While the
primary focus of research was to study the olfactory tubercle, a recording was done from the
hippocampus for sake of comparison with a region of cells less responsive to odors. These
varying frequencies can be attributed to the tuning of the individual cells. A variety of odorants
produces a variety of tuning breadths to distinct odorants, with cells in close proximity generally
tuned to the same frequency (12). The single unit recordings, coupled with the local field
potential (LFP) data, aid in observation of the general trend of odor representation in a particular
region of the brain. Data using the LFP utilizes electrodes to monitor the electrical signal of
thousands of different neurons in one region of the brain, enabling easy recognition of areas of
[3-10]
high and low electric activity. Subsequent receptive field analysis was conducted to determine if
any of the cells recorded were odor responsive or odor selective. By observing the activity of
specific neurons, it is possible to understand how individual cells represented the stimulus
information. Furthermore, the single unit recording enables specific analysis of how information
is represented in the brain and how small clusters of cells (neural circuits) function in response to
varying stimuli. Recording the LFP signal in urethane-anesthetized rats enables easy observation
of how general populations of neurons in a specific region of the temporal lobe are affected by
different stimuli. The correlation between the peaks in the LFP and the firing of action potentials
by individual cells shows the network properties of neural circuits.
Materials and Methods
Surgical Procedures
In order to access the brain, a surgical procedure was completed. The logistics of the
procedure were altered slightly depending on the region of the brain being analyzed, as the
hippocampus is accessed from the top of the head while the olfactory tubercle is accessed from
the side of the head. Before the procedure, the rat was injected with 3 milligrams of urethane per
kilogram of body weight. Isoflurane was used as a supplementary anesthetic when deemed
necessary during the surgery. Once the rat was fully anesthetized, it was placed in the stereotaxic
instrument and held in place with ear bars and an incisor bar. An electric razor was used to shave
the rat’s fur from the ear line forward. To record from the hippocampus, an incision was made
along the midline of the rat’s skull from the incisor bar to the ear line. The skin was pulled aside
by clamps to reveal the skull. An “X” shape was cut into the connective tissue on the skull with a
scalpel, and the connective tissue was then scraped away and cleaned with hydrogen peroxide.
To find the target location for the probe, an electrode was positioned at Bregma, where the
coronal suture meets the sagittal suture. This was used as a reference along with measurements
from the rat atlas to locate the target position. This location was marked with a drill and a 2 mm
square of the skull around it was removed. A saline solution was then used to wash away any
remaining bone pieces and clean the area.
Electrophysiological Recording Procedures
After the surgery, the rat was placed in a Faraday cage with grounding wires to reduce
electrical noise. A thermocouple (# 5TC-TT-K-36-36, Omega Engineering, Inc., Stamford, CT)
was inserted into the rat’s nostril to detect respiration rate. Multiple wires were connected to
transmit the signal from the electrode to an amplifier (A-M Systems Models 1800/3600) that
filtered between 500 and 3000 Hz and amplified 10,000x and to a digitizer (32 kHz, Power14013, Cambridge Electronic Design, Cambridge, UK). The LFP signal was also filtered between 0.1
and 100 Hz and digitized at 1 kHz. For the single channel recording, a blunted reference
electrode and a tungsten microelectrode (12 MOhm, A-M Systems, Sequim, WA) were attached
to the stereotaxic apparatus. A sixteen-channel probe from NeuroNexus was also used to record
data from multiple sites. The electrodes were positioned at the target location within the
craniotomy window by adjusting the knobs on the stereotaxic instrument corresponding to
movement along all three axes, utilizing Bregma as a reference. Then, the electrode was
[3-11]
positioned at the surface of the brain and lowered slowly through the piriform cortex, pausing
every few seconds to allow the electrode to settle in place. Once a clear signal was detected at
the correct depth, a template was made for the action potential in Spike2 (Cambridge Electronic
Design, Cambridge, UK). When recording began after a 2-minute baseline session, six neutral
odors, 1-heptanol, 2-heptanone, isoamyl acetate, limonene, 1,7-octadiene, and propyl butyrate,
were presented in a pseudo-random order for 60 seconds each. Odors were diluted to 350 ppm in
mineral oil and presented from a flow-dilution olfactometer in nitrogen gas.
Data Analysis with Offline Sorter and NeuroExplorer
Once the data was collected, it was analyzed using several programs. Offline Sorter
(Plexon, Inc., Dallas, TX) was used to group similar waveforms and discriminate between action
potentials from different cells. The data was sorted using a graph of the action potential signals
according to principle component (PC) analysis and timestamp. The data points of small
amplitude near the threshold are generally considered electrical noise and are not included in the
groupings. The remaining points are cluster cut into groups, distinguishing specific cells. The file
was then exported to NeuroExplorer (Nex Technologies, Madison, AL), which created figures of
neural activity, including perievent rasters and trial bin counts. A program on Matlab (The
Mathworks, Natick, MA) assigned odor timestamps to the data.
Results
Cells
After analyzing and cluster cutting the data points in Offline Sorter using the variables
PC 1, PC 2, PC 3, and timestamp, a total of five cells were found. In the 7/15 recording, the
target area for the recording was the anterior OT. Two cells were found during the OT recording.
In the 7/22 recording, the target area for the recording was the hippocampus. Three cells were
found during the hippocampus recording.
Olfactory Tubercle Recording 7/15
In the 7/15 recording, there were no clear patterns of activity observed in either of the
cells found in response to the presentation of the odors. Neither cell changed its firing pattern
consistently in reaction to the odors, implying that the cells were firing randomly. In addition,
they had a low firing rate overall and thus, the odors did not affect the cells significantly.
Figure 4: Trial bin counts were graphed for the second cell of the 7/15 recording correlating the
local field potential (LFP) signal with the second cell’s action potentials. The x-axis indicates the
[3-12]
time the cell fired an action potential. The y-axis plots each individual action potential. The
colors on the graph indicate the LFP signal before, while, and after the action potential was fired.
Although the correlation between the LFP and the action potentials was not the focus of
the recording, an interesting pattern was observed on the screen recording the cell potentials. It
appeared that whenever an action potential was fired, there was a spike in the LFP. When
analyzed using trial bin counts, the pattern indicated a clear correlation between the firing of the
action potential and the LFP signal. Based on the red-yellow strip in the center of the graph,
which indicates the LFP signal was at a peak, a correlation between action potentials and LFP
can be inferred by the pattern of the coloration. A peak in LFP indicates that cumulative effect of
the inhibitory postsynaptic potentials and excitatory postsynaptic potentials was high relative to
the rest of the recording (Figure 4).
Hippocampus Recording 7/22
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Figure 5: Perievent rasters and firing rate histograms showing pre-odor and odor periods for 6
monomolecular odorants at 350 ppm were created for the hippocampus recording. Each odor was
presented for two seconds. The triangles represent the time of odor onset. Each row shows a
different cell and each column shows a different odor (L to R: 1-heptanol, 2-heptanone, isoamyl
acetate, limonene, 1,7-octadiene, propyl butyrate).
In Figure 5, which shows the first cell found in the 7/22 hippocampal recording, there is a
slight pattern in the cell’s response to 1-heptanol and limonene. During some trials, the cell was
not firing before the presentation of the odor, but after the presentation of the odor, the cell began
to fire. However, in other trials, the same cell did not appear to respond to the odor, firing at
similar rates before and after the odor introduction. For the second cell identified during this
recording, there is too little data to see any pattern. For the last cell identified in this recording,
there are too many marks to clearly discern a pattern.
Discussion
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Two data sets were collected over the course of this experiment: one from the olfactory
tubercle and the other from the hippocampus, both of which are involved in the processing of
odorous stimuli. In the first recording from 7/15, two groupings of cell were observed with a
third grouping of clear electrical noise acquired from the environment. Two actively firing
neurons were found and separated from the nonessential data that was collected.
Though these neurons were recorded, an in-depth analysis (Figure 5) reveals a random
distribution of action potentials, independent of variations in odors. They were identified and
sorted, allowing for the study of individual cells. These graphs, in which frequency of action
potential is plotted against time, demonstrate that while the odor is presented, there is no pattern
or significant difference in electrical activity. Had there been an increase in firing rate, further
scrutiny would reveal data similar to that of Figure 6, taken from a prior study on olfaction in the
brain. A large, consistent rise in firing rate was observed upon presentation of the odors,
demonstrating that the neuron analyzed was responding to those specific stimuli, but this did not
occur in the recordings of the olfactory tubercle. Thus, the two neurons observed were untuned,
had a low firing rate, and may not have been directly connected to the odor pathway during the
recording. The overall electrical activity in the region, however, measured as local field potential
(LFP), is similar to the single-unit firing pattern of the two neurons observed in the 7/15
recording, indicating that other neurons in the region may be incorporated in the same pathway.
Figure 6: This figure shows the clear response of a cell to various odors. The pattern of
increased cell activity after the introduction of the odor is clear in the high density of spikes. This
data was retrieved from the “Impact of Olfactory Fear Conditioning on Olfactory Tuning in
Cortical and Medial Amygdaloid Nuclei” poster created for the Faculty for Undergraduate
Neuroscience convention.
The hippocampal recording revealed two clearly distinguishable cells and one cell which
was unable to be separated from electrical noise (Figure 5). The graphs in the first two rows
plotted the single unit data collected from the identifiable neurons. Although the second neuron
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was found towards the end of the session allowing for insufficient observation time, and the third
cell was indecipherable from electrical noise, there was a slight spike in electrical activity after
odor presentation to the first cell. In most cases, however, there was no viable pattern, as
demonstrated by the positive data set example in Figure 6, above. A comparison with this
positive analysis suggests that the first neuron in the hippocampal study may have been tuned to
the first odor, but the remainder of the results was inconclusive. The probe was either incorrectly
placed in the anterior section of the olfactory tubercle or the identified neurons did not
discriminate between various odors or are uninvolved in olfactory processing, due to the small
sample size. Further research must be done to investigate whether the first hippocampal cell was
tuned or if it was simply a matter of chance. In the Xu-Wilson experiment on olfactory activity in
the mouse cortex, 27 mice were recorded from in total, and there was a very obvious trend in
electrical activity in response to a variety of odors (3). The observed neurons were tuned, and the
data was numerous and consistent enough to provide reliable results. Ideally, with a larger
sample size, possible connections between odor discrimination and neurons in the olfactory
tubercle or hippocampus can be determined.
GENERAL CONCLUSIONS
Research conducted in these three studies has contributed to ongoing projects involving
olfaction in rats. The successfully trained subjects from the operant learning project can be used
in future experiments to elucidate the effect of such learning on neural pathways. For example,
they could be used in future electrophysiology studies to determine the effect of associative
learning on the firing rate of cells. In addition, the Acoustic Startle Response study showed that
the auditory stimuli paired with the pheromone odorants did not have an effect on the rats.
Overall, the behavioral component of the study was successful. Furthermore, while the neural
study was inconclusive, the results could be attributed to a small sample size. However, the study
allowed the lab to gain familiarity with using sixteen channel probes and recording procedure for
future work.
Future implications for this project, in addition to contributing to ongoing research in the
lab of Dr. Graham Cousens, include determining more about olfactory processing in both rats
and humans. Due to homologies in the neural functioning of mammals, in-depth understanding
about olfactory pathways and the complex relationships between different regions of the brain in
rats can aid in human studies and allow for innovations in the fields of medicine and
biotechnology.
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