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The Effects of an Odorous Diluent

Permanent Link: http://ufdc.ufl.edu/UFE0022527/00001

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Title: The Effects of an Odorous Diluent
Physical Description: 1 online resource (32 p.)
Language: english
Creator: Gamble, Katherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Psychology -- Dissertations, Academic -- UF
Genre: Psychology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Odorant diluents are generally chosen because of their odorless qualities, allowing them to dilute a target odorant without affecting its perception. Unpublished results from my laboratory, suggest that the commonly-used diluent mineral oil (MO) is not odorless. To test this possibility, I used four brands of MO, a common diluent for oil-based odorants, in a discrimination paradigm with mice, to test for odor detection and brand discrimination. Studies showed that mice were able to both detect and discriminate among all four MO brands. In a second discrimination experiment, I diluted cineole to identical suprathreshold concentrations in two brands of MO, such that the only difference between the S+ and S- cineole stimuli was the brand of MO used as the diluent. Again, mice were able to discriminate between the two stimuli, responding to cineole diluted in one brand of MO and not responding to the identical concentration of cineole in a different brand of MO. Thus, this discrimination task showed that mice were discriminating between the two MOs despite the presence of cineole. These results suggest that mice are able to discriminate among MOs when presented alone and also when in a mixture with an intense odorant. MO is therefore, not an odorless diluent and MO from different sources possess varying notes and must be used with caution in olfactory experiments, as the perception of the odors being diluted may be altered in unknown ways.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Katherine Gamble.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Smith, David W.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022527:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022527/00001

Material Information

Title: The Effects of an Odorous Diluent
Physical Description: 1 online resource (32 p.)
Language: english
Creator: Gamble, Katherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Psychology -- Dissertations, Academic -- UF
Genre: Psychology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Odorant diluents are generally chosen because of their odorless qualities, allowing them to dilute a target odorant without affecting its perception. Unpublished results from my laboratory, suggest that the commonly-used diluent mineral oil (MO) is not odorless. To test this possibility, I used four brands of MO, a common diluent for oil-based odorants, in a discrimination paradigm with mice, to test for odor detection and brand discrimination. Studies showed that mice were able to both detect and discriminate among all four MO brands. In a second discrimination experiment, I diluted cineole to identical suprathreshold concentrations in two brands of MO, such that the only difference between the S+ and S- cineole stimuli was the brand of MO used as the diluent. Again, mice were able to discriminate between the two stimuli, responding to cineole diluted in one brand of MO and not responding to the identical concentration of cineole in a different brand of MO. Thus, this discrimination task showed that mice were discriminating between the two MOs despite the presence of cineole. These results suggest that mice are able to discriminate among MOs when presented alone and also when in a mixture with an intense odorant. MO is therefore, not an odorless diluent and MO from different sources possess varying notes and must be used with caution in olfactory experiments, as the perception of the odors being diluted may be altered in unknown ways.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Katherine Gamble.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Smith, David W.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022527:00001


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1 THE EFFECTS OF AN ODOROUS DILUENT By KATHERINE GAMBLE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Katherine Gamble

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3 ACKNOWLEDGMENTS I thank m y family for standing behind me the past two years. I thank my advisor, Dr. David Smith, for all of his support and guidance. I thank all of the undergraduates who made everything possible.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................3 LIST OF FIGURES.........................................................................................................................5 LIST OF FIGURES.........................................................................................................................5 ABSTRACT.....................................................................................................................................6 CHAP TER 1 ODORANT MIXTURES.........................................................................................................7 2 METHODS.............................................................................................................................14 Animals...................................................................................................................................14 Apparatus................................................................................................................................14 Experimental Design............................................................................................................ ..16 Odorants..................................................................................................................................16 Procedure................................................................................................................................17 Training....................................................................................................................... ....17 Threshold Procedures...................................................................................................... 18 Mineral Oil Discrimination............................................................................................. 18 Cineole in Mineral Oil Discrimination............................................................................ 19 3 RESULTS...............................................................................................................................21 Cineole Threshold.............................................................................................................. .....21 Mineral Oil Discrim ination..................................................................................................... 21 Cineole in Mineral Oil Discrimination................................................................................... 21 4 DISCUSSION.........................................................................................................................26 5 CONCLUSIONS AND I MPLICATIONS............................................................................. 29 LIST OF REFERENCES...............................................................................................................30 BIOGRAPHICAL SKETCH.........................................................................................................32

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5 LIST OF FIGURES Figure page 1-1 2-deoxuglucose images for propionic acid and (+)-lim onene........................................... 12 1-2 Habituation trials for MO and three com plex odorants..................................................... 13 2-1 Knosys liquid-dilution olfactometer.................................................................................. 20 3-1 Cineole threshold for give C57Bl/6J m ice in my laboratory............................................. 23 3-3 Mineral oil discrimination trials be tween four different brands of MO. ........................... 24 3-4 Discriminations of cineole concentra tions in two different brands of MO ....................... 25

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6 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Masters of Science THE EFFECTS OF AN ODOROUS DILUENT By Katherine Gamble August 2008 Chair: David Smith Major: Psychology Odorant diluents are generally chosen because of their odorless qualiti es, allowing them to dilute a target odorant without affecting its perception. Unpublis hed results from my laboratory, suggest that the commonly-used dilu ent mineral oil (MO) is not odorle ss. To test this possibility, I used four brands of MO, a co mmon diluent for oil-based odorants, in a discrimination paradigm with mice, to test for odor detection and brand discrimination. Studies showed that mice were able to both detect and discriminate among all four MO brands. In a second discrimination experiment, I diluted cineole to identical suprathreshold concentr ations in two brands of MO, such that the only difference between the S+ and Scineole stimuli was the brand of MO used as the diluent. Again, mice were able to discri minate between the two stimuli, responding to cineole diluted in one brand of MO and not resp onding to the identical co ncentration of cineole in a different brand of MO. Thus, this discrimination task showed that mice were discriminating between the two MOs despite the presence of cineole. These results suggest that mice are able to discriminate among MOs when presented alone and also when in a mixture with an intense odorant. MO is therefore, not an odorless diluent and MO from di fferent sources possess varying notes and must be used with caution in olfactory experime nts, as the perception of the odors being diluted may be altered in unknown ways.

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7 CHAPTER 1 ODORANT MIXTURES Proper control of stim uli is an essential component of psychophysical experimentation, and adequate control can only be obtained with a full understanding of all aspects of the stimulus being applied. In olfactory rese arch it is necessary to rigor ously control the odorants being tested, but it is equally important to control the s ubstances in which the test odorants are diluted. Most olfactory stimuli used in experimental st udies are soluble in water, alcohol, or oil, and are often diluted with deionized water, di ethyl phthalate, or mineral oil, respectively, substances with properties similar to that of th e odorant. Because a dilu ent is mixed with, and thus perceived simultaneously wit h, a target odorant, diluent choi ce is critical. A diluent is required to be inherently odorle ss, serving the purpose of dilu ting an odorant or mixture of odorants without altering the origin al perceptual characteristics. If a diluent has an odor, the way in which the target odorant, or odorants, are perceived will be altered due to a phenomenon called mixture interaction (Laing et al. 1989; Smith 1998; Rospars et al. 2008). Mixture interaction occurs when the res ponse to an odorant in a mixture is different from the response to that odorant when presented indi vidually. Derby et al. (1989) suggested th at because sensory information is carried by olfactory neurons, a mixture interaction may alter the message being sent by individual odorants, and thus affect c oding within the olfact ory bulb. Thus, when investigating mixtures, it must be considered ho w odorants interact in the mixture as well as how this mixture is being perceived by the animal. In the context of the present work, interactions between the odorant of interest ma y interact within an odorous diluent and result in unintended stimulus properties. Binary mixtures are generally said to have one of two perceptu al qualities, either configural or elemental (Livermore and La ing 1998; Kay et al. 2005). A mixture has a

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8 configural perception when two odorants with sim ilar molecular structures are combined to form a mixture that does not smell like either of its two components, but has a mixture-unique synthetic quality (Smith 1998; Kay et al. 2005 ). Elemental mixtures, on the other hand, contain two odorants with dissimilar molecular stru ctures that, when combined, are perceived as the sum of the two odorants (Kay et al. 2005). Overshadowing is another phenomenon that occurs in binary mixtures, where the concentrati on level of one of two odor ants is increased and the perception of the second odoran t is decreased, having been overshadowed by the first (Smith 1998; Kay et al. 2005). Oversha dowing is described in a similar way to odor masking, another phenomenon that can occur when two odors are presented simultaneously (Laing et al. 1989; Livermore and Laing 1998; Wiltrout et al. 2003). In masking, the perception of one odorant is decreased in the presence of a nother odorant, and an increase in threshold level of the masked odorant is necessary in order for it to be detected. Though masking has been described independently from overshadowing, the processes involved seem identical; two odors are present simultaneously, and as the concentration of odor A is increased, it covers up the perception of odor B, so that in order to be detected, odor B must be presented at higher levels in the presence of odor A than would be necessary in a null background. Odor masking in the rat, as described by Lai ng et al. (1989), is virt ually identical to a phenomenon described in Bell et al. (1987) in both humans and rats, which they correctly termed mixture suppression. Bell et al. found that wh en propionic acid (vineg ar) was unable to be perceived by humans in the presence of limonene in a mixture, 2-deoxyglucose (2-DG) uptake in rats showed suppression of the ol factory receptors typically activ ated by propionic acid. Imaging results showed that activation to propionic acid was not covered up, or masked by limonene, but was simply suppressed, making levels of the acid in the presence of limonene elicit less

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9 receptor excitation than would th e same levels in a null background. Masking is a phenomenon where the presence of one odor (odor A) masks, or covers up the receptors neural responses to the second odor (odor B), the one being masked (Moore 1997). Suppression, also occurring at the periphery, is seen when one odor (odor C) suppresses the response to a second odor (odor D), lowering the amount of receptor excitation to th e odor being suppressed (odor D). Though Laing et al. (1989) conducted only behavi oral studies, the resu lts obtained were the same as those in Bell et al. (1987), and thus likely showed mixture suppression and not masking as they claimed. In both experiments, limonene levels were sim ilar, while propionic acid levels were 3-4 magnitudes higher in the Bell et al. than the La ing et al. study. Although the behavioral portion of the Bell et al. study was done in humans, who typically have higher odor thresholds than rodents, this higher level of acid was used in th eir 2-DG experiment with rats where they showed propionic acid receptor suppressi on when limonene was present. Thus, the extension of these studies that Laing et al make to their experiment when acid detection levels were lower, is incorrectly termed masking. They are showing the same thing behavior ally in rats that Bell et al. showed in humans, where receptor excitation to propionic acid is decr eased, or suppressed, by limonene and thus perceived with less intensity than when presented alone. 2-DG images of the olfactory bulb for propionic acid and limonene s how that activity levels have little overlap, suggesting that limonene could not cover up, or mask receptors responding to the acid (Figure 1-1; Leon and Johnson). The intensity of odors in a mixture can al so affect how the mixture is perceived (Livermore and Laing 1998; Wiltrout et al. 2003; McNamara et al. 2007). Perception is dependent on both the concentration and the rati o of components within a mixture, but it is generally the more intense odor within a mixt ure that is perceive d over the less intense

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10 component or components. Laing (1989) found that an odorant only had to have an intensity slightly higher (one to two log units) than a second odorant in the mixture in order to be perceived as more intense. McNamara et al (2007) found that binary mixtures with low intensities combined to form a configural per ception, whereas mixtures with components of high intensity were perceived elementa lly, regardless of the perceptual similarity of the two odors. Wiltrout et al. (2003) showed that when a re ward was associated with components of a binary mixture containing perceptually dissimilar odorants, the rats did not discriminate the mixture from its components, suggesting that the animals detected th e individual components within the mixture and were able to identify th e one for which they were rewarded, both in a mixture and individually. This suggests that dissimilar odorants in a binary mixture can be detected simultaneously, and one can be ignored so that the other can be perceived similarly to how it would if presented independently. Th ese findings support the elemental phenomenon previously described (Livermore and Laing 1998; Kay et al. 2003). Regardless of the way in which two substances interact in a mixture, however, if two odorous substances are combined, the way in which the mixture is perceived, or at least the cues an animal uses to respond, may be affected. Mineral oil (MO), a common diluent for oil-ba sed odorants, is genera lly described as an odorless substance and has been used in ma ny olfaction experiments (Bodyak and Slotnick 1999; Wiltrout et al. 2003; Abraham et al. 2004; Kay et al. 2005; Li et al. 2006; Mandairon et al. 2006; McNamara et al. 2007). In unpublished work from my laborat ory, I replicated a previously published experiment (Linster et al. 2001) in which odorant control was largely based on the assumption that MO was odor less. I conducted a habituation experiment to measure the amount of time mice spent investigating various odorants over a number of 3 minute exposures.

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11 In the experiment performed by Linster et al., MO was used in a habituation test as an odorless control before testing an odorant. Their results showed a very short habituation time, though an exact number of investigations wa s not reported (Linster et al. 2001). In my performance of this same study, I observed that mice took as long, if not longer, to habituate to MO than they did to other, more complex odorants (unpublishe d results; Figure 1-2). Through personal communications with the published reports author (Dr. Christiane Linster), I became aware that the use of MO as a diluent is not always well controlled, with some researchers using multiple brands even within an experiment In addition, most published studies fail to report the source of MO used (Bodyak and Slotnick 1999; Wiltrout et al. 2003; Kay et al. 2005; Li et al. 2006; Mandairon et al. 2006; McNamara et al. 2007; Yoshida and Mori 2007). The possibility that MO may be odorous and, wh en used as a diluent, might affect the perception of a target odor, c ould create unintended changes in target odorants which could complicate comparisons of results. Based on findings from previous resear ch in this laboratory using the habituation paradigm described above, I sought to determine whether various brands (Sigma-Aldrich, CVS, Fisher Scientific and Wa l-Mart) of odorless MO did contain an odor, and if they could be discriminated from one a nother, which would suggest they have their own distinct odors. The purpose of the present study was to compare various brands of MO to one another to determine whether or not some odorless MOs contain an odor, and whether or not those odors and can be discriminated from one a nother by mice. My findings show that MO has properties that may affect the pe rception of odors which it is used to dilute, and this finding should be taken into consideration when disc ussing mixtures or singl e odorants using MO.

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12 A B Figure 1-1. The 2-deoxyglucose images of activation in the olfactory bulb. A) For propionic acid, B) for limonene.

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13 Investigations of Odorants in a Habituation Paradigm0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 123456789101112 Investigation TrialsTime Spent Investigating (s) Mineral Oil Appleblossom Corn oil Tangerine oil Figure 1-2. Habituation trials fo r MO and three complex odorants. Mice investigated MO in 11 three minute trials, while they investigated for fewer trials for the other three complex odorants that should have prompted more investigation than the odorless MO.

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14 CHAPTER 2 METHODS Animals Five C57Bl/6J m ice were used in this study. Mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and a colony main tained at the McKnight Brain Institute, University of Florida. All of the animals were male, 15 to 21 months old and had been previously used in similar behavioral studies. Mice had ad libitum access to dry LabDiet mouse chow ( LLC/PMI Nutrition International, St. Louis, MO ) and were maintained on a 23-hour water restriction schedule. They were weighed daily and were kept at 85% to 90% of their free-body weight (26.5 to 31.5 g). During te sting, mice received no more than 3 ml of a liquid reward of vanilla Ensure (Abbott Laboratorie s, Columbus, OH), a nutritional en ergy drink. At the end of a testing day, they also received a certain am ount of supplementary water based on how much Ensure they consumed during testing, up to a total of 3 ml per day ( National Research Council, 1996). All animals were individually housed in order to regulate the consumption of supplementary water given after testing. All testing was done with the approval and oversight of the University of Florida Institutional Animal Care and Use Committee. Apparatus All training and testing was performed using Knosys comm ercial liquid-dilution olfactometers (Lutz, FL; Figure 2-1). The olfactometers had eight channels controlled by a compressor which delivered air thro ugh a charcoal filter system before entering the olfactometer. Air flow was divided and fed th rough two glass manifolds, one c ontinuously carrying a clean air stream, the other carrying the st imulus stream containing the odor ant to be delivered to the animal. Flow meters were calibrated before each animal was tested, ensuring that the clean air

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15 manifold had a constant stream of 1.95 liters/minute, and the stimul us air stream was constant at 0.05 liters/minute. The clean air manifold delivered air through 3/16 inch ID tubing (C-flex, Cole Palmer, Vernon Hills, IL) into 250 mL plastic saturation bottles containing 10 mL of liquid odorant. The odorant at the bottom of the bottle saturated the air in the head space of the bottle, so that air was able to flow out of a second piece of tubing into the stimulus stream manifold. The stimulus stream then entered a glass mixing ball which created turbulence in th e air flow, mixing the odorant before its final delivery to the animal. Two ports left the mixing ball, and the attached tubing then passed through a final solenoid pinch valve. One of th e tubes was evacuated into an in-line exhaust fan to a room-evacuation syst em, while the tube from the second port was controlled by the final valve, allowing precise delivery of the stimulus to the animal. Airflow from the manifolds into the plasti c bottles was controlled by QBASIC software which opened or closed automate d solenoid pinch valves, allowing precise timing of air into and out of specified bottles. The olfactometer was ab le to deliver up to eight stimuli to the animal, but for these experiments only two stimuli were used at a time. Mice were trained to respond to a target stimulus, S+, while ignor ing the diluent alone, or control stimulus, S-. The odorant was delivered to the animal after the final valve into the base of a glass cylinder, the sampling port, into which the animal was able to nose poke. The sampling port was attached to a Plexiglas behavioral chamber 15 cm deep, 20 wide, and 13 cm tall in which the mice were tested. The odorant was delivered from the final valve into th e base of the sampling port. The sampling port had an opening which fit into a hole in the Plex iglas box through which the animal could poke its head in order to sniff the airflow to dete ct the odorant being delivered. The odor was immediately evacuated through a vent at the top of the cylinder c onnected to the room-

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16 evacuation system. The Plexiglas chamber also contained a fan at the opposite side of the box from the sampling port in order to maintain pos itive pressure within the box, thus keeping the odorant stream in the sampling port and preventing it from entering the behavioral chamber. Experimental Design Each discrim ination block included 20 trials, with the target odorant (S+) and the diluent (S-) each being delivered 10 times in a pseudoran domized order. The behavioral chamber had a conductive metal floor, so that when the mouse licke d a metal lick tube in side the nose port, an electrical circuit was completed. When the animal poked its nose into the sampling port, it broke an infrared photo beam, initiating a trial sequence. The mouse was required to keep its head in the sampling port for at least 200 ms, at which time e ither an S+ or an Sstimulus was delivered to the animal, allowing it to sni ff and respond. The animals received 5 l of liquid Ensure when they responded correctly to the presence of the S+ by licking the lick tube inside the sampling port. If the animal licked the lick tube in response to an Spr esentation, a Failure, or did not respond to an S+ trial, a Miss, the animal r eceived a 5 s time out during which it was not able to initiate a new trial. When the animal responded correctly to an Strial by withdrawing its head from the sampling port following the Sodo rant presentation, it was allowed to initiate a new trial 1.5 s later, once the final valve closed. When a diluted odorant wa s used as an S+, the Sstimulus was always the substance with which the S+ was diluted. This was done so that if there were to be any contaminants or odors in the diluent, it always being present in the form of the Sshould have allowed any of t hose cues to be ignored in the S+. Odorants Four brands of MO were obtai ned from different distributors: CVS (C VS Pharm acy, Inc., Woonstock, RI), Wal-Mart (Cumberland Swan, Sm yrna, TN), Sigma-Aldrich (light; St. Louis,

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17 MO), and Fisher Scientific ( light; Hanover Park, IL). Cine ole was purchased from SigmaAldrich and was of 99% purity. Procedure Training Nave m ice were initially trai ned to place their nose into the sampling port where the odorants were delivered. The port contained the li ck tube from which mice were able to receive a liquid reward for correct responses. Mice were first placed on a training regimen during which they were presented with cineole alone. For the first 20 trials, they were reinforced for each nose poke that broke the photo beam. In the second st age, the animals were required to sample the odor for incrementally longer intervals, progr essing from 0.2 to 1.2 s requirements over 140 trials. They were trained in such a way that, wh en they licked the lick tube in response to a presentation of the target odorant, they received a reward of 5 l of Ensure. If the mouse failed to respond to the odorant presenta tion, they received a 5 s time out during which they could not initiate a new trial. After successful completion of the initial training program, the mice were run on a twobottle discrimination program. The first 40 trials of the discrimination program were used as an extension of the initial training, with the presenta tion of the target odorant alone (100% cineole). For the remainder of the discrimination program, the animals were presented with the target odorant (S+; cineole) for half of the trials, and with the diluent (S-; CV S MO) for the other half of the trials, in a pseudorandom order. It took the mice approximately one week to become fully trained on this program, reaching 85% or higher accuracy in responding to the target odorant and not responding to the diluent.

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18 Threshold Procedures Mice were considered to have passed a con centration level when they pass ed three consecutive blocks of 20 trials with 85% or higher accuracy. Because initial training was done with 100% cineole as the S+ and CVS brand MO as the S-, the same s timuli in the threshold experiment, the animals were able to reach pa ssing levels on the discrimination program with 100% cineole fairly rapidly. Upon successful comp letion of three consecutive blocks with 100% cineole, mice were then tested on 10% cineole di luted in CVS MO as the S+ and CVS MO alone as the S-. As the mice passed a concentrati on level with 85% accuracy on three consecutive blocks, they were moved down by serial dilutions until they were no longer able to discriminate between the cineole S+ and the MO S-. Once mi ce failed a concentration level two days in a row, I determined their threshold. In this experiment, threshold was considered to be the lowest serial dilution at which each mouse was able to pass three consecutive blocks with 85% accuracy. Mineral Oil Discrimination Mice were trained to d etect CVS MO as the S+, in the same way that they were previously trained to detect cineol e. For the training portion of this experiment, an empty jar containing clean air served as the S-. Once the animals su ccessfully completed training, they began running on the two-bottle discrimination program, where CVS MO (S+) was sequentially tested against three different brands of MO (S-). As with the cineole threshold test where mice were discriminating between a cineole dilution (S+) and the MO diluen t (S-), mice were required to pass three consecutive blocks of 20 trials of di fferent MO brand discri minations with 85% or higher accuracy in order to pass a discrimination pair.

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19 Cineole in Mineral Oil Discrimination Discrim ination among different types of MO c ould be a compelling argument for different MOs having distinct olfactory no tes, but this finding would hold the most practical significance if MO were shown to affect the perception of odors for which it was used as a diluent. To address this point, I compared identical suprathreshold levels of cineole using two different brands of MO as diluents by pair ing them in a discrimination tas k. Levels of cineole used in these trials were three to five log concentrations above previously determined threshold levels. The only difference between the S+ and the Sstimuli was the MO diluent, either CVS brand or Sigma-Aldrich MO (i.e., I was rein forcing discrimination of the dilu ent, not the odorant). As with the previous discrimination trials, mice ha d to pass three blocks of 20 trials with 85% accuracy in order to pass the discrimination task.

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20 Figure 2-1. Knosys liquid-dilu tion olfactometer (Bodyak and Slotnick 1999). Manifold 1 contains the clean air stream and Manifold 2 contains the stimulus air stream.

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21 CHAPTER 3 RESULTS Cineole Threshold Three of the five m ice used in this study had a threshold of 10-8% cineole, and two had a threshold of 10-9% (Figure 3-1). As previously describe d, threshold was defined as the lowest concentration at which the animal was able to detect cineole with 85% or higher accuracy on three consecutive blocks. Three mice failed at 10-9% and two failed at 10-10%, making their thresholds 10-8% and 10-9%, respectively. Published data sup port these values; mice of the same strain had a threshold of 10-8% for cineole (Kelli her et al. 2003). Mineral Oil Discrimination All three MO pairs were discrim inated from one a nother by at least two of five mice with 85% or higher accuracy on three consecutive blocks (Figur e 3-3). CVS MO was able to be discriminated from Sigma-Aldrich and Wal-mart MOs by four of the five mice, and from Fisher Scientific MO by two of five mice. Cineole in Mineral Oil Discrimination Identical cineole concentrations three to five log steps above threshold were created using two different brands of MO as diluents. Concentrations of 10-5% and 10-4% cineole were made with either CVS (S+) or Sigma-Aldrich (S-) MO, and were tested against one another in discrimination trials. Thus, mice were required to use the MO diluents to discriminate the two stimuli, as cineole levels were identical in both the S+ and the Sstimuli. All five mice were able to discriminate 10-5% cineole diluted with CVS MO from 10-5% cineole diluted with SigmaAldrich MO with 85% or higher acc uracy on three consecutive blocks This level of cineole was three to four levels above the th reshold for all mice, depending on individual thresholds. Cineole levels of 10-4% were then made, again using either CVS or Sigma-Aldrich MO as a diluent, and

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22 were compared. At this concentr ation, four of the five mice were able to discriminate between the two stimuli with 85% or higher accuracy on three consecu tive blocks (Figure 3-4). Depending on the threshold of individual mice, this level of cineole was four to five levels above the threshold for all mice.

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23 Cineole Threshold0 20 40 60 80 100 1201% 0.10% 0.01% 10-3% 10-4% 10-5% 10-6% 10-7% 10-8% 10-9% 10-10%Percent CorrectCineole Concentration AG2 AG4 AB4 AB3 AR4 Figure 3-1. Cineole threshold for five C57Bl/6J mice in my laboratory. Two mice have a threshold of 10-8% and three mice have a threshold of 10-9%.

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24 Mineral Oil Discrimination0 20 40 60 80 100 120C VS v Sig m a-Ald r ic h CVS v Fis h e r Scientific CVS v W al m artMineral Oil PairsPercent Correct AG2 AG4 AB4 AB3 AR4 Figure 3-3. Mineral oil discrimina tion trials between four different brands of MO are shown. Four of five mice were able to discrimi nate Sigma-Aldrich and Wal-mart MO from CVS MO, and two of five mice were able to discriminate Fisher Scientific MO from CVS MO.

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25 Cineole in CVS MO v. Cineole in Sigma-Aldrich MO0 10 20 30 40 50 60 70 80 90 100 10-5% 10-4% Cineole ConcentrationPercent Correct AG2 AG4 AB4 AB3 AR4 Figure 3-4. Discriminations of ci neole concentrations in two di fferent brands of MO, CVS and Sigma-Aldrich are shown here. All five mice were able to discriminate 10-5% cineole in CVS MO from the same concentration in Sigma-Aldrich MO, a nd four of the five mice were able to discriminate 10-4% cineole in CVS MO from the same concentration in Sigma-Aldrich MO.

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26 CHAPTER 4 DISCUSSION Mineral oil is a comm on diluen t in olfactory research and is typically referred to as odorless. Many studies that use MO as a dilu ent and/or an Sstim ulus in discrimination studies do not mention the vendor or types of MO used, but merely call it, odorless (Bodyak and Slotnick 1999; Wiltrout et al. 2003; Kay et al. 2005; Li et al. 2006 ; Mandairon et al. 2006; McNamara et al. 2007; Yoshida and Mori 2007). Th e results presented here clearly demonstrate that all four brands of MO tested can easily be discriminated from one another, suggesting that each has a distinct odor. Animals were originally trained to respond to the presentation of CVS MO as an S+ compared to an Sof filtered air, suggesting that it had an odor, where mice would not have been able to discriminate it from a clean bottle if it was truly odorless. Once trained to CVS MO, they were also able to discriminate it from the three other brands of MO against which it was tested. The use and description of MOs in the literature suggests that many researchers consider all MOs to be the same, odorless substance. Howeve r, the ability of mice to easily discriminate between MO brands in this study suggests that they not only have an odor, but each brand or lot may have a distinct odor. Pruns et al. (2003) used laser desorp tion/ionization time-of-flight mass spectrometry to characterize various samples of paraffin oil and petrolatum, two substances synonymous with MO. Paraffin oil is largely dependent upon both the origin and the processing of the petroleum from which it is derived. Analyses showed that it contains alkanes, isoalkanes, cycloalkanes, as well as many of their isomers, and may contain hundreds of variations of these substances. Petrolatum cont ains paraffin oil (with all of its compounds) as well as microcrystalline wax. Samples vary consider ably, by both their molecular weight and the composition of compounds they contain. Fisher Scientific cites MOs molecular formula as,

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27 hydrocarbons, while Sigma-Aldrich has no fo rmula for it; neither manufacturer shows a molecular structure as they do fo r most of their products. Togeth er, this suggests that MO is a substance with a variable composition containing many compounds that are di fficult to control in the distillation process from petroleum. Kay et al. (2005) suggested that contaminants within a mixture may affect its perception, perhaps due to a higher vapor pressure causing th em to be more salient than the mixtures components. In addition to possible contaminan ts, impurities, which exist to some extent in almost all odorants, may also affect the percepti on of MOs and give them a more distinct odor. As suggested previously, it is po ssible that there are contaminants within MO that may vary, not only from brand to brand, but possibly from bottle to bottle, that allow them to be recognized and readily discriminated from one a nother. Regardless of what is being perceived within MO, it is clear that MO is not odorless as it has been previously assumed. This finding is problematic not only for studies in which MO is used as a null control, but also when MO is used as an odorless diluent. A binary mixture is rarely perceived in the sa me way as one of its components alone. If one of its components is at a leve l where it suppresses the other co mponent so that it is unable to be detected, the mixture may smell like the dom inant component; however most mixtures are affected by both components. Whether a binary mi xture has elemental or configural perceptual qualities, the mixture is still perceived differently from when its components are presented independently (Livermore and Laing 1998; Kay et al. 2005). In a configural mixture, two odorants combine to form a new odorant that smells different from either of its components. The two molecularly similar components are no longer distinguishable from on e another in mixture, and the mixture is perceived as a synthetic, unique odor. An elemental mixture, on the other

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28 hand, allows two molecularly diss imilar odorants in a mixture to be perceived separately from one another simultaneously, maintaining their dis tinct odors independently even when combined. Despite the ability to separately perceive each component, the act ual perception of the mixture is different from smelling the two odors independe ntly. Because two components are present simultaneously in a binary mixture, one must be ignored for the other to be perceived as independent. My results suggest one of two mixture possibi lities, based on the way the mice responded. Cineole and MO could be combining to form an odor different from the two individually, a configural mixture, combining differently for each type of MO. The second, more likely possibility, is that the identical concentrations of cineole are bei ng ignored, and the MO alone is being used for discrimination. This would be the case if it was an elemental mixture as I suggest, with each component being detected individually. Thus, the diluent, no matter how it is being detected, is altering the percep tion of the target odorant. Cineole diluted to 10-4% and 10-5% in MO likely represents an elemental mixture in this experiment. Previous reports in the literature (Livermore a nd Laing 1998; Wiltrout et al. 2003; McNamara et al. 2007) suggest that the more inte nse odorants in a mixture are recognized above less intense odorants. In this experiment, cineole, an intense and trigeminal odor, was diluted to identical concentrations in two different type s of MO, so that MO was the only difference between the two stimuli. Despite the fact that cineole is intense, it was the diluent, MO, that was being detected in the discrimination of cineole in CVS MO from cineole in Sigma-Aldrich MO.

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29 CHAPTER 5 CONCLUSIONS AND IMPLICATIONS These findings suggest that di fferent MOs have distinct, discrim inable notes and, when mixed with odorants, can influence their perception. When research is pe rformed using specific odorants and MO is involved, it should be recogn ized that MO is not odo rless, and may affect the perception of stimuli with which it is mixed. A single odorant dilute d with MO is no longer simple, but becomes a mixture. In the same wa y, then, when a binary mixture is diluted with MO, it is no longer a binary mixture, but becomes complex with multiple components. Likewise, MO should be used with caution if it is used as an odorless or null control for any experiments, as it contains a dist inct odor. For discrimination trials such as the ones used in this study, using the diluent as the Sis a good way to compensate for any odor that may exist in the diluting substance. Consistency of both brands and bottles of MO w ithin an experiment is essential. It is my recommendation that, if more than one bottle of MO is necessary for an experiment, multiple bottles should be mixed together and aliquots taken from the mixed batch to avoid changing bottles mid-experiment. It is not recommende d to ever switch brands of MO during an experiment, as it is more likely for MO to diffe r more between brands/distributors than between bottles.

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30 LIST OF REFERENCES Bell, GA, Laing, DG, Panhuber, H. 1987. Odour m i xture suppression: evid ence for a peripheral mechanism in human and rat. Brain Res. 17, 8-18. Bodyak, N, Slotnick, B. 1999. Performance of mice in an automate d olfactometer: odor detection, discrimination and odor memory. Chem. Senses, 24, 63745. Derby, CD, Girardot, M-N, Da niel, PC, Fine-Levy, JB. 1989. Olfactory discrimination of mixtures: behavioral, electrophys iological and theoretical stud ies using the spiny lobster Panulirus argus. In Laing, DG, Cain, WS, McBr ide, RL, and Ache, BW. (eds), Perception of Complex Smells and Tastes. Academic Press, Australia. Kay, LM, Crk, T, Thorngate, J. 2005. A Redefi nition of Odor Mixture Quality. Behav. Neurosci., 119, 726-33. Kelliher, KR, Ziesmann, J, Munger, SD, Reed, RR, Zufall, F. 2003. Importance of the CNGA4 channel gene for odor discrimination and ad aptation in behaving mice. Proc Natl Acad Sci USA. 100, 4299-304. Laing, DG 1989. The role of physicochemical and neural factors in the perception of odor mixtures. In Laing, DG, Cain, WS, McBride, RL, and Ache, BW. (eds), Perception of Complex Smells and Tastes. Academic Press, Australia. Laing, DG, Panhuber, H, Slotnick, BM. 1989. Odor masking in the rat. Physiol. Behav. 46, 80914. Leon, M, Johnson, BA. Glomerular Activity Res ponse Archive. Retrieved May 30, 2008. From the Department of Neurobiol ogy and Behavior, University of California, Irvine. http://leonserver.bio.uci.edu/index.jsp Linster, C, Johnson, BA, Yue, E, Morse, A, Xu, Z, Hingco, EE, Choi, Y, Choi, M, Messiha, A, Leon, M, 2001. Perceptual Correlates of Ne ural Representations Evoked by Odorant Enantiom ers. The Journal of Neurosci., 21, 9837-9843. Livermore, A, Laing, D. 1998. The influence of odor type on the discrimination and identification of odorants in multicomponent odor mixtures. Physiol. Behav. 65, 311-20. Mandairon, N, Stack, C, Kiselycznyk, C, Lins ter, C. 2006. Enrichment to Odors Improves Olfactory Discrimination in Adu lt Rats. Behav. Neurosci. 120, 173-79. Mandairon, N, Stack, C, Linster, C. 2006. Olfact ory enrichment improves the recognition of individual components in mixt ure. Physiol. Behav. 89, 379-84. McNamara, AM, Magidson, PD, Linster, C. 2007. Binary Mixture Perception Is Affected by Concentration of Odor Compone nts. Behav. Physiol. 121, 1132-36.

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31 Moore, BCJ. An Introduction to the Psychology of Hearing, 4th Ed. 1997. Academic Press, London. National Academies of Science. 1996. Guide for the Care and Use of Laboratory Animals. Institute of Laboratory Animal Resources Commission on Life Sciences. National Academy Press, Washington, D.C. Pruns, JK, Rapp, C, Hintze, U, Wittern, K-P, K nig, WA. 2003. Characterization of paraffin oils and petrolatum using LDS-TOF MS and princi ple component analysis. Eur. J Lipid Sci. Technol. 105, 275-80. Rospars, J-P, Lansky, P, Chaput, M, Duchamp-Viret, P. 2008. Competitive and Noncompetitive Odorant Interactions in the Early Neural Coding of Odorant Mixt ures. J Neurosci. 28, 2659-66. Smith, BH. 1998. Analysis of interaction in bi nary odorant mixtures. Physiol Behav. 56, 397407. Smith, DW, Thach, T., Marshall, E., Mendoza, M-G, Kleene, SJ. 2008. Mice lacking NKCC1 have normal olfactory sensitivity. Physiol. Behav., 29, 44-9. Wiltrout, C, Dogra, S, Linster, C. 2003. Confi gurational and nonconfigurational interactions between odorants in binary mixtures. Behav. Neurosci. 117, 236-45. Yoshida, I, Mori, K. 2007. Odoran t Category Profile Selectivity of Olfactory Cortex Neurons. J Neurosci. 27, 9105-14.

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BIOGRAPHICAL SKETCH Katherine Gam ble graduated from Gettysburg College, Gettysburg, PA in 2006. She majored in psychology with minors in neuroscience and biology. She worked in a cognitive neuroscience laboratory for two years looking at attention and object recognition using saccadic movement and eye tracking. She has worked for the past two years in an ol faction laboratory in the behavioral neuroscience area of the Department of psychology at the University of Florida. Her current work is in behavioral and psyc hophysical studies with both people and mice looking at sensory phenomenon in olfaction, such as adaptation and masking.