The Pennsylvania State University
The Graduate School
Department of Biobehavioral Health
CONDITIONING DISCRETE VISUAL CUES TO AVERSIVE INTEROCEPTIVE
STIMULI IN THE MOUSE
A Dissertation in
Biobehavioral Health
by
Sezen Kislal
 2015 Sezen Kislal
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
August 2015
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The dissertation of Sezen Kislal was reviewed and approved* by the following:
David Blizard
Senior Research Associate of Biobehavioral Health
Dissertation Adviser
Co-Chair of Committee
Byron C. Jones
Professor Emeritus of Biobehavioral Health
Co-Chair of Committee
David J. Vandenbergh
Associate Professor of Biobehavioral Health
Frederick Brown
Associate Professor of Psychology
Sonia Angele Cavigelli
Associate Professor of Biobehavioral Health
Victoria Braithwaite
Professors of Biology
Robert Turrisi
Professor of Biobehavioral Health
Chair of Graduate Program, Department of Biobehavioral Health
*Signatures are on file in the Graduate School
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ABSTRACT
According to the principle of selective associative learning, first described by Garcia
(1966), learning occurs more easily when specific classes of stimuli are paired with particular
reinforcers (as discussed in Chapter 1). For example, when an internal stimulus, such as taste, is
paired with illness ( an internally applied reinforcer ), rats develop strong conditioned aversion.
However, pairing an external stimulus like the size of the food pellet with illness results in only
weak or no aversion. Conversely, pairing an internal stimulus such as taste with an externally
applied reinforcer, such as shock, results in weak or ineffective conditioning but pairing an
external stimulus such as the size of the pellet, with an externally applied reinforcer, such as
shock, results in strong conditioning (Chapter 1). These findings led Garcia to propose that
evolution has shaped the nervous system so that certain kinds of stimuli are more easily
associated with certain classes of reinforcers (selective associative learning). Nevertheless,
subsequent studies have shown that pairing of large contextual changes (External stimulus) with
illness can cause conditioned context aversions in rats, raising questions about the limits of
selective associative learning. The aim of present studies was to discover if conditioned aversion
can be seen when discrete visual cues are paired with illness using mice rather than rats as
subjects (Chapters 3, 4 and 5). We paired visual cues with illness (produced by injection of
lithium chloride) using genetically heterogeneous mice and obtained strong aversion to a novel
container (CS) after a single conditioning trial (Chapters 3, 4 and 5). Moreover, strong
conditioned context aversion was also demonstrated even when there was a 30- minute delay
between the presentation of the CS and the UCS, just as occurs in conditioned taste aversion
experiments. In Chapter 4, we compared duration of retention in conditioned context aversion
(CCA) and conditioned taste aversion (CTA). The results provide very little evidence that
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conditioned taste aversion is retained for longer than conditioned context aversion. In our
experiments, we only investigated one part of the theory of selective associative learning. More
specifically, we examined whether pairing an external stimulus (such as a visual cue) with an
internally applied reinforcer (illness) would result in strong conditioned aversion. We did not
examine whether pairing an internal stimulus (such as taste) with an externally applied reinforcer
(such as shock) would likewise result in strong aversion. Although our experiments are thus
limited to one particular form of learning (pairing an external stimulus to an internally applied
reinforcer), many previous experiments have also dealt with one aspect of selective associative
learning (Garcia, Ervin, & Koelling, 1966; Gemberling & Domjan, 1982; Rescorla, 2008). In
addition, the results of Chapter 6 show that a conditioned context preference can be formed when
sucrose solution is used as a positive reinforcer. However, the retention is short-lived (only 6
hours). Development of contextual aversion conditioning protocols for mice will enable the
molecular resources available for this species to be exploited in studies of this kind of learning
and representation of the CS by discrete (e.g. a visual stimulus) rather than multi-modal stimuli
offers more focus when considering relevant brain regions to explore.
v
TABLE OF CONTENTS
List of Figures .......................................................................................................................... vii
List of Tables ........................................................................................................................... xi
Acknowledgements .................................................................................................................. xiii
Chapter 1 Conditioning Discrete Visual Cues to Aversive Interoceptive Stimuli in the
Mouse ............................................................................................................................... 1
Classical (Pavlovian) Conditioning.................................................................................. 3
Conditioned Taste Aversion (CTA) ................................................................................. 7
Selective-Associative Learning........................................................................................ 11
Experiments with Compound Stimuli .............................................................................. 14
Context Aversion Learning .............................................................................................. 17
Two common techniques to reduce conditioned context aversion: ................................. 20
Overshadowing and Latent inhibition .............................................................................. 20
Conditioned Place Aversion and Conditioned Place Preference...................................... 24
Conclusion ....................................................................................................................... 25
Chapter 2 Methods common to many experiments ................................................................. 27
Subjects ............................................................................................................................ 27
Maintenance Conditions .................................................................................................. 28
Assignments of animals to experimental groups.............................................................. 28
Colony Room (Light Cycle, Temperature, Food) ............................................................ 29
General Experimental Considerations.............................................................................. 29
I. Adaptation and Training ....................................................................................... 30
II. Conditioning Procedures ..................................................................................... 31
III. Recovery Period ................................................................................................. 33
IV. Retention ............................................................................................................ 33
Statistical issues ............................................................................................................... 33
Suppression index .................................................................................................... 33
Data Analyses........................................................................................................... 34
Conditioning ............................................................................................................. 34
Retention .................................................................................................................. 34
Appendix .......................................................................................................................... 35
Visual system of mouse ................................................................................................... 35
Anatomy of the retina............................................................................................... 36
Composition of photoreceptors: human vs mice ...................................................... 37
Visual acuity............................................................................................................. 38
Chapter 3 Exploratory Studies with B6D2 mice ...................................................................... 41
Experiment 3-1 ................................................................................................................. 41
Experiment 3-2 ................................................................................................................. 47
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Experiment 3-3 ................................................................................................................. 52
Discussion ........................................................................................................................ 59
Chapter 4 Comparison of CCA and CTA in pigmented LGSM mice ..................................... 63
Experiment 4-1 ................................................................................................................. 63
Experiment 4-2 ................................................................................................................. 72
Experiment 4-3 ................................................................................................................. 81
Discussion ........................................................................................................................ 90
Chapter 5 Comparison CCA in albino LGSM mice ................................................................ 92
Experiment 5-1 ................................................................................................................. 92
Experiment 5-2 ................................................................................................................. 101
Discussion ........................................................................................................................ 110
Chapter 6 Conditioned Context preference using positive reinforcement in B6D2 mice ........ 112
Discussion ........................................................................................................................ 114
Chapter 7 General Discussion .................................................................................................. 116
Selective Associative Learning ........................................................................................ 116
Features of CTA and CCA ............................................................................................... 117
Acquisition ....................................................................................................................... 118
Comparing duration of retention in CCA and CTA ......................................................... 118
Non-specific Suppression ................................................................................................ 120
Conditioned Context Preference ...................................................................................... 124
Other concerns ................................................................................................................. 124
Historical Note ................................................................................................................. 125
References ................................................................................................................................ 126
vii
LIST OF FIGURES
Figure 1-1. Chemotherapy in cancer patients. Adapted from Ursula Stockhorst, HansJoachim Steingrueber , Paul Enck, Sibylle Klosterhalfen, Pavlovian conditioning of
nausea and vomiting. Autonomic Neuroscience, 129 (1), 50-57 (2006). ........................ 2
Figure 1-2. Pavlov`s dog experiment. Adapted from “The method of Pavlov in animal
psychology”, by Robert M. Yerkes and Sergius Morgulis, 1909, Psychological
Bulletin. ............................................................................................................................ 3
Figure 1-3 (left). Figure 1-4 (right). Effects of the CS-UCS Interval on the strength of the
CR. Adapted from “Principles of learning and behavior”, (p.84), Domjan, M.
(2009). .............................................................................................................................. 4
Figure 1-5 (right). Conditioned Taste Aversion. Adapted from “Conditioned aversion to
saccharin resulting from exposure to gamma radiation”, by Garcia, J., Kimeldorf, D.
J., & Koelling, R. A. (1955), Science. .............................................................................. 8
Figure 1-6. Long-delay taste aversion learning. Adapted from “Principles of learning and
behavior”, (p.82), Domjan, M. (2009). ............................................................................ 10
Figure 2-1. Plastic bottles were presented before starting the experiment. ............................. 30
Figure 2-2.The experimental set up is illustrated from the back of the cage in order to
clearly demonstrate the tube arrangement. ....................................................................... 31
Figure 2-3(left). and Figure 2-4 (right). The CS tubes that we have used during the
conditioning trials. ........................................................................................................... 32
Figure 2-5. Shows the rods and cons in the human retina. Small circular cells are rod
photoreceptors, whereas larger cells are cones.Scale bar is 10 μm (Curcio, Sloan,
Kalina, and Hendrickson, 1990)....................................................................................... 36
Figure 2-6. Shows the mosaic of rods and cones in the mouse (C57BL/6). Dark mosaics
show the cones, lighter mosaic shows the rods. Scale bar is 10 μm (Jeon et al.,
1998). ............................................................................................................................... 37
Figure 2-7. Shows the visible light spectrum for humans. ....................................................... 38
Figure 2-8.Red and yellow tapes are shown together white and black tape as reference. ....... 40
Figure 2-9.The different degrees of brightness of the four tapes. ............................................ 40
Figure 3-1. B6D2 AI mice maintained on plastic bottles (PB) were given 3 conditioning
trials in which drinking from glass bottles (GB) was paired with NaCl (controls) or
LiCl. PB-GBTW/LiCL and PB-GB PW/LiCl groups showed high suppression to the
glass bottles (CS) after a single trial which was sustained for 7 days. ............................. 46
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Figure 3-2. B6D2 AI mice maintained on glass bottles (GB) were exposed to 3
conditioning trials when they drank from graduated tubes with dark-colored tape
paired with injections of LiCl or NaCl (controls). Strong suppression was found with
a single conditioning trial for GB-DT LiCl group but 2 trials were required for LT-DT
LiCl
group. During retention tests both experimental groups showed high suppression
to CS tubes during which was sustained for at least 13 days after CCA3. There was
no difference in consumption between experimental and control groups in the
specificity test when animals drank from their maintenance containers. ......................... 52
Figure 3-3. B6D2 AI mice maintained on graduated tubes with light-colored tape (LT)
were exposed to 3 conditioning trials when they drank from graduated tubes with
dark-colored tape (DT) paired with either injections of LiCl or NaCl immediately or
30 minutes later (delay). The suppression was clearly stronger on CCA3 for both
immediate and delay groups............................................................................................. 57
Figure 3-4. Both total and non-specific retention were tested at weekly intervals after
CCA 3. There was specific suppression (total greater than non-specific suppression)
in both week 1 and 2 for the LT-DT LiCl/Immed, however, specific suppression had
disappeared by week 2 for LT-DT LiCl/Delay group. By week 3, there was no evidence
for both total or nonspecific suppression for both immediate and delay groups.............. 58
Figure 4-1(left). Maintenance bottle. Figure 4-2(right). CS tube. ........................................... 65
Figure 4-3. LGSM AI mice maintained on glass bottles (GB) were exposed to 3
conditioning trials when they drank from graduated tubes with dark-colored tape
paired with injections of LiCl or NaCl (controls). Strong suppression was found with
a single conditioning trial for GB-DT LiCl/Immed group. During retention tests,
GB-DT LiCl/Immed group showed high suppression to CS tubes than their
maintenance tubes until week 4 (CCA3+28 days). .......................................................... 69
Figure 4-4. LGSM AI mice maintained on glass bottles (GB) were exposed to 3
conditioning trials when they drank from graduated tubes with dark-colored tape
paired with injections of LiCl or NaCl (controls). Strong suppression was found with
a single conditioning trial for GB-DT LiCl/Delay group. During retention tests, GBDT LiCl/Delay group showed high suppression to CS tubes than their maintenance
tubes until week 4 (CCA3+ 28 days). .............................................................................. 70
Figure 4-5 (left). Maintenance tube. Figure 4-6(right). Brightness differences between
tape on CS and Maintenance tubes .................................................................................. 74
Figure 4-7. LGSM AI mice maintained on graduated tubes with light-colored tape (LT)
were exposed to 3 conditioning trials when they drank from graduated tubes with
dark-colored tape paired with injections of LiCl or NaCl (controls). Strong
suppression was found with a single conditioning trial for LT-DT LiCl/Immed
group. During retention tests, LT-DT LiCl/Delay group showed high suppression to
CS than their maintenance bottles until week 3 (CCA3+ 21days). .................................. 78
Figure 4-8. LGSM AI mice maintained on graduated tubes with light-colored tape (LT)
were exposed to 3 conditioning trials when they drank from graduated tubes with
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dark-colored tape paired with injections of LiCl or NaCl (controls). Strong
suppression was found with a single conditioning trial for LT-DT LiCl/Delay group.
During retention tests, LT-DT LiCl/Delay group showed high suppression to CS
than their maintenance bottles until week 7 (CCA3+ 49 days)........................................ 79
Figure 4-9. Maintenance tube .................................................................................................. 82
Figure 4-10. LGSM AI mice maintained on water in graduated tubes were exposed to 3
conditioning trials when they drank sodium saccharin in their maintenance tubes
paired with injections of LiCl or NaCl (controls). Strong suppression was found with
a single conditioning trial for SS LiCl/Immed group. During retention tests, SS LiCl/Immed
group showed high suppression to CS (sodium saccharin) than water until week 6
(CCA3+ 42 days). ............................................................................................................ 87
Figure 4-11. LGSM AI mice maintained on water in graduated tubes were exposed to 3
conditioning trials when they drank sodium saccharin in their maintenance tubes
paired with injections of LiCl or NaCl (controls). Strong suppression was found with
a single conditioning trial for SS LiCl/Delay group. During retention tests, SS LiCl/Delay
group showed high suppression to CS (sodium saccharin) than water until week 3
(CCA3+ 21 days). ............................................................................................................ 88
Figure 5-1. (left) Maintenance Bottle
Figure 5-2. (right) CS tube .................................. 94
Figure 5-3. LGSM AI mice maintained on glass bottles (GB) were exposed to 3
conditioning trials when they drank from graduated tubes with dark-colored tape
paired with injections of LiCl or NaCl (controls). Strong suppression was found with
a single conditioning trial for GB-DT LiCl/Immed group. During retention tests, GB-DT
LiCl/Immed
group showed high suppression to CS than their maintenance bottles until
week 3 (CCA3+ 21 days). ................................................................................................ 98
Figure 5-4. LGSM AI mice maintained on glass bottles (GB) were exposed to 3
conditioning trials when they drank from graduated tubes with dark-colored tape
paired with injections of LiCl or NaCl (controls). Strong suppression was found with
a single conditioning trial for GB-DT LiCl/Delay group. During retention tests, GB-DT
LiCl/Delay
group showed high suppression to CS than their maintenance bottles until
week 3 (CCA3+ 21 days). ................................................................................................ 99
Figure 5-5. (left) Maintenance Tubes, Figure 5-6. (right) Difference brightness between
maintenance and CS tubes ............................................................................................... 103
Figure 5-7. LGSM AI mice maintained on graduated tubes with light-colored tape (LT)
were exposed to 3 conditioning trials when they drank from graduated tubes with
dark-colored tape paired with injections of LiCl or NaCl (controls). Strong
suppression was found with a single conditioning trial for LT-DT LiCl/Immed group.
During retention tests, LT-DT LiCl/Immed group showed high suppression to CS than
their maintenance bottles until week 3(CCA3+ 21 days). ............................................... 108
Figure 5-8. LGSM AI mice maintained on graduated tubes with light-colored tape (LT)
were exposed to 3 conditioning trials when they drank from graduated tubes with
x
dark-colored tape paired with injections of LiCl or NaCl (controls). Strong
suppression was found with a single conditioning trial for LT-DT LiCl/Delay group.
During retention tests, LT-DT LiCl/Delay group showed high suppression to CS than
their maintenance bottles until week 3 (CCA3+ 21 days). .............................................. 108
Figure 6-1. B6D2 AI mice maintained on water in graduated tubes without tape. During
the retention tests, two graduated tubes (one with tape, the other without tape) were
presented to each mouse. Conditioned context preference could be formed up to 6
hours when sucrose solution is used as a positive reinforce in the experimental
group. ............................................................................................................................... 115
Figure 7-1. Results of specific suppression in Immediate experimental groups (LGSM AI
pigmented mice). Specific suppression was calculated by subtracting non-specific
suppression from total suppression (Specific Suppression = Total Suppression Non-Specific Suppression). Both context groups (LT-DT LiCl/Immed and GB-DT
LiCl/Immed
) showed similar extinction to the CS, and the duration that conditioned taste
aversion was retained slightly longer (SS LiCl/Immed ). ........................................................ 121
Figure 7-2.Results of specific suppression in Delay experimental groups (LGSM AI
pigmented mice). Specific suppression was calculated by subtracting non-specific
suppression from total suppression (Specific Suppression = Total Suppression Non-Specific Suppression). The Taste group (SS LiCl/Delay) and one of the context
groups (GB-DT LiCl/Delay) showed quicker extinction to the CS, whereas LT-DT
LiCl/Delay
was retained for longer. ....................................................................................... 122
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LIST OF TABLES
Table 1-1 (left). Conditioned Taste Aversion. Adapted from “Conditioned aversion to
saccharin resulting from exposure to gamma radiation”, by Garcia, J., Kimeldorf, D.
J., & Koelling, R. A. (1955), Science. .............................................................................. 8
Table 1-2. Experimental Setup ................................................................................................ 16
Table 1-3.Experimental Setup ................................................................................................. 16
Table 1-4. Experimental Setup. Adapted from “Overshadowing and latent inhibition of
context aversion conditioning in the rat”(42-49), by Hall, G. and M. Symonds, 2006,
Autonomic Neuroscience.................................................................................................. 19
Table 1-5.Experimental Setup, Adapted from “Overshadowing and latent inhibition of
context aversion conditioning in the rat” (42-49), by Hall, G. and M. Symonds,
2006, Autonomic Neuroscience. ...................................................................................... 21
Table 1-6. Experimental Setup, Adapted from “Overshadowing and latent inhibition of
context aversion conditioning in the rat” (42-49), by Hall, G. and M. Symonds,
2006, Autonomic Neuroscience........................................................................................ 22
Table 2-1. Shows the general procedure that we used in our experiments. The experiment
consisted of five phases: adaptation, training, conditioning, recovery period, and
retention tests. Top row: The number of days devoted to retention phase varied by
experiment........................................................................................................................ 30
Table 2-2. Design of ANOVA ................................................................................................. 35
Table 3-1. Experimental Setup ................................................................................................ 43
Table 3-2. Water Intakes and percent suppression for Experiment 3- 1 .................................. 45
Table 3-3. Experimental Setup ................................................................................................ 48
Table 3-4. Water Intakes and percent suppression for Experiment 3- 2 .................................. 50
Table 3-5. Experimental Setup ................................................................................................ 53
Table 3-6. Water Intakes and percent suppression for Experiment 3- 3 .................................. 56
Table 4-1. Experimental Setup ................................................................................................ 66
Table 4-2. Design of ANOVA ................................................................................................. 68
Table 4-3. Water Intakes and Percent suppression for Experiment 4-1 .................................. 72
Table 4-4. Experimental Setup ................................................................................................ 75
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Table 4-5. Design of ANOVA ................................................................................................. 77
Table 4-6a, 4-6b and 4-6c. Water Intakes and Percent suppression for Experiment 4-2. ........ 81
Table 4-7. Experimental Setup ................................................................................................ 83
Table 4-8. Design of ANOVA ................................................................................................. 85
Table 4-9a, 4-9b and 4-9c. Water Intakes and Percent suppression for Experiment 4-3 ......... 90
Table 5-1. Experimental Setup. ............................................................................................... 95
Table 5-2. Design of ANOVA ................................................................................................. 96
Table 5-3a, 5-3b and 5-3c. Water Intakes and Percent suppression for Experiment 5-1. ........ 101
Table 5-4 Experimental Setup ................................................................................................. 104
Table 5-5. Design of ANOVA ................................................................................................. 106
Table 5-6a, 5-6b and 5-6c. Water Intakes and Percent suppression for Experiment 5-2. ........ 110
Table 6-1. Experimental Setup ................................................................................................ 113
Table 7-1. Duration of retention in LGSM AI pigmented mice. ............................................. 123
Table 7-2. Duration of retention in LGSM AI albino mice. .................................................... 123
xiii
ACKNOWLEDGEMENTS
I would like to express my deepest gratitude to my advisor, Dr. David Blizard, whose
patience, expertise, and wisdom know no end. I appreciate both your guidance and mentoring
during my completion of this dissertation. I know that without your help, I could not have
finished this dissertation successfully.
I would also like to thank the other members of my dissertation committee: Dr. Byron C.
Jones, Dr. David J. Vandenbergh, Dr. Frederick Brown, Dr. Sonia Angele Cavigelli, and Dr.
Victoria Braithwaite, all of whom have enriched this dissertation with their helpful comments and
thoughtful guidance. I have learned much though our conversations.
I owe sincere thanks to Dr. Joseph Gyekis, who has supported me from the beginning of
my graduate career. Your suggestions have shaped much of the discussion in this dissertation.
Thank you to the professors who allowed me to serve as a teaching assistant for them and
to all the students whom I had the privilege of teaching.
Thank you to the administrative staff of the Biobehavioral Health Department. You have
been exceptionally helpful in explaining the many nuanced policies and procedures associated
with graduate studies. Thanks especially to Shannon Seiner-Anthony who patiently answered my
complicated questions.
Thanks also to all of my fellow BBH graduate students for providing much-needed
support and friendship. I have always felt lucky to be part of such a great cohort.
xiv
Many thanks to my family members, who have supported and encouraged me with their
best wishes throughout my graduate studies. Even though we are thousands of miles apart, you
have always been there when I needed you. I am especially grateful to my dearest sister, Esin
Yertutanol Guven, without whose support I could not have completed my degree. You have
always been with me, and your love has never wavered.
Finally, I would like to thank the love of my life, Orhan Kislal. Thank you for always
supporting me and always letting me know that you believe in me no matter what. I am truly
thankful to have you in my life.
1
Chapter 1
Conditioning Discrete Visual Cues to Aversive Interoceptive Stimuli in the
Mouse
The phenomena known as conditioned taste aversion (CTA) and conditioned context
aversion (CCA) have had very different histories. A conditioned taste aversion can occur when
ingestion of a novel taste is followed by illness (induced by a variety of procedures). CTA was
first shown by Garcia and colleagues (Garcia, Kimeldorf, & Koelling,1955) to result in strong
and long-lasting aversions with a single conditioning trial, even to solutions that are usually
strongly preferred (e.g., saccharin, sucrose ). Subsequently, many investigators (Smith & Roll,
1967; Domjan, 1980, Domjan & Galef, 1983) confirmed these original findings. CCA, on the
other hand, was more difficult to establish. Garcia and many other investigators were unable to
demonstrate conditioned aversion by pairing illness and exteroceptive cues. Selective associative
learning was first described by Garcia and his colleagues (Garcia, Ervin & Koelling; 1966).
According to the principle, one can condition strong aversion to internal cues such as taste to
sickness and external cues such as size of the food pellet to externally applied reinforcers such as
electric shock. However, reciprocal pairings (internal stimuli/external reinforcer, external
stimuli/internal reinforcer) resulted in weak or ineffective conditioning.
Subsequent studies (Revusky & Parker, 1976; Boakes, Westbrook, Elliott, &
Swinbourne, 1997; Rodriguez, Lopez, Symonds, & Hall, 2000) have shown that context aversion
learning can be demonstrated reliably when the CS involves changes in the environment in the
absence of taste cues. The aim of this review is to consider the evidence for and against the
2
principle of selective associative learning and to examine the reasons for the different outcomes
described above.
The issue of selective associative learning has important practical as well as theoretical
relevance. For example, in a study by Andrykowski and Redd (1987), after cancer patients
experienced a number of chemotherapy and radiation sessions (UCS), the chemotherapy
equipment and other physical environmental cues, such as a clock on the wall or interactions with
nurses, became conditioned stimuli (CS) that triggered a sickness response (Figure 1-1).
Figure 1-1. Chemotherapy in cancer patients. Adapted from Ursula Stockhorst, HansJoachim Steingrueber , Paul Enck, Sibylle Klosterhalfen, Pavlovian conditioning of nausea and
vomiting. Autonomic Neuroscience, 129 (1), 50-57 (2006).
Thus, conditioned aversion is observed when an environmental cue is paired with
sickness in cancer patients (Andrykowski & Redd, 1987; Stockhorst et al., 1998; Rodriguez et al.,
2000; Symonds & Hall, 2000, 2002). An improved understanding of selective associative
learning can help patients in the clinical arena. In addition, this principle allows us to better
3
understand which environmental stimuli in chemotherapy rooms are more likely to be
conditioned and how we can reduce conditioned nausea in patients by using learning theory.
Classical (Pavlovian) Conditioning
Conditioned taste aversion is an example of classical conditioning and it will be helpful
to briefly review experimental protocols of classical conditioning that are used in this area.
Pavlovian conditioning theory was first developed as a result of experiments on dogs by Ivan
Pavlov (1927) (Figure 1-2). Initially, the dogs did not produce any salivation to a neutral stimulus
(the sound of the bell). During the conditioning, the neutral stimulus (the sound of the bell) was
repeatedly presented with unconditioned stimulus (UCS; a piece of meat). After several
conditioning trials, the dogs showed a conditioned response (CR; salivation) to the bell (now, the
conditioned stimulus or CS) even it was presented alone. In addition, experiments using sourtaste (an unpleasant taste) as a UCS and light as a CS in his experiments provided additional
evidence of conditioning. Thus, Pavlov (1927) claimed that any CS could readily become
associated with any UCS (an assertion that is challenged by the idea of selective associative
learning).
Figure 1-2. Pavlov`s dog experiment. Adapted from “The method of Pavlov in animal
psychology”, by Robert M. Yerkes and Sergius Morgulis, 1909, Psychological Bulletin.
4
Depending on the procedure used in classical conditioning, learning may occur quickly or
slowly. Commonly used procedures are short and long delayed conditioning (Figure1-3).
According to the short delayed procedure, the CS starts and the UCS is presented after a brief
delay. The CS might continue during the UCS or end when the UCS begins. However, the longdelayed conditioning differs in that the CS is presented for a long time before the UCS begins. In
trace conditioning, the CS precedes the UCS but there is always a gap in time between the two
stimuli (Figure 1-3). According to the so-called simultaneous conditioning procedure, the CS and
UCS are presented at the same time (Figure1-3). Finally, the slowest learning occurs in backward
conditioning when the UCS is presented before presentation of the CS (Figure 1-3). Thus, the
interval between CS and UCS presentation has an important influence on the acquisition of a CR
(Figure 1-4). The longer the CS-UCS interval the more difficult it is to demonstrate conditioning
(Rescorla & Cunningham, 1979) (Figure1-4). A typical protocol in CTA resembles either long
delay conditioning or trace conditioning and these features will be discussed in more detail later.
Figure 1-3 (left). Figure 1-4 (right). Effects of the CS-UCS Interval on the strength of the
CR. Adapted from “Principles of learning and behavior”, (p.84), Domjan, M. (2009).
5
Since Pavlov`s original work, many features of CS and UCS have been shown to
influence the effectiveness of classical conditioning. First, the novelty of conditioned and
unconditioned stimuli affect the learning process (pre-exposure to CS or UCS often inhibits or
disrupts learning). Domjan and Wilson (1972) showed that if saccharin has been presented a
number of times without being followed by any UCS, more trials are necessary for it to become a
CS during the conditioning (known as the “latent-inhibition effect). In addition, similar to the
latent-inhibition effect, developing a conditioned response to the CS is more difficult when
subjects are familiarized with a UCS before it is paired with a CS, a phenomenon called UCS preexposure effect (Randich & LoLordo, 1979). Moreover, the intensity and significance
(noticeability) of the CS and UCS are also important features for the learning process (Kamin,
1965; Scavio & Gormezano, 1974). For example, sexual conditioning has been studied by Cusato
and Domjan (1998) to emphasize the importance of the significance of the stimulus. Two
different stimuli were presented to a male quail. One of the CS’s was made from terrycloth and
had no cues associated with a female quail, while the other stimulus contained several features of
a female quail, such as a representation of the eye and the bill. These CS’s signaled a copulatory
opportunity for the male quail. The study showed that the more rapid learning occurs when a CS
consisting of the kinds of stimuli an animal is likely to encounter in its natural environment. The
issue of noticeability is very relevant to understanding the ability of mice to develop associations
with contextual cues as will be discussed later.
So far, we have emphasized that subjects are able to learn an association between a
specific CS and the UCS. When the association has been learned, the subjects use their
experiences to guide their future behavior. If they use what they learn in very specific way, then
they will only respond to the stimulus that is exactly the same as that used in conditioning.
Discrimination is the ability to differentiate between a conditioned stimulus and other stimuli that
have not been paired with an unconditioned stimulus. To analyze this idea further, Campolattaro,
6
Schnitker and Freeman (2008, Experiment 3) used a discrimination training procedure in eyeblink
conditioning using rats. Eyeblink conditioning is a simple associative task in which a CS after
being paired with an UCS (a puff of air) elicits an eye blink. In their experiment, a low pitched
tone (2000 cycles per second) and a high pitched tone (8000 cycles per second) served as the
conditioning stimuli. For half of the trials one of the tones (A+) was paired with the UCS, on the
remaining trials, the other tone (B-) was presented without the UCS. Results showed that subjects
can easily discriminate the two stimuli. They showed increases in eyeblink responding to the A+
tone that was paired with UCS whereas that response was not observed to B-. On the other hand,
in the absence of discrimination training, Pavlov proposed that subjects may not discriminate
among the cues present in an experimental situation. In other words, subjects show the same CR
to different stimuli that resemble the CS that was presented during training (generalization). In
this case, more salivation would occur if a tone was close to the frequency of training tone, less
salivation would occur if the tone was very different from the training tone. More recently,
Guttman and Kalish (1956) reinforced pigeons to peck a key illuminated by a light with a
wavelength of 580 nanometers (nm). The highest amount of pecking was observed with a light of
580 nm during the test trials. The pigeons also made substantial number of pecks when lights of
570-nm and 590-nm wavelengths were tested, whereas, as the color of the test stimuli became
very different from the color of original training stimulus, fewer responses occurred. Responding
to 580 nm color generalized to the 570 and 590 nm stimuli. Taken together, the phenomena of
stimulus discrimination and stimulus generalization offer insight into some of the ways in which
behavior comes under the control of environmental stimuli, which may be relevant to
understanding the phenomenon involved in conditioned context aversion.
7
Conditioned Taste Aversion (CTA)
As noted, a typical conditioned taste aversion (CTA) protocol consists of a CS-UCS
pairing when a novel taste is paired with a stimulus that results in sickness. CTA was first
demonstrated by John Garcia and his colleagues using laboratory rats (Garcia et al., 1955).
During World War II, the use of atomic weapons by the US government against Japan stimulated
a lot of research to understand the effects of radiation on living systems, including the research by
Garcia (1954) for the US navy. In his experiments, rats were exposed to radiation in a testing
chamber, and by coincidence water was presented in plastic bottles during the radiation exposure.
Garcia noticed that the intake of water from the plastic bottles decreased while they were in the
testing chamber. However, when they went back to the colony room, the food and water intake
was normal. He hypothesized that the water in the plastic bottle had a distinct flavor so that it was
somehow being associated with radiation-induced sickness. To test this hypothesis, Garcia and
his colleagues paired the ingestion of saccharin solution with radiation exposure (Garcia et al.,
1955). Specifically, during the conditioning trial, experimental groups were exposed to one of
two doses of radiation (30 or 57 roentgens) while they were drinking saccharin, one control group
received water from a glass bottle and was exposed to radiation just like the experimental rats,
and another control group was not exposed to radiation at all (Table 1-1).
8
Table 1-1 (left). and Figure 1-5 (right). Conditioned Taste Aversion. Adapted from
“Conditioned aversion to saccharin resulting from exposure to gamma radiation”, by Garcia, J.,
Kimeldorf, D. J., & Koelling, R. A. (1955), Science.
The effects of conditioning after radiation were evaluated using two bottle preference
tests that compared intake of saccharin solution to water. Retention test results demonstrated that
both control groups showed the predicted strong preference to saccharin over water (Figure 1-1).
In addition, there was no evidence that rats receiving water paired with radiation showed any
decrease in fluid consumption (water or saccharin) compared to the non-radiated controls (Table
1-1). In contrast, experimental groups that received saccharin paired with radiation showed a
radiation-dependent reduction in saccharin preference, whereas they did not show a reduction in
water intake (Table 1-1). These results show that a decrease in consumption was not a direct
effect of radiation, because the animals receiving radiation but not paired with saccharin
continued to consume the saccharin at control levels.
9
In discussing these findings, Garcia made the prediction that taste sensations may be
especially suitable for conditioning by internal sensations such as nausea because there is a
relatively close relationship between consuming behaviors and gastric function. Later, this
interpretation led Garcia to work on selective associative learning.
Conditioned taste aversion has three key features:
First, a single pairing of a novel taste with illness causes conditioned aversion to the
novel taste (Garcia et al., 1955, Garcia, Ervin, & Koelling, 1966, Bernstein & Webster, 1980).
Although, one-trial learning is also observed in fear conditioning (it is a type of classical
conditioning in which people and animals learn to fear certain objects or situations), such rapid
learning is not easily observed in eyeblink and salivary conditioning (two very frequently used
response measures in classical conditioning paradigm).
Second, an effective CTA can be demonstrated even when a long delay is introduced
between CS and UCS (Garcia et al. 1966; Smith & Roll, 1967, Revusky & Garcia, 1970). For
example, Smith and Roll (1967) conducted an experiment in which experimental rats were given
saccharin solution (CS) and exposed to radiation at various intervals (0-24 hours) after
presentation of saccharin. In retention tests, rats exhibited conditioned aversion when delay was
as long as 12 hours after presentation of saccharin (Figure 1-6)
10
Figure 1-6. Long-delay taste aversion learning. Adapted from “Principles of learning and
behavior”, (p.82), Domjan, M. (2009).
Thus, it can be seen that this form of CTA protocol fits the paradigm of trace
conditioning described in previous section.
Third, CTA responses can be retained for a long period of time (Garcia et al., 1955;
Houpt, Philopena, Joh, & Smith, 1996; Martin & Timmins, 1980; Steinert, Infurna, & Spear,
1980). For instance, when Garcia, Kimeldorf and Koelling (1955) paired a distinctive flavor with
radiation exposure on a single conditioning trial, the resulting aversion was sustained for up to 30
days after conditioning. All of these features support the idea that taste aversion learning
represents a very important survival mechanism: it prevents the repeated ingestion of poisonous
substances or spoiled foods.
Conditioned taste aversion has also been studied in humans. For example, Bernstein and
Webster (1980) conducted an experiment on cancer patients to explore illness-induced taste
aversion learning. All experimental subjects were exposed to one of two distinctly flavored ice
creams before a receiving chemotherapy drugs (UCS). The control group sampled two ice creams
but was not given any drug treatment. In subsequent choice tests, flavor preference and amount
consumed were recorded. Results showed that patients who consumed the specific flavor prior to
treatment developed aversion to its flavor, however this effect was not observed in the control
group.
11
Selective-Associative Learning
As noted, different views on the ability of animals to associate specific CSs with
particular reinforcers have been voiced. Pavlov claimed that a CS could be associated with any
reinforcer, whereas Garcia found limitations in the ability of particular classes of CS to be
associated with a particular UCS. For example, Garcia, Ervin and Koelling (1966) presented a
compound stimulus consisting of a distinctive taste (saccharin) and an audiovisual cue (a click
and a flash of a light) to different groups of rats which were reinforced by either an electric shock
to the feet (externally applied reinforcer) or radiation (internally applied reinforcer) during the
conditioning trials. During retention tests, subjects were given either saccharin or audiovisual cue
(a click and a flash of a light). Rats conditioned with radiation avoided drinking saccharin, but
they did not respond to audiovisual cues. On the other hand, rats conditioned with shock
exhibited a reduction in water intake during the audiovisual cue, but they drank as much
saccharin as the controls. According to this result, an audiovisual CS can be associated with an
externally applied reinforcer, such as shock, and an internally applied reinforcer, such as sickness,
can be easily associated with an internal cue, such as taste, but the reciprocal pairings are not
easily learned. This particular experimental design, using presentation of a compound stimulus,
has generated some evidence that supports the concept of selective associative learning but we
must be careful to consider that simultaneously presentation of internal and external stimuli as a
compound CS may limit the ability of animals to attend to both equally at the same time.
In a later experiment, Garcia and his colleagues (Garcia, McGowan, Ervin, & Koelling,
1968) used a different methodology in which interoceptive and exteroceptive CSs were presented
separately. In the experiment, the CSs were either the size of the pellet (exteroceptive cue) or the
taste of the pellet (interoceptive cue) and were paired with either foot shock (externally applied
reinforcer) or X-ray (internally applied reinforcer) at the testing chamber. Results showed that
12
one can condition strong aversion to internal cues, such as taste to sickness, and external cues,
such as size of the food pellet to externally applied reinforcers, such as electric shock, but not in
opposite direction. This finding led Garcia propose that evolution has shaped the nervous system
so that specific classes of stimuli (e.g., taste) can be more easily associated with certain kinds of
reinforcers (e.g., illness). For example, Garcia suggested that interoceptive stimuli were perceived
through nerves that were connected to different parts of brain than nerves that perceived
exteroceptive cues. These parts of the brain regulate different behaviors. This statement is very
general and did not provide specific suggestions as to the nature of the nervous system changes
relevant to this hypothesis.
Since Garcia`s experiments, many other studies have also supported the principle of
selective associative learning (Gemberling & Domjan, 1982; Rescorla, 2008). The study by
Gemberling and Domjan (1982) found evidence for selective associative learning, even in infant
rats. The CS was either the flavor of saccharin or the texture of the cardboard surface, and two
kinds of CS were followed by either by the injection of LiCl or the administration of a shock.
Results were consistent with the hypothesis of selective associative learning: aversions occurred
only when the LiCl treatment immediately followed taste exposure or when the shock was
concurrent with exposure to the tactile stimulus. The fact that these observations were obtained
from one-day-old rats provided evidence that taste and internal sensation (cue-consequence
correlations) are present at an early age, because it is unlikely for such young rats to experience
relevant learning. It is also important to point out that these two kinds of stimuli (taste and tactile)
may not have been equally noticeable.
Seligman (1970) also supported the principle of selective associative learning. He
claimed that each species learns some associations more easily than others (continuum
preparedness). He also suggested that behavior is influenced by the morphology, nervous system,
physiology and physiological function. This means that particular species are prepared to
13
associate certain stimuli and responses but unprepared to learn other associations. According to
Seligman, there are three degrees of preparedness. First, behavior that occurs with few or no
previous experiences are called “prepared,” and the subject is already structured (nervous system,
physiology and physiological) to produce this behavior. Second, behavior that seems to develop
with experience (by classical or operant conditioning) is called “unprepared,” and subjects do not
have any hereditary predisposition to perform this behavior. Third, behavior that never develops
even if we use classical and operant conditioning and it is called “contraprepared”.
As noted, Pavlov (1927) suggested that the selection of CSs and UCSs was entirely
arbitrary to obtain a conditioned response. Sixty years after Pavlov`s demonstration, the issue of
selective associative learning challenges of Pavlov’s assumption.
The concept of selective associative learning asserts that the appropriate matching of
UCS and CS has a major effect on the efficacy of conditioned learning (Garcia et al., 1966, 1968,
Rozin & Kalat, 1971; Smith & Roll; 1967).
1- Evolutionary history of a particular species determines the relative associability of events
for members of that species (Garcia et al., 1968, Rozin & Kalat, 1971). For example,
Mineka and her colleagues (Mineka, Watson, & Clark, 1998) presented two objects (a toy
snake and a toy flower) to rhesus monkeys. After monkeys had experience with these two
objects only once, they exhibited strong fear reactions to the snake but not to the flower.
They claimed that the evolutionary history of monkeys (like humans) made them more
attuned to danger in some situations which provides recurrent survival threats in
mammalian evolution.
2-
The persistence of either CS or UCS plays role in selective association learning. For
example, the effects of LiCl and taste are not short lived but an auditory stimulus and
shock are less likely to leave a persisting aftereffect (Garcia et al., 1966, 1968, Smith &
Roll; 1967). Effects of CS-UCS intervals on CTA by varying the interval between CS
14
(saccharin) and UCS (apomorphine; which produces gastrointestinal disturbances) on rats
were investigated by Garcia and his colleagues (Garcia et al., 1968). In this paper, they
claimed that CTA learning was an adaptive specialization because there was evidence
that this associative mechanism was further refined and safeguarded by a system that
enabled the association to form over extended delays (toxicity was likely to follow
consumption of a toxin after some delay because of natural function of digestion).
Experiments with Compound Stimuli
As noted above (Garcia, Ervin and Koelling,1966), the use of compound stimuli as the
CS have produced evidence in favor of the principle of selective association, on the other hand
there are a number of other experiments using compound stimuli as the CS, which has produced
evidence inconsistent with selective associative learning. For example, Archer and his colleagues
(Archer, Sjödén, Nilsson, & Carter, 1979) conducted an experiment where a compound stimulus
consisting of a novel taste (saccharin) and exteroceptive cues (variations in odor, cage and bottle)
were paired with a LiCl injection (UCS). Then, saccharin aversion was extinguished either in the
same context or in a different exteroceptive context (different from conditioning context;
transparent cage, standard bottle, no odor). Extinction that involved a change of context from the
one employed during conditioning resulted in a stronger conditioned response in retention tests in
the conditioning context, whereas the conditioned response was weaker during retention tests
conducted in the same context as that used during extinction. In a similar experiment, Archer and
his colleagues (Archer, Sjödén, Nilsson, & Carter, 1980) typically conditioned aversions to a
variety of external (e.g. type of cage, water container and drinking tube) and internal stimuli
(gustatory and olfactory) and then compared aversions to the taste stimulus following the
extinction of groups of rats to a component of stimulus configuration present during conditioning
15
trials. In general, this series of experiments provided evidence that, when presented with a
compound stimulus consisting of exteroceptive and interoceptive CSs, rats develop aversions to
both kinds of CSs.
More recently, Symonds and his colleagues (Symonds, Hall, Lopez, Loy, Ramos, &
Rodriguez, 1998) have used rats to examine the role of drinking response itself in the
development of context aversion. They used two distinctive cages different from each other
(Context A and B), both different from the home cage. First, all rats were exposed to Context A
which was followed by an injection of LiCl. In this context, subjects are divided into two groups:
the water group (W) was permitted to drink water, whereas for the other group (NW) water was
not available. Then, all subjects were exposed to Context B which was not followed by injection
of LiCl. In Context B, all subjects were allowed to drink water before being returned to the home
cage. A test period followed in which all subjects had access to a sucrose solution in both
contexts. Group W showed suppressed consumption of sucrose in the conditioned context
(Context A) but not in the control context (Context B). This effect was not observed in Group
NW because they did not differ in sucrose intake in the two contexts. This shows that both
drinking a solution and contexts have an effect on learning. Group W showed suppression to
sucrose which is also a consequence of generalization from one substance (water) to another
(sucrose).
Many researchers have used a blocking procedure in their experiments to examine the
associative strength of the context. In a blocking procedure, during Phase 1, Stimulus A (CS) is
conditioned with a UCS in the experimental group; however, the control group only receives
stimulus A without an UCS. During Phase 2, Stimulus A is presented simultaneously with
Stimulus B and is paired with the UCS in both the experimental and control groups. A later
retention test of stimulus B alone shows that less conditioned responding occurs to stimulus B in
the experimental group compared to the control group (Table 1-2).
16
Groups
Phase 1
Phase 2
Test
Experimental
A is paired with UCS
A+B is paired with UCS
B
Control
A is unpaired UCS
A+B is paired with UCS
B
Table 1-2. Experimental Setup
If contextual cues really have acquired aversive features, then they should block further
aversive conditioning. For example, Symonds and Hall (1997, Experiment 2) used two groups of
rats in which the experimental group received LiCl in Context A (different than home cage).
However, the control group spent time in Context B (also different than home cage) without any
injection of LiCl. In the second phase of training, all groups received sucrose in the home cage
and were transferred to the context in which they received pre-training. After they spent about 30
minutes in the context, subjects were injected with LiCl. During the retention test, a sucrose
solution was given to all subjects in their home cages. The experimental group did not show any
suppression to sucrose, whereas the control group drank considerably less (Table 1-3).
Groups
Phase 1
Phase 2
Test
Results (ml)
Experimental
Context A + LiCl
Sucrose + Context A + LiCl
Sucrose
13.3
Control
Context B
Sucrose + Context B + LiCl
Sucrose
5
Table 1-3.Experimental Setup
17
In this section, we have reviewed the experiments which have paired toxicosis with a
combination of gustatory and exteroceptive cues. To summarize a large number of findings, it
was found that environmental stimuli can be more closely associated with toxicosis when a taste
stimulus is present during conditioning than when it is not (Best, Brown, & Sowell, 1984; Best,
Batson, Meachum, Brown, & Ringer, 1985, Archer et al.1979). In comparison, there has been
evidence that conditioned aversion resulting from the pairing of environmental stimuli and illness
(without using any taste as a CS) is hard to develop (Domjan & Wilson, 1972; Garcia & Koelling,
1967; Garcia, Kimeldorf & Hunt, 1961; and Garcia et al., 1966). This seems to suggest that
somehow taste stimuli enhance conditioned context aversion.
Context Aversion Learning
Aside from studies of compound stimuli (e.g. taste and context) a number of studies have
paired illness with substantive alterations in the housing environment in the absence of gustatory
cues to study context aversion learning. For example, Revusky and Parker (1976) demonstrated
that rats can develop conditioned aversion to novel drinking cups when paired with toxicosis. In
Experiment 1, all rats had free access to deionized water in glass spouts before the experiment
started. During two conditioning trials, deionized water or a sucrose solution was presented to
experimental groups in either steel cups or glass spouts for 15 minutes, and an injection of LiCl
was given immediately or 30 minutes after drinking in both groups. Two control groups drank
deionized water or sucrose solution from the steel cup without injection of LiCl. Conducted 16
days after the last conditioning trial, retention test results showed that a strong aversion to
deionized water in either glass spouts or steel cup was observed only if the rats consumed
deionized water prior to toxicosis, whereas rats exposed to a sucrose solution in either glass bottle
or steel cup during the conditioning trial did not show any aversion to deionized water in a steel
18
cup. Surprisingly, it was also observed that the container in which the deionized water was
presented was a factor. If the container used during training was the same as that used during
conditioning trial (in this case glass spout), the aversion was stronger.
In Experiment 2, they used different methods to study the effects of variations in delay.
For instance, nine conditioning trials were conducted rather than two, tap water was used rather
than deionized water, and steel spouts were used rather than glass spouts as maintenance tubes.
During conditioning, rats were exposed to LiCl at different delays after drinking tap water from a
steel cup. On days when training trials were not scheduled, the rats were allowed to drink tap
water from a glass spout for 15 minutes. Retention test results showed that the reduction in water
intake from the steel cup was inversely related to the delay of toxicosis. Only immediate and 30minute delay groups exhibited significant aversion. Moreover, in Experiment 2, there was no
effect that could be attributed to the sensitization when rats were given water in glass spouts on
days when training was not scheduled. Revusky and Parker concluded that aversion was obtained
by pairing illness with the appearance of the cup. However, it could not be explained by taste of
water because no aversions to drinking tap water from glass spouts were observed. As later
discussed by Nachman and his colleagues (Nachman, Rauschenberger, & Ashe, 1977),
conditioned aversion occurred in the cited study either to visual cues (different appearance of
containers) or to somatosensory stimulation (different body positions or oral sensations)
depending on if the rats drank from bottles vs. cups.
More recently, a number of studies have paired toxicosis with substantive alterations in
the housing environment. For instance, Boakes and his colleagues (Boakes et al., 1997) examined
if a conditioned aversion can be established dependent on context. During three conditioning
trials, two distinctive contexts (Context1 and 2) different than the rats’ home cages (variations in
cage size, illumination level, and floor composition (wire mesh vs. grid) were paired with either
LiCl or NaCl. All subjects were given discrimination training in which Context 1 was followed
19
by an injection of LiCl but Context 2 was followed by injection of NaCl. Rats showed decreased
water intake in the context paired with LiCl compared to the context paired with NaCl.
In a related study, Rodriguez and his colleagues (Rodriguez et al., 2000) conducted an
experiment. They used two phases: during the first phase, rats received 30-minute exposure trials
in each of two novel contexts (contexts A and B; both different from the home cages). In context
A, animals received LiCl, and in context B, they received saline injections. Then, they were tested
for consumption of sucrose during phase 2 (Table 1-4). The experimental group was tested in
context A and the control group was tested in context B. Results showed that the experimental
group (in context A) did not consume as much sucrose as the control group (in context B) (Table
1-4). In other words, rats consumed less sucrose in the LiCl-paired environment than in the
environment associated with NaCl. This result shows that contextual cues became conditioned
stimuli, which results in a conditioned response (decreased consumption).
Table 1-4. Experimental Setup. Adapted from “Overshadowing and latent inhibition of
context aversion conditioning in the rat”(42-49), by Hall, G. and M. Symonds, 2006, Autonomic
Neuroscience.
Context aversion learning has been also studied in the drug administration arena. Drug
administration paraphernalia in medical environment (e.g. syringe, white uniform, needle) or in
specific location (e.g. doctor`s office where the drug is administrated) can serve as conditioned
20
stimuli that, when paired with the drug (UCS), come to elicit conditioned responses
(Andrykowski & Redd, 1987).
Two common techniques to reduce conditioned context aversion:
Overshadowing and Latent inhibition
As previously mentioned, anticipatory nausea is caused by an association between cues in
the clinic and the experience of nausea (the side effects of chemotherapy or radiation treatment).
It has been shown that this association (development of context aversion) can be reduced by two
techniques: overshadowing (presenting a novel salient flavor during context conditioning) and
latent inhibition (prior exposure to the context).
Overshadowing is a technique that was first described by Pavlov and his colleagues
(Pavlov & Anrep; 1960), and it is used to reduce the association of CS with UCS. It consists of
presenting a compound stimulus as a CS and pairing it with a UCS. A compound stimulus
consists of two separate elements, one of which is more salient than other. This procedure results
in more intense stimuli becoming conditioned more easily than weaker stimuli. Thus, a novel
salient cue that is presented during chemotherapy sessions can overshadow the context and
prevent the development of anticipatory nausea and vomiting (ANV) in humans.
Latent inhibition is similar to overshadowing. If a stimulus is very familiar, it will not as
easily be associated with a UCS as a novel stimulus. Thus, CS pre-exposure inhibits learning. For
instance, a simple pre-exposure to the context may be a very good way to reduce ANV in cancer
patients.
21
Symonds and Hall (1999) conducted experiments to demonstrate the overshadowing
procedure. During the first phase, both the experimental and control groups remained in context
A (differed from home cage) and were injected with LiCl (UCS). The experimental group was
given a salient flavor stimulus (H; a weak acid solution with a sour taste), whereas the control
group was given water. To equalize the experiences of the two groups in the first phase, the
control group was then given a salient flavor stimulus in context B (differed from home cage) and
the experimental group was then given water in context B, followed by an injection of LiCl
(Table 1-5). The experiment then proceeded to the compound conditioning phase, where both
groups received sucrose in context A paired with LiCl. During the test period, the sucrose intake
of the rats was measured in their home cages. The results showed that context A was less
effective in blocking the conditioning to sucrose in the experimental group because the salient cue
overshadowed context A in the first phase of the conditioning. Thus, the experimental group had
learned an association between sucrose and LiCl during the compound conditioning phase,
leading them to consume less sucrose than the control group. However, for the control group,
during context conditioning there was no salient cue to overshadow the context. Instead, context
A was much more effective at blocking the conditioning to sucrose, leading the control group to
be less averse to sucrose (Table 1-5).
Table 1-5.Experimental Setup, Adapted from “Overshadowing and latent inhibition of
context aversion conditioning in the rat” (42-49), by Hall, G. and M. Symonds, 2006, Autonomic
Neuroscience.
22
Hall and Symonds (2006) conducted an experiment on rats to observe the effects of
context pre-exposure (latent inhibition). Experimental groups had stayed in the experimental
context (A) for 30 minutes per day over eight days before the context conditioning phase started.
However, the control group was not exposed to the experimental context before the context
conditioning phase. Later, during the context conditioning phase, both groups were exposed to the
experimental context and were injected with LiCl. During the test phase, sucrose intake was
measured in the experimental context (A) for both groups. The control group drank significantly
less sucrose compared to the experimental group, suggesting that pre-exposure to the
experimental context produced a latent inhibition effect (Table 1-6).
Table 1-6. Experimental Setup, Adapted from “Overshadowing and latent inhibition of
context aversion conditioning in the rat” (42-49), by Hall, G. and M. Symonds, 2006, Autonomic
Neuroscience
These examples of obtaining information from an animal model help us develop methods
to study human patients exhibiting nausea and vomiting. However, various researchers have
suggested that specialized methods are needed for humans because they have different
requirements than other animals (Yates, Miller and Lucot., 1998; Stockhorst, Steingrueber, Enck,
& Klosterhalfen, 2006). For instance, we cannot simply inject healthy people with LiCl to induce
nausea, as we can with rodents. Moreover, when we use rodents, we can control the
23
environmental changes (floor texture, cage size and shape, wall pattern, olfactory cues) and pair
them with illness to generate conditioned aversion (Boakes et al., 1997). However, cancer
patients are exposed to more substantial environmental changes (the hospital, places near the
hospital, the chemotherapy room) in their life.
Stockhorst and her colleagues (Stockhorst et al., 1998) conducted a study to induce
overshadowing using a rotation technique in humans. 24 healthy subjects (12 males-12 females)
were divided into two groups. In the acquisition phase, the experimental group received a salient
taste (elderberry, sallow-thorn, sloe) before rotation started on three subsequent days. The control
group received water instead and the other conditions were the same as experimental group. To
control for different taste experiences, 12 hours later in their home environments, the
experimental group drank water, while the control group drank the salient beverage. Later, both
groups drank water before being rotated in the rotation environment on fourth day. Results
showed that the experimental group (who drank the salient beverage) experienced reduced ANV.
Klosterhalfen and his colleagues (Klosterhalfen et al., 2005) used a latent-inhibition
procedure in one of their experiments in healthy subjects. Subjects were exposed to a rotation
context (CS) before the first rotation. Later, subjects were divided into three groups. Subjects in
Group LI0 were placed in a natural environment for 3 days. Subjects in group LI1 were placed in
a natural environment for 2 days and then were exposed to the rotation context for 1 day. Subjects
in group LI3 were placed in the rotation environment for three days without any rotation. Results
showed that since LI3 and LI1 groups were exposed to the rotation context more often compared
to LI0 group, ANV symptoms were significantly reduced in these LI3 and LI1 groups.
These two techniques are obviously very important to reduce conditioned aversion in
both animal and human models. In addition, animal models have been extremely beneficial for
24
understanding these learning techniques and are suitable models to study ANV in cancer patients.
Furthermore, animal models also allow us to better understand which environmental stimuli are
more likely to condition. With this improved understanding, we can create environments and
procedures to better avoid conditioned aversion.
Conditioned Place Aversion and Conditioned Place Preference
In addition to studies of context aversion learning, there are well established protocols in
which drugs of abuse are associated with contextual stimuli, and their effects are evaluated. In
mouse research today, every year at least 100 papers are published about conditioned place
aversion (CPA) and conditioned place preference (CPP). Both of these procedures use context in
their experiments to examine the positive and negative effects of drugs. CPP occurs when a
subject comes to prefer one place more than others because the preferred location has been paired
previously with rewarding events (Bardo, Bevins, 2000; Prus, James, and Rosecrans, 2009). At
the beginning, the animal is allowed to explore two different environments without restrictions.
During the conditioning phase, the rewarding drug is paired with a specific environment that
serves as a conditioned stimulus. Thereafter, the animals are returned to their home cages.
Subsequently, when animals have the choice of freely exploring the drug-paired (CS) and nondrug-paired compartments, they prefer the CS environment, which indicates the development of
conditioned placed preference (Itzhak and Martin, 2002).
Conditioned Place Aversion (CPA) is another type of learning that evaluates aversions to
a specific environment that has been associated with a negative reward (Grillon, Baas, Cornwell,
Johnson, 2006; Azar, Jones, and Schulteis, 2003; Stewart, Grupp, 1986). Frisch and colleagues
(Frisch, Hasenöhrl, Mattern, Häcker, & Huston, 1995) designed an experiment in which rats were
exposed to an injection of LiCl in the compartment that they had preferred over three baseline
25
trials. During the test period, subjects were returned to an apparatus where they could freely move
between a compartment where they were conditioned with an aversive stimulus (LiCl) and a
compartment with neutral cues. Results showed that animals treated with LiCl spent less time in
the treatment compartment.
In conclusion, rewarding or aversive effects of drugs are examined by these procedures.
It is very important for context learning because this protocol shows that contextual cues can
control behavior if serving as a signal for a UCS or a reinforcer.
Conclusion
Different views on the ability of animals to associate specific CS with particular
reinforcer have been presented in the cited papers. After Garcia reported that only particular
classes of CS can be associated with a particular UCS (the principle of selective associative
learning), a number of subsequent studies supported his theory (Gemberling & Domjan, 1982;
Rescorla, 2008). On the other hand, several researchers have shown that rats developed
conditioned aversions when illness is induced in a particular environmental context.
Most of the cited studies conducted their experiments by making large alterations in the
environment and inducing illness to cause context aversion learning (Boakes et al., 1997;
Rodriguez et al., 2000). In the literature, only one experiment paired small alterations in the
environment with illness (Revusky & Parker, 1976).They showed that rats can show aversion to
novel cups if these are paired with toxicosis during conditioning trials. However, as later
discussed by Nachman and his colleagues (Nachman et al., 1977), conditioned aversion might
have occurred in the cited study as a response either to visual cues (the appearance of different
containers) or to somatosensory stimulation (different body positions or oral sensations) related to
drinking from bottles versus cups. Moreover, there is no previous research that evaluates the
26
development of context aversion to small changes in the environment in mice, as opposed to rats.
Thus, the aim of the present study is to augment the evidence that animals are able to learn
aversion to contexts paired with aversive stimuli.
Rats have been the species of choice in the study of conditioned learning. In my
experiments, I will use mice to confirm that pairing illness with environmental context can result
in strong aversion. I will use advanced intercross mice in an attempt to make my results relevant
to laboratory mice as a species. In addition, inbred mice are idiosyncratic, which means that they
have unique responses and are genetically identical, so that it is very hard to generalize their
behaviors to laboratory mice. Therefore, if we take the average behavior of advanced intercross
mice, we are more likely to understand their general response to stimuli.
The literature emphasized three key features with regard to CTA. First, a single pairing of
a novel taste with illness causes conditioned aversion to the novel taste. Second, even when a
long delay is introduced between CS and UCS, an effective CTA can still be demonstrated
(Garcia et al. 1966; Revusky & Garcia, 1970) .Third, CTA learning can last for months (Houpt et
al., 1996; Martin & Timmins, 1980; Steinert, Infurna, & Spear, 1980). In this thesis, I will look at
rates of acquisition across three conditioning trials, CS-UCS delay, and how long mice retain
context aversion.
I am especially interested in applying the principles of learning theory in clinical
situations to reduce nausea symptoms when patients are undergoing cancer treatment. It will help
us to understand how environmental cues in chemotherapy rooms can act as conditioned stimuli
and how we can reduce conditioned nausea in patients by using learning theory.
27
Chapter 2
Methods common to many experiments
The following methods and procedures are common to several experiments in this thesis.
The animal care and use procedures have been approved by the IACUC (Institutional Animal
Care and Use Committee) of The Pennsylvania State University.
Subjects
We have used advanced intercross mice for most of our experiments. Advanced
intercross mice are created by crossing two or more strains and continuing to intercross the litters
in subsequent generation (F2, F3,F4, F5 etc) in such a way as to avoid inbreeding. In most of our
experiments, either genetically heterogeneous mice from an advanced intercross (AI) of C57BL/6
J (B6) and DBA/2J (D2) (B6D2) strains or the LG/J (LG) and SM/J (SM) (LGSM) strains have
been used. We have used mice produced by the thirteenth generation of crossing for the B6D2
strains and the eightieth generation for the LGSM strains.
The B6D2 (both male and female) and LGSM male mice were obtained from Dr.
Abraham Palmer at The University of Chicago. The LGSM female mice were obtained from Dr.
James Cheverud at Washington University in St. Louis.
Inbred mice are idiosyncratic—meaning they have unique responses and are genetically
identical—it becomes hard to generalize about their behaviors. Collecting data regarding the
typical behavior of AI mice makes it more likely that we can generalize our results to the
laboratory mouse as a species.
28
Maintenance Conditions
AI mice were mated in pairs. Mice were weaned and housed in groups with samesex
littermates on postnatal day (PND) 28. Before each experiment they were housed individually for
at least one week.
Assignments of animals to experimental groups
Each mother/father breeding pair was unique, and their offspring are more similar to one
another than they are to offspring of different litters. Thus, mice from each litter were randomly
assigned to different experimental conditions, so as to control for potential variations among the
litters. More specifically, the reason that we assigned mice from different litters to different
groups is that animals from the same litter tend to be more similar than animals from different
litters (Lazic & Essioux, 2013). For instance, the number of offspring might be higher in one litter
than another litter; this means that the animals are often raised in dissimilar environments. In
addition, there is an inverse relationship between litter size and offspring body weight that
develops during the lactation period in mice (Tanaka & Ichikawa, 1995). Furthermore, there is a
high correlation between litter size and behavioral development in mice (Tanaka, 1998). For
example, swimming direction (skills), which requires the development of coordinated movement,
is significantly higher in smaller litters (Tanaka, 1998).
In addition, coat colors were balanced across the groups to prevent any genetic influence
related to coat color (linked genes, etc.) from biasing the results. Even though we were unable to
consistently regulate the proportion of males and females in each group, due to limited
availability of mice, we generally assigned an equal proportion of males and females to each
29
experimental group. We usually used mice aged approximately 3 to 5 months old at the beginning
of the experiments.
Colony Room (Light Cycle, Temperature, Food)
The colony room that we used had a 12/12h light/dark cycle (lights on 0500-1700 hrs),
and its temperature was maintained at 22.2 °C +/-1. There was no natural light in the room. Body
weights were recorded at both the beginning and end of the experiments. Either the Rodent Lab
Diet 5001 (PMI International; Brentwood, MO) or the Rodent Irradiated Lab Diet 5053 (PMI
International; Brentwood, MO) (they are nutritionally equivalent), as well as tap water, were
available ad libitum during the experiments, except when animals were water restricted as
described below.
General Experimental Considerations
Before the experiments were conducted, all mice were routinely given tap water in pintsize translucent plastic bottles with blue plastic lids and metal spouts. Mice were housed in
plastic shoe-box cages (12 cm x18 cm x 29.5 cm) (Figure 2-1). Nesting materials obtained from
Ancare in Belimore, NY, were provided to all mice.
30
Figure 2-1. Plastic bottles were presented before starting the experiment.
Table 2-1. Shows the general procedure that we used in our experiments. The experiment
consisted of five phases: adaptation, training, conditioning, recovery period, and retention tests.
Top row: The number of days devoted to retention phase varied by experiment.
I. Adaptation and Training
Before we started our experiments, we acclimated the mice to their maintenance
bottles/tubes for about one week. We used different kinds of maintenance tubes depending on the
type of experiment that we were conducting. For instance, one of the maintenance tubes that we
used was: 25 mL graduated tube with a piece of light-colored tape near its stainless steel (SS)
spout, complete with a ball bearing and a rubber stopper (Figure 2-2).
In situations in which we have used graduated tubes for our experiments, we have made
appropriate cage configurations. We have reversed the lids and put the graduated tubes at a 45degree angle on the right-hand side of the food. This is a more reliable angle for presenting water
in graduated tubes to mice (Figure 2-2).
31
Figure 2-2.The experimental set up is illustrated from the back of the cage in order to
clearly demonstrate the tube arrangement.
We trained the mice to drink promptly water from their maintenance tubes during the
light phase of the circadian cycle by depriving the mice of water for 16 hours and giving them
access to water several times per day during the light phase of the light/dark cycle. Mice had 30
minutes access to water on 3 occasions starting at 9.00 a.m. and then were allowed to drink for 3
hours until the beginning of the dark phase of the cycle. This procedure was repeated on 3
separate days.
II. Conditioning Procedures
Following adaptation and training, context conditioning aversion trials (CCA) were
carried out at two-day intervals. After 16 hours of water deprivation, the mice had water
presented in their CS tubes for 15 minutes. These CS tubes were located as described above in
their home cages. We used different kinds of CS tubes depending on the experiment that we were
doing. Two types of CS tubes that we commonly used during the conditioning trials were 25 mL
graduated tubes, each with a piece of dark-colored tape near its stainless steel (SS) spout (Figure
32
2-3) and pint-size regular glass bottles, each with a pin-hole SS spout and a rubber stopper
(Figure 2- 4).
Figure 2-3(left). and Figure 2-4 (right). The CS tubes that we have used during the
conditioning trials.
15 minutes after being presented with the CS tubes, the mice in the control groups were
injected intraperitoneally (IP) with sodium chloride (NaCl) (0.15 M, 0.3 mL /10 grams body
weight), whereas mice in the experimental groups were injected with lithium chloride (LiCl) (at
the same molar concentration and dose as NaCl). Then water intake was measured. On the
intervening days, the animals had ad libitum access to their maintenance tubes.
Delay conditioning
In some of our experiments, we wanted to examine if CCA developed with a 30-minute
delay. In those specific experiments, 30 minutes after the CS tubes were removed from the cages,
the mice were injected with LiCl or NaCl based on their assigned groups.
33
III. Recovery Period
After the last conditioning trial, the mice were allowed a recovery period. During this
period, the mice were given their regular tubes for two or three days without any water
deprivation.
IV. Retention
Following completion of the recovery period, we conducted retention tests: we presented
the mice with their CS tubes for 42 minutes and recorded intake.
We observed that the mice showed slight suppression of intake when presented with their
maintenance tubes during the retention tests. Therefore, in some experiments, if a mouse had
access to water from the CS tubes during the retention test, on the following day, the mouse was
presented with the maintenance tube, or vice versa. This provided us with an opportunity to
assess both the total (the CS tube) and non-specific suppression (the maintenance tube). Thus,
using retention analyses, we were also able to examine the effects of the day of presentation of
the CS tubes (CS Day) on the results.
Statistical issues
Suppression index
A suppression index was calculated in which each individual animal’s intake was
subtracted from the control group mean for that trial and that difference score was divided by
mean control group intake, subtracted from 1 and expressed as a %. ((1 - [intake of the individual
34
animal] / [mean intake of the control group]) X 100). For example, 0% suppression indicates the
same intake as control, 100% suppression indicates 0 mL intake (Nowlis et al., 1980).
Data Analyses
Conditioning
We conducted two separate conditioning analyses of variance for CCA 2 and CCA 3: the
first analysis was of intake, and the second, suppression. For intake, there were two betweensubject factors, Groups (Control vs. Experimental) and CS-UCS Interval (Immediate vs. Delay)
as well as a within-subjects measure (Trials). In the second analysis (suppression), there was one
between-subject factor, CS-UCS Interval (Immediate vs. Delay) as well as a within-subjects
measure (Trials).
Retention
We assessed retention on one or several weeks (starting 1 week after conditioning) in
order to compare duration of retention in Immediate and Delay groups.
In some experiments, we evaluated when extinction occurred by using post-hoc paired ttests comparing total to non-specific suppression within each conditioned group. To adjust for
multiple comparisons, we adjusted the p-value by Bonferroni correction.
There were two between-subject factors, CS-UCS Interval (Immediate vs. Delay) and CS
Day (the day of the presentation of the CS), as well as two within-subjects measures that were
Weeks (6) and Tubes (CS vs. Regular).
35
In addition, in some of our experiment, a t-test was used to determine statistical
significance for the retention test. 1 or 2 tail tests were used as appropriate. Alpha level was set at
p<0.05.
Table 2-2. Design of ANOVA
Appendix
Visual system of mouse
As our experiments progressed, we tested the ability of mice to learn more subtle
variations in the home cage environment. The most subtle were the experiments evaluating the
ability of mice to respond to the brightness of tape on drinking tubes. Since this is a visual
stimulus, we need to discuss the visual system of a mouse. Therefore, I will briefly summarize the
anatomy of the retina, light spectrum detection, and visual acuity in both mice and humans.
36
Anatomy of the retina
The retina is a light-sensitive layer of cells at the back of the eye. Rods and cones are two
types of photoreceptors cells; in humans, cone cells are densely packed in the fovea centralis and
rods are concentrated at the outer edges of the retina.
These photoreceptors convert light into action potentials which are carried to the brain by
the optic nerve. Rods are specialized for low-light vision but are not sensitive to color. Cones, on
the other hand, are specialized for daytime vision, much less sensitive to light than rods and can
detect color. Both mice and humans have more rods than than cones in their retinas. On average,
rods constitute 97.2 % of mouse retinal photoreceptors, and cones make up the remainder (2.8
%) (Carter- Dawson & Lavail, 1979). This proportion of cones is about half as many as humans
(~5%,) (Curcia & Allen, 1990), and may be lower due to their need for more rods to support their
nocturnal habits.
Figure 2-5. Shows the rods and cons in the human retina. Small circular cells are rod
photoreceptors, whereas larger cells are cones.Scale bar is 10 μm (Curcio, Sloan, Kalina, and
Hendrickson, 1990).
37
Figure 2-6. Shows the mosaic of rods and cones in the mouse (C57BL/6). Dark mosaics
show the cones, lighter mosaic shows the rods. Scale bar is 10 μm (Jeon et al., 1998).
Composition of photoreceptors: human vs mice
Most primates and humans have three types of cones in their retinas. These are blue,
green and red (trichromatic vision) color cones. The visible spectrum range for human eye is from
780 nanometers (nm) to 390 nanometers.
However, mice have dichromatic vision, which means that they have two types of color
cones in their retinas: blue-violet and green. This enables them to see greens, with a maximal
spectral sensitivity of 508 nm, and blue-ultraviolet, with a maximal spectral sensitivity 360 nm
(Wang et al., 2011). Thus, mice have better vision at short wave lengths than humans. However,
they have a poor ability to differentiate reds from other colors because they lack cones specific to
the longer wavelengths.
38
Ultraviolet
Figure 2-7. Shows the visible light spectrum for
humans.
<380 nm
Violet
380-450
Blue
450-500
Blue-green
500-520
Green
520-550
Yellow-green
550-570
Yellow
570-600
Orange
600-630
Red
630-680
Infra-red
>680
Visual acuity
Visual acuity is measured in cycles per degree (c/deg) and it gives information about the
clarity or sharpness of vision. Acuity of humans is about 30 cpd (Prusky et al. 2002). Among
common laboratory mouse strains, visual acuity varies widely.
Brown and Wong (2007) examined two commonly used strains to measure visual acuity.
Results showed that the visual acuity is 0.38 c/deg for C57BL/6J mice and it is 0.54 c/deg for
DBA/2J. Although young DBA/2J mice have higher visual acuity compared to C57BL/6J, they
develop glaucoma when they get old (onset at about one year of age) which results in loss of
visual acuity (Chang et al., 1999). However, C57BL/6J mice retain their visual ability at 0.38
c/deg to two years of age (Brown and Wong, 2007). Wong and Brown (2006) also demonstrated
39
that the visual acuity was below the 0.17 c/deg for several albino strains (A/J, BALB/cByJ, and
BALB/Cj).
In general, it has been shown that mice that have no visual defects have a visual acuity
between 0.38 and 0.5 c/deg, whereas albino mice have visual acuity below 0.17 c/deg (Wong &
Brown, 2006; Gianfranceschi et al.,1999). Thus, pigmented mice are about 60 to 80 times weaker
in acuity compared to human and albinos,~ 180 times. This variation in visual capacity is relevant
to behavior: the mice of 129S1/SvlmJ, C57BL/6J and DBA/2J and AKR/J (albino strain)
performed better on visual tasks than the mice of albino strains A/J, BALB/cByJ and BALB/Cj
(Wong and Brown, 2006). Although albinism in mice is associated with poor vision, it is not
known why the performance of AKR/J mice on visual task is comparable to the performance of
mice with normal vision (Wong and Brown, 2006).
Even in behavioral studies that are not intending to evaluate vision, but instead to
evaluate other psychophysical attributes, vision may be relevant. A series of behavioral tasks are
needed to examine the mouse’s behavioral phenotypes (Bailey at al., 2006). Performance on these
behavioral tasks often depends on the combined motor, sensory, cognitive abilities of the mouse.
Behavioral tests of learning frequently use visual stimuli as cues for performance tasks (such as
the radial arm maze, the Morris water maze, etc.). Our experiments also rely on visual ability in
learning to discriminate tape brightness during the conditioning trials. Thus it is important to
understand visual capacity in different strains of mice.
In the experiments described in this dissertation, B6D2 AI or LGSM AI mice were used.
In our experiments (Chapter 3, 4 and 5), we used two different colored tapes: lightcolored (yellow) tape for the maintenance tubes and dark-colored (red) tape for the CS tubes
(Figure 2-8). As previously discussed, mice do not possess the retinal cells needed to discriminate
red from yellow. As shown in Figure 2-9, there is a difference in brightness between the light-
40
colored and dark-colored tapes that we used and it is the brightness dimension that we think mice
are responding to.
Figure 2-8.Red and yellow tapes are shown together white and black tape as reference.
Figure 2-9.The different degrees of brightness of the four tapes.
In conclusion, even though, mice are considered as nocturnal animals which mean that
they use their olfaction, audition and vibrissae for sensing their environment, it is also important
to consider them as visual animals. We should carefully choose the strain of mice that we are
going to use in experiments to measure the visual ability of mice. It is very clear that variations in
visual capacity of the mice will affect the task performance in behavior tests.
41
Chapter 3
Exploratory Studies with B6D2 mice
Experiment 3-1
During the course of studies of CTA in our laboratory, we observed that control group
mice maintained on water in plastic bottles, presented with distilled water in a novel graduated
tube and injected with LiCl in conditioning procedures, drank little or no water during retention
trials. However, if animals were adapted for several days to the novel tubes used during
conditioning these aversions to tubes in retention trials (among control mice) were no longer
seen. Interestingly, some of Garcia`s earliest studies provided evidence consistent with our
observations. Rats were exposed to radiation in a testing chamber while they were drinking from
novel plastic bottles and drank less from these containers than from their regular glass bottles.
Garcia (1954) hypothesized that the pairing of radiation with water from plastic bottles resulted in
a conditioned aversion to the novel taste of the water from plastic bottles. However, another
hypothesis can also account for the reduction in fluid intake from plastic bottles: that rats formed
an association between radiation-induced sickness and the novel features of the plastic bottle
(e.g., size, shape, and opacity).
In Experiment 3-1, we tested this hypothesis by maintaining mice on tap water from
opaque plastic bottles (with blue plastic stoppers and metal spouts) and then pairing illness
(induced by LiCl injections) while receiving water from glass bottles. This is the opposite
configuration to that used by Garcia (plastic to water rather than water to plastic) but it was
adopted because it is very difficult to measure water intake accurately from plastic bottles. In any
case, it was predicted that mice would develop conditioned aversions to one or more features that
42
differ between plastic and glass bottles and this aversion would be reflected in reduced water
intakes when drinking from glass bottles. An experimental group was also included that drank
from glass bottles containing water recently decanted from plastic bottles. If, as suggested by
Garcia, the difference between taste of water in the glass vs plastic bottles was a key feature
underlying any conditioning, this group should not develop a conditioned aversion.
Subjects
45 AI B6D2 mice (mean age 93 days, range; 87-100) from 20 different litters were used.
Sex (24M, 21F) and litter membership was evenly distributed across the 3 experimental groups
(15/group).
Methods
All mice were routinely maintained on tap water in pint size translucent plastic bottles
with blue plastic lids and metal spouts. Mice were then trained to drink water promptly from
these bottles in the light phase of the circadian cycle by depriving them of water for 16 h and
giving them access to water for 30 minutes access on 3 occasions between 9.00 a.m. and 2 p.m.
and then were allowed to drink for 3 hour until the beginning of the dark cycle. This procedure
was repeated on 3 separate days.
Conditioning procedure
Following adaptation training, 3 context aversion conditioning (CCA) trials were carried
out at 2 days intervals. After 16 hour of water deprivation, water was presented in pint size glass
bottles with rubber stoppers and stainless steel (SS) spouts with ball-bearings to all mice for 10
minutes in their home cage. Mice in the control group (PB-GB TW/NaCl) drank tap water and were
injected intraperitoneally (IP) with NaCl (0.15 M, 0.3 mL /10 grams body weight; see Table 1).
One experimental group (Glass bottle tap water: PB-GB TW/LiCl) also drank tap water and was
injected with LiCl (same concentration and dose as NaCl). A second experimental group (also
43
injected with LiCl) was presented with glass bottles containing water decanted from plastic
bottles (the water had been held in the plastic bottles for at least 48 hours before being decanted
into glass bottles) immediately before the trial (PB-GB PW/LiCl; see Table 3- 1). 10 minutes after
the injection, the CS bottles were removed from the cages; water consumption of each mouse was
determined by weighing the water bottles at the start and finish of the conditioning. 20 minutes
after the CS tubes were removed from cages their regular plastic bottles were returned to the
home-cage. On the intervening days, the animals had ad libitum access to their regular plastic
bottles.
After CCA3, animals were maintained on ad libitum food and water in plastic bottles
until a retention test (CCA3+7 days) conducted.
Following our completion of the recovery period, retention test was carried out after the
last conditioning trial (CCA3+7 days). We presented the mice with their CS bottles for 42
minutes after 16 hour water deprivation; we then calculated water intake from their CS bottles.
Table 3-1. Experimental Setup
44
Data Analyses
The following suppression index was calculated: ((1 - [intake of the individual animal] /
[mean intake of the control group]) X 100) (Nowlis, Frank, & Pfaffmann, 1980). 0% suppression
indicates the same intake as control, 100% suppression indicates no intake.
Conditioning Analyses
We conducted two analyses of variance for CCA 2 and CCA 3: the first analysis was of
intake, and the second, suppression. For intake, there was one between-subject factor, Groups
(Control, TW and PW) and a within-subjects measure (Trials). In the second analysis
(suppression), there was one between-subject factor, Groups (TW vs. PW) as well as one withinsubjects measure (Trials).
Retention Analyses
A t-test was used to determine statistical significance for the retention test. 1 or 2 tail
tests were used as appropriate. Alpha level was set at p<0.05.
Results
The raw intakes for Experiment 3-1 are shown in Table 3-2 and suppression ratios in
Figure 3-1.
45
Table 3-2. Water Intakes and percent suppression for Experiment 3- 1
Conditioning Results
As anticipated, there was no significant difference in water intake between the LiCl and
NaCl groups during the first conditioning trial.
The analysis of variance showed a highly significant reduction in intake on CCA2 and
CCA3 in conditioned mice compared to the controls (F1, 42=75.9, P<0.001). In addition, there
was no significant Group X Trial interaction across CCA2 and CCA3.
Our analysis of suppression shows that suppression was not significantly stronger on
CCA3 than on CCA2 (Trial, n.s.). In addition, there were no significant differences between the
experimental groups in their responses to suppression during CCA2 and CCA3 (Groups X Trial
interaction, n.s.).
46
Retention Results
The retention test conducted 7 days after CCA3 showed that both experimental groups
drank significantly less than the control group (Control group vs. PB-GB TW/LiCl, t=11.104, df=27,
p<0.001; vs. PB-GB PW/LiCl ,t=8.476, df=28, p<0.001; Figure 3-1). Moreover, the analyses of
suppression showed that there were no significant differences between PB-GB PW/LiCl and PB-GB
TW/LiCl
experimental groups in retention.
The fact that there were no statistically significant difference between the experimental
groups in either conditioning trials or during the retention test is not consistent with a role for the
flavor of water from plastic bottles influencing the strength of conditioning.
Suppression Index (%)
Development of aversion to glass bottles by mice maintained on plastic bottles
PB-GB TW/LiCl
100
90
80
70
60
50
40
30
20
10
0
PB-GB PW/LiCl
CCA1
CCA2
CCA3
Conditioned Context Aversion: Acquisition and Retention
Retention
CCA3+7
Days
Figure 3-1. B6D2 AI mice maintained on plastic bottles (PB) were given 3 conditioning
trials in which drinking from glass bottles (GB) was paired with NaCl (controls) or LiCl. PBGBTW/LiCL and PB-GB PW/LiCl groups showed high suppression to the glass bottles (CS)
after a single trial which was sustained for 7 days.
47
Experiment 3-2
The findings of Experiment 3-1 provided good support for the hypothesis that minor
alterations in the cage environment (plastic vs glass water bottles of similar size and shape) can
serve as CS’s in an aversion conditioning paradigm. The present experiment was carried out to
provide more evidence that a change in the taste of water is not necessary to cause context
aversion in mice. In this study, we used either glass bottles or graduated tubes with light-colored
tape, both containing tap water, at all phases of the experiment during maintenance and graduated
tubes (ball bearing spout) with a piece of dark –colored (DT) tape attached to the tube near the
spout were presented to the mice during conditioning. In addition, we explored the nature of the
changes in context that were necessary to produce context aversion: The control group (GB-DT
NaCl
) and one experimental group (GB-DT LiCl) were maintained on pint size regular glass bottles
with rubber stoppers and a pin-hole SS spout; the experimental group drank from graduated tubes
with a piece of light-colored (LT) tape near the spout, a rubber stopper and a SS spout with a
ball-bearing to prevent leakage (LT-DT LiCl). During conditioning graduated tubes (ball bearing
spout) with a piece of dark–colored tape attached to the tube near the spout were presented to the
mice. Thus, there were large differences (glass bottle vs a graduated tube) distinguishing
maintenance conditions and those present during conditioning in one comparison and small
differences (graduated tube with light-colored tape vs graduated tube with dark-colored tape), in
the other.
It was predicted that mice would develop aversions to graduated glass tubes with darkcolored tape (after maintenance on pint size glass bottles) thus ruling out a major influence of the
taste of water in context conditioning (because both tubes had rubber stoppers and contained tap
water). Less certainly, we predicted that animal maintained on graduated tubes with light-colored
tape would develop aversions to tubes with dark-colored tape. Finally, we predicted that there
48
would be evidence for more effective conditioning when the differences between maintenance
containers and those used during conditioning were greater.
Methods
51 B6D2 AI mice (mean age 145 days, range; 140-150) from 17 different litters were
used. Sex (24M, 27F) and litter membership was evenly distributed across the 3 experimental
groups (17/group).
Before the experiment, mice were routinely maintained on pint size translucent plastic
bottles with a blue plastic lids and metal spouts. Mice were then habituated to two different kinds
of water containers for 5 days. 17 mice drank from 25 mL graduated tubes with a piece of lightcolored tape near the spout, a rubber stopper and a stainless steel (SS) spout with a ball-bearing to
prevent leakage; 34 mice drank from pint size regular glass bottles with rubber stoppers and a
pin-hole SS spout (Table 3-3).
Table 3-3. Experimental Setup
49
Conditioning procedure
During conditioning trials, graduated tubes (ball bearing spout) with a piece of darkcolored tape attached to the tube near the spout were presented to the mice. Injection procedures
were the same as described for Experiment 3-1. Experimental groups (LT-DT LiCl and GB-DT
LiCl
) were injected intraperitoneally (IP) with LiCl and the control group (GB-DT NaCl ) was
injected with NaCl during 3 conditioning trials (Table 3-3).
Retention tests (42 minutes in duration) were carried out twice (CCA3+3 and CCA3+13
days) after conditioning. In addition, as a test of specificity, the maintenance containers were
presented in a trial conducted on CCA3+10 days.
Data analyses
Conditioning Analyses
We conducted two analyses of variance for CCA 2 and CCA 3: the first analysis was of
intake, and the second, suppression. For intake, there was one between-subject factor, Groups
(Control, GB and LT) and a within-subjects measure (Trials). In the second analysis
(suppression), there was one between-subject factor, Experimental groups (GB vs. LT) as well as
a within-subjects measure (Trials).
Retention Analyses
In Experiment 3-2, we assessed retention 3 and 13 days after the last conditioning trial.
There was a between-subject factor, Experimental groups (GB vs.LT), as well as a withinsubjects measure (Trials, 2).
50
Results
The raw intakes for Experiment 3-2 are shown in Table 3- 4 and suppression ratios in
Figure 3-2.
Table 3-4. Water Intakes and percent suppression for Experiment 3- 2
Conditioning Results
The analysis of variance showed a highly significant reduction in intake on CCA2 and
CCA3 in conditioned mice compared to the controls (F1, 49=30.79, P<0.001).
Our analysis of suppression shows that there were significant differences between the
experimental groups in their suppression during CCA2 and CCA3 (Groups X Trials interaction,
F1, 32= 5.89, P<0.05). LT-DT LiCl group provided a weaker basis for conditioning on the second
conditioning trial compared to GB-DT LiCl group (LT-DT LiCl vs. GB-DT LiCl; t= -3.44,df=32,
p<0.01), however this difference between experimental groups was not observed on the third
conditioning trial (n.s.).
51
Retention Results
Our analyses of suppression also showed that there were differences between GB-DT LiCl
and LT-DT LiCl groups in their suppression (F1,32= 7.6, P<0.05).
The results of the specificity test (CCA3+10 days) showed that there was no difference in
consumption between experimental and control groups when animals drank from their
maintenance bottles (n.s.). In addition, there were no differences in their suppression between
GB-DT LiCl and LT-DT LiCl groups on the specificity test (n.s.). Thus, this finding shows that mice
were responding to CS’s encountered during conditioning and were able to distinguish their own
tubes from the novel tubes used during conditioning.
Garcia and colleagues found that CTA’s were retained for months after conditioning
(Garcia, Kimeldorf, & Koelling, 1955). In Experiment 1, we tested for retention 7 days after
CCA3. In the present experiment, retention was demonstrated to be present at least 13 days after
CCA3 in both experimental groups (Figure 3-2).
Development of aversion to graduated tubes with dark-colored tape
by mice maintained on either glass bottles or graduated tubes with light -colored tape
LT-DT LiCl
Suppression Index (%)
100
GB-DT LiCl
80
60
40
20
0
-20
CCA1
CCA2
CCA3
1st Retention Maintenance
2nd
CCA3+3 days
Tubes
Retention
CCA3+10
CCA3+13
days
days
Conditioned Context Aversion: Acquisition, Retention and Specificity Trials
52
Figure 3-2. B6D2 AI mice maintained on glass bottles (GB) were exposed to 3
conditioning trials when they drank from graduated tubes with dark-colored tape paired with
injections of LiCl or NaCl (controls). Strong suppression was found with a single conditioning
trial for GB-DT LiCl group but 2 trials were required for LT-DT LiCl group. During retention tests
both experimental groups showed high suppression to CS tubes during which was sustained for at
least 13 days after CCA3. There was no difference in consumption between experimental and
control groups in the specificity test when animals drank from their maintenance containers.
Experiment 3-3
The results of Experiments 3-1 and 3-2 demonstrated that mice developed strong
aversions to both large and small alterations in their home cages when these were paired with
illness. Furthermore, Experiment 3-2 showed that the conditioning was sustained for 13 days. The
present experiment was designed to discover if context aversion could be formed when there is a
substantial delay between CS and UCS, a characteristic known to be an important feature of
conditioned taste aversion (Revusky & Garcia, 1970). An additional aim was to evaluate the
duration of retention.
Methods
40 B6D2 AI mice (mean age 125 days, range; 120-130) from 17 different litters were
used. Sex (15M, 25F) and litter membership was evenly distributed across the 3 experimental
groups (10/group).
Before the experiment, mice were maintained with pint size translucent plastic bottles
with a blue plastic lids and metal spouts. They were then habituated to graduated tubes with a
piece of light-colored tape near the spout for 7 days. After this habituation phase, experimental
53
subjects were trained to drink water promptly from those tubes as previously described in
Experiment 3-1 and 3-2.
Table 3-5. Experimental Setup
Conditioning procedure
The conditioning procedure was as previously described. During conditioning trials,
water was presented to all mice in graduated tubes with a piece of dark-colored tape attached to
the tube near the spout for 15 minutes. Two experimental groups were injected with LiCl
immediately or 30 minutes after the graduated tubes were removed from cages: LT-DT LiCl/Immed
and LT-DT LiCl/Delay. Control groups were injected with NaCl in the same manner as the LiCl
groups: LT-DT NaCl/Immed and LT-DT NaCl/Delay (Table 3-5).
54
Retention tests (42 minutes in duration) were carried out on three occasions (CCA3+7,
+14, +21 days). In each weekly trial, two consecutive daily tests were conducted: on the first day,
half of the animals had access to water from a graduated tube with dark-colored tape (the CS), on
the following day, these mice were presented with a graduated tube with a piece of light-colored
tape (the maintenance tube), the other half were presented CS and maintenance tubes in the
opposite order. This provided us with an opportunity to assess the total (dark-colored tape tube)
and non-specific suppression (light-colored tape tube).
Data analyses
Conditioning Analyses
We conducted two conditioning analyses of variance: the first analysis was of intake,
and the second, suppression. For intake, there were two between-subject factors, Groups (Control
vs. Experimental) and CS-UCS Interval (Immediate vs. Delay) and a within-subjects measure
(Trials).In the second analysis (suppression), there was one between-subject factor, CS-UCS
Interval (Immediate vs. Delay) as well as a within-subjects measure (Trials).
Retention Analyses
We conducted two analyses of variance for retention tests: the first analysis was of
intake, and the second, suppression. For intake, there were three between-subject factors, Groups
(Control vs. Experimental), CS Day (CS presented first or second within each week) and CS-UCS
Interval (Immediate vs. Delay) and as well as two within subjects measures that were Weeks (3)
and Tubes (CS vs. Regular).
55
In the second analysis (suppression), there were two between-subject factors, CS-UCS
Interval (Immediate vs. Delay) and CS Day (the day of the presentation of the CS), as well as two
within-subjects measures that were Weeks (3) and Tubes (CS vs. Regular).
Results
The raw intakes for Experiment 3-3 are shown in Table 3-6a, 3-6b and suppression ratios
in Figure 3-3 and 3-4.
56
Table 3-6. Water Intakes and percent suppression for Experiment 3- 3
Conditioning Results
The analysis of variance showed a highly significant reduction in intake on CCA2 and
CCA3 in conditioned mice compared to the controls (F1, 36=34.08, P<0.001). In addition, there
were no significant differences between the response of immediate and delay groups to
conditioning (Trials X Groups X CS-UCS Interval interaction, n.s.).
Our analysis of suppression shows that suppression was significantly stronger on CCA3
than on CCA2 (F1, 18=33.74, P<0.001). In addition, there were no significant differences
between the experimental groups in their responses to suppression during CCA2 and CCA3
(Groups X Trial interaction, n.s.).
57
These findings also support the previous finding (Experiment 3-2) that switching from
graduated tubes with light-colored to dark-colored tape (smaller difference between novel and
regular tubes) required 2 conditioning trials to obtain strong suppression.
Development of aversion to graduated tubes with dark-colored tape by mice
maintained on graduated tubes with light-colored tape
100
LT-DT (LiCl/Immed)
80
60
LT-DT (LiCl/Delay)
40
20
0
-20
1st cond
2nd cond
3rd cond
-40
Figure 3-3. B6D2 AI mice maintained on graduated tubes with light-colored tape (LT)
were exposed to 3 conditioning trials when they drank from graduated tubes with dark-colored
tape (DT) paired with either injections of LiCl or NaCl immediately or 30 minutes later (delay).
The suppression was clearly stronger on CCA3 for both immediate and delay groups.
Retention Results
Analyses of intake of all retention results showed that there was a large difference
between experimental and control groups in their specific aversion to the CS (Groups X Tubes
interaction, F 1, 68 = 5.18 , p<0.05). Control group intakes were similar from CS and
maintenance tubes throughout the experiment, whereas the difference in intake between
maintenance and CS tubes for experimental mice were initially very large, but then greatly
reduced during the retention tests, indicating a gradual extinction of the learned aversion across
the three week periods (Groups X Tubes X Weeks interaction, F 2, 68 =3.37, p< 0.05). For this
latter analysis, control immediate and delay groups were combined because there was no
58
evidence that they responded differently during retention (Groups X CS-UCS Interval X Tubes X
Weeks interaction, n.s.)
Analyses of suppression of all retention results showed that there was a statistically
significant Tubes X Weeks interaction indicating that LT-DT LiCl/Delay group showed a decrease in
suppression to CS tubes during retention (F 2, 34=6.45, p<0.01). In addition, we compared the
total and non-specific suppression for all retention trials. We saw a significant difference between
total and non-specific suppression until week 2 for the LT-DT LiCl/Immed (Total vs. Non-specific;
1st week; t=2.36, df=9, p<0.05 and 2nd week; t=4.75, df=9, p<0.001) however, specific
suppression had disappeared by week 2 for LT-DT LiCl/Delay group (Total vs. Non-specific; 1st
week; t=5.76, df=8, p<0.001). In weeks 3, consistent with extinction, there was no significant
difference between total and non-specific suppression within either of the conditioned groups.
Suppression Index (%)
Total (dark-colored tape) and Non-specific (light-colored tape) suppression following
three conditioning trials with B6D2 mice
90
80
Dark (CS Tape)
70
Light (Maintenance
Tape)
60
50
40
30
20
10
0
Immediate
Week 1
Delay
Immediate
Week 2
Delay
Immediate
Delay
Week 3
Figure 3-4. Both total and non-specific retention were tested at weekly intervals after
CCA 3. There was specific suppression (total greater than non-specific suppression) in both week
1 and 2 for the LT-DT LiCl/Immed, however, specific suppression had disappeared by week 2 for LT-
59
DT LiCl/Delay group. By week 3, there was no evidence for both total or nonspecific suppression for
both immediate and delay groups.
Discussion
In Experiment 3-1, both PB-GB PW/LiCl and PB-GB TW/LiCl groups showed strong
suppression to the glass bottles after a single conditioning trial when there was a large difference
between regular and novel tubes (plastic bottle vs glass bottle). The results of Experiment 3-2
confirmed that when there was a large difference between maintenance and novel tubes
(switching from glass bottle to a graduated tube with dark-colored tape) strong suppression was
also found after a single conditioning trial. The results of Experiments 3-2 and 3-3 also showed
that the number of conditioning trials required to show strong suppression depends on the
magnitude of differences between regular and novel tubes. For example, switching from
graduated tubes with light-colored to dark-colored tape (smaller difference between novel and
regular tubes) required 2 conditioning trials to obtain strong suppression.
The results of retention tests conducted in Experiment 3-1 and 3-2 showed that when
there was a large difference between maintenance and novel tubes (e.g., Experiment 3-1, plastic
bottle vs glass bottle or Experiment 3-2, glass bottle vs graduated tubes with dark-colored tape)
strong suppression to CS tubes was well retained for at least 13 days. In contrast, the results of
Experiment 3-2 and 3-3 showed that when the difference between novel and maintenance tubes
was smaller (switching from graduated tubes with light-colored to dark-colored tape), it was
retained for a shorter time and non-specific suppression was higher. The retention periods
obtained with the large differences between maintenance and CS cues are not as long as those
60
shown by Garcia for CTA (Garcia et al., 1955) but they at least raise the possibility of long-term
retention.
The effects of introducing a delay between CS and UCS were mixed. Both LT-DT
LiCl/Immed
and LT-DT LiCl/Delay groups required 2 conditioning trials to develop strong suppression
to novel tubes. In contrast, while there was specific suppression (total suppression greater than
non-specific suppression) in both week 1 and 2 for the LT-DT LiCl/Immed group, specific
suppression had disappeared by week 2 for LT-DT LiCl/Delay group. In this experiment, there were
minor changes from maintenance to novel tubes (switching from graduated tubes with lightcolored to dark-colored tape), it would be interesting to see the effects of delay when large
alterations in the home-cage of a mouse are paired with illness.
As mentioned earlier, Garcia (1954) hypothesized that rats developed conditioned
aversions to the taste of water from plastic bottles. However, in Experiment 3-1, we hypothesized
that conditioned aversion may occur because of the different appearance of bottles (plastic bottle
vs glass bottle) rather than the role of taste. According to the result of Experiment 3-1, PB-GB
PW/LiCl
group which drank from glass bottles into which water from plastic bottles had been
decanted showed less suppression than PB-GB TW/LiCl (glass bottle/ tap water) after a single
conditioning trial. This may be weak evidence that the taste of water from plastic bottles can
serve as a CS for mice. However, during the 3rd conditioning trial and the subsequent retention
test, both of the experimental groups showed similar strong suppression to the novel bottles,
providing no support for a role for taste. Without repeating Garcia’s experiment in exactly the
same way by using the same material and apparatus, it is impossible to conclude that Garcia`s rats
were responding to the appearance of plastic bottles rather than the taste of water in them.
However, it is intriguing to speculate that Garcia may have missed evidence of CCA in one of his
earliest studies and that CCA may have helped lead him to the discovery of CTA.
61
Although all animals had access to maintenance tubes for 7 days after CCA3 and might
have been expected to show no response to those tubes during the retention tests, we conducted
specificity tests to see whether there was any difference in consumption between LiCl and NaCl
groups when they drank from their maintenance bottles. Supporting the idea that mice were able
distinguish the differences between CS and maintenance tubes, the results of a specificity test
conducted in Experiment 3-2 (CCA3+10 days) showed that when there was a large difference
between CS and maintenance tubes (glass bottle vs graduated tubes with dark-colored tape), there
was no significant difference in consumption between experimental and control groups when
animals drank from their maintenance bottles. On the other hand, the magnitude of differences
between maintenance and CS tubes appears to influence the result of specificity tests. For
example, when there was a small difference between maintenance and CS tubes as in Experiment
3-3 (switching from graduated tubes with light-colored to those with dark-colored tape), both LTDT LiCl/Immed and LT-DT LiCl/Delay groups showed significant strong suppression to their
maintenance tubes in the first retention test. Subjects may have generalized aversion to their
maintenance tubes when they were presented following water deprivation, even though they
drank normally from the same tubes during recovery and inter-retention trial periods.
A very significant finding of Experiment 3-3 was that non-specific components of
conditioning are considerable and need to be taken into account in this area of research. In LT-DT
LiCl/Immed
group, non-specific suppression appears to explain 50% of the 80% reduction in fluid
intake by the mice conditioned to avoid graduated tubes with dark-colored tape tubes on the first
retention test and 20% of the 50% reduction on the second retention test. However, there was an
important difference between immediate and delay groups. For instance, in LT-DT LiCl/Delay group,
non-specific suppression appears to explain 25% of the 70% reduction in fluid intake by the mice
conditioned to avoid graduated tubes with dark-colored tape tubes on the first retention test but
62
they did not show greater suppression to the CS tubes than they did to the maintenance tubes on
the second retention test. On the third trial when admittedly the aversion appeared to be
extinguished non-specific and total suppression were similar in both Immediate and Delay
experimental groups. The issue of non-specific suppression is sometimes dealt with in
experimental contexts by administering several exposures to neutral stimuli under the same
conditions that CS is presented. Then, CS tubes are presented assuming that non-specific
responses have been extinguished. The present results raise the question of whether non-specific
tests should be routinely incorporated into retention trials to permit accurate side by side
comparisons with the response to CS.
63
Chapter 4
Comparison of CCA and CTA in pigmented LGSM mice
Experiment 4-1
The findings of Chapter 3 using B6D2 AI mice provide good support for the hypothesis
that minor alterations in cage environment can serve as CSs when paired with LiCl (illness). For
instance, in the previous experiments, we showed that pairing a glass bottle with illness (produced
by injection of LiCl) resulted in strong suppression (80%) in the experimental group as compared
to the control group after a single conditioning trial; this suppression was well retained for at least
13 days. However, switching from graduated tubes with light-colored to dark-colored tape
(smaller difference between novel and regular tubes) required 2 conditioning trials to obtain
strong suppression and the retention of the aversion was weaker. Moreover, in the experiments
described in the previous chapter, context aversion was shown to be formed when there was a 30
minutes delay between the presentation of the CS and the UCS. However, the aversion was
weaker and was retained for a shorter period of time.
As we discussed in the introductory chapter, different views on the ability of animals to
associate a specific CS with a particular reinforcer have been presented. A number of studies has
shown that animals cannot develop conditioned aversions when illness is induced in a particular
environmental context. However, these studies were able to demonstrate conditioned aversion by
pairing illness and taste (selective associative learning). On the other hand, a variety of studies
have paired illness with a combination of gustatory and exteroceptive cues and made inferences
about the ability of animals to form an association between illness and the external environment.
The present experiments were performed to provide further evidence that taste is not necessary to
64
induce context aversion in mice. In addition, we sought to identify the duration of retention for
both CCA and CTA using the same experimental procedures. Experiments in this chapter were
conducted using pigmented LGSM AI mice. We also wanted to compare the magnitude of CCA
when we performed alterations between CSs and regular tubes (switching from graduated tubes
with light-colored tape to graduated tubes with dark-colored tape or switching from glass bottles
to graduated tubes with dark-colored tape) using these specific advanced intercross mice.
Before performing these experiments, we predicted that the mice would show similar
duration of retention and extinction for CTA and CCA. In our previous experiments, we observed
that the mice showed slight suppression to their regular tubes as well as to the CS tubes during the
retention tests. Thus, within these experiments, we examined the issue of specificity more
carefully. Finally, we hoped to provide more evidence that context aversion could be formed
when there was a substantial delay between the presentation of the CS and the UCS.
Subjects
48 LGSM AI male mice (mean age 93 days, range; 87-100) from 18 different litters were
used. Litter membership was evenly distributed across the 4 experimental groups (12/group).
Methods
Maintenance conditions were similar to those described in the Chapter 2. Before the
experiment, mice were routinely maintained using pint-size translucent plastic bottles with blue
plastic lids and metal pin-hole spouts. The mice were then habituated to drinking water from pintsize regular glass bottles with rubber stoppers and stainless steel (SS) spouts with ball-bearings
(GB) for 7 days (Figure 4-1).
65
Mice were then trained to drink water promptly from these bottles in the light phase of
the circadian cycle by depriving them of water for 16 h and giving them access to water several
times per day during the light phase of the light/dark cycle. Mice had 30 minutes access to water
on 3 occasions and then were allowed to drink for 3 hour until the beginning of the dark cycle.
This procedure was repeated on 3 separate days.
Figure 4-1(left). Maintenance bottle. Figure 4-2(right). CS tube.
Conditioning Procedures
During the conditioning trials, tap water in graduated tubes (with ball-bearing spouts)
with pieces of dark tape attached to the tubes near their spouts (DT) were presented to water –
deprived mice for 15 minutes in their home cage (Figure 4-2). Two experimental groups were
injected with LiCl (0.15 M, 0.3 Ml/10g body weight) immediately or 30 minutes after the
graduated tubes had been removed from the cages: GB-DT LiCl/Immed and GB-DT LiCl/Delay. The
control groups were injected with NaCl in the same manner as the LiCl groups (same
concentration and dose as LiCl): GB-DT NaCl/Immed and GB-DT NaCl/Delay (Table 4-1). After the
last conditioning trial, the mice were allowed a recovery period of 7 days. During this period, the
animals had ad libitum access to their regular bottles.
66
Table 4-1. Experimental Setup
Retention tests (42 minutes in duration) were administered over two days in each week
after recovery (e.g., CCA3+7 days, CCA3+14 days). If a mouse had access to water from a
graduated tube with dark tape (the CS) on the initial day, on the following day, a glass bottle (the
maintenance tube) was presented, or vice versa. This provided us with an opportunity to assess
the total (dark-tape tube) and non-specific suppression (glass bottle).
Data Analyses
Conditioning Analyses
We conducted two conditioning analyses of variance for CCA 2 and CCA 3: the first
analysis was of intake, and the second, suppression. For intake, there were two between-subject
factors, Groups (Experimental vs. Control) and CS-UCS Interval (Immediate vs. Delay), as well
as a within-subjects measure (Trials). In the second analysis (suppression), there was one
between-subject factor, CS-UCS Interval (Immediate vs. Delay), as well as a within-subjects
measure (Trials).
67
Except the places where we specified Bonferroni `s correction was used, p<0.05 was
accepted as significant.
Retention Analysis
We assessed retention on 6 consecutive weeks (starting 1 week after conditioning) in
order to compare duration of retention in Immediate and Delay groups.
There were two between-subject factors, CS-UCS Interval (Immediate vs. Delay) and CS
Day (the day of the presentation of the CS), as well as two within-subjects measures that were
weeks (6) and tubes (CS vs. maintenance suppression).
We evaluated which week extinction occurred by using post-hoc paired t-tests comparing
total to non-specific suppression within each conditioned group. To adjust for multiple
comparisons across the 6 weeks, we adjusted the p-value according to Bonferroni`s correction
(p<.008 accepted as significant).
Results
The raw intakes for Experiment 4-1 are shown in Table 4- 3a, 3b, 3c and suppression
ratios in Figure 4-3 and 4-4.
Table 4-2 shows the ANOVA design that we used to calculate intake and suppression
results for the conditioning and retention tests.
68
Table 4-2. Design of ANOVA
Conditioning
The analysis of variance showed a highly-significant reduction in intake on CCA2 and
CCA3 in conditioned mice compared to the controls (F 1, 44 = 206.7, p< 0.001,). There were no
significant differences between the Immediate and Delay groups in their responses to
conditioning (Trials X Groups X CS-UCS Interval interaction, n.s.; Table 4-3 a).
Figure 4-3 shows the suppression ratios calculated from the intake data. Our analysis of
suppression shows that suppression was significantly stronger on CCA3 than on CCA2 (F 1, 22
=17.3, p< 0.001). There were no significant differences between the Immediate and Delay
groups in their responses to suppression during CCA2 and CCA3 (CS-UCS Interval X Trial
interaction, n.s.; Table 4-3 a).
Retention Suppression
Analyses of suppression of all retention results showed that all experimental groups
experienced greater suppression to the CS tubes than they did to the maintenance tubes overall (F
1, 100 =201, p< 0.001). In addition, we showed that there was a statistically significant Tubes X
69
Weeks interaction indicating that mice showed declined suppression to CS tubes during the
retention, while non-specific suppression seems to stay about the same across weeks (F 5, 100
=17.6, p<0.001). In addition, the CS day had no significant effect on the results (CS day, n.s.).We
also compared the total and non-specific suppression for all retention trials. We saw a significant
difference between total and non-specific suppression until week 4 in both the Immediate and
Delay experimental groups. In weeks 5 and 6, consistent with extinction, there was no significant
difference between total and non-specific suppression within the conditioned groups.
Analyses of suppression of all retention results showed that there were no differences in
suppression between the Immediate and Delay groups (CS-UCS Interval, n.s). In addition, there
was no statistically significant CS-UCS Interval X Tubes X Weeks interaction indicating that
there was no difference between Immediate and Delay groups in the gradual disappearance of the
specific conditioned aversion.
Figure 4-3. LGSM AI mice maintained on glass bottles (GB) were exposed to 3
conditioning trials when they drank from graduated tubes with dark-colored tape paired with
70
injections of LiCl or NaCl (controls). Strong suppression was found with a single conditioning
trial for GB-DT LiCl/Immed group. During retention tests, GB-DT LiCl/Immed group showed
high suppression to CS tubes than their maintenance tubes until week 4 (CCA3+28 days).
Figure 4-4. LGSM AI mice maintained on glass bottles (GB) were exposed to 3
conditioning trials when they drank from graduated tubes with dark-colored tape paired with
injections of LiCl or NaCl (controls). Strong suppression was found with a single conditioning
trial for GB-DT LiCl/Delay group. During retention tests, GB-DT LiCl/Delay group showed high
suppression to CS tubes than their maintenance tubes until week 4 (CCA3+ 28 days).
71
72
Table 4-3. Water Intakes and Percent suppression for Experiment 4-1
Experiment 4-2
The results of Experiment 4-1 suggest that alterations in cage environment (switching
from glass bottle to graduated tubes with dark-tape and pairing with illness) can serve as CS`s
within the standard aversion conditioning paradigm. In other words, mice can discriminate
between the appearance of the CS and the appearance of the regular tubes (e.g., the shape and size
of the containers). In addition, Experiment 4-1 suggests that GB-DT LiCl/Immed and GB-DT LiCl/Delay
groups experience similar rates of extinction (up to 28 days after the last conditioning trial;
CCA3+28 days), regardless of whether they are injected immediately or after a 30 minute delay
between the presentation of the CS and the UCS.
73
In this subsequent study, we made even smaller alterations in the mice’s cages (the only
difference between the maintenance tubes and the CS tubes was the brightness of the tape).
During this experiment, we carefully examined whether conditioned aversion could also be
developed when a 30 minute CS-UCS delay was used.
It was predicted that these mice would develop conditioned aversion to minor visual cues
and they would respond to brightness differences between the light and dark tape. Less certainly,
we predicted that animals maintained on graduated tubes with light tape would develop similar
magnitudes of aversion to CS tubes as were observed in Experiment 4-1 (when we performed
large alterations; glass bottles vs. graduated tubes with dark tape). Finally, we predicted that both
the immediate and the delayed groups would develop similar magnitudes of aversion during the
conditioning and the rates of extinction to the CS tubes during retention.
Subjects
48 LGSM AI male mice (mean age 93 days, range; 87-100) from 12 different litters were
used. Litter membership was evenly distributed across the 4 experimental groups (12/group).
Methods
Maintenance conditions were similar to those described in the Chapter 2. Before the
experiment, the mice were routinely maintained on pint-size translucent plastic bottles with blue
plastic lids and pin-hole metal spouts. The mice were then habituated to drinking water from 25
mL graduated tubes with pieces of light tape near the spouts, rubber stoppers, and stainless steel
(SS) spouts with ball bearings for 7 days (Figure 4-5).
74
Mice were then trained to drink water promptly from these bottles in the light phase of
the circadian cycle by depriving them of water for 16 h and giving them access to water several
times per day during the light phase of the light/dark cycle. Mice had 30 minutes access to water
on 3 occasions and then were allowed to drink for 3 hour until the beginning of the dark cycle.
This procedure was repeated on 3 separate days.
Figure 4-5 (left). Maintenance tube. Figure 4-6(right). Brightness differences between
tape on CS and Maintenance tubes
Conditioning Procedures
During the conditioning trials, tap water in graduated tubes (with ball-bearing spouts)
with pieces of dark-colored tape attached to the tubes near the spouts (DT) were presented to the
water-deprived mice for 15 minutes in their home cage (Figure 4- 6). The two experimental
groups were injected with LiCl (0.15 M, 0.3 mL/10g body weight) immediately or 30 minutes
after the graduated tubes were removed from the cages: LT-DT LiCl/Immed and LT-DT LiCl/Delay. The
control groups were injected with NaCl in the same manner as the LiCl groups (the same
concentration and dose of NaCl as of LiCl): LT-DT NaCl/Immed and LT-DT NaCl/Delay (Table 4-4).
75
After the last conditioning trial, the mice were allowed a recovery period of 3 days.
During this period, the animals had ad libitum access to their regular bottles.
Table 4-4. Experimental Setup
Retention tests (42 minutes in duration) were administered over two days in each week
after the recovery period (e.g., CCA3+7 days, CCA3+14 days). If a mouse had access to water
from a graduated tube with dark tape (the CS) on the initial day, on the following day, a
graduated tube with light tape (the maintenance tube) was presented, or vice versa. This provided
us with an opportunity to assess the total (dark-tape tube) and non-specific suppression (light-tape
tube).
Data Analyses
Conditioning Analyses
76
We conducted two conditioning analyses for CCA 2 and CCA 3: the first analysis was of
intake, and the second, suppression. For intake, there were two between-subject factors, Groups
(Experimental vs. Control) and CS-UCS Interval (Immediate vs. Delay), as well as a withinsubjects measure (Trials). In second analysis (suppression), there was one between-subject factor,
CS-UCS Interval (Immediate vs. Delay), as well as a within-subjects measure (Trials).
Retention Analysis
We assessed retention on 6 consecutive weeks (starting 1 week after conditioning) in
order to compare duration of retention in Immediate and Delay groups.
There were two between-subject factors, CS-UCS Interval (Immediate vs. Delay) and CS
Day (the day of the presentation of the CS), as well as two within-subjects measures that were
weeks (6) and tubes (CS vs. Maintenance).
We evaluated which week extinction occurred by using post-hoc paired t-tests comparing
total to non-specific suppression within each conditioned group (p<.05 accepted as significant).
Results
The raw intakes for Experiment 4-2 are shown in Table 4-6 and suppression ratios in
Figure 4-7 and 4- 8.
Table 4-5 shows the ANOVA design that we used to calculate intake and suppression
results for the conditioning and retention tests.
77
Table 4-5. Design of ANOVA
Conditioning
Analysis of variance showed a highly significant reduction in intake on CCA2 and CCA3
in conditioned mice compared to controls (F 1, 43 =156.68, p< 0.001). There were no significant
differences between the response of immediate and delay groups in their responses to
conditioning (Groups X CS-UCS Interval X Trial interaction, n.s).
Figure 4-7 shows the suppression ratios calculated from the intake data. Analysis of
suppression showed that the suppression was significantly stronger on CCA3 than CCA2 (F 1, 22
=19.4, p< 0.001). There were no significant differences between the response of immediate and
delay groups to suppression during CCA2 and CCA3 (CS-UCS Interval X Trial interaction, n.s.).
Retention Suppression
Analyses of suppression of all retention results showed that all experimental groups
experienced greater suppression to the CS tubes than they did to the maintenance tubes overall (F
1, 95 =116.5, p< 0.001). In addition, we showed that there was a statistically significant Tubes X
Weeks interaction (F 5, 95= 3.79, p<0.01) reflecting that mice showed declined suppression to
78
CS tubes during the retention, while non-specific suppression seems to stay about the same across
weeks (F 5, 95=3.79, p<0.01). In addition, the CS day had no significant effect on the results (CS
day, n.s.).We also compared the total and non-specific suppression for all retention trials. We saw
a significant difference between total and non-specific suppression until week 3 in the LT-DT
LiCl/Immed
group, and until week 7 in LT-DT LiCl/Delay group.
Analyses of suppression showed that there were no differences in suppression between
the Immediate and Delay groups (CS-UCS Interval, n.s). However, there was a statistically
significant CS-UCS Interval X Tubes X Weeks interaction (F 5,95=2.86, p<0.05), indicating a
difference between the Immediate and Delay groups in the gradual disappearance of the specific
suppression in conditioned aversion. The Immediate group showed quicker extinction to the CS
tubes compared to the Delay group.
Suppression Index (%)
100
80
Development of aversion to CS and regular tubes by mice maintained on graduated tubes with light-colored tape
(Immediate Injection)
GTDT= CS
GTLT = Regular Tubes
60
40
20
0
-20
GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT
CCA1 CCA2 CCA3 CCA3 + 7
Days
CCA3+14
Days
CCA3+21
Days
CCA3+28
Days
CCA3+35
Days
CCA3+ 42
Days
Conditioned Context Aversion: Acquisition and Retention Trials
Figure 4-7. LGSM AI mice maintained on graduated tubes with light-colored tape (LT)
were exposed to 3 conditioning trials when they drank from graduated tubes with dark-colored
tape paired with injections of LiCl or NaCl (controls). Strong suppression was found with a single
79
conditioning trial for LT-DT LiCl/Immed group. During retention tests, LT-DT LiCl/Delay group
showed high suppression to CS than their maintenance bottles until week 3 (CCA3+ 21days).
Suppression Index (%)
Development of aversion to CS and regular tubes by mice maintained on graduated tubes with
light-colored tape (Delay Injection)
100
GTDT= CS Tubes
GTLT= Regular Tubes
80
60
40
20
0
-20
GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT
CCA1 CCA2 CCA3 CCA3 + 7
Days
CCA3+14
Days
CCA3+21
Days
CCA3+28
Days
CCA3+35 CCA3+ 42 CCA3+ 49
Days
Days
Days
Conditioned Context Aversion: Acquisition and Retention Trials
Figure 4-8. LGSM AI mice maintained on graduated tubes with light-colored tape (LT)
were exposed to 3 conditioning trials when they drank from graduated tubes with dark-colored
tape paired with injections of LiCl or NaCl (controls). Strong suppression was found with a single
conditioning trial for LT-DT LiCl/Delay group. During retention tests, LT-DT LiCl/Delay group
showed high suppression to CS than their maintenance bottles until week 7 (CCA3+ 49 days).
80
81
Table 4-6a, 4-6b and 4-6c. Water Intakes and Percent suppression for Experiment 4-2.
Experiment 4-3
Our results from Experiments 4-1 and 4-2 with pigmented LGSM AI mice show that
pairing illness with small environmental changes can result in strong aversion. It is also clear that
conditioned context aversion can be developed when a 30 minute delay between CS and UCS is
implemented.
The aim of the following experiment, Experiment 4-3, was to compare the duration of
retention for both CCA and CTA using the same experimental procedures as those in
Experiments 4-1 and 4-2. In Experiment 4-3 aversions were established to sodium saccharin (CS)
by pairing its ingestion with an injection of LiCl during the conditioning phase delivered
immediately or after a 30 minute delay.
82
Subjects
46 LGSM AI male mice (mean age 125 days, range; 120-130) from 10 different litters
were used. Litter membership was evenly distributed across the 4 experimental groups.
Methods
Maintenance conditions were similar to those described in previous experiments. Before
the experiment, the mice were routinely maintained on pint-size translucent plastic bottles with
blue plastic lids and metal spouts. The mice were then habituated to drinking water from 25 mL
graduated tubes, rubber stoppers, and stainless steel (SS) spouts with ball bearings for 7 days
(Figure 4-9).
Mice were then trained to drink water promptly from these graduated tubes in the light
phase of the circadian cycle by depriving them of water for 16 h and giving them access to water
several times per day during the light phase of the light/dark cycle. Mice had 30 minutes access
to water on 3 occasions and then were allowed to drink for 3 hour until the beginning of the dark
cycle. This procedure was repeated on 3 separate days.
Figure 4-9. Maintenance tube
83
Conditioning Procedures
During the conditioning trials, sodium saccharin (5mM) (SS) was presented to water
deprived mice for 15 minutes in their home cage. The two experimental groups were injected
with LiCl (0.15 M, 0.3 mL/10g body weight) immediately or 30 minutes after the graduated tubes
were removed from their cages: (SS LiCl/Immed and SSLiCl/Delay). The control groups were injected
with NaCl in the same manner as the LiCl groups (the same concentration and dose used for
LiCl): SS NaCl/Immed and SSNaCl/Delay (Table 4-7).
After the last conditioning trial, the mice were allowed a recovery period of 3 days.
During this period, the animals had ad libitum access to water in their regular bottles.
Table 4-7. Experimental Setup
Retention tests (42 minutes in duration) were administered over two days in each week
after the recovery period (e.g., CCA3+7 days, CCA3+14 days). If a mouse had access to sodium
84
saccharin (CS) on the initial day, on the following day, water was presented, or vice versa. This
provided us with an opportunity to assess the total (saccharin in regular tubes) and non-specific
suppression (water in regular tubes).
Data Analyses
Conditioning Analyses
We conducted two conditioning analyses of variance for CCA 2 and CCA 3: the first
analysis was of intake, and the second, suppression. For intake, there were two between-subject
factors, Groups (Experimental vs. Control) and CS-UCS Interval (Immediate vs. Delay), as well
as a within-subjects measure (Trials). In second analysis (suppression), there was one betweensubject factor, CS-UCS Interval (Immediate vs. Delay), as well as a within-subjects measure
(Trials).
Retention Analysis
We assessed retention across 6 consecutive weeks (starting 1 week after conditioning) in
order to compare duration of retention in Immediate and Delay groups.
We evaluated which week extinction occurred by using post-hoc paired t-tests comparing
total to non-specific suppression within each conditioned group (p<.05 accepted as significant).
There were two between-subject factors, CS-UCS Interval (Immediate vs. Delay) and CS
Day (the day of the presentation of the CS), as well as two within-subjects measures that were
Weeks (6) and Solution (Sodium Saccharin vs. Water).
Results
The raw intakes for Experiment 4-3 are shown in Table 4-9 and suppression ratios in
Figure 4-10 and 4-11.
85
Table 4-8 shows the ANOVA design that we used to calculate intake and suppression
results for the conditioning and retention tests.
Table 4-8. Design of ANOVA
Conditioning
The analysis of variance showed a highly-significant reduction in intake on CCA2 and
CCA3 in conditioned mice compared to the controls (F 1, 41 = 61.87, p< 0.001). In addition,
there were no significant differences between the Immediate and Delay groups in their responses
to conditioning (Trials X Groups X CS-UCS Interval interaction, n.s.; Table 4-9 a).
Figure 4-10 and 4-11 shows the suppression ratios calculated from the intake data. Our
analysis of suppression shows that suppression was significantly stronger on CCA3 than on
CCA2 (F 1, 20 = 10.98, p< 0.01). There were no significant differences between the Immediate
and Delay groups in their responses to suppression during CCA2 and CCA3 (CS-UCS Interval X
Trial interaction, n.s).
Retention Suppression
86
We compared the total and non-specific suppression for all retention trials. Analyses of
suppression of all retention tests showed that both experimental groups experienced greater
suppression to the sodium saccharin than they did to the water throughout the experiment
(Solution effect; F 1, 90 =41.28, p< 0.001). In addition, analyses of suppression of all retention
results showed that we showed that there was a statistically significant Solution X Weeks
interaction indicating that experimental mice showed a decrease in suppression to sodium
saccharin (CS) during the retention while non-specific suppression seems to stay about the same
across weeks (F 5, 90 =3.39 , p<0.01). In addition, the CS day had no significant effect on the
results (CS day, n.s.)We saw a significant difference between total and non-specific suppression
until week 6 in the Immediate experimental group and week 3 in the Delay group. Consistent
with extinction, there was no significant difference between total and non-specific suppression in
Delay group in the following weeks.
There were no overall differences in suppression between the Immediate and Delay
groups (CS-UCS Interval, n.s). However, there was a statistically significant CS-UCS Interval X
Solution X Weeks interaction (F 5, 90=3.16, p<0.05), indicating a difference between the
Immediate and Delay groups in the gradual disappearance of the specific conditioned aversion.
The Immediate group retained longer than the Delay group.
87
Figure 4-10. LGSM AI mice maintained on water in graduated tubes were exposed to 3
conditioning trials when they drank sodium saccharin in their maintenance tubes paired with
injections of LiCl or NaCl (controls). Strong suppression was found with a single conditioning
trial for SS LiCl/Immed group. During retention tests, SS LiCl/Immed group showed high suppression to
CS (sodium saccharin) than water until week 6 (CCA3+ 42 days).
88
Figure 4-11. LGSM AI mice maintained on water in graduated tubes were exposed to 3
conditioning trials when they drank sodium saccharin in their maintenance tubes paired with
injections of LiCl or NaCl (controls). Strong suppression was found with a single conditioning
trial for SS LiCl/Delay group. During retention tests, SS LiCl/Delay group showed high suppression to
CS (sodium saccharin) than water until week 3 (CCA3+ 21 days).
89
90
Table 4-9a, 4-9b and 4-9c. Water Intakes and Percent suppression for Experiment 4-3
Discussion
Our studies with B6D2 AI mice (Chapter 3) showed that pairing illness with minor
environmental changes (switching from glass bottle to graduated tubes with dark tape or
switching from graduated tubes with light tape to dark tape) resulted in strong aversion.
The results from our experiments on pigmented LGSM (AI) mice (Chapter 4) support
this finding. Mice showed strong conditioned aversions to changes in the environment in both
conditions (switching from graduated tubes with light-colored tape to graduated tubes with darkcolored tape or glass bottles to graduated tubes with dark-colored tape) when discrete visual cues
were paired with illness.
The results of Experiments 4-1 and 4-2 (Chapter 4) showed a highly significant reduction
in intake of CCA2 and CCA3 in conditioned mice, compared to the controls in both conditions
(switching from graduated tubes with light-colored tape to graduated tubes with dark-colored tape
or glass bottles to graduated tubes with dark-colored tape). We were also able to show that, as in
Experiment 3-3, there were no significant differences LT-DT LiCl/Immed and LT-DT LiCl/Delay
conditioning in this experiment.
Moreover, as we discussed in the introduction, a single pairing of a novel taste with
illness has been shown to cause conditioned aversion to the novel taste (Garcia et al., 1955;
Garcia, Ervin, & Koelling, 1966; Bernstein & Webster, 1980), and an effective CTA can be
demonstrated even when a long delay is introduced between the CS and the UCS (Garcia et al.
1966; Smith & Roll, 1967, Revusky & Garcia, 1970). In Experiment 4-3, we were able to
corroborate these original findings, as conditioned LGSM AI mice showed a highly significant
reduction in intake on CCA2 and CCA3, compared to controls when sodium saccharin was paired
91
with illness. In addition, we showed that conditioned context aversion, just like conditioned taste
aversion, could be developed when a 30-minute CS-UCS delay was used.
The results of Chapter 4 show that conditioned aversions due to changes in the
environment are retained for a similar duration in both conditions using LGSM pigmented mice
(switching from graduated tubes with light-colored tape to graduated tubes with dark-colored tape
or glass bottles to graduated tubes with dark-colored tape). In Experiment 4-1, we saw a
significant difference between total and non-specific suppression for the first three weeks in both
the Immediate and Delay experimental groups. In Experiment 4-2, surprisingly, the LT-DT
LiCl/Delay
group retained longer than the LT-DT LiCl/Immed group. A significant difference between
total and non-specific suppression was also seen for the first three weeks in the LT-DT LiCl/Immed
group; this difference was retained until Week 7 in the LT-DT LiCl/Delay group. Based on our
finding in Experiment 3-3 that the Delay experimental group retained for a shorter period of time,
we did not expect that the LT-DT LiCl/Delay group would retain longer than the LT-DT LiCl/Immed
group.
In this chapter, we were also able to compare the duration of CCA and CTA. We
observed that conditioned context aversions and conditioned taste aversions are retained for
comparable durations.
In Chapter 4, like in Chapter 3, the experimental groups showed non-specific suppression
to their regular tubes. Thus, we conclude that the non-specific components of conditioning are
considerable and merit further consideration by researchers working in this field.
92
Chapter 5
Comparison CCA in albino LGSM mice
Experiment 5-1
The results from our experiments on pigmented LGSM (AI) mice (Chapter 4) show that
conditioned aversions to changes in the environment (switching from graduated tubes with lightcolored tape to graduated tubes with dark-colored tape or glass bottles to graduated tubes with
dark-colored tape) are retained for a similar duration . The results of the experiments conducted in
Chapter 3 also suggest that in both cases, CCA can be developed when a 30-minute CS-UCS
delay is implemented.
Moreover, we observed that CCAs and conditioned taste aversions are retained for
comparable durations. Furthermore, the experimental groups also showed some non-specific
suppression to their regular tubes. Thus, we conclude that the non-specific components of
conditioning are considerable and merit further consideration by researchers working in this field.
In this chapter, we describe similar experiments using albino mice. A review of recent
literature suggests that most albino mice have weaker visual abilities than most pigmented mice
(Wong and Brown, 2006). The aim of our experiments, then, was to examine whether albino mice
would develop conditioned context aversions comparable to those of pigmented mice.
In Experiment 5-1, the magnitude of CCA was examined by performing large alterations
in the environment (switching from glass bottles to graduated tubes with dark-colored tape). We
again compared retention and extinction of conditioned aversions by injecting the mice either
immediately or after a 30-minute delay between the presentation of CS and the UCS.
93
Subjects
36 LGSM AI male mice (mean age 70 days, range; 67-80) from 12 different litters were
used. Litter membership was evenly distributed across the 4 experimental groups.
Methods
Maintenance conditions were similar to those described in the Chapter 2. Before the
experiment, mice were routinely maintained using pint-size translucent plastic bottles with blue
plastic lids and metal pin-hole spouts. The mice were then habituated to drinking water from pintsize regular glass bottles with rubber stoppers and stainless steel (SS) spouts with ball-bearings
(GB) for 7 days (Figure 5-1).
Mice were then trained to drink water promptly from these bottles in the light phase of
the circadian cycle by depriving them of water for 16 h and giving them access to water several
times per day during the light phase of the light/dark cycle. Mice had 30 minutes access to water
on 3 occasions and then were allowed to drink for 3 hour until the beginning of the dark cycle.
This procedure was repeated on 3 separate days.
94
Figure 5-1. (left) Maintenance Bottle
Figure 5-2. (right) CS tube
Conditioning Procedures
During the conditioning trials, tap water in graduated tubes (with ball-bearing spouts)
with pieces of dark-colored tape attached to the tubes near their spouts (DT) were presented to
water –deprived mice for 15 minutes in their home cage (Figure 5-2). Two experimental groups
(12 mice per group) were injected with LiCl (0.15 M, 0.3 Ml/10g body weight) immediately or 30
minutes after the graduated tubes had been removed from mice’s cages: GB-DT LiCl/Immed and
GB-DT LiCl/Delay. The control groups (6 mice per group) were injected with NaCl in the same
manner as the LiCl groups (the same concentration and dose of NaCl as of LiCl): GB-DT
NaCl/Immed
and GB-DT NaCl/Delay (Table 5-1).
After the last conditioning trial, the mice were allowed a recovery period of 7 days.
During this period, the animals had ad libitum access to their regular bottles.
95
Table 5-1. Experimental Setup.
Retention tests (42 minutes in duration) were administered over two days in each week
after the recovery period (e.g., CCA3+7 days, CCA3+14 days). If a mouse had access to water
from a graduated tube with dark-colored tape (the CS) on the initial day, on the following day, a
glass bottle (the maintenance tube) was presented, or vice versa. This provided us with an
opportunity to assess the total (dark-colored tape tube) and non-specific suppression (glass
bottle).
Data Analyses
Conditioning Analyses
We conducted two conditioning analyses for CCA 2 and CCA 3: the first analysis was of
intake, and the second, suppression.
For intake, there were two between-subject factors, Groups (Experimental vs. Control)
and CS-UCS Interval (Immediate vs. Delay), as well as a within-subjects measure (Trials). In
96
second analysis (suppression), there was one between-subject factor, CS-UCS Interval
(Immediate vs. Delay), as well as a within-subjects measure (Trials).
Except the places where we specified Bonferroni `s correction was used, p<0.05 was
accepted as significant.
Retention Analysis
We assessed retention on 6 consecutive weeks (starting 1 week after conditioning) in
order to compare duration of retention in Immediate and Delay groups.
We evaluated when extinction occurred by using post-hoc paired t-tests comparing total
to non-specific suppression within each conditioned group. To adjust for multiple comparisons
across the 6 weeks, we adjusted the p-value according to Bonferroni`s correction (p<.008
accepted as significant).
There were two between-subject factors, CS-UCS Interval (Immediate vs. Delay) and CS
Day (the day of the presentation of the CS), as well as two within-subjects measures that were
weeks (6) and tubes (CS vs. Maintenance tubes).
Table 5-2. Design of ANOVA
97
Results
The raw intakes for Experiment 5-1 are shown in Table 5-3 and suppression ratios in
Figure 5-3 and 5-4.
Table 5-2 shows the ANOVA design that we used to calculate intake and suppression
results for the conditioning and retention tests.
Conditioning
The analysis of variance showed a highly-significant reduction in intake on CCA2 and
CCA3 in conditioned mice compared to the controls (F 1, 32 =179.22, p< 0.001,). There were no
significant differences between the Immediate and Delay groups in their responses to
conditioning (Trials X Groups X CS-UCS Interval interaction, n.s).
Figure 5-3 and 5-4 shows the suppression ratios calculated from the intake data. Our
analysis of suppression shows that suppression was significantly stronger on CCA3 than on
CCA2 (F 1, 22 =5.56, p< 0.05). There were no significant differences between the Immediate
and Delay groups in their responses to suppression during CCA2 and CCA3 (CS-UCS Interval X
Trial interaction, n.s.; see Table 5-3a).
Retention Suppression
Analyses of suppression of all retention tests showed that both experimental groups
exhibited greater suppression to the CS tubes than to the maintenance tubes overall (F 1, 100
=133.53, p< 0.001). In addition, we showed that there was a statistically significant Tubes X
Weeks interaction indicating that mice showed declined suppression to CS tubes during the
retention while non-spesific suppression seems to stay about the same across weeks (F 5, 100
=19.81, p<0.001). In addition, the CS day had no significant effect on the results (CS day, n.s.).
98
We saw a significant difference between total and non-specific suppression until week 3
in both the Immediate and Delay groups. In weeks 4, 5 and 6, consistent with extinction, there
was no significant difference between total and non-specific suppression within each conditioned
groups.
There were no differences in suppression between the Immediate and Delay groups (CSUCS Interval, n.s). In addition, there was no statistically significant CS-UCS Interval X Tubes X
Weeks interaction indicating that there was no difference between Immediate and Delay groups
in the gradual disappearance of the specific conditioned aversion.
Suppression Index (%)
100
Development of aversion to CS and regular tubes by mice maintained on glass bottle
(Albino mice/Immediate Injection)
80
60
40
20
0
GTDT GB GTDT GB GTDT GB GTDT GB GTDT GB GTDT GB
-20
-40
CCA1 CCA2 CCA3
CCA3 + 7
Days
CCA3+14
Days
CCA3+21
Days
CCA3+28
Days
CCA3+35
Days
CCA3+ 42
Days
Conditioned Context Aversion: Acquisition and Retention Trials
Figure 5-3. LGSM AI mice maintained on glass bottles (GB) were exposed to 3
conditioning trials when they drank from graduated tubes with dark-colored tape paired with
injections of LiCl or NaCl (controls). Strong suppression was found with a single conditioning
trial for GB-DT LiCl/Immed group. During retention tests, GB-DT LiCl/Immed group showed high
suppression to CS than their maintenance bottles until week 3 (CCA3+ 21 days).
99
Suppression Index (%)
Development of aversion to CS and regular tubes by mice maintained on graduated tubes with glass bottle
(Albino mice/Delay Injection)
100
80
60
40
20
0
-20
GTDT GB GTDT GB GTDT GB GTDT GB GTDT GB GTDT GB
CCA1 CCA2 CCA3
CCA3 + 7
Days
CCA3+14
Days
CCA3+21
Days
CCA3+28
Days
CCA3+35
Days
CCA3+ 42
Days
Conditioned Context Aversion: Acquisition and Retention Trials
Figure 5-4. LGSM AI mice maintained on glass bottles (GB) were exposed to 3
conditioning trials when they drank from graduated tubes with dark-colored tape paired with
injections of LiCl or NaCl (controls). Strong suppression was found with a single conditioning
trial for GB-DT LiCl/Delay group. During retention tests, GB-DT LiCl/Delay group showed high
suppression to CS than their maintenance bottles until week 3 (CCA3+ 21 days).
100
101
Table 5-3a, 5-3b and 5-3c. Water Intakes and Percent suppression for Experiment 5-1.
Experiment 5-2
Experiment 5-1 showed that aversions to changes in the environment (switching from
glass water bottles to graduated tubes with dark-colored tape) could serve as CS’s within the
aversion conditioning paradigm when using albino mice. Experiment 5-1 likewise demonstrated
that both experimental groups (immediate and delay) showed similar magnitudes of retention and
rates of extinction in relation to the CS tubes.
In Experiment 5-2, we examined whether conditioned aversion could also be developed
when we made small alterations in the home-cage of a mouse (switching from graduated tubes
with light-colored tape to graduated tubes with dark-colored tape tubes).We predicted that
animals maintained on graduated tubes with light-colored tape would develop aversions to tubes
102
with dark-colored tape similar to those observed in the pigmented LGSM (AI) mice. We also
carefully examined whether conditioned aversion could also be developed when a 30 minute CSUCS delay was used. We predicted that the immediate-injection and delayed-injection groups
would develop similar magnitudes of aversion and extinction to the CS tubes.
Subjects
36 LGSM AI (mean age 70 days, range; 67-80) from 10 different litters were used. Sex
(21M, 14F) and litter membership was evenly distributed across the 4 experimental groups
(9/group).
Methods
Maintenance conditions were similar to those described in the methods chapter. Before
the experiment, the mice were routinely maintained on pint-size translucent plastic bottles with
blue plastic lids and metal pin-hole spouts. The mice were then habituated to drinking water from
25 mL graduated tubes with pieces of light-colored tape near the spouts, rubber stoppers, and
stainless steel (SS) spouts with ball bearings for 7 days (Figure 5-5).
Mice were then trained to drink water promptly from these bottles in the light phase of
the circadian cycle by depriving them of water for 16 h and giving them access to water several
times per day during the light phase of the light/dark cycle. Mice had 30 minutes access to water
on 3 occasions and then were allowed to drink for 3 hour until the beginning of the dark cycle.
This procedure was repeated on 3 separate days.
103
Figure 5-5. (left) Maintenance Tubes, Figure 5-6. (right) Difference brightness between
maintenance and CS tubes
Conditioning procedure
During the conditioning trials, tap water in graduated tubes (with ball-bearing spouts)
with pieces of dark-colored tape attached to the tubes near their spouts (DT) were presented to
water –deprived mice for 15 minutes in their home cage (Figure 5-6). The two experimental
groups were injected with LiCl (0.15 M, 0.3 mL/10g body weight) immediately or 30 minutes
after the graduated tubes were removed from the cages: LT-DT LiCl/Immed and LT-DT LiCl/Delay. The
control group was injected with NaCl in the same manner as the LiCl groups (the same
concentration and dose of NaCl as of LiCl): LT-DT NaCl/Immed and LT-DT NaCl/Delay (Table 5-4).
104
Table 5-4 Experimental Setup
After the last conditioning trial, the mice were allowed a recovery period of 7 days.
During this period, the animals had ad libitum access to their regular bottles.
Retention tests (42 minutes in duration) were administered over two days in each week
after the recovery period (e.g., CCA3+7 days, CCA3+14 days). If a mouse had access to water
from a graduated tube with dark-colored tape (the CS) on the initial day, on the following day, a
graduated tube with light-colored tape (the maintenance tube) was presented, or vice versa. This
provided us with an opportunity to assess the total (dark-colored tape tube) and non-specific
suppression (light-colored tape tube).
105
Data Analyses
Conditioning Analyses
We conducted two conditioning analyses for CCA 2 and CCA 3: the first analysis was of
intake, and the second, suppression. For intake, there were two between-subject factors, Groups
(Experimental vs. Control) and CS-UCS Interval (Immediate vs. Delay), as well as a withinsubjects measure (Trials). In the second analysis (suppression), there was one between-subject
factor, CS-UCS Interval (Immediate vs. Delay), as well as a within-subjects measure (Trials).
Except the places where we specified Bonferroni `s correction was used, p<0.05 was
accepted as significant.
Retention Analysis
We assessed retention on 7 consecutive weeks (starting 1 week after conditioning) in
order to compare duration of retention in Immediate and Delay groups.
We evaluated which week extinction occurred by using post-hoc paired t-tests comparing
total to non-specific suppression within each conditioned group.
There were two between-subject factors, CS-UCS Interval (Immediate vs. Delay) and CS
Day (the day of the presentation of the CS), as well as two within-subjects measures that were
weeks (7) and tubes (CS vs. maintenance suppression).
Results
The raw intakes for Experiment 5- 2 are shown in Table 5-6 and suppression ratios in
Figure 5-7 and 5-8.
106
Table 5-5 shows the ANOVA design that we used to calculate intake and suppression
results for the conditioning and retention tests.
Table 5-5. Design of ANOVA
Conditioning
The analysis of variance showed a highly-significant reduction in intake on CCA2 and
CCA3 in conditioned mice compared to the controls (F 1, 31 =98.62, p< 0.001). There were no
significant differences between the Immediate and Delay groups in their responses to
conditioning (Trials X Groups X CS-UCS Interval interaction, n.s.; see Table 5-6 a).
Figure 5-7 and 5- 8 shows the suppression ratios calculated from the intake data. Our
analysis of suppression shows that suppression was significantly stronger on CCA3 than on
CCA2 (F 1, 22 =12.84, p< 0.01). There were no significant differences between the Immediate
and Delay groups in their responses to suppression during CCA2 and CCA3 (CS-UCS Interval X
Trial interaction, n.s.; see Table 5-6 a).
107
Retention Suppression
Analyses of suppression of all retention tests showed that all experimental groups
experienced greater suppression to the CS tubes than they did to the maintenance tubes overall (F
1, 120 =179.02, p< 0.001). In addition, we showed that there was a statistically significant Tubes
X Weeks interaction indicating that mice showed declined suppression to CS tubes during the
retention while non-specific suppression seems to stay about the same across weeks (F 6, 120
=10.29, p<0.001). In addition, the CS day had no significant effect on the results (CS day, n.s.).
We also compared the total and non-specific suppression for all retention trials. We saw a
significant difference between total and non-specific suppression until week 3 in both Delay and
Immediate experimental groups. In weeks 4, 5 and 6, consistent with extinction, there was no
significant difference between total and non-specific suppression within each conditioned groups.
There were no significant differences in suppression between the Immediate and Delay
groups (CS-UCS Interval, n.s). In addition, there was no statistically significant CS-UCS Interval
X Tubes X Weeks interaction indicating that there was no difference between Immediate and
Delay groups in the gradual disappearance of the specific conditioned aversion.
Development of aversion to CS and regular tubes by mice maintained on graduated tubes with light-colored tape
(Immedidate Injection)
Suppression Index (%)
120
100
80
60
40
20
0
GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT
CCA1 CCA2 CCA3 CCA3 + 7
Days
CCA3+14
Days
CCA3+21
Days
CCA3+28
Days
CCA3+35
Days
CCA3+ 42 CCA3+ 49
Days
Days
Conditioned Context Aversion: Acquisition and Retention Trials
108
Figure 5-7. LGSM AI mice maintained on graduated tubes with light-colored tape (LT)
were exposed to 3 conditioning trials when they drank from graduated tubes with dark-colored
tape paired with injections of LiCl or NaCl (controls). Strong suppression was found with a single
conditioning trial for LT-DT LiCl/Immed group. During retention tests, LT-DT LiCl/Immed group
showed high suppression to CS than their maintenance bottles until week 3(CCA3+ 21 days).
Development of aversion to CS and regular tubes by mice maintained on graduated tubes with light-colored tape
(Delay Injection)
100
Suppression Index (%)
80
60
40
20
0
GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT
CCA1 CCA2 CCA3 CCA3 + 7
Days
CCA3+14 CCA3+21 CCA3+28 CCA3+35 CCA3+ 42 CCA3+ 49
Days
Days
Days
Days
Days
Days
Conditioned Context Aversion: Acquisition and Retention Trials
Figure 5-8. LGSM AI mice maintained on graduated tubes with light-colored tape (LT)
were exposed to 3 conditioning trials when they drank from graduated tubes with dark-colored
tape paired with injections of LiCl or NaCl (controls). Strong suppression was found with a single
conditioning trial for LT-DT LiCl/Delay group. During retention tests, LT-DT LiCl/Delay group showed
high suppression to CS than their maintenance bottles until week 3 (CCA3+ 21 days).
109
110
Table 5-6a, 5-6b and 5-6c. Water Intakes and Percent suppression for Experiment 5-2.
Discussion
A review of recent literature suggests that most albino mouse strains mice have weaker
visual abilities than most pigmented mice (Wong & Brown, 2006). However, in this chapter, we
showed that like pigmented LGSM AI mice, LGSM albino mice can develop aversion to changes
in the environment (either switching from graduated tubes with light-colored tape to graduated
tubes with dark-colored tape or glass bottles to graduated tubes with dark-colored tape) when
discrete visual cues are paired with illness.
In addition, the results of Chapter 5 support those of our previous experiments (Chapters
3 and 4). There was a highly significant reduction in intake of CCA2 and CCA3 in conditioned
111
mice as compared to the controls in both conditions (switching from graduated tubes with lightcolored tape to graduated tubes with dark-colored tape or glass bottles to graduated tubes with
dark-colored tape). There were no significant differences between the Immediate and Delay
groups in their responses to conditioning as we found in Chapters 3 and 4.
The results from our experiments with albino LGSM AI mice in this chapter suggest that
conditioned aversions to changes in the environment are retained for a similar duration in both
conditions (switching from graduated tubes with light-colored tape to graduated tubes with darkcolored tape or glass bottles to graduated tubes with dark-colored tape), including when using a
30-minute delay between the presentation of the CS and the UCS. Both experimental groups (LTDT LiCl/Immed and LT-DT LiCl/Delay) showed higher total suppression than non-specific suppression for
the first three weeks in both conditions in Experiments 5-1 and 5-2. The experimental groups also
showed some non-specific suppression to their regular tubes, as we expected based on our
previous experiments’ results.
112
Chapter 6
Conditioned Context preference using positive reinforcement in B6D2 mice
The aim of this experiment was to examine whether a contextual cue could be formed
when it is paired with a positive reinforcer. In this experiment, sucrose was used as the positive
reinforcer.
Subjects
43 (21 male, 12 female) B6D2 AI mice were used. An equal proportion of males and
females were assigned to each group (mean age 125 days, range; 120- 130). 10 different litters
were used for this study and mice from each litter were assigned to different experimental
conditions in a balanced way.
Adaptation to Restricted Water
Before the experiment began, all of the mice were routinely maintained on tap water
using pint-size translucent plastic bottles with blue plastic lids and metal spouts. The mice were
then adapted to drink water from regular tubes over the course of 7 days. Graduated tubes without
tape (with ball-bearing spouts) were used as regular tubes.
Conditioning Procedure
Following the adaptation phase conditioning took place over 6 days and lasted for 20
minutes each day. The mice were divided into two groups: the experimental group and the control
group. The experimental group was presented with sucrose solution (0.1 M) in graduated tubes
with dark-colored tape and water in graduated tubes without tape on alternate days (Table 6-1). In
113
other words, the sucrose solution was associated with graduated tubes with dark-colored tape and
water with graduated tubes no tape (Table 6-1). This was repeated three times (6 days in total). In
contrast, the control group had water during all of the conditioning trials. The water was
presented in graduated tubes with or without dark-colored tape (Table 6- 1).
Table 6-1. Experimental Setup
During the retention tests, two graduated tubes (one with tape, the other without tape)
were presented to each mouse. We recorded the water intake from each of the two tubes at 6
hours and 24 hours.
114
Results
At 6 hours (during the light phase), the mice in the experimental group had drunk more
from their graduated tubes with dark-colored tape than from their tubes without tape
(experimental group; dark-colored vs. no tape; p<0.05; Figure 6-1). Yet the mice in the control
group exhibited no significant difference in water intake between the two tubes.
We also calculated water consumption from each of the two types of tubes between 6 and
24 hours (this period included a dark phase: 18 hours, and also a light phase: 6 hours). There was
no significant difference in water intake between the graduated tubes with dark-colored tape and
their tubes without tape in the experimental group of mice. However, surprisingly, we found a
significant difference in water intake between the graduated tubes with dark-colored tape and
their tubes without tape in the control group of mice; they had drunk more from their graduated
tubes without tape than graduated tubes with dark-colored tape (control: dark-colored tape vs. no
tape; p<0.05, Figure 6-1).
Discussion
The results thus show that a positive conditioned context preference can be formed for up
to 6 hours when measurements were taken in the light phase. On the other hand, there was no
difference in water intake between the two tubes when measurements were primarily taken in a
dark phase.
The control group did not show any preference for the graduated tubes with or without
tape at 6 hours. The control group did, however, drink more from the graduated tubes without
tape than tubes with tape between 6 and 24 hours. To better understand the reasons for this, we
should have considered each mouse’s intake from both types of tubes before the experiment
115
began to see if the mouse had preexisting position preferences.
6 HOURS
Experimental
NoTape
Experimental
Tape
Control
NoTape
Control
Tape
Experimental
NoTape
Experimental
Tape
Control
NoTape
Control
Tape
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
6- 24 HOURS
Figure 6-1. B6D2 AI mice maintained on water in graduated tubes without tape. During
the retention tests, two graduated tubes (one with tape, the other without tape) were presented to
each mouse. Conditioned context preference could be formed up to 6 hours when sucrose solution
is used as a positive reinforce in the experimental group.
116
Chapter 7
General Discussion
Selective Associative Learning
Selective associative learning was first described by John Garcia (1966). According to
this principle, evolution has shaped the nervous system so that learning occurs more easily when
specific classes of stimuli are paired with particular reinforcers. For example, when an internal
stimulus (taste) was paired with illness (an internally applied reinforcer), rats developed strong
conditioned aversion, however pairing of an external stimulus (audiovisual) with illness resulted
in weak or no conditioned aversion. After Garcia reported that only particular classes of CS are
able to be associated with a particular UCS (the principle of selective associative learning), a
number of researchers went on to corroborate his theory (Domjan & Gemberling, 1982; Rescorla,
2008). Even his interpretation of selective associative learning was considered to be
unimpeachable (Davis & Buskist, 2008). As such, contradictory findings were ignored. For
example, it has been established that rats can develop conditioned aversion resulting from the
pairing of illnesses with environmental changes. Pairing of illness with substantive alterations in
compound stimuli (visual, acoustic or tactile) cause conditioned aversion to these stimuli in rats
(Boakes et al., 1997; Revusky & Parker, 1976; Rodriguez et al., 2000). However, it is difficult to
know which of the stimuli that the animals are responding to in these experiments because these
studies rely on substantial, multi-modal exteroceptive changes in the rats’ environment. Our
present observations extend current knowledge of contextual conditioning by providing evidence
that laboratory mice can develop aversion to small alterations in their home cages. These kinds of
117
experiments are helpful because they focus attention on the specific cues to which subjects
respond.
It is also important to point out that in our experiments; we only investigated one part of
the theory of selective associative learning. More specifically, we examined whether pairing an
external stimulus (such as a visual cue) with an internally applied reinforcer (in this case, illness)
would result in strong conditioned aversion. We did not examine whether pairing an internal
stimulus (such as taste) with an externally applied reinforcer (such as shock) would likewise
result in strong aversion. Although our experiments are thus limited to one particular form of
learning (pairing an external stimulus to an internally applied reinforcer), many previous
experiments have also dealt with one aspect of selective associative learning (Garcia, Ervin, &
Koelling, 1966; Domjan & Gemberling, 1982; Rescorla, 2008).
Features of CTA and CCA
Three key features have been emphasized with regard to CTA: First, a single pairing of a
novel taste with illness causes conditioned aversion to the novel taste (Garcia et al., 1955).
Second, an effective CTA can be demonstrated even when a long delay is introduced between CS
and UCS (Garcia, Ervin, & Koelling, 1966; Revusky & Garcia, 1970). Third, CTA learning can
last for months (Houpt, Philopena, Joh, & Smith, 1996; Martin & Timmins, 1980; Steinert,
Infurna, & Spear, 1980). The literature suggests that the first two features of conditioned taste
aversion constitute an unusual form of classical conditioning (Pavlov, 1927). However, our
studies suggest that these features are not unique for conditioned taste aversion. Strong
conditioned context aversion can also be demonstrated after a single trial and even after a 30 min
CS-UCS delay. Moreover, CCA is retained for comparable durations as CCA.
118
Acquisition
Based on the findings presented in Chapter 3, we conclude that the number of
conditioning trials required to show strong suppression in context aversion protocols depends on
the magnitude of differences between customary and novel stimuli. For example, switching from
(maintenance) graduated tubes with light-colored tape to (novel) graduated tubes with darkcolored tape (tubes with a small difference) required two conditioning trials to obtain strong
suppression in B6D2 mice, whereas switching from (maintenance) glass bottles to (novel)
graduated tubes with dark-colored tape (a larger difference), strong suppression was found after a
single conditioning trial. On the other hand, in Chapters 4 and 5, we found that regardless of
whether switching from glass bottles to graduated tubes with dark-colored tape or switching from
graduated tubes with light-colored tape to graduated tubes with dark-colored tape, only one
conditioning trial was required for LGSM AI mice to develop aversion to the CS. These results
are consistent with one of two possible explanations: on the one hand, since we used different AI
mice, they may have had different responses to acquisition; on the other hand, as the experiments
continued, we may have become better at conducting them, and thus have obtained better
conditioning results. Finally, the results of Experiment 4-3 supported one of the features of CTA;
mice developed aversion to sodium saccharin (CS) after a single pairing of a novel taste with
illness.
Comparing duration of retention in CCA and CTA
When we compared the duration of retention in our conditioned context experiments, we
observed that in both conditions (switching from graduated tubes with light-colored tape to
graduated tubes with dark-colored tape or from glass bottles to graduated tubes with dark-colored
119
tape), mice showed similar durations of retention: about 3 to 4 weeks (Table 7-1 and 7-2). The
only unusually long retention (7 weeks) was found in Experiment 4-2 in the group of LT-DT
LiCl/Delay
mice. We are still investigating the cause of this outcome (Figure 7-1 and Table 7-1).
The results of the experiments in Chapters 3, 4, and 5 show that context aversion is able
to be formed when there is a substantial delay between the presentation of the CS and the UCS.
The results also showed that there were no differences between the Immediate and Delay
experimental groups in their responses to the conditioning and retention tests (Table 7-1 and 7-2).
In Chapter 4, we compared duration of retention to CCA and CTA. The results provide
very little evidence that conditioned taste aversion is retained for longer than conditioned context
aversion (Table 7-1).
An important observation is that overall the LGSM AI mice did show longer retention of
extinction as compared to the B6D2 AI mice. The results of Experiment 3-3 with B6D2 AI mice
indicate that the LT-DT LiCl/Immed group retained for 2 weeks; however, the LT-DT LiCl/Delay group
retained for only 1 week. However, experiments with LGSM AI mice showed that both groups
(LT-DT LiCl/Immed and LT-DT LiCl/Delay ) retained for about 3 to 4 weeks (Table7-1 and 7-2). These
results may be due to one of two reasons: on one hand, since they are different AI mice, they may
have different responses to retention and extinction; on the other hand, as the experiments
continued, we may have been better at conducting them, and thus have obtained better retention
results.
The conditioned taste aversion was retained for longer (approximately 3 more weeks) in
Garcia`s experiment than our experiment (Experiment 4-3). In Garcia et al’s experiment (Garcia
et al., 1955) the ingestion of saccharin solution was paired with radiation exposure. The effects of
120
conditioning after radiation were evaluated using two bottle preference tests that compared intake
of saccharin solution to water and in non-deprived rats. In other words, rats always had a chance
to avoid the CS (saccharin solution) and could always drink water in their experiments. The
results of experiment showed that it took about 7 weeks for rats to show extinction to the taste. In
our cases, mice always had water restriction for 16 hours before retention tests. In addition, only
one tube, that includes saccharin solution (the CS), was presented during the retention tests. In
other words, in our experiments, mice were exposed only to the CS tube because they were
thirsty. Thus, mice had opportunity to develop quicker extinction to the CS in our experiments.
That might be the reason that aversion was retained for longer in Garcia`s experiment.
Non-specific Suppression
The experimental groups showed significant non-specific suppression to their regular
tubes. Thus, we conclude that the non-specific components of conditioning are considerable and
merit further consideration by researchers working in this field. The issue of non-specific
suppression is sometimes dealt with in experimental contexts by administering several exposures
to neutral stimuli under the same conditions that CS is presented (before retention is tested).
Then, CS tubes are presented assuming that non-specific responses have been extinguished. The
present results raise the question of whether non-specific tests should be routinely incorporated
into retention trials to permit accurate side by side comparisons with the response to CS (Figure
3-5, 4-3, 4-4, 4-7, 4-8, 5-3, 5-4, 5-7 and 5-8).
121
Specific Suppression to CS
Suppression Index (%)
100
GB-DT IMMED
LT-DT IMMED
80
TASTE IMMED
60
40
20
0
WK1SPEC WK2SPEC WK3SPEC WK4SPEC WK5SPEC WK6SPEC
-20
Figure 7-1. Results of specific suppression in Immediate experimental groups (LGSM AI
pigmented mice). Specific suppression was calculated by subtracting non-specific suppression
from total suppression (Specific Suppression = Total Suppression - Non-Specific Suppression).
Both context groups (LT-DT LiCl/Immed and GB-DT LiCl/Immed ) showed similar extinction to the CS,
and the duration that conditioned taste aversion was retained slightly longer (SS LiCl/Immed ).
122
GB to DT DELAY
80
LT to DT DELAY
70
TASTE DELAY
60
50
40
30
20
10
0
-10
WK1SPEC WK2SPEC WK3SPEC WK4SPEC WK5SPEC WK6SPEC
-20
Figure 7-2.Results of specific suppression in Delay experimental groups (LGSM AI
pigmented mice). Specific suppression was calculated by subtracting non-specific suppression
from total suppression (Specific Suppression = Total Suppression - Non-Specific Suppression).
The Taste group (SS LiCl/Delay) and one of the context groups (GB-DT LiCl/Delay) showed quicker
extinction to the CS, whereas LT-DT LiCl/Delay was retained for longer.
123
GROUPS
SUBJECTS
ACQUISITION
CS-UCS
INTERVAL
DURATION OF
RETENTION
GB- DT
LGSM AI
(Pigmented)
1 Trial
Immediate
4 weeks
LT-DT
LGSM AI
(Pigmented)
1 Trial
Immediate
3 weeks
Sodium
Saccharin
LGSM AI
(Pigmented)
1 Trial
Immediate
6 weeks
GB- DT
LGSM AI
(Pigmented)
1 Trial
Delay
4 weeks
LT-DT
LGSM AI
(Pigmented)
1 Trial
Delay
7 weeks
Sodium
Saccharin
LGSM AI
(Pigmented)
1 Trial
Delay
3 weeks
Table 7-1. Duration of retention in LGSM AI pigmented mice.
GROUPS
SUBJECTS
ACQUISITION
CS-UCS
INTERVAL
DURATION OF
RETENTION
GB- DT
LGSM AI(Albino)
1 Trial
Immediate
3 weeks
LT-DT
LGSM AI(Albino)
1 Trial
Immediate
3 weeks
GB- DT
LGSM AI(Albino)
1 Trial
Delay
3 weeks
LT-DT
LGSM AI(Albino)
1 Trial
Delay
3 weeks
Table 7-2. Duration of retention in LGSM AI albino mice.
124
Conditioned Context Preference
The results of Chapter 6 show that a conditioned context preference can be formed when
sucrose solution is used as a positive reinforcer with B6D2 AI mice. However, the retention is
short-lived (only 6 hours).
Other concerns
Previous studies (Boakes et al., 1997; Rodriguez et al., 2000) were conducted by making
large alterations in the environment and inducing illness to cause context aversion learning. In the
literature, only one experiment paired small alterations in the environment with illness (Revusky
& Parker, 1976). They showed that rats can show aversion to novel cups if these are paired with
toxicosis during conditioning trials. As later discussed by Nachman, Rauschenberger and Ashe
(1977), conditioned aversion occurred in the cited study either to visual cues (different
appearance of containers) or to somatosensory stimulation (different body positions or oral
sensations related to drinking from bottles vs cups). In our experiment, somatosensory stimulation
is unlikely to be important because we presented water in the same way in both maintenance and
novel containers so that mice used similar drinking behavior patterns/postures during the
experiments.
Rats have been the species of choice in the study of conditioned context learning.
Researchers show that rats developed conditioned aversions by pairing illness with large
environmental changes. In our experiments, we showed that using animals produced from a cross
between the B6 and D2 or LG and SM strains (i.e. genetically heterogeneous mice) minor
alterations in cage environment can serve as CSs using an illness conditioning model.
125
In conclusion, development of context aversion conditioning paradigms for use with mice
will enable the powerful molecular tools available for use in this species (knockouts, transgenics,
etc.) to be applied to this type of learning. Furthermore, the ability to demonstrate this form of
conditioning using small contextual changes as the CS provides a focus for neuroanatomic studies
which is more difficult or impossible to arrive at when multi-modal changes are used as CS’s. In
addition, the results provided by the present experiments will help us understand how
environmental cues in chemotherapy rooms may act as conditioned stimuli and how we can
reduce conditioned nausea in humans by using learning theory.
Historical Note
Garcia (1954) hypothesized that rats developed conditioned aversions to the taste of
water from plastic bottles. However, based on findings in Experiment 3-1, we suggest that
Garcia`s rats were conditioned to the appearance of plastic bottles rather than the taste of water in
them. Without repeating Garcia’s experiment in exactly the same way using the same material
and apparatus, it is impossible to conclude that Garcia`s rats were responding to the appearance of
plastic bottles rather than the taste of water in them. However, it is intriguing to speculate that
Garcia may have missed evidence of CCA in one of his earliest studies and that CCA may have
helped lead him to the discovery of CTA.
126
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Curriculum Vitae
Sezen Kislal
EDUCATION
2010-2015
Ph.D. Biobehavioral Health
The Pennsylvania State University; University Park, Pennsylvania, USA
2006-2009
B.S. Biology
Hacettepe University; Ankara, Turkey
PEER-REVIEWED PUBLICATIONS
Ishii, A., Koide, T., Takahashi, A., Shiroishi, T., Hettinger, T. P., Frank, M. E., Savoy, L. D.,
Formaker, B. K., Yertutanol, S., Lionikas, A., & Blizard, D. A. (2011). B6-MSM consomic
mouse strains reveal multiple loci for genetic variation in sucrose octaacetate aversion. Behavior
Genetics 41(5), 716-723.
Yertutanol, S., & Blizard, D. A. (Resubmitted). Aversions conditioned by pairing illness with
visual cues in the home cage of a mouse.
PRESENTATIONS
September 2012
“Aversion to Sucrose octaacetate by Laboratory Mice is controlled
by a Polygenic System”
Penn State Graduate Exhibition
The Pennsylvania State University; State College, PA
September 2007
“Telomeres and Regulation”
Department of Molecular Biology and Genetics
Istanbul Technical University; Istanbul, Turkey
WORK EXPERIENCE
June-July 2010
Behavioral research on bitter taste
Mouse Genomics Resource Laboratory
National Institute of Genetics; Mishima, Japan
June-September 2008
Internship with cardiovascular medicine group
Erasmus Internship Program
Department of Molecular and Cellular Medicine
Katholieke University of Leuven; Leuven, Belgium