The Pennsylvania State University. The Graduate School. Department of Biobehavioral Health CONDITIONING DISCRETE VISUAL CUES TO AVERSIVE INTEROCEPTIVE

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1 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

2 ii 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

3 iii 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

4 iv 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.

5 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 Experiments with Compound Stimuli Context Aversion Learning Two common techniques to reduce conditioned context aversion: Overshadowing and Latent inhibition Conditioned Place Aversion and Conditioned Place Preference Conclusion Chapter 2 Methods common to many experiments Subjects Maintenance Conditions Assignments of animals to experimental groups Colony Room (Light Cycle, Temperature, Food) General Experimental Considerations I. Adaptation and Training II. Conditioning Procedures III. Recovery Period IV. Retention Statistical issues Suppression index Data Analyses Conditioning Retention Appendix Visual system of mouse Anatomy of the retina Composition of photoreceptors: human vs mice Visual acuity Chapter 3 Exploratory Studies with B6D2 mice Experiment Experiment

6 vi Experiment Discussion Chapter 4 Comparison of CCA and CTA in pigmented LGSM mice Experiment Experiment Experiment Discussion Chapter 5 Comparison CCA in albino LGSM mice Experiment Experiment Discussion Chapter 6 Conditioned Context preference using positive reinforcement in B6D2 mice Discussion Chapter 7 General Discussion Selective Associative Learning Features of CTA and CCA Acquisition Comparing duration of retention in CCA and CTA Non-specific Suppression Conditioned Context Preference Other concerns Historical Note References

7 vii LIST OF FIGURES Figure 1-1. Chemotherapy in cancer patients. Adapted from Ursula Stockhorst, Hans- Joachim Steingrueber, Paul Enck, Sibylle Klosterhalfen, Pavlovian conditioning of nausea and vomiting. Autonomic Neuroscience, 129 (1), (2006) 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 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) 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 Figure 1-6. Long-delay taste aversion learning. Adapted from Principles of learning and behavior, (p.82), Domjan, M. (2009) Figure 2-1. Plastic bottles were presented before starting the experiment Figure 2-2.The experimental set up is illustrated from the back of the cage in order to clearly demonstrate the tube arrangement Figure 2-3(left). and Figure 2-4 (right). The CS tubes that we have used during the conditioning trials 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) 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) Figure 2-7. Shows the visible light spectrum for humans 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 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

8 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 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 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 Figure 4-1(left). Maintenance bottle. Figure 4-2(right). CS tube 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) 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) Figure 4-5 (left). Maintenance tube. Figure 4-6(right). Brightness differences between tape on CS and Maintenance tubes 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) 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 viii

9 ix 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) Figure 4-9. Maintenance tube Figure 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) Figure 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) Figure 5-1. (left) Maintenance Bottle Figure 5-2. (right) CS tube 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) 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) Figure 5-5. (left) Maintenance Tubes, Figure 5-6. (right) Difference brightness between maintenance and CS tubes 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) 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

10 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) 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 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 ) 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

11 xi 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 Table 1-2. Experimental Setup Table 1-3.Experimental Setup 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 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 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 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 Table 2-2. Design of ANOVA Table 3-1. Experimental Setup Table 3-2. Water Intakes and percent suppression for Experiment Table 3-3. Experimental Setup Table 3-4. Water Intakes and percent suppression for Experiment Table 3-5. Experimental Setup Table 3-6. Water Intakes and percent suppression for Experiment Table 4-1. Experimental Setup Table 4-2. Design of ANOVA Table 4-3. Water Intakes and Percent suppression for Experiment Table 4-4. Experimental Setup... 75

12 xii Table 4-5. Design of ANOVA Table 4-6a, 4-6b and 4-6c. Water Intakes and Percent suppression for Experiment Table 4-7. Experimental Setup Table 4-8. Design of ANOVA Table 4-9a, 4-9b and 4-9c. Water Intakes and Percent suppression for Experiment Table 5-1. Experimental Setup Table 5-2. Design of ANOVA Table 5-3a, 5-3b and 5-3c. Water Intakes and Percent suppression for Experiment Table 5-4 Experimental Setup Table 5-5. Design of ANOVA Table 5-6a, 5-6b and 5-6c. Water Intakes and Percent suppression for Experiment Table 6-1. Experimental Setup Table 7-1. Duration of retention in LGSM AI pigmented mice Table 7-2. Duration of retention in LGSM AI albino mice

13 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.

14 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.

15 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

16 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, Hans- Joachim Steingrueber, Paul Enck, Sibylle Klosterhalfen, Pavlovian conditioning of nausea and vomiting. Autonomic Neuroscience, 129 (1), (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

17 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.

18 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).

19 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,

20 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.

21 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).

22 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.

23 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)

24 Figure 1-6. Long-delay taste aversion learning. Adapted from Principles of learning and behavior, (p.82), Domjan, M. (2009). 10 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.

25 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

26 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

27 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

28 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

29 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).

30 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

31 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

32 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 30- minute 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

33 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

34 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.

35 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.

36 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

37 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

38 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

39 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

40 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.

41 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.

42 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

43 experimental group. We usually used mice aged approximately 3 to 5 months old at the beginning of the experiments. 29 Colony Room (Light Cycle, Temperature, Food) The colony room that we used had a 12/12h light/dark cycle (lights on 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.

44 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 45- degree 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).

45 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

46 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.

47 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

48 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). 34 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 t- tests 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).

49 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.

50 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).

51 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.

52 38 Ultraviolet <380 nm Violet Blue Blue-green Green Yellow-green Yellow Orange Figure 2-7. Shows the visible light spectrum for humans. Red 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

53 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-

54 colored and dark-colored tapes that we used and it is the brightness dimension that we think mice are responding to. 40 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.

55 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

56 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; ) 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

57 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

58 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 Figure 3-1. The raw intakes for Experiment 3-1 are shown in Table 3-2 and suppression ratios in

59 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.).

60 Suppression Index (%) 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. Development of aversion to glass bottles by mice maintained on plastic bottles CCA1 CCA2 CCA3 Retention CCA3+7 Days Conditioned Context Aversion: Acquisition and Retention PB-GB TW/LiCl PB-GB PW/LiCl 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.

61 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

62 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; ) 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

63 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).

64 50 Results Figure 3-2. The raw intakes for Experiment 3-2 are shown in Table 3-4 and suppression ratios in 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.).

65 Suppression Index (%) 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 GB-DT LiCl CCA1 CCA2 CCA3 1st RetentionMaintenance CCA3+3 days Tubes CCA3+10 days Conditioned Context Aversion: Acquisition, Retention and Specificity Trials 2nd Retention CCA3+13 days

66 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; ) 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

67 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).

68 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).

69 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.

70 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.).

71 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 st cond 2nd cond 3rd cond LT-DT (LiCl/Immed) LT-DT (LiCl/Delay) -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

72 Suppression Index (%) 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; 1 st week; t=2.36, df=9, p<0.05 and 2 nd 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; 1 st 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. Total (dark-colored tape) and Non-specific (light-colored tape) suppression following three conditioning trials with B6D2 mice Dark (CS Tape) Light (Maintenance Tape) Immediate Delay Immediate Delay Immediate Delay Week 1 Week 2 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-

73 DT LiCl/Delay group. By week 3, there was no evidence for both total or nonspecific suppression for both immediate and delay groups. 59 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

74 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 3 rd 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.

75 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 LT- DT 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

76 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.

77 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

78 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; ) 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).

79 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.

80 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).

81 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.

82 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

83 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

84 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).

85 71

86 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.

87 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; ) 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).

88 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).

89 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

90 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.

91 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

92 Suppression Index (%) 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. 100 Development of aversion to CS and regular tubes by mice maintained on graduated tubes with light-colored tape 80 (Immediate Injection) GTDT= CS 60 GTLT = Regular Tubes CCA1 CCA2 CCA3 CCA3 + 7 Days GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT 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

93 Suppression Index (%) 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). 79 Development of aversion to CS and regular tubes by mice maintained on graduated tubes with 100 light-colored tape (Delay Injection) GTDT= CS Tubes 80 GTLT= Regular Tubes CCA1 CCA2 CCA3 CCA3 + 7 Days GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT GTDT GTLT CCA3+14 Days CCA3+21 Days CCA3+28 Days CCA3+35 Days CCA3+ 42 Days CCA3+ 49 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).

94 80

95 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.

96 82 Subjects 46 LGSM AI male mice (mean age 125 days, range; ) 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

97 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 SS LiCl/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 SS NaCl/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

98 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.

99 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

100 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.

101 87 Figure 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).

102 88 Figure 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).

103 89

104 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

105 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.

106 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.

107 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.

108 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.

109 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

110 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

111 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.).

112 Suppression Index (%) 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 (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. 100 Development of aversion to CS and regular tubes by mice maintained on glass bottle (Albino mice/immediate Injection) CCA1 CCA2 CCA3 CCA3 + 7 Days GTDT GB GTDT GB GTDT GB GTDT GB GTDT GB GTDT GB CCA3+14 Days CCA3+21 Days CCA3+28 Days CCA3+35 Days Conditioned Context Aversion: Acquisition and Retention Trials CCA3+ 42 Days 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).

113 Suppression Index (%) 99 Development of aversion to CS and regular tubes by mice maintained on graduated tubes with glass bottle (Albino mice/delay Injection) 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 Conditioned Context Aversion: Acquisition and Retention Trials CCA3+ 42 Days 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).

114 100

115 101 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

116 102 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 CS- UCS 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.

117 103 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).