The Neural Substrates of Incidental Sensory Experience. Senior Thesis. Presented to

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1 The Neural Substrates of Incidental Sensory Experience Senior Thesis Presented to The Faculty of the School of Arts and Sciences Brandeis University Undergraduate Program in Neuroscience Don Katz, Advisor In partial fulfillment of the requirements for the degree of Bachelor of Science by Max Bernstein May 2015 Copyright by Max Bernstein

2 Introduction In the 1890s, an accidental discovery led to a hypothesis that would change science forever. Ivan Pavlov, a Russian scientist studying the salivation of dogs in response to being fed, found that the dogs began salivating when he entered the room, even when he was not bringing food. He extrapolated that it might be possible to condition a stimulus to evoke a specific response, and when he proved it he gave birth to the study of what became known as classical conditioning (or, for obvious reasons, Pavlovian conditioning). In classical conditioning, a natural stimulus (the unconditioned stimulus, or US), such as the presence of food, evokes a reflexive, unconditioned response (UR), like salivation in preparation for said food. However, when the UR is paired with an otherwise neutral stimulus (the conditioned stimulus, or CS), such as the ringing of a bell when food is presented, eventually the CS presented alone will be sufficient to evoke the response, which is now called the conditioned response, or CR. The discovery of this basic process was the beginning of a massive revolution in the study of learning and behavior. Later (specifically, in the mid-1950s), Dr. John Garcia designed experiments to observe the effects that radiation has on various behaviors using rats as a model organism (Garcia, Kimeldorf, and Koelling, 1955). He found that, following exposure to radiation, the rats came to treat recently-consumed tastes as if they were aversive. Garcia hypothesized that the rats were associating the malaise caused by the radiation with the recently-consumed stimuli, then actively avoiding it in the future. Garcia tested this hypothesis by running an experiment where all rats were given saccharin, which is inherently palatable, after which he gave the rats either no radiation exposure, mild exposure, or strong exposure. The results of this experiment were that a huge decrease in saccharin consumption followed radiation exposure a decrease that scaled with the amount of exposure the rat had endured. This effect came to be known as Conditioned Taste Aversion, or CTA (or, for obvious reasons, the Garcia effect), which is now one of the most widely used and studied learning paradigms in behavioral neuroscience (Bouton, 1994; Bures, Bermúdez-Rattoni, & Yamamoto, 1998; Reilly & Schachtman, 2009; Welzl, D'Adamo, & Lipp, 2001). As with other forms of classical conditioning, CTA can be affected and altered by a wide variety of factors. One factor that has been widely used to manipulate CTA is exposure to the novel taste (CS) prior to pairing with malaise a procedure, known as latent inhibition, that results in an attenuation of the aversion (Lubow, 1973). This attenuation most likely reflects the fact that the CS has already become associated with safe/no outcome, such that the aversion must first overcome that outcome. If this benign taste experience has a such a powerful effect on taste learning, the next question to ask is whether even incidental exposure to other tastes could affect the formation of the aversion to a novel CS. However, little work has been done on this topic, though a few studies (Capretta, Petersik, & Stewart, 1975; Franchina & Gilley, 1986; Kwok, Livesey, &

3 Boakes, 2012; Miller & Holzman, 1981a, 1981b; Tarpy & McIntosh, 1977) have suggested that exposure to one taste make a rat less fearful of a future novel taste. Furthermore, a recent study in our lab found, though it was not the focus of the paper, that if a rat was exposed to a diverse array of tastes, extinction of the aversion (the process whereby the taste-illness relationship is forgotten) occurs faster (Moran & Katz, 2014). While it was not studied further, these studies offer the tantalizing possibility that mere experience with one taste might change perception of other tastes. This possibility is important to study because the results of such studies have frightening implications regarding the generalizability of rodent laboratory experiments. Laboratory rats are traditionally deprived of all taste stimuli so as to be naïve. However, humans naturally have extensive prior experience with tastes, a difference that has not been accounted for in the customary CTA paradigm and which has not been studied in great detail. If innocuous experience affects later learning, then naïve laboratory rats provide a poor model of human consumption. The work described in this report was conducted to directly determine the effects of innocuous incidental exposure of taste arrays on future conditioned taste aversion associations. This work reveals that previous innocuous exposure to a diverse array of tastes before conditioning induces conditioned aversions of greater magnitude to novel sucrose, which we have called the Diverse Pre-conditioning Exposure effect, or DPE. Furthermore, the strength of these aversions scales appropriately with the number of tastes presented and the number of preconditioning sessions. Finally, we determined that this effect was independent of the method by which the tastes were presented. The table below summarizes the experiments conducted over the course of our manuscript, currently being revised, Pre-exposure to a diverse array of tastes enhances later conditioned taste aversion to novel sucrose (Lee, Moran, Bernstein, & Katz, in revision). WITH MY ASSISTANCE INDEPENDENT WTHOUT MY ASSISTANCE Array of tastes during conditioning Aversion is not caused by LiCl injection itself DPE trials with IOC-only presentation Array of tastes during preconditioning IOC-only presentation with water only Sucrose in array, aversion to NaCl DPE scales with number of tastes DPE scales with number of conditioning sessions Increasing conditioning sessions of water-only trials

4 Methods and Materials Subjects All subjects were adult female Long Evans rats obtained from Charles River Laboratories. Rats were housed individually with ad libitum exposure to food and water prior to experiments and were on a strict 7am-7pm light schedule. Subjects were removed from the experiment if they displayed lethargy, lack of grooming, or 15% or greater loss of pre-surgery weight. Surgery Rats were between grams at the time of surgery. A ketamine/xylazine mixture (1ml ketamine,.05 ml xylazine/kg body weight) was intraperitoneal injection for anaesthetization. The head was shaved and cleaned before being stabilized in a stereotaxic device. Lidocaine injection in the scalp (.15ml) was followed by an incision and cleaning of the skull to allow for the insertion of four support screws in the skull. Intra oral cannulae (IOC), small flexible polyurethane tubes, were bilaterally inserted through the mouth posterolaterally to the first maxillary molar and parallel to the masseter muscle (Phillips & Norgren, 1970). Dental acrylic was used to make a stable head cap encasing the IOCs utilizing the screws as an anchor. Post-operational injections included saline (3 ml), an analgesic called meloxicam (0.04 mg/kg), and an antibiotic (Pro-Pen- G 150,000 U/kg), and additional antibiotic and analgesic were administered every 24 hours for 3 days. Rats were given 7 days post-surgery to recover. Experimental Design Each experiment had a very similar base design. Rats underwent surgery and were allowed to recover for a full week. On the last 2 days of recovery, the rats were placed in the testing chamber, separate from their own home cage, with a water bottle present to familiarize themselves with the testing apparatus. For consistency, all experiments were carried out around 1pm every day following a 20 hour water derivation to ensure that the rats would drink. Each experiment spanned 4-5 days, including 2-3 days of pre-exposure, 1 day of conditioning and 1 testing day. The stimuli we used reflected the concentrations of stimuli found broadly in the literature: 0.01M sodium chloride (NaCl), 0.02M citric acid, and double distilled water. Generally, the pre-conditioning sessions consisted of 5 minutes of free bottle access to a fluid in the testing chamber, a 5 minute break with no bottle access, followed by 15 minutes of fluid delivery through the IOC. These deliveries were made in 40 μl bursts across the tongue using pressure from a nitrogen tank through polymide tubes inserted as a manifold into the IOC. A total of 60 deliveries of tastes, pseudo-randomly selected from the tastes in the specific experiment, were ejected into the rat s mouth in 15 second intervals, ultimately resulting in 2.4 ml of fluid. The conditioning sessions was identical in structure to the pre-conditioning sessions except that all rats received only 0.3M sucrose solution in the bottle, as well as sucrose alone or with an array of tastes through the IOCs. Immediately following the conditioning, the rats were

5 subcutaneously injected with lithium chloride (LiCl, 0.3 M, 0.5% of current weight) to induce malaise. The concentration of LiCl we used is lower than the commonly used dosage (Nachman & Ashe, 1973) to avoid a floor-effect and allow us to observe any possible enhancements of CTA learning (Stone et al, 2005). The rats were then apologized to and returned to their home cages. The amount of sucrose during the conditioning stage became the baseline for the consumption measurement of each animal. After 24 hours following the conditioning, rats were tested only by lick spout. They were presented with sucrose for 5 minutes, followed by a 5 minute break, after which they were exposed to 5 minutes of water. The amount of sucrose consumed on test day was divided by the amount consumed on conditioning day to create a normalized aversion index, wherein a smaller aversion index implies a stronger aversion. After the first attempt at publication, the reviewers returned the paper with some concerns, one of which is that the results may have been affected by the delivery method of the taste stimuli. To ameliorate the concerns of presenting both in a bottle and through the IOCs (Schafe, G. E., Thiele, T. E., and Bernstein, I. L., 1998), the experiments were repeated using only presentation of tastes through the IOCs. During these experiments, the rat was placed in the testing chamber and presented with tastes through the IOCs for 100 trials, amounting to an approximate total volume of 5 ml. Another of these concerns was addressed by adding a control experiment to ensure that it was the lithium chloride, not the injection itself, which causes the aversion. This concern was accounted for by running a standard CTA paradigm using saline injections instead of LiCl. Lastly, the question was raised over whether these enhancements were generalizable to other palatable tastes, not only sucrose. Thus, an experiment was designed where sucrose was included in the array and the malaise-induced aversion was paired with NaCl. Results Exposure to an array of tastes The first experiment conducted was to confirm the expectation that experiencing a variety of tastes during the conditioning stage would attenuate the aversion. The full taste array (citric acid, NaCl, water) was presented through the IOCs with sucrose (CS) before exposure to LiCl while control subjects received only sucrose through the IOCs. As expected, diverse exposure during conditioning resulted in an attenuation of the aversion to novel sucrose, which is reflected in the significantly higher aversion index. Following this experiment, we wanted to determine whether pre-exposure to a variety of tastes would have an effect on the strength of the aversion. Rats in the second experiment were exposed to the full array of tastes as well, but during the preconditioning stage. During three habituation sessions, the rats were presented with the full battery of tastes through the IOCs while the control rats received only water. Following conditioning, we saw a significant decrease in sucrose consumption when the animals received the full taste array compared to the animals that received only water. The strength of the aversion to sucrose was much higher followed by experience with citric acid and NaCl, implying that previous experiences enhance future associative learning, which we called the diverse pre-conditioning exposure effect, or DPE. Manipulating DPE Having established that DPE results in stronger aversive associations, the next question to investigate was what manipulations in the paradigm were the result of the increase in magnitude.

6 If the results of the first experiment were accurate, then it stands to reason that enhancement of CTA should be linked to the amount of tastes that are experienced prior to conditioning. To test this, a block of experiments was conducted that varied the number of tastes given during the preconditioning stage: only 1 (water), 2 (water and NaCl), and 3 (water, NaCl, and citric acid). As expected, the magnitude of CTA to sucrose increased in a linear fashion with the addition of each taste in the presented array. The increase in aversive association supports the hypothesis that additional experience increases the strength of the aversion. If increasing the number of tastes experienced before aversive learning increases the the strength of CTA, then increasing the number of days of exposure to these tastes should likewise increase the magnitude of aversion. Rats were given the full taste array over the course of either 2 or 3 days of habituation. Once again, our hypothesis was borne out and the results showed that aversions were significantly stronger when the rats were given 3 days of exposure compared to those who only received 2. However, this raises the question of whether it is merely the number of days of exposure that leads to the increase in aversion magnitude rather than the diversity of tastes. To test this, rats were exposed to only water for either 2 or 3 days before being exposed to novel sucrose. Unsurprisingly, the results showed that there was no significant difference between having 2 or 3 days of water-only exposure on CTA magnitude, implying that the increased magnitude of aversion reflects an impact of experience with diversity of tastes and not just the number of days of exposure. Injection itself does not cause aversion Upon submission, the reviewers were curious to know whether our unconditioned stimulus, lithium chloride (LiCl, 0.3M) was causing the CTA to novel sucrose or whether it was merely the discomfort of receiving an injection after exposure. I conducted this control experiment independently to prove that the LiCl injection but not a saline injection would induce CTA. To simplify matters, that rats received only water through the IOCs for 3 days followed by conditioning to novel sucrose. Rats in the experimental group (n=7) received an injection of LiCl on conditioning day while rats in the control group (n=7) were given an injection of saline of identical volume. Due to lithium chloride s pervasive use in the literature as a malaise-inducing unconditioned stimulus, we were certain that we would find that LiCl does in fact induce CTA. This fact is reflected in our analysis which shows a significant decrease in sucrose consumption in the experimental group (p<0.05). From these results, it is quite clear that the aversion is induced through association of malaise induced by lithium chloride, not simply the injection. Influence of the method of taste exposure on DPE The reviewers were similarly unsatisfied with the method by which tastes were presented to the animals. Up to this point, we presented the rats with tastes through the IOC followed by 5 minutes of free exposure to a bottle. The two methods of taste exposure, IOC and bottle, are passive and active taste exposure respectively. The reviewers postulated that the ability for the rat to choose to drink freely from the bottle could be influencing the aversive learning. To determine whether or not our observed results would be attenuated if we removed the aspect of choice from the paradigm, we eliminated the period of free-drinking and presented taste solely through the IOCs.

7 The first control experiment necessary was to show that a conditioned taste aversion could still take place in the absence of bottle exposure. Evidence in the literature, as well as logical sense, implied that the aversion should still take place regardless of the delivery method of tastes. I conducted this experiment to prove that the removal of the free-access bottle would not impact the induction of CTA. All rats were exposed solely to water through the IOCs for 3 days of habituation, followed by exposure to novel sucrose. We found that our results were once again in line with our hypothesis and that the rats were still able to develop CTA without the free-drinking aspect of the paradigm (p<0.05 ). Having established that delivery method does not influence the development of CTA, our next line of inquiry regarded whether DPE would still be observed with only passive exposure to the full taste array. The rats received either the full array of tastes through the IOCs or water only during the habituation sessions leading up to exposure to novel sucrose. Based on the results previously seen in our experiments, we expected to see significantly stronger aversions in the rats passively exposed to the full taste array. As portrayed by the results, the rats that received the full taste array drank significantly less novel sucrose compared to those that received only water. These results imply strongly that DPE is still evident when the only taste exposure is passive. The results of both of the experiments I conducted can be seen in figure 1. Figure 1: The type of unconditioned stimulus dictates the amount of sucrose consumed during condition Incidence of DPE with less palatable tastes One final concern raised by the reviewers was that sucrose, of all taste modalities, is inherently the most palatable and most desirable of all tastes. The reviewers believed it was possible that this effect might not be as prominent with a less palatable, but still highly desired, taste. To test whether this hypothesis was true, we gave the rats a full battery of tastes during habituation that included citric acid, water, and sucrose rather than NaCl. NaCl acted as the conditioned stimulus in place of sucrose during the conditioning day. The results showed that DPE was generalizable to NaCl as well as sucrose, implying that DPE is not dependent on the sweet modality. Discussion In our experiments, our goal was to determine the effects of innocuous taste experience and the timing of exposure to these tastes on later conditioned taste aversion. Based on previous work with associative learning (McLaren and Mackintosh, 2002), we assumed that exposure to a

8 diverse set of stimuli and attempting to induce CTA to a novel taste simultaneously would result in the diverse taste array interfering with the aversive association, which was reflected in our results. We were surprised to find, however, that exposure to the tastes prior to the conditioning phase significantly increased the magnitude of the aversions. We also found that this effect was correlated with the number of tastes in the array presented as well as the number of exposure sessions that the animals received. Furthermore, this effect occurred whether the exposure to the tastes were presented passively through intraoral cannulae or actively through a bottle. These results strongly imply that even entirely innocuous taste experience influences how a novel taste will be responded to in future events of associative learning. The manner of experiments that we conducted bear resemblance to previous work regarding neophobia, which is the initial avoidance of a novel taste. The argument has been raised that exposure to a diverse array of tastes serves to attenuate neophobic behavior (Braveman and Jarvis, 1978), which aligns with the findings in the current work. However, in their paradigm they found that the CTAs remained intact but did not report any of the reinforcement of associative learning that we report here. A possible explanation for this difference is the strength of the US used to form the association. In our study, we used a weaker solution of LiCl than that used prolifically throughout the literature (Smith, 1968) to enable us to observe slight and subtle changes in the magnitude of the aversion. This weaker US has been used previously to induce weaker aversions than usually seen in the literature (Stone, Grimes, and Katz, 2005). Therefore, it is possible that the strength of the stimulus used for associative learning in previous studies prevented the alterations in aversion magnitude from being seen in analysis. If the pre-exposure to a diverse taste array did in fact reduce the magnitude of the neophobic response, we would expect the rats to be less wary of ingesting sucrose before conditioning and we should see a higher consumption volume. Upon further analysis, however, we observed no correlation between higher preconditioning sucrose consumption and the magnitude of the aversion, which lends support to our current results. Additional evidence can be found in previous work in our lab which shows that neophobia only occurs under very distinct circumstances (Monk, Rubin, Keene, Katz, 2014). Furthermore, it has been shown that neophobia does not always occur when a rat is exposed to novel sucrose (Franchina & Gilley, 1986; Miller & Holzman, 1981a, 1981b), leading us to believe that the effects we have seen in this paper are due to the experience gained with pre-exposure rather than an attenuation of neophobic behavior. Another possible explanation for these effects is an increase in the novelty of the CS due to recent induction of taste experience (Kutlu and Schmajuk, 2012). This is supported by the plethora of studies conducted on latent inhibition (Lubow, 1973; Lubow and More, 1959; McLaren and Mackintosh, 2002). During a latent inhibition trial, the animals would be exposed to a novel CS before the conditioning occurs, which later results in weaker aversions to the previously experienced CS. This weakened aversion most likely occurs due to the fact that a safe association has already been formed with the CS, hindering the re-association of the taste with a

9 negative outcome. If, however, this safe association has been made with several tastes in the battery of tastes experienced during pre-exposure, the novel taste may be brought into more eminent prominence, enabling it to be more associable and resulting in the observed increased magnitude in CTA. This hypothesis, while compelling, does not quite resonate with the observed results. The idea calls for the perceived novelty of sucrose to be extremely heightened, which would logically then increase, not decrease, the neophobic response. As mentioned before, previous studies (Capretta et al., 1975; Franchina & Gilley, 1986; Miller & Holzman, 1981b; Tarpy & McIntosh, 1977), as well as the results of our current study, seem to indicate the opposite to be true, implying that an increase in perceived novelty does not quite explain the DPE we observed. Future work is necessary to determine the neural underpinnings and mechanisms that ultimately cause the effect of pre-exposure that we observed in this paper. It is necessary to take into account the implications our results have on the current treatment of rats in other studies. We have shown here that previous experience with a diverse taste array strongly impacts future associative learning. Laboratory rats are almost exclusively raised naïve with the belief that having had little to no experience with tastes beforehand will remove the possibility of future confounding effects. However, rats are used as a transferable animal model to understand humans, which have a vast reservoir of gustatory experience to draw from. Our work may show that, to have a more successfully transferable model, it may be beneficial to provide all laboratory rats with gustatory experience before using them in experiments.

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