RESEARCH FEATURE: The Memory Trace of Addiction Raena E. Greenbaum, Steven J. Simmons, William R. Haury, Amelia J. Eisch

RESEARCH

THE MEMORY TRACE OF ADDICTION: HOW DRUG-

LINKED MEMORIES IMPACT EMOTIONALITY AND DRUG-SEEKING IN AN EXPERIMENTAL MODEL OF OPIOID ABUSE

Raena E. Greenbaum, Steven J. Simmons, William R. Haury, Amelia J. Eisch

Children’s Hospital of Philadelphia Research Institute; Abramson Pediatric Research Center; 3615 Civic Center Boulevard; Philadelphia, PA, USA.

ABSTRACT

While efforts have been made to interpret the emotional effects of drug-taking and drug-seeking in humans, there is currently a gap in research on these affective changes in animal models for opioid use disorders (OUDs). Indeed, one way to measure emotional changes in a model of OUD is to study ultrasonic vocalizations (USVs) from the drug-taking rat, as two main frequency ranges are interpreted to refl ect positive (50-kHz) and negative (22-kHz) affect. While used in studies on other drugs of abuse like cocaine, this study is the fi rst to use USVs as a measure in the self-administration of a prescription opioid and during recall of drug-linked memories. We put rats (n=66) through intravenous self-administration of the prescription opioid oxycodone by training them to press a lever for intravenous drug infusions during behavioral sessions. This was followed by an extinction of the drug-taking behavior in either the drug-linked context or a novel context. This paper examines the results of two studies. The fi rst was an analysis of emotionality over the course of the acquisition phase, which involved recording drug infusions earned, lever presses made, and USVs emitted by self-administering rats to characterize affective states associated with the development of addiction. We found that rats will experience greater negative affect early in acquisition, and greater positive affect in later acquisition sessions, and that a positive anticipatory affect will be experienced when the rats are exposed to the drug-paired context before the start of the session commences. The second study involved recording USVs emitted by rats during one single extinction probe test with different levels of drug-memory linkage to assess whether context and new action-outcome learning impact emotionality. The analysis of this extinction data is in progress, results of which hold important insights for understanding the neurobiology of drug-linked memory retrieval.

INTRODUCTION

There has been a sharp increase in the prescription rate of opioids due to their unmatched capability to relieve pain. However, their highly addictive qualities have led to severe consequences that have proven diffi cult to address. Roughly 21-29% of patients prescribed opioids for pain have reported misusing them (SAMHSA 2017), and 75% of heroin users reported that their fi rst opioid was a prescription drug (Cicero et al. 2014). Each day over 130 people in the United States die due to overdose on opioids (SAMHSA 2017) and as of October 16, 2017, the United States government declared the opioid epidemic a public health emergency.

It is clear that people do not take prescription opioids with the intention to become dependent; rather, the effect of the drugs includes intense emotional experiences that are craved after the high wanes. Because much is still unknown about the dynamic subjective experiences typifi ed in opioid addiction, preventing such dependence has proven to be a serious clinical challenge and is an important research focus in the face of the current epidemic.

When patients were given a list of prescription opioids and asked to identify the one most desirable to themselves, the one most desirable among drug-using communities, and the one they deemed most addictive, oxycodone was ranked most highly in all categories (Remillard et al. 2019). Due to its immense “likability” in users, oxycodone has one of the highest potentials for abuse and dependence compared with other prescription opioids. Subjective effects of the high of a drug like oxycodone may contribute to the extent to which the drug is sought after initial use and thus informs measures such as proclivity to opioid abuse as well as relapse propensity. Because relapse is one of the main challenges in combating addiction, an important next step in research is to understand the subjective effects of prescription opioid use.

Experimentally, opioid abuse disorder (OUD) can be modeled in rats by training them to “self-administer” intravenous oxycodone infusions following a behavior such as lever pressing as an operant response. We know that when given the opportunity, rats willingly and continually self-administer oxycodone (Bossert et al. 2018, Mavrikaki et al. 2017), which is known as the acquisition phase. Once drug is no longer available, as rats learn that a lever press will not lead to an infusion, a behavioral process described as extinction will occur. This refers to the elimination of the target response when its reinforcing consequence, in our case the oxycodone infusion, is no longer presented (Rey et al. 2020). The extinction occurs as a result of new action-outcome learning, which is the realization and learning that pressing the active lever will no longer lead to an infusion of oxycodone. Animal models are vital in the development of novel therapies to manage OUDs, but while efforts have been made to interpret the emotional effects of drug-taking in humans (Remillard et al. 2019), there is currently a gap in research on these affective changes in animal models for opioid use disorders (OUDs). One way to measure emotional changes in animals is to analyze the animals’ ultrasonic vocalizations (USVs) as has been performed in prior work (Burgdorf et al. 2000). In rats, USVs are emitted in two frequency ranges that are interpreted to refl ect positive (50-kHz; e.g., “tickling” [Panksepp and Burgdorf 2000], electrical stimulation of brain reward structures [Burgdorf et al. 2000]) and negative (22-kHz; e.g., electrical footshock [Tonoue et al. 1986], sight of predator [Blanchard et al. 1991]) mood states.

USVs have been used in several ways to study substance abuse, including the response to the presentation of drug-related cues, experimenter- and self-administered drug, drug withdrawal, and relapse tests (Barker et al. 2015). Emotionality in response to other drugs of abuse such as heroin, cocaine has been studied through the use of USVs (Avvisati et al. 2016, Simmons et al. 2018, Mahler et al. 2013), and other studies have demonstrated that rats become dependent on oxycodone in a self administration model of abuse (Bossert et al. 2018, Mavrikaki et al. 2017). Yet, oxycodone has never been a focus of acquisition and extinction USV studies, which is an essential gap in knowledge given oxycodone’s essential contribution to the opioid crisis in the US (CDC, 2017). Our study uniquely contributes an understanding of the emotionality behind oxycodone dependence which further drives opioid-taking behavior. We performed two studies to address these knowledge gaps in the fi eld by recording and analyzing USVs during drug acquisition and an extinction probe trial. Study 1 will add important information by characterizing the subjective effects of acquisition of oxycodone dependence in a self-administration animal model of abuse. Through Study 1, we had four main goals. The fi rst was to compare positive and negative affect in early acquisition (day 1), the second was to compare positive and negative affect later in acquisition (days 7 and 13), the third was to examined a positive anticipatory affect before later sessions (days 7 and 13) commenced, and the fourth was to check for the presence of a correlation between the number of 50kHz calls and total number of infusions. Study 1 holds important insights for creating effective preventative screening methods for OUD as well as clinical treatments for diminishing opioid dependence and protecting against relapse.

Study 2 will test how drug linked memories impact emotionality during the extinction of self-administration behavior by recording and analyzing USVs during a single-session extinction probe test, where drug is not available. Through Study 2, we had two main goals. The fi rst was to examine the emotional experience of undergoing extinction in the drugpaired context compared to a novel context. The second was to examine the emotional effect of new action-outcome learning, represented by the pressing of the previously active lever not leading to a drug infusion. This study holds important implications for understanding the neurobiology of drug-linked memory retrieval in order to develop mechanisms targeted to mitigate the salience of relapse-promoting druglinked memories.

METHODS

Animals

The subjects were male Long-Evans rats (N=66) (Charles River Laboratories; Wilmington, MA, USA) aged 6-8 weeks upon arrival. Rats were kept on a 12h:12h light cycle (lights on at 7:00AM), with humidity and temperature controlled, and access to food and water ad libitum. Rats were initially pair-housed and given 5-7 days to acclimate before receiving jugular vein catheterization surgeries. After surgery, rats were switched to single housing. Two groups of rats were used for these studies, Study 1 rats (n=14; Eisch Lab designation M2.1&3.1) were used for Acquisition data, Study 2 rats (n=52, Eisch Lab designation M3.1, 3.2, 4.1, 4.2, 8.1) were used for Extinction probe trial data. All procedures were approved by the Institu-

Surgery

A catheter (SAI Infusion Technologies) was surgically implanted into each rat’s right external jugular vein. Each catheter was connected to a dorsal stainless steel exit port. The rats were initially anesthetized with isofl urane gas anesthesia (5% induction, 1-3% maintenance) per the Anesthesia and Analgesia guidelines of Institutional Animal Care and Use Committee (IACUC) at a fl ow rate of 1.2 – 1.5 L/ min mixed with O2. Meloxicam (2 mg/kg, 10 mL/kg, s.c.) was used as a preoperative analgesic. The rat’s dorsal neck area and underarm were shaved and sterilized using alcohol and iodine wipes. Then, with the rat lying on its ventral surface, an incision was made on the rat’s mid-scapular region using stainless steel surgical scissors. Blunted scissors were used to separate connective tissue. Then, the rat was fl ipped onto its dorsal surface, and an incision was made along the underarm area. The catheter tubing was pushed under the skin from the exit port at the dorsal insincision until reaching the jugular vein, where a needle (22-gauge) was used to puncture the vein and the end of the catheter was inserted. Wounds were closed using surgical staples (9-mm, Braintree Scientifi c), and rats were given 8-10 days to recover before beginning behavioral sessions.

Self-administration

All rats (both Studies 1&2) were taught to self administer oxycodone. Intravenous oxycodone self-administration sessions were conducted in operant chambers (MED Associates) consisting of a plexiglas enclosure (29.53 H cm x 24.84 W cm x 18.67 H cm) with metal bars as fl ooring. Inside, there are two levers (one retractable, one stationary), a house light, cue lights above the levers, a fan, an automated syringe pump, and an auditory tone generator (Sonalert). Outside, syringes containing oxycodone solution are connected to tubing run through a metal spring leash for protection against chewing, which attach to the catheter exit ports on the back of the rats. The retractable lever was used as the active lever and the stationary lever was used as the inactive lever. All elements are controlled by MED-IV software (Med Associates; Fairfax, VT, USA) on a desktop computer next to the chambers. The physical context of the chamber used for acquisition was metal bar fl ooring, house light on, fan on, and a start time of 9:00AM. At the start and end of each session, a saline fl ushing solution containing heparin and enrofl oxacin (~0.1 mL) was infused through the catheter of each rat to maintain catheter patency. All rats acquired self-administration behavior for 3 hours sessions, 6 days per week for 18 days, similar to a prior study (Bossert et al. 2018). Catheter exit ports attached by PE-50 tubing to the oxycodone-dispensing syringe. Each active lever (retractable) press would result in a dose of oxycodone (0.10 mg/kg/inf during acquisition days 1-12, 0.05 mg/kg/inf during acquisition days 13-18), delivered over 3 s, paired with a ~7-kHz auditory tone. Directly after the infusion the active lever would retract for 20 s to prevent overdose. Presses on the inactive lever resulted in no drug infusion but were recorded.

Extinction Probe Test

The extinction probe test used in Study 2 was one single hour-long session, occurring in the same operant chambers as acquisition. A new cohort of rats (Cohort 2) was used that had also underwent the same self-administration procedure. Two different contexts (A and B) were used, half the rats acquired drug in each. Context A included stainless steel bar fl ooring, a house light, fan on, and start time was 9:00am. Context B included solid plexiglass fl oors, above-lever lights, fan off, and start was time 2:00pm. Half the rats extinction probe test in the same context in which they acquired drug (context sequence of AA or BB), and half experienced the test in the novel context (context sequence of AB or BA). Rats assigned to the drug-context group had their catheters tethered to the original tubing, whereas rats assigned to the novel-context group did not.

Ultrasonic Vocalizations (USVs)

Condenser microphones (UltraMic200K; Dodotronic; Italy) were placed on top of the plexiglass, facing down into the operant chambers. USVs were recorded at a sampling rate of 192-kHz (twice the maximum frequency detectable by the microphone). During acquisition, USVs of Cohort 1 were recorded of the entire 3 hour session once during week 1 (day 1 or 2 - referred to as “day 1”), once during week 2 (day 7 or 8 - referred to as “day 7”), and once during week 3 (day 13, 14, or 15 - referred to as “day 13”). USV recording began 5-10 min prior to the start of the self-administration session to constitute an “anticipation” time epoch. During the extinction probe trial, USVs of Cohort 2 were recorded during the duration of the one hour session.

USVs were analyzed using RavenPro (Cornell Laboratory of Ornithology) sound analysis software program which creates spectrograms of audio recordings

for manual scoring of USVs and provides details such as frequency range, duration, and power of each manually scored USV. USVs greater than 15 ms in duration were included in analysis. USVs with a mean frequency from 18- to 32-kHz were considered “22-kHz calls, while USVs with a mean frequency of 38 kHz or greater were considered “50-kHz calls”. “50-kHz calls” that took place within the fi rst 10 minutes of each session were considered “anticipatory calls”.

Statistics

All data were analyzed using Prism 8 software. Behavioral data (infusions, active presses, inactive presses) were initially analyzed using a repeated-measure mixed effects linear model to test for a main effect of “Session”, reported in F values. Study 1 (Acquisition) dependent variables were: Time (Self Administration, D1-D18); Time within Session (Self Administration, D1, 7, 13); Time (USVs, D1, 7, 13). If a main effect was detected, Sidak-adjusted post-hoc pairwise comparisons proceeded against the respective Day 1 value, reported in t values. Quartile analyses of within-session infusion data compared four 45 minute time bins on session days 1, 7, and 13. USV data were non-parametric, violating the assumption of normality. Thus, we used repeated-measures Friedman tests, reported in �2 values, followed by post hoc pairwise comparisons, reported in Z values. Familywise alpha (Type I error) was established at 0.05 for all analyses. Study 2 (Extinction) dependent variables were: Time (Self Administration, D1-D18), Context (USVs, DrugCx Vs. NovelCx), and Lever Availability (USVs, Lvr Vs.NoLvr). Statistic analysis of Study 2 (the extinction data) is in progress.

RESULTS

STUDY 1: Infusion and lever press data over the course of acquisition

As seen in past studies (Bossert et al. 2018, Mavrikaki et al. 2017), we observed a main effect on all three measures of oxycodone self administration: infusions [F(2.359, 28.450) = 13.18, p < 0.0001], active lever presses [F(2.520, 30.38) = 11.21, p < 0.0001], and inactive lever presses [F(4.154, 49.84) = 2.868, p < 0.0309] earned during the 18 days of acquisition. The average number of infusions ranged from lowest 10.64 ± 1.868 (95% CI: 6.607, 14.86) on Day 2 to highest 42.45 ± 4.862 (95% CI: 31.62, 53.29) on Day 16. Post hoc pairwise comparisons showed a statistical difference in infusions earned on day 15-18 compared with day 1 (p < 0.05). [all t(12) > 2.50, p < 0.05] (Figure 2A). The 3 hour session of each acquisition day was then divided into 4 quartiles, each composed of 45 minute time bins of infusions to characterize drug taking over time within individual acquisition sessions which had not been shown in this model before. We found a main effect of quartile on infusions earned on all 3 days we looked at: day 1 [F(2.250, 27.00) = 4.330, p < 0.0200], day 7 [F(1.738, 20.85) = 2.998, p < 0.0778], and day 13 [F(2.263, 29.42) = 4.904, p < 0.0118]. On day 1, post hoc pairwise comparisons showed a statistical difference in infusions earned in quartile 1 and quartile 3 (p < 0.05), [t(12) > 2.480, p > 0.0171]. On day 7, post hoc pairwise comparisons showed a statistical difference in infusions earned in quartile 1 and quartile 3, (p < 0.05), [t(12) > 0.7482, p < 0.0319] and quartile 2 and quartile 3, (p < 0.05), [t(12) > 4.464, p < 0.0361]. On day 13, post hoc pairwise comparisons showed a statistical difference in infusions earned in quartile 1 and quartile 2, (p < 0.05), [t(13) > 4.896, p < 0.0193] and quartile 2 and quartile 3 (p < 0.05), [t(13) > 4.734, p < 0.0237] (Figure 2B).

STUDY 1: USV data at 3 timepoints during acquisition

When examining the effect of session number on number and type of USVs produced, there was a main effect observed of session on 50-kHz USVs [�2(24) = 8.000, p = 0.0183] (Figure 3A), and of session on 22kHz USVs [�2(24) = 13.000, p = 0.0015] (Figure 3B). Post hoc pairwise comparison showed that there were signifi cantly more 50-kHz USVs detected on day 7 compared to day 1 [Z =2.746, p = 0.0181] (Figure 3A), and signifi cantly more 22-kHz USVs detected on day 7 compared to day 1 [Z = 0.1890, p = >0.9999] and day 13 compared to day 1 [Z = 3.213, p = 0.0039] (Figure 3B). The mean value for 50-kHz USVs across all rats for day 1 was 186.2±64.35, for day 7 was 461.8±115.4 with a standard deviation of 416.2, and for day 13 was 399.6±106.2 (Figure 3A). The mean value for 22-kHz USVs across all rats for day 1 was 57.25±9.850 with a standard deviation of 36.86, for day 7 was 65.79±12.75 with a standard deviation of 47.71, and for day 13 was 21.69±6.018 with a standard deviation of 22.52 (Figure 3B).

There was also a phenomenon of anticipatory USVs seen beginning after the fi rst day of acquisition, characterized by 50-kHz calls made during the fi rst 5 minutes the rats are placed in the chambers but before the lever extends and drug becomes available. On day 1, we see very few anticipatory USVs, with a mean of only 31.00±17.03 with a standard deviation of 35.04. On day 7, this increases to a mean of 242.1±51.36 with a standard deviation of 39.5. On day 13, the mean

number of anticipatory calls remains similar to day 7 at 202.0±73.73 with a standard deviation of 195.1. When looking for overall effect, we see that session number does have a main effect on number of anticipatory USVs [�2(24) = 10.29, p = 0.0036]. Post hoc pairwise comparison showed that there were signifi cantly more anticipatory USVs detected on day 7 compared to day 1 [Z = 3.207, p = 0.0040] (Figure 3C).

STUDY 1: Correlation between USVs and number of infusions

From a scatter plot of number of infusions vs. number of USVs, no signifi cant effect was observed from the linear regression for either 50-kHz calls (P = 0.2572, r = 0.3390, 95% CI -0.2607, 0.7499)(Figure 4A) or 22-kHz calls (P = 0.1771, r = 0.3825, 95% CI -0.1858, 0.7590) (Figure 4B).

STUDY 2: Extinction probe test 50 kHz and 22 kHz USV data

Analysis of extinction probe test data remains in progress (Figure 7A&B).

DISCUSSION

STUDY 1: Lever Presses and Infusions

These data indicate that there is an increase in drug seeking-behavior in rats over the course of oxycodone self-administration acquisition, as the rats are clearly differentiating between the active and inactive levers as one being drug-linked and the other not. The signifi cant difference between infusions earned on the fi rst day compared with later days indicates that the acquisition was successful and that the rats are taking substantially more drug after 3 weeks of self-administration. These fi ndings are consistent with prior work with IV self administration of oxycodone (Mavrikaki et al. 2017, Bossert et al. 2019). Additionally, we found that there is a distinct pattern of drug taking within the individual sessions (Figure 2B-E). The signifi cantly higher number infusions are earned in quartile 1 compared to later quartiles indicates the presence of a “load up” period, where the rats take a large number of infusions, and then are more satiated and have a lower, more consistent level of drug seeking throughout the session - just enough to “maintain” the high. This pattern has been consistently observed with cocaine self-administration in rodents (Ettenberg et al. 1982, Wise et al. 1995), and is characterized by “an initial burst of drug intake (loading) followed by more stable infusion rates (maintenance)” (Angarita et al. 2009). This phenomenon was not observed for cocaine use in humans (Angarita et al. 2009), and future

STUDY 1: Ultrasonic Vocalizations (USVs)

To my knowledge, this is the fi rst study to assess and characterize the subjective effects of acquisition of oxycodone self-administration by recording and analyzing USVs. Other studies have been done examining USVs with other drugs of abuse, such as heroin (Avvisati et al. 2016), cocaine (Avvisati et al. 2016, Simmons et al. 2018), methamphetamine (Mahler et al. 2013), and bath salts (Simmons et al. 2018). Our study adds to this subset of knowledge by examining USVs with oxycodone addiction, which is an essential gap in knowledge given that oxycodone is one of the most commonly prescribed opioids in the U.S. and one of the most common drugs involved in prescription opioid overdose deaths (CDC, 2017). Through Study 1, we aimed to test four main hypotheses. First, we expected that rats to experience greater negative affect early in acquisition, when fi rst exposed to the experience of oxycodone. Second, we expected to see increased positive affect over the course of acquisition as the operant self-administration behavior is developed and learned. Third, we expected to see an even further positive anticipatory affect when exposed to the drug-paired context before the start of the session commences. More specifi cally, we hypothesized that predominantly 22-kHz USVs will be observed during day 1, followed by predominantly 50-kHz USVs during the rest of intravenous oxycodone infusions, and that anticipatory 50-kHz USVs will be observed in week 2 and most robustly week 3. Fourth, we expected to see a correlation between the number of 50-kHz calls and total number of infusions, which would inform questions about the correlation between the initial affective experience of taking opioids and proclivity for abuse.

Our data support our fi rst two hypotheses that rats will experience greater negative affect early in acquisition, and greater positive affect as the acquisition sessions go on. The main effect observed of session on USVs and session-session comparisons indicates that negative affect, represented by 22-kHz USVs is most prevalent on day 1 and day 7 and decreases in the later sessions, while positive affect, represented by 50-kHz USVs, is low on day 1 and increases after the initial session. When fi rst exposed to the experience of oxycodone, the rats are more likely to have an adverse reaction compared to when they are familiar with the effects and the operant self-administration behavior is developed and solidifi ed. This is an inter-

esting result, as typically 22-kHz USVs are known to emerge during drug withdrawal in models of self-administration (Barker et al. 2015), but are not thought of as being an integral part of acquisition as well. A similar result of some initial negative affect early in acquisition followed by increasing positive affect has been shown in the self-administration of methamphetamine in rodents (Mahler et al. 2013).

Our data also support our third hypothesis, that a positive anticipatory affect will be experienced when the rats are exposed to the drug-paired context before the start of the session commences. On day 1 there are almost no anticipatory 50-kHz calls, while there is a high number of anticipatory calls in later acquisition sessions due to the context serving as a conditioned stimulus to elicit the positive affect before drug is even presented. These fi ndings show that anticipation of oxycodone taking elicits positive affective reactions due to learned associations with drug-paired contexts, which is different from what was found when a similar study was done with cocaine, in which both positive and negative affective anticipatory responses were observed when exposed to the drug-paired chamber (Coffey et al. 2013).

In contrast, our data did not support our last hypothesis, that we expected to see a correlation between number of 50 kHz calls and total number of infusions. This indicates that whether an individual animal is a particularly high caller or low caller does not necessarily have a relationship with the amount of drug they take. It remains unclear whether this is due to individual differences in call rate or individual differences in affective experience.

Going forward, it would be interesting to use similar data to compare USVs/emotional state during drug use relative to USVs emitted during tests of relapse, as such studies hold important insights for creating clinical treatments for diminishing opioid dependence and protecting against relapse. Additionally, it would be interesting to look further at what the difference in call rate between animals means, and whether different levels of sensitization to the drug can be represented by USV rates, and if so, how different levels of sensitization impact acquisition, extinction, and relapse rates. I believe that such fi ndings may hold important insights for creating preventative screening methods for OUD, as well as clinical treatments for diminishing opioid dependence and protecting against relapse A reasonable criticism of Study 1 is that it does not address the effect of the drug, given that all rats took oxycodone, and were not compared to a saline-taking control group. However, studies have already shown that cocaine self-administration induces more 50kHz USVs than saline self administering (Barker et al. 2015), and we know that rats self-administering cocaine show a similar acquisition curve to that of oxycodone. Because of this, we decided to focus on a more novel concept: the patterns of emotionality over time throughout the acquisition period. Future studies comparing the USVs of oxycodone-self administering rats and saline self-administering rats are welcomed and needed to address this gap.

STUDY 2: Lever Presses and Infusions

Incorporating lever presses and infusion data in Study 2 aimed to demonstrate and confi rm that rats that underwent the extinction probe trial had acquired self administration behavior as shown in Study 1 and in past studies with Oxycodone (Bossert et al. 2018, Mavrikaki et al. 2017). These data once again indicate that there is an increase in drug seeking-behavior in rats over the course of oxycodone self-administration acquisition, as the rats are clearly differentiating between the active and inactive levers as one being drug-linked and the other not. This is important for the new action-outcome learning we sought to examine through comparing USV data in the presence vs. absence of the active lever. Our fi ndings indicate that the acquisition was successful and that the rats were taking substantially more drug after 3 weeks of self-administration, once again consistent with prior work (Mavrikaki et al. 2017, Bossert et al. 2019).

STUDY 2: Ultrasonic Vocalizations (USVs)

While the extinction USV data analyses are still in progress, it is instructive to think about what the possible outcomes mean for our study. Through Study 2, we aimed to test two main hypotheses. Our fi rst hypothesis was that we expected that extinction in the drug-paired context will also have an increased emotional experience compared to extinction in a novel context, and expected to see a higher number of both 50 kHz and 22 kHz USVs. It is clear that the physiological effects of drugs of abuse can become paired with contextual information, contributing to continued future drug-seeking behavior (Le Foll et al. 2006, Tropea et al. 2008, Kutlu and Gould 2016). In humans, we know that relapse to non-medical use of prescription opioids often occurs after exposure to places previously associated with drug use and it is more challenging to wean off an addiction when constantly exposed to those places (Wikler 1973, O’Brien et al. 1992). In rats, many studies have demonstrated

context-induced relapse using a contextual renewal paradigm which involves a reinstatement probe trial in the acquisition context after extinction in a druglinked or alternate context (ABA vs AAA contextual sequence) (Bouton and Bolles 1979, Crombag and Shaham 2002, Crombag et al. 2008). Thus, investigating the emotional effect of undergoing extinction in the drug-paired context compared to a novel context holds important insights for understanding the neurobiology behind this behavior.

Behaviors measured during drug self-administration paradigms such as lever presses have been shown to depend on interactions between several interconnected brain regions. Given that the hippocampus is the primary brain region associated with learning and memory, the perforant path from the entorhinal cortex to the dentate gyrus has garnered a growing body of evidence implicating these areas in coding information related to reward and generating contextual memories of drug-induced reward (Guo et al. 2016, Hernández-Rabaza et al. 2008). Our preliminary data (Figure 7A) suggests that DrugCx rats may produce more 50k kHz calls (positive affect) compared to NovelCx rats. Our interpretation of this result so far is that drug-linked contextual memory retrieval engages the dentate gyrus and associated brain areas to drive a positive anticipatory subjective state and promote drug-seeking behavior. Future research will explore how biochemical and molecular adaptations in these brain regions might play important roles in the behavioral manifestations of emotional affect and drug taking.

Our second hypothesis was that that new action-outcome learning, represented by the pressing of the previously active lever not leading to a drug infusion, would have negative emotional affect. We predicted that the presence of the active lever would thus lead to diminished 50 kHz USVs and increased 22kHz USVs, compared to when the previously active lever is not present at all and the new action-outcome learning never occurs. In humans, studies on the role of action-outcome learning on affect indicate that positive events following a specifi c response are represented as desired outcomes by showing that anticipating positive events triggers the associated response. On the other hand, when that learned association is challenged, negative affect is elicited. We know that rodents, when given the opportunity, will be able to learn and form new action-outcome relationships, although the behavioral affect of such learning is yet to be explored (Laurent and Balleine 2015). Several brain structures have been implicated in the memory encoding of action–outcome learning, and studies have shown that the perforant path once again has ties to such behavior as part of an extended circuitry critical for maintaining habitual responding and responding to new stimuli (Corbit et al. 2002, Lex and Hauber 2010). Our preliminary data suggests that new action-outcome learning may lead to increased negative emotional affect, as seen by a possible increase in 22 kHz USVs with the lever present (Figure 7B). We suspect that the extinguishing of the memory association of drug-linked lever and the reward engages the dentate gyrus to drive a negative affective state, which would in turn cause a decline in drug-seeking behavior in response to the new learning. Future research would involve examining the explicit role of the perforant path in promoting this negative effect in order to understand the precise neurological mechanisms behind emotionality in drug-seeking behavior and how such pathways can be modifi ed to diminish these tendencies and protect against relapse.

CONCLUSION

Study 1 data collection, analysis, fi gure creation, and drafting components of the manuscript were completed during BIBB 399. Study 2 data collection, fi gure creation, discussions, and completion and fi nalization of the manuscript were completed during BIBB 499. Recommendations for future work on the Study 2 extinction USV dataset (Figures 7A&B) involves the statistical analysis of this data to confi rm or deny my given hypotheses. This includes a normality and lognormality tests followed by two-way ANOVA without a repeated measure (given that data are normal) due to the presence of 2 variables (Context and Lever). Then, a post-hoc analysis is required consisting of either a Bonferroni or Tukey test. If signifi cance is found in the interaction, the uncorrected Fisher test should be as a post-hoc analysis. If data are not normal in any test, a non-parametric test such as a rankbased test should be used.

ACKNOWLEDGEMENTS

I would like to thank everyone in the Eisch lab for making BBB499 a great experience. Special thanks to Dr. Steven Simmons, my mentor, and Dr. Amelia Eisch, my PI, for all of their help and support in bringing this project to fruition. I am so appreciative for all that I am learning, and am grateful to be a part of the research being done in the lab.

Figure 1. Study 1: experimental timeline. A er arrival and catheterization surgery, oxycodone self-administration training consisted of 18 3-hour sessions, 6 days per week for 3 weeks. During the rst 2 weeks (12 sessions), lever pressing at the active lever under a xed ratio 1 (FR-1) of response was reinforced by 0.10 mg/ kg/infusion of oxycodone. en, for the remaining 6 sessions, FR-1 was reinforced by 0.10 mg/kg/infusion.

Figure 2. Study 1: Infusion and lever press data over the course of acquisition. (A) Number of infusions (black triangles), active lever presses (black circles), and inactive lever presses (open circles) over 18 days of acquisition of intravenous oxycodone self-administration in male rats. Black solid arrows indicate days of ultrasonic vocalization (USV) recordings. Gray dashed arrows indicate oxycodone concentration change. Plotted data are means. N=14. (B) Average number of infusions divided into 4 time bins (0-45 min, 45-90 min, 90-135 min, and 135-180 min) for days 1, 7 and 13 of acquisition. (C-E) Time of each oxycodone infusion over the 180 minute session on days 1, 7, and 13 for each rat in Mischief 2 and 3. Name of individual rat represented by mischief number, cohort number, and box number.

Figure 3. Study 1: USV data at 3 timepoints during acquisition. (A) Number of 50 kHz (N=14), (B) 22 kHz (N=14), and (C) anticipatory 50 kHz (N=7) USVs emitted by individual rats during acquisition day 1 (circles), 7 (squares) and 13 (triangles). Means ± standard error are shown. ** indicates a signi cant di erence (p ≤ 0.05), * indicates a signi cant di erence (p ≤ 0.0015) in a One-Way ANOVA Friedman test.

Figure 4. Study 1: Correlation between 50 kHz calls and infusion #. Scatterplots of number of infusions vs. number of (A) 50-kHz calls and (B) 22-kHz calls on day 1 of acquisition. Each fi lled circle represents an individual animal (n=14).

Figure 5. Study 2: experimental timeline and groups. (A) After arrival and catheterization surgery, oxycodone self-administration training consisted of 18 3-hour sessions, 6 days per week for 3 weeks. During the fi rst 2 weeks (12 sessions), lever pressing at the active lever under a fi xed ratio 1 (FR-1) of response was reinforced by 0.10 mg/kg/infusion of oxycodone. Then, for the remaining 6 sessions, FR-1 was reinforced by 0.10 mg/kg/infusion. Directly following the 18 days of acquisition, the extinction probe trial consisted of a 1 hour single-session event during which no infusions were given. (B) During the extinction probe test, the active lever was either available or unavailable (Lvr Vs. NoLvr), and rats were either placed in the same context in which they acquired drug or a novel context (DrugCx Vs. NovelCx). In total, there were 4 experimental groups: DrugCx-Lvr, DrugCx-NoLvr, NovelCx-Lvr, NovelCx-NoLvr. (C) To suffi ciently differentiate between the two contexts, context A included stainless steel bar fl ooring, a house light, fan on, and start time was 9:00am. Context B included solid plexiglass fl oors, above-lever lights, fan off, and start was time 2:00pm. (D) Rats were divided up into two groups and acquired self-administration in either context A or context B. Rats in the DrugCx group extinguished in the same context that they acquired in (context sequence of AA or BB), rats in the Novel Cx group extinguished in the alternate context (context sequence of AB or BA).

Figure 6. Study 2: Infusion and lever press data over the course of acquisition. Number of infusions (open triangles), active lever presses (black circles), and inactive lever presses (open circles) over 18 days of acquisition of intravenous oxycodone self-administration in male rats. Gray arrows indicate oxycodone concentration change. Plotted data are means. N=66.

Figure 7. Study 2: Extinction probe test 50 kHz and 22 kHz USV data (A) Number of 50 kHz (N=66), (B) 22 kHz (N=66) USVs emitted by individual rats during the extinction probe test (equivalent to extinction day 1) in each context sequence with the active lever available (Open Circles) or unavailable (black squares). Means ± standard error are shown.

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Contributions: Study conception and methodology: Simmons, Eisch. Investigation: Greenbaum, Simmons. Data acquisition: Greenbaum, Simmons, Haury. Data visualization/presentation: Greenbaum. Writing of manuscript: Greenbaum. Supervision and critical review: Simmons, Eisch. Keywords: Opioid, Oxycodone, Self-administration, Ultrasonic Vocalizations, Affect, Acquisition, Extinction, Memory, Context, Action-Outcome.

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