Ketamine and Phencyclidine Reward and Sensitization in Adult and Adolescent Rats

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CALIFORNIA STATE UNIVERSITY SAN MARCOS

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THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE

MASTER OF ARTS

PSYCHOLOGICAL SCIENCE

THESIS TITLE: KETAMINE AND PHENCYCLIDINE REWARD AND SENSITIZATION IN ADULT

AND ADOLESCENT RATS

AUTHOR: Talal Javed Zafar

DATE OF SUCCESSFUL DEFENSE: 12/05/2017

THE THESIS HAS BEEN ACCEPTED BY THE THESIS COMMITTEE IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS IN PSYCHOLOGICAL SCIENCE.

Running head: SENSITIZATION AND DISSOCIATIVES 1

Ketamine and Phencyclidine Reward and Sensitization in Adult and Adolescent Rats

Talal J. Zafar

California State University San Marcos

SENSITIZATION AND DISSOCIATIVES 2

Abstract

Dissociative drugs, a class that includes ketamine, phencyclidine and related drugs, are popularly abused, especially by teens and young adults in club and rave settings. Despite use by young people, little is known of the effects of these drugs in adolescents, and potential differences between adolescents and adults. The current thesis examined the effects of dissociatives in adolescents and adults in an animal model. Two behavioral responses, locomotor stimulation and ultrasonic vocalizations (USVs) were examined simultaneously following administration of ketamine or phencyclidine in adolescent and adult Sprague-Dawley rats. The effects of repeated administration were examined to determine if behavioral sensitization, a phenomenon that is characterized by an increase in the effect of a drug after repeated adminstration, occurs. Behavioral sensitization has been linked to drug addiction due to neural plasticity. USVs represent a new approach to measuring positive (reward) and negative (aversion) affective states. It was hypothesized that animals treated with ketamine or phencycline would show increases in locomotor activity and 50 kHz vocalizations, and that they will develop sensitization to both. Furthermore, it was hypothesized that adolescent animals would develop more rapid sensitization than adults, and the adolescents would show long-lasting effects of exposure to dissociatives when tested as adults. In Experiment 1, which examined ketamine, the first treatment induced a short-lived stimulant response that was greater in adolescents. Activity increased across days in adults and adolescents, reflecting the development of sensitization, with adolescents showing more rapid sensitization compared to adults. For 50 kHz USVs on Day 1 ketamine induced a short-lived response in adolescents, but not adults, and the adolescent group showed an upward trend in USVs across days, reflecting sensitization. In contrast, adults showed little evidence of sensitization to the USVs. Both adolescents and adults demonstrated persistent sensitization to locomotor behavior when tested 23 days later; only the adolescents showed persistent sensitization to USVs. In Experiment 2, which examined phencyclidine, the first treatment induced a stimulant response that was greater in adolescents. Locomotor activity increased across days for adults and adolescents, reflecting sensitization in both groups, with adolescents showing more rapid sensitization than adults. For 50 kHz USVs on Day 1 phencyclidine induced an increase in adolescents, but not adults. In contrast to our hypothesis USVs in adolescents decreased across days, reflecting the development of tolerance instead of sensitization. Both adolescents and adults demonstrated persistent sensitization in locomotor behavior. For USVs there was persistent tolerance in adolescents when tested 23 days later, but not in adults. The results demonstrate that adolescents are more sensitive to the stimulant and rewarding effects of dissociatives, and that changes following repeated use are dependent on the specific dissociative examined.

SENSITIZATION AND DISSOCIATIVES 3

Ketamine and Phencyclidine Reward and Sensitization in Adult and Adolescent Rats

Drug abuse is prevalent in the USA and around the world and is typically initiated in adolescence (Johnston, O’Malley, & Bachman, 2013; SAMHSA 2015). Adolescence is a transitional time between childhood and adulthood that is characterized by complex modifications of behavior, brain function and anatomy that mature at different ages. Therefore, the adolescent brain may be affected by drugs of abuse in ways that differ from the adult brain.

Exposure to drugs during the adolescent period may increase vulnerability to drug abuse and neuropsychiatric disorders because of the immature brain development (Anthony & Petronis

1995; Ellgren, Spano, & Hurd, 2007). Drug abuse in adolescence is a cause for concern. For instance, opioid drug abuse among adolescents has led to thousands of hospitalizations annually

(Unick, Rosenblum, Mars, & Ciccarone, 2013). Adolescent response to drugs of abuse is therefore a growing area of research (Spear & Brake, 1983; Belluzzi, Lee, Oliff, & Leslie, 2004;

Zakharova, Leoni, Kichko, & Izenwasser, 2009; Spear & Varlinskaya 2010; Wiley, & Spear,

2013).

An effective way to study the effects of drugs of abuse in a controlled scientifically rigorous manner is to utilize animal models. Human subjects have much variability that complicates research on drug responses. Moreover, conducting research on humans with drugs of abuse has ethical implications, particularly in adolescence. If the goal is to find out the long term effects of drugs on the brain, animal models are essential. Importantly, laboratory rats have behavioral, neurochemical, neuroanatomical and developmental parallels to humans, making them excellent tools for neuroscientific research (Lynch, Nicholson, Dance, Morgan, & Foley,

2010).

The main goal of this proposed study is to compare the effects of repeated administration of a class of drugs known as dissociatives (including ketamine and phencyclidine) in adolescents SENSITIZATION AND DISSOCIATIVES 4 and adults. Because brain development is incomplete during adolescence it is possible that introducing drugs during this stage of development will differ from adults. For this study, ketamine and phencyclidine were administered repeatedly to compare the changes that take place in adolescence and adulthood.

Ketamine and PCP

Ketamine and phencyclidine are known as dissociatives because of their distinct effects, including distortion of perception of sight and sound, an experience of separation between the environment and one’s self, and out of body experiences (Dillon, Copeland, & Jansen, 2003;

Jansen 2000). These drugs were developed initially as anesthetics, however their use has expanded (Jansen, 2000; Rudgley, 1998). Recreational use of ketamine started to increase in the

1970s and peaked in the 1990s when the drug became popular in the club and rave scene.

Common routes of administration for recreational use of ketamine are injection, snorting, smoking (powder added to tobacco or marijuana cigarettes), or swallowing (NIDA, 2015). At low doses, ketamine users claim that the drug is rewarding and has the ability to produce stimulation and excitation, feelings of euphoria, lucid intoxication, and increased empathy

(Dillon et al., 2003; Jansen, 2000; Jansen and Darracot-Cankovic, 2001). At higher doses people experience a sometimes frightening experience known as the K-hole, with distortion of space and time, derealization, depersonalization, dissociation, hallucinations, and near-death experiences

(Dillon et al., 2003; Muetzelfeldt et al., 2008; Stone & Pilowski, 2006).

PCP is also known recreationally as angel dust, sheet, synthetic marijuana and rocket fuel. Routes of administration for PCP are smoking in marijuana or tobacco cigarettes dipped in

PCP solution, snorting, or injecting the drug. Once widely used on college campuses during the SENSITIZATION AND DISSOCIATIVES 5

1970s PCP is now relatively rare among college populations and used chiefly in large cities and urban areas (Domino, 1991; Johnston, O’Malley, & Bachman (2014). Its effects are very similar to those of ketamine, but longer lasting.

Adolescence

Adolescence is a transitional time between childhood and adulthood that is characterized by complex changes in physiology and behavior. Compared to people of other ages, adolescents have higher sensation seeking, risk taking, and reckless behavior. Such behaviors are beneficial to the adolescent’s development of autonomy (Spear, 2000). One of the difficulties that arises from the increased sensation seeking and risk taking is that adolescents may experiment with drugs of abuse. For example, 15-20% of teenagers in the US and several European regions have used marijuana (Cannabis sativa) in the past month (Ellgren et al., 2007). In addition, more than

90% adult smokers first started smoking during adolescence (Faraday, Elliott, & Grunberg,

2001; U.S. Department of Health and Human Services [USDHHS], 2014).

The changes in behavior seen during adolescence are accompanied by changes in the brain that are thought to be responsible for the behavioral changes. These changes in brain anatomy and function peak at different ages. For instance, frontal lobe gray matter reaches its maximal volume around 11-12 years of age, temporal lobe cortical gray matter peaks around 16-

17 years, and parietal lobe cortical gray matter peaks between 10-12 years (Lenroot & Giedd,

2006; Spear, 2000). Additionally, myelenation occurs throughout the young adult’s life until roughly age 25 when myelin sheaths are fully developed (Lenroot et al., 2006). Adolescents go through marked changes in the brain and the normal trajectory of brain development could be affected if drugs of abuse are taken during this phase. SENSITIZATION AND DISSOCIATIVES 6

An area of the brain that has received considerable attention for developmental change is the prefrontal cortex, which is the last brain region to develop in its adult form (Lenroot et al.,

2006; Giedd, 2008; Kolb, Mychasiuka, Muhammad, Li, Frost & Gibb, 2012). The prefrontal cortex is associated with impulse control and sound decision-making skills (Lenroot et al., 2006;

Giedd, 2008; Kolb et al., 2012; Schramm-Sapyta, Walker, Caster, Levin, & Kuhn, 2009). The prefrontal cortex is not fully developed in adolescents and this underdeveloped prefrontal cortex is thought to contribute to the reduced impulse control that can lead to adolescents experimenting with alcohol and other drugs (Kolb et al., 2012).

Recent research has led to a better understanding of adolescence in laboratory animals, and the recognition that behaviors seen in humans can also be similarly observed in laboratory rats. In rats, adolescence begins around postnatal day 28 and the transition into adulthood around postnatal day 60 (Spear, 2000). To recall, human adolescents tend to engage in increased levels of sensation-seeking, risk-taking, and reckless behavior. As adolescents transition into adulthood, risk-taking behavior declines. Similarly, in adolescent rats compared to adult rats there is an increase in reward-seeking behavior (Anker, Zlebnik, Navin, & Carroll, 2011; Burton, Noble, &

Fletcher, 2011; Friemel, Spanagel, & Schneider, 2010; Shahbazi, Moffett, Williams, & Frantz,

2008; Zakharov et al., 2009). Futhermore, reseachers have demonstrated age differences in novelty-seeking behavior with adolescent rodents showing elevated novelty-seeking compared to their adult counterparts (Adriani, Chiarotti, & Laviola, 1998; Wooters, Dwoskin, & Bardo, 2006;

Stansfield, & Kirstein, 2006). Adolescent mice spend more time and have more activity in a novel environment compared to adult mice. As mice grow older their novelty-seeking behavior declines (Adriani et al., 1998; Wooters, et al., 2006). In addition to these behavioral changes, there are also changes in neurochemistry through adolescence, with different neurotransmitters SENSITIZATION AND DISSOCIATIVES 7 and neurotransmitter receptors maturing at different stages (Pinilla, Lee, & Cotman, 1994; Kolb et al., 2012).

With regard to drugs of abuse, adolescent rats self-administer more nicotine than adult rats (Levin, Rezvani, Montoya, Rose, & Swartzwelder, 2003; Belluzzi et al., 2004; Faraday,

Elliot, & Grunberg, 2001). A parallel to this in humans is that individuals are less likely to abuse drugs if they delay drug use until age 20 (Spear, 2000). Conversely, if drug use starts by age 14, there is a higher chance that the person will develop addiction in adulthood (Spear, 2000). There are lasting effects to drug exposure that occurs during adolescence (Spear, 2000).

Animal Models: Locomotor Activity and Behavioral Sensitization

Locomotor activity in laboratory rodents has long been regarded as a means for studying addictive substances, with increases in activity being seen in response to abused drugs across all classes (Wise & Bozarth, 1987). The Psychomotor Stimulant Theory of Addiction, which posits that addictive substances have the propensity to elicit psychomotor activity, has provided an animal model for studying reward by measuring increases in rodent locomotion (Wise et al.,

1987). Locomotor activity is a simple and straightforward behavior and it is widely used in the study of drugs of abuse, however it is not specific to reward so it is often supplemented with other approaches.

Behavioral sensitization is a phenomenon that is characterized by an increase in the effect of a drug after repeated adminstration and is often assessed in laboratory rodents via increases in the locomotor stimulant effects. Also referred to as “reverse tolerance,” sensitization is thought be involved in the development of addiction. According to this idea, repeated adminstration of a drug leads to an increase in the desire for the drug that contributes to addiction, beginning with wanting and escalating to craving (Robinson & Berridge, 1993). For instance, many addicts SENSITIZATION AND DISSOCIATIVES 8 crave a particular drug days, months, or years after abstaining from the drug. It is thought that this craving is a result of sensitization.

Locomotor sensitization arising from drugs of abuse occurs when the neural substrates associated with reward are altered (Robinson and Berridge, 2003, 2008). The area of the brain associated with this reward pathway is known as the mesocorticolimbic circuit. The principal structures in this circuit are the ventral tegmental area (VTA), the nucleus accumbens (NAcc), and the prefrontal cortex (Meyer & Quenzer, 2005). Among the drugs that have been shown to activate this neural circuit are nicotine, caffeine, ethanol, tetrahydrocannabinal (THC), cocaine, amphetamines, PCP, and ketamine (Koob, 1992; Wise et al., 1987).

In terms of dissociatives, locomotor research has shown that ketamine, phencyclidine and related dissociatives increase locomotor activity and the activity escalates with repeated administration (Castellani & Adams, 1981; Wolf, 1991; Xu & Domino, 1999; Uchihashi,

Kuribara, Morita, & Fujita, 1999; Trujillo, Zamora, & Warmoth, 2008). Thus, behavioral sensitization is evident with dissociatives, as it is with other drugs of abuse. Furthermore, locomotor sensitization to the dissociatives differs in adolescents and adults. Following the first administration adolescents show increased locomotor activity compared to adults. In addition, sensitization in adolescents persists into adulthood, demonstrating the long-lasting impact of the drug-induced changes (Bates & Trujillo, 2010; Zafar, Rocha, Escobedo, & Trujillo, 2016; see also preliminary results, below).

Animal Models: Ultrasonic Vocalization

The investigation of vocalization and its relation to affect in vertebrate species has a long history starting back when Charles Darwin was trying to understand emotions (Panksepp, and SENSITIZATION AND DISSOCIATIVES 9

Burgdorf, 2000). Some species, including laboratory rodents, can vocalize outside the range of human hearing. These ultrasonic vocalizations (USVs) have been found to reflect the affective state of the animals (Brudzynski, 2009). USVs used to assess affect in laboratory rats occur predominantly in two frequency ranges: 22 kHz and 50 kHz. The first, 22 kHz USVs, are emitted in distressing situations; for example, when a predator is detected, in response to pain or during intra-species conflict (Knutson et al., 2002). The other major type is 50 kHz calls, which occur predominantly during rewarding stimuli such as rough and tumble play in adolescents, during mating behavior and in response to drugs of abuse. They have been defined as “happy” calls (Knutson et al., 2002). To better understand the neurobiological basis of reward, aversion and other emotions, researchers wish to identify the positive and negative affective states of rats, including those induced by drugs of abuse. USVs appear to be an excellent tool for this task.

Drugs of abuse such as cocaine and amphetamines have been shown to elicit increased 50 kHz calls in a manner similar to natural rewards (Barker, Simmons, & West, 2015; Wright,

Gourdon, & Clarke, 2010; Simola et al., 2012). Furthermore, microinjections of amphetamine

(AMPH) into the nucleus accumbens (NAcc) of rats evokes 50 kHz calls (Burgdorf, Knutson,

Panksepp, & Ikemoto, 2001). In contrast, 22 kHz calls are emitted during withdrawal from cocaine, opiates or ethanol (Brudzynski, 2015; Mutschler, & Miczek, 1998). This demonstrates that rodents tend to have a natural unconditioned response to the effects of these compounds by producing 50 kHz USVs during drug reward and 22 kHz USVs during drug withdrawal.

Curiously, 50 kHz USVs do not appear to be evoked by all drugs of abuse, as MDMA, morphine, and nicotine do not produce 50 kHz calls even though these three drugs have rewarding properties (Barker et al., 2015; Brudzynski, 2015; Simola, 2015). The reasons are yet to be understood. SENSITIZATION AND DISSOCIATIVES 10

Most of the research on USVs and drugs of abuse has looked at the psychostimulants, with dissociatives largely ignored. Preliminary work in our laboratory demonstrates that at moderate doses of ketamine the animals elicit both 50 kHz and 22 kHz USVs, suggesting that the drug produces both rewarding and aversive effects (Gonzalez, Altamirano, Rocha, & Trujillo,

2015; Roos & Trujillo, 2013; Roos & Trujillo, 2014a). Further research in our laboratory demonstrates that 50 kHz USVs are potently increased after ketamine sensitization (Zafar et al.,

2016; see also preliminary results, below). Curiously PCP did not increase 50 kHz calls following a single injection, but we have not yet explored repeated injections (Roos & Trujillo,

2014). These findings are consistent with those of others who also found that PCP did not increase 50 kHz USVs (Boulay, Ho-Van, Bergis, Avenet, & Griebel, 2013). Further studies are needed to definitively determine whether dissociatives produce positive or negative affect as reflected by USVs, and whether sensitization to USVs develops with repeated administration.

The current thesis examined the effects of ketamine and phencyclidine, in locomotor behavior and ultrasonic vocalizations simultaneously, in adolescents and adults following repeated administration. It was hypothesized that animals treated with ketamine or phencycline would show increases in locomotor activity and 50 kHz vocalizations, and that they would develop sensitization in both paradigms. Furthermore, it was hypohtesized that adolescent animals would develop more rapid sensitization than adults, and the adolescents would show long-lasting effects of exposure to dissociatives when tested as adults.

Preliminary Study 1: Differences Between Adult and Adolescent Rats in Locomotor

Sensitization to Codeine SENSITIZATION AND DISSOCIATIVES 11

Introduction.

I began my research in the laboratory investigating differences between adolescents and adults in response to opioids. This work helped to establish protocols and pointed toward differences between adolescents and adults in the response to specific drugs of abuse following repeated administration.

Codeine (COD) is an opioid analgesic that is often used by teens for recreational purposes (Johnston et al., 2013). According to the US Drug Enforcement Administration, codeine mixtures sometimes referred to as “sizzurp” or “purple drank,” have been increasingly abused in the hip-hop culture (NDIC, 2011; DEA, 2012). Evidence suggests that abuse of COD can lead to compulsive use and abuse (Burns & Boyer, 2013). While there are reports of behavioral sensitization to opioids in adult rats, little has been done to elucidate sensitization in adolescents. Some experiments have been conducted and showed greater sensitization to opiates in adolescents compared to adults (White & Holtzman, 2005; Doherty & Frantz, 2013), however codeine has not been examined in this regard. The present study examined the locomotor effects of COD following repeated administration to adolescent and adult Sprague-Dawley rats. We hypothesized that COD would induce an increase in locomotor activity across days reflective of behavioral sensitization and that adolescent rats would differ in sensitization when compared to adults.

Methods.

Animals.

Adult (approximately 60 days of age) and adolescent (30 days of age) Sprague-Dawley rats were used in this experiment. Animals were habituated to the vivarium for one week prior to the start of the study. The animals were divided into four treatment groups (6 animals/group): SENSITIZATION AND DISSOCIATIVES 12

Codeine Adult (COD AD), Saline Adult (Sal AD), Codeine Adolescent (COD JV), and Saline

Adolescent (Sal JV).

Drugs.

Codeine hydrochloride (10.0 mg/kg; NIDA Drug Supply Program) drug was dissolved in

0.9% saline and was administered subcutaneously. This dose was selected based on previous work to avoid ceiling or floor effects allowing for the drug response to shift upward or downward with repeated administration. Saline was used for control injections in the same volume (1 ml/kg).

Apparatus.

A Kinder Scientific Open Field Motor Monitor System was used to assess locomotor activity. This system consists of eight Plexiglas enclosures (16” X 16” X 15”). Each enclosure has two arrays of photocells (16 in each dimension): one placed 5 cm above the floor (used to measure horizontal activity) and one placed 12.5 cm above the floor (to measure vertical rearing activity). The apparatus is interfaced with a personal computer for the collection of data. The computer has the ability to provide several different measures of locomotor behavior including photocell beam breaks, ambulations, fine movements, time-spent active, distance traveled, rearing and time spent rearing. Ambulations, which represent horizontal locomotion, were used to calculate activity.

Procedure.

Each animal was handled for five minutes each on the three days preceding the study.

During these days, the animals were also habituated to a mock injection but not injected. The animals received codeine or saline once daily, for seven (7) consecutive days. On each testing day during the study, the animals were weighed in the vivarium and transported in their home SENSITIZATION AND DISSOCIATIVES 13 cage with their cage mates to the testing room. The entire time the animals were in the testing room white noise was played to mask background noise. Animals were given 15 minutes habituation to the room, and then were individually placed in the locomotor apparatus to habituate for 30 minutes. Upon completion of the habituation phase, the animals were injected and locomotor activity was assessed for 180 minutes in order to assess their acute (day 1) effects of the drugs, and to determine if the drug treated animals developed sensitization (days 2-7).

Statistical Analyses.

Activity across days was analyzed using a two-way repeated measures ANOVA (group x day of treatment). Fisher’s LSD test was used to determine any pairwise differences post hoc. A probability of p < .05 was considered significant. To analyze development of sensitization across days in each group a within-subjects t-test was used comparing the first day of treatment to the final day of treatment.

Results.

On Day 1 of treatment, codeine (10.0 mg/kg) induced a short-lived stimulant effect that was not different in adolescents and adults. Activity escalated across days of treatment, reflecting the development of sensitization, and sensitization was more rapid in adolescents than adults.

Because the maximum stimulant effect was observed in the first 30 minutes post-injection, this timeframe was used for analysis. A two-way repeated measures ANOVA of activity across days revealed significant effect of treatment group, day of treatment and an interaction [Treatment

Group: F(3,15) = 41.11, p < .0001; Day: F(6,30) = 9.39, p < .0001; Interaction: F(18,90) = 9.90, p < .0001]. A Fisher’s LSD multiple comparison test revealed that the adolescent codeine group produced more locomotor activity than the adult codeine group and the adolescent saline group at multiple timepoints (Fig. 1). The adolescent group showed a significant increase in activity by SENSITIZATION AND DISSOCIATIVES 14 day 2 of treatment, however an increase in the adults was not seen until day 4. In addition, the codeine-induced activity was greater in adolescents than adults on days 2-5 of treatment (Fig 1).

Within subjects t-tests showed that there was a significant increase in activity for adolescents from day 1 to day 7, t(10) = 5.24, p = .0004 and for adults from day 1 to day 7, t(10) = 3.96, p =

.0027, reflecting the development of sensitization in both groups. )

M 3 0 0 0 A D C O D E J V C O D # S * # * # $ A D S A L

 J V S A L ( * # s 2 0 0 0 #

t * # # #$ n

u * # #

y 1 0 0 0

t c

A 0 1 2 3 4 5 6 7

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Figure 1. Locomotor activity across the 7 days of treatment during the first 30 minutes post- injection. Note the increases in activity across days, reflecting the development of sensitization. Values expressed as mean +/- SEM. * indicates significant differences between adolescent codeine and adult codeine groups. # indicates differences between the adolescent or adult codeine group and the respective saline control group. $ indicates significant increase from day 1 to day 7. Juvenile Codeine (JV COD) and Juvenile Saline (JV SAL) are adolescent groups.

Discussion.

The most important finding from this study is that codeine (COD) induces more rapid locomotor sensitization in adolescents than in adults. COD induced a small increase in locomotor activity on day 1 and activity escalated across days of treatment, reflecting the development of sensitization. Although there were only modest differences between adolescents and adults early in treatment, the adolescents escalated more rapidly than adults. These results are similar to work SENSITIZATION AND DISSOCIATIVES 15 on opioids from other labs, which also found that adolescents showed greater sensitization compared to adults (White et al., 2005; Doherty et al., 2013).

Sensitization is thought to be related to the development of addiction. The fact that sensitization occurred more rapidly in adolescents may help to explain why drug use in adolescence is more likely to lead to addiction (Crews et al., 2007). Further research will help to determine the factors involved in COD sensitization in adults and adolescents, and may lead to better prevention and treatment for COD abuse and addiction in young people. The results of this study, demonstrating differences between adolescents and adults in the acute and chronic effects of codeine, prompted us to examine other drugs of abuse.

Preliminary Study 2: Differences Between Adult and Adolescent Rats in to Ketamine:

Effects of Acute and Repeated Administration

Introduction.

As discussed above, little published research has explored the differences in response to dissociatives in adolescents and adults. Based on the preliminary results of the codeine study we wanted to extend the work to dissociatives to determine if differences between adults and adolescents would also been seen in this drug class. The present study examined the effects of repeated exposure to ketamine. Given earlier work by our laboratory (Trujillo et al., 2008) we predicted that repeated administration of the drug would produce progressively greater increases in response, reflective of sensitization. Furthermore, we predicted that adolescents would show a greater stimulant effect of the drug and greater sensitization than adults (Zafar et al., 2016; Bates et al., 2010). Finally, we predicted that ketamine would induce 50 kHz vocalizations in rats, and SENSITIZATION AND DISSOCIATIVES 16 that animals sensitized to the locomotor stimulant effects of ketamine as adolescents would show increased USVs in response to ketamine as adults.

Methods.

Animals.

Ketamine Adult (KET AD), Saline Adult (Sal AD), Ketamine Adolescent (KET JV), and Saline

Adolescent (Sal JV).

Drugs.

Ketamine hydrochloride (10.0 mg/kg; Sigma/Aldrich) was dissolved in 0.9% saline and was administered subcutaneously. Saline was used for control injections in the same volume (1 ml/kg).

Apparatus.

Locomotor Activity: A Kinder Scientific Open Field Motor Monitor System was used to assess locomotor activity. See above for description.

Sound Attenuating Chambers: For the recording of USVs 4 sound attenuating chambers (Med Associates) were used. Each chamber was equipped with sound-attenuating foam attached to the inside walls to minimize background noise and maximize clarity of USV recording. A single Avisoft microphone was hung 10 cm from the floor in the center ceiling of each chamber. On test days, animals were carted in standard plastic cages with fresh bedding and wire lids and each cage was placed into a sound-attenuating chamber for recording USVs. SENSITIZATION AND DISSOCIATIVES 17

Ultrasonic Vocalizations (USV) recording software: An Avisoft Ultrasoundgate 416H recording device and Avisoft SASLabpro software were used. The Avisoft SASLabpro software has the ability to create spectrograms of the recorded files.

Measurement:

Ultrasonic Vocalizations (USV):

Based on the literature, USVs were identified as fixed frequency 50 kHz, frequency modulated 50 kHz, and frequency modulated with trills 50 kHz, as well as short and long 22 kHz

(Brudzynski, 2009; Wright et al., 2010). Flat 22 kHz USVs were defined as falling within the range of 0-35 kHz with a generally flat or slightly sloping quality. Short flat 22kHz are short duration calls lasting between 0-300 ms. Long 22 kHz calls have longer duration of greater than

300 ms (although short and long 22 kHz USVs were assessed, since there were very few they were not included in the results). Fixed frequency 50 kHz USVs fall within the range of 35-70 kHz and are characterized by a flat or slightly sloping quality which ranges between -.2 kHz and

.2 kHz. Frequency modulation of the vocalizations also fall within the range of 35-70 kHz calls but usually have varying shapes such as inverted U-shape, a step up, step down (Wright et al.,

2010). The frequency modulated with trills 50 kHz vocalization category refers to all calls that fall within the 50 kHz range and are frequency modulated but also contain multiple modulations that have elongated wavelengths considered trills (Brudzynski, 2009; Wright et al., 2010).

Procedure.

On each of the seven days of treatment, following 15 minutes of habituation to the room and 15 minutes of habituation to the apparatus, the animals were injected with saline or ketamine

(10 mg/kg) and locomotor activity was assessed for 90 minutes in order to assess acute (day 1) SENSITIZATION AND DISSOCIATIVES 18 effects of the drugs, and to determine if the drug treated animals develop sensitization (day 2-7).

The animals were then given a washout period during which the adolescent animals developed into adulthood (at this point the former adolescents were 60 days of age and adults were 90 days of age). On this day all animals were given a challenge treatment of ketamine (10 mg/kg) to assess response in the ultrasonic vocalization paradigm. Animals were weighed in the vivarium, and taken to the recording room. They were then habituated for 15 minutes in the USV recording chambers and baseline USVs were recorded. After the 15-minute habituation animals were removed from the USV recording chambers and injected subcutaneously. They were then placed back into the USV recording chambers and USVs were recorded continuously for 60 minutes.

Animals were returned to their home cages following the conclusion of the study.

Statistical Analyses.

Ambulations across days were analyzed using a two-way repeated measures ANOVA

(group x day of treatment). Fisher’s LSD test was used to determine any pairwise differences post hoc. A probability of p < .05 was considered significant. To analyze development of sensitization across days in each group a within-subjects t-test was used comparing day 1 to day

7. To assess the long lasting effects of sensitization on USVs, 50 kHz USVs were scored, including fixed frequency, frequency modulated, and frequency modulated with trills. To determine whether there were differences among groups in the number of 50 kHz calls a one- way ANOVA was performed followed by a Fisher’s LSD multiple comparison post hoc test.

Results.

On Day 1 of treatment, ketamine (10.0 mg/kg) induced a short-lived stimulant effect that was considerably greater in adolescents than adults (Fig. 2A). Examination of the response to ketamine (Fig. 2A) revealed the maximum stimulant effect in the first 10 minutes post-injection, SENSITIZATION AND DISSOCIATIVES 19 therefore this timeframe was used for analysis across days. Activity escalated across days of treatment, reflecting the development of sensitization, and sensitization was greater in adolescents than adults (Fig. 2B). A two-way repeated measures ANOVA test revealed significant effect of treatment group, day of treatment and an interaction [Treatment Group:

F(3,15) = 42.82, p < .0001; Day: F(6,30) = 16.08, p < .0001; Interaction: F(18,90) = 7.92, p <

.0001] (Fig. 2B). A Fisher’s LSD multiple comparison test revealed that the adolescent ketamine group produced more locomotor activity than the adult ketamine group on days 2-7. In addition, the adult ketamine produced more locomotor activity than the adult saline group on days 2-7.

Within subjects t-tests showed that there was a significant increase in activity for adolescents from day 1 to day 7, t(10) = 4.85, p = .0007 and a significant increase in activity for adults from day 1 to day 7, t(10) = 4.52, p = .0017, reflecting the development of sensitization in both groups.

To examine the persistent effects of ketamine treatment, the USV response to ketamine was determined 23 days later, when the adolescent group reached adulthood (60 days of age) and the adult group was older (90 days of age). At this timepoint, animals that had been sensitized to ketamine showed greater ketamine-induced 50 kHz USVs than animals that had received repeated saline treatment, with the former adolescent group showing the greatest effect. A one- way ANOVA of total 50 kHz USVs revealed significant differences among groups, F(3,20) =

4.43, p =.0152 (Fig. 2C). A Fisher’s LSD multiple comparison test revealed that the ketamine- pretreated groups showed a greater response than their saline-pretreated counterparts, and the group that received repeated ketamine as adolescents produced significantly greater 50 kHz USV calls than animals that received repeated ketamine as adults, t(20) = 2.24, p = .036. SENSITIZATION AND DISSOCIATIVES 20

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Figure 2. A) Timecourse of activity on Day 1 of ketamine treatment. The adolescent ketamine- treated group shows increased activity compared to the adult ketamine-treated group. B) Total activity across 7 days of treatment during the first 10 minutes post-injection. Note the increases in activity across days, reflecting the development of sensitization. Values expressed as mean +/- SEM. * indicates significant differences between adolescent ketamine and adult ketamine groups. # indicates differences between the adolescent or adult ketamine group and the respective saline control group. $ indicates significant increase from day 1 to day 7. C) Total 50 kHz USVs in response to ketamine challenge after 23 days without treatment. Animals exposed to ketamine as adolescents showed the greatest response to ketamine. * indicates significant difference between adolescent ketamine and adult ketamine groups. # indicates differences between the adolescent or adult ketamine group and the respective saline control group. Juvenile Ketamine (JV KET) and Juvenile Saline (JV SAL) are adolescent groups.

Discussion.

As has been seen previously in our laboratory (Bates et al., 2010; Rocha, Hart, & Trujillo,

2017), adolescents showed an increased response to ketamine when compared to adults. SENSITIZATION AND DISSOCIATIVES 21

According to The Psychomotor Stimulant Theory of Addiction, rewarding effects of drugs are reflected in increased locomotor behavior (Wise et al., 1987). What this means in terms of our study is that adolescents may be experiencing greater reward than adults. Because of this, adolescents may be more attracted to ketamine than adults. This could help to explain the greater use of ketamine by young people when compared to older individuals.

In addition to increased locomotor behavior following the first injection, adolescents showed increased sensitization to ketamine. Both adolescents and adults had increased ketamine- induced stimulation across days of treatment, reflecting sensitization, but sensitization developed more rapidly and to a higher degree in the adolescent group. According to current theories, sensitization contributes to increases in desire for the drug, which escalates to compulsive craving (Robinson et al., 1993). For adolescents, what this means is that with repeated use they may experience more desire for the drug because of drug-induced changes in the brain.

To determine the long-term impact of exposure to ketamine, the animals were given a prolonged washout period during which the adolescent group developed into adults. We found in the ultrasonic vocalization paradigm that both adolescents and adults showed evidence of sensitization, but the response was far greater in adolescents. These results demonstrate that the rewarding effects of ketamine are enhanced by repeated use, and the effects are long-lasting. In the group exposed to ketamine as adolescents, rewarding effects were dramatically increased when the animals were challenged with ketamine as adults compared to the groups exposed to saline as adolescents. The increased rewarding response to ketamine in the ketamine pretreated group may help to explain the phenomenon of relapse, in which users fall back to problematic use, even after long-term abstinence (Robinson et al., 2008). SENSITIZATION AND DISSOCIATIVES 22

The fact that sensitization occurred more rapidly in adolescents may help to explain why drug use in adolescence is more likely to lead to addiction. To the extent that these findings can be applied to human adolescents, there are important implications. First, use of certain drugs at an earlier age can change the brain in a way that leads to more rapid development of addiction.

Second, these effects produce long term changes, which can increase the likelihood of later use.

Further research will help to determine the factors involved in ketamine sensitization in adults and adolescents, and may lead to better prevention and treatment for ketamine abuse and addiction in young people.

Thesis Studies: Differences Between Adult and Adolescent Rats in Locomotor and

Ultrasonic Vocalization Sensitization to Dissociatives

The current thesis is founded on preliminary experiments that showed that adolescents differ from adults in response to drugs of abuse, including codeine and ketamine. We were particularly interested in following up with the research on ketamine by refining the work and determining if the findings would extend to another dissociative drug. Strikingly, adolescents showed more rapid locomotor sensitization in response to repeated administration of ketamine than adults. In addition, sensitization in the adolescents persisted into adulthood. To expand on these findings, we chose to examine development of sensitization in locomotor activity and ultrasonic vocalizations simultaneously and compare adolescents and adults. This allowed us to follow the progression of changes in both behaviors over time at the two developmental stages.

To determine whether the findings generalized to other dissociatives, we examined phencyclidine. As with the preliminary experiment, we also examined the persistence of sensitization in adolescents and adults. SENSITIZATION AND DISSOCIATIVES 23

This thesis fills important gaps in the literature. Little published research has explored the rewarding response to dissociatives and almost no work has been published on the effects of dissociatives on ultrasonic vocalizations (Swalve, Mullholland, Schulz, & Li, 2015). In addition, little is known about the differences in response to dissociatives in adolescents and adults or on the long-lasting effects of dissociatives in adolescents or adults. Finally, more research is needed directly comparing USVs and locomotor activity.

Hypotheses.

The current thesis examined the effects of ketamine and PCP on USVs and locomotor behavior across days of repeated administration in adolescents and adults. Two identical experiments were performed, one examining ketamine and the other examining phencyclidine.

The following hypotheses were based on the preliminary studies.

1. Ketamine and phencyclidine will stimulate locomotor activity and USVs, and behavioral

sensitization will develop with repeated administration.

2. Adolescents will show more rapid sensitization in both locomotor activity and USVs when

compared to adults.

3. Adolescents will show persistent sensitization when tested in adulthood in both locomotor

activity and USVs.

Methods.

Two identical experiments were performed, the first examining ketamine and the second examining phencyclidine.

Animals. SENSITIZATION AND DISSOCIATIVES 24

Twenty-four male Sprague-Dawley rats were used in each experiment, including 12 adult

(approximately 60 days of age) and 12 adolescent (30 days of age). The animals for each experiment had free access to food and water at all times except during experimental sessions.

The animals were housed two per cage. The vivarium was on a 12-hour light/dark cycle with a humidity range of 50 to 70 percent and a temperature range of 68 to 72 F. Animals were allowed to acclimate to the vivarium for one week prior to experiments. For each experiment the animals were divided into four treatment groups (6 animals/group): Adult Drug, Adult Saline, Adolescent

Drug, and Adolescent Saline.

Drugs.

Ketamine hydrochloride (KET; Sigma-Aldrich) 10.0 mg/kg was used in the first experiment and phencyclidine hydrochloride (PCP; NIDA Drug Supply Program) 3.0 mg/kg was used in the second experiment. Doses of the drugs were selected based on previous research in the laboratory demonstrating that these were in the moderate range for locomotor stimulation.

KET and PCP were dissolved in physiological saline (0.9%) in a volume of 1 ml/kg and was administered subcutaneously. The animals received drug or vehicle subcutaneously, once daily, for 7 days (PND 30-37 in adolescents and approximately 60-67 in adults). Following a washout period of 23 days during which the adolescents aged into adulthood, the animals received one additional treatment day. On this day, all animals (including those that were previously administered repeated saline) received a drug injection and locomotor behavior and USVs were assessed. In this manner, we had the opportunity to assess differences between animals that received drug for the first time (the animals that received saline during the first 7 days of the study) and animals that received drug during the first 7 days. For Experiment 1, the animals SENSITIZATION AND DISSOCIATIVES 25 received a ketamine challenge and for Experiment 2, the animals received a phencyclidine challenge at the same doses used earlier.

Apparatus.

Locomotor and USV apparatus.

During testing, the locomotor chambers and the USV microphones were used in combination in order to allow for simultaneous measurements of both locomotor activity and ultrasonic vocalizations. Avisoft microphones were attached above the locomotor chambers roughly 15 cm from the floor. The open field locomotor chambers were left untouched with the exception of the addition of the microphones to record the USVs.

Procedure.

Each animal was handled for five minutes for the three days preceding the study. During these days, the animals were habituated to the injection procedure but were not injected. The animals received treatment once daily for 7 days. On each testing day the animals were given 15 minutes habituation to the room. Then they were individually placed in the locomotor/USV apparatus to habituate for 15 minutes. Upon completion of the habituation phase, the animals were injected with saline, ketamine or phencyclidine in accordance with the appropriate experiment. Locomotor activity and ultrasonic vocalizations were assessed for 60 minutes post- injection simultaneously (Fig. 3). The same procedure was followed each day for 7 days to assess acute (day 1) effects of the drugs, and to determine if the drug treated animals develop sensitization. The animals were then given a washout period of 23 days. This extended washout period was adopted in order to allow the adolescent animals to fully develop into adulthood (60 SENSITIZATION AND DISSOCIATIVES 26

PND). On this day all animals were challenged with ketamine or phencyclidine to determine if sensitization persisted (Fig. 3).

Figure 3. Timeline of Experiments: Drug treatment and testing occurred daily for 7 days then again 23 days later.

Statistical Analyses.

To assess locomotor activity, ambulations (reflecting horizontal activity) were measured by the Kinder Scientific Motor Monitor (described above). The timecourse of activity was examined to follow the response to treatment and then total ambulations during the period post- injection were used for statistical analyses. Ultrasonic vocalizations were examined by Avisoft

SASLabpro software, with 50 kHz USVs as the key measurement. These call types are emitted by animals during positive affect and can be classified into three different call types: fixed frequency (FF), frequency modulated (FM), and frequency modulated with trills (FM w/Trills).

For the current studies, frequency modulated 50 kHz USVs were used as the primary measure as these were greatest in number and because they have been identified as reflective of positive affect (Wright et al., 2010). Locomotor activity and USVs across days were analyzed using a two-way repeated measures ANOVA (group x day of treatment). Since the response to ketamine lasted approximately 10 minutes, total counts during this timeframe were used for analysis. Since

PCP lasted for the duration of the experimental session, total counts for the session were used.

Fisher’s LSD test was used to determine any pairwise differences post hoc. A probability of p < SENSITIZATION AND DISSOCIATIVES 27

.05 was considered significant. To analyze development of sensitization across days in each group a within-subjects t-test was used, comparing day 1 to day 7. To assess differences on the challenge day a one-way ANOVA was used to compare groups followed by Fisher’s LSD multiple comparison post hoc test.

Thesis Study 1: Ketamine: Locomotor Behavior and Ultrasonic Vocalizations

Results.

Locomotor Behavior.

On Day 1 of treatment, ketamine (10.0 mg/kg) induced a short-lived stimulant effect that was considerably greater in adolescents than adults (Fig. 4A). Because the maximum stimulant effect was observed in the first 10 minutes post-injection, this timeframe was used for analysis across days. Activity escalated across days of treatment, reflecting the development of sensitization, and there appeared to be more rapid sensitization in adolescents than adults. A two- way repeated measures ANOVA test revealed significant effect of treatment group, day of treatment and an interaction [Treatment Group: F(3,20) = 39.5, p < .0001; Day: F(6,120) =

10.69, p < .0001; Interaction: F(18,120) = 3.85, p < .0001] (Fig. 4B). A Fisher’s LSD multiple comparison test revealed that the adolescent ketamine group produced more locomotor activity than both the adult ketamine group and the adolescent saline group on days 1-7. In addition, the adult ketamine group produced more locomotor activity than adult saline group on days 2-7. The adolescent group showed an increase in activity on day 1 and continued to escalate through of treatment across days. The escalation across days of treatment in the adolescent ketamine group was greater than that seen in adults, especially on days 1-5. Within subjects t-tests showed that there was a significant increase in activity for adolescents from day 1 to day 7, t(10) = 3.51, p = SENSITIZATION AND DISSOCIATIVES 28

.005 and a significant increase in activity for adults from day 1 to day 7, t(10) = 10.19, p =

.0001.

Ultrasonic Vocalization.

On Day 1 of treatment, ketamine (10.0 mg/kg) induced an increase in USVs that was considerably greater in adolescents than adults (Fig. 4C). Because the maximum calls were observed in the first 10 minutes post-injection, this timeframe was used for analysis across days.

USVs showed variability across days of treatment, but overall escalated, reflecting the development of sensitization, and sensitization was greater in adolescents than adults. A two-way repeated measures ANOVA test revealed significant effect of treatment group, but not time (day of treatment) nor interaction [Treatment Group: F(3,20) = 17.89, p < .0001; Day: F(6,120) =

1.50, p < .18; Interaction: F(18,120) = 124 6, p < .23] (Fig. 4D). A Fisher’s LSD multiple comparison test revealed that the adolescent ketamine group produced more USVs than the adult ketamine group and the adolescent saline group on days 1-7. The adolescent group showed an increase in USVs from day 1 to day 2 and variability across subsequent days, with an upward trend on days 6-7. Within subjects t-tests showed that there was a significant increase in USVs for adolescents from day 1 to day 7, t(5) = 2.43, p = .05 but not for adults, t(10) = 1.29, p = .23. SENSITIZATION AND DISSOCIATIVES 29

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Figure 4. A) Timecourse of activity on Day 1 of ketamine treatment. The adolescent ketamine- treated group shows increased activity compared to the adult ketamine-treated group. B) Total activity across 7 days of treatment during the first 10 minutes post-injection. Note the increases in activity across days, reflecting the development of sensitization. Values expressed as mean +/- SEM. * indicates significant differences between adolescent ketamine and adult ketamine groups. # indicates differences between the adolescent or adult ketamine group and the respective saline control group. $ indicates significant increase from day 1 to day 7. C) Timecourse of USVs on Day 1 of ketamine treatment. The adolescent ketamine-treated group shows increased vocalizations compared to the adult ketamine-treated group. D) Frequency modulated 50 kHz of USVs across 7 days of ketamine treatment during the first 10 minutes post- injection. Note that adolescent’s activity counts increase Day 1 to Day 7, reflecting the development of sensitization. Values expressed as mean +/- SEM. * indicates significant differences between adolescent ketamine and adult ketamine groups. # indicates differences between the adolescent or adult ketamine group and the respective saline control group. $ indicates significant increase from day 1 to day 7. Juvenile Ketamine (JV KET) and Juvenile Saline (JV SAL) are adolescent groups.

SENSITIZATION AND DISSOCIATIVES 30

Ketamine Challenge.

Locomotor Behavior.

In order to determine if residual sensitization was present in the ketamine-treated animals in this study, 23 days following repeated ketamine administration all animals (including those pretreated with saline) were challenged with 10.0 mg/kg of ketamine and locomotor activity and

USVs were assessed. The saline control animals demonstrated relatively low levels of activity after administration of ketamine. However, animals that previously received ketamine showed an increase in locomotor activity after ketamine administration, with animals receiving ketamine as adolescents showing a slightly greater response. One-way ANOVA on ambulations revealed that there was a significant difference among groups [F(3,20) = 8.75 p < .0007] (Fig. 5A-B). Fisher’s

LSD revealed that the age at which the animals were treated with ketamine did not affect residual sensitization. Animals that received KET as adults (t(10)= 4.77, p = .0008) and animals that received KET as adolescents (t(10) = 3.24, p =.009) were more active than their age-matched controls, and there was no difference between the adult and adolescent KET groups in activity, t(10) = .02, p = .984. Therefore, residual sensitization was evident in KET-treated animals but did not differ between adolescents and adults.

Ultrasonic Vocalization.

In examining the response to the ketamine challenge, the saline control animals demonstrated few USVs after administration of the drug. However, animals that previously received ketamine showed an increase in 50 kHz USVs after ketamine administration. One-way

ANOVA revealed that there was a significant difference among groups [F(3,20) = 4.07, p =.022]

(Fig. 5C-D). Fisher’s LSD multiple comparison post hoc test revealed that the response to ketamine was dependent on the age at which the animals were treated. Animals that received SENSITIZATION AND DISSOCIATIVES 31 repeated KET as adults (t(10) = 1.37, p = .20) did not significantly differ from their age-matched controls. However, animals that received repeated KET as adolescents (t(10) = 2.86, p = .019) displayed more 50 kHz USVs than their age-matched controls. There was no significant difference between the adults and adolescents ketamine treated groups (t(10) = 0.87, p = .396).

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Figure 5. A-B) Total Activity Counts in response to ketamine challenge after 23 days without treatment. Animals exposed to ketamine as adolescents continued to show sensitization, but the differences between ketamine treated animals was not different. # indicates differences between the adolescent or adult ketamine group and the respective saline control group. C-D) Total Frequency Modulated 50 kHz USVs in response to ketamine challenge after 23 days without treatment. Animals exposed to ketamine as adolescents showed the greatest response to ketamine. # indicates differences between the adolescent or adult ketamine group and the respective saline control group. Juvenile Ketamine (JV KET) and Juvenile Saline (JV SAL) are adolescent groups. SENSITIZATION AND DISSOCIATIVES 32

Table 1. Summary of Ketamine Findings.

Locomotor Behavior USVs

Acute Greater in adolescents Increase in adolescents, but not adults Repeated Sensitization in adolescents Sensitization in adolescents, and adults but not adults Persistent Persistent sensitization in Persistent sensitization in adolescents and adults adolescents, but not adults

Summary.

Overall, the results of this study were consistent with our hypotheses (see Table 1 for a summary of the results). With regard to locomotor stimulation, as hypothesized, we found that adolescents showed a greater stimulant response to the first injection of ketamine than adults.

Similarly, as hypothesized, following repeated treatment of ketamine both adolescents and adults showed sensitization to the locomotor stimulant effects and there was more rapid sensitization in the adolescents. With regard to USVs, similar to the results found in locomotor testing, on Day 1 the adolescent ketamine treated group showed greater USVs than adults. In addition, although there was variability in response to repeated administration of ketamine across days, the adolescents showed sensitization. However, sensitization did not develop to ketamine-induced

USVs in adults.

Following a washout period of 23 days during which the adolescents aged into adulthood all animals received treatment with ketamine to determine the persistence of drug induced changes in behavior. We found that adolescents and adults that received ketamine previously continued to show sensitization to locomotor behavior on the challenge day, indicating that there was a long term persistence in both adolescents and adults. Furthermore, sensitization to ketamine-induced USVs persisted, however this effect was seen only in adolescents, not adults.

Thus, animals that received ketamine as adolescents showed an increased rewarding response to SENSITIZATION AND DISSOCIATIVES 33 the drug when tested as adults. These results replicate and extend the findings of Preliminary

Study 2.

Taken together, the results demonstrate that adolescents have stimulant and rewarding effects of ketamine that are greater than adults, adolescents show more rapid sensitization than adults with repeated use, and adolescents show persistence of sensitization into adulthood.

Implications of these findings are discussed below.

Thesis Study 2: PCP: Locomotor Behavior and Ultrasonic Vocalizations

Results.

Locomotor Behavior.

Some technical problems arose during the study, such that on selected days some animals did not receive a PCP injection. Specifically, animal 2 on day 4, animal 4 on day 4, and animal 6 on day 5. Therefore, the data were omitted for these animals on these days with the final sample size on each of the aforementioned days being 22 and 23, respectively.

On Day 1 of treatment, phencyclidine (3.0 mg/kg) induced a stimulant effect that was evident in adolescents but not in adults and that lasted for the duration of the session (Fig. 6A).

Activity showed variability across days of treatment, but overall increased across days, reflecting the development of sensitization. In addition, there appeared to be more rapid sensitization in adolescents when compared to adults. A two-way repeated measures ANOVA on activity across days revealed significant effect of treatment group, day of treatment and an interaction

[Treatment Group: F(3,20) = 225.8, p < .0001; Day: F(6,120) = 5.13, p < .002; Interaction:

F(18,120) = 1.85, p < .02] (Fig. 6B). A Fisher’s LSD multiple comparison test revealed that the adolescent PCP group produced more locomotor activity than the adult PCP group and the SENSITIZATION AND DISSOCIATIVES 34 adolescent saline group on days 2-7. In addition, the adult PCP group produced more locomotor activity than adult saline group on days 2-7. The adolescent group showed an increase in activity on day 1-2 and then came down from days 3-5 then went back up again from days 6-7. Within subjects t-tests showed that there was a significant increase in activity for adolescents from day 1 to day 7, t(10) = 2.79, p = .02 and a significant increase in activity for adults from day 1 to day 7, t(10) = 3.78, p = .004.

Ultrasonic Vocalization.

On Day 1 of treatment, PCP (3.0 mg/kg) induced a short-lived increase in USVs that was considerably greater in adolescents than adults (Fig. 6C). USVs in adolescents decreased across days of treatment, reflecting the development of tolerance instead of sensitization. A two-way repeated measures ANOVA revealed significant effect of treatment group, time (day of treatment) and interaction [Treatment Group: F(3,20) = 4.08, p < .02; Day: F(6,120) = 2.29, p <

.04; Interaction: F(18,120) = 2.42, p < .002] (Fig. 6D). A Fisher’s LSD multiple comparison test revealed that the adolescent PCP group produced more USVs than the adult PCP group and the adolescent saline group on days 1-2. The adolescent group showed a decrease in USVs from day

1 to day 7. Within subjects t-tests showed that there was a trend towards significant decrease in

USVs for adolescents from day 1 to day 7, t(10) = 1.61, p = .069. SENSITIZATION AND DISSOCIATIVES 35

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5 20 5 0 0 0 5 10 15 20 25 30 35 40 45 50 55 60 1 2 3 4 5 6 7 Minutes Post-Injection Treatment Day Figure 6. A) Timecourse of activity on Day 1 of phencyclidine treatment. The adolescent phencyclidine -treated group shows increased activity compared to the adult phencyclidine - treated group. B) Total activity across 7 days of treatment. Note the increases in activity across days, reflecting the development of sensitization. Values expressed as mean +/- SEM. * indicates significant differences between adolescent phencyclidine and adult phencyclidine groups. # indicates differences between the adolescent or adult phencyclidine group and the respective saline control group. $ indicates significant increase from day 1 to day 7. C) Frequency Modulated 50 kHz of USVs on Day 1 of phencyclidine treatment. The adolescent phencyclidine- treated group shows increased vocalizations compared to the adult phencyclidine-treated group. * indicates significant differences between adolescent phencyclidine and adult phencyclidine groups. # indicates differences between the adolescent or adult phencyclidine group and the respective saline control group. D) Frequency Modulated 50 kHz across 7 days of treatment. Note the decrease in USVs across days, reflecting the development of tolerance. Values expressed as mean +/- SEM. * indicates significant differences between adolescent phencyclidine and adult phencyclidine groups. # indicates differences between the adolescent or adult phencyclidine group and the respective saline control group. Juvenile PCP (JV PCP) and Juvenile Saline (JV SAL) are adolescent groups.

PCP Challenge. SENSITIZATION AND DISSOCIATIVES 36

Locomotor Behavior.

In order to determine the persistent effects of phencyclidine in this study, 23 days following repeated treatment all animals were challenged with 3.0 mg/kg of phencyclidine and locomotor activity and USVs were assessed. The saline control animals, which received phencyclidine for the first time, showed low levels of locomotor activity in response to the drug challenge. However, animals that previously received phencyclidine showed an increase in locomotor activity after phencyclidine administration. One-way ANOVA on horizontal activity revealed that there was a significant difference among groups [F(3,20) = 3.92, p < .02] (Fig. 7A-

B). Fisher’s LSD revealed that the age at which the animals were treated with phencyclidine did not affect residual sensitization. Animals that received PCP as adults (t(10)= 2.24, p = .049) and animals that received PCP as adolescents (t(10) = 2.93, p =.015) were more active than their age-matched controls. Also, there was no difference between the adolescent and adult drug- treated groups in activity (t(10) = .18, p = .86). Therefore, residual sensitization was evident in

PCP-treated animals and there was no effect of age.

Ultrasonic Vocalization.

After the washout period, animals that had become tolerant to PCP-induced USVs as adolescents appeared to show a reduced response to PCP compared to all other groups. A one- way ANOVA of total 50 kHz USVs did not reveal significant differences among groups,

[F(3,20) = 1.01, p =.410] (Fig. 7C-D). Although the ANOVA was not significant the trends were strong so a follow-up planned t-test was conducted. Between subjects t-tests showed that there was a significant decrease in USVs for adolescents compared to their saline control group, t(10)

= 2.87, p = .008, but not for adults. SENSITIZATION AND DISSOCIATIVES 37

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Figure 7. A-B) Timecourse and bar graph of Total Activity Counts in response to phencyclidine challenge after 23 days without treatment. Animals exposed to phencyclidine as adolescents and adults continued to show sensitization, and the differences between phencyclidine treated animals was not different. # indicates differences between the adolescent or adult phencyclidine group and the respective saline control group. C-D) Timecourse and bar graph Total Frequency Modulated 50 kHz USVs in response to phencyclidine challenge after 23 days without treatment. Animals exposed to phencyclidine as adolescents showed the greatest response to phencyclidine. Juvenile PCP (JV PCP) and Juvenile Saline (JV SAL) are adolescent groups.

SENSITIZATION AND DISSOCIATIVES 38

Table 2. Summary of Phencyclidine Findings.

Locomotor Behavior USVs

Acute Greater in adolescents Increase in adolescents, but not adults Repeated Sensitization in adolescents Tolerance in adolescents, but and adults not adults Persistent Persistent sensitization in Persistent tolerance in adolescents and adults adolescents, but not adults

Summary.

The results of this study were similar to those of Experiment 1 in locomotor behavior, but strikingly different for USVs (see Table 2 for a summary of the results). As in Experiment 1, adolescents showed a greater locomotor stimulant response to the first injection of phencyclidine than adults and more rapid sensitization. However, the results diverged for USVs. On Day 1, the adolescent phencyclidine treated group did show greater USVs than adults, however in response to repeated administration of phencyclidine across days, the adolescents showed decreases in effect, reflective of tolerance, rather than sensitization. Neither sensitization nor tolerance developed in adults.

Following a washout period of 23 days during which the adolescents aged into adulthood all animals received treatment with phencyclidine to determine the persistence of drug induced changes in behavior. We found that both groups that received phencyclidine previously continued to show sensitization to locomotor behavior on the challenge day, indicating that there was a long term persistence in both adolescents and adults. Furthermore, tolerance to phencyclidine-induced USVs persisted, however this effect was seen only in adolescents, not adults. Thus, animals that received phencyclidine as adolescents showed a decreased rewarding response to the drug when tested as adults. SENSITIZATION AND DISSOCIATIVES 39

Therefore, similar to ketamine, adolescents have a stimulant effect and a rewarding effect to phencyclidine that is greater than adults. However, the effects of repeated treatment differ depending on the behavior that is being tested. In locomotor behavior adolescents show more rapid sensitization that persists into adulthood. In contrast, in USVs adolescents show tolerance that persists into adulthood, while adults show no changes with repeated use. Implications of these findings are discussed below.

General Discussion

Present Findings.

The primary goals of the present thesis were to determine if exposure to ketamine or phencyclidine during adolescence would induce a stimulant and a rewarding effect upon acute treatment, induce behavioral sensitization with repeated treatment, and induce long-lasting effects that persist in adulthood. We also wanted to compare the effects in adolescents to those in adults. We measured locomotor behavior and ultrasonic vocalizations to obtain information about drug-induced reward. Whereas ultrasonic vocalizations are a direct measure of positive affect and reward, increased locomotor activity is an indirect and non-specific measure of reward.

Overall, the results in locomotor behavior were consistent with our hypotheses. Ketamine and phencyclidine induced stimulant effects that were greater in adolescents than adults.

Sensitization occurred across days for both adults and adolescents, and the sensitization was more rapid in adolescents. Finally, ketamine and phencyclidine sensitization persisted into adulthood. SENSITIZATION AND DISSOCIATIVES 40

Interestingly, the findings for USVs showed striking differences between the two dissociatives. As hypothesized, both ketamine and phencyclidine induced increases in USVs in adolescents, but not adults following the first injection. For ketamine, sensitization occurred across days for adolescents but not adults, and sensitization in the adolescents persisted into adulthood. In contrast, for phencyclidine, instead of sensitization, tolerance developed across days in adolescents, and this tolerance persisted into adulthood. Thus, whereas repeated administration of ketamine led to sensitization to USVs in adolescents, repeated administration of phencyclidine produced tolerance to USVs. In addition, sensitization and tolerance in the adolescents persisted into adulthood.

The results in locomotor behavior are consistent with previous work that shows that PCP and ketamine produce locomotor stimulation and that repeated administration of these drugs produces locomotor sensitization in animal models (Abekawa et al., 2002; Iwamoto, 1986; Parise et al., 2013; Phillips et al., 2001; Scalzo and Burge, 1992; Scalzo and Holson, 1992; Trujillo et al., 2008; Uchihashi et al., 1993; Wily et al., 2007; Xu and Domino, 1994a, 1994b; Xu and

Domino, 1999).

Research conducted by Rocha, Hart, & Trujillo, 2017 has found similar results showing greater locomotor stimulation to ketamine and PCP in adolescents. The current results and those of Rocha et al. overlap with those of Parise et al. (2013) and Pesic et al., (2010), who showed that adolescents had greater locomotor response compared to adults to ketamine and MK-801 (a potent and selective NMDA receptor blocker), respectively. Furthermore, earlier work in our lab showed an increased response to PCP and other NMDA receptor antagonists in adolescents when compared to adults (Trujillo, & Warmoth, 2005; Hartfield, & Trujillo, 2008; Sullivan et al.,

2009; Klumph et al., 2010; Oviedo, & Trujillo, 2012). However, some studies have reported SENSITIZATION AND DISSOCIATIVES 41 reduced response to ketamine in adolescents (Wiley et al., 2008) or little differences between adults and adolescents (Wilson et al., 2007). Taken together, there is increasing work demonstrating greater effects of ketamine and related drugs in adolescents, when compared to adults.

Researchers have demonstrated that adolescents and adults both show differences in response to other drugs of abuse in locomotor behavior. In several studies, cocaine, methamphetamine, amphetamine, and ethanol exposure produced increased stimulant effects in adolescents compared to adults, however, in other studies these same drugs showed no differences or an increased response in adults (Rezvani and Levin, 2004; Lopez et al., 2003;

Lanier et al., 1977; Bolanos et al., 1998; Collins et al., 2004; Laviola et al. 1999; Zombeck et al.

2009; Spear and Brick, 1979; Laviola et al. 1995). More work is necessary to determine behavioral differences between adolescents and adults in response to different classes of drugs of abuse.

There has been little published work on differences in locomotor sensitization to ketamine or PCP in adolescents and adults, and there has been almost no work on ultrasonic vocalizations. Thus, the present work is among the first to examine these phenomena.

Differences Between Ketamine and Phencyclidine.

Despite results that supported our hypotheses for ketamine, an unexpected pattern emerged for USVs when examining the response to repeated phencyclidine. Phencyclidine did produce an increase in USVs in adolescents, but not adults, but tolerance was seen with repeated administration in the adolescents. Previous research in our lab has shown that adult animals do not vocalize in response to PCP (Roos et al., 2014), as seen in the current study. Other studies have similarly reported a lack of USV response to PCP, and furthermore that 50 kHz USVs in SENSITIZATION AND DISSOCIATIVES 42 response to tickling (Boulay et al., 2013) or nicotine (Swalve et al., 2015) were reduced by PCP in adult animals. To our knowledge, the current work is the first study to examine USVs in adolescents and in response to repeated injections of ketamine or PCP. The differences in response to ketamine and PCP are surprising, but may result from differences in the neurobiology of these drugs.

Ketamine and phencyclidine (PCP) belong to a class of drugs known as dissociative anesthetics. These drugs produce similar responses in terms of out of body experiences, depersonalization, euphoria, etc., and their primary mechanism of actions is blockade of NMDA receptors. There are, however, some marked differences between the two. Ketamine is less potent than PCP, has a shorter duration of action (Lodge & Mercier, 2015), and binds with lower affinity to NMDA receptors (Roth & Giarman, 1969). This means that ketamine will generally produce less intoxicating effects compared to PCP and the symptoms will resolve quicker for ketamine compared to PCP. It is possible that differences in the duration of action contributed to the differences between ketamine and PCP. It has been shown that repeated intermittent administration of cocaine and amphetamine, with wide spacing between injections, leads to sensitization, but that continuous infusion or closely spaced dosing leads to drug tolerance

(Izenwasser and French, 2002; King et al., 1994a,b; Kunko et al., 1998; Nelson and Ellison,

1978; Nielsen, 1981; Post, 1980; Reith et al., 1987; Robinson and Becker, 1986; Stewart and

Badiani, 1993). The long duration of action of PCP may have led to a tendency for tolerance, rather than sensitization, while the shorter duration of action for ketamine led to sensitization. If this is the case, then longer spacing of doses may lead to sensitization to PCP-induced USVs.

However, this doesn't explain why sensitization developed to PCP-induced locomotor SENSITIZATION AND DISSOCIATIVES 43 stimulation while tolerance developed to PCP-induced USVs in the adolescents. Further research is needed to further explore this interesting dissociation.

There are other important differences between ketamine and PCP that may have contributed to the current results, most notably in their interactions with other neurotransmitters systems. For example, ketamine has been found to have similar affinity at NMDA receptors and dopamine D2 receptors (and lower affinity for 5HT2 serotonin receptors), while PCP shows similar affinity for the NMDA and 5-HT2 receptors (and lower affinity for D2 receptors) (Kapur

& Seeman, 2002). Thus, differing interactions with NMDA receptors, dopamine, serotonin (and perhaps other neurotransmitter systems) may have contributed to the differences between the drugs. Together, differences in pharmacokinetics (duration of action) and pharmacodynamics

(interactions with neurotransmitter receptors) likely contributed to the differences seen between ketamine and PCP in the current research.

Differences Between Adolescents and Adults.

We hypothesized that differences in the acute response to ketamine and PCP would occur in adolescents and adults, and we found some clear differences. Consistent with these findings, studies have shown that adults respond differently to ketamine than adolescents for other behaviors. For example, when wakening from ketamine anesthesia adult patients often show an

‘emergence reaction’ consisting of psychotomimetic symptoms, but adolescents are less prone to have these effects (Cho et al., 2014; Green et al., 2009; Green et al., 2011). In addition, in rodent models, adults tend to have prolonged antidepressant effects that are not seen in adolescents

(Nosyreva et al., 2014). Furthermore, adult laboratory rats show increased neurotoxic effects to SENSITIZATION AND DISSOCIATIVES 44 high doses of drugs compared to adolescents (Farber et al., 1995; Farber and Olney, 2003;

Noguchi et al., 2005; Olney and Farber, 1995).

One potential explanation of differences between adolescents and adults in response to ketamine and phencyclidine is pharmacokinetic factors (i.e., differences in drug metabolism).

For instance, if adolescents are unable to metabolize the dissociatives as quickly as adults there may be a higher concentration of the drug inside the brain and this would lead to increased locomotor behavior and potentially increased USVs (Rocha et al., 2017). There is currently no published research on potential differences in plasma concentrations of ketamine or PCP in adolescents compared to adults. However, human research and animal research both lean in the direction of more rapid metabolism and lower plasma levels of a variety of drugs in adolescents

(Grant et al., 1983; Lockhart and Nelson, 1974). Having lower plasma levels for adolescents means that there is less drug in the body due to rapid elimination from the system. This pattern would, in fact, predict that adolescents should show a reduced effect when compared to adults.

Since we found greater effects in adolescents, rather than reduced effects, differences in drug metabolism are unlikely to explain the current results. More broadly considering differences between adolescents and adults in response to dissociatives, because some behaviors are increased in adolescents when compared to adults (locomotor behavior, USVs), while others are reduced (antidepressant effects, neurotoxicity), differences between the age groups cannot be explained by differences in drug metabolism. More studies are needed to look at differences between adolescents and adults in response to dissociatives (Rocha et al., 2017; Vitiello &

Jensen, 1995).

Much of the work on differences in response to psychoactive drugs between adolescents and adults has focused on the neurobiological systems that mediate the effects of the drugs. For SENSITIZATION AND DISSOCIATIVES 45 ketamine and PCP, it is important to consider differences in NMDA receptors in the two age groups. These receptors reach a peak in concentration in adolescence, around 30 days of age, and then decline in adulthood, around 60 days of age (Colwell, 1998; Insel, Miller, & Gelhard,

1990). At PND 30 adolescents tend to respond greater to dissociatives than adult animals at

PND 60, and this could be due to higher concentration of NMDA receptors in the adolescent brain.

Another important neurobiological difference between adults and adolescents is in dopamine systems in the brain. The mesolimbic dopamine pathway, consisting of the ventral tegmental area (VTA) and nucleus accumbens (NAcc) plays a crucial role in drug reward. The dopamine levels and the D1 dopamine receptor concentration in adolescents are significantly higher than in adults and decline as the animal ages (Andersen et al., 2000; Philpot and Kirstein,

2004; Tarazi & Baldessarini, 2000; Teicher et al., 1995). In addition, researchers have shown that electrophysiological parameters related to NMDA receptor/dopamine interactions are not fully developed during adolescence (Flores-Barrera et al., 2014; O'Donnell, 2011; Tseng and

O'Donnell, 2005; Tseng et al., 2007; Wahlstrom et al., 2010). As a result, differences in NMDA receptor concentration, dopamine systems and their interactions may have mediated the differences seen between adolescents and adults in the present studies.

Differences Between USVs and Locomotor Behavior.

Our results show some clear differences between USVs and locomotor behavior. For instance, for PCP there was locomotor sensitization and USV tolerance for adolescents. were used since they both offer information on drug reward. The Psychomotor Stimulant Theory of

Addiction predicts an association between increased locomotor activity and drug reward (Wise et SENSITIZATION AND DISSOCIATIVES 46 al., 1987). However, locomotor activity is not specific to reward, as it can be enhanced in non- rewarding situations, and it is only indirectly associated with reward. On the other hand, 50 kHz

USVs seem to be relatively direct assessment of reward in laboratory rats (Panskepp et al., 2010;

Knutson, Burgdorf, & Panksepp, 2002). When looking at locomotor behavior, ketamine and PCP produced sensitization in adolescents and adults. However, when examining USVs, adolescents showed sensitization to ketamine and tolerance to PCP. Other research has also found a lack of correlation between locomotor behavior and USVs (Garcia & Cain, 2016; Kaniuga et al., 2016;

Roos & Trujillo, 2014; Simola & Morelli, 2015; Wright, Deng, & Clarke, 2012). For instance, 50 kHz USVs were produced by a cued reward while the locomotor activity remained constant

(Knutson, Burgdorf, & Panksepp, 2002). In another example, microinjections of amphetamine into the nucleus accumbens produced increases in 50 kHz USVs without increasing locomotor activity (Burgdorf, Knutson, Panksepp, & Ikemoto, 2001). In terms of our current findings, what this means is that to the extent that the behaviors are measures of reward, locomotor behavior and USVs are distinct.

Conclusions

The current research demonstrated that ketamine and PCP induced a greater response in adolescents when compared to adults regardless of the behavior or the drug. However, the consequences of repeated administration depended on the specific drug and the behavior measured with both ketamine and PCP inducing locomotor sensitization, but ketamine inducing sensitization to USVs and PCP inducing tolerance. Furthermore, repeated administration led to sensitization or tolerance that persisted even after a 23-day period of abstinence. SENSITIZATION AND DISSOCIATIVES 47

In humans, the initial exposure to the drug can increase the risk of drug use. Individuals seek drugs that are rewarding, but do not seek drugs that are aversive or neutral. Greater rewarding and lower aversive effects of drugs of abuse in adolescents can lead to increased risk of drug use leading to a greater opportunity for addiction (Schramm-Sapyta et al., 2011). In the present studies, the greater rewarding effects of ketamine and PCP in adolescents could contribute to increased risk of addiction. In addition, drug-induced plasticity, including tolerance and sensitization, are important contributors to addiction. We showed that after chronic administration of PCP and ketamine adolescents showed tolerance and sensitization that persisted into adulthood. This may help to explain why early use of drugs of abuse is more likely to lead to addiction than use that begins later in life.

PCP and ketamine are popular with teenagers and young adults, so it is critical we understand how these drugs affect adolescents differently from adults. Further research should identify the neural mechanisms of differences between adolescents and adults, and that underlie chronic NMDA receptor blockade.

SENSITIZATION AND DISSOCIATIVES 48

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