Honors Thesis

Extinction of Cocaine CPP & Basolateral Amygdala
Effects of temporary inactivation of the basolateral amygdala during extinction of cocaine-
conditioned place preference in rats
Nicholas Fasolt
Submitted in partial fulfillment for a Bachelors of Arts Degree with College Honors in
Neuroscience at Knox College, Galesburg, IL
May 21st
, 2009
College Honors Committee:
Heather Hoffmann (Chair), Professor of Psychology
Linda Dybas, Professor of Biology
Esther Penick, Assistant Professor of Biology
Pamela Skoubis (External Examiner), Adjunct Professor of Biology, DePaul University

Extinction of Cocaine CPP & Basolateral Amygdala 2
Abstract
Acquisition of cocaine conditioned place preference (CPP) has been shown to depend on
cue-reward associations established by the basolateral amygdala, yet little research has
investigated the specific role of the amygdala in re-learning of cue-reward associations after
extinction of cocaine-CPP. In order to assess the role of the amygdala in re-association of
cocaine-paired cues during extinction trials, animals were outfitted with cannulae, trained for
cocaine-CPP, and received either temporary inactivation of the basolateral amygdala using 2%
lidocaine solution or no inactivation (PBS vehicle) during extinction trials. No significant
difference between control and experimental groups was observed. This could suggest that the
role of the basolateral amygdala may be limited to initial cue-reward associations but not re-
association of such links during extinction of cocaine-CPP.

The use of chemical, and specifically psychoactive, compounds for medical and spiritual
purposes is one of the oldest of practices recorded in human history. Surely, ancient cultures and
civilizations realized the recreational potential of perception- and/or mood-altering compounds, a
practice continued for millennia and still very much in existence today. Along with the discovery
of modern chemical methods and the emergence of extremely concentrated natural or potent
synthetic compounds, the recreational use of such chemicals has become more prominent and
widespread. In fact, in 2007 it was estimated that approximately 8% of the United States
population above age twelve had used an illicit drug in the past 30 days (National Survey of
Drug Use and Health [NSDUH], 2007). As a consequence of recognized widespread drug usage,
the negative effects of these drugs, including dependence or addiction, have become an
increasingly prevalent medical and societal concern. However, not all people who use drugs
recreationally become addicted. For example, it was estimated in 2004 that only 27.8% of
reported cocaine users in the U.S. were classified as addicted, compared to 17.6% of marijuana
users, 11.9% of alcohol users, and 67.8% of heroin users (NSDUH, 2004). Thus, it is important
to clearly differentiate between recreational drug use and addiction. Addiction, as defined in the
Diagnostic and Statistical Manual of Mental Disorders IV, is a state of periodic or chronic
intoxication resulting from using a drug. Addiction is characterized by 1) a developed tolerance
to the effects of the drug, leading to 2) to an increase in drug concentrations necessary to elicit
the previous effect and 3) compulsive efforts to obtain the drug with an overpowering desire or
continue using the drug, often followed by 4) unpleasant to painful symptoms of withdrawal
from the substance, and 5) the persistent desire and unsuccessful efforts to cut down substance
use (American Psychiatric Association, 2000). Not only does addiction affect the addict, the
consequences can be far reaching and leave a significant impact on society. In fact, the National

Center on Addiction and Substance Abuse at Columbia University estimates approximately $80
billion are spent annually as result of drug related crime, incarceration, medical and addiction
treatment, welfare programs, and accidents (CASA Report, 1998). Although a wealth of
information on the societal impact of addiction exists, this does not begin to describe the
devastation and hopelessness felt by addicts and their families.
As our knowledge of addiction has increased, it has become clear that we must begin to
approach addiction from a neurological perspective. The neural systems regulating motivation,
reward, and decision-making are of primary interest, as interactions between these systems are
most prominently affected in addicts (Wise, 2002). In order to provide viable treatments, we
must first understand how addictive substances affect neural systems and cause them to become
dysfunctional. Furthermore, the high incidence of relapse after years of abstinence underlines the
importance of understanding lasting changes in the function of neural systems that mediate
reward caused by drug abuse. Accordingly, the prominent and long-lasting vulnerability to
relapse has been has been identified as a primary point for pharmacological intervention in
addiction (Kalivas & Volkow, 2005).
Characteristics of Addiction
Often, when an individual experiments with a new drug (e.g. stimulants, opiates, alcohol)
in a typically recreational setting, a novel and pleasurable subjective drug experience can be
generated. In the case that the individual enjoys the initial drug exposure, repeated use of the
drug becomes likely. However, as noted previously, this does not necessarily indicate that the
individual will become addicted. Nonetheless, clear distinctions between the addict and
recreational drug user develop early. A key difference relates to the experience of craving for the

drug, driving compulsive and detrimental drug use as outlined in the DSM-IV. While a plethora
of different and sometimes conflicting models that attempt to explain craving exist, it is
generally agreed that drug craving constitutes a major component of drug addiction and is
thought to maintain addiction. Research has demonstrated on multiple occasions that exposure to
the drug (Jaffe, Cascella, Kumor, & Sherer, 1988) and more importantly the drug-related stimuli
(e.g. drug-paraphernalia, contextual, and environmental cues) can trigger self-reports of cue-
induced craving (Bonson et al., 2002). This occurs in addition to measurable physiological
responses including decreases in skin temperature and increases in heart rate (Ehrman, Robbins,
Childress, & O‟Brien, 1992). Furthermore, neuroimaging research has suggested a pattern of
distributed activation throughout cortical areas, specifically in prefrontal cortex and limbic
regions believed to underlie cognitive and affective processes involved in the state of craving
(Garavan et al., 2000). In addition to cue-induction, craving can also be triggered by stress
(Sinha, Catapano, & O‟Malley, 1999) and withdrawal (Kalivas & Volkow, 2005). Withdrawal is
typically characterized by mild to extreme discomfort manifested as somatic (or peripheral)
symptoms including tremors, increased heart rate, sweating, nausea and vomiting and/or
psychological (or central) symptoms such as anxiety, depression, fatigue, irritability, and
hostility (Koob & LeMoal, 2005; Stewert & Brown, 1995). Often, when addicts decide that the
costs of addiction outweigh the benefits of continued drug use and attempt to rehabilitate,
abstinence is ended prematurely as a result of [perceived] inability to cope with withdrawal
symptoms (Koob & LeMoal, 1997). This phenomenon is referred to as relapse and considered to
be another significant component of drug addiction by many models (Milkman, Weiner, &
Sunderwirth, 1983). Relapse is defined as the reinstatement of drug use after any period of
abstinence. Recovered addicts often remain quite vulnerable to this phenomenon, even after

multiple years or decades after the cessation of drug use (O‟Brien, 2001). While it is very likely
that addiction is influenced by a multitude of additional factors, motivation for drug use
(pleasure-seeking, stress relief, self medication, etc.) and psychological health (depression,
anxiety, schizophrenia, etc.) are difficult to accurately measure in laboratory settings, especially
when dealing with non-human subjects. Thus, a majority of addiction research has focused on
cue-induced craving, which benefits from being measurable in both human (as self-report) and
animals (as measurable, instrumental behaviors). However, it is important to note that addiction
is currently understood as a human phenomenon, thus any measures of drug-seeking in animals
can only approximate craving in humans.
Models of Addiction
A fundamental approach in addiction research has concentrated on elucidating how
intense cravings for drugs like heroin, morphine, or cocaine develop. On one hand research has
suggested addiction is the result of positive reinforcement of drug-seeking behavior, i.e. the
desire to re-experience the hedonic effects of the drug (Markou, Weiss, Gold, Caine, Schulteis,
& Koob, 1993). However, this approach fails to appreciate the facts that craving can persist
directly after drug administration and that drugs are pursued in spite of the lack of a positive
subjective experience to the drug. For example, Lamb et al. (1991) showed that recovered heroin
addicts would continue to self-administer intravenous morphine at doses failing to register a
subjective experience but would not continue to self-administer saline. Conversely, a negative
reinforcement model has been proposed that suggests addicts experience negative affective states
including anxiety and dysphoria as a result of withdrawal, thus they seek drugs to alleviate these
negative effects (Koob & Le Moal, 1997). Congruent with this theory are implications that

withdrawal severity and the associated negative affective states are reliable indicators of relapse,
and that treatments ameliorating affective components of withdrawal are effective in preventing
relapse (Baker, 2004). While this theory accounts for addiction to drugs that do not readily
induce somatic withdrawal effects, such as stimulants (Meyer & Quenzer, 2005), it fails to
provide rationale for why drug use would be initiated to begin with. Furthermore, neither model
adequately accounts for relapse; particularly relapse resulting from cue-induced craving or after a
long bout of abstinence.
A third approach described in Berridge & Robinson‟s “incentive-sensitization” theory of
addiction (1993) provides a model that circumvents deficits observed in the traditional
reinforcement theories already described. This theory also incorporates an explanation for the
effects of cue-induced craving. Here, two functionally distinct processes are proposed to mediate
reward. Specifically there is an appetitive system that directs behavior toward obtaining a reward
upon activation by a stimulus and a hedonic system, which is activated when the reward is
received. In other words, „wanting‟ can be described as the system that directs behavior in
response to reward-predicting cue; the affective response generated upon reception of reward is
considered „liking‟. The incentive-sensitization theory is an experimentally validated theory of
addiction (Berridge & Robinson, 2003; Robinson & Berridge, 1993; 2000; 2003) that suggests
classical conditioning as a means by which abused drugs become transiently paired with
environmental and contextual stimuli, thus acquiring „incentive salience‟. Incentive salience
refers to the degree to which the stimulus becomes sought after, attractive or attention grabbing.
As drug use continues and becomes predicted by specific cues, dopamine in the reward circuit no
longer increases in response to the drug, but rather to cues predicting it (Schultz, Dayan &
Montague, 1997). Specifically, long-term changes induced by drugs cause an increased

responsiveness, or sensitization to the drug and associated stimuli, causing both to attain high
incentive salience and [implicitly] driving drug-seeking behavior further. Importantly, both
„wanting‟ and „liking‟ appear to have identifiable neural correlates, which in line with being
functionally distinct are also thought to somewhat anatomically separated. The mesolimbic
dopamine system is thought to mediate „wanting‟ (Robinson & Berridge, 1993) and is described
in detail below, while opioid transmission in „hedonic hotspots‟ -- the nucleus accumbens and
ventral pallidum, are believed to be involved in mediating „liking‟ (for a review, see Peciña,
Smith, Berridge 2006). While these systems are functionally and anatomically distinct, it is
proposed that they interact, operating in tandem to produce responding to needed incentives. For
example, appetite modulates the potency of a food reward by altering „wanting‟ and „liking‟
systems together. If an organism is hungry the „wanting‟ system directs behavior towards a
preferred food reward. Once the organism is satiated, the „liking‟ system signals the „wanting‟
system to cease motivating food-directed behavior (Berridge, 1996)
Reward: Structure and Function of the Mesolimbic Dopamine System
As alluded to above, the mesolimbic dopamine system is seems to represent the neural
correlate of motivation for biological rewards, and thus thought to mediate appropriate responses
to natural rewards important for survival such as food and sexual activity (Kelley & Berridge,
2002). Accordingly, the mesolimbic dopamine system is involved identifying and motivating
behaviors that benefit the organism, thus ensuring survival and a continued state of well-being.
Additionally, in higher species with complex social structures this system could influence control
of more cognitively or experientially based rewards that provide less evolutionarily necessary,
but nonetheless enjoyable experiences such as social engagement (Kalivas & Volkow, 2005;

Young & Wang, 2004). In addition to mediating reward, it has recently been suggested that the
mesolimbic dopamine system may also be involved in responding to and learning about aversive
stimuli (Faure, Reynolds, Richard, & Berridge, 2008).
The circuit imbues a neural representation of the stimulus with relevant motivational
information to be recalled upon future exposure to similar (or identical) stimuli, thus directing
future behavioral responses. The circuit (Figure 2) is composed of a complex network of neural
projections between cortical and subcortical structures including the nucleus accumbens (NAc),
the basolateral complex (basolateral and lateral amygdala) and central nuclei of the amygdala
(BLC and CeN respectively), the prefrontal cortex (PFC), the ventral tegmental area (VTA), and
the ventral pallidum (for a review see Kalivas & Volkow, 2005). Ventral tegmental cells
projecting to the NAc are thought to release dopamine (DA) in response to a motivationally
relevant event (Robinson & Berridge, 1993). This signal is believed to prompt an adaptive
behavioral response resulting in cellular changes such as long-term potentiation that are thought
to establish connections among environmental stimuli, the event and subsequent behavior (Jay,
2003; Nestler 2001). These adaptations allow the organism to more readily emit an effective
behavioral response in case of similar events. As the event becomes familiar from repeated
exposure and the associated behavior becomes well learned, further neuroplastic changes are
likely no longer necessary and DA release in the NAc may cease to occur in response to similar
events (Schultz, 1998). Thus, it can be thought that the dopaminergic dependent activation of the
NAc by the VTA occurs in order to 1) alert the organism of novel and emotionally salient stimuli
to promote learning, and 2) indicate the presence of cues associated with similar previous events.
Indeed the NAc and VTA are heavily interconnected, however it is important to distinguish
between two anatomically and functionally distinct sub-compartments of the NAc, namely the

shell and core (Kelley, 2004). The shell has been shown to be strongly interconnected with the
VTA and hypothalamus and is thought to be involved in ingestive behaviors (Robinson &
Berridge, 1993; Kelley, 2004). Reciprocal dopaminergic projections between these structures are
thought to modulate motivational salience, contributing to the association of current
environmental perceptions with motivationally relevant stimuli (Di Ciano & Everitt, 2001). For
example, associating the previously neutral sound of splashing water (stimulus) with the
perception that water (reward) is nearby. The core compartment is anatomically associated with
prefrontal cortex and appears to be involved in mediating behavioral responses to motivationally
relevant stimuli leading to the expression of learned behaviors (Di Ciano & Everitt, 2001;
Kelley, 2004), such as following the sound that is associated with water reward. It has been
shown that increased activity of dopaminergic neurons in the VTA leads to increased activity in
prefrontal cortices associated with the NAc core, specifically the anterior cingulate (ACC) and
orbitofrontal (OFC) cortices (Breiter et al., 1997). Further, neuroimaging studies have implicated
the OFC as likely candidates in linking hedonic experience to food reward (Kringelbach, 2005).
It is thought that these structures contribute to the initiation and intensity (i.e. motivational
magnitude) of ensuing behavioral responses by relaying information about the hedonic value of
the reward via glutamatergic efferents to the NAc core (Di Ciano, Cardinal, Cowell, & Everitt,
2001). In fact, one study showed that activation of the OFC in response to rewarding stimuli was
shown to be greatest when the stimulus was unpredictable (Berns, McClure, Pagnoni, &
Montague, 2001), suggesting glutamate release by the PFC relays information about the
predictability of the reward (Volkow et al., 2003). Additionally, Schultz (1998) argues DA
released into the NAc might represent expected reward, producing a form of prediction error of
expected reward vs. obtained reward. These findings illustrate a possibility for DA released in

the NAc from the VTA to act as a representation of potential reward while glutamate released
from the PFC represents information about the predictability and hedonic value of the reward. In
this way, it can be postulated that the network establishes motivation for a goal-directed
behavior, or a „wanting‟ consisting of expected reward in combination with information about
likelihood of reward reception. Finally, such a goal-directed response is initiated in the ventral
pallidum (VP), innervated by dopaminergic VTA and GABAergic NAc projections (Austin &
Kalivas, 1990), thereby comprising an interface between reward system and basal ganglia, where
voluntary motor responses are initiated.
Addiction: Maladaptations in the Mesolimbic Dopamine System
As outlined above, the availability of natural rewards such as food and sex readily
activate the mesolimbic dopamine system (Kelley & Berridge, 2002). Unfortunately, potent
drugs of abuse can effectively “hijack” this system, resulting in long lasting changes in its neural
connectivity (Robinson & Berridge, 1993; Kalivas & Volkow, 2005). Just as all drugs of abuse
increase DA transmission to the NAc after acute administration, common adaptations in DA
function are apparent after chronic exposure and can be understood as a homeostatic response to
repeated drug activation of the system, or tolerance (Koob & LeMoal, 1997). After chronic drug
use, baseline DA function is reduced and as a result normal rewarding stimuli may become less
effective at eliciting DA transmission in the mesolimbic dopamine circuit, which may contribute
to the negative affective state experienced during drug withdrawal. Further, down-regulation of
D2 dopamine receptor expression in the NAc due to constant overstimulation resulting from
chronic drug exposure has been shown to occur (Volkow et al., 1993). Conversely, chronic drug
exposure is also thought to sensitize the circuit, with greater DA transmission in response to the

drug of abuse and associated stimuli (Berridge & Robinson, 2003; Wise, 2004). This
sensitization is long lasting and thought to participate in drug craving and relapse. Perhaps the
longest-lived molecular adaptation of the mesolimbic dopamine circuit known to occur in
response to addictive drugs involves the up-regulation of the transcription factor ΔFosB
(McClung & Nestler, 2003). Unlike other members of the Fos transcription factor family, which
appear upon acute drug exposure, ΔFosB uniquely accumulates in the NAc as a result of chronic
exposure to drugs or natural reward and evidence suggests this as an underlying mechanism of
sensitization, (Nestler, Barrot, & Self, 2004). In fact over-expression of ΔFosB has been shown
to increase behavioral responses to cocaine (McClung & Nestler, 2003; Nestler, 2005; Hyman,
Malenka, & Nestler, 2006).
Structure and Function of the Amygdala
The well-established role of the amygdala in the association of neutral stimuli with fear
responses (for a review see Fanselow & Poulos, 2006; Phelps, 2006) suggests the amygdala as a
likely candidate in associating neutral stimuli with motivationally relevant events. Indeed,
research has documented associative amygdalar function extending beyond formation of fear-
invoking stimuli, asserting involvement of the amygdala in associations pertaining to appetitive
events as well (Everitt et al., 2003). Congruent with this assertion, multiple neuroimaging studies
have shown increased amygdala activation in response to cocaine-related cues but not neutral
ones (Bonsen et al., 2002; Childress, Mozley, McElgin, Fitzgerald, Relvich, & O'Brien, 1999;
Grant et al., 1996; Kilts et al., 2001).
While some doubt exists, the amygdala is generally thought to consist of a diffuse
collection of multiple interconnected nuclei (Figure 3) organized into groupings: the basolateral

complex (BLC) consists of the lateral and basolateral nuclei; the extended amygdala consists of
central (CeN) and medial nuclei in addition to the distant bed nucleus of the stria which lies in
the basal forebrain near the NAc shell (for a review, see LeDoux, 2007). The basolateral
complex is thought to be the primary source of sensory input. Specifically, the lateral nucleus
receives auditory, visual, olfactory, taste, and somamtosensory information from cortical and
thalamic inputs (LeDoux, 2007) of which information about auditory stimuli has been most
extensively examined (LeDoux, Farb, & Romanski, 1991). Information about contextual cues
from the hippocampus and entorhinal cortices arrive at both nuclei of the BLC via the ventral
angular bundle (Maren & Fanselow, 1995), while the insula and posterior thalamus provide
additional information about painful stimuli (Jasmin, Granato, & Ohara, 2004; Lanuza, Nader, &
LeDoux, 2004). Glutamatergic afferents from the BLC projecting to the PFC and NAc are
thought to be necessary for learned associations to influence behavioral responses (Cardinal et
al., 2002). In order for sensory information arriving in the BLC to influence behavior,
intercalated neurons of the amygdala carry information between the BLC and CeN (Marowsky,
Yanagawa, Obata, & Vogt, 2005; Royer, Martina, & Paré, 1999), establishing a connection
between what can be seen as the input (BLC, specifically lateral nucleus) and output (CeN)
stations of the amygdala. The CeN receives information about the homeostatic state of the body
from the brainstem viscero-sensory cortex and is thought to be involved in expression of
emotional and associated physiological responses (LeDoux, 2007). Neurons of the CeN project
to the modulatory systems of brainstem nuclei (LeDoux, Iwata, Cicchetti, & Reis, 1988) thought
to be involved in the arousal while projections to the lateral hypothalamus may influence
endocrine responses (Ehrman et al., 1992; LeDoux, 2007). Additionally, research has shown
activation of VTA neurons innervated by the CeN in response to a footshock (McFarland,

Davidge, Lapish, & Kalivas, 2004). This finding suggests a means by which the amygdala may
couple information about sensory and contextual stimuli with an unconditioned stimulus (the
footshock) and subsequently relay that information to the mesolimbic dopamine system,
suggesting a role for the amygdala in appetitive conditioning (Gallagher & Holland, 1994).
Gaining an intricate understanding of how such stimulus-reward associations are generated,
influenced, and extinguished by the amygdala, and how such processes further influence the
reward system are of paramount concern if the runaway action of potent drugs on the mesolimbic
dopamine system are to be clearly elucidated.
Basolateral Amygdaloid Complex and Addiction: Cue and Reward
Numerous studies indicate the BLC as a very likely neural substrate critically involved in
associative learning of the drug-reward associations involved in addiction. Bilateral pre-training
excitotoxic NMDA lesions of the basolateral amygdala have been shown to disrupt acquisition of
cocaine-conditioned place preference (CPP) in rats (Fuchs, Weber, Rice, & Neisewander, 2002),
whereas a similar study showed that both acquisition and expression of amphetamine-CPP to be
dependent on the lateral amygdala (Hiroi & White, 2000; White & McDonald, 1992). Post
training intra-basolateral infusions of scopolamine, a cholinergic antagonist impaired
consolidation of both food and amphetamine induced CPP (Schroeder & Packard, 2002) and
post-training infusions of glucose facilitated extinction of amphetamine-CPP (Schroeder &
Packard, 2003). Furthermore, BLC lesions disrupt conditioned reinstatement on a cocaine-paired
lever (Meil & See, 1997; Grimm & See, 2000) and permanent excitotoxic quinolinic acid lesions
of the BLC were shown to impair acquisition of cocaine-seeking behavior under a second-order
schedule of reinforcement in rats (Whitelaw, Markou, Robbins, & Everitt, 1996) as well as

responding in rats and monkeys (Everitt, Morris, O‟Brien, & Robbins, 1991; Everitt & Robbins,
2000). Temporary inactivation of the rostral and caudal basolateral amygdala using lidocaine
during reinstatement training have been shown to block relapse to addicted or cocaine-seeking
behavior in rats (Kantak, Black, Valencia, Green-Jordan, & Eichenbaum, 2002) and direct
electrical stimulation of the BLC has been shown sufficient to reinstate cocaine self-
administration after extinction (Hayes, Vorel, Spector, Liu, & Gardner, 2003). Interestingly, one
study suggested inhibition of BLC protein synthesis using asinomycin had no effect on
morphine-CPP while it clearly impaired consolidation of contextual fear conditioning (Yim,
Moraes, Ferreira, & Oliveira, 2006). Clearly, a range of studies has examined the role of BLC in
both formation and expression of cue-reward associations underlying drug-seeking behavior.
However, only very few studies have directly examined the involvement of the BLC in
extinction, and none have specifically examined the role of the BLC during trials of cocaine-CPP
extinction. Understanding the neural mechanisms underlying extinction of drug-predicting cues
will greatly contribute to understanding of learning and addiction.
Justification for Current Approach
Conditioned place preference (CPP), a common behavioral paradigm utilized to measure
reward, generally involves confining an animal to two distinct environments, one paired with a
drug injection and the other with a control, typically a saline vehicle injection (Bardo & Bevins,
2000). Often, after even a single pairing or training session, an animal left to roam the
conditioning apparatus freely will spend more time in the drug-paired context than previously,
and the animal is said to display a conditioned place preference. It should be noted that the
environment-drug pairing extinguishes if the animal spends too much time in or multiply visits

the paired context without reinforcement. Based on the assumption that the animal prefers the
drug-paired environment because it associates the context with drug-induced reward, expecting it
to occur upon subsequent visits, this forced-choice task provides a useful and simple means for
demonstrating rewarding properties of the paired drug. Given that the BLC has been shown to
play a crucial role in establishing cue-reward associations and that cocaine is well known as a
highly rewarding drug, capable of consistently reinforcing drug-seeking behavior (Wise &
Rompre, 1989), it follows that cocaine-CPP should provide a valid means of testing whether
amygdalar function is necessary for cue-reward associations to be established. The aim of the
present study was to investigate whether amygdala function is crucial for the extinction of
previously drug-paired context. It was hypothesized that temporary bilateral lidocaine
inactivation of the basolateral amygdala complex would prevent extinction compared to controls,
and that cocaine-CPP would remain unchanged after extinction trials.
Method
Subjects
Three sets of animals totaling twenty-three male Harlan Spague-Dawley rats (Set A:
240-310g, Set B: 310-360g, Set C: 350-410g) were housed individually in a climate-controlled
colony on a 12h light-dark cycle, with food and water available ad libitum. Set A (n = 10) was
excluded from analysis due to loss of implanted cannulae, two subjects from set B (n=10) died in
surgery and one lost implanted cannulae, no animals in Set C (n=3) were excluded. A total of ten
subjects remained, comprised of six experimental and four control subjects from sets B and C.
All animals were treated in accordance with the guidelines for animal care and use issued by the

Office of Laboratory Animal Welfare and administered by the IACUC at Knox College.
Experimental Procedure
Figure 1. graphically displays the temporal progression of the experiment. In brief, rats
were trained for cocaine conditioned place preference (CPP) on three consecutive days, followed
by one day of rest to allow for consolidation. Following training, bilateral cannulae aimed at the
basolateral amygdala complex (BLC) were surgically implanted and the rats were allowed six
days of rest to recover from surgery. On post-surgery day 7 (PS 7) rats received CPP reminder
training, identical to a single day of training prior to surgery, and were assessed for CPP on PS 8.
Extinction training occured on PS 9-11, animals recieved intracranial perfusions of either
phosphate buffered saline (PBS; control) or lidocaine (experimental) while confined to the
chamber previously paired with cocaine. Rats were assessed for CPP-extinction on PS 12.
Cocaine-Conditioned Place Preference Training
A Plexigas alley was divided into two equal sized black and white opaque
compartments (75cm x 10cm x 19cm each) with one neutral translucent area between the two
compartments (21cm x 10cm x 19cm). Prior to training, rats were placed in the apparatus for 15
minutes with unrestricted access to both areas in order to establish baseline preferences, with
total time in each compartment and movement between compartments recorded. To control for
directional preferences, rats were placed in the neutral area facing either black or white
compartments at the start of testing, and returned to this position facing the opposite
compartment after 7.5 min. Rats were trained for cocaine-conditioned place preference (CPP)
using intraperitoneal (IP) injections of cocaine hydrochloride (Sigma-Aldrich, St. Louis, MI) in

PBS buffer (7.5mg/kg) and confined to the white compartment (cocaine-paired) for 30 min daily
on three consecutive days. Rats were injected with PBS (0.05ml/100g) and confined to the black
compartment (unpaired) for 30 min prior to daily CPP training to equalize exposure across both
sides of the alley. CPP-training was completed prior to surgery and rats were given one day of
rest after training to promote consolidation. Rats received one additional training session on post-
surgery day seven to act as a reminder prior to CPP-testing. Cocaine-conditioned place
preference was measured on PS 8 using the same procedure to assess baseline preference.
Surgery and Cannula Implantation
Rats were deprived of food 24h prior to surgery. Animals were anesthetized using an IP
injection of a ketamine (85mg/kg, Sigma-Aldrich, St. Louis, MI) and xylazine (8mg/kg Sigma-
Aldrich, St. Louis, MI) mixture. Bilateral stainless steel guide cannulae (22 gauge, Plastics One,
Roanoke, VA) were stereotaxically implanted 1mm above the BLA using coordinates derived
from the Paxinos and Watlas Atlas (AP -4.8 mm, L ±5.0 mm, DV -8.5 mm relative to Bregma).
Cannulae were secured with 3-5 jeweler‟s screws and dental acrylic cement (Snap self cure resin
S429 and solvent S441, Parkell, Farmingdale, NY), capped following surgery. Cannulae
uncapped daily to prevent blockage from clotting blood as a large artery ran near the
implantation site. Following surgery, rats were given oral antibiotic (Cefadroxil, Fort Dodge, IA)
and allowed to recover for six days.
Cocaine-Conditioned Place Preference Extinction
Following CPP testing, rats were randomly divided into experimental and control
groups. Extinction consisted of 3 days of confinement to both black and white compartments for

30 minutes each. Prior to confinement in the white compartment, an injection cannula (28 gauge,
Plastics One, Roanoke, VA) attached to a Hamilton syringe via PE-20 tubing was used to
administer intracranial infusions (5µL) of either 2% lidocaine (Sigma-Aldrich, St. Louis, MI)
solution or PBS at a rate of 2.5µL/30 seconds. Subjects received one infusion per cannula and
were tested five minutes following the second infusion to allow time for diffusion. Prior to
confinement in the black compartment subjects were handled by inserting injection cannulae
without performing infusions in order to avoid increased intracranial pressure and prevent
damage. Subjects received five minutes of rest prior to confinement to maintain consistency
across all tests. Post-extinction CPP was measured by allowing rats free access to the entire
apparatus. Time spent in either compartment and movements between compartments were
recorded in a fashion similar to tests to assess baseline and CPP.
Histology
After post-extinction CPP testing, rats received intracranial infusions (5µL) of black ink
to aid in identifying the affected area while sectioning, followed by an overdose IP injection of
sodium pentobarbital (Fort Dodge Laboratories, Fort Dodge, IA). Immediately following
anesthetization, rats were decapitated, their brain extracted and frozen on dry ice (-80°C) until
sectioning. The region extending 2mm rostral and caudal from the amygdala was coronally
sectioned (8μm) on a Cryostat, and sections were then stained using a standard Cresyl Violet
(Sigma-Aldrich, St. Louis, MI) stain as described in the Armed Forces Institute of Pathology
Manual of Histology. Due to a discrepancy in the researched protocol, brains were not perfused
with formalin prior to removal of cannulae and extraction and as thus, were not fixed. Finally
sections were photographed under a microscope and analyzed to determine cannula placement.

Results
Cocaine Conditioned-Place-Preference Training
In order to assess cocaine conditioned place preference, time in white and time in black
difference scores (t[w] and t[b], respectively) were created by subtracting pre-training measures
of time spent in white cocaine-paired and black unpaired compartments (t[w]base and t[b]base,
respectively) from post-training measures t[w]cpp and t[b]cpp (Table 1). Thus, positive scores for
each measure indicate that more time was spent in the respective compartment post-training, one
control subject received a negative difference score for t[w] and was subsequently excluded from
further analysis. In addition to measures of time, difference scores for movement between
compartments pre- and post-training (mcpp - mbase) were also created. A paired t-test showed that
for all rats with a positive difference scores, mean time spent in the cocaine-paired compartment
was significantly higher following CPP training (t(8) = 2.31, p < .001; Figure 4), indicating that
animals had indeed acquired a cocaine-CPP. Additionally, rats spent significantly less time in the
black compartment following training (t(8) = 2.31, p < .001), while a trend for increased
movement between compartments after conditioning arose, it did not reach significance (t(8) =
2.31, p = 0.064), indicating that the increased time spent in the cocaine-paired compartment may
have resulted out of increase in duration of each visit in addition to an increase in the number of
times the compartment was visited.
Cocaine Conditioned-Place-Preference Extinction
To examine the effects of temporary lidocaine inactivation of the BLA on cocaine

conditioned place preference, difference scores similar to those originally used to assess CPP
were created. Difference scores provided a comparison of pre- and post-extinction parameters
such as time spent in the white cocaine-paired compartment (t[w]ext - t[w]cpp), time spent in the
black unpaired compartment (t[b]ext - t[b]cpp), and movement between both compartments (mext -
mcpp) across experimental and control groups. Note that negative scores indicate less time spent
in the respective compartment, or less movement between compartments following extinction.
Both experimental (n=6) and control groups (n=3) attained negative difference scores for time
spent in the cocaine-paired compartment and positive difference scores for time spent in the
unpaired compartment and movement between compartments. Paired t-tests supported these
findings, showing that mean time spent in the cocaine-paired compartment was significantly
lower (t(8) = 2.31, p = 0.0068) following CPP extinction for both control and experimental
groups, while time spent in the unpaired compartment (t(8) = 2.31, p = 0.0093) and movement
between compartments (t(8) = 2.31, p = 0.019) increased, indicating a general trend for
extinction across both groups. However, as can be seen in Figure 5, while experimental animals
appeared to spend less average time in the cocaine-paired compartment following extinction
training compared to control, a t-test showed both groups to be relatively homogenous (t(7) =
2.37, p = 0.48). Congruently, experimentals and controls did not differ in the amount of time
they spent in the black chamber (t(7) = 2.37, p = 0.47). Additionally, while movement between
compartments increased significantly for both groups following extinction training, a t-test
showed that difference scores for movement did not differ significantly between groups (t(7) =
2.37, p = 0.25).
Extinction and Baseline

In order to determine the extent of the extinction training, post extinction data obtained
was compared to original baseline data. A paired t-test showed no difference between baseline
preferences and post-extinction preferences for time spent in the white chamber (t(8) = 2.37, p =
0.61) or black chamber (t(8) = 2.31, p = 0.60) for each group, while movement between
chambers increased significantly compared to baseline (t(8) = 2.37, p = 0.010; Figure 6). In order
to determine whether experimental treatment had an effect in the baseline post-extinction
comparison, difference scores similar to those described above were generated for time in the
white compartment (t[w]ext - t[w]base), time in the black compartment (t[b]ext - t[b]base), and
movement between compartments, (mext - mbase). An independent t-test confirmed the
homogeneity of both groups observed above as no significant differences were found (t[w]: t(8)
= 2.37, p = 0.93, t[b]: t(8) = 2.37, p = 0.84, m: t(8) = 2.37, p = 0.33).
Histology
In order to assess whether cannulae were accurately positioned relative to the basolateral
amygdala ink was perfused through injection cannulae and the injection site successfully
identified during sectioning. Slides of the injection site, including areas rostral and caudal to the
site, were successfully stained using a Cresyl Violet stain (Figure 7). The dark color
charracteristic of cell bodies stained with Creysl Violet was used to successfully determine the
location of the densely packed basolateral amygdala. However, no clear or consistent evidence of
cell bodies tracts typical of scar tissue that develops around cannulae was visible in the region
upon examination. Even so, some sections did display irregularities within a 1-2mm region
dorsal of the basolateral amygdala and nearby ventricles. Specifically, the wall of the right
ventricle dorsal to the BLC appears to be disrupted while the left ventricle appears unaltered

(Figure 7).
Discussion
Following cocaine-CPP training rats spent significantly more time in the cocaine-paired
compartment, indicating cocaine-CPP was readily acquired and suggesting that cannula
implantation following such training will not affect subsequent expression of cocaine-CPP.
However, it is worth noting that this study included neither non-surgery or pre-training surgery
groups, and as such the current finding should be interpreted with caution. In fact, in the case of
a single subject, CPP was not acquired, indicating the possibility of a confound that the current
experimental design was not sensitive enough to detect. Nonetheless, all other subjects, including
those not represented in the final results due to lost or blocked cannulae, readily acquired CPP.
These findings echo research suggesting that excitotoxic post-training lesions of the BLC
increased resistance of cocaine-seeking behavior and cocaine-CPP (Fuchs et al., 2002), and
while rats did extinguish cocaine-CPP in later extinction trials, further investigation will be
necessary to clarify and fully characterize the extent of post-training BLC cannula implantation
effects on CPP and extinction training.
Contrary to previous research (Schroeder & Packard, 2002, 2003), the current findings
suggest that temporary lidocaine-induced inactivation of the BLC prior to cocaine-CPP
extinction sessions has no effect on post-extinction expression of CPP compared to controls. This
study differed from past research as it examined temporary inactivation of the BLC prior to
extinction training whereas past studies that have demonstrated involvement of the BLC in CPP
consolidation have used methods of temporary inactivation after completion of extinction trials
(Kantak et al., 2002; Schroeder & Packard, 2002). It is possible that the BLC is involved in

initial pairing of cue-reward associations but is not recruited in the re-association of drug-related
cues, implying that a separate process is involved in the extinction and re-association of
previously cocaine-paired cues in a drug-free setting. Conversely, the BLC may play an
important role in the consolidation of cue-reward associations that require amygdala activity
after, but not during the contextual pairing of cue and reward.
Methodological considerations may have significantly impacted the current findings and
must also be taken into account. The procedure and techniques were adjusted slightly halfway
through the surgeries in order to improve duration of the cannula implants which may have also
affected outcomes. Lack of a proper syringe pump often resulted in inconsistent infusions that
may have caused solution to drain back into the guide cannulae after injection cannulae were
removed or caused diffusion into nearby ventricles, possibly introducing further confounds.
Additionally, the rapid influx of solution may have led to increased intracranial pressure,
possibly resulting in pain or other adverse effects on cue re-association learning. Due to the
labile nature of the infusion procedure used, both experimenter and subject may have
experienced infusions as aversive, further comfounding the current findings. The large variance
in both experimental and control groups suggests the possibility that further studies accounting
for the aformentioned confounds may yield different results.
In order to assess cannulae positioning relative to the basolateral amygdala, histology
slides of tissue surrounding the amygdala were examined. Scar tissue develops around implants
leaving visible tracts of dark violet cell bodies characteristic of Creysl Violet stains along the
length of the cannulae. Upon examination no clear or consistent cannula tracts were found. It is
possible that the lack of a tissue fixation procedure may have allowed scar tissue to disperse
following brain extraction while the specimens were handled. This may have distorted or

eliminated tracts that typically form around cannula implants. However, some irregularities were
visible, the wall of the right ventricle dorsal to the BLC appears to damaged. When compared to
the continuous wall of the ventrical, the wall tissue of the right ventricle is disrupted, possibly
indicating damage left behind by the insertion or presence of a cannula. Without tissue fixation it
is difficult to accurately verify the injection site relative to the region of interest, and thus
difficult to determine whether lidocaine actually reached the intended target and subsequent
lidocaine inactivation occurred.
Interestingly, an unexpected trend of increased movement between compartments was
observed throughout the progression of the experiment. When initially tested for baseline
preference, movement between compartments was minimal. Upon testing for cocaine-CPP
acquisition the observed increase in movement bordered on reaching statistical significance.
Further, in post-extinction CPP assessment, movement between chambers increased significantly
relative to movement during both baseline and CPP tests. It is possible that the cannulae may
have had an unforseen effect, inducing hyperactivity expressed as an increase in movement
through the conditioning chamber. However, this does not explain why movement increase more
between post-surgery tests than pre-surgery tests. Additionally, this observation has not been
reported in previous research, raising the possibility of a procedural cause. Specifically, limited
initial exposure to the conditioning apparatus may be one explanation for this finding. The rats‟
first exposure to the apparatus was during the baseline preference test, and animals may not have
been comfortable enough with the apparatus to determine accurate measurements of preference.
As the rats spent more time in the apparatus, they may have grown more comfortable and
engaged in search for novel stimuli, thus increasing movement between compartments. Being
uncomfortable with the apparatus may have had confounding effects on the observed baseline

preference or had an effect on learning of cocaine-paired cues in the earlier phases of the
experiment. Further studies would do well to avoid this problem by allowing subjects to become
somewhat accustomed to the conditioning apparatus before any targeted conditioning is initiated.

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Figure Captions
Figure 1. Temporal progression of different phases of the experiment. Displays the different
phases of the experiment in order of completion moving from left to right.
Figure 2. Schematic of reward circuit. Schematic shows brain regions linked to „wanting‟
(yellow), „liking‟ (green), and cognitive incentive processing (blue). (Berridge & Robinson,
2003)
Figure 3. Schematics of amygdala nuclei. a) Schematic shows sensory inputs to diffuse nuclei
of the amygdala, note thalamic and cortical sensory and memory (hippocampal) inputs to the
lateral and basal amygdala. b) Schematic of outputs of the diffuse nuclei of the amygdala,
including basal outputs to motor and cognitive centers and central outputs to subcortical
structures. (LeDoux, 2007)
Figure 4. Time spent in black and white compartments: baseline vs CPP training. Black
bars represent time spent in the black compartment pre- and post-training, white bars represent
time spent in the white compartment. Subjects spent significantly more time in the white
cocaine-paired compared compartment following training (t(8) = 2.31, p < .0010).
Figure 5. Time spent in black and white compartments: difference scores (extinction -
CPP). Represents average of calculated difference-scores for time spent in white and black
compartments after extinction training. Negative scores indicate less time spent in the respective
compartment following training. Both control and experimental groups spent less time in the
cocaine-paired white compartment following training (t(8) = 2.31, p = 0.0068).
Figure 6. Movements between compartments. Represents average number of movements
between compartments per rat observed during each phase of testing. A significant increase in
movements was observed during final extinction testing (t(8) = 2.31, p = 0.010).
Figure 7. Histological sections of region surrounding amygdala. a) Section including
basolateral amygdala and ventricle above, notice disruption in the dorsal region of ventricle wall,
includes magnification to the right. c) Section including basolateral amygdala and intact ventricle
for comparison, includes magnification to the right.

Figure 1.

Figure 2.

Figure 3.
a)
b).

Figure 4.
0
100
200
300
400
500
600
700
800
900
Black White
Time(seconds)
Baseline
CPP

Figure 5.
-500
-400
-300
-200
-100
0
100
200
300
400
500
Control Experimental
DifferenceScore(seconds)
White
Black

Figure 6.
0
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10
15
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Base CPP Extinction
NumberofMovementsbetween
Compartments

Figure 7.
a)
b)

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