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THE EFFECTS OF SIMULTANEOUS COCAINE AND ALCOHOL SELF-ADMINISTRATION ON STRIATAL GLUTAMATE
By
BETHANY A. STENNETT
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2018
© 2018 Bethany A. Stennett
To those affected by addiction
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ACKNOWLEDGMENTS
I give immense thanks to Lizhen Wu for teaching countless skills and being with
me from the very beginning. I would also like to think my friends for their support and
encouragement. I would also like to thank my committee for their guidance.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 8
LIST OF FIGURES .......................................................................................................... 9
LIST OF ABBREVIATIONS ........................................................................................... 11
ABSTRACT ................................................................................................................... 12
CHAPTER
1 GENERAL INTRODUCTION .................................................................................. 14
Substance Use Disorder and Relapse .................................................................... 14 Cocaine Use ........................................................................................................... 15
Alcohol Use ............................................................................................................. 16 Prevalence and Patterns of Combined Alcohol and Cocaine Use .......................... 16
Cocaethylene .......................................................................................................... 18 Animal Models of Drug Administration and Relapse ............................................... 20
Neurocircuitry of Drug-Seeking and Relapse .......................................................... 22 The Role of Nucleus Accumbens Glutamate in Reinstatement of Cocaine
Seeking................................................................................................................ 24 Glutamate Homeostasis .......................................................................................... 25
The Effect of Cocaine on Glutamate Homeostasis ................................................. 27 Glutamate and Alcohol............................................................................................ 28
Ceftriaxone ............................................................................................................. 31 Ceftriaxone and Cocaine .................................................................................. 31
Ceftriaxone and Alcohol ................................................................................... 33 Rationale ................................................................................................................. 34
2 EXPERIMENT 1: USING A RODENT MODEL OF SIMULTANEOUS COCAINE AND ALCOHOL USE TO SCREEN THE ABILITY OF CEFTRIAXONE TO PREVENT COCAINE RELAPSE ............................................................................ 36
Introduction ............................................................................................................. 36
Materials and Methods............................................................................................ 37 Subjects............................................................................................................ 37
Intermittent Drinking Paradigm (IDP) ................................................................ 38 Surgical Procedures ......................................................................................... 38
Drugs ................................................................................................................ 39 Operant Cocaine and Oral Alcohol Self-Administration .................................... 39
Blood Plasma Cocaine and Cocaethylene Levels ............................................ 41 Blood Alcohol Assay ......................................................................................... 41
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Statistical Analyses .......................................................................................... 42 Results .................................................................................................................... 42
Cocaine and Alcohol Intake .............................................................................. 42 Extinction Training ............................................................................................ 44
Cue-Primed and Cocaine-Primed Reinstatement Tests. .................................. 44 Blood Plasma Levels of Cocaine and Cocaethylene and Blood Alcohol
Levels ............................................................................................................ 44 Discussion .............................................................................................................. 45
3 EXPERIMENT 2: THE ROLE OF GLUTAMATE RELEASE IN THE NUCLEUS ACCUMBENS CORE DURING COCAINE REINSTATEMENT IN RATS WITH A HISTORY OF BOTH ALCOHOL AND COCAINE SELF-ADMINISTRATION ......... 62
Introduction ............................................................................................................. 62
Materials and Methods............................................................................................ 64 Subjects............................................................................................................ 64
Intermittent Drinking Paradigm (IDP) ................................................................ 65 Surgical Procedures ......................................................................................... 65
Drugs ................................................................................................................ 66 Operant Cocaine and Oral Alcohol Self-Administration .................................... 67
Microdialysis and Reinstatement ...................................................................... 67 HPLC-ECD for Glutamate Quantification.......................................................... 68
Statistical Analyses .......................................................................................... 68 Correlations ...................................................................................................... 69
Results .................................................................................................................... 69 Behavioral Results ........................................................................................... 69
Cocaine consumption prior to Ceftriaxone treatment ................................. 69 Alcohol consumption prior and during Ceftriaxone treatment ..................... 70
Self-administration lever presses ............................................................... 70 Extinction training. ...................................................................................... 71
Cue+Cocaine–prime reinstatement during microdialysis. .......................... 71 Percent Change of Glutamate in the NAc Core during Reinstatement to
Cocaine Seeking. .......................................................................................... 72 pMol Glutamate in the NAc Core During Reinstatement to Cocaine Seeking .. 74
Correlations ...................................................................................................... 74 Discussion .............................................................................................................. 75
4 EXPERIMENT 3: THE NUCLEUS ACCUMBENS CORE IS LESS ACTIVE DURING REINSTATEMENT TO COCAINE SEEKING IN ANIMALS WITH A HISTORY OF ALCOHOL USE AS COMPARED TO COCAINE USE ONLY .......... 96
Introduction ............................................................................................................. 96
Materials and Methods............................................................................................ 97 Subjects............................................................................................................ 97
Tissue Preparation ........................................................................................... 98 Tissue Slicing ................................................................................................... 98
Immunohistochemistry for C-Fos ...................................................................... 99
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Imaging Brain Regions ..................................................................................... 99 Statistical Analyses .......................................................................................... 99
Correlations .................................................................................................... 100 Results .................................................................................................................. 100
Nucleus Accumbens Core .............................................................................. 100 Nucleus Accumbens Shell .............................................................................. 101
Dorsomedial shell .................................................................................... 101 Ventromedial shell ................................................................................... 102
Lateral shell.............................................................................................. 102 Prefrontal Cortex ............................................................................................ 102
Prelimbic cortex ....................................................................................... 103 Infralimbic cortex ...................................................................................... 103
VTA ................................................................................................................ 103 Correlations .................................................................................................... 103
Discussion ............................................................................................................ 104
5 GENERAL DISCUSSION ..................................................................................... 122
Summary of Results.............................................................................................. 122 Cocaine and Alcohol Effects on Glutamate Homeostasis ..................................... 123
Role of the NAc in Mediating Reinstatement of Cocaine Seeking ........................ 125 NAc Core ........................................................................................................ 125
NAc Shell ........................................................................................................ 127 PFC ................................................................................................................ 128
VTA ................................................................................................................ 129 Conclusions .......................................................................................................... 130
Future Directions .................................................................................................. 131
LIST OF REFERENCES ............................................................................................. 132
BIOGRAPHICAL SKETCH .......................................................................................... 145
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LIST OF TABLES
Table page 4-1 Number (n) of tissue samples per group. ......................................................... 100
4-2 Correlation of AUC Glutamate p value. ............................................................ 104
4-3 Correlation of EtOH Consumption p value. ....................................................... 104
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LIST OF FIGURES
Figure page 2-1 Timeline .............................................................................................................. 38
2-2 Cocaine infusions during self-administration ...................................................... 49
2-3 Cocaine infusions (mg/kg) during self-administration ......................................... 50
2-4 Ethanol consumed during self-administration. .................................................... 51
2-5 Total cocaine intake mg/kg during self-administration. ....................................... 52
2-6 Total ethanol intake during self-administration ................................................... 53
2-7 Active lever presses during self-administration................................................... 54
2-8 Inactive lever presses during self-administration. ............................................... 55
2-9 Lever presses on the previously active lever during extinction training .............. 56
2-10 Inactive lever presses during extinction training. ................................................ 57
2-11 Cue-Primed and cocaine-primed reinstatement tests ......................................... 58
2-12 Plasma Cocaine levels ....................................................................................... 59
2-13 Plasma cocaethylene levels ............................................................................... 60
2-14 Blood alcohol levels. ........................................................................................... 61
3-1 Cocaine infusions during self-administration ...................................................... 83
3-2 Cocaine infusions (mg/kg) during self-administration ......................................... 84
3-3 Ethanol consumed during self-administration ..................................................... 85
3-4 Total cocaine intake (mg/kg) during self-administration ...................................... 86
3-5 Total ethanol intake during self-administration ................................................... 87
3-6 Active lever presses during self-administration................................................... 88
3-7 Inactive lever presses during self-administration ................................................ 89
3-8 Lever presses on the previously active lever during extinction training. ............. 90
3-9 Inactive lever presses during extinction training ................................................. 91
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3-10 Cue+Cocaine–prime reinstatement during microdialysis .................................... 92
3-11 Inactive lever presses during Cue+Cocaine-prime reinstatement testing ........... 93
3-12 Percent change of glutamate in the NAc core during reinstatement to cocaine seeking ............................................................................................................... 94
3-13 pMol glutamate in NAc core during reinstatement testing .................................. 95
4-1 NAc core Fos expression.................................................................................. 113
4-2 NAc shell total Fos expression ......................................................................... 114
4-3 Dorsomedial NAc shell Fos expression ............................................................ 115
4-4 Ventromedial NAc shell Fos expression ........................................................... 116
4-5 Lateral NAc shell Fos expression ..................................................................... 117
4-6 Prefrontal cortex total Fos expression .............................................................. 118
4-7 Prelimbic cortex total Fos positive cells ............................................................ 119
4-8 Infralimbic cortex Fos expression ..................................................................... 120
4-9 VTA Fos expression ......................................................................................... 121
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LIST OF ABBREVIATIONS
Cef Ceftriaxone
CE Cocaethylene
CFZ Cefazolin
Coc Cocaine
DA Dopamine
EtOH Ethanol
EXT Extinction
GLU Glutamate
H2O Water
HPLC High Pressure Liquid Chromatography
IDP Intermittent Drinking Paradigm
NAc Nucleus Accumbens
PFC Prefrontal Cortex
SAL Saline
Veh Vehicle
VTA Ventral Tegmental Area
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
THE EFFECTS OF SIMULTANEOUS COCAINE AND ALCOHOL SELF-
ADMINISTRATION ON STRIATAL GLUTAMATE
By
Bethany A. Stennett
August 2018
Chair: Lori A. Knackstedt Major: Psychology
Cocaine addiction is a significant public health problem in the United States
today. One of the difficulties in successful treatment of cocaine use disorder is in
reducing the high risk of relapse that exists even after long periods of abstinence.
Relapse can be modeled in animals using the extinction-reinstatement paradigm. This
paradigm involves training animals to lever-press for cocaine reinforcement in an
operant chamber. The operant response is then extinguished and reinstated with either
cues previously paired with the response made to attain cocaine delivery. Previous
research has established the role of nucleus accumbens (NAc) glutamate transmission
in the reinstatement of cocaine seeking and has shown that the drug Ceftriaxone (Cef)
prevents relapse to cocaine seeking in rats. However, it is estimated that 60% to 90% of
cocaine addicts use alcohol with cocaine. The combination of alcohol and cocaine
potentially produces unique neuroadaptations that differ from those produced by either
drug alone. Therefore, we used a model of poly-drug use in which rats self-administer
cocaine for two hours in an operant chamber and subsequently drink alcohol (20% v/v)
from bottles in the home cage for 6 hours. Following two weeks of drug consumption,
animals underwent extinction training. Our data reveal that chronic Ceftriaxone (100 or
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200 mg/kg) does not alter cue- or cocaine-primed reinstatement of cocaine seeking in
animals that consumed alcohol in addition to cocaine. We then utilized microdialysis to
examine changes in glutamate efflux in the nucleus accumbens core during
reinstatement. Following chronic cocaine and alcohol self-administration, glutamate
efflux in the nucleus accumbens did not accompany reinstatement of cocaine seeking,
whereas it did for those rats self-administering cocaine alone. Ceftriaxone did not
prevent relapse in animals that consumed both alcohol and cocaine. Fos activation was
in agreement with glutamate release into the NAc core. Interestingly, rats with a history
of Coc+EtOH use showed more Fos expression than the saline control group but less
than vehicle treated Coc+H2O animals.
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CHAPTER 1 GENERAL INTRODUCTION
Substance Use Disorder and Relapse
Substance use disorder is a chronic disorder that is marked by periods of abuse,
dependence, abstinence, and relapse. It can be further defined by several criteria
including maladaptive behaviors, neurological and psychological changes such as
uncontrollable motivation to seek out drugs while losing motivation to seek non-drug
rewards (Goldstein and Volkow, 2002). The neuronal pathways subserving memory,
motivation, and reward are altered by chronic drug use and have resulted in addiction
being classified as a brain disorder (Cadet et al., 2014).
Although there are behavioral and pharmacological approaches to addiction
treatment, the rate of relapse to both illegal and legal drug use remains high (O’Brien
and Gardner, 2005). Studies investigating relapse rates and chronology support the
idea that addiction is a chronic and reoccurring disorder. For example, a longitudinal
study followed heroin users on methadone treatment and found that 86% of users
relapsed to heroin use within 5 years of treatment (Termorshuizen et al., 2005). Though
it may seem like a respectable amount of time, the individuals essentially still relapse to
drug use. Despite these studies, the relapse rates for opiate and psychostimulant
addiction are between 50-80% within the first year after treatment (Bailey and
Husbands, 2014). Another study indicates that 60-80% of abstinent alcoholics will
relapse in their lifetime at least once (Barrick and Conners, 2002). Given the harmful
effects of drug use on the user’s health as well the ineffectiveness of current addiction
treatments, there is a pressing need for novel and effective treatments for addiction.
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Cocaine Use
Cocaine use disorder is classified as a chronic, compulsive, and uncontrollable
disease affecting over a million individuals every year (NIDA, 2014). In the 2011
National Survey on Drug Use and Health, approximately 1.4 million Americans reported
active use of cocaine (SAMHSA, 2011). Cocaine itself is a quick acting, highly addictive
psychostimulant drug due to its short half-life and multiple ways of administration, but
mostly because of its effects on brain reward pathways. Within the brain, cocaine has
two means of action resulting in strong reinforcing effects that can lead to compulsive
use. One mechanism of action is blocking the re-uptake of noradrenaline, serotonin,
and dopamine allowing longer effects of their post-synaptic actions (Ritz et al., 1990).
Elevated extracellular dopamine concentrations within the brain can result in increased
locomotor activity as well as it’s reinforcing properties. Cocaine can also block voltage-
dependent sodium channels that can alter glutamate levels in the synapse (Reith, Kim,
and Lajtha, 1986).
Routes of administration for cocaine include intranasal, insufflation, and
intravenous (IV) use (Gossop et al., 2006). Intranasal is the most commonly used route
of administration for cocaine and is pharmacokinetically similar to that of IV
administered cocaine. The importance of this fact will be discussed later in regards to
animal models of drug abuse. The acute effects of cocaine include increased energy,
alertness, and feelings of intense euphoria. Cardiovascular effects of cocaine include
increased heart rate and blood pressure (Andrews, 1997). An overdose of cocaine can
result in strokes, headaches, cardiovascular effects, and seizures (Nnadi et al., 2005).
Side effects from long-term use of cocaine include depression, anxiety, and craving
(Cadet et al., 2014).
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Alcohol Use
Alcohol use and abuse is a serious public health concern. According to the
National Institute of Alcohol Abuse and Alcoholism (2015), approximately 15.1 million
adults have an alcohol use disorder (AUD) in the United States. Alcohol use disorders
are the third leading cause of preventable deaths in the United States, resulting in over
100,000 annual deaths (Mokdad et al., 2004). Currently, there are only three FDA
approved medications to treat alcoholism: disulfiram, naltrexone, and acamprosate.
Sadly, these medications are minimally effective in maintaining abstinence and
preventing relapse (Heilig and Egli, 2006; Soyka and Roesner, 2006). Alcohol alters
extracellular glutamate transmission throughout the reward pathway (for review see:
Gass et al., 2010). Taking that into account, medications targeting the glutamate
transmitter system hold promise for reducing alcohol intake as well as use of other
addictive drugs (for review see: Olive et al., 2012).
Prevalence and Patterns of Combined Alcohol and Cocaine Use
The majority of cocaine users are polydrug users, reporting use of more than one
drug at one time (Rounsaville et al., 1991; Jatlow, 1993). Polydrug use produces
different and more complex maladaptive behavioral and physiological changes than
using one drug alone. Two general patterns of polydrug use have been documented;
the first pattern is to use two or more drugs simultaneously. The second pattern involves
a sequential or staggered administration of two or more drugs at multiple points in time
over the course of a day to longer periods of time (Leri et al., 2003). In fact, studies
surveying cocaine users found that they report using alcohol in close proximity to
cocaine and in an intermingled fashion (Barrett et al., 2016; Gossop et al., 2006;
Macdonald et al., 2015).
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There is a greater association between alcohol and cocaine dependence than
with any other drug combination (Helzer and Pryzbeck, 1988), and it has been
estimated that 62% to 90% of cocaine users co-abuse alcohol with cocaine (Rounsaville
et al., 1991; Brookoff et al., 1996; Weiss et al., 1988). Lifetime prevalence for co-morbid
alcohol use within cocaine users is 62% (Brookoff et al., 1996; Rounsaville et al., 1991).
In an American sample of 302 cocaine dependent individuals admitted for addiction
treatment, an analysis revealed that 75% of patients reported using both cocaine and
alcohol (Heil et al., 2001). In a nonclinical sample, the rates of alcohol co-use at the time
of the most recent cocaine use were estimated between 79 and 87% (Barrett et al.,
2016; Licht et al., 2012).
An important reason for attention to this issue is patients with cocaine and
alcohol co-dependence have poorer clinical outcomes than those with cocaine
dependence alone (Brady et al., 1995; Schmitz et al., 1997). A placebo-controlled study
of modafinil to decrease cocaine use in 210 outpatient treatment-seekers was
conducted over a 12-week treatment period with a 4-week follow-up. This study
observed a significant difference in the days of non-cocaine use between individuals
with a history of only cocaine use compared to those with a history of alcohol use in
addition to cocaine. Modafinil was successful in individuals that did not have a history of
comorbid alcohol and cocaine use, but not in those that did (Anderson et al., 2009).
Modafinil has shown some success with decreasing cocaine use and cravings, but upon
further investigation, that study had excluded individuals with a history of alcohol use
(Dackis et al., 2005). This pattern exemplifies why it is important to address comorbid
cocaine and alcohol use compared to each drug individually. Treatments that work on
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one drug alone, does not mean it will be able to produce the same effect if a person has
a history of polydrug use. Given the prevalence of high cocaine and alcohol-combined
use (62-90%), further research needs to address the chronic effects of both drugs in
order to cater towards a larger population of substance abusers.
Thus, the use of an animal model of simultaneous/sequential cocaine and EtOH
use is necessary in order to generate more accurate pre-clinical data regarding the
neuroadaptations produced by this drug combination and the efficacy of medications to
attenuate cocaine relapse in comorbid addicts. Currently, few studies have assessed
alcohol and cocaine in combination, let alone while both drugs are being self-
administered. Outside of our group, the effects of experimenter-administered alcohol on
cocaine self-administration have been evaluated in rhesus monkeys (Aspen and
Winger, 1997). Two of the four monkeys increased their responding for cocaine after
being pretreated with 1 g/kg of alcohol (Aspen and Winger, 1997). In awake, rhesus
monkeys that have a history of cocaine self-administration, there was an increase in
mesolimbic extracellular dopamine following ethanol administration (Bradberry, 2002).
Currently, no research has evaluated simultaneous cocaine and alcohol use followed by
a period of extinction and relapse.
Cocaethylene
When cocaine and alcohol are consumed simultaneously, the liver forms the
metabolite cocaethylene (CE). Cocaethylene is the ethyl ester of the cocaine
metabolite, benzoylecgonine (Pan and Hedaya, 1999; McCance-Katz et al., 1993;
McCance et al., 1995). In fact, cocaethylene has been found to be pharmacologically
active (Pan and Hedaya, 1999) and displays psychomotor stimulant characteristics
similar to that of cocaine (Jatlow et al., 1991).
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A group of non-treatment seeking, active cocaine and alcohol abusers
participated in a study where they consumed low and high doses of CE, cocaine, or
placebo via intranasal insufflation. Upon consuming CE, participants in this study
reported feelings of being “high” and experiencing “euphoria” mirroring the effects of
cocaine. In fact, many participants were unable to distinguish the pleasant effects of CE
from cocaine. Other similarities between CE and cocaine were observed including
increased diastolic and systolic blood pressure and increased heart rate (McCance et
al., 1995).
In rodents, CE has been used in many drug abuse models such as self-
administration (Jatlow et al., 1991) and conditioned place preference (Knackstedt et al.,
2002) exemplifying the rewarding properties of consuming cocaethylene. In addition,
cocaethylene is capable of inducing increased motor movement, stereotypy, and
behavioral sensitization in rodents. These effects last roughly 30 minutes (Horowitz et
al., 1997). Taken together, the effects of CE on behavior in rodents are equivalent to
that of cocaine (for review see Horowitz and Torres, 1999).
Within the brain, cocaethylene blocks the reuptake of dopamine in the synapse
by binding to presynaptic dopamine transporters similar to cocaine (Jatlow et al., 1991).
Interestingly, the half-life of cocaethylene is longer than that of cocaine in both humans
(McCance-Katz et al., 1993; McCance et al., 1995; Perez-Reyes et al., 1994) and
rodents (Pan and Hedaya, 1999). In rodents the plasma half-life of cocaine is 15.8 ± 1.5
min, whereas the CE plasma half-life is 25 ± 3.1 minutes (Pan and Hedaya, 1999).
Therefore, similar to cocaine, cocaethylene acts on the dopaminergic system within the
brain and has equivalent effects in both humans and rodents.
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Summarizing what has been discussed thus far, cocaine and alcohol are
commonly abused together by humans and this has been replicated in non-human
primate samples. One difficulty of substance use disorder and dependence treatment
for individuals with a history of co-morbid cocaine and alcohol abuse is poorer observed
outcomes compared to individuals that use only one of the drugs. Both drugs work in
conjunction on the dopamine system within the brain, including the psychoactive
metabolite cocaethylene. Procuring a better understanding of neurological actions
produced by concurrent use of both cocaine and alcohol will allow for better treatment
options for the human population. In order to discover these effects, rodent models of
polydrug use need to be utilized. By using a rodent model, we can gain a better
understanding of comorbid cocaine and alcohol use from the behavioral to the
neurotransmitter level.
Animal Models of Drug Administration and Relapse
In order to shed light unto the cellular, molecular, and neurological mechanism
behind substance abuse, various animal models of addiction have been created. Using
these models, we are able to gain a better understanding of the acquisition,
maintenance, and the inability to stop use of drugs (Epstein et al., 2006). The
reinstatement model of relapse has been developed to study relapse in experimental
animals. In the reinstatement model, animals are first trained to self-administer drugs by
pressing a lever, for example, in order to receive intravenous drug infusions in operant
chambers. Lever presses are often paired with conditioned stimuli such as an
illumination of a light above the lever and the playing of a tone. An alternate lever is
present, opposite the lever that results in a drug infusion, and is referred to as an
“inactive lever.” The purpose of the inactive lever is to demonstrate the animal’s
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preference for the active lever and to show that lever pressing is not as result of general
locomotor activity.
After stable levels of responding for the drug-reward have been achieved, the
animal goes through ‘extinction’ of the response made to obtain drug. Presses upon the
previously active lever no longer result in drug delivery and the paired light and tones.
As a result, pressing on the once active lever declines. Once levels of responding are
minimal, animals can then be presented with the drug-paired cues (cue-primed
reinstatement) such as the light and tone that was previously paired with drug delivery,
stressors, or the actual drug (drug-primed reinstatement) in order to ‘reinstate’ drug-
seeking behavior (Epstein et al., 2006). To be defined as reinstated to drug seeking,
animals must resume presses on the active lever during testing and reach a significantly
higher level of responses than during the extinction sessions (Katz and Higgins, 2003;
O’Brien and Gardener, 2005). The reinstatement model appears to have good
predictive validity because conditions that provoke drug relapse and craving in humans
(drug re-exposure, drug cues, and stress) also reinstate, for example, heroin and
cocaine seeking following prolonged withdrawal periods in laboratory animals
(Knackstedt and Kalivas, 2009; Shalev et al., 2002).
Other animal paradigms are also useful for the study of drug use like conditioned
place preference (CPP) or behavioral sensitization. CPP is thought to measure “reward”
rather than “reinforcement” and thereby does not fall as synonymously with human
substance use disorder as the drug self-administration paradigm (For review see: Bardo
and Bevins, 2000). Behavioral sensitization is commonly used to assess drug-induced
increases in locomotor activity, which is associated with the enhancement of the
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rewarding effect of drugs (Kauer and Malenka, 2007). Though CPP and behavioral
sensitization are widely used to assess varying aspects of substance use disorder, we
utilize self-administration and will therefore focus on studies using the drug self-
administration and reinstatement model of drug use. The benefit of using a self-
administration model over other models such as CPP or non-contingent administration
is because self-administration better models human patterns of drug use. In a self-
administration model, the rodent must “work” to get the drug. Therefore, we can infer
the animals are actively seeking out drug use on their own accord. As I will discuss in
the next section, neurobiology differs based on whether the animal chooses to take the
drug rather than passively receiving the drug.
Neurocircuitry of Drug-Seeking and Relapse
Compulsive drug seeking involves complex underlying neural circuitry involving
multiple neurotransmitter systems. The above mentioned extinction-reinstatement
model has been used to identify brain areas involved in relapse (McFarland and
Kalivas, 2001). For example, limbic system circuitry has long been studied in regards to
drug seeking behavior and relapse. The limbic system is comprised of the prefrontal
cortex (PFC), the basolateral amygdala (BLA), the nucleus accumbens (NAc) (or
striatum), and the ventral tegmental area (VTA) (Kalivas, 2009).
This knowledge has been gained via use of animal models of cue-induced and
drug-induced reinstatement as well as clinical studies of drug abuse (Kalivas et al.,
2009; McFarland and Kalivas, 2001; Goldstein and Volkow, 2002). Briefly, cue induced
reinstatement can be blocked by inactivation of the dorsal prefrontal cortex via
pharmacological (McLaughlin and See, 2003) and optogenetic manipulations (Stefanik
et al., 2016). Optogenentic inhibition of the PrL afferent neurons to the NAc prevent cue-
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primed reinstatement to cocaine seeking (Stefanik et al., 2016), thus furthering the
argument that the PFC, more specifically, the PrL projections to the NAc core are
necessary for reinstatement to drug seeking (Kalivas and McFarland, 2003; McFarland
et al., 2014). Intracranial microinjections of GABAA (muscimol) and GABAB (baclofen)
agonists into differing brain regions can be used to isolate direct connections in the
limbic circuitry. Inactivation of the PrL projections to the NAc core attenuated cocaine-
primed reinstatement to cocaine seeking (McFarland and Kalivas, 2001). More
specifically McFarland and Kalivas (2001) found the flow of this pathway to be from the
VTA to the PrL to the NAc core by contralateral activation of these brain regions using
the GABA agonist cocktail, Baclofen Muscimol (McFarland and Kalivas, 2001).
The sodium channel blocker tetrodotoxin (TTX) can be infused into the brain as a
reversible targeted inactivation tool. After a period of extinction, bilateral infusion of TTX
(5 ng/ 0.5 ul/side) in the BLA or PrL impaired reinstatement to cue-primed cocaine
seeking. However, TTX inactivation of the IL had no effect on cue-primed reinstatement
(McLaughlin and See, 2003). The difference in PrL and IL findings is likely given the PrL
has projections going to the NAc core, whereas the IL sends projections to the NAc
shell.
Inactivation studies have also been used to isolate the role of the BLA. For
example, inactivation of the rostral and caudal BLA via a high dose of lidocaine (56 –
100 ug) prevented cue+cocaine-primed reinstatement to cocaine seeking following a
period of extinction and abstinence (Kantak et al., 2002). A low dose of lidocaine (10 ug)
in the rostral BLA prevented cue-prime reinstatement to cocaine seeking, but not in the
caudal (Kantak et al., 2002). In addition, responding to cues was prevented by lesions
24
to- and inactivation via TTX to the BLA (Meil and See, 1997; Grimm and See, 2000).
More recently, optogenetic inhibition of the BLA inhibited cue-primed reinstatement to
cocaine seeking (Stefanik and Kalivas, 2013). Taken together, these findings display
the role of the BLA in cued reinstatement to cocaine seeking.
It is generally accepted that changes in the nucleus accumbens and its
associated circuitry have an influential role in reward-dependent learning, impulsive
activity, and addiction. The reinforcing effects from drug abuse are powerful enough to
commandeer the reward circuitry and produce the maladaptive and pathological
behaviors that define addiction (Kauer and Malenka, 2007; Koob and Volkow, 2010).
Anatomically, the nucleus accumbens can be divided into two regions, the core and the
shell. Both the NAc shell and core share multiple qualities including mediating forms of
Pavlovian conditioning and learned behaviors (Voorn et al., 2004). However, certain
functional distinctions can be made between these two sub regions of the NAc.
The Role of Nucleus Accumbens Glutamate in Reinstatement of Cocaine Seeking
The NAc can be seen as a gateway through which information is processed in
the limbic system. In fact, cocaine-primed reinstatement to cocaine seeking is
consistently found to be accompanied by glutamate release into the NAc core
(McFarland et al., 2003; Lutgen et al., 2012; Trantham-Davidson et al., 2012). After two
weeks of cocaine self-administration and 6 days of extinction training, an increase in
NAc glutamate was observed during a cocaine-primed reinstatement test via
microdialysis (Trantham-Davidsion et al., 2012). Specifically, the corticostriatal
glutamate projections (PFC to NAc) are responsible for cocaine-primed reinstatement of
cocaine seeking (McFarland et al., 2003). Indeed, pharmacological deactivation of the
PFC prevented cocaine-primed reinstatement following a period of extinction
25
(McFarland et al., 2003). The necessity of glutamate binding in the NAc core to facilitate
relapse-like behavior is evidenced by blocking reinstatement behaviors with an intra-
accumbens infusion of the AMPA/kianate receptor antagonist CNQX (Cornish and
Kalivas, 2000).
The importance of glutamate transmission in reinstatement to cocaine seeking
has also been demonstrated in cue-primed reinstatement models (Hotsenpillar et al.,
2001; Sari et al., 2009; Smith et al., 2017). Mirroring the effects on cocaine-primed
reinstatement to cocaine seeking, blocking the same AMPA/kainate ionotropic receptors
in the NAc core prevented cue-induced reinstatement to cocaine seeking (Di Ciano and
Everitt, 2001). Optogenetic inhibition of the glutamatergic PrL neuron terminals in the
NAc core prevented cue-primed reinstatement to cocaine seeking (Stefanik et al.,
2016). Inactivation of the glutamatergic amygdala afferent projections to the NAc core
via optogenetic mechanisms successfully prevented cue-primed reinstatement to
cocaine seeking (Stefanik and Kalivas, 2013). Utilizing DREADDs technology by
administration of a GFAP-Gq-DREADD infused bilaterally into the NAc core, a systemic
injection of CNO prior to a cue-prime reinstatement test inhibited responding in rats
compared to vehicle treated counterparts (Scofield et al., 2015). Taking the above-
mentioned research into account, glutamate transmission in the NAc is critical for both
cue and cocaine-primed reinstatement to cocaine seeking.
Glutamate Homeostasis
Glutamate is the major excitatory neurotransmitter found in the CNS. Too much
glutamate in the synapse can act as a neurotoxin and cause excitatory neurotoxicity
(Rothstein et al., 1996). To prevent this from happening, there are several mechanisms
involved with maintaining a healthy level of glutamate throughout the brain. There are
26
two methods of glutamate release into the extracellular space: synaptic or nonsynaptic
(glial). The balance between release and reuptake is what can be referred to as
glutamate homeostasis (Baker et al., 2002).
Basal extrasynaptic glutamate concentrations are sourced primarily from
nonsynaptic glial release. In the nucleus accumbens core, ~60% of the basal
extracellular glutamate is derived from the cystine-glutamate exchanger, system xC-.
This system is responsible for removing cystine molecules from the extracellular space
and outputs intracellular glutamate (Bannai and Ishii, 1982). xCT is a subunit of system
xC- which exchanges an extracellular cysteine for an intracellular glutamate and is
expressed predominantly on glia (Baker et al., 2002). The cystine uptake and glutamate
release occurs in a 1/1 stoichiometry (McBean and Flynn, 2001). This exchanger is
sodium independent, meaning it does not require energy (McBean, 2002).
Once released into the extracellular space, glutamate needs to be eliminated in
order to maintain homeostasis. Glutamate transporters are located on glial cells and
neurons throughout the brain (Tzingounis and Wadiche, 2007). Glutamate transporter 1
(GLT-1) is a major glial glutamate transporter accounting for 90% of total brain
glutamate uptake. Being the primary mediator of glutamate transport into astrocytes,
GLT-1 plays a substantial role in preventing excitatory neurotoxicity (Haugeto et al.,
1996; Danbolt, 2001). More specifically, GLT-1 partitions the non-vesicular (arising from
system xC-) and vesicular pools of glutamate (Danbolt, 2001). Within the NAc, GLT-1 is
vastly expressed and is concentrated near the synaptic cleft (Danbolt, 2001). After
removal from the synapse via GLT-1, glutamate is converted into glutamine and
subsequently released by the astrocyte into the extracellular space. Once released,
27
glutamine is taken up by neurons and the enzyme glutaminase then converts it back
into glutamate. Afterwards, glutamate is packaged into vesicles via the vesicular
glutamate transporter, vGluT, and is ready to be released into the synapse, completing
the cycle (Albrecht et al., 2010).
Another mechanism contributing to glutamate homeostasis is the Gi/Go -coupled
group II metabotropic glutamate receptor (mGlu2/3) located on presynaptic neurons in
the NAc (Cartmell and Schoepp, 2000). The function of mGlu2/3 is as an autoreceptor.
This means that once enough glutamate has filled the synaptic space to “overflow”,
glutamate diffuses away from the cleft and thereby activating the perisynaptic mGlu2/3
site, which results in an inhibition of glutamate release from the presynaptic neuron
(Cartmell and Schoepp, 2000). Subsequently, there is attenuation of synaptic glutamate
release. To be more specific, glutamate release can be inhibited presynaptically by
stimulating the mGlu2/3 release regulating autoreceptors. In fact, administration of
mGlu2/3 agonist, LY379268, inhibits the reinstatement of cocaine seeking (Peters and
Kalivas, 2006; Baptista et al., 2004). In addition, activation of mGlu2/3 inhibits relapse to
cocaine seeking when given systemically during a cue-primed reinstatement test
(Cannella et al., 2013). An agonist of mGlu2/3 also prevents drug seeking when
administered via the NAc core during a cocaine-primed reinstatement test (Peters and
Kalivas, 2006). Therefore, mGlu2/3 receptor stimulation is capable of preventing both
relapse to cocaine seeking and the glutamate efflux that accompanies it.
The Effect of Cocaine on Glutamate Homeostasis
Basal extrasynaptic glutamate in the NAc core is decreased following chronic
self-administration of cocaine (Baker et al., 2003). This decrease in basal glutamate is a
result of reductions in the protein, xCT (Knackstedt et al., 2010). The release regulating
28
autoreceptor, mGlu2/3, also has reduced tone following prolonged cocaine
administration (Xi et al., 2002; Moussawi and Kalivas, 2010). GLT-1 expression is also
downregulated following cocaine use. A reduction in GLT-1 expression results in a
reduction in glutamate uptake (Knackstedt et al., 2010). This poses a problem given the
overflow of glutamate into the synapse during reinstatement to cocaine seeking.
During reinstatement, the additional overflow of glutamate outside of the synapse
is a product of GLT-1 downregulation. In fact, self-administration of many drugs of
abuse including cocaine and alcohol, results in a down-regulation of GLT-1 (Alhaddad
et al., 2014b; Reissner et al., 2014; Sari et al., 2011) and the subsequent spillover of
glutamate released into the NAc core during reinstatement. Reduced GLT-1 function
after cocaine (Knackstedt et al., 2010) likely results in spillover of vesicular glutamate to
extrasynaptic receptors. In support of this idea, intra-NAc antagonism of the
extrasynaptic mGlu5 receptors attenuates the reinstatement of cocaine seeking (Wang
et al., 2012). To summarize, cocaine-induced reinstatement to drug seeking is
associated with synaptic NAc glutamate release and basal levels of NAc glutamate are
decreased following chronic cocaine use.
Glutamate and Alcohol
As mentioned above, a substantial factor contributing to treatment failure in
cocaine addicts is the comorbid use of alcohol with cocaine. To better understand
treatment mechanisms for these cocaine addicts, we must understand the role alcohol
plays in glutamate homeostasis following chronic use of alcohol. We have established
that glutamate homeostasis in the NAc is disrupted following chronic cocaine use. In
sum, the basal level of glutamate in the NAc is decreased after chronic cocaine and
glutamate release accompanies relapse to cocaine seeking. In fact, prolonged alcohol
29
use also results in glutamate homeostasis disruption. The assessment of alcohol’s
ability to alter the well-characterized glutamate adaptations produced by cocaine is
essential in order to develop treatments for addicts who abuse both cocaine and
alcohol.
Both non-contingent and contingent alcohol administration are accompanied by
increased extracellular glutamate concentration in the NAc. We previously found that
voluntary intermittent alcohol consumption in outbred male rats increased basal
extracellular glutamate in the NAc core when assessed 24 hours after the removal of
alcohol access (Pati et al., 2016). Increased NAc glutamate transmission also
accompanies operant self-administration of alcohol (Li et al., 2010) and relapse to
operant alcohol-seeking after a withdrawal period (Gass et al., 2010). Thus, basal
glutamate in the NAc is found consistently as increased in early abstinence from
alcohol. Given that chronic cocaine results in decreased basal glutamate after a period
of withdrawal and alcohol is an increase, it is plausible that a history of both drugs would
leave basal glutamate levels different than the level of either drug alone.
While decreases in NAc basal extrasynaptic glutamate following cocaine have
been attributed to decreased expression of xCT and function of system xC- (Baker et al.,
2003; Knackstedt et al., 2010), increases in basal extrasynaptic glutamate following
intermittent access to alcohol have not been associated with increased xCT expression
(Pati et al., 2016) or function (Griffin et al., 2015). Interestingly, continuous access to
alcohol results in downregulation of xCT expression in the NAc of male P rats, but not
when a one-week period of alcohol abstinence is implemented (Alhaddad et al., 2014a).
Thus, the role of xCT/system xC- in mediating changes in NAc basal extracellular
30
glutamate levels following alcohol is currently not well understood. Pulling this together
with the knowledge of a downregulation in xCT following chronic cocaine use, a history
of both alcohol and cocaine use could either result in a further decrease in xCT
expression or a level where the effects of cocaine and alcohol on xCT counteract each
other.
Following continuous alcohol access (5 weeks), there are consistent reports of
downregulated GLT-1 expression in the NAc of alcohol-preferring rats (Sari et al.,
2013a; Alhaddad et al., 2014a, 2014b; Hakami et al., 2016). Therefore, GLT-1
downregulation is hypothesized to mediate the increase in basal glutamate levels.
However, we have previously shown that outbred Sprague-Dawley rats with access to 7
weeks of intermittent access to alcohol display no changes in NAc GLT-1 surface
expression, but still an increase in basal glutamate (Pati et al., 2016). Therefore, similar
to the inconsistent findings on the influence alcohol has on xCT, changes in GLT-1
expression are also not well understood at this time.
Given the above-mentioned information about chronic cocaine and alcohol
separately, it is plausible that in animals consuming both drugs, basal levels of
glutamate may be different than after either drug alone. In fact, the decreased basal
glutamate levels following chronic cocaine use could counterbalance the increased
basal glutamate levels resulting from chronic alcohol use. If that is the case, this
counterbalance could result in NAc glutamate homeostasis resembling that of a drug
naïve animal. However, this result would theoretically only be possible if changes in
basal glutamate levels equally changed from chronic use of both drugs. At this time, we
31
do not know the magnitude of glutamate homeostasis change in the NAc that would
ensue following repeated use of both drugs.
Ceftriaxone
Ceftriaxone, a beta-lactam antibiotic, induces up-regulation and activation of
GLT-1 in the brain (Rothstein et al., 2005). Following administration of daily Ceftriaxone
(200 mg/kg) treatment for five to seven days, tissue samples collected from rats showed
a three-fold increase in GLT-1 expression (Rothstein et al., 2005). In addition,
Ceftriaxone induces expression of system xc- in multiple brain regions (Lewerenz et al.,
2009).
Ceftriaxone and Cocaine
A commonly used animal model to study the ability of Ceftriaxone to reduce drug
seeking consists of cocaine self-administration for two weeks followed by a 2-3 week
period of extinction. These studies have reliably found administration of Ceftriaxone
(200 mg/kg IP) for 5-7 days during extinction training attenuates relapse to cocaine
seeking (Fischer et al., 2013; Lacrosse et al., 2016, 2017; Knackstedt et al., 2010; Sari
et al., 2009; Sondheimer and Knackstedt, 2011). One such study tested rodent for
separate cue-prime and cocaine-primed reinstatement to cocaine seeking. Ceftriaxone
(200 mg/kg) successfully attenuated both types of reinstatement while upregulating xCT
in the NAc (Knackstedt et al., 2010). Another study utilized microdialysis during cocaine-
primed reinstatement and observed no increase of NAc glutamate in animals treated
with Ceftriaxone. Also, Ceftriaxone was reported to attenuate cue-primed cocaine
relapse in a dose dependent manner with the higher doses (100 mg/kg and 200 mg/kg)
as more efficacious (Sari et al., 2009).
32
In these same animal models of cocaine self-administration and extinction,
Ceftriaxone restores GLT-1 and system xC- function (Knackstedt et al., 2010). It thereby
increased basal glutamate levels and prevented the efflux of glutamate in the NAc that
drives reinstatement (Trantham-Davidson et al., 2012). In addition, Ceftriaxone was
found to specifically increase GLT-1 expression in the PFC (Sari et al., 2009) and the
NAc (Rothstein et al., 2005; Knackstedt et al., 2010). Digging further into the
mechanism of Ceftriaxone’s action, a recent study sought to parse out the independent
roles of xCT and GLT-1 (LaCrosse et al., 2017), as they are both restored following
chronic Ceftriaxone treatment (Knackstedt et al., 2010; Trantham-Davidson et al.,
2012). Using the same model as this experiment, following a period of cocaine self-
administration, animals went through a period of extinction training where the last 6
days were paired with Ceftriaxone treatment. In addition to Ceftriaxone, rats received an
intra-accumbens injection of either an antisense targeting xCT or GLT-1. The
knockdown of xCT paired with Ceftriaxone treatment, prevented Ceftriaxone from
preventing reinstatement. In other words, knocking down xCT made Ceftriaxone
ineffective and the rats reinstated to cocaine seeking. The GLT-1 antisense had the
same effect on reinstatement behavior has the xCT knockdown. Ceftriaxone was unable
to prevent reinstatement. Interestingly, the knockdown of xCT prevented Ceftriaxone
from normalizing GLT-1 levels, whereas GLT-1 knockdown had no effect on expression
of xCT (LaCrosse et al., 2017). Taken together, this study demonstrates the crucial role
of GLT-1 in facilitating the effects of Ceftriaxone on preventing reinstatement to cocaine
seeking.
33
Overall, chronic cocaine use results in a reliable downregulation of the important
mechanisms, GLT-1 and xCT, responsible for maintaining glutamate homeostasis.
Treatment with Ceftriaxone during the last 5-7 days of extinction training normalizes
GLT-1 and xCT and thereby prevents an overflow of glutamate and subsequently
attenuates reinstatement to cocaine seeking.
Ceftriaxone and Alcohol
Glutamate efflux in the NAc also accompanies the reinstatement of alcohol
seeking (Gass et al., 2010). Ceftriaxone also attenuates the reinstatement of operant
alcohol-seeking in outbred rats (Weiland et al., 2015). In alcohol preferring rats,
Ceftriaxone decreases continuous alcohol consumption in the home cage (Sari et al.,
2011; Alhaddad et al., 2014a; Das et al., 2015; Rao et al., 2015) and diminishes relapse
to continuous home cage drinking after an alcohol-free period (Qrunfleh et al., 2013;
Rao and Sari, 2014). Although a low dose of Ceftriaxone (50 mg/kg) attenuates alcohol
consumption in alcohol preferring rats (Sari et al., 2011), only higher doses (100 mg/kg
and 200 mg/kg) result in an upregulation of GLT-1 in the nucleus accumbens (Qrunfleh
et al., 2013; Sari et al., 2011). Ceftriaxone increases xCT and GLT-1 expression in the
NAc of alcohol preferring rats that had continuous access to alcohol (Alhaddad et al.,
2014a; Rao et al., 2015). Thus, in paradigms where rats have continuous access to
alcohol, resulting in decreased NAc GLT-1 and xCT expression, Ceftriaxone is capable
of increasing expression of these proteins and reducing alcohol consumption.
In a recent study we utilized the intermittent access to alcohol (IAA) paradigm on
outbred rats whereby 20% alcohol was available for 24 hours followed by 24 hours of
abstinence. This study is different than others based on the use of outbred rats rather
than alcohol preferring rats. Ceftriaxone (200 mg/kg) was administered following 17 IAA
34
sessions for a total of 5 days. A decrease in alcohol consumption was observed in
following 2 days of Ceftriaxone treatment and continued for 72 hours following the fifth
(final) Ceftriaxone treatment. This decrease in alcohol consumption mediated by
Ceftriaxone was accompanied by an increase in NAc core xCT expression, but not
GLT-1 (Stennett et al., 2017). The lack of change in GLT-1 is in agreement with our
previous work (Pati et al., 2016). Given that we did not observe and increase in GLT-1
expression, it is likely that xCT is necessary to decrease alcohol consumption, not GLT-
1 (Stennett et al., 2017). In summary, Ceftriaxone is capable of attenuating
reinstatement to and decreasing alcohol consumption in both alcohol preferring and
outbred rats. The changes in GLT-1 and xCT are not as clearly defined as with cocaine
use research, but it seems GLT-1 may not be as involved as xCT.
Rationale
In summary, reinstatement to drug seeking is facilitated by glutamatergic
transmission in the NAc core from regions of the PFC, BLA, and VTA. Glutamate
homeostasis in the NAc core is disrupted following a history of either cocaine
(decreased basal glutamate) or alcohol (increased basal glutamate) use alone. This
disruption of NAc glutamate homeostasis is due to changes in GLT-1 and system xc-.
Ceftriaxone restores levels of xCT and GLT-1 expression in the NAc core, thereby
restoring glutamate homeostasis and preventing the efflux of glutamate typically paired
with reinstatement to cocaine or alcohol seeking. In doing so, Ceftriaxone effectively
attenuates reinstatement to cue- or drug-primed cocaine- or alcohol seeking.
Ceftriaxone also decreases alcohol consumption in continuous and intermittent
paradigms.
35
Given that the majority of human cocaine abusers also have a history of alcohol
consumption, pharmacological therapies need to understand the long-term changes
resulting from use of both drugs. In doing so, treatments have the potential to be more
efficacious for an increased number of cocaine addicts. Hence, there is a strong need to
discover the changes to NAc core glutamate resulting from a history of both cocaine
and alcohol use. The development of the above-mentioned drug self-administration
animal models presents us the means to investigate these changes.
In this study, we used a rodent model of cocaine and alcohol co-abuse where
animals daily self-administer IV cocaine for 2 hours followed by access to 20% alcohol
for 6 hours. After a period of extinction, we tested animals for cue-, cocaine-, or
cue+cocaine primed reinstatement to cocaine seeking. Subsets of animals were given
differing doses of Ceftriaxone (100 or 200 mg/kg) or vehicle during extinction training in
attempts to prevent reinstatement to cocaine seeking. Based on previous research
where Ceftriaxone effectively prevents glutamate efflux in the NAc core and the
subsequent reinstatement to either cocaine or alcohol seeking, we hypothesized that
Ceftriaxone will also be effective in animals with a history of cocaine and alcohol co-
abuse. We secondly evaluated glutamate release into the NAc core during
reinstatement to cue+cocaine seeking via microdialysis. Because we did not find that
glutamate release in the NAc accompanied reinstatement in rats with a history of both
cocaine and alcohol self-administration, our third experiment evaluated Fos expression
in other parts of the reward circuitry during reinstatement testing.
36
CHAPTER 2 EXPERIMENT 1: USING A RODENT MODEL OF SIMULTANEOUS COCAINE AND
ALCOHOL USE TO SCREEN THE ABILITY OF CEFTRIAXONE TO PREVENT COCAINE RELAPSE
Introduction
Cocaine use disorder is a significant public health problem in the United States
today. One of the difficulties in successful treatment of cocaine use disorder is the high
risk of relapse that exists even after long periods of abstinence. Currently, there are no
FDA-approved drugs in existence for cocaine use disorder. In addition, an estimated
60% to 90% of cocaine abusers use alcohol with cocaine simultaneously. The
combination of alcohol and cocaine is expected to produce unique neuroadaptations
that differ from those produced by either drug alone. These potential unique
neuroadaptations could confound treatment to cocaine use disorder and thus, hinder
treatment success further. In order to develop more effective treatments and potential
pharmacotherapies, an animal model of concurrent alcohol and cocaine abuse should
be utilized to screen such pharmacotherapies. It has previously been shown that
Ceftriaxone prevents relapse to cocaine seeking in animals self-administering cocaine
alone (Fischer et al., 2013; Knackstedt et al., 2010; Sari et al., 2009; Sondheimer and
Knackstedt, 2011; Trantham-Davidson et al., 2012).
Previous research has established the role of nucleus accumbens glutamate
transmission in the reinstatement of cocaine seeking (McFarland et al., 2003) and has
shown that the antibiotic Ceftriaxone prevents relapse to cocaine seeking in rats
(Fischer et al., 2013; Knackstedt et al., 2010; Sari et al., 2009; Sondheimer and
Knackstedt, 2011; Trantham-Davidson et al., 2012). In this experiment we used the
reinstatement model of relapse to test Ceftriaxone for its efficacy at reducing cue- and
37
cocaine-primed reinstatement in animals that consumed alcohol after daily cocaine self-
administration sessions. We predict that Ceftriaxone will reduce cocaine-primed
reinstatement regardless of whether alcohol is consumed. Repeated evidence supports
the ability of Ceftriaxone to attenuate reinstatement to drug seeking for both cocaine
and alcohol use separately. This agrees with the finding that increased glutamate
release into the NAc core during reinstatement to both cocaine- and alcohol-seeking.
Therefore, even if basal levels of NAc glutamate are altered following a history of both
cocaine and alcohol use, we still suspect there to be glutamate efflux during
reinstatement testing regardless of glutamate homeostasis prior to testing. Thus, in this
experiment, we use Ceftriaxone as a potential pharmacotherapy for cocaine use
disorder in animals with a history of both cocaine and alcohol use.
Materials and Methods
Subjects
Adult male Sprague-Dawley rats (n = 30; Charles Rivers Laboratories, Raleigh,
NC) were housed in a temperature- and humidity-controlled vivarium. Rats were single-
housed under a reverse light cycle with lights off at 7 am and on at 7 pm. Animals were
food restricted to 20 g/day, which still yielded an increase in body weight that was within
the standard deviation of the growth curve for male Sprague-Dawley rats. Water was
provided ad libitum for the duration of the experiment. All procedures were approved by
the University of Florida Institutional Animal Care and Use Committees and were in
accordance with National Institutes of Health Guide for the Care and Use of Laboratory
Animals. Four rats were eliminated from the study due to catheter failure.
38
Intermittent Drinking Paradigm (IDP)
The intermittent drinking paradigm (IDP) paradigm has been shown to be
effective at inducing alcohol drinking in outbred rats without sucrose-fading (Simms et
al., 2008). This method provides rats with 24-hr access to alcohol on alternating days
without water-deprivation. Animals were weighed and then presented with alcohol (20%
v/v) in graduated bottles with sipper tubes within the first hour of the dark cycle. The
daily allotment of food (20 g) was also given at this time, such that even if rats did not
eat the entire allotment from the previous day, the total available for one day was 20 g.
Food-restriction began the night prior to the first alcohol presentation. The amount of
alcohol consumed was recorded 6- and 24- hour post-presentation. Rats received
alcohol via the IDP for 5 sessions prior to surgery (see Figure 2-1).
Figure 2-1. Timeline
Surgical Procedures
Animals were anesthetized using a mixture of ketamine (87.5 mg/kg, IP) and
xylazine (5 mg/kg, IP) and surgically implanted with catheters in the jugular vein.
Ketorolac (2 mg/kg, IP) was administered post-operatively and 4 days following surgery
for analgesia. Catheters (SILASTIC silicon tubing, ID 0.51 mm, OD 0.94 mm, Dow
Corning, Midland, MI) were implanted in the jugular vein, secured with 4-0 silk sutures,
and then passed subcutaneously between the shoulder blades and exited through the
skin on the back. Catheter tubing was then connected to a stainless-steel cannula
(Plastics One, Roanoke, VA, USA) embedded in a rubber harness (Instech, Plymouth
Meeting, PA, USA). The harness was worn for the duration of cocaine self-
39
administration. The antibiotic Cefazolin (100 mg/kg) was administered IV (0.1 mL) for 4
days post-surgery. Catheters were flushed with 0.1 mL of heparinized saline (100 U/mL)
before and after each self-administration to ensure prolonged catheter patency.
Catheter patency was tested periodically with methohexital sodium (10 mg/mL; Eli Lilly,
Indianapolis, IN, USA), which results in a temporary loss of muscle tone.
Drugs
Cocaine hydrochloride was acquired from NIDA Controlled Substance Program
(Research Triangle Institute, NC, USA) and was dissolved in saline (0.9% sodium
chloride) for intravenous self-administration (4 mg/mL; 0.35 mg/infusion) and
intraperitoneal (IP) injection during reinstatement sessions (10 mg/kg).
Ceftriaxone (Sigma Aldrich, St. Louis, MO) was prepared in sterile 0.9% saline
and administered intraperitoneally (IP) at a dose of 100 mg/kg or 200 mg/kg (in 1
mg/mL). This dose was chosen because we have previously shown it to be effective at
reducing the reinstatement of both cocaine- and alcohol-seeking in our lab (Knackstedt
et al., 2010; Weiland et al., 2015) and others and we have shown it to reduce alcohol
consumption in outbred (Stennett et al., 2017) and alcohol preferring rats (Sari et al.,
2011). Vehicle (0.3 mL of 0.9% sterile saline) was administered IP. Injections were
administered immediately following the 2-hour extinction training sessions.
Operant Cocaine and Oral Alcohol Self-Administration
Animals were first trained to drink alcohol using the Intermittent Access to
Alcohol paradigm (described above and in Simms et al., 2008) for 5 sessions. Following
surgery to implant a jugular catheter, the subjects were trained to self-administer
cocaine in a standard two-lever operant chamber (Med Associates, St. Albans, VT),
whereby presses on the active lever resulted in the delivery of 0.2 mg cocaine/infusion.
40
An FR-1 schedule of reinforcement was employed and each drug infusion was paired
with auditory (a 2900 Hz tone played for 5 seconds) and visual cues (the illumination of
a light over the active lever). Throughout the 2-hour self-administration session, presses
on the inactive lever (left) were recorded but had no programmed consequence. Each
infusion of cocaine was followed by a 20 second time-out period where no drug could
be delivered. Animals continued with daily self-administration until a criterion of 10 or
more infusions of cocaine per session for 12 sessions was attained. A subset of cocaine
animals received access to an oral ethanol solution (20% v/v) in the homecage
immediately following conclusion of daily cocaine self-administration sessions. Upon
completion of the operant self-administration portion of the experiment, animals began
extinction training during which presses on the previously active lever no longer
produced drug infusions or cue presentation. Once animals experienced a minimum of
12 extinction sessions and lever pressing had reached 20% of self-administration levels,
reinstatement testing began.
Subjects were first tested for cue-primed reinstatement during a 2-hour test,
wherein presses on the active lever produced the cues associated with drug delivery.
Animals then underwent extinction procedures for a minimum of 3 days prior to a
cocaine-primed reinstatement test. During the cocaine-primed reinstatement test,
animals were administered 10 mg/kg cocaine (IP) then immediately placed into the
operant chamber. During both types of reinstatement tests, cocaine was not delivered
upon active lever presses. Ceftriaxone (200 mg/kg, 100 mg/kg IP) or vehicle (saline; 0.3
mL) was administered for at least 6 days prior to the first reinstatement test. Injections
were administered following removal from the operant chamber and continued to be
41
administered immediately after the extinction sessions that followed the cue-primed
reinstatement test.
Blood Plasma Cocaine and Cocaethylene Levels
Following a two hour cocaine self-administration session rats were given access
to 20% EtOH. Blood was collected from the jugular catheter port at 30, 60 and 120 min
post access to alcohol. Samples were collected in order to evaluate cocaine and
cocaethylene blood plasma levels. Blood samples were collected into sterile BD
Vacutainers (BD, Franklin Lakes, NJ) pretreated with K3 EDTA (12 mg) to prevent
clotting. Blood samples were centrifuged for 15 min at 4000 rpm. Blood serum was
collected and frozen at minus 80°C for later analyses. Samples were then sent to the
University of Florida Chemistry Core for quantification of cocaine and cocaethylene
plasma levels via gas chromatography-mass spectrometry (GC-MS) as done in
(Jagerdeo et al., 2008).
Blood Alcohol Assay
Blood alcohol levels were determined in a subset of rats following a cocaine self-
administration session, during the beginning of their access to alcohol. Blood was
sampled from the jugular catheter at 30, 60 and 120 min post-alcohol access. We
collected blood from another subset of rats only at the 120 min mark to control for
drinking disruption due to the 30 and 60 min checks in the alternate group. Blood
samples were collected into sterile BD Vacutainers (BD, Franklin Lakes, NJ) pretreated
with K3 EDTA (12 mg) to prevent clotting. Blood samples were centrifuged for 15 min at
4000 rpm. Blood serum was collected and frozen at minus 80 °C for later analyses. The
alcohol content of each sample was determined using the alcohol dehydrogenase assay
(Peris et al., 2006). A standard curve was prepared containing a range of standards (0–
42
25 mM ethanol) added to 100 μL 0.6M glycine buffer (pH 9.2) to ensure that ethanol
evaporation was minimal. Samples (10 μL) were mixed with glycine buffer and enzyme
solution (0.88 mg NAD and 0.29 mg ADH per mL of 0.6M glycine buffer) and incubated
at 37 °C in a shaking water bath for 20 min. Samples were then kept on ice for at least 5
min prior to transfer into a 96-well plate on ice. Absorbance was read at 340 nm using a
Synergy HT platereader (BioTek, Winooski, VT), converted to mg% alcohol by the use
of a polynomial equation and fits were better than R2=0.98.
Statistical Analyses
GraphPad Prism (version 7, GraphPad Software, La Jolla, CA, USA) was used to
analyze data. For all statistical analysis used, the alpha level was set at p<0.05. For
self-administration data, infusions of cocaine, active and inactive lever presses during
both self-administration and extinction training were analyzed with repeated measure
(RM) 2-way analysis of variance (ANOVAs), with group and day as factors and repeated
measures conducted on day. Lever presses during reinstatement tests were analyzed
with RM-ANOVAs with group and test as factors and repeated measures on test.
Significant main effects and/or interactions were followed by Tukey’s post-hoc analysis
to examine differences of group or day/test. All data is presented as mean±SEM.
Results
Cocaine and Alcohol Intake
The number of cocaine infusions (F (2, 23) = 0.372, p = 0.693; see Fig. 2-2) or
cocaine infusions (mg/kg) (F (2, 23) = 0.538, p = 0.591; see Fig. 2-3) during self-
administration did not differ between groups later assigned to receive Ceftriaxone 100
mg/kg, Ceftriaxone 200 mg/kg, or vehicle treatments. There was a significant effect of
time on cocaine infusions (F (11, 253) = 6.701, p < 0.0001) and cocaine infusions
43
(mg/kg) (F (11, 253) = 10.340, p < 0.0001) indicating an increase of cocaine intake over
the course of self-administration sessions. The total amount of cocaine infusions during
self-administration did not differ between groups later assigned to treatment groups of
Ceftriaxone 100 mg/kg, Ceftriaxone 200 mg/kg, or vehicle treatments (F (2, 23) = 0.372,
p = 0.693; see Fig. 2-5). Active lever presses during cocaine self-administration did not
differ between groups later assigned to receive Ceftriaxone 100 mg/kg, Ceftriaxone 200
mg/kg, or vehicle treatments (F (2, 22) = 0.308, p = 0.738; see Fig. 2-7). There was no
effect of time on the number of active lever presses over the course of cocaine self-
administration (F (11, 242) = 1.596, p = 0.100). Inactive lever presses during cocaine
self-administration did not differ between groups later assigned to receive Ceftriaxone
100 mg/kg, Ceftriaxone 200 mg/kg, or vehicle treatments (F (2, 22) = 0.291, p = 0.751;
see Fig. 2-8). There was an effect of day (F (11, 242) = 4.476, p < 0.0001) indicating
that rats decreased responding on the inactive lever throughout the self-administration
sessions.
The amount of alcohol consumed each day (mg/kg) during self-administration
sessions did not differ between groups later assigned to receive Ceftriaxone 100 mg/kg,
Ceftriaxone 200 mg/kg, or vehicle treatments (F (2, 23) = 1.213, p = 0.316; see Fig. 2-
4). As opposed to the increase in cocaine intake across days, there was no significant
effect of day on alcohol consumption (F (11, 253) = 1.474, p = 0.142). Prior to the start
of Ceftriaxone (100 mg/kg or 200 mg/kg) and vehicle injections, total alcohol intake did
not differ between groups later assigned to treatment groups (F (2, 23) = 0.372, p =
0.693; see Fig. 2-6).
44
Extinction Training
During extinction training, the number of lever presses on the previously active
lever did not differ between groups of rats prior to receiving Ceftriaxone/vehicle or after
treatment began (F (2, 23) = 1.056, p = 0.364; see Fig. 2-9). There was a significant
effect of day across extinction training sessions (F (11, 253) = 31.070, p < 0.0001), as
lever pressing declined. The number of inactive lever presses during extinction did not
differ between groups of rats prior to receiving treatment or after treatment began (F (2,
23) = 0.916, p = 0.414; see Fig. 2-10). There was a significant effect of day across
extinction training sessions (F (11, 253) = 6.966, p < 0.0001).
Cue-Primed and Cocaine-Primed Reinstatement Tests.
Animals significantly reinstated to both cue- and cocaine-primed reinstatement
tests despite pretreatment with Ceftriaxone. Two-way ANOVAs were conducted on the
(average of last three days) Extinction-1 to Cue-prime reinstatement to cocaine seeking
and Extinction-2 to Cocaine-prime reinstatement (see Fig. 2-11). For cue-primed
reinstatement there was a significant effect of test (F (1, 22) = 56.890, p < 0.0001).
Sidak’s post hoc analysis revealed significant reinstatement (difference between
extinction and test) during the cue-primed test for all groups: Veh (p = 0.002), Cef-100
(p = 0.002), and Cef-200 (p < 0.0001). There was also a significant effect of test for
cocaine-primed reinstatement (F (1,22) = 28.350, p < 0.0001). Again, Sidak’s post hoc
analysis revealed significant reinstatement during the cocaine-primed test for all groups:
Veh (p = 0.003), Cef-100 (p = 0.033), and Cef-200 (p = 0.039).
Blood Plasma Levels of Cocaine and Cocaethylene and Blood Alcohol Levels
Blood serum levels were taken via jugular catheter at 30, 60, and 120 minutes
during access to 20% alcohol following a cocaine self-administration. Cocaine levels
45
peaked early and decreased over time (see Fig. 2-12), as expected. Cocaethylene was
detected, however was at very low levels, (see Fig. 2-13). Blood alcohol levels were
detected in rats repeatedly sampled as well as the rats only sampled at the 120 min
time point (see Fig. 2-14). Though BALs steadily decreased in the repeated sampling
rats, we can attribute this to multiple handlings of the animals interrupting normal
drinking patters. Taking this into account, we analyzed BALs of rats at 120 min that had
not been interrupted prior. This group of rats had higher BALs at the 120 min time point
than the repeated sampling group.
Discussion
This experiment was completed using the reinstatement model of relapse to test
Ceftriaxone for its efficacy at reducing cue- and cocaine-primed reinstatement in
animals that consumed alcohol after daily cocaine self-administration sessions. Based
on the literature discussed above, we predicted that Ceftriaxone would prevent cue-
primed and cocaine-primed reinstatement of cocaine seeking in Coc+EtOH animals.
However, our data did not support this hypothesis and Ceftriaxone did not attenuate the
reinstatement of cocaine seeking in animals that consumed alcohol with cocaine. The
groups later assigned to receive Veh, Ceftriaxone (100 mg/kg) or Ceftriaxone (200
mg/kg) did not initially differ in mean number of cocaine infusions or mg/kg of cocaine
self-administered. Animals in all treatment conditions consumed similar amounts of
EtOH (g/kg). Mean active lever presses during extinction training did not differ between
groups. Animals treated with IP Ceftriaxone (100 or 200 mg/kg) showed no attenuation
of cue- or cocaine-primed reinstatement compared to animals pretreated with Veh (IP
saline). Reinstatement is defined as a significant increase in lever pressing from
46
extinction to test. All treatment groups significantly reinstated active lever pressing
during both cue-prime and cocaine-prime testing compared to extinction training.
Based on the literature discussed above, we predicted Ceftriaxone would
suppress reinstatement in cocaine animals that have a history of alcohol consumption,
with 200 mg/kg having a greater effect than the lower dose (100 mg/kg). We also
predicted that Ceftriaxone would prevent both cue-prime and cocaine-primed
reinstatement to cocaine seeking in these animals. These hypotheses were based on
previous research demonstrating the efficacy of Ceftriaxone to prevent reinstatement to
cocaine seeking and alcohol seeking separately (Knackstedt et al., 2010; Sari et al.,
2009; Sari et al., 2011; Trantham-Davidson et al., 2012; Weiland et al., 2015). We did
not utilize positive controls for this study, cocaine intake without EtOH access and
Ceftriaxone treatment, nor EtOH intake with Ceftriaxone treatment as we have
completed studies within our lab using Ceftriaxone on these conditions (LaCrosse et al.,
2017; Stennett et al., 2017; Weiland et al., 2015).
However, neither dose of Ceftriaxone attenuated neither cue- nor cocaine-primed
reinstatement in animals with a history of cocaine and alcohol co-abuse. Two different
cohorts of animals were used for this experiment at two different time points. Therefore,
the effects of Ceftriaxone quality did not affect reinstatement behavior. The combination
of cocaine and alcohol seem to alter neurobiological underpinnings of relapse to
cocaine differently than cocaine use alone, thereby rendering Ceftriaxone ineffectual. In
consequence of these findings, Ceftriaxone is unlikely to be an effectual treatment for
cocaine addicts who are comorbid for alcohol abuse.
47
In a subset of animals, we collected blood to analyze cocaethylene levels in
blood plasma. We found lower levels than previous studies in these animals. In previous
studies, animals were injected with a substantial dose of alcohol rather than consuming
it on their own. The low levels of CE could be because rats did not consume a large
amount of alcohol following the cocaine self-administration session. The half-life of
cocaine in humans is 30 minutes, whereas the half-life in rats is only 15 minutes. Thus,
cocaine may not be present at high enough concentrations in order for CE to be formed
in the current model.
The blood alcohol levels in rats that we collected blood at multiple time points
started out high but tapered off, despite the availability of alcohol during the sampling.
This finding could be due to the repeated experimenter disruption of their normal
drinking pattern in order to collect blood samples. Therefore, we collected blood from a
second cohort of rats that were not disturbed in the first two hours of access to alcohol.
The blood alcohol levels in the latter group resemble what we would expect the BAL to
be after two hours of an uninterrupted drinking session.
In summary, both high (200 mg/kg) and low (100 mg/kg) doses of Ceftriaxone did
not attenuate cue- nor drug-primed reinstatement to cocaine seeking when animals had
consumed alcohol as well as cocaine. These findings indicate that Ceftriaxone, despite
its repeated demonstration of effectiveness in blunting cocaine reinstatement and
decreasing alcohol consumption, will not be effective in animals with a history of both
cocaine and alcohol use. These findings should be taken into consideration in the event
that Ceftriaxone moves into clinical trials. In addition, these findings are also important
48
because it strongly suggests that the neurobiology underlying cocaine relapse is altered
when animals have a history of alcohol use.
49
Figure 2-2. Cocaine infusions during self-administration. Infusions of cocaine during self-administration did not differ between groups later assigned to receive Ceftriaxone 100 mg/kg, Ceftriaxone 200 mg/kg, or vehicle treatments (F (2, 23) = 0.372, p = 0.693). There was a significant effect of time on cocaine infusions (F (11, 253) = 6.701, p < 0.0001).
1 2 3 4 5 6 7 8 9 10 11 120
10
20
30
40
Day
Co
cain
e In
fusio
nVeh (n=9)
Cef-100 (n=7)
Cef-200 (n=10)
50
Figure 2-3. Cocaine infusions (mg/kg) during self-administration. Infusions of cocaine
(mg/kg) during self-administration did not differ between groups later assigned to receive Ceftriaxone 100 mg/kg, Ceftriaxone 200 mg/kg, or vehicle treatments (F (2, 23) = 0.538, p = 0.591). There was a significant effect of time on cocaine infusions (F (11, 253) = 10.340, p < 0.0001).
1 2 3 4 5 6 7 8 9 10 11 120
10
20
30
40
Day
Co
cain
e In
fusio
nVeh (n=9)
Cef-100 (n=7)
Cef-200 (n=10)
51
Figure 2-4. Ethanol consumed during self-administration. As measured by g/kg of body
weight, the amount of EtOH consumed each day during self-administration did not differ between groups later assigned to receive Ceftriaxone 100 mg/kg, Ceftriaxone 200 mg/kg, or vehicle treatments (F (2, 23) = 1.213, p = 0.316). There was no significant effect of time (F (11, 253) = 1.474, p = 0.142).
1 2 3 4 5 6 7 8 9 10 11 120
2
4
6
Day
g/k
g E
tOH
Veh (n=9)
Cef-100 (n=7)
Cef-200 (n=10)
52
Figure 2-5. Total cocaine intake mg/kg during self-administration. The total amount of
cocaine infusions did not differ between groups later assigned to receive Ceftriaxone 100 mg/kg, Ceftriaxone 200 mg/kg, or vehicle treatments (F (2, 23) = 0.538, p = 0.591). Numbers inside bars indicate number of animals per group.
Veh Cef-100 Cef-2000
100
200
300C
ocain
e m
g/k
g
9 7 10
53
Figure 2-6. Total ethanol intake during self-administration. The total amount of ethanol
consumed (g/kg) did not differ between groups later assigned to receive Ceftriaxone 100 mg/kg, Ceftriaxone 200 mg/kg, or vehicle treatments (F (2, 23) = 1.213, p = 0.316). Numbers inside bars indicate number of animals per group.
Veh Cef-100 Cef-2000
20
40
60E
tOH
g/k
g
9 7 10
54
Figure 2-7. Active lever presses during self-administration. Active lever presses during
cocaine self-administration did not differ between groups later assigned to receive Ceftriaxone 100 mg/kg, Ceftriaxone 200 mg/kg, or vehicle treatments (F (2, 22) = 0.308, p = 0.738). There was no effect of time (F (11, 242) = 1.596, p = 0.100).
1 2 3 4 5 6 7 8 9 10 11 12
0
10
20
30
40
50
60
70
Day
Acti
ve L
ever
Pre
sses
Veh (n=9)
Cef-100 (n=7)
Cef-200 (n=10)
55
Figure 2-8. Inactive lever presses during self-administration. Inactive lever presses
during cocaine self-administration did not differ between groups later assigned to receive Ceftriaxone 100 mg/kg, Ceftriaxone 200 mg/kg, or vehicle treatments (F (2, 22) = 0.291, p = 0.751). There was an effect of time (F (11, 242) = 4.476, p < 0.0001).
1 2 3 4 5 6 7 8 9 10 11 12
0
10
20
30
40
50
60
70
Day
Inacti
ve L
ever
Pre
sses
Veh (n=9)
Cef-100 (n=7)
Cef-200 (n=10)
56
Figure 2-9. Lever presses on the previously active lever during extinction training. The
number of lever presses during extinction training on the previously active lever did not differ between groups of rats prior to receiving treatment or after treatment began (F (2, 23) = 1.056, p = 0.364). There was a significant effect of time across extinction training sessions (F (11, 253) = 31.070, p <0.0001).
1 2 3 4 5 6 7 8 9 10 11 12
0
20
40
60
80
100
120
140
Day
Acti
ve L
ever
Pre
sses
Veh (n=9)
Cef-100 (n=7)
Cef-200 (n=10)
57
Figure 2-10. Inactive lever presses during extinction training. The number of lever
presses during extinction training on the inactive lever did not differ between groups of rats prior to receiving treatment or after treatment began (F (2, 23) = 0.916, p = 0.414). There was a significant effect of time across extinction training sessions (F (11, 253) = 6.966, p < 0.0001).
1 2 3 4 5 6 7 8 9 10 11 12
0
20
40
60
80
100
120
140
Day
Inacti
ve L
ever
Pre
sses Veh (n=9)
Cef-100 (n=7)
Cef-200 (n=10)
58
Figure 2-11. Cue-Primed and cocaine-primed reinstatement tests. Two-way ANOVAs
were conducted on the (average of last three days) Extinction-1 to Cue-prime reinstatement to cocaine seeking and Extinction-2 to Cocaine-prime reinstatement. There was a significant effect of test for both cue-primed (F (1,22) = 56.890, p < 0.0001) and cocaine-primed (F (1,22) = 28.350, p < 0.0001) reinstatement tests. Sidak’s post hoc analysis revealed significant reinstatement during the cue-primed test for all groups: Veh (p = 0.002), Cef-100 (p = 0.002), and Cef-200 (p < 0.0001) as indicated by “*”. Sidak’s post hoc analysis revealed significant reinstatement during the cocaine-primed test for all groups: Veh (p = 0.003), Cef-100 (p = 0.033), and Cef-200 (p = 0.039) as indicated by “*”.
Ext-1 Cue Ext-2 Coc0
50
100
150A
cti
ve L
ever
Pre
sses
Veh (n=9)
Cef-100 (n=7)
Cef-200 (n=10)*
*
*
* * *
59
Figure 2-12. Plasma Cocaine levels. Following a 2-hour cocaine self-administration
session, cocaine levels in blood plasma were evaluated for 2 hours.
0 30 60 90 120-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
Time (min)
Pla
sm
a c
ocain
e (n
g/m
L)
60
Figure 2-13. Plasma cocaethylene levels. Following a two-hour session of cocaine self-
administration, levels of Cocaethylene in blood plasma were evaluated for two hours post access to alcohol.
30 60 90 1200
5
10
15
20
Time
Pla
sm
a C
E (
ng
/mL
)
61
Figure 2-14. Blood alcohol levels. Blood was sampled at 30, 60, and 120 post access to 20% EtOH. Another group of rats had blood samples taken at 120 min only.
0 30 60 90 1200
20
40
60
80
100
Time
BA
L (
mg
%)
Multiple collections
Single collection
62
CHAPTER 3 EXPERIMENT 2: THE ROLE OF GLUTAMATE RELEASE IN THE NUCLEUS
ACCUMBENS CORE DURING COCAINE REINSTATEMENT IN RATS WITH A HISTORY OF BOTH ALCOHOL AND COCAINE SELF-ADMINISTRATION
Introduction
Ceftriaxone is an FDA-approved antibiotic that increases the expression of xCT
and GLT-1, thereby normalizing both basal glutamate levels and glutamate release
during reinstatement (Trantham-Davidson et al., 2012). Ceftriaxone has been
repeatedly demonstrated to attenuate the reinstatement of cocaine seeking (Fischer, et
al., 2013; Knackstedt et al., 2010; Sari et al., 2009; Sondheimer and Knackstedt, 2011;
Trantham-Davidson et al., 2012). The therapeutic effects of Ceftriaxone continue for
weeks after the cessation of Ceftriaxone treatment (Sondheimer and Knackstedt, 2011).
This finding indicates that Ceftriaxone produces a long-term reversal of cocaine-induced
neuroadapations. However, the co-use of alcohol with cocaine may produce
neuroadaptations distinct from those produced by cocaine alone, and thus render
Ceftriaxone an ineffective clinical approach for treatment of cocaine use disorder. In
fact, Experiment 1 indicates that Ceftriaxone is no longer effectual at attenuating
reinstatement to cocaine seeking when animals have a history of both cocaine and
alcohol use. This finding is the first indication that the consumption of alcohol and
cocaine together produces different neuroadaptations than cocaine alone and the
following experiment will characterize these differences.
In the model proposed here, alcohol access will occur after operant cocaine self-
administration. Cocaine-induced anxiety is attenuated by oral alcohol self-administration
occurring after the cocaine session, which in turn increases the motivation for rats to
self-administer a single infusion of cocaine (Knackstedt and Ettenberg, 2005). Animals
63
have also shown a preference for alcohol solution over water when pre-treated with
experimenter-administered intravenous cocaine (Knackstedt et al., 2006). These studies
provide valuable information regarding the ability of alcohol to modulate cocaine self-
administration and vice versa. However, published information does not exist regarding
neuroadaptations produced by simultaneous self-administration of the two drugs (which
occurs in the majority of human cocaine users).
In Experiment 1 we found that Ceftriaxone does not attenuate cue- or cocaine-
primed reinstatement of cocaine seeking when animals consume both alcohol and
cocaine. Cocaine-primed and cue-primed reinstatement is driven by glutamate release
in the NAc (McFarland et al., 2003; Knackstedt et al., 2010; Trantham-Davidson et al.,
2012; Lutgen et al., 2012; Sari et al., 2009). Here we will use the same cocaine and
alcohol self-administration paradigm as in Experiment 1 and measure glutamate efflux
in the NAc during cue+cocaine primed reinstatement, to determine whether alcohol
consumption alters cue+cocaine-induced increases in glutamate release, and how
Ceftriaxone modulates this effect.
In this experiment, animals will self-administer either cocaine alone or cocaine
followed by alcohol in the home cage as described in Experiment 1. During extinction
training, half the animals in each group will receive Ceftriaxone (200 mg/kg) and half will
receive vehicle. After animals have undergone at least 12 days of extinction training and
lever pressing has reached extinction criteria, they will be implanted with microdialysis
probes and sample collection for glutamate quantification will begin the next day.
Animals will be tested for Cue+Cocaine-prime reinstatement. The amount of glutamate
release during reinstatement will be compared between the groups. In doing so, we can
64
determine whether alcohol consumption alters cocaine-induced increases in glutamate
release, and how Ceftriaxone modulates this effect. The assessment of the ability of
alcohol to alter the well-characterized glutamate adaptations produced by cocaine is
essential in order to develop treatments for addicts who abuse both cocaine and
alcohol.
In Experiment 1, rats that self-administered cocaine and ethanol reinstated lever
pressing following cue and cocaine primes. Because glutamate release has been found
to underlie the reinstatement of cocaine (Knackstedt et al., 2010; McFarland et al.,
2003) and alcohol (Gass et al., 2010), we predict that we will continue to observe an
increase in glutamate release in the NAc in these animals. However, we also predict
that since Ceftriaxone does not attenuate relapse in these animals. Therefore, it is likely
Ceftriaxone will not dampen glutamate levels in animals with a history of cocaine and
alcohol self-administration but will in animals that self-administered cocaine alone as
others have previously demonstrated (Trantham-Davidson et al., 2012). Such a finding
implies different mechanisms of glutamate dysregulation following cocaine and alcohol
compared to cocaine alone. Potential underlying adaptations that cause this failure of
Ceftriaxone to attenuate glutamate levels during reinstatement will be examined in
Experiment 3.
Materials and Methods
Subjects
Adult male Sprague-Dawley rats (n = 50; Charles Rivers Laboratories, Raleigh,
NC) were housed in a temperature- and humidity-controlled vivarium. Rats were single-
housed under a reverse light cycle with lights off at 7 am and on at 7 pm. Animals were
food restricted to 20 g/day, which still yielded an increase in body weight that was within
65
the standard deviation of the growth curve for male Sprague-Dawley rats. Water was
provided ad libitum at all times. All procedures were approved by the University of
Florida Institutional Animal Care and Use Committees and were in accordance with
National Institutes of Health Guide for the Care and Use of Laboratory Animals. Within
each treatment and drug condition, several animals were eliminated from the study for
various reasons: catheter failure during cocaine self-administration (n = 5); failure to
acquire cocaine self-administration (n = 2); failure to extinguish drug seeking behavior
(n = 1); complications during microdialysis (i.e., broken probe, chewed tubing; n = 4);
reinstatement active lever pressing above two standard deviations away from the mean
(n = 2).
Intermittent Drinking Paradigm (IDP)
As in experiment 1, animals were weighed and then presented with alcohol (20%
v/v) in graduated bottles with sipper tubes within the first hour of the dark cycle. The
daily allotment of food (20 g) was also given at this time, such that even if rats did not
eat the entire allotment from the previous day, the total available for one day was 20 g.
Food-deprivation began the night prior to the first alcohol presentation. The amount of
alcohol consumed was recorded 6- and 24- hours post-presentation. Rats experienced
5 IDP sessions prior to surgical procedures.
Surgical Procedures
Surgical procedures for implantation of the jugular catheter are the same as in
Experiment 1. Briefly, animals were anesthetized using a mixture of ketamine (87.5
mg/kg, IP) and xylazine (5 mg/kg, IP) and surgically implanted with catheters in the
jugular vein. Ketorolac (2 mg/kg, IP) was administered post-operatively and 4 days
following surgery for analgesia. Catheter tubing was then connected to a rubber
66
harness (Instech, Plymouth Meeting, PA, USA). The harness was worn for the duration
of cocaine self-administration. The antibiotic cefazolin was administered postoperatively
and catheters were flushed daily with heparin to maintain patency throughout self-
administration.
Immediately following jugular catheter implantation, guide cannula (22 gauge,
Synaptech, Marquette, MI, USA). Using a stereotaxic frame (Stoelting, Wood Dale, IL,
USA), cannulas were aimed at the NAc core using the following coordinates (AP +1.2
mm. ML +1.6 mm, DV -5.5 mm; Paxinos and Watson, 2007). Guide cannulas were
secured to the skull with skull screws and dental acrylic (Co-Oral-Ite Dental MFG. Co.,
Diamond Springs, CA, USA).
Drugs
Cocaine hydrochloride was acquired from NIDA Controlled Substance Program
(Research Triangle Institute, NC, USA) and was dissolved in saline (0.9% sodium
chloride) for intravenous self-administration (4 mg/mL; 0.35 mg/infusion) and
intraperitoneal injection during reinstatement sessions (10 mg/kg).
Ceftriaxone (Sigma Aldrich, St. Louis, MO) was prepared in sterile 0.9% saline and
administered intraperitoneally (IP) at a dose of 200 mg/kg (in 1 mg/mL). This dose was
chosen because we have previously shown it to be effective at reducing the
reinstatement of both cocaine- and alcohol-seeking in our lab (Knackstedt et al., 2010;
Weiland et al., 2015) and others have shown it to reduce alcohol consumption (Sari et
al., 2011). We chose to use 200 mg/kg dosage because we are seeking out the
maximum effect of Ceftriaxone on glutamate levels in the NAc core. Vehicle (0.3 mL of
0.9% sterile saline) was administered IP. Injections were administered immediately
following the 2-hour extinction training sessions.
67
Operant Cocaine and Oral Alcohol Self-Administration
The methods of this experiment are identical to that of experiment 1 in alcohol
access and cocaine self-administration. Briefly, animals were placed in operant
chambers whereby active lever (right) presses resulted in an infusion of cocaine.
Following the two-hour cocaine self-administration, a subset of animals was allowed
access to 20% oral alcohol for 6 hours in their home cages. Once animals completed a
minimum of 12 self-administration sessions with 10 or more cocaine infusions, animals
began extinction training. During the last 6 days of extinction training, animals received
either Ceftriaxone (200 mg/kg) or vehicle (saline; 0.3 mL) IP. Minimums of 12 extinction
sessions were completed prior to reinstatement testing.
Microdialysis and Reinstatement
On the night prior to reinstatement testing, rats were implanted with a
microdialysis probe (24 gauge; 2 mm of active dialysis membrane; Synaptech,
Marquette, MI, USA) and remained in their home cages overnight with food and water.
Home cages were placed adjacent to operant boxes equipped with liquid swivels
mounted onto counterbalanced lever arms (Instech Laboratories, Plymouth Meeting,
PA). Probes were perfused overnight with artificial cerebrospinal fluid (aCSF) containing
(125 mM NaCl, 2.5 mM KCl, 1 mM MgCl26H2O. 5 mM D-glucose, 1.2 mM CaCl2H20,
0.75 mL 10 x phosphate buffered saline) at a flow rate of 0.2 µL/min. The next morning,
animals were moved into the operant chambers and the flow rate was increased to 2.0
µL/min for two hours. Subsequently, twelve 10-minute baseline samples were collected.
Rats then received an injection of cocaine (10 mg/kg IP) and levers were extended for a
cue+cocaine-primed reinstatement test, wherein presses on the active lever resulted in
the cue light and tone played, however no drug was delivered. Samples were collected
68
in 10-minute increments during the 2-hour reinstatement test, resulting in 12 test
samples.
HPLC-ECD for Glutamate Quantification
Glutamate levels were analyzed using an Ultimate 3000 (ThermoSci/Dionex,
Waltham, MA, USA), a high-pressure liquid chromatography system with
electrochemical detection (HPLC-ECD). Microdialysis samples were derivatized with o-
pthalaldehyde (Sigma-Aldrich, St. Louis, MO, USA) by an autosampler (Thermo Fisher
Scientific) immediately prior to injection onto a CAPCELL PAK C18 column (5 µm,
2.0mm I.D. X 50mm; Shiseido Inc., Tokyo, Japan). The mobile phase consisted of 2.5%
acetonitrile (v/v), 100 mM dibasic sodium phosphate (Na2HPO4), 8% (v/v) methanol,
and pH = 6.5. Glutamate levels in the dialysis samples were quantified by comparing
computer-integrated peak areas of samples with an external glutamate standard curve
(10, 5, 2.5, 1.25, 0.625 µM).
Statistical Analyses
Behavioral data for this experiment was analyzed using SPSS (IBM, Amorak,
NY). Comparisons of the dependent measures of cocaine infusions, active/inactive lever
presses during self-administration, active/inactive lever presses during extinction
training were conducted using repeated measures ANOVAs with Day as the within
subject factor and Treatment (Ceftriaxone or Vehicle) and Drug (EtOH or H2O) as
between subject factors. For all analyses, significant main effects and/or interactions
were followed by Sidak’s post-hoc analysis to examine differences of group or
session/test. For microdialysis samples, all glutamate concentrations were converted to
a percent change based on the animal’s individual baseline percent value of glutamate.
All data is presented as mean±SEM.
69
Correlations
Correlations were conducted with Pearson r tests comparing active lever presses
during reinstatement tests, alcohol consumed during self-administration sessions,
cocaine consumed during self-administration sessions, and glutamate levels during
reinstatement. For the purposes of these correlations, the number for total alcohol
consumption was calculated by adding the total amount (g/kg) of alcohol consumed
during the self-administration sessions. Total cocaine consumed was calculated from
adding the total number of cocaine infusions in mg/kg across all self-administration
sessions. The area under the curve (AUC) of glutamate concentration during
reinstatement was divided by AUC glutamate during baseline samples to yield a single
value for each rat.
Results
Behavioral Results
Cocaine consumption prior to Ceftriaxone treatment
Mean infusions of cocaine during self-administration did not differ between
Coc+EtOH and Coc+H2O groups later assigned to receive Ceftriaxone 200 mg/kg or
vehicle treatment, as there were no main effects of Drug or Treatment, and no
interactions between any of the variables on cocaine infusions across the 12 days (F
(33, 341) = 0.985, p = 0.460). Cocaine infusions (mg/kg) did not differ between groups
later to receive Ceftriaxone or vehicle treatment (F (33, 341) = 0.979, p = 0.470). There
was a significant effect of Time on cocaine infusions (F (11, 341) = 13.851, p = 0.000;
see Fig. 3-1) and mg/kg cocaine infusions (mg/kg) (F (11, 341) = 12.518, p < 0.0001;
see Fig. 3-2) indicating that rats escalated their cocaine intake over time. There was a
significant effect of time on cocaine infusions The total cocaine intake (mg/kg) during
70
self-administration also did not differ between Coc+EtOH and Coc+H2O groups later
assigned to receive Ceftriaxone 200 mg/kg or vehicle treatment (F (3, 32) = 0.820, p =
0.372; see Fig. 3-4).
Alcohol consumption prior and during Ceftriaxone treatment
Similar to cocaine self-administration, the amount of EtOH consumed (g/kg)
during self-administration did not differ between groups later assigned to receive
Ceftriaxone 200 mg/kg or vehicle (F (1, 13) = 0.086, p = 0.774; see Fig. 3-3). There was
a significant effect of Time (F (11, 143) = 3.62, p < 0.0001) as rats increased their
alcohol intake across self-administration sessions. A group x time interaction was
detected (F (11, 143) = 2.130, p = 0.022) indicating that each group changed intake
across time in a different pattern. However the total amount of ethanol consumed (g/kg)
did not differ between groups later assigned to vehicle or Cef (t (13) = 0.225, p = 0.825;
see Fig. 3-5).
Self-administration lever presses
Active lever presses during cocaine self-administration did not differ between
Coc+EtOH and Coc+H2O groups which were later assigned to receive Ceftriaxone 200
mg/kg or vehicle treatment (F (33, 341) = 1.043, p = 0.408 see Fig. 3-6). There was no
effect of time (F (11, 341) = 1.600, p = 0.097). A main effect of Drug (EtOH vs. H2O) on
inactive lever presses during cocaine self-administration (F (33, 341) = 4.354, p = 0.005;
see Fig. 3-7). This likely occurred because the Coc+H2O+Cef group displayed greater
lever pressing on this lever during the first few days of self-administration. However,
there was no Time x Drug x Treatment interaction (F (33, 341) = 0.720, p = 0.719).
71
Extinction training.
After animals met the criteria of self-administration, they were subsequently put
into extinction training wherein presses on the active lever no longer result in drug
infusion and light and tone cues. No differences in lever presses between groups were
observed during extinction sessions. There were no main effects of Treatment or Drug.
There was also no Time x Drug x Treatment interaction (F (33, 341) = 0.147, p = 0.999;
see Fig. 3-8). There was a significant effect of Time across extinction training sessions
(F (11, 341) = 57.693, p < 0.0001). A significant effect of time is to be expected because
there will be a burst in lever pressing given that the drug and cues are no longer
presented. Rats then learn that presses on the active lever no longer result in a drug
infusion and therefore decrease responding. The number of lever presses during
extinction training on the inactive lever did not differ between groups of rats prior to
receiving treatment nor after treatment began (F (33, 341) = 0.711, p = 0.728; see Fig.
3-9). Similar to the previously active lever, there was a significant main effect of Time
across extinction training sessions (F (11, 314) = 18.208, p < 0.0001).
Cue+Cocaine–prime reinstatement during microdialysis.
All groups significantly reinstated to cue+cocaine-prime reinstatement to cocaine
seeking except for the Coc+H2O+Cef group in which Ceftriaxone attenuated
reinstatement. A three-way ANOVA revealed a significant main effect of Test (F (1,33) =
88.210, p < 0.0001; see Fig. 3-10). Post hoc analyses controlling for repeated measures
found that lever presses during the reinstatement test were significantly greater than
those during extinction for all groups (p < 0.010 for all) except for the Coc+H2O+Cef
group. A significant interaction between Test and Drug use (EtOH or H2O) was also
detected (F (3, 33) = 8.425, p = 0.007). There were no Test x Drug x Treatment or Test
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x Treatment interactions. We did not detect a main effect of Ceftriaxone on active lever
presses during reinstatement. This is plausible because while Ceftriaxone did attenuate
reinstatement in the cocaine group, (post-hoc test p < 0.500 comparing Ceftriaxone to
Vehicle); the group with a history of alcohol and cocaine use had no effect of
Ceftriaxone on reinstatement lever presses. No significance was observed comparing
inactive extinction lever presses with inactive lever presses during reinstatement testing
(See Fig. 3-11). There was no significant main effect of Test (F (1, 33) = 0.913, p =
0.346). There were no significant differences between groups in reinstatement lever
presses (F (3, 33) = 0.035, p = 0.853).
Percent Change of Glutamate in the NAc Core during Reinstatement to Cocaine Seeking.
Glutamate significantly increased in the Coc+H2O vehicle treated group. No
change in glutamate was observed in both Coc+EtOH group despite significant
reinstatement lever pressing. Along with attenuated reinstatement lever pressing, the
Coc+H2O+Cef group did not display a significant increase in NAc core glutamate. A
three-way RM-ANOVA revealed significant main effects of Drug (EtOH vs. H2O) (F (1,
27) = 24.164, p < 0.0001) and Treatment (Cef vs. Veh) (F (1,27) = 20.243, p < 0.0001;
see Fig. 3-12). There was also a main effect of Time (F (11, 297) = 1.980, p = 0.030).
There was also a significant Drug x Time interaction (F (11, 297) = 5.104, p < 0.0001).
There was also a Treatment x Time interaction (F (11, 197)= 4.215, p < 0.0001). There
was no Time x Drug x Treatment interaction. Tukey’s multiple comparisons of time
points between groups revealed no significant differences in baseline samples. The first
sample collected after baseline (10 min post I.P. cocaine injection and cue presentation
upon lever press) Coc+H2O+Veh glutamate levels were significantly different from the
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other three groups: Coc+EtOH+Veh (p = 0.006), Coc+EtOH+Cef (p = 0.0001),
Coc+H2O+Cef (p = 0.004). Glutamate levels for the Coc+H2O+Veh group were also
significantly different than the other three groups for all other time points [20 min:
Coc+EtOH+Veh (p < 0.0001), Coc+EtOH+Cef (p < 0.0001), Coc+H2O+Cef (p < 0.0001);
30 min: Coc+EtOH+Veh (p = 0.001), Coc+EtOH+Cef (p < 0.0001), Coc+H2O+Cef (p =
0.000); 40 min: Coc+EtOH+Veh (p = 0.000), Coc+EtOH+Cef (p < 0.0001),
Coc+H2O+Cef (p = 0.001); 50 min: Coc+EtOH+Veh (p = 0.031), Coc+EtOH+Cef (p <
0.0001), Coc+H2O+Cef (p = 0.021); 60 min: Coc+EtOH+Veh (p = 0.001),
Coc+EtOH+Cef (p < 0.0001), Coc+H2O+Cef (p = 0.002); 70 min: Coc+EtOH+Veh (p =
0.001), Coc+EtOH+Cef (p = 0.000), Coc+H2O+Cef (p = 0.001); 80 min: Coc+EtOH+Veh
(p < 0.0001), Coc+EtOH+Cef (p < 0.0001), Coc+H2O+Cef (p = 0.000); and 90 min:
Coc+EtOH+Veh (p = 0.003), Coc+EtOH+Cef (p < 0.0001), Coc+H2O+Cef (p = 0.008).
Glutamate levels significantly increased from baseline and remained increased
throughout the reinstatement test in the Coc+H2O+Veh group [baseline vs. 10 (p =
0.004); vs. 20 (p < 0.0001); vs. 30 (p < 0.0001); vs. 40 (p < 0.0001); vs. 50 (p = 0.001);
vs. 60 (p < 0.0001); vs. 70 (p < 0.0001); vs. 80 (p < 0.0001); vs. 90 (p = 0.027). These
findings agree with previous research showing an increase in nucleus accumbens
glutamate from baseline during the reinstatement of cocaine seeking in animals that
only have a history with cocaine use. The Coc+EtOH+Cef group glutamate levels were
significantly different from baseline only during the 80 min (p = 0.044) and 90 min (p =
0.020) time points. Computing AUC glutamate during the reinstatement test revealed a
significant main effect of Drug (F (1,27) = 28.85, p < 0.0001). There was also a main
effect of Treatment (F (1,27) = 12.590, p=0.001).
74
pMol Glutamate in the NAc Core During Reinstatement to Cocaine Seeking
Baseline levels of glutamate as measured by pMol in 10 minute samples,
significantly changed during reinstatement testing (see Fig. 3-13). Coc+H2O+Veh group
pMol glutamate increased over time. No change was observed in Coc+EtOH+Veh and
Coc+H2O+Cef groups. The Coc+EtOH+Cef group pMol glutamate decreased over time.
A three-way RM-ANOVA revealed a significant effect of time (F (17, 459) = 5.555, p =
0.000). There were also significant Time x Drug (H2O v EtOH) interaction (F (17, 459) =
11.506, p = 0.000) and Time x Treatment (Cef v Veh) interaction (F (17, 459) = 11.233,
p = 0.000). There was no Time x Drug x Treatment interaction (F (17, 459) = 1.518, p =
0.084). There was no main effect of Drug (F (1, 27) = 0.092, p = 0.764) and no main
effect of Treatment (F (1, 27) = 3.627, p = 0.068). There was a significant Drug x
Treatment interaction (F (1, 27) = 17.409, p = 0.000).
Summing together the total pMol of glutamate during baseline samples
compared to the sum of test pMol glutamate samples revealed significant changes in
the Coc+H2O+Veh and Coc+EtOH+Cef groups. A two-way RM-ANOVA revealed no
effect of time, but a significant difference in groups (F (3, 27) = 7.444, p = 0.001; see
Fig. 3-14). There was a significant interaction (F (3, 27) = 11.09, p < 0.0001). Sidak’s
post hoc analysis revealed a significant difference in Baseline and Test pMol glutamate
levels in the Coc+H2O+Veh group (p = 0.009) and the Coc+EtOH+Veh group (p =
0.0002).
Correlations
Pearson r tests were used to compare total cocaine intake, total alcohol intake,
reinstatement lever presses, and AUC glutamate with each other. In addition, the effects
of alcohol use and treatment with Ceftriaxone were evaluated individually. Taking into
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account all groups (Coc+H2O+Veh, Coc+H2O+Cef, Coc+EtOH+Veh, Coc+EtOH+Cef),
the total amount of cocaine consumed did not correlate with the total amount of EtOH
consumed (R2 = -0.32, p = 0.090) as we expected. Total EtOH consumption correlated
with active lever presses during reinstatement (R2 = 0.40, p = 0.027) and percent
change in AUC glutamate (R2 = -0.41, p = 0.021). Evaluating only animals treated with
Veh, there is a significant correlation between EtOH intake and change in AUC
glutamate (R2 = -0.78, p = 0.001).
Discussion
In Experiment 2, rats were tested for cue+cocaine–primed reinstatement to
cocaine seeking. There was a significant increase in NAc glutamate release
corresponding to reinstatement to cocaine seeking in Coc+H2O animals treated with
vehicle. Ceftriaxone attenuated reinstatement and prevented an efflux of NAc glutamate
in Coc+H2O+Cef animals. Both groups with a history of alcohol consumption
(Coc+EtOH+Veh and Coc+EtOH+Cef) significantly reinstated to cocaine seeking,
however there was no increase in glutamate from baseline samples.
Prior to treatment with Ceftriaxone, all groups had similar cocaine intake. Rats
that drank alcohol self-administered the same amount of cocaine as the rats that only
had access to water after the daily self-administration sessions. Given that all rats
received comparable amounts of cocaine, we would not expect the underlying
neurobiology to differ between groups based on cocaine consumption. Therefore, the
changes we observed in glutamate release during reinstatement can be attributed to the
history of alcohol consumption. Similarly, the two groups of rats that consumed alcohol
in addition to cocaine did not differ in the total amount of alcohol consumed. The
76
difference in glutamate that we observed between the two alcohol groups is credited to
the effects of Ceftriaxone.
The total amount of cocaine consumed did not correlate with the total amount of
alcohol consumption. This lack of correlation is important because that would mean that
EtOH consumption altered the amount of cocaine rats consumed during the self-
administration sessions compared to rats with no access to alcohol. If alcohol lessened
the amount of cocaine intake, then this would compromise the integrity of the model of
poly-drug use and specific relapse to cocaine seeking behavior. For example, rodents
could favor alcohol intake over cocaine and therefore not show the same amount of
desire to reinstate to cocaine seeking. Oppositely, if alcohol increased the amount of
cocaine intake compared to rats that only had access to water, this could also confound
the reinstatement test. Previous research showed that the self-administration of cocaine
in an operant task is facilitated on the following day by post-session consumption of oral
EtOH (Knackstedt and Ettenberg, 2005). Conversely, pre-treatment with experimenter
administered cocaine led to animals preferring to drink alcohol to water (Knackstedt et
al., 2006). In both Experiments 1 and 2, we saw no differences in the amount of cocaine
or alcohol consumed throughout self-administration. However, a correlation between the
total amount of EtOH consumed and reinstatement lever pressing was observed.
Greater alcohol intake was associated with a greater number of lever presses during
reinstatement to cocaine seeking. Thus, while alcohol intake did not alter cocaine self-
administration, it does increase later reinstatement to cocaine seeking.
After self-administration of cocaine alone, treatment with Ceftriaxone inhibits both
cue and drug-primed reinstatement (Knackstedt et al., 2010). Ceftriaxone reduces
77
alcohol intake in alcohol preferring rats (Sari et al., 2013) and outbred rats (Stennett et
al., 2017). Ceftriaxone inhibits the reinstatement to alcohol seeking (Alhaddad et al.,
2014a; Qrunfleh et al., 2013). Taking this information together, we hypothesized that
Ceftriaxone would be effectual at preventing reinstatement to cocaine seeking despite a
history of alcohol use. However, Experiment 1 demonstrated that Ceftriaxone is no
longer effective in preventing drug seeking when animals have a history of cocaine and
alcohol use.
As expected, in Experiment 2, animals with a history of cocaine and alcohol use
significantly reinstated to cocaine seeking. However, neither alcohol-consuming group
displayed an increase in glutamate in the NAc core during the reinstatement test. The
group of rats with a history of only cocaine use and saline treatment (Coc+H2O+Veh)
showed a significant increase in glutamate during reinstatement in agreement with
previous work (Trantham-Davidson et al., 2012; McFarland et al., 2003; LaCrosse et al.,
2016; Smith et al., 2017). The cocaine use only group treated with Ceftriaxone
(Coc+H2O+Cef) did not significantly reinstate to cocaine seeking and did not display an
increase in glutamate, which is also in agreement with previous research (Knackstedt et
al., 2010; Trantham-Davidson et al., 2012). Interestingly, the Coc+H2O+Cef group’s
glutamate levels mimicked those of the animals with a history of cocaine and alcohol
use.
In order to understand findings from this study, it is important to remember what
cocaine and alcohol individually do to the NAc core glutamatergic system. Following
cocaine self-administration, basal NAc core extrasynaptic glutamate is decreased
(Baker et al., 2003; Lutgen et al., 2012; Madayag et al., 2007; Trantham-Davidson et al.,
78
2012). Oppositely, NAc core extracellular glutamate levels are increased following
contingent and non-contingent alcohol use (Pati et al., 2016; Griffin et al., 2014;
Melendez et al., 2005; Das et al., 2015). Thus, it is possible that animals that consume
both alcohol and cocaine do not show the decrease in basal glutamate observes in rats
that only self-administer cocaine. In fact, it is entirely possible that the combined effects
of past cocaine and alcohol use could negate each other and render glutamate levels in
the NAc core to resemble that of a drug naïve animal, neither raised nor decreased. In
support of a direct causal relationship between alcohol consumption and the role of
glutamate in mediating reinstatement, the total amount of EtOH consumed was
negatively correlated with the percent change in area under the curve (AUC) glutamate
during reinstatement.
These data indicate that in rats with a history of cocaine and alcohol self-
administration, reinstatement of cocaine-seeking is no longer accompanied by the
increase in nucleus accumbens glutamate efflux that has previously been observed in
rats with a history of cocaine self-administration alone (Trantham-Davidson et al., 2012;
McFarland et al., 2003). These findings are novel and unexpected because of previous
research evaluating reinstatement to cocaine alone (Smith et al., 2017; McFarland et
al., 2003; Lacrosse et al., 2016) and alcohol alone (Backstrom and Hyytia, 2004; 2005;
Gass et al., 2010) showed consistently increased glutamate levels in the NAc core
during reinstatement. Indeed, cocaine-seeking primed by context, cocaine-associated
cues, and cocaine itself is accompanied by glutamate release in the NAc core,
regardless of whether rats experienced extinction training or abstinence, which allows
79
for context-primed relapse (LaCrosse et al., 2016; McFarland et al., 2003; Smith et al.,
2017).
Animals with extended access to cocaine seeking (6 hours/day) significantly
reinstate to cue-induced cocaine seeking (Cannella et al., 2016). Animals also increase
their cocaine intake over time in an extended access model (Cannella et al., 2016). No
differences in cocaine seeking reinstatement behavior are observed between long and
short access self-administration groups after two to three weeks of extinction training
(De Vries et al., 2005). In a study comparing short-access (2 hours/day) to long access
(6 hours/day) cocaine self-administration, no differences are observed in lever pressing
during extinction training (Knackstedt and Kalivas, 2005). In this same study, both short
and long access groups significantly reinstate to cocaine seeking following a 10 mg/kg
dose of cocaine.
Glutamate levels in the NAc core do not increase following acute injections of
cocaine (1 or 2 mg/kg) (Miguens et al., 2008). However, following 20 consecutive days
of cocaine self-administration, glutamate levels are decreased in the NAc core as seen
by microdialysis (Miguens et al., 2008). One such study was completed comparing the
role of NAc glutamate efflux during reinstatement to cocaine seeking based on short
versus long access cocaine and varying durations of extinction training (Lutgen et al.,
2012). Though the high dose/long access to cocaine self-administration condition
results in significantly greater cocaine intake than the low dose/short access condition,
basal glutamate levels are equally decreased compared to drug naïve controls. In
addition, baseline levels of glutamate do not differ between high and low intake
conditions based on 1, 21, or 60 days of extinction training (Lutgen et al., 2012). This is
80
in agreement with other work showing decreased baseline glutamate on days 1 and 5 of
extinction training (Miguens et al., 2008). During a cocaine primed reinstatement test,
both high and low intake conditions display the same magnitude of increased glutamate
efflux into the NAc. Again, glutamate increases in the NAc do not differ based on the
length of drug free periods (Lutgen et al., 2012). Therefore, more self-administration
sessions (20) as compared to the present study (12), results in the similar decrease of
resting glutamate in the NAc core. Also, shorter periods of extinction than used in the
present study and a priming dose of cocaine, produce increased glutamate efflux in the
NAc core (Miguens et al., 2008).
Another factor that does not change glutamate release in the NAc during
reinstatement to cocaine seeking is the schedule of reinforcement for which was
required during the self-administration period. A study utilizing similar methodology
started animals at an FR1 schedule with a higher dose (1 mg/kg) of cocaine and then
switched to an FR2 schedule with half the dose (0.5 mg/kg) of cocaine. During cocaine
prime reinstatement testing, glutamate efflux was observed in the NAc similar to our
work (Xi et al., 2006). Taking these findings into consideration, we do not attribute any
of our microdialysis findings to be influences by the length of cocaine access, the drug
free period during extinction training, or the fixed ratio schedule of reinforcement.
Though we present amount of NAc core glutamate in pMol, we do not believe
this accurately represents basal levels of glutamate as described prior. If our findings
were in agreement with previous literature, the Coc+H2O+Veh group’s pMol glutmate
levels prior to testing should be lower than the Coc+H2O+Cef group. Briefly, prolonged
cocaine use results in a decrease level of basal glutamate in the NAc core. Treatment
81
with Ceftriaxone returns the glutamate homeostatic mechanism to normal levels and
thereby returning basal glutamate to a drug naïve level. However, we observed the
opposite. This finding can be attributed to the method we used as not being suitable to
address basal levels of glutamate. Our method does not account for the specific probe
extraction fraction (recovery rate). In addition, we used several cohorts of rats over an
extended period of time. As a result, our probes were made at different times from
different batches from the manufacturer and therefore could have very different
recovery rates. Taking this into consideration, we utilized a percent change of glutamate
from each rats’ respective baseline to control for probe variation.
This finding is consistent with many other studies showing that Ceftriaxone
attenuates the reinstatement to cocaine seeking (Knackstedt et al., 2010; Trantham-
Davidson et al., 2012). Allowing us to evaluate the history of alcohol use versus water
only intake, we investigated if there were any correlations between the two groups
treated with Ceftriaxone. In fact, between the Coc+H2O+Cef and Coc+EtOH+Cef
conditions, reinstatement lever presses positively correlated with the history of alcohol
use. This finding is very important because it displays the effect of Ceftriaxone on
preventing reinstatement in animals that only have a history of cocaine use while having
no effect on animals with a history of both alcohol and cocaine use.
Given that the Coc+EtOH rats all significantly reinstated to cocaine seeking but
did not display an increased level of glutamate in the NAc core leads us to believe other
brain regions may be mediating reinstatement due to the history of alcohol consumption
with cocaine use. In summary, glutamate transmission in the nucleus accumbens core
does not mediate relapse to cocaine seeking in animals that consume ethanol with
82
cocaine. These findings indicate that medications targeting glutamate may not be
effective therapies for preventing relapse in humans that drink alcohol with their
cocaine.
83
Figure 3-1. Cocaine infusions during self-administration. Infusions of cocaine during self-administration did not differ between Coc+EtOH and Coc+H2O groups later assigned to receive Ceftriaxone 200 mg/kg or vehicle treatment (F (33, 341) = 0.985, p = 0.460). There was a significant effect of time on cocaine infusions (F (11, 341) = 13.851, p < 0.0001).
1 2 3 4 5 6 7 8 9 10 11 120
5
10
15
20
25
30
Day
Co
cain
e In
fusio
ns
Coc+H2O+Veh (n=11)
Coc+EtOH+Veh (n=9)
Coc+EtOH+Cef (n=7)
Coc+H2O+Cef (n=9)
84
Figure 3-2. Cocaine infusions (mg/kg) during self-administration. Infusions of cocaine
(mg/kg) during self-administration did not differ between Coc+EtOH and Coc+H2O groups later assigned to receive Ceftriaxone 200 mg/kg or vehicle treatment (F (33, 341) = 0.979, p = 0.470). There was a significant effect of time on cocaine infusions (mg/kg) (F (11, 341) = 12.518, p < 0.0001).
1 2 3 4 5 6 7 8 9 10 11 120
5
10
15
20
25
30
Day
Co
c In
fusio
ns m
g/k
gCoc+H2O+Veh (n=11)
Coc+EtOH+Veh (n=9)
Coc+EtOH+Cef (n=7)
Coc+H2O+Cef (n=9)
85
Figure 3-3. Ethanol consumed during self-administration. As measured by g/kg of body weight, the amount of EtOH consumed each day during self-administration did not differ between groups later assigned to receive Ceftriaxone 200 mg/kg or vehicle treatment (F (1, 13) = 0.0863, p = 0.774). There was a significant effect of time (F (11, 143) = 3.620, p = 0.0002). There was a group x time interaction (F (11, 143) = 2.130, p = 0.022).
1 2 3 4 5 6 7 8 9 10 11 120
1
2
3
4
5
Day
EtO
H g
/kg
Coc+EtOH+Veh (n=9)
Coc+EtOH+Cef (n=7)
86
Figure 3-4. Total cocaine intake (mg/kg) during self-administration. The total amount of cocaine infusions did not differ between Coc+EtOH and Coc+H2O groups later assigned to receive Ceftriaxone 200 mg/kg or vehicle treatment (F (3, 32) = 0.819, p = 0.372).
0
100
200
300C
ocain
e m
g/k
g
11 99 7
Coc+H2O+Veh
Coc+H2O+Cef
Coc+EtOH+Veh
Coc+EtOH+Cef
87
Figure 3-5. Total ethanol intake during self-administration. The total amount of ethanol consumed (g/kg) did not differ between groups later assigned to vehicle or Cef (t (13) = 0.225, p = 0.825).
Coc+EtOH+Veh Coc+EtOH+Cef0
10
20
30
40E
tOH
g/k
g
9 7
88
Figure 3-6. Active lever presses during self-administration. Active lever presses during cocaine self-administration did not differ between Coc+EtOH and Coc+H2O groups, which were also later assigned to receive Ceftriaxone 200 mg/kg or vehicle treatment (F (33, 341) = 1.043, p = 0.408). There was no effect of time (F (11, 341) = 1.600, p = 0.097).
1 2 3 4 5 6 7 8 9 10 11 12
0
10
20
30
40
50
Day
Acti
ve L
ever
Pre
sses
Coc+H2O+Veh (n=11)
Coc+H2O+Cef (n=9)
Coc+EtOH+Veh (n=9)
Coc+EtOH+Cef (n=7)
89
Figure 3-7. Inactive lever presses during self-administration. Inactive lever presses during cocaine self-administration differed between Coc+EtOH and Coc+H2O groups, which were also later assigned to receive Ceftriaxone 200 mg/kg or vehicle treatment (F (3, 31) = 4.354, p = 0.005). There was no Time x Drug x Treatment interaction (F (33, 341)= 0.720, p=0.719).
1 2 3 4 5 6 7 8 9 10 11 12
0
10
20
30
40
50
Day
Inacti
ve L
ever
Pre
sses
Coc+H2O+Veh (n=11)
Coc+H2O+Cef (n=9)
Coc+EtOH+Veh (n=9)
Coc+EtOH+Cef (n=7)
90
Figure 3-8. Lever presses on the previously active lever during extinction training. The number of lever presses during extinction training on the previously active lever did not differ between groups of rats prior to receiving treatment nor after treatment began (F (33, 341) = 0.147, p = 0.999). There was a significant effect of time across extinction training sessions (F (11, 341) = 57.693, p > 0.0001).
1 2 3 4 5 6 7 8 9 10 11 12
0
20
40
60
80
100
120
Day
Acti
ve L
ever
Pre
sses
Coc+H2O+Veh (n=11)
Coc+EtOH+Veh (n=9)
Coc+EtOH+Cef (n=7)
Coc+H2O+Cef (n=9)
91
Figure 3-9. Inactive lever presses during extinction training. The number of lever
presses during extinction training on the inactive lever did not differ between groups of rats prior to receiving treatment nor after treatment began (F (33, 341) = 0.711, p = 0.728). There was a significant effect of time across extinction training sessions (F (11, 341) = 18.208, p < 0.0001).
1 2 3 4 5 6 7 8 9 10 11 12
0
20
40
60
80
100
120
Day
Inacti
ve L
ever
Pre
sses
Coc+H2O+Veh (n=11)
Coc+EtOH+Veh (n=9)
Coc+EtOH+Cef (n=7)
Coc+H2O+Cef (n=9)
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Figure 3-10. Cue+Cocaine–prime reinstatement during microdialysis. A three-way ANOVA revealed a significant main effect of Test (F (1, 33) = 88.21, p < 0.0001). Post hoc analyses controlling for repeated measures found that lever presses during the reinstatement test were significantly greater than those during extinction for all groups (p < 0.01 for all as indicated by “ * “) except for the Coc+H2O+Cef group. A significant interaction between Test and Drug use (EtOH or H2O) was also detected (F (3, 33) = 8.425, p = 0.007). There were no Test x Drug x Treatment or Test x Treatment interactions.
Extinction RL0
50
100
150L
ever
Pre
sses
Coc+H2O+Veh
Coc+EtOH+Veh
Coc+EtOH+Cef
Coc+H2O+Cef
*
*
*
11 9 9 7
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Figure 3-11. Inactive lever presses during Cue+Cocaine-prime reinstatement testing. No
significant differences were observed comparing inactive extinction lever presses with inactive lever presses during reinstatement testing. There was no significant main effect of Test (F (1, 33) = 0.913, p = 0.346). There were no significant differences between groups in reinstatement lever presses (F (3, 33) = 0.035, p = 0.853).
Extinction LL0
50
100
150L
ever
Pre
sses
Coc+H2O+Veh
Coc+EtOH+Veh
Coc+EtOH+Cef
Coc+H2O+Cef
94
Figure 3-12. Percent change of glutamate in the NAc core during reinstatement to
cocaine seeking. A three-way RM-ANOVA revealed significant main effects of Drug (EtOH vs. H2O) (F (1, 27) = 24.164, p < 0.0001) and Treatment (Cef vs. Veh) (F (1, 27) = 20.243, p < 0.0001). There was also a main effect of Time (F (11, 297) = 1.98, p = 0.030). There was also a significant Drug x Time interaction (F (11, 297) = 5.1038, p < 0.0001). There was also a Treatment x Time interaction (F (11,197)= 4.215, p=0.000). Glutamate levels significantly increased from baseline and remained increased throughout the reinstatement test in the Coc+H2O+Veh group (p < 0.0001). Glutamate levels in this group are also significantly higher than the other three groups (p < 0.001). * = significantly different from respective baseline. # = Coc+H2O+Veh is significantly different than other three groups.
Bas
elin
e
Bas
elin
e
Bas
elin
e 10 20 30 40 50 60 70 80 90
60%
80%
100%
120%
140%
160%
Time
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tam
ate
(%
Baselin
e) Coc+H2O+Veh (n=8)
Coc+EtOH+Veh (n=8)
Coc+EtOH+Cef (n=7)
Coc Inj.
** * *
** * *
*
* *
#
# # ##
# ##
#Coc+H2O+Cef (n=8)
95
Figure 3-13. pMol glutamate in NAc core during reinstatement testing. A three-way RM-
ANOVA revealed a significant effect of Time (F (17, 459) = 5.555, p = 0.000). There was also a significant Time x Drug (H2O v EtOH) interaction (F (17, 459) = 11.506, p = 0.000) and a Time x Treatment (Cef v Veh) interaction (F (17, 459) = 11.233, p = 0.000). There was no Time x Drug x Treatment interaction (F (17, 459) = 1.518, p = 0.084). There was no main effect of Drug (F (1, 27) = 0.092, p = 0.764) and no main effect of Treatment (F (1, 27) = 3.627, p = 0.068). There was a significant Drug x Treatment interaction (F (1, 27) = 17.409, p = 0.000).
-90 -70 -50 -30 -10 10 30 50 70 90
1
3
5
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Time
pM
ol G
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Coc+H2O+Veh
Coc+EtOH+Veh
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Coc Inj.
#
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##
* * * * * * **
* *++
++
++
Coc+H2O+Cef
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CHAPTER 4 EXPERIMENT 3: THE NUCLEUS ACCUMBENS CORE IS LESS ACTIVE DURING
REINSTATEMENT TO COCAINE SEEKING IN ANIMALS WITH A HISTORY OF ALCOHOL USE AS COMPARED TO COCAINE USE ONLY
Introduction
We are interested in identifying the neurocircuitry that mediates cocaine
reinstatement in rats that also consuming alcohol, as our results from Experiment 2
indicate that a history of alcohol and cocaine use no longer involves glutamate release
in the NAc. The immediate early gene c-Fos is a marker of trans-synaptic neuronal
activation. Upon stimulation, the c-Fos gene encodes for a transcription factor, the Fos
protein. c-Fos has been used in addiction research as a marker to evaluate activity in
reward circuitry brain regions including the NAc core, PFC, hippocampus, and the
amygdala. Signaling through NMDA and D1 receptors induces Fos expression
(Horowitz et al., 1997a). Stimuli including cocaine itself (Graybiel et al., 1990; Young et
al., 1991) and cocaine related stimuli (Brown et al., 1992; Crawford et al., 1995) induce
c-Fos and its product Fos. Cocaethylene also induces Fos expression (Torres and
Horowitz, 1999; Horowitz et al., 1997b). The IEG expression here is mediated by
dopamine D1 receptors in the brain (Horowitz et al., 1997a). Cocaine priming injections
in rodents increased Fos expression in multiple brain regions including the VTA and
amygdala (Neisewander et al., 2000). Other brain regions have demonstrated Fos
expression immediately following a cue-primed reinstatement of cocaine-seeking test,
including the NAc core, NAc shell, and the PFC. In fact, there was a correlation between
reinstatement and Fos in the BLA, the more cocaine-seeking behavior presented, the
more Fos expression. In addition, the PFC was broken down into the PrL and IL where
there was significantly more Fos expression in the cue-prime reinstatement group
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compared to the group that received no cues. This pattern was also observed in the
NAc core, VTA and BLA (Kufahl et al., 2009).
Given the findings from these studies, we sought to evaluate the activity of the
NAc core, NAc shell, PrL, IL, and VTA during cue+cocaine prime reinstatement.
However, based on the findings from Experiment 2 where the NAc core did not show a
significant increase in glutamate for groups that had a history of alcohol use with
cocaine and for all groups receiving Ceftriaxone treatment, we do not expect to see a
strong presence of c-Fos positive cells in the NAc core of these groups. We do expect
to see large amounts of c-Fos expression in the NAc core of animals that have a history
of cocaine use only and were treated with saline (Coc+H2O+Veh).
Materials and Methods
Subjects
Animals from experiment 2 were used for this experiment of quantifying brain
region activation via c-Fos expression. As described in experiment 2, animals
underwent a cue+cocaine primed reinstatement test to cocaine seeking that was a
duration of 90 min. As strong Fos expression is evident 90 minutes to 2 hours following
neuronal activation (Young et al., 1991), analysis of Fos positive cells immediately
following the 2 hour reinstatement session will reflect activity near the beginning of the
reinstatement session. This time frame is the period of highest active lever pressing
(Mahler and Aston-Jones, 2012). Centered on the findings of c-Fos expression following
cues to drug seeking and cocaine itself, we analyzed several brain regions following cue
+ cocaine – primed reinstatement to cocaine seeking. In addition to the rats used from
Experiment 2, we utilized a control saline group of rats to control for random or baseline
brain activity. This group received yoked saline infusions during self-administration
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sessions. They were treated the same through extinction and cue+cocaine prime
reinstatement sessions.
This time was chosen because c-Fos expression is maximal 60-90 minutes after
a behavior, which corresponds with peak reinstatement behavior in the beginning of the
testing session (Kufhal et al., 2009; Neisewander et al., 2000). Animals removed from
Experiment 2 due to microdialysis sample collection issues (n = 4) were treated the
same as all other animals during reinstatement procedures. In doing so, we were able
to use these rats for c-Fos quantification in this experiment.
Tissue Preparation
Animals from Experiment 2 were used for this study. Immediately following
microdialysis and reinstatement testing, animals were deeply anesthetized with
pentobarbital (100 mg/kg, IP) and were transcardially perfused with phosphate buffered
saline (PBS) followed by 4% paraformaldehyde (PFA). Brains were extracted and
preserved in 4% PFA for 24 hours then transferred to 20% sucrose solution for 48
hours. Brains were then frozen and stored at -80°C until sliced.
Tissue Slicing
Brains were sliced on a cryostat in 30 µm coronal sections. Slices were collected
for probe placement within the nucleus accumbens core and for immunohistochemistry
of c-Fos staining. Slices for probe placement verification were stored in PBS-azide until
they were transferred to slides for cresyl violet staining. For c-Fos staining, slices were
collected from the PFC, NAc core, NAc shell, amygdala, and VTA. Bregma coordinates:
PFC (– 3.24 mm), NAc Core and Shell (– 1.80 mm), VTA (- 6.72).
Areas analyzed include the prelimbic cortex (PrL), Infralimbic cortex (IL), Nucleus
accumbens core (NAc Core), Nucleus accumbens dorsomedial shell (DM), nucleus
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accumbens ventromedial shell (VM), and nucleus accumbens lateral shell (L) as
depicted in Mahler and Aston-Jones, 2012. Also, the VTA was analyzed.
Immunohistochemistry for C-Fos
Sections were stained for Fos expression. Fos was visualized by incubating free-
floating sections in rabbit anti-Fos (1:10000, Santa Cruz Biotechnology) overnight,
incubation in biotinylated donkey anti-rabbit secondary antibody (1:500, Jackson
ImmunoResearch Laboratories) for 2 h, and incubation in avidin-biotin complex (ABC,
1:500) for 1.5 h. Finally, sections were incubated in 3,3 -diaminobenzidine (DAB,
Sigma), producing a brown reaction product in the nucleus. The sections were then
mounted onto slides, air-dried, and protected with a coverslip.
Imaging Brain Regions
Brain regions and boundaries of each location were determined with reference to
anatomical landmarks using a rat brain atlas (Paxinos and Watson, 2007, 6th Edition).
Brain regions were imaged via Tuscan imaging software IS Capture on an Olympus
BX51 MF5 with a 4x objective lens. Images were reconstructed/compiled into mosaics
manually in Adobe Illustrator. Fos expression was analyzed using ImageJ (NIH). Data is
represented as c-Fos positive cells divided by the area of the brain region measured
and reported as c-Fos positive cells per mm2.
Statistical Analyses
GraphPad Prism (Version 7, GraphPad Software, La Jolla, CA, USA) was used
to analyze all data. For all statistical analysis used, the alpha level was set at p<0.05.
One-way ANOVAs were used to compare the amount of c-Fos expression per mm2 in
each structure between groups. Tukey’s post hoc analysis was used to identify
significant main effects between groups. Across brain regions, the number of animals
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per group varies due to numerous reasons including problems with sectioning and
damage to tissue during immunohistochemistry. The numbers of rats per group are
listed in Table 4-1.
Correlations
Correlations were conducted with Pearson r tests comparing active lever presses
during reinstatement tests, c-Fos expression in the NAc core, NAc shell, PFC, VTA,
alcohol consumed during self-administration sessions, cocaine consumed during self-
administration sessions, and glutamate levels during reinstatement as expressed as the
area under the curve (AUC) of glutamate during reinstatement divided by AUC
glutamate during baseline samples.
Table 4-1. Number (n) of tissue samples per group.
Coc+H2O+Veh Coc+H2O+Cef Coc+EtOH+Veh Coc+EtOH+Cef SaL+H2O+Veh
NAc Core 6 7 8 7 6
NAc Shell 6 7 8 7 6
PFC 6 8 8 7 6
VTA 6 8 5 7 4
Results
Nucleus Accumbens Core
The number of c-Fos positive cells in the nucleus accumbens core were counted
and represented as c-Fos positive cells per mm2. All conditions have significantly more
c-Fos positive cells than the saline control group indicating that the NAc core is active
during reinstatement testing. Analysis using a one-way ANOVA showed a significant
effect of group (F (4, 29) = 13.670, p < 0.0001). All groups showed significantly more c-
Fos positive cells than the saline control group [vs. Coc+H2O+Cef (p = 0.001); vs.
Coc+H2O+Veh (p < 0.0001); vs. Coc+EtOH+Veh (p = 0.001); Coc+EtOH+Cef (p =
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0.013)]. The Coc+H2O+Veh group showed significantly more c-Fos than all other
groups [Coc+H2O+Cef (p = 0.023), Coc+EtOH+Veh (p = 0.013), Coc+EtOH+Cef (p =
0.003)]. This finding indicates that Ceftriaxone prevented activity in the NAc core during
the reinstatement test in animals that only have a history of cocaine use.
Nucleus Accumbens Shell
Analysis using a one-way ANOVA of total c-Fos expression in the whole NAc
shell showed a significant effect of group (F (4, 29) = 10.050, p < 0.0001). All groups
show significantly more c-Fos positive cells compared to the Sal+H2O+ Veh group [vs.
Coc+H2O+Veh (p = 0.000); vs. Coc+H2O+Cef (p < 0.0001); Coc+EtOH+Veh (p =
0.001); Coc+EtOH+Cef (p = 0.036)]. There were no significant differences between
other groups indicating all drug treatment groups showed equal activity throughout the
shell, but more activation than if they were not reinstating to drug-seeking. The nucleus
accumbens shell was subdivided into the dorsomedial, ventromedial, and lateral shell,
as these sections receive differing projections from the prefrontal cortex (Mahler and
Aston-Jones, 2012; Voorn et al., 2004).
Dorsomedial shell
Dorsomedial nucleus accumbens shell c-Fos expression analysis using a one-
way ANOVA showed significant differences between groups (F (4, 29) = 10.560, p <
0.0001). Tukey’s post hoc analysis showed that all drug treated groups had significantly
higher c-Fos expression than the Sal+H2O+Veh group [vs. Coc+H2O+Veh (p = 0.000);
vs. Coc+H2O+Cef (p < 0.0001); Coc+EtOH+Veh (p = 0.001); Coc+EtOH+Cef (p =
0.037)]. In addition, animals in the Coc+H2O+Cef group displayed significantly more c-
Fos than the Coc+EtOH+Cef group, (p = 0.036). Because both groups received
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Ceftriaxone treatment, it is likely that the history of alcohol use is responsible for the
differences in dorsomedial NAc shell c-Fos expression.
Ventromedial shell
Significant differences between groups were revealed by a one-way ANOVA (F
(4, 29) = 4.923, p = 0.004). Tukey’s post hoc analysis showed that three of the four
groups had significantly higher c-Fos expression than the Sal+H2O+Veh group [vs.
Coc+H2O+Veh (p = 0.007); vs. Coc+H2O+Cef (p = 0.006); Coc+EtOH+Veh (p = 0.047)],
whereas the Coc+EtOH+Cef (p = 0.318) group did not.
Lateral shell
Significant differences between groups were revealed by a one-way ANOVA (F
(4, 29) = 6.854, p = 0.001). Tukey’s post hoc analysis showed that all drug treated
groups displayed significantly higher c-Fos expression than the Sal+H2O+Veh group
[vs. Coc+H2O+Veh (p = 0.000); vs. Coc+H2O+Cef (p = 0.006); Coc+EtOH+Veh (p =
0.008); Coc+EtOH+Cef (p = 0.025)]. No other differences between groups were
observed.
Prefrontal Cortex
One-way ANOVA revealed a significant effect of group (F (4, 31) = 13.070, p <
0.000). Tukey’s post hoc analysis showed that all drug treated groups displayed
significantly higher c-Fos expression than the Sal+H2O+Veh group [vs. Coc+H2O+Veh
(p = 0.000); vs. Coc+H2O+Cef (p = 0.002); Coc+EtOH+Veh (p = 0.001); Coc+EtOH+Cef
(p = 0.012)]. The Coc+H2O+Veh group also displayed significantly more c-Fos
expression than the other three drug treatment groups [vs. Coc+H2O+Cef (p = 0.019);
vs. Coc+EtOH+Veh (p = 0.041); Coc+EtOH+Cef (p = 0.005)]. No other differences
between groups were observed.
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Prelimbic cortex
A one-way ANOVA revealed a significant effect of group (F (4, 31) = 9.917, p =
0.000). Tukey’s post hoc analysis showed that all drug treated groups displayed
significantly higher c-Fos expression than the Sal+H2O+Veh group [vs. Coc+H2O+Veh
(p = 0.000); vs. Coc+H2O+Cef (p = 0.003); Coc+EtOH+Veh (p = 0.005); Coc+EtOH+Cef
(p = 0.044)]. In addition, the Coc+H2O+Veh group displayed significantly more c-Fos
expression than the Coc+EtOH+Cef group (p = 0.016).
Infralimbic cortex
A one-way ANOVA revealed a significant effect of group (F (4, 31) = 8.745, p <
0.0001). Tukey’s post hoc analysis showed that all drug treated groups displayed
significantly higher c-Fos expression than the Sal+H2O+Veh group [vs. Coc+H2O+Veh
(p = 0.000); Coc+EtOH+Veh (p = 0.010)] except for the Coc+H2O+Cef group (p = 0.117)
and the Coc+EtOH+Cef group (p = 0.061), which showed a trend. In addition, the
Coc+H2O+Veh group displayed significantly more c-Fos expression than the
Coc+H2O+Cef group (p = 0.009) and the Coc+EtOH+Cef group (p = 0.031).
VTA
A one-way ANOVA revealed significant differences between groups (F (4, 25) =
4.278, p = 0.009). The Coc+H2O+Cef group displayed significantly more c-Fos
expression than the Coc+H2O+Veh (p = 0.050) and the Sal+H2O+Veh group (p =
0.007). No other group differences were found.
Correlations
Brain regions mentioned above were correlated with total cocaine intake, total
EtOH intake, and percent change in AUC glutamate during cue+cocaine prime
reinstatement testing. Pearson r tests showed significant correlations of total EtOH
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intake in all groups (Coc+H2O+Veh, Coc+H2O+Cef, Coc+EtOH+Veh, Coc+EtOH+Cef)
with, NAc shell (R2 = -0.38, p = 0.040) Fos expression. In vehicle treated groups,
alcohol intake correlated with NAc core (R2 = -0.60, p = 0.030) and PFC (R2 = -0.55, p =
0.040) Fos expression.
The percent change in AUC glutamate during reinstatement significantly
correlated with total PFC (R2 = 0.480, p = 0.009) c-Fos expression when comparing all
groups. Taking into consideration only the Coc+H2O+Veh and Coc+H2O+Cef groups,
VTA c-Fos expression and percent change in AUC glutamate significantly correlated (R2
= 0.630, p = 0.016). Evaluating only Veh-treated groups, there is a significant correlation
between AUC glutamate with PFC (R2 = 0.570, p = 0.034) total c-Fos expression.
Groups that received EtOH showed AUC glutamate correlating with NAc shell (R2 =
0.520, p = 0.040) Fos expression.
Table 4-2. Correlation of AUC Glutamate p value.
All Groups Coc+H2O Only
Coc+EtOH Only
Veh Only Cef Only
NAc Core 0.077 0.373 0.418 0.400 0.609
NAc Shell 0.160 0.998 0.047 0.745 0.100
PFC 0.009 0.016 0.929 0.034 0.672
VTA 0.119 0.009 0.538 0.158 0.838
Table 4-3. Correlation of EtOH Consumption p value.
All Groups Coc+H2O Only
Coc+EtOH Only
Veh Only Cef Only
NAc Core 0.067 n/a 0.997 0.032 0.400
NAc Shell 0.048 n/a 0.743 0.688 0.017
PFC 0.069 n/a 0.328 0.043 0.418
VTA 0.472 n/a 0.626 0.419 0.196
Discussion
Glutamate transmission in the nucleus accumbens does not mediate relapse to
cocaine seeking in animals that consume ethanol with cocaine. These findings indicate
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that medications targeting glutamate may not be effective therapies for preventing
relapse in humans that drink alcohol with their cocaine.
The findings from Experiment 2 show no increase in NAc core glutamate levels
during cue+cocaine primed reinstatement to cocaine seeking if an animal has a history
of co-morbid alcohol and cocaine use. Given that all animals with a history of alcohol
use and the vehicle treated cocaine group reinstated to cocaine seeking, we conclude
that the nucleus accumbens core is less active in animals with a history of polydrug use.
Therefore, we sought out to investigate potential active brain regions from the addiction
neurocircuitry including the NAc core, NAc shell and its three subregions, the PFC and
two of its subregions, and the ventral tegmental area. In addition, correlations were
performed between total cocaine intake, total EtOH intake, reinstatement lever presses,
percent change in AUC glutamate with total c-Fos expression in the NAc core, NAc
shell, PFC, and VTA.
Here, the expression of immediate early gene c-Fos, was quantified in order to
assess what brain regions were active during reinstatement to cocaine seeking. In
addition to the groups from Experiment 2, we utilized a group of rats that were yoked-
saline controls, only had access to water, and were treated with vehicle but placed back
into the operant chamber prior to perfusion in order to control for baseline c-Fos
expression. This group did not receive a cocaine-priming injection; instead they
received a saline injection IP.
Much of the previous research involves only cue or cocaine primed reinstatement
as compared to this experiment where we used both types on reinstatement. Following
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extinction training, the NAc core, prelimbic cortex, and the VTA are vital for cocaine-
primed reinstatement (McFarland and Kalivas, 2001; Sun and Rebec, 2003).
In the NAc core, rats with a history of cocaine use, cocaine and alcohol use, and
pretreatment with saline vehicle or Ceftriaxone 200 mg/kg all display significantly more
c-Fos expression per mm2 than the yoked-saline control group. Given that all groups
significantly reinstated to cocaine seeking, activation in the NAc core is involved with
drug-seeking behavior. Interestingly, the groups with a history of cocaine use only
differed significantly in c-Fos expression based of whether they were pre-treated with
Ceftriaxone 200 mg/kg or vehicle. The Coc+H2O+Cef group showed significantly less c-
Fos positive cells than the Coc+H2O+Veh indicating that Ceftriaxone did in fact,
normalize glutamate levels in the NAc core similar to previous findings by our labs and
others (Fischer et al., 2013; LaCrosse et al., 2016; Knackstedt et al., 2010; Sari et al.,
2009; Sondheimer and Knackstedt, 2011).
We also evaluated the NAc shell based on previous research showing distinct
differences in the shell and core of the nucleus accumbens in cue induced relapse to
drug seeking. For example the NAc core and not the shell, is essential for cue-primed
cocaine seeking behavior (Fuchs et al., 2004a; Ito et al., 2004; Kufhal et al., 2009; Di
Ciano and Everitt, 2001). Following the methods of Mahler and Aston-Jones (2012), we
divided the NAc shell into the following three subregions: dorsomedial, ventromedial,
and lateral shell. Our findings regarding total NAc Shell area showed all groups
expressing significantly more c-Fos positive cells than the saline control vehicle
treatment group. However, there were no significant differences between groups
whether or not they had cocaine only, cocaine and alcohol, or Ceftriaxone treatment.
107
In the dorsomedial NAc shell, all drug treated groups showed significantly more
c-Fos expression than the saline control group. In addition, the Coc+H2O+Cef group
had significantly more c-Fos expression than the Coc+EtOH+Cef group. The difference
between these groups is the history of alcohol consumption in addition to cocaine use.
Since both groups were treated with Ceftriaxone, the lesser amount of dorsomedial
shell activation could be attributed to alcohol’s effects. This finding is in agreement with
the difference in c-Fos expression in the NAc core and the lack of glutamate release if
the rat has a history of alcohol use. In the lateral shell, all groups had significantly more
c-Fos expression than the Sal+H2O+Veh group similar to the dorsomedial portion of the
NAc shell. No other group differences were observed lateral shell c-Fos expression.
The ventromedial NAc shell had interesting findings that differ than the other two
regions of the shell. Not all groups showed significantly more c-Fos expression than the
saline control group. The Coc+H2O+Cef, Coc+H2O+Veh, and the Coc+EtOH+Veh group
displayed more c-Fos positive cells than the saline control group. However, the
Coc+EtOH+Cef group did not significantly differ from the saline control group.
Interestingly, there were no other group differences found indicating a low amount of c-
Fos activation across all groups of animals with a history of drug use. Therefore, the
ventromedial NAc shell may not be heavily involved in the reinstatement to cue+coc-
primed drug seeking.
The NAc receives glutamatergic projections for the PFC and these projections
have long been implicated in drug-seeking behavior of cocaine and alcohol (Childress et
al., 1999; Rao and Sari, 2012). The PFC itself is instrumental in drug reinforcement and
reinstatement to drug seeking (Goldstein and Volkow, 2002). This involvement of the
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PFC has been discovered by the use of inactivation studies blocking reinstatement to
drug seeking (McFarland et al., 2003; McLaughlin and See, 2003; Stefanik et al., 2016).
For example, the inactivation of the medial PFC by TTX infusions attenuates cue-
primed reinstatement to cocaine seeking (Fuchs et al., 2005; See, 2002). Given the
continuously proven importance of the prefrontal cortex in drug use and relapse, we
investigated the activation of the infralimbic and prelimbic subregions of the PFC
separately, as well as the additive activation of the two PFC regions. In fact, the PrL
sends projections to the NAc core that promote drug seeking (McFarland et al., 2003;
Stefanik et al., 2013). Likewise, PrL inactivation via lidocaine attenuates cue-primed
reinstatement (Di Pietro et al., 2006; Fuchs et al., 2006).
Investigation of the total c-Fos expression of the PrL and IL cortex showed a
significant difference between groups. All groups had significantly more c-Fos positive
cells than the Sal+H2O+Veh group. Also, the Coc+H2O+Veh showed significantly more
c-Fos expression than the other three groups. This finding is in agreement with others
work showing the effects of cues increasing Fos expression in the PrL and IL of animals
reinstating to cocaine seeking after extinction training (Kufahl et al., 2009). Also in
agreement with previous research, we observed more c-Fos activation in the PrL cortex
than in IL in the Coc+H2O+Veh group (Zavala et al., 2008). Given that the
Coc+H2O+Veh was the only group to show an increase in glutamate during
reinstatement in Experiment 2, and this group has significantly more c-Fos expression
than the other 3 groups, the PFC could be the major source of activity for cue+coc-
primed reinstatement to cocaine seeking. The use of alcohol and/or treatment with
Ceftriaxone could shift activation to a different brain region.
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Further investigation into the prefrontal cortex showed different levels of
activation between the infralimbic and prelimbic cortex. In the prelimbic cortex, all
groups had significantly more c-Fos expression than the saline control group. In
addition, the Coc+H2O+Veh showed significantly more c-Fos expression than the
Coc+EtOH+Cef group. This difference could be an example of the additive effects of
previous alcohol use and Ceftriaxone treatment on PFC activation. Alternately, the
infralimbic cortex had unique activation as compared to the other brain regions. Both
vehicle treated groups, Coc+H2O+Veh and Coc+EtOH+Veh, showed significantly more
c-Fos expression compared to the Sal+H2O+Veh group. However, both Ceftriaxone
treated groups did not have significantly more activation than the control group.
Following the pattern of increased PFC activation in the Coc+H2O+Veh group,
this group showed significantly more than the Ceftriaxone treated groups
(Coc+H2O+Cef and Coc+EtOH+Cef). This finding further supports the effect of
Ceftriaxone in reducing PFC activation compared to a control vehicle treatment. In
summary, these findings indicate that Ceftriaxone reduces activation of the NAc core
and PFC following extinction training and a cocaine-primed reinstatement test in
animals with only a history of cocaine use. Indeed, the glutamatergic projection from the
PFC to the NAc core is heavily implicated in cocaine-reinstatement.
Interestingly, cue-primed cocaine seeking activates PrL neurons projecting to the
NAc core (McGlinchey et al., 2016) but the PrL projections to the VTA are not (Mahler
and Aston-Jones, 2012). Inconsistencies in c-Fos activation have been observed. One
study showed VTA Fos expression in response to cue-prime reinstatement (Kufahl et
al., 2009), whereas similar research did not see the same pattern of VTA activation
110
(Neisewander et al., 2000). In this experiment, the VTA of the Coc+H2O+Cef group
displayed significantly more c-Fos expression than the Coc+H2O+Veh and the
Sal+H2O+Veh group. Animals with a history of alcohol use in addition to cocaine did not
display significantly greater amounts of c-Fos expression than the saline control group.
The history of alcohol use, despite Ceftriaxone treatment, also displayed less activation
than the cocaine only group treated with Ceftriaxone.
One of the major influences correlating to other factors is the history of alcohol
consumption (total EtOH). In fact, the total amount of EtOH consumed correlated with c-
Fos expression in the NAc shell. In the NAc shell, there is a correlation between c-Fos
positive cells and total alcohol consumed. The amount of c-Fos positive cells observed
between drug treated groups did not significantly differ from each other, though there
was less activation in the groups that consumed alcohol in addition to cocaine during
self-administration.
The percent change in AUC glutamate positively correlated with the PFC c-Fos
activation. Like the NAc core, only the Coc+H2O+Veh displayed significantly more c-Fos
expression in the PFC compared to the other groups, (Coc+EtOH+Veh,
Coc+EtOH+Cef, Coc+H2O+Veh, and Sal+H2O+Veh). This finding is important because
glutamatergic synapses from the PFC to the NAc core stimulate the release of
glutamate in the core during reinstatement. The cocaine group treated with vehicle
(Coc+H2O+Veh) reinstated to cocaine seeking so the AUC is positively correlated with
c-Fos expression in the PFC.
In order to isolate the effects of a polydrug history with that of cocaine only, we
ran correlations on the Coc+H2O+Veh and Coc+EtOH+Veh groups. In the NAc core,
111
there is an inverse correlation between c-Fos expression and a history of alcohol use.
This is in agreement with the finding in Experiment 2 that greater alcohol consumption
was correlated with less glutamate efflux during reinstatement. Thus, the Fos
expression is in agreement with the dialysis data. In animals that had a history of both
drugs, there were significantly less c-Fos positive nuclei in the core compared to
animals that only had a history of cocaine use. Therefore, the history of only cocaine
consumption correlates with more c-Fos expression, which means more activity in the
NAc core during reinstatement. Inversely, the history of alcohol use means less c-Fos
expression in the nucleus accumbens core. This finding is key because an increase in
glutamate during the reinstatement to cocaine seeking was not observed even though
they did, in fact, reinstate. If there were no glutamate release into the core, we would
not expect c-Fos activation.
Again, we correlated the Coc+H2O+Veh and Coc+EtOH+Veh groups to obtain
the effects of alcohol use compared to cocaine use only without the interruption of
Ceftriaxone activity. Exhibiting the same pattern as the NAc core correlation with total
EtOH is the amount of c-Fos positive cells in the PFC Glutamatergic projections from
the PFC to the NAc core would not be as active because activation in the core was not
observed in these groups. Therefore, it stands to reason that the source of many
projections, the PFC, is not as active in the groups with a history of alcohol use
compared to the Veh treated cocaine condition (Coc+H2O+Veh). In fact, the
Coc+H2O+Veh group displayed significantly more c-Fos positive cells in the PFC
compared to the other groups indicating significantly more activation during
reinstatement.
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Interestingly, when comparing Coc+H2O groups (Cef vs. Veh), there was a
significant correlation between change in AUC glutamate and Fos expression in the
VTA. AUC glutamate significantly increased during reinstatement to cocaine seeking in
the Veh treated Coc+H2O group. However, there is little Fos expression in the VTA of
this group. Intriguingly, though there was no significant change in AUC glutamate of the
Coc+H2O+Cef group, there was significantly more Fos expression in the VTA of this
group. Therefore, there is an inverse correlation comparing these groups together.
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Figure 4-1. NAc core Fos expression. The number of c-Fos positive cells in the nucleus accumbens core were counted and represented as c-Fos positive cells per mm2. Statistical analysis using a one-way ANOVA showed a significant effect of group (F (4, 29) = 13.67, p < 0.0001). All groups showed significantly more c-Fos positive cells than the Sal+H2O+Veh group (vs. Coc+H2O+Cef, p = 0.001; vs. Coc+H2O+Veh, p < 0.0001; vs. Coc+EtOH+Veh, p = 0.001; Coc+EtOH+Cef, p = 0.013) as indicated by “+”. The Coc+H2O+Veh group showed significantly more Fos than Coc+H2O+Cef (p = 0.023) as indicated by “ * ”.
0
25
50
75
100
c-F
os c
ells / m
m2
Coc+H2O+Veh
Coc+H2O+Cef
Coc+EtOH+Veh
Coc+EtOH+Cef
Sal+H2O+Veh
+
++
+ **
*
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Figure 4-2. NAc shell total Fos expression. One-way ANOVA of total Fos expression in
the NAc shell showed a significant effect of group (F (4, 29) = 10.05, p < 0.0001). All groups show significantly more c-Fos positive cells compared to the Sal+H2O+ Veh group (vs. Coc+H2O+Veh, p = 0.0001; vs. Coc+H2O+Cef, p < 0.0001; Coc+EtOH+Veh, p = 0.001; Coc+EtOH+Cef, p = 0.036) as indicated by “+”. There were no significant differences between other groups.
0
25
50
75
100c-F
os c
ells / m
m2
Coc+H2O+Veh
Coc+H2O+Cef
Coc+EtOH+Veh
Coc+EtOH+Cef
Sal+H2O+Veh
+ +
+
+
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Figure 4-3. Dorsomedial NAc shell Fos expression. Significant differences between
groups were revealed by a one-way ANOVA (F (4, 29) = 10.56, p < 0.0001. Tukey’s post hoc analysis showed that all drug treated groups had significantly higher Fos expression than the Sal+H2O+Veh group (vs. Coc+H2O+Veh, p = 0.0003; vs. Coc+H2O+Cef, p < 0.0001; Coc+EtOH+Veh, p = 0.001; Coc+EtOH+Cef, p = 0.037) as indicated by “+”. Animals in the Coc+H2O+Cef group displayed significantly more Fos than the
Coc+EtOH+Cef group, (p = 0.036) as indicated by “ ⌃ ”.
0
25
50
75
100
125c-F
os c
ells / m
m2
Coc+H2O+Veh
Coc+H2O+Cef
Coc+EtOH+Veh
Coc+EtOH+Cef
Sal+H2O+Veh
++
+
^ +
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Figure 4-4. Ventromedial NAc shell Fos expression. Significant differences between
groups were revealed by a one-way ANOVA (F (4, 29) = 4.923, p = 0.004). Tukey’s post hoc analysis showed that all drug treated groups had significantly higher Fos expression than the Sal+H2O+Veh group (vs. Coc+H2O+Veh, p = 0.007; vs. Coc+H2O+Cef, p = 0.006; Coc+EtOH+Veh, p = 0.047) as indicated by “+”, except for the Coc+EtOH+Cef group (p = 0.318).
0
25
50
75
100
125c-F
os c
ells / m
m2
Coc+H2O+Veh
Coc+H2O+Cef
Coc+EtOH+Veh
Coc+EtOH+Cef
Sal+H2O+Veh
++
+
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Figure 4-5. Lateral NAc shell Fos expression. Significant differences between groups
were revealed by a one-way ANOVA (F (4, 29) = 6.854, p = 0.0005). Tukey’s post hoc analysis showed that all drug treated groups displayed significantly higher Fos expression than the Sal+H2O+Veh group (vs. Coc+H2O+Veh, p = 0.0002; vs. Coc+H2O+Cef, p = 0.006; Coc+EtOH+Veh, p = 0.008; Coc+EtOH+Cef, p = 0.025) as indicated by “+”.
0
25
50
75
100
125c-F
os c
ells / m
m2
Coc+H2O+Veh
Coc+H2O+Cef
Coc+EtOH+Veh
Coc+EtOH+Cef
Sal+H2O+Veh +
+
+ +
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Figure 4-6. Prefrontal cortex total Fos expression. A one-way ANOVA revealed a
significant effect of group (F (4, 31) = 13.07, p < 0.0001). Tukey’s post hoc analysis showed that all drug treated groups displayed significantly higher Fos expression than the Sal+H2O+Veh group (vs. Coc+H2O+Veh, p < 0.0001; vs. Coc+H2O+Cef, p = 0.002; Coc+EtOH+Veh, p = 0.001; Coc+EtOH+Cef, p = 0.012) as indicated by “+”. The Coc+H2O+Veh group also displayed significantly more Fos expression than the other three drug treatment groups (vs. Coc+H2O+Cef, p = 0.019; vs. Coc+EtOH+Veh, p = 0.041; Coc+EtOH+Cef, p = 0.005) as indicated by “ * “.
0
25
50
75
100
125c-F
os c
ells / m
m2
Coc+H2O+Veh
Coc+H2O+Cef
Coc+EtOH+Veh
Coc+EtOH+Cef
Sal+H2O+Veh
+
++
+ **
*
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Figure 4-7. Prelimbic cortex total Fos positive cells. A one-way ANOVA revealed a
significant effect of group (F (4, 31) = 9.917, p < 0.0001). Tukey’s post hoc analysis showed that all drug treated groups displayed significantly higher Fos expression than the Sal+H2O+Veh group (vs. Coc+H2O+Veh, p < 0.0001; vs. Coc+H2O+Cef, p = 0.003; Coc+EtOH+Veh, p = 0.005; Coc+EtOH+Cef, p = 0.044) as indicated by “+”. In addition, the Coc+H2O+Veh group displayed significantly more Fos expression than the Coc+EtOH+Cef group (p = 0.016) as indicated by “ * “.
0
25
50
75
100
125c-F
os c
ells / m
m2
Coc+H2O+Veh
Coc+H2O+Cef
Coc+EtOH+Veh
Coc+EtOH+Cef
Sal+H2O+Veh
+
+ ++ *
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Figure 4-8. Infralimbic cortex Fos expression. A one-way ANOVA revealed a significant
effect of group (F (4, 31) = 8.745, p < 0.0001). Tukey’s post hoc analysis showed that the Coc+H2O+Veh (p = 0.0001) and Coc+EtOH+Veh (p = 0.010) groups displayed significantly higher Fos expression than the Sal+H2O+Veh group as indicated by “+”. The Coc+H2O+Cef group (p = 0.117) and the Coc+EtOH+Cef group (p = 0.061), which displayed a trend, did not significantly differ from the Sal+H2O+Veh group. In addition, the Coc+H2O+Veh group displayed significantly more Fos expression than the Coc+H2O+Cef group (p = 0.009) and the Coc+EtOH+Cef group (p = 0.031) as indicated by “ * “.
0
25
50
75
100
125c-F
os c
ells / m
m2
Coc+H2O+Veh
Coc+H2O+Cef
Coc+EtOH+Veh
Coc+EtOH+Cef
Sal+H2O+Veh
*
+
+
*
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Figure 4-9. VTA Fos expression. A one-way ANOVA revealed significant differences
between groups (F (4, 25) = 4.278, p = 0.009). The Coc+H2O+Cef group displayed significantly more Fos expression than the Coc+H2O+Veh (p = 0.050) as indicated by “ * “, and the Sal+H2O+Veh group (p = 0.007) as indicated by “+”. No other group differences were observed.
0
100
200
300
c-F
os c
ells / m
m2
Coc+H2O+Veh
Coc+H2O+Cef
Coc+EtOH+Veh
Coc+EtOH+Cef
Sal+H2O+Veh
+ *
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CHAPTER 5 GENERAL DISCUSSION
Summary of Results
In Experiment 1 we sought to determine if the beta-lactam antibiotic, Ceftriaxone,
would attenuate the reinstatement of cocaine seeking in animals that also have a history
of alcohol self-administration. However, we found that Ceftriaxone did not attenuate
cue- or cocaine-primed reinstatement to cocaine seeking in rats with a history of alcohol
consumption in addition to cocaine use.
In Experiment 2, we found that glutamate efflux occurred during reinstatement
only in rats that did not consume alcohol with cocaine, in agreement with previous work
by our group and others (Knackstedt et al., 2010; McFarland et al., 2003; Trantham-
Davidson et al., 2012; Lutgen et al., 2012). However, in rats that self-administered both
cocaine and alcohol, cue+cocaine-primed reinstatement of cocaine seeking was not
accompanied by glutamate efflux in the nucleus accumbens. Also in agreement with
previous work, Ceftriaxone treatment prevented glutamate efflux during reinstatement
testing in the Coc+H2O group (Knackstedt et al., 2010; Trantham-Davidson et al., 2012).
Chronic Ceftriaxone was not effective in attenuating cue+cocaine-primed reinstatement
in animals that consumed both alcohol and cocaine. The lack of an effect of Ceftriaxone
on reinstatement and the absence of glutamate efflux during reinstatement strongly
indicate that EtOH co-administration alters the neurobiology underlying cocaine relapse.
Based on the lack of glutamate efflux in the NAc core during reinstatement to
cocaine seeking in animals with a history of cocaine and alcohol use, we sought to
determine brain regions potentially involved with relapse outside of the NAc. We found
that increased glutamate efflux in the NAc core was accompanied by increased Fos
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expression in the NAc core and PFC in the Coc+H2O+Veh group compared to all other
groups. Interestingly, we observed more c-Fos activation in the VTA of the only group
with attenuated reinstatement behavior, the Coc+H2O+Cef group. The observed Fos
expression in the VTA may be a result of GABAergic activity inhibiting downstream NAc
core glutamate release and thereby attenuating reinstatement to cocaine seeking.
Cocaine and Alcohol Effects on Glutamate Homeostasis
In the reinstatement model of relapse to drug seeking, animals consistently
display increased glutamate release into the synapse as they are actively seeking drug.
This holds true for alcohol (Gass et al., 2010; Gass et al., 2014) and cocaine
(Knackstedt et al., 2010; McFarland et al., 2003; Trantham-Davidson et al., 2012;
Lutgen et al., 2012). Our lab and others have reliably found that chronic Ceftriaxone
(200 mg/kg IP for 5-7 days) attenuates relapse to cocaine seeking (Fischer et al., 2013;
LaCrosse et al., 2016; Knackstedt et al., 2010; Sari et al., 2009; Sondheimer and
Knackstedt, 2011; Trantham-Davidson et al., 2012). Ceftriaxone prevents reinstatement
to drug seeking by normalizing basal and synaptic glutamate release within the NAc
core. Ceftriaxone exhibits this effect by normalizing the expression and function of
glutamate transport systems including GLT-1 and system xc-/xCT. In fact, Ceftriaxone
attenuated relapse to EtOH drinking and upregulated GLT-1 levels in the PFC and NAc
in alcohol-preferring P rats (Qrunfleh et al., 2013; Sari et al., 2011, 2013a, 2013b). A
more recent study using P rats in a relapse-like paradigm of ethanol drinking, showed
that treatment with Ceftriaxone upregulated both glutamate transporter isoforms, GLT-
1a and GLT-1b, in the PFC and the NAc (Alhaddad et al., 2014; Qrunfleh et al., 2013).
Cue-primed reinstatement to operant alcohol seeking after a period of extinction has
also been prevented with Ceftriaxone treatment (Weiland et al., 2015). Similar to
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reinstatement to alcohol, once-daily treatment with Ceftriaxone for five days attenuates
cue- and cocaine- primed reinstatement of cocaine seeking following an extinction
period (Knackstedt et al., 2010; Sari et al., 2009). Ceftriaxone upregulates xCT/system
xc- expression and function, restores glutamate uptake, and attenuates glutamate efflux
during cocaine-primed reinstatement (Knackstedt et al., 2010; Trantham-Davidson et
al., 2012). We found the underlying cause for Ceftriaxone’s inability to prevent cue- and
cocaine-primed reinstatement when animals have a history of both cocaine and alcohol
consumption use lies in the fact that no glutamate increase is observed in the NAc core
during reinstatement in these animals.
Interestingly, both alcohol administration alone and cocaine administration alone
alter basal glutamate levels in the NAc. After cocaine self-administration and 10-21 days
of extinction training, basal glutamate levels in the NAc core are decreased (Baker et
al., 2003; Lutgen et al., 2012; Madayag et al., 2007; Trantham-Davidson et al., 2012).
Alternately, basal glutamate levels are increased in the same brain region following
continuous alcohol consumption with no withdrawal (Das et al., 2015; Griffin et al.,
2014) and following 24 hours of withdrawal (Pati et al., 2016). Decreasing glutamate
levels in the NAc core decreases EtOH intake, whereas increasing glutamate release
increases EtOH consumption (Cozzoli et al., 2009; Griffin et al., 2014; Kapasova and
Zsumlinski, 2008).
The increase in basal glutamate after chronic EtOH does not last past 14 days. In
the present experiments, rats were tested for reinstatement after 2-3 weeks of extinction
training. Even if the increased basal glutamate from EtOH did not persist as long as the
changes from cocaine, basal glutamate levels in these rats may be close to a drug
125
naïve animal or only slightly decreased from the longer lasting cocaine effects.
Ceftriaxone does not alter glutamate homeostasis in animals that did not have a history
of drug consumption. Therefore, it is likely the animals with a history of both cocaine
and EtOH did not benefit from Ceftriaxone treatment like they would have in a single
drug use condition.
However, we do not know the status of basal glutamate levels when animals
have a history of both cocaine and alcohol use. We do know the drug combination does
in fact, alter glutamate systems based on our finding that Ceftriaxone is no longer
effectual. Indeed, this finding is a strong indicator of distinct neuroadaptations following
cocaine and alcohol relative to cocaine alone. Future studies should evaluate basal NAc
core glutamate levels in rodents with a history of both cocaine and alcohol use. The
most informative time points to examine will be immediately following chronic self-
administration of both drugs and then during reinstatement to cocaine seeking.
Determining basal glutamate levels during these time points will better explain why
Ceftriaxone was ineffectual in Coc+EtOH animals and in the Coc+EtOH+Veh animals
given there was no glutamate release into the NAc core during reinstatement testing.
Depending on the basal glutamate levels in these conditions, it would also be beneficial
to examine the status of GLT-1 isoforms and the xc-/xCT system.
Role of the NAc in Mediating Reinstatement of Cocaine Seeking
NAc Core
In experiment 3 we observed significantly more Fos protein expression in the
NAc core of the Coc+H2O+Veh group than the other groups. This increased expression
was paired with an increase in glutamate release during reinstatement testing.
Interestingly, an increase in glutamate was not observed in the Coc+EtOH groups
126
despite robust reinstatement behavior. While significantly less than the Coc+H2O+Veh
group, the Coc+EtOH groups had significantly more Fos expression than the saline
control group, indicating increased neuronal activation (D1 receptor (Herrera and
Robertson, 1996; Ming, 2008) and NMDA signaling (Horowitz et al., 1997a) in the NAc
core.
Fos expression in the NAc core is to be expected in all groups that received a
cocaine injection. As studied by in vivo microdialysis, DA is released in the NAc core
even in a saline group yoked to animals that had previously self-administered cocaine
(McFarland et al., 2003). Given the response to cocaine injections mentioned above,
some level of neuronal activity should be present in the NAc core for all groups that
received a systemic cocaine injection compared to the saline control group. However, to
our knowledge, no one has observed dopamine levels during reinstatement in animals
with a history of both cocaine and alcohol. Taken together, it is likely our findings of c-
Fos activation observed in the NAc core of drug treated groups is because of the i.p.
cocaine injection priming the reinstatement test, but we cannot be certain.
Our findings point to the potential role of DA in reinstatement (as compared to
glutamate) in rats with a history of alcohol consumption. Increases in NAc core DA have
been observed during cue-primed reinstatement of cocaine seeking (McFarland et al.,
2003; Madayag et al., 2010). In addition, a direct infusion of DA in the NAc elicited
reinstatement to cocaine seeking, as did an AMPA agonist (Cornish and Kalivas, 2000).
Conversely, administration of intra-accumbens CQNX (an AMPA/kainate receptor
antagonist) is capable of inhibiting this same reinstatement to cocaine seeking (Cornish
and Kalivas, 2000). However, when fluphenazine, a DA receptor antagonist, was
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microinjected directly into the NAc, it failed to reduce cocaine-primed reinstatement
(Cornish and Kalivas, 2000). In addition, infusions of DA antagonists into the NAc core
had no effect on cocaine seeking triggered by a cue presentation in a second order
schedule of reinforcement (Di Ciano and Everitt, 2001). Taken together, DA release in
the NAc accompanies reinstatement. However, DA antagonists administered into the
NAc or core fail to prevent reinstatement to drug seeking.
A D1 receptor antagonist, SCH-23390, failed to attenuate cocaine primed
reinstatement (Anderson et al., 2003). In addition, the D2 receptor antagonist, sulpiride
also did not attenuate reinstatement (Anderson et al., 2003). Similar to the work with
antagonists, administration of these D1 and D2 receptor agonists into the NAc core
failed to produce reinstatement to cocaine seeking (Schmidt et al., 2006).
NAc Shell
In contrast to the NAc, Fos expression patterns in the NAc shell do not
significantly differ between groups. Ceftriaxone does not reduce Fos expression in the
Coc+H2O rats even though reinstatement was attenuated in this group. In fact, there is
significantly more Fos expression in all groups compared to the saline control group
indicating shell involvement with drug seeking. The comparable levels of Fos in the NAc
shell of the Coc+H2O-Cef group and the Coc+EtOH groups may be a response to DA
release, not glutamate.
An extensive literature exists investigating the role of DA transmission in the NAc
shell as it pertains to cocaine seeking during reinstatement. Cocaine-primed
reinstatement can be successfully attenuated by administration of the D1 receptor
antagonist, SCH-23390, into the NAc shell (Anderson et al., 2003). Similarly, when the
D2 receptor antagonist, sulpiride, is administered into the NAc shell, reinstatement to
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cocaine seeking induced by a cocaine prime injection is attenuated (Anderson et al.,
2006). Administration of a D1 receptor agonist (SKF-81297) or a D2 receptor agonist
(quinpirole) into the NAc shell induced reinstatement (Schmidt et al., 2006). Taken
together, D1 and D2 receptor activation in the NAc shell, but not in the NAc core,
mediates cocaine-primed reinstatement to drug seeking. In summary, DA release in the
NAc shell may be a mediator in reinstatement to cocaine seeking.
Continuing with our hypothesis that DA may be involved with reinstatement in
animals with a history of both alcohol and cocaine as opposed to glutamate, the
presence of c-Fos activation in the NAc shell could be due to D1 and D2 receptor
binding. It is also important to note that treatment with Ceftriaxone did not alter Fos
expression in both Coc+H2O and Coc+EtOH group. Had there been a difference in the
Ceftriaxone groups compared to their respective vehicle group, it would be likely a result
of glutamate transmission rather than DA.
PFC
Mirroring the c-Fos pattern we observed in the NAc core, the Coc+H2O+Veh
group had significantly more Fos expression in the PFC than the other groups and all
groups had more expression than the saline control group. Greater amounts of Fos
expression in the PFC and NAc core of Coc+H2O+Veh animals support our
microdialysis data showing a significant increase of NAc core glutamate release during
reinstatement to cocaine seeking. The PrL neurons of the PFC project to the NAc and
mediate cocaine-primed (McFarland and Kalivas, 2001) and cue induced (McLaughlin
and See, 2003) reinstatement to cocaine seeking. Our observed activation of the PFC
during reinstatement to drug seeking is supported by previous literature. Prefrontal
cortex neurons are activated during cue-primed reinstatement to drug seeking (Zavala
129
et al., 2008). In fact, pharmacological inactivation (Kalivas et al., 2005) and optogenetic
inhibition (Stefanik et al., 2013) of the PrL cortex or the NAc core prevents
reinstatement to cocaine seeking.
The Coc+H2O+Cef group had significantly less Fos expression than the
Coc+H2O+Veh group correlating with a lack of glutamate release in the NAc core and
attenuation of reinstatement. This observation is in agreement with previous literature
indicating the importance of the PFC to NAc core glutamatergic projections in
reinstatement to drug seeking. Considering the rats with a history of alcohol with
cocaine, the amount of Fos activation in the PFC is comparable to the Coc+H2O+Cef
group. This finding is not surprising given the lack of glutamate level change in the NAc
of the Coc+EtOH groups during reinstatement. Taken together, the similar finding of
greater c-Fos activation in the PFC and NAc core of the Coc+H2O+Veh group
compared to other groups is supported by the robust literature on the importance of the
glutamatergic projection between these two regions during relapse.
VTA
The VTA is involved with drug seeking and reward. An estimated 60-65% of VTA
neurons are dopaminergic and project to the PFC, BLA, and the NAc. Afferent
glutamatergic projections to the VTA come from the PFC. Another important role of the
VTA is its GABA-ergic projections to the NAc (Sesack and Grace, 2010; van Zessen et
al., 2012). In our c-Fos activation experiment, the only group with significantly more VTA
Fos expression than the saline control group is the Coc+H2O+Cef group. Given that
reinstatement to cocaine seeking in this group was attenuated by the use of
Ceftriaxone, the VTA GABA-ergic projections could be actively preventing the release of
glutamate and DA into the NAc core. This potential action agrees with the lack of
130
glutamate release observed by the microdialysis results in experiment 2. The VTA
sends glutamatergic, dopaminergic, and GABA-ergic projections to the NAc core. There
are also GABA-ergic interneurons within the VTA that can modulate activity of these
projection neurons. It is possible that the GABA-ergic interneurons are inhibiting
glutamate and DA release from the VTA to the NAc core in the Coc+H2O+Cef group.
Conclusions
In conclusion, Ceftriaxone does not attenuate cue- nor cocaine-primed
reinstatement to cocaine seeking in rats that consumed 20% EtOH after operant
cocaine session. The findings of these experiments are important for two main reasons:
1) they imply that Ceftriaxone, despite its repeated demonstration of effectiveness in
blunting cocaine reinstatement, is not effective in animals with a history of alcohol
consumption and thus is not a strong candidate for moving forward in clinical trials to
prevent relapse; and 2) the neurobiology underlying cocaine relapse is altered when
animals have a history of alcohol use.
Previous research has established the role of glutamate increase in
reinstatement to alcohol or cocaine individually. However, when these two drugs are
combined, other brain regions and neurotransmitters may be involved when the drugs
are used together. Much is still unknown about the effects of simultaneous alcohol and
cocaine use on glutamatergic and dopaminergic transmission within the brain. Fos
expression has helped us understand which brain regions are more active during the
process of reinstatement to cocaine seeking. This research is the first indication that
reinstatement to cocaine seeking is not independently controlled by glutamate release
within the NAc core. In fact, this research points to the role of the VTA mediating
reinstatement via various reactions from glutamate, DA, and GABA mechanisms. Future
131
research would benefit from investigating Fos expression in the amygdala and
hippocampus among other brain regions involved in the reward circuitry.
Future Directions
Our studies provided data regarding the glutamatergic neuroadaptations
occurring in the NAc following the combined use of alcohol and cocaine. Because
Ceftriaxone is ineffective at attenuating the reinstatement of cocaine-seeking, data
generated here indicates that strategies to increase basal glutamate during withdrawal
will not be effective treatments for those with a history of both alcohol and cocaine
consumption. In this case, future work will need to find mechanisms for attenuating
cocaine reinstatement either by targeting the glutamate system with a different
approach or with pharmacological interventions aimed at a different neurotransmitter
system.
132
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Alhaddad, H., Kim, N.T., Aal-Aaboda, M., Althobaiti, Y.S., Leighton, J., Boddu, S.H.S., Wei, Y., Sari, Y., 2014b. Effects of MS-153 on chronic ethanol consumption and GLT1 modulation of glutamate levels in male alcohol-preferring rats. Front. Behav. Neurosci. 8, 366.
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BIOGRAPHICAL SKETCH
Bethany Stennett was born in Fayetteville, NC on Fort Bragg Army base. She
grew up in the military and went to high school in north Alabama. In 2007, Bethany
began her undergraduate degree at Auburn University in Auburn, Alabama where she
was on the rowing team. She graduated in 2011 with a Bachelor of Arts degree in
psychology. That August, she began a master’s degree at University of North Florida in
Jacksonville, Florida. It was there that she joined the Neuropsychopharmacology
Laboratory at The Mayo Clinic research and teaching hospital. She conducted her
master’s thesis research supervised by Dr. Elliott Richelson and Dr. Mona Boules. The
thesis was titled “Novel Therapy of Nicotine Addiction in Alcohol Dependent Rats.” In
2013, Bethany graduated from UNF and began her doctoral studies at University of
Florida under the supervision of Dr. Lori Knackstedt. Bethany is awarded her Ph.D. in
psychology from the University of Florida in 2018.