Department of Physics, Chemistry and Biology
Master Thesis
Dependence-induced
changes in opioid-receptor gene expression
Anna Johansson
LiTH-IFM- Ex—
12/2708
—SE
Supervisor: Annika Thorsell, IKE, Linköping University
Co Supervisor: Susanne Hilke IKE, Linköping University
Examiner: Bengt Harald “Nalle” Jonsson, IFM, Linköping
University
Department of Physics, Chemistry and Biology
Linköpings universitet
Rapporttyp Report category Examensarbete D-uppsats Master Thesis Språk/Language Engelska/English Titel/Title:
Dependence induced changes in opioid receptor gene expression
Författare/Author:
Anna Johansson
Sammanfattning/Abstract:
Using drugs such as alcohol and morphine among others can be addictive in some individuals, and progress into a substance abuse disorder. The mesolimbic dopaminergic system (MD-system) is involved in the reward process during the development of drug addiction. The MD-system is critical for survival and affects different behaviors in both man and animal. Neurochemical pathways drive for instance physical activity, food intake, love and reproduction and are part of the natural reward process involved partly in the release of dopamine (DA) into frontal lobes. Within the MD-system opioid receptors throughout the brain are affected by drug intake, and activation of these receptors modulate DA-release in brain regions involved in reward-behavior. The aim of this study was to evaluate gene expression of MOR and DOR within the endogenous opioid system (EO-system) in relation to voluntary physical activity, a natural reinforcer. Further on investigations of the drug alcohol was compared to the natural reinforcer sucrose using voluntary consumption.
For both experiments qRT-PCR was used to measure mRNA levels of MOR and DOR from brain areas of interest. We found a small significant up regulation in NAc, PFC and VTA but for DOR in VTA a down regulation in gene expression of physical exercising mice. Additionally these two different genes OPRM1- and the OPRD1- gene are down regulated in VTA and NAc due to alcohol- and sugar-intake. This implicate that the natural reward system and their ORs point in the direction of earlier findings; the opioid receptors have a key role in regulate alcohol intake and the natural rewarding stimuli as food intake.
ISBN
LITH-IFM-A-EX—12/2708—SE
__________________________________________________ ISRN
__________________________________________________ Serietitel och serienummer ISSN
Title of series, numbering
Handledare/Supervisor: Annika Thorsell
Bihandledare/Co supervisor: Susanne Hilke
Ort/Location: Linköping
Nyckelord/Keyword:
Neuropeptides, Alcohol, Gene expression, C57/BL6 mice model, Addiction, VTA, Opioid Receptors, Reward, Quantitative RT-PCR
Datum/Date
2013-02-12
URL för elektronisk version
Institutionen för fysik, kemi och biologi
Department of Physics, Chemistry and
Biology
Avdelningen för biologi
Content
1
Abstract ... 2
2
List of abbrevations ... 2
3
Introduction ... 3
4
Material & methods ... 8
4.1
Experiment and choice of method ... 8
4.2
General procedures and behavioral studies ... 8
4.2.1
Voluntary exercise ... 9
4.2.2
Two bottle choise test: EtOH and sucrose ... 9
4.3
Tissue collection and cryostat ... 9
4.4
Genetic Analysis ... 9
4.4.1
RNA isolation and cDNA conversion ... 9
4.4.2 Quantitative RT-PCR ... 10
4.5
Statistical analyses ... 10
5
Results ... 11
5.1
Voluntary exercise and its impact on MOR and DOR gene
expression ... 11
5.2 Alcohol and sucrose consumption and its impact on
MOR and DOR gene expression ... 13
5.3
Consumption of alcohol- and sugar-solution ... 15
6
Discussion ... 18
6.1
Conclusions ... 20
7
Acknowledgement ... 20
1 Abstract
Using drugs such as alcohol and morphine among others can be addictive in some individuals, and progress into a substance abuse disorder. The mesolimbic dopaminergic system (MD-system) is involved in the reward process during the development of drug addiction. The MD-system is critical for survival and affects different behaviors in both man and animal. Neurochemical pathways drive for instance physical activity, food intake, love and reproduction and are part of the natural reward process involved partly in the release of dopamine (DA) into frontal lobes. Within the MD-system opioid receptors throughout the brain are affected by drug intake, and activation of these receptors modulate DA-release in brain regions involved in reward-behavior. The aim of this study was to evaluate gene expression of MOR and DOR within the endogenous opioid system (EO-system) in relation to voluntary physical activity, a natural reinforcer. Further on investigations of the drug alcohol was compared to the natural reinforcer sucrose using voluntary consumption.
For both experiments qRT-PCR was used to measure mRNA levels of MOR and DOR from brain areas of interest. We found a small significant up regulation in NAc, PFC and VTA but for DOR in VTA a down regulation in gene expression of physical exercising mice. Additionally these two different genes OPRM1- and the OPRD1- gene are down regulated in VTA and NAc due to alcohol- and sugar-intake. This implicate that the natural reward system and their ORs point in the direction of earlier findings; the opioid receptors have a key role in regulate alcohol intake and the natural rewarding stimuli as food intake.
Keywords: Voluntary wheel exercise, Alcohol, Gene expression, C57/BL6 mice model, Dependence, VTA Opioid Receptors, Reward, qRT-PCR
2 List of abbrevations
AA – Amygdala OR − opioid receptor
DOR − delta opioid receptor OPRD1 − delta opioid receptor gene type 1
EOS – endogenous opioid system OPRM1 − mu opioid receptor gene type 1
EtOH – ethanol/alcohol OPRK1 − kappa opioid receptor gene type 1
MD-system–mesolimbic PFC – prefrontal cortex
dopaminergic PCR – Polymerase chain reaction
System VTA − ventral tegmental area
MOR − mu opioid receptor qRT– Quantitative Real-Time
3 Introduction
Addiction & society
Worldwide, there is a huge and expanding problem of abuse of alcohol morphine, heroine and other drugs. Alcohol or drug-intake has a negative effect on society as it leads to tolerance, and to addictive behavior. Every year, violence, car accidents, burglaries, and health problems give society enormous problems in terms of cost and lead to approximately 2.5 million deaths each year, according to the WHO. “It also causes harm far beyond the physical and psychological health of the drinker. It harms the well-being and health of people around the drinker” (WHO, 2012).
Historically, it has been known that drugs and alcohol exert their effects within the central nervous system and increased use or abuse may cause brain damage [1, 2]. The desire for drugs i.e. a compulsion to take the drug results in increased intake of the drug, which in turn could lead to drug dependence. According to WHO addiction is classified as a neurobiological disease, characterized by behaviors of compulsive
use, using the drug despite it causes harm and craving for the drug (WHO, 2012) [3].
Alcohol and drugs may be more commonly used by individuals with mental illnesses such as schizophrenia, depression or bipolar disorders [4]. It has also been found that use of alcohol or drugs during pregnancy can cause, for example, fetal alcohol syndrome (FAS) or fetal alcohol spectrum disorders (FASD), and the risk of cerebral palsy is to some extent increased [5-7].Alcohol dependence develops only when exposure to alcohol occurs and genetic factors [8], contribute with approximately 55 percent to this end-stage disease. Additionally, recently epigenetic modifications of DNA have been studied and have been demonstrated to affect this physical disability and shown to affect the transition into drug addiction, as well as the maintenance of an addicted phenotype. Moreover addiction is a disease highly involving the reward system [8, 9].
Addiction & Reward system-Dopamine
During development of dependence, reprogramming of the brain occurs and neuronal
function is altered0F
1
[10]. When abusing drugs or alcohol, a gradual escalation of drug intake occurs due to increased tolerance. Intake of high doses of alcohol results ultimately in sedation and also loss of coordination. The development of dependence related to alcohol can be divided into three stages; 1.the effect as a positive effect, 2. Withdrawal symptoms when that intake is limited and 3. an emotional state of expectation [11]. As abuse of alcohol develops into an addiction, an adaptation of the brain’s signal system where neurons die occurs and the individual suffers from memory loss and cognitive disorders. This is due to that after an abrupt removal of alcohol during withdrawal or abstinence, a release of glutamate follows which gives hyperactivity of neurons in turn leading to apoptosis of brain neurons [12].
Biological studies on the reward system have previously shown that it is involved in the neurophysiological mechanisms related to different types of drug abuse. The
1Neurogenesis; process by which neurons are generated from neural stem and progenitor cells.
reward system and the endogenous opioid system (EO-system) are thus involved in the development of an addiction and is of interest to further investigate with regards to the biological mechanisms underlying development of drug and alcohol addiction [13, 14]. Individual differences in these systems may be the reason for individuals’ different susceptibility to developing addictions. Individuals who have not developed alcohol dependence have shown that a low intake of alcohol causes feelings of euphoria and reduce the discomfort of feelings of anxiety more than in developed alcohol dependence [15]. At a higher alcohol intake, sedating effects on the brain occur and also a reduction to control body movements [16].
The limbic system contributes to our learning and memory formation, escape and defence (“fight or flight”), motivation, and emotions which are connected to the organism’s ability to survive. The limbic system consists of the hippocampus (H), amygdala (AA), parts of the cortex and thalamus. According to Paul D MacLean, in his theory, "The Triune Brain in Evolution", already in 1960, the limbic system developed long before other parts of the brain. [17, 18]. Thus, from an evolutionary perspective, the limbic system is an old structure present in reptiles and mammals, as well as in tetrapods. From an evolutionary perspective, man and mice have a comparable conserved biological structure in the limbic system’s ability to perceive positive emotions such as pleasure, enhancing our ability to survive [19]. The limbic system within the human brain is critical for our existence and contains the reward system [13], where different regions regulate and control a behavior, due to different feelings of pleasure. The motivation required to perform an act is modulated by a region called the nucleus accumbens (NAc), and the brain's frontal cortical structures. The prefrontal cortex (PFC) is a significant relay in the reward circuit. Information from the limbic system to enable a motorical action is converted in NAc.
Natural rewarding stimuli, such as food or voluntary exercise, exert their positive effect through actions within the reward system. Natural rewarding stimuli such as food affect the human brain through different feelings having an impact on behavior [13]. Activation of this ancient brain-structure rewards the individual and reinforces specific behaviors such as food intake [19]. The NAc is the most important brain region involved in regulation of reward-related behavior and, together with the ventral tegmental area (VTA), composes parts of the so called mesolimbic dopamine system (MD). Additionally, the MD system is closely interconnected with the endogenous opioid system where opioid receptors (ORs) occur.
These systems have important roles in response to rewarding stimuli, both natural and artificial. The reward system is mediated by artificial opioid peptides and natural endogenous opioid peptides. Natural rewards such as food, physical activity and also response to stress and pain triggers endogenous peptides to be released [13, 20, 21] Drugs have similar, but more pronounced, effect on the EO-System [22].
Activation of MD system occurs when a rewarding stimuli triggers the neurotransmitter dopamine (DA) to be released. Increased firing of DA-containing neurons in VTA releases DA into the NAc from the projection neurons [23, 24]. Release of DA from nerve terminals into NAc creates an emotional state of
well-being and reward, acting as a feedback loop which increases DA levels. Further on the DA-neurons project into the PFC, another structure belonging to the mesolimbic dopamine-system [21]see figure 1.
Figure 1.The mesolimbic dopamine system within the human brain. Brain structures involved in mediating the rewarding properties of drugs of abuse include VTA, NAc, PFC and also the hippocampus [21].
It has been seen that the MD system pathway and the EO-system are significantly affected during the development of drug abuse. Drugs and alcohol effectively activate the MD system. Drugs conquer the brain's natural reward system by taking over the mechanisms that evolved to facilitate our survival [13, 14, 21, 25] Addictive drugs can activate the MD system to a greater extent and faster than natural rewards such as food.
The endogenous opioid-system and regulation of reward
Opioid receptors are expressed in the brains limbic regions. These membrane-associated receptor types are to be found throughout the central nervous system and are located within structures such as the NAc, amygdala (AA), VTA of the midbrain and frontal cortical regions [13, 14]. Within the VTA, one region where ORs regulate reward through mediation of dopamine-release, mu opioid- (MOR) and delta opioid receptors (DOR) are present on GABAergic interneurons. GABA is an inhibitory neurotransmitter which affects DA levels within NAc [26]. GABA interneurons have an effect on DA-projection neurons, which use DA as a neurotransmitter through dopaminergic nerve pathways from VTA. DA-neurons is projected further to NAC that modulates release of DA levels in NAc [27, 28]. κ- receptors (KOR) are primarily found in the hypothalamus and in the spinal cord. In studies on the development of alcohol dependence it has been seen that the receptors can be down regulated. When this occurs, it requires an increased intake of the drug to produce the same cellular response of the receptor. There are three different subtypes: µ-, δ-, and κ -opioid receptor. The genes that encodes for these different ORs are genes from the OR gene family [29, 30] named OPRM1, OPRD1, and OPRK1 respectively [31].
Opioid receptors have a seven-trans membrane topology and µ- receptor, δ- receptor and κ-receptor are all G-protein coupled receptors[29, 32]. There are three families of endogenous opioid peptides divided into endorphins, enkephalins and dynorphins. These three different types of peptides have in common the N-terminus part with the same amino acid sequence (Tyr-Gly-Gly-Phe) that is responsible for the rise of the opioid effect that occurs. The first pentapeptides where identified in 1975, named Leu- and Met-enkephalin after the binding site to ORs within the brain was identified in 1973. The first opioid receptor gene was isolated by expression cloning in 1992 [33].
Table 1 displaying an overview of opioid receptors in man and rodents, preferred ligands (note 1) and effects in different biological system [22, 34-36]
OR- gene OR- class Endogenous peptide Function Exogenous agonist
OPRD1 δ- receptor Leu-Enkephalin 1 Cardiovascular vascular DADLE depression Met- Enkephalin1 Pain relief
Β- Endorphin
OPRK1 κ-receptor Dynorphin A, B 1 Hallucination, reward Dynorphin1a Sedation
OPRM1 µ- receptor Endomorphin 1 Pain relief, euphoria DAMGO, Morphine Β- Endorphin Physical dependence M6G
MOR is a receptor that plays a central role within the reward system in the brain and partakes in regulation of physiological functions such as food, cardiovascular function, learning, memory, locomotor activity and immune functions [19]. In both mice and humans the natural endogenous opioid ligand that binds selectively to MOR is β-endorphin [36]. Physiologically endogenous endorphins are produced within hypothalamus during pain, exercise, and excitement [37, 38]. Endorphins have a natural analgesic effect in response to severe pain in the body, see table 1. Pain also causes an altered state of mind that instinctively produces a fear- and anxiety-related behavior as seen through the early evolution. During stressful events endorphins are released from limbic area and thereby reduce the individual’s stress and anxiety [20, 39].
ORs have a central role in the regulation of reward-related behavior and experience. When endogenous opioid peptides such as endorphins are triggered to be released, which occurs in natural rewarding stimuli as well in rewarding stimuli from drugs and alcohol, endorphins will trigger release of DA. This occurs through endorphins that bind to ORs in NAc and VTA, mostly the subtype µ-receptor (MOR). As previously mentioned, opiate drugs activate the endogenous opioid system consisting of VTA and NAc where opiates mimic endorphins thereby hijacking the reward system [13, 14, 21, 25]. The opioid peptides enkephalin and dynorphin are endogenous ligands with high affinity of the ORs subtype δ- receptor (DOR) and κ-receptor (KOR)
respectively [40]. DOR are distributed in NAc, olfactory bulb1F
2
and neocortex2F
3 . The receptors also occur in lower levels in the hypothalamus and the brainstem. The ORs in VTA are activated indirectly via ethanol (EtOH) and cocaine ingestion while drug rewarding effects of morphine are mediated via binding to ORs. The rewarding effect of alcohol is primarily regulated through an activation of MOR via endorphins and for DOR mainly via enkephalin [22]. In general agonists’ preferred for MOR or DOR receptors are analgesic and enhance the rewarding effects of drugs of abuse [41] . A target for drug therapy is mu opioid receptor antagonist Naltrexone® and has previously been used for treatment of alcohol addiction [42].
Exercise & reward
Physical activity in mice and man have been studied and workout in an abnormal manner could develop into an addiction, similar to that for alcohol and drugs [43, 44]. A method for developing addiction-like behavior to physical activity in laboratory animals is to give them free access to a running wheel in their cages [45]. Wheel running in mice is rewarding [46] and rodents have high preference for do it more likely on a voluntary basis [47] Wheel running has been shown to reduce depression-like behavior in mice and also to have a protective effect on neurons [48]. Interesting one has also found that exercise is stress-relieving, mood-enhancing and this is modulated through the reward system [47]. Rats given access to running wheels in their cages have been developing a compulsive behavior to persist with the running-behavior at the expense of other running-behaviors. Other studies have found that exercise could as well act as enrichment or “environmental modifications” for rodents in a laboratory environment [49]. It has been seen that exercise also can reduce the negative cognitive symptoms caused by alcohol abuse and age [50], as well as cognitive symptoms related to post-traumatic stress disorder (PTSD) [51]. Symptoms of PTSD in humans have been shown to change behavior in response to an emotional stage, also this supported by animal models with free access to exercise [52, 53].
Aim of the Study
The aim was to study how natural rewards such as physical activity and sugar intake as a natural affects expression of components of the endogenous opioid system, primarily the opioid receptors, and how these effects relate to the effect of alcohol intake on the ORs. Moreover investigation of drug alcohol-intake will be compared with the natural reinforcement sucrose and the effects on the reward system and the EO- system.
2
Olfactory bulb; part of the brain that sense odours
3 Neocortex is part of the cerebral cortex involved in the limbic system and is mainly responsible for
4 Material & methods
4.1 Experiments and choice of method
In order to identify and analyze different underlying changes of gene expression, due to for example wheel running or alcohol consumption behavior in mice, genetic tools like real-time PCR and microarrays can be used[54]. The important difference is that the microarrays show several thousands of genes expressed simultaneously and may lead to identification of new gene expression changes putatively involved in regulation of the measured behavior [54]. In real-time PCR, which is a quantitative method, one can only see the gene expression changes of known genes where one estimates the RNA present in the sample from the start [55]. Different underlying behaviors are a result of (epi-) genetic variations and changes in gene-expression. The inbred mouse-strain C57BL/6 is an excellent animal model for use when examining anxiety- and stress- related behaviors and alcohol intake since this strain has an anxiogenic phenotype and readily consumes high levels of alcohol. The use of mice in these studies is also appropriate since many molecular and genetic assays exist commercially [13, 54, 55]. In the first experiment we examined whether we could find differences in gene expression of MOR (i.e. OPRM1 gene levels) and DOR (OPRD1 gene levels) correlated to physical activity. Regions in the brain that are primarily of interest were PFC, VTA, and NAc. In the following experiment, the gene expression of the same receptors was examined, but following prolonged intake of alcohol or -sugar solutions in the so-called two bottle choice test.
4.2 General procedure behavioral studies
Two behavioral studies were performed. In Experiment 1, animals were given access to a running wheel for three weeks where after brain tissue was collected. In Experiment 2, drinking behavior in an intermittent access paradigm was performed. Here, the animals were divided into three groups; control (water access only), ethanol (20% ethanol and water) and sucrose (5% sucrose and water), and given intermittent access to their respective liquids (3 x 24 h per week). Following the five-week 4.2.1 Animals
Male C57Bl/6 mice were used in all studies (age 5-6 weeks at start of experiments). The animals were held at the animal facility, University of Linköping during the spring and summer of 2012. Protocols for all animals were approved by the Animal Care and Use Committee at Linköping University and in accordance with the European Communities Council Directive guidelines. Animals were kept in a temperature (20+/-2˚C) and humidity (30% to 45%) controlled environment with lights on 12 h/day (on at 6:00, off at 18:00). Food and water were provided every day ad libitum. During the experiments the animals were kept single housed except during the initial one-week habituation to the animal facility when they were housed in groups of four.
4.2.1 Voluntary exercise
In Experiment 1, a total of 20 naïve, 6 -week-old male mice (C57Bl/6) were split into two groups. The experiment group (n = 10) was given access to a running wheel during three weeks and the other control group (n = 10) was not. The mice were
housed individually, in Macrolon-cages measuring 777 cm2 (37*21*18;
length*width*height cm). Normally, rats use these cages but space was required to provide space for the running wheel (∅ 13 cm).
4.2.2Two bottle choice test: EtOH and sucrose
In Experiment 2, 30 male mice 6-weeks old (C57Bl/6), were split into three groups and given access to different drinking solutions in a two-bottle choice model. One experiment group (n = 10) was given access to one bottle containing 20% ethanol and one bottle containing water. A second experimental group (n=10) had access to 5% sucrose and water. The control group had access to two bottles of water with the same number of animals as the experiment groups. The experiment with ethanol 20% and sucrose 5% was a two bottle choice test and position of the drinking-bottle with ethanol or sucrose was changed from one side to the other every 24 hour access period. The alcohol-bottle was changed once a week and the sucrose tubes more often. Drinking sessions lasted for 24 hours three times a week, every other day, and the bottles were weighed for calculations of ethanol and sucrose-intake of the mice. Animals were weighed once per week in order to calculate intake in grams (ethanol or sucrose) per kilogram bodyweight.
4.3 Tissue collection and cryo-dissection
All animals were euthanized using a prefilled container with CO2, followed by
cervical dislocation. The brain was hastily removed, placed in cold NaCl 9 mg/ml (B Braun Melsungen AG Germany), frozen on dry-ice within 5 minutes following euthanasia, wrapped in foil, and stored at -80 ˚C until sliced and punched in the cryostat. Accurate punch-area was taken from correct brain region within cryostat (Microm HM 560) using punch needle (1 mm diameter) chilled on dry ice before each section was collected. Control sections for every brain were taken and stained with the following method; Cresyl violet 1-2 minutes, rinse with distilled water, followed by EtOH 70%, then EtOH 99,5% and fixation with Xylene 1 minute. Sections were collected from NAc, PFC, BNST, AA, and VTA, and varied in thickness between 100-300 µm. For every animal, all punched samples left and right from the brain slice were combined and collected in tubes, directly placed on dry ice and stored at - 80˚C.
4.4 Genetic Analysis
4.4.1 RNA isolation and cDNA conversion
Samples from the first experiment were taken from micro-punched brain regions NAc, PFC, AA, and VTA (Franklin & Paxinos 2007) [21]. The tissues were homogenized with Fast Prep tubes containing beads in Tissue Lyser (Lysing Matrix D, MP Biomedicals) and RNA was extracted using AllPrep DNA/RNA Mini Kit Part 1 and Part 2 (QIAGEN®). The RNA samples were handled with great care; using
RNase Zap (Invitrogen®) during all procedures in order to clean the lab bench and all important areas. RNA samples were stored at -80 ᵒC awaiting further treatment. For experiment two, tissues were homogenized with beads and RNA was extracted according to protocol RNeasy Micro Kit (QIAGEN®).
Conversion of RNA into cDNA was done by using the High Capacity cDNA Reverse Transcription kit (Applied Biosystem®) according to the instructions manual. All samples were run in Thermal Cycler; 25°C for 10 min, 37°C for 60 min, 85°C for 5 sec using the Veriti PCR instrument 9700 GeneAmp® (Applied Biosystems®, Foster City, Calif, USA). Quality check of RNA, and cDNA was done by Nano Drop™ 1000 Spectrophotometer (Thermo scientific). Before further analysis of different brain regions using Quantitative real time PCR; cDNA was diluted 1:10 using 20 μL cDNA and 180 μL RNase free water (Biotech®) and stored in a -80 freezer.
4.4.2 qRT-PCR
Quantitative PCR was carried out on a real-time detection instrument 7900 Fast Sequence Detection Instruments (Applied Biosystem®) in 96-well optical plates using TaqMan Universal PCR Master Mix (2X) and assay on demand primers and probes (Applied Biosystems®) according to manufacturer’s instructions. The following gene expression assays were used; MOR: Mm01188089_m1, DOR: Mm01180757_m1 and housekeeping gene β-actin: Mm00607939_s1. qRT-PCR program was run; activation 5min at 95°C, 45 cycles denaturation 10s at 95 °C, annealing at 30s 53-57 °C (primer dependent) and finally extension 10s at 72 °C. Mean expression of the MOR and DOR genes were normalized to β-actin using the Livak method.
4.5 Data analyses -Statistical analyses
For normally distributed samples the Student's t-test was used to compare mean values of experimental and control groups. The significance level was p < 0.05. Statistical tests were performed in STATISTICA 10 and all data are presented as mean ± SEM.
5 Results
5.1 Voluntary exercise and its impact on MOR and DOR gene
expression
mRNA levels of MOR and DOR were studied in relation to voluntary exercise within brain regions of the endogenous reward system. C57Bl/6 mice were subjected to voluntary wheel running during three weeks. Our hypothesis was that voluntary exercise would be rewarding and therefore modify MOR- and DOR-expression. Our gene expression study showed a significant 2- fold decrease of the DOR expression in NAc compared to controls (p=0,00340; Fig.2A) while the expression of the MOR was up regulated ~2.5-fold (p=0,050; Fig. 2 B). Within the VTA, DOR expression was up regulated about 3-fold after exposure to running wheel. (p=0, 0216; Fig 2 C). Within another region of importance in reward-related behavior, the PFC, MOR expression was up regulated 2.5-fold following exercise (p=0, 0339; Fig 2
D)
Figure 2 also visualizes the specific brain regions according to Franklin & Paxinos: The Mouse Brain in Stereotaxic Coordinates. NAc (2 A-B) blue circles, VTA (2 C) blue circles and PFC (2 D) blue square (Franklin and Paxinos, 2007).
Figure 2.Graphs displaying gene expression of MOR (B, D) and DOR (A, C) in different brain regions after three weeks of voluntary exercise. Gene expression of DOR in NAc (A) demonstrates a significant 2-fold down regulation. MOR gene in NAc (B) significantly displays a 2-fold up regulation. QRT-PCR of DOR in VTA showed a ~3-fold up regulation (C) and in PFC region a ~2-fold change expression of MOR, also significant, compared to controls. (D) Each value represents mean ± SEM and *P-value ≤ 0, 05 versus control, and **P≤ 0, 01 significant.
Nucleus Accumbens F o ld c h a n g e (G e n e e x p r e ss io n ) DOR DOR 0.0 0.5 1.0 1.5 2.0 2.5 Control Exercise F o ld c h a n g e ( G e n e e x p r e ss io n ) MOR MOR 0.0 0.5 1.0 1.5 2.0 2.5 Nucleus Accumbens Control Exercise DOR DOR 0.0 0.5 1.0 1.5 2.0 2.5 F ol d c h a n ge ( G e n e e x p r e ss ion )
Ventral Tegmental Area
Control Exercise Prefrontal Cortex F o ld c h a n g e (G e n e e x p r e ss io n ) MOR MOR 0 1 2 3 Control Exercise
A
B
C
D
5.2 Alcohol and sucrose consumption and its impact on MOR and
DOR gene expression
Drugs of abuse are more potent in triggering the pathways of the reward system and the endogenous pathways than natural rewards. We exposed mice to an intermittent access paradigm using 20% alcohol as a drug for five weeks in order to examine the effect on mRNA expression of the opioid receptors subtypes MOR and DOR in NAc and VTA. In addition, we evaluated a natural reward and its influence on the EO-system, and the natural reinforcer sucrose solution (5%) was used for this purpose. Expression of the opioid receptors was evaluated by real-time PCR.
Intermittent access to an alcohol solution or to a sucrose solution had similar effects on gene-expression of OPRM1 and OPRD1 in the VTA and NAc. MOR and DOR mRNA levels in NAc and VTA followed by 5 weeks of continuous access to alcohol- or sucrose-intake were all down-regulated. QRT-PCR of MOR demonstrated a significant 2-fold down regulation followed by access to alcohol and a 3-fold down regulation of MOR in response to alcohol and sugar intake in NAc (p=; 0.0005; 0.0059 Fig 3 A). As for RNA expression of DOR in NAc we did find a significantly
~2-fold down regulation in response to both alcohol and sugar consumption (p= 0,
046878; 0.029842 Fig 3 B). In VTA there was a ~2 fold change in response to alcohol and also significant fold change of DOR gene expression in response to sugar and ethanol, compared to controls (p=0, 00121; 0, 02 Fig 3 C). As for MOR in VTA a 3-fold- and a ~5-fold down regulation for ethanol respectively sucrose were observed (sucrose p= 0,021 Fig 3 D).
Figure 3 also visualizes the specific brain regions according to Franklin & Paxinos: The Mouse Brain in Stereotaxic Coordinates. NAc (3 A-B) blue circles and VTA (3
Nucleus Accumbens F o ld c h a n g e ( G e n e e x p r e ss io n )
MOR MOR MOR
0.0 0.5 1.0 1.5 Control Ethanol Sucrose Nucleus Accumbens F o ld c h a n g e ( G e n e e x p r e ss io n )
DOR DOR DOR
0.0 0.5 1.0 1.5 Control Ethanol Sucrose
Ventral Tegmental Area
F o ld c h a n g e ( G e n e e x p r e ss io n )
DOR DOR DOR
0.0 0.5 1.0 1.5 Control Ethanol Sucrose
Ventral Tegmental Area
F o ld c h a n g e ( G e n e e x p r e ss io n )
MOR MOR MOR
0.0 0.5 1.0 1.5 Control Ethanol Sucrose A B C D
Figure 3. Gene expression by qRT-PCR of MOR and DOR mRNA in nucleus accumbens (A-B) and ventral tegmental area (C-D) followed by 5 weeks of continuous access to ethanol or sucrose. QRT-PCR of MOR demonstrates a significant 2-fold down regulation followed by access to ethanol and a 3-fold down regulation in response to sugar in NAc (A). Similar expression of DOR in NAc showed a significant ~2-fold down regulation in response to both ethanol and sugar (B). In VTA a ~2 fold change in response to ethanol and also significant fold change of DOR gene expression of sugar, compared to controls(C). For MOR (D) in VTA a ~2.5-fold- and a ~5-fold down regulation for ethanol respectively sucrose occurs. Each value represents mean ± SEM and *P-value ≤ 0, 05 versus control, **P≤ 0, 01 significant and ***P≤ 0, 001.
5.3 Consumption of alcohol- and sugar-solution
During five weeks the animals were subjected to “the two bottle choice” method. The animals were introduced to 20% concentration of alcohol from the beginning of the experiment. C57Bl/6 male mice had access to alcohol or sucrose solution during five weeks and the animals had also free access to water continuous. [56, 57]. The two bottle choice test is well known and used for several years in attendance to avoid “place preference” for a specific bottle in the cage, one bottle is filled with water and the other with test-solution [58, 59].
The results from the self-administration of ethanol consumption (Figure 4 A) display an average higher intake 50-65 gram/kg/day during week three and week four than the first week. The average drink intake is quite high over the test-period albeit decreasing during the two last weeks of testing to 25-36 gram/kg/day.
For consumption of sucrose intake (Figure 4 B), during week 1-3 the average of intake varied between 15-25 gr/kg/day. Week four there was a peak up to approximately 45 gram/kg/day. The last week of sucrose intake, drinking declines and values are almost the same as for the start of the drinking period.
In addition solution-preference for ethanol and sucrose over tap water were calculated (Figure 4 C, D). Our results for sucrose show that the preference is higher for sucrose (5%) than for tap water while the preference for ethanol (20%) is less than 50% on average.
E tO H i n ta k e ( g r /k g /2 4 h )
week 1 week 2 week 3 week 4 week5 0 10 20 30 40 50 60 70 80 90 100 S u c r o s e i n ta k e ( g r /k g /2 4 h )
Week 1 Week 2 Week 3 Week 4 Week 5 0 10 20 30 40 50 60 70 80 90 100 E tO H p r e fe r e n c e o v e r w a te r ( % )
Week 1 Week 2 Week 3 Week 4 Week 5
0 10 20 30 40 50 60 70 80 90 100 S u c r o s e p r e fe r e n c e o v e r w a te r ( % )
Week 1 Week 2 Week 3 Week 4 Week 5
0 10 20 30 40 50 60 70 80 90 100
A
B
C
D
Figure 4. Chart displaying the intake in the two bottle choice tests during five weeks; drinking intake of 20% ethanol (A) and 5% sucrose (B) in C57Bl/6 mice. Each dot represents an average of n= 10 C57Bl/6 mice/group where drink intake was measured every second 24 hour. Also chart displaying consumption of ethanol and sucrose preference over tap water (C-D). Preference of drinking is represented as percentage (y-axes) over tap water in the two bottle choice experiment. Each dot represents n=10 and mean ± SEM bars.
6 Discussion
It is established that the rewarding effects that drugs of abuse are mediated via the endogenous opioid system and the mesolimbic dopamine-system. Within the opioid system different receptor subtypes, specifically the mu- (MOR), delta- (DOR) and kappa-opioid receptors (KOR) participate in the regulation of drug intake, for such drugs as alcohol and morphine among others [31, 60-62]. These three different ORs, MOR, DOR and KOR, are encoded by three different genes OPRM1, OPRD1 and OPRK1 [31]. Both alcohol and drugs of abuse affect this system and the opioid receptors in various ways during the development of addiction (e.g. Koob GF, 2005) [14, 25, 61, 63]. However, studies on the reward system and the endogenous opioid system are limited for voluntary exercise. We, therefore, aimed to investigate the effects of natural rewards such as wheel running and sugar intake on the gene-expression of MOR and DOR and comparing the gene-gene-expression changes to those induced by alcohol intake. We examined MOR and DOR gene-expression in the nucleus accumbens, prefrontal cortex and ventral tegmental area.
Effect of voluntary exercise on OPRM1 and OPRD1 expression
In the studies presented here we show that voluntary exercise up- regulates MOR mRNA expression in the NAc and PFC. DOR mRNA expression in the VTA is also up regulated for exercising animals, whereas it is reduced within the NAc. These findings are consistent with the fact that endogenous release of opioids is known to play a key role in regulation of the activity of mesolimbic circuits originating in the VTA and projecting to the NAc [13]. During exercise the neuropeptide B-endorphin is peripherally and centrally released from the pituitary and hypothalamus respectively and binds to mu-opioid receptors [64, 65] that trigger the reward system (MD-system). Within the MD-system activation of neurons within the VTA leads to increased dopamine-release within the NAc, which in turn has projections to the PFC. Voluntary exercise triggers thus natural release of dopamine resulting in a feeling of euphoria and well-being, and as supported by other studies (e.g. Boecker H et al, 2008) [66]. Therefore, physical exercise is associated with opioid activation. Our finding of the MOR mRNA expression in PFC and NAc is probably due different levels of endorphin peptides and ORs that modulate dopamine levels released upon rewarding stimuli. Based on previous studies (e.g. Gago, B., 2007; Julie Le Merrer et al 2009; Simmons D et al 2009) [13, 67, 68] we know that dopamine modulates locomotor and motivated behaviors and have rewarding effects. One can speculate if the up regulation of MOR in NAc might be due to that exercise enhances the rewarding effects and also increases DA-levels within this area kept in mind that dopamine levels was not measured in the study.
Mice and other rodents with access to running wheels have been seen in other studies to develop an obsession- or an addiction-like behavior, and voluntary exercise also exert antidepressant-like effects [43, 44].The reason for that might be that the
MD-system upon activation, through release of endogenous opioid neuropeptides, acts
rewarding and thereby reinforces a behavior making it compulsive and later transitioning into an addiction. MOR has been observed to affect DA levels in the NAc and alternating levels of DA levels enhances or reduce the rewarding effects which obtained via the MD system.
DOR mRNA level in NAc is significantly down regulated. This is not consistent with other findings (e.g. Greenwood et.al. 2011) that demonstrate exercised animals increase their mRNA levels on OPRD1 gene transcription [69]. How come that our findings are so dissimilar in NAc? An explanation might be that in Greenwood´s findings DOR mRNA expression of accumbens shell were only investigated and in our study we had both the shell and the core of accumbens. Additionally delta opioid receptors are located on GABA expressing neurons in NAc and physical activity might have an influence on this neurotransmission system. The effect could be a GABAergic inhibition [70] and decreased DA neurotransmission and thereby also a reduction of neuropeptide release. This might be the reason that the DOR is significant down regulated in NAc.
For the DOR mRNA expression in VTA voluntary exercise up regulates the receptor as well. This finding suggests that activation of DOR triggers the MD-pathways upon natural rewarding stimuli such as physical activity. These results are also consist with the fact that endogenous release of opioid peptides are known to play a key role in regulating anterior limbic circuits originating in the VTA and projecting to the NAc. As mentioned before GABA-interneurons within VTA synapse onto dopaminergic neurons that extend to NAc (e.g. Rainer Spanagel al 1999) [71].
On the other hand, it is possible that DOR is less involved in reward circuit compared to MOR. As mentioned before, different neuropsychiatric disorders and stress affect the outcome of a behavior [72]. One cannot discard the fact that in response to stress or anxiety the rewarding effects of wheel running enhance or modulating the mechanisms of the EO-system. We did not make any further behavior tests for e.g. anxiety after three weeks of wheel running. Although with this kept in mind, findings in PFC and NAC might as well depend on wheel running that acts as an anti-depressant factor and also rewardingly (e.g. Rhodes, J.S., T 2003).
Effect of alcohol and sucrose-consumption on OPRM1 and OPRD1
expression
Alcohol and sugar are two dissimilar reward- stimuli thus affecting the MD-system and the opioid peptides and their receptors. However, alcohol- and sugar intake may differ with regards to effects on components of the EO-system. While alcohol is a small-molecule compound it has effect as a drug of abuse and is strongly reinforcing in the early stages of addiction development. Sugar most likely does not have “drug-like” direct effects, but it does act rewarding and is highly palatable for rodents.
Our results from alcohol- and sugar-consumption show significantly reduced mRNA expression of OPRM1 and OPRD1 in the NAc as well as the VTA. We found that a 3-week intake of the drug alcohol in a choice-model leads to a reduction in gene expression of MOR and DOR in VTA. We also observed that the mice have high alcohol consumption, but the preference for alcohol over water was in general below 50%. In earlier studies of 10% ethanol, made by Maclearn (1968) and Rogers (1972) they find that intake differs between genetic mouse strains. Also C57BL/6J in two-bottle choice voluntary consume more of 10% ethanol solution than water, which partly support our study of quite a high intake [73, 74]. Altough keept in mind we did
not take account of spillage, i.e. that the bottles could leak when the animals were fed. The average alcohol intake is quite high compared to other findings, whereas EtOH (10%) self-administration C57/BL6 mice have a consumption of ~10 gram/kg/day [75]. The overall consumption varies in our study with support of Warner Wolstenholme findings of that individual variation in the inbred strain C57Bl6/J occurs.
Reinforced drugs e.g. alcohol are more potent in triggering the pathways of the reward system than natural rewards. The explanation might be that dopamine level increases up to ten times more following drug-exposure than in response to natural reward. According to earlier studies, one found that opiate injection into the VTA increased DA release into the NAC e.g., Leone et al., 1991; Koob, 1992 [76, 77]. MOR and DOR in VTA have both in common, that upon activation whether it is from drugs of abuse or natural reward they both exert their effect in the brain by activating the reward-system [13]. In this case, we observed gene expression in the VTA, where both receptors significant are down regulated. As for DOR and MOR gene expression in NAc our results of the natural reinforcement sugar and the drug alcohol, both genes are also down regulated. Sugar intake acts rewarding trough same pathways as for alcohol and the molecular entrance for addiction might be MOR e.g. Julie Le Merrer 2009 [13]. During these five weeks the constant intake of sugar varies between 20-30 gram/kg/day. In the pathway of activation via opioids receptor subtype µ-1, the receptors do not need to have a direct binding ligand for activation [78, 79]. Alcohol acts this way for activation of MOR and thereby affects the reward system indirectly via the mesolimbic pathway. This is the equal with sugar intake and activation of the reward system.
With this kept in mind it can be assumed that alcohol compared to natural reward such as exercise ought to be considered to have a stronger effect on the MD system through stronger activation of dopamine release [13]. From VTA, as mentioned previously, neuron originates and transmits information to different parts of the brain. It is known that alcohol interacts with the opioid system through its various receptors (e.g. Koob, G.F 2005 [14]). Primarily the neuropeptides B-endorphins and enkephalin binds to MOR and DOR. Important to mention is the rewarding peptide B-endorphin does not bind KOR and activation of this OR subtypes has been determined to have opposing effects on the brain reward-system compared to MOR and DOR. One can also assume that during the time an addiction develops a reduction of endogenous opioid activity is seen. All together it is unlikely during an addiction a neuro adaption of the brain develops e.g. Koob GF, Le Moal, 2005 [80] and mechanisms of EO-system such as dezensitation of ORs might also be the reason for our findings in response to alcohol and sugar.
Overall the effect of alcohol differs significant for gene transcription as for our findings in the first study where we investigated the natural reward exercise. For exercise we found a general up regulation of MOR and DOR except in NAC in which DOR was down regulated. Our study of ORs mRNA expression following exercise is dissimilar according to Greenwood, 2011 and needs to be further evaluated. In the second study alcohol and sugar, we found opposite results except for mRNA expression of DOR in VTA. In order to verify our results, it requires subsequent studies which can generate a greater comprehensive picture of EO-system and its mechanisms. One can look at neuropeptides of the ORs using qRT-PCR and also look at the actual protein levels using ELISA. Furthermore, it would be interesting at the
same time to enlarge the study with increased number of samples and also extend the study to measure DA-levels in the animals if possible. Following this, another interesting aspect is to investigate MOR and also DOR with different opioid antagonists in pharmacological studies. Here, the receptor MOR is most interesting There is already a treatment for alcohol dependence (Naltrexone®). This drug has proved to be effective and ineffective in different studies as already stated by Kimura M, 2011 [81].
The study of Spanagel, R et al (1999) [71], and Chang, G. Q. et al. (2010) show that μ
antagonist3F
4
naltrexone upon binding decrease craving for alcohol. This may be due to a decreased reward-value of the alcohol following naltrexone-treatment. That might be interpreted in different ways, such as the alcohol itself is not as satisfying in terms of taste, or that the positive response that alcohol usually provide alcoholics diminishes. Naltrexone® does not cure an addiction, but reduce craving for alcohol or drugs for individuals that suffer from alcohol or drug addiction. Evolutionary from a biological perspective, conserved genes and underlying mechanisms are of interest as mentioned before. Appetitive motivation is associated with motivation that has benefit for the species themselves. Additional for the EO-system and natural reinforcement, sugar and exercise, it is difficult to evaluate as we do not have the whole picture. Further on some considerations must be made according to that the levels of endorphin or enkephalin peptides mRNA expression never were investigated. Therefore conclusion can only be made from the receptors mRNA levels and only thoughts of theoretic binding of neuropeptides.
6.1 Conclusions
Evaluation of gene-expression may give us the possibility to discover novel targets for the development of new treatments for targeting different human diseases. Different diseases such as Parkinson, anorexia, drug abuse, anxiety and PTSD among many others are under investigation in different animal models. They all have in common that dopamine effects and neuropeptides are contributing factors to the disorders, and influences these systems within the brain. Epigenetic effects might influence gene function of the EO-system as well and further contribute to the individual differences seen in these complex phenomena.
7 Acknowledgement
Ending this master thesis I would like to thank my supervisor Annika Thorsell for all her guiding in this project together with Susanne Hilke. Moreover, I would like to thank, Daniel Nätt in the lab, my very good friend Josefine Jonsson for all the happy times in the Health University and in “Valla”, Annexet, and Gert Nilsson IMK also
Gareth Morgan, Department of Laboratory Medicine (Labmed), KI. Thanks to the
whole lab group Lovisa Holm, Abdul Maruf Asif Aziz for help and support and also my examiner Bengt Harald “Nalle” Jonsson.
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