2
TABLE OF CONTENTS
FOREWORD………7
AKNOWLEDGEMENTS..………...…8
GENERAL SUMMARY………...9
RIASSUNTO ANALITICO………12
RESUME GENERAL……….………14
INTRODUCTION……….15
1 STRESS: CONCEPT AND NEUROPHYSIOLOGY………15
1.1 STRESS: definition………...15
1.2 Stress response: the hypothalamus-pituitary-adrenal axis………..16
1.3 Stress neurobiology: sex differences……….19
2 STRESS-RELATED DISORDERS……….21
2.1 Comorbidity and statistics………22
2.2 Stress and immune system………22
2.3 Early programming of stress-related disorders………...23
2.4 Anxiety………...26
2.5 Depression……….26
3
2.7 Addiction and other stress related-disorders: comorbidity in animals and
hu-mans..…….……….27
3 FOCUS ON ADDICTION………....30
3.1 Clinical studies : epidemiology and symptoms………32
3.2 Substances and non- substances use disorders………...33
3.3 Addiction neurobiology: reward pathway………...39
3.4 Factors of vulnerability………....42
3.5 The impact of stress on addiction...………....43
3.6 Males and Females differences in addiction………...44
4 PRENATAL RESTRAINT STRESS AS A MODEL OF EARLY
PROGRAMMING OF STRESS-RELATED DISORDERS…………....46
4.1 Fetal and perinatal programming of adult diseases…..………..…………...46
4.2 Interest and validity of animal model….………..50
4.3 Prenatal stress model in the rat………...52
4.4 Maternal factors and long term effects on the offspring…….…...………...54
4.5 PRS: An animal model to study the anxious/depressive behaviors….…...….….56
4.6 PRS as an animal model of addiction: neural substrates implicated in PRS- induced addiction behavior……….………...58
4.7 Prenatal stress as a model of comorbidity between addiction and other stress-related disorders……..………...61
4
5 SELF-MEDICATION HYPOTHESIS IN PSYCHOLOGY AND
PSYCHIATRY………...64
5.1
Specific mechanisms………..67
5.2 Effectiveness……….………...68
SPECIFIC AIMS OF THE THESIS ………...70
MATERIALS AND METHODS………..72
1 REPRODUCTION………....72
1.1 Animals……….72
1.2 Reproduction……….………....72
2 PRENATAL RESTRAINT STRESS………...74
2.1 PRS Protocol……….74
2.2 Weaning………....76
2.3 Maternal Behavior and nest score………...77
3 TREATMENTS……….78
3.1 Drug Treatments: Cocaine injection and chronic sensitization……….79
4 ANALYSIS………..80
4.1 Behavioral analysis………..80
5
4.1.2 Activating effect of cocaine………82
4.1.3 Sensitiveness to cocaine effects……….………....82
4.2 Biochemical analysis………87
4.2.1 Sacrifices and dissections…...………...87
4.2.2 Subcellular fractionation………88
4.2.3 Protein assay: BCA dosage………90
4.2.4 Western Blot……….……….91
4.2.5 Densitometry……….92
RESULTS………...93
1 BEHAVIOR….………..93
1.1 Maternal behavior………93
1.2 Conditioned place preference paradigm………..94
1.3 Locomotor activity………....95
1.4 Light and dark test………....97
1.5 Porsolt forced swim test……….………...99
2 NEUROCHEMISTRY………..102
2.1 mGluR2/3 and mGluR5………..…102
2.2 DAD2R………...………..105
DISCUSSION AND CONCLUSION ………....107
PERSPECTIVES ……….………..118
6
APPENDIXES ….………128
KEYWORDS:
Prenatal restraint stress, HPA axis, drug abuse ,cocaine, addiction, depression, anxiety, self-medication, metabotropic glutamate receptors, dopamine, Nu-cleus Accumbens, Hippocampus.7
FOREWORD
This work has been possible, thanks to my thesis internship in the Neuroplasticity Team in Lille 1, France. The Neuroplasticity team is part of the CNRS in the Unit of Structural and Functional Glycobiology, University Lille1, France and coordinates the LIA, (International Associated Laboratory “Prenatal stress and neurodegenerative diseases”) together with University of Rome “La Sapienza” and Neuromed in Italy.
The team, of which I have been a member during this academic year, works on a model of prenatal stress in rats, and studies the various effects that this type of stress induces on the offspring. The intra-utero period is a delicate window during which there is a high neuronal plasticity that leads to a greater vulnerability of the offspring towards stress related disorders. Prenatal Restraint Stress (PRS) in rats is a well-documented model of early stress known to induce long-lasting neurobiological and behavioral alterations including impaired feedback mechanisms of the HPA axis metabolic pathways and immune system, enhanced novelty seeking and increased sensitiveness to psychostimulants as well as anxiety/depression-like behavior.
Therefore, the research in this laboratory covers a wide range of aspects all aimed to find the hormonal and neurobiological mechanisms behind prenatal stress. Prenatally stressed animals have proven particularly useful animal models to study psychiatric disorders such as depression, anxiety and addictive behaviours. Studies in the group have shown standing evidence that the PRS rat model has a depressive and anxious type behaviour. Thus, this particular animal model has proven to be useful to study antidepressant and anxiolytic drugs. PRS rats have also showed to be particularly vulnerable, towards the rewarding ef-fects of psychostimulant drugs. Together with the HPA axis, functional alterations of the mesolimbic dopamine system and of the metabotropic glutamate receptors system appear to be involved in the addiction-like profile of PRS rats.
However, there is a clear gender difference in this addictive behaviour, and the present study focuses on the differences in cocaine addiction between PRS males and females compared to their unstressed counterparts in the putative anxiolytic and antidepressant ef-fects of cocaine mediated by a self-medication hypothesis.
8
AKNOWLEDGEMENTS
First of all, I would like to strongly thank Professor Bagnoli Paola, who gave me the opportunity and the honor to be my thesis supervisor of my University of Pisa.
Then I would also sincerely thanks Professor Cammalleri Maurizio and Professor Scarpato Roberto for their great kindness and helpfulness as co-supervisors.
At last but not least I thank my family for all their constant support during these years of study and especially during this period of hard work for my thesis.
9
General summary
BACKGROUND It is recognized that exposure to an adverse environment during fetal
pe-riod may have lifelong programming effects on different body functions with a considera-ble impact on disease susceptibility. Prenatal restraint stress (PRS) in rat is a well-documented model of early life stress (Maccari et al., 1995) known to induce long-lasting neurobiological and behavioral alterations (reviewed by Darnaudéry and Maccari, 2008). This model is a clear example of early programming of vulnerability to stress -related dis-orders, such as anxiety, depression or drug abuse (Zuena et al., 2008; Morley-Fletcher et
al., 2011; Marrocco et al., 2012; Deminière et al., 1992; Koehl et al., 2000;
Morley-Fletcher et al., 2004; Kippin et al., 2008). An impairment in the glutamatergic machinery and metabotropic glutamate receptors system lies at the core of the PRS phenotype (Mar-rocco et al., 2012; Morley-Fletcher et al., 2011) and antidepressant (ATD) treatment was able to correct behavioral abnormalities and alterations in glutamatergic system (Marrocco
et al., 2014). Interestingly, PRS induces a clear-cut sex effect for anxiety-like behavior
with PRS male rats being more anxious and females less anxious in comparison to control animals (Zuena et al., 2008), while an enhancement in depressive-like profile was ob-served both in male and female PRS animals. We have also recently shown a sex effect in the response of PRS rats to natural reward (Reynaert et al., in revision).
AIMS Here, we investigate a sex effect in rats response to the psychostimulant drug co-caine in a conditioned place preference (CPP) paradigm and in a locomotor activity test.
We also aimed to address whether behavioral sensitization with cocaine could have a ben-eficial impact on the anxious/depressive phenotype in PRS rats and be responsible for in-creased preference for the drug.
MATERIALS AND METHODS Behavioral and neurochemical analysis were performed
on Control and PRS male and female rats treated or not with cocaine. Preference for a
co-caine-paired environment and psychomotor activation inducted by cocaine administration
were studied, as well as anxiety- and depressive-like phenotypes.
10 Behavioral analysis:
CPP: To measure cocaine reinforcing properties. On D1 (20 min, pre-test) spontaneous preference for a one of the two compartments of the apparatus (one grey, one white) is evaluated. During daily conditioning sessions (D2 – D9), rats are alternatively injected intraperitoneally with vehicle (saline) or cocaine and placed in the appropriate chamber for 30 min (cocaine is paired to least preferred side). During the test (D10), re-alized like the pretest (D1), rats preference is measured.
Locomotor activity: Animals during a chronic sensitization protocol with increasing doses of cocaine were tested for the psychomotor effects of cocaine on day1(15 mg/Kg) and on day 6 (30 mg/Kg). Locomotor activi-ty in standard rat cages was registered over a period of 90 min.
Light and dark test : An apparatus, composed of a white and a black boxes, separated by a small door allowing the rat to visit the 2 boxes, al-lows an evaluation of the anxious-like behavior. Rats are injected with vehicle or cocaine on day 7 of chronic sensitization with cocaine and the time spent in the light box, anxiogenic, and the latency to enter this box are recorded.
Forced swim test : Animals are subjected to two trials during which they are placed in an inescapable situation represented by a cylinder filled with water. The first trial lasts 15 min. Then, after 24 h, a 2nd trial is performed for 5 min. The time spent in immobility and the latency to immobility are recorded. This test is classically performed for the screening of ATD drugs (Porsolt, 1978). Rats are injected with cocaine before being placed in the test (pretest: D9 of chronic sensitization pro-tocol; test on D10).
11 Biochemical Analysis: 24 h after FST test, sacrifices are done, and Nucleus
Ac-cumbens (NAc) are dissected and keep at -80°C until use. A subcellular fractioning is performed on NAc samples. The effect of PRS, sex and cocaine on the expres-sion of neurobiological key markers such as mGlu receptors and DAD receptors is analyzed in synaptosomal fractions by Western Blot.
RESULTS We report that PRS enhances cocaine-induced CPP and locomor activity in
both sexes with respect to unstressed rats. Remarkably, cocaine exerts an anxiolytic effect in PRS males and has anti-depressive properties in both males and female PRS rats. Inter-estingly, the reversal of the PRS behavioral phenotype induced by chronic cocaine treat-ment is mainly associated to neurobiological changes in metabotropic glutamate receptors in the NAc. Enhanced response to cocaine would be thus a self-medication strategy to counteract the PRS-induced anxious/depressive phenotype.
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Riassunto analitico
Lo scopo di questo lavoro è di investigare se le differenze sessuali e lo stress possono co-stituire rilevanti fattori di rischio in comportamenti di dipendenza. E’ ben noto, infatti, che lo stress può condurre a molti disturbi come l'ansia, la depressione e la dipendenza. I dati clinici che sostengono queste scoperte evidenziano anche un certo grado di comorbi-dità tra il fenomeno della dipendenza alle droghe e gli altri disordini emotivi indotti dallo stress.
Nel presente lavoro si è voluto verificare se una particolare forma di stress, lo stress pre-natale, unitamente al sesso potessero giocare un ruolo chiave nei fenomeni di abuso e di-pendenza alla cocaina relativamente anche ai suoi effetti ansiolitici ed antidepressivi mediati dal fenomeno della amministrazione intesa come forma di auto-medicazione.
La ricerca sperimentale di questo progetto di lavoro è stata articolata in due approcci sinergicamente interdipendenti e correlati: una fase di indagine comportamentale e una seconda fase di analisi neurochimiche, intesa a complementare la prima.
Nella prima parte di questo studio è stata misurata la preferenza nei confronti del-la cocaina usando il conditional pdel-lace preference test. I dati raccolti hanno rivedel-la- rivela-to che ratti adulti di sesso femminile presentano un punteggio di preferenza mag-giore rispetto ai maschi, e che lo stress aumenta la percentuale di preferenza. Nella seconda parte dello studio comportamentale gli animali sono stati sottoposti ad un protocollo di sensibilizzazione cronica mediante incrementate dosi di
cocai-na (15 mg/kg-30 mg/kg) e nei giorni 1 e 6, 7, 10 sono state rispettivamente testate
l’attivazione psicomotoria mediata da tale droga e le sue proprietà ansiolitiche (me-diante il light and dark test) e antidepressive (me(me-diante il Porsolt forced swim test).
In questo caso, i dati raccolti supportano e confermano l’ipotesi di un effetto ansio-litico e antidepressivo della cocaina ed evidenziano un dimorfismo sessuale ad es-so correlato, particolarmente marcato relativamente alla sintomatologia ansiosa.
Nella seconda parte del presente lavoro sono stati sacrificati (24 ore dopo il FST) i ratti maschi e femmine PRS e controlli precedentemente sottoposti ai test
compor-13 tamentali per dissezionare i NAc dalle loro strutture cerebrali. E’ stato effettuato un frazionamento subcellulare sui differenti campioni dei NAc prelevati con lo scopo di analizzarne tramite Western Blot la frazione sinaptosomale per investiga-re gli effetti del PRS, del sesso e della cocaina sull’espinvestiga-ressione di markers protei-ci chiave (i.e. recettori metabotropiprotei-ci del Glutammato e recettori della Dopamina) individuati come il substrato neurobiologico dei cambiamenti e delle differenze comportamentali osservati nella prima fase dell’approccio sperimentale. Le analisi condotte hanno fatto emergere il dato interessante che la reversione del fenotipo comportamentale PRS indotto dal trattamento cronico con la cocaina è associato principalmente a modifiche neurochimiche dei recettori metabotropici del Glu-tammato.
In conclusione i dati ottenuti, nel loro complesso, suggeriscono che i ratti di sesso femmi-nile presentano una vulnerabilità maggiore rispetto a quelli di sesso maschile, e che lo stress e le differenze sessuali costituiscono i principali fattori di rischio nello sviluppo della dipendenza alle droghe, alla cocaina in questo specifico caso, e che l’effetto antidepressivo di questa droga sintetica d’abuso è associato ad una sua aumentata auto-amministrazione in ratti PRS sia di sesso maschile che femminile.
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RESUME GENERAL
Le modèle de stress prénatal chez le rat (PRS : prenatal restraint stress) est un exemple de programmation précoce d’un phénotype vulnérable pour le développement d’un certain nombre d’altérations comportementales et neurobiologiques. Ce modèle récapitule les symptômes d’anxiété, de dépression et de vulnérabilité aux drogues.
Concernant les paramètres d’anxiété, un dimorphisme sexuel a toutefois été mis en éviden-ce : les mâles PRS apparaissent plus anxieux que les mâles contrôle tandis que les femelles PRS s’avèrent moins anxieuses que leurs homologues contrôles. Au contraire, ce profile n’est plus observé pour un paramètre de dépression. En effet, les tests permettant de mesurer le niveau de dépression de l’animal ont permis de montrer un état de dépression plus accru chez les rats PRS des deux sexes.
Concernant l’addiction, il semblerait que de nature, les femelles soient plus sensibles à la cocaïne que les mâles. Aussi de manière générale, les rats PRS sont plus vulnérables aux psychostimulants, et nous avons montré qu’ils présentaient une sensibilité accrue pour la cocaïne dans un protocole de préférence de place conditionnée.
Ici, nous vérifions si sexe et stress puissent constituer facteurs considérables de risque en comportements de dépendance et l’hypothèse de l’automédication dans la sensibilité aux drogues dans le modèle PRS en mettant en évidence un effet anxiolytique et antidépresseur de la cocaïne par l’utilisation de tests comportementaux. Ces résultats sont corroborés par des analyses sur des marqueurs neurobiologiques dans le noyau accumbens, ayant un rôle dans l’addiction et les symptômes anxiodépressifs, i.e., les récepteurs métabotropiques au glutamate et les récepteurs à la dopamine.
15
INTRODUCTION
1
STRESS: CONCEPT AND NEUROPHISIOLOGY
1.1 STRESS: definition
Stress is a term that is commonly used in every-day life and has a wide range of meanings. The term stress comes from the Latin word “stringere” “to draw tight.” At first it was used in physics to define the internal distribution of a force exerted on a material body, resulting in strain - or stress -such as a rubber band pulled tightly. To understand the scientific meaning of “stress” we should date back to Claude Bernard and his concept of “Mileu interior”. The concept of an internal equilibrium was first suggested by a French physiologist, Claude Bernard (1813-1878). He coined the “Milieu intérieur”, to refer to the extracellular fluid environment and it’s capacity to isolate internal tissues and organs. He probably was influenced by the histologist Charles Robin (1743- 1824), who used the phrase “milieu de l’intérieur” as a synonym for the ancient hippocratic idea of humors. Gross, 1998 reports that in "Claude Bernard and the constancy of the internal environment" the concept of the “mileu interior” is summed up,:
“The fixity of the milieu supposes a perfection of the organism such that the external variations are at each instant compensated for and equilibrated.... All of the vital mechanisms however varied they may be, have always one goal, to maintain the uniformity of the conditions of life in the internal environment.... The stability of the internal environment is the condition for the free and independent life”.
This concept was later developed by Walter Canon, an American physiologist (1871-1945), who called it “Homeostasis”. Homeostasis (from Greek: ὅμοιος, "hómoios", "similar", and στάσις, stásis, "standing still". He described it in his book “The Wisdom of the Body” (Cannon, 1939), as a process in which a system, constantly works in order to maintain a stable internal environment. However, the true pioneer of the scientific meaning of the word “Stress”, is the Austrian endocrinologist Hans Selye (1907-1982). In "A Syndrome Produced by Diverse Nocuous Agents," (1936) he observed that after injecting mice with various irritating substances, they all produced similar symptoms (swelling of
16 the adrenal cortex, atrophy of the thymus, gastric and duodenal ulcers). According to him the word “stress”, in his book “The Stress of Life” (1956) is “the non-specific response of the body to any demand placed upon it” Every stressful event, either positive or negative, is able to affect our body’s natural equilibrium, or homeostasis. The balance of our body system may be disturbed due to a wide range of events such as a life-threatening situation or even a simple insult. Stress activates a cascade of biological reactions as the body attempts to bring the body/mind back to equilibrium. The body’s fight or flight response recruits the body’s energy stores and focuses all its resources in order to restore homeostasis. The intensity and duration of stress changes from person to person according to circumstances and emotional conditions of the individual.
1.2 Stress response : the hypothalamus-pituitary-adrenal axis
Our body responds to stress thanks to the cooperation of three important systems: the
nervous system, the immune system, and the endocrine system. The heart of the response starts from the brain and reaches every single part of our body across the vast network of glands, that mediate their responses through hormones. The leader of the stress response is the hypothalamus, a small part of the brain, part of the limbic system, which is located above the brainstem. When it perceives a stressful stimulus it carries out four important functions:
1. Activates the autonomous nervous system (ANS). The ANS (made up of the sympathetic nervous system and the parasympathetic nervous system) regulates the vegetative functions of our body. When a stressful event occurs the sympathetic nervous system, is immediately activated and releases catecholamines (norepinephrine and epinephrine) that activate the fight and flight response.
2. Releases CRH (coticotropin-releasing-hormone) that activates the HPA (hypothalamus-pituitary gland-adrenal cortex) axis. CRH reaches the pituitary gland and stimulates the production of ACTH (adrenocorticotropic hormone). ACTH travels to the adrenal cortex and stimulates the release of corticoids (cortisol and aldosterone).
17 3. Produces vasopressin or anti-diuretic hormone (ADH) that is released through a special portal system that travels to the kidney. This hormone regulates water absorption and increases blood pressure.
4. Produces TRH (thyrotropin releasing hormone). TRH travels to the anterior part of the pituitary gland and stimulates the production of TTH (thyrotropic hormone), which travels to the thyroid where it stimulates the production of thyroxin and triiodothyroxin. Both hormones, generally increase basal metabolic rate.
Figure 1: (A) Major components of the HPA axis and connected brain structures. (B) Con-nections between the hypothalamus, pituitary, and adrenal glands in the HPA axis and hip-pocampus, medial prefrontal cortex (mPFC), and DRN. Activation of the HPA axis is initiat-ed by stimulation of neurons in the minitiat-edial parvocellular region of the paraventricular nucle-us (PVN) of the hypothalamnucle-us and secretion of corticotropin-releasing hormone (CRH, or corticotropin-releasing factor, CRF) and arginine vasopressin (AVP) that amplifies the effect of CRH, in the portal vein. The pituitary gland secretes adrenocorticotropic hormone (ACTH), initiating the release of glucocorticoids (GC) from the adrenal cortex (AC), and adrenaline (Ad) and noradrenaline (NAd) from the adrenal medulla (AM) into the blood stream. This cascade is transient, and upon termination or removal of the stimulus, the HPA axis returns to a baseline state by the action of several negative feedback loops. In these loops,
18 GC act directly to shut down the response of the hypothalamus and pituitary, and the release of CRH then ACTH, and indirectly by activating glucocorticoid receptors (GRs) in the hip-pocampus and frontal cortex, that project back to the hypothalamus. GC also activate miner-alocorticoid receptors (MRs). The hypothalamus and mPFC have reciprocal projections with the dorsal raphe nucleus (DRN). Neurogenesis occurs in the dentate gyrus and yields new neurons from neural progenitor cells (NPCs). (From Franklin et al., 2012).
Although all the pathways are a response to stress, not all act at the same speed (Table1). The hypothalamus immediately stimulates the sympathetic nervous system to release catecholamines, but at the same time induces the adrenal medulla to secrete the same substances. These catecholamines, differ from those released by the sympathetic nervous system, as they are released directly into the blood and therefore last longer and are responsible for the intermediate effects. Our body has developed further more efficient mechanisms to back up the temporary effects produced by the catacholamines. The activation of the adrenal cortex, the ADH and thyroxin axis are responsible for the prolonged effects of the stress response.
Effects Reaction Time
Immediate effects
Epinephrine and
norepinephrine from the
sympathetic nervous system.
2-3 seconds
Intermediate effects
Epinephrine and
norepinephrine from the
adrenal medulla
20-30 seconds
Prolonged effects HPA axis, ADH axis, thyroxin
axis.
Minutes, hours, days and weeks.
Table 1: Different components of the stress response have different speeds. Source: Allen, Human Stress: It’s Nature and control (Minneapolis, MN Burgess, 1983).
As mentioned before, the HPA (Hypothalamus- Pituitary gland- Adrenal cortex axis), is the most important axis in regulating the stress response. The anterior hypothalamus produces CRH (Corticotrophin Releasing Hormone) This hormone signals the anterior pituitary gland to release adrenocorticotropic hormone (ACTH), which travels through the
19 blood to the adrenal glands, a small pair of glands that sit above each kidney. ACTH simulates the adrenal cortex to release glucocorticoids (cortisol) and mineralcorticoids (aldosterone) Aldosterone’s effects are on the kidney where it increases reabsorption. Instead glucocorticoids, have the function of generating glucose. They achieve this by degrading proteins, breaking down fats, and by a process called gluconeogenesis, which consists of the generation of new glucose in the liver.
Termination of the stress response occurs via a negative feedback of the HPA axis. At the level of the hypothalamus and the pituitary gland, inhibits the synthesis and/or release of (CRH), vasopressin (AVP), and (ACTH), thus terminating the stress response. Glucocorticoids bind to intracellular receptors, which function as ligand-activated transcription factors. There are two receptor subtypes: the lower and higher affinity glucocorticoid (GR) and (MR) receptor.
The stress response is essential for survival but is intended to be temporary and has to be shut down. Therefore, when it is prolonged, it represents an “allostatic load” for the body, the “cost” the body pays for an inefficient stress response (McEwen, 2007). Therefore, prolonged secretion of hormones is dangerous and can lead to stress-related disorders, such as hypertension and increased risk of cardiovascular diseases.
1.3 Stress neurobiology: sex differences
In both humans and rodents, several brain regions are involved in stress and corticotropin-releasing factor (CRF)-mediated HPA axis regulation and emotional affect, such as the Nucleus Accumbens, prefrontal cortex, amygdala, and hippocampus, continue to mature well into adolescence (Fig. 2) (Crews et al., 2007; Sowell et al., 2007). These areas also demonstrate significant sex differences in both structure and function into adulthood (Ca-hill, 2006). Recent studies have begun to clarify whether the onset of these possible sex differences are related to varying rates of maturation between males and females. Neu-roimaging studies in humans show that amygdala volume increases significantly more in adolescent boys, whereas hippocampal volume increases faster in girls (Giedd et al., 1997). Moreover, the thinning of frontal cortical gray matter associated with adolescence occurs earlier in females (Giedd et al., 1999), which may be associated with their earlier onset of puberty. Interestingly, these same limbic and forebrain regions are extremely sensitive to hormones released during stress, especially glucocorticoids (McEwen, 2005). Because
glu-20 cocorticoids are potent modulators of synaptic function and plasticity (Sapolsky, 2003), it is likely that exposure to stressors during adolescence or impaired CRF pathway function would alter the shaping of these
areas critical to emotionality, possibly in a sex-dependent manner (Goel and Bale, 2007). Unfortunately, little is known regarding the effects of stress during puberty on the devel-opmental trajectory of these areas in males and females (Romeo and McEwen, 2006). It will be imperative to better identify and delineate the interaction between adolescent de-velopment, sex, and stress to determine whether there is a female-biased sex difference in susceptibility to stress-related psychopathologies.
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2 STRESS-RELATED DISORDERS
As reviewed in McEwen 2005, several clinical studies have shown that stress can lead to mood disorders . Hippocampus amygdale and prefrontal cortex are targets of stress hor-mones, and stress is known to precipitate and exacerbate mood disorders. In long-term de-pressive illness, the hippocampus and prefrontal cortex undergo atrophy, whereas the amygdale is hyperactive in anxiety and mood disorders and may undergo a biphasic change in structure increasing in size in acute depression and shrinking on long-term de-pression (McEwen, 2003). Furthermore there has been evidence that stress induces simpli-fication and retraction of the dendrites in the hippocampus. (McEwen, 1999). This reflects many interactions with neurochemical systems in the hippocampus, including serotonin, gamma-aminobutyric acid (GABA), and excitatory amino acids. Probably, the most im-portant interactions are those with excitatory amino acids such as glutamate.
Naturally the magnitude of a stressor determines the impact it can have in triggering a cer-tain disorder. Many findings have shown that impairments of HPA axis and cortisol levels are the cause of stress-related disorders. Under chronic stress conditions, some studies sug-gest an exaggerated activation of the HPA-axis with a hypersecretion of cortisol (hypercor-tisolism) while a few others find a reduced adrenocortical activity (hypocor(hypercor-tisolism). While hypercortisolism is a well-known biological marker in melancholic depression, anorexia nervosa (Gold et al., 1988), and alcoholism (Inder et al., 1995), hypocortisolism has been discussed as a biological marker of posttraumatic stress disorder (PTSD) and stress-related bodily disorders (Chrousos and Gold, 1992). Impairment of the HPA axis and alteration of CRH release is speculated to be involved in many psychiatric disorders.
Stress related neuropsychiatric disorders characterized by insufficient glucocorticoid sig-nalling, impaired glucocorticoid responsiveness are often associated to hypersecretion of CRH. Hyperactivity of these stress-responsive systems, especially inflammation, in turn, may contribute to the behavioural features of these disorders, including depressed mood, anhedonia, fatigue, pain, and cognitive dysfunction, as well as neurobiological, metabolic, and immunologic consequences of stress.
22
2.1 Comorbidity and statistics
The greatest part of the epidemiological studies underline that around the 35% of the pop-ulation had or will have a psychic trouble during its life , and among these, the most dif-fused are anxiety and depressive-like troubles.
The ESEMeD (European Study on the Epidemiology of Mental Disorders) [Acta Psychiatr Scand 2004: 109 (Suppl. 420): 21.27] is the first epidemiological study about the preva-lence of stress related disorders done on a representative sample of the adult general Ital-ian population and of other 5 European (Belgium, France, Germany, Holland and Spain) countries.
Data show that the most vulnerable categories are people who are already in mental illness conditions , young and not married people, unemployed, who lives in the city and the housewives. The women have a triple probability to develop a trouble of anxiety in comparison to the men and they are very more susceptible to PTSD demonstrations.
2.2 Stress and immune system
Virtually all stressors, including infections, physical trauma, and even psychological in-sults, are associated with immune activation and the release of proinflammatory cytokines such as tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1) and Interleukin-6 (IL-6). Through their inhibitory effects on nuclear signaling pathways, glucocorticoids are the most potent anti-inflammatory hormones in the body and thereby serve to suppress the production of pro-inflammatory cytokines during stress exposure and return the organism back to baseline (homeostasis) after cessation of the stressor (McKay, 2000). Indeed, in the absence of glucocorticoids, the body quickly succumbs to the action of the inflammatory response.
Inadeguate glucocorticoid-mediated feedback inhibition of immune responses, has been reported in a number of stress-related neuropsychiatric disorders. Best characterized is in major depression. The inflammatory changes reported include increased levels of acute-phase reactants, increased plasma and CNS concentrations of proinflammatory cytokines (especially IL-1 in CNS and IL-6 in plasma), increased levels of prostaglandins, and an in-crease in stimulated in vitro peripheral blood monocyte production of proinflammatory
cy-23 tokines (Sluzewska, 1999). Moreover, increased concentrations of plasma IL-6 have been found to positively correlate with post-DST cortisol levels, suggesting that DST non sup-pression identifies a group of depressed subjects most likely to demonstrate immune acti-vation. Moreover depression has been shown to predict exacerbations in diseases with an inflammatory component, including both rheumatoid arthritis (Parker et al., 1992) and multiple sclerosis (Foley et al., 1992). Immune activation has also been reported in stress-related disorders characterized by hypocortisolism. These disorders include PTSD (Post traumatic Stress disorder), chronic fatigue syndrome, and fibromyalgia. (Spivak et al., 1997; Cannon et al., 1999).
2.3 Early programming of stress-related disorders
Several epidemiological studies on pregnant women have shown a high positive correla-tion between stressful events during the pregnancy and dramatic effects at birth, such as spontaneous abortion, preterm birth, low body weight, developmental delays and long-term behavioral abnormalities (Stott, 1973; Blomberg, 1980; Meijer, 1985; Homer et al., 1990; Holmes, 2001; Weinstock, 2001). However, the investigation of the effects of early stress in humans results very difficult to perform, due to ethical concerns and retrospective ap-proaches. Accordingly, during the last decades, scientists have developed specific animal models, especially in rats, where the stress is applied to the pregnant mother or early in life and the study is performed in the adult offspring. In rats, prenatal glucocorticoid exposure reduces birth weight in the new-born (Seckl, 2001), and small doses of the synthetic gluco-corticoid dexamethasone prenatally given alter neuropsychological parameters such as emotionality (Trautman et al., 1995; Lajic et al., 1998). Also, prenatal exposure to dexa-methasone reduces birth weight, affects brain development (Slotkin et al., 1993) and pro-grams hypertension and hyperglycaemia in adult offspring (Nyirenda and Seckl, 1998). Postnatal stress is also detrimental, in particular in early infancy which is a critical period during which the offspring almost entirely depends on parents or caregivers. Remarkably, while high level of active maternal behaviors such as licking-grooming and nursing has beneficial effects throughout life and in adulthood, low level can lead to depressive-like symptoms, anxiety, and altered synaptic plasticity and cognitive and social behaviors (Champagne et al., 2008; Zhang et al., 2010; Meaney, 2010; Bagot et al. 2009; Myers-Schulz and Koenigs, 2012). However the use of animal models of psychopathologies such
24 as depression is risked with a singular anthropomorphic drift consisting in studying non-human-animal disorders defined by cognitive and emotional processes that are typically human (for example: depressed mood or self-esteem reduction in major depression). None-theless, by taking into account the necessary limitations, it remains possible to use animal models and, in particular, rodents for the investigation of cognition and behavioral disturb-ances. Indeed the following considerations are important to approach the research in ani-mal models:
1. The assumptions on the psychological processes which cannot be measured in rodents must be eliminated from the field of investigation.
2. It is preferable to focus on the study of the symptoms instead of the syn-drome. Rather than to observe the human syndrome overall and to compare it with the disorder presented by the model by making them correspond to a common etiology, one should focus on the analysis of each symptom and its origin.
3. A neurobehavioral mechanistic approach, in which neurobiological hypothe-sis rather than psychological hypothehypothe-sis are posed as mechanisms for discrete symptoms, will yield more useful information concerning the nature and the treatment of Stress-related disorders. (Holmes, 2003).
Here following a short presentation of the most studied animal models of perinatal stress, i.e. stress occurring during the prenatal and postnatal period.
Early handling
Early handling is a simple paradigm that consists in subjecting pups to short periods of separation from their mother during the first week(s) of life. This manipulation decreases overall stress responsiveness and favors a rapid surge and return to baseline of glucocorti-coids immediately after stress (Levine, 1957; Cirulli et al., 2003; Meaney et al., 1996). Such fast adaptive response minimizes the risk of damage to the nervous system due to prolonged glucocorticoids exposure. It also reduces anxiety and enhances exploratory ac-tivity across life (Levine, 1957; Weinberg et al., 1978). Early handling in rodents increases active maternal behaviors, which reduces HPA axis activity and can elicit stress resilience in the offspring when adult (Meaney et al., 1996; Pryce et al., 2001).
25
Maternal separation
Extended periods of maternal separation during postnatal life can persistently interfere with neurochemical, hormonal, and behavioral responses and induce stress vulnerability even in the absence of any direct manipulation of the pups (Moles et al., 2004; Kikusui and Mori, 2009, Lucassen et al., 2010). In rodents, 3 hours of daily separation from birth to 2 weeks postnatal can result in depressive-like behaviors upon re-exposure to stress later in life (Franklin et al., 2011; Uchida et al., 2010). At the behavioral level, male and female rats deprived from their mother at PND3 exhibit enhanced tone-cue fear conditioning sponses (Oomen et al., 2010, Oomen et al., 2011 and Champagne et al., 2008). These re-sponses are mediated by the amygdala that is critically involved in fear and anxiety (Le-Doux, 2007). Curiously, maternal deprivation do not affect the total branch length, number of branch points and primary dendrites or dendritic complexity index in the basolateral amygdala of male and female offspring (Krugers et al., 2012). Maternal separation can have a strong or mild impact depending on its duration, frequency, and predictability. Nonetheless in some conditions, maternal separation can also be beneficial and promote stress resilience later in life. In rats, prolonged separation (6 hours) can lower emotional response and risk assessment and decrease anxiety in adverse conditions in adults (Roman
et al., 2006). Likewise, in mice, pups exposed to chronic unpredictable separation
com-bined with maternal stress develop some resilience to social stress when adult (Franklin et
al., 2011). Prenatal stress
Different procedures have been adopted to submit the pregnant rat to a stress condition (Weinstock, 2001, Baier et al., 2012). The most frequent ones are the noise and flashing light stress applied on unpredictable basis three times a week throughout gestation (Fride and Weinstock, 1984), and prenatal restraint stress (PRS) applied three times a day during the last week or the last 11 days of gestation (Fig. 10) (Ward and Weisz, 1984; Maccari et
al., 1995; Alonso et al., 1991). Interestingly, during this period, the fetal HPA axis begins
to release its own ACTH and corticosterone (Boudouresque et al., 1988). PRS paradigm constitutes the stress procedure that has been adopted in all of the studies described in the present thesis.
26
2.4 Anxiety
Anxiety is a normal reaction to stress and can actually be beneficial in some situations. For some people, however, anxiety can become excessive. There are a wide variety of anxiety disorders and collectively, they are among the most common mental disorders experienced by the population.
The most common anxiety disorders are:
Obsessive-compulsive disorder (OCD),
Panic disorder (PD)
Post-traumatic stress disorder (PTSD), and
Social phobia (or social anxiety disorder).
Generalized anxiety disorder (GAD).
There is evidence that anxiety is related to impairments with the HPA axis. Patients who have PD have been reported to exhibit increased baseline plasma cortisol concentration, which is positively correlated with the risk for a panic attack after lactate administration. Moreover increased overnight plasma cortisol concentrations corresponding to sleep dis-ruption have been noted in subjects who have PD. (Kent et al., 2002).
2.5 Depression
One of the best-documented psychiatric disorders related to hypercortisolism is major depression. (Board et al., 1957) for the first time reported elevated cortisol levels in depressed patients. Since then, hypersecretion of cortisol in major depression has been confirmed in a large number of studies (Gold et al., 1988). In addition to the assessment of basal cortisol levels, (Carroll et al., 1968). reported a failure to suppress endogenous cortisol secretion following the administration of dexamethasone (an exogenous steroid that provides a negative feedback and thus suppresses the HPA axis) in patients with major depression.
Referring to the actual differentiation of major depression in the melancholic and atypical, according to the DSM-IV (American Psychiatric Association, 1994), hypercortisolism and non-suppression of dexamethasone is related to melancholic depression, while atypical depression (e.g. chronic fatigue syndrome, CFS) seems to be associated with a hypofunctional HPA-axis.
27 In melancholic depression hypercortisolemia could be caused by a lower feedback sensitiv-ity, which is the consequence of a central CRH hyperactivity.
2.6 Addiction
Several preclinical studies have shown the ability of stressors to alter the acquisition of drug self-administration in rats (Goeders, 2002; Piazza and Le Moal, 1998). The acquisi-tion of amphetamine and cocaine self-administraacquisi-tion is enhanced in rats exposed to tail pinch (Demeniére et al., 1992), social defeat (Haney et al., 1995; Tidey and Miczek,1997; Kabbaj et al., 2001) or neonatal isolation (Kosten et al., 2000). Exposure to electric foot-shock also increases the subsequent reinforcing efficacy of heroin (Shaham and Stewart, 1994) in rats. The ability of stressors to facilitate the acquisition of drug self-administration may therefore result from a similar sensitization phenomenon, perhaps involving dopamine (Goeders, 1997; Piazza and Le Moal, 1998). Although exposure to the stressor itself may be aversive, the net result is reflected as an increased sensitivity to the drug. Therefore, if certain individuals are more sensitive to stress (Piazza and Le Moal, 1998) and/or if they find themselves in an environment where they do not feel that they have adequate control over this stress (Levine, 2000), then these individuals may be more likely to engage in sub-stance abuse.
From a neurobiological point of view CRH and other neuropeptides dynorphin (Bruchas et
al., 2010) and neuropeptide Y (NPY) (Schank et al., 2010) have established roles in
link-ing stress and addiction-related behaviour. More recently, however, additional neuropep-tides including the urocortins (Ucns), neuropeptide S (NPS), nociceptin/orphanin FQ (N/OFQ), and neurokinins (NKs), have been implicated in processes that link stress re-sponses with drug seeking, drug taking, and long-term neuroadaptations.
2.7 Addiction and other stress-related disorders: comorbidity in animals and humans
A high prevalence of substance use disorders is observed in patients with major depressive disorders (Davis et al., 2005) or other psychiatric conditions (Kessler et al., 2005) and most patients admitted for drug addiction disorders have a concomitant use of antidepressant
28 treatment. Women are more sensible to the effects of drugs and are more vulnerable to re-lapse. This would be due to an important comorbidity between psychiatric, mood, anxiety or depression disorders and addiction in women (reviewed by Zilbermann et al., 2003), and an important sensitiveness to the symptoms associated to withdrawal induces a high pro-pensity to relapse in women (Ambrose-Lanci et al., 2008; Back et al., 2005).
Some psychoanalysts see addiction as a “protection” against several psychopathological states (depression, psychosis, severe neurosis) (Véléa, 2005).
As example, it has been shown that ethanol intake during adolescence prevented the “depressive behavior” otherwise seen in adult PRS rats. Perhaps, the increase in ethanol preference observed in adolescent PRS female rats might reflect a strategy of
self-medication aimed at preventing the onset of depressive disorders later in life. (Van Waes et al., 2011b).Remarkably, the reverse was also true, i.e. ethanol intake during adolescence induced depression-like effects in the adult life. These effects were observed in rats subjected to PRS.
There is a debating on the addictive power of food; it’s interesting to see how food can influence mood. In adult rats, the effect of “cafeteria diet” has been assessed on anxiety-like behavior. It has been shown that this kind of diet was able to modify the anxious-anxiety-like profile of rats, but, remarkably, the dietary effects on behavior were different between males and females. The effects in adult males were increased grooming and fewer entries into the aversive open arms of the EPM apparatus, consistently with another work showing that a cafeteria diet increases the response of male rats to chronic variable stress (Zeeni et al., 2013). This would suggest a potential anxiogenic effect in males. An anxiolytic effect in females was on the contrary expressed by the increased time spent on the aversive open arms. In addition, the decreased grooming in females would be in line with an anxiolytic profile in adult females (Warneke et al., 2013). Grooming is a self-directed behavior. When shown upon exposure to aversive situations, as for example the plus maze or the open field, grooming can be interpreted as a de-arousing activity. Anxious rats groom more often and anxiolytic drugs reduce grooming behavior (Dunn et al., 1981; Voigt et al., 2005).
From an epidemiological perspective, there is a higher degree of comorbidity between depression and drug dependence, indicating that the rates of depression among drug abusers and the rates of drug abuse among depressed patients are substantially higher than
29 expected from the individual rates of these disorders (for review, Markou et al., 1998). This leads to suppose that the anhedonic symptoms of depression, which constitute the core feature of this illness, would be due to a dysfunctional brain reward system. Thus, alterations in reward and motivational processes at both the behavioral and neurobiological levels may constitute the defining characteristics of both depression and drug dependence. Nevertheless, it is not clear if drug dependence and depression are different behavioral expressions of the same neurobiological abnormalities, or whether one psychiatric disorder leads the other. An extension of this concept made by Markou and colleagues (1998) is known as the “self-medication hypothesis” and takes into account the possibility that drug dependence may involve self-medication to reverse some of the abnormalities associated with depression. Hence, it is possible that through the simultaneous use of multiple drugs, people determine the drug or drugs combination that best normalize their behavior.
So, many risk factors can predispose an individual to initiate and maintain drug use, such as preexisting depression or bipolar disorders. Thus, drug abusers are attempting to medi-cate themselves for a range of psychiatric disorders and painful emotional states. People who experience major trauma in their life may self-medicate with drugs or alcohol to re-lieve the symptoms of post-traumatic stress disorders and depression (Danstsky, 1994).
30
3 FOCUS ON ADDICTION
The American Psychiatric Association, defines addiction as a "chronically relapsing disor-der that is characterized by three major elements: (i) compulsion to seek and take the drug, (ii) loss of control in limiting intake, and (iii) emergence of a negative emotional state when access to the drug is prevented". This definition implies a loss of control regarding drug use, and the addict will continue to use a drug despite serious medical and/or social consequences.
Although we often associate the word addiction with drugs and alcohol, there are new findings that suggest that non substance addictions are as much addictive as cocaine. (Holden et al., 2010) have proposed behavioural addictions, such as gambling, over-eating, sex, compulsive shopping. To sustain these arguments scientists have used fMRIs and have seen for example that a compulsive gambler and a cocaine addict have similar brain alterations. Apart from gambling, the others haven’t quite yet made it into the DSM VI (Diagnostic and statistic manual of mental disorders).
However, the classical addictive disorders that are classily considered such by the DSM VI are drug and alcohol addictions. The criteria for addiction to alcohol and drugs are typically diagnosed using the criteria for substance dependence. There are seven criteria for substance dependence. To be diagnosed, the person would have to have at least three of the criteria within the same year.
The seven criteria for substance dependence are:
(1) Tolerance, as defined by either of the following: (a) A need for markedly increased amounts of the substance to achieve intoxication or desired effect. (b) Markedly diminished effect with continued use of the same amount of the substance.
(2) Withdrawal, as manifested by either of the following: (a) The characteristic withdrawal syndrome for the substance (refer to Criteria A or B of the criteria sets for Withdrawal from specific substances) (b) The same (or a closely related) substance is taken to relieve or avoid withdrawal symptoms.
31 (3) The substance is often taken in larger amounts or over a longer period than was intended.
(4) There is a persistent desire or unsuccessful efforts to cut down or control substance use.
(5) A great deal of time is spent in activities necessary to obtain the substance (such as visiting multiple doctors or driving long distances), use the substance (such as chain smoking) or recover from its effects.
(6) Important social, occupational, or recreational activities are given up or reduced because of substance use.
(7) The substance use is continued despite knowledge of having a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by the substance.
Drug addiction has major consequences on the brain, on mood, and on a person’s behav-iour. Drugs within a class are often compared with each other with terms like potency and efficacy. Potency refers to the amount of a drug that must be taken to produce a certain ef-fect, while efficacy refers to whether or not a drug is capable of producing a given effect regardless of dose. Both the strength and the ability of a substance to produce certain ef-fects play a role in whether that drug is selected by the drug abuser. It is important to keep in mind that the effects produced by any drug can vary significantly and is largely depend-ent on the dose and route of administration. Concurrdepend-ent use of other drugs can enhance or block an effect, and substance abusers often take more than one drug to boost the desired effects or counter unwanted side effects.
32 Figure 3 : Phases of drug abuse
3.1 Clinical studies: epidemiology and symptoms
In 2008 155 to 250 million people or 3.5% to 5.7% of the world's population aged 15-64, used psychoactive substances, such as cannabis, amphetamines, cocaine, opioids,
and non-prescribed psychoactive prescription medication.
(http://www.who.int/substance_abuse/facts/en/).
The use of psychoactive substances causes significant health and social problem for the people who use them and also for others in their families and communities. In 2004 WHO estimated that 0.7% of the global burden of disease was due to cocaine and opi-oid use.
Not only psychostimulant drugs but also the use of alcohol is a global problem. It re-sults in 2.5 million deaths each year. Alcohol is the world’s third largest risk factor for premature mortality, disability and loss of health; it is the second largest risk factor in Europe. Alcohol is associated with many serious social and developmental issues, in-cluding violence, child neglect and abuse, and absenteeism in the workplace. It harms the well-being and health of people around the drinker. An intoxicated person can harm
33 others or put them at risk of traffic accidents or violent behaviour, or negatively affect co-workers, relatives, friends or strangers. Thus, the impact of the harmful use of alco-hol reaches deep into society.
Harmful drinking is a major determinant for neuropsychiatric disorders, such as alcohol use disorders and epilepsy and other non-communicable diseases such as cardiovascular diseases, cirrhosis of the liver and various cancers. The harmful use of alcohol is also associated with several infectious diseases like HIV/AIDS, tuberculosis and sexually transmitted infections. This is because alcohol consumption weakens the immune sys-tem and has a negative effect on patients’ adherence to antiretroviral treatment.
Figure 4 : Percentage of consumers that develops an addiction to the substance they con-sume.
3.2 Substances and non- substances use disorders
Natural addiction: Addictive drugs act on brain reward systems, although the brain evolved to respond not to drugs but to natural rewards, such as food and sex. Appropriate responses to natural rewards were evolutionarily important for
34 survival, reproduction, and fitness. In a quirk of evolutionary fate, humans dis-covered how to stimulate this system artificially with drugs. Both natural addic-tions such as sex, food-binging and drugs increase dopamine in the NAc. DA is also involved as an important mediator in primary food motivation or appetite similar to drugs of abuse. Many findings in literature provides a number of pa-pers that show the importance of DA in food craving behaviour and appetite mediation (Blumenthal et al., 2010). Blum and Gold 2013, have reviewed the concept of food addiction. (Avena et al., 2008) say that because addictive drugs activate the same neurological pathways as natural rewards like food, addiction to food seems plausible. Moreover, sugar is noteworthy as a substance that re-leases opioids and DA and thus might be expected to have addictive potential. Moreover findings have showed that sugar can surpass the rewarding effects of drugs like cocaine. (Lenoir et al., 2007).
Addiction to substances : According to the molecular pathways each type of drug activates, we can classify addictive substances in three classes:
Table 2 : The mechanistic classification of drugs of abuse (Lüscher. & Ungless and Ungless, 2006). Drugs fall into one of three categories that target either G protein–coupled receptors, ionotropic receptors/ion channels, or biogenic amine transporters. Note that drugs with RR = 1 are readily abused but will not induce addiction.
35 Class I
Morphine and other opioids. These strongly increase the release of mesolimbic
dopamine by their action on μ-opioid receptors (MORs), which are expressed on inhibitory GABAergic interneurons of the VTA (Pickel et al., 2002)
THC: Delta-9-tetrahydrocannabinol (THC) binds to type 1-cannabinoid
recep-tors (CB1Rs) in the brain. In the VTA, these receprecep-tors are expressed on GABA neurons and on terminals of glutamatergic synapses on dopamine neurons (Melis et al., 2004)
GHB. This is an increasingly popular club drug that is readily self-administered
and induces conditioned place preference in animal models, and leads to addic-tion in humans (Snead, 2005). GHB binds to GABA receptors. Although GABAB receptors are expressed on both GABA and dopamine neurons of the VTA, GHB affects almost exclusively GABA neurons at concentrations typi-cally obtained with recreational use.
Class II
Nicotine. This drug targets nicotinic acetylcholine receptors (nAChRs) in the
brain. When nicotine binds nAChRs they become cation-permeable and depolarise the cell. Nicotine increases dopamine through a complex interplay of actions at these ionotropic receptors on GABA and dopamine neurons, and glutamatergic inputs to dopamine neurons (Fagen et al., 2003).
It is evident that β2-containing nAChRs are responsible for the rewarding effects of nicotine because β2 knockout mice do not selfadminister nicotine and do not show nicotine-evoked dopamine release.
Benzodiazepines. Benzodiazepines (BZD) increase mesocorticolimbic
dopamine and can lead to addiction. BZD are positive modulators of the GABAA receptor. When injected into the VTA, the GABAA receptor agonist muscimol seems to inhibit interneurons more effi ciently compared to
dopamine neurons, which may lead to a net disinhibition of the dopamine neurons (Kalivas, 1990).
Ethanol. This drug has a complex pharmacology. No single receptor mediates
all the effects of alcohol (Koob et al., 1998). On the contrary, alcohol alters the function of a number of receptors and cellular functions, including GABAA
36 receptors, Kir3/GIRK and other K channels, Ih, N-methyl-D-aspartate
(NMDA) receptors, nAChRs , and 5-HT3 receptors as reviewed byLüscher &
Ungless, 2006 . In addition, ethanol also interferes with adenosine re-uptake by inhibiting the equilibrative nucleoside transporter ENT1, although it is not clear if this plays a role in ethanol-induced dopamine release.
Class III
Focus on Cocaine
Figure 4: Cocaine structure.
In the central nervous system, cocaine blocks dopamine, noradrenaline, and serotonin uptake through inhibition of their respective transporters. Blocking of the dopamine transporter (DAT) leads to an increase of dopamine concentrations in the nucleus accumbens. The firing rate of DA neurons of the
37 VTA actually decreases with cocaine application, which is due to the effects of dopamine on D2 autoreceptors on DA neurons (Brodie, 1990). In mice lacking DAT, dopamine still increases in response to cocaine, which, could be the result of inhibition of dopamine uptake by other monoamine transporters. Consistent with this suggestion, DAT knockout mice still self-administer
cocaine, and this behaviour is abolished in combined DAT– serotonin
transporter (SERT) knock-out mice (Rocha, 2003). SERT-mediated re-uptake of dopamine only occurs in situations where dopamine levels are already high, as in DAT knockout mice. This is confirmed by a study that used a knock-in mouse line carrying a functional DAT that was insensitive to cocaine. In these mice, cocaine did not elevate extracellular dopamine in the nucleus accumbens, and did not produce reward, as measured by conditioned place preference (Chen et al., 2006).
Finally, it is important to point out that selective SERT inhibition in humans (e.g., fluoxetine to treat depression) does not carry any addiction liability.
Amphetamine, methamphetamine, and their many derivates. These exert their
effects by reversing the action of biogenic amine transporters at the plasma membrane (Sulzer et al., 2005). Amphetamines are substrates of these transporters and are taken up into the cell. Every molecule that is taken up generates a current causing a depolarisation of the dopamine neurons, which could contribute to enhanced dopamine release (Ingram et al., 2002). In addition, once in the cell, amphetamines interfere with the vesicular monoamine transporter, depleting synaptic vesicles. As a consequence, dopamine increases in the cytoplasm from where it is released by plasma membrane transporters working in reverse. In other words, normal vesicular release of dopamine decreases Similar mechanisms apply for other biogenic amines such as serotonin and norepinephrine.
Methylenedioxymetamphetamine (ecstasy). As for the amphetamines, MDMA
causes the release of biogenic amines by reversing the action of their respective transporters. MDMA has a preferential affinity for SERTs and therefore increases the extracellular concentration of serotonin, and increases dopamine (Morton, 2005).
38 There are many other commonly used drugs like LSD, NMDA, and ketamine, that are not considered addictive because they do not cause the release of dopamine.
Figure 5: Annual deaths by drugs
39
3.3 Addiction neurobiology: reward pathway
Figure 6: The brain reward system: Dopaminergic efferents from the VTA and Glutama-tergic afferents to the Nucleus Accumbens
Drugs of abuse have very different acute mechanisms of action but converge on the brain’s reward pathways by producing a series of common functional effects after both acute and chronic administration. There is considerable evidence, from animal models and more re-cently from humans, that all drugs of abuse converge on a common circuitry in the brain’s limbic system as reviewed in (Nestler, 2005). The main circuit is the ventral tegmental ar-ea- nucleus accumbens (VTA-NAc), which includes dopaminergic neurons in the ventral tegmental area (VTA) of the midbrain and their targets in the limbic forebrain, especially the nucleus accumbens (NAc).
The main neurotransmitter involved in reward processing is dopamine (DA). Although dif-ferent substances of abuse use difdif-ferent pathways, all have the common objective to in-crease the release of DA in the NAc (Figure 7). DA binds to its receptor (DA1-DA5). The DA receptors are G coupled proteins. They ultimately activate AC (adenylate cyclise) and increase the intracellular levels of cAMP.
40
Figure 7: (from Nestler, 2005) common molecular pathways of addictive substances. All ad-dictive substances finally increase dopamine in the NAc with different mechanisms.
The VTA-NAc pathway is part of a series of parallel, integrated circuits, which involve several key brain regions. Other structures are also involved in the reward pathway such as amygdale, hippocampus, hypothalamus, frontal regions of cerebral cortex, locus coeruleus and dorsal raphe.
The VTA is the site of dopaminergic neurons, which tell the organism whether an envi-ronmental stimulus (natural reward, drug of abuse, stress) is rewarding or aversive. The NAc, is a principle target of VTA dopamine neurons. This region mediates the rewarding effects of natural rewards and drugs of abuse. The amygdale is particularly important for conditioned forms of learning. It helps an organism establish associations between envi-ronmental cues and whether or not that particular experience was rewarding or aversive, for example, remembering what accompanied finding food or fleeing a predator. It also in-teracts with the VTA-NAc pathway to determine the rewarding or aversive value of an en-vironmental stimulus (natural reward, drug of abuse, stress). The hippocampus is critical for declarative memory, the memory of persons, places, or things. Along with the amygda-la, it establishes memories of drug experiences, which are important mediators of relapse. The hypothalamus is important for coordinating an individual's interest in rewards with the body's physiological state. This region integrates brain function with the physiological
