Monoamines and Anhedonia old targets, new concepts Layout Tjeerd Schuitemaker Cover art Roberto Saporito (www.123rf.com) Cover design Ridderprint Printed by Ridderprint

ISBN: 978-90-393-5857-3

All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical (including photocopying, recording, or information storage and retrieval system) without written permission from the author.

© 2012, Jolanda Prins, Utrecht, the Netherlands Monoamines and Anhedonia old targets, new concepts

Monoamines en Anhedonie oude aangrijpingspunten, nieuwe concepten

(met een samenvatting in het Nederlands)

Proefschrift

ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op woensdag 28 november 2012 des middags te 2.30 uur

door

Jolanda Prins

geboren op 2 mei 1984 te Almelo Promotoren: Prof. dr. B. Olivier Prof. dr. D. A. J. P. Denys

Co-promotoren: Dr. S. M. Korte Dr. R. S. Oosting

Printing of this thesis was financially supported by:

Antec B.V., Zoeterwoude, the Netherlands Med Associates Inc., St. Albans, VT, USA PsychoGenics Inc., Tarrytown, NY, USA

Table of contents chapter 1 General introduction 9 chapter 2 Triple reuptake inhibitors for treating subtypes of major 17 depressive disorder: The monoamine hypothesis revisited chapter 3 The putative DOV 216,303, a triple reuptake 47 inhibitor, increases monoamine release in the prefrontal cortex of olfactory bulbectomized rats chapter 4 The potential and limitations of DOV 216,303 as a triple 61 reuptake inhibitor for the treatment of major depression: A microdialyis study in olfactory bulbectomized rats chapter 5 The triple reuptake inhibitor DOV 216,303 induces long- 81 lasting enhancement of brain reward activity as measured by intracranial self-stimulation in rats chapter 6 The 5-HT1A/1B receptor agonist eltoprazine alters brain 93 monoamine levels without enhancing brain stimulation reward chapter 7 Acute and chronic monoamine reuptake inhibitors differently 113 affect brain stimulation reward and monoamine release in the nucleus accumbens and prefrontal cortex in rats chapter 8 Discussion 133

References 145

Author affiliations 163

Samenvatting in het Nederlands 167

Dankwoord 175

About the author 181

List of publications 185

General introduction

c h a p t e r 1 Chapter 1

10 General introduction

Major depressive disorder is one of the most prevalent forms of psychiatric disorders and is among the leading causes of disability. According to the World Health Organization, 1 within 20 years, depression will become the biggest health burden on society, both economically and sociologically. c h a p t e r

Anhedonia Anhedonia is one of the core symptoms of major depressive disorder. It is defined as the inability to experience pleasure from normal daily activities that where once enjoyable, such as going to the movies, working in the garden and doing sports. Patients experiencing anhedonia do not feel pleasure anymore from such activities. The other core symptom of major depression is depressed mood. Whereas it is very difficult, if not impossible, to study depressed mood in animals, anhedonia and the corresponding reward-related deficits can be studied in animals.

Monoamines Monoamines are neurotransmitters that are released from axonal terminals into the synapse to transmit signals to postsynaptic neurons. Monoamines include the catecholamines (DA) and (NE) and the indoleamine (5-HT). DA and NE are both synthesized from the common precursor by tyrosine hydroxylase. Upon release DA can bind to several receptors; D1- like receptors (D1 and D5), which are excitatory, and D2-like receptors (D2, D3 and

D4), which are inhibitory. The DA signal is terminated via reuptake into presynaptic terminals via the DA transporter (and in cortical brain areas via the NE transporter) and packed into storage vesicles via the vesicular 2 (VMAT2) for re-use or DA is intracellular metabolized by monoamine oxidase (MAO)-A or MAO-B into 3,4-dihydroxyphenylacetic acid (DOPAC), which is released into the extracellular fluid. Extracellular DOPAC can be further metabolized to HVA via catechol-O- methyltransferase (COMT). Extracellular DA can be metabolized to 3-methoxytyramine (3-MT) by COMT and will be further metabolized by MAO to homovanillic acid (HVA).

NE can bind to α- adrenoceptors (α1, and α2) or β- adrenoceptors (β1, β2 or β3). NE is taken up into the synapse via the NE transporter or degraded preferentially by MAO-A to 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) and some other metabolites. Serotonin (5-hydroxytryptamine, 5-HT) is synthesized from the precursor , upon release it can bind pre- and postsynaptically to 14 different 5-HT receptors divided over 7 classes of receptors, of which the 5-HT1 and 5-HT2 class are the best studied. The 5-HT signal is terminated by reuptake of 5-HT into the synapse via 5-HT transporters (SERT). Intracellular, 5-HT is re-packed into vesicles or metabolized preferentially by MAO-A to 5-hydroxyindoleacetic acid (5-HIAA), which will subsequently be released into the extracellular space.

11 Chapter 1

Depression and The original monoamine hypothesis of depression proposes that depression is associated with decreased concentrations of 5-HT, NE and DA (Schildkraut 1965). Other theories followed including the monoaminergic receptor hypothesis, the hypothesis of signaling adaptation and the involvement of neurogenesis and neuroplasticity in antidepressant treatment (McEwen and Chattarji 2004; Pittenger and Duman 2008). However, the underlying neurobiological mechanisms behind depression are still not very well understood and are mainly derived from studying the mode of action of available antidepressants. Antidepressant therapy mainly targets monoaminergic systems in the brain. The first-line treatment for major depression is the use of selective serotonin reuptake inhibitors (SSRIs; e.g. , , , , ) or serotonin/norepinephrine reuptake inhibitors (SNRIs or NRIs; e.g. , , , ), which enhance both 5-HT and NE transmission. A limited number of antidepressants also target the DA system (e.g. ). However, a large number of patients fail to respond to the first-line antidepressant therapy or encounter severe side effects, including sexual dysfunction (Waldinger et al 1998) or have to contend with residual symptoms like fatigue and sleeping problems (Demyttenaere et al 2005). Another limitation of the current antidepressants is the delay of therapeutic onset. Whereas side effects appear almost immediately after taking the drugs, the therapeutic effects only emerge after at least 2-4 weeks of treatment. A role for DA in the pathophysiology of depression has been postulated and is extensively reviewed. Targeting the dopaminergic system in antidepressant treatment is thought to give a faster therapeutic onset of antidepressant action and might be particularly effective in the treatment of anhedonia (Dunlop and Nemeroff 2007; Nestler and Carlezon 2006; Nutt et al 2007). This is why triple reuptake inhibitors (TRIs) are in development. These compounds simultaneously block the 5-HT-, NE- and DA-transporters (SERT, NET and DAT) (Guiard et al 2009b; Skolnick et al 2003b).

Intracranial self-stimulation In the present thesis we performed intracranial self-stimulation (ICSS) experiments to study the rewarding effects of monoaminergic compounds. ICSS is a powerful tool that provides a sensitive measure of the activity of brain reward systems. It was discovered by Olds and Milner in 1954 that electrical stimulation in certain brain areas act as positive reinforcement (brain stimulation reward, BSR) and that rats will work to obtain rewarding stimulation (Olds and Milner 1954). The strong rewarding properties of BSR are reflected by the facts that ICSS is easily learned by animals, that animals will withstand punishing foot shocks in order to obtain BSR and that BSR is preferred over natural reinforcers such as food, water or sex (Olds 1958). BSR directly activate neuronal circuits that are also activated by the conventional reinforcers and by drugs of abuse. ICSS is an operant paradigm in which rats turn a wheel to obtain BSR directly

12 General introduction into their brain, through an implanted electrode targeted at the lateral hypothalamus (LH) in the medial forebrain bundle (MFB) (Fig 1.). The MFB is part of the mesolimbic 1 dopamine reward pathway and consist of a large bundle of axons running between the ventral tegmental area and the nucleus accumbens, which play an important role in reward and pleasure. The intensity of the stimulation can be varied in order to detect the c h a p t e r minimal electrical intensity (reward threshold) at which an animal responds. Lowering of ICSS thresholds can be explained by increased brain reward signaling resulting in a potentiating of the reward perceived by ICSS. On the contrary, elevations of ICSS thresholds can be explained by decreased activity of reward circuitry and desensitization of the rewarding effects of ICSS (Carlezon and Chartoff 2007; Kenny 2007; Kornetsky 1985; Markou and Koob 1992).

Microdialysis In the present thesis we measured extracellular monoamine concentrations in several brain areas of conscious freely moving animals with the use of microdialysis. Microdialysis is a process in which a probe with a semi-permeable membrane is inserted into a brain area of interest. Ringer’s solution is pumped through the system, from inlet to outlet and small molecules (including monoamines and metabolites) diffuse through the membrane of the probe and are collected in the dialysate, which can be measured with high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). In microdialysis experiments, samples are collected from a relatively large region. Since the membranes are 1-3 mm in length, the dialysate contains

Figure 1. A simplistic overview of rat brain areas on which is focused in this thesis. NAc: nucleus accumbens; PFC: prefrontal cortex; DH: dorsal hippocampus; VTA: ventral tegmental area, midbrain nucleus, origin of dopaminergic cell bodies; DR: dorsal raphe nucleus, midbrain nucleus, origin of serotonergic cell bodies; MR: median raphe nucleus, midbrain nucleus, origin of serotonergic cell bodies; LC: locus coeruleus, midbrain nucleus, origin of norepinephrinergic cell bodies; LH: lateral hypothalamus, place of electrical intracranial self-stimulation (ICSS); The dopaminergic neurons in the VTA project via the medial forebrain bundle (MFB) to the NAc. The MFB runs through the LH; OB: olfactory bulbs, part of the brain removed in the OBX studies.

13 Chapter 1 overall extracellular monoamine concentrations of the brain areas of interest. In the experiments discussed in this thesis we measured DA, NE, 5-HT and the metabolites DOPAC, HVA and 5-HIAA in the nucleus accumbens (NAc), the prefrontal cortex (PFC) and dorsal hippocampus (DH) of rats (Fig. 1). These brain areas where chosen because of their role in depression and reward. The NAc is part of the mesolimbic dopamine system and plays a key role in reward processes. The PFC is involved in overall cognitive functioning and the suppression of negative affect, whereas the DH is involved in reward-related processes via contextual cues with projections to the NAc (Everitt and Robbins 2005).

Aim and outline of the thesis The aim of this thesis was to study the role of monoamines in brain stimulation reward and anhedonia.

In chapter 2, the monoamine hypothesis of depression is revisited and the potential use of triple reuptake inhibitors in the treatment of subtypes of major depressive disorder; atypical and melancholic depression is reviewed. In chapter 3, extracellular monoamine concentrations are measured in the prefrontal cortex of olfactory bulbectomized (OBX) rats and the effect of the triple reuptake inhibitor DOV 216,303 was studied. In chapter 4, the acute and chronic effects of DOV 216,303 treatment are compared in OBX rats on monoamine concentrations in the prefrontal cortex and dorsal hippocampus. Because of the concerning abuse liability and reinforcing effects of dopaminergic compounds, in chapter 5, the rewarding properties of DOV 216,303 were measured with use of intracranial self-stimulation (ICSS). To further investigate the role of DA in reward, in chapter 6, we performed a microdialysis study in which the effects of the 5-HT1A/1B receptor agonist eltoprazine on extracellular monoamine concentrations were studied in the prefrontal cortex and nucleus accumbens. Furthermore, the effects of eltoprazine on reward were measured in an ICSS paradigm. In order to study the contribution of the separate monoamine systems to reward, in chapter 7, we investigated the effects of escitalopram, reboxetine and on brain stimulation reward and performed a microdialysis study with probes in the prefrontal cortex and nucleus accumbens to measure the effects of chronic and acute treatment of these compounds on monoamine release. Finally, in chapter 8, the main findings are summarized and discussed with perspectives for future research.

14

Jolanda Prins Jolanda Berend Olivier S. Mechiel Korte Mechiel S. revisited for treating subtypes of for treating Triple reuptake inhibitors reuptake inhibitors Triple major depressive disorder: major depressive The monoamine hypothesis hypothesis The monoamine Expert Opinion on Investigational DrugsExpert 2011, 20(8): 1107-1130 Opinion on Investigational

c h a p t e r 2 Chapter 2

Abstract Major depression is one of the most prevalent forms of mental illnesses and among the leading causes of disability, affecting about 121 million people worldwide. Approximately 30% of patients fail to respond to present therapies. Therefore, the search for novel antidepressant drugs continues. The most prescribed antidepressants are serotonin reuptake inhibitors and/or noradrenaline reuptake inhibitors, which only indirectly affect dopaminergic neurotransmission. As a consequence residual symptoms remain, including impaired motivation and impaired pleasure. This article reviews the development of new broad spectrum antidepressants, the triple reuptake inhibitors, which also increase brain dopamine levels. In this review a distinction is made between the subtypes melancholic and atypical depression and their associated brain abnormalities and dysfunctions in neurotransmitter systems. Subsequently, we propose a hypothetical model: “the monoamine hypothesis revisited” to predict what kind of pharmacological treatment will be effective in the different subtypes of depression. It is expected that the triple reuptake inhibitors, inhibiting the reuptake of all three monoamines, can produce a greater efficacy than traditional antidepressants especially in atypical depression. Since triple reuptake inhibitors may also dampen states of hyperglutamatergic activity and subsequent excitotoxity, it is suggested that these new drugs have a considerable neuroprotective potential in major depression, especially in melancholic depression.

18 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited

Introduction Major Depressive Disorder (MDD) is one of the most prevalent forms of mental illnesses and is among the leading causes of disability, affecting about 121 million people worldwide (Andrews et al 2000). Although a large number of antidepressants are available, approximately 30% of patients fail to respond to this therapy. Therefore, the search for novel antidepressant drug continues (Kulkarni and Dhir 2009). 2 There are several reasons why the present antidepressants do not, or not fully, cure all depressive patients. First, the most prescribed antidepressants are selective serotonin reuptake inhibitors (SSRI) and/ or noradrenaline reuptake inhibitors (SNRI), c h a p t e r which indirectly affect dopaminergic neurotransmission. Lesion and pharmacological studies have described the influence of both monoamines and their receptors in the ventral tegmental area (VTA) (Di Giovanni et al 2010; El Mansari et al 2010).

Remarkably, the fact that both serotonin (5-HT) through 5-HT2C receptors and noradrenaline (NA) through α2 receptors inhibit dopaminergic neurons might explain why residual symptoms (including impaired motivation and impaired pleasure (Dunlop and Nemeroff 2007) remain or may be accentuated by SSRIs/SNRIs (Millan 2009). Therefore, the development of new broad-spectrum antidepressants, the triple reuptake inhibitors (TRIs), which directly increase brain dopamine (DA) levels is an attractive strategy (Skolnick et al 2006; Skolnick et al 2003b). It is expected that drugs inhibiting the reuptake of all three monoamines can produce a faster onset of action and greater efficacy than traditional antidepressants (Skolnick et al 2003b). Second, patients suffering from MDD may differ in their neurobiological pathophysiology, suggesting the existence of different MDD subtypes. The Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) (APA 1994; Chen et al 2008) describes MDD as a condition in which an individual experiences at least two weeks of depressed mood and/or loss of interest and pleasure (anhedonia) accompanied by at least four additional symptoms of depression. The latter include significant changes in appetite, whether or not accompanied by weight gain or weight loss, depending on an increased or decreased appetite, respectively. Other additional symptoms are insomnia or hypersomnia, psychomotor agitation or retardation, fatigue or loss of energy, feelings of worthlessness or guilt, diminished ability to think or concentrate and recurrent thoughts of death. Remarkably, these additional symptoms of depression are sometimes extreme opposites and lead to subdivisions in different subtypes of depression (Gold and Chrousos 2002; Horwath et al 1992; Lamers et al), the most prominent subtypes being melancholic depression and atypical depression. The efficacy of a variety of compounds for the treatment of depression hasbeen extensively studied. Unfortunately, most studies ignore a possible division in subtypes of depression. However, antidepressant efficacy may rely heavily on the symptom profile of the patients. This may lead to missing some essential information about the real effectiveness of antidepressants for different subtypes. In the present review a distinction is made between subtypes of depression and their associated brain abnormalities and dysfunctions in neurotransmitter systems. Subsequently, we propose a simplistic and hypothetical model: “the monoamine

19 Chapter 2 hypothesis revisited” to show that monoamines are differently affected in atypical and melancholic depression and that monoaminergic neurotransmission is ‘out of tune’, rather than deficient. Evidence is presented that the novel TRIs might be helpful as first line antidepressant treatment.

Major depressive disorder

Subtypes of MDD: different underlying pathophysiological mechanisms Patients diagnosed with MDD have depressed mood and/or diminished interest or pleasure, nearly every day (APA 1994). Either one of these two core symptoms must be present to be diagnosed as MDD together with some other sub symptoms, listed in Table 1. Although no further clustering or distinguishing has been made between the opposite symptoms of depression, it is most likely that these symptoms can be categorized and classified into subtypes as either melancholic or atypical depression, with putatively different underlying neurobiological mechanisms (Gold and Chrousos 1999; Gold and Chrousos 2002). Melancholic patients experience loss of appetite and accompanied weight loss, insomnia and psychomotor agitation, while in contrast atypical depression is associated with an increased appetite, weight gain, fatigue, hypersomnia and psychomotor retardation (Baldwin and Papakostas 2006; Gold and Chrousos 1999; Gold and Chrousos 2002). Additional symptoms are also associated with MDD and can be subdivided over the melancholic and atypical subtypes (Table 1). Melancholic depression is furthermore associated with increased anxiety, less responsiveness to environmental cues, an increased sympathetic activity, a hyperactive hypothalamus- pituitary-adrenal (HPA) axis and corticotropin-releasing factor (CRF) system, leading to an inhibition of growth and reproduction cycles and altered immune function, leading to enhanced infection susceptibility. Atypical depression, on the other hand, is associated with lethargy, increased reactivity to environmental cues (i.e. depressed mood can lift during the depressive phase, though only temporarily), decreased sympathetic activity, down-regulation of the HPA-axis, CRF-deficiency and an increased immune function, making someone more vulnerable for inflammation (Horwath et al 1992; Lamers et al; Levitan et al 1997). The DSM-IV TR (APA 1994) defines atypical depression as a subtype of major depressive disorder with atypical features, characterized by: i) mood reactivity (i.e., mood brightens in response to actual or potential positive events); ii) At least two of the following: significant weight gain or increase in appetite; hypersomnia (sleeping too much); heavy, leaden feelings in arms or legs; and long-standing pattern of interpersonal rejection sensitivity that results in significant social or occupational impairment. Most depressed patients experience mixed symptoms from either subtype. These patients are mostly diagnosed with moderate depression (Levitan et al 1997) and do often not respond adequately to antidepressants (Fournier et al 2010; Kirsch et al 2008). Patients with pure melancholic (25-30%) or atypical features (15-30%) generally have a more severe course of disease and antidepressant treatment is most effective in these most severely depressed patients (Fournier et al 2010; Kirsch et al 2008; Levitan

20 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited et al 1997). The DSM-IV TR (APA 1994) has classified depression, besides melancholic and atypical depression, into other types such as bipolar depression with a history of mania or hypomania; dysthymia; seasonal depression; rapid cycling depression; postpartum depression; psychotic depression; agitated depression and so on. Recently, also additional subtypes have been suggested such as adult depression after childhood trauma; depressive reaction to separation stress; late-life depression and depression secondary to substance abuse or to a medical condition (Lichtenberg and Belmaker 2 2010).

Different brain areas involved in atypical and melancholic depression c h a p t e r Patients are often characterized and diagnosed for depression based on a mixture of symptoms listed in the DSM-IV (APA 1994). Irrespective of the more melancholic or atypical character of depression, first line antidepressant treatment is often similar for all patients, the selective serotonin reuptake inhibitors (SSRIs). It is not very logic to assume that depressive subtypes with such a different symptom profile (Table 1) have the same underlying disturbances in brain functioning and will benefit from similar treatment. Figure 1 represents a schematic representation of different brain areas and their connections involved in MDD. The different roles of monoamines in different subsets of symptoms of MDD have been extensively discussed and reviewed, but most studies do not distinguish between the opposite subsymptoms of depression (e.g. they mention sleep disturbances, but do not distinguish between insomnia and hypersomnia). In the following sections specific symptoms are (if possible) linked to particular brain abnormalities. However, it should be noted that it is not easy to link one particular symptom to a specific disturbance in a specific brain area, as most brain areas participate in more complex interacting brain systems and networks and rely closely on connections with other structures (Fuster 2001).

Depressed mood The establishment of correlations between subtypes of pathology and specific associated abnormalities in circuitry is often tempting and is derived from preclinical studies. Depressed mood has been associated with dysfunctional neuronal activation of the prefrontal cortex (PFC), including the anterior cingulate and orbitofrontal cortex (Davidson et al 2002; Mayberg et al 1999; Price and Drevets). The PFC is involved in overall cognitive functioning that allows an organism to get things done, problem solving and time planning, reinforcement, reappraisal and suppression of negative affect (Koenigs and Grafman 2009; Koob and Volkow 2010; Robbins and Arnsten 2009). The frontal cortex is highly innervated by inhibitory serotonergic fibers from the midbrain raphe nucleus (RN) (Molliver 1987; Sastry and Phillis 1977), with the dorsal raphe nucleus (DR) projecting to the PFC and striatum and the median raphe nucleus (MR) innervating hippocampus and striatum. It has been suggested that the DR is more involved in affective disorders, because its fibres are, in contrast to MR projections, vulnerable to psychostimulants (Molliver 1987). Moreover, the cingulate cortex and PFC are inhibited by noradrenergic (NA) fibers from the locus coeruleus (LC) (Berridge and Waterhouse 2003; Dillier et al 1978; Gold and Chrousos 2002). During stress, the

21 Chapter 2

Table 1. Major Depressive Disorder categorized into the melancholic and atypical subtype of depression based on DSM-IV criteria and neurobiological symptoms (APA 1994; Gold and Chrousos 1999; Gold and Chrousos 2002; Lamers et al 2010). Major Depressive Disorder Melancholic depression Atypical depression DSM-IV Core symptoms: (either one, or both should be present) - Dysphoria (depressed mood) - Anhedonia (loss of pleasure)

DSM-IV Subsymptoms per subtype: - Weight loss - Weight gain - loss of appetite - Increase in appetite/hyperphagic - Insomnia/less sleep - Hypersomnia/more sleep - Psychomotor agitation - Psychomotor retardation - Suicidal thoughts - Fatigue/energy loss

DSM-IV Subsymptoms present in both subtypes: - Feelings of worthlessness and guilt - Diminished ability to think or concentrate

Neurobiological symptoms per subtype Melancholic depression Atypical depression - Anxious - Lethargic - less responsive to environment - Reactive to environment - Symptoms at worst in morning - Symptoms at best in morning - Increased sympathetic activity - Decreased sympathetic activity - Hyperactive HPA-axis - Hypoactive HPA-axis - Activated CRF-system - CRF-deficiency - Increased susceptibility for infection - Increased susceptibility for inflammation CRF: Corticotropin-releasing factor; HPA: Hypothalamus-pituitary-adrenal.

HPA-axis becomes activated and subsequently activates the LC-noradreniergic systems which in turn further inhibit the PFC, thereby favouring more rapid and basal responses over more complex ones (Arnsten 2000). In terms of melancholic depression, in which a prolonged and intensified stress system (Table 1) causes a hyperactive NA system, because of the excitatory connection of the LC with the CRF-system (Wong et al 2000b), the NA projections to the PFC are strengthened and the PFC more inhibited. This contrasts to atypical depression in which the HPA-CRF system is hypoactive and the PFC receives less inhibitory innervation by the LC, which leads to a hyperactive PFC (Gold and Chrousos 2002). Furthermore, the PFC receives dopaminergic projections from the ventral tegmental area (VTA) (Williams and Goldman-Rakic 1998). Although

22 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited

DA transporters are absent in the PFC, the dopaminergic signal in the PFC can be terminated by dopamine reuptake via the noradrenaline transporter (NAT) (Sesack et al 1998). Here it must be mentioned that emerging literature suggests that also “non- traditional” transporters such as organic cation transporters (OCT) and the plasma membrane monoamine transporter (PMAT) are capable of clearing biogenic amines from extracellular fluid and may serve to buffer the effects of antidepressants, such as SSRIs (Daws 2009). 2

Anhedonia

One key regulatory pathway for motivation and experience of pleasure and reward is c h a p t e r the dopaminergic mesolimbic pathway arising in the VTA and projecting to the nucleus accumbens (NAc), bed nucleus of stria terminalis (BNST), amygdala and septum (Dunlop and Nemeroff 2007). Hedonic hotspots furthermore include the shell of wthe NAc (NAcs) and ventral pallidum (Berridge and Kringelbach 2008). During stress, glucocorticoids (GCs) may facilitate DA transmission especially in the NAcs, but not in the core (NAcc) (Marinelli and Piazza 2002). GCs are part of the feedback mechanism in the immune system as well (Rhen and Cidlowski 2005). So, in a hypoactive stress system, present in atypical depression (Table 1), less GCs are present, the immune system is hyperactive (with an increased risk of autoimmunity) and dopaminergic transmission in the NAc may be decreased, leading to deficiencies in experiencing pleasure. On the other hand, in the melancholic subtype, a chronic activated CRF- system and a subsequent increased DA system might lead to desensitization of the reward system, leading to anhedonia as well, but via different mechanisms (Table 1) (Dunlop and Nemeroff 2007; Leshner and Koob 1999).

Weight/ appetite changes Changes in eating patterns might also be a result of a disturbance in reward perceived from food, regulated by the mesolimbic reward centres. Severely depressed patients experience greater reward after -intake and had altered brain activation of the ventrolateral PFC, orbitofrontal cortex, caudate and putamen, compared with mild depressed patients and healthy controls (Tremblay et al 2002; Tremblay et al 2005). Furthermore feeding in itself is rewarding (Saper et al 2002; Zheng and Berthoud 2007) and an excessive consumption of palatable food over-stimulates brain reward systems and induces a state of reward hyposensitivity and compulsive-like eating (Johnson and Kenny 2010), which might represent the hyperphagic symptom in atypical depression. Besides the mesolimbic dopaminergic reward pathway, another key player regulating interest and ‘drive’ is the ventromedial nucleus of the hypothalamus which receives important innervations from noradrenergic and serotonergic fibres. This area is thought to be more important to regulate appetite drives and vegetative functions, including sexual functioning (Adamec 1976; Dunlop and Nemeroff 2007; Gold and Chrousos 2002). A disturbance in this area may also underlie appetite disturbances in MDD and into less appetite in melancholic depression in particular.

23 Chapter 2

Sleep disturbances The neurophysiology of sleep is very complex and its role in MDD is even more complicated (Mignot 2001; Saper et al 2001; Wisor et al 2001). It is, therefore, beyond the scope of this review to extensively discuss the neurobiology of sleep disturbances in the different subtypes of MDD. Problems with sleep not only are common symptoms of MDD and are difficult to treat, but also remain as residual symptoms in patients ‘successfully’ treated with antidepressants (Baldwin and Papakostas 2006). In general, states of arousal are regulated by the hypothalamic sleep-wake switch, which consist of sleep-promoting neurons in the ventrolateral preoptic area and wake-promoting neurons in the tuberomamillary nucleus (TMN) (Saper et al 2001). Noradrenergic and serotonergic projections from the LC and dorsal and median raphe nuclei respectively, run through the hypothalamus where they are combined with histaminergic projections from the TMN. Orexin/hypocretin (Peyron et al 1998) and melanin-concentrating hormone (Bittencourt et al 1992) also join this projection. Histaminergic projections from the TMN must be activated for normal wakefulness to occur (Strecker et al 2002). Disturbances in these projections might lead to an altered sleep-wake cycle, which will result in insomnia in melancholic depression and excessive sleep and hypersomnia in atypical depression (Gold and Chrousos 2002; Lamers et al). In atypical depression might be helpful to encounter hypersomnia (Dunlop et al 2007), due to decreased hypothalamic histamine concentrations or at least a disturbed histaminergic neurotransmission. Modafinil is a wake-promoting agent and activates orexin-containing neurons in the hypothalamus and enhances histaminergic neurotransmission in the hypothalamus (Ishizuka et al 2003). Moreover, modafinil is a weak DA reuptake inhibitor. Furthermore, there was a greater improvement in hypersomnia scores among bupropion-treated than SSRI-treated depressed patients, suggesting the involvement of a noradrenergic and/or dopaminergic component in hypersomnia (Papakostas et al 2006).

Fatigue Fatigue can be divided into physical and mental fatigue, which both may have different neurobiological pathways involved, but are both also associated with other symptoms of depression, which makes it difficult to distinguish them from other symptoms. Physical fatigue, leaden paralysis, tiredness and exhaustion of the body might be associated with psychomotor retardation and are partly driven by a dysfunctional DA signalling in the striatum (Flint et al 1993) and other motor controlling brain areas such as the cerebellum which is innervated by noradrenergic fibers (Stahl et al 2003). Mental fatigue might be related to cognitive dysfunction and lack of motivation. These symptoms might be linked to cortical brain areas. Fatigue and energy loss are also associated with atypical depression (Gold and Chrousos 2002).

Psychomotor disturbances Psychomotor agitation can be primarily linked to the melancholic subtype of depression, while the complete opposite symptom, psychomotor retardation, belongs to the atypical depression. Studies in depressed patients with particular psychomotor retardation

24 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited showed lower concentrations of the metabolite of DA, homovanillic acid (HVA), in cerebrospinal fluid (Banki 1977; Korf and van Praag 1971; Praag et al 1975). It is not surprising that motor controlling brain areas such as dopaminergic mesocortical and nigrostriatal pathways are hypoactive and may underlie these symptoms (Stein 2008). Symptoms of retardation of motor functions are probably regulated by dysfunctional striatal dopaminergic transmission, as patients with Parkinson’s disease experience similar symptoms as depressed patients (Flint et al 1993). On the other hand, hyperactivity of 2 the HPA-axis might be associated with psychomotor agitation, linking psychomotor agitation to melancholic depression (Carroll et al 1981; Mitchell et al 1996). In addition, it has been shown that dopaminergic functioning in the left caudate, bilateral putamen c h a p t e r and NAC, left parahippocampus and dorsal brainstem is lower in depressed patients with affective flattening and psychomotor retardation (Martinot et al 2001), while dopaminergic functioning in impulsive depressives is decreased in the anterior cingulate and hypothalamus, but increased in the right parahippocampal (Bragulat et al 2007).

Suicidal thoughts Post mortem studies examining brains of suicide victims indicated lower 5-HT concentrations in the ventromedial PFC (Mann 2003; Mann et al 1996). Lowered serotonergic functioning in humans and animals is correlated with both aggression and impulsivity, which may lead to the actual act of committing suicide; however a causal relationship between serotonergic neurotransmission and suicidal behaviour is not easy to make (Olivier 2004). Lower 5-HT concentrations may indicate a lower tonic serotonergic state of the brain; phasic 5-HT bursts in that situation may have a higher impact because of the upregulated and thereby hypersensitive serotonergic system (Gonzalez et al 2004). Here it is hypothesized that when these patients are treated with SSRIs, the first phasic 5-HT burst as a consequence of the drug will lead to a hyperactive response, which in turn may lead to enhanced anxiety. Chronic SSRIs shift the tonic state of the brain and more 5-HT is available at steady-state conditions, leading to downregulation of receptors and transporters, and to a less sensitive 5-HT system. Besides 5-HT, NA has also been implicated in suicidal behaviour. Lowered LC densities of noradrenergic neurons were found in suicide victims together with increased prefrontal NA concentrations and decreased α-adrenergic receptor binding, indicating an overactive noradrenalinergic system. Severe anxiety is also associated with a noradrenergic hyperactivity, a hyperactive HPA-axis and increased suicide (Brown et al 1988; Fawcett et al 1997), suggesting that suicidal behaviour is closely linked to melancholic depression. Because the mean growth hormone (GH) responses to apomorphine is significantly lower in suicide completers than in controls it has been concluded that reduced dopaminergic activity is present in depressed suicides (Pitchot et al 2001). There are, however, difficulties associated with the use of GH as an output measure of DA function, because corticosteroids inhibit GH release directly at the pituitary level. Since HPA-axis function is dysregulated in violent suicidal behaviour (Roy 1992), the changes in dopaminergic neurotransmission might also be the consequence of altered HPA-axis functioning (Watson and Young 2001).

25 Chapter 2

Feelings of worthlessness or guilt and cognitive dysfunction Feelings of worthlessness or guilt as well as cognitive dysfunction, including concentration problems and problem solving disabilities are all linked to hypo-functioning of the dorsolateral PFC (DLPFC) and regulated by multiple neurotransmitters projecting to the PFC (Li et al ; Rypma and D’Esposito 1999). It can be expected that similar areas, which are responsible for depressed mood, are involved in cognitive dysfunction. However, as the functions of the PFC and surrounding cortical areas rely closely on connections with a lot of other neuronal structures, none of its cognitive functions can be fully understood if taken out of context of its wide-ranging networks (Fuster 1991; Fuster 2000; Fuster 2001).

Psychosis

Research over the last four decades has revealed the importance of DA, D2 receptors, and the basal ganglia in psychotic thinking (Morrison and Murray 2009). It has been hypothesized that psychotic symptoms in depression may be due to increased DA activity secondary to HPA- axis overactivity. However, in contrast to schizophrenia, psychotic symptoms in depression seem not to be related to DA function dysregulation (Duval et al 2000). There is a large body of evidence that HPA-axis hyperactivity is a causal factor in depression and the increased risk for conditions, such as diabetes, dementia, coronary heart disease, and osteoporosis. Such a link is the strongest among older inpatients who display melancholic depression (including psychotic features) (Stetler and Miller 2011).

Pain Descending pathways, from monoaminergic nuclei to the spinal cord, are specifically implicated in the inhibition of nociception providing rationale for the use of 5-HT and/or norepinephrine reuptake inhibitors, in the relief of pain (Hache et al 2011). Recently, it was shown that is a functional triple reuptake inhibitor (TRI) with activity in acute, persistent, and chronic pain models, with activation of dopaminergic pathways contributing to its antihyperalgesic actions (Basile et al 2007). Therefore, it is concluded that TRIs might represent new promising therapeutic tools in the relief of painful symptoms with depression (Hache et al 2011).

Antidepressants

Short history: Back to the future The understanding that mental disorders are brain diseases came with the development of neuropharmacological compounds including antidepressants. Before antidepressants were discovered and became available in the mid-1950s, various opiates and were used as antidepressants. One major drawback was their addictive properties (Weber and Emrich 1988). The early antidepressants were all discovered by serendipity; by studying their mechanism of action a better understanding of neurotransmission became possible on which newer classes of antidepressant are all

26 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited based. In 1951, isoniazid and iproniazid were developed and appeared very effective in treating tuberculosis. Interestingly, clinicians noticed overall mood improvements in these patients. Isoniazid and similar compounds are monoamine oxidase inhibitors (MAOIs) inhibiting the enzyme breaking down monoamines; this results in increases in brain concentrations of 5-HT, NA and DA. Simultaneously compounds with a different mechanism of action were developed; tricyclic antidepressants (TCAs). TCAs were originally developed to treat psychosis; 2 one of those compounds was the drug ; however, they were not effective in psychotic disorders. The clinical psychiatrist, Ronald Kuhn, saw the potential of imipramine as antidepressant, because of its sedating effect comparable to , c h a p t e r Imipramine appeared very effective in relieving depressive symptoms, especially symptoms of the atypical subtype, such as mental and motor retardation, fatigue, hypersomnia and feelings of guilt and despair (Kuhn 1957; Kuhn 1958). Although antidepressant drugs were very welcome at that moment, as the only methods to relieve depressive symptoms were electroshock and cognitive therapy, with associated disadvantages, the MAOIs and TCAs appeared far from perfect. MAOIs were problematic because of the strict diet people need to follow to prevent toxic consequences of the drug. TCAs also induced adverse side effects and had the disadvantage to be easily overdosed with possible life- threatening consequences. Julius Axelrod studied the mechanism by which MAOIs and TCAs act and it became clear that monoamines are not only metabolized after release, but also ‘taken up’ presynaptically (Axelrod et al 1961a; Axelrod et al 1961b). The alleviation of symptoms by MAOIs and TCAs was assigned to their potential to elevate monoamines, while the adverse effects of TCAs were ascribed to their action on histamine and acetylcholine. These developments led to drugs that selectively blocked monoamine transporters leading to increased extracellular monoamine levels. The monoamine of choice became 5-HT, triggered by the lower 5-HT levels found in brains of depressed suicide victims. In 1982 the first selective reuptake inhibitor (SSRI), zimilidine came on the market, developed by the Swedish pharmaceutical company Astra, but it was withdrawn in 1983 because several patients developed Guillain-Barré syndrome. Fluvoxamine appeared on the market in Switzerland in 1984, followed by other European countries in 1985. Fluoxetine followed in 1987 and was followed by many others such as paroxetine, sertraline, and escitalopram (the s-enantiomer of citalopram). Although other antidepressants followed, the older drugs never disappeared. They are still used in treating resistant depression. MAOIs are still considered the most effective antidepressant medications presently available, and are effective in patients non-responsive to other classes (such as SSRIs and TCAs) and also very effective in atypical depression (Thase et al 1992). Because of the potentially fatal interactions between MAOIs and certain foods, this class of drugs has become less popular. But MAO also breaks down potentially toxic dietary monoamines, such as tyramine, in the gut mucosa. The inhibition of gut MAO by MAOIs, coupled with ingestion of food containing tyramine (e.g. cheese), may produce life-threatening toxicity. Recently, however, it was shown that the transdermal administered MAO-B drug, selegiline, which by by-passing the first pass effect in gut and liver, appears to produce antidepressant

27 Chapter 2 effects with significantly reduced risk for dietary-induced toxicity (Nandagopal and DelBello 2009). Nevertheless, by inhibiting the breakdown of all three monoamines (5-HT, DA and NA) an increased risk for hypertension, tachycardia, tremors, seizures, and hyperthermia will remain.

Monoaminergic regulation of the different depressive symptoms Unravelling the working mechanism of the first antidepressants led to the simple hypothesis that depression is caused by inadequate monoaminergic neurotransmission and that antidepressants act by increasing monoamine availability. Moreover, the classical monoamine theory of depression was derived from observations that drugs that deplete extracellular monoamine levels, such as , lead to depressive symptoms (Schildkraut 1965). However, the fact that antidepressants need several weeks to relieve symptoms of depression, while monoaminergic neurotransmission is enhanced immediately after administration, does not comply with this hypothesis. It cannot be denied however, that monoamines play an important role in depression, but the therapeutic effects probably derive from long term adaptations in monoaminergic signal transduction, both pre- and postsynaptically (Guiard et al 2008; Guiard et al 2009b). A cluster of symptoms seems insufficiently addressed by serotonergic antidepressants, including fatigue, loss of pleasure, interest, energy, and sleep disturbances. These symptoms appear more difficult to treat and respond more slowly to the existing therapy (Boyer et al 2000; Demyttenaere et al 2005; Kopta et al 1994; Opdyke et al 1996). Moreover, when symptoms as fatigue, loss of energy and pleasure are part of the diagnosed depression, remission probably fails using first line antidepressant therapy (Moos and Cronkite 1999). In the case of residual symptoms, these are probably regulated by other neurobiological pathways than the improved symptoms and are not targeted by that kind of treatment. That some antidepressants are more effective than others might completely depend on the expressed symptoms and based on corresponding neurobiological deficits. Phenelzine, an irreversible MAOI, was more effective in treating atypical depression than the TCA imipramine. In patients lacking atypical symptoms, imipramine was equally effective to phenelzine (Thase et al 1995). Besides SSRIs, compounds selective for the NAT became also available. The selective noradrenergic reuptake inhibitor (NRI) reboxetine was more effective in severely depressed patients, compared to fluoxetine (Massana 1998; Montgomery 1997). However, a meta-analysis study indicated reboxetine equally effective to TCAs and SSRIs in MDD (Chuluunkhuu et al 2008). Although efficacy was similar, the side-effect profile differed. Where fluoxetine treated patients are more likely to experience nausea, hypersomnia and fatigue, reboxetine-treated patients experienced more constipation, painful urination (dysuria) and insomnia (Papakostas et al 2008). Venlafaxine, and duloxetine are dual-acting drugs, blocking both serotonin and the noradrenaline transporters (SNRIs). Although individual studies report differences in efficacy and onset of remission between SNRIs and SSRIs, no real differences are present (Clerc et al 1994; Entsuah et al 2001; Kasper et al 1996; Lopez-Ibor et al 1996; Thase et al 2001). The main differences come from side effects

28 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited

(Lopez-Ibor et al 1996). Since SNRIs have also a serotonergic action these drugs have similar side-effects as SSRIs (e.g., sexual dysfunction, i.e. decreased ejaculation time, gain in body weight, suicidal thoughts/action in adolescents etc.), but their additional noradrenergic action sometimes causes anxiety and elevated blood pressure. Therefore people who are susceptible to hypertension, heart disease, or stroke have to be careful with taking SNRIs (Stahl et al 2005). Beyond the serotonergic, noradrenergic and dual-acting agents, a role for DA in 2 the treatment of depression has been postulated as well (Dunlop and Nemeroff 2007; Nestler and Carlezon 2006; Nutt et al 2007). Dopaminergic agents such as amisulpride, , pramipexole and ropinirole exhibit antidepressant properties (Boyer c h a p t e r et al 1999; Cassano et al 2005; Corrigan et al 2000). The dopamine D2/D3 receptor antagonist, amisulpride, blocks presynaptic autoreceptors, resulting in increased DA levels and inducing hedonic effects in animals (Guyon et al 1993) and relieve anhedonia in schizophrenic patients (Boyer et al 1999). Pramipexole, a dopamine D2/D3 receptor agonist, is effective as augmentation in antidepressant treatment (Goldberg et al 2004; Gupta et al 2006) and can also relieve depressive symptoms when given alone (Corrigan et al 2000). Furthermore, pramipexole reduced anhedonia in Parkinson’s patients when given together with l-DOPA (Lemke et al 2006). Recently, it was suggested that bupropion and , both noradrenergic and dopaminergic reuptake inhibitors (NDRIs) can alleviate therapeutically depressive symptoms associated with Parkinson’s Disease in primates and humans (Hansard et al 2002; Raskin and Durst 2010). Clearly, it is not easy to predict what kind of antidepressant will be effective in the different subtypes of MDD. One of the reasons is that studies often do not mention which symptoms were initially present, which symptoms were relieved by antidepressant treatment and which symptoms are residual. Moreover, it is unknown whether different classes of antidepressants act on different kind of depressive symptoms. Therefore a clear picture of how different monoaminergic reuptake inhibitors can be linked to different symptoms cannot be provided. What we do know is that all these drugs alter monoaminergic neurotransmission, but at the same time it is evident that the pathophysiology of depression cannot be easily explained by any shortage of any monoamine in the brain and that monoamine transporter blockers do not solve the problem by simply increasing monoamine concentrations in the synaptic cleft. However, in our view, the pathophysiology of depression should make a distinction between the ‘state’ of the diseased brain, compared with a healthy brain. It is more accurate to hypothesize that in MDD, neurotransmission is ‘out of tune’, rather than deficient. For example, normal neurotransmission can be explained as a balance between tonic levels of extrasynaptic monoamines that are present at steady-state concentrations in the synaptic cleft and phasic monoamine bursts in response to neuronal firing (Schultz 2007; Schultz 2010). For example, when tonic DA levels are high, the phasic DA response will be attenuated (Floresco et al 2003; Schultz 2007), whereas low tonic DA facilitates phasic DA postsynaptic functioning (Grace 1991; Leknes and Tracey 2008).

29 Chapter 2

The glutamatergic and GABA-ergic system

General Although the antidepressant drugs MAOI, TCAs, SSRIs, NRIs, SNRIs, NDRIs and TRIs have well-established effects on monoamines, evidence is emerging that they may also affect other neurotransmitter systems. Special attention should be given to the main excitatory and inhibitory neurotransmitters, respectively glutamate and GABA, that are involved in the pathophysiology of major depression (Brambilla et al 2003; Cheetham et al 1988; Cryan and Kaupmann 2005; Holemans et al 1993; Krystal et al 2002; McEwen et al 2010; Merali et al 2004; Noga et al 1997; Nowak et al 1995; Sanacora et al 2004; Stewart and Reid 2002; Trullas and Skolnick 1990). Recently, it was shown that the cingulate and/or PFC displayed up-regulation of several glutamate and

GABAA receptor subunits, of which GABAAα1, α3, α4, δ and GABAAβ3 expression were changed in individuals who had died by suicide (Choudary et al 2005; Merali et al 2004; Sequeira et al 2009). Also glutamatergic pathways play an important role in anhedonia and impaired emotion processing. For example, Horn and collaborators (2010) nicely demonstrated that glutamatergic mechanisms are involved in patients with major depression, which are related to the functional connectivity between pregenual anterior cingulate cortex and anterior insula and depression severity. The demonstration that glutamatergic neurotransmission and glutamate are elevated in cortical and limbic brain areas of depressed patients (Drevets 2000; Sanacora et al 2004) and radioligand binding to N-methyl-D-aspartic acid (NMDA) glutamate receptors is also altered in frontal cortex of suicide victims compared with controls (Nowak et al 1995; Trullas and Skolnick 1990) is consistent with the hypothesis that the glutamatergic system is involved in the pathophysiology of major depression. Altogether, these findings support the hypothesis that the ratio of excitatory-inhibitory neurotransmitter functioning is increased in the brain of depressed patients. It is well known that most monoaminergic antidepressants are effective only after following several weeks of administration. Since monoamine reuptake inhibition is almost immediate, slowly developing events must be involved. Chronic treatment with monoaminergic reuptake inhibitors down-regulates monoamine autoreceptors with consequent disinhibition of respectively the release of monoamines, which will depress glutamate exocytosis through monoaminergic presynaptic heteroreceptors (see below in more detail) in conditions in which these receptors do not seem to undergo down- regulation (Pittaluga et al 2007). Since the insertion into the membranes of ionotropic glutamate receptors is regulated by glutamate inputs, the decreased glutamate exocytosis caused by the monoamines during chronic monoaminergic reuptake inhibitors could lead to local down-regulation of NMDA glutamate receptor function and increased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor phosphorylation and trafficking in limbic and cortical circuits (see below in more detail) (Sanacora et al 2008; Zarate et al 2003). This combined effect would produce a dampening of glutamate neurotransmission and an enhancement of AMPA-

30 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited vs NMDA-mediated neurotransmission and thereby would increase neuroplasticity, cellular resilience and synaptic plasticity (Racagni and Popoli 2008). It is quite well possible that these monoamine-induced changes in glutamatergic system restore neuroplasticity, cellular resilience and synaptic plasticity, which may underly the delayed onset of therapeutic efficacy of monoaminergic antidepressants.

Antidepressants reduce glutamate release during basal conditions 2 In cerebrocortical synaptosomes, depolarization–stimulated glutamate release is reduced both by the SSRI fluoxetine via a protein kinase (PKC)-sensitive signaling pathway (Wang et al 2003), and by the NDRI bupropion via mitogen activated/extracellular c h a p t e r signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) (Bonanno et al 2005). In hippocampal synaptosomes, depolarization– stimulated glutamate release is reduced by chronic (not acute) treatment with the SSRI fluoxetine, the NRI reboxetine, and the TCA (Lin et al 2011b). In rat prefrontal cortical and cerebrocortical slices chronic treatment with the TCA imipramine results in reduced glutamate release (Michael-Titus et al 2000; Tokarski et al 2008).

Antidepressants dampen stress-induced glutamate release In prefrontal cortical and in frontal cortical synaptosomes, acute stress-induced increase in depolarization-evoked release of glutamate is dampened by chronic treatment with the antidepressants fluoxetine, desipramine, venlafaxine, and agomelatine (Musazzi et al 2010).

Antidepressants increase the expression of the vesicular glutamate transporter1 Repeated treatment with SSRIs fluoxetine, paroxetine or the TCA desipramine increases vesicular glutamate transporter1 (VGLUT1, but not VGLUT2 or VGLUT3) mRNA abundance in frontal, orbital, cingulate and parietal cortices, and regions of the hippocampus and these antidepressants also increase VGLUT1 protein expression (Moutsimilli et al 2005; Tordera et al 2005).

Antidepressants alter expression and function of glutamate receptors Chronic treatment with the TCA imipramine, and the SSRIs fluoxetine and citalopram reduce the expression or function of NMDA receptors in the cerebral cortex of rodents. These changes develop slowly, persist for some time after cessation of treatment, and are dose dependent (Nowak et al 1993; Paul et al 1993; Skolnick et al 1996). Recently, it has been shown that both chronic reboxetine and fluoxetine treatments markedly reduced the expression level of NMDA receptor subunit NR1 in synaptic membranes of hippocampal synaptosomes (Pittaluga et al 2007). Lower synaptic expression of NR1 subunit of the NMDA receptor has also been found in a genetic animal model of depression (Flinders Sensitive Line rats) (Ryan et al 2009). Dopaminergic antidepressants may also affect NMDA receptors, because stimulation of D1 DA receptors increases NMDA currents at the postsynaptic level in

31 Chapter 2 the PFC, which appears to be mediated through both a protein kinase A (PKA) and Ca2+-dependent mechanisms (Jay 2003; Jay et al 1998; Kruse et al 2009; Lavergne and

Jay 2010). In addition, DA D1 receptor stimulation also increases NR2B-containing NMDA receptor surface expression in postnatal rat PFC neuronal cultures, and tyrosine phosphorylation plays an important role in the trafficking of NR2B-containing NMDA receptors in PFC neurons and the regulation of their trafficking by DA receptors (Gao and Wolf 2008). Remarkably, recently it has been demonstrated that the NMDA receptor antagonist rapidly activates the mammalian target of rapamycin (mTOR) pathway, leading to increased synaptic signaling proteins and increased number and function of new spine synapses in the PFC of rats (Li et al 2010). This might explain why ketamine has a very rapid onset of clinical antidepressant action. Besides NMDA receptors, also AMPA receptors are affected by monoaminergic antidepressants. Chronic fluoxetine and desipramine treatment exerted moderate but selective effects on glutamate receptor (GluR) expression and editing, while the NRI reboxetine appeared to be the drug that affects glutamate receptors most, that is, a decrease of the AMPA subunit GluR3 expression both in hippocampus, prefrontal and frontal cortex (Barbon et al 2006). Chronic treatment with the SSRI fluoxetine increases phosphorylation (with a critical role for the phosphoprotein DARPP-32) of the AMPA receptor subunit GluR1 at Ser-845 (Svenningsson et al 2002). The SSRI Paroxetine and the TCA desipramine enhance membrane expression of AMPA receptors in the hippocampus probably due to interactions of GluR1 and GluR2/3 AMPA receptor subunits with proteins implicated in AMPA receptor trafficking and with scaffolding (Martinez-Turrillas et al 2007). Remarkably, previously it has been shown that AMPA receptor activation increases the expression of brain-derived neurotrophic factor (BDNF) in hippocampal and cortical neurons (Lauterborn et al 2000). Dopaminergic mechanisms may also be involved in AMPA receptor regulation. Stimulation of D1 DA receptors increases surface expression of GluR1-containing AMPA receptors through a PKA-dependent mechanism, thereby creating a receptor pool available for synaptic insertion (Sun et al 2005).

Triple reuptake inhibitors

General As has been discussed in section ‘Major depressive disorder’, a role for DA can be recognized in several subsymptoms of depression. Moreover, dopaminergic pathways are involved in reward and hedonic processes, thereby hypothetically affecting anhedonic symptoms in depression (Dunlop and Nemeroff 2007; Guiard et al 2009b; Nestler and Carlezon 2006; Nutt et al 2007). DA is also involved in motor responses, via the mesocortical projection pathways, thereby presumably also involved in psychomotor disturbances. The rationale for developing a TRI came from indications that augmentation of antidepressant therapy with either the noradrenaline-dopamine

32 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited reuptake inhibitor (NDRI), bupropion (Rush et al 2006; Trivedi et al 2006), or the DA receptor agonists (Goldberg et al 2004; Gupta et al 2006) was clinically effective. Moreover, enhancement of dopaminergic neurotransmission might give rise to a rapid onset of antidepressant action (Willner 1997a). Drugs acting on all three monoamine neurotransmitter systems simultaneously should hypothetically cover a larger range of depressive symptoms (Guiard et al 2009b). 2

Binding potential of triple reuptake inhibitors (Figure 2, Table 2) In the past 15 years many TRIs were developed in order to treat mood disorders, , Parkinson’s disease and chronic pain, some of them still in preclinical phases, c h a p t e r but others already made it through phase II clinical trials. The only clinically effective TRI thus far is DOV 216,303. This drug has appeared to be safe, tolerable and effective in a phase II and reduced Hamilton Rating Scale for Depression scores of depressed patients (Skolnick et al 2006). NS-2359 showed no significant differences in efficacy between the compound and the placebo in a phase II clinical trial (press release NeuroSearch). Many TRIs are still in development and many new compounds are synthesized (Table 2). The main strategy for detecting new potential TRIs starts with examining the binding profiles of novel compounds. Many studies provide Kd values for their compounds, or Ki values, which are sometimes used interchangeably.

However, whereas Kd/Ki values give information about binding affinity, IC50 values give information about the potency of the inhibitor and give more functional information about the drug. Ratios in which different TRIs block the 5-HT transporter (SERT), NA transporter (NAT) and DA transporter (DAT) are provided in figure 3. In Table

2 it can be seen that ratios when provided by Kd values differ from ratios obtained from IC50 values for the same compounds. This makes it doubtful to focus only on

Kd values when screening novel compounds for their potential effects. Furthermore, it is not known how much inhibition or occupancy for each transporter is required for antidepressant action. It is known that in humans SSRIs typically inhibit 80% of the SERT binding sites at minimally effective doses (Meyer et al 2004), whereas bupropion produces its antidepressant effects at approximately 22% to 26% DAT occupancy (Learned- Coughlin et al 2003; Meyer et al 2002). But how relevant these transporter occupancies are is questionable. There is apparently no clear causal relationship between binding, occupancy and efficacy, because in patients not responding to SSRIs a comparable transporter occupancy is achieved as in patients responding to treatment (Meyer et al 2004). More biologically relevant information comes from in vivo studies, for example microdialysis studies in animals, in which the biological effect of a compound can be examined on neurotransmitter reuptake or release in different brain areas. Remarkably, when looking at microdialysis data from DOV 216,303 and JZAD-IV-22 it can be seen that the ratio of extracellular levels of 5-HT, NA and DA does not correlate with ratios of IC50 values (Caldarone et al 2010; Prins et al 2010) and that this also differs between different brain areas (Prins et al 2011b), which is of course not surprising regarding the different interactions between monoaminergic innervated brain areas (Figure 1). This

33 Chapter 2

l a r t u P u s o i p t v u G u a o h d y + m e a b l a e l r a h + T + r u o i v a h e b

m d r u P a s t t i w a G b e i a r v r

h t s e t S s a l s d u s e d c u o o r m p p

y r t m

o n A d a e 1 e m c T c n e m o e H c i o - m c t

i / r 5 p

A d o t f n x p n N o e i i t c e 2 n r H o α c l o r t t n n 2 e o - - c s α m

e e c 2 c v i r D t A o u f c n N i e e x r C E

& F A

N 2 g P - T n D i

S - / k

V - 2 a α m - 2

n α 1 o i C A r + α 2 2 s a i T T e c f T

y H H e t 2 - - d D e S 5 5 e i α n x C i N n - + a A t a L 2 s B T u s H - 5 + - + - 2 + D 1 r A α + a e + + e f 1 C α - 1 + α R R s t D e M u u c p

t d u A e C o n

2 + L

o / e

A A i t n 1 1 A B i i 2

T T r d /

c n H H A - - - - o o N 1 d c 5 5 T n V e H

- P & 5

c i m o n o - Schematic overview of Schematic In red, serotonergic projections. In green, noradrenergic projections. in MDD and their (monoaminergic) connections. brain areas involved t u a Figure 1. of nucleus bed BNST, excitatory;inhibitory;+, -, prefrontal cortex; amygdala; central CeA, basolateral amygdala; BLA, PFC, terminalis; stria dopaminergic projections. In blue, of nucleus paraventricular coeruleus;locus PVN, dorsal striatum; dStriatum, core; LC, accumbens dorsal DR, nucleus hypothalamus; NAcc, shell; accumbens nucleus NAcs, raphe nucleus; MR, median raphe nucleus; VTA, ventral tegmental area; SN, substantia nigra;ventral globus dorsal vGP, pallidus; globus dGP, pallidus. Many publications support the finding that a high degree of connectivity exists between monoaminergic neurons (Everitt andRobbins 2005; Guiard et al 2008; Guiard et al 2009b;Koob and 2010; Millan 2006; 2009) Volkow

34 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited

      

 2  

 c h a p t e r  

 

  





         

Figure 2. The half maximal inhibitory concentration (IC50) of novel triple reuptake inhibitors (TRIs) and reference drugs: IC50 represents the relative concentration of a drug that is required for 50% transporter inhibition. This quantitative measure indicates how much of a particular drug is needed to inhibit a given biological process by half. In other words, lower IC50 values indicate greater inhibitory potency. 5-HT: 5-Hydroxytryptamine, NA: Noradrenaline, DA: Dopamine raises important questions about the relevance of Kd or Ki and even IC50 values in the search for novel compounds. Another explanation for this discrepancy is the possibility that the capacity of TRIs to enhance extracellular levels of monoamines may depend, at least in part, on the density of monoamine transporters in the different brain areas involved. When looking at all the novel TRIs listed in Table 2 it appeares that irrespective of binding efficacies (IC50 or Ki values) or ratios between bindings to different transporters (Figure 2), most compounds are effective in the forced swim test (FST) and tail suspension test (TST). Although in the past it has been shown that positive results (especially for SSRIs) in these behavioural tests are often predictive of clinical antidepressant properties. From the present observation it can be concluded that the in vitro binding profiles do not have predictive validity for biological activity (e.g.in vivo neurotransmitter release) per se. One of the explanations for this discrepancy is the high degree of connectivity between the monoaminergic systems, which may produce counter-productive effects on the potency of TRIs to enhance brain dopaminergic neurotransmission (Figure 1). For example, as suggested in Figure 1, 5-HT through

5-HT2C receptors and NA through α2 receptors may inhibit dopaminergic VTA neurons

(Millan 2009), but in contrast 5-HT through 5-HT2A receptors and NA through α1 receptors may stimulate dopaminergic VTA neurons (Guiard et al 2009b; Millan 2006).

35 Chapter 2 value, the higher affinity ofaffinity higher the value, the d K Reference (Lee et al 2010) (Marks et al 2008; McMillen 2007) (Caldarone et al 2010) (Beer et al 2004; Breuer 2008; Caldarone et al 2010; Chen and 2007; Marks et al 2008) Skolnick and (Guiard et al 2011; Skolnick et al 2006; Basile 2007; Skolnick et al 2003) Skolnick (Hansen et al 2010; Hauser 2007; Larsen et al Rascol al 2008) (Basile et al 2007) (Aluisio et al 2008; Guiard 2011; Maryanoff et al 1987) et al 2007) (Guiard et al 2011; Shaw et al 2007) (Guiard et al 2011; Shaw values depend on experimental conditions and, therefore, cannot be easily compared between compared between easily cannot be depend and, therefore, conditions on experimental values 50 Preclinical/Clinical after unsuccessful Phase II trial in MDD discontinued Development patients reduction ofIncrease in 5-HTP induced stereotypy mice, immobility of Reduction and TST (3 mg/kg p.o.) in FST mice (10 mg/kg p.o.) ofnumber buried writhes and marbles in mice (30 mg/kg). Decrease of consumption of volitional alcohol. in Phase Ia trial for alcohol abuse. Active in TST. in FST mice (30 and 60 mg/kg) effective Effective in FST mice and rat OBX paradigm (Breuer et al 2008) Effective (Prins et al). Reduction otherwise not effective in the body, when available et al 2006). in HAM-D score Phase II study (Skolnick for MDD and alcohol drug discontinued Development abuse. in FST rats (5 mg/kg) and dose-dependent reduction in Effective immobility in TST (5 mg/kg) Currently in Phase II clinical trial patients with depression. Disease and in treating symptoms ofEffective Parkinson’s obesity, in system. Active the Indirectly stimulates Disease. Alzheimer’s Phase II Clinical trial properties. Antinociceptive after unsuccessful Phase III trial for pain development Discontinuation treatment. in TST (0.3 mg/kg i.p.). Effective in FST rats; decreased immobility and increased swimming, Effective in TST (5 and 10 mg/kg) and effective in FST rats; decreased immobility and increased swimming, Effective in TST (5 and 10 mg/kg) and effective (nM), DAT 2 58 129 120 80 78 96 65 910 2.4 100 120 values of values for monoamine transportersTRIs ofare a measure of effectiveness the particularthat transportersinhibit the to TRI 50 values of values ofaffinity the describe TRIs the smaller the transporters, different the to compound the d K NAT 4 19.8 103 84 45 21 23 1.7 55 0.09 19 0.4 IC50 (nM) ED50 (mg/kg), Kd ­ SERT 14 143 133 15 30 14 12 11 117 0.18 6 6

IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 ED50 Kd Kd DAT 222 190 213 5200 5.2 53 43 NAT 1030 380 262 5000 17 10 1.2 value (nM) value In vitro inhibition Ki values are absolute values for each compound. The compound. IC for each values are absolute values SERT d 740 190 99 2400 0.9 6 12 K values give more functional information give about a drug. values 50 Binding affinities, preclinical data and status reports from clinical trials from novel triple reuptake inhibitors (TRIs) and reference compounds. triple reuptake preclinical data and status reports from clinical trials novel Binding affinities, values are often used interchangeably. The or constant dissociation interchangeably. used are often values d K . In other words, it is the concentration ofconcentration the is it In other words, . of50% block to needed TRI the IC monoamine transporters. the and i Table 2. Table Compound SEP-225289 3-aryl-3-azolylpropan-1 amine 19r 102,677 DOV JZAD-IV-22 216,303 DOV 21,947 DOV EB-1010 NS 2330 () Bicifadine JNJ-7925476 025 PRC 050 PRC K drug transporter. for the is in vitro IC studies. Olfactory OBX: disorder; depressive Major MDD: Depression; for Scale Rating Hamilton HAM-D: 5-Hydroxytryptophan; 5-HTP: (Porsolt); test swim Forced FST: disorder; hyperactivity deficit attention ADHD: in mice) suspension test (always Tail inhibitor; TST: serotonin reuptake SSRI: Selective bulbectomy;

36 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited 2 value, the higher affinity ofaffinity higher the value, the d K Reference (Guiard et al 2011; Liang 2008) (Bannwart et al 2008) (Carter et al 2010) et al 2010a) (Micheli et al 2010b) (Micheli (Hill et al 2009) et al 2003) (Sanchez and (Guiard et al 2009; Sanchez Hyttel 1999) (Millan et al 2001) et al 1997) (Tatsumi c h a p t e r values depend on experimental conditions and, therefore, cannot be easily compared between compared between easily cannot be depend and, therefore, conditions on experimental values 50 Preclinical/Clinical no self-administration. in FST rats and TST, Dose-dependently effective in Phase I clinical trial for now for ADHD, development Discontinued MDD and neuropathic pain. in TST (30 mg/kg i.p.). Effective (10 mg/kg) in TST and locomotor assay Effective Dose-dependent increase in 5-HTP induced stereotypy and reduced immobility time in FST mice (3, 10 and 30 mg/kg). Dose-dependent increase in 5-HTP induced stereotypy and reduced Increased locomotor 10 and immobility time in (3, in 30 mg/kg). FST mice at 10 mg/kg. activity high abuse potential. Inhibitor, Reuptake Triple antidepressant and anxiolityic SSRI, effective SSRI, treatment in MDD NRI, treatment in MDD NDRI, treatment in ADHD (nM), DAT 18 450 40000 5000 >10000 82 24 values of values for monoamine transportersTRIs ofare a measure of effectiveness the particularthat transportersinhibit the to TRI 50 values of values ofaffinity the describe TRIs the smaller the transporters, different the to compound the d K NAT 0.6 670 2500 370 2 440 234 IC50 (nM) ED50 (mg/kg), Kd ­ SERT 2 680 2.1 6.8 794 7600 44000

Kd IC50 IC50 IC50 IC50 Kd DAT 61 11 8.2 8 8.1 3764 NAT 1.5 1 8.1 8.1 9.3 600 value (nM) value In vitro inhibition Ki values are absolute values for each compound. The compound. IC for each values are absolute values SERT d 2.1 6 7.9 9.2 9.8 6.6 1.1 K values give more functional information give about a drug. values 50 Binding affinities, preclinical data and status reports from clinical trials from novel triple reuptake inhibitors (TRIs) and reference compounds. triple reuptake preclinical data and status reports from clinical trials novel Binding affinities, values are often used interchangeably. The or constant dissociation interchangeably. used are often values d K . In other words, it is the concentration ofconcentration the is it In other words, . of50% block to needed TRI the IC monoamine transporters. the and i Table 2. Table Compound PRC200-SS SEP-228432 3,3-disubstituted pyr- rolidines compound (+16) 2-substituted N-aryl (5) 6-(3,4-Dichlorophenyl)- 1-[(Methyloxy)]-3- azabicyclo[4.1.0] heptane -17 1-(aryl)-6-[alkoxyalkyl]-3- azabicyclo[3.1.0]hexane – 15 compounds Reference Escitalopram Fluoxetine Reboxetine Methylphenidate K drug transporter. for the is in vitro IC studies. Olfactory OBX: disorder; depressive Major MDD: Depression; for Scale Rating Hamilton HAM-D: 5-Hydroxytryptophan; 5-HTP: (Porsolt); test swim Forced FST: disorder; hyperactivity deficit attention ADHD: in mice) suspension test (always Tail inhibitor; TST: serotonin reuptake SSRI: Selective bulbectomy;

37 Chapter 2

This might explain why some SSRIs/SNRIs under certain conditions might increase extracellular DA levels (Lavergne and Jay 2010). Moreover, even biological efficacy in animal models can not predict whether these compounds will be active in clinical trials. Even if animal models do duplicate clinical symptoms of depression in humans, the affective dimension of the pathology remains difficult to evaluate in animals. Until now, many clinical trials with TRIs appeared to be unsuccessful and only a few are still in active Phase I, II or III trials for many disorders such as MDD, Parkinson’s, Alzheimer’s, obesity, or pain (Table 2).

Selection of TRIs for treatment of MDD based on depressive symptom profile The next step in developing potential antidepressants is testing the compound with certain binding affinities in an animal model. The most frequently used depression tests are the TST and the FST. In these tests, rodents are suspended by their tails or placed in a cylinder with water, respectively, and changes in mobility are measured. Antidepressants typically decrease the immobility time of the animal. In the modified FST a further distinction can be made in swimming and climbing; climbing behaviour (which is affected by the escapability of the situation) is increased by noradrenergic drugs, without affecting swimming, and SSRIs increase swimming without affecting climbing (Lucki 1997). The same distinction can be made in humans, where drug-induced alterations in the serotonergic system have been associated with anxiolytic effects and changes in the noradrenergic systems with reducing psychomotor symptoms (Katz et al 2004). Acquired immobility behaviour in the FST is often taken as a state of despair in which the rat or mouse has learned that escape is impossible and this immobility time is reduced by antidepressants (Porsolt et al 1977). However, doubts have risen about this interpretation of the FST (Borsini and Meli 1988). An alternative explanation would be that the immobility posture can be seen as an energy-conserving strategy (West 1990). According to this line of reasoning immobility (floating) behaviour in the FST is a successful passive coping style (Korte et al 1996). The activity-increasing effects induced by enhancement of serotonergic or noradrenergic neurotransmission can be seen as a shift in coping style and might hypothetically be explained by an altered hippocampal and PFC functioning. Here it must be mentioned that altered glutamatergic and GABA-ergic neurotransmission are, at least partly, responsible for these altered hippocampal and PFC functioning (Hasler). The hippocampus plays a crucial role in the expression of acquired immobility in the FST (Korte 2001; Korte et al 1996). The hippocampus is most likely required to integrate incoming sensory stimuli and process the kinds of complex stimulus representations that make up the context. These contextual factors are also an essential part of what gives a stimulus its stressful meaning (LeDoux 1993). It is known that 5-HT via post- synaptic 5-HT1A receptors induces hyperpolarization of hippocampal neurons (Joels et al 1991), consequently a less active hippocampus may lead to an altered interpretation of the given environment, that is, inescapability in the FST. Furthermore, acute rises in 5-HT levels might induce anxiogenic behavioural effects due to activation of post- synaptic 5-HT1A receptors in the dorsal hippocampus (File et al 1996). In agreement,

38 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited it is well known that after starting antidepressant medication initial exacerbation of anxiety may occur (Flint 2003). Thus, it can not be excluded that by adopting the FST, one has been selecting drugs that increase anxiety after acute treatment, rather than an antidepressant effect per se. Furthermore, selecting novel compounds on their action in the FST or TST would result in compounds with similar working mechanisms as the existing antidepressants (me-too drugs), because that is what the tests are validated on. When novel compounds are rejected based on their inactivity in these tests, potentially 2 useful compounds might be thrown away (Drugan et al 2010). Especially with regard to the different symptomatology of melancholic and atypical depression which probably both need a different pharmacological treatment, c h a p t e r it is unlikely that only one test in animals can cover all the complex depressive aspects. Ideally, novel compounds should be tested in several behavioural tests and animal models to address their putative mechanism of action on a wide variety of symptoms of MDD (Willner 1984). For example anhedonia can be induced in animals by exposure to a chronic mild stress paradigm (Willner et al 1992), or other stressors, such as social defeat (Der-Avakian and Markou 2010) followed by individual housing (Ruis et al 1999). Reward-related deficits can in turn be measured with an intracranial self-simulation (ICSS) paradigm (Markou and Koob 1992) or the (less sensitive) sucrose preference test (Willner et al 1987). Other tests for anhedonia or libido might be to measure the sexual activity in rodent females (Snoeren et al 2011) as well as males (Chan et al 2010) and examine the effects of different pharmacological compounds. Another animal model that is associated with psychomotor agitation and often used to predict antidepressant

     



 

     

       Figure 3. A simplistic and hypothetical model: “the monoamine hypothesis revisited“ is presented to show that monoamines are differently affected in atypical and melancholic depression and that monoaminergic neurotransmission is ‘out of tune’, rather than deficient. The circles represent the increased or decreased monoaminergic functioning and capacity. Depression may develop when the relationship between serotonin (5-HT), noradrenaline (NA) and dopamine (DA) is thrown off balance. TRIs are thought to restore the delicate balance between monoaminergic neurotransmitters, transporters, receptors and pre- and postsynaptic processes.

39 Chapter 2 activity is the olfactory bulbectomy (OBX) paradigm in which chronic, but not acute, antidepressants, decrease OBX-induced hyperactivity (Breuer et al 2007). This brings up another important drawback in the development of antidepressants, which is the screening of compounds on their acute effects rather than their chronic use. Depressed patients often take antidepressants for months-years, therefore chronic administration effects should be investigated on all levels (e.g. binding affinity, neurotransmitter reuptake and/or release and behaviour). For example, it has been shown that chronic administration of the TRI DOV 216,303 resulted in blunted extracellular neurotransmitter levels compared with an acute injection (Prins et al 2010; Prins et al 2011b). In the search for new drugs not only chronic administration induced changes in activity of liver enzymes, but also adaptations in receptor and transporter density pre- and post-synaptically should be investigated.

Combination strategies It is important to realize that complementary agents (extensively and thoroughly reviewed by Millan (2006)) may also be needed to successfully treat major depression by decreasing both co-morbid symptoms and onset of action and by increasing efficacy and tolerance (de Bodinat et al 2010; Millan 2006; Millan 2009). Since melancholic depression is associated with both a hyperactive CRF-system and HPA-axis, and consequently increased plasma cortisol levels it is suggested that corticotropin-releasing factor 1 receptor (CRF1) antagonists or glucocorticoid receptor (GR) antagonists can be used as interesting tools in combination strategies to treat the “hyperactive stress system” (including features of anxiety and psychosis) in melancholic depression (Korte 2001). Furthermore, N-methyl-d-aspartate (NMDA) receptor antagonists and neurotrophic factor (e.g. BDNF) enhancers might be used to counteract the increased neurotoxicity in chronic re-occuring melancholic depression. Sleep disturbances can be observed in both melancholic depression and atypical depression, therefore it is speculated that combination therapy with 5-HT1A receptor/5-HT2C receptor antagonists and melatoninergic (MT1/MT2) receptor agonists, respectively improve sleep and produce resynchronisation of circadian rhythms (Millan 2006). The side effect of sexual dysfunctioning due to SSRIs treatment in melancholic depression might be counteracted by α2-adrenergic receptor and 5-HT2C receptor antagonists (Millan 2006).

Expert Opinion and Conclusion As has been discussed in this review, it is important to realize that subsymptoms of depression may be regulated by disturbances in different brain circuits and that disturbed monoaminergic neurotransmission may or may not be accountable for these symptoms. It should be clear that subtypes of depression such as melancholia and atypical depression may have a complete opposite symptom profile and that it is not surprising that the current first line of antidepressant treatment, the SSRIs, is not equally effective in both subtypes. It should be strongly emphasized that the pathogenesis of MDD does not rely on only one impaired system, whether that is an impaired HPA-axis

40 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited

/CRF-system (Holsboer 2000), a dysregulated immune system (Dantzer et al 2008) or a hypofunctional monoaminergic neurotransmission (Schildkraut 1965). Depending on symptoms, different systems are more or less involved, but in this review we primarily focused on a dysregulation in monoamines. Based on the different symptom profiles of atypical and melancholic depression discussed in section two (Figure 1), we suggest adaptations in the early monoamine hypothesis, which fit the different depressive subtypes (Figure 3). 2 In this revisited monoaminergic hypothesis of depression we propose, under healthy condition, three balanced monoaminergic systems, 5-HT, NA, and DA, which all have more or less interactions with each other and have stable steady-state concentrations. In c h a p t e r melancholic depression, a highly active stress system causes a hyperactive noradrenergic system (Gold and Chrousos 2002; Wong et al 2000b); furthermore, symptoms of anxiety in melancholic depression are associated with increased serotonergic neurotransmission, whereas dopaminergic neurotransmission is often decreased. The alterations in NA and 5-HT, however, cannot be explained as simple increased or decreased neurotransmitter availability. More importantly in melancholic depression, the tonic ‘resting state’ of 5-HT in the brain may be decreased compared with a healthy brain. This lowered serotonergic state has as consequence that critical 5-HT receptors and transporters are upregulated, to maximally increase the capacity of the system to transmit serotonergic signals. When melancholic depression is treated with SSRIs, an acute phasic response in 5-HT levels is present, which has a high impact on the hypersensitive system, leading to immediate adverse side effects (e.g. anxiety). When drugs are given chronically, the tonic 5-HT levels will shift upwards and the serotonergic system will adapt by downregulation of critical receptors and transporters, leading to a normal balanced system. Moreover, agitation and an increased sympathetic activity in melancholic depression are associated with high phasic response NA levels. So, in melancholic depression the capacities of these two monoamine systems are high and drugs which can normalize these disturbed neurotransmission pathways may have the highest probability of success to relieve symptoms. In atypical depression, symptoms as fatigue, psychomotor retardation and decreased sympathetic activity can be mainly attributed to disturbances in dopaminergic and noradrenergic systems, while serotonergic systems are less affected. Prolonged stress or pain is known to result in increased tonic DA levels, which will result in an attenuated phasic DA response (Wood 2006). Here it is hypothesized that in atypical depression, DA transporters and receptors are down regulated, thereby decreasing the capacity of the system to regulate dopaminergic signalling. An improved treatment of MDD should start with a better diagnosis, based on the different subtypes of MDD present. Treatment should be based on the symptoms patients encounter as disturbances in different neurotransmitter system capacity may underlie the different symptom profiles. It is known, especially if atypical symptoms as fatigue and energy loss are part of the diagnosis, chances of successful treatment decrease (Moos and Cronkite 1999). It could be imaginable that the first line of treatment will become TRIs, because they promise to be beneficial in a lot of symptoms of especially atypical depression, but also melancholic depression. An alternative would be to use dopaminergic agents as augmentation on existing therapies. However, it should

41 Chapter 2 be realized that TRIs will not be able to treat all kinds of symptoms, as histaminergic pathways are highly involved in sleep disturbances, and the CRF-system in stress and anxiety. Furthermore, heightened awareness for potential abuse and dependence is needed, because drugs that increase DA in the NAc have the potential for abuse. For instance, looking at IC50 values (Table 2 and Figure 2), cocaine is a very good TRI, including having a strong dopaminergic component. Thus, DA reuptake inhibition is not without danger. Recently, it was shown that the psychostimulant modafinil blocked DA transporters and increased DA in the human brain, including the NAc (Volkow et al 2009). In addition, two preclinical assays (locomotor sensitization and discriminative stimulus) indicated potential abuse liability of modafinil (Paterson et al 2010). Bupropion also binds selectively to the DA transporter and also has inhibiting properties which might be responsible for bupropion’s amphetamine-like abuse potential as observed in preclinical studies in which animals substituted bupropion for amphetamine in a drug-discrimination task (Bevins et al 2006), and in other studies in which it was self-administered intravenously by monkeys (Bergman et al 1989) and rats (Nicholson et al 2009). In humans it was reported that bupropion and amphetamine increased arousal, mood, and euphoria measured by self-reports (Cousins et al 2001). However, in humans, it was shown in a very small study (n=13) that it is unlikely that bupropion will give rise to amphetamine-like patterns of abuse (Griffith et al 1983), although by others it was suggested that bupropion might be of some abuse liability in a small subgroup of the investigated smokers (6%) (Zernig et al 2004). Recently, it was shown that the TRI tesofensine in humans (n=52 of recreational users) did not have abuse potential 48 h after drug administration and the effects of tesofensine were either lower than or not different from those of bupropion (Schoedel et al 2010). Why does tesofesine have no abuse potential, given the fact that the chemical structure is so similar to cocaine? The answer is almost certainly that it is just too slow in its onset of action. After taking it orally, it takes approximately six hours for blood levels to peak, and persists in the body for weeks (half-life of approximately 220 h). With cocaine, peak blood levels and peak brain (striatum) levels are achieved in minutes and the half-life is less than one hour (Volkow et al 1997). Previously, it has been reported that cocaine is believed to produce highs by blocking the DAT and increasing the availability of free DA within the brain. The time course for the high paralleled that of cocaine in the striatum. Remarkably, at least 47% of the DAT sites had to be blocked for subjects to perceive highs (Volkow et al 1997). Recently, only partial cocaine-like discriminative stimulus properties were observed for either DOV 216,303 or JZAD- IV-22, similar to non-abused compounds such as the TCA desipramine (Caldarone et al 2010). Therefore, only a low abuse potential for both TRIs is anticipated. Thus, it can be concluded that TRIs can be developed that have no, or very minimal, abuse potential, but it is important to take slow-onset and long-lasting properties into account during drug development (Gardner et al 2006). Better antidepressants are needed; however, it is not reasonable to think that we will find better drugs when these novel compounds are discovered with easy screening methods. Furthermore, there is an increased risk that in vitro binding studies and some

42 Triple reuptake inhibitors, subtypes of depression and the monoamine hypothesis revisited easy in vivo tests (e.g., FST) will leave us with similar compounds as the existing drugs. On the one hand, underlying brain mechanisms in the different subtypes of depression and the different symptoms separately should be investigated in several animal models for depression (including reward-related animal models) and in atypical and melancholic depressed humans as well. On the other hand, it could be beneficial to examine existing drugs on their acute and chronic effects to regulate neurotransmitter systems or other systems and use this knowledge in the better understanding of MDD. 2 In the past fifty years there has been an evolution of hypotheses onthe pathophysiology/pharmacotherapy of major depression (Racagni and Popoli 2008): monoaminergic hypothesis (1960s-1970s); monoaminergic receptor hypothesis (1980s); c h a p t e r hypothesis of signaling adaptation (1990s); and finally, hypothesis of neuroplasticity (2000s). This does not mean that earlier ideas were plain false, but they were incomplete. Therefore, to fully understand the pathophysiology of major depression one has to measure stress- and depression-related changes in in vivo neurotransmitter reuptake or release; (hetero) receptor trafficking, expression and functioning; post-receptor signaling cascades, gene transcription, translation and expression (Drevets et al 1997; Manji et al 2001; Manji et al 2003; McEwen and Chattarji 2004; Pittenger and Duman 2008); and cellular resilience, neuroplasticity (e.g. volumetric changes, dendritic remodeling, glial loss, neurogenesis impairment), and synaptic plasticity. The hypothesis of neuroplasticity is in accordance with modern ideas that major depression is not influenced by “nature or nurture”, but by both “nature and nurture”. It is well known that stressful life events may trigger a depressive episode (Brilman and Ormel 2001) and an anxious personality trait (Sandi and Richter-Levin 2009) makes a person more vulnerable to develop major depression. Thus, here it is suggested that stress-mechanisms during “nature and nurture” may play an important role in disturbed neuroplasticity processes in major depression. In this light, melancholic depression needs extra attention. Melancholic depression is associated with a hyperactive CRF-system and HPA- axis, consequently hypersecretion of glucocorticoids may decrease neuroplasticity in frontal cortex, PFC and hippocampus, e.g., neurogenesis in the dentate gyrus, dendritic remodeling in the CA3 region of the hippocampus (McEwen 2001; Sapolsky 2000). Chronic exposure to glucocorticosteroids may even cause a selective loss of hippocampal volume and neuron loss (Sapolsky 2000). It has been postulated that such an allostatic load of glucocorticosteroids via prolonged elevated levels of glutamate is responsible for these neurological changes (McEwen 2001). Different paradigms of stress or corticosteroid administration induce a rapid and transient increase in extracellular glutamate in PFC and hippocampus (Moghaddam et al 1994; Stein-Behrens et al 1994; Venero and Borrell 1999). Moreover, it has been shown that acute stress may rapidly increase the level of circulating corticosteroids that, by binding to membrane-located mineralocorticoid receptors and rapid non-transcriptional action, induces the release of glutamate in hippocampus (Karst et al 2005; Olijslagers et al 2008), and by binding to GRs induces the release of glutamate in PFC and frontal cortex (Musazzi et al 2010; Yuen et al 2009). If new monoaminergic antidepressants, including TRIs, can dampen states of hyperglutamatergic activity and the subsequent excitotoxicity, their chronic use may have a considerable neuroprotective potential in major depression, especially

43 Chapter 2 melancholic depression (Michael-Titus et al 2000). Furthermore, it is expected that TRIs, like MAOIs, are especially effective in atypical depression (Thase et al 1992; Thase et al 1995), but with a better safety profile.

44

Jolanda Prins Jolanda Berend Olivier 633(1-3): 55-61 Rudy Schreiber Rudy S. Mechiel Korte Mechiel S. Maria S. Quinton Maria S. Damiaan A. Denys Lucianne Groenink Koen G. C. Westphal C. G. Koen Gerdien A. H. Korte-Bouws DOV216,303, a triple monoamine release in monoamine the prefrontal cortex of the prefrontal European of Journal Pharmacology 2010, The putative antidepressant antidepressant The putative reuptake inhibitor, increases inhibitor, increases reuptake olfactory bulbectomized rats olfactory bulbectomized

c h a p t e r 3 Chapter 3

Abstract The first line of antidepressant treatment nowadays are selective serotonin reuptake inhibitors. Although they are relatively safe to use, selective serotonin reuptake inhibitors (SSRIs) can induce severe side effects. New promising antidepressants may be the triple monoamine reuptake inhibitors, which not only enhance serotonin and norepinephrine neurotransmission, but also increase brain dopamine levels. Recently it has been shown that one of the triple reuptake inhibitors, DOV 216,303 has antidepressant-like effects in the olfactory bulbectomy (OBX) model of depression, but the alterations in monoaminergic neurotransmission in these animals are still unknown. In the present study we investigated not only the effect of acute, but also chronic treatment of DOV 216,303 in OBX rats on monoamine and metabolite levels. The main results are decreased baseline dopamine levels in the prefrontal cortex one day after OBX, while 38 days after OBX no difference could be observed in monoamine levels after vehicle treatment. Treatment with DOV 216,303 leads to increased extracellular levels of serotonin and norepinephrine neurotransmission, but also increased dopamine levels in OBX animals as well as their controls. This increase could be observed after one single administration, but also after chronic treatment. However, a DOV 216,303 challenge in chronically treated animals resulted in lower monoamine concentrations than the same challenge in untreated animals. More research is needed to investigate this seemingly hyporesponsivity to chronic DOV 216,303 treatment.

48 DOV 216,303 increases extracellular monoamines in the PFC of olfactory bulbectomized rats

Introduction Major depressive disorder is one of the most prevalent psychiatric disorders. Its core symptoms are depressed mood and loss of interest or pleasure in activities that were once enjoyed (i.e., anhedonia) (Torpey and Klein 2008). Selective serotonin reuptake inhibitors (SSRIs) are the most frequently prescribed antidepressants, because they are relatively effective and safe to use. Nevertheless, SSRIs can induce adverse side effects, including sexual dysfunction (Waldinger et al 1998), agitation (Henry and Demotes- Mainard 2006), weight gain and sleeplessness (Croom et al 2009), causing patients to withdraw from treatment (Demyttenaere and Jaspers 2008). Although the exact mechanisms underlying depression remain unclear, an important theory explaining the symptoms of depression, already for more than 30 years, is the 3 monoamine hypothesis (Schildkraut 1965). This hypothesis proposes that a deficiency of central monoamines is crucial for depression. Despite the dysfunction of mesolimbic dopaminergic system in depression, causing anhedonia, research on norepinephrine- c h a p t e r and serotonin-containing circuits has largely overshadowed the role of dopamine in depression. (Dunlop and Nemeroff 2007; Kinney et al 2000; Nestler and Carlezon 2006). Therefore the development of new broad spectrum antidepressants that also increase brain dopamine levels is an attractive strategy (Skolnick et al 2006; Skolnick et al 2003a). One of these new putative antidepressants is the triple monoamine reuptake- inhibitor (TRI) DOV 216,303. Previously it has been shown that DOV 216,303 has antidepressant-like effects in the forced swim test (both rats and mice) and the mouse tail suspension test (Skolnick et al 2003a) as well as in humans (Skolnick et al 2006). Recently, in our lab it was demonstrated that two weeks of treatment with DOV 216,303 had antidepressant-like behavioural effects, without showing sexual side effects, in the olfactory bulbectomy (OBX) model of depression (Breuer et al 2008). In the OBX rat model, the olfactory bulbs are removed resulting in neurochemical changes resembling those of depressed patients (Song and Leonard 2005), namely decreased striatal dopamine (Masini et al 2004) and decreased serotonin levels in the hippocampus and basolateral amygdala (van der Stelt et al 2005). Removal of the bulbs also leads to hyperactivity in a novel stressful environment such as an open field. This hyperactivity can be reversed by chronic, but not acute, antidepressant treatment (Kelly et al 1997; Mar et al 2000; van Riezen and Leonard 1990), lasting up to 10 weeks after cessation of treatment (Breuer et al 2007). Although DOV 216,303 showed antidepressant-like effects in the OBX animal model of depression, it is not known how monoaminergic neurotransmission is altered in these animals after acute and chronic DOV 216,303 treatment. Therefore, in the present study we investigated the effect of acute and chronic treatment of DOV 216,303 in olfactory bulbectomized rats on monoamine levels (serotonin, norepinephrine and dopamine) and their metabolites in the medial prefrontal cortex using in vivo microdialysis in freely moving rats. In this study we used the only dose of DOV 216,303 (20 mg/kg) shown to be effective in reducing behavioural hyperactivity in the OBX animal model (Breuer et al 2008). The aim of this study was to determine the contribution of the different monoaminergic systems in OBX rats

49 Chapter 3 after acute and chronic treatment with a triple reuptake inhibitor. The prefrontal cortex is innervated by serotonergic, norepinephrinerergic and dopaminergic fibers and plays a major role in major depressive disorder (Koenigs and Grafman 2009).

Materials and methods

Animals Male Sprague Dawley rats (Harlan, Zeist, the Netherlands) weighing between 290 and 350 g at time of OBX or Sham surgery were socially housed, two or four per cage on a 12h/12h light/dark cycle with lights on at 6:00h and off at 18:00h. Food and water were available ad libitum. Animals had one week to acclimate to their environment and were subjected to an open field test before receiving surgery. After microdialysis probe implantation animals were housed individually until the end of the experiment. For Experiment I (acute drug treatment), 25 animals were used and 36 animals for Experiment II (chronic drug treatment). The care and use of laboratory animals and all the experimental procedures were in accordance with the governmental guidelines and approved by the Ethical Committee for Animal Research of the Faculties of Pharmaceutical Sciences, Chemistry and Biology at Utrecht University, the Netherlands.

Olfactory bulbectomy For the olfactory bulbectomy (OBX) surgery animals were anesthetized by inhalation of isoflurane gas (2-3%), mixed with nitrous oxide and oxygen and placed ina stereotaxic instrument (Kopf, David Kopf Instruments). Lidocaine hydrochloride (2%) + adrenaline (0.001%) were applied in the incision as a local anesthetic. Two burr holes with a diameter of 2 mm were drilled bilaterally, 8 mm anterior to bregma and 2 mm from the midline of the frontal bone overlying the olfactory bulbs. The bulbs were aspirated with a blunt hypodermic needle attached to a vacuum pump. Burr holes were paced with haemostatic sponge to prevent blood loss. Sham operated animals underwent the same procedure except that their olfactory bulbs were not removed. All incisions were closed with use of 4-0 vicryl suture material and animals received Rimadyl (5 mg/kg, subcutaneously) for pain relief.

Microdialysis probe implantation For Experiment I, a cuprofane microdialysis probe (MAB 4.7.2 CU) was implanted in the prefrontal cortex (PFC), immediately after OBX/Sham surgery. The coordinates of the PFC were tooth bar set at -3.3, A: +3.2 mm, ML: +0.8 mm, and DV: -4.0 mm from bregma and skull. Probes were anchored in place with three screws and dental cement on the skull. For Experiment II, the probe was implanted on day 16 of DOV 216,303 treatment, the experimental set-up for Experiment II is shown in Fig. 1

50 DOV 216,303 increases extracellular monoamines in the PFC of olfactory bulbectomized rats

Microdialysis experiment There was no significant difference in weight between the different groups at time of the microdialysis day, weights of the animals in the different groups ranged from 365 till 410 grams. The day after implantation, microdialysis experiments were performed in conscious freely moving animals. The system was perfused with Ringer solution (147 mM NaCl,

2.3 mM KCl, 2.3 mM CaCl2, and 1 mM MgCl2) with use of a KdScientific Pump 220 series (USA) at constant flow rate of 1 ml/min. Animals were connected to a dual channel swivel (type 375/D/22QM) which allowed them to move relatively unrestricted. During microdialysis, the pump rate was set at 0.09 ml/h. Two hours after connection of the animals to the system, ten 30-minute samples were manually collected in vials 3 containing 15 µl of 0.1 M acetic acid and frozen at -80 ºC until analysis with HPLC. After two hours of baseline samples DOV 216,303 (synthesized by Sepracor Inc.,

Marlborough, USA) (20 mg/kg, 2 ml/kg, i.p.) or vehicle (0.9% NaCl) was administered c h a p t e r intraperitoneally to the animals. At the end of the microdialysis test day all animals were sacrificed and their brains were removed and examined to verify complete olfactory bulb ablation and probe placement accuracy.

HPLC analysis Microdialysis samples were stored at −80 °C until analysis. Neurotransmitters, norepinephrine, dopamine and serotonin and the metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were detected simultaneously by HPLC with electrochemical detection using an Alexys 100 LC-EC system (Antec Leyden, the Netherlands) (Korte-Bouws et al 1996; Verhagen et al 2009). The system consisted of two pumps, one autosampler with a 10 port injection valve, two columns and two detector cells. Column 1 (ALF 105 C18 1×50mm, 3 μm particle size) in combination with detector cell 1, separated and detected dopamine and serotonin. Column 2 (ALF 115 C18 1×150 mm, 3 μm particle size) in combination with detector cell 2, separated and detected norepinephrine and the metabolites. The mobile phase for column 1 consisted of 50mM phosphoric acid, 8mM KCl, 0.1mM EDTA

Microdialysis test day OBX/sham surgery Probe implantation

wk 1 wk 2 wk 3 wk 4 wk 5 wk 6 wk 7

DOV 216,303 (20 mg/kg)

Figure 1. Design of Experiment II with OBX and microdialysis during chronic drug treatment. Fig. 1. Design of Experiment II with OBX and microdialysis during chronic drug treatment

51 Chapter 3

(pH 6.0), 12% Methanol and 500 mg/L 1-Octanesulfonic acid, sodium salt (OSA). The mobile phase for column 2 consisted of 50mM phosphoric acid, 50 mM citric acid, 8mM KCl, 0.1mM EDTA (pH 3.2), 10% methanol and 500 mg/L OSA. Both mobile phases were pumped at 50 μl/min. Samples were kept at 8 °C during analysis. From each microdialysis sample 5 μl was injected simultaneously onto each column. The neurotransmitters were detected electrochemically using μVT-03 flow cells (Antec Leyden, the Netherlands) with glassy carbon working electrodes. Potential settings were for dopamine and serotonin +0.30 V versus Ag/AgCl and for norepinephrine and metabolites +0.59 V versus Ag/AgCl. The columns and detector cells were kept at 35 °C in a column oven. The chromatogram was recorded and analyzed using the Alexys data system (Antec Leyden, the Netherlands). The limit of detection was 0.03 nM (S/N ratio 3:1).

Drug treatment In Experiment I, animals received only one injection during the microdialysis day with either DOV 216,303 (+/-1-(3,4-dichlorophenyl)-3-azabicyclo-[3.1.0]hexane hydrochloride) (20 mg/kg, i.p.) or vehicle (0.9 % NaCl). For Experiment II, drug treatment started 3 weeks after OBX/Sham surgery. Animals received one injection per day for 17 days, with the last injection on the microdialysis test day. OBX and Sham animals were randomly assigned to DOV 216,303 (20 mg/kg, i.p.) or vehicle (0.9% NaCl) treatment groups (n=9 per group).

Histology All the animals were sacrificed at the end of the microdialysis test day. For the acute treatment experiment brains were fixed in 4% paraformaldehyde and later on transferred to a 30% sucrose solution and after three days frozen slices of 60 µm were made and stained with a cresyl violet staining for probe track verification. For the chronic treatment experiment brains were quickly frozen in isopentane and stored at -80 ºC, verification of probe track was done. Data of animals were discarded if olfactory bulbs were not completely ablated, or if the microdialysis probe was not in the PFC.

Statistical analysis The area under the curve of the monoamine response was calculated using the trapezoid algorithm. To detect significant differences in area under the curve data analysis of variance (one-way ANOVA) was used. Data from Experiment I as well as Experiment II, were analyzed by two-way ANOVA with the between group factors ‘drug’ (2 levels: vehicle and DOV 216,303) and ‘lesion’ (OBX and Sham). Subsequent repeated analysis of variance (RM-ANOVA) was performed for the OBX and Sham-treated group with time as ‘within’ and drug treatment as ‘between’ factor. The level of significance was set a priori at p<0.05. In case the ANOVAs were significant they were followed by post-hoc multiple comparisons with the use of Tukey’s test. Data are given as mean ± S.E.M.

52 DOV 216,303 increases extracellular monoamines in the PFC of olfactory bulbectomized rats

Results

Baseline monoamine levels No animals were excluded because all olfactory bulb ablations were complete. In total six animals from Experiment II were excluded from the analysis based on incorrect microdialysis probe placement. In two animals the microdialysis sampling during the experiment stopped and therefore these incomplete data were excluded from analysis. For Experiment I, the number of animals used in each treatment group was Sham- vehicle (n=7), OBX-vehicle (n=6), Sham-DOV 216,303 (n=6), OBX-DOV 216,303

(n=6). For Experiment II, the number of animals used in each treatment group was 3 Sham-vehicle (n=5), OBX-vehicle (n=7), Sham-DOV 216,303 (n=9), OBX-DOV 216,303 (n=7). In Experiment I, a microdialysis experiment was performed to measure monoamine and metabolite levels in the prefrontal cortex, one day after removal of the olfactory c h a p t e r bulbs. Baseline dopamine levels in the prefrontal cortex were significantly lower one day after OBX when compared to Sham operated animals [F(1, 21) = 8.8, p = 0.007], while baseline levels of norepinephrine, serotonin or metabolites were not affected (Fig. 2). In Experiment II, a microdialysis study was carried out after chronic treatment with DOV 216,303. After 17 days of treatment with DOV 216,303, only significantly increased baseline serotonin levels [F(1, 22) = 5.7, p = 0.026] were found in both OBX and Sham treated animals. A trend was observed for increased dopamine baseline

      



       

       

Figure 2. Baseline levels of monoamines and metabolites in the prefrontal cortex one day after OBX/Sham surgery. * P<0.01, significant difference between OBX and Sham.

Table 1. Baseline extracellular monoamine and metabolite concentrations after long term treatment with DOV 216,303 or vehicle. Data represented as mean ± S.E.M. Sham vehicle Sham DOV 216,303 OBX vehicle OBX DOV 216,303 (nM) (nM) (nM) (nM) Dopamine 0.171 ± 0.064 0.382 ± 0.082 0.176 ± 0.053 0.237 ± 0.075 Norepinephrine 0.341 ± 0.065 0.351 ± 0.07 0.242 ± 0.05 0.338 ± 0.093 Serotonin 0.09 ± 0.021 0.128 ± 0.012a 0.061 ± 0.012 0.120 ± 0.025a DOPAC 21.53 ± 9.54 25.53 ± 5.98 18.07 ± 5.97 18.58 ± 4.20 HVA 31.98 ± 12.4 44.39 ± 7.82 27.38 ± 7.02 35.45 ± 7.55 5-HIAA 115.34 ± 24.12 153.45 ± 12.88 104.14 ± 14.97 119.80 ± 17.66 ap < 0.05 compared to vehicle treated corresponding controls.

53 Chapter 3 levels in the DOV 216,303 treated groups [F(1, 22) = 3.2, p = 0.085) (Table 1). The concentrations of norepinephrine and the metabolites DOPAC, HVA and 5-HIAA were not significantly altered by surgery or treatment after 17 days (Table 1).

Experiment I: acute drug treatment After a single dose of DOV 216,303, significant increases in dopamine, norepinephrine and serotonin levels were measured in both OBX and Sham animals. The change in dopamine, norepinephrine and serotonin levels over time were dependent of the group animals were assigned to, for dopamine [F(18, 126) = 27.8, p < 0.001], for norepinephrine [F(18, 126) = 24.2, p < 0.001], and for serotonin [F(18, 120) = 19.5, p

     

                       

                              

     

 

 

    

 

                           

 

  

 

                         

Figure 3. Monoamine levels in the prefrontal cortex after acute (A, B and C) or chronic (D, E and F) treatment with vehicle or DOV 216,303 in OBX and Sham animals. Timepoints -90 till 0 minutes represents baseline measurements. At t=0 minutes a single injection was given with either DOV 216,303 or vehicle. [DA] is concentration dopamine, [NE] is concentration norepinephrine and [5-HT] is concentration serotonin.

54 DOV 216,303 increases extracellular monoamines in the PFC of olfactory bulbectomized rats

* 25 Sham vehicle 10 Sham DOV 216,303 ) ) # ) ) 8 0 OBX vehicle h h * * * M M * OBX DOV 216,303 n n ( ( 6

* -25 * s s b b a a ( (

4 C C * U U * * -50 A A 2

0 -75 * * Dopamine Norepinehprine Serotonin DOPAC HVA 5-HIAA

Figure 4. Area under de curve (AUC) (absolute concentrations) of monoamines and metabolites after acute treatment with DOV 216,303 or vehicle in OBX and Sham animals. *p < 0.001 compared to vehicle treated controls, # p < 0.001 effect of surgery.

< 0.001]. Apart from this time × group interaction there was also a highly significant 3 effect of time, for dopamine [F(6, 126) = 78.2, p < 0.001], for norepinephrine [F(6, 126) = 74.6, p < 0.001], for serotonin [F(6, 120) = 64.1, p < 0.001] and group, for dopamine [F(3, 21) = 34.6, p < 0.001], norepinephrine [F(3, 21) = 12.3, p < 0.001] and c h a p t e r serotonin [F(3, 20) = 39.2, p < 0.001]. Furthermore, dopamine levels of Sham animals were significantly higher than those of DOV 216,303 treated OBX rats (Fig. 3A, B and

C). These results are also represented by a higher area under the curve [F(1, 21) = 225, p < 0.001] (Fig. 4) and a significant increase in absolute concentrations of all monoamines is shown in DOV 216,303 treated Sham and OBX animals. This effect was stronger in Sham animals than in OBX rats [F(1, 21) = 28.8, p < 0.001]. Acute DOV 216,303 treatment also significantly decreased the area under the curve of DOPAC [F(1, 21) = 19.6, p < 0.001] and the area under the curve of 5-HIAA [F(1,21) = 17.8, p < 0.001]. These differences were independent of surgery. Sixty minutes after a challenge with DOV 216,303 on the microdialysis day DOPAC levels of DOV 216,303 treated OBX animals were significantly lower than those of vehicle treated Sham animals. From 90 till 180 min after treatment DOPAC levels of both DOV 216,303 treated groups are significantly lower than those of both vehicle treated groups. No significant interaction between surgery and treatment was found for norepinephrine, serotonin and DOPAC, HVA and 5-HIAA. A summary of the statistics for the area under the curve data for Experiment I is represented in Table 2.

Table 2. Summary statistics of area under the curve (AUC) of monoamine response after acute DOV 216,303 treatment. Surgery Treatment Surgery × Treatment Dopamine F(1, 21) = 24.2a F(1, 21) = 225a F(1, 21) = 28.8a Norepinephrine F(1, 21) = 3.6 F(1, 21) = 87.4a F(1, 21) = 1.6 Serotonin F(1, 21) = 0,4 F(1, 21) = 152a F(1, 21) = 0.4 DOPAC F(1, 21) = 2.0 F(1, 21) = 19,6a F(1, 21) = 0.84 HVA F(1, 21) = 1.0 F(1, 21) = 0.39 F(1, 21) = 0.59 a 5-HIAA F(1, 21) = 0.01 F(1, 21) = 17.8 F (1, 21) = 0.003 a p ≤ 0.001

55 Chapter 3

Experiment II: chronic drug treatment An injection with DOV 216,303 in chronically treated OBX and Sham animals significantly increased dopamine, norepinephrine and serotonin (Fig. 3D, E and F) in the prefrontal cortex and significantly decreased DOPAC levels in both OBX and Sham animals (Table 3). Dopamine, norepinephrine and serotonin levels altered over time, for dopamine [F(6, 132) = 23.2, p < 0.001], for norepinephrine [F(6, 120) = 24.2, p <

0.001] and for serotonin [F(6, 132) = 26.9, p < 0.001]. This effect was dependent on the group animals were in, for dopamine [F(18, 132) = 7.3, p < 0.001], norepinephrine

[F(18, 120) = 9.6, p < 0.001] and serotonin [F(18, 132) = 6.7, p < 0.001]. There was also an overall difference between groups, dopamine [F(3, 22) = 12.9, p < 0.001], norepinephrine [F(3, 20) = 8.9, p < 0.001] and serotonin [F(3, 22) = 24.1, p < 0.001]. There was no effect of surgery or an interaction between surgery and treatment for all monoamines and metabolites. An injection of DOV 216,303 in animals chronically treated with DOV 216,303 resulted in a significantly higher area under the curve of dopamine, norepinephrine and serotonin. DOV 216,303 treatment also significantly decreased the area under the curve for DOPAC (Fig. 5, Table 3), while no significant alterations were observed on HVA and 5-HIAA concentration. The differences were independent of the surgery. No significant interactions between surgery and treatment were found for all monoamines and metabolites. A summary of the statistics for the area under the curve data for Experiment II is given in Table 3.

Discussion The present study demonstrates that one day after olfactory bulbectomy (OBX), baseline dopamine levels are significantly decreased in the prefrontal cortex of OBX animals as compared to Sham operated controls, while levels of norepinephrine, serotonin and metabolites remain unaltered. This difference in dopamine concentration was no longer present 38 days after OBX surgery, nor could any difference be observed in baseline norepinephrine, serotonin or metabolite levels between OBX and Sham at that time point. Another finding of this study is that chronic treatment with the triple reuptake inhibitor DOV 216,303 increases extracellular concentrations of dopamine, norepinephrine and serotonin in the medial prefrontal cortex of both OBX and Sham

                                     

        Figure 5. Area under de curve (AUC) (absolute concentrations) of monoamines and metabolites 17 days after chronic treatment with DOV 216,303 or vehicle in OBX and Sham animals. *p ≤ 0.001 compared to vehicle treated control.

56 DOV 216,303 increases extracellular monoamines in the PFC of olfactory bulbectomized rats

Table 3. Summary statistics of area under the curve (AUC) of monoamine response after chronic DOV 216,303 treatment. Surgery Treatment Surgery × Treatment Dopamine F(1, 22) = 1.9 F(1, 22) = 41.2b F(1, 22) = 2.4 Norepinephrine F(1, 19) = 0.11 F(1, 19) = 14.9b F(1, 19) = 0.004 Serotonin F(1, 22) = 0.4 F(1, 22) = 76b F(1, 22) = 2.59 DOPAC F(1, 21) = 3 F(1, 21) = 15.9b F(1, 21) = 0.11 HVA F(1, 21) = 2.39 F(1, 21) = 4.4a F(1, 21) = 0.20 5-HIAA F(1, 22) = 0.55 F(1, 22) = 5.8a F(1, 22) = 0.69 ap < 0.05, bp ≤ 0.001 3 animals and significantly increased extracellular baseline serotonin concentrations. A single injection of DOV 216,303 one day after OBX results in elevated extracellular

levels of dopamine, norepinephrine and serotonin in the medial prefrontal cortex of c h a p t e r OBX and Sham animals, only for dopamine this increase was significantly higher in Sham animals than OBX animals. An injection of DOV 216,303 in chronically treated DOV 216,303-animals also resulted in an increase in extracellular dopamine, norepinephrine and serotonin. These monoamine concentrations, however, do not reach the same levels as for the acute treatment as can be seen in Fig. 3. The findings that there were no differences in baseline extracellular monoamine or metabolite levels between OBX and Sham animals 38 days after OBX surgery are in contrast with previous microdialysis studies in the dorsal hippocampus and basolateral amygdala. One of these studies demonstrated decreased extracellular concentrations of serotonin, but not of dopamine two weeks after surgery, lasting up for five months for the dorsal hippocampus (van der Stelt et al 2005). Another study showed elevated dopamine levels in the ventral striatum and dorsal striatum two weeks after OBX (Masini et al 2004). Most studies examined extracellular monoamine release two weeks after OBX, arguing that at that time point behavioural changes that characterize OBX emerge. However, changes in basal locomotor activity, body temperature, heart rate and heart rate variability already occur immediately after OBX (Vinkers et al 2009). This study is to our knowledge the first study analyzing extracellular monoamine levels one day after olfactory OBX. The fact that OBX acutely leads to decreases in dopamine is consistent with the finding that OBX leads to anhedonia measured with intracranial self-stimulation (ICSS) (Slattery et al 2007). OBX leads to increased ICSS thresholds up to seven days after surgery. After seven days, thresholds went back to baseline, which is consistent with our findings that 38 days after OBX no difference in dopamine could be detected anymore, assuming that lower dopamine levels are reflected in higher ICSS thresholds and an anhedonic state of the animal. Chronic treatment (17 days) with the triple reuptake inhibitor DOV 216,303 increases extracellular levels of dopamine, norepinephrine and serotonin in the medial prefrontal cortex of OBX as well as Sham animals. This is in line with the antidepressant-like behavioural effects of DOV 216,303 in the OBX animal model of depression (Breuer et al 2008). It fits the profile of the compound, because DOV 216,303 is a potent reuptake

57 Chapter 3 blocker of the (DAT), norepinephrine transporter (NET) and (SERT). Chronic DOV 216,303 treatment also increased baseline extracellular serotonin concentrations in both OBX and Sham animals, whereas the baseline levels of the other monoamines and metabolites remained unaffected. It is important to realize that DOV 216,303 is a racemate, and its enantiomers DOV 21,947 [(+)-1-(3,4-dichlorophenyl)-3-azabicyclo-[3.1.0]hexane hydrochloride] and DOV 102,677 [(–)-1-(3,4-dichlorophenyl)-3-azabicyclo-[3.1.0]hexane hydrochloride] have been characterized (Skolnick et al 2003a). The optically active compound DOV 21,947 is almost twice as biologically active in the Porsolt swim test than DOV 216,303 [(±)-1-(3,4-dichlorophenyl)-3-azabicyclo-[3.1.0]hexane hydrochloride] (Skolnick et al 2006; Skolnick et al 2003a). In line with our microdialysis study, it has been shown previously that acute treatment with DOV 102,677 (20 mg/kg, i.p.) increases extracellular levels of dopamine, norepinephrine and serotonin in the prefrontal cortex, and also increases extracellular levels of dopamine and serotonin in the rat nucleus accumbens (Popik et al 2006). DOV 102,677 (20 mg/kg, p.o.) was the minimal effective dose in the Porsolt swim test (Popik et al 2006). By blocking the SERT, DOV 216,303 causes an acute increase in serotonin levels which in turn act on the somatodendritic 5-HT1A autoreceptors, inhibiting a further release of serotonin. Long term blockade of the serotonin transporter causes downregulation of the 5-HT1A autoreceptor, thereby disinhibiting serotonin release and causing sustained elevated extracellular serotonin levels (Blier and de Montigny 1994). Moreover, serotonin release in the prefrontal cortex is under the influence of postsynaptic 5-HT1A receptors (Casanovas et al 1999) These postsynaptic 5-HT1A receptors directly influence serotonergic activity of the dorsal raphe nuclei projecting to the prefrontal cortex and have been functionally localized in the medial prefrontal cortex (Martin-Ruiz and Ugedo 2001). Systemic administration of 5HT1A receptor agonists resulted in a greater reduction in 5-HT release in the frontal cortex than in the dorsal raphe nucleus (Casanovas et al 1997). We cannot exclude the involvement of postsynaptic 5-HT1A receptors in the elevated serotonin levels found after chronic DOV 216,303 administration. In this study we also looked at the effect of a DOV 216,303 challenge on monoamine release in the prefrontal cortex in long term DOV 216,303-treated animals as well as one day after OBX surgery. As expected, a single injection of the triple reuptake inhibitor one day after OBX results in an elevation of extracellular dopamine, norepinephrine and serotonin in the prefrontal cortex of OBX as well as Sham animals. However, only the increase in dopamine was significantly higher for Sham animals when compared to the OBX group. This can be explained by the finding that OBX animals had lower dopamine levels one day after OBX, suggesting that less dopamine was available. A DOV 216,303-challenge in chronically treated DOV 216,303-animals also resulted in an increase in extracellular dopamine, norepinephrine and serotonin. However, as can be seen in Fig. 3 the monoamine concentrations in these groups do not reach the same levels as for the acute treatment. This difference in response can be explained by a developed tolerance of the system to DOV 216,303 after chronic use. However, we cannot exclude the possibility of interference with surgery the day before and therefore cannot

58 DOV 216,303 increases extracellular monoamines in the PFC of olfactory bulbectomized rats appropriately compare these two groups, because of differences in experimental set-up. Therefore further experiments are needed to explain this difference in concentrations. Recently, antidepressant effects of DOV 216,303 were observed in the OBX model (Breuer et al 2008). The elevated monoamine levels found in our microdialysis study can be a possible explanation for this antidepressant effect. However, since a single DOV 216,303 administration elicits less monoamine release in the chronically treated group compared to the acutely treated group, it cannot be excluded that, on the long term it is necessary to have DOV 216,303 systemically available to elicit a behavioural effect. In summary, this study showed that one day after OBX, dopamine concentration levels in the prefrontal cortex are significantly decreased in OBX animals, which can be a possible explanation for the anhedonic state of the OBX animals (Slattery et al 2007). 3 A single administration of the triple reuptake inhibitor DOV 216,303 in these animals resulted in a significant increase in all three monoamines, however, for dopamine, this increase was less in OBX than Sham animals. Chronic treatment with DOV 216,303 leads to increased baseline levels of serotonin in the prefrontal cortex, partly explaining c h a p t e r the antidepressant effects found in previous studies (Breuer et al 2008). A challenge with DOV 216,303 elicits elevated levels of serotonin, norepinephrine and dopamine concentrations in OBX and Sham animals. This increased release profile seemed to be lower in the chronically treated DOV 216,303 group compared to an acute injection, but more research is needed to investigate this seemingly hyporesponsivity to chronic DOV 216,303 treatment.

59

S. Quinton S. Jolanda Prins Jolanda Berend Olivier Rudy Schreiber Rudy S. Mechiel Korte Mechiel S. Maria Koen G. C. Westphal C. G. Koen Gerdien A. H. Korte-Bouws study in olfactory bulbectomized rats reuptake inhibitor for reuptake the treatment of major the treatment of DOV as a triple 216,303 depression: A microdialyis depression: Pharmacology, Biochemistry and Behavior 2011, 97: 444-452 The potential and limitations and limitations The potential

c h a p t e r 4 Chapter 4

Abstract DOV 216,303 belongs to a new class of antidepressants, the triple reuptake inhibitors (TRIs), that blocks serotonin, norepinephrine and dopamine transporters and thereby increases extracellular brain monoamine concentrations. The aim of the present study was to measure extracellular monoamine concentrations both in the prefrontal cortex (PFC) and dorsal hippocampus (DH) after chronic administration of DOV 216,303 in the OBX animal model of depression and to compare the effects with acute drug treatment. OBX animals showed lower dopamine levels in PFC upon acute administration of DOV 216,303 than sham animals for up to five weeks after surgery. No such changes were observed in the DH. Unexpectedly, a DOV 216,303 challenge in chronic DOV 216,303 treated sham animals resulted in a blunted dopamine response in the PFC compared to the same challenge in vehicle treated animals. This blunted response probably reflects pharmacokinetic adaptations and/or pharmacodynamic changes, since brain and plasma concentrations of DOV 216,303 were significantly lower after chronic administration compared to acute administration. Surprisingly, and in contrast to what we have reported earlier, chronic DOV 216,303 treatment was unable to normalize the hyperactivity of the OBX animals. Interestingly, by measuring the drug plasma and brain levels, it was demonstrated that at the time of behavioral testing (24 h after last drug treatment) DOV 216,303 was not present anymore in either plasma or brain. This seems to indicate that this putative antidepressant drug has no lasting antidepressant-like behavioral effects in the absence of the drug in the brain.

62 The potential and limitations of DOV 216,303 for the treatment of major depression

Introduction Selective serotonin reuptake inhibitors (SSRIs) are the first line of treatment for major depression. However, 30% of the depressed patients do not respond to these drugs (Steffens et al 1997). Efficacy of SSRIs depends on the severity of the depression (Kirsch et al 2008) and on the specific depressive symptoms (Katz et al 1994). Anhedonia is one of the core symptoms of depression and characterized by loss of pleasure and motivation. It has been postulated that a hypodopaminergic state causes anhedonia (Dunlop and Nemeroff 2007; Nestler and Carlezon 2006). Therefore, drugs that increase dopaminergic activity may be of interest for the treatment of depression. One such class of molecules are triple monoamine reuptake inhibitors (TRIs), such as DOV 216,303, that not only block serotonin and norepinephrine transporters, but also dopamine transporters, thereby increasing synaptic concentrations and function of all three monoamines. TRIs are believed to consist of a novel class of antidepressants because DOV 216,303 and/or its enantiomer(s) increase extracellular monoamines in vitro (Skolnick et al 2006) as well as in vivo (Popik et al 2006; Prins et al 2010), 4 and are effective in animal models of depression (Breuer et al 2008; Skolnick et al 2003a) and in depressed patients (Skolnick et al 2006), thereby supporting the classical monoamine theory of depression. In this theory, depression is associated with decreased synaptic concentrations of the monoamines dopamine, norepinephrine and serotonin c h a p t e r (Schildkraut 1965). Moreover, animals subjected to chronic stress, leading to anhedonia (Willner et al 1992; Willner et al 1987), also show a reduced release of dopamine in the prefrontal cortex in response to palatable food compared to non-stressed rats. This blunted dopamine response could be restored with chronic antidepressants (Di Chiara et al 1999). But a note of caution: it cannot be assumed that every drug that inhibits the three monoamine transporters in vitro will possess clinically efficacious antidepressant activity as illustrated by the recent studies of two putative TRIs, NS2359 and SEP 225289, which did not meet their endpoints in phase 2 depression studies. The olfactory bulbectomized (OBX) rat is considered an animal model of depression (Song and Leonard 2005). Removal of the olfactory bulbs results in hyperactive behavior in a novel stressful environment (Klein and Brown 1969), which can be normalized by chronic, but not acute, antidepressant treatment (Breuer et al 2007; Kelly et al 1997). In a recent microdialysis study in OBX rats, animals had lower extracellular dopamine levels in the prefrontal cortex than their sham counterparts one day after bulbectomy. Furthermore, acute administration of DOV 216,303 lowered extracellular dopamine levels in OBX animals (Prins et al 2010). Such decreased dopamine levels might cause the anhedonic state of these OBX animals as reflected by increased ICSS thresholds (Slattery et al 2007). Moreover, the blunted response to amphetamine on ICSS thresholds and a long-lasting reduction in sucrose intake (Romeas et al 2009), further supported the suggestion that OBX animals have dysfunctional reward circuitries. These findings make this animal model an attractive model to study new antidepressant agents and justify the use of the OBX model to investigate brain mechanisms underlying anhedonia.

63 Chapter 4

Because antidepressants require several weeks before they relieve symptoms of depression in patients, animal studies should ideally run over multiple weeks. Several studies examined the behavioral effects that characterize OBX only ten till fourteen days after surgery (Giardina and Radek 1991; Richman et al 1972) and start antidepressant treatment after three weeks (Breuer et al 2009b). We aimed to assess the effect of an acute DOV 216,303 challenge and repeated drug administration on monoamine release a few weeks after OBX surgery. Monoamines were measured in two brain areas simultaneously, the medial prefrontal cortex (PFC) and the dorsal hippocampus (DH) because disturbed neural mechanisms in these brain areas are both associated with symptoms of depression (Hjorth and Auerbach 1994; Malagie et al 1996). The DH is involved in reward-related processes via contextual cues which are processed via the hippocampus with projections to the nucleus accumbens (Everitt and Robbins 2005), while the PFC is involved in overall cognitive “executive” functioning that allows organisms to get things done, reinforcement, reappraisal and suppression of negative affect (Koenigs and Grafman 2009; Koob and Volkow 2010; Robbins and Arnsten 2009). Moreover, the PFC and DH are both innervated by fibres from the dopaminergic ventral tegmental area (Gasbarri et al 1997; Romanides et al 1999) and the serotonergic raphe nuclei (Acsady et al 1996). Furthermore, we measured the concentration of DOV 216,303 in blood plasma and whole brain after acute and chronic DOV 216,303 administration in OBX and sham animals.

Experimental procedures

Animals Forty-eight male Sprague Dawley rats (Harlan, Horst, the Netherlands) weighing between 290 and 350 g at the time of OBX or sham surgery were socially housed, two or four per cage on a 12 h light-dark cycle with lights on at 6:00h and off at 18:00h. Food and water were available ad libitum. Animals had one week to acclimate to their environment and were subjected to an open field test in week one, four or five days before receiving surgery (Fig. 1). All animal experimental procedures were carried out in accordance with the governmental guidelines and approved by the Ethical Committee for Animal Research of the Faculties of Pharmaceutical Sciences, Chemistry and Biology at Utrecht University, the Netherlands.

Open Field Locomotion was measured in grey open field chambers measuring 70×70×45 cm with use of EthoVision® 3.1 (Noldus). All testing was done during the light period, under 20 lx lighting in the open field. After a 30-minute period, during which the animals acclimated to the test room in their home cage, each animal was placed in a corner of the open field and allowed to explore for 15 min. Animals were tested four times in the open field. The first time was four or five days before OBX/sham surgery. Based on these results animals were randomized across surgery groups. The first post-surgery

64 The potential and limitations of DOV 216,303 for the treatment of major depression

       



         

   

               Figure 1. Experimental design, with time of surgeries, open field tests, chronic drug administration and microdialysis, OF is open field. OF1 is a pre-surgical open field test, OF2 is postsurgery. OF3 is 30 min after start 4 drug treatment, and OF4 is performed two weeks after start of treatment, 30 min before injection.

open field took place two weeks after surgery; based on these results animals were c h a p t e r assigned to treatment groups. Drug treatment was initiated 21 days after surgery and animals were tested 30 min after the first injection. The final open field test took place on the fourteenth day after the start of treatment, 24 h after daily drug administration (Fig. 1).

Olfactory bulbectomy Olfactory bulbectomy (OBX) surgery was performed in animals which were anesthetized by inhalation of isoflurane gas (2-3%), mixed with nitrous oxide and oxygen and animals were placed in a stereotaxic instrument (Kopf, David Kopf Instruments). Lidocaine hydrochloride (2%) + adrenaline (0.001%) were applied in the incision as a local anesthetic. Two burr holes with a diameter of 2 mm were drilled bilaterally, 8 mm anterior to bregma and 2 mm from the midline of the frontal bone overlying the olfactory bulbs. The bulbs were aspirated with a blunt hypodermic needle attached to a vacuum pump. Burr holes were filled up with haemostatic sponge to prevent blood loss. Sham operated animals underwent the same procedure except that their olfactory bulbs were not removed. All incisions were closed with use of 5-0 vicryl rapide suture material and animals received Rimadyl (5 mg/kg, subcutaneously) for pain relief.

Drug treatment Drug treatment started 21 days after OBX/sham surgery. All animals received one injection every day for 17 days. All injections were given by oral gavage. Treatment groups consisted of either vehicle (sterile water) or 20 mg/kg DOV 216,303 [(±)-1-(3,4-dichlorophenyl)-3-azabicyclo-[3.1.0]hexane hydrochloride] synthesized by Sepracor Inc., Marlborough, USA], administered in a volume of 2 ml/kg.

65 Chapter 4

The vehicle-vehicle group received 17 daily vehicle injections in total with the last injection on the microdialysis day. The vehicle-DOV 216,303 group received 16 vehicle injections and one DOV 216,303 (20 mg/kg, p.o.) injection during microdialysis. The DOV 216,303-DOV 216,303 group received 17 injections with the DOV 216,303 compound (20 mg/kg, p.o.), the last injection occurring during microdialysis. The experimental set up is shown in Fig. 1.

Microdialysis probe implantation After 14 days of drug treatment, cuprofane microdialysis probes (MAB 4.7.3 CU) were implanted in the medial prefrontal cortex (PFC) and dorsal hippocampus (DH). The coordinates of the PFC were incisor bar lowered at -3.3, AP: +3.2 mm, ML: ±0.8 mm, DV: -4.0 mm from bregma and dura. For the DH, the coordinates were incisor bar lowered at -3.3, AP: -3.8 mm, ML: ±2.8 mm, DV: -4.0 mm from bregma and skull. Probes were anchored with three screws and dental cement on the skull. After microdialysis probe implantation animals were housed individually until the end of the experiment.

Microdialysis experiment Two days after implantation, microdialysis experiments were performed in conscious freely moving animals. The system was perfused with Ringer solution (147 mM NaCl,

2.3 mM KCL, 2.3 mM CaCl2, and 1 mM MgCl2) with use of a KdScientific Pump 220 series (USA) at constant flow rate of 1 ml/min. Animals were connected to a dual channel swivel (type 375/D/22QM) which allowed them to move relatively unrestricted. During microdialysis, the pump rate was set at 0.09 ml/h. Two hours after connection ten 30-minute samples were manually collected in vials containing 15 µl of 0.1 M acetic acid and frozen at -80 ºC until analysis with HPLC. After 2 h of baseline samples animals were injected orally with either DOV 216,303 (20 mg/kg, 2 ml/kg, p.o.) or vehicle (sterile water), and samples were collected for an additional 3 h. After a drug washout period of three days, all animals were sacrificed and their brains were removed and examined to verify complete olfactory bulb ablation and probe placement accuracy.

HPLC-ECD Microdialysis samples were stored at −80 °C until analysis. The following neurotransmitters and metabolites were measured: norepinephrine (NE), dopamine (DA) and serotonin (5-HT) and 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were detected simultaneously by HPLC with electrochemical detection using an Alexys 100 LC-EC system (Antec Leyden, the Netherlands) (Korte-Bouws et al 1996; Verhagen et al 2009). The system consisted of two pumps, one auto sampler with a 10 port injection valve, two columns and two detector cells. Column 1 (ALF 105 C18 1×50mm, 3 μm particle size) in combination with detector cell 1, separated and detected DA and 5-HT. Column 2 (ALF 115 C18 1×150 mm, 3 μm particle size) in combination with detector cell 2, separated and detected NE and the metabolites. The mobile phase for column 1 consisted of

66 The potential and limitations of DOV 216,303 for the treatment of major depression

50mM phosphoric acid, 8mM KCL, 0.1mM EDTA (pH 6.0), 12% Methanol and 500 mg/L 1-Octanesulfonic acid, sodium salt (OSA). The mobile phase for column 2 consisted of 50mM phosphoric acid, 50 mM citric acid, 8mM KCl, 0.1mM EDTA (pH 3.2), 10% methanol and 500 mg/L OSA. Both mobile phases were pumped at 50 μl/ min. Samples were kept at 8 °C during analysis. From each microdialysis sample 5 μl was injected simultaneously onto each column. The neurotransmitters were detected electrochemically using μVT-03 flow cells (Antec Leyden, the Netherlands) with glassy carbon working electrodes. Potential settings were for DA and 5-HT +0.30 V versus Ag/AgCl and for NE and metabolites +0.59 V versus Ag/AgCl. The columns and detector cells were kept at 35 °C in a column oven. The chromatogram was recorded and analyzed using the Alexys data system (Antec Leyden, the Netherlands). The limit of detection was 0.03 nM (S/N ratio 3:1).

Histology All animals were sacrificed three days after the microdialysis test day. Brains were quickly frozen in isopentane and stored at -80 ºC until verification of probe track. Data were 4 discarded if olfactory bulbs were not completely ablated, or if the microdialysis probe was not in the PFC or DH. c h a p t e r Determination of DOV 216,303 in blood plasma and whole brain A separate group of twenty-four animals was subjected to the same procedures as described above; including open field studies, OBX/sham surgery and drug treatment, except for the microdialysis experiment. Two weeks after OBX/sham surgery, animals were distributed across three different groups (n=8 each) and received either DOV 216,303 (20 mg/kg, p.o) or vehicle (sterile water). One group was treated with vehicle for 14 days and injected with DOV 216,303 on the last day 30 min before sacrifice. One group was treated with DOV 216,303 for 14 days and received a last DOV 216,303 injection on the 15th day, 30 min before sacrifice. A third group of animals was treated with DOV 216,303 for 14 days and sacrificed on the last day, 24 h after the final injection. 5 ml of trunk blood of each animal was collected in a tube containing 280 µl 0.21 M EDTA. After collection, tubes were shaken and kept on ice until centrifugation for 15 min at 4ºC at 3000 RPM. Plasma was collected for measurement of DOV 216,303 and brains were extracted and quickly frozen in isopentane and stored at -80ºC until further use.

Measurement of DOV 216,303 concentrations in plasma and whole brain. In order to determine plasma and brain concentrations, brain aliquots (~200 mg) were diluted to 40 mg/mL with 95:5 water:acetonitrile containing 0.1% formic acid. The mixture was then homogenized using an ultrasonic homogenizer (Sonics model VCX 130; Newtown, CT) for 1 min. A 60 µL aliquot of plasma or homogenized rat brain was placed into a 96 deep-well plate and 200 µL of acetonitrile was added to precipitate the proteins. Samples were vortexed for 1 min, and centrifuged at 4500 RPM for 15 min at 4 °C. 200 µL aliquots of supernatant were transferred to a 96 deep-

67 Chapter 4 well plate, and evaporated to dryness. The samples were then reconstituted in 150 µL 90:10 5 water:acetonitrile with 0.1% formic acid, and centrifuged at 4500 RPM for 15 min at 4°C. A 45 µL aliquot from the supernatant was injected into the LC/MS/ MS instrument. The LC/MS/MS analysis was performed using an Applied Biosystems MDS Sciex API-2000 (Concord, ON, Canada) triple quadruple mass spectrometer using a Turboion® spray source. The MS was coupled to an Agilent 1200 series HPLC system (Santa Clara, CA) and a Leap HTS PAL auto-sampler (Carrboro, NC). DOV 216,303 was optimized by infusion of the 1.0 mg/mL solutions in positive ion mode. Multiple reaction monitoring was used for quantitation using a dwell time of 150 ms. Nitrogen was used as the collision gas at a setting of 5.0 for the collision-induced dissociation (CID). The source temperature was 500 °C. Chromatographic separation was accomplished using an ACE C-8 column (50 mm × 2.1 mm, i.d.; 5 mm; Mac Mod, Chadds Ford, PA). Mobile phases A and B consisted of HPLC grade water or acetonitrile with 0.1% formic acid added, respectively.

Statistical analysis Pre- and post surgical open field data were analyzed by a one-way ANOVA with ‘surgery’ as between factor. Open field data after acute (OF3) and chronic (OF4) treatment were analyzed by a multivariate analysis of variance with locomotion data of OF3 and OF4 as dependent variables and ‘treatment’ and ‘surgery’ as fixed factors. The area under the curve of the dopamine response was calculated using the trapezoid algorithm. To detect significant differences in area under the curve data analysis of variance (one-way ANOVA) was used with Tukey post-hoc comparisons. Baseline microdialysis data were analyzed with use of multivariate analysis of variance, and in case of significant effects followed by one-way ANOVA with post-hoc Tukey tests. Microdialysis data were analyzed with use of repeated measures ANOVA with ‘time’ as within and ‘surgery’ and ‘treatment’ as between factors. When a significant surgery × treatment interaction was found, each timepoint was analyzed by one-way ANOVA, in case of significant ANOVA effects, post-hoc Tukey tests were performed. Where appropriate, all reported results were corrected by the Greenhouse-Geisser procedure. Blood and brain concentrations of DOV 216,303 were analyzed by multivariate analysis of variance with ‘surgery’ and ‘treatment’ as between factors, in case of significant effects post-hoc Tukey tests were performed. All statistical analyses were carried out with the Statistical Package SPSS 16.0. The level of significance in all tests was set a priori at p < 0.05.

Results

Locomotion in open field (Fig. 2) No animals were excluded based on incomplete olfactory bulb ablation. Olfactory bulbectomy increased locomotion compared to sham-operated controls two weeks after

68 The potential and limitations of DOV 216,303 for the treatment of major depression

    







        

   

   4         c h a p t e r





       

Figure 2. Total distance travelled in an open field in 15 min. A. Locomotion before and after OBX/sham surgery. B. Locomotion 30 min after DOV 216,303 injection (acute) and 24 h after the 14th injection in chronically treated animals (chronic). ***p < 0.001; *p < 0.05 compared to their sham counterparts. surgery [F(1, 42) = 32.5, p < 0.001] (Fig. 2A). The OBX animals remained hyperactive during the complete duration of the experiment. After acute treatment, vehicle treated

OBX animals were more active than vehicle treated shams [F(1, 44) = 6.791, p < 0.05]. No effect of acute DOV 216,303 administration could be observed on locomotion in the OBX animals compared to vehicle-treated OBX rats (Fig. 2B). Two weeks after treatment, vehicle-treated OBX animals were more active than their vehicle-treated shams [F(1, 44) = 15.298, p < 0.001]. After 14 days of treatment no difference could be observed in locomotion in the DOV 216,303-treated OBX animals when compared with vehicle-treated OBX rats (Fig. 2B), suggesting that DOV 216,303 administration, both acute and chronic, did not reduce hyperactivity.

69 Chapter 4

Baseline monoamine levels in PFC and DH (Fig. 3) Baseline monoamine levels in PFC were not different between treatment groups. In the DH a significant surgery × treatment interaction was found for baseline serotonin levels [F(1, 32) = 10.798, p < 0.01], while baseline levels for dopamine and norepinephrine were not affected. (Fig. 3). One-way ANOVA with post-hoc Tukey tests revealed significantly higher baseline serotonin levels in the DH of chronic DOV 216,303-treated shams compared to the vehicle-treated sham, vehicle-treated OBX and the DOV 216,303-treated OBX [F(3, 38) = 12.612, p < 0.001].

Extracellular monoamine levels in PFC after drug challenge (Fig. 4)

Histology Some animals were excluded from analysis based on several reasons. Either on incorrect microdialyis probe placement, or because of technical problems during microdialysis sampling or because of drop-out earlier in the experiment, twelve animals in the PFC group and eleven animals in the DH group were excluded. The number of animals used in each treatment group was sham vehicle-vehicle (PFC, n=7; DH, n=8), OBX vehicle- vehicle (PFC, n=6; DH, n=6), sham vehicle-DOV 216,303 (PFC, n=5; DH, n=7), OBX vehicle-DOV 216,303 (PFC, n=5; DH, n=5), sham DOV 216,303-DOV 216,303 (PFC, n=7; DH, n=6), OBX DOV 216,303-DOV 216,303 (PFC, n=6; DH, n=5).

              





       

     





       Figure 3. Absolute baseline concentrations in prefrontal cortex (upper panel) and dorsal hippocampus (lower panel) in sham and OBX animals chronically treated with either vehicle or DOV 216,303. ***p < 0.001 compared to DOV 216,3030-treated OBX and vehicle treated groups.

70 The potential and limitations of DOV 216,303 for the treatment of major depression

Dopamine in PFC An acute challenge of DOV 216,303 increased extracellular dopamine levels in the prefrontal cortex over time [F (4, 54) = 9.503, p < 0.001, ε = 0.215] (Fig. 3A, 3B). A significant surgery × treatment interaction was found [F(2, 28) = 4.853, p < 0.05]. One- way ANOVA analysis per time point with Tukey post-hoc analysis revealed significantly lower dopamine levels at T 60 min and T 90 min in the acute DOV 216,303-treated OBX animals compared to their sham counterparts (p < 0.001) (Fig. 4A). Analysis also

           

               4  

    

 

                    c h a p t e r

   

    

                     

    

 

 

       

                         

Figure 4. Extracellular monoamine concentrations in prefrontal cortex five weeks after OBX/sham surgery, after an acute challenge of DOV 216,303 in chronic vehicle treated animals (A, C and E). And after a DOV 216,303-challenge in animals chronically treated with DOV 216,303 (B, D and F). Timepoints -90 till 0 min represents baseline measurements. At T=0 a single injection with either DOV 216,303 or vehicle was given. Data is represented as percentage of baseline. *p < 0.01, sham vehicle–DOV 216,303 compared to OBX vehicle–DOV 216,303. #p < 0.01 sham DOV 216,303–DOV 216,303 compared to sham vehicle–DOV 216,303.

71 Chapter 4 revealed significantly lower extracellular dopamine concentrations in the chronically DOV 216,303 treated shams with DOV 216,303 challenge when compared to the sham animals with an acute DOV 216,303 challenge at timepoints T 30 min, T 60 min, and T 90 min (p < 0.01). (Fig. 4B). Sham animals, acutely treated with DOV 216,303 showed significant increased area under the curve (AUC) data for dopamine when compared to OBX animals after acute treatment with DOV 216,303 [F(5, 30) = 11.858, p < 0.05] (Fig. 5). A trend was observed towards a decreased AUC for dopamine in chronic DOV 216,303-treated shams, compared to an acute DOV 216,303 administration in sham animals (p = 0.85) (Fig. 5).

Norepinephrine in PFC Administration of DOV 216,303 during microdialysis resulted in significantly elevated extracellular norepinephrine levels over time [F(7, 91) = 15.450, p < 0.001, ε = 0.363] (Fig 4C, 4D). No effect of surgery [F(1, 28) = 0.001, p = 0.975] or surgery × treatment interaction could be observed [F(2, 28) = 1.006, p = 0.358].

Serotonin in PFC DOV 216,303-challenge in vehicle treated animals as well as in chronic DOV 216,303-treated animals resulted in significantly elevated extracellular serotonin levels over time [F(3, 48) = 30.902, p < 0.001, ε = 0.180] (Fig. 4E, 4F). No effect of surgery [F(1, 30) = 0.106, p = 0.747] or surgery × treatment interaction could be found [F(2, 30) = 1.146, p = 0.332].

     



         

Figure 5. Area under the curve (AUC) data from the dopamine response in prefrontal cortex of OBX and sham animals after treatment with vehicle–vehicle, vehicle–DOV 216,303 (acute) or DOV 216,303–DOV 216,303 (chronic). *p < 0.05.

72 The potential and limitations of DOV 216,303 for the treatment of major depression

           

         

 

      

  4                    

    c h a p t e r  

 

      

                     

     

 

 

        

                         

Figure 6. Extracellular monoamine concentrations in dorsal hippocampus five weeks after OBX/sham surgery, after an acute challenge of DOV 216,303 in chronic vehicle treated animals (A, C and E). And after a DOV 216,303-challenge in animals chronically treated with DOV 216,303 (B, D and F). Timepoints -90 till 0 min represents baseline measurements. At T=0 a single injection with either DOV 216,303 or vehicle was given. Data is represented as percentage of baseline.

73 Chapter 4

Extracellular monoamine levels in DH after drug challenge (Fig. 6)

Dopamine in DH Administration of DOV 216,303 in vehicle-treated animals as well as in chronic DOV 216,303-treated animals resulted in significantly elevated extracellular dopamine levels in the dorsal hippocampus over time [F(4,54) = 3.884, p < 0.01, ε = 0.215] (Fig 6A, 6B). Neither a significant effect of surgery [F (1, 28) = 3.636, p = 0.067], nor surgery × treatment interaction could be observed [F (2, 28) = 1.724, p = 0.197].

Norepinephrine in DH Administration of DOV 216,303 during microdialysis resulted in significantly elevated extracellular norepinephrine levels over time [F (4, 57) = 6.752, p < 0.001, ε = 0.218)] (Fig. 6C, D). No effect of surgery [F(1, 29) = 0.368, p = 0.549] and a trend towards a surgery × treatment interaction could be observed [F(2, 2) = 3.236, p = 0.054].

Serotonin in DH One single injection of DOV 216,303 in vehicle treated animals as well as in chronic DOV 216,303-treated animals resulted in significantly elevated extracellular serotonin levels over time [F(3, 46) = 18.341, p < 0.001, ε = 0.171] (Fig 6E, 6F). No effect of surgery [F (1,30) = 0.318, p = 0.577] or surgery × treatment interaction could be found [F (2,30) = 0.871, p = 0.429].

DOV 216,303 plasma and whole brain levels (Fig. 7) A highly significant effect was found of treatment on DOV 216,303 concentrations in brain [F(2, 21) = 119,972, p < 0.001] (Fig 7A) and plasma [F (2,21) = 66.173, p < 0.001] (Fig 7B). DOV 216,303 brain and plasma levels, measured 30 min after drug administration, were significantly lower in the group that received chronic DOV 216,303 when compared to the group that received chronic vehicle. No difference between

                                      

                  

Figure 7. Concentration of DOV 216,303 in whole brain (A) and blood plasma (B) of OBX and sham animals. 30 min are animals treated chronic with vehicle (veh/dov) or DOV 216,303 (dov/dov) and received a DOV 216,303 challenge 30 min before sacrifice. 24 h are animals chronic treated with DOV 216,303 (dov/dov) and sacrificed 24 h after the last injection of DOV 216,303. *p < 0.001.

74 The potential and limitations of DOV 216,303 for the treatment of major depression sham and OBX could be observed. Post-hoc analysis revealed significant differences in DOV 216,303 concentrations between all treatment groups (p < 0.001).

Body weights The weights of the different treatment groups (mean ± S.E.M.) at the start and the end of treatment were as followed: Sham-vehicle 375 ±5 g and 394 ± 5 g, Sham-DOV 216,303 376 ± 4 g and 380 ± 7 g, OBX-vehicle 362 ± 4 g and 381 ± 5 g, OBX-DOV 216,303 356 ± 6 g and 360 ± 4 g. At the start of treatment, three weeks after surgery, sham animals significantly weighed more that OBX animals [F(1, 41) = 7.894, p <0.01]. All animals increased in weight during treatment, however chronic DOV 216,303-treated OBX animals gained less weight than vehicle-treated OBX animals [F(1, 19) = 6.945, p < 0.05].

Discussion 4 Acute administration of the triple reuptake inhibitor (TRI) DOV 216,303 in olfactory bulbectomized (OBX) rats, five weeks after surgery, results in an increase in dopamine (DA), norepinephrine (NE) and serotonin (5-HT) in the prefrontal cortex (PFC) and dorsal hippocampus (DH). Only for DA in the PFC this increase was lower in OBX c h a p t e r than sham animals. For the other monoamines (NE and 5-HT) no difference was found between sham and OBX. Thus, a hypodopaminergic state may be responsible for the long term behavioral depressive-like effects in the OBX model. The same challenge with DOV 216,303 in chronic DOV 216,303-treated animals also leads to a significant increase in the same three neurotransmitters. In the chronically treated animals no differences were found between sham and OBX on monoamine release. However, the same challenge of DOV 216,303 elicits higher increases in DA levels in the PFC of sham animals when given for the first time than in shams chronically treated with DOV 216,303 at the same time point after surgery. Our microdialysis data are in accordance with our previous results which showed that an acute DOV 216,303 challenge leads to a lower DA response in the prefrontal cortex of OBX rats when compared to sham animals (Prins et al 2010). However, in our previous study, we measured this DA release one day after the OBX surgery. It appears that the same DOV 216,303 challenge five weeks after surgery leads to a similar blunted response in these OBX animals. A recent study found that three weeks after OBX, amphetamine-induced lowered ICSS thresholds were more profound in sham animals than in OBX animals. They also showed that OBX animals had a long lasting reduction in sucrose intake, suggesting that these animals have a dysfunctional reward circuitry and respond less to normal pleasurable stimuli (Romeas et al 2009). Our finding that OBX animals can release less DA than their sham counterparts five weeks after surgery is a possible explanation for this blunted response to rewarding stimuli. Another study reported increased ICSS thresholds in rats following OBX surgery, suggesting an anhedonic state of the animals (Slattery et al 2007). Remarkably, this effect only lasted for eight days, therefore it may have been due to a surgery effect rather than a disrupted

75 Chapter 4 reward circuitry. Nevertheless, in our previous microdialysis study we found lower baseline DA levels one day after OBX, which is consistent with the data from Slattery and co-workers, assuming that lower DA levels in the prefrontal cortex correlate with higher ICSS thresholds and thus an anhedonic state. In our previous study a challenge with DOV 216,303 seemed to result in lower extracellular monoamine concentrations in chronically DOV 216,303 treated animals compared to the same challenge in untreated animals (Prins et al 2010). However, a definitive conclusion could not be made at the time, due to limitations inthe experimental design and the absence of specific controls. In the current experiment we reproduced our previous findings. A single administration of DOV 216,303 in chronic DOV 216,303-treated animals led again to a blunted response on DA release compared to the same challenge given to chronic vehicle-treated shams. This was only significant for DA release in the prefrontal cortex. For the other monoamines and in the dorsal hippocampus this blunted response to a DOV 216,303 challenge was also marginally present in sham animals, albeit not significant. One possible explanation could be that tolerance to the effects of the drug has developed (Cohen and Baldessarini 1985) or that less drug is available in chronic treated animals, due to an increased drug metabolism in chronic treated animals (Brosen and Naranjo 2001). Our results contrast with a previous study in which Van der Stelt and co-workers showed that OBX animals had permanent deficits in serotonergic function; OBX rats showed lower 5-HT levels in the basolateral amygdala (BLA) and dorsal hippocampus two weeks and five months after surgery. When they blocked the 5-HT transporter (SERT) with fluvoxamine, the increase in 5-HT was smaller in OBX animals than in shams (van der Stelt et al 2005). We did not see such a difference between OBX and sham animals on extracellular 5-HT concentrations in DH and PFC after blockade of the SERT with DOV 216,303. However, this difference could be explained by different binding occupancies of the SERT by fluvoxamine (Suhara et al 2003) and DOV 216,303 (Chen and Skolnick 2007; Micheli et al 2010). To further explore why chronically treated animals display a blunted monoamine response, the effects of chronic and acute administration of DOV 216,303 on the concentrations of this compound in blood plasma and whole brain were examined. A major finding of the present study is that 30 min after DOV 216,303 administration, concentrations of DOV 216,303 in whole brain and plasma were significantly reduced in chronic treated DOV 216,303 animals, when compared to rats that were presented with DOV 216,303 for the first time. Moreover, very little DOV 216,303 was still present in the brain or plasma 24 h after an injection with DOV 216,303 in chronic DOV 216,303-treated animals. Therefore we conclude that in chronic treated animals, DOV 216,303 is cleared more rapidly from the body than when given for the first time. However, this finding may be species specific, In a human study, DOV 216,303 was given once and daily for ten days and the plasma concentrations did not differ between day one and ten (Beer et al 2004). The mean elimination half-time after one single dose was 3.3 to 4.4 h for six different doses, which means that after 24 h the compound should have been cleared from the body. This is confirmed by our data that 24 h after injection almost no DOV 216,303 is present in chronic treated animals. A reason for

76 The potential and limitations of DOV 216,303 for the treatment of major depression the increased elimination of DOV 216,303 after chronic use could be changes in the pharmacokinetic properties of the drug such as liver enzymes upregulation leading to faster rates of elimination from the body (Brosen and Naranjo 2001), a phenomenon which can occur after treatment with SSRIs or other antidepressants (DeSanty and Amabile 2007; Solomons et al 2005). The consequence of these findings in patients is that DOV 216,303 should be given twice or three times a day in order to be clinically effective (Beer et al 2004; Skolnick et al 2006). However, these are only speculations; based on the present study we cannot conclude anything about an induction of liver enzymes. Further experiments are needed to confirm this hypothesis. An increased elimination of DOV 216,303 might also explain an apparent discrepancy between the present behavioral data and previous work from our lab. Here, we also looked at the effect of DOV 216,303 on OBX-induced hyperactive locomotion in the open field test. Neither acute, nor chronic treatment with DOV 216,303 could normalize OBX-induced hyperactivity. In a previous study in our lab chronic drug treatment, but not acute administration of DOV 216,303, had antidepressant effects

in the OBX animal model of depression (Breuer et al 2008). An important difference 4 between our study and the study from Breuer and co-workers is that in the present study the chronic open field was performed 24 h after the last injection, while in the study from Breuer and colleagues, the animals had received the last injection 30 min c h a p t e r before the chronic open field. These behavioral data are completely in line with the DOV 216,303 concentrations we measured in blood and brain of chronically DOV 216,303 treated animals. Thirty min after the DOV 216,303 injection DOV 216,303 was still systemically available, while 24 hours after the DOV 216,303 injection no DOV 216,303 was present any longer. Although, in our hands DOV 216,303 does not seem to be the perfect alternative candidate for SSRI antidepressant treatment, the important role of dopaminergic function in the pathophysiology of major depressive disorder should not be underestimated (D’Aquila et al 2000; Guiard et al 2009a; Naranjo et al 2001). Recently it has been shown that the D2/D3 receptor agonist pramipexole acted by augmenting the antidepressant effect while given together with regular antidepressant pharmacological treatment (Goldberg et al 2004; Gupta et al 2006). Chronic administration of pramipexole showed antidepressant effects in the OBX animal model (Breuer et al 2009a). Furthermore, pramipexole seemed to be effective in reducing anhedonia in Parkinson’s patients as an additive treatment to L-dopa (Lemke et al 2006) giving further evidence for a role for DA in anhedonia. Several antidepressants, each acting on one or more monoamines, are involved in the therapeutic effect of different behavioral aspects of depression (Katz et al 2004). Drug-induced changes in the serotonergic system seem to be associated with anxiolytic and antidepressant effects, while changes in NE are primarily involved in reducing psychomotor symptoms in humans diagnosed for depression (Katz et al 1994). In animals, the same distinction can be found in that SNRIs increase climbing behavior without affecting swimming in the forced swim test, whereas SSRIs increased swimming without affecting climbing (Lucki 1997). In summary, the present study showed that even five weeks after surgery, OBX animals showed lower DA levels upon acute administration with the TRI DOV 216,303.

77 Chapter 4

This is consistent with the notion that this animal model possesses an anhedonia component. Furthermore, chronic treatment with DOV 216,303 resulted in a blunted extracellular monoamine response in sham animals after a DOV 216,303 challenge, most likely due to the lower brain and plasma drug levels following repeated treatment. Surprisingly, chronic DOV 216,303 treatment did not have antidepressant behavioral effects in the present OBX animals. Interestingly, by measuring the drug plasma and brain levels, it was demonstrated that at the time of behavioral testing (24 h after last drug treatment) DOV 216,303 was not present anymore in either plasma or brain. This seems to indicate that this putative antidepressant drug has no lasting antidepressant- like behavioral effects in the absence of the drug in the brain.

78

Jolanda Prins Jolanda Paul J. Kenny J. Paul Berend Olivier Rudy Schreiber Rudy Ivo DoomernikIvo S. Mechiel Korte Mechiel S. self-stimulation in rats brain reward activity as brain reward lasting enhancement of lasting enhancement measured by intracranial measured DOV216,303 induces long- European of Journal Pharmacology 2012, 693(1-3): 51-56 The triple reuptake inhibitor reuptake inhibitor The triple

c h a p t e r 5 Chapter 5

Abstract Triple reuptake inhibitors (TRIs) are potential new antidepressants, which not only enhance brain serotonin and norepinephrine concentrations but also increase dopamine levels. Therefore TRIs are believed to have faster therapeutic onset than SSRIs, and may be particularly useful for the treatment of anhedonia (i.e. inability to experience pleasure), one of the core symptoms of major depression. The current study aimed at getting better insight into the rewarding properties of DOV 216,303, which is a TRI, regarding its possible use to treat anhedonia. It is known that psychostimulant drugs lower intracranial self-stimulation (ICSS) reward thresholds, reflecting enhanced brain reward activity, whereas withdrawal from those compounds mostly results in increased ICSS thresholds. Therefore we assessed the effects of DOV 216,303 on ICSS thresholds in rats. Animals were trained in the discrete-trial current- threshold procedure and after stable ICSS reward thresholds were established, animals received one injection per day of DOV 216,303 (20 mg/kg) or amphetamine (5 mg/ kg) for four consecutive days. ICSS thresholds were assessed 3, 6, and 23 h after each injection. DOV 216,303 decreased ICSS thresholds up to 6 h after drug treatment. To our knowledge this is the first time that a triple reuptake inhibitor, DOV 216,303, induces relatively long-lasting enhancement of brain reward activity. Elevated ICSS thresholds were found after amphetamine administration, which is consistent with previously reported reward deficits induced after amphetamine-withdrawal.

82 DOV 216,303 induces long-lasting enhancement of brain reward activity in rats

Introduction Pharmacological treatment of Major Depressive Disorder is mainly based on increasing serotonin and/or norepinephrine levels by selective serotonin reuptake inhibitors (SSRIs) and norepinephrine reuptake inhibitors (NRIs) or dual acting drugs, acting on both serotonin and norepinephrine (SNRIs). Nevertheless, it takes a relatively long time before the therapeutic effects of SSRIs are established. Moreover, anhedonia (one of the core symptoms of major depression), which is defined as the inability to experience pleasure, is often not alleviated with currently available antidepressants. A role for dopamine in the pathophysiology of depression has been postulated and extensively reviewed (Dunlop and Nemeroff 2007; Kapur and Mann 1992; Naranjo et al 2001; Nestler and Carlezon 2006). Evidence for disturbed dopaminergic reward function in depression comes from preclinical research in which the dopamine response after palatable food is blunted in the chronic mild stress animal model for depression (Di Chiara et al 1999). Moreover, clinical research also demonstrated a role for a dysfunctional reward system in depression (Tremblay et al 2002; Tremblay et al 2005). Co-targeting of the dopaminergic system has been proven to be effective in antidepressant treatment. Bupropion, which is a dopamine reuptake inhibitor (DRI) and NRI, augments antidepressant treatment with SSRIs (Trivedi et al 2006). Moreover, bupropion alone shows antidepressant effects (Dhillon et al 2008b) and even enhances

brain reward function in rats (Cryan et al 2003a). Furthermore, the D2/D3 receptor 5 agonist pramipexole has antidepressant-like effects in an animal model of depression (Breuer et al 2009a), and can also augment the effects of SSRI-based antidepressants (Goldberg et al 2004; Gupta et al 2006). These insights have led to the development of triple reuptake inhibitors (TRIs). TRIs c h a p t e r are considered as a new class of antidepressants (Guiard et al 2009a; Marks et al 2008b; Millan 2009; Skolnick and Basile 2007). DOV 216,303 is such a TRI, which enhances brain serotonin, norepinephrinergic and dopaminergic neurotransmission (Prins et al 2010; 2011b; Skolnick et al 2006). By impacting dopaminergic neurotransmission, TRIs are believed to have faster therapeutic onset than SSRIs (Willner 1997a), and may be particularly useful for the treatment of anhedonia (Marks et al 2008a). The current study aimed at getting better insight into the rewarding properties of DOV 216,303. In this study only one dose of DOV 216,303 (20 mg/kg, p.o.) was used which had shown to have a clear monoamine release profile, including an increase in dopaminergic neurotransmission, even after repeated administration (Prins et al 2010; 2011b). A similar monoamine release profile has been observed after DOV 216,303 (20 mg/kg, i.p.). To assess the potential of DOV 216,303 to activate brain reward systems we examined the effects of acute, sub-chronic and after cessation of treatment with DOV 216,303 and amphetamine on intracranial self-stimulation (ICSS) thresholds in rats. ICSS is considered as a direct measure of brain reward (Carlezon and Chartoff 2007; Kenny 2007; Markou and Koob 1992). The hypothesis of the current study is that DOV 216,303, which is also known to increase dopaminergic neurotransmission, increases brain reward functioning as reflected by decreasing ICSS thresholds.

83 Chapter 5

Material and methods

Animals Twenty-four male Wistar rats (Harlan, Horst, the Netherlands) weighing between 290 – 350 grams at the time of surgery were socially housed, four per cage on a 12h light- dark cycle with lights on at 6:00h and off at 18:00h. Food and water were available ad libitum, except for the first 3 days of ICSS training where animals were given 20 g food per animal per day in order to have a more optimized training of the animals (Carr 1990). All animal experimental procedures were carried out in accordance with the government guidelines and approved by the Ethical Committee for Animal Research at Utrecht University, the Netherlands.

Surgery Animals were anesthetized by inhalation of isoflurane gas (2-3%), mixed with nitrous oxide and oxygen and placed in a stereotaxic instrument (Kopf, David Kopf Instruments). Lidocaine hydrochloride (2%) + adrenaline (0.001%) were applied in the incision as local anesthetic. Bipolar ICSS electrodes (cut to a length of 11 mm) were implanted into the lateral hypothalamus (LH). The coordinates of the LH were AP: -0.5 mm from bregma; ML: ±1.7 mm from bregma; DV: -8.3 mm from dura. The incisor bar was adjusted to 5 mm above the interaural line (Pellegrino et al 1979). Electrodes were anchored with four screws and dental acrylic on the skull. All animals received Rimadyl (5 mg/kg, subcutaneously) for pain relief twice daily, up to a total of four injections.

ICSS apparatus All ICSS experiments were performed in eight sound-attenuating operant chambers (30.5 × 30 × 17 cm) with a grid floor and a wheel manipulandum in one of the sides. The implanted electrode was connected to the electrical stimulator through a swivel and a bipolar connector cable (Plastics One), ensuring unrestrained movement throughout the ICSS procedure. The electrical stimulations were delivered by a constant current stimulator (Med Associates Inc.). The stimulator was connected to a computer running MED-PC IV (Med Associates Inc.) controlling all stimulation settings, programs and recording of data.

ICSS procedure In order for the animals to make the association that turning the wheel results in electrical stimulation, all animals were initially trained to turn the wheel manipulandum on a fixed ratio 1 schedule of reinforcement. In this training phase each quarter turn of the wheel resulted in an electrical stimulus with train duration of 500 ms. After several successful training sessions (more than 1000 turnings in 30 minutes), the rats were trained on a discrete-trial current-threshold procedure according to the procedure described by Markou and Koob (1992). At the start of a trial, rats received a free, non- contingent stimulus and had 7.5 s to react and turn the wheel a quarter turn to obtain

84 DOV 216,303 induces long-lasting enhancement of brain reward activity in rats a second, contingent stimulus (positive response) of the same current intensity and duration (100 ms). In case no response occurred during the 7.5 s period, a negative response was recorded. The 7.5 s period in which a positive or negative response occurred was followed by an inter trial interval (ITI) with a duration range from 7.5 to 12.5 s. Responses during the ITI resulted in a delay of onset of the next trial of 12.5 s. Turning the wheel in the 2 s after a positive response did not have further consequences. Responses during this period often reflect the force with which the animal turned the wheel, because a powerful pull will result in more than a quarter turn of the wheel. Animals were subjected to alternating descending and ascending series of current intensities starting with a descending series. The stimulus intensity of the first series was set 40 µA above each animal’s own baseline. Current levels were presented in sets of five trials of the same current intensity and altered by steps of 5µA.

Parameters

ICSS thresholds The current threshold for a series was defined as the midpoint between two consecutive current intensities for which animals responded in at least three of the five trials and two consecutive current intensities for which animals did not respond in three or more of the five trials. The overall threshold of the session was defined as the mean of the

thresholds of the four alternating descending and ascending series. Stable thresholds 5 were defined as less than 10% change in threshold over three consecutive days after at least 10 days of testing.

Response latencies c h a p t e r The response latency is the time between the presentation of the non-contingent stimulus and the turning of the wheel by the animal. The overall response latencies were defined as the mean response latency of all trials during which a positive response occurred.

Drugs Animals were divided into three treatment groups. Treatment groups consisted of vehicle (n=4, sterile water, administered p.o. by oral gavage and n=3, 0.9% saline, administered i.p.), 5 mg/kg d-amphetamine (dissolved in 0.9% saline administered i.p. in a volume of 1 ml/kg, n=6), or 20 mg/kg DOV 216,303 [(±)-1-(3,4-dichlorophenyl)- 3-azabicyclo-[3.1.0]hexane hydrochloride] synthesized by Sepracor Inc., Marlborough, USA, (dissolved in sterile water and administered p.o. in a volume of 2 ml/kg by oral gavage, n=7).

Experimental design An overview of the experimental set-up is pictured in figure 1. After stable ICSS thresholds were established (less than 10% change in threshold over three consecutive days), animals were randomly assigned to their treatment groups. Either vehicle,

85 Chapter 5

ICSS tests (hours after injection)

3 6 23 3 6 23 3 6 23 3 6 23

1st 2nd 3rd 4th

Injection with either - vehicle - D-amphetamine (5 mg/kg, i.p.) or - DOV 216,303 (20 mg/kg, p.o.) Figure 1. Experimental design of the study. After ICSS surgery, training and until stable ICSS thresholds were established (not shown), animals received one injection per day of d-amphetamine, DOV216,303 or vehicle for four consecutive days. ICSS thresholds were assessed 3, 6, and 23 hours after each injection. d-amphetamine or DOV 216,303 was administered and ICSS thresholds were measured 3, 6 and 23 hours after drug treatment. At 24 hours, the second drug treatment was given and again ICSS thresholds were measured at 3, 6 and 23 hours after administration. In total four injections were given on four consecutive days and 3, 6 and 23 h after each drug treatment ICSS thresholds were measured. Furthermore ICSS thresholds were regularly measured from day three up to 10 days after the last drug treatment.

Statistical analysis Mean absolute baseline thresholds and response latencies (± S.E.M.) were measured for each experimental group. All ICSS current-threshold were expressed as a percentage of the baseline data. Response latencies were expressed as absolute values (s). ICSS current-thresholds and response latencies were analyzed by repeated measures ANOVA with time (three levels: 3, 6 and 23h) and day (four levels: day 1-4) as within subject factors and treatment (vehicle, d-amphetamine and DOV 216,303) as between subject factor. In case of significant overall effects or interaction effects, effects of treatment individual time points were analyzed with a one-way ANOVA following post-hoc Bonferroni comparisons. When the assumption of sphericity was violated, reported results were corrected by the Greenhouse-Geisser correction factor.

Results

Baseline ICSS thresholds Mean (± S.E.M.) baseline ICSS thresholds were calculated by taking an average from thresholds 10 days prior to the start of treatment. Mean absolute baseline ICSS thresholds were 104.1 ± 12.7 µA, 108.6 ± 12.2 µA and 93.6 ± 8.4 µA for the control, amphetamine and DOV 216,303 treated groups respectively. As expected, no significant

86 DOV 216,303 induces long-lasting enhancement of brain reward activity in rats difference could be found between the two different vehicle treatments [F(1, 5) = 0.302], therefore the water and saline groups were taken together and in further analysis considered as one vehicle group.

ICSS thresholds after treatment Repeated measures ANOVA with time and day as within factors and treatment as between subjects factor revealed a significant effect of treatment [F(2, 17) = 5.850, p=0.012]. Furthermore, an interaction between time and the three different treatments [F(4, 34) = 7.997 p<0.001] was found. One-way ANOVA per time point revealed significant differences between treatment groups. Although a large variation in individual data points 3 hours after amphetamine treatment was observed (see also Fig. 4). Post-hoc bonferroni comparisons between amphetamine-treated and DOV 216,303-treated groups revealed significant differences at time point t3, t6 and t23 of

                                        5           c h a p t e r                                           

   

            

  

             

Figure 2. Percentage change in ICSS thresholds after treatment. A. ICSS thresholds 3, 6, and 23 hours after each injection for four consecutive days. B. Average ICSS thresholds per time point. # p < 0.05 compared to other treatment group (DOV 216,303 or d-amphetamine) * p < 0.05 compared to vehicle group. Bars represent mean ± S.E.M.

87 Chapter 5 day 1, and differences at t6 and t23 of day 2, 3 and 4 (Fig. 2A). No effect of day [F(2, 31) = 1.967, ε = 0.607] or day × treatment interaction [F(4, 31) = 1.646 ε = 0.607] could be observed. Therefore, the 3, 6 and 23 hours measurements of all days were averaged and visualized in figure 2B. One way ANOVA on these averaged data revealed significant effects at time point t3 [F(2, 19) = 3.818, p = 0.043], t6 [F(2, 19) = 7.671, p = 0.004] and t23 [F(2, 19) = 8.812, p = 0.002]. Post-hoc bonferroni comparisons revealed a significant decrease in thresholds 6 and 23 hours after DOV216,303 administration compared to the same time points after amphetamine administration. Compared to the vehicle group, post-hoc bonferroni comparisons showed significant elevations in ICSS thresholds at t23 after amphetamine administration, reflecting decreased reward values of the stimulation. Significant decreases in ICSS thresholds were observed until 6 hours after DOV 216,303 administration when compared to vehicle. Since ICSS thresholds showed no significant differences between the treatment groups from day three up to 10 days after the last injection, these data are not presented.

ICSS thresholds vehicle group As can be seen in figure 2A and 2B, ICSS thresholds of the vehicle group increase during the day, where t23 is in the morning, at the beginning of the light cycle and t6 is in the afternoon, 2h before the lights went off. One way ANOVA revealed significant differences between time points at day 2 [F(2, 20) = 7.219, p < 0.01] and day 3 [F(2, 20) = 4.810, p < 0.05]. Post-hoc bonferroni comparisons revealed a trend towards an increased threshold at t3 compared to t23 at day 2 (p = 0.055) and day 3 (p = 0.053) and significant increases in thresholds at t6 compared to t23 at both days.

Response latencies Mean (± S.E.M.) baseline response latencies were calculated by taking an average from response latencies 10 days prior to the start of treatment. Mean absolute baseline ICSS response latencies were 2.86 ± 0.10 s, 2.73 ± 0.17 s and 2.44 ± 0.16 s for the control, amphetamine and DOV 216,303-treated groups respectively. Repeated measures ANOVA with ‘time’ and ‘day’ as within factors and treatment as between subjects factor

      

                                 

                                          

Figure 3. Absolute response latencies (s) 3, 6, and 23 hours after each injection for four consecutive days. B = baseline (average response latencies ten days prior to the start of treatment).

88 DOV 216,303 induces long-lasting enhancement of brain reward activity in rats revealed only a significant main effect of day [F(2, 28) = 4.289 p=0.031] (Fig. 3). No significant difference could be observed between treatment groups [F(1, 17) = 0.718] (see Fig. 3).

Discussion The most important finding of the present study is that the triple reuptake inhibitor (TRI) DOV 216,303 leads to long-lasting enhancement of brain reward activity, reflected by long-lasting decreased intracranial self-stimulation (ICSS) thresholds. Such long-lasting hedonic effects were not observed in the amphetamine-treated animals. Moreover, amphetamine-treated animals showed withdrawal-like reward deficits (elevated ICSS thresholds) 23 hours after administration. No significant differences between response latencies of the different treatment groups were observed (see Fig. 3), indicating that a possible hyperactivity or behavioral impairment due to drug treatment do not attribute to the observed changes in ICSS thresholds (see Fig. 2). ICSS thresholds are considered as a direct measure of brain reward function in rats and mice (Carlezon and Chartoff 2007; Kenny 2007; Markou and Koob 1992). Many psychostimulant drugs (e.g. amphetamine, cocaine, and heroin) amplify reward signals in the brain reflected in lowering of ICSS thresholds (Kenny 2007). Drug-induced lowering of ICSS thresholds can be explained by increased reward signaling resulting in potentiating the reward perceived by ICSS. On the contrary, withdrawal-induced 5 elevations of ICSS thresholds can be explained by decreased activity of reward circuitry and desensitization of the rewarding effects of ICSS (Carlezon and Chartoff 2007; Kenny 2007; Markou and Koob 1992). Previously, it was shown that rats increased their c h a p t e r levels of heroin self-administration to avoid this withdrawal-associated state of negative reward, suggesting that withdrawal-induced reward dysfunction serves as a substrate for













    

        

Figure 4. Individual data points of percentage change in ICSS thresholds 3 hours after amphetamine treatment on day 1, 2, 3 and 4. The horizontal dashed line represents the 0% baseline ICSS threshold.

89 Chapter 5 negative reinforcement that also contributes to the development of habitual drug use (Kenny et al 2006). The relatively long-lasting stimulatory effects (up to 6 hours) on brain reward by DOV 216,303 observed in the current study are quite different from the transient effects of psychostimulant drugs like amphetamine (Leith and Barrett 1976; Lin et al 2000) and cocaine (Kenny et al 2003), which only decrease ICSS thresholds for a relatively short period (from 15 minutes up to 3 hours). In the present study we focused especially on long-lasting drug effects on brain reward systems and we therefore might have missed the early short-term hedonic effects of amphetamine. Although many amphetamine- treated animals did show decreases in ICSS thresholds (see Fig. 4), this is not reflected in the mean value because some individuals already show strong withdrawal effects, i.e. increased ICSS thresholds, 3 hours after amphetamine treatment. This explanation is in agreement with earlier findings in which it was shown that after acute amphetamine administration short-lasting decreased ICSS thresholds were observed followed in time by an increase in ICSS thresholds, reflecting drug withdrawal effects (Cryan et al 2003b; Kornetsky and Esposito 1979; Lin et al 1999; 2000). Remarkably, no such withdrawal effects were detected after DOV 216,303 treatment. Even 23 hours after DOV 216,303 treatment, no withdrawal-associated reward deficits were present, since no elevated ICSS thresholds were observed. However, to be absolutely sure that no withdrawal effects take place, in the future ICSS thresholds also have to be measured for more time-points (between 24 and 48 hours) after DOV 216,303 treatment. Our data with DOV 216,303 are in accordance with other studies in which monoamine reuptake inhibitors have an effect on brain reward sensitivity. The norepinephrine/dopamine reuptake inhibitor (NDRI) bupropion dose-dependently enhanced brain reward function 30 minutes after treatment. But this effect was relatively short-term, because 24 hours after treatment ICSS thresholds were back to baseline levels (Cryan et al 2003a). Data points in between were not reported, so nothing is known about the time-course of this effect. Furthermore, the NRI desipramine lowered ICSS thresholds 30 minutes after administration, but had no effect after chronic treatment (Paterson et al 2008). Neither acute nor chronic treatment nor withdrawal from the SSRI fluoxetine altered ICSS thresholds. Although chronic fluoxetine did not have an effect themselves on brain reward systems, they did alter the ability of amphetamine to increase ICSS thresholds (Lin et al 1999). However, other studies showed opposite effects in that acute as well as chronic treatment with fluoxetine elevated reward thresholds (Lee and Kornetsky 1998). Moreover, another study showed that chronic blockade of the serotonin transporter by fluoxetine, or a deletion of this transporter in SERT knockout rats, results in reduced responding for natural food reward (Sanders et al 2007), suggesting that the serotonin system is involved in reward-related processes (Kranz et al 2010). So, our data showed an increased sensitivity of brain reward systems by DOV 216,303 for a longer period than reported in these studies. The combination of increasing all three monoamines at the same time might be a possible explanation for this enhancement of brain reward function, which might be a key therapeutic advantage in the treatment of anhedonia.

90 DOV 216,303 induces long-lasting enhancement of brain reward activity in rats

Since DOV 216,303 enhances dopaminergic neurotransmission, its possible use as an antidepressant drug raises concerns related to its abuse potential. Cocaine (which is like DOV 216,303 also a TRI) or amphetamine also increase dopamine neurotransmission, and are known for their intrinsic abuse potential and reinforcing effects (Kuhar et al 1991). It is known that rats titrate their patterns of self-administration of intravenous cocaine (Kenny et al 2003), heroin (Kenny et al 2006) or nicotine (Kenny and Markou 2006) at a level that achieves maximal drug-induced lowering of ICSS thresholds. Thus, the stimulatory effects of these drugs on brain reward systems represent an important source of positive reinforcement that motivates habitual consumption (Kenny 2007). Before it can be concluded that DOV 216,303 does not have abuse potential, similar self-administration experiments as described above have to be performed. Recently, a preclinical study assessing the potential abuse liability of DOV 216,303 showed that the compound only partially substituted cocaine in a drug-discrimination assay in rats and produced locomotor sensitization in mice at doses that are at least six times higher than the minimally effective dose in antidepressant tests (Caldarone et al 2010). Furthermore, a clinical trial did not mention adverse effects as abuse potential of DOV 216,303 (Beer et al 2004; Skolnick et al 2006). Moreover, a recent study showed that (DOV 21,947 or EB-1010, the active enantiomer of DOV 216,303) significantly improved symptoms (including anhedonia), in patients with major depressive disorder without observing adverse side effects (Tran et al 2012). Nevertheless, more experiments have to be performed before it can be concluded that DOV 216,303 has no addictive properties 5 at all. In conclusion, DOV 216,303 (20 mg/kg, p.o.), which has previously shown to

have an antidepressant-like action in the forced swim test in rats and mice (Caldarone c h a p t e r et al 2010), can activate brain reward systems for a relatively long period. Therefore it is hypothesized that TRIs, especially due to their dopaminergic component, can be used to treat anhedonia. Enhancement of dopaminergic neurotransmission in antidepressant treatment, however, should be carefully investigated with regards to abuse potential.

Acknowledgements The authors would like to thank Koen Westphal, Gerdien Korte-Bouws and Hans Sturkenboom for their excellent technical assistance and animal upkeep.

91

Jolanda Prins Jolanda Rudy Dupree Rudy reward Berend Olivier S. Mechiel Korte Mechiel S. Ronald S. Oosting S. Ronald Damiaan A. Denys Koen G. C. Westphal C. G. Koen Gerdien A. H. Korte-Bouws receptor agonist receptor 1A/1B eltoprazine alters brain eltoprazine monoamine levels without monoamine enhancing brain stimulation brain stimulation enhancing The 5-HT

c h a p t e r 6 Chapter 6

Abstract Selective serotonin (5-HT) reuptake inhibitors are the most prescribed antidepressants. Disadvantages of these agents are the adverse side effects and long therapeutic lag- time. Recently the dopamine (DA) system has been postulated as therapeutic target for the treatment of depression. Increasing DA transmission is believed to be particularly useful for the treatment of anhedonia, one of the core symptoms of depression. It has also been shown that 5-HT-DA interactions are critical for a fast onset of action of antidepressants in animal models for depression. The current study aimed

at linking the monoaminergic neurotransmission profile of eltoprazine, a 5-HT1A/1B receptor agonist, with possible concurrent changes in ICSS thresholds. Rats were treated with vehicle or eltoprazine (1, 3, 10 or 30 mg/kg, i.p.) and in vivo microdialysis in the prefrontal cortex (PFC) or in the nucleus accumbens (NAc) was carried out. In a separate study, rats were trained in a discrete-trial current-threshold ICSS procedure. Once stable ICSS thresholds were established, eltoprazine (1 and 3 mg/kg, i.p.) was administered using a within-animal design. The microdialysis data showed that eltoprazine dose-dependently increased, both in the NAc and the PFC, DOPAC and HVA levels and decreased 5-HT and 5-HIAA concentrations. Furthermore, in the PFC, but not in the NAc, significant increases in DA and NE were found. Surprisingly, eltoprazine increased ICSS thresholds, suggesting a lowered brain reward functioning.

In conclusion, this study demonstrated that the 5-HT1A/1B receptor agonist eltoprazine was able to elicit a clear 5-HT-DA and 5-HT-NE interaction, confirming the already existing evidence that the serotonergic, dopaminergic and norepinephrinergic systems are heavily modulated by each other through a high degree of connectivity between monoaminergic projection areas. Furthermore, our study confirmed recent evidence that enhancement of dopaminergic activity alone is not enough to enhance brain reward systems.

94 Eltoprazine alters brain monoamine levels without enhancing brain stimulation reward

Introduction The most prescribed antidepressant drugs are selective serotonin (5-HT) reuptake inhibitors (SSRIs). Unfortunately, this class of drugs do not work in all patients, they have a long therapeutic lag-time and induce severe side effects. Increasing dopamine (DA) transmission is believed to be particularly useful for the treatment of anhedonia, one of the core symptoms of depression (Dunlop and Nemeroff 2007; Nestler and Carlezon 2006; Prins et al 2011a). Therefore, the DA system has been postulated as a therapeutic target for effective antidepressant drugs (Millan 2009). 5-HT-induced DA release (i.e. 5-HT-DA interaction) was shown to be critical for a fast onset of action of antidepressants in an animal model for depression (Dremencov et al 2004). A role for mesocorticolimbic dopaminergic pathways and increased prefrontal cortical DA concentrations have been hypothesized to contribute to the mechanism of action of antidepressants (Tanda et al 1994). Furthermore, combining an SSRI with the dopamine/ norepinephrine reuptake inhibitor buproprion enhances antidepressant efficacy (Trivedi et al 2006). Buproprion shows even antidepressant effects when given alone (Dhillon et al 2008a). Moreover, it enhances brain reward systems (Cryan et al 2003a).

The 5-HT1A receptor is important in this process, since drug-induced increases in extracellular DA levels in the PFC could be blocked by pretreatment with the 5-HT1A receptor antagonist WAY 100635. The extracellular DA levels in the PFC are increased by the 5-HT1A receptor agonists flibanserin (Invernizzi et al 2003), ipsapirone (Wedzony et al 1996) and buspirone (Gobert et al 1999), but also by mirtazapine, a ‘dirty’ drug acting on several 5-HT (but not 5-HT1 receptors) and adrenergic receptors (Nakayama et al 2004) and the SSRI fluoxetine (Gobert et al 1999; Sakaue et al 2000). Moreover, augmentation of SSRI action with the 5-HT1A receptor agonist buspirone is effective in reducing depressive symptoms (Trivedi et al 2006) 6

5-HT1A receptors are located both pre- and postsynaptically within the brain. Presynaptically they can be found as somatodendritic autoreceptors in the dorsal (DRN) and median raphe nuclei (MRN), where they cause neuronal membrane c h a p t e r hyperpolarization, leading to a decreased firing rate of serotonergic neurons. Limbic and cortical brain areas express high densities of postsynaptic 5-HT1A receptors, where they also attenuate the firing rate of the cells (Blier and de Montigny 1990; Nichols and

Nichols 2008; Pytliak et al 2011). 5-HT1B receptors are also located presynaptically as autoreceptors and postsynaptically where they serve as heteroreceptors on terminals of non-serotonergic neurons, respectively. In the frontal cortex, 5-HT1B heteroreceptors inhibit the release of dopamine whereas 5-HT1B autoreceptors in the striatum and basal ganglia are thought to inhibit 5-HT release (Nichols and Nichols 2008; Pytliak et al

2011). Based on early ligand binding studies, it was claimed that the 5-HT1B receptor only exists in rodents and was absent in humans (Pedigo et al 1981), however more recent molecular and pharmacological studies discovered human and primate homologues as well (Sari 2004), making it more interesting and clinically relevant to study this receptor subtype.

As shown above, 5-HT1A receptors may play an important role in antidepressant mechanisms. However, less is known about the role of these 5-HT1 receptors in reward

95 Chapter 6 mechanisms. Therefore the aim of the present study was to investigate the monoamine neurotransmission profile of the 5-HT1A/1B receptor agonist, eltoprazine, and its effect on brain stimulation reward. We performed an in vivo microdialysis study to investigate the dose-related effects of this serotonergic drug on extracellular concentrations of 5-HT, NE, DA and their metabolites, 5-HIAA, HVA and DOPAC in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). These brain areas have been implicated as critical neural substrates underlying depression and reward (Koenigs and Grafman 2009; Nestler et al 2002). The PFC is especially involved in overall cognition and executive functions. It is active in decision-making processes and is involved in reinforcement, reappraisal and suppression of negative affect (Koob and Volkow 2010; Robbins and Arnsten 2009). The NAc is the most important area for reward processing and is implicated in the pathophysiology of depression and especially anhedonia (Nestler et al 2002; Nestler and Carlezon 2006). To correlate the eltoprazine-induced changes in monoamine levels with brain reward, we also performed an intracranial self-stimulation (ICSS) experiment.

Material and Methods

Animals Eighty male Wistar rats (microdialysis PFC: n=40; microdialysis NAc: n=24; ICSS experiment: n=16) were used for the experiment. Animals weighed between 125 – 175 grams on arrival and between 250 – 360 grams at the time of microdialysis surgery and were socially housed, four per cage. For the microdialysis experiments, animals were single housed after surgery. All animals were kept on a 12h light-dark cycle with lights on between 6:00h and 18:00h and rooms were controlled for temperature (21 ± 2°C) and humidity (50 ± 10 %). Food and water were available ad libitum except during ICSS training. After arrival animals had at least one week to acclimate to their environment before subjected to experimental procedures. All animal experimental procedures were carried out in accordance with the governmental guidelines and approved by the Ethical Committee for Animal Research of Utrecht University, the Netherlands.

Drugs Eltoprazine (1-[2,3-dihydro-1,4-benzodioxin-5-yl]- hydrochloride; synthesized by PsychoGenics Inc, USA) has high affinity for the 5-HT1A and 5-HT 1B subtypes (Ki = 40 and 52 nM, respectively) (Gommans et al 1997; Olivier et al 1994). Eltoprazine was dissolved in 0.9% NaCl and administered intraperitoneally (i.p.) in a volume of 2 ml/kg. For the microdialysis experiments in the PFC, animals received drug doses of 0, 1, 3, 10 and 30 mg/kg. For the microdialysis study in the NAc, animals received drug doses of 0, 1 and 3 mg/kg. Due to loss of animals during surgery or training in the ICSS study, seven animals were used in a cross-over design. Each animal was injected with vehicle, 1 mg/kg and 3 mg/kg eltoprazine with a one-week interval.

96 Eltoprazine alters brain monoamine levels without enhancing brain stimulation reward

On a testing day, a pre-drug test was carried out at 9:00 A.M. Animals received injections at 10:30 A.M. and were tested at 11:00 A.M. and 1:30 P.M.

Surgery For the microdialysis experiment cuprofane microdialysis probes were implanted in the PFC (MAB 4.7.3.CU) and the NAc (MAB 4.7.2.CU) in two separate cohorts of animals. The coordinates of the medial prefrontal cortex (mPFC), coordinates were with the incisor bar lowered at -3.3 mm, AP: +3.2 mm, ML: +0.8 mm, DV: -4.0 mm from bregma and skull. The coordinates of the nucleus accumbens (NAc) were with the incisor bar lowered at -3.3 mm, AP: +1.5 mm, ML: -1.8 mm, DV: -8.4 mm from bregma and skull (Paxinos and Watson 1998). Probes were anchored with three screws and dental cement on the skull. After this surgery, animals were housed individually until the end of the experiment. For the ICSS experiments bipolar ICSS electrodes (Plastics One, cut to 11 mm in length) were implanted into the lateral hypothalamus (LH), coordinates were AP: -0.5 mm from bregma; ML: ±1.7 mm; DV: -8.3 mm from dura. The incisor bar was adjusted to 5 mm above the interaural line (Pellegrino et al 1979). Electrodes were anchored with four screws and dental acrylic on the skull. All animals received Rimadyl (5 mg/kg, subcutaneously) for pain relief.

Microdialysis experiment One day after surgery, microdialysis experiments were carried out in awake, freely moving animals, as described previously (Prins et al 2010; Prins et al 2011b). During microdialysis, the pump rate was set at 0.09 ml/hr. Two hours after connection ten 30-minute samples were manually collected in vials containing 15 µl of 0.1 M acetic acid and frozen at -20 ºC. At the end of the test day samples were transferred to -80 ºC until 6 analysis with HPLC. After two hours of baseline sampling animals were injected with eltoprazine (1, 3, 10 or 30 mg/kg, 2 ml/kg i.p.) or vehicle (0.9% NaCl), and 30-minute samples were collected for an additional 3 hours. Immediately after the microdialysis experiment, animals were decapitated under gas anesthesia; brains were removed and c h a p t e r kept in 4% paraformaldehyde for at least three days. For probe placement verification, brains were transferred from paraformaldehyde to 30% sucrose for three days, brains were frozen to -20 °C and cut to coronal sections of 60 µm and placed on gelatine- coated glass slides. Sections underwent cresyl violet staining and were microscopically analyzed to verify microdialysis probe placement.

HPLC analysis Samples were analyzed with HPLC with electrochemical detection for norepinephrine (NE), dopamine (DA) and serotonin (5-HT) and 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) simultaneously by an Alexys 100 LC-EC system (Antec Leyden, the Netherlands) (Korte-Bouws et al 1996; Prins et al 2011b; Verhagen et al 2009). The system consisted of two pumps, one auto sampler with a 10 port injection valve, two columns and two detector cells. DA and 5-HT were separated and detected by column 1 (ALF 105 C18

97 Chapter 6

1×50 mm, 3 μm particle size) in combination with detector cell 1. Column 2 (ALF 115 C18 1×150 mm, 3 μm particle size) in combination with detector cell 2, separated and detected NE, DOPAC, HVA and 5-HIAA. The mobile phase for column 1 consisted of 50mM phosphoric acid, 8mM KCl, 0.1mM EDTA (pH 6.0), 12% Methanol and 500 mg/L 1-Octanesulfonic acid, sodium salt (OSA). The mobile phase for column 2 consisted of 50mM phosphoric acid, 50 mM citric acid, 8mM KCl, 0.1mM EDTA (pH 3.2), 10% methanol and 500 mg/L OSA. Both mobile phases were pumped at 50 μl/ min. Samples were kept at 8 °C during analysis. From each microdialysis sample 5 μl was injected simultaneously onto each column. The neurotransmitters were detected electrochemically using μVT-03 flow cells (Antec, the Netherlands) with glassy carbon working electrodes. Potential settings were for DA and 5-HT +0.30 V versus Ag/AgCl and for NE and metabolites +0.59 V versus Ag/AgCl. The columns and detector cells were kept at 35 °C in a column oven. The chromatogram was recorded and analyzed using the Alexys data system (Antec, the Netherlands). The limit of detection was 0.03 nM (S/N ratio 3:1).

ICSS Apparatus All ICSS experiments were performed in eight sound-attenuated operant chambers with a grid floor and a wheel manipulandum in one of the sides. The implanted electrode was connected to an electrical stimulator through a swivel and bipolar connector cable (Plastics One), ensuring unrestrained movement throughout the ICSS procedure. Electrical stimulation was delivered by a constant current stimulator (Med Associates Inc.). The stimulator was connected to a computer running MED-PC IV (Med Associates, Inc.), controlling all stimulation settings, programs and recording of data.

ICSS procedure All the animals were initially trained to turn the wheel manipulandum on a fixed ratio 1 schedule of reinforcement, in order for the animals to make the association that turning the wheel results in electrical stimulation. In this training phase each quarter turn of the wheel resulted in an electrical stimulus with train duration of 500 ms. After several successful training sessions, the rats were trained on a discrete-trial current- threshold procedure according to the procedure described by Markou and Koob (1992). At the start of each trial rats receive a free, non- contingent stimulus and have 7.5 s to react and turn the wheel a quarter turn to obtain a second, contingent stimulus (positive response) of the same current intensity and duration. The time between the first free stimulus and the time of turning the wheel was measured as response latency. The number of responses in the 2 s after a positive response was counted as extra responses; turning the wheel during that period did not have further consequences. Responses during this period often reflect the force with which the animal turned the wheel, because a powerful pull will result in more than a quarter turn of the wheel. If no response occurred during the 7.5 s period, a negative response was recorded. The inter trial interval (ITI) following the 7.5 s response period had a duration range from 7.5 to 12.5 s. Responses during the ITI resulted in a delay of onset of the next stimulus

98 Eltoprazine alters brain monoamine levels without enhancing brain stimulation reward of 12.5 s. Animals were subjected to alternating descending and ascending series of current intensities starting with a descending series. The stimulus intensity of the first series was set 40 µA above each animal’s own baseline. Current levels were presented in sets of five trials altered by steps of 5 µA and presented in sets of five trials of the same current intensity.

ICSS Parameters

ICSS Thresholds The current threshold for each series was defined as the midpoint between two consecutive current intensities for which animals responded in at least three of the five trials and two consecutive current intensities for which animals did not respond in three or more of the five trials. The overall threshold of the session was defined as the mean of the thresholds for the four individual series. Stable thresholds were defined as less than 10% change in threshold over three consecutive days after at least ten days of testing.

Response latency The response latency is the time between the presentation of the non-contingent stimulus and the positive response of the animal. The overall response latency was defined as the mean response latency of all trials during which a positive response occurred.

Extra responses Extra responses are the amount of responses in the 2 s after a positive response. The overall extra responses were calculated as the total number of extra responses per test session divided by the total number of positive responses of that test session. 6

Statistical analysis

Mean (±S.E.M.) absolute extracellular baseline concentrations are presented for each c h a p t e r monoamine and metabolite in the PFC and NAc. Four baseline samples were averaged and set at 100% and all data was presented as percentage change from baseline. Microdialysis data were analyzed with a repeated measures ANOVA with ‘time’ as within-subjects factor (10 levels) and ‘treatment’ as between-subject factors. When a significant time × treatment interaction was found, each time point was analyzed by one-way ANOVA. In case of a significant main effect, a two-sided Dunnett’s test was performed with the vehicle group as control. All statistical outcomes from our repeated measures ANOVA were corrected by the Greenhouse-Geisser correction factor, because the assumption of sphericity was violated. The area under the curve was calculated using the trapezoid algorithm. Area under the curve data was analyzed with a one-way ANOVA and post-hoc Dunnett’s test comparisons. When the Levene’s test of homogeneity of variances gave a significant outcome (assumption of equality of variances was violated), Games-Howell tests were performed to make post-hoc comparisons and Welch F-statistics are presented.

99 Chapter 6

Table 1. Time × treatment interactions microdialysis experiments. Degrees of freedom and F-values presented here are corrected by the Greenhouse-Geisser correction (ε). In case of significant time x treatment interaction, individual timepoints were analyzed by one-way ANOVAs with post-hoc Dunnett’s comparisons when appropriate. N.S. is not significant. PFC NAc DA F(10, 70) = 18.549, p < 0.001 ε = 0.285 F(5, 54) = 1.444, N.S. ε = 0.285 NE F(13, 90) = 5.489, p < 0.001 ε = 0.368 F(6, 36) = 0.890, N.S. ε = 0.329 5-HT F(22, 138) = 4.046, p < 0.001 ε = 0.612 F(10, 100) = 9.564, p < 0.001 ε = 0.534 DOPAC F(12, 75) = 20.461, p < 0.001 ε = 0.322 F(5, 36) = 5.307, p < 0.001 ε = 0.265 HVA F(9, 59) = 30.414, p < 0.001 ε = 0.241 F(7, 70) = 18.646, p < 0.001 ε = 0.366 5-HIAA F(17, 116) = 15.658, p < 0.001 ε = 0.476 F(8, 87) = 9.095, p < 0.001 ε = 0.463

Mean absolute ICSS baseline thresholds and response latencies (± S.E.M.) are presented for each experimental group. All ICSS current-thresholds and response latencies were expressed as a percentage of baseline data. Thresholds, response latencies and extra responses were analyzed independently, using a repeated measures ANOVA with ‘time’ as the within-subject factor and ‘dose’ as between-subject factor. The baseline stability was analyzed using a repeated measures ANOVA with ‘time’ as the within-subject factor. In case of non-sphericity of the data, reported results were corrected by the Greenhouse-Geisser correction. Significant interaction effects were analyzed with a one-way ANOVA as post hoc. A post hoc for significant main effects was carried out using a two-sided Dunnett’s test when applicable.

Results

Microdialysis

Monoamine and metabolite concentrations in PFC (Fig. 1 and 2) Mean (± S.E.M.) absolute extracellular baseline concentrations in the PFC were for DA: 0.61 ± 0.04 nM, for NE: 0.69 ± 0.03 nM, for 5-HT: 0.16 ± 0.01 nM, for DOPAC: 17.36 ± 1.30 nM, for HVA: 31.29 ± 2.66 nM, and for 5-HIAA: 127.26 ± 6.18 nM. Baseline was set at 100% and all drug effects are presented as percentage change from baseline. A significant time × treatment interaction was found for all monoamine and metabolite concentrations in the PFC (see Table 1 for statistical details). One-way ANOVAs on each time point revealed overall significant differences between treatment groups at all time points after eltoprazine administration, except for t=30 min in the HVA measurements. All doses of eltoprazine induced significant and dose-dependent increases in extracellular DA, NE, DOPAC and HVA and a decrease in extracellular 5-HIAA concentrations when compared to the vehicle. Except for the highest (30 mg/ kg) dose, eltoprazine caused a decrease in extracellular 5-HT concentrations compared to vehicle. (Figures 1 and 2; see Table 2 for significance values per time point). Area under

100 Eltoprazine alters brain monoamine levels without enhancing brain stimulation reward the curve (AUC) was calculated to better visualize the overall change in monoamine and metabolite level after eltoprazine administration. Analysis of the AUC was analyzed with a one-way ANOVA (statistical details see Table 3). Post-hoc Dunnett’s comparisons against the vehicle condition showed significant increases in DA concentrations at all doses, an increase in extracellular NE concentrations at the two highest doses of 10 and 30 mg/kg. Significant increases in DOPAC and HVA at the 3, 10 and 30 mg/kg doses in the PFC. Furthermore, eltoprazine administration significantly decreased extracellular

           

       

      

           

                    

   

 

   6

      

  c h a p t e r                   

  

 

 

                              

Figure 1. Eltoprazine significantly and dose-dependently increased concentrations of dopamine (DA) and norepinephrine (NE) in microdialysate from the medial PFC and decreased serotonin (5-HT), except for the highest dose. Left graphs; time points -90 till 0 represent baseline samples. At t = 0 a single injection of eltoprazine was given and 30-minute samples were taken up to 3 hours after injection. Right panels; bars represent the area under the curve (AUC) of the corresponding graphs on the left. AUC was calculated with use of a trapezoid algorithm. *p < 0.05, ***p < 0.001

101 Chapter 6

5-HIAA and 5-HT concentrations at all doses in the PFC, except at the 30 mg/kg dose for 5-HT (Figures 1 and 2).

Monoamine and metabolite concentrations in NAc (Fig. 3 and 4) Mean (± S.E.M.) absolute extracellular baseline concentrations in the NAc were for DA: 2.82 ± 0.15 nM, for NE: 0.32 ± 0.05 nM, for 5-HT: 0.11 ± 0.01 nM, for DOPAC: 607.77 ± 37.82 nM, for HVA: 278.12 ± 20.73 nM, and for 5-HIAA: 234.41 ± 9.62 nM.

           

       

     

           

                    

                

                    

    

                

                    

Figure 2. Eltoprazine significantly and dose-dependently increased concentrations of DOPAC and HVAin microdialysate from the medial PFC and decreased 5-HIAA. Left graphs; time points -90 till 0 represent baseline samples. At t = 0 a single injection of eltoprazine was given and 30-minute samples were taken up to 3 hours after injection. Right panels; bars represent the area under the curve (AUC) of the corresponding graphs on the left. AUC was calculated with use of a trapezoid algorithm. *p < 0.05, **p < 0.01, ***p < 0.001

102 Eltoprazine alters brain monoamine levels without enhancing brain stimulation reward

Baseline was set at 100% and all drug effects are presented as percentage change from baseline. A significant time × treatment interaction was found for 5-HT, DOPAC, HVA and 5-HIAA concentrations in the NAc (see Table 1 for statistical details). One-way ANOVAs on each time point revealed overall significant differences between treatment groups at almost all time points after eltoprazine administration, except for t=30 min in the HVA and 5-HIAA measurements and t=180 min for the DOPAC measurements. 5-HT and 5-HIAA concentrations were decreased at both doses of eltoprazine when compared to vehicle. Concentrations of DOPAC and HVA in the NAc were increased after both 1 and 3 mg/kg eltoprazine (Figures 3 and 4; see Table 2 for significance values per time point). Analysis of the AUC with one-way ANOVAs gave similar results; significant overall differences in 5-HT, 5-HIAA, DOPAC and HVA (statistical details see Table 3), with increases in the metabolites of DA; DOPAC and HVA and a decrease in 5-HT and 5-HIAA after both doses of eltoprazine (Figures 3 and 4).

Table 2. In bold significant difference of tested dose (1, 3, 10 or 30 mg/kg) compared to vehicle condition with one-way ANOVA and post-hoc Dunnett’s t-test. When box is left blank, no post-hoc comparisons were made, because there was no significant main effect in the ANOVA. For NAc, the highest (10 and 30 mg/kg) were not tested. PFC NAc Dose T30 T60 T90 T120 T150 T180 T30 T60 T90 T120 T150 T180 1 .129 .042 .104 .297 .364 .994 3 .001 .001 .001 .003 .001 .012 DA 10 .001 .001 .001 .001 .001 .001 6 30 .001 .001 .001 .001 .001 .001 1 .376 .118 .053 .382 .573 .996 3 .249 .047 .015 .066 .075 .284 NE

10 .003 .005 .001 .016 .027 .089 c h a p t e r 30 .005 .001 .001 .001 .001 .003 1 .006 .086 .006 .073 .001 .001 .015 .001 .001 .001 .001 .004 3 .001 .003 .001 .055 .001 .001 .001 .001 .001 .001 .001 .001 5-HT 10 .001 .049 .002 .085 .001 .001 30 .227 .996 .994 .860 .351 .311 1 .213 .174 .236 .646 .929 1.00 .423 .001 .001 .053 .159 3 .016 .007 .003 .010 .121 .658 .012 .001 .001 .001 .014 DOPAC 10 .111 .004 .001 .001 .001 .001 30 .004 .001 .001 .001 .001 .001 1 .052 .096 .428 .630 .997 .002 .003 .001 .024 .061 3 .001 .001 .012 .050 .216 .001 .001 .001 .001 .001 HVA 10 .001 .001 .001 .001 .001 30 .001 .001 .001 .001 .001 1 .827 .142 .001 .001 .001 .001 .002 .001 .001 .001 .001 3 .002 .001 .001 .001 .001 .001 .002 .001 .001 .001 .001 5-HIAA 10 .003 .001 .001 .001 .001 .001 30 .029 .001 .001 .001 .001 .001

103 Chapter 6

       

       

                

                

  

 

 

       

                

   

 

  

   

                       

Figure 3. Eltoprazine (1 and 3 mg/kg) significantly decreased 5-HT concentrations in microdialysate from the NAc. Left graphs; time points -90 till 0 represent baseline samples. At t = 0 a single injection of eltoprazine was given and 30-minute samples were taken up to 3 hours after injection. Right panels; bars represent the area under the curve (AUC) of the corresponding graphs on the left. AUC was calculated with use of a trapezoid algorithm. ***p < 0.001

104 Eltoprazine alters brain monoamine levels without enhancing brain stimulation reward

       

       

         

    

     

                

     

 

     6       

                 c h a p t e r

   

  

                             

Figure 4. Eltoprazine (1 and 3 mg/kg) significantly increased DOPAC and HVA concentrations and decreased 5-HIAA concentrations in microdialysate from the NAc. Left graphs; time points -90 till 0 represent baseline samples. At t = 0 a single injection of eltoprazine was given and 30-minute samples were taken up to 3 hours after injection. Right panels; bars represent the area under the curve (AUC) of the corresponding graphs on the left. AUC was calculated with use of a trapezoid algorithm. *p < 0.05, ***p < 0.001

105 Chapter 6

Table 3. Summary statistics area under the curve. Area under the curve was analyzed with one-way ANOVA, in case of main significant effect, post-hoc Dunnett’s test were performed with vehicle condition as the control. N.S. is not significant PFC (AUC) NAc (AUC) DA F(4, 27) = 26.478, p < 0.001a F(2, 21) = 1.735, p = 0.201 N.S. NE F(4, 27) = 17.476, p < 0.001a F(2, 12) = 1.922, p = 0.335 N.S.a 5-HT F(4, 25) = 18.899, p < 0.001 F(2, 21) = 53.259, p < 0.001 DOPAC F(4, 26) = 26.744, p < 0.001a F(2, 15) = 16.666, p < 0.001 HVA F(4, 27) = 36.451, p < 0.001a F(2, 21) = 37.941, p < 0.001a 5-HIAA F(4, 27) = 38.190, p < 0.001 F(2, 21) = 23.723, p < 0.001 aWhen equal variances could not be assumed (significant outcome in the Levene’s test of homogeneity of variances), post-hoc Games-Howell tests were performed and Welch F-statistics are presented. Reward-related behavior

Baseline thresholds The 9:00 A.M. pre-drug ICSS thresholds did not differ significantly throughout the experiment compared to the baseline threshold (calculated as the mean thresholds over five days immediately prior to the first testing day). Mean (± S.E.M.) absolute baseline ICSS thresholds (9:00 A.M. pre-drug) were 163.0 ± 17.5 µA, 150.4 ± 19.6 µA and 159.9 ± 16.4 µA for the control, 1 mg/kg and 3 mg/kg dose groups respectively.

ICSS Thresholds Eltoprazine administration significantly increased ICSS thresholds as shown bya significant main effect of treatment [F(2, 16) = 3.962, p < 0.05]. Furthermore, post hoc analysis revealed that the increase in thresholds was only significantly increased at the lowest dose (1 mg/kg; p < 0.05) compared to vehicle, while the highest dose (3 mg/kg) did not differ significantly from vehicle. No significant time × treatment interaction was

        

                  Figure 5. Eltoprazine (1 mg/kg) increased intracranial self stimulation (ICSS) thresholds, suggesting that it did not enhance brain reward systems. *p < 0.05

106 Eltoprazine alters brain monoamine levels without enhancing brain stimulation reward found [F(4, 32) = 1.785, n.s.]. A significant main effect of time [F(2, 32) = 24.427, p < 0.001] was found, indicating an increase in thresholds over time (Figure 5).

Response latencies No significant differences were found between the response latencies of the different treatment groups, indicating that eltoprazine administration did not influence the reaction time of the animals (data not shown).

Extra responses Both a significant time × treatment interaction [F(3, 24) = 6.323, p < 0.05] as well as a significant main effect of time [F(2, 24) = 46.136, p < 0.001] was observed. Subsequent post hoc analysis with a one-way ANOVA for each time point revealed no significant difference between the different doses at the time points pre-drug and 3 hours after treatment. However, a significant effect of treatment was found 90 minutes after drug treatment [F(2, 18) = 4.679, p < 0.05]. Subsequent post hoc analysis showed that administration of 3 mg/kg eltoprazine significantly decreased extra responses (p < 0.05), while administration of 1 mg/kg eltoprazine showed a trend towards a decreased amount of extra responses (p = 0.07) (Figure 6).

Discussion

The present study showed that acute systemic administration of the 5-HT1A/1B receptor agonist eltoprazine results in decreased levels of serotonin (5-HT) and its metabolite 5-HIAA in the prefrontal cortex (PFC) and the nucleus accumbens (NAc). Furthermore, dopamine (DA) and norepinephrine (NE) levels were increased in the PFC, but not in the NAc. However, because the levels of DA’s metabolites DOPAC and HVA were 6

    c h a p t e r   

   

          

Figure 6. Eltoprazine (3 mg/kg) decreased the number of extra responses, suggesting an increased efficiency in the ICSS task by increased ability to withhold inappropriate responding. *p < 0.05

107 Chapter 6 increased in the NAc following eltoprazine treatment, it is likely that the dopaminergic activity in the NAc was increased. Moreover, the enhancement of dopaminergic activity was not paralleled by activation of brain reward systems, but on the contrary by a deficit in brain reward functioning, reflected by increased ICSS thresholds. The decreased 5-HT concentrations following eltoprazine administration can be explained by activation of somatodendritic 5-HT1A autoreceptors, because acute activation of these receptors in the raphe nuclei (RN) leads to an inhibition of 5-HT release in the projection areas of the RN, like the NAc and PFC (Blier and Ward 2003; Sprouse and Aghajanian 1987). Moreover, 5-HT release in the PFC is also under influence of postsynaptic 5-HT1A receptors (Casanovas et al 1999), which are present on non-serotonergic cells (Pazos and Palacios 1985) in the PFC were they directly influence serotonergic activity of the dorsal raphe nucleus (DRN) projecting to the PFC (Martin-Ruiz and Ugedo 2001). Casanovas and co-workers (1997) have demonstrated that systemic administration of 5-HT1A receptor agonists was followed by reductions in 5-HT release, which was more prominent in the frontal cortex than in the DRN. The effect of eltoprazine on extracellular 5-HT levels is probably not only mediated by 5-HT1A receptors, but also by 5-HT1B receptors. Activation of 5-HT1B autoreceptors reduces 5-HT release (Adell et al 2001; de Groote et al 2002), while a 5-HT1B receptor antagonist potentiates the fluoxetine-induced increase in cortical 5-HT release (Gobert et al 1997). It is not clear why the highest dose of eltoprazine (30 mg/kg) did not result in changes in 5-HT concentrations. However, it should be noted that all animals that receive that dose (and to a lesser extent also the 10 mg/kg dose) elicited the so- called serotonin syndrome, which consist of lower-lip retraction, flat body posture and loss of coordination (Izumi et al 2006). This undesirable profile is known to emerge by activation of postsynaptic 5-HT1A receptors with high potency 5-HT1A receptor agonists (Hjorth 1985; Izumi et al 2006). In the present study we found an eltoprazine-induced dose-dependent increase in extracellular DA concentrations in the mPFC, but not in the NAc and a dose-dependent increase in DOPAC and HVA in both brain areas. Several studies have shown complex, region-specific, effects of 5-HT1A receptor agonists on dopaminergic neurotransmission.

The 5-HT1A receptor agonist 8-OH-DPAT reduced 5-HT levels and increased DA levels in the mPFC, but not in striatum (Rollema et al 2000). The 5-HT1A receptor antagonist WAY 100635 by itself had no effect on monoamine levels, but completely blocked the 8-OH-DPAT-induced cortical DA release, suggesting that this increase in DA release in the PFC is indeed mediated by the 5-HT1A receptor (Di Matteo et al 2008; Rollema et al 2000). Administration of the highly potent 5-HT1A receptor agonist MKC-242 increased dopamine release in the PFC and hippocampus by activating postsynaptic

5-HT1A receptors (Sakaue et al 2000), but not in striatum and NAc. This observation may be explained by the differences in 5-HT1A receptor density, which is high in the PFC and hippocampus, but low in striatum and NAc (Pazos and Palacios 1985). Similar results were obtained by Diaz-Mataix and co-workers (2005). They found an increased firing activity of DA neurons in the ventral tegmental area (VTA) and increased DA release in the VTA and mPFC after systemic 5-HT1A receptor agonist administration (Diaz-Mataix et al 2005). Furthermore, local infusion of a low dose of the selective 5-HT1A receptor

108 Eltoprazine alters brain monoamine levels without enhancing brain stimulation reward agonist BAYx3702 in the mPFC increases extracellular DA concentrations, which can be blocked by the GABAA receptor antagonist bicuculline (Diaz-Mataix et al 2005). 5-HT1A receptors are present on GABAergic interneurons in the mPFC (Santana et al 2004) and low doses of BAYx3702 are thought to preferentially activate these 5-HT1A receptors on GABAergic interneurons, leading to disinhibition of cortical pyramidal neurons, which project to the VTA (Diaz-Mataix et al 2006). Higher doses of BAYx3702 decreased DA release in the mPFC, most likely because the higher dose activates 5-HT1A receptors on the pyramidal cells directly and thereby reducing prefrontal excitatory output to DA neurons (Diaz-Mataix et al 2005).

5-HT1B receptor activation may also play a role in increased dopaminergic activity in the PFC and NAc following eltoprazine administration. 5-HT1B receptor agonists are able to increase extracellular DA concentrations in the PFC (Iyer and Bradberry 1996), indicating a functional interaction between DA and 5-HT pathways in the PFC. However,

5-HT1B receptors are in particular involved in dopaminergic neurotransmission in the mesolimbic DA system. Local administration of 5-HT1B receptor agonists in the VTA or NAc increases DA release in the NAc (Yan and Yan 2001). This agonist is thought to activate inhibitory 5-HT1B receptors on GABAergic interneurons in the VTA leading to a reduction of the negative feedback of the DA release (Yan et al 2004). Summarizing, the effects of eltoprazine on DA levels in the PFC presented in the current study, might be an effect of the 5-HT1A receptor, because this receptor is known for its effects on mesocortical dopaminergic systems and concurrent increases of DA in the mPFC. Whereas the 5-HT1B receptor is more implicated in the dopaminergic mesolimbic system with DA increases in the NAc and striatum after 5-HT1B receptor activation. Although we did not find significant changes in increased DA levels, a tendency towards increased DA levels was found together with significant increases in

DOPAC and HVA, suggesting an activated mesolimbic dopaminergic system, which 6 might be explained by increased 5-HT1B receptor activation. The increased eltoprazine-induced NE levels in the PFC found in the present study might also be explained by the activation of the 5-HT1A receptor. Activation of the c h a p t e r

5-HT1A receptor has been shown to result in increases in NE in the VTA (Chen and Reith 1995) and in the PFC (Gobert et al 1998). However, little research has focused on the role of serotonergic drugs on NE neurotransmission and it is therefore not yet clear whether the 5-HT1A receptors involved are located on (nor)epinephrinergic neurons or on interneurons. Surprisingly, the observed increased dopaminergic activity in the PFC and NAc following eltoprazine were not accompanied by decreases in ICSS thresholds. On the contrary, ICSS thresholds increased after eltoprazine administration, suggesting an anhedonic state of the animals. Thus, increases in brain DA levels, are not necessarily followed by activation of brain reward systems as often is suggested. Psychostimulant drugs (e.g. amphetamine and cocaine) increase brain DA levels (Kuhar et al 1991) and amplify reward signals in the brain (Kenny 2007) and it is generally assumed that increases in DA are needed for facilitation of ICSS behavior. In our study we found a concomitant decrease in 5-HT levels, which suggest that 5-HT is also needed for mediating reward. Recent studies confirm that 5-HT is implicated in the rewarding

109 Chapter 6 effects of psychostimulant drugs (Kranz et al 2010; Muller et al 2007). More specifically, the role of 5-HT receptors in reward have been extensively reviewed (Hayes and Greenshaw 2011). Stimulating 5-HT neurotransmission resulted in increased reward sensitivity, whereas 5-HT depletion had the opposite effect and made animals less sensitive to positive feedback (Bari et al 2010). Our results with eltoprazine are in agreement with published studies, which demonstrated in particular a role for 5-HT1B receptors and to a lesser extent the 5-

HT1A receptor in ICSS behavior. For instance, administration of the 5-HT1A/1B receptor agonist RU 24969 increased ICSS thresholds, which could be reversed by pre-treatment with a 5-HT1B receptor antagonist. The 5-HT1B receptor agonist CP 94253 caused a dose-dependent rightward shift in VTA-ICSS thresholds (indicating a decrease in reward), which could be reversed by pretreatment with a 5-HT1B receptor antagonist (Hayes et al 2009).

On the other hand, the 5-HT1A receptor agonist 8-OH-DPAT has been shown to decrease ICSS thresholds at low doses and increase thresholds at high doses (Montgomery et al 1991). Low doses of 8-OH-DPAT were thought to activate somatodendritical

5-HT1A autoreceptors leading to a reduction in 5-HT inhibitory activity and to increases in DA transmission, while higher doses were thought to stimulate postsynaptic 5-HT1A receptors, leading to inhibition of self-stimulation (Montgomery et al 1991). It is important to note that we only studied acute effects of eltoprazine administration. Prolongation of treatment probably may result in desensitization of the 5-HT1A autoreceptor in the DRN and a dose-dependent inhibition of the firing rate of 5-HT neurons (Blier and de Montigny 1990). Postsynaptic 5-HT1A receptors (in the dorsal hippocampus) are more resistant to desensitization and therefore long- term treatment with a 5-HT1A receptor agonist will increase the activity at postsynaptic

5-HT1A receptors and presumably enhances 5-HT function (Blier and de Montigny

1994; De Vry 1995). Since downregulation of 5-HT1A receptors is suggested to mediate the effects of antidepressant treatment (Blier and de Montigny 1994; Millan 2006), it might be of particular interest to investigate the chronic effects of eltoprazine on neurotransmitter release and on ICSS behavior. In the present study, eltoprazine (3 mg/kg) decreased the amount of extra responses, measured in the ICSS paradigm. Extra responses are the rotations of the wheel during the two seconds following the contingent stimulus (positive response) and reflect the vigor in turning the wheel. Decreases in extra responses can be interpreted as proper inhibition in inappropriate responding (i.e. measurement for impulsivity) (Amitai et al 2009). Our results are in agreement with previous studies which indicate that eltoprazine lowers impulsive aggression in animals (Olivier and van Oorschot 2005) and aggressive behavior has clear ties to certain types of impulsivity, as reflected by correlations in humans (Solanto et al 2001) and in animals (Van den Bergh et al 2006). Our findings are also in line with other studies in which lower extracellular 5-HT concentrations in the PFC are associated with lower levels of premature responding in the 5-choice serial reaction time task (Dalley et al 2002a; Dalley et al 2002b) and that low levels of 5-HT are favorable in the ability to wait for large rewards (Liu et al 2004).

110 In conclusion, the present study demonstrates that the 5-HT1A/1B receptor agonist eltoprazine was able to elicit a profound 5-HT-DA and 5-HT-NE interaction, confirming the already existing evidence that the serotonergic, dopaminergic and norepinephrinergic systems are heavily modulated by each other through a high degree of connectivity between monoaminergic projection areas. Furthermore, our study confirmed recent evidence that enhancement of dopaminergic activity alone is not enough to enhance brain reward systems.

r e t p a h c

Acute and chronic monoamine reuptake inhibitors differently affect brain stimulation reward and monoamine release in the nucleus accumbens and prefrontal cortex in rats

Jolanda Prins Anne M. Krajnc Ronald S. Oosting Gerdien A. H. Korte-Bouws Koen G. C. Westphal Berend Olivier Damiaan A. Denys S. Mechiel Korte 7 Submitted Chapter 7

Abstract Serotonin (5-HT) and/or norepinephrine (NE) reuptake inhibitors are widely used as first line treatment for major depressive disorder, but patients often encounter residual symptoms or do not respond at all. Moreover, evidence exists for an impaired brain reward system in depressed patients. In order to investigate the role of the separate monoamines and their role in reward, 5-HT, NE and dopamine (DA) transporters in rats were acutely and chronically blocked with escitalopram (5 and 10 mg/kg, i.p.), reboxetine (5 and 10 mg/kg, i.p.) or methylphenidate (0.1 and 5 mg/kg, i.p.) respectively. The rewarding properties of these compounds were investigated in an intracranial self-stimulation (ICSS) paradigm. In parallel, a microdialysis study, with probes in the prefrontal cortex (PFC) and nucleus accumbens (NAc) was performed in which the highest dose of each compound was administered acutely and chronically. One single escitalopram injection increased 5-HT levels in the PFC and increased ICSS thresholds, whereas, unexpectedly, chronic escitalopram administration did not affect ICSS thresholds or 5-HT levels (both baseline levels and the levels after a drug challenge did not differ from vehicle). A methylphenidate challenge in animals chronically treated with methylphenidate led to enhanced brain reward systems, as expected. The methylphenidate challenge led to increased NE levels in the PFC and increased DA levels in the NAc, but not in the PFC. Chronic reboxetine administration led to increased DA and NE in the PFC. From the present study it becomes clear that the mesolimbic dopamine system is an important target modulating anhedonia.

114 Monoamine reuptake inhibitors, ICSS and in vivo microdialysis in the NAc and PFC

Introduction Depression is one of the most prevalent forms of psychiatric disorders and is among the leading causes of disability. According to the World Health Organization affecting about 121 million people worldwide. The most prescribed antidepressants are selective serotonin or norepinephrine reuptake inhibitors (SSRIs/SNRIs). Although these compounds are effective in many patients suffering from severe major depression, a large number of patients fail to respond to therapy, probably depending on the type of their depression (Fournier et al 2010; Prins et al 2011a), or encounter residual symptoms, including impaired motivation and pleasure. Besides serotonin (5-HT) and norepinephrine (NE) a role for dopamine (DA) in the treatment of depression and in particular anhedonia (loss of pleasure) has been postulated (Dunlop and Nemeroff 2007; Nestler and Carlezon 2006). Interestingly, when the dopaminergic system is co- targeted, antidepressant treatment becomes more effective (Gupta et al 2006; Trivedi et al 2006). These insights have led to the development of triple reuptake inhibitors (TRIs) (Skolnick and Basile 2006; Skolnick and Basile 2007), which simultaneously block the 5-HT transporter (SERT), the NE transporter (NET) and the DA transporter (DAT). Previous studies in our group have shown that the TRI DOV 216,303 induces long-lasting enhancement of brain reward systems (Prins et al 2012) and increases extracellular brain concentrations of DA, 5-HT and NE (Prins et al 2010; Prins et al 2011b). The question remains what the contribution is of the separate monoamines in reward-related processes. Therefore we decided to study the effects of a low and a high dose of a SSRI (escitalopram), a NRI (reboxetine) and a DNRI (methylphenidate) on intracranial self-stimulation (ICSS). Moreover, it is known that prolonged treatment with SSRIs causes a flattened effect in the processing of aversive and rewarding stimuli in humans (McCabe et al 2010) and that chronic fluoxetine causes an attenuated response to amphetamine in rats (Lin et al 1999). In the latter study, an amphetamine challenge was given in order to investigate the ability of amphetamine to alter ICSS responding after chronic blockade of the transporters. In a second study we performed a microdialysis experiment simultaneously in the prefrontal cortex (PFC) and nucleus accumbens (NAc) to evaluate 7 regional differences in response to acute and chronic administration of the same 5-HT, NE and/or DA reuptake inhibitors. We focused on the PFC and NAc because of

their involvement in depression and reward. These brain areas are part of two main c h a p t e r dopaminergic tracts in the brain, the mesocortical and mesolimbic dopamine system, both originating in the ventral tegmental area (Nestler and Carlezon 2006). Moreover, the NAc is an effective target for deep brain stimulation in treatment-resistant depression (Bewernick et al 2012). Furthermore, the PFC is innervated by serotonergic, norepinephrinergic and dopaminergic fibers from the raphe nuclei, locus coeruleus and ventral tegmental area respectively, and is therefore an interesting brain area in the context of the present study.

115 Chapter 7

Material and Methods

Animals 96 Male Wistar rats (n=40 for ICSS experiment, n=56 for the microdialysis experiments) were used. At the start of treatment animals weighed between 370 and 535 gram (ICSS) and between 247 and 318 gram (microdialysis) and were socially housed, four per cage, on a 12 h light-dark cycle with lights on at 6 A.M. and lights off at 6 P.M. After implantation of the microdialysis probe, the animals were housed individually until the end of the microdialysis experiment. Food and water were available ad libitum, except for the first three days of ICSS training, when animals were given 20 g food per animal per day in order to facilitate the training of the animals. All animal experimental procedures were carried out in accordance to the governmental guidelines and approved by the Ethical Committee for Animal Research of Utrecht University, the Netherlands.

Surgery For surgeries, animals were anesthetized with isoflurane 2-4% and oxygen. Rimadyl (5 mg/kg, subcutaneously) was administered for pain relief. Bipolar ICSS electrodes (PlasticsOne) were cut to 11 mm in length and implanted in the lateral hypothalamus (LH), coordinates were AP: -0.5 mm, ML: ±1.7 mm from bregma and DV: -8.3 mm from dura. In half of the animals, an electrode was placed on the left side of the brain, the other half received an electrode on the right side of the brain, to counterbalance for possible brain asymmetries. The incisor bar was adjusted to 5 mm above the interaural line (Pellegrino et al 1979). In a separate cohort of animals two cuprofane microdialysis probes (MAB 4.6.3.CU and MAB 4.7.2.CU) were respectively implanted in the PFC and the NAc in the same animal. The coordinates of the medial prefrontal cortex (mPFC), were with the incisor bar lowered at -3.3 mm, AP: +3.2 mm, ML: +1.0 mm, DV: -4.0 mm from bregma and skull. The coordinates of the nucleus accumbens (NAc) were with the incisor bar lowered at -3.3 mm, AP: +1.6 mm, ML: -1.8 mm, DV: -8.2 mm from bregma and skull (Paxinos and Watson 1998). Electrodes or probes were anchored with screws and dental cement on the skull.

ICSS apparatus ICSS experiments were performed in eight sound-attenuating operant chambers with a grid floor and a wheel manipulandum in one of the sides. The animal’s electrode was connected to an electrical stimulator with a bipolar cable and through a swivel (Plastics One) to ensure unrestricted movement throughout the ICSS procedure. The electrical stimulations were delivered by a constant current stimulator (Med Associates, Inc.), which was connected to a computer running MED-PC IV (Med Associates, Inc.) controlling all stimulation settings, programs and recording of data.

116 Monoamine reuptake inhibitors, ICSS and in vivo microdialysis in the NAc and PFC

ICSS procedure ICSS training and testing was performed as described previously (Kenny and Markou 2006; Markou and Koob 1992; Prins et al 2012). Briefly, animals were trained on a fixed ratio 1 schedule of reinforcement to make the association that turning the wheel results in electrical stimulation. After several successful training sessions, animals were trained on a discrete-trial current-threshold procedure. Each trial starts with a free, non- contingent stimulus and a quarter turn of the wheel within 7.5 s results in a second, contingent stimulus (positive response) of the same current and duration (100 ms). Current intensities can be altered to determine the threshold at which an animal reacts on the non-contingent stimulus. Animals were subjected to alternating descending and ascending series of current intensities starting with a descending series.

ICSS parameters

ICSS Thresholds The current threshold for each series was defined as the midpoint between two consecutive current intensities at which the animal responded in at least three out of five trials and two consecutive intensities at which the animal did respond in less than three out of five trials. After four turning points the overall average threshold could be determined. Stable ICSS thresholds were defined as less than 10% change in threshold over three consecutive days after at least ten days of testing. At that moment baseline thresholds could be measured and calculated and treatment could be started.

Response latencies The time between the presentation of the non-contingent stimulus and the rotation of the wheel is the response latency. Overall response latencies were defined as the mean response latency of all trials in which the animals performed a positive response.

Microdialysis experiments Microdialysis experiments were carried out as described previously (Prins et al 2010; 7 Prins et al 2011b) In short, microdialysis experiments were performed the next day after surgery in awake, freely moving animals. At the beginning of the test day, animals were connected to the microdialysis system, which was rinsed with Ringer solution, via a dual channel swivel (type 375/D/22QM) with one of the channels connected to the c h a p t e r probe in the PFC and the other channel to the probe in the NAc. During microdialysis, the pump rate (KdScientific Pump 220 series, USA) was set at 0.09 ml/h. Three hours after connection ten 30-minute samples were manually collected in vials containing 15 µl of 0.1 M acetic acid and immediately frozen at -20°C. After four baseline samples drugs were administered and samples were collected for another three hours. At the end of the day samples were transferred to -80 °C until HPLC analysis. Animals were decapitated immediately after the microdialysis experiment and brains were removed and stored for verification of probe localization later on.

117 Chapter 7

HPLC analysis Microdialysis samples were analyzed with HPLC with electrochemical detection as described previously (Prins et al 2010; Prins et al 2011b). In short, the Alexys 100 LC- EC system (Antec, the Netherlands) separated and detected DA and 5-HT by column 1 (NeuroSep C18, 50 mm × 1 mm id × 3 µm particle size)/detector cell 1 (V = 0.30 V vs Ag/AgCl) and NE, DOPAC, HVA and 5-HIAA by column 2 (NeuroSep C18 150 mm × 1mm id × 3 µm particle size)/detector cell 2 (V = 0.59 V vs Ag/AgCl). All measurements were performed simultaneously (i.e. one sample injection resulted in two chromatograms). The mobile phase for column 1 consisted of 50 mM phosphoric acid, 8 mM KCl, 0.1 mM EDTA (pH 6.0), 17.5% Methanol and 400 mg/L 1-Octanesulfonic acid, sodium salt (OSA). The mobile phase for column 2 consisted of 50 mM phosphoric acid, 50 mM citric acid, 8 mM KCl, 0.1mM EDTA (pH 3.3), 19% methanol and 700 mg/L OSA. The columns and detector cells were kept at 35 °C in a column oven. The chromatogram was recorded and analyzed using the Alexys data system (Antec, the Netherlands). The limit of detection was 0.03 nM (S/N ratio 3:1).

Drugs and treatment The SSRI escitalopram (Lexapro®, Lundbeck) and the NRI reboxetine (Edronax®, Pfizer) were suspended in 0.9% NaCl and administered intraperitoneally (i.p.) ina volume of 10 ml/kg. The DNRI methylphenidate hydrochloride was dissolved in 0.9% NaCl and administered i.p. in a volume of 2 ml/kg. The drugs were obtained from a local pharmacist. The selected doses of the three drugs were based on literature, for escitalopram (Breuer et al 2007; Reed et al 2009); reboxetine (Rogoz 2009; Wong et al 2000); methylphenidate (Kuczenski and Segal 1997 2001; Valvassori et al 2007). In the ICSS experiments, first the low dose treatment (5 mg/kg for escitalopram and reboxetine and 0.1 mg/kg for methylphenidate) was given to the animals for 14 days. After a washout period of 6 days, the high dose treatment was started (10 mg/kg for escitalopram and reboxetine and 5 mg/kg for methylphenidate). The drugs were given immediately after ICSS testing, except for days 1, 8 and 14 when the drugs were given 1 hour before the ICSS test. On day 14 D-amphetamine (5 mg/kg, dissolved in 0.9% NaCl, i.p. in a volume of 1 ml/kg) was given after the ICSS test and ICSS tests were performed 6 hours, 1 day and 4 days after injection. In the microdialysis experiments, the control group received 14 daily vehicle injections; the last injection was given during microdialysis. The acute treatment groups received 13 daily vehicle injections prior to one dose of escitalopram (10 mg/kg), reboxetine (10 mg/kg) or methylphenidate (5 mg/kg) on the microdialysis test day. The chronic treatment groups received 14 daily injections of one of the compounds with the last injection occurring during microdialysis. Drug solutions/suspensions were prepared fresh at the beginning of each testing day.

Statistical analysis Mean absolute ICSS thresholds are presented with the standard error of the mean (S.E.M.). ICSS thresholds are expressed as a percentage of baseline data. Changes in ICSS thresholds were analyzed with repeated measures ANOVA with ‘time’ as within-

118 Monoamine reuptake inhibitors, ICSS and in vivo microdialysis in the NAc and PFC subjects factor and ‘treatment’ as between-subjects factor. In case of significant overall group differences (p < 0.05), one-way ANOVA and post-hoc Dunnett’s tests were used to assess differences in treatment groups at a specific time point. Microdialysis data were analyzed with use of a repeated measures ANOVA with ‘time’ as within and ‘treatment’ as between factors. To detect significant differences between experimental groups and the vehicle group, one-way ANOVA and post-hoc Dunnett’s test were used. Post-hoc Tukey tests were performed to detect significant differences between acute and chronic treatment. All statistical outcomes from our microdialysis data were corrected by the Greenhouse-Geisser correction factor, because the assumption of sphericity was violated. Sphericity is a mathematical assumption and relates to the equality of the variances for each set of differences scores in the repeated measures ANOVA. Sphericity requires that the variances for each set of difference scores are equal. When the assumption of sphericity is violated (significant outcome in Mauchly’s Test of Sphericity in SPSS) the Greenhouse-Geisser correction can be used. To correct for sphericity, these corrections alter the degrees of freedom, thereby altering the significance value of the F-ratio. The Greenhouse-Geisser correction is denoted with ε (epsilon). The closer this value reaches 1, the more homogeneous the variances of differences and therefore the closer the data to being spherical (Keselman et al 2001).

Results

ICSS behavior

Baseline ICSS thresholds Mean (± S.E.M) baseline ICSS thresholds were calculated by averaging the thresholds from ten days prior to the start of the low dose treatment. The mean absolute baseline values of the pre-treatment period for the vehicle, methylphenidate, reboxetine and

escitalopram group did not differ and were respectively 99.5 ± 12.1 µA, 107.1 ± 10.6 7 µA, 95.8 ± 8.8 µA and 114.8 ± 9.0 µA. All changes in ICSS thresholds were calculated based on pre-treatment baseline thresholds. After a washout period of 6 days, ICSS thresholds were still around baseline value, therefore we were allowed to calculate change in thresholds after high dose treatment on original pre-treatment baseline thresholds. c h a p t e r

ICSS thresholds after treatment Significant changes in ICSS thresholds were found only after treatment with the high doses of methylphenidate (5 mg/kg) and escitalopram (10 mg/kg), while the high dose of reboxetine was without effect (Fig. 1). The low dose treatments with the 3 drugs had no significant effect on ICSS thresholds. Repeated measures ANOVA with ‘time’ as within factor and ‘treatment’ as between subjects factor revealed a significant effect of treatment [F(3, 26) = 5.251, p = 0.006] and a time × treatment interaction [F(6, 52) = 2,927, p = 0.016] at days where treatment was given 1 hour before the ICSS test, so on

119 Chapter 7

  

  

    

     



  

    

      



  

    

     

 

     

    

Figure 1. Percentage change in ICSS thresholds (± S.E.M.) of high dose treatments. The 3 data points on the left side of the x-axis show the last three days of the washout period between the low and high dose treatment which lasted 6 days in total. Animals were treated with either vehicle, methylphenidate (5 mg/kg), reboxetine (10 mg/kg) or escitalopram (10 mg/kg) for 14 days and ICSS tests were performed before drug treatment, except for day 1, 8 and 14 when animals received drug treatment 1 hour before ICSS testing (black arrows). An acute injection with escitalopram increased ICSS thresholds. When methylphenidate was given 1 hour before the test, ICSS thresholds decreased, which was only significant at day 14. *p < 0.05 compared to vehicle with Dunnett’s test.

120 Monoamine reuptake inhibitors, ICSS and in vivo microdialysis in the NAc and PFC day 1, day 8 and day 14 of treatment. One-way ANOVA revealed significant changes in ICSS thresholds at day 1 [F(3, 29) = 7.445, p < 0.001] and day 14 [F(3, 29) = 3.205, p < 0.05] of treatment. Post-hoc analysis with the Dunnett’s test, in which all treatment groups where compared with the vehicle group, indicated that methylphenidate significantly decreased ICSS thresholds at day 14 and that acute escitalopram treatment significantly increased ICSS thresholds on day 1 (see Fig. 1).

ICSS response latencies Mean absolute (± S.E.M.) baseline response latencies were calculated by taking the mean response latencies ten days preceding the start of treatment. Mean absolute response latencies for the different treatment groups were 2.62 ± 0.20 s, 2.57 ± 0.08 s, 2.52 ± 0.13 s and 2.58 ± 0.18 s for the vehicle-, methylphenidate-, reboxetine- and escitalopram- treated groups, respectively. Treatments did not significantly change response latencies (data not shown).

Amphetamine challenge Immediately after the ICSS test performed on day 14, amphetamine (5 mg/kg, i.p.) was administered to all animals. The amphetamine challenge had severe impact, especially on the escitalopram and methylphenidate-treated animals. These animals showed pilo- erection, excessive salivation, lifelessness and even self-mutilation (two animals), leading to death of all escitalopram and 3 out of 7 methylphenidate-treated animals within hours after injection. The vehicle- and reboxetine-treated animals displayed behavior typical for amphetamine, like stereotypy and hyperactivity. ICSS behavior was assessed 6 hours after amphetamine challenge in these two groups but this led to high individual differences in groups as shown before (Prins et al 2012) and no significant differences between groups. 24 Hours after the amphetamine injection there was a tendency of elevated ICSS thresholds in all treatment groups (data not shown).

Microdialysis results

Monoamine and metabolite concentrations in NAc A significant time × treatment interaction was found for DA, DOPAC, HVA and 7 5-HIAA (for statistical details see Table 1) (Fig. 2 and 3). Neither acute nor chronic

Table 1. Statistical details repeated measures ANOVA time × treatment interactions microdialysis experiment. c h a p t e r Presented F-values and degrees of freedom are corrected by the Greenhouse-Geisser correction (ε). In case of significant time × treatment interactions, timepoints were analyzed by one-way ANOVAs, and when appropriate, comparisons between treatment groups were made with post-hoc analysis to detect for significant differences. PFC NAc DA F(19.44; 136.11) = 6.086, p < 0.001, ε = 0,360 F(14.46; 101.21) = 6.828, p < 0.001 ε = 0.268 NE F(14.04; 98.25) = 11.520, p < 0.001, ε = 0.260 - 5-HT F(11.33; 81.23) = 8.288, p < 0.001, ε = 0.210 F(8.02; 56.11) = 1.217, N.S. ε = 0.148 DOPAC F(21.06; 150.95) = 2.667, p < 0,001, ε = 0.390 F(9.40; 65.78) = 2.654, p = 0.01 ε = 0.174 HVA F(13.68; 98.05) = 2.474, p = 0.005, ε = 0.253 F(16.97; 118.77) = 3.061, p < 0.001 ε = 0.314 5-HIAA F(15.74; 112.81) = 8.658, p < 0.001, ε = 0.291 F(13.62; 95.37) = 5.607, p < 0.001 ε = 0.252

121 Chapter 7

                  

   

    

      

    

 

                     

 

 

 

     

                         

Figure 2. Absolute monoamine concentrations (DA and 5-HT) in microdialysate from the nucleus accumbens (NAc) after a challenge (i.p.) with vehicle, escitalopram (10 mg/kg), reboxetine (10 mg/kg) or methylphenidate (5 mg/kg) in chronic vehicle treated animals (left panels) or chronically drug-treated animals (right panels). Only methylphenidate, acute and chronic, increased DA concentrations in the NAc. Time points -90 till 0 represents baseline measurements. At t = 0 min a single injection was given. And six 30-minute samples were taken up to 3 hours after the challenge. NE levels were below detection limit. *p < 0.05 compared to vehicle, **p < 0.01 compared to vehicle escitalopram and reboxetine treatment had an effect on monoamine and metabolite concentrations in the NAc. Methylphenidate caused an increase in extracellular DA levels in the NAc. A methylphenidate challenge in chronically treated animals seemed to result in a higher increase in DA than an acute challenge, although this did not reach significance. Chronic treatment with methylphenidate increased baseline values of DOPAC, HVA and 5-HIAA. For 5-HIAA these increased levels remained high even after the challenge, while the concentrations of DOPAC and HVA dropped to vehicle values after methylphenidate challenge (Fig. 2 and 3).

Monoamine and metabolite concentrations in PFC A significant time × treatment interaction was found for DA, NE, 5-HT, DOPAC, HVA and 5-HIAA (for statistical details see table 1). Acute treatment with escitalopram led to significant increases in extracellular 5-HT-concentrations in the PFC. Chronic

122 Monoamine reuptake inhibitors, ICSS and in vivo microdialysis in the NAc and PFC

                  

   

    

 

 

    

                              

 

    

 

                                   

 

      

                      7    

Figure 3. Absolute metabolite concentrations (DOPAC, HVA and 5-HIAA) in microdialysate from the nucleus

accumbens (NAc) after a challenge (i.p.) with vehicle, escitalopram (10 mg/kg), reboxetine (10 mg/kg) or c h a p t e r methylphenidate (5 mg/kg) in chronic vehicle treated animals (left panels) or chronically drug-treated animals (right panels). Acute treatments were without effects. Chronic methylphenidate increased baseline levels of all metabolites. Time points -90 till 0 represents baseline measurements. At t=0 min a single injection was given. And six 30-minute samples were taken up to 3 hours after the challenge. *p < 0.05 compared to vehicle **p < 0.01 compared to vehicle treatment with escitalopram did not lead to changes in baseline 5-HT, nor did a challenge with escitalopram affect 5-HT concentrations in chronically treated animals (Fig. 4). Escitalopram decreased 5-HIAA levels when compared to its own baseline values in acute as well as chronically treated animals. Post-hoc tests indicated that chronic treatment with reboxetine led to increased baseline levels of DA and NE. A challenge with reboxetine significantly increased DA and NE concentrations in

123 Chapter 7

                   

   

            

            

 

                     

         

                           

                                   

 

   

                         

Figure 4. Absolute monoamine concentrations (DA, NE and 5-HT) in microdialysate from the prefrontal cortex (PFC) after a challenge (i.p.) with vehicle, escitalopram (10 mg/kg), reboxetine (10 mg/kg) or methylphenidate (5 mg/kg) in chronic vehicle treated animals (left panels) or chronically drug-treated animals (right panels). Escitalopram increased 5-HT concentrations, only after acute treatment. Chronic escitalopram-treated animals neither had changed baseline 5-HT concentrations, nor did react on an escitalopram challenge. Acute and chronic methylphenidate increased NE concentrations, but not DA concentrations. Reboxetine had an effect on DA and NE concentrations. Time points -90 till 0 represents baseline measurements. At t = 0 min a single injection was given. And six 30-minute samples were taken up to 3 hours after the challenge. *p < 0.05 compared to vehicle, **p < 0.01 compared to vehicle, ##p < 0.01 acute compared to chronic treatment.

124 Monoamine reuptake inhibitors, ICSS and in vivo microdialysis in the NAc and PFC

                   

   

 

 

 

     

    

 

                     

            

 

    

 

                                       

      

  7                        

Figure 5. Absolute metabolite concentrations (DOPAC, HVA and 5-HIAA) in microdialysate from the prefrontal

cortex (PFC) after a challenge with vehicle, escitalopram (10 mg/kg), reboxetine (10 mg/kg) or methylphenidate c h a p t e r (5 mg/kg) in chronic vehicle treated animals (left panels) or chronically drug-treated animals (right panels). Reboxetine increased baseline HVA and 5-HIAA concentrations in the PFC. Escitalopram decreased 5-HIAA concentrations when compared to its own baseline values. Time points -90 till 0 represents baseline measurements. At t=0 min a single injection was given. And six 30-minute samples were taken up to 3 hours after the challenge. *p < 0.05 compared to vehicle, ** p<0.01 compared to vehicle, #p < 0.05 acute compared to chronic treatment. *R p < 0.05 reboxetine group vs. vehicle.

125 Chapter 7 chronically reboxetine-treated animals when compared to the vehicle-challenged group and when compared to an acute reboxetine challenge. Reboxetine treated animals also had significantly higher levels of HVA and 5-HIAA when compared to the vehicle treated group. Methylphenidate increased NE, but not DA concentrations in the PFC in acute as well as chronic treated animals (Fig. 4 and 5).

Body weights During the first days of treatment the escitalopram and reboxetine-treated animals lost weight compared to the vehicle, with the highest weight loss in the reboxetine-group (Fig. 6). After 2-3 days of treatment all animals gained weight again, but the escitalopram and reboxetine-treated animals stayed behind in weight during the whole duration of treatment [(F9.8; 153.4) = 21.91, p < 0.0001 ε = 0.326)].

                                    

  

Figure 6. Change in body weights from the start of drug treatment in the microdialysis experiment. Escitalopram and reboxetine treated animals significantly lost weight at the start of treatment compared to the vehicle treated group. The weight loss was significantly higher in the reboxetine treated animals. *p < 0.001 compared to vehicle.

Discussion In the present study, rats were treated with the selective serotonin reuptake inhibitor (SSRI) escitalopram, the norepinephrine reuptake inhibitor (NRI) reboxetine and the dopamine-norepinephrine reuptake inhibitor (DNRI) methylphenidate. Although a methylphenidate-challenge showed a tendency to decrease intracranial self-stimulation (ICSS) thresholds, at day 1 and day 8 of the chronic treatment, only a methylphenidate-challenge at day 14 of the chronic treatment resulted in enhancement of brain reward systems as reflected by decreased ICSS thresholds. We conclude from this that 1) methylphenidate has to be “on board” and 2) that chronic administration of methylphenidate sensitizes the brain reward system for a next challenge with methylphenidate. The rewarding and reinforcing properties of methylphenidate in

126 Monoamine reuptake inhibitors, ICSS and in vivo microdialysis in the NAc and PFC

Wistar rats are also apparent from studies that show conditioned place preference (dela Pena et al 2012a) and enhanced self-administration (dela Pena et al 2012b) after repeated methylphenidate administration. Increased extracellular levels of DA in the NAc paralleled the observed rewarding effects of methylphenidate. Acute as well as chronic administration of methylphenidate led to an increase in extracellular levels of DA in the NAc, This is in accordance with the known action of methylphenidate that, like other psychostimulants, immediately increases mesolimbic DA concentration in rodents (Di Chiara and Imperato 1988) and humans (Volkow et al 2001). The rewarding effects, measured by the facilitation of ICSS behavior together with the increase in DA in the NAc of healthy rats, might be an explanation why this drug is gaining popularity for its hedonic properties (Teter et al 2006). Interestingly, administration of the NRI reboxetine did not have an impact on ICSS, which contrasts with the finding that nomifensine, a NET-blocker, facilitates ICSS behavior and enhances motivation for ICSS (Katz et al 1977; Sagara et al 2008). The antidepressant desipramine also possess NET blocking capacity and lowers ICSS thresholds 30 minutes after treatment (Paterson et al 2008), whereas the NRI has no effect on ICSS thresholds (Izenwasser and Kornetsky 1989). These discrepancies might be explained by differences in transporter binding profiles and in ICSS protocols. The interpretation of older ICSS studies are hampered by the fact that rate-dependent measures of self-stimulation were used, which makes it difficult to differentiate between drug-induced changes resulting from reward alterations or motor performances. In our study, no differences in response latencies were found, indicating that motor dysfunctions could not be an explanation for the observed effects on ICSS thresholds. Finally, reboxetine did not result in alterations of brain reward circuitry, at doses that induce behavioral changes (Rogoz 2009; Wong et al 2000) and which showed a clear increase in NE and DA in the PFC (Fig. 4). The observed increase in DA can be explained by the fact that DA is taken up via NET, since DA transporters are absence in the PFC (Moron et al 2002). Chronic treatment with reboxetine led to increased baseline concentrations of DA and NE in the PFC, which is in agreement with studies in which desipramine and reboxetine increased basal levels of DA and NE in the PFC (Invernizzi 7 et al 2001; Seo et al 1999; Tanda et al 1996), which might be explained by desensitization of terminal α2-adrenoceptors which normally inhibit NE release (Invernizzi et al 2001).

Chronic treatment with the SSRI escitalopram did not alter ICSS thresholds, c h a p t e r but remarkably, acute administration of escitalopram did show an increase in ICSS thresholds. Although the SSRI fluoxetine has previously been used in ICSS experiments (Cazala 1980; Katz and Carroll 1977), to our knowledge, this is the first study in which the effects of escitalopram on ICSS behavior are investigated. The present results are in agreement with these earlier findings in which the rate of responding for reward was decreased by acute administration of fluoxetine both in rats (Katz and Carroll 1977) and mice (Cazala 1980), suggesting an inhibitory influence of 5-HT on reward systems. However, Lee and Kornetsky (1998) showed that acute as well as chronic administration of fluoxetine increased ICSS thresholds, while in our study, chronic treatment with escitalopram failed to alter ICSS thresholds. We can only speculate about the reason

127 Chapter 7 for these differences between the two SSRIs. Differences in metabolism between the drugs may be one of the reasons. The metabolite of fluoxetine, norfluoxetine, is an active SSRI itself (Caccia et al 1990) which may contribute to the long-lasting effects of fluoxetine, whereas the main metabolite of escitalopram, S-demethylcitalopram, does not contribute to the therapeutic effects of escitalopram (Rao 2007). Acute administration of escitalopram led to an increase in extracellular 5-HT levels in the PFC, while this was not longer observed after chronic administration of the drug (Fig 4), suggesting that increased levels of prefrontal 5-HT inhibit brain stimulation reward. However, this may be a too simplistic conclusion, since many contradictory studies exist (Kranz et al 2010). Moreover, the rewarding or non-rewarding properties of 5-HT may also depend on the (an)hedonic state of the animal at the time of drug treatment (Harrison et al 2001), which is a very important aspect in our study as well. Since we did not test the different drugs in (chronically stressed) anhedonic animals, we cannot conclude whether the same compounds would produce similar results in anhedonic animals. This is important to realize because healthy persons and patients might respond differently to these drugs. Our finding that chronic escitalopram treatment does not change extracellular 5-HT levels in the PFC contrasts the leading hypothesis of how SSRIs execute their antidepressant action. The leading hypothesis is based on the fact that chronic treatment with SSRIs increase 5-HT concentrations in the synaptic cleft (Fuller 1994). However, serotonergic activity is controlled by somatodendritic autoreceptors in the raphe nuclei (RN), which upon binding by 5-HT inhibit the activity of 5-HT neurons (Aghajanian et al 1990). Many studies suggest that chronic SSRI treatment leads to an enhanced exposure of 5-HT to 5-HT1A autoreceptors in the RN, which will consequently downregulate, thereby disinhibiting the serotonergic firing activity and sustained increases in extracellular 5-HT levels because of chronic SERT blockade, extensively demonstrated by electrophysiological studies (Blier et al 1987). The question remains to what extent the increase in extracellular 5-HT levels by blockade of the 5-HT transporter is counteracted by a reduction in 5-HT release by the serotonergic cells in the RN. Our observation that a challenge with escitalopram does not lead to an increase in extracellular 5-HT levels in rats chronically treated with this drug suggest that the

5-HT1A autoreceptors in the RN are still able to reduce serotonergic firing and decrease 5-HT release. Our results are supported by a study in mice reported by Popa and colleagues (2010) showing that 5-HT levels were still increased in the RN following 28 days of fluoxetine treatment. However, at this time point extracellular levels of 5-HT in the hippocampus were no longer different from those in vehicle-treated animals. These results can be explained by assuming that 5-HT1A autoreceptors are still functional after prolonged blockade of the 5-HT transporter. This hypothesis is supported by various pharmacological studies. For instance, the 5-HT1A receptor agonist 8-OH-DPAT gave the same response in vehicle and chronic citalopram treated animals in decreasing 5-HT levels (Hjorth and Auerbach 1994). Moreover, autoreceptors still respond to a 5-HT1A/1B receptor antagonist after chronic citalopram treatment by increasing 5-HT in the frontal cortex and dorsal hippocampus (Hjorth and Auerbach 1999).

128 Monoamine reuptake inhibitors, ICSS and in vivo microdialysis in the NAc and PFC

Short-term SSRI treatment (7 days) induces emotional blunting in healthy humans and causes a decreased neural response to both rewarding and aversive stimuli (McCabe et al 2010). Moreover, animals chronically treated with fluoxetine showed a blunted response to an amphetamine challenge as measured with ICSS (Lin et al 1999). We therefore decided to challenge all the animals with amphetamine at the end of our ICSS study. Unfortunately and unexpectedly within hours after this amphetamine challenge all animals of the escitalopram group and half of the animals of the methylphenidate group showed severe toxicity. A major difference between our study and the above mentioned study by Lin and co-workers is that they administered amphetamine 24 hours after the last fluoxetine injection, whereas in our study amphetamine was given approximately 3 hours after the last drug treatment. We can only speculate about the mechanism of the increased toxicity of amphetamine in the escitalopram and methylphenidate groups compared to vehicle groups. Studies have shown that under certain conditions amphetamine can have neurotoxic effects (Carvalho et al 2012). Exposure to 3,4-methylenedioxymethylamphetamine (MDMA) in relatively high ambient temperatures can reach life-threatening hyperthermia in rodents (Capela et al 2009). This effect appeared also under influence of group size, because single housed animals were able to tolerate higher doses of MDMA (Miller and O’Callaghan 1995). This however does not explain the high lethality in our escitalopram and methylphenidate group and the absence of neurotoxic effects in the vehicle- treated groups. A possible explanation might lie in the synergestic interaction between serotonergic and dopaminergic systems following the stimulant effects of amphetamine (Kuczenski and Segal 1989) and methylphenidate (Breese et al 1975). Whatever the underlying mechanism is, our study clearly indicates that caution is required when amphetamine (®) is combined with SSRIs. Especially when this SSRI belongs to the CYP2D6 inhibitor subclass of SSRIs and therefore an inhibitor in the metabolism of amphetamine, which could lead to elevated amphetamine levels, intoxications and eventually to severe side effects (Preskorn et al 2007; Spina et al 2008). It is important to mention that in the present study we measured monoamine concentrations only in healthy, non-stressed animals. Administering drugs to healthy individuals might result in different responses than depressive patients. This is supported 7 by findings that antidepressants have different effects on moderate and severe depressive disorders (Fournier et al 2010; Kirsch et al 2008). The observation that healthy Wistar rats respond differently to a methylphenidate challenge than spontaneous hypertensive c h a p t e r rats does also suggest a different mode of action in different rat strains (dela Pena et al 2012a; dela Pena et al 2012b). In the future we are planning to perform experiments similar to the present study, but then in anhedonic animals. It is very important to have an animal model with a depressive-like phenotype, like insensitivity to reward, in which we can study the pathophysiology of depression and search for therapeutic targets to treat anhedonia. Summarizing, in the current study we targeted specific monoamine transporter sites and thereby studied the role of single neurotransmitters systems in reward. The present study shows that the DNRI methylphenidate increased DA in the NAc and in parallel increased brain reward. Chronic treatment with the SSRI escitalopram and

129 Chapter 7

NRI reboxetine did not affect changes in brain reward. Although SSRIs are the most prescribed psychopharmacological drugs, we still do not fully understand how they work. From this study and many other studies from the scientific literature (Goldberg et al 2004; Gupta et al 2006; Trivedi et al 2006), it has become clear that the mesolimbic dopamine system is an important target to treat anhedonia.

130

Discussion

c h a p t e r 8 Chapter 8

134 Discussion

Anhedonia is one of the core symptoms of major depressive disorder. It is defined as the inability to experience pleasure from activities that where once enjoyable. Anhedonia is difficult to treat with the current antidepressants. A novel class of antidepressants, the triple reuptake inhibitors (TRIs), is believed to act as broad-spectrum drug with an early-onset antidepressant effect and might be particularly useful for the treatment of anhedonia. TRIs block the transporters of all three monoamines simultaneously: serotonin (5-HT), norepinephrine (NE) and dopamine (DA). In particular the effects of TRIs on the DA transporter are thought to be important for the treatment of anhedonia.

The aim of this thesis was to study the role of the serotonergic, norepinephrinergic and dopaminergic systems in anhedonia and on brain stimulation reward in rats.

We therefore performed microdialysis studies (chapter 3, 4, 6 and 7) and intracranial self-stimulation studies (chapter 5, 6 and 7) in healthy and “depressed/anhedonic” (olfactory bulbectomized) rats (chapter 3 and 4) in which we examined the effects of the novel TRI DOV216,303 (chapter 3, 4 and 5), the 5-HT1A/1B receptor agonist eltoprazine (chapter 6) and selective inhibitors for different transporters (SERT: escitalopram, NET: reboxetine, DAT/NET: methylphenidate) (chapter 7).

Subtypes of depression A large number of patients do not respond to the first-line antidepressant treatment with selective serotonin reuptake inhibitors (SSRIs). In chapter 2 we made a distinction between different subtypes of depression; melancholic and atypical depression, which both have their own symptom profile. Melancholic depression is associated with loss of weight due to loss of appetite, insomnia, psychomotor agitation and increased sympathetic activity with a subsequent hyperactive HPA-axis and increased CRF. Atypical depression on the other hand is characterized by hyperphagia associated with weight gain, hypersomnia, psychomotor retardation, decreased sympathetic activity, a hypoactive HPA-axis and decreases in CRF (Gold and Chrousos 1999; Gold and Chrousos 2002; Prins et al 2011a). We hypothesize that disturbances in different monoaminergic mechanisms are partly associated with these opposite symptom profiles. Anhedonia is one of the core symptoms and can therefore be present in both melancholic as well as atypical depression, but its etiology might be completely different 8 in nature. Stressful life events can be a predisposing factor to develop anhedonia. During stress, glucocorticoids (GCs) may facilitate DA transmission in the nucleus accumbens (NAc) (Marinelli and Piazza 2002) and have anti-inflammatory actions c h a p t e r (Rhen and Cidlowski 2005). It is hypothesized that in melancholic depression, an activated HPA system will lead to increased GCs levels, which via its negative feedback mechanisms might cause lower levels of pro-inflammatory cytokines and increased DA neurotransmission. This increase in dopaminergic activity might lead to desensitization of the reward system and anhedonia (Dunlop and Nemeroff 2007). In contrast, atypical

135 Chapter 8 depression is characterized by a less active HPA-axis and consequently lower GCs levels causing a more pro-inflammatory immune response pattern (Korte et al 2005) and decreased DA transmission in the NAc, which may lead to anhedonia. It is not surprising that the classical SSRIs are not equally effective in both atypical and melancholic depression (Thase et al 1992; 1995; 2001) given the fact that the symptoms present in both subtypes are on opposite ends of the spectrum. A major group of depressed patients experiences mixed symptoms of either subtype and are diagnosed with moderate depression (Levitan et al 1997). These patients do often not respond adequately to antidepressant treatment (Fournier et al 2010; Kirsch et al 2008) and the presence of anhedonia in depressed patients appears to be a predictor of poor response to treatment in general (Spijker et al 2001). Moreover, a recent review by Treadway and Zald (2011) criticized the current clinical definition of anhedonia. They argued that the definition of anhedonia is not specific enough and that subdivisions should be made into consummatory (pleasure) anhedonia, motivational anhedonia and decisional anhedonia in order to better investigate underlying brain mechanisms. Strikingly, whereas clinical research on anhedonia mainly focuses on the hedonic capacity of patients (consummatory anhedonia), in preclinical research the focus is on motivation and reinforcement (motivational anhedonia) (Treadway and Zald 2011), which again stresses the importance of subdividing different symptom profiles in major depression in order to better study the underlying disturbances in related brain areas.

Monoaminergic neurotransmission in olfactory bulbectomized rats - ‘anhedonia’ In chapter 3 and chapter 4 we investigated the effects of the TRI DOV 216,303 on the olfactory bulbectomy animal model (OBX). Over the past years, many researchers have used the OBX model to predict putative antidepressant activity of novel compounds. The removal of the olfactory bulbs in rodents leads to a variety of behavioral and neurochemical changes (Song and Leonard 2005). Besides that, several lines of evidence suggest that OBX animals have disrupted reward processing. OBX animals showed increased ICSS thresholds up to 8 days after bulbectomy (Slattery et al 2007). A diminished response to amphetamine on ICSS thresholds was found 3 weeks after OBX (Romeas et al 2009) together with a long-lasting reduction in sucrose intake (Chambliss et al 2004; Romeas et al 2009). OBX animals did not show cocaine-induced conditioned place preference (Calcagnetti et al 1996) and have a blunted reactivity to emotional stimuli (Bijlsma et al 2010). Furthermore, they showed decreased sexual behavior (Chambliss et al 2004). These findings made us initially think that the OBX animal was an interesting animal model to study anhedonia. Previous findings in our lab demonstrated that DOV 216,303 had antidepressant properties reflected by the normalized hyperactivity in the OBX animals after chronic treatment without showing sexual side effects (Breuer et al 2008). However, how monoaminergic neurotransmission was modulated in these animals after acute and chronic treatment with DOV 216,303 was not known. We

136 Discussion therefore performed microdialysis studies in OBX and Sham animals. In chapter 3 we showed that one day after ablation of the olfactory bulbs, OBX animals had lower baseline DA levels in the PFC compared to their Sham counterparts. This finding is consistent with the finding that OBX animals have reward deficits up to 8 days after OBX (Slattery et al 2007), assuming that lower DA levels in the PFC correlate with an anhedonic state. However 38 days later, we did not find differences any longer between OBX and Sham animals on baseline DA levels. Again, in a subsequent microdialysis study in OBX animals (chapter 4), we did not see an effect on baseline monoamines several weeks after surgery. This may suggest that this short-lasting effect on DA is due to a surgery-induced increase in pro-inflammatory cytokines (Anisman et al 2008). It is known that systemically administered lipopolysaccharide (LPS) provokes sickness behavior and induces the release of several pro-inflammatory cytokines (Konsman et al 2002), which affect monoaminergic regulation (Borowski et al 1998; van Heesch et al submitted).

Monoamine concentrations in olfactory bulbectomized rats after DOV 216,303 In both microdialysis studies, DOV 216,303 increased 5-HT, NE and DA levels in the PFC and dorsal hippocampus (Prins et al 2010; Prins et al 2011b). In the PFC, the increase in DA was blunted in OBX animals, consistent with findings that OBX animals have a blunted ICSS threshold lowering effect in response to amphetamine and a reduced preference for sucrose compared to Shams, three weeks after surgery (Romeas et al 2009). A remarkable finding from our microdialysis studies with DOV 216,303 was that the monoamine levels in the PFC did not increase to the same extent following chronic treatment, when compared to an acute challenge. Whereas in chapter 3, this finding could be due to differences in time of treatment after surgery, in the later microdialysis study in chapter 4 (with a better experimental design for the comparison of chronic and acute treatment) we showed that indeed, chronic pretreatment with DOV 216,303 caused a blunted response on extracellular monoamine levels following a DOV 216,303-challenge. This effect was most pronounced for the DA levels in the PFC of sham animals. Moreover, in chapter 4, we also assessed the locomotor activity of the animals in an open field to check whether we could replicate the finding of Breuer et al (2008) that DOV 216,303 exerts antidepressant activity in the OBX animal. To our 8 surprise, we did not see a normalization of the OBX-induced hyperactivity by chronic DOV 216,303, which may be explained by a difference in experimental design. The last injection in the study of Breuer et al was given 30 minutes before the last open c h a p t e r field, whereas we measured the locomotor activity 24 hours after the last injection of the chronic administration regimen. Interestingly, HPLC analysis showed that DOV 216,303 was absent in the brain and plasma in our experimental set-up, whereas in the study design of Breuer and co-workers the drug was still present to elicit a behavioral effect. Moreover, we found that brain and plasma levels of DOV 216,303 in chronically

137 Chapter 8 treated animals were lower 30 minutes after administration when compared to an acute first challenge. These findings suggest that DOV 216,303 does not have a very suitable pharmacokinetic profile, that the drug has to be ‘on board’ to give antidepressant-like behavioral effects and that DOV 216,303 will need extra attention before it can be developed as an effective antidepressant treatment. Amitifadine (also known as DOV 21,947 or EB-1010, the + enantiomer of DOV 216,303) was more effective than placebo in treating symptoms of major depressive disorder in patients (Tran et al 2012), but was given twice daily. Amitifadine is currently in a phase 2-3 clinical trial.

Chronic antidepressant treatment and the serotonergic system Long term treatment with DOV 216,303 leads to increased baseline levels of 5-HT in the PFC in Sham as well as OBX animals; there was no difference between the two groups (chapter 3). In chapter 4 we were unable to replicate these findings; no difference in monoamine baseline levels (including 5-HT) were observed after 14 days of treatment with DOV 216,303 in the PFC. However, in the dorsal hippocampus (DH) an increase in 5-HT concentration was measured, but only in Sham and not in OBX animals. This later finding is consistent with previous findings from our laboratory, which showed that OBX animals had long-lasting deficits in serotonergic functioning in the DH and a blunted 5-HT response following fluvoxamine administration (van der Stelt et al 2005). The increased baseline levels in 5-HT in the PFC (chapter 3) and in the DH of Sham animals (chapter 4) were consistent with the leading hypothesis of how antidepressants are thought to execute their function. This hypothesis is based on the fact that chronic treatment with SSRIs lead to enhanced exposure and activation of 5-HT1A autoreceptors in the raphe nuclei (RN), which will therefore downregulate, leading to disinhibited serotonergic cell firing and increased extracellular 5-HT levels (Blier et al 1987). Chronic treatment with DOV 216,303 indeed led to increased baseline 5-HT concentrations, although not consistently (difference between chapter 3 and 4). In chapter 7, we chronically treated animals with the SSRI escitalopram and measured monoamine levels in the PFC and NAc. We observed an increase in 5-HT concentrations in the PFC after acute treatment with escitalopram, but remarkably baseline 5-HT concentrations were not altered after chronic treatment, nor did 5-HT levels change in response to an escitalopram challenge in chronically treated animals. This was rather unexpected, and cannot be explained by the hypothesis described above. However, it is consistent with a study by Popa and colleagues (2010) showing that chronic SSRI treatment leads to increased 5-HT concentrations in the RN, but not in the dorsal hippocampus (DH)

(i.e. projection areas of RN) at moments when 5-HT1A receptors were desensitized. Subchronic treatment on the other hand did lead to increased 5-HT concentrations. The study of Popa and colleagues suggests that prolonged SSRI treatment does not completely inactivate all somatodendritic 5-HT1A autoreceptors in the RN. We therefore hypothesize now that the absence of altered 5-HT levels in the PFC following chronic treatment with an SSRI in our study might be explained by a dampened serotonergic

138 Discussion activity. Due to long-lasting blockade of the 5-HT transporter by chronic SSRIs, high concentrations of 5-HT are present in the RN, thereby intensely stimulating the few autoreceptors available. These autoreceptors cause an increased inhibitory effect on serotonergic activity, leading to decreased 5-HT release in projection areas such as DH and PFC and thus leading to low baseline 5-HT concentrations after chronic SSRI treatment. This hypothesis is in line with studies that 5-HT1A autoreceptors were still functional after prolonged SERT blockade (Hjorth and Auerbach 1994; Hjorth and Auerbach 1999). However, a note of caution is required. Although we measured extracellular baseline values in all experiments, we have to be very careful to compare these baseline values, since they are just above detection limit. Furthermore, the amount of neurotransmitter in the dialysate is determined by several in vitro as well as in vivo parameters, like flow rate, membrane characteristics and exact location of the active region of the microdialysis probe. Therefore, changes in observed baselines should be carefully interpreted and can only serve as indication of the activity of the system, whereas the response to a (pharmacological) challenge gives more reliable information.

Limitations to the olfactory bulbectomy paradigm as depression model OBX animals have a blunted DA response in the PFC after an acute challenge with DOV 216,303. This seems to correspond with studies demonstrating an anhedonic state of OBX animals, immediately (Slattery et al 2007) and after several weeks (Romeas et al 2009) after surgery. However, we have to be careful with the interpretation of these studies, as the observed behavioral and biochemical effects following olfactory bulbectomy might be explained in a completely different way. It might well be possible that the findings in disrupted conditioned place preference by OBX (Calcagnetti et al 1996) are the result of the cognitive impairments such as learning and memory deficits in OBX animals (Borre et al 2012; Douma et al 2011; Hendriksen et al 2012). Since removal of the olfactory bulbs results in a disability to smell and probable alterations in taste, assessing anhedonia in OBX animals by measuring there preference for sweetened solutions might be questionable. Moreover, the increases in ICSS thresholds following the first days after surgery might well be a consequence of inflammation-induced alterations in monoamine neurotransmission. Taken together, the OBX paradigm might be of good value to predict antidepressant activity of novel compounds, however, too much value might have been given to OBX animals as induction model for depressive- 8 like behavior in the past by ascribing depression symptoms to this animal model. New insights have led to the idea that the OBX animal can be better used as animal model for neurodegeneration rather than depression, since removal of the olfactory bulbs causes c h a p t e r severe cognitive deficits (Borre et al 2012; Douma et al 2011; Hendriksen et al 2012). To study anhedonia, it is important to use an animal model that resembles more closely the lower brain reward activity of anhedonic patients. It is likely that such a model will have better validity to study the underlying mechanisms of anhedonia in depression. At the end of this discussion, we will come back to this point.

139 Chapter 8

Rewarding properties and abuse liability of dopaminergic compounds The above mentioned reasons together with our own experience in trying to replicate the findings of OBX-induced changes on ICSS behavior led us decide to continue performing experiments in healthy rats. The development of dopaminergic compounds for the treatment of psychiatric illnesses raises concerns about their intrinsic abuse potential and reinforcing effects (Kuhar et al 1991). In chapter 5 we therefore assessed the rewarding effects of the DOV 216,303; we measured ICSS thresholds in rats after acute and sub-chronic treatment with DOV 216,303 and amphetamine. It was shown that DOV 216,303 induces relatively long-lasting enhancement of brain reward activity, without anhedonia-like reward deficits after cessation of treatment, whereas amphetamine induced withdrawal-associated reward deficits. It is known that drugs of abuse, which increase DA levels in the brain, like cocaine and amphetamine, lower ICSS reward thresholds, whereas withdrawal from those compounds results in increased ICSS thresholds (Barr et al 2002; Cryan et al 2003b; Kenny et al 2003; Markou and Koob 1991). Furthermore, withdrawal from addictive drugs in humans results in symptoms like anxiety and anhedonia (Gawin and Kleber 1986; Weddington et al 1990). From our study we can conclude that it is unlikely that DOV 216,303 has strong abuse potential, because of the absence of long-term compensatory adaptations in reward circuitries similar to those induced by amphetamine. This conclusion is supported by the fact that DOV 216,303 only partly substituted for cocaine in a drug-discrimination assay (Caldarone et al 2010). Although, we did not measure the effects of DOV 216,303 on monoamine release in the NAc of rats in the present experiments, recently we administered DOV 216,303 to mice, which resulted in increased DA levels in the NAc, the most important dopaminergic brain area involved in reward (van Heesch et al unpublished data). Moreover a recent study showed that 10 mg/kg of DOV 21,947 (the + isomer of DOV 216,303) increased dopamine levels in the NAc of rats (Golembiowska et al 2012). Although ICSS is not an exclusive test for studying abuse potential, it can definitely measure some aspects of the addiction cycle, like binge/intoxication (brain stimulation reward thresholds) and withdrawal/negative affect (drug withdrawal-induced elevated reward thresholds) (Koob and Le Moal 2010). However, intravenous self-administration experiments should give more information to what extent DOV 216,303 and other TRIs have addictive properties. In chapter 7, we acutely and chronically blocked simultaneously the NE and DA transporter with methylphenidate and measured monoamines in the NAc and PFC. We observed that decreased ICSS thresholds were accompanied by increased DA concentrations in the NAc. However, increased DA concentrations by chronic reboxetine (NRI) treatment in the PFC did not lead to alterations in brain reward thresholds, indicating that brain reward is much more than simply increased DA levels.

140 Discussion

5-HT-DA interaction The observation that an increase in DA concentrations not necessarily leads to activation of brain reward circuitry became also clear in chapter 6. We investigated the effects of the 5-HT1A/1B receptor agonist eltoprazine on ICSS behavior and in an in vivo microdialysis study in the NAc and PFC. Eltoprazine induced an increase in DA in the PFC and increased metabolites of DA in the NAc. Unexpectedly, this increased dopaminergic activity caused by eltoprazine was not accompanied by a decreased threshold, but on the contrary, by an increased threshold, reflecting decreased sensitivity of brain reward. This effect can be explained by the involvement of 5-HT and its different receptors (e.g. the 5-HT1A and 5-HT1B subtype) in reward-related processes (Hayes and Greenshaw 2011; Kranz et al 2010). Eltoprazine elicited a clear 5-HT-DA and a 5-HT-NE interaction in the PFC, and a 5-HT-DA interaction in the NAc, which all might be indirectly mediated by disinhibition of GABAergic interneurons with a probable major role for the 5-HT1A receptors and a minor role for 5-HT1B receptors in the regulation of extracellular monoamine concentrations in the PFC and the other way around in the NAc (Di Matteo et al 2008; Millan et al 2000).

5-HT and reward Besides an increase in DA, eltoprazine also induced decreased 5-HT concentrations in NAc and PFC (chapter 6), from which we concluded that 5-HT is also important in reward-processing. This is also supported by recent literature (Hayes and Greenshaw 2011; Kranz et al 2010). SSRIs do elevate mood in depressed patients, but they sometimes have troubles relieving anhedonia symptoms, which might be reflected by the fact that the SSRI escitalopram in our hands does not enhance brain stimulation reward (chapter 7). On the contrary, this drug increased ICSS thresholds after acute treatment. Acute rises in 5-HT may be anxiogenic due to activation of postsynaptic

5-HT1A receptors (File et al 1996) and although it is somewhat speculative, because changes in ICSS thresholds do not directly reflect anxiety, the observed increase in thresholds might be explained by an acute SSRI-induced negative state of the animal. Escitalopram also increased 5-HT concentrations in PFC and NAc (chapter 7), whereas eltoprazine increased ICSS thresholds at doses where 5-HT levels in PFC and NAc were decreased. Although a role of 5-HT in valuation (reward and aversion) and

emotional processing is well known, it is clear that 5-HT does not play a unitary role 8 in reward. The multiple receptor subtypes that are targeted by 5-HT may account for the diverse findings with 5-HT and reward. Activation of 1Bthe 5-HT receptor, for example, is known to have an inhibitory effect on reward (Hayes et al 2009), whereas c h a p t e r activation of the 5-HT1A receptor has ambiguous effects on brain stimulation reward (Montgomery et al 1991). However, based on the results presented in this thesis we can only speculate about the role of 5-HT in reward.

141 Chapter 8

Improvement of preclinical research: need for better animal models In chapter 6 and chapter 7, we demonstrated the effects of several drugs on monoamine neurotransmission profile and reward in healthy animals. However, monoamine neurotransmission might be completely different in depressed patients compared to healthy humans. It is known that the role of 5-HT in reward is depending on the anhedonic state of the animal at the time of drug treatment (Harrison et al 2001). Furthermore, methylphenidate is self-administered after chronic pre-exposure to methylphenidate in healthy adult Wistar rats, but not in spontaneous hypertensive rats, suggesting a different mode of action in different rat strains (dela Pena et al 2012b). Exposure to drugs during development also has influence on the response to these drugs later in life. Exposure to methylphenidate during pre-adolescence leads to alterations in brain reward systems in adulthood apparent from a reduction in cocaine- induced potentiating of brain stimulation reward (Mague et al 2005), a blunted response to the rewarding effects of cocaine in place conditioning tests (Carlezon et al 2003) and a reduction in the rewarding effects of sucrose and sex (Bolanos et al 2003). As we suggested in chapter 2, the resting states of the monoaminergic systems in the brain may be out of balance in depressed patients. The drugs tested in this thesis, might therefore give different behavioral and neurochemical responses in a depressed individual. In melancholic depression for example, the tonic 5-HT levels might be reduced, 5-HT receptors and transporters might be upregulated to maximally transmit serotonergic signals. Because of this increased sensitivity of the system, an SSRI in melancholic depression might have a completely different response then in atypical depression or in healthy individuals, where serotonergic functioning is relatively unaffected. To screen novel compounds for their antidepressant activity, animal tests with high predictive validity are available (e.g. open field test with hyperactive OBX-rats, (modified) forced swim test, tail suspension test,differential reinforcement of low rates of responding). Unfortunately and inevitably, this will likely lead to antidepressants with similar mode of actions as the existing antidepressant drugs, because that is where the tests have been validated on. To better understand the biological mechanisms underlying anhedonia, an animal model with predictive validity alone is not sufficient. We therefore need an animal model of depression which resembles more closely the clinical features also present in depression like desensitized brain reward systems. Attempts have been made to induce anhedonia in rats by subjecting them to various stress paradigms. Rats exposed to chronic unpredictable stress procedures showed long-term increases in ICSS thresholds which could be normalized by treatment with the atypical antidepressant (Moreau et al 1994). Moreover, the use of stress paradigms to induce anhedonia has good face validity (resembles symptoms of clinical depression), but more important it shows construct validity (decreased sensitivity to reward) (Willner 1997b). Moreover, social defeat has shown to induce anhedonic symptoms in rats with concurrent disturbances in the dopaminergic reward system (Nocjar et al 2012). And therefore, in future research projects we want to induce long term anhedonia by social defeat with electric simulation-

142 Discussion induced (in the hypothalamus) aggression of the intruder in order to standardize the severity of the stressor (Lin et al 2011a). We may even combine the social defeat with an immunological challenge (Dantzer et al 2008) and individual housing (Ruis et al 1999; Von Frijtag et al 2000), which may result in robust and long term anhedonic symptoms which can be measured by alterations in ICSS thresholds. Currently available animal models and animal tests are not suitable for studying brain mechanisms underlying anhedonia. A good animal model for anhedonia does not necessarily need to have predictive validity (i.e. current, available antidepressants do not necessarily need to relieve anhedonic symptoms in the animal). A large group of patients, especially those experiencing anhedonia, do not sufficiently recover from depressive symptoms after treatment (Spijker et al 2001). To find new targets for antidepressant treatment it is important to first understand which brain mechanisms are disturbed in those patients.

Concluding remarks A substantial body of evidence implicates that the original monoamine hypothesis of depression is too simplistic and that low levels of monoamines alone do not give rise to the diverse variety of depressive symptoms in major depression. However, there is no doubt that monoamines play a role in depression, but the importance of the serotonergic, norepinephrinergic and dopaminergic systems might be different in the different subtypes of depression. Therefore, we proposed an alternative monoamine hypothesis of depression, which supposes a disbalanced monoaminergic system in different subtypes of depression, where a hyperactive 5-HT and NE system might be responsible for melancholic depression and a hypoactive and decreased functioning of the NE and DA system might be more prominent in atypical depression. Anhedonia and other symptoms in depression should therefore be better distinguished in the diagnosis of depression and antidepressant treatment should be better adjusted to the different types of depression. Moreover, monoaminergic systems do not act on their own, but are heavily modulated by each other (and other systems) and do not have a single uniform function in the large brain networks. Triple reuptake inhibitors might be a good first line of antidepressant treatment as they target the 5-HT, NE and DA system simultaneously thereby highly interacting with each other (e.g. 5-HT- DA interaction), but also modulating other neurotransmitter systems, like the GABA, glutamate and systems. Moreover, neuroplasticity processes (including changes 8 in gene-expression and alterations in synaptic transmission, plasticity and neurogenesis) may also play an important role in depression and antidepressant mechanisms of action. The mode of action of current antidepressants is still not fully understood, c h a p t e r as prolonged treatment with SSRIs in our hands does not lead to the hypothesized elevated 5-HT levels. The mechanism of antidepressant drugs should be better investigated in anhedonic as well as healthy animals in order to better understand their action in depressed patients. Animal models are needed in which the neurobiological mechanisms of anhedonia can be studied. ICSS is a sensitive readout for the functionality

143 Chapter 8 of the brain reward system of animals and microdialysis is a reliable way to measure neurotransmission processes in brain areas of interest. The combination of these techniques and the use of animal models displaying chronic anhedonia will give us more insight in the neurobiology of reward and anhedonia. In the mean time, triple reuptake inhibitors might serve as good first-line antidepressants, until an appropriate diagnosis of major depressive disorder will be made, regarding the different subtypes in depression and with special attention to anhedonia.

144 References 146 References

Acsady L, Arabadzisz D, Katona I, Freund TF (1996): Topographic distribution of dorsal and median raphe neurons with hippocampal, septal and dual projection. Acta Biol Hung 47:9-19. Adamec RE (1976): Hypothalamic and extrahypothalamic substrates of predatory attack. Suppression and the influence of hunger.Brain Res 106:57-69. Adell A, Celada P, Artigas F (2001): The role of 5-HT1B receptors in the regulation of serotonin cell firing and release in the rat brain. J Neurochem 79:172-182. Aghajanian GK, Sprouse JS, Sheldon P, Rasmussen K (1990): Electrophysiology of the central serotonin system: receptor subtypes and transducer mechanisms. Ann N Y Acad Sci 600:93-103; discussion 103. Amitai N, Semenova S, Markou A (2009): Clozapine attenuates disruptions in response inhibition and task efficiency induced by repeated administration in the intracranial self-stimulation procedure. Eur J Pharmacol 602:78-84. Andrews G, Sanderson K, Corry J, Lapsley HM (2000): Using epidemiological data to model efficiency in reducing the burden of depression*. J Ment Health Policy Econ 3:175-186. Anisman H, Merali Z, Hayley S (2008): Neurotransmitter, peptide and cytokine processes in relation to depressive disorder: comorbidity between depression and neurodegenerative disorders. Prog Neurobiol 85:1-74. APA (1994): Diagnostic and Statistical Manual of Mental Disorders, Fourth edition ed. Washington, DC: American Psychiatric Association. APA (2000): Diagnostic and Statistical Manual of Mental Disorders. Text Revision, Fourth edition ed. Washington, DC: American Psychiatric Association. Arnsten AF (2000): Through the looking glass: differential noradenergic modulation of prefrontal cortical function. Neural Plast 7:133-146. Axelrod J, Whitby LG, Hertting G (1961a): Effect of psychotropic drugs on the uptake of H3- norepinephrine by tissues. Science 133:383-384. Axelrod J, Whitby LG, Hertting G, Kopin IL (1961b): Studies on the metabolism of catecholamines. Circ Res 9:715-720. Baldwin DS, Papakostas GI (2006): Symptoms of fatigue and sleepiness in major depressive disorder. J Clin Psychiatry 67 Suppl 6:9-15. Banki CM (1977): Correlation between cerebrospinal fluid amine metabolites and psychomotor activity in affective disorders. J Neurochem 28:255-257. Barbon A, Popoli M, La Via L, Moraschi S, Vallini I, Tardito D, et al (2006): Regulation of editing and expression of glutamate alpha-amino-propionic-acid (AMPA)/kainate receptors by antidepressant drugs. Biol Psychiatry 59:713-720. Bari A, Theobald DE, Caprioli D, Mar AC, Aidoo-Micah A, Dalley JW, et al (2010): Serotonin modulates sensitivity to reward and negative feedback in a probabilistic reversal learning task in rats. Neuropsychopharmacology 35:1290-1301. Barr AM, Markou A, Phillips AG (2002): A ‘crash’ course on psychostimulant withdrawal as a model of depression. Trends Pharmacol Sci 23:475-482. Basile AS, Janowsky A, Golembiowska K, Kowalska M, Tam E, Benveniste M, et al (2007): Characterization of the antinociceptive actions of bicifadine in models of acute, persistent, and chronic pain. J Pharmacol Exp Ther 321:1208-1225. Beer B, Stark J, Krieter P, Czobor P, Beer G, Lippa A, et al (2004): DOV 216,303, a “triple” reuptake inhibitor: safety, tolerability, and pharmacokinetic profile.J Clin Pharmacol 44:1360-1367. Bergman J, Madras BK, Johnson SE, Spealman RD (1989): Effects of cocaine and related drugs in nonhuman primates. III. Self-administration by squirrel monkeys. J Pharmacol Exp Ther 251:150- 155. Berridge CW, Waterhouse BD (2003): The locus coeruleus-noradrenergic system: modulation of behavioral state and state-dependent cognitive processes. Brain Res Brain Res Rev 42:33-84. Berridge KC, Kringelbach ML (2008): Affective neuroscience of pleasure: reward in humans and animals. Psychopharmacology (Berl). Bevins RA, Wilkinson JL, Palmatier MI, Siebert HL, Wiltgen SM (2006): Characterization of nicotine’s ability to serve as a negative feature in a Pavlovian appetitive conditioning task in rats. Psychopharmacology (Berl) 184:470-481. Bewernick BH, Kayser S, Sturm V, Schlaepfer TE (2012): Long-term effects of nucleus accumbens deep brain stimulation in treatment-resistant depression: evidence for sustained efficacy. Neuropsychopharmacology 37:1975-1985. Bijlsma EY, Oosting RS, Olivier B, Groenink L (2010): Disrupted startle modulation in animal models for affective disorders. Behav Brain Res 208:383-390. Bittencourt JC, Presse F, Arias C, Peto C, Vaughan J, Nahon JL, et al (1992): The melanin-concentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization. J Comp Neurol 319:218-245.

Blier P, de Montigny C (1990): Differential effect of gepirone on presynaptic and postsynaptic serotonin r efer ences

147 receptors: single-cell recording studies. J Clin Psychopharmacol 10:13S-20S. Blier P, de Montigny C (1994): Current advances and trends in the treatment of depression. Trends Pharmacol Sci 15:220-226. Blier P, de Montigny C, Chaput Y (1987): Modifications of the serotonin system by antidepressant treatments: implications for the therapeutic response in major depression. J Clin Psychopharmacol 7:24S-35S. Blier P, Ward NM (2003): Is there a role for 5-HT1A agonists in the treatment of depression? Biol Psychiatry 53:193-203. Bolanos CA, Barrot M, Berton O, Wallace-Black D, Nestler EJ (2003): Methylphenidate treatment during pre- and periadolescence alters behavioral responses to emotional stimuli at adulthood. Biol Psychiatry 54:1317-1329. Bonanno G, Giambelli R, Raiteri L, Tiraboschi E, Zappettini S, Musazzi L, et al (2005): Chronic antidepressants reduce depolarization-evoked glutamate release and protein interactions favoring formation of SNARE complex in hippocampus. J Neurosci 25:3270-3279. Borowski T, Kokkinidis L, Merali Z, Anisman H (1998): Lipopolysaccharide, central in vivo biogenic amine variations, and anhedonia. Neuroreport 9:3797-3802. Borre Y, Bosman E, Lemstra S, Westphal KG, Olivier B, Oosting RS (2012): Memantine partly rescues behavioral and cognitive deficits in an animal model of neurodegeneration. Neuropharmacology 62:2010-2017. Borsini F, Meli A (1988): Is the forced swimming test a suitable model for revealing antidepressant activity? Psychopharmacology (Berl) 94:147-160. Boyer P, Lecrubier Y, Stalla-Bourdillon A, Fleurot O (1999): Amisulpride versus amineptine and placebo for the treatment of dysthymia. Neuropsychobiology 39:25-32. Boyer P, Tassin JP, Falissart B, Troy S (2000): Sequential improvement of anxiety, depression and anhedonia with sertraline treatment in patients with major depression. J Clin Pharm Ther 25:363-371. Bragulat V, Paillere-Martinot ML, Artiges E, Frouin V, Poline JB, Martinot JL (2007): Dopaminergic function in depressed patients with affective flattening or with impulsivity: [18F]fluoro-L-dopa positron emission tomography study with voxel-based analysis. Psychiatry Res 154:115-124. Brambilla P, Perez J, Barale F, Schettini G, Soares JC (2003): GABAergic dysfunction in mood disorders. Mol Psychiatry 8:721-737, 715. Breese GR, Cooper BR, Hollister AS (1975): Involvement of brain monoamines in the stimulant and paradoxical inhibitory effects of methylphenidate. Psychopharmacologia 44:5-10. Breuer ME, Chan JS, Oosting RS, Groenink L, Korte SM, Campbell U, et al (2008): The triple monoaminergic reuptake inhibitor DOV 216,303 has antidepressant effects in the rat olfactory bulbectomy model and lacks sexual side effects. Eur Neuropsychopharmacol 18:908-916. Breuer ME, Groenink L, Oosting RS, Buerger E, Korte M, Ferger B, et al (2009a): Antidepressant effects of pramipexole, a dopamine D3/D2 receptor agonist, and 7-OH-DPAT, a dopamine D3 receptor agonist, in olfactory bulbectomized rats. Eur J Pharmacol 616:134-140. Breuer ME, Groenink L, Oosting RS, Westenberg HG, Olivier B (2007): Long-term behavioral changes after cessation of chronic antidepressant treatment in olfactory bulbectomized rats. Biol Psychiatry 61:990-995. Breuer ME, van Gaalen MM, Wernet W, Claessens SE, Oosting RS, Behl B, et al (2009b): SSR149415, a non-peptide vasopressin V(1b) receptor antagonist, has long-lasting antidepressant effects in the olfactory bulbectomy-induced hyperactivity depression model. Naunyn Schmiedebergs Arch Pharmacol 379:101-106. Brilman EI, Ormel J (2001): Life events, difficulties and onset of depressive episodes in later life. Psychol Med 31:859-869. Brosen K, Naranjo CA (2001): Review of pharmacokinetic and pharmacodynamic interaction studies with citalopram. Eur Neuropsychopharmacol 11:275-283. Brown RP, Stoll PM, Stokes PE, Frances A, Sweeney J, Kocsis JH, et al (1988): Adrenocortical hyperactivity in depression: effects of agitation, delusions, melancholia, and other illness variables. Psychiatry Res 23:167-178. Caccia S, Cappi M, Fracasso C, Garattini S (1990): Influence of dose and route of administration on the kinetics of fluoxetine and its metabolite norfluoxetine in the rat.Psychopharmacology (Berl) 100:509- 514. Calcagnetti DJ, Quatrella LA, Schechter MD (1996): Olfactory bulbectomy disrupts the expression of cocaine-induced conditioned place preference. Physiol Behav 59:597-604. Caldarone BJ, Paterson NE, Zhou J, Brunner D, Kozikowski AP, Westphal KG, et al (2010a): The novel triple reuptake inhibitor JZAD-IV-22 exhibits an antidepressant pharmacological profile without locomotor stimulant or sensitization properties. J Pharmacol Exp Ther 335:762-770. Capela JP, Carmo H, Remiao F, Bastos ML, Meisel A, Carvalho F (2009): Molecular and cellular mechanisms of ecstasy-induced neurotoxicity: an overview. Mol Neurobiol 39:210-271. Carlezon WA, Jr., Chartoff EH (2007): Intracranial self-stimulation (ICSS) in rodents to study the

148 References

neurobiology of motivation. Nat Protoc 2:2987-2995. Carlezon WA, Jr., Mague SD, Andersen SL (2003): Enduring behavioral effects of early exposure to methylphenidate in rats. Biol Psychiatry 54:1330-1337. Carr KD (1990): Effects of antibodies to dynorphin A and beta-endorphin on lateral hypothalamic self- stimulation in ad libitum fed and food-deprived rats. Brain Res 534:8-14. Carroll BJ, Feinberg M, Greden JF, Tarika J, Albala AA, Haskett RF, et al (1981): A specific laboratory test for the diagnosis of melancholia. Standardization, validation, and clinical utility. Arch Gen Psychiatry 38:15-22. Carvalho M, Carmo H, Costa VM, Capela JP, Pontes H, Remiao F, et al (2012): Toxicity of amphetamines: an update. Arch Toxicol. Casanovas JM, Hervas I, Artigas F (1999): Postsynaptic 5-HT1A receptors control 5-HT release in the rat medial prefrontal cortex. Neuroreport 10:1441-1445. Casanovas JM, Lesourd M, Artigas F (1997): The effect of the selective 5-HT1A agonists alnespirone (S- 20499) and 8-OH-DPAT on extracellular 5-hydroxytryptamine in different regions of rat brain. Br J Pharmacol 122:733-741. Cassano P, Lattanzi L, Fava M, Navari S, Battistini G, Abelli M, et al (2005): Ropinirole in treatment- resistant depression: a 16-week pilot study. Can J Psychiatry 50:357-360. Cazala P (1980): Effects of Lilly 110140 (flouxetine) on self-stimulation behavior in the dorsal and ventral regions of the lateral hypothalamus in the mouse. Psychopharmacology (Berl) 71:143-146. Chambliss HO, Van Hoomissen JD, Holmes PV, Bunnell BN, Dishman RK (2004): Effects of chronic activity wheel running and imipramine on masculine copulatory behavior after olfactory bulbectomy. Physiol Behav 82:593-600. Chan JS, Waldinger MD, Olivier B, Oosting RS (2010): Drug-induced sexual dysfunction in rats. Curr Protoc Neurosci Chapter 9:Unit 9 34. Cheetham SC, Crompton MR, Katona CL, Parker SJ, Horton RW (1988): Brain GABAA/benzodiazepine binding sites and glutamic acid decarboxylase activity in depressed suicide victims. Brain Res 460:114-123. Chen NH, Reith ME (1995): Monoamine interactions measured by microdialysis in the ventral tegmental area of rats treated systemically with (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin. J Neurochem 64:1585-1597. Chen Z, Skolnick P (2007): Triple uptake inhibitors: therapeutic potential in depression and beyond. Expert Opin Investig Drugs 16:1365-1377. Chen Z, Yang J, Tobak A (2008): Designing new treatments for depression and anxiety. IDrugs 11:189-197. Choudary PV, Molnar M, Evans SJ, Tomita H, Li JZ, Vawter MP, et al (2005): Altered cortical glutamatergic and GABAergic signal transmission with glial involvement in depression. Proc Natl Acad Sci U S A 102:15653-15658. Chuluunkhuu G, Nakahara N, Yanagisawa S, Kamae I (2008): The efficacy of reboxetine as an antidepressant, a meta-analysis of both continuous (mean HAM-D score) and dichotomous (response rate) outcomes. Kobe J Med Sci 54:E147-158. Clerc GE, Ruimy P, Verdeau-Palles J (1994): A double-blind comparison of venlafaxine and fluoxetine in patients hospitalized for major depression and melancholia. The Venlafaxine French Inpatient Study Group. Int Clin Psychopharmacol 9:139-143. Cohen BM, Baldessarini RJ (1985): Tolerance to therapeutic effects of antidepressants. Am J Psychiatry 142:489-490. Corrigan MH, Denahan AQ, Wright CE, Ragual RJ, Evans DL (2000): Comparison of pramipexole, fluoxetine, and placebo in patients with major depression. Depress Anxiety 11:58-65. Cousins MS, Stamat HM, de Wit H (2001): Acute doses of d-amphetamine and bupropion increase cigarette smoking. Psychopharmacology (Berl) 157:243-253. Croom KF, Perry CM, Plosker GL (2009): Mirtazapine: a review of its use in major depression and other psychiatric disorders. CNS Drugs 23:427-452. Cryan JF, Bruijnzeel AW, Skjei KL, Markou A (2003a): Bupropion enhances brain reward function and reverses the affective and somatic aspects of nicotine withdrawal in the rat. Psychopharmacology (Berl) 168:347-358. Cryan JF, Hoyer D, Markou A (2003b): Withdrawal from chronic amphetamine induces depressive-like behavioral effects in rodents. Biol Psychiatry 54:49-58. Cryan JF, Kaupmann K (2005): Don’t worry ‘B’ happy!: a role for GABA(B) receptors in anxiety and depression. Trends Pharmacol Sci 26:36-43. D’Aquila PS, Collu M, Gessa GL, Serra G (2000): The role of dopamine in the mechanism of action of antidepressant drugs. Eur J Pharmacol 405:365-373. Dalley JW, Theobald DE, Eagle DM, Passetti F, Robbins TW (2002a): Deficits in impulse control associated with tonically-elevated serotonergic function in rat prefrontal cortex. Neuropsychopharmacology 26:716-728.

Dalley JW, Theobald DE, Pereira EA, Li PM, Robbins TW (2002b): Specific abnormalities in serotonin r efer ences

149 release in the prefrontal cortex of isolation-reared rats measured during behavioural performance of a task assessing visuospatial attention and impulsivity. Psychopharmacology (Berl) 164:329-340. Dantzer R, O’Connor JC, Freund GG, Johnson RW, Kelley KW (2008): From inflammation to sickness and depression: when the immune system subjugates the brain. Nat Rev Neurosci 9:46-56. Davidson RJ, Lewis DA, Alloy LB, Amaral DG, Bush G, Cohen JD, et al (2002): Neural and behavioral substrates of mood and mood regulation. Biol Psychiatry 52:478-502. Daws LC (2009): Unfaithful neurotransmitter transporters: focus on serotonin uptake and implications for antidepressant efficacy.Pharmacol Ther 121:89-99. de Bodinat C, Guardiola-Lemaitre B, Mocaer E, Renard P, Munoz C, Millan MJ (2010): Agomelatine, the first melatonergic antidepressant: discovery, characterization and development. Nat Rev Drug Discov 9:628-642. de Groote L, Olivier B, Westenberg HG (2002): Extracellular serotonin in the prefrontal cortex is limited through terminal 5-HT(1B) autoreceptors: a microdialysis study in knockout mice. Psychopharmacology (Berl) 162:419-424. De Vry J (1995): 5-HT1A receptor agonists: recent developments and controversial issues. Psychopharmacology (Berl) 121:1-26. dela Pena I, Lee JC, Lee HL, Woo TS, Lee HC, Sohn AR, et al (2012a): Differential behavioral responses of the spontaneously hypertensive rat to methylphenidate and : lack of a rewarding effect of repeated methylphenidate treatment. Neurosci Lett 514:189-193. dela Pena I, Yoon SY, Lee JC, dela Pena JB, Sohn AR, Ryu JH, et al (2012b): Methylphenidate treatment in the spontaneously hypertensive rat: influence on methylphenidate self-administration and reinstatement in comparison with Wistar rats. Psychopharmacology (Berl) 221:217-226. Demyttenaere K, De Fruyt J, Stahl SM (2005): The many faces of fatigue in major depressive disorder. Int J Neuropsychopharmacol 8:93-105. Demyttenaere K, Jaspers L (2008): Review: Bupropion and SSRI-induced side effects. J Psychopharmacol 22:792-804. Der-Avakian A, Markou A (2010): Neonatal maternal separation exacerbates the reward-enhancing effect of acute amphetamine administration and the anhedonic effect of repeated social defeat in adult rats. Neuroscience 170:1189-1198. DeSanty KP, Amabile CM (2007): Antidepressant-induced liver injury. Ann Pharmacother 41:1201-1211. Dhillon S, Yang LP, Curran MP (2008a): Bupropion: a review of its use in the management of major depressive disorder. Drugs 68:653-689. Dhillon S, Yang LP, Curran MP (2008b): Spotlight on bupropion in major depressive disorder. CNS Drugs 22:613-617. Di Chiara G, Imperato A (1988): Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc Natl Acad Sci U S A 85:5274- 5278. Di Chiara G, Loddo P, Tanda G (1999): Reciprocal changes in prefrontal and limbic dopamine responsiveness to aversive and rewarding stimuli after chronic mild stress: implications for the psychobiology of depression. Biol Psychiatry 46:1624-1633. Di Giovanni G, Esposito E, Di Matteo V (2010): Role of serotonin in central dopamine dysfunction. CNS Neurosci Ther 16:179-194. Di Matteo V, Di Giovanni G, Pierucci M, Esposito E (2008): Serotonin control of central dopaminergic function: focus on in vivo microdialysis studies. Prog Brain Res 172:7-44. Diaz-Mataix L, Artigas F, Celada P (2006): Activation of pyramidal cells in rat medial prefrontal cortex projecting to ventral tegmental area by a 5-HT1A receptor agonist. Eur Neuropsychopharmacol 16:288-296. Diaz-Mataix L, Scorza MC, Bortolozzi A, Toth M, Celada P, Artigas F (2005): Involvement of 5-HT1A receptors in prefrontal cortex in the modulation of dopaminergic activity: role in atypical action. J Neurosci 25:10831-10843. Dillier N, Laszlo J, Muller B, Koella WP, Olpe HR (1978): Activation of an inhibitory noradrenergic pathway projecting from the locus coeruleus to the cingulate cortex of the rat. Brain Res 154:61-68. Douma TN, Borre Y, Hendriksen H, Olivier B, Oosting RS (2011): Simvastatin improves learning and memory in control but not in olfactory bulbectomized rats. Psychopharmacology (Berl) 216:537-544. Dremencov E, Gispan-Herman I, Rosenstein M, Mendelman A, Overstreet DH, Zohar J, et al (2004): The serotonin-dopamine interaction is critical for fast-onset action of antidepressant treatment: in vivo studies in an animal model of depression. Prog Neuropsychopharmacol Biol Psychiatry 28:141- 147. Drevets WC (2000): Neuroimaging studies of mood disorders. Biol Psychiatry 48:813-829. Drevets WC, Price JL, Simpson JR, Jr., Todd RD, Reich T, Vannier M, et al (1997): Subgenual prefrontal cortex abnormalities in mood disorders. Nature 386:824-827. Drugan RC, Macomber H, Warner TA (2010): Intermittent and continuous swim stress-induced behavioral depression: sensitivity to norepinephrine- and serotonin-selective antidepressants.

150 References

Psychopharmacology (Berl) 212:85-91. Dunlop BW, Crits-Christoph P, Evans DL, Hirschowitz J, Solvason HB, Rickels K, et al (2007): Coadministration of modafinil and a selective serotonin reuptake inhibitor from the initiation of treatment of major depressive disorder with fatigue and sleepiness: a double-blind, placebo- controlled study. J Clin Psychopharmacol 27:614-619. Dunlop BW, Nemeroff CB (2007): The role of dopamine in the pathophysiology of depression. Arch Gen Psychiatry 64:327-337. Duval F, Mokrani MC, Crocq MA, Bailey PE, Diep TS, Correa H, et al (2000): Dopaminergic function and the cortisol response to dexamethasone in psychotic depression. Prog Neuropsychopharmacol Biol Psychiatry 24:207-225. El Mansari M, Guiard BP, Chernoloz O, Ghanbari R, Katz N, Blier P (2010): Relevance of norepinephrine- dopamine interactions in the treatment of major depressive disorder. CNS Neurosci Ther 16:e1-17. Entsuah AR, Huang H, Thase ME (2001): Response and remission rates in different subpopulations with major depressive disorder administered venlafaxine, selective serotonin reuptake inhibitors, or placebo. J Clin Psychiatry 62:869-877. Everitt BJ, Robbins TW (2005): Neural systems of reinforcement for drug addiction: from actions to habits to compulsion. Nat Neurosci 8:1481-1489. Fawcett J, Busch KA, Jacobs D, Kravitz HM, Fogg L (1997): Suicide: a four-pathway clinical-biochemical model. Ann N Y Acad Sci 836:288-301. File SE, Gonzalez LE, Andrews N (1996): Comparative study of pre- and postsynaptic 5-HT1A receptor modulation of anxiety in two ethological animal tests. J Neurosci 16:4810-4815. Flint AJ, Black SE, Campbell-Taylor I, Gailey GF, Levinton C (1993): Abnormal speech articulation, psychomotor retardation, and subcortical dysfunction in major depression. J Psychiatr Res 27:309- 319. Flint J (2003): Animal models of anxiety and their molecular dissection. Semin Cell Dev Biol 14:37-42. Floresco SB, West AR, Ash B, Moore H, Grace AA (2003): Afferent modulation of dopamine neuron firing differentially regulates tonic and phasic dopamine transmission. Nat Neurosci 6:968-973. Fournier JC, DeRubeis RJ, Hollon SD, Dimidjian S, Amsterdam JD, Shelton RC, et al (2010): Antidepressant drug effects and depression severity: a patient-level meta-analysis. JAMA 303:47-53. Fuller RW (1994): Uptake inhibitors increase extracellular serotonin concentration measured by brain microdialysis. Life Sci 55:163-167. Fuster JM (1991): The prefrontal cortex and its relation to behavior. Prog Brain Res 87:201-211. Fuster JM (2000): Memory networks in the prefrontal cortex. Prog Brain Res 122:309-316. Fuster JM (2001): The prefrontal cortex--an update: time is of the essence. Neuron 30:319-333. Gao C, Wolf ME (2008): Dopamine receptors regulate NMDA receptor surface expression in prefrontal cortex neurons. J Neurochem 106:2489-2501. Gardner EL, Liu X, Paredes W, Giordano A, Spector J, Lepore M, et al (2006): A slow-onset, long-duration indanamine monoamine reuptake inhibitor as a potential maintenance pharmacotherapy for psychostimulant abuse: effects in laboratory rat models relating to addiction. Neuropharmacology 51:993-1003. Gasbarri A, Sulli A, Packard MG (1997): The dopaminergic mesencephalic projections to the hippocampal formation in the rat. Prog Neuropsychopharmacol Biol Psychiatry 21:1-22. Gawin FH, Kleber HD (1986): Abstinence symptomatology and psychiatric diagnosis in cocaine abusers. Clinical observations. Arch Gen Psychiatry 43:107-113. Giardina WJ, Radek RJ (1991): Effects of imipramine on the nocturnal behavior of bilateral olfactory bulbectomized rats. Biol Psychiatry 29:1200-1208. Gobert A, Rivet JM, Audinot V, Newman-Tancredi A, Cistarelli L, Millan MJ (1998): Simultaneous quantification of serotonin, dopamine and noradrenaline levels in single frontal cortex dialysates of freely-moving rats reveals a complex pattern of reciprocal auto- and heteroreceptor-mediated control of release. Neuroscience 84:413-429. Gobert A, Rivet JM, Cistarelli L, Melon C, Millan MJ (1999): Buspirone modulates basal and fluoxetine- stimulated dialysate levels of dopamine, noradrenaline and serotonin in the frontal cortex of freely moving rats: activation of serotonin1A receptors and blockade of alpha2-adrenergic receptors underlie its actions. Neuroscience 93:1251-1262. Gobert A, Rivet JM, Cistarelli L, Millan MJ (1997): Potentiation of the fluoxetine-induced increase in dialysate levels of serotonin (5-HT) in the frontal cortex of freely moving rats by combined blockade of 5-HT1A and 5-HT1B receptors with WAY 100,635 and GR 127,935. J Neurochem 68:1159-1163. Gold PW, Chrousos GP (1999): The endocrinology of melancholic and atypical depression: relation to neurocircuitry and somatic consequences. Proc Assoc Am Physicians 111:22-34. Gold PW, Chrousos GP (2002): Organization of the stress system and its dysregulation in melancholic and atypical depression: high vs low CRH/NE states. Mol Psychiatry 7:254-275.

Goldberg JF, Burdick KE, Endick CJ (2004): Preliminary randomized, double-blind, placebo-controlled r efer ences

151 trial of pramipexole added to mood stabilizers for treatment-resistant bipolar depression. Am J Psychiatry 161:564-566. Golembiowska K, Kowalska M, Bymaster FP (2012): Effects of the triple reuptake inhibitor amitifadine on extracellular levels of monoamines in rat brain regions and on locomotor activity. Synapse. Gommans J, Hijzen TH, Maes RA, Olivier B (1997): Discriminative stimulus properties of eltoprazine. Life Sci 61:11-19. Gonzalez LE, Quinonez B, Rangel A, Pino S, Hernandez L (2004): Tonic and phasic alteration in amygdala 5-HT, glutamate and GABA transmission after prefrontal cortex damage in rats. Brain Res 1005:154-163. Grace AA (1991): Phasic versus tonic dopamine release and the modulation of dopamine system responsivity: a hypothesis for the etiology of schizophrenia. Neuroscience 41:1-24. Griffith JD, Carranza J, Griffith C, Miller LL (1983): Bupropion: clinical assay for amphetamine-like abuse potential. J Clin Psychiatry 44:206-208. Guiard BP, El Mansari M, Blier P (2008): Cross-talk between dopaminergic and noradrenergic systems in the rat ventral tegmental area, locus ceruleus, and dorsal hippocampus. Mol Pharmacol 74:1463- 1475. Guiard BP, El Mansari M, Blier P (2009a): Prospect of a dopamine contribution in the next generation of antidepressant drugs: the triple reuptake inhibitors. Curr Drug Targets 10:1069-1084. Guiard BP, El Mansari M, Blier P (2009b): Prospect of a Dopamine Contribution in the Next Generation of Antidepressant Drugs: The Triple Reuptake Inhibitors. Curr Drug Targets. Gupta S, Vincent JL, Frank B (2006): Pramipexole: augmentation in the treatment of depressive symptoms. CNS Spectr 11:172-175. Guyon A, Assouly-Besse F, Biala G, Puech AJ, Thiebot MH (1993): Potentiation by low doses of selected neuroleptics of food-induced conditioned place preference in rats. Psychopharmacology (Berl) 110:460-466. Hache G, Coudore F, Gardier AM, Guiard BP (2011): Monoaminergic Antidepressants in the Relief of Pain: Potential Therapeutic Utility of Triple Reuptake Inhibitors (TRIs). Pharmaceuticals 4:285- 342. Hansard MJ, Smith LA, Jackson MJ, Cheetham SC, Jenner P (2002): Dopamine, but not norepinephrine or serotonin, reuptake inhibition reverses motor deficits in 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine-treated primates. J Pharmacol Exp Ther 303:952-958. Harrison AA, Liem YT, Markou A (2001): Fluoxetine combined with a serotonin-1A receptor antagonist reversed reward deficits observed during nicotine and amphetamine withdrawal in rats. Neuropsychopharmacology 25:55-71. Hasler G Pathophysiology of depression: do we have any solid evidence of interest to clinicians? World Psychiatry 9:155-161. Hayes DJ, Graham DA, Greenshaw AJ (2009): Effects of systemic 5-HT(1B) receptor compounds on ventral tegmental area intracranial self-stimulation thresholds in rats. Eur J Pharmacol 604:74-78. Hayes DJ, Greenshaw AJ (2011): 5-HT receptors and reward-related behaviour: a review. Neurosci Biobehav Rev 35:1419-1449. Hendriksen H, Meulendijks D, Douma TN, Bink DI, Breuer ME, Westphal KG, et al (2012): Environmental enrichment has antidepressant-like action without improving learning and memory deficits in olfactory bulbectomized rats. Neuropharmacology 62:270-277. Henry C, Demotes-Mainard J (2006): SSRIs, suicide and violent behavior: is there a need for a better definition of the depressive state?Curr Drug Saf 1:59-62. Hjorth S (1985): Hypothermia in the rat induced by the potent serotoninergic agent 8-OH-DPAT. J Neural Transm 61:131-135. Hjorth S, Auerbach SB (1994): Lack of 5-HT1A autoreceptor desensitization following chronic citalopram treatment, as determined by in vivo microdialysis. Neuropharmacology 33:331-334. Hjorth S, Auerbach SB (1999): Autoreceptors remain functional after prolonged treatment with a serotonin reuptake inhibitor. Brain Res 835:224-228. Holemans S, De Paermentier F, Horton RW, Crompton MR, Katona CL, Maloteaux JM (1993): NMDA glutamatergic receptors, labelled with [3H]MK-801, in brain samples from drug-free depressed suicides. Brain Res 616:138-143. Holsboer F (2000): The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology 23:477- 501. Horn DI, Yu C, Steiner J, Buchmann J, Kaufmann J, Osoba A, et al (2010): Glutamatergic and resting-state functional connectivity correlates of severity in major depression - the role of pregenual anterior cingulate cortex and anterior insula. Front Syst Neurosci 4. Horwath E, Johnson J, Weissman MM, Hornig CD (1992): The validity of major depression with atypical features based on a community study. J Affect Disord 26:117-125. Invernizzi RW, Parini S, Sacchetti G, Fracasso C, Caccia S, Annoni K, et al (2001): Chronic treatment with reboxetine by osmotic pumps facilitates its effect on extracellular noradrenaline and may

152 References

desensitize alpha(2)-adrenoceptors in the prefrontal cortex. Br J Pharmacol 132:183-188. Invernizzi RW, Sacchetti G, Parini S, Acconcia S, Samanin R (2003): Flibanserin, a potential antidepressant drug, lowers 5-HT and raises dopamine and noradrenaline in the rat prefrontal cortex dialysate: role of 5-HT(1A) receptors. Br J Pharmacol 139:1281-1288. Ishizuka T, Sakamoto Y, Sakurai T, Yamatodani A (2003): Modafinil increases histamine release in the anterior hypothalamus of rats. Neurosci Lett 339:143-146. Iyer RN, Bradberry CW (1996): Serotonin-mediated increase in prefrontal cortex dopamine release: pharmacological characterization. J Pharmacol Exp Ther 277:40-47. Izenwasser S, Kornetsky C (1989): The effect of or nisoxetine in combination with morphine on brain-stimulation reward. Pharmacol Biochem Behav 32:983-986. Izumi T, Iwamoto N, Kitaichi Y, Kato A, Inoue T, Koyama T (2006): Effects of co-administration of a selective serotonin reuptake inhibitor and monoamine oxidase inhibitors on 5-HT-related behavior in rats. Eur J Pharmacol 532:258-264. Jay TM (2003): Dopamine: a potential substrate for synaptic plasticity and memory mechanisms. Prog Neurobiol 69:375-390. Jay TM, Gurden H, Yamaguchi T (1998): Rapid increase in PKA activity during long-term potentiation in the hippocampal afferent fibre system to the prefrontal cortex in vivo. Eur J Neurosci 10:3302- 3306. Joels M, Hesen W, de Kloet ER (1991): Mineralocorticoid hormones suppress serotonin-induced hyperpolarization of rat hippocampal CA1 neurons. J Neurosci 11:2288-2294. Johnson PM, Kenny PJ (2010): Dopamine D2 receptors in addiction-like reward dysfunction and compulsive eating in obese rats. Nat Neurosci 13:635-641. Kapur S, Mann JJ (1992): Role of the dopaminergic system in depression. Biol Psychiatry 32:1-17. Karst H, Berger S, Turiault M, Tronche F, Schutz G, Joels M (2005): Mineralocorticoid receptors are indispensable for nongenomic modulation of hippocampal glutamate transmission by corticosterone. Proc Natl Acad Sci U S A 102:19204-19207. Kasper S, Pletan Y, Solles A, Tournoux A (1996): Comparative studies with milnacipran and tricyclic antidepressants in the treatment of patients with major depression: a summary of clinical trial results. Int Clin Psychopharmacol 11 Suppl 4:35-39. Katz MM, Maas JW, Frazer A, Koslow SH, Bowden CL, Berman N, et al (1994): Drug-induced actions on brain neurotransmitter systems and changes in the behaviors and emotions of depressed patients. Neuropsychopharmacology 11:89-100. Katz MM, Tekell JL, Bowden CL, Brannan S, Houston JP, Berman N, et al (2004): Onset and early behavioral effects of pharmacologically different antidepressants and placebo in depression. Neuropsychopharmacology 29:566-579. Katz RJ, Baldrighi G, Carroll BJ (1977): Effects of nomifensine (HOE 984) upon psychomotor activity and intracranial self-stimulation in the rat. Pharmacol Biochem Behav 7:269-272. Katz RJ, Carroll BJ (1977): Intracranial reward after Lilly 110140 (fluoxetine HCl): evidence for an inhibitory role for serotonin. Psychopharmacology (Berl) 51:189-193. Kelly JP, Wrynn AS, Leonard BE (1997): The olfactory bulbectomized rat as a model of depression: an update. Pharmacol Ther 74:299-316. Kenny PJ (2007): Brain reward systems and compulsive drug use. Trends Pharmacol Sci 28:135-141. Kenny PJ, Chen SA, Kitamura O, Markou A, Koob GF (2006): Conditioned withdrawal drives heroin consumption and decreases reward sensitivity. J Neurosci 26:5894-5900. Kenny PJ, Markou A (2006): Nicotine self-administration acutely activates brain reward systems and induces a long-lasting increase in reward sensitivity. Neuropsychopharmacology 31:1203-1211. Kenny PJ, Polis I, Koob GF, Markou A (2003): Low dose cocaine self-administration transiently increases but high dose cocaine persistently decreases brain reward function in rats. Eur J Neurosci 17:191- 195. Keselman HJ, Algina J, Kowalchuk RK (2001): The analysis of repeated measures designs: a review. Br J Math Stat Psychol 54:1-20. Kinney GG, Taber MT, Gribkoff VK (2000): The augmentation hypothesis for improvement of antidepressant therapy: is pindolol a suitable candidate for testing the ability of 5HT1A receptor antagonists to enhance SSRI efficacy and onset latency?Mol Neurobiol 21:137-152. Kirsch I, Deacon BJ, Huedo-Medina TB, Scoboria A, Moore TJ, Johnson BT (2008): Initial severity and antidepressant benefits: a meta-analysis of data submitted to the Food and Drug Administration. PLoS Med 5:e45. Klein D, Brown TS (1969): Exploratory behavior and spontaneous alternation in blind and anosmic rats. J Comp Physiol Psychol 68:107-110. Koenigs M, Grafman J (2009): The functional neuroanatomy of depression: distinct roles for ventromedial and dorsolateral prefrontal cortex. Behav Brain Res 201:239-243. Konsman JP, Parnet P, Dantzer R (2002): Cytokine-induced sickness behaviour: mechanisms and

implications. Trends Neurosci 25:154-159. r efer ences

153 Koob GF, Le Moal M (2010): Neurobiological mechanisms for opponent motivational processes in addiction. In: Robbins TW, Everitt BJ, Nutt D editors. The Neurobiology of Addiction: New Vistas. New York: Oxford University Press, pp 7-23. Koob GF, Volkow ND (2010): Neurocircuitry of addiction. Neuropsychopharmacology 35:217-238. Kopta SM, Howard KI, Lowry JL, Beutler LE (1994): Patterns of symptomatic recovery in psychotherapy. J Consult Clin Psychol 62:1009-1016. Korf J, van Praag HM (1971): Retarded depression and the dopamine metabolism. Psychopharmacologia 19:199-203. Kornetsky C (1985): Brain-stimulation reward: a model for the neuronal bases for drug-induced euphoria. NIDA Res Monogr 62:30-50. Kornetsky C, Esposito RU (1979): Euphorigenic drugs: effects on the reward pathways of the brain. Fed Proc 38:2473-2476. Korte SM (2001): Corticosteroids in relation to fear, anxiety and psychopathology. Neurosci Biobehav Rev 25:117-142. Korte SM, De Kloet ER, Buwalda B, Bouman SD, Bohus B (1996): Antisense to the glucocorticoid receptor in hippocampal dentate gyrus reduces immobility in forced swim test. Eur J Pharmacol 301:19-25. Korte SM, Koolhaas JM, Wingfield JC, McEwen BS (2005): The Darwinian concept of stress: benefits of allostasis and costs of allostatic load and the trade-offs in health and disease. Neurosci Biobehav Rev 29:3-38. Korte-Bouws GA, Korte SM, De Kloet ER, Bohus B (1996): Blockade of corticosterone synthesis reduces serotonin turnover in the dorsal hippocampus of the rat as measured by microdialysis. J Neuroendocrinol 8:877-881. Kranz GS, Kasper S, Lanzenberger R (2010): Reward and the serotonergic system. Neuroscience 166:1023- 1035. Kruse MS, Premont J, Krebs MO, Jay TM (2009): Interaction of dopamine D1 with NMDA NR1 receptors in rat prefrontal cortex. Eur Neuropsychopharmacol 19:296-304. Krystal JH, Sanacora G, Blumberg H, Anand A, Charney DS, Marek G, et al (2002): Glutamate and GABA systems as targets for novel antidepressant and mood-stabilizing treatments. Mol Psychiatry 7 Suppl 1:S71-80. Kuczenski R, Segal D (1989): Concomitant characterization of behavioral and striatal neurotransmitter response to amphetamine using in vivo microdialysis. J Neurosci 9:2051-2065. Kuczenski R, Segal DS (1997): Effects of methylphenidate on extracellular dopamine, serotonin, and norepinephrine: comparison with amphetamine. J Neurochem 68:2032-2037. Kuczenski R, Segal DS (2001): Locomotor effects of acute and repeated threshold doses of amphetamine and methylphenidate: relative roles of dopamine and norepinephrine. J Pharmacol Exp Ther 296:876-883. Kuhar MJ, Ritz MC, Boja JW (1991): The dopamine hypothesis of the reinforcing properties of cocaine. Trends Neurosci 14:299-302. Kuhn R (1957): [Treatment of depressive states with an iminodibenzyl derivative (G 22355)]. Schweiz Med Wochenschr 87:1135-1140. Kuhn R (1958): The treatment of depressive states with G 22355 (imipramine hydrochloride). Am J Psychiatry 115:459-464. Kulkarni SK, Dhir A (2009): Current investigational drugs for major depression. Expert Opin Investig Drugs 18:767-788. Lamers F, de Jonge P, Nolen WA, Smit JH, Zitman FG, Beekman AT, et al Identifying depressive subtypes in a large cohort study: results from the Netherlands Study of Depression and Anxiety (NESDA). J Clin Psychiatry 71:1582-1589. Lauterborn JC, Lynch G, Vanderklish P, Arai A, Gall CM (2000): Positive modulation of AMPA receptors increases neurotrophin expression by hippocampal and cortical neurons. J Neurosci 20:8-21. Lavergne F, Jay TM (2010): A new strategy for antidepressant prescription. Front Neurosci 4:192. Learned-Coughlin SM, Bergstrom M, Savitcheva I, Ascher J, Schmith VD, Langstrom B (2003): In vivo activity of bupropion at the human dopamine transporter as measured by positron emission tomography. Biol Psychiatry 54:800-805. LeDoux JE (1993): Emotional memory systems in the brain. Behav Brain Res 58:69-79. Lee K, Kornetsky C (1998): Acute and chronic fluoxetine treatment decreases the sensitivity of rats to rewarding brain stimulation. Pharmacol Biochem Behav 60:539-544. Leith NJ, Barrett RJ (1976): Amphetamine and the reward system: evidence for tolerance and post-drug depression. Psychopharmacologia 46:19-25. Leknes S, Tracey I (2008): A common neurobiology for pain and pleasure. Nat Rev Neurosci 9:314-320. Lemke MR, Brecht HM, Koester J, Reichmann H (2006): Effects of the dopamine agonist pramipexole on depression, anhedonia and motor functioning in Parkinson’s disease. J Neurol Sci 248:266-270. Leshner AI, Koob GF (1999): Drugs of abuse and the brain. Proc Assoc Am Physicians 111:99-108. Levitan RD, Lesage A, Parikh SV, Goering P, Kennedy SH (1997): Reversed neurovegetative symptoms of

154 References

depression: a community study of Ontario. Am J Psychiatry 154:934-940. Li CT, Lin CP, Chou KH, Chen IY, Hsieh JC, Wu CL, et al Structural and cognitive deficits in remitting and non-remitting recurrent depression: a voxel-based morphometric study. Neuroimage 50:347-356. Li N, Lee B, Liu RJ, Banasr M, Dwyer JM, Iwata M, et al (2010): mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists. Science 329:959-964. Lichtenberg P, Belmaker RH (2010): Subtyping major depressive disorder. Psychother Psychosom 79:131-135. Lin D, Boyle MP, Dollar P, Lee H, Lein ES, Perona P, et al (2011a): Functional identification of an aggression locus in the mouse hypothalamus. Nature 470:221-226. Lin D, Koob GF, Markou A (1999): Differential effects of withdrawal from chronic amphetamine or fluoxetine administration on brain stimulation reward in the rat--interactions between the two drugs. Psychopharmacology (Berl) 145:283-294. Lin D, Koob GF, Markou A (2000): Time-dependent alterations in ICSS thresholds associated with repeated amphetamine administrations. Pharmacol Biochem Behav 65:407-417. Lin TY, Yang TT, Lu CW, Wang SJ (2011b): Inhibition of glutamate release by bupropion in rat cerebral cortex nerve terminals. Prog Neuropsychopharmacol Biol Psychiatry 35:598-606. Liu YP, Wilkinson LS, Robbins TW (2004): Effects of acute and chronic buspirone on impulsive choice and efflux of 5-HT and dopamine in hippocampus, nucleus accumbens and prefrontal cortex. Psychopharmacology (Berl) 173:175-185. Lopez-Ibor J, Guelfi JD, Pletan Y, Tournoux A, Prost JF (1996): Milnacipran and selective serotonin reuptake inhibitors in major depression. Int Clin Psychopharmacol 11 Suppl 4:41-46. Lucki I (1997): The forced swimming test as a model for core and component behavioral effects of antidepressant drugs. Behav Pharmacol 8:523-532. Mague SD, Andersen SL, Carlezon WA, Jr. (2005): Early developmental exposure to methylphenidate reduces cocaine-induced potentiation of brain stimulation reward in rats. Biol Psychiatry 57:120- 125. Malagie I, Trillat AC, Douvier E, Anmella MC, Dessalles MC, Jacquot C, et al (1996): Regional differences in the effect of the combined treatment of WAY 100635 and fluoxetine: an in vivo microdialysis study. Naunyn Schmiedebergs Arch Pharmacol 354:785-790. Manji HK, Drevets WC, Charney DS (2001): The cellular neurobiology of depression. Nat Med 7:541-547. Manji HK, Quiroz JA, Sporn J, Payne JL, Denicoff K, N AG, et al (2003): Enhancing neuronal plasticity and cellular resilience to develop novel, improved therapeutics for difficult-to-treat depression. Biol Psychiatry 53:707-742. Mann JJ (2003): Neurobiology of suicidal behaviour. Nat Rev Neurosci 4:819-828. Mann JJ, Henteleff RA, Lagattuta TF, Perper JA, Li S, Arango V (1996): Lower 3H-paroxetine binding in cerebral cortex of suicide victims is partly due to fewer high affinity, non-transporter sites. J Neural Transm 103:1337-1350. Mar A, Spreekmeester E, Rochford J (2000): Antidepressants preferentially enhance habituation to novelty in the olfactory bulbectomized rat. Psychopharmacology (Berl) 150:52-60. Marinelli M, Piazza PV (2002): Interaction between glucocorticoid hormones, stress and psychostimulant drugs. Eur J Neurosci 16:387-394. Markou A, Koob GF (1991): Postcocaine anhedonia. An animal model of cocaine withdrawal. Neuropsychopharmacology 4:17-26. Markou A, Koob GF (1992): Construct validity of a self-stimulation threshold paradigm: effects of reward and performance manipulations. Physiol Behav 51:111-119. Marks DM, Pae CU, Patkar AA (2008a): Triple reuptake inhibitors: a premise and promise. Psychiatry Investig 5:142-147. Marks DM, Pae CU, Patkar AA (2008b): Triple reuptake inhibitors: the next generation of antidepressants. Curr Neuropharmacol 6:338-343. Martin-Ruiz R, Ugedo L (2001): Electrophysiological evidence for postsynaptic 5-HT(1A) receptor control of dorsal raphe 5-HT neurones. Neuropharmacology 41:72-78. Martinez-Turrillas R, Del Rio J, Frechilla D (2007): Neuronal proteins involved in synaptic targeting of AMPA receptors in rat hippocampus by antidepressant drugs. Biochem Biophys Res Commun 353:750-755. Martinot M, Bragulat V, Artiges E, Dolle F, Hinnen F, Jouvent R, et al (2001): Decreased presynaptic dopamine function in the left caudate of depressed patients with affective flattening and psychomotor retardation. Am J Psychiatry 158:314-316. Masini CV, Holmes PV, Freeman KG, Maki AC, Edwards GL (2004): Dopamine overflow is increased in olfactory bulbectomized rats: an in vivo microdialysis study. Physiol Behav 81:111-119. Massana J (1998): Reboxetine versus fluoxetine: an overview of efficacy and tolerability.J Clin Psychiatry 59 Suppl 14:8-10. Mayberg HS, Liotti M, Brannan SK, McGinnis S, Mahurin RK, Jerabek PA, et al (1999): Reciprocal limbic- cortical function and negative mood: converging PET findings in depression and normal sadness.

Am J Psychiatry 156:675-682. r efer ences

155 McCabe C, Mishor Z, Cowen PJ, Harmer CJ (2010): Diminished neural processing of aversive and rewarding stimuli during selective serotonin reuptake inhibitor treatment. Biol Psychiatry 67:439-445. McEwen BS (2001): Plasticity of the hippocampus: adaptation to chronic stress and allostatic load. Ann N Y Acad Sci 933:265-277. McEwen BS, Chattarji S (2004): Molecular mechanisms of neuroplasticity and pharmacological implications: the example of tianeptine. Eur Neuropsychopharmacol 14 Suppl 5:S497-502. McEwen BS, Chattarji S, Diamond DM, Jay TM, Reagan LP, Svenningsson P, et al (2010): The neurobiological properties of tianeptine (Stablon): from monoamine hypothesis to glutamatergic modulation. Mol Psychiatry 15:237-249. Merali Z, Du L, Hrdina P, Palkovits M, Faludi G, Poulter MO, et al (2004): Dysregulation in the suicide brain: mRNA expression of corticotropin-releasing hormone receptors and GABA(A) receptor subunits in frontal cortical brain region. J Neurosci 24:1478-1485. Meyer JH, Goulding VS, Wilson AA, Hussey D, Christensen BK, Houle S (2002): Bupropion occupancy of the dopamine transporter is low during clinical treatment. Psychopharmacology (Berl) 163:102-105. Meyer JH, Wilson AA, Sagrati S, Hussey D, Carella A, Potter WZ, et al (2004): Serotonin transporter occupancy of five selective serotonin reuptake inhibitors at different doses: an [11C]DASB positron emission tomography study. Am J Psychiatry 161:826-835. Michael-Titus AT, Bains S, Jeetle J, Whelpton R (2000): Imipramine and phenelzine decrease glutamate overflow in the prefrontal cortex--a possible mechanism of neuroprotection in major depression? Neuroscience 100:681-684. Micheli F, Cavanni P, Arban R, Benedetti R, Bertani B, Bettati M, et al (2010): 1-(Aryl)-6-[alkoxyalkyl]-3- azabicyclo[3.1.0]hexanes and 6-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes: a new series of potent and selective triple reuptake inhibitors. J Med Chem 53:2534-2551. Mignot E (2001): A commentary on the neurobiology of the hypocretin/orexin system. Neuropsychopharmacology 25:S5-13. Millan MJ (2006): Multi-target strategies for the improved treatment of depressive states: Conceptual foundations and neuronal substrates, drug discovery and therapeutic application. Pharmacol Ther 110:135-370. Millan MJ (2009): Dual- and triple-acting agents for treating core and co-morbid symptoms of major depression: novel concepts, new drugs. Neurotherapeutics 6:53-77. Millan MJ, Lejeune F, Gobert A (2000): Reciprocal autoreceptor and heteroreceptor control of serotonergic, dopaminergic and noradrenergic transmission in the frontal cortex: relevance to the actions of antidepressant agents. J Psychopharmacol 14:114-138. Miller DB, O’Callaghan JP (1995): The role of temperature, stress, and other factors in the neurotoxicity of the substituted amphetamines 3,4-methylenedioxymethamphetamine and . Mol Neurobiol 11:177-192. Mitchell P, Hadzi-Pavlovic D, Parker G, Hickie I, Wilhelm K, Brodaty H, et al (1996): Depressive psychomotor disturbance, cortisol, and dexamethasone. Biol Psychiatry 40:941-950. Moghaddam B, Bolinao ML, Stein-Behrens B, Sapolsky R (1994): Glucocorticoids mediate the stress- induced extracellular accumulation of glutamate. Brain Res 655:251-254. Molliver ME (1987): Serotonergic neuronal systems: what their anatomic organization tells us about function. J Clin Psychopharmacol 7:3S-23S. Montgomery AM, Rose IC, Herberg LJ (1991): 5-HT1A agonists and dopamine: the effects of 8-OH- DPAT and buspirone on brain-stimulation reward. J Neural Transm Gen Sect 83:139-148. Montgomery SA (1997): Reboxetine: additional benefits to the depressed patient.J Psychopharmacol 11:S9-15. Moos RH, Cronkite RC (1999): Symptom-based predictors of a 10-year chronic course of treated depression. J Nerv Ment Dis 187:360-368. Moreau JL, Bourson A, Jenck F, Martin JR, Mortas P (1994): Curative effects of the atypical antidepressant mianserin in the chronic mild stress-induced anhedonia model of depression. J Psychiatry Neurosci 19:51-56. Moron JA, Brockington A, Wise RA, Rocha BA, Hope BT (2002): Dopamine uptake through the norepinephrine transporter in brain regions with low levels of the dopamine transporter: evidence from knock-out mouse lines. J Neurosci 22:389-395. Morrison PD, Murray RM (2009): From real-world events to psychosis: the emerging neuropharmacology of delusions. Schizophr Bull 35:668-674. Moutsimilli L, Farley S, Dumas S, El Mestikawy S, Giros B, Tzavara ET (2005): Selective cortical VGLUT1 increase as a marker for antidepressant activity. Neuropharmacology 49:890-900. Muller CP, Carey RJ, Huston JP, De Souza Silva MA (2007): Serotonin and psychostimulant addiction: focus on 5-HT1A-receptors. Prog Neurobiol 81:133-178. Musazzi L, Milanese M, Farisello P, Zappettini S, Tardito D, Barbiero VS, et al (2010): Acute stress increases depolarization-evoked glutamate release in the rat prefrontal/frontal cortex: the dampening action of antidepressants. PLoS One 5:e8566. Nakayama K, Sakurai T, Katsu H (2004): Mirtazapine increases dopamine release in prefrontal cortex by

156 References

5-HT1A receptor activation. Brain Res Bull 63:237-241. Nandagopal JJ, DelBello MP (2009): Selegiline transdermal system: a novel treatment option for major depressive disorder. Expert Opin Pharmacother 10:1665-1673. Naranjo CA, Tremblay LK, Busto UE (2001): The role of the brain reward system in depression. Prog Neuropsychopharmacol Biol Psychiatry 25:781-823. Nestler EJ, Barrot M, DiLeone RJ, Eisch AJ, Gold SJ, Monteggia LM (2002): Neurobiology of depression. Neuron 34:13-25. Nestler EJ, Carlezon WA, Jr. (2006): The mesolimbic dopamine reward circuit in depression. Biol Psychiatry 59:1151-1159. Nichols DE, Nichols CD (2008): Serotonin receptors. Chem Rev 108:1614-1641. Nicholson KL, Balster RL, Golembiowska K, Kowalska M, Tizzano JP, Skolnick P, et al (2009): Preclinical evaluation of the abuse potential of the analgesic bicifadine. J Pharmacol Exp Ther 330:236-248. Nocjar C, Zhang J, Feng P, Panksepp J (2012): The social defeat animal model of depression shows diminished levels of orexin in mesocortical regions of the dopamine system, and of dynorphin and orexin in the hypothalamus. Neuroscience 218:138-153. Noga JT, Hyde TM, Herman MM, Spurney CF, Bigelow LB, Weinberger DR, et al (1997): Glutamate receptors in the postmortem striatum of schizophrenic, suicide, and control brains. Synapse 27:168-176. Nowak G, Ordway GA, Paul IA (1995): Alterations in the N-methyl-D-aspartate (NMDA) receptor complex in the frontal cortex of suicide victims. Brain Res 675:157-164. Nowak G, Trullas R, Layer RT, Skolnick P, Paul IA (1993): Adaptive changes in the N-methyl-D-aspartate receptor complex after chronic treatment with imipramine and 1-aminocyclopropanecarboxylic acid. J Pharmacol Exp Ther 265:1380-1386. Nutt D, Demyttenaere K, Janka Z, Aarre T, Bourin M, Canonico PL, et al (2007): The other face of depression, reduced positive affect: the role of catecholamines in causation and cure. J Psychopharmacol 21:461-471. Olds J (1958): Self-stimulation of the brain; its use to study local effects of hunger, sex, and drugs. Science 127:315-324. Olds J, Milner P (1954): Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain. J Comp Physiol Psychol 47:419-427. Olijslagers JE, de Kloet ER, Elgersma Y, van Woerden GM, Joels M, Karst H (2008): Rapid changes in hippocampal CA1 pyramidal cell function via pre- as well as postsynaptic membrane mineralocorticoid receptors. Eur J Neurosci 27:2542-2550. Olivier B (2004): Serotonin and aggression. Ann N Y Acad Sci 1036:382-392. Olivier B, Mos J, Raghoebar M, de Koning P, Mak M (1994): Serenics. Prog Drug Res 42:167-308. Olivier B, van Oorschot R (2005): 5-HT1B receptors and aggression: a review. Eur J Pharmacol 526:207-217. Opdyke KS, Reynolds CF, 3rd, Frank E, Begley AE, Buysse DJ, Dew MA, et al (1996): Effect of continuation treatment on residual symptoms in late-life depression: how well is “well”? Depress Anxiety 4:312-319. Papakostas GI, Nelson JC, Kasper S, Moller HJ (2008): A meta-analysis of clinical trials comparing reboxetine, a norepinephrine reuptake inhibitor, with selective serotonin reuptake inhibitors for the treatment of major depressive disorder. Eur Neuropsychopharmacol 18:122-127. Papakostas GI, Nutt DJ, Hallett LA, Tucker VL, Krishen A, Fava M (2006): Resolution of sleepiness and fatigue in major depressive disorder: A comparison of bupropion and the selective serotonin reuptake inhibitors. Biol Psychiatry 60:1350-1355. Paterson NE, Fedolak A, Olivier B, Hanania T, Ghavami A, Caldarone B (2010): Psychostimulant-like discriminative stimulus and locomotor sensitization properties of the wake-promoting agent modafinil in rodents.Pharmacol Biochem Behav 95:449-456. Paterson NE, Semenova S, Markou A (2008): The effects of chronic versus acute desipramine on nicotine withdrawal and nicotine self-administration in the rat. Psychopharmacology (Berl) 198:351-362. Paul IA, Layer RT, Skolnick P, Nowak G (1993): Adaptation of the NMDA receptor in rat cortex following chronic electroconvulsive shock or imipramine. Eur J Pharmacol 247:305-311. Paxinos G, Watson C (1998): The rat brain in stereotaxic coordinates, 4th edition ed. Pazos A, Palacios JM (1985): Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors. Brain Res 346:205-230. Pedigo NW, Yamamura HI, Nelson DL (1981): Discrimination of multiple [3H]5-hydroxytryptamine binding sites by the neuroleptic spiperone in rat brain. J Neurochem 36:220-226. Pellegrino L, Pellegrino A, Cushman A (1979): A stereotaxic Atlas of the Rat Brain. New York: Plenum Press. Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, et al (1998): Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 18:9996-10015. Pitchot W, Reggers J, Pinto E, Hansenne M, Fuchs S, Pirard S, et al (2001): Reduced dopaminergic activity in depressed suicides. Psychoneuroendocrinology 26:331-335.

Pittaluga A, Raiteri L, Longordo F, Luccini E, Barbiero VS, Racagni G, et al (2007): Antidepressant r efer ences

157 treatments and function of glutamate ionotropic receptors mediating amine release in hippocampus. Neuropharmacology 53:27-36. Pittenger C, Duman RS (2008): Stress, depression, and neuroplasticity: a convergence of mechanisms. Neuropsychopharmacology 33:88-109. Popa D, Cerdan J, Reperant C, Guiard BP, Guilloux JP, David DJ, et al (2010): A longitudinal study of 5-HT outflow during chronic fluoxetine treatment using a new technique of chronic microdialysis in a highly emotional mouse strain. Eur J Pharmacol 628:83-90. Popik P, Krawczyk M, Golembiowska K, Nowak G, Janowsky A, Skolnick P, et al (2006): Pharmacological profile of the “triple” monoamine neurotransmitter uptake inhibitor, DOV 102,677. Cell Mol Neurobiol 26:857-873. Porsolt RD, Bertin A, Jalfre M (1977): Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 229:327-336. Praag HM, Korf J, Lakke J, Schut T (1975): Dopamine metabolism in depressions, psychoses, and Parkinson’s disease: the problem of the specificity of biological variables in behaviour disorders. Psychol Med 5:138-146. Preskorn SH, Shah R, Neff M, Golbeck AL, Choi J (2007): The potential for clinically significant drug- drug interactions involving the CYP 2D6 system: effects with fluoxetine and paroxetine versus sertraline. J Psychiatr Pract 13:5-12. Price JL, Drevets WC Neurocircuitry of mood disorders. Neuropsychopharmacology 35:192-216. Prins J, Denys DA, Westphal KG, Korte-Bouws GA, Quinton MS, Schreiber R, et al (2010): The putative antidepressant DOV 216,303, a triple reuptake inhibitor, increases monoamine release in the prefrontal cortex of olfactory bulbectomized rats. Eur J Pharmacol 633:55-61. Prins J, Kenny PJ, Doomernik I, Schreiber R, Olivier B, Korte SM (2012): The triple reuptake inhibitor DOV 216,303 induces long-lasting enhancement of brain reward activity as measured by intracranial self-stimulation in rats. Eur J Pharmacol 693:51-56. Prins J, Olivier B, Korte SM (2011a): Triple reuptake inhibitors for treating subtypes of major depressive disorder: the monoamine hypothesis revisited. Expert Opin Investig Drugs 20:1107-1130. Prins J, Westphal KG, Korte-Bouws GA, Quinton MS, Schreiber R, Olivier B, et al (2011b): The potential and limitations of DOV 216,303 as a triple reuptake inhibitor for the treatment of major depression: A microdialyis study in olfactory bulbectomized rats. Pharmacol Biochem Behav 97:444- 452. Pytliak M, Vargova V, Mechirova V, Felsoci M (2011): Serotonin receptors - from molecular biology to clinical applications. Physiol Res 60:15-25. Racagni G, Popoli M (2008): Cellular and molecular mechanisms in the long-term action of antidepressants. Dialogues Clin Neurosci 10:385-400. Rao N (2007): The clinical of escitalopram. Clin Pharmacokinet 46:281-290. Raskin S, Durst R (2010): Bupropion as the treatment of choice in depression associated with Parkinson’s disease and it’s various treatments. Med Hypotheses 75:544-546. Reed AL, Anderson JC, Bylund DB, Petty F, El Refaey H, Happe HK (2009): Treatment with escitalopram but not desipramine decreases escape latency times in a learned helplessness model using juvenile rats. Psychopharmacology (Berl) 205:249-259. Rhen T, Cidlowski JA (2005): Antiinflammatory action of glucocorticoids--new mechanisms for old drugs. N Engl J Med 353:1711-1723. Richman CL, Gulkin R, Knoblock K (1972): Effects of bulbectomization, strain, and gentling on emotionality and exploratory behavior in rats. Physiol Behav 8:447-452. Robbins TW, Arnsten AF (2009): The neuropsychopharmacology of fronto-executive function: monoaminergic modulation. Annu Rev Neurosci 32:267-287. Rogoz Z (2009): Potentiation of the antidepressant-like effect of desipramine or reboxetine by metyrapone in the forced swimming test in rats. Pharmacol Rep 61:1173-1178. Rollema H, Lu Y, Schmidt AW, Sprouse JS, Zorn SH (2000): 5-HT(1A) receptor activation contributes to -induced dopamine release in the rat prefrontal cortex. Biol Psychiatry 48:229-237. Romanides AJ, Duffy P, Kalivas PW (1999): Glutamatergic and dopaminergic afferents to the prefrontal cortex regulate spatial working memory in rats. Neuroscience 92:97-106. Romeas T, Morissette MC, Mnie-Filali O, Pineyro G, Boye SM (2009b): Simultaneous anhedonia and exaggerated locomotor activation in an animal model of depression. Psychopharmacology (Berl) 205:293-303. Roy A (1992): Hypothalamic-pituitary-adrenal axis function and suicidal behavior in depression. Biol Psychiatry 32:812-816. Ruis MA, te Brake JH, Buwalda B, De Boer SF, Meerlo P, Korte SM, et al (1999): Housing familiar male wildtype rats together reduces the long-term adverse behavioural and physiological effects of social defeat. Psychoneuroendocrinology 24:285-300. Rush AJ, Trivedi MH, Wisniewski SR, Stewart JW, Nierenberg AA, Thase ME, et al (2006): Bupropion-SR, sertraline, or venlafaxine-XR after failure of SSRIs for depression. N Engl J Med 354:1231-1242.

158 References

Ryan B, Musazzi L, Mallei A, Tardito D, Gruber SH, El Khoury A, et al (2009): Remodelling by early-life stress of NMDA receptor-dependent synaptic plasticity in a gene-environment rat model of depression. Int J Neuropsychopharmacol 12:553-559. Rypma B, D’Esposito M (1999): The roles of prefrontal brain regions in components of working memory: effects of memory load and individual differences. Proc Natl Acad Sci U S A 96:6558-6563. Sagara H, Kitamura Y, Sendo T, Araki H, Gomita Y (2008): Motivational effect of nomifensine in the intracranial self-stimulation behavior using a runway method. Biol Pharm Bull 31:1036-1040. Sakaue M, Somboonthum P, Nishihara B, Koyama Y, Hashimoto H, Baba A, et al (2000): Postsynaptic 5-hydroxytryptamine(1A) receptor activation increases in vivo dopamine release in rat prefrontal cortex. Br J Pharmacol 129:1028-1034. Sanacora G, Gueorguieva R, Epperson CN, Wu YT, Appel M, Rothman DL, et al (2004): Subtype-specific alterations of gamma-aminobutyric acid and glutamate in patients with major depression. Arch Gen Psychiatry 61:705-713. Sanacora G, Zarate CA, Krystal JH, Manji HK (2008): Targeting the glutamatergic system to develop novel, improved therapeutics for mood disorders. Nat Rev Drug Discov 7:426-437. Sanders AC, Hussain AJ, Hen R, Zhuang X (2007): Chronic blockade or constitutive deletion of the serotonin transporter reduces operant responding for food reward. Neuropsychopharmacology 32:2321-2329. Sandi C, Richter-Levin G (2009): From high anxiety trait to depression: a neurocognitive hypothesis. Trends Neurosci 32:312-320. Santana N, Bortolozzi A, Serrats J, Mengod G, Artigas F (2004): Expression of serotonin1A and serotonin2A receptors in pyramidal and GABAergic neurons of the rat prefrontal cortex. Cereb Cortex 14:1100-1109. Saper CB, Chou TC, Elmquist JK (2002): The need to feed: homeostatic and hedonic control of eating. Neuron 36:199-211. Saper CB, Chou TC, Scammell TE (2001): The sleep switch: hypothalamic control of sleep and wakefulness. Trends Neurosci 24:726-731. Sapolsky RM (2000): The possibility of neurotoxicity in the hippocampus in major depression: a primer on neuron death. Biol Psychiatry 48:755-765. Sari Y (2004): Serotonin1B receptors: from protein to physiological function and behavior. Neurosci Biobehav Rev 28:565-582. Sastry BS, Phillis JW (1977): Inhibition of cerebral cortical neurones by a 5-hydroxytryptaminergic pathway from median raphe nucleus. Can J Physiol Pharmacol 55:737-743. Schildkraut JJ (1965): The catecholamine hypothesis of affective disorders: a review of supporting evidence. Am J Psychiatry 122:509-522. Schoedel KA, Meier D, Chakraborty B, Manniche PM, Sellers EM (2010): Subjective and objective effects of the novel triple reuptake inhibitor tesofensine in recreational stimulant users. Clin Pharmacol Ther 88:69-78. Schultz W (2007): Multiple dopamine functions at different time courses. Annu Rev Neurosci 30:259-288. Schultz W (2010): Dopamine signals for reward value and risk: basic and recent data. Behav Brain Funct 6:24. Seo DO, Shin CY, Lee CJ, Dailey JW, Reith ME, Jobe PC, et al (1999): Effect of alterations in extracellular norepinephrine on adrenoceptors: a microdialysis study in freely moving rats. Eur J Pharmacol 365:39-46. Sequeira A, Mamdani F, Ernst C, Vawter MP, Bunney WE, Lebel V, et al (2009): Global brain gene expression analysis links glutamatergic and GABAergic alterations to suicide and major depression. PLoS One 4:e6585. Sesack SR, Hawrylak VA, Matus C, Guido MA, Levey AI (1998): Dopamine axon varicosities in the prelimbic division of the rat prefrontal cortex exhibit sparse immunoreactivity for the dopamine transporter. J Neurosci 18:2697-2708. Skolnick P, Basile A (2006): Triple reuptake inhibitors as antidepressants. Drug Discovery Today: Therapeutic Strategies 3. Skolnick P, Basile AS (2007): Triple reuptake inhibitors (“broad spectrum” antidepressants). CNS Neurol Disord Drug Targets 6:141-149. Skolnick P, Krieter P, Tizzano J, Basile A, Popik P, Czobor P, et al (2006): Preclinical and clinical pharmacology of DOV 216,303, a “triple” reuptake inhibitor. CNS Drug Rev 12:123-134. Skolnick P, Layer RT, Popik P, Nowak G, Paul IA, Trullas R (1996): Adaptation of N-methyl-D-aspartate (NMDA) receptors following antidepressant treatment: implications for the pharmacotherapy of depression. Pharmacopsychiatry 29:23-26. Skolnick P, Popik P, Janowsky A, Beer B, Lippa AS (2003a): Antidepressant-like actions of DOV 21,947: a “triple” reuptake inhibitor. Eur J Pharmacol 461:99-104. Skolnick P, Popik P, Janowsky A, Beer B, Lippa AS (2003b): “Broad spectrum” antidepressants: is more better for the treatment of depression? Life Sci 73:3175-3179.

Slattery DA, Markou A, Cryan JF (2007): Evaluation of reward processes in an animal model of depression. r efer ences

159 Psychopharmacology (Berl) 190:555-568. Snoeren EM, Chan JS, de Jong TR, Waldinger MD, Olivier B, Oosting RS (2011): A new female rat animal model for hypoactive sexual desire disorder; behavioral and pharmacological evidence. J Sex Med 8:44-56. Solanto MV, Abikoff H, Sonuga-Barke E, Schachar R, Logan GD, Wigal T, et al (2001): The ecological validity of delay aversion and response inhibition as measures of impulsivity in AD/HD: a supplement to the NIMH multimodal treatment study of AD/HD. J Abnorm Child Psychol 29:215-228. Solomons K, Gooch S, Wong A (2005): Toxicity with selective serotonin reuptake inhibitors. Am J Psychiatry 162:1225. Song C, Leonard BE (2005): The olfactory bulbectomised rat as a model of depression. Neurosci Biobehav Rev 29:627-647. Spijker J, Bijl RV, de Graaf R, Nolen WA (2001): Determinants of poor 1-year outcome of DSM-III-R major depression in the general population: results of the Netherlands Mental Health Survey and Incidence Study (NEMESIS). Acta Psychiatr Scand 103:122-130. Spina E, Santoro V, D’Arrigo C (2008): Clinically relevant pharmacokinetic drug interactions with second- generation antidepressants: an update. Clin Ther 30:1206-1227. Sprouse JS, Aghajanian GK (1987): Electrophysiological responses of serotoninergic dorsal raphe neurons to 5-HT1A and 5-HT1B agonists. Synapse 1:3-9. Stahl SM, Grady MM, Moret C, Briley M (2005): SNRIs: their pharmacology, clinical efficacy, and tolerability in comparison with other classes of antidepressants. CNS Spectr 10:732-747. Stahl SM, Zhang L, Damatarca C, Grady M (2003): Brain circuits determine destiny in depression: a novel approach to the psychopharmacology of wakefulness, fatigue, and executive dysfunction in major depressive disorder. J Clin Psychiatry 64 Suppl 14:6-17. Steffens DC, Krishnan KR, Helms MJ (1997): Are SSRIs better than TCAs? Comparison of SSRIs and TCAs: a meta-analysis. Depress Anxiety 6:10-18. Stein DJ (2008): Depression, anhedonia, and psychomotor symptoms: the role of dopaminergic neurocircuitry. CNS Spectr 13:561-565. Stein-Behrens BA, Lin WJ, Sapolsky RM (1994): Physiological elevations of glucocorticoids potentiate glutamate accumulation in the hippocampus. J Neurochem 63:596-602. Stetler C, Miller GE (2011): Depression and hypothalamic-pituitary-adrenal activation: a quantitative summary of four decades of research. Psychosom Med 73:114-126. Stewart CA, Reid IC (2002): Antidepressant mechanisms: functional and molecular correlates of excitatory amino acid neurotransmission. Mol Psychiatry 7 Suppl 1:S15-22. Strecker RE, Nalwalk J, Dauphin LJ, Thakkar MM, Chen Y, Ramesh V, et al (2002): Extracellular histamine levels in the feline preoptic/anterior hypothalamic area during natural sleep-wakefulness and prolonged wakefulness: an in vivo microdialysis study. Neuroscience 113:663-670. Suhara T, Takano A, Sudo Y, Ichimiya T, Inoue M, Yasuno F, et al (2003): High levels of serotonin transporter occupancy with low-dose in comparative occupancy study with fluvoxamine using positron emission tomography. Arch Gen Psychiatry 60:386-391. Sun X, Zhao Y, Wolf ME (2005): Dopamine receptor stimulation modulates AMPA receptor synaptic insertion in prefrontal cortex neurons. J Neurosci 25:7342-7351. Svenningsson P, Tzavara ET, Witkin JM, Fienberg AA, Nomikos GG, Greengard P (2002): Involvement of striatal and extrastriatal DARPP-32 in biochemical and behavioral effects of fluoxetine (Prozac). Proc Natl Acad Sci U S A 99:3182-3187. Tanda G, Carboni E, Frau R, Di Chiara G (1994): Increase of extracellular dopamine in the prefrontal cortex: a trait of drugs with antidepressant potential? Psychopharmacology (Berl) 115:285-288. Tanda G, Frau R, Di Chiara G (1996): Chronic desipramine and fluoxetine differentially affect extracellular dopamine in the rat prefrontal cortex. Psychopharmacology (Berl) 127:83-87. Teter CJ, McCabe SE, LaGrange K, Cranford JA, Boyd CJ (2006): Illicit use of specific prescription among college students: prevalence, motives, and routes of administration. Pharmacotherapy 26:1501-1510. Thase ME, Entsuah AR, Rudolph RL (2001): Remission rates during treatment with venlafaxine or selective serotonin reuptake inhibitors. Br J Psychiatry 178:234-241. Thase ME, Frank E, Mallinger AG, Hamer T, Kupfer DJ (1992): Treatment of imipramine-resistant recurrent depression, III: Efficacy of monoamine oxidase inhibitors.J Clin Psychiatry 53:5-11. Thase ME, Trivedi MH, Rush AJ (1995): MAOIs in the contemporary treatment of depression. Neuropsychopharmacology 12:185-219. Tokarski K, Bobula B, Wabno J, Hess G (2008): Repeated administration of imipramine attenuates glutamatergic transmission in rat frontal cortex. Neuroscience 153:789-795. Tordera RM, Pei Q, Sharp T (2005): Evidence for increased expression of the vesicular glutamate transporter, VGLUT1, by a course of antidepressant treatment. J Neurochem 94:875-883. Torpey DC, Klein DN (2008): Chronic depression: update on classification and treatment. Curr Psychiatry

160 References

Rep 10:458-464. Tran P, Skolnick P, Czobor P, Huang NY, Bradshaw M, McKinney A, et al (2012): Efficacy and tolerability of the novel triple reuptake inhibitor amitifadine in the treatment of patients with major depressive disorder: a randomized, double-blind, placebo-controlled trial. J Psychiatr Res 46:64-71. Treadway MT, Zald DH (2011): Reconsidering anhedonia in depression: lessons from translational neuroscience. Neurosci Biobehav Rev 35:537-555. Tremblay LK, Naranjo CA, Cardenas L, Herrmann N, Busto UE (2002): Probing brain reward system function in major depressive disorder: altered response to . Arch Gen Psychiatry 59:409-416. Tremblay LK, Naranjo CA, Graham SJ, Herrmann N, Mayberg HS, Hevenor S, et al (2005): Functional neuroanatomical substrates of altered reward processing in major depressive disorder revealed by a dopaminergic probe. Arch Gen Psychiatry 62:1228-1236. Trivedi MH, Fava M, Wisniewski SR, Thase ME, Quitkin F, Warden D, et al (2006): Medication augmentation after the failure of SSRIs for depression. N Engl J Med 354:1243-1252. Trullas R, Skolnick P (1990): Functional antagonists at the NMDA receptor complex exhibit antidepressant actions. Eur J Pharmacol 185:1-10. Valvassori SS, Frey BN, Martins MR, Reus GZ, Schimidtz F, Inacio CG, et al (2007): Sensitization and cross-sensitization after chronic treatment with methylphenidate in adolescent Wistar rats. Behav Pharmacol 18:205-212. Van den Bergh F, Spronk M, Ferreira L, Bloemarts E, Groenink L, Olivier B, et al (2006): Relationship of delay aversion and response inhibition to extinction learning, aggression, and sexual behaviour. Behav Brain Res 175:75-81. van der Stelt HM, Breuer ME, Olivier B, Westenberg HG (2005): Permanent deficits in serotonergic functioning of olfactory bulbectomized rats: an in vivo microdialysis study. Biol Psychiatry 57:1061-1067. van Heesch F, Prins J, Konsman JP, Korte-Bouws GA, Westphal KG, Olivier B, et al (submitted): Lipopolysaccharide-induced anhedonia is abolished in male serotonin transporter knockout rats: an intracranial self-stimulation study. submitted to Brain, Behavior and Immunity. van Riezen H, Leonard BE (1990): Effects of psychotropic drugs on the behavior and neurochemistry of olfactory bulbectomized rats. Pharmacol Ther 47:21-34. Venero C, Borrell J (1999): Rapid glucocorticoid effects on excitatory amino acid levels in the hippocampus: a microdialysis study in freely moving rats. Eur J Neurosci 11:2465-2473. Verhagen LA, Luijendijk MC, Korte-Bouws GA, Korte SM, Adan RA (2009): Dopamine and serotonin release in the nucleus accumbens during starvation-induced hyperactivity. Eur Neuropsychopharmacol 19:309-316. Vinkers CH, Breuer ME, Westphal KG, Korte SM, Oosting RS, Olivier B, et al (2009): Olfactory bulbectomy induces rapid and stable changes in basal and stress-induced locomotor activity, heart rate and body temperature responses in the home cage. Neuroscience 159:39-46. Volkow ND, Fowler JS, Logan J, Alexoff D, Zhu W, Telang F, et al (2009): Effects of modafinil on dopamine and dopamine transporters in the male human brain: clinical implications. JAMA 301:1148-1154. Volkow ND, Wang G, Fowler JS, Logan J, Gerasimov M, Maynard L, et al (2001): Therapeutic doses of oral methylphenidate significantly increase extracellular dopamine in the human brain.J Neurosci 21:RC121. Volkow ND, Wang GJ, Fischman MW, Foltin RW, Fowler JS, Abumrad NN, et al (1997): Relationship between subjective effects of cocaine and dopamine transporter occupancy. Nature 386:827-830. Von Frijtag JC, Reijmers LG, Van der Harst JE, Leus IE, Van den Bos R, Spruijt BM (2000): Defeat followed by individual housing results in long-term impaired reward- and cognition-related behaviours in rats. Behav Brain Res 117:137-146. Waldinger MD, Berendsen HH, Blok BF, Olivier B, Holstege G (1998): Premature ejaculation and serotonergic antidepressants-induced delayed ejaculation: the involvement of the serotonergic system. Behav Brain Res 92:111-118. Wang SJ, Su CF, Kuo YH (2003): Fluoxetine depresses glutamate exocytosis in the rat cerebrocortical nerve terminals (synaptosomes) via inhibition of P/Q-type Ca2+ channels. Synapse 48:170-177. Watson S, Young AH (2001): Reduced dopaminergic activity in depressed suicides. Psychoneuroendocrinology 26:757, 759-760. Weber MM, Emrich HM (1988): Current and historical concepts of opiate treatment in psychiatric disorders. Int Clin Psychopharmacol 3:255-266. Weddington WW, Brown BS, Haertzen CA, Cone EJ, Dax EM, Herning RI, et al (1990): Changes in mood, craving, and sleep during short-term abstinence reported by male cocaine addicts. A controlled, residential study. Arch Gen Psychiatry 47:861-868. Wedzony K, Mackowiak M, Fijal K, Golembiowska K (1996): Ipsapirone enhances the dopamine outflow via 5-HT1A receptors in the rat prefrontal cortex. Eur J Pharmacol 305:73-78.

West AP (1990): Neurobehavioral studies of forced swimming: the role of learning and memory in the r efer ences

161 forced swim test. Prog Neuropsychopharmacol Biol Psychiatry 14:863-877. Williams SM, Goldman-Rakic PS (1998): Widespread origin of the primate mesofrontal dopamine system. Cereb Cortex 8:321-345. Willner P (1984): The validity of animal models of depression. Psychopharmacology (Berl) 83:1-16. Willner P (1997a): The mesolimbic dopamine system as a target for rapid antidepressant action. Int Clin Psychopharmacol 12 Suppl 3:S7-14. Willner P (1997b): Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology (Berl) 134:319-329. Willner P, Muscat R, Papp M (1992): Chronic mild stress-induced anhedonia: a realistic animal model of depression. Neurosci Biobehav Rev 16:525-534. Willner P, Towell A, Sampson D, Sophokleous S, Muscat R (1987): Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a . Psychopharmacology (Berl) 93:358-364. Wisor JP, Nishino S, Sora I, Uhl GH, Mignot E, Edgar DM (2001): Dopaminergic role in stimulant-induced wakefulness. J Neurosci 21:1787-1794. Wong EH, Sonders MS, Amara SG, Tinholt PM, Piercey MF, Hoffmann WP, et al (2000a): Reboxetine: a pharmacologically potent, selective, and specific norepinephrine reuptake inhibitor.Biol Psychiatry 47:818-829. Wong ML, Kling MA, Munson PJ, Listwak S, Licinio J, Prolo P, et al (2000b): Pronounced and sustained central hypernoradrenergic function in major depression with melancholic features: relation to hypercortisolism and corticotropin-releasing hormone. Proc Natl Acad Sci U S A 97:325-330. Wood PB (2006): Mesolimbic dopaminergic mechanisms and pain control. Pain 120:230-234. Yan QS, Yan SE (2001): Activation of 5-HT(1B/1D) receptors in the mesolimbic dopamine system increases dopamine release from the nucleus accumbens: a microdialysis study. Eur J Pharmacol 418:55-64. Yan QS, Zheng SZ, Yan SE (2004): Involvement of 5-HT1B receptors within the ventral tegmental area in regulation of mesolimbic dopaminergic neuronal activity via GABA mechanisms: a study with dual-probe microdialysis. Brain Res 1021:82-91. Yuen EY, Liu W, Karatsoreos IN, Feng J, McEwen BS, Yan Z (2009): Acute stress enhances glutamatergic transmission in prefrontal cortex and facilitates working memory. Proc Natl Acad Sci U S A 106:14075-14079. Zarate CA, Jr., Du J, Quiroz J, Gray NA, Denicoff KD, Singh J, et al (2003): Regulation of cellular plasticity cascades in the pathophysiology and treatment of mood disorders: role of the glutamatergic system. Ann N Y Acad Sci 1003:273-291. Zernig G, De Wit H, Telser S, Nienhusmeier M, Wakonigg G, Sturm K, et al (2004): Subjective effects of slow-release bupropion versus as determined in a quasi-naturalistic setting. Pharmacology 70:206-215. Zheng H, Berthoud HR (2007): Eating for pleasure or calories. Curr Opin Pharmacol 7:607-612.

162 Author affiliations 164 Author affiliations

Damiaan A. Denys Academic Medical Center, Department of Psychiatry, University of Amsterdam, Amsterdam, the Netherlands

Ivo Doomernik Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Faculty of Science, Utrecht University, the Netherlands

Rudy Dupree Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Faculty of Science, Utrecht University, the Netherlands

Lucianne Groenink Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Faculty of Science, Utrecht University, the Netherlands

Paul J. Kenny Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA

S. Mechiel Korte Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Faculty of Science, Utrecht University, the Netherlands

Gerdien A. H. Korte-Bouws Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Faculty of Science, Utrecht University, the Netherlands

Anne M. Krajnc Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Faculty of Science, Utrecht University, the Netherlands

Berend Olivier Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Faculty of Science, Utrecht University, the Netherlands affiliations

165 Ronald S. Oosting Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Faculty of Science, Utrecht University, the Netherlands

Maria S. Quinton Sepracor Inc., Marlborough, MA 01752, USA

Rudy Schreiber Sepracor Inc., Marlborough, MA 01752, USA

Koen, G. C. Westphal Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Faculty of Science, Utrecht University, the Netherlands

166 Samenvatting in het Nederlands 168 Samenvatting in het Nederlands

Depressie is een van de meest voorkomende psychiatrische aandoeningen. Een van de kernsymptomen van een depressie is anhedonie, het onvermogen om plezier te beleven aan activiteiten die normaal gesproken wel als plezierig worden ervaren. Een ander kernsymptoom van depressie is een neerslachtige stemming. In de DSM-IV, het handboek voor de psychiatrie, wordt gesproken van een depressie als een persoon gedurende een periode van minstens twee weken een van deze twee kernsymptomen, of beide, ervaart en daarnaast nog te kampen heeft met een aantal subsymptomen zoals gewichtsverlies of -toename, veranderde eetlust, toegenomen slaapbehoefte of juist slapeloosheid, een geagiteerde of een vertraagde motoriek, energieverlies en moeheid, verminderde concentratie, gevoelens van schuld en waardeloosheid en terugkerende suïcidale gedachten. Een aantal van deze symptomen zijn nogal tegenstrijdig en het is dan ook aannemelijk dat verschillende hersenmechanismen een rol spelen in de totstandkoming van deze symptomen. In hoofdstuk 2 worden de subsymptomen geclusterd en ondergebracht onder verschillende typen depressie; de melancholische depressie en de atypische depressie. Melancholische depressie gaat onder meer gepaard met gewichtsverlies, verminderde eetlust, slapeloosheid en geagiteerdheid, terwijl gewichtstoename, toegenomen eetlust, meer slapen en een vertraagde motoriek vallen onder een atypische depressie. Bovendien wordt in hoofdstuk 2 een overzicht gegeven van de hersenmechanismen die een rol zouden kunnen spelen bij deze twee typen depressie en daarbij wordt voornamelijk gefocust op de veranderingen in monoamine systemen in het brein. Monoamines zijn neurotransmitters die worden uitgescheiden aan het uiteinde van de axonen in de synaps waar ze zorgen voor signaaloverdracht naar postsynaptische neuronen. In dit proefschrift focussen we ons vooral op de monoamines serotonine (5-HT), noradrenaline (NE) en dopamine (DA) en de metabolieten (afbraakproducten) van 5-HT en DA; 5-HIAA, DOPAC en HVA.

Het doel van dit proefschrift was om in proefdieren de rol van 5-HT en in het bijzonder DA in hersenmechanismen betrokken bij anhedonie te bestuderen.

In dit proefschrift zijn voornamelijk in vivo microdialyse experimenten en intracraniale zelf-stimulatie (ICSS) studies uitgevoerd in ratten. Microdialyse is een techniek waarbij in levende dieren de afgegeven monoamines en hun metabolieten kunnen worden gemeten in een hersengebied naar keuze. ICSS is een techniek waarbij de functionaliteit van het beloningsgebied kan worden gemeten in levende dieren.

De meeste antidepressiva grijpen aan op bovengenoemde monoaminesystemen en dan vooral op 5-HT en in mindere mate ook op NE. Ze zorgen ervoor dat monoamines niet worden afgebroken, of dat monoamines niet heropgenomen kunnen worden in het presynaptisch neuron. Beide manieren resulteren er in dat monoamines langer in de synaps aanwezig blijven. Een simpele hypothese die hier in het verleden uit volgde veronderstelt dat een depressie komt door een tekort aan monoamines. In hoofdstuk 2 veronderstellen we een herziene versie van de monoamine hypothese. We suggereren dat een depressie niet zozeer gepaard gaat met een vermindering in monoamines, als samenvatting

169 wel met het uit balans zijn van monoaminerge systemen. Daarbij gaat het niet alleen om de monoamines op zich, maar ook om veranderingen in gevoeligheid van de pre- en postsynaptische aanwezige transporters en receptors. Ook hierbij maken we weer een onderscheid tussen een melancholische depressie, waarbij het serotonerge en noradrenerge systeem hyperactief functioneren en waarbij het dopaminerge systeem minder goed functioneert en de atypische depressie, waarbij niet zozeer het serotonerge systeem is verstoord, maar waarbij er een verminderde werking is van het dopaminerge en noradrenerge systeem. De meest voorgeschreven antidepressiva zijn de selectieve serotonine heropname remmers (SSRIs). Deze psychofarmaca blokkeren de 5-HT transporters, waardoor 5-HT niet meer opgenomen kan worden en langer in de synaps aanwezig blijft. Gezien bovengenoemde herziene monoamine hypothese is het niet vreemd dat SSRIs niet dezelfde mate van effectiviteit hebben in een atypische en melancholische depressie. Vooral wanneer anhedonie aanwezig is in een depressie is de kans op een succesvolle behandeling met SSRIs klein. Ondanks het feit dat SSRIs relatief veilig zijn in gebruik, zijn er toch een aantal nadelen aan deze klasse psychofarmaca. Gebruikers ervan hebben vaak te kampen met vervelende bijwerkingen, zoals verminderd libido, gewichtstoename, geagiteerdheid en slapeloosheid. Daarnaast reageert een grote groep mensen niet of nauwelijks op SSRIs. Een nieuwe en veelbelovende klasse antidepressiva zijn de triple heropname remmers (TRIs). Deze medicijnen zorgen naast een verhoging van serotonerge en noradrenerge neurotransmissie, ook voor een verhoogde dopaminerge neurotransmissie. Door aan te grijpen op het DA systeem wordt gedacht dat TRIs met name belangrijk zullen zijn in de behandeling van anhedonie. DA is belangrijk bij de regulering van plezierbeleving, beloning en motivatie in het brein. Door verhoging van DA concentraties is er echter wel reden tot voorzichtigheid wat betreft het verslavende aspect die deze stoffen met zich mee zouden kunnen brengen.

In hoofdstuk 3 en hoofdstuk 4 hebben we het effect van de TRI DOV 216,303 getest in OBX ratten. Bij OBX dieren wordt de bulbus olfactorius aan beide zijden van het brein operatief verwijderd. Hierdoor worden de dieren hyperactief, deze hyperactiviteit kan worden genormaliseerd door chronische toediening van SSRIs en tricyclische antidepressiva. Het OBX model is vaak gebruikt om van nieuwe stoffen hun mogelijke antidepressieve werking te voorspellen. Er zijn ook onderzoeken die aantonen dat OBX dieren een verstoring hebben in de gevoeligheid van hun beloningscircuit. Eerdere studies in ons lab hebben aangetoond dat DOV 216,303 een antidepressieve werking heeft in het OBX model, maar wat acute en chronische toediening van deze stof op monoamine afgifte doet was onbekend. In hoofdstuk 3 laten we zien dat één dag na verwijdering van de bulbi olfactorius, OBX dieren lagere baseline DA concentraties hebben vergeleken met Sham geopereerde dieren, wat een verklaring zou kunnen zijn voor de eerder gevonden verstoring in het beloningssysteem van OBX ratten. Echter, 5 weken later waren er geen verschillen meer te ontdekken tussen OBX en Sham dieren. Een behandeling met DOV 216,303 zorgde voor een verhoging van extracellulaire concentraties van 5-HT, NE, DA en hun

170 Samenvatting in het Nederlands metabolieten in de prefrontale cortex (PFC) van ratten. Deze verhoging leek lager te zijn in chronische behandelde dieren, vergeleken met acuut behandelde dieren, maar vanwege tekortkomingen in de experimentele opzet in hoofdstuk 3 konden we dit niet met zekerheid zeggen. In hoofdstuk 4 hebben we opnieuw een OBX studie uitgevoerd waarbij we het verschil in monoamine concentraties in de PFC en ook de dorsale hippocampus hebben gemeten na acute en chronische toediening van DOV 216,303. We hebben voor deze hersengebieden gekozen omdat ze betrokken zijn bij depressie. In deze studie zagen we dat een acute behandeling met DOV 216,303 resulteerde in lagere DA concentraties in OBX dieren dan in Sham dieren. Een ander belangrijk resultaat uit deze studie was dat een behandeling met DOV 216,303 in een chronisch behandeld dier leidde tot een lagere DA afgifte dan in een acuut behandeld dier. Deze verminderde respons is waarschijnlijk te wijten aan farmacokinetische of farmacodynamische veranderingen ten gevolge van chronische toediening, aangezien concentraties DOV 216,303 in het brein en het bloedplasma na chronische toediening lager waren dan na acute toediening. Dit is waarschijnlijk ook de reden waarom we in deze studie geen normalisatie zagen van de OBX-geïnduceerde hyperactiviteit door DOV 216,303, terwijl dat in een eerdere studie in onze groep wel was aangetoond (met een iets andere experimentele opzet). In eerste instantie leek het OBX model een goed model te zijn om anhedonie in te bestuderen, maar bij nader inzien zouden de gedragsveranderingen in OBX dieren ook wel eens verklaard kunnen worden door cognitieve stoornissen door neurodegeneratie of ontstekingsgeïnduceerde veranderingen in neurotransmissie. Daarnaast bleek het heel moeilijk om anhedonie te meten in OBX dieren met behulp van ICSS. Daarom hebben we er voor gekozen om verder onderzoek te doen in gezonde dieren. TRIs verhogen DA concentraties in de hersenen, cocaine is bijvoorbeeld ook een TRI. Een reden tot bezorgdheid in de ontwikkelingen van TRIs voor depressie is dan ook dat ze, door aan te grijpen op het dopaminerge systeem, mogelijk verslavend zouden kunnen werken. Het is bekend dat verslavende stoffen acuut zorgen voor een verlaging in ICSS thresholds (drempelwaarden), dit reflecteert het euforische gevoel dat ervaren wordt bij het nemen van deze drugs. Wanneer de drug is uitgewerkt ontstaan er onthoudingsverschijnselen, dat gemeten kan worden als verhoogde ICSS thresholds. Daarom hebben we in hoofdstuk 5, het effect van DOV 216,303 op ICSS thresholds onderzocht, we hebben dit vergeleken met ratten die amphetamine toegediend kregen. Het belangrijkste resultaat uit dit experiment was dat DOV 216,303 een relatief langdurig stimulerend effect had op beloningscircuits in de hersenen, zonder dat onthoudingsverschijnselen optraden, terwijl amphetamine wel zorgde voor onthouding-geïnduceerd verminderd functioneren van beloningsgebieden. Met ICSS kunnen belonende effecten van stoffen worden gemeten, het kan echter niet volledig uitsluitsel geven over de mogelijke verslavende werking van de stof. Daarvoor zullen experimenten moeten worden uitgevoerd waarin ratten de mogelijkheid krijgen om de stof aan zich zelf toe te dienen. De mate waarin ze dat willen doen geeft meer een indicatie over de verslavende werking en mogelijk misbruikgevoeligheid van deze stof. samenvatting

171 We zagen dat DOV 216,303 een verhoging in DA concentraties veroorzaakt en daarnaast ook ICSS thresholds verlaagd. Dat verhoging in DA en een verlaging in ICSS thresholds niet persé samen hoeft te gaan zagen we in hoofdstuk 6. Daarin hebben we een microdialyse studie uitgevoerd in de PFC en nucleus accumbens (NAc, belangrijk hersengebied in beloning) en een ICSS studie met eltoprazine, een 5-HT1A/1B receptor agonist. Opmerkelijk was dat de serotonerge stof eltoprazine zorgde voor een verhoging in DA concentraties in de PFC en een verhoging in de metabolieten van DA in de NAc. Bovendien ging deze verhoging in dopaminerge activiteit niet gepaard met een verlaging in ICSS thresholds. Het tegenovergestelde bleek het geval, eltoprazine zorgde voor een verhoging in ICSS thresholds, wat neerkomt op een verminderd functionerend beloningscircuit in het brein. Dit effect kan verklaard worden door stimulatie van de

5-HT1A en 5-HT1B receptoren, die elk ook weer een effect hebben op monoaminerge neurotransmissie en beloning. In deze studie zagen we dat, zoals verwacht, 5-HT concentraties daalden. Het zou dus zo kunnen zijn dat ook 5-HT een belangrijke rol speelt in beloning. In hoofdstuk 7 hebben we de afzonderlijke rollen van 5-HT, NE en DA bestudeerd met stoffen die specifiek de 5-HT, NE en DA transporter blokkeren, namelijk de SSRI escitalopram, de NRI reboxetine en de DNRI methylphenidate. We hebben een microdialyse experiment uitgevoerd met probes in de PFC en NAc en hebben het effect van acute en chronische toediening van deze stoffen op monoamine afgifte in deze hersengebieden bestudeerd. Daarnaast hebben we een ICSS experiment uitgevoerd waarin we het acute en chronische effect van deze stoffen op het beloningscircuit hebben onderzocht. Dit experiment heeft een aantal belangrijke resultaten opgeleverd. Een opmerkelijke vinding is dat een langdurige toediening van escitalopram niet zorgde voor verhoogde baseline concentraties in 5-HT. We hadden dit wel verwacht, omdat de werking van SSRIs vaak wordt verklaard door een verhoging in 5-HT op de lange termijn die ze zouden veroorzaken. De verhoging van 5-HT na acute toediening met een

SSRI activeert 5-HT1A autoreceptoren in de raphe nucleus (RN), die vervolgens zorgen voor een remming van de 5-HT afgifte in de projectiegebieden van de RN (o.a. NAc en PFC). Door chronische activering van deze autoreceptoren zullen ze downreguleren (minder worden in aantal) waardoor de remming op de serotonerge neuronen wegvalt en de 5-HT concentratie zal toenemen. In het licht van onze resultaten kan deze hypothese niet kloppen. Wij denken dat 5-HT1A autoreceptoren, zelfs na langdurige blootstelling aan 5-HT, niet volledig downreguleren. Door de chronische blokkade van de 5-HT transporter zullen er hoge concentraties 5-HT aanwezig zijn in de RN, die een constante remming geven op de weinige autoreceptoren die beschikbaar zijn, deze constante remming legt de serotonerge neurotransmissie naar projectiegebieden plat, wat een verklaring kan zijn voor de afwezigheid van verhoogde baseline concentraties van 5-HT in de PFC. In hoofdstuk 7 zien we bovendien dat een stof die de DA verhoogt in de NAc, zoals methylphenidate, ICSS thresholds verlaagd, maar dat een langdurig gegeven SSRI geen effect heeft op het beloningssysteem, het zorgt na acute toediening zelfs voor een verhoging van ICSS thresholds. Dit geeft te denken over de effectiviteit van SSRIs in de behandeling van anhedonie. We kunnen in ieder geval vaststellen, dat ondanks het feit

172 Samenvatting in het Nederlands dat wereldwijd miljoenen mensen SSRIs slikken en dat deze stoffen al bijna 40 jaar op de markt zijn, we nog steeds niet precies weten wat hun werkingsmechanisme is. Wat belangrijk is om te vermelden is dat we in deze studies stoffen getest hebben in gezonde dieren. Het is aannemelijk dat monoaminerge regulatie verschilt tussen een gezond en een depressief brein, zelfs binnen verschillende subtypen van een depressie verschillen de onderliggende verstoringen in neurotransmitters (hoofdstuk 2). Het is dan ook niet te verwachten dat een monoamine heropname remmer hetzelfde effect heeft in zowel een depressieve als een gezonde toestand. De belangrijkste resultaten van dit proefschrift worden besproken in hoofdstuk 8. Daarin wordt ook het belang van een goed diermodel voor anhedonie benadrukt. De huidige diermodellen en diertesten zijn niet geschikt om neurobiologische processen te bestuderen die verstoord zijn in anhedonie. Een veelbelovend diermodel (waarbij dieren een minder gevoelig beloningssysteem hebben) zou verkregen kunnen worden door een rat bloot te stellen aan een dominante rat. De rat ondergaat een verlies en ondervind daarvan een hevige stress. Door sociaal verlies te combineren met bijvoorbeeld een immunologische challenge en individuele huisvesting zou de rat wel eens langdurig verhoogde ICSS thresholds kunnen ontwikkelen. Deze ratten zouden zeer geschikt kunnen zijn om verstoringen in (o.a. monoaminerge) hersenenmechanismen bij anhedonie te onderzoeken. Ook hebben we aanwijzingen dat voor een goede behandeling van anhedonie, het belangrijk zou kunnen zijn om farmaca voor te schrijven die naast serotonine, ook dopamine verhogen. Welk antidepressivum goed is hangt onder meer af van de soort depressie die mensen hebben, maar zolang daar in de diagnose geen duidelijk onderscheid in gemaakt wordt zou een triple heropname remmer een goede eerstelijns behandeling kunnen zijn. samenvatting

173

Dankwoord 176 Dankwoord

Dan is het nu tijd om het meest gelezen stukje van dit proefschrift te schrijven. Toch wil ik de lezer er op wijzen dat de rest van m’n boekje minstens zo interessant is…

De afgelopen jaren heb ik met heel veel plezier gewerkt aan de totstandkoming van dit proefschrift. Gelukkig heb ik dat niet alleen hoeven doen en waren er op allerlei vlakken mensen om mij te helpen, om vreugdemomenten mee te vieren en om mijn stressmomentjes te relativeren. Op deze plek wil ik iedereen daar hartelijk voor bedanken. Mechiel, co-promotor, bedankt dat je mij ‘gespot’ hebt toen ik nog als student over de afdeling rondhuppelde. Jouw enthousiasme heeft me destijds over de streep getrokken en heeft me in mijn project vaak aangezet om toch maar weer die ‘berg’ te beklimmen. Bedankt dat je altijd vertrouwen in me hebt gehad, dat ik mijn eigen keuzes mocht maken en dat je me gevormd hebt tot de onderzoeker die ik nu ben. Ronald, ik ben blij dat jij ook als co-promotor betrokken bent geweest bij dit proefschrift. Bedankt voor je aardige woorden en je gemene grapjes. Bovendien wil ik je bedanken voor de snelle en grondige revisies van mijn manuscripten. Mijn promotoren, Berend Olivier en Damiaan Denys wil ik bedanken voor hun enthousiasme bij de bespreking van resultaten. De verhalen uit de industrie en kliniek waren altijd goed om alles in een kader te plaatsen, bedankt daarvoor. Berend, dank je wel voor de mogelijkheid om in jouw lab te promoveren. Damiaan, dank je wel dat je betrokken wilde zijn bij mijn project en dat je ook promotor wilt zijn. I would like to thank Prof dr. Adan, Prof. dr. Den Boer, Prof. dr. Ohl, Prof. dr. Robbins and Prof. dr. Vanderschuren for their participation in the reviewing committee of my manuscript and for their useful comments. Er zijn twee mensen die bijzonder veel voor me hebben gedaan. Koen en Gerdien, wat zijn jullie harde werkers! Koen, zonder jouw hulp zouden de microdialyse experimenten nooit zo goed verlopen zijn en zou Gerdien het niet zo druk hebben gehad. Gerdien, bedankt voor het analyseren van al die honderden, nee duizenden monsters (ik overdrijf niet) die je voor dit proefschrift hebt bepaald. I am very grateful to Paul Kenny from the Scripps Research Institute in Florida for his hospitality to welcome me in his lab and teach me all the necessary things I need to know for using the ICSS technique (surgery, training and providing programs). Back in our lab, the set up went very smooth and thanks to you, three chapters of this thesis contain very nice ICSS data. Ook ben ik Ruud van Oorschot zeer erkentelijk voor het wegnemen van een hoop injectiestress (bij mij!) door de vele trainingen die hij mij en veel van mijn studenten heeft gegeven in het oraal injecteren. Liesbeth en Eelke, collega’s, maatjes en vriendinnen, ik ben heel blij dat jullie mijn paranimfen willen zijn. Eelke, bedankt voor de heerlijke discussies in de Vooghel, ik mis ze wel hoor! Bedankt dat je mijn kamergenootje was op congressen en bedankt voor het leuke contact dat we, ondanks de duizenden kilometers die tussen ons liggen, nog steeds hebben. Liesbeth, het is al even geleden, maar ik zie mezelf nog staan op de rand van cabine B1, zweet op m’n rug, tranen in m’n ogen. Gelukkig was jij daar ook. Jij was er altijd. Bedankt voor je steun de afgelopen jaren en voor de gezelligheid binnen en buiten het lab. da nk woor d

177 Ik wil alle collega’s van farmacologie en in het bijzonder toch het psycho-clubje bedanken voor de gezellige dagelijkse koffie- en lunchpauzes met discussies over wetenschap en niet-wetenschap, voor de gezelligheid in het lab en de dierstallen, voor de karaoke- en Wii-avondjes in het Went, voor de Vooghelavonden tot in de vroege uurtjes en voor de volleybaltoernooien (toen die nog bestonden), voor de labuitjes, de spareribs en de AIO-etentjes. Tessa, het is fijn om het met een andere jonge moeder ongegeneerd vaak over de combi baby-promoveren te kunnen hebben. Floor en Marjolein, voormalig kamergenootjes, jullie komst maakte de ‘Korte-groep’ tot een echte groep, twee compleet verschillende karakters, maar dat maakt de groep dynamisch en extra gezellig. Erik, sinds we het Went hebben verruild voor het DDW zien we elkaar niet meer zo vaak, maar ik vond de seriebesprekingen op onze kamer altijd erg gezellig. Lucianne, bedankt voor je altijd aanwezige interesse en scherpe opmerkingen. Monika, de lunchgesprekken fleuren op door jouw aanwezigheid. Marga en Lidija, bedankt voor jullie hulp bij allerlei administratieve zaken, Christiaan, Ed V, Filip, Herman, Jan V, Johnny, Joris, Lydia, Meg B, Meg v B, Monique, Yuliya, jullie dragen allemaal bij (of hebben bijgedragen) aan een ontspannen en gezellige werksfeer. Al bevat dit proefschrift geen neuroimmun werk, ik ben ook nog betrokken (geweest) bij het neuroimmuun onderzoek. Graag wil ik Sofia, Aletta, Mechiel, Floor, Caroline, Jiangbo, Carmen en Paula bedanken voor de introductie van een voor mij nieuw, maar interessant onderzoeksveld. Aan de basis van goed proefdieronderzoek staat een goede dierverzorging. Hans, bedankt voor de prettige samenwerking en voor de goede zorg voor mijn dieren. De proefdierdeskundigen Harry en Fred wil ik bedanken voor hun goede raad en adviezen om de dierexperimenten goed te laten verlopen. Het begeleiden van studenten is een voorrecht dat hoort bij het opleidingstraject als AIO en ik ben dan ook erg blij dat er de afgelopen jaren zoveel studenten stage bij mij hebben gelopen. Rasielle, Jacoline, Ivo, Jagoda, Annika, Rudy, Lydia, Anne en Mohammed, bedankt voor het vele werk dat jullie voor me hebben gedaan, bedankt dat ik jullie wat mocht leren, bedankt dat jullie mij zoveel hebben geleerd. Gelukkig is er ook nog een leven naast het werk. Een grote groep familie en vrienden hebben er voor gezorgd dat ik mijn werk kon relativeren en me bezig kon houden met dingen die echt/ook belangrijk zijn. Allereerst wil ik graag alle leden van de (oud-)Biltkring en Overvechtkring bedanken voor de regelmatige samenkomsten. De dinsdagavonden zijn altijd een heerlijk rustpunt in de week. Kringers, bedankt voor de ongedwongen sfeer, de gezelligheid, voor de etentjes, de goede gesprekken en voor de vriendschappen. Twee vriendinnen wil ik in het bijzonder bedanken. Anne, een dagje samen met jou is altijd heerlijk ontspannen. Hylkje, BMW-vriendinnetje vanaf het eerste uur, kringgenootje vanaf later en nu vriendin op afstand, bedankt dat ik bij jou altijd mijn ei kwijt kan. Lieve schoonfamilie, bedankt voor de belangstelling in mijn onderzoek en het promotietraject in het algemeen. Het is nu af, maar ik ga door. Opa en oma, Ria en Engelbert, Jelle en Henrike, Marijn en Bertina, bedankt voor het gezellig samenzijn.

178 Dankwoord

Sandra, Leonard en Maria, Werner en Renske, bedankt dat jullie altijd voor me klaar staan. In dit boekje kunnen jullie lezen waar jullie kleine zusje nu zo druk mee bezig is geweest de afgelopen jaren. Ik wil het best nog wel eens toelichten hoor. Mijn ouders wil ik bedanken voor de manier waarop ze me hebben opgevoed. Lieve pa en ma, bedankt dat jullie me altijd de vrijheid hebben gegeven om te kunnen ontdekken wat ik leuk vindt. Bedankt voor jullie steun en altijd aanwezige betrokkenheid en interesse. En bedankt voor alle goede zorgen (ook in 2011). Lieve Tjeerd, bedankt voor de vele uren die je, zonder ook maar een moment te mopperen, in de opmaak van dit proefschrift hebt gestopt. Het is onvoorstelbaar wat jij allemaal voor me doet en hebt gedaan. Wat er ook allemaal nog op ons pad komt, ik ben blij dat ik het avontuur samen met jou aan kan gaan. Evi, lieve, kleine meid, je bent nog te klein om het allemaal te beseffen, maar wat zijn we ongelooflijk gelukkig met jou. Het is het allemaal waard geweest… da nk woor d

179

About the author 182 About the author

Jolanda Prins was born on the 2nd of May 1984 in Almelo. She passed her final exams at the C.S.G. Reggesteyn in Nijverdal and obtained her athenaeum certificate in 2002. She started her studies Biomedical Sciences at the University of Utrecht. After graduating for her bachelor’s degree in 2005 she started the master Neuroscience and Cognition in the Experimental and Clinical Neuroscience track. During her master program she conducted a research project at the department of psychopharmacology under supervision of Dr. Ronald Oosting and Prof. dr. Berend Oliver and performed molecular and behavioral studies in animal models for affective disorder. Her second research project was conducted at the Netherlands Institute for Neuroscience in Amsterdam under supervision of Dr. Bas Blits and Prof. dr. Joost Verhaagen where she studied the possibility of neutralization chemorepellent molecules in spinal cord injury. She graduated in August 2007 and started her PhD project in September 2007 at the department of psychopharmacology under supervision of Dr. Mechiel Korte and Dr. Ronald Oosting and Prof. dr. Berend Olivier in collaboration with Prof. Dr. Damiaan Denys of the Academic Medical Center in Amsterdam. The research performed during her PhD project is described in this thesis au t h o r

183

List of publications 186 List of publications

Full length articles 2012 Prins, J., Krajnc, A.M., Oosting, R.S., Korte-Bouws, G.A., Westphal., K.G., Olivier, B., Denys, D.A., Korte, S.M., Acute and chronic monoamine reuptake inhibitors differently affect brain stimulation reward and monoamine release in the nucleus accumbens and prefrontal cortex in rats. Submitted. van Heesch, F., Prins, J., Konsman, J.P., Westphal., K.G.C., Olivier, B., Kraneveld, A.D., Korte, S.M., Lipopolysaccharide-induced anhedonia is abolished in male serotonin transporter knockout rats: an intracranial self-stimulation study. Submitted. van Heesch, F., Prins, J., Korte-Bouws, G.A.H., Westphal, K.G.C., Olivier, B., Kraneveld, A.D., Korte, S.M., 2012, Tumor necrosis factor-alpha (TNF-α) elevates serotonin metabolism in the nucleus accumbens and increases anhedonia in mice. Submitted.

Prins, J., Kenny, P.J., Doomernik, I., Schreiber, R., Olivier, B., Korte, S.M., 2012. The triple reuptake inhibitor DOV 216,303 induces long-lasting enhancement of brain reward activity as measured by intracranial self-stimulation in rats. Eur J Pharmacol 693, 51-56.

2011 Prins, J., Olivier, B., Korte, S.M., 2011a. Triple reuptake inhibitors for treating subtypes of major depressive disorder: the monoamine hypothesis revisited. Expert Opinion on Investigational Drugs 20, 1107-1130.

Prins, J., Westphal, K.G., Korte-Bouws, G.A., Quinton, M.S., Schreiber, R., Olivier, B., Korte, S.M., 2011c. The potential and limitations of DOV 216,303 as a triple reuptake inhibitor for the treatment of major depression: A microdialyis study in olfactory bulbectomized rats. Pharmacol Biochem Behav 97, 444-452.

2010 Prins, J., Denys, D.A., Westphal, K.G., Korte-Bouws, G.A., Quinton, M.S., Schreiber, R., Groenink, L., Olivier, B., Korte, S.M., 2010a. The putative antidepressant DOV 216,303, a triple reuptake inhibitor, increases monoamine release in the prefrontal cortex of olfactory bulbectomized rats. Eur J Pharmacol 633, 55-61.

Caldarone, B.J., Paterson, N.E., Zhou, J., Brunner, D., Kozikowski, A.P., Westphal, K.G., Korte-Bouws, G.A., Prins, J., Korte, S.M., Olivier, B., Ghavami, A., 2010. The novel triple reuptake inhibitor JZAD-IV-22 exhibits an antidepressant pharmacological profile without locomotor stimulant or sensitization properties. J Pharmacol Exp Ther 335, 762-770. publications

187 Hendriksen, H., Prins, J., Olivier, B., Oosting, R.S., 2010. Environmental Enrichment Induces Behavioral Recovery and Enhanced Hippocampal Cell Proliferation in an Antidepressant-Resistant Animal Model for PTSD. PLoS One 5.

Blits, B., Derks, S., Twisk, J., Ehlert, E., Prins, J., Verhaagen, J., 2010. Adeno-associated viral vector (AAV)-mediated gene transfer in the red nucleus of the adult rat brain: comparative analysis of the transduction properties of seven AAV serotypes and lentiviral vectors. J Neurosci Methods 185, 257-263.

2009 Korte, S.M., Prins, J., Vinkers, C.H., Olivier, B., 2009. On the origin of allostasis and stress-induced pathology in farm animals: celebrating Darwin’s legacy. Vet J 182, 378- 383.

Abstracts (*oral presentation) 2012 Prins, J., Kranjc, A.M., Korte-Bouws, G.A., Westphal., K.G., Olivier, B., Korte, S.M. The role of monoaminergic neurotransmission in reward: A microdialysis study in the prefrontal cortex and nucleus accumbens and intracranial self-stimulation in rats. Program. No. 74.23. 2012 Neuroscience Meeting Planner. New Orleans, LA: Society for Neuroscience, 2012. Online.

2011 Prins, J., van Heesch, F., Krajnc, A.M., de Haan, L., Olivier, B., Kraneveld, A.D., Korte, S.M., Endotoxin-induced anhedonia: increases in brain stimulation reward thresholds and alterations in cytokine-profile, ENP 2011, Dutch Endo-Neuro-Psycho Meeting, Lunteren

Prins, J., van Heesch, F., de Haan, L., Krajnc, A.M., Kenny, P.J., Olivier, B., Kraneveld, A.D., Korte, S.M., 2011b. Lipolysaccharide-induced changes in brain stimulation reward: anhedonia or sickness behaviour? ECNP workshop Nice European Neuropsychopharmacology 21, S39-S40.

Prins, J., van Heesch, F., de Haan, L., Olivier, B., Kraneveld, A.D., Korte, S.M., Lipopolysaccharide induces anhedonia-like reward deficits, reflected by long-term increases in brain stimulation reward thresholds, European Journal of Pharmacology, Volume 668, Supplement 1, September 2011, Pages e27-e28

2010 Prins, J., Oosting, R.S., Dupree, R., Korte-Bouws, G.A., Kenny, P.J., Olivier, B., Korte,

S.M., The effects of eltoprazine, a 5-HT1A/1B receptor agonist, on monoamine release

188 List of publications in the prefrontal cortex, on brain reward and impulsivity. Program No. 168.21. 2010 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2010. Online.

Prins J., Dupree R., Korte-Bouws G. A., Olivier B., Korte S. M., Effects of the partial 5-HT1A/1B receptor agonist eltoprazine on monoamine release in the prefrontal cortex and on brain reward as measured by intracranial self-stimulation in rats, FENS Abstr., vol.5, 017.32, 2010

Prins, J., Oosting, R.S., van den Bergh, F.S., Dupree, R., Korte-Bouws, G.A.H., Olivier,

B., Korte S.M., Eltoprazine, a 5-HT1A/1B receptor agonist, increased dopamine in the prefrontal cortex and decreased brain reward functioning, Figon, Dutch Medicine Days 2010, Lunteren

Prins, J.*, Dupree, R., Korte-Bouws, G.A.H., Olivier, B., Korte, S.M., 2010. Serotonin- dopamine interaction in reward: microdialysis and intracranial self-stimulation. ECNP workshop Nice, European Neuropsychopharmacology 20, S50-S50.

2009 Prins, J., Kenny, P.J., Doomernik, I., Olivier, B., Korte, S.M., 2009 The triple reuptake inhibitor DOV216,303, a putative new antidepressant, decreases ICSS thresholds without producing withdrawal effects. Program No. 549.11. Neuroscience Meeting Planner, Chicaco, IL: Society for Neuroscience, 2009. Online

Prins, J., Kenny, P.J., Doomernik, I., Olivier, B., Korte, S.M., The putative new antidepressant DOV216,303, a triple reuptake inhibitor, increases hedonia without producing withdrawal effects, ENP 2009, Dutch Endo-Neuro-Psycho Meeting, Doorwerth

Prins, J., Breuer, M.E., Westphal, K.G.C., Korte-Bouws, G.A.H., Oliver, B., Korte, S.M., 2009. Triple reuptake inhibitors: behavioral and microdialysis studies in the olfactory bulbectomy (OBX) model of depression. Naunyn-Schmiedebergs Archives of Pharmacology 380, 265-265. publications

189