u Ottawa L'UniversiUi canadienne Canada's university run FACULTE DES ETUDES SUPERIEURES FACULTY OF GRADUATE AND ET POSTOCTORALES U Ottawa POSDOCTORAL STUDIES
I.'University oanadierme Canada's university
Noam Katz AUTEUR DE LA THESE / AUTHOR OF THESIS
M.Sc. (Neuroscience) GRADE/DEGREE
Department of Neuroscience FACULTE, ECOLE, DEPARTEMENT / FACULTY, SCHOOL, DEPARTMENT
Effects of the Sustained Administration of the Catecholamine Reuptake Inhibitor Nomifensine on the Firing Activity of Norepinephrine, Dopamine and Serotonin Neurons
TITRE DE LA THESE / TITLE OF THESIS
Pierre Blier DIRECTEUR (DIRECTRICE) DE LA THESE/ THESIS SUPERVISOR
CO-DIRECTEUR (CO-DIRECTRICE) DE LA THESE / THESIS CO-SUPERVISOR
EXAMINATEURS (EXAMINATRICES) DE LA THESE/THESIS EXAMINERS
Stefany Bennet
.Gary.W. Slater Le Doyen de la Faculte des etudes superieures et postdoctorales / Dean of the Faculty of Graduate and Postdoctoral Studies Effects of the Sustained Administration of the Catecholamine Reuptake Inhibitor Nomifensine on the Firing Activity of Norepinephrine, Dopamine and Serotonin Neurons
Noam Katz
Department of Neuroscience Supervisor: Dr. Pierre Blier University of Ottawa
This thesis is submitted to the Faculty of Graduate and Postdoctoral Studies as a partial fulfillment of the M.Sc. program in Neuroscience
Noam Katz, Ottawa, Canada, 2008
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The effects of acute and sustained administration of the catecholamine reuptake inhibitor nomifensine were investigated on the firing properties of monoaminergic neurons in order to understand the mechanism of action of nomifensine in vivo.
In vivo extracellular recordings of monoaminergic neurons were obtained from male Sprague-Dawley rats. Acute dose-response curves (i.v.) were constructed and subcutaneously implanted osmotic minipumps were used to investigate the effects of sustained administration.
Nomifensine acted directly on both NE and DA neurons. Sustained administration potently inhibited DA neurons after 2 days, with a complete recovery after
14 days, due to D2 autoreceptor desensitization. NE neuronal firing rate was potently inhibited after 2 and 14 days of administration, while 5-HT neurons were increased at both times, partly due to 5-HTIA autoreceptor desensitization after 2 days.
This medication likely treated depression by acting on DA, NE and 5-HT neurons, highlighting the importance of the functional connectivity between these systems.
11 ABSTRACT
FIGURES
ABBREVIATIONS
ACKNOWLEDGMENTS
INTRODUCTION 1. Major Depressive Disorder
2. Neuroanatomy
2.1. The Catecholamines 2.1.1. Neuroanatomy of the Norepinephrine System 2.1.1.1. Adrenergic Receptor Subtypes 2.1.1.1.1. a-Adrenergic Receptors
2.1.1.1.1.1. ar Adrenoceptors
2.1.1.1.1.2. a2-Adrenoceptors 2.1.1.1.1.3. p-Adrenoceptors 2.1.1.2. Electrophysiological Properties of NE Neurons 2.1.2. Neuroanatomy of the Dopamine System 2.1.2.1. Dopamine Receptor Subtypes
2.1.2.1.1. D2 Receptors 2.1.2.2. Electrophysiological properties of DA Neurons 2.1.3. Synthesis, Storage, Release and Metabolism of DA an 2.1.4. The NE and DA Transporters
2.2. The 5-Hydroxytryptamine System 2.2.1. Neuroanatomy of the 5-HT System 2.2.2. 5-HT Receptor Subtypes 2.2.2.1. 5-HTIA Receptors 2.2.3. 5-HT Synthesis, Storage, Release and Metabolism 2.2.4. The Serotonin Transporter (SERT) 2.2.5. Electrophysiological Properties of 5-HT Neurons 3. Evidence Implicating the Monoamines in the Pathophysiology of MDD 33
3.1. History of Antidepressants 33 3.2. Evidence of 5-HT Involvement in Depression 35 3.3. Evidence of Catecholamine Involvement in Depression 37 3.3.1. NE in Depression 37 3.3.2. DA in Depression 38
4. Reciprocal Interactions Between the Monaminergic Systems 39
4.1. NE - DA Interactions 39 4.2. 5-HT - NE Interactions 39 4.3. 5-HT - DA Interactions 41 4.4. Clinical Relevance of Interactions 42
5. Nomifensine - A Catacholamine Reuptake Inhibitor 44
5.1. Pharmacological Profile 44 5.2. Animal and Human Studies 45
MATERIALS AND METHODS 47
RESULTS 53
DISCUSSION 66
REFERENCES 75
iv Figures
Introduction Figure 1: The major pathways of the 5-HT system Figure 2: Synthesis and metabolism of 5-HT Figure 3: The major pathways of the NE system Figure 4: The major pathways of the DA system Figure 5: Synthesis of the catecholamines DA and NE Figure 6: Major routes for the catabolism of DA Figure 7: Major routes for the catabolism of NE Figure 8: Functional interactions between monoaminergic neurons Figure 9: Chemical structure of nomifensine and sertraline
Results Figure 10: Acute dose-response curves of i.v. administration of nomifensine in NE and DA neurons Figure 11: Average firing rate of DA neurons in the VTA Figure 12: Burst analyses of DA cells in the VTA Figure 13: Acute dose-response curve of DA autoreceptor sensitivity through i.v. administration of the DA agonist apomorphine Figure 14: Average firing rate of NE neurons Figure 15: Burst analysis of NE cells in the LC Figure 16: Average firing rate of 5-HT neurons in the DRN Figure 17: Burst analyses of 5-HT cells in the DRN Figure 18: Acute dose-response curve of the 5-HT autoreceptor sensitivity through i.v.
administration of the 5-HTIA agonist LSD Figure 19: Measurement of whole blood 5-HT levels Figure 20: Effect of single- or dual-acting catecholaminergic reuptake inhibitors on escitalopram-induced decrease in DRN 5-HT neuronal activity
v Abbreviations
5-HT - 5-hydroxytryptamine; serotonin 5-HTR - 5-HT receptors AR - adrenergic receptor CMS - chronic mild stress CNS - central nervous system COMT - catechol-O-methyl transferase DA - dopamine DAT - dopamine transporter DRN - dorsal raphe nucleus GPCR- G-protein coupled receptors ISI - interspike analysis KO - knockout LC - locus coeruleus MAO - monoamine oxidase MAOI - monoamine oxidase inhibitor MDD - major depressive disorder MFB - medial forebrain bundle MRN - median raphe nucleus NAcc - Nucleus Accumbens NE - norepinephrine NET - norepinephrine transporter NF - nomifensine NRI - norepinephrine reuptake inhibitor PCPA - para-chlorophenylalanine PLC - phospholipase C SERT - 5-HT transporter SN - substantia nigra TCA - tricyclic antidepressant TH — tyrosine hydroxylase TTX - tetrodotoxin VMAT - vesicular monoamine transporter VTA - ventral tegmental area
vi Acknowledgments
First and foremost I would like to thank my supervisor, Dr. Pierre Blier, not only for the incredible opportunity I have been afforded in being able to work in his laboratory for the past two years, but for the encouragement and guidance he has constantly provided along the way. I also wish to express my appreciation for each member that has been a part of my time at the IMHR, including lab-mates, office-mates and others, who all ensured that there was never a dull moment. A special thank you goes to Jonathan James for putting time and energy into helping analyze blood samples, and of course for his random stories.
I would be remiss not to mention Dr. Gabriella Gobbi, who for the past three years has been heavily involved in my academic career, and has afforded me many opportunities during that time. It has all been greatly appreciated, and I know I would not be where I am today if it were not for her.
There are far too many people who have been a major part of my life to mention here, but to all my friends who have been there along the way, both past and present, from western Canada to the Middle East, my most sincere thanks for being part of the journey. To SM and AF, two people with a very special place in my heart, thank you both for putting up with me, and for becoming such a big part of my life. You make life fun every single day, and it certainly would not be the same without you in it.
Of course, I would in no way be the person I am today if it were not for my family. To Miriam, Jerry and Ari Katz, thank you for always believing in me and letting me know that no matter what, I will always have somewhere to turn in both the best and worst of times. Your constant love and support means more to me than I am able to express, so all I can say is thank you and I love you.
vii Introduction
1. Major Depressive Disorder
Major depressive disorder (MDD, unipolar depression) is a debilitating psychiatric disorder, affecting approximately 6.6% of the population in a 12 month period, with a lifetime prevalence of 16.2% in the United States, affecting nearly twice as many women as men (Kessler et al, 2003). It is characterized by: depressed mood, loss of interest in previously enjoyed activities (anhedonia), alterations in eating and/or sleeping patterns and other changes in thoughts such as suicide ideation (DSM IV, 2000).
In order for a diagnosis of major depressive disorder (MDD) to be applied to an individual, they must display either a depressed mood or anhedonic symptoms nearly every day for a period of greater than two weeks, while also being accompanied by a number of psychophysiological changes, such as changes in sleep, appetite and concentration. Furthermore, this depressed mood must not be due to physical illness, drug or alcohol use or the normal bereavement period caused by loss, and must cause a substantial interference with a persons work and family life.
MDD has been shown to be a highly complex disease likely involving many genes. Twin studies have demonstrated that the heritability is only about 37 %, indicating that this is not a simple genetic illness, and that a number of factors are required for the manifestation of the disorder (Sullivan et al, 2000). Although linkage studies have implicated various chromosomal regions, no distinct loci have been identified as being involved in all cases of familial depression. A functional
1 polymorphism in the promoter region of the serotonin transporter has found that individuals possessing the short allele of this gene have an elevated risk of developing depression, especially when subjected to a high degree of life stresses (Caspi et al, 2003).
This indicates that gene-by-environment interactions play a significant role in the pathophysiology of this disorder, again highlighting the complexity that exists in the development of MDD. The heterogeneous nature of MDD makes it extremely complicated in elucidating precisely how this disease comes to manifest itself in certain individuals but not others, and why there is a large discrepancy in the response of depressed individuals to various antidepressant drugs.
2. Neuroanatomy
An understanding of the neuroanatomical features of the monoaminergic systems is necessary to fully grasp many theories of depression and antidepressant treatments used to date. As such, a brief review of the neuroanatomy of the serotonin (5-HT), norepinephrine (NE) and dopamine (DA) systems will follow.
2.1. The Catecholamines
2.1.1. Neuroanatomy of the Norepinephrine System
Initial investigations of the NE system involved using fluorescence histochemistry to visualize these neurons in the central nervous system (CN)gpcS, while gradually
2 immunohistochemical methods were employed for more precise identification of the location and projections of these neurons. As was done for the raphe nuclei, Dahlstrom and Fuxe (1964) labeled the prominent NE bundles (i.e. Al, A2, etc.), although this terminology was again modified once superior anatomical labeling techniques were developed. Currently, the NE system is divided into two major clusters of neurons which innervate numerous regions throughout the neuraxis; the locus coeruleus (LC), which contains almost half of all NE producing neurons in the brain (Swanson and Hartman,
1975), and the lateral tegmental noradrenergic neurons.
There are both rostral and caudal projections emanating from the lateral tegmental system; the former forms inhibitory synaptic connections to sympathetic preganglionic neurons in the spinal cord while the latter terminates in the hypothalamus and other diencephalic structures. The inhibitory connections within the spinal cord act as a mechanism by which the central NE system regulates physiological homeostasis, such as heart rate and visceral function (Guyenet and Cabot, 1981). The rostral connections act as a bridge between the central and peripheral sympathetic systems, mediating communication between these two regions.
The LC is widely considered to be the most important structure within the NE system, despite the fact that this nucleus contains only 1600 neurons on both sides of the rat brain (Swanson, 1976), while in the human brain there are around 16 000 neurons per side (Mouton et al, 1994). Both ascending and descending projections emanate from the
LC along well-defined tracts, with some axons bifurcating near the cell body projecting in both directions. Contrary to the original belief that neurons of the LC innervate multiple brain areas, studies using retrograde tracers have shown that at least some of these neurons project nearly exclusively to precise postsynaptic structures. These axons
can however become very diffuse in these regions, with one axon branching up to 100
000 times (Moore and Bloom, 1979).
There are five major tracts that project from the LC, three of which ascend to
nearly the entire telencephelon and diencephalon (the central tegmental tract, the central
grey dorsal longitudinal fasiculus tract and the
ventral tegmental-medial forebrain bundle tract),
including the neocortex, hippocampus, amygdala,
septum, thalamus and hypothalamus. The fourth
tract ascends to the superior cerebellar peduncle,
innervating the cerebellar cortex, while the fifth
major tract descends into the spinal cord,
terminating in the ventral-lateral column. Nearly all
physiological studies have found that activation of Figure 3: The Major Pathways of the NE System
these erierent pathways extending from the LC
inhibits spontaneous discharge in the postsynaptic structures, such as the cerebellum,
hippocampus and cerebral cortex; an effect due to a hyperpolarization induced by an
increased membrane resistance. Importantly, the LC is the sole source of NE in the
hippocampus and neocortex (Foote et al, 1983), once again reinforcing the imperative
role the LC plays in the regulation of postsynaptic regions.
4 2.1.1.1. Adrenergic Receptor Subtypes
The two major types of adrenergic receptors (adrenoceptors) were originally differentiated based on their responses to sympathomimmetic drugs such as isoproterenol
(ISO). These drugs were labeled as a- and p-adrenoceptors (a-AR, |3-AR) in the first half of the 20l century (Ahlquist, 1948), based on the former showing a greater potency to
NE than ISO and being blocked by phentolamine, while the latter showed a greater potency to ISO and was antagonized by propanolol. As was the case with 5-HT receptors, the development of more specific pharmacological agents, coupled with advances in molecular biology demonstrated that this simple designation of a and P- adrenoceptors was not sufficient as subtypes of both families clearly existed based on amino acid sequence, brain localization and other factors. It is however clear that all adrenoceptors are G-protein coupled receptors (GPCR), acting via second-messenger systems to elicit their effects in the CNS, with the receptor subtypes being differentiated based on their major G-protein. Currently, these subtypes have been categorized as oti, (X2 and p-adrenergic receptors (see Wozniak et al, 1995).
2.1.1.1.1. a-Adrenergic Receptors
Based on pharmacological evidence, it was initially postulated that all postsynaptic adrenoceptors, which were responsible for the actions of the effector organ, were of the a 1-subtype, while all presynaptic receptors, thought to be responsible for transmitter release, were of the ct2-subtype (Langer, 1974). This was however discovered
5 not to be the case, as (^-receptors were found to exist, centrally as well as in the periphery, both pre- and postsynaptically. Although the classification of these two subtypes remained, the precise knowledge of their actions became more refined as a more intimate understanding of this system was accrued.
2.1.1.1.1.1. ou-Adrenoceptors
The excitatory action of NE in various neural regions is due to activation of the oii-adrenergic receptor (ai-AR). Upon activation, this receptor stimulates phospholipase
C (PLC) action via a Gq protein, although this mechanism is still poorly understood.
Further subclassification of the AR, which was originally believed to be of the aiA-AR subtype, is now known to be a distinct in its properties, and is found in a variety of tissues, including vascular smooth muscle, cerebral cortex and the rat lung (Graham et al, 1996). Although beneficial to the understanding of the adrenergic system, molecular cloning of these receptors created confusion as the nomenclature for cloned receptors did not necessarily coincide with the properties of the pharmacologically defined receptors sharing the same name. As such, opinions differ as to the precise number of ai-ARs which truly exist. 6 The most prominent effects of ai-AR activation tend to be in the periphery, where they stimulate smooth muscle contraction as well as venous and arteriolar constriction leading to an increase in blood pressure. As such, majority of drugs targeting these receptors have been peripheral in nature, and have mainly antagonized these receptors, acting as hypertensive drugs. These receptors have only recently begun to be targeted by researchers as potential CNS drug targets, as they may play a role in neural dysfunction, such as affective disorders. This is proving difficult, as oti-agonists, although potentially beneficial when 'active in the brain, also increase blood pressure when binding peripherally, making the side-effect profile of these drugs difficult to control. 2.1.1.1.1.2. a?-Adrenoceptors ai-ARs can be distinguished from 012-ARs by the chemical compound prazosin, which antagonizes ai-ARs but has little binding affinity for 012-AR. The 012-AR is negatively coupled to adenylyl cyclase via a G; protein, ultimately inhibiting the generation of cAMP. Pharmacological and molecular cloning studies have revealed three subtypes of the (X2-AR, denoted as O,2A-C, with all three subtypes being activated by agonists such as clonidine, and blocked by plant-derived antagonists such as yohimbine. The LC was found to mainly contain mRNA for the 012A-AR subtype (Scheinin et al, 1994), suggesting that this subtype acts as the main autoreceptor on the cell body of NE neurons. Additional evidence for this theory was demonstrated by the fact that application of 012 agonists such as clonidine potently inhibits the firing of NE neurons in the LC by inducing a membrane hyperpolarization through a K+-channel (Aghajanian and 7 VanderMaelen, 1982a). However, mRNA for the (X2A-AR was also found to be widely distributed in the brain stem, cerebral cortex, septum, hypothalamus, hippocampus and amygdala, suggesting that this receptor is also involved in mediating postsynaptic effects (Scheinin et al, 1994). Furthermore, it was found that 012-ARs existed not only as autoreceptors on NE neurons, but as well as heteroreceptors on 5-HT terminals, where they act to inhibit 5-HT release (Maura et al, 1982). In recent years, the ot2-ARs have become a site of heightened interest in the treatment of clinical disorders such as depression. Drugs such as mirtazepine and mianserine, which have antagonistic activity at the 012-AR and have been widely used clinically as antidepressants, are often being used in combination with other antidepressant medications (Maes et al, 1999; Blier et al, 2008) 2.1.1.1.1.3. ^-Adrenoceptors As with the a-ARs, two subtypes of the [3-ARs were pharmacologically characterized based on differential responses to various agonist and antagonist compounds, and were denoted as Pi- and P2-ARS. Recently, a third type was discovered (P3), which appears to mainly be present in white adipose tissues where it is responsible for lipolysis and in brown adipose tissue (found mainly in rodents) where it controls thermogenesis (Wozniak et al, 1995). The distinction between the Pi and P2-AR was initially proposed in the late 1960s based on the differential responses to peripheral sympathomimetic phenylethylamine compounds in various peripheral tissues (Lands et al, 1967a, b). Importantly, it was also 8 found that the binding of the NE and epinephrine (EPI) were equipotent at the pVAR, while the binding of EPI was nearly 100-fold greater at the pVAR.. Thus, it has been suggested that the latter receptor is more prominent as a hormonal receptor in the periphery, while the Pi-AR is mainly located in the CNS, regulating NE released from sympathetic nerve endings. Despite this potential difference, all three types of (3-AR are GPCRs, and are believed to activate a Gs protein, which stimulates adenylyl cyclase ultimately generating cAMP leading to various intracellular changes which are still not completely known (Cooper et al, 2003). In vitro autoradiographic studies initially found P-ARs located in many brain regions with a wide-spread distribution, including the NAcc, caudate-putamen, layers I- III of the cerebral cortex, the dentate gyrus and CA1 fields of the hippocampus and in various thalamic nuclei among others (Palacios and Kuhar, 1980). Regional differences between Pi- and P2-ARS was further delineated by Rainbow et al (1984), which found the Pi-AR to be most prominent in many layers of the cerebral cortex, dentate and CA1 hippocampal regions and the caudate-putamen (among other regions), while the P2-AR was nearly exclusively found in the cerebellum and thalamus. Later studies looking at mRNA localization in these regions provided additional evidence for this discrepant pattern of receptor distribution (i.e. mainly Pi mRNA in cortical regions and mainly p2 mRNA in the cerebellum), while also finding effectively no mRNA in other regions with had previously autoradiographically located P-ARs (i.e. caudate-putamen; Nicholas et al, 1993). This effect may be due to ligands used in binding studies interacting with a non receptor site in the region; an explanation quite plausible as there is sparse noradrenergic innervation in the striatum, and thus likely few NE receptors. 9 As |3-ARs seem to be much more prominent in regulating the PNS, majority of all drugs which target these receptors are for the amelioration of peripheral disorders. 2.1.1.2. Electrophysiological Properties of NE Neurons The firing pattern of LC NE neurons has been found to be largely dependent on vigilance-states, firing most prominently during active waking at a rate of approximately 2 Hz (Aston-Jones and Bloom, 1981a), decreasing by nearly half of this rate during slow- wave sleep, and being nearly absent during REM sleep. The firing rate in anesthetized rats is about 2 Hz, similar to the rate seen in quiet waking rats. Additionally, NE neurons have been found to respond to noxious stimuli, such as a contralateral paw pinch, with a brief burst of spikes followed by a quiescent period. Evidence suggests that this electrophysiological effect is mediated via collateral inhibition of the NE neurons emanating from the LC (Aghjanian et al, 1977). Neurons of the LC were also found to predictably elicit short-latency, biphasic and transient changes in discharge in response to non-noxious auditory, visual and somatosensory stimuli, with this sensory responsiveness being decreased during grooming and sweet water consumption, similar to what is seen during sleep (Aston-Jones and Bloom, 1981b). It was thus proposed that NE LC discharge may enhance activity within target cell systems primarily concerned with processing salient external stimuli and suppress central nervous system activity related more to tonic, vegetative functions. It has further been shown that weak electrotonic coupling exists between neurons of the LC, and that such coupling can induce both synchronous and asynchronous states, depending on the frequency of the network, and 10 may lead to coordinated activation of widespread regions of the CNS (Alvarez et al, 2002). 2.1.2. Neuroanatomy of the Dopamine System For a great deal of time, it was thought that DA was simply a metabolic precursor to NE and had no distinct physiological function of its own. This is obviously not the case as the DA system has become one of the most highly studied systems in the brain, and is now known to modulate motor activity, reward and affective states among many others. Although catecholaminergic cell bodies and pathways were originally mapped using histochemical fluorescence, this technique proved inadequate in the precise mapping of individual DA neurons. As seen with the previous monoaminergic systems, immunocytochemistry proved to be a far superior method in the delineation of the DA system using antibodies against tyrosine hydroxylase (TH) - the precursor to catecholamine synthesis - and eventually to DA itself. Dahlstrom and Fuxe (1964) provided the initial designation of the DAergic bundles as A8-A15, which has been subsequently altered to reflect the greater understanding of the DA nuclei. The DA system is more complex than the NE system as it contains a far greater number of neurons than NE, while also having several DA-containing nuclei (as opposed to mainly the LC for NE neurons). In fact, DA is thought to comprise nearly 80% of all catecholamine content in the brain, despite having at most 1 000 000 neurons in the entire brain (Feldman et al, 1997). 11 Numerous pathways have been mapped out for the DA system, however three main pathways emanating from the mesencephalon stand out; the nigrostriatal, the mesocortical and the mesolimbic pathways. The nigrostriatal pathway is mainly composed of DA projections stemming from the substantia nigra pars compacta (SNpc) to the caudate, putamen and globus pallidus via the MFB. This pathway is crucial in proper motor functioning, and it is the degeneration of SN DA neurons that is thought to cause the major extrapyramidal symptoms of Parkinson's disease. The mesocortical and mesolimbic pathways both emanate from neurons in the mesencephalic nucleus known as the ventral tegmental area (VTA), the former pathway projecting mainly to the medial prefrontal and cingulate cortices, with the latter projecting to subcortical limbic structures such as the nucleus accumbens, amygdaloid complex, olfactory tubercle and piriform cortex. Dysfunctions in both of these pathways have been implicated in many Figure 4: The Major Pathways of the DA System psychiatric disorders such as schizophrenia and depression, demonstrating the crucial role that proper DAergic function plays in the development of mental disorders. Over the years, it has become clear that these pathways form heterogeneous connections, with projections from the SN and VTA creating overlap in many of the postsynaptic DAergic regions (Fallon and Moore, 1978), thus suggesting that dysfunction of multiple DA pathways may be involved in a single disorder. 12 Recently, it has been discovered that there is significant DAergic innervation of the thalamus in primates, which is largely absent in rodents (Sanchez-Gonzalez et al, 2005), and unlike the other DA pathways, this innervations stems from several structures, including the periaqueductal gray matter, ventral mesencephelon and the hypothalamic nuclei. This newly discovered pathway may be involved in the gating of information transmitted from the thalamus to various postsynaptic regions, including the neocortex and striatum; regions often implicated in neuropsychiatric disorders. However the hypothetical role of this pathway in such disorders has yet to be clarified. 2.1.2.1. Dopamine Receptor Subtypes Investigations into DA receptors did not begin until the early 1970s with the discovery that DA could stimulate adenylyl cyclase activity (Kebabian et al, 1972); more than twenty years after Ahlquist (1948) had initially proposed the existence of distinct receptor subtypes for NE. The initial designation of the two distinct DA receptor subtypes as Di and D2 was not, however, made until years later by Kebabian and Calne (1979) who elaborated on previous works that found anatomical, physiological and pharmacological evidence of more than one DA receptor (Titeler et al, 1978; Garau et al, 1978). As with nearly all other monaminergic receptors, the two DA receptors act as GPCRs; the Di receptor is positively coupled to adenylyl cyclase via a Gs protein, while the D2 receptor interacts with a Gj protein to inhibit cAMP production. Molecular techniques eventually led to the cloning of the D2 receptor in the late '80s (Bunzow et al, 1988), and the subsequent discovery of additional DA receptors which became known as 13 D3-D5. Despite* these new receptors subtypes, it was found that the Di and D5 receptors shared a common homology and became known as the Di-like receptors, while D2, D3 and D4 receptors, which shared similar structure and function, became known as the D2- like receptors. The distribution of the Dj receptor has been found through autoradiography to be highest in regions where mesencephalic DA terminal regions such as the caudate-putamen, NAcc, and olfactory tubercle, with relatively lower levels in cortical regions (Boyson et al, 1986). This finding indicates that majority of the Di-like receptors are likely postsynaptic in nature, and have little effect, if any, on DA neurons themselves. 2.1.2.1.1. D? Receptors Similar to the Di receptors, the distribution of the D2 receptors has been found to be widespread throughout the brain in a heterogeneous fashion, being detected in the caudate-putamen, NAcc and olfactory tubercle (Boyson et al, 1986). However, unlike the Di receptors, mRNA for the D2 receptors is found at high levels in the midbrain neurons of the SNpc and VTA, suggesting that it is likely this receptor which acts as a presynaptic autoreceptor in somatodendritic regions (Meador-Woodruff et al, 1991) while also acting as an autoreceptor on DA terminals (Roth, 1984). Indeed, it has been found that stimulation of somatodendritic autoreceptors decreases the firing rate of midbrain DA neurons, while stimulation of the terminal autoreceptors inhibits DA synthesis and release, showing how the two work in concert to exert feedback regulation on DA transmission (Roth and Elsworth, 1995). 14 Soon after the cloning of the D2 receptor by Bunzow et al (1988), the human receptor was cloned and found to share a 96% sequence homology with that of the rat, while also revealing that the D2 receptor existed in two different isoforms - long (D2O and short (D2s) - through alternative splicing (Dal Toso et al, 1989). It was unclear for some time whether or not there was any significant difference that existed between these two isoforms; however, a recent knock-out study found that the D2s constitutes the main somatodendritic autoreceptor on DA neurons (Centonze et al, 2002) suggesting a clear difference in the alternatively spliced receptors. The pharmacological targeting of the D2 receptor has been instituted for many years in the treatment of schizophrenia. Thus, a large number of the 'typical' antipsychotics such as haloperidol and chlorpromazine have been used for many years in order to attempt to alleviate symptoms of this disorder. The targeting of the D2 receptor for the treatment of mental disorders such as depression has classically been ignored until very recently. 15 2.1.2.2. Electrophysiological properties of DA Neurons Extracellular studies of anesthetized rats have found that DA neurons possess the unique property of alternating from a state of spontaneously active firing, to a period of quiescence due to hyperpolarization or hyperexcitation resulting in a depolarization blocked state (Bunney and Grace, 1978). Additionally, when DA neurons axe in the spontaneously active state, they may fire either in a single-spike pattern (Grace and Bunney, 1984a) or a bursting pattern in which 3-10 spikes of decreasing amplitude are elicited with an initial interspike interval (ISI) of less than 80 ms, and no subsequent ISI greater than 160 ms (Grace and Bunney, 1984b). In either state, the firing pattern of DA neurons tends to be irregular, and fire at an average frequency of 4-6 Hz. Neighbouring DA neurons often fire synchronously with a spontaneously active DA neuron nearby, a characteristic indicative of electronic coupling between these neurons (Komendantov and Canavier, 2002; Vandecasteele et al, 2005), which is mediated by intercellular gap junctions (Grace and Bunney, 1983). The majority of evidence has found that the release of DA is spike-dependent, as inactivation of DA neurons effectively eliminates release (Keefe et al, 1992). Furthermore, when DA neurons fire in burst mode there is a greater amount of DA released (Gonon and Buda, 1985), suggesting that burst firing plays a significant role in mediating DA transmission, and subsequent DA-related behaviours. 16 2.1-3. Synthesis, Storage, Release and Metabolism of DA and NE The precursor for both DA O and NE synthesis is the amino acid ^*\ OH L-Tyrosine X J NH2 HO" ^ 02. Tetrahy^rp tyrosine derived from dietary b*op-1enn Tyrosine hydroxylases HyO, Dihydfo- proteins, while also less substantially hi-op serin *' O being synthesized in the liver by T^ OH L-Dihydroxyprwnylalanine X L> «t-DOPA) altering the amino acid DOPA decarboxylase phenylalanine. The first step in the Aromatic L-amino acid decarboxylase CO; • synthesis of catecholamines is the ^ ^""\ Dopamine II 'I ^ NH2 hydroxylation of L-Tyrosine into HO CV Ascorbic *\ what is commonly referred to as L- Dopamine p-hydroxylase ascorbic acid J 1 DOPA (DOPA) by the enzyme OH HO\.-- "'I *" ~~~\ Norepinephrine tyrosine hydroxylase (TH) - the rate NHZ HO > limiting enzyme for the production Figure 5: Synthesis of the catecholamines DA and NE of all catecholamines. As such, TH is a primary target in studies investigating catecholamine depletion, often with the TH inhibitor a-methyl-paratyrosine (AMTP), leading to marked reductions of DA and NE in the brain. The second and final step in the biosynthesis of DA is the decarboxylation of DOPA, catalyzed by the enzyme aromatic L-amino acid decarboxylaxe (AADC) at a rate so rapid that levels of DOPA in the brain are quite negligible. Inhibition of this enzyme typically leads to a large accumulation in brain DOPA levels, and is therefore a useful 17 tool in measuring the level of TH activity in vivo. When present in DAergic neurons, the enzyme DA-P-hydroxylase (DBH) subsequently alters DA into NE. Following the synthesis of the catecholamines, they are packaged into granules or vesicles for subsequent release. The mechanism underlying the transport of the neurotransmitters into the granule membrane is via an ATPase located on the granule membrane which translocates protons from the cytoplasm into the interior of the cell. An electrochemical gradient is thus induced via a pH gradient across the membrane and a transmembrane electric potential, thereby allowing the catecholamine vesicular transporter to take up the catecholamine into the granule. This protein acts as an antiporter, and is so efficient that it is capable of producing a 135 000:1 gradient of catecholamines across the chromaffin cell membrane (Johnson, 1987). The accumulation of DA, NE and 5-HT in synaptic vesicles all appear to be under control of the same protein, now known as the vesicular monoamine transporter (VMAT2), indicating minimal variation in storage mechanisms (Erickson et al, 1992). It was found that reserpine binds to sites on the vesicle transporter, thereby making it a good chemical marker for said vesicles, while also explaining the monoaminergic depletion induced by its application (Henry and Scherman, 1989). The release of catecholamines has been found to occur via stimulus-evoked exocytosis through many line of evidence including electron microscopy showing fusion of vesicles with the nerve terminal membrane (Thureson-Klein, 1983) as well as the fact that application of reserpine drastically decreases release. Multiple in vitro and in vivo studies have determined that DA release possesses the classical characteristics of Ca2+- dependent release, and is sensitive to the tetrodotoxin (TTX)-induced blockade of Na+ 18 channels. However, a second type of Ca -independent release has also been found, in which there is a reversal of the catecholamine membrane transport proteins allowing for the release of neurotransmitter stores in the nerve terminal cytoplasm (Ratieri et al, 1979). Evidence of this mode of release has been seen in VMAT2 knock-out mice, in which there is no vesicular storage of the monoamines, however, release can be induced through amphetamine application, increasing the survival rates of these animals (Fon et al, 1997). The enzymatic degradation of the catecholamines occurs primarily through two enzymes; catechol-O-methyltransferase (COMT), which is believed to exist extrasynaptically and monoamine oxidase (MAO), which is believed to be mainly intraneuronal, although does exist extraneuronally as well (Fowler et al, 1984). COMT is Mg2+-dependent, catalyzing the transfer of a methyl group on catecholaminergic compounds leading to their eventual degradation. On the other hand, MAO catalyzes the oxidative deamination of the monoamines, including DA, NE and 5-HT, eventually producing an aldehyde product. Two forms of MAO - MAO-A and MAO-B - have been identified; MAO-A being primarily responsible for the breakdown of NE, while DA is broken down equally by both. The primary metabolites of DA depend on whether it is broken down by COMT or MAO, however as estimates as high as 90% of striatal DA catabolism occurs via MAO, this will be what is focused on here. The immediate product of MAO deamination is 3,4-dihydroxyphenylacetaldehyde (DHPA), which is rapidly oxidized to 3,4- dihydroxyphenylacetic acid (DOPAC), considered to be the primary DA metabolite. Around 40% of DOPAC is eliminated from the brain, while the other 60%o is altered by 19 COMT, leading to the production of homovanillic acid (HVA). Both DOPAC and HVA enter the CSF, and are measures of DA metabolism, although DOPAC is considered to be a far more accurate index of intraneuronal DA metabolism. NE metabolism occurs via COMT or MAO through various interconnected pathways, producing multiple intermediates and two final products; 3-methoxy-4- hydroxyphylglycol (MHPG) and vanillymandelic acid (VMA). The major urinary metabolite in humans is VMA, while MHPG is the primary brain metabolite, suggesting that they are good measures of brain and peripheral NE metabolism, respectively. However, MHPG is in fact generated in peripheral tissues, as well as evidence that MHPG is converted to VMA prior to excretion, therefore making this an oversimplification in the measurement of NE metabolism. 20 HD HO'-/~V -^ 7-CHo j — €H2 — 'NHj CHj — CHj — JSfHj \ywM.y IDHPAJ 4S-MT) FAD •deb MAO HO. HO -/ V- CHj — COOH HO CHfe—CHD 3j*Dityd*&^lvniytaiHifc mid 3- Meth<«yw44^m{^p|MjYy|M%^ fa j,^. (DOPAJCJ CGMT Aldehyde CH.j — COOH HcwKfvuiiUte add Figure 6: Major routes for catabolism of DA. 21 HO \ HO -4 V-CH—CHj—NHj OH CQMT HO-/ V-CH —CHO HO -/ V-CH- CHj ~nnt OH OH 3,*-Dlhyditm'ptienylglyeii*W*hj AMehyctc Aldehyde FAP MAO reductase HO CHjO( HO-f V-CH—CM2OH HO-i V-;H—p CHjrOH HO CH3 "^'CnO CDHTC) iddMDHMA) (MHJGA) Aldehyde SAM, SAM, dehyclrogefiis* COMT COMT Aldehyde dehydrogenase- / GUjA CH|Q, CHjQ HO CH-CHjQK HO~f VCH-COOH HO-/ V-CH— CHjOH OH OH OH >Wrt*a*y-4- V*nSlllyraajndit« arid hydHXjrptMnylgtyMl 1VMA) frydto^feenyfglyvdl (MHFQ Figure 7: Major routes for catabolism of NE. 22 2-1.4. The NE and DA Transporters The primary mechanism in the removal of both synaptic DA and NE is by Na+/Cl—dependent transport proteins, known as the DA transporter (DAT) and NE transporter (NET), respectively. Contrary to the vesicular packaging of catecholamines, these 12 membrane-spanning protein transporters require an electrochemical gradient in order to reuptake these neurotransmitters, which is accomplished by creating a Na+ gradient through the Na+-K+ ATPase (sodium-potassium pump). It has been shown that in mice lacking the DAT gene (knock-out; KO mice), the synaptic clearance rate for DA in tissues slices was nearly 100 times slower than that seen in control animals, following stimulus-evoked release (Giros et al, 1996). Furthermore, in these KO animals, there was found to be a reduction of both D] and D2 receptors, as well as a decrease in DA synthesis, indicating that drastic adaptive changes occur in response to a lack of neurotransmitter reuptake. It is quite interesting that despite DA and NE neurons producing only mRNA for their respective transporters, there is a certain degree of promiscuity that these transporters exhibit. More specifically, it has been found that the NET is able to reuptake DA as well as NE (Raiteri et al, 1977) and in fact has a greater affinity for DA than does the DAT itself. Furthermore, it was shown through knocking out either the NET or DAT gene, that although the DAT is crucial for DA uptake in the caudate and NAcc, it is the NET that is primarily responsible for DA reuptake in the frontal cortex; an area with a much higher NE innervation than DA (Moron et al, 2002). 23 Therapeutically, a number of reuptake inhibitors have been developed in order to attempt to alleviate symptoms of various neurological disorders, although majority have been targeted at NE rather than DA, as increasing striatal DA levels can have serious addiction potential. However, these proteins do still remain prominent sites for potential pharmacological targeting in the treatment of various disorders, including depression. 2.2. The 5-Hydroxytryptamine System 2.2.1. Neuroanatomy of the 5-HT System A variety of morphological techniques have provided the means to allow for the characterization of the 5-HT system. Initially, the renowned neuroanatomist Ramon y Cajal discovered the nature of the serotonergic neurons along the midline of the brainstem. The anatomical architecture of the 5-FIT system was further advanced using histochemical techniques by Dahlstrom and Fuxe (1964), who divided these cell clusters into nine groups (B1-B9), now known collectively as the raphe nuclei. These nuclei are further subdivided into two groups: the rostral or superior raphe nuclei and inferior or caudal raphe nuclei. Innervation of the forebrain arises nearly entirely from two nuclei contained within the rostral system; the dorsal raphe nucleus Figure 1: The Major Pathways of the 5-HT System 24 (DRN) and the medial raphe nucleus (MRN; Moore et al, 1978). In the human brain, the DRN contains 50-60 % of all serotonergic nuclei (Baker et al, 1990) making this a crucial region for the proper function of the 5-HT system as a whole. However, serotonergic cells may make up as little as 25-50 % of the DRN and 20-30 % in the MRN, indicating that the majority of cells in raphe nuclei are non-5-HT in their nature (Pineyro and Blier, 1999). The brainstem raphe nuclei have one of the most diffuse efferent systems, innervating essentially every region in the CNS. Electrolytic and chemically induced lesioning found that the caudal raphe nuclei project predominantly to the medulla and spinal cord regions. The inferior nuclei have three known pathways by which they innervate the spinal cord via the medial longitudinal fasiculus (MLF), tectospinal tract (TST) and reticulospinal tract (RST). Every spinal level of the autonomic system, including both sensory and motor nuclei, are affected by the 5-HT fibers generated from these pathways as they innervate the dorsal, lateral and ventral horn neurons of the spinal cord (see Azmitia and Whitaker-Azmitia, 2000 for review). Immunocytochemical studies determined that there is an extensive innervation of the cerebral cortex in what appears to be a relatively patternless orientation, as well as indicating a relatively high degree of overlapping terminal fields from most of the raphe nuclei. At least 5 separate ascending pathways projecting to the forebrain have been described, the largest of which in the rat is the medial forebrain bundle (MFB) carrying fibers from the DRN and MRN to various forebrain regions. The DRN and MRN are somewhat of an exception to the terminal overlap found in the forebrain regions, with the MRN appearing to preferentially innervate limbic regions such as the cingulate cortex, septal nuclei and the hippocampus, 25 while the DRN largely innervates the substantia nigra, corpus striatum, amygdala and nucleus accumbens. Despite the preferential innervation demonstrated by these two structures, the selectivity of the postsynaptic regions to which they project is not absolute, and some overlap does exist. Nevertheless, this is a prime example of the potential for an exceptionally large number of projections emanating from a single neuron or group of neurons to numerous terminal regions, demonstrating the widespread control that the serotonergic system exerts within the entire CNS. The main serotonergic fiber pathways give rise to a vast number of branches which follow along other neuronal pathways, as well as along the ventricular system and blood vessels among other structures, making this system extremely complex to describe as the branches reach every region of the brain. Furthermore, the terminal regions of these fibers end in both a junctional and non- junctional fashion depending on the region, indicating that 5-HT can act both in a highly- specific manner as well is in a more diffuse, global manner (Beaudet and Descarries, 1981). The advent of immunocytochemistry permitted the use of antibodies to directly target endogenous 5-HT in the brain, thereby no longer relying on the uptake of radiolabeled 5-HT or 5-HT analogues to attempt to characterize 5-HT containing terminals throughout the brain. This technique gave rise to distinct morphological types of 5-HT axon terminals: fine axons and beaded axons. Using an anterograde tracer, it was found that 'beaded' axons arising from the MRN are characterized by large, spherical varicosities (type M axons) and by variations in axonal diameter, while the 'fine' fibers from the DRN are quite fine with small, pleomorphic varicosities that are granular or fusiform in shape (type D axons; Kosofsky and Molliver, 1987). This 26 technique also confirmed the topographic distributional differences between the DRN and MRN, with the MRN mainly projecting to limbic regions. Furthermore, these two types of cells were found to be differentially vulnerable to neurotoxicity by the amphetamine like compounds methylenedioxyamphetamine (MDA), and p-chloroamphetamine (PCA), with the beaded axons being markedly resistant to degeneration, indicating a functional difference between efferents stemming from the different raphe regions (Mamounas et al, 1991). 2.2.2. 5-HT Receptor Subtypes The first suggestions of multiple subtypes of 5-HT receptors (5-HTRs) were put forth by Gaddum and Picarelli (1957), with the further classification of three distinct types of 5-HTRs (5-HT1.3) occurring in the late 80's essentially via operational pharmacology, employing selective receptor agonists and antagonists (Bradley et al, 1986). Since this time, an explosion in the field of 5-HTR research due to a vast increase in the availability of specific pharmacological agents, as well as advances in the field of molecular biology have provided definitive evidence of at least 15 subtypes of 5-HTRs (5-HTi.y; Hoyer et al, 1994). It is now known that all of the 5-HTRs are 7- transmembrane spanning G-protein coupled receptors (GPCRs) acting metabotropically via second messengers, aside from the 5-HT3 subtype which acts as a ligand-gated ion channel. Although most of the 5-HTRs are metabotropic in nature, there still exist differences in the mechanism of action of the various superfamilies. Receptors of the 5- HTj family are all are all negatively coupled to adenylyl cyclase via the Gj family of G- 27 proteins, while receptors of the 5-HT2 family, all of which share a similar homology and aniino-acid sequence, are of the Gq-protein family, inducing the hydrolysis of phosphoinositide through phospholipase C activation. The receptors positively coupled to adenylyl cyclase are heterogeneous in nature, encapsulating the 5-HT4, 5-HT6 and 5- HT7 families. 2.2.2.1. 5-HTJA Receptors 5-HTIA receptors where initially defined by their high affinity to [ H] spiperone relative to the other 5-HTi receptors, but are now characterized by their affinity for 8- hydroxy-2-(di-n-propylamino) tetralin (8-OH DPAT), a more specific serotonergic compound. Postsynaptically, these receptors are found with the highest density in the hippocampus, septum, amygdala, and cortical limbic areas. Importantly, they are also present in the raphe nuclei where they act as somatodendritic autoreceptors controlling the firing rate (Blier et al, 1987) and local release (Pineyro et al, 1996; Davidson and Stamford, 1995) of 5-HT from the soma of DRN neurons, as well as the terminal release of 5-HT in regions such as the striatum and hippocampus (Kreiss and Lucki, 1994; Casanovas et al, 1999). Corresponding to the decrease in adenylyl cyclase levels, activation of 5-HTIARS induces a depression of neuronal firing in both pre- and postsynaptic regions caused by a hyperpolarization elicited by an increased K+-channel conductance. Due to the relative abundance of the 5-HTi receptors throughout the CNS, this indicates that the main effect of 5-HT is inhibitory in nature, albeit not nearly exclusively. 28 The behavioural functions of the 5-HTIA receptors are the source of constant study, and have consistently become the targets in the treatment of numerous disorders. Systemic administration of specific agonists such as 8-OH DPAT as well as partial agonists of the azapirone family (i.e. buspirone, gepirone, ipsapirone) elicit behavioural effects predictive of an anxiolytic effect such as hyperphagia and hypothermia (Lucki 1992). This evidence indicates that activation of these receptors may be therapeutic in the treatment of anxiety disorders, and in fact, drugs such as buspirone are currently used in clinical settings for this purpose. However, one of the consequences of an optimal 5- HTIA receptor activation is the occurrence of "serotonin syndrome"; a condition in humans that causes myoclonic tremors (muscle spasms), hyperreflexia, diaphoresis and delirium. This condition is induced through activation of postsynaptic 5-HT]A receptors in the lower brain stem or spinal cord, as well as 5-HT2 receptors (which are responsible for the head-twitch), and in extreme cases may even lead to death (see Sternbach 1991 for review). 2.2.3. 5-HT Synthesis, Storage, Release and Metabolism The synthesis of 5-HT occurs from the dietary amino acid tryptophan. The initial, and rate-limiting step of 5-HT synthesis involves the hydroxylation of L-tryptophan in the 5-position to form 5-hydroxytryptophan (5-HTP). This alteration is catalyzed by the enzyme tryptophan-5'-monoxygenase (commonly referred to as tryptophan hydroxylase), which has been found to be exclusively localized to serotonergic neurons making it a good marker for these neurons. Pharmacological inhibition of this enzyme by para- 29 chlorophenylalanine (PCPA) is used frequently to study the effects of central 5-HT depletion. The subsequent decarboxylation of 5-HTP into the final product of 5-HT occurs very rapidly via the enzyme 5-HTP decarboxylase (see Cooper and Bloom, 2003). The majority of 5-HT is stored in vesicles located in 5-HT cell bodies and nerve terminals, although small amounts do exist in cytoplasmic regions. Both large (70-120 nm) and small (40-60 nm) diameter vesicles have been found in 5-HT nerve terminals (Maley et al, 1990), although majority of 5-HT is thought to exist in the large dense-core granules, leaving many questions regarding the function of the smaller ones. As is the case for the catecholamines, 5-HT enters these vesicles for storage and presumed exocytotic release via the VMAT2 protein (see catecholamine section). COOH I The release of 5-HT is -CHjCH NM, •^-, generally accepted to be through Tryptophan ibydfoxfo&e exocytosis, as both in vitro and in I COOH HO (^ ^, rr-CHjCH $*hyd!'t>xyrrypEc^hai vivo studies have found 5-HT release IXI L aromatic add decarbox^as to be sensitive to Na+-blockade by (^ck^pi J-IO/^- -CHjCHjhiHj TTX, as well as dependent on the presence of Ca2+ ions. Although >/ F-laiwsafnl ne oxidase (MAO) convincing, these factors do not / necessarily confirm that the CHjO^^N p-CHjCHjNH; € v-,A,J Akffchyrfi; 30 are required. Additionally, it has been shown that 5-HT release can occur through a reverse transport through the SERT induced by drugs such as MDMA and fenfluramine, as this type of release is attenuated by both fluoxetine and cocaine; two compounds known to block the SERT (Berger et al, 1992). The breakdown of 5-HT is catalyzed the enzyme monoamine oxidase (MAO) through a process of oxidative deamination. The primary metabolite of 5-HT is 5- hydroxyindoleacetic acid (5-HIAA), which diffuses out of the nerve cell and into the CSF, providing an indirect measure of 5-HT turnover in the brain. It appears that the metabolism of 5-HT occurs by the MAO-A isozyme as opposed to MAO-B, as the selective monoamine oxidase inhibitor (MAOI) clorglyine, which inhibits MAO-A, decreases extracellular levels of 5-HIAA in the rat striatum, while treatment with deprenyl, an MAO-B inhibitor, does not (Kato et al, 1986; Azzaro et al, 1988). Despite the fact that MAO-B has a Km nearly ten-fold greater than MAO-A for 5-HT, 5-HT neurons in the rat appear to largely contain MAO-B (Willoughby et al, 1988). Provided the in vitro affinities are similar to what occurs in vivo, this may in fact be a method to promote the reuptake of 5-HT rather than degradation, thereby requiring minimal synthesis of new neurotransmitter. 2.2.4. The Serotonin Transporter (SERT) One of the main therapeutic drug targets in the 5-HT system is the 12 membrane- spanning, Na+/Cl" dependent transport protein located on presynaptic terminals (Kuhar et al, 1972) commonly referred to as the serotonin transporter (SERT). It is this protein that 31 removes exocytotically released 5-HT from synaptic regions back into intracellular regions for repacking into vesicles and subsequent re-release. Therefore, it is the SERT that plays a prominent role in regulating the amount of 5-HT present in synaptic and extrasynaptic regions and consequently the degree to which pre- and postsynaptic receptors are activated. Radiolabelling studies have found regional differences in the binding of pharmacological agents such as the tricyclic antidepressant [ H] imipramine (Langer et al, 1980) and SSRIs such as [3H]citalopram (D'Amato et al, 1987) and [ H]paroxetine (Habert et al, 1985), with the TCA compound showing a higher density of binding in postsynaptic regions such as the cortex and hippocampus. This discrepancy has been attributed to imipramine binding to both the high and low affinity SERT, while SSRIs bind only to the high affinity SERT (Moret and Briley, 1986), which is believed to be the type that is mainly involved in 5-HT reuptake. 2.2.5. Electrophysiological Properties of 5-HT Neurons 5-HT cells of the DRN are electrophysiologically characterized in anesthetized rats by their regular, slow (0.5-2.5 Hz) firing rate and long duration action potential (2-5 ms) (Aghajanian and Vandermaelen, 1982b). This monotonic, nearly clock-like activity is manifested as early as 3-4 days before birth, demonstrating the crucial role of 5-HT in global physiological functioning. Electrophysiological studies in awake, freely moving rats has found that activity of 5-HT neurons is highest during periods of waking arousal, slightly less active during quiet waking, greatly reduced during slow-wave sleep, and effectively inactive during REM sleep (Jacobs and Fornal, 1993). It was therefore 32 postulated that the main function of brain 5-HT is to facilitate motor output and suppress the continual processing of sensory input, thereby explaining the vigorous firing of DRN 5-HT neurons during behavioural activation and the relative quiescence when the motor system is inactive (i.e. REM sleep). However, this theory falls short in explaining the involvement of 5-HT in a vast array of physiological processes such as nociception, thermoregulation, aggression, reproduction and stress, among others, excitingly leaving many questions to be answered regarding the precise function of this extremely diffuse system. Interestingly, it has been shown that there exists an electrotonic coupling between 5-HT neurons, and that 5-HT release in the DRN may be controlled independently of 5-HTIA autoreceptors (Pineyro et al, 1996). Such multimodal release characteristics may in fact play differing roles in the various behaviours affected by 5- HT. 3. Evidence Implicating the Monoamines in the Pathophysiology of MDD 3.1. History of Antidepressants The monoamine-deficiency hypothesis of depression has been one of the central theories of depression for nearly 5 decades. This biochemical basis of depression began to develop in the 1950s when a number of clinical observations led researchers to believe that dysregulation of brain neurotransmitter levels, specifically 5-HT and NE were responsible for the manifestation of depressive symptoms. Reserpine, one of the first clinically used antihypertensive drugs, was found to induce depressive symptoms in 33 individuals being treated with this drug (Muller et al, 1955). It was later found that reserpine nearly irreversibly depleted catecholamine levels in presynaptic neurons by blocking the vesicular transporter VMAT, causing the unprotected monoamines to be metabolized by MAO. Additional evidence implicating the monoamines in depression came from the serendipitous discovery of two drugs in the same decade - iproniazid and imipramine. Iproniazid, initially developed to treat tuberculosis, was found to improve mood in patients suffering concurrent depression, and later to be effective in depressed individuals who were not suffering from tuberculosis (Pare and Sandler, 1959). Further study of this drug found it to be an MAO inhibitor (MAOI), effectively increasing brain levels of the monoamines whilst also being associated with behavioural excitement. Many side effects such as weight gain, headaches and impotence, as well as the more severe hepatotoxicity were caused by iproniazid, but this was the initial step in the development of future generations of MAOIs. Similar to iproniazid, imipramine was discovered to be an effective antidepressant while it was being developed as a putative antipsychotic (Kuhn, 1958). The discovery of this drug spawned the future development of the class of drugs which came to be known as tricyclic antidepressants (TCAs), as they contain three rings in their chemical structure. The precise mechanism of action of TCAs in the treatment of depression is still not entirely known, however, it is likely that they exert their therapeutic effect by blocking the reuptake of 5-HT and NE, effectively increasing the synaptic levels of these monoamines in various brain regions. Additionally, it has been shown that TCA treatment sensitizes postsynaptic 5-HT receptors, which may partially underlie the therapeutic effect of these drugs (Blier et al, 1987). TCAs also however interact with a 34 number of other receptors including histamine, acetylcholine and adrenaline, which causes a great deal of side-effects such as drowsiness, sedation, hypotension, dry mouth, constipation, blurred vision and arrhythmia among others. Most importantly, an overdose of TCAs can be fatal, therefore producing a risk in the prescription of these drugs to depressed patients, especially those at risk of suicide. Antidepressants such as TCAs and MAOIs provided the first evidence that drugs which increased synaptic concentrations of the monoamines, specifically 5-HT and NE, produced a therapeutic effect in depressed individuals. However, it wasn't until the availability of selective-serotonin reuptake inhibitors (SSRIs) that the putative functional role of 5-HT could be elucidated. Due to the discovery of SSRIs, NE became somewhat neglected, while majority of the focus was shifted towards 5-HT (Brunello et al, 2002). 3.2. Evidence of 5-HT Involvement in Depression One of the most compelling lines of evidence for the involvement of 5-HT in the pathophysiology of MDD came about in the mid-1970s with the use of 5-HT depletion experiments. It was discovered that certain individuals who had recently recovered from MDD through treatment with either the MAOI tranylcypromine or the TCA imipramine relapsed into a state of depression when treated with low levels of PCPA (Shopsin et al, 1976). Those individuals who did relapse recovered from these symptoms after stopping PCPA treatment for 2-7 days, indicating that 5-HT is crucial for these pharmacological agents to be effective. 35 The discovery and development of selective serotonin reuptake inhibitors (SSRIs) was a positive step in the treatment of depression, and was based on the fact that TCAs ameliorated depressive symptoms by increasing extracellular neurotransmitter levels. Although there is an increase in synaptic 5-HT upon treatment with SSRIs, there is a decrease in the presynaptic firing rate of DRN cells in rats, and thus a relatively low increase in extracellular 5-HT levels. It was found however, that the therapeutic effect elicited by SSRI application was due to an eventual desensitization of 5-HTIA and 5- HTIB autoreceptors, and an increase in the amount of synaptic 5-HT (Pineyro and Blier, 1999). It is this desensitization period that is seen in animals that may account for the delayed onset of SSRI antidepressant action in humans. Additionally, this class of drugs brought great benefits to the treatment of depression as these medications are not toxic in overdose, and produce fewer side effects than other classes of antidepressant drugs such as TCAs and MAOIs. This is largely attributable to SSRIs being less "pharmacologically dirty", as most of them lack potent action on other neurotransmitter receptors, such as histamine, cholinergic and adrenergic receptors (Westenberg and Sander, 2006). Unfortunately, SSRIs are not effective in 100% of individuals; only 2/3 of individuals who take SSRIs show any decrease in depressive symptoms, and only 1/3 of actually enter into a lasting remission of symptoms. This therefore suggests that the pathophysiology of depression is not solely due to a flawed 5-HT system, but likely involves other neurotransmitters such as NE and DA. 36 3-3. Evidence of Catecholamine Involvement in Depression 3.3.1. NE in Depression Inducing depression in animals through the validated chronic mild stress (CMS) paradigm (Willner, 1997) has been shown to lead to a number of alterations within the NE system, including an increase in a2-adrenoceptors. As these receptors act as autoreceptors on the cell body and terminals of NE neurons, it suggests an ultimate decrease in NE release (Leonard, 1997). Additionally, it was found that there was a significant decrease in NET levels in the LC of post-mortem brain samples of depressed patients relative to age matched controls, suggesting a compensatory downregulation of the NET due to decreased extracellular NE levels (Klimek et al, 1997). Such evidence led to the development of antidepressant drugs which specifically targeted the NE system. The antidepressant drugs mianserin and mirtazepine target the NE system by preferentially antagonizing presynaptic a2-adrenoceptors, although these two drugs in this class are not completely specific and act on various 5-HT receptors as well (Haddjeri et al, 1997). In addition, a class of drugs akin to SSRIs such as reboxetine, were developed to act on the NE transporter, thereby inhibiting the reuptake of NE (NRIs) and producing positive clinical results. It was found that not only did synaptic levels of NE increase, but a desensitization of a2-autoreceptors on NE terminals as well as a2-heteroreceptors on 5- HT terminals occurred, with no desensitization of the a2-somatodendritic receptors (Szabo and Blier, 2001a; Parini et al, 2005). It has also been found that co-administration of an a2-adrenoceptor antagonist along with NRIs enhanced NE levels in various regions 37 of the brain (Sacchetti et al, 1999). This lends credit to the theory that desensitization of the terminal (^-adrenoceptor contributes to the enhancing effect of NRIs after chronic treatment, and may explain the delayed onset seen in patients being treated with NRIs (see Invernizzi and Garratini, 2004 for review). 3.3.2. DA in Depression Until recently, the role of DA in depression was generally overlooked, despite a DAergic theory of depression being proposed over 30 year ago (Randrup et al, 1975). The involvement of DA in MDD seems somewhat intuitive, as a markedly diminished interest in previously enjoyed activities - anhedonia -, reduced motivation and decreased energy, all of which are affected by the DA reward pathway, are key symptoms of MDD. Many lines of evidence have demonstrated that: 1) decreased DA or DA metabolite levels; 2) increased D2/D3-receptor binding/sensitivity and, 3) decreased DA transporter (DAT) activity exists in depressed individuals, although there have also been neuroimaging studies showing no greater D2 receptor binding or decreased DAT activity (See Papakostas 2004 for review). Additionally, minor to severe depression often exists comorbidly in individuals with Parkinson's Disease (PD), with close to 50% of these individuals exhibiting anhedonia, a core symptom of depression. Treatment of these individuals with the D2/D3 agonist pramipexole significantly reduced both depression and anhedonia, indicating that it is likely a DA depletion that is inducing the depressed state of those afflicted by PD (Lemke et al, 2005). In rats, subjection to CMS induced a reversal of response to DA in the NAcc and PFC when exposed to aversive and 38 rewarding stimuli, thereby potentiating the stimulatory DA response to aversive stimuli and diminishing the response to rewarding stimuli (Di Chiara et al, 1999). Furthermore, it was found that DA-related neuroanatomical substrates are involved in the altered reward processing seen in depressed individuals (Tremblay et al, 2005). Interestingly, an increased sensitivity of D2 receptors in depressed individuals has been linked to resistance of SSRI treatment for depression, suggesting that the DA system is important in mediating the antidepressant response (Healy and McKeon, 2000). The evidence of DAergic involvement in MDD is nearly impossible to ignore, and there is a great deal of potential in creating new pharmaceuticals targeting this system for the treatment of affective disorders such as MDD. 4. Reciprocal Interactions Between the Monaminergic Systems Based on the evidence, it appears impossible to claim that the etiopathology of depression is due to alterations in only one of the monoaminergic systems, and likely that the 5-HT, NE and DA systems all play a role. 4.1. NE - DA Interactions DA neurons projecting to NE neurons of the LC produce an inhibitory effect, demonstrated by the suppressant activity of DA on the firing of LC neurons in anesthetized rats (Elam et al, 1986). Further evidence documented the reciprocal result, with the systemic injection of the D2 antagonist haloperidol causing an increase in the 39 firing rate of NE neurons (Piercey et al, 1994). On the contrary, projections from the LC produce an excitation of DA neurons. Single-pulse LC stimulations cause an excitation of DA neurons, an effect blocked by pretreatment with reserpine, and the ai-receptor antagonist prazosin (Grenhoff et al, 1993). Moreover, systemic administration of the NRI reboxetine induced an increase in burst-firing in DA neurons, indicative of a stimulatory action of NE on DA neuronal firing (Linner et al, 1991). However, contrary to this notion of the LC projections being able to enhance DA release, a recent study by Guiard et al (2008) found that lesioning the LC neurons with 6-OHDA actually enhanced DA firing, including firing rate, burst firing and spikes per burst. This suggests that the net effect of LC neurons on the VTA is in fact inhibitory. 4.2. 5-HT - NE Interactions Reciprocal innervations are known to exist between the noradrenergic nuclei of the LC and the serotonergic dorsal raphe nuclei. 5-HT neurons exert an inhibitory effect on the NE neurons of the LC, mediated by 5-HT2A receptors on GABAergic neurons (Haddjeri et al, 1997; Szabo and Blier 2001a; 2002). The LC neurons conversely have an excitatory effect on the 5-HT neurons, mediated by a direct pathway to ai-adrenoceptors on 5-HT neurons (Vandermaelen and Aghajanian, 1983). 40 4.3. 5-HT - DA Interactions Evidence suggests that DA neurons of the VTA send excitatory projections to the DRN 5-HT neurons. It was found that local application of the non-selective DA agonist apomorphine on 5-HT neurons, as well as a specific D2 receptor agonist, produced an increase in extracellular 5-HT levels, an effect blocked by the D2 antagonist raclopride but not by a specific Di antagonist (Ferre and Artigas, 1993). Furthermore, it was found that bath application of the D2/D3 agonist quinpirole induced a concentration-dependant membrane depolarization of 5-HT neurons in vitro, and effect that was blocked by the D2 antagonist haloperidol (Haj-Dhamane, 2001; Aman et al, 2007). In contrast to the excitation of the 5-HT system produced by the DA neurons, 5-HT projections effectively Figure 8: Functional interactions between monoaminergic neurons. (-) and (+) represent inhibitory and excitatory interactions, respectively, mediated via the various receptors located on and around VTA DA, LC NE and DRN 5-HT neurons 41 inhibit DA release. Systemic injection of the 5-HTiA agonist 8-OH-DPAT increases firing rate and burst activity of VTA cells, while this effect is not seen with iontophoretic application of 8-OH-DPAT in the VTA (Prisco et al, 1994). These observations indicate that the excitatory effect of 8-OH-DPAT is due to a decrease in 5-HT availability resulting from a suppression of firing of 5-HT neurons. Further investigations determined that constitutive activity of the 5-HT2C receptors produced tonic inhibition of striatal and accumbal DA release (De Deurwaerdere et al, 2004). In agreement with such reciprocal connectivity of this system, lesioning of 5-HT neurons was found to enhance the firing of VTA DA neurons, and lesioning of the VTA neurons produced a pronounced decrease in spontaneous 5-HT firing (Guiard et al, 2008). 4.4. Clinical Relevance of Interactions Clinically, the interactions seen above are relevant, as has been shown through depletion studies. Individuals in antidepressant-induced remission from depression being treated with serotonergic antidepressants (i.e. SSRIs) readily relapse in a tryptophan depletion challenge in which 5-HT is effectively decreased, but do not relapse in NE depletion, and vice versa for individuals being treated with noradrenergic antidepressants (Delgado and Moreno, 2000). Moreover, when individuals being treated with SNRIs undergo a full acute tryptophan depletion, they do not display the decreased mood seen with SSRI treatment, but on the contrary show an improvement in mood and other psychiatric symptoms (Booij et al, 2005). This result demonstrates that there is certainly 42 interplay between multiple systems, and altering one will ultimately cause an effect on the other, and quite possibly other, non-monoaminergic systems. As summarized in the above evidence, there is a significant amount of interplay within the monoaminergic systems. Research into the pathology of depression requires the investigation of a multitude of regions and systems. As such, elucidating the mechanism of action of catecholamine reuptake inhibition with drugs such as nomifensine is essential in understanding and treating various forms of MDD. 43 5. Nomifensine - A Catacholaminc Reuptake Inhibitor A) Figure 9: Chemical structure of A) nomifensine and B) sertraline. Although the two compounds share structural similarities, only nomifensine demonstrated potent reuptake inhibition of DA 5.1. Pharmacological Profile Nomifensine (l,2,3,4-tetrahydro-2-methyl-4-phenylisoquinolin-8-amine; trade name: Merital) is an isoquinoline antidepressant distinct from all other available antidepressants on the market. It has been shown to be a potent reuptake inhibitor of both NE (Schacht and Heptner, 1974) and DA (McKillop and Bradford, 1981; Church et al, 1987; Carboni et al, 1989), producing an increase in extracellular levels, while only minimally affecting their release, unlike substances such as amphetamine (Kd values in nM: 1010±30-SERT, 15.6±0.4-NET, 56±3-DAT; Tatsumi et al, 1997). Nomifensine is equipotent to desipramine in inhibiting NE reuptake, and over 100 times more potent than desipramine in regards to DA reuptake inhibition (Coccaro and Siever, 1985) making it a potent catecholaminergic reuptake inhibitor. Therefore, nomifensine is the only antidepressant ever used routinely in the clinic with a high potency to block DA reuptake, aside from amineptine. Only minimal acute effects on 5-HT accumulation were found in response to nomifensine, with levels comparable to desipramine (Schacht and Heptner, 44 1974). Interestingly, pharmalogical testing using the metabolites of nomifensine showed a very active Ml, which is equipotent to nomifensine in inhibiting DA, twice as active in inhibiting NA uptake, and displaying potent inhibition of 5-HT uptake, unlike nomifensine (Kruse et al, 1977). Thus, it is possible that nomifensine may be mislabeled as a dual reuptake-inhibitor, but is rather a triple reuptake-inhibitor, effectively acting on all three monoaminergic systems. 5.2. Animal and Human Studies Drug-naive rats were found to readily acquire and sustain self-administration of high doses of nomifensine (1 or 3 mg/kg), and showed a similar response for low doses (0.1 or 0.3 mg/kg), although the low-dose group gradually declined to levels similar to controls. All groups readily extinguished responses with water substitution, and restored the behavioural response upon resubstitution of nomifensine, indicating that it does in fact contain reinforcing properties (Telia et al, 1996). Furthermore, it was found that persistant use of nomifensine did not produce any upregulation of the DAT in striatal regions, unlike cocaine, demonstrating a differential regulation of various compounds which act on the DAT. Despite the reinforcing properties of nomifensine (NF) in animals, there is limited addiction potential in humans, seemingly being problematic only in individuals with "addictive personalities" on high doses of the drug (Rothman, 1990; Yakabowetal, 1996). Intravenous injection of NF in human volunteers caused an increased heart-rate, an increase in plasma levels of NE and its metabolite MHPG and powerfully stimulated 45 growth hormone release and inhibited prolactin release (Scheinin et al, 1987). However, these effects were not seen when NF was delivered orally over a two week period, initially making NF a model compound for a new class of antidepressants, devoid of many side effects seen with TCAs while maintaining a profound efficacy in the treatment of depression (Gillings et al, 1984). Unfortunately, despite the apparent efficacy of NF in treating even severely retarded depression, it was removed from the market due to cases of hemolytic anemia. To our knowledge, no studies have been done on the sustained administration of a catecholamine reuptake inhibitor as powerful as NF, and the effect on the monoaminergic systems. This is crucial in understanding the mechanism of action of a drug such as NF, which was very beneficial in the treatment of even severe depression with minimal side effects in the CNS. By elucidating such a mechanism, it will be beneficial in the creation of new antidepressant drugs which may induce its effect by a similar mechanism. This research endeavour is all the more relevant as several pharmaceutical firms are now developing triple-reuptake inhibitors, drugs that inhibit the reuptake of 5-FIT, NE and DA. 46 Materials and Methods Animals Adult male Sprague-Dawley rats (Charles River, Saint-Constant, QC, Canada) weighing 260-320 g at the time of the experiments were used. Animals were housed 2 per cage at standard experimental conditions (12h light/dark cycle, with lights on at 7:00 am; temperature 21°C ± 1, 40-50% relative humidity) with access to food and water ad libitum. All procedures were approved by the local Animal Care Committee and were in accordance with the guidelines set by the Canadian Council for Animal Care. Sustained Treatment Rats were anesthetized with an inhalant mixture of isofluorane and oxygen. Subcutaneously implanted osmotic minipumps (Alzet, Durect Corporation, Cupertino, CA) were preloaded with nomifensine (5 mg/kg/day), GBR 12909 (7.5 mg/kg/day), reboxetine (2.5 mg/kg/day) or 20% hydroxypropyl-beta-cyclodextrin (P-OH) which provided delivery of each drug for 2- or 14-days. Minipumps remained in situ throughout the recordings in order to minimize duress to the body of the subjects, as well as to ensure the steady-state levels of the drug were maintained, thereby mimicking clinical conditions. In-vivo Electrophysiological Recordings Single-unit, extracellular recordings of presumed monoaminergic neurons were performed in order to examine the firing rates of NE, DA and 5-HT neurons in the LC, 47 VTA and DRN, respectively. Recordings were taken using single-barreled glass micropipettes (Stoelting Co., Wood Dale, IL, USA) pulled on a Narashige pipette puller (Tokyo, Japan) and preloaded with fiberglass strands to promote filling with a 2 M NaCl solution. Pipette tips were broken to 1-3 urn, with impedances ranging from 2.5-5 MQ. Rats were anesthetized with chloral hydrate (400 mg/kg i.p.) and mounted on a stereotaxic frame. Additional anesthesia (50-100 mg/kg i.p.) was given as necessary to maintain a full anesthetic state, characterized by the absence of response to a nociceptive paw or tail pinch. Stereotaxically defined coordinates were used to locate the appropriate neural regions and neurons were identified by their spike shape, duration and frequency. Electrodes were lowered using a hydraulic micropositioner (Kopf Instruments), and all neuronal activity was recorded in real-time using Spike2 software (Cambridge Electronic Design, Cambridge, UK), which was also used to analyze neurons offline. Body temperature was maintained at 37°C throughout the experiments using a thermistor- controlled heating pad (Seabrook Medical Instruments, Saint-Hyacinthe, Quebec, Canada). Prior to recordings, a catheter was inserted into the lateral tail vein for systemic injections of any drugs required. For all dose-response curves, only one neuron was used from each animal. Recording ofLC NE Neurons The following coordinates were used in order to record from the LC (in mm from lambda): AP - 1.0 to - 1.2, L 1.0 to 1.3, V 5-7. NE neurons were identified by the following criteria: regular firing rate (1-5 Hz), a positive action-potential of long duration (0.8-1.2 ms), and a robust response to a nociceptive contralateral pinch of the hindpaw, 48 eliciting a burst of spikes followed by a brief quiescent period (Aghajanian and Vandermaelen, 1982a). Recording of VTA DA Neurons The following coordinates were used for micropipette descent into the VTA (in mm from lambda): AP + 3.2 ± 0.4, L 0.9-1.1, V 7-9. DA neurons were identified by the well-established in vivo properties of spontaneously firing neurons: biphasic spikes (often with an inflection or 'notch' in the rising phase) with a prominent negative phase, long spike duration (2.75 ± 0.75 ms), low-pitched sound and irregular spontaneous firing rate (2-9 Hz) often demonstrating bursts of 3-10 spikes with decreasing amplitude within the burst (Grace and Bunney, 1984a,b). A criterion of duration of > 1.1 ms from the beginning of the action potential to the negative trough was also used for identification of DA neurons (Ungless et al, 2004). Recording ofDRN 5-HT Neurons In order to record from 5-HT neurons in the DRN, a burr hole was drilled in order to descend the glass micropipette at the following coordinates (in mm from lambda): AP + 1.0 to + 1.2, L 0 ± 0.1, V 5-7. Presumed 5-HT neurons were identified by their characteristic slow, regular firing rate (0.5-2.5 Hz) and long duration action potential (2- 5ms) (Aghajanian and Vandermaelen, 1982b). 49 Firing Rate and Burst Analysis DA and NE Neurons The bursting activity of both DA and NE neurons were measured using interspike interval (ISI) analysis as previously described (Grace and Bunney, 1984b). Briefly, the onset of a 'burst' was defined as two spikes occurring < 80 ms apart, with the termination of the spike being defined as 2 bursts occurring > 160 ms apart. 5-HT Neurons The characteristics for a distinct population of bursting 5-HT neurons have been known to exist for some time (Hajos et al, 1995). As such, this was the criterion used to measure bursting activity in 5-HT neurons of the DRN. Briefly, the onset of a spike was considered to be an interspike interval (ISI) of < 12 ms, with the termination of the burst as an ISI > 24 ms, with spikes occurring as a doublet or triplet (two or three spikes, respectively) of decreasing amplitudes. Whole-Blood 5-HT Measurement In order to measure 5-HT reuptake, blood samples from control and 14-day treated animals were collected following recording, and frozen in tubes containing 7.5 mg/ml ascorbic acid. Whole blood 5-HT levels were measured by HPLC using a previously described method (Blier et al, 2006). In brief, 5-HT reuptake inhibition produces a depletion of whole-blood 5-HT levels, due to the blockade of the SERT located on blood platelet cells. As platelet cells lack the ability to synthesize 5-HT, any 50 5-HT present in these cells has been taken up the SERT, and provides an indirect indication of neuronal 5-HT reuptake activity. Drugs Nomifensine and the selective DA reuptake inhibitor GBR 12909 (Sigma-Aldrich Canada, Oakville, ON, Canada) were dissolved in a 20% (2 g / 10 ml dH^O) solution of hydroxypropyl-beta-cyclodextrin, 97% (P-OH), and sonicated until completely dissolved. The D2 receptor agonist apomorphine hydrochloride, the D2 receptor antagonist haloperidol, the a2-adrenoceptor antagonist idazoxan hydrochloride the NE reuptake inhibitor reboxetine mesylate hydrate, the 5-HT]A receptor agonist lysergic acid diethylamide (LSD) and the 5-HTu receptor antagonist WAY100635 (Sigma-Aldrich), were all dissolved in distilled H2O. Escitalopram, provided by Lundbeck (Lundbeck, Copenhagen, Denmark) was dissolved in 0.9% NaCl. Statistical A n alysis All data are reported as mean values ± SEM obtained from a single neuron for the following parameters: firing rate expressed in Hz (number of spikes/second), percentage of total spikes in 60 second intervals occurring as bursts (% of spikes as bursts), percentage of neurons displaying any burst activity (% neurons in burst mode), the number of spikes occurring per burst, and the number of cells found in each electrode descent (number cells/track). 51 For the construction of dose-response curves, the assessment of neuronal responsiveness was taken as the percent of baseline firing rate measured 60 seconds after the systemic administration of the drug. A two-way ANOVA was conducted for the firing rate of each monaminergic neuron population, with 'drug treatment' and 'duration of treatment' used as factors. The Bonferonni corrected post-hoc analysis was conducted when significant ANOVA results were obtained. A one-way ANOVA was conducted in order to determine significant differences in the burst activity and the number of cells per track. The drug treatment was used as the factor in this case, and separate AN OVA s were run for the 2-day and 14-day treatment groups. Again, the Bonferonni corrected post-hoc analysis was conducted when significant ANOVA results were obtained. All statistical analyses were completed using SigmaStat software (Systat, Chicago, IL, USA), aside from dose-response curves which were completed using GraphPad Prism 5.0 (GraphPad Software Inc, La Jolla, CA). 52 Results Acute Dose-Response Curves in Response to Nomifensine In order to assess the potency of nomifensine to inhibit NE and DA neurons in vivo, acute dose-response curves (i.v.) were constructed in the LC and VTA respectively. As previously reported, the in vitro receptor affinities for nomifensine on the NE, DA and 5-HT transporters are 15.6 ± 0.4 nM, 56 ± 3 nM and 1010 ± 30 nM, respectively (Tatsumi et al, 1997), indicating the high affinity for nomifensine at both the NE and DA transporter sites. Nomifensine inhibited the NE neurons in the LC with an ED5o of 40 ± 1 ug/kg, and a dose of 100 ug/kg required to produce a complete, lasting inhibition in all neurons (EDioo; Fig 10A, B). In the VTA, the ED5o was much greater at 450 ± 41 ug/kg, with an extremely high dose of 10 mg/kg required to induce a nearly complete inhibition (Fig 10C, D). The nomifensine-induced inhibition was reversed with 1 mg/kg of the 0.2- adrenoceptor antagonist idazoxan for NE neurons, and 200 jJ-g/kg of the D2 receptor antagonist haloperidol for DA neurons (Fig 10A, C). As expected, due to the low affinity of nomifensine for the 5-HT transporter, acute administration of nomifensine produced no significant change in the firing rate of DRN 5-HT neurons (see Fig 20B, 5 mg/kg, n = 5). Sustained Administration of Nomifensine, GBR 12909 and Reboxetine Upon completion of the acute dose-response curves, sustained treatment was examined in all three monoaminergic neuronal types using subcutaneously implanted osmotic minipumps for 2 or 14 days, delivering 5 mg/kg of nomifensine per day. Due to 53 1 mg/kg 1 mg/kg 1 mg/kg 1 mg/kg nomifensine idazoxan tdazoxan idazoxan B I CO log[nomifensine] 10 mg/kg 0.2 mg/kg D nomifensine haloperidol «) 45 c • o 100n 3 0> ! z O 75H 50- 0) ' 1 i , ' Ji ' i M ,i ' ' a '[ i 1,1, c V) ' \ 25H !i , ... ' S i i o — i c ~* •-,-•' % ,v IHW| I IIMWI * ' -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 log[nomifensine] Figure 10: Acute dose-response curves of i.v. administration of nomifensine in NE and DA neurons. A) Representative histogram of the firing rate of a NE neuron in the LC. Each bin represents the number of spikes / 10s. Arrows indicate the compounds administered and the time at which the injection of the specified doses was completed. B) Dose-response curve of NE neurons in the LC in response to nomifensine. C) Representative histogram of the firing rate of a DA neuron in the VTA. D) Dose-response curve of DA neurons in the VTA in response to i.v. administration of nomifensine. nomifensine being both a NE and DA reuptake inhibitor, the effects of sustained administration of GBR 12909 - a selective DA reuptake inhibitor - and reboxetine - a selective NE reuptake inhibitor - were also examined. As the effect of sustained administration of reboxetine had previously been studied in both the DRN and LC (Szabo and Blier, 2001b), only the response to the drug in the VTA was examined. The effect of sustained administration of GBR 12909 was examined in all three monoaminergic regions. 54 Ventral Tegmental Area A two-way ANOVA elicited significant main effects for both drug treatment (F(3,266) = 8.78, p < 0.001), as well as treatment duration (F(i,266) = 9.81, p < 0.01), while = also eliciting a significant drug X duration interaction (FQ,266) 4.36, p < 0.01). For the drug treatment, post-hoc analysis revealed significant differences between the control group and all three treatment groups (reboxetine and GBR vs controls: p < 0.001, nomifensine vs controls: P < 0.01), while none of the treatment groups significantly differed from one another. A robust decrease of 39 % was seen following the 2-day administration of nomifensine in the firing rate of DA neurons in the VTA relative to controls (p < 0.001). However, a complete recovery in the firing rate of these neurons to control levels was observed after 14-days (p > 0.05; Fig 11). Burst analysis elicited a 52 % decrease in the percentage of total spikes occurring as bursts in the 2-day treatment group relative to controls (p < 0.05), again recovering to control levels after the 2 week treatment (p > 0.05; Fig 12A). Although no significant difference was found in the percentage of cells exhibiting burst activity (burst mode), nor in the number of spikes elicited per burst (Fig 12B), a non-significant trend was apparent for both parameters as a decrease was seen in the 2-day treated group, with a recovery to near control levels after 14 days. No significant difference was found in the number of cells recorded per track (Fig 12C). The previously established effective dose of reboxetine of 2.5 mg/kg/day (Szabo and Blier, 2001b) was used in order to examine the effect of the drug on DA neurons in 55 5 • Controls a Nomifensi ne 5 rng/kg • GBR 7.5 mg/kg • Reboxetine 2.5 mg/kg • 33 30 33 32 ' 33 33 Controls 2-day Treated 14-day Treated Figure 11: Average firing rate of DA neurons in the VTA. A significant decrease in firing rate of DA neurons was found in the group treated with nomifensine for 2-days, with a complete recovery to control levels after 14-days. Both reboxetine and GBR 12909 produced a significant decrease in firing after 2-days which no longer remained significant at 14-days. n = number of neurons. ** p < 0.01, ***p< 0.001 the VTA. Following the 2-day treatment, a significant decrease of 31 % in the firing rate of these neurons was found relative to controls (p < 0.01; Fig 11). Following the 14-day treatment with reboxetine, the average firing rate of these neurons was decreased by 22 % relative to controls, thus no longer remaining significantly different from control levels (p > 0.05). A decrease of 39 % and 56 % was found in the percentage of spikes occurring as bursts in both 2 and 14-day treated animals, respectively, however only the 14-day treated group was significantly different from control levels (p < 0.05; Fig 12A). Although the one-way ANOVAs showed a significant (~ 40 %) decrease in the number of cells exhibiting burst activity between the 14-day treated group relative to controls (p < 0.05), this difference did not remain significant following post-hoc analysis. No 56 difference was found in the number of spikes per burst (Fig 12B), nor the number of cells found per track (Fig 12C) in any of the groups. A * 1 ; ,,,.;_ , : B 2-etey Tro=rt0rf 14Ht;iy Tt«*« Figure 12: A, B) Burst analysis of DA cells in the VTA. A) Percentage of total spikes occurring as bursts in 60s, n = neurons. (B) Number of spikes occurring per burst, n = bursting neurons. C) Number of spontaneously active cells found per track, n = tracks. Error bars; ± SEM, * p < 0.05, **p<0.01. K-tfay Trout « As the ED5o of GBR 12909 to inhibit DA neurons of the VTA had previously been found to be ~ 7.5 mg/kg (Einhorn et al, 1988), this was the first dose tested. The administration of GBR 12909 for 2-days produced a 26 % decrease in the firing rate of DA neurons in the VTA (p < 0.01) recovering to a decrease of only 22 % after 14-days, no longer being significantly different to control levels (p > 0.05; Fig 11). When coupled with the approximately 30 % decrease seen in firing following the 2-day treatment of reboxetine, it was determined that 7.5 mg/kg/day was an effective dose of GBR 12909 for sustained treatment, therefore this dose was used for all future experiments. A significant decrease of 66 % was also found in the number of the percentage of total spikes occurring as bursts after the 2-day treatment relative to controls (p < 0.01), which did not remain significant after treatment for 14-days (p > 0.05; Fig 12A). None of the other 57 burst parameters, nor the number of cells found per track were significantly different from control levels (Fig 12B, C). However, as previously mentioned the one-way ANOVA within the 14-day treated group produced a significant result in the number of neurons firing in burst mode, although this difference did not remain significant following post-hoc analysis. Assessment ofD2 Autoreceptor Sensitivity As a complete recovery in firing rate was observed following the 14-day treatment with nomifensine, the sensitivity of the D2 autoreceptor was tested in the VTA using the D2 agonist apomorphine. In control animals, a complete attenuation of DA neuronal firing was achieved with a dose of 30 ug/kg, with and ED50 of 12 ± 0.5 u-g/kg (Fig 13). Following the 14-day treatment with nomifensine, the ED50 increased to 21 ±3 |ug/kg, with administration of a more than three-fold increase in the ED100 of control animals to 100 ug/kg unreliably inducing a complete firing inhibition. In addition, the slopes of the dose-response curves were significantly different at 2.4 ± 0.4 versus 0.7 ± 0.1 for controls and the 14-day nomifensine group, respectively; F(]ji5) = 15.02, p = 0.001). This rightward shift of the dose-response curve indicated that a significant desensitization of the D2 autoreceptor occurred with sustained treatment of nomifensine. c 1001 Figure 13: Acute dose-response curve of DA 8 80< autoreceptor sensitivity through i.v. administration z < of the DA agonist apomorphine. Inhibition of" the a < 60 neurons by apomorphine was reversed with the D2 antagonist haloperidol. The rightward shift of the 40 A o dose-response curve clearly indicates a • Controls desensitization of the D2 autoreceptor has occurred. c 20 H • 14d Treated Controls: n = 11,14-day treated: n = 8 XL c 25 50 75 100 Apomorphine ((Jg/kg) 58 Locus Coeruleus A two-way ANOVA elicited a significant main effect of drug treatment (Fp, 269) = 85.32, p < 0.001) as well as a significant drug X duration interaction (Fp, 269) = 4.358, p < 0.01). Post-hoc analyses revealed significant differences between nomifensine and both the control group as well as GBR (p < 0.001), with no difference between GBR and controls. A significant decrease of 71 % in the firing rate of NE neurons in the LC was found following the 2-day treatment and remained decreased by 62 % after 14-days relative to controls (p < 0.001 for both groups; Fig 14). Accordingly, no recovery in the firing rate of NE LC neurons occurred following sustained treatment with 8 Controls UJ 1.6 ^Nomifensine 5 mg/kg V) E)GBR7.5rng/kg +1 N 14 in 2 1 3 HI Z 1 UJ z O 0.8 _l <*- l.\ Wi 0 jLs& »-£*$: *:;#? (U 0.6 §S?l\;i ?'. J'::-f.fej-'• jjfc IS 0 ft EjViji fcfcWfc^S !p D) 0.4 im 'jZ m 52 &.$ 46 "" 0.2 fefe #jMfe# Lil DJ_ tit Controls 2-day Treated 14-day Treated Figure 14: Average firing rate of NE neurons in the LC. Treatment with nomifensine potently inhibited firing of neurons relative to controls after both 2-day and 14-day treatment. GBR 12909 did not affect the firing rate of NE neurons after 2-day treatment, but produced a potent decrease in firing rate relative to controls after 14 days, n = neurons. ** p < 0.01, *** p < 0.001 59 nomifensine, contrary to what was observed in DA cells of the VTA. No significant differences were observed for any of the bursting parameters (Fig 15 A, B), while only 44 % of the number of cells per track was found in the 14-day treated group relative to controls (p< 0.05; Fig 15C). Following treatment with GBR 12909 for 2-days, no significant deviation from control levels for any of the measured parameters in NE neurons in the LC were observed. After 14-day treatment with GBR 12909, a 26 % decrease was seen in the firing rate of LC NE neurons relative to controls (p < 0.01; Fig 14). Despite this decrease, the firing rate of the LC NE neurons remained significantly different from the firing rate of nomifensine treated animals (p < 0.001) within both the 2- and 14-day groups (i.e. nomifensine X'2 days vs GBR X 2-days), as well as a significant difference within the GBR 12909 treated groups (p < 0.001). None of the burst parameters significantly differed from control levels (Fig 15A, B), while a 45 % decrease in the number of cells found per track was observed in the 14-day GBR 12909 treated group relative to controls (p < 0.05; Fig 15C). SB Controls ©Nomifensine 5 mg/kg • GBR 7.5 mg/kg ! 2.J-? 1 I ! 1MI , *i* fiHH&SI * - i^Hnl Ijjjj IM—n^^^H• WIrlffl i"" <* fir,-.; - 7- 3 mmmUmm * It* *k* 1 -u».A*u 2-day Treated Controls 2-tJay Treat ed 14~day Treated Figure 15: A, B) Burst analysis of NE cells in the LC. A) Percentage of total spikes occurring as bursts in 60s, n = neurons. (B) Number of spikes occurring per burst, n = bursting neurons. C) Number of spontaneously active cells found per track, n = tracks. All error bars; ± SEM, * p < 0.05 1* 60 Dorsal Raphe Nucleus A two-way ANOVA showed a significant main effect of drug treatment (F(2,44i) = 19.47, p < 0.001), as well as a significant drug X duration interaction (Fp, 449) = 6.715, p < 0.01). Post-hoc analyses revealed significant differences between nomifensine and both the control group as well as GBR (p < 0.001), with no difference found between GBR and controls. Contrary to the other regions examined, a significant increase in the firing rate of 5-HT neurons in the DRN by 50 % was observed following the 2-day nomifensine treatment (p < 0.001) and remained significantly elevated by 32 % after 14-days (p < 0.01) relative to controls (Fig 16). None of the burst analyses .~.~ -v 2-1 ~~~. ~ ...~~—„™_™ ,.„— ... __—„„ —-— •• - • Controls *** SE 1.B- ^ Nomifensine 5 mg/kg .1 .. 0GBR 7.5 mg/kg 8 J •" * ' " ' ** +1 1.6- ..-, r, J.• „.-,w; - -M„* N "•• X •w ' -.' J. .:• 1,4- ; * ? v...'.:«",{,:':*. •r. c '• ! v.V-s-l;.^ 0 : ! '*'-"' "~ *' * i * 1 -. i .... -.. •.'.;..*; I ••-••• ,.;" .i.' 5 1.2 ^^_M^^_ , * 1 • v .!'..• z JHVBHHVJ . • • . .-• . :%VA K- ** .. i • '-.'j:- * 1 - HBVBVBVJ . •'" * j 10 i HHVHVHV^^^^^|fJl fB * i -;•:.$:$. Q.fi- '. , g ' •'•': ' ^^^^H . . "»*. t r/.-'Svfr O 0.6- HHHHHHH^^^^HJ ' ', ; : ^.?;«''•'* < tu 1 i . ^'r.-- <*i ^^^^^^^^^^^^B .*» ':• Jt- (5 1 1 '- „."•'-& .J: * 04- •••••••J t-- ': '- -'•'*'.•'':. •£ ."*f •BVBVBVJ J U) H^H^H^H^HJ :• :> '-•'..."s * E •BVBVBVJ I1' • V; * u. • , " •»• "r, .-';* ... il 0.2- ^^^^^^H V: 70 <:m-i 0- .•••••••.... - - ... :", ..£ Controls 2-day Treated 14-day Treated Figure 16: Average firing rate of 5-HT neurons in the DRN. A significant increase in the firing rate of 5-HT neurons was found in the group treated with nomifensine for 2- and 14-days. GBR 12909 did not significantly alter firing rates., n = number of neurons. ** p < 0.01, *** p < 0.001 elicited any significant differences within the 2-day or 14-day treated groups (Fig 17A, B), although the increase in total burst activity nearly reached significant levels in the 2- 61 day treated group (p = 0.08). No significant difference was observed in the number of cells per track in any group (Fig 17C). Following treatment with GBR 12909 for 2-days, no significant deviation from control levels for any of the measured parameters in 5-HT neurons in the DRN occurred. Although a 23 % increase in the firing rate of DRN 5-HT neurons in the 14-day treated GBR 12909 group was observed, this increase did not quite reach significance relative to controls (p = 0.10), but did significantly differ from the firing rate of the 2-day GBR 12909 treated group (p < 0.001; Fig 16). No significant differences were detected for any of the burst parameters (Fig 17A, B) or the number of cells per track (Fig 17C) relative to controls. < B »• I a *.*>• i -* « 2.0s 1 «.', *it • 'i* 5 - 't*' *" * *JI '•'• J .' . * «i .* ,' i -1 I 3d «* » at I 2-rfsiy Treatod 14-day Treated Figure 17: A, B) Burst analysis of 5-HT cells in the DRN. A) Percentage of total spikes occurring as bursts in 60s, n = neurons. (B) Number of spikes occurring per burst, n = bursting neurons. C) Number of spontaneously i. M . active cells found per track, n = tracks. All error bars; ± SEM, * p < 0.05. Sfei - 2- 62 Assessment of 5-HTi A Autoreceptor Sensitivity As a significant increase in firing rate was observed following the 2-day treatment with nomifensine, the sensitivity of the 5-HTIA autoreceptor was tested in the DRN using the 5-HT autoreceptor agonist LSD. LSD is a reliable indicator of 5-HT]A autoreceptor sensitivity as it produces the same effect when applied systemically, or locally onto 5-HT neurons via iontophoresis, unlike other 5-HTiA receptor agonists (Blier and de Montigny, 1987) In control animals, a complete attenuation of 5-HT neuronal firing was achieved with a dose of 10 ug/kg, with and ED50 of 6 ± 0.2 ug/kg (Fig 18). Following the 2-day 100 Figure 18: Acute dose-response curve of 5-HTu autoreceptor sensitivity through i.v. 80 H administration of the 5-HT1A agonist LSD. The LSD-induced inhibition was reversed with the 5- z HTIA antagonist WAY100635. The rightward a a shift of the dose-response curve clearly indicates 40 a desensitization of the 5-HT1A autoreceptor has occurred. Due to steepness of the curve, the • Controls 20 confidence intervals for the slope of the control A 2d Treated curve were removed. Controls: n = 10, 2d treated: n = 8. 50 100 150 LSD fug/kg) treatment with nomifensine, the ED50 increased to 36 ± 2 ug/kg, while administration of an increased dose of 150 ug/kg unreliably induced a complete suppression in firing. In addition, the slopes of the dose-response curves were significantly different at 14.2 ± 2.7 versus 0.5 ± 0.1 for controls and the 14-day nomifensine group, respectively (F(i;i4) = 22.36, p = 0.001). This rightward shift of the dose-response curve coupled to the significant difference between the two slopes indicated that a significant desensitization of the 5-HTJA autoreceptor occurs after only 2 days of the nomifensine treatment. 63 Whole Blood 5-HTLevels Analysis of whole blood 5-HT levels indicated a significant increase in platelet levels in the 14-day treated group (1743 ± 113 ng/ml) relative to controls (1217 ± 67 ng/ml; p < 0.001). This indicates that rather than a decrease in 5-HT reuptake occurring, as would be expected if brain levels of the Ml metabolite reached effective levels in the brain, an increase in 5-HT reuptake is occurring in the brains of nomifensine treated rats, Measurement of Whole Blood 5-HT Levels . 1 . Figure 19: Measurement of whole blood 5-HT levels. [ A significant difference was observed between control animals and the 14-day nomifensine treated 1 group (p < 0.01). This was the reverse result than what was expected, as a decrease in platelet levels of V- • J^v. A • * - j-^aew ; 5-1 IT would have been observed in the 14-day , ' , •3|'*£i; treated group if the metabolite of nomifensine did [ ' '£?- ^i 3 indeed reach effective levels in the brain. :'.":;.tt'f9; j n= II . 14-day NF Treated Catecholaminergic Regulation of 5-HT Neuronal Firing Rate in the DRN In order to determine the effect of single- or dual-catecholaminergic reuptake inhibition on the escitalopram induced inhibition of DRN 5-HT neuronal firing rates, dose-response curves were created in response to escitalopram in rats pretreated (i.v.) with the NE reuptake inhibitor reboxetine, the DA reuptake inhibitor GBR 12909, or the NE/DA reuptake inhibitor nomifensine. A two-way ANOVA for the percentage of baseline firing rate of DRN 5-HT neurons indicated an overall significant inhibitory effect of pretreatment (F^^s) = 16.4, p < 0.001) and dose of escitalopram (F(2,28) = 10.5, p < 0.001) factors, while no significant interaction between the independent variables was detected (F(6,28) = 0.4, p > 0.05). 64 At each dose tested, the inhibitory effects of escitalopram on DRN 5-HT basal firing rates were not significantly different from those obtained in rats pretreated with the selective DA reuptake inhibitor GBR12909, or the selective NE reuptake inhibitor reboxetine relative to vehicle (Fig 20A). However, in rats administered with the dual DA/NE reuptake inhibitor nomifensine, a significant attenuation of the effect of escitalopram was detected (Fig 5B and C). Alone, none of the reuptake inhibitors tested modified the spontaneous firing activity of DRN 5-HT neurons (1.3 ± 0.4 Hz vs 1.2 ± 0.4; p > 0.05; before and after the administration of GBR12909, respectively; 1.5 ± 0.3 Hz vs 1.3 ± 0.2; p > 0.05; before and after the administration of reboxetine, respectively; 1.5 ± 0.2 Hz vs 1.7 ± 0.3; p > 0.05; before and after the administration of nomifensine, respectively). These results put into evidence that an elevation of both catecholamines in the DRN was a prerequisite to counterbalance the inhibitory action of the SSRI escitalopram. WAYM0835 DO •loo am Mrts; f* 000 t»S*|j; iv) 1 "*^•^-v! ^ **> w i TS - s>Nv IS ip " Jk. "^V, ft- * » I *"*< '& *j HHHHHHBHHHK~ .wittn ,, imlHIB SO - T"N 2 x ©ftGiUiopr&m f^-~^ J*A * UGrt>iffmsin© 100 209 300 400 25- • v B 1 \.,^"-^.-.._.K*- ~--0." $*>-l 0 • ! . . 1 -\,r_^ "*> Cumulative dose of escftatopram Figure 20: Effect of single- or dual-acting catecholaminergic reuptake inhibitors on escitalopram- induced decrease in DRN 5-HT neuronal activity. A, B: Examples of integrated firing histograms showing the effects of cumulative intravenous doses of escitalopram on the spontaneous activity of DRN 5-HT neurons in presence of vehicle (A), or of the DA/NE reuptake inhibitor nomifensine (5 mg/kg; iv) (B). The arrows indicate the compounds administered and the time at which the injection of the specified doses was completed with the numbers representing the total cumulative dose at that point. The escitalopram-induced inhibition of firing rate was reversed with the 5-HTu antagonist WAY100635. C) Symbols represent the mean (± SEM) of percent of baseline firing rate of DRN 5- HT neurons observed at each dose of escitalopram after the administration of vehicle (-O-; n=9); GBR 12909 (-•-; n=5), reboxetine (-A-; n=5) and nomifensine (-•-; n=5). These means were calculated on the 60 second-period preceding each drug administration. ***p<0.001. 65 Discussion The results of the present study showed that sustained administration of the NE and DA reuptake inhibitor nomifensine induced robust alterations in the firing properties of all three types of monoaminergic neurons. Acute, i.v. administration of nomifensine produced a robust inhibition in the firing rate of both NE and DA neurons of the LC and VTA, respectively. This suggests that nomifensine acts directly on both the NET and DAT in vivo, corresponding to previous data demonstrating that nomifensine has a high affinity for both of these transporters in vitro (Tatsumi et al, 1997). Additionally, the lack of any acute effect of nomifensine in vivo was observed on 5-HT neurons of the DRN, again correlating to the relative low affinity of nomifensine in vitro for the SERT, indicating that this drug does not directly affect these neurons. Nomifensine markedly decreased the firing rate of NE neurons in the LC after 2- days, with no recovery in the firing rate of these neurons after 14 days similar to previous results found with the NRI reboxetine (Szabo and Blier, 2001b). The absence of a recovery in the firing rate of these neurons is likely mediated by a lack of desensitization of somatodendritic (X2-adrenoceptors, which are known to inhibit NE neuronal firing (Svensson et al, 1975; Mateo et al, 1998; Szabo et al, 2000). Furthermore, nomifensine significantly decreased the number of spontaneously active NE cells found in the LC, although it had little effect on the burst-firing of these neurons, which when increased is known to correlate to an increase in neurotransmitter release (Florin-Lechner et al, 1996). The lack of recovery in the firing rate of these NE neurons is dissimilar to another antidepressant, bupropion, which also has a dual mechanism affecting both NE and DA. 66 Current experiments in our lab demonstrate that bupropion attenuates NE neuronal firing in the LC after 2 days, similar to other NRIs such as desipramine and reboxetine (Szabo et al, 2000; Szabo and Blier, 2001b), although unlike what is seen with these drugs, the firing rate of these neurons recover to control levels after 14-day treatment (El Mansari et al, in press). This may in fact be due to the differing mechanisms of action of the bupropion relative to nomifensine, as bupropion has been postulated to be a NE releaser rather than a reuptake inhibitor (Dong and Blier, 2001) with the increase in extracellular levels being action-potential-dependent as it is blocked by TTX (Nomikos et al, 1989). In support of this probability, the SSRI/5-HT2A antagonist YM-992, which increases NE release (Hatanaka et al, 1997), produces like bupropion an initial decrease of NE firing that is followed by a full recovery due to 012-adrenergic autoreceptor desensitization. Similar to NE neurons, the firing rate of DA neurons in the VTA elicited a very robust decrease with the short-term treatment, however, unlike the NE neurons there was also a significant decrease in the percentage of the total burst activity. As well, contrary to the NE neurons both the firing rate and burst activity of the DAergic neurons completely recovered to control levels following the 14-day treatment. Through assessing the sensitivity of the somatodendritic D2R, the primary DA autoreceptor, it was found that the recovery of the firing rate of these neurons is due to the desensitization of this receptor. This is similar to what was previously described by Pitts et al (1995) after 14-day repeated i.p. administration of the D2-like receptor agonist quinpirole, and confirmed by autoradiographic study which found a decreased D2 receptor density following 14-day quinpirole administration (Subramaniam et al, 1992). 67 One of the primary concerns of using a drug that acts on the DA system is the potential for such compounds to have addictive properties, leading to adverse effects and negative symptoms upon discontinuation of the medication. Initial investigations found that nomifensine possessed psychostimulant-like effects, as there was a significant increase in intracranial self-stimulation (Katz et al, 1977) as well as intravenous self- administration (Spyraki and Fibiger, 1981) in rats, suggesting a potential for nomifensine abuse in humans. Additionally, Telia et al (1996) found that rats readily acquire and sustain self administration of nomifensine at both high and low doses, providing further evidence of the addiction potential that nomifensine possesses. However, what is interesting to note is that nomifensine, unlike cocaine, does not upregulate the DAT in striatal regions, indicating heterogeneity exists in the mechanism of action of drugs which act on the DA system. As nomifensine was found to have very low addiction potential in humans, aside from limited cases of people with "addictive problems" on high doses (400-600 mg) of the drug (Boning and Fuchs, 1986; Rothamn, 1990), the lack of DAT upregulation may help explain why this may be the case. Additionally, nomifensine has a relatively short elimination half-life of about 4 hours, regardless of the route of administration, with this half-life being shortened by nearly 50 % after 2-weeks of oral administration (Lindberg et al, 1986). This suggests the induction of metabolizing enzymes for nomifensine, which may further contribute to the lack of addiction seen with nomifensine in humans. The fact that nomifensine acts on the DAT without the reinforcing properties seen with cocaine, which also acts on the DAT, suggests that this class of DAergic drugs may not only be useful in the treatment of affective disorders, but may be beneficial as a treatment for cocaine addiction (Rothman, 1990). 68 Nomifensine induced a significant increase in the firing rate of 5-HT neurons after 2 days, which remained significantly elevated after the 14-day treatment contrary to the catecholaminergic neurons. It is well documented that increases in synaptically available NE and DA cause an excitation of DRN 5-HT neurons via a\-adrenoceptors (Vandermaelen and Aghajanian, 1983) and fVreceptors (Ferre and Artigas, 1993; Haj- Dhamane, 2001; Aman et al, 2007), respectively. Although it has been previously shown that acute administration of the NRI reboxetine increases 5-HT firing (Linner et al, 2004), sustained treatment with the reboxetine does not in fact alter 5-HT neuronal firing rates (Szabo and Blier, 2001b). Regardless, the robust increase in 5-HT firing following nomifensine treatment indicates that the DAergic alterations likely play a major role in increasing 5-HT neuronal firing. Additionally, there is a desensitization of the 5-HT|A autoreceptors on DRN 5-HT neurons after only the 2-day treatment with nomifensine as observed by the blunted response of 5-HT neurons to systemic application of LSD similar to what was found with sustained administration of bupropion (El Mansari et al, in press). This desensitization generally occurs after sustained SSRI administration, but only after a much longer period of 14-days, which has been correlated to the delayed onset of these drugs. Again, this may occur in such a short timeframe with nomifensine due to the substantial excitation that 5-HT neurons receive via the activation of the ai-adrenergic and D2 heteroreceptors. Furthermore, activation of the 69 Contrary to what is seen with SSRI administration, following treatment with nomifensine for 14 days, a significant increase in whole blood 5-HT levels was observed, suggesting that there is no inhibition of 5-HT reuptake occurring in the brain. Therefore, despite the fact that the Ml metabolite of nomifensine is a potent 5-HT reuptake inhibitor, this metabolic compound is unlikely to reach levels within the brain to induce any such effect. Furthermore, there is a significant increase in 5-HT neuronal firing and after only 2-days, whereas a decrease would have been expected in the presence of 5-HT reuptake inhibition. The robust increase in 5-HT neuronal firing following 2-day nomifensine treatment again suggests a powerful synergistic effect of the dual action of this drug since GBR 12909 produced no such effect. The increase in synaptically available NE and DA simultaneously induces robust alterations within 5-HT neurons, which is not induced by either single reuptake inhibitor alone, and certainly not in 2 days as seen with nomifensine. It is possible that the concurrent activation of both ai-receptors and D2- receptors on 5-HT neurons leads to a much more pronounced excitation of these neurons. To further explore the mechanism of action of DA and NE in the DRN the effects of single- or dual-acting DA/NE reuptake inhibitors such as GBR12909, reboxetine or nomifensine were examined on SSRI (escitalopram)-induced inhibition of DRN 5-HT neuronal activity. It was reported that neither pre-treatment with the NRI reboxetine nor pre-treatment with the DRI GBR12909 at doses previously shown to elevate extracellular NE (Page and Lucki, 2002) or DA levels (Baumann et al., 1994), attenuated the escitalopram-induced decrease in DRN 5-HT firing rates. However, when NE and DA levels were simultaneously increased by systemic administration of the dual-acting 70 reuptake inhibitor nomifensine, an upward shift of the dose-response curve of escitalopram was observed demonstrating that both catecholamines were required to counteract the inhibitory electrophysiological effects of escitalopram. These results suggested that dual reuptake inhibitors such as nomifensine enhanced brain NE and DA transmission which in turn stimulated 5-HT neurons. Since the DRN receives noradrenergic and DAergic innervation and expresses both the NET (Kung et al 2004 ; Tejani-Butt 1992; Javitch et al 1985 ; Biegon and Rainbow 1983), and DAT (Fujita et al, 1994), it can be anticipated that the acute systemic administration of nomifensine does effectively increase catecholamine levels around 5-HT neuron cell bodies which would potentiate their neurochemical effects at nerve terminals. Consistent with this hypothesis, microdialysis data have recently shown that the increase in cortical 5-HT levels induced by citalopram is enhanced by the DA/NE reuptake inhibitor methylphenidate (Weikop et al, 2007a,b). Following the 2-day GBR 12909 treatment, a significant decrease was observed in both the firing rate and total burst activity of VTA DA neurons, as was expected due to the high affinity of GBR 12909 to block the DAT. Neither of these parameters remained significantly different from control levels after 14 days similar to nomifensine. No significant deviations from control levels for any of the measured parameters in LC NE neurons or DRN 5-HT neurons were observed in GBR 12909 2-day treated animals. This indicates that in the short term, the primary site of action of GBR 12909 is solely in the DA neurons of the VTA, and produces no effect in either of the other two monoaminergic systems. After the 14-day treatment with GBR 12909, a significant decrease was observed in the firing rate of LC NE neurons, as well as a trend towards an 71 increase in the firing rate of the DRN 5-HT neurons. This was not surprising as DA is known to both induce an inhibition and excitation of neuronal firing via D2 receptors located on the cell-body of LC NE and DRN 5-HT neurons, respectively. This again demonstrates that the dual action of nomifensine rapidly induces alterations directly within the DAergic system and indirectly in the serotonergic system. An interesting result of this study was the decrease in DAergic firing following the 2-day treatment with the NE reuptake inhibitor reboxetine. In a recent study by Guiard et al (2008), it was found that selective lesioning of LC NE neurons effectively increased the firing rate of VTA DA neurons by 70 %, indicating that the noradrenergic projections from the LC to the VTA have a net inhibitory effect. Despite this, in vitro application of NE has been found to be excitatory on midbrain DA neurons inducing a membrane depolarization via ai-adrenoceptors, an effect that was blocked by the ai- adrenoceptor antagonist prazosin (Grenhoff et al, 1995). As well, electrical stimulation of the LC induced a period of excitation in DA neurons followed by a period of inhibition. Acute systemic injection of reboxetine was also reported to induce an increase in burst activity of VTA DA neurons in vivo, while having no net effect on the mean firing rate (Linner et al, 2001). Nevertheless, repeated studies have demonstrated that local application of NE on VTA DA neurons generally induces an inhibition of the firing rate of these neurons (Aghajanian and Bunney, 1977; White and Wang, 1984), including recent data from Guiard et al (unpublished) in which this inhibition is significantly attenuated by both the a2-adrenoceptor antagonist idazoxan and the D2R antagonist raclopride. Based on this evidence, it is not surprising to have observed a decrease in the 72 firing rate of VTA DA neurons following sustained reboxetine treatment via (X2- adrenoceptors present on these neurons. Nomifensine was one of the first non-tricyclic antidepressant drugs to selectively inhibit the reuptake of multiple neurotransmitters, proving to not only be effective in allev/ating depressive symptoms, but also possessing a side effect profile far superior to TCAs such as imipramine, as well as other often prescribed antidepressants (Meredith et al, 1984; Yakabow et al, 1984). The relative lack of side-effects was likely due to the fact that nomifensine was less pharmacologically "dirty" than majority of the TCAs, acting directly on the NET and DAT with little affinity for adrenergic, cholinergic or histamine receptors (Kinney, 1985). Despite the newer reuptake inhibitors, such as SSRIs, NRIs and SNRIs, being effective in the treatment of depression, these drugs are still not ideal due to the their delayed onset of action (> 3 weeks in most double-blind, placebo-controlled trials; Nemeroff and Owens, 2002), the fact that only about 65 % of patients respond to antidepressant therapy, with about 15 % resistant to all forms of known therapy (reviewed in Richelson, 2001) and the fact that they still have negative side-effects, such as sexual dysfunction characteristic of "serotonin based" antidepressants (Skolnick and Basile, 2007). Taking into account the contributory role DA plays in the pathophysiology of depression and the relative lack of antidepressant drugs which act on the DA system, current development of antidepressant medications is focusing on the creation of triple reuptake inhibitors (TRI); drugs which act on the SERT, NET and DAT, effectively inhibiting reuptake of all three monoamines. The rationale for developing such drugs is highlighted by a recent STAR*D trial which demonstrated that adding 73 buproprion, a drug known to affect both DA and NE, to the treatment of individuals failing to reach remission on citalopram increased remission rates by ~ 30% (Rush et al, 2006). Preclinical behavioural studies have found a number of novel TRI compounds to be effective in reducing immobility times in both the forced swim test and tail-suspension test, which are predictive of these compounds possessing antidepressant action (Skolnick et al, 2003; Shaw et al, 2007). As this class of drugs is quite new, very little clinical data is available regarding their efficacy in humans. However, it has been shown that depression rating scores of individuals being treated with the TRI DOV 216,303 significantly improve following a 2-week treatment (reviewed in Skolnick and Basile, 2007). This is unfortunately limited by a number of factors, including limited sample size, short-term period of patient response and importantly the lack of a placebo-group to ensure that the effect seen was not in fact simply an artifact due to artificially mimicking true clinical conditions. Despite this class of drug requiring a great deal more research pertaining to their efficacy in clinical settings, TRIs present a novel approach to the treatment of depression and may in fact become the treatment of choice in the near future. 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