published_or_final_version / Zoology / Master / Master of Philosophy

This dissertation is composed of five major chapters. The first is a comprehensiveliterature review, followed by three chapters of research findings, and a final concludingchapter. Major Depressive Disorder (MDD) is a phenotypically complex andheterogeneous syndrome. This challenge and others faced when investigating the geneticbasis for the susceptibility to MDD are discussed, as are tools used to address andovercome these challenges. Included in this review of the literature is a discussion onfindings of genome-wide analyses of MDD, as well as candidate genes that may play arole in the susceptibility to major depression. Following the literature review, threechapters of studies are presented. The first one demonstrates that in humans, the actualnumber of risk genotypes in the serotonin system accounts for over half of the variance inmood response to tryptophan depletion. There was no association between the dopaminesystem and mood response. The main conclusion from that study is that using a pathwayanalysis, rather than a single gene approach, may lead to more informative results whenstudying the genetics of a complex behavior. The next study demonstrates a similarconclusion, however, is not pathway specific. It is shown that in a group of de pressedsubjects not capable of treatment response, the mean number of risk genotypes is greaterthan in a group without depression. This supports the thought that treatment resistancemay be a more severe form of MDD. This study also presents data on single gene resultswhich demonstrate that the genetic basis for susceptibility to major depression may bedifferent and independent from the genetic basis for the capacity to respond to treatment.Several individual polymorphisms are implicated in each case. The final investigation is apublished manuscript refuting the findings of a previously published article onpolymorphisms in the TPH2 gene and association with treatment resistance. Many otherresearch groups have also been able to replicate the results demonstrated here. A finalchapter discusses the overall conclusions about the three research studies, as well as thefield of psychiatric genetics with a focus on the continuing search for the genetic basis ofsusceptibility to major depressive disorder.

A better understanding of the neural mechanisms associated with Attention Deficit Hyperactivity Disorder (ADHD) and related cognitive deficits can potentially clarify the neural circuits involved in ADHD symptoms, help define neurobiologically informed subtypes and aid in developing more refined treatments. Two neurotransmitter (NT) systems have been implicated in ADHD: Dopamine (DA), and Norepinephrine (NE), and the primary cognitive deficits associated with ADHD are in working memory, response inhibition, reaction time variability, and reward processing. Frank et al. (2007a) proposes, based on computational models, that DA is associated with deficits in reward-based learning and updating of working memory, while NE is associated with deficits in response inhibition and greater response variability. Therefore, it might be possible to learn more about the NT systems' specific roles in ADHD by studying the associated cognitive deficits. The primary goal of this study was to assess performance in adolescents with and without ADHD on a number of cognitive tasks. We expected that the Attention Deficit Hyperactivity Disorder - Inattentive Subtype (ADHD-I) group would perform the worst on NE tasks and that the Attention Deficit Hyperactivity Disorder - Combined Subtype (ADHD-C) group would perform the worst on DA tasks, and that both groups would perform worse than controls on all tasks. Instead, we found that the ADHD-I group performed the most poorly on updating of working memory, while the ADHD-C group performed the best on this variable. However, the ADHD-C group performed worst on overall working memory. Dimensional analyses revealed that hyperactivity/impulsivity is positively correlated with updating of working memory, while inattention is negatively correlated with updating of working memory. In addition, hyperactivity/impulsivity was positively correlated with reaction time variability. In conclusion, it is likely that the roles of these NT systems are not as mutually exclusive as initially expected. It is also possible that our ADHD group was performing more like control groups in other studies, which might be due to a more 'pure' ADHD sample with less comorbid Oppositional Defiant Disorder (ODD) and Conduct disorder (CD), or could be due to a less symptomatic ADHD group.

Environmental stimuli associated with drug use are related to drug craving and relapse. The mechanism of cue-induced craving is thought to involve the release of dopamine (DA) in brain regions associated with reward and habit formation. The aim of the study was to investigate the role of DA in cue-induced craving in tobacco-dependent smokers using Positron Emission Tomography (PET) and a picture cue paradigm. Tobacco-associated cues were capable of eliciting significantly greater subjective reports of craving relative to neutral cues in tobacco smokers (n=6) in a neuroimaging environment. Using this cue paradigm and [11C]-(+)-PHNO PET (n=6), a non-significant trend towards a greater decrease in binding potential, indicative of dopamine release, was shown in selected brain regions of interest. These findings are similar to findings in cocaine-dependent individuals and suggest the involvement of dopamine in the response to smoking-associated cues in tobacco-dependent individuals.

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Dysregulation of dopaminergic homeostasis has been established as the primary source of numerous neurological disorders including Parkinson’s and drug addiction. A tonic increase of dopamine (DA) in the nucleus accumbens is required for associating everyday events and behaviors with rewards. Yet many addictive exogenous compounds such as amphetamine (AMPH) and cocaine (COC) produce a much greater augmentation of synaptic DA levels that are linked to euphoria and a shift in behavior towards drug seeking. The protein responsible for maintaining extracellular levels of DA is the dopamine transporter (DAT). It is primarily located in the perisynaptic area at terminals of pre-synaptic neurons where its main function is to sequester DA from the extracellular space and to transport it back into the cell, a process that is electrogenic. AMPH and COC directly interact with DAT and alter its ionic currents. Not much is known about the effect of psychostimulant-induced DAT currents on neuronal excitability and neurotransmitter release. We use synthetic chemistry, molecular biology, and biophysics in heterologous expression systems to decipher the actions of drugs of abuse on DAT. Furthermore we demonstrate drug-induced DAT currents can activate Ca2+ channels associated with dopaminergic excitability. Lastly, we focused on investigating drug effects on excitability in a human midbrain dopaminergic cell line. Understanding how psychostimulants interact with DAT to produce the dysfunctional states of the transporter may facilitate the development of unique therapeutic strategies to treat psychostimulant dependence.

The high affinity, selective dopamine transporter ligand GBR 12909 has served as a template for the design of two novel classes of dopamine transporter ligands. A series of 3-[2- (diarylmethoxyethyidenyl)]-N-substituted tropane derivatives were synthesized and the binding affinities of these compounds were determined at the dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in rat brain tissue preparations. The tropane derivatives were found to exhibit more potent affinity and selectivity for DAT than GBR 12909. From the SAR of the tropane analogues and GBR 12909, a novel series of 3-[2-(diarylmethoxyethylidenyl)]-Nsubstituted azetidine derivatives has been developed.

In an effort to search for potential therapeutic agents for cocaine addiction, a novel class of compounds was synthesized and evaluated for in vitro dopamine and serotonin transporter affinities. These unique 3ƒÀ-aryl-3ƒ¿-arylmethoxytropane analogues incorporated the structure of dopamine selective 2-substituted-3-phenyltropanes and the design of serotonin selective meperidine derivatives. In general, the 3ƒÀ-aryl-3ƒ¿-arylmethoxytropane analogues exhibited greater potency for the serotonin transporter than the dopamine transporter. The most potent compounds of this series were 3ƒÀ-phenyl-3ƒ¿.(3, 4-dichlorophenyl)methoxy-8.azabicyclo [3.2.1]nortropane (Ki = 0.06 nM) and 3ƒÀ-(4Œ-chlorophenyl)-3ƒ¿.(4-chlorophenyl)methoxy-8. azabicyclo[3.2.1]nortropane (Ki = 0.09 nM) at the serotonin transporter and their binding affinities were equipotent with paroxetine and fluoxetine (Prozac). A series of 8-azabicyclo[3.2.1]oct-2-ene derivatives were synthesized from 3-tropinone based on the structure of triple re-uptake inhibitor, DOV 216, 303. The compounds were designed as potential triple re-uptake inhibitors which could exhibit equipotent affinities at the monoamine transporters for dopamine, serotonin and norepinephrine. A short and efficient synthetic methodology was developed for the synthesis of unique compounds which could exhibit potency for both the dopamine and serotonin transporters. The 3ƒÀ-aryl-3ƒ¿-(4Œ, 4-disubstituteddiphenylmethoxy)tropane analogues were designed as hybrid structures of the dopamine transporter selective benztropines and the serotonin transporter selective meperidine derivatives.

A placebo effect is a real and beneficial psychobiological phenomenon following the administration of a substance or procedure that has no inherent power to produce an effect. Nocebo effects, on the other hand are genuine and detrimental psychobiological phenomenon following the administration of and inert substance or procedure. These effects have been extensively studied but are not well understood. Central to the development of a placebo effect is the anticipation of benefit or the anticipation of harm. Indeed, expectancy and conditioning are thought to be the two primary mechanisms involved in the acquisition of the placebo effect. The neurotransmitter Dopamine (DA) is integral to expectancy and reward and as such has recently been considered a key player in the mechanisms of the placebo effect. Based on this line of inquiry this study sought to investigate the role DA might have in the development of the placebo effect as observed in pain using an animal (mouse) model. It was proposed that DA is involved in the acquisition and maintenance of the placebo effect. Specifically it was proposed that the DA agonist cocaine would enhance the magnitude and duration of the placebo analgesia and that the DA antagonists SCH23390 and eticlopride would together or separately block the acquisition of the placebo analgesia. These proposals were assessed by utilizing supra-spinal (hotplate) and spinal (tail flick latency) protocols. Results indicated that cocaine enhanced placebo analgesia in spinal but not supra-spinal measures and that the DA antagonists SCH23390 and eticlopride each contributed to the acquisition, rather than the blockade, of placebo analgesia in both spinal and supra-spinal models. In fact, the most profound effect was observed when both antagonists were administered together rather than separately on supra-spinal measures but not spinal measures resulting in an enduring nocebo effect contradicting all predictions. The novel results presented in this study raises more questions than they answer, warranting more detailed exploration of the mechanisms of DA and its relationship with placebo effects.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The overall goal of this dissertation was to determine the role of the subthalamic nucleus (STN) in regulating the release of dopamine in the substantia nigra (SN). Experiments first established the existence of a direct connection between subthalamic neurons and SN dopaminergic cells. Further experiments showed that this connection triggers the dopamine release in the SN, and the mechanisms involved in this release were determined. Whole-cell current clamp recordings were performed in parasagittal brain slices obtained from 10 to 16 day-old rat pups. Electrical stimulations of the STN reliably triggered excitatory post-synaptic potentials (EPSPs) in dopaminergic neurons of the SN pars compacta (SNc). Pharmacological experiments with specific receptor antagonists indicated that this EPSP was mediated by NMDA, non-NMDA and metabotropic glutamate receptors. Stimulations of the subthalamic input triggered the release of dopamine. In a subset of neurons in the SN pars reticulata (SNr), repetitive stimulations of the STN produced a summating EPSP that was followed by an inhibitory postsynaptic potential (IPSP). A D2 receptor antagonist blocked this IPSP suggesting that it represents the D2 receptor-mediated response of the recorded cell to dopamine released upon stimulation of the STN. Pharmacological experiments using this assay indicated that NMDA, non-NMDA or metabotropic glutamate receptors were individually not required for dendritic release of dopamine; however, each contributed to this release. In dopaminergic neurons located in the SN pars compacta, the inhibitory effect of dopamine was revealed following block of L-type Ca channels, NMDA and non-NMDA glutamate receptors. These results indicated that dopaminergic neurons located both in the SNc and SNr respond to the dendritic release of dopamine triggered upon stimulations of the STN. Finally, a specific blocker of the dopamine transporter (GBR12935) blocked the IPSP reversibly in both SNr and SNc dopaminergic neurons. If release occurred by exocytosis, block of the transporter should increase extracellular levels of dopamine and produce an increase in the size of the recorded IPSP. Therefore, these results suggest that dopamine dendritic release triggered by activation of the subthalamic input was mediated by reversed transport of dopamine rather than by exocytosis. / 2031-01-01