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

Este trabalho tem como objetivo principal a utilização da técnica “bottom-up" para a construção de nanoestruturas de ouro por meio de técnicas voltamétricas sobre uma superfície de Carbono Vítreo. Estas estruturas foram caracterizadas por métodos microscópicos e voltamétricos. A dimensão crítica das nanoestruturas foi obtida por meio de uma curva Gausseana de distribuição de alturas e diâmetros, fornecendo um valor médio de 35 nm por 150 nm de diâmetro. As nanoestruturas obtidas foram modificadas pela deposição de uma camada automontada de cistamina (um tiól), resultando num substrato adequado para futuras aplicações como a imobilização de enzimas. Os comportamentos eletroquímicos da dopamina e do ácido ascórbico, sobre eletrodos de ouro, Carbono Vítreo e Nanoestruturado, foram avaliados para a caracterização das modificações dos eletrodos. A diferença dos potenciais de pico de oxidação do AA sobre eletrodos de Carbono Vítreo e de ouro, de cerca de 300 mV, possibilitou avaliar o recobrimento do Carbono Vítreo com nanoestruturas pela diminuição da corrente de pico. Já a dopamina foi utilizada neste trabalho para determinar a presença da camada automontada de cistamina sobre a superfície das nanoestruturas de ouro, uma vez que sua resposta voltamétrica mostra um deslocamento do potencial de pico quando realizada sobre Au e sobre Au modificado com a camada automontada. / The subject of this work is focused in the utilization of a “bottom-up" approach to develop an Au nanostructured electrodeposits on the glassy carbon surface by a voltammetric procedure. These Au nanostructures have been characterized either by microscopic and voltammetric techniques. The critical dimensions of nanostructures has been evaluated by a Gausean normal data distribution curve and presented, as results a mean height of 35 nm and a mean diameter of 150 nm. The Au nanostructures were further modified by the deposition of a cystamine (thiol) self-assembled monolayer, yielding a suitable substrate for enzyme immobilization, as a possible future application. The electrochemical behaviour of dopamine and ascorbic acid on gold, Glassy Carbon and nanostructures was studied aiming to characterize the modified electrode. The 300 mV shift in the oxidation peak potential for ascorbic acid on gold in relation to Glassy Carbon allows to calculate the nanostructure coverage factor by the peak current inhibition as 43%. Finally, dopamine was employed to detect the self assembled monolayer formation on the Au nanostructure through its voltammetric behaviour. The variation in the oxidation potential for DA on gold and self assembled monolayer was used as an indicator for such modification.

The measurement of dopamine neurotransmission in the brain has evolved alongside techniques for measuring neuronal activity. This evolution has progressed from coarse physical inspection (using lesions or dialysis approaches) to measuring electrical signatures of the phenomena of interest. Most recently, an optical revolution has taken hold within the neurosciences. We present an optical dopamine measurement technique as a companion to burgeoning neural activity monitors such as GCaMP. The electrical consequences of individual glutamate molecules impinging upon a postsynaptic membrane can be captured in an electrophysiological trace. On the other hand, dopamine has no consistent, measureable postsynaptic effects and therefore cannot easily be measured electrophysiologically. Researchers have instead used the electrochemical features of dopamine to measure samples of it in physical space. This approach, termed cyclic voltammetry, has generated nearly all of the existing knowledge about the precise characteristics of dopamine release. While a reliable method for measuring dopamine release from heavily innervated areas, cyclic voltammetry lacks the resolving power to establish release from dopamine terminals in other areas of interest. One such area is the external globus pallidus (GPe), the focus of this work. Research performed nearly thirty years prior to this thesis established the presence of sparsely distributed dopamine varicosities within the GPe. Here, we leverage FFN102, a newly developed optical method used as a proxy for dopamine release, to measure dopamine release in this area. Based on previous literature showing that dopamine varicosities were present in the GPe and that dopamine receptors exist on principal cells in the area, we hypothesized that these dopamine varicosities were capable of releasing FFN102. Moreover, previous work had shown that anatomically, the most prominent dopaminergic innervation to the GPe came from the substantia nigra. By validating the FFN102 method in the GPe, we showed that a substance is released which likely reflects dopamine vesicle release. Moreover, the use of two dopamine depletion mouse models allowed us to conclude that FFN102 is released exclusively from dopamine terminals. Finally, we advanced the understanding of dopamine release in the area by examining whether pharmacological manipulation could alter the amount of FFN102 released from dopamine terminals.

The ‘synaptic tagging-and-capture’ (STC) theory of cellular memory consolidation holds that memory persistence can be altered by prior or subsequent patterns of neural activity (Redondo & Morris 2011). The aim of this thesis was to develop a realistic model of everyday memory for mice and use the optogenetic toolbox to investigate the neuromodulatory circuitry that modulates persistence of everyday spatial memories. The task involved learning a win-stay rule with the daily goal of finding the location of food in the event arena. Using the developed task, it was confirmed that unrelated novel experiences can facilitate the persistence of spatial memory in a manner sensitive to pharmacological blockade of hippocampal dopamine D1/D5 receptors. Further analysis focused on identifying the specific neuromodulatory systems that mediate this effect. An influential model called the ‘hippocampus- VTA loop’ (Lisman & Grace 2005) points to the critical role of dopaminergic neurons in the ventral tegmental area (VTA), but recent evidence also implicates locus coeruleus (LC) as a potential source of dopamine in the hippocampus (Smith & Greene 2012). In order to identify the dopaminergic structure(s) that may mediate the novelty effect on memory persistence, single unit activity of optogenetically identified catecholaminergic (CAergic) neurons in mouse VTA and LC was recorded in a novelty exploration paradigm. Using tyrosine hydroxylase-Cre knock-in mice and a Cre-dependent adeno-associated viral vectors, CAergic neurons in VTA and LC were selectively tagged with channelrhodopsin-2 (ChR2). Conditional ChR2 expression made it possible to reliably identify CAergic neurons during unit recording sessions in freely moving animals. The main conclusion of the study is that that CAergic neurons in both VTA and LC selectively increase their firing rates in novel environments, relative to both a familiar environment and a home cage baseline. When normalised to their average baseline firing rates, LC neurons are more strongly activated by novelty than VTA neurons. In the final experiment outlined in this thesis, another cohort of Th-Cre mice, in which ChR2 was expressed in CAergic neurons of both VTA and LC using a Cre-dependent adeno-associated virus, was trained on the everyday appetitive spatial memory task. ChR2-mediated photoactivation of CAergic neurons in LC but not in VTA 30 min after encoding, substituting for novelty, was successful in enhancing the persistence of memory. Paradoxically, the effect of optogenetic LC activation was blocked by hippocampal microinfusion of dopamine D1/D5 receptor antagonist but not β-adrenergic receptor antagonist. Results of experiments described in this thesis support the principle of STC theory and collectively indicate that dopamine released from hippocampal terminals of LC neurons mediates the novelty effect on memory persistence. Importantly, they also point to a more general of role of LC in gating of entry to long-term memory.

The nuclei of the midline thalamus are an important part of the brain’s limbic system. Previous work has described the presence of dopamine D2 receptors in the midline thalamus in humans, non-human primates, and rodents. A similar body of literature has also demonstrated dopaminergic innervation of the midline thalamus across these species. However, little is known regarding a) the source of dopaminergic innervation to the midline thalamus in rodents and b) the function of D2R in the midline thalamus in any species. I begin this thesis with a review of the literature examining the anatomy, electrophysiological properties, and role in behavior of the paraventricular nucleus of the thalamus (PVT), a region where D2R mRNA and protein is expressed. I next describe a series of three sets of experiments aimed toward examining the anatomical, electrophysiological, and behavioral role of D2R in the PVT in mice. In the first set of experiments, I used anatomical methods to show that D2R are particularly enriched in neurons of the PVT. I focused on D2R-expressing PVT neurons specifically and show their afferent and efferent projections throughout the brain. In addition, I describe a set of experiments aimed to establish a dopaminergic innervation to the PVT. In the second set of experiments, I used electrophysiological methods to study D2R-expressing PVT neurons. Here, I establish that tonic firing in D2R-expressing thalamic relay neurons in the PVT is inhibited by quinpirole, a D2R/D3R agonist, and increased by sulpiride, a D2R/D3R antagonist. In the third set of experiments, I assessed the behavioral function of D2R in PVT neurons since this has never been studied in any species. I directly manipulated PVT D2R in two directions: a) by overexpressing D2R, and b) by downregulating D2R. Here I show PVT D2R plays a role in both cocaine locomotor sensitization as well as contextual fear expression. Our findings demonstrate for the first time the role of D2R in the PVT and add to literature suggesting that the PVT is an important component of the neural circuitry underlying fear behavior and drug reward. I conclude this thesis with a discussion of the findings described in the three sets of experiments as well as a proposal for future experiments.

The hallmark of an adult mammalian central nervous system is the inability to regenerate after an injury. Zebrafish, on the other hand, have an astounding regenerative capacity. After a spinal cord lesion, zebrafish can re-establish the damaged neuronal network and regain their swimming ability within weeks. This is partly due to the presence of the ependymal radial glia (ERGs), which line the wall of the central canal and act as the stem/progenitor cells of the spinal cord. Under homeostatic conditions the ERGs are largely quiescent, however, the lesion triggers them to proliferate and replace cells that have been lost due to the damage. Previous studies have shown that the regeneration of the motor neurons is affected by the signalling pathways similar to those governing the first development of these cells during embryogenesis, such as Sonic hedgehog, Notch and dopamine signalling. Serotonin (5-HT), similar to dopamine, is a monoaminergic neurotransmitter with a wide range of physiological and behavioural functions. It has also been shown to play a role during development of the nervous system. In this doctoral thesis I address the hypothesis that 5-HT has a positive effect on the development and adult regeneration of motor neurons. In addition, I expand on the previously discovered augmenting effect of dopamine on motor neuron development, by analysing the downstream pathways of its action. I show that during the development, incubating embryos in 5-HT increases the proliferation of the motor neuron progenitor (pMN) cells, which leads to augmented motor neuron production. RT-PCR on FAC sorted pMN cells highlights a number of serotonergic receptors that might be responsible for this effect. Although the downstream pathways are still unknown, 5-HT appears not to act on the sonic hedgehog canonical pathway, as shown by the unchanged expression of the hedgehog effector gene, patched2 after 5-HT treatment. I show that 5-HT does not affect the generation of vsx1+ or pax2a+ interneurons, suggesting that it has a predominant effect on motor neuron production. In the intact spinal cord of an adult zebrafish, the pMN-like ERGs express serotonergic receptors, indicating they are responsive to 5-HT stimulation. After a lesion, 5-HT administration enhances the proliferation of the pMN-like ERGs caudal to the lesion site resulting in an increase in the number of newborn motor neurons. Rostral to the lesion site, administration of exogenous 5-HT does not have an effect on the ERG proliferation, possibly due to the fact that the endogenous source of 5-HT, in the form of the descending axons, is still present and might already elicit a maximal response of the progenitor cells. 5-HT does not have an effect on the proliferation of the progenitor cells dorsal or ventral to the pMNlike domain, nor does it affect the regeneration of the serotonergic interneurons. These results suggest that 5-HT from the brain preferentially contributes to the regeneration of the motor neurons. Dopamine is another monoamine shown to enhance motor neuron production during the development and regeneration. To investigate the downstream pathways of dopamine signalling on motor neuron production during embryogenesis, RNA-sequencing was performed on FAC sorted pMN cells after a treatment with a dopamine agonist, pergolide. The results yielded 14 differentially expressed genes (FDR < 0.05) with diverse functions in the cell, indicating that dopamine might act on multiple targets to promote motor neuron production. Taken together, these results demonstrate the positive effect of monoaminergic stimulation on motor neuron development and regeneration. They provide an insight into the pathways that govern the proliferation of stem/progenitor cells in the embryonic and adult spinal cord, which might contribute to the research working on enhancing adult neurogenesis in mammals.

Methylphenidate (trade name: Ritalin) resulted in a conditioned place preference, but in contrast to work in juveniles, there were no sex differences. In addition, methylphenidate produced a significant decrease in the dopamine transporter compared to controls that may have implications towards development and plasticity of the dopamine system.

The difficulty with movement initiation, or akinesia, is a cardinal symptom of Parkinson�s disease (PD) and the loss of dopaminergic cells, affecting the function of the basal ganglia, the thalamus and the motor cortex, has long been documented. From a broader perspective, it has been proposed that akinesia is caused by impaired function in different brain areas, inside and outside the basal ganglia, operating as a �behavioural arrest control system� (Klemm, 2001). Several neurotransmitters seem to modulate the activity of this system and, contrasting the well-known effects of dopamine, the involvement of adenosine has only recently emerged, particularly via A2A receptors. Adenosine plays an opposite role to dopamine in the brain: adenosine stimulation at A2A receptors inhibits movement (Ferre et al., 1991a; Hauber and Munkle, 1995; Rimondini et al., 1997), whereas A2A antagonists seem to promote movement (Kanda et al., 2000; Bara-Jimenez et al., 2003; Pinna et al., 2005). Although specific adenosine A2A and dopamine D2 receptors are known to antagonistically interact (Ferre et al., 1997; Fuxe et al., 1998; Ferre et al., 2001), little is known of the involvement of A2A receptors in regulating neural activity in the basal ganglia, a crucial point for the future use of A2A antagonists as adjuvant therapy in Parkinson�s disease. In fact, although it is generally accepted that akinesia results from altered function in the cortico-basal ganglia-cortical loop, as confirmed in several studies reporting changes in basal ganglia activity following dopamine depletion (Blandini et al., 2000; Bevan et al., 2002; Boraud et al., 2002), no study to date has systematically investigated electrophysiological changes in the basal ganglia during akinesia induced by adenosine receptor stimulation. Starting from a common behavioural effect, this study tries to bridge this gap by investigating and comparing, in two basal ganglia structures, the neural substrate of akinesia after acute dopamine D2 receptor blockade and adenosine A2A receptor stimulation. The external segment of the globus pallidus (GP, or simply globus pallidus in the rat) and the substantia nigra pars reticulata (SNr) were chosen as the recording sites because both nuclei are included into the �behavioural arrest control system� and seem to express somewhat complementary functions, as a respective key integrative station and main output of the basal ganglia. Dopamine function was manipulated by acute decrease in availability of dopamine binding sites in the brain, through specific dopamine D2 receptor blockade with systemic injections (1.0 and 1.5 mg/kg) of raclopride(3,5-dichloro-N-[(1-ethylpyrrolidin-2-y)methyl]-2-hydroxy-6-methoxy-benzamide), resulting in akinesia. Conversely, movement was inhibited by specific adenosine A2A receptor stimulation with systemic injections (2.5 and 5.0 mg/kg) of the drug CGS21680 (sodium-2-p-carboxyethylphenylamino-5-N-carboxamidoadenosine). In both situations, behaviour was assessed through specific akinesia tests. Single neuron activity before injection and changes in the firing frequency and firing pattern occurring after injection have been analysed and compared for each cell recorded from GP and SNr, during periods of behavioural rest. Synchronised firing between cell pairs has also been assessed. However, the small number of cell pairs showing correlated firing in each structure after systemic injection of drugs was not statistically relevant for further analysis and interpretation of synchronised firing during drug induced akinesia. In our experiments, both drugs inhibited movement, albeit somewhat differently, with lack of rigidity and �flat� body position after adenosine stimulation. Dopamine blockade decreased mean firing rate and dramatically altered the firing pattern in both investigated structures, generally increasing burst activity (increased percentage of spikes in bursts, mean number of bursts, mean number of spikes per burst, mean intra-burst firing frequency) and decreasing regularity of firing (increased coefficient of variation of the inter-spike intervals). Increased burst activity in the rat basal ganglia in an acute model of parkinsonian akinesia, following systemic raclopride injections, confirmed the importance of changes in the firing pattern in PD. The only electrophysiological effect of systemic A2A stimulation was decreased mean firing rate in the GP, a weak effect that could not propagate towards output stations of the basal ganglia. The lack of changes in the firing pattern, at both input and output levels of the basal ganglia, suggests a correlation with the lack of rigidity in adenosine-stimulation-induced akinesia.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.