Cathinones are psychostimulant compounds heavily implicated as drugs of abuse. They exert their physiological actions at the monoamine transporters, which are responsible for maintaining synaptic neurotransmitter homeostasis. Monoamine transporters produce currents during transport and have been shown to depolarize cell membranes and activate voltage-gated calcium channels in mammalian expression systems. This phenomenon is harnessed in an assay which measures these induced calcium transients, allowing for quantification of pharmacodynamic effects of compounds at monoamine transporters. It is unknown if this electrical coupling occurs in neurons, but the implications if it does are significant. In the current work, fluorescent resonance energy transfer studies of HEK cells expressing hDAT suggest that a subpopulation of monoamine transporters and calcium channels may be interacting directly. Additionally, this work presents calcium assay data comparing several novel methcathinone analogs. Of the compounds tested, a single α-methyl substituent at the α-carbon yields the greatest potency at hDAT. The implications of these results shed light on future psychostimulant studies and further define the physiological relationship of the components of a system used to study these compounds.

Reward has been shown to improve multiple forms of learning. However, many of these studies do not distinguish whether reward directly benefits learning or if learning is boosted by modulation of top-down factors such as attention and motivation. The work outlined in this dissertation explores the modulatory effects of reward and punishment without directly manipulating top-down factors such as attention or motivation. We achieved this goal by studying the effects of reward and punishment on incidental learning – a branch of procedural learning where learning occurs without intention and through repetition. Our results reveal that reward is able to bolster incidental learning during the performance and learning of an associative task, even when awareness of how to achieve the reward is minimized (Experiments 1 and 2). However, a similar benefit was not observed in an analogous set of experiments examining the effect of punishment on incidental learning (Experiments 3 and 4). A direct comparison between the effect of reward and punishment on incidental learning revealed a significant advantage for rewarded combinations over punishment. However, this advantage was only observed when high cognitive (associative) demands were emphasized (Experiment 6), as opposed to high motor demands (Experiment 5). Finally, we explored the role of dopamine in the effect of reward on incidental learning. Because dopamine neuron dynamics have been implicated in both reward processing and in various forms of learning, we hypothesized that patients with Parkinson's disease (PD), who experience an accelerated rate of death of dopamine neurons, would experience impaired learning from rewards compared to healthy older adults. Experiment 7 revealed a significant impairment in reward-related incidental learning for patients with Parkinson's disease relative to comparisons. The amount of levodopa medication taken by PD patients predicted the effect of reward, demonstrating a potential link between dopamine levels and the effect of reward on incidental learning. Together, this dissertation demonstrates that 1) reward improves incidental learning, 2) reward may be an exceptional form of feedback, as opposed to punishments, and 3) dopamine levels may potentially drive the effect of reward on incidental learning

Steroid hormones are known to have significant effects on a wide variety of biological processes. In particular, they serve as critical modulators of neural function and behavior and play critical roles in stress responses and neurologic disorders. Until recently the biological actions of steroid hormones were believed to operate primarily through activation of cognate nuclear hormone receptors or the allosteric modulation of ion channels (Majewskaet al., 1986). However, new signaling pathways involving G-protein coupled receptors (GPCRs) for steroid hormones have been recently identified in multiple different species, implicating steroid hormones in direct fast modulation of intracellular signaling and in turn behavior (Thomas et al., 2006, Gabor et al., 2015). In mammals G protein-coupled estrogen receptor 1 (GPER), also known as G protein-coupled receptor 30 (GPR30), is expressed throughout the body including in the nervous system and has been suggested to play a variety of roles in health and behavior (Prossnitz and Barton, 2011). Despite recent progress in this area from studies using rodent models, the mechanisms underlying "non-genomic” actions of steroids remain largely elusive. This gap in our understanding presents a significant scientific and clinical challenge to a comprehensive view of the role of steroid hormones in regulating both neural function, behavior and overall health of the organism. To understand the mechanisms for this unconventional steroid signaling we sought to use a simpler system to explore the functions of GPCR’s for steroid hormones. In 2005, Peter Evans’s group identified DopEcR, a unique GPCR in Drosophila melanogaster, which responds to ecdysone—the major steroid hormone in insects (Srivastava et al. 2005). This unconventional GPCR for steroid hormones is particularly interesting because it is a dual receptor that also responds to a structurally dissimilar compound, dopamine. DopEcR is preferentially expressed in the nervous system and has recently been implicated in modulating multiple behaviors including starvation-induced enhancement of sugar sensitivity (Inagaki et al., 2012), experience-dependent courtship suppression, habituation of the giant fiber pathway (Ishimoto et al., 2013) and ethanol-induced sedation (Petruccelli et al. 2016) in flies. DopEcR also plays a role in perception of sex pheromones in moths (Abrieux et al., 2013). More recently the mammalian GPCR for estrogen GPER has also been found to bind dopamine indicating that this unique attribute may be more prevalent among these novel GPCRs for steroids (Evans et al. 2013). Despite these previous findings, we still know little about how GPCRs for steroids modulate neurons at the cellular level and how they modulate behaviors. Therefore we sought to forge a more comprehensive understanding of the function of steroid signaling by characterizing DopEcR function in neuronal and behavioral modulation through GPCR’s. To characterize DopEcR’s function we looked at the consequences of DopEcR signaling at three levels: behavior, neuronal morphology and finally physiology. Because changes steroid hormones levels are often associated with environmental stressors we assayed the role of DopEcR in a stress related behavior: starvation-induced sleep suppression and hyperactivity. To look at DopEcR’s role in neuronal physiology we used bioluminescent calcium imaging to measure its effect on the stimulated calcium response in a brain structure critical for behavior. Finally we used principal clock neurons in the brain (PDF+ l-LNv neurons) as a model to examine DopEcR’s role in modulating plasticity and neuronal structure. In our present work described in Chapter 2, we found that the D1-like receptor, DopR1, modulates sleep and activity independent of starvation while DopEcR plays a role in mediating starvation-induced sleep suppression and enhanced activity. We found that knocking down EGFR in a DopEcR mutant background restored starvation induced changes in behavior, suggesting that DopEcR normally suppresses EGFR signaling to suppress sleep under starvation. In Chapter 4, we show that the nicotine-induced Ca2+-response was selectively enhanced in the medial lobes either in DopEcR mutant or in flies with DopEcR selectively knocked down within the MBs. Using a pharmacological approach, we show that the endogenous ligands of DopEcR mediated two different responses in the MBs: the steroid ligand ecdysone enhances activity in the calyx and cell body region, whereas monoaminergic ligand dopamine reduced activity in the medial lobes. In Chapter 5, we find that reducing DopEcR in PDF neurons results in reduced basal levels of bouton numbers. The reduction in bouton number is independent of cAMP signaling but instead relies on inhibition of EGFR signaling. Signifying that DopEcR may modulate EGFR associated signaling to make changes in the in the brain. These results demonstrate that DopEcR is able to modulate neuronal excitability, physical structure of neurons and the behavior of the organism. Interestingly it also indicates that DopEcR’s different ligands, dopamine and ecdysone, may have unique and spatially distinct effects on different brain structures or within the same structure. Overall, this study provides a solid foundation for understanding the roles and action mechanisms of GPCR-mediated steroid signaling in regulation of neural development, physiology and behavior.

Resurgence refers to the reappearance of an extinguished operant behavior when reinforcement for an alternative behavior is also discontinued. It is especially relevant to the reappearance of problem behavior because many behavioral interventions discontinue reinforcement for aberrant behavior while simultaneously reinforcing an appropriate response. Existing information about the neuropharmacology of resurgence is scarce, but suggests overlap between drug seeking observed in the resurgence model and drug seeking observed in the more widely studied reinstatement and renewal models. The aim of this dissertation was to explore additional neural systems relevant to reinstatement and renewal preparations within a resurgence paradigm to assess further overlap. The neuropharmacology of resurgence was examined in two studies via administration of two drugs that have proven effective in blocking drug seeking in reinstatement and renewal preparations. In two experiments, rats earned food pellets for pressing a target lever in Phase I. In Phase II, lever pressing no longer produced food, but food was delivered contingent on an alterative nose poke response. Finally in Phase III, neither response produced food deliveries. Prior to these Phase III sessions, separate groups of rats were injected with 0, 50, or 100 mcg/kg of the dopamine D-2 receptor antagonist raclopride in Experiment 1 or 0, 20, or 40 mcg/kg of alpha-2 agonist clonidine in Experiment 2. Both doses of raclopride were effective in blocking resurgence, but there was strong evidence that the higher dose did so via motor rather than motivational impairment. Furthermore, the lower dose significantly suppressed the alternative nose poke, which suggests motor impairment, as well. Only the higher dose of clonidine blocked resurgence, but did so with no evidence of motor impairment. Raclopride significantly impacted extinction of the alternative poke at both doses tested, whereas clonidine had no effect at either dose. The results of the present studies provide additional information about the neuropharmacology of resurgence, as well as additional evidence of overlap between resurgence, reinstatement, and renewal. The present results may also have implications regarding underlying neural mechanisms and for pharmacotherapies to attenuate relapse when alternative sources of reinforcement are thinned or discontinued.

The mesolimbic dopamine (DA) system has been implicated in providing the basis of pleasure, guiding the general mechanism of reinforcement as well as motivation. Support for these roles have grown from neurochemical research in the field of addiction. It is now well known that DA activity increases in the nucleus accumbens septi (NAcc) with exposure to addictive substances. Moreover, pharmacological manipulation of this system produces predictable changes in the administration of drugs of abuse, as well as natural reinforcers. This system is responsive to natural reinforcers and addiction may be the transference of routine mesolimbic function to environmental stimuli predictive of drug administration. The role of the NAcc in addiction specifically appears to be the facilitation of attention to drug-paired stimuli and addiction may be the behavioral manifestation of conditioned NAcc DA reactivity to the presence of drug-related stimuli. Although these findings have been reported in adults, few studies have focused on adolescence, the time when drug use/abuse begins. Adolescents may be particularly susceptible to addiction when considered in the light of this hypothesis. Recent research has revealed that the mesolimbic system of periadolescent animals is undergoing dramatic transition in functional tone. DA receptor and transporter levels are up regulated, synthesis rates are altered, and innervation from prefrontal cortex (PFC), involved in regulating tonic and phasic DA activity, is increasing. Consequently, during adolescence there is a dramatic change in tonic DA levels, variations in phasic responses to acute drug administration and alterations in how the system adapts to repeated drug exposure. The present study utilizes the procedures of conditioned place preference, Novelty preference and in vivo microdialysis to determine how this conditioning process changes during the period of adolescence. The results indicate that adolescents are different from adults not only on behavioral measures associated with drug abuse, but in their neurochemical responsiveness to alcohol, and that these differences are related to a general developmental aspect of adolescence that renders them susceptible to addiction.

No description available.

The computational ability of principal neurons and interneurons in the brain and their ability to work together in concert are thought to underlie higher order cognitive processes such as learning, memory, and attention. Dendrites play a very important role in neuronal information processing because they receive and integrate incoming input and can undergo experience-dependent changes that will alter the future output of the neuron. Here, I have used whole-cell patch clamp recordings and fluorescent Ca2+-imaging to examine the modulation of dendritic excitability in principal neurons of the rat and human hippocampus and neocortex. First, I determined that dendrites of dentate granule cells of the hippocampus are tuned to high frequencies of both afferent input and backpropagating action potentials. Under these conditions they are also capable of generating regenerative dendritic activity that can propagate to the soma, which is prone to modulation. In particular, Neuropeptide Y (NPY) Y1 receptors can decrease frequency-dependent dendritic Ca2+ influx. Dopamine D1 receptors (D1Rs) have an opposite effect; they potentiate frequency-dependent dendritic excitability. These two neuromodulators also have an opposing effect on plasticity, with dopamine acting to induce, and NPY acting to inhibit long-term potentiation (LTP). Parallel observations of D1-induced LTP and an NPY-mediated decrease in dendritic excitability in rodents were complemented by findings in human dentate granule cells. Second, I examined the role of NPY receptors on dendrites of layer 5 pyramidal neurons. In these neurons I found that NPY acts post-synaptically on distal dendrites via the Y1 receptor to inhibit frequency-dependent Ca2+-currents, similar to the findings in dentate granule cells. NPY also decreased regenerative Ca2+ currents caused by the appropriate pairing of pre- and post-synaptic input. Together, these observations demonstrate that the role of NPY in the hippocampus and neocortex is not solely as an anti-epileptic agent. NPY release, likely to occur during high frequency oscillatory activity, can act locally to limit dendritic excitability, which can have a profound effect on plasticity. In the dentate gyrus, NPY can inhibit a D1R induced increased dendritic excitability and resultant changes in synaptic strength. These findings will further the understanding of dendritic information processing in the hippocampus and neocortex.

Parkinson’s disease (PD) is a progressive neurodegenerative disease and characterized by the loss of dopaminergic (DA) neurons in the substantia nigra in the midbrain. The causes of the disease are still unknown. The most commonly used treatment is administration of L-DOPA, however, another possible treatment strategy is to transplant DA neurons to the striatum of PD patients to substitute the loss of neurons. Clinical trials have demonstrated beneficial effects from transplantation, but one obstacle with the grafting trials has been the variable outcome, where limited graft reinnervation of the host brain is one important issue to solve. To improve and control the graft DA nerve fiber outgrowth organotypic tissue cultures can be utilized. Cultures of fetal ventral mesencephalon (VM) have been used to investigate astrocytic migration and dopamine nerve fiber formations at different time points and under varying conditions to study how to control nerve fiber formation. The early appearing DA nerve fibers as revealed by tyrosine hydroxylase (TH) –immunoreactivity, form their fibers in the absence of glial cell bodies, are not persistent over time, and is called non-glial-associated TH-positive nerve fiber outgrowth. A monolayer of astrocytes guides a second persistent subpopulation of nerve fibers, the glial-associated TH-positive nerve fiber formation. Investigations of the interactions between the astrocytic migration and nerve fiber formations were made. In embryonic (E) day 14 VM cultures the mitosis of the astrocytes was inhibited with the antimitotic agent β-D-arabinofuranoside. The results revealed decreased astrocytic migration, reduced glial-associated TH-positive outgrowth, and enhanced presence of the non-glial-associated TH-positive outgrowth in the cultures. Thus, astrocytes affect both the non-glial- and the glial-associated growths by either its absence or presence, respectively. The astrocytes synthesize proteoglycans. Therefore the nerve fiber formation was studied in VM or spinal cord cultures treated with the proteoglycan blockers chondroitinase ABC (ChABC), which degrades the proteoglycans, or methyl-umbelliferyl-β-D-xyloside (β-xyloside), which blocks the proteoglycan synthesis. β-xyloside inhibited the migration of the astrocytes and the outgrowth of the glial-associated TH-positive nerve fibers in both VM and spinal cord cultures, whereas ChABC treatment had no effect in E14 VM or spinal cord cultures. E18 VM and spinal cord cultures were evaluated to investigate how the different developmental stages influence astrocytes and the two nerve fiber formations after 14 DIV. No nerve fiber formation was found in E18 VM cultures, while the non-glial-associated nerve fiber outgrowth was obvious as long and robust fibers in E18 spinal cord cultures. The astrocytic migration was similar in VM and spinal cord cultures. β-xyloside and ChABC did not affect nerve fiber growth but astrocytic migration in E18 VM cultures, while no effects was found in the spinal cord cultures. However, the neuronal migration found in control cultures was abolished in both VM and spinal cord cultures after both ChABC and β-xyloside. Neuroinflammation plays a critical role in the development of PD. Increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) are observed in postmortem PD brains and the levels of TNFα receptors on circulating T-lymphocytes in cerebrospinal fluid of PD patients are increased. The effects of TNFα were studied on E14 VM cultures. The outgrowth of the non-glial-associated TH-positive nerve fibers was inhibited while it stimulated astrocytic migration and glial-associated TH-positive nerve fiber outgrowth at an early treatment time point. Furthermore, blocking the endogenous levels of TNFα resulted in cell death of the TH-positive neurons. Furthermore, cultures of E14 mice with gene deletion for the protein CD47 were investigated. CD47 is expressed in all tissues and serves as a ligand for the signal regulatory protein (SIRP) α, which promotes e.g migration and synaptogenesis. CD47-/- cultures displayed massive and long non-glial-associated TH-positive nerve fiber outgrowth despite a normal astrocytic migration and the presence of glial-associated TH-positive nerve fiber outgrowth. For the first time, it was observed that the non-glial-guided TH-positive nerve fiber outgrowth did not degenerate after 14 DIV. Taken together, there is an interaction between astrocytes and TH-positive nerve fiber formations. Both nerve fiber formations seem to have their task during the development of the DA system.

Recent approaches in depression therapeutics include triple reuptake inhibitors, drugs that target three monoamine systems. Using in vivo electrophysiological and microdialysis techniques, the effects of 2- and 14-day treatments of escitalopram, nomifensine and the co-administration of these two drugs (TRI) were examined in male Sprague-Dawley rats. Short- and long-term TRI administration decreased NE firing and had no effect on DA neurons. Normal 5-HT firing rates were maintained after 2-day TRI administration compared to the robust inhibitory action of selective serotonin reuptake inhibitors (SSRIs). Escitalopram treatment enhanced the tonic activation of the 5-HT1A receptors given the increase in firing observed following WAY100635 administration. Nomifensine treatment enhanced tonic activation of the α2–adrenoceptors following idazoxan administration. TRI treatment caused a robust increase in extracellular DA levels that was in part mediated by a serotonergic contribution. Therapeutic effects of the drugs examined in this study may be due to the enhancement of 5-HT, NE and/or DA neurotransmission.

Schizophrenia (SCZ) is a severe neuropsychiatric disorder with a genetic component. Most candidate gene association studies have given mixed results. We investigated the GABAA receptor gamma2 subunit gene GABRG2, the dopamine receptor gene DRD3, and the Brain-derived neurotrophic factor gene BDNF that is required for D3 expression by genotyping polymorphisms spanning and surrounding these genes for association with SCZ, as well as suicidal behaviour. We also examined the BDNF, DRD3, as well as the dopamine receptor gene DRD2 and Protein Kinase B gene AKT1 for association with Tardive Dyskinesia (TD), a potentially irreversible motor side effect of long-term antipsychotic medication. Our analysis included single-marker tests, haplotype tests, and gene-gene interactions. We found a haplotype in the 5’ region of GABRG2 to be associated with SCZ in both families and matched case-control samples. We also found two synonymous DRD2 polymorphisms, rs6275 (C939T) and rs6277 (C957T), and their haplotypes, as well as a polymorphism 5’ of DRD3, rs905568, to be associated with TD. Further, we reviewed two putative functional DRD2 polymorphisms, -141C Ins/Del and TaqIA, in TD and found TaqIA 3’ of the gene to be associated with TD in a meta-analysis. Lastly, we found a significant interaction between AKT1 rs3730358 and DRD2 C939T in TD. Though replication studies are required, these results contribute to the future development of genetic tests to assess for the risks of SCZ and TD, leading to better outcome for patients suffering from these debilitating conditions.