Bryan K. Yamamoto - US grants

The nigrostriatal dopamine (DA) system has been traditionally considered a homogeneous entity necessary for movement. Recent studies have shown that the striatum may be topographically organized in an anatomical and neurochemical manner. Furthermore, there has been some evidence that subregions of the caudate-putamen contribute differentially to a variety of DA-dependent behaviors. Previous studies have inferred a functional heterogeneity by observing sensorimotor alterations resulting from microinjections of drugs or lesions of specific striatal areas. However, they may be confounded by the risk of non-specific damage and chemical diffusion to surrounding regions. Although important, these studies do no directly address the dynamic relationship between in vivo subregional DA function and voluntary locomotor behavior in the unlesioned animal. The recent development of in vivo voltammetry has made it possible to study, in a relatively non-invasive and specific manner, DA release in discrete brain regions of awake, moving animals. The specific aims of this proposal will investigate and characterize the neurochemical heterogeneity of 9 striatal subregions by observing in vivo DA release in response to specific dopaminergic drugs. DA content and turnover will also be measured in vitro by HPLC with electrochemical detection. In addition, a functional profile of these subregions will include DA release during voluntary circling and in response to specific manipulations of posture, speed and direction. To further assess a heterogeneity of the striatum, the nigrostriatal path of rat will be unilaterally lesioned with 6-hydroxydopamine. DA release and turnover and L-dopa will be measured within subregions during amphetamine and L-dopa-induced turning. The long term objectives of this research will be a higher level of sophistication and an improved understanding of the anatomical, neurochemical and functional relationships between striatal subregions during normal behavior and hopefully add insight into the pathophysiology of striatal dysfunctions such as Parkinson's Disease.

[unreadable] DESCRIPTION (provided by applicant): High doses of METH produce long-term consequences indicative of neurotoxicity as revealed by cognitive deficits in humans and long-term decreases in markers of dopamine (DA) and 5HT neurotransmission in humans and animals. Our studies during the previous funding period and findings by others revealed that high levels of striatal glutamate (GLU) play an important role in METH toxicity. Nevertheless, there is no evidence of how striatal GLU transmission is increased by METH and if this produces excitotoxic damage. Moreoever, despite the neurochemical similarities between METH, environmental stress, and drug abuse, it is unknown if and how stress might enhance the excitotoxic effects of METH. The hippocampus is also vulnerable to the toxic effects of METH and is particularly sensitive to stress and excitotoxic insult due the dense composition of GLU neurons and glucocorticoid receptors in this region. Despite numerous studies demonstrating that the hippocampus is involved in cognition and human METH abusers exhibit cognitive deficits, it is surprising that little is known about how METH damages the hippocampus or how stress affects the excitotoxic effects of METH. The proposed project is a novel extension of our prior studies and will elucidate the neurochemical determinants and consequences of GLU-mediated excitotoxicity to the striatum and hippocampus and how they are affected by prior exposure to chronic unpredictable stress. The overarching hypothesis that will be tested by the proposed specific aims is that excitotoxicity in the striatum is produced by METH, augmented by prior exposure to chronic stress, and mediated differentially by D1 and D2 receptors. Excitotoxicity will be paralleled by increased presynaptic storage and extracellular concentrations of striatal GLU resulting in oxidative stress to the vesicular monoamine transporter (VMAT2) and the mitochondrial electron transport chain to culminate in proteasomal inhibition and spectrin proteolysis. In addition, a stress-induced enhancement of GLU transmission in the hippocampus will be similarly evidenced by increased synaptic and extracellular GLU and consequently, decreased cellular bioenergetics, decreased proteasomal activation, and spectrin proteolysis. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): There is a significant overlap with the neurobiology of stress and abuse of drugs such as amphetamine and cocaine. 3,4-methylenedioxymethamphetamine (MDMA), is an amphetamine derivative whose abuse has dramatically increased worldwide since the 1980s. Although MDMA produces a selective neurotoxicity to serotonin (5HT) terminals, the long-term functional consequences of MDMA use are less understood. Moreover, the interactions between prior exposure to MDMA and chronic stress have not been examined. Our preliminary studies indicate that 5HT, at the level of the ventral tegmental area (VTA), dampens MDMA-induced dopamine (DA) release in n. accumbens (NAcc). Consequently, prior MDMA-induced 5HT depletions may disinhibit psychostimulant-induced NAcc DA release, disrupt the normal 5HTergic modulation of this system, and enhance the stress-associated DAergic and rewarding aspects of abused drugs. It also is known that chronic stress markedly affects the hippocampus (HIPP). Since 5HT inhibits hippocampal glutamate transmission, exposure to chronic stress after MDMA-induced damage to 5HT terminals could enhance the vulnerability of the HIPP to the excitotoxic effects of glutamate. The resultant excitotoxicity to the HIPP may underlie the reported memory deficits observed in human MDMA abusers. While neither MDMA-induced damage to 5HT terminals nor stress alone may be sufficient to produce marked consequences, they may' interact synergistically to cause dramatic neurochemical and functional changes associated with enhanced drug abuse and cognitive deficits. The overarching hypothesis is that MDMA-induced neurotoxicity to 5HT terminals disinhibits DA release in the NAcc and glutamate release in the HIPP, each of which synergizes with chronic stress to enhance drug reward and produce excitotoxicity to the HIPP, respectively. This hypothesis will be tested by 2 Specific aims: (1) examine hippocampal glutamate release as well as learning and memory in MDMA pre-treated rats subsequently exposed to a mild chronic unpredictable stress regimen and (2) assess the neurotoxic effects of MDMA on chronic stress-induced changes in DA release during cocaine and MDMA self-administration. These studies have significant implications for the possible synergistic interactions between prior MDMA exposure, chronic stress, and the vulnerability to subsequent drug self-administration.

[unreadable] DESCRIPTION (provided by applicant): The widely abused amphetamine analogue, 3 4-methylenedioxymethamphetamine (MDMA, Ecstasy) selectively damages the axon terminals of 5-HT neurons in the brain. Several lines of evidence suggest that dopamine (DA) contributes to this toxicity. Although DA clearly produces oxidative by-products such as reactive oxygen species and quinones, it remains unclear as to how DA-derived oxidative species produce selective damage to 5HT terminals, a hallmark of MDMA-induced neurotoxicity. More specifically, the mechanism as to how DA accumulates within 5-HT terminals to produce its selective damage is unknown. The overaraching hypothesis of the current proposal is that L- tyrosine, the amino acid precursor of DA, contributes to the neurodegenerative process. This hypothesis is based on our recent preliminary data indicating a 5-fold increase in the extracellular concentration of tyrosine measured in vivo after MDMA. Unlike DA, tyrosine is actively transported from the periphery and into the brain and neurons. While tyrosine is the natural precursor for DA synthesis within DA neurons, high concentrations of tyrosine in 5HT neurons may have deleterious consequences. The hypothetical framework of this proposal is that the oxidative environment produced by MDMA and hyperthermia in 5-HT neurons causes the non-enzymatic oxidation of tyrosine to the DA precursor, DOPA. Aromatic amino acid decarboxylase (AADC), within the 5-HT terminal then would decarboxylate DOPA to DA, leading to an accumulation of DA and consequently, DA-derived reactive oxygen species and oxidative damage within 5HT terminals. This hypothesized role of tyrosine as a mediator of MDMA-induced toxicity is a novel mechanism that effectively synthesizes current existing hypotheses and to some extent, discrepant observations and apparent caveats into a cohesive, theoretical and testable framework. [unreadable] The Specific Aims are (1) to demonstrate the non-enzymatic oxidation of tyrosine to DOPA and DA within 5HT neurons, (2) to characterize the contributions of L-tyrosine and tyramine to MDMA and hyperthermia-induced oxidative stress and (3) to assess subsequent neuronal damage. The use of cultured RN46A 5HT cells is a novel approach that is uniquely suited to address the hypothesized mechanism of MDMA-induced damage. These experiments will directly measure the individual and relative effects of MDMA, tyrosine, hyperthermia, and the formation of intracellular dopamine on oxidative processes within 5-HT neurons and how these variables affect cell viability. The testing of this model in conjunction with in vivo microdialysis studies, provides a unique and powerful approach to address enigmatic issues previously related to MDMA-induced neurodegeneration of 5HT systems [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The rampant abuse of methamphetamine (Meth) and its documented toxicity to brain neurotransmitter systems are well known but its potential damage to other targets such as the brain microvascular endothelium has been overlooked. Moreover, because Meth is highly co-morbid with other health concerns such as stress and post-traumatic stress disorder, it is imperative that the mechanistic underpinnings between stress and Meth are understood so that effective therapeutic strategies can be developed to effectively treat the scope of Meth abuse and overdose. The proposal examines a new consequence associated with the co-morbidity of stress and Meth abuse that is evidenced by long-term damage to the blood-brain barrier (BBB) and brain microvascular endothelium. The long term goal is to identify the comprehensive effects associated with this co-morbidity and assess the risk to human health produced by stress-induced augmentation of brain injury resulting from the abuse of Meth. Our working model provides the basis for the hypothesis that chronic stress-induced neuroinflammation is a contributory factor to the BBB damage observed after Meth exposure and that this damage is manifested as large molecule extravasation into the brain parenchyma and phosphorylation-dependent decreases in endothelial tight junction proteins. The translational rationale is to develop a novel and feasible neuroprotective strategy that targets neuroinflammation and is either prophylactic or can rescue the BBB from the harmful consequences resulting from the combined exposures to stress and Meth. Three distinct but complementary aims will address our hypothesis. Specific Aim 1 will identify the duration and degree of BBB permeability after the serial exposure to chronic stress and the self administration of Meth. Specific Aim 2 will examine the underlying causes of BBB permeability and will elucidate the time-dependent neuroinflammatory mechanisms responsible for the permeability changes. Specific Aim 3 will determine the consequences of the increased permeability of the BBB produced by the serial exposure to stress and Meth by examining the augmentation of neuroinflammation caused by entrance of the oral bacterium associated with Meth mouth, p. gingivalis, into the brain. The findings will have an overall positive impact because the determination of the causes and consequences of a breach in the BBB can guide the design of future therapeutic strategies for the treatment of METH neurotoxicity and overdose. The hope is to fundamentally advance the field of drug abuse-induced brain injury in general, by broadening the significance of Meth toxicity to include the long-term impact on the cerebral vasculature endothelium and thereby begin to understand the far reaching neurobiological consequences associated with this effect.