Margaret E. Rice - US grants

DESCRIPTION (Investigator's Abstract); The long term objective of the proposed studies is to determine mechanisms that dynamically regulate ascorbic acid (Asc) compartmentalization in the brain. Asc functions as an antioxidant and free radical scavenger to protect the brain from reactive species generated during aerobic metabolism. Preliminary data suggest that the anoxic injury-resistant turtle brain may synthesize Asc, suggesting a protective value of the vitamin. The planned experiments provide basic information about Asc regulation in the brain including: (1) uptake, (2) release and (3) utilization. These will be compared in hippocampal slices from the guinea pig, which, like humans, cannot synthesize Asc, and the turtle. Specific aims are: (1) To characterize Asc uptake in brain tissue using HPLC for tissue analysis. Experiments will determine Michaelis-Menten uptake parameters, ionic dependence and energy requirements. Whether turtle brain can synthesize Asc will also be verified. (2) To determine the mechanisms of Asc release from brain cells. [Asc]o will be monitored using carbon fiber microelectrodes and high-speed voltammetry. Release and extracellular volume change will be distinguished by co-monitoring the extracellular marker tetramethylammonium using ion-selective microelectrodes. (3) To examine Asc utilization during depolarization and hypoxia/reoxygenation by measuring tissue Asc and its oxidation product, dehydroascorbate, using HPLC. [Asc]o will be monitored. The effects of therapeutic agents known to affect free radical production will be tested to evaluate the mechanisms of Asc action as a protective agent. The protective action of Asc in brain is important during normal aerobic metabolism, but may be crucial during the pathological conditions of stroke or ischemia. The strength of this proposal lies in the estimation of intra- and extracellular compartmentalization of Asc under various conditions by coupling total tissue analysis with direct extracellular Asc measurements.

Cerebral antioxidants are neuroprotective agents that prevent oxidative injury during normal aerobic metabolism. Ascorbic acid (ascorbate) and glutathione (GSH) are the most abundant antioxidants of low molecular weight in the CNS. At the onset of ischemic injury, energy-dependent mechanisms that maintain cellular levels of ascorbate and GSH fail, so that they and other small molecules are lost brain cells. The underlying premise of this proposal is that loss of intracellular ascorbate and GSH during ischemia leaves brain cells vulnerable to oxidative damage. Despite the central role of ascorbate and GSH in the antioxidant network, surprising little is known about the mechanisms that regulate their homeostasis in the CNS. The long-term objective of the studies is to determine the mechanisms that regulate antioxidant homeostasis in the CNS, which is a necessary first step in the development of appropriate therapies. Preliminary data indicate ascorbate and GSH levels decrease with age and vary with gender. These data are consistent with human epidemiology, which indicate that stroke incidence and severity increase with age and are greater in males than in females. Proposed studies, therefore, will necessarily address factors that govern age and sex differences. Experiments will use HPLC to determine total tissue ascorbate and GSH, and histological methods to evaluate tissue damage and antioxidant neuroprotection. In addition, tools are available to monitor the extracellular levels of ascorbate ([Asc]o). Using voltammetry with carbon fiber microelectrodes, we will detect [Asc]o in brain slice in vitro and after middle cerebral artery occlusion (MCAO) in vivo, then combine these data with total tissue ascorbate content to calculate [Asc]i. Specific aims are: 1. To determine the differential localization of ascorbate and GSH in the CNS, with emphasis on the hypothesis that ascorbate is in neurons and GSH is in glia. We will confirm antioxidant loss in the aging brain and extend gender studies to include antioxidant enzymes and iron. II. To evaluate the mechanism of intra-and extracellular ascorbate homeostasis. In vitro experiments will test the hypothesis that intra- and extracellular ascorbate concentrations ([Asc]i and [Asc]o) are regulated homeostatically and that the dynamics of this process change with age. III. To evaluate where and how ascorbate and GSH protect. In vitro experiments will test the hypotheses that ascorbate and GSH act intracellularly. IV. To determine mechanisms of ascorbate and GSH loss in ischemia. We will extend preliminary data indicating that loss of antioxidants during ischemia is greater in male rats than in females. V. To determine the relationship between loss of intracellular ascorbate and in focal ischemia. Experiments will test the hypothesis that regions with the greatest loss of [Asc]i during ischemia have the greatest extent of damage.

[unreadable] DESCRIPTION (provided by applicant): Dopamine (DA) is a key transmitter in motor and emotive pathways of the brain. Dysfunction of dopaminergic neurotransmission underlies a variety of brain disorders that have a critical impact on society, including Parkinson's and Huntington's diseases, schizophrenia, and addiction. DA cell groups in the midbrain provide the primary source of DA to the CNS. Cells of the substantia nigra pars compacta (SNc) project rostrally to innervate the dorsal striatum (nigrostriatal DA system) whereas those of the adjacent ventral tegmental area (VTA) project to the nucleus accumbens and other limbic structures (mesolimbic DA system). A potentially unique characteristic of DA neurons is that they release DA from their dendrites in midbrain, as well as from distant axon terminals. Dendritic, as well as terminal release of DA is critical for DA-mediated behaviors, yet little is known about factors that regulate DA release In midbrain. Using carbon-fiber microelectrodes and fast-scan cyclic voltammetry to detect evoked DA release in real-time in brain slices, we have found significant differences in the Ca2+-dependence and Ca2+-channels required for dendritic vs. terminal release. Further, we have found distinct patterns of regulation by glutamate and GABA acting at ionotropic receptors in SNc, VTA and striatum. Significantly, we also discovered a novel, endogenous regulator of the nigrostriatal DA release: the reactive oxygen species (ROS), H202. In this continuation, we will investigate the distinct Ca2+ dependence of dendritic DA release, with emphasis on contributions from synaptic input to DA cells and release of Ca2+ from intracellular stores (Aim I). We will also elucidate the role of ROS as modulators of dendritic and terminal DA overflow (Aim ll-IV). Aim II will examine the involvement of H202 and other ROS in nigrostriatal vs. mesolimbic DA systems. Aim Ill will investigate sources of ROS, including terminals and cells adjacent to DA release sites. Aim IV will test whether H202 inhibits dendritic DA overflow by causing hyperpolarization of DA cells; effects on membrane properties will be indicated by whole cell recording.

DESCRIPTION (provided by applicant): Dopamine (DA) is a key modulator of motor and emotive pathways in the brain. Forebrain structures receive DA input exclusively from midbrain DA neurons, with cells of the substantia nigra pars compacta (SNc) projecting via the nigrostriatal pathway to dorsal striatum and those of the adjacent ventral tegmental area (VTA) project via the mesolimbic pathway to nucleus accumbens and other limbic structures. DA cells in both systems share common physiological properties, including somatodendritic release of DA. A significant difference, however, is that nigrostriatal DA cells degenerate in Parkinson's disease, whereas mesolimbic DA cells are spared. Several biochemical differences may contribute to greater SNc vulnerability, including weaker regulation of reactive oxygen species (ROS) in SNc than in VTA. This difference may be crucial, because oxidative stress has been proposed as a causal factor in Parkinson's disease. In addition to being potentially neurotoxic, however, ROS can act as signaling agents. Preliminary data implicate one ROS, hydrogen peroxide (H202), as a modulator of DA release. When applied exogenously, H202 inhibits release in the SNc and VTA as well as in dorsal striatum and nucleus accumbens. Additional data suggest that endogenous H202 generated during local stimulation inhibits DA release in the SNc and striatum, but not in the VTA. These data may reveal a normal physiological process in the nigrostriatal DA system that could contribute to oxidative stress, if regulation of H202 became disrupted. It is not clear, however, whether H202 per se is the modulator or whether it acts via related ROS. The goal of this R21 proposal is to determine specific ROS involved in modulating somatodendritic and synaptic DA release in the nigrostriatal vs. mesolimbic DA systems. Evoked DA release will be monitored using evoked using carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Experiments in Aim t will compare regulation of somatodendritic DA release by ROS in the SNc and VTA, whereas those in Aim 2 will compare regulation in the dorsal striatum and the shell of the nucleus accumbens. Surprisingly, the studies proposed here would be among the first to investigate functional differences in ROS regulation between vulnerable and resistant DA systems. Consistent with the goals of the R21 program, these data should provide ground-breaking new information about underlying mechanisms in nigrostriatal degeneration.

[unreadable] DESCRIPTION (provided by applicant): Dopamine (DA) is a key transmitter in motor, cognitive, and reward pathways of the brain, with dysfunction of DA transmission linked to significant disorders, including Parkinson's disease, schizophrenia, and addiction. The long-term goal of this project is to identify local factors that regulate somatodendritic DA release from DA neurons in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) and axonal DA release in striatum. In the previous funding period, we focused on DA release regulation by endogenous glutamate, GABA, and Ca2+ entry, using real-time voltammetric recording of evoked DA release. We discovered that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is an intracellular messenger in SNc DA neurons that both modulates cell firing rate and inhibits somatodendritic DA release. By contrast, in dorsal striatum, H2O2 is a diffusible messenger that mediates regulation of axonal DA release by glutamate and GABA. These effects of H2O2 are mediated by the activation of ATP-sensitive K+ (KATP) channels. Proposed work will provide mechanistic insight into regulation of DA transmission by H2O2, as well as indicate functional consequences of H2O2 signaling on somatodendritic and axonal DA release. Aim 1 will test the hypothesis that H2O2 activates KATP channels by decreasing channel sensitivity to ATP; Aim 2 will determine the ionic dependence of H2O2 generation; Aim 3 will investigate the role of H2O2 generation in the regulation of somatodendritic DA release and DA cell physiology by glutamatergic NMDA receptors; and Aim 4 will evaluate the temporal and spatial characteristics of glutamate-dependent H2O2 signaling in dorsal striatum. Methods include voltammetric detection of DA release, whole-cell and excised patch recording, and fluorescence imaging of H2O2, intracellular ions, and mitochondrial membrane potential. Experimental systems include isolated DA neurons, transfected cells, and brain slices from guinea pigs and from mice lacking a specific KATP channel subtype. [unreadable] [unreadable] Several brain disorders that involve DA dysfunction, including Parkinson's disease and schizophrenia, have also been linked to oxidative stress. Proposed studies will clarify how endogenous H2O2 normally regulates DA release. Because unregulated H2O2 can lead to oxidative stress, however, the findings may also point to possible targets for therapeutic intervention in these debilitating disorders. [unreadable] [unreadable] This project is based on our novel finding that hydrogen peroxide is an endogenous factor that regulates the nigrostriatal dopamine pathway. Understanding factors that regulate this pathway is important, since it is nigrostriatal dopamine that is lost in Parkinson's disease, leaving individuals unable to move. Proposed studies will clarify how endogenous hydrogen peroxide normally regulates dopamine release and dopamine neuron activity in this pathway. Additionally, because unregulated peroxide can lead to oxidative stress, which is a causal factor in Parkinson's disease, the findings may also point to possible new targets for therapeutic intervention, consistent with one aspect of the mission of NINDS. [unreadable] [unreadable] [unreadable]

DESCRIPTION: Dopamine (DA) neurons in the substantia nigra pars compacta (SNc) play a crucial role in basal- ganglia mediated motor function. Indeed loss of SNc DA neurons leads to the motor deficits in Parkinson's disease. However, the role of these neurons in the normal control of movement is poorly understood. Eventual understanding of mechanisms underlying Parkinson's disease and development of treatment strategies largely depends on understanding DA neuron properties. One of the most critical mechanisms that controls neuronal firing pattern is the prolonged afterhyperpolarization (AHP) that follows a single action potential or burst of action potentials. In DA neurons the AHP contributes to the regulation of the frequency and pattern of spontaneous and evoked discharges and is mediated in part by apamin-sensitive Ca2+-activated K+ channels. In addition, however, an apamin-insensitive component is mediated by an as yet unidentified Ca2+- insensitive channel. Our previous studies showed that membrane properties and firing frequency of DA neurons in the SNc are modulated by endogenous hydrogen peroxide (H2O2) and this modulation occurs via ATP-sensitive K+ (KATP) channels. In DA neurons burst-like firing induced by depolarizing current injection is accompanied by an increase in the production of reactive oxygen species, like H2O2, as well as a prolonged AHP. Excitingly, new data presented in this application shows that the H2O2-metabolyzing enzyme, catalase, and the selective KATP channel blocker, glibenclamide, significantly decrease the amplitude and duration of the AHP in DA neurons, suggesting an involvement of both H2O2 and KATP channels. Proposed studies will therefore test the involvement of these factors in the AHP in DA neurons. I will use patch-clamp recording coupled with fluorescence imaging of H2O2 in SNc DA neurons in guinea pig midbrain slices. Current-clamp mode will be used to study AHP amplitude and duration and hybrid-clamp recording to assess the AHP tail current. Experiments in Aim 1 will address the question whether the AHP in DA neurons has an H2O2- sensitive component and assess its sensitivity to apamin. The involvement of H2O2-sensitive KATP channels in the AHP in SNc DA neuron will be assessed in Aim 2. Overall, this project will provide unique insights into dynamic regulation of AHP and consequently DA neuron activity by H2O2 and KATP channels. PUBLIC HEALTH RELEVANCE This project is focused on testing a novel hypothesis concerning the regulation of the activity of dopaminergic neurons in the substantia nigra pars compacta (SNc). These cells play a crucial role in basal ganglia mediated function. Indeed loss of SNc dopamine neurons leads to motor deficits in Parkinson's disease. This study could therefore potentially lead to the development of novel antiparkinsonian drugs. Thus, it is of direct relevance to NINDS which supports projects focused on the discovery of mechanisms of neurodegenerative disorders.

DESCRIPTION (provided by applicant): Chronic food restriction (FR) increases the rewarding and locomotor-activating effects of drugs of abuse. By contrast, high fat, obesity-promoting diets (OB) decrease sensitivity to psychostimulants compared to ad libitum feeding (AL) of standard rodent chow. These findings suggest a role for endocrine adiposity hormones such as insulin in regulating brain reward pathways. Indeed, there is evidence for such a role of insulin, but mechanisms Novel preliminary data obtained using in vitro voltammetry show that nM levels of insulin increase evoked extracellular dopamine (DA) concentration ([DA]o) in caudate/putamen (CPu) and nucleus accumbens (NAc). Notably, this response is enhanced in FR rats, but blunted in OB rats. The goal of this multi-PI project is to test the hypothesis that insulin-dependent changes in DA neurotransmission contribute significantly to the influence of diet on brain reward circuitry. Pilot data suggest that insulin-enhanced evoked [DA]o is mediated by insulin receptors (InsRs), not on DA axons, but on cholinergic interneurons, and show a pivotal role for DA release regulation by nicotinic acetylcholine (ACh) receptors (nAChRs) located on DA axons. Previous studies of effects of insulin on feeding behavior and reward have emphasized the anorexigenic effect of insulin in the medial hypothalamus, implying a role in satiety. However, our data provide evidence for a novel role of acute insulin elevation in reward by enhancing DA release. Consistent with this hypothesis, other pilot data indicate lower evoked [DA]o in CPu in slices from FR rats, which is restored by exogenous insulin. Interestingly, evoked [DA]o is also lower in OB CPu. Mechanistic experiments in Aim 1 will evaluate signaling pathways and circuitry underlying the effects of insulin on DA release using voltammetry in brain slices and test the hypotheses that hypoinsulinemia contributes to low evoked [DA]o in FR rats, whereas decreased InsR sensitivity may underlie low evoked [DA]o in OB. Aim 2 will combine quantitation of DA release and uptake dynamics with assessment of the effect of physiologically relevant (low nM) levels of insulin on dopamine transporter (DAT) expression and activity in axon terminal regions and in DA cell bodies in the ventral tegmental area (VTA) in synapto(neuro)somes. To translate the effects of insulin from these in vitro studies to in vivo behavior, experiments in Aim 3 will determine the effect of intrastriatal insulin and insulin receptor (InsR) antagonist injections on behavioral responses to rewarding brain stimulation and food- and drug-paired environments. The increasing prevalence of insulin resistance and obesity and the high comorbidity of disordered eating and substance abuse indicate that understanding the connections among diet, insulin, DA and reward will have important implications for prevention and treatment of addictive disorders. Our preliminary finding that insulin promotes transmitter release, including that of DA and ACh, indicates a completely new role for insulin in brain function. The marriage of mechanistic in vitro studies and in vivo behavioral assays in this multi-PI, multi-disciplinary project has high potential to help decompartmentalize pathological eating and drug addiction and to drive the development of crossover therapies are poorly understood. PUBLIC HEALTH RELEVANCE: Eating disorders and substance abuse are significant health risks. The pathology of both involves brain reward circuitry, but whether the same pathways are affected is unclear, in part because of segregation of research on diet vs. drugs. This multi-PI, multi-disciplinary project is built around our preliminary evidence that insulin, a recognized regulator of satiety and metabolism, is also a reward signal in the brain. This project has the potential to change the way that insulin is viewed, and thereby lead to novel crossover therapies to treat both obesity and addiction.

Project Summary Dopamine (DA) is an established neuromodulator in brain reward pathways. It is increasingly recognized that regulatory factors acting within the striatum sculpt local DA release, complementing the role of midbrain DA neuron activity in supplying DA to target regions. We have reported that glucose-induced increases in levels of the metabolic hormone insulin acts as a reward signal in the nucleus accumbens (NAc). Using ex vivo striatal slices, we discovered that insulin enhances DA release in the NAc and dorsal striatum, and that insulin responsiveness is lost in rodents that are obese from a high-fat high-sugar (HF-HS) diet. Our companion behavioral studies show that insulin action in the NAc is necessary for flavor-nutrient learning with glucose- containing solutions, and for the escalation of glucose intake seen during training. Although understanding of this reward pathway is far from complete, we have established the pivotal role of a striatal microcircuit involving DA axons and striatal cholinergic interneurons (ChIs), both of which express insulin receptors (InsRs). An abundant literature indicates that acetylcholine (ACh) from ChIs promotes DA release via ?2-subunit containing nicotinic ACh receptors (?2-nAChRs) on DA axons. We showed previously that insulin acting at InsRs increases ChI excitability, and that the DA-boosting effect of insulin is prevented by ?2-nAChR-selective antagonism, and is absent in mice that lack striatal ACh synthesis (ChAT KO mice). However, key elements required to understand and harness this pathway are missing, including: 1) cellular mechanisms by which insulin increases ChI activity; 2) specific components of flavor-nutrient learning influenced by insulin and whether they are blocked by ?2- nAChR antagonists and impaired by a HF-HS diet; and 3) patterns of insulin-dependent synaptic plasticity in NAc medium spiny output neurons (MSNs) that underlie flavor-nutrient learning. These missing elements will be addressed in three specific aims that capitalize on the complementary expertise of the PIs. Overall, our previous and pilot data show that NAc insulin signaling is necessary for flavor-nutrient learning, which guides food choice and consumption based on predicted nutritive yield. This insulin-dependent reward learning is impaired in subjects with InsR subsensitivity induced by HF-HS feeding, leading to maladaptive consumption. By identifying cellular mechanisms and pathway-specific plasticity that drive nutritive learning, this project will not only answer key questions about NAc insulin, but also indicate targets that might bypass subsensitive NAc InsRs and restore healthy eating.

DESCRIPTION (provided by applicant): The transmitter dopamine (DA) is critical for motivation, reward, movement, and cognition. Originating in the ventral tegmental area (VTA), the mesolimbic DA pathway projects to the nucleus accumbens (NAc) core and shell where DA transmission contributes to the rewarding effects of food and drugs, and promotes goal- directed behavior and reward-related learning. Operating in parallel with the mesolimbic system, the nigrostriatal DA pathway originates in the substantia nigra pars compacta (SNc) and projects to the caudate- putamen (CPu). This pathway contributes to motivated behavior and movement, and is involved in driving behaviors that have transitioned from goal-directed and intentional to automatic and compulsive. Given the key roles of these pathways, dysfunction of DA transmission plays a key role in neuropsychiatric disorders including addiction to psychostimulant drugs and the motor deficits of Parkinson's disease. Elucidating causal factors in these disorders, and developing corresponding treatments, requires an understanding of factors that govern DA release. It is often assumed that DA signaling is governed solely by DA neuron firing rate and pattern, with homogeneous activity-dependent changes in extracellular DA concentration ([DA]o) throughout the striatum. However, this is not the case: studies using fast-scan cyclic voltammetry (FCV) show that DA release is regulated locally within the striatum, with temporally and spatially discrete [DA]o transients detected in vivo and in ex vivo brain slices. Preliminary data suggest that a novel source of dynamic local DA release regulation is mediated by glutamate and GABA that are co-released from striatal DA axons. This project will determine autoregulatory roles of co-released glutamate and GABA on axonal DA release in NAc (Aim 1) and CPu (Aim 2), and somatodendritic DA release in VTA and SNc (Aim 3), in ex vivo slices from mice that express channelrhodopsin 2 (ChR2) in DA neurons. Companion studies will assess the presence of glutamate and GABA receptor subunits on striatal DA axons using immuno-electron microscopy. Based on preliminary data showing enhanced AMPA receptor-dependent regulation of striatal DA release in ex vivo slices 24 h after a single cocaine injection, each aim will include examination of the effect of acute cocaine exposure on DA release regulation by co-released transmitters. Overall, this project will define the roles of co-released glutamate and GABA in sculpting how changes in DA neuron activity translate into DA release, and how this regulation can be disrupted by cocaine.