John A. Dani - US grants

The overall objective is to understand the structure, function, and regulation of integral membrane proteins that serve as ion channels in excitable cells. Recently all the acetylcholine receptor (AChR) subunits were cloned and the amino acid sequences were determined and used to model the subunits organization into a transmembrane channel structure. Giga-seal patch techniques in conjunction with streaming potential measurements will be used to learn about the internal structure of the AChR channel to provide a basis for judging the four proposed structural models. Streaming potential measurements will determine the number of water molecules coupled to the transport of different size cations. The water coupled to the largest cation that can fit through will indicate the length of the narrowest portion of the channel. The length of the narrow region and ion-water interaction will be related directly to permeation studies. This electrical method of obtaining structure has been used with artificial membranes, but this will be the first time it is extended to study bio-channels in their native membranes. The Na channel and AChR are extensively glycosylated. The carbohydraate contributes 30% of the weight and about 100 negative charges to the Na channel from Electrophorus electricus, and the AChR has a glycosidic linkage near the proposed ACh binding site. However, little is knonw about the function of the carbohydrate. Electrical and standard biochemical methods with radioactive and fluorescent labels will be used to study Na channel and AChR glycosylation. These modification studies address the importance of glycosylation in AChR and Na channel function, ACh binding, channel maturation and lifetime, and events related to synaptogenesis. This should provide a step toward understanding post-translational modifications, which regulate ion channel properties during differentiation, development, and learning. The proposed studies provide insights into molecular mechanisms that are altered by pathological conditions affecting the nervous system and neuromuscular transmission.

The purpose of the proposed research is to study the function and structure of the nicotinic acetylcholine receptor (AChR) and then to describe the results cohesively in terms of the amino acid sequences of the receptor subunits. A better understanding of single AChRs will then serve as a background for exploring the AChR's involvement in complex processes at the neuromuscular junction. Electrophysiological techniques, theoretical approaches, fluorescent ion indicators, and biophysical studies of structure will provide a body of information to guide site-directed mutagenesis experiments with the AChR. The mutation studies allow direct comparison of structure and function to test existing hypotheses and to generate better ones. Calcium's permeability, interactions with other permeants, and influence on protein phosphorylation will be examined as a possible unique self-regulating mechanism of AChRs at the neuromusclar junction. Quantifying and determining the biological importance of calcium entry through AChRs is not only important at cholinergic synapses; it also could suggest regulatory roles of calcium that enters the cell through other synaptic receptors not known primarily as Ca channels. The proposed research directly relates the structure and function of the AChR to its overall roles at the neuromuscular endplate. In addition, the studies will contribute to the general understanding of possible mechanisms involved in regulating synapses.

The goal of this proposal is to develop a working understanding and ion permeation models that account for the structure and function of glutamate receptors (GluRs) and nicotinic acetylcholine receptors (nAChRs). The ongoing work is at different stages for the two receptor types. -1- For the peripheral (P) nAChR, biochemical, biophysical and ultrastructural data have provided a picture that relates structure and function. Our research has focused on how the open channel structure governs ion permeation, and for the first time, we presented an ion permeation model that describes permeation data, based explicitly on the general structure of the channel. The working understanding and physically based permeation model resulting from the research now serve as predictive guides into the study of synaptic processes. For example, the results and model guided our characterization of MK -801 inhibition of nAChRs, and provided a prediction of the synaptic Ca 2+ influx through PnAChRs. The fundamental biophysical work also will serve as a basis for the interpretation of results with neuronal (N) nAChRs. In vitro transcribed mRNAs from the cDNAs encoding different subunits of NnAChRs will be expressed in oocytes. The functions of different subunit combinations will be characterized, and then, they will be compared with our working picture of the PnAChR. The objective is to understand the functional diversity that can arise from different NnAChR subunit combinations. This work is important because the heterogeneous distribution of subunits in the CNS suggests that alterations in the NnAChR's compositions play a role in modulating cholinergic signals that influence synaptic transmission. -2- Structure-function studies of GluRs are proposed because glutamate synapses are the most important excitatory synapses in the brain, but there is little biochemical or biophysical information and practically no structural data for the channels. The proposed research is the first step along the same path we have taken with the PnAChR. The work will progress through a series of experiments that each contribute to the final goal of associating function to the general structure of the channel. Experiments on ion permeation, open channel blockade and ion-water interactions will provide the following information about the pore: the minimum cross section, the length of the minimum cross section, the size of the entrance vestibules, the general charge, the number and position of binding sites, and the volume change associated with channel gating. Because methods that resolve structures of water soluble proteins cannot be applied at high resolution to ion channels in membranes, the proposed research, at present, is the best way to answer questions about the open channel structure and function of the GluRs. If crystals for the channels become available, these basic structural results should guide the interpretation of the crystals. The results also are important for relating the amino acid sequences of the cloned channels to their functions. For example, these types of results guided the development of the currently accepted structural model based on the primary sequences for the PnAChR. As with the PnAChR, the permeation models and the general working knowledge gained from these experiments should lead to predictions that direct research into the GluRs' involvement in synaptic processes.

The goal of this proposal is to develop a working understanding and ion permeation models that account for the structure and function of glutamate receptors (GluRs) and nicotinic acetylcholine receptors (nAChRs). The ongoing work is at different stages for the two receptor types. -1- For the peripheral (P) nAChR, biochemical, biophysical and ultrastructural data have provided a picture that relates structure and function. Our research has focused on how the open channel structure governs ion permeation, and for the first time, we presented an ion permeation model that describes permeation data, based explicitly on the general structure of the channel. The working understanding and physically based permeation model resulting from the research now serve as predictive guides into the study of synaptic processes. For example, the results and model guided our characterization of MK -801 inhibition of nAChRs, and provided a prediction of the synaptic Ca 2+ influx through PnAChRs. The fundamental biophysical work also will serve as a basis for the interpretation of results with neuronal (N) nAChRs. In vitro transcribed mRNAs from the cDNAs encoding different subunits of NnAChRs will be expressed in oocytes. The functions of different subunit combinations will be characterized, and then, they will be compared with our working picture of the PnAChR. The objective is to understand the functional diversity that can arise from different NnAChR subunit combinations. This work is important because the heterogeneous distribution of subunits in the CNS suggests that alterations in the NnAChR's compositions play a role in modulating cholinergic signals that influence synaptic transmission. -2- Structure-function studies of GluRs are proposed because glutamate synapses are the most important excitatory synapses in the brain, but there is little biochemical or biophysical information and practically no structural data for the channels. The proposed research is the first step along the same path we have taken with the PnAChR. The work will progress through a series of experiments that each contribute to the final goal of associating function to the general structure of the channel. Experiments on ion permeation, open channel blockade and ion-water interactions will provide the following information about the pore: the minimum cross section, the length of the minimum cross section, the size of the entrance vestibules, the general charge, the number and position of binding sites, and the volume change associated with channel gating. Because methods that resolve structures of water soluble proteins cannot be applied at high resolution to ion channels in membranes, the proposed research, at present, is the best way to answer questions about the open channel structure and function of the GluRs. If crystals for the channels become available, these basic structural results should guide the interpretation of the crystals. The results also are important for relating the amino acid sequences of the cloned channels to their functions. For example, these types of results guided the development of the currently accepted structural model based on the primary sequences for the PnAChR. As with the PnAChR, the permeation models and the general working knowledge gained from these experiments should lead to predictions that direct research into the GluRs' involvement in synaptic processes.

DESCRIPTION (Investigator's Abstract): The hippocampus has a high density of nicotinic acetylcholine receptors(NnAChRs) and rich cholinergic innervation, but the roles of the NnAChRs are not well understood. Using patch-clamp techniques, fluorescent Ca2+ measurements, and NnAChR labelling methods, we have obtained compelling preliminary results to support a working hypothesis for investigation of nicotinic mechanisms in the hippocampus. The applicants hypothesize that a Ca2+ influx initiated by presynaptic NnAChRs enhances the synaptic release of glutamate. Furthermore, intense synaptic activity decreases external Ca2+ and, consequently, diminishes the efficacy of NnAChRs. The aims are to investigate the hypothesis by assessing the roles of NnAChRs and by examining the underlying hippocampal slices are used to provide a more biologically relevant preparation that offers greater experimental versatility. They will determine whether NnAChR activity alters glutamatergic synaptic transmission and synaptic plasticity. Timed local applications of nicotinic agonists will be used to judge whether presynaptic NnAChRs help regulate intra-terminal Ca2+ and, thereby, modulate glutamate release. They will directly test whether NnAChRs can mediate a presynaptic Ca2+ influx by monitoring presynaptic NnAChR currents and intra-terminal Ca2+ in tissue culture and at an intact mossy-fiber terminal in the hippocampal slice. Because other presynaptic terminals in the hippocampus are small, they can only use the relatively large mossy-fiber terminals to monitor intracellular Ca2+ with fura-2 while simultaneously measuring NnAChR currents with patch-clamp techniques. Another influence on the action of NnAChRs occurs because the high current density at an active synapse not only elevates intracellular Ca2+ but also locally lowers extracellular Ca2+. Our preliminary results indicate that physiological decreases in extracellular Ca2+ diminish the efficacy of NnAChRs. In summary, a presynaptic Ca2+ influx initiated by NnAChRs could spur glutamate release. Intense synaptic stimulation, however, decreases external Ca2+, which may turn off the NnAChRs, producing a negative feedback onto glutamatergic activity.

DESCRIPTION: (Applicant's Abstract) In the United States, tobacco use causes 400,000 deaths a year, making it the leading cause of premature death. Although multiple psychopharmacological effects contribute to tobacco abuse, nicotine is accepted as the primary component that reinforces the habit. Nicotine binds to nicotinic acetylcholine receptors (nAChRs). The rate of nicotine delivery and the time of exposure dictates how the populations of nAChRs distributes among functional states that are closed, ion conducting, desensitized, or long term inactivated. Smokers take in a small pulse of nicotine with each cigarette. These peaks of nicotine ride on a maintained low level of nicotine that increases with repeated cigarettes during the day. The research will test the following hypothesis: The pulse of nicotine could activate nAChRs that evoke dopamine release and produce "rewarding" effects mediated mainly via the mesolimbic dopaminergic system. The sustained low levels of nicotine, however, could cause significant desensitization or longer-term inactivation of nAChRs that may underlie tolerance or aspects of withdrawal symptoms mediated by non-reward pathways. Patch-clamp electrophysiology, calcium measurements, and immunocytochemistry will be used to investigate the hypothesis. Microexplants of ventral tegmental area (VTA) neurons synapsing onto low density cultures of nucleus accumbens neurons will be examined to determine how nicotine evokes dopamine release. VTA brain slices and tissue cultures of VTA and habenula neurons will be used to understand the time scales and effects of nAChR desensitization and long-term inactivation as well as recovery from those states. Nicotinic AChR activation, leading to DA release by mesolimbic neurons, may provide the reinforcing reward that initiates tobacco abuse. A further drive to smoke may arise because smokers are medicating themselves with nicotine on longer time scales to control the degree to nAChR desensitization and inactivation that may underlie aspects of tolerance and withdrawal symptoms.

Tobacco use is a major health problem in the United States, where over 400,00 deaths and $50 billion in medical costs yearly are directly attributed to smoking (Epping-Jordan et al., 1998). Teenagers smoke 1.1 billion packs of cigarettes annually and will account for more than $200 billion in future health care costs (Woolf, 1997). Nicotine is the major component of tobacco that leads to addiction, but the underlying mechanisms remain unclear. This Program Project application takes a multi-level approach to understand nicotinic cholinergic systems and nicotine's effect on synaptic events, cardiovascular function, and behavior. Overall the Program will investigate the hypothesis that different nicotinic acetylcholine receptor (nAChR) subunits have special importance in the individual properties that contribute to nicotinic cholinergic functions in the nervous system. Three Projects have arisen from the production of mutant mice for nAChR subunits. The Mouse Core, headed by Dr. Beaudet, has already produced null mice lacking alpha3, alpha5, alpha7, beta3, or beta 4, and a mouse expressing in alpha7 L247T mutation that diminishes desensitization. The Morphology Core, headed by Dr. Armstrong, will do a systematic anatomical and histological screen of each mutant mouse, and will pursue further morphological investigations as warranted. The three Projects could not exist without the mutant mice, which are the fundamental tool used in each investigation. The Dani Project will directly study the altered nAChRs and the synaptic basis for nicotine's actions. The work is intended to provide basic biophysical and synaptic information that will be helpful when interpreting the results from all of the Projects. The De Biasi Project will use radio-telemetry to examine the effects of nicotine on the autonomic nervous system and cardiovascular functions. Among the specific goals is to understand how tolerance to nicotine develops more rapidly for increases in blood pressure than for increases in heart rate. The Paylor Project will use a battery of behavioral tests to examine the mutant mice with the aim of understanding how nAChR subunits regulate the sensitivity to nicotine and the development of tolerance.

DESCRIPTION: (Applicant's Abstract) Cholinergic projections are present throughout the brain, and nicotinic receptors (nAChRs) modulate multiple neurotransmitter systems. Because of these properties, nicotinic mechanisms modulate neuronal activity in broad areas of the brain. These abilities are best exemplified by the recent finding that two different single amino acid mutations in a nAChR produce epilepsy. Further support arises from a wide range of evidence suggesting that the alpha7 nAChR subunit is linked to sensory gating deficits, as observed in schizophrenic patients. Guided by these findings, our working hypothesis is that nicotinic systems modulate circuit excitability and, consequently, influence observable and testable responses. We will study nicotinic modulation of neuronal excitability with brain slices and invivo recordings from the hippocampus. Our long-standing investigations into new nicotinic synaptic mechanisms will continue, and we will examine the influence of nAChR-mediated calcium signals in common forms of hippocampal synaptic plasticity. From the same areas of the brain, we will examine circuit activity using invivo recordings from multielectrode assemblies that can isolate the firing of many individual neurons and simultaneously provide detailed multiple EEG records. The study of synaptic mechanisms and invivo recordings will provide different levels of information about the neuronal activity of the hippocampus. Because the invivo recordings are made in wake, freely moving rats or mice, we can examine neuronal activity arising from experimental tests or behavioral tasks. Cholinergic activity influences the sleep/wake cycle, spatial tasks, and sensory gating, as quantified by models of acoustic startle, but the nicotinic contributions have not been studied with combined slice physiology and multi-unit invivo recordings. Because alpha7 and beta2 are the most common nAChRs in the hippocampus, we will determine nicotinic contributions by comparing wild-type, alpha7-null, heterozygous alpha7L250T-mutant, and beta2-null mice. We have all these mice backcrossed for six generations.

Tobacco use in developed countries has been estimated to cause nearly 20% of all deaths, making it the largest single contributor to premature death (Peto et al., 1992). Nicotine is the primary component of tobacco that supports continued use, presumably exerting its behavior effects by initially acting upon nicotinic acetylcholine receptors (nAChRs). This project investigates basic properties of nAChRs at synapses, with the expectation that those properties underlie some of the systems physiology and behaviors investigated in the other two projects. The application focuses on how nAChRs respond to the changing concentrations of nicotine experienced by a smoker. As the nicotine concentrations changes, the population of nAChRs distributes among functional states (i.e. activated, desensitized, and long-term inactivated states) that may underlie the reward mediated by nicotine as well as contributing to aspects of tolerance and withdrawal. After determining quantitatively the calcium signals mediated by nAChRs, we will determine how those calcium signals may modulate hippocampal synapses. Finally, we will determine how the changing activity of nAChRs modulates the responses of ventral tegmental area neurons. Patch clamp electrophysiology, quantitative calcium measurements, and fluorescence techniques will be used to investigate the hypothesis that nicotine delivered by smoking activates and desensitizes nAChRs, which are constantly distributing among functional states. Microisland cultures and brain slices will be used to study tissue from mutant mice, which serve as the fundamental and unifying tool for this Program Project application. The work will initially utilize mutant mice lacking alpha3, alpha5, alpha7, or beta2, and the mouse containing the alpha7 (L247T) subunit, which exhibits diminished desensitization. All of these mice are presently available. Because the number of nAChRs is increased in the brains of smokers, it has been argued that addicted smokers medicate themselves with nicotine to control the level nAChR desensitization and inactivation. Thus, our studies of the changing functional states of nAChRs may have direct importance for understanding issues fundamental to nicotine addiction.

[unreadable] DESCRIPTION (provided by applicant): Nicotine from tobacco is addictive, and it is a major health problem. In developed countries, smoking is the leading cause of premature deaths. In the United States, smoking-related illness causes more than 430,000 deaths and $50 billion in medical costs annually. Nicotine addiction has consequences on the health of the US population that are greater than the combined effects of all other addictive drugs (8,63,149,150). Dopamine (DA) systems are thought to play an important role in reinforcing rewarding behaviors, and drugs of addiction, such as nicotine, can alter or commandeer those systems. We will employ a combination of in vitro and in vivo techniques to study nicotinic cholinergic mechanisms that influence DA release both at the source and the target. We also will investigate how nicotine, as obtained from tobacco, alters the normal nicotinic cholinergic mechanisms that regulate the DA systems. Aim 1 will use patch-clamp electrophysiology applied to midbrain slices (VTA/SNc) to investigate nicotinic cholinergic mechanisms that modulate synaptic plasticity in the VTA/SNc (the DA source). Examining these issues should help us to understand the short-term and longer-term nicotinic influences over the firing of the midbrain neurons that supply DA. Aim 2 will use fast cyclic voltammetry and HPLC in striatal slices to investigate nicotinic influence over DA release in a target (the nucleus accumbens, NAc). Aim 3 will use in vivo unit recording and microdialysis with HPLC to examine the relationship between VTA/SNc activity and NAc activity or DA concentration. The relationship between VTA/SNc activity and downstream consequences in the NAc is a vital component of the addiction process. The experiments will investigate how endogenous nicotinic cholinergic mechanisms and exogenous nicotine modulate the activity of DA neurons in the midbrain and influence DA release in the striatum (particularly the NAc). The studies directly investigate developing theories of "reward" arising from new results. There is a complex (and not yet understood) relationship between the activity of midbrain DA neurons and the downstream consequences at the targets. The proposed research directly addresses DA's ro1e in reward mechanisms and examines the specific actions of nicotine that contribute to addiction

DESCRIPTION (provided by applicant): Cholinergic systems provide diffuse innervation to nearly every area of the brain, and drive or modulate a wide variety of behaviors. Nicotinic acetylcholine receptors (nAChRs) have been implicated in many diseases, such as epilepsy, addiction, schizophrenia, Parkinson's disease, vascular dementia, dementia with Lewy bodies, and Alzheimer's disease. In this application, we propose experiments that are interactively coupled to computer models of synaptic function. The computer models are intended to be generalized tools that enhance the experimentalist's insights into basic synaptic mechanisms, pharmacology, and disease. We are initiating this approach by focusing on cholinomimetic drugs at nicotinic synapses. Such drugs are now the only approved treatments for mild to moderate Alzheimer's disease (AD). The working hypothesis is that the cholinergic drugs have varied mechanistic effects at nicotinic cholinergic synapses, and by affecting nAChRs, these drugs also influence the release of other neurotransmitters. Our aim is to implement this approach with three levels of interactive modeling and experimentation. At each level we have used data from the literature to develop preliminary computer models. The models produce simulations that guide the design and interpretation of the experiments. At the first level of interaction between simulations and experimentation, we apply patch-clamp electrophysiology in tissue culture and slice to determine activation and desensitization parameters for the nAChRs. These basic data supplement those from the literature, enabling us to develop reliable models of nAChR kinetics that will be used throughout this research. At the second level of interaction, we use electrophysiology to examine the pharmacology of cholinomimetic drugs at nicotinic synapses. The experiments are guided and the interpretation assisted by a model of a CNS nicotinic synapse. At the third level, we use cyclic voltammetry in striatal brain slices to examine cholinergic/dopaminergic interactions guided by a model of interacting CNS synapses. The work proposed here will serve as the basis for future extensions to network interactions among neurotransmitter systems and future applications to other neurological diseases.

DESCRIPTION (provided by applicant): Nicotinic receptor density and nicotinic function decline beyond normal aging in schizophrenia, dementia with Lewy bodies, Parkinson's disease, and Alzheimer's dementia. Evidence indicates that especially in degenerative diseases, impaired nicotinic function contributes to cognitive deficits, and nicotinic therapies have their main impact on cognitive dysfunction. Behavioral studies have identified nicotinic roles in memory, learning, and attention;and in particular, decreased nicotinic activity in the hippocampus has been linked to impaired memory. Recent advances indicate that addiction shares many commonalities with the synaptic plasticity normally attributed to learning and memory. Drugs subvert normal memory mechanisms, leading to long-lasting changes in behavior that accrue with the ongoing progression of addiction. Subsequently, environmental cues that elicit memories linked to addictive behaviors motivate cravings and relapse. The hippocampus is among the areas associated with internally generated craving. The perforant path is particularly important, relaying information that is rich in contextual and spatial content. Although behavioral and synaptic studies have examined nicotinic roles, there have been practically no in vivo physiological studies that directly link nicotinic mechanisms to the synaptic changes underlying memory. The main goal of this study is to reveal the in vivo link between nicotinic function and synaptic mechanisms of the memory process. A combination of in vivo recording approaches and in vitro physiology will examine the following working hypothesis: nicotinic cholinergic systems modulate circuit excitability, and nicotinic mechanisms influence local and long-range circuits causing synaptic changes that underlie memory. We have direct preliminary measures of in vivo perforant path synaptic plasticity arising from nicotinic action. Furthermore, our preliminary results indicate that long-range signaling from dopamine centers enables this synaptic plasticity, which arises from local nicotinic modulation of dentate gyrus circuits. The experiments monitor the strength of perforant path transmission in vivo. In addition, long-term tetrode implants are used to measure in vivo neuronal activity underlying local interactions between GABAergic interneurons and granule cells as well as long-range signaling from dopamine neurons. To gain more experimental control, cellular and synaptic mechanisms are being investigated using in vitro brain slices. These studies directly assess in vivo controls that are the basis for nicotinic involvement in cognition. The results immediately apply to associative memory of addiction, and more broadly indicate the synaptic mechanisms that likely underlie the nicotinic component of normal memory and memory dysfunction during degenerative diseases.

DESCRIPTION (provided by applicant): Cholinergic systems provide diffuse innervation to nearly every area of the brain, and drive or modulate a wide variety of behaviors. Nicotinic acetylcholine receptors (nAChRs) have been implicated in many diseases, such as epilepsy, addiction, schizophrenia, Parkinson's disease, vascular dementia, dementia with Lewy bodies, and Alzheimer's disease. In this application, we propose experiments that are interactively coupled to computer models of synaptic function. The computer models are intended to be generalized tools that enhance the experimentalist's insights into basic synaptic mechanisms, pharmacology, and disease. We are initiating this approach by focusing on cholinomimetic drugs at nicotinic synapses. Such drugs are now the only approved treatments for mild to moderate Alzheimer's disease (AD). The working hypothesis is that the cholinergic drugs have varied mechanistic effects at nicotinic cholinergic synapses, and by affecting nAChRs, these drugs also influence the release of other neurotransmitters. Our aim is to implement this approach with three levels of interactive modeling and experimentation. At each level we have used data from the literature to develop preliminary computer models. The models produce simulations that guide the design and interpretation of the experiments. At the first level of interaction between simulations and experimentation, we apply patch-clamp electrophysiology in tissue culture and slice to determine activation and desensitization parameters for the nAChRs. These basic data supplement those from the literature, enabling us to develop reliable models of nAChR kinetics that will be used throughout this research. At the second level of interaction, we use electrophysiology to examine the pharmacology of cholinomimetic drugs at nicotinic synapses. The experiments are guided and the interpretation assisted by a model of a CNS nicotinic synapse. At the third level, we use cyclic voltammetry in striatal brain slices to examine cholinergic/dopaminergic interactions guided by a model of interacting CNS synapses. The work proposed here will serve as the basis for future extensions to network interactions among neurotransmitter systems and future applications to other neurological diseases.

[unreadable] DESCRIPTION (provided by applicant): Nicotine is the main addictive component of tobacco that motivates continued use despite the harmful effects. In developed countries, tobacco use is estimated to be the largest single cause of premature death, causing approximately 440,000 deaths and more than $75 billion in direct medical costs annually in the USA. Although many areas of the brain participate, the midbrain dopamine (DA) systems serve a vital role in the acquisition of behaviors that are inappropriately reinforced by psychostimulant drugs, including nicotine. The proposed studies examine the following broad hypothesis: during the progression from casual tobacco (nicotine) use to addiction the DA systems change, contributing to the transition to nicotine abuse. The studies specifically examine the physiological changes in the DA systems that evolve as the nicotine exposure continues through time. The changes within the DA systems often use the mechanisms of synaptic plasticity that normally underlie learning and memory. Thus, an ancillary hypothesis is that nicotine induces synaptic changes of the kind that underlie drug-linked memory. Recent advances indicate that addiction shares many commonalities with the synaptic plasticity normally attributed to learning and memory. Drugs subvert normal memory mechanisms, leading to long-lasting changes in behavior that accrue with the ongoing progression of addiction. Subsequently, environmental cues that elicit memories linked to addictive behaviors motivate cravings and relapse. The three aims examine the physiological changes induced by nicotine exposure that progresses from an acute single administration to a short-term chronic exposure and, finally, to a long-term chronic exposure. In each of the three specific aims, we will use in vivo unit recordings to examine nicotine-influences over DA neuron firing patterns and to examine the relationship between DA neurons and the neighboring circuitry. The firing patterns of the DA neurons measured in vivo will guide detailed studies of nicotine modulation of DA release measured using microdialysis and fast cyclic voltammetry. In addition, for each nicotine exposure we will follow the time-course for nicotine-induced synaptic potentiation of the glutamatergic afferents onto DA neurons of the ventral tegmental area. The time course of the synaptic plasticity suggests a window of vulnerability during which DA signaling is altered by drug-associated memory. Our strategy is to investigate DA signaling in vivo, which preserves the overall intact biological systems. Then, we sacrifice some of the pertinence provided by the in vivo studies to pursue greater experimental control and greater detail using in vitro brain slices. A novel combination of physiological studies applied at multiple levels of neuronal integration will provide complementary data sets that are presently lacking within the field of nicotine addiction. PUBLIC HEALTH RELEVANCE Addiction to nicotine motivates tobacco use, which causes approximately 440,000 deaths and more than $75 billion in direct medical costs annually in the USA. This study will determine the nicotine induced short-term and long-term neuronal changes that underlie nicotine addiction. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cognitive dysfunction and decline associated with disease and aging are serious health problems. Nicotinic cholinergic and dopaminergic neurotransmitter systems contribute to the impairments that prevent many patients from entering the work force and fully socializing. For example, these neurotransmitter systems are major contributes to schizophrenia and Alzheimer's dementia, which are estimated to cost > $62 billion and > $100 billion a year respectively in the United States. Ultimately, therapeutic approaches to decrease cognitive impairments must influence pertinent neural circuits. Identifying critical neural pathways, neurotransmitters, and mechanisms will provide rational targets for therapeutic approaches to decrease cognitive impairments, produce memory enhancement, cause forgetting (e.g. in post traumatic stress disorder), or prevent forgetting (e.g. in dementia). Our preliminary results show that cholinergic activity dose-dependently induces in vivo hippocampal long-term synaptic potentiation correlated with mice learning spatial tasks or novel objects. The earlier work showed that induction of in vivo synaptic plasticity requires local disinhibition of excitatory circuits coupled with an afferent dopamine signal arriving from the midbrain. The results are consistent with the view that dopamine signals into the hippocampus lower the threshold for synaptic plasticity that underlies learning. In the proposed studies, we will examine the basis for the nicotinic/dopaminergic influences, and will investigate the following working hypothesis: The dopamine signal lowers the threshold and regulates the magnitude of synaptic plasticity underlying learning. Furthermore, the dopamine signal enhances learning associations among environmental events because dopamine causes a broader timing window for the induction of synaptic plasticity. Dopamine normally contributes to the efficient learning of appropriate behavioral responses motivated by environmental cues. The working hypothesis, however, also helps to explain the cognitive dysfunctions that arise during dopamine signaling imbalances found in diseases where inappropriate sensory gating, attention, and learning produce maladaptive behavior. A multidisciplinary approach applied to wild-type and strategically prepared mutant mice will cross neural levels of integration to understand the synaptic mechanisms underlying performance of behavioral tasks. While mice run behavioral tasks, endogenous cholinergic and dopaminergic signals will be controlled and in vivo synaptic plasticity will be measured in real time. Guided by the in vivo results, brain slices will be cut from these same pertinent neural areas to understand the mechanisms that control synaptic plasticity and learning. The delineated mechanisms within these critical neural circuits will provide targets for developing therapeutic strategies that diminish cognitive impairments. Future research will examine therapeutic interventions targeted to the characterized mechanisms using this mouse model platform.

DESCRIPTION (provided by applicant): Tobacco and alcohol are the most frequently used and co-abused drugs, with smokers being ten times more likely to develop alcohol-related disorders. Despite the consistent positive correlation between smoking and subsequent alcohol abuse, the physiology that underlies the interaction between tobacco and alcohol use remains remarkably under studied. Individuals disposed to alcohol abuse often exhibit less sensitivity and less intoxication after moderate alcohol drinking. Based on dopamine's crucial role in drug reinforcement, it has been hypothesized that individual differences in dopamine signaling contribute to variations in alcohol sensitivity. Our preliminary data show that pre-exposure to nicotine decreases the dopamine response arising from alcohol (i.e., lower sensitivity). Correlated with the smaller alcohol-induced dopamine signals, acute pre-exposure to nicotine increases alcohol self-administration. The literature and our preliminary results led us to the following working hypothesis: exposure to nicotine at a concentration obtained from tobacco decreases the responsiveness of the mesolimbic dopamine system to alcohol and increases the vulnerability to alcohol abuse. We will measure the consequences of nicotine pre-exposure on alcohol-induced dopamine responses, particularly during alcohol self-administration. We use a powerful array of in vivo and in vitro techniques to measure the consequences of nicotine pre-exposure upon the dopamine system during non-contingent alcohol administration and during operant alcohol self-administration. After nicotine pretreatment, we measure the responsiveness of the mesolimbic system, including dopamine neuron activity, dopamine release, and dopamine re-uptake. We also examine the underlying cellular and synaptic events. The methodology includes in vivo microdialysis and multi-tetrode unit recordings during alcohol self-administration. In that way, midbrain neural activity and dopamine signaling are being precisely linked to the complete repertoire of behaviors during alcohol self-administration. To obtain greater experimental control, brain slices are cut from those same neural areas to investigate synaptic level mechanisms. The proposed studies cross neural levels of integration, from synaptic and cellular to systems and behavior, providing novel kinds of data that have not previously been obtained during the biologically relevant protocol of alcohol self-administration. Understanding the neural circuits and the mechanisms that drive those circuits will provide a valuable foundation for developing strategies that diminish the propensity for alcohol abuse caused by smoking. The aim is to examine the circuit elements and their interactions as freely-moving rats self-administer alcohol. Upon completing this study, this highly relevant animal model (monitored with our powerful array of techniques) will serve as an ideal testing platform to understand how therapeutic interventions influence the circuitry that drives alcohol abuse. Future therapeutic strategies can be rationally developed and tested based on biological processes that underlie nicotine's influence upon alcohol use.

DESCRIPTION (provided by applicant): Genome-wide association studies identified a nonsynonymous SNP (rs16969968) within the CHRNA5 gene that encodes for the ¿5 nicotinic receptor (nAChR) subunit. This SNP produces a twofold higher risk for heavy smoking and increases the risk for lung cancer. Consistent with the human genetics, our preliminary studies showed that the ¿5 nAChR is necessary for the expression of the nicotine withdrawal syndrome. The ¿5 nAChR also regulates sensitive to nicotine-induced behaviors and controls nicotine self-administration at high doses. In these proposed studies, we will investigate ¿5's mechanistic action on the midbrain dopamine (DA) systems that reinforce rewarding and addictive behaviors. We will examine the effect of the rs16969968 SNP on DA signaling from its source in the midbrain to its main targets in the striatum, including the nucleus accumbens (NAc) which is important for processing reward. Our in vivo multi-tetrode recordings show that nicotine increases the phasic burst firing of DA neurons, and our cyclic voltammetry and in vivo microdialysis data show that nicotine-induced changes in DA neuron firing are translated in a target-specific manner in areas that process reinforcement and reward, including the NAc core and shell. However, little is known about the role of the ¿5 subunit or the rs16969968 SNP and about the role of the ¿5-subunit in regulating the relationship between DA neuron firing and DA release in targets. Our working hypothesis is that nicotine acts via the ¿5-nAChR subunit to modulate DA signaling both at the source (i.e., DA neurons in the midbrain) and at the targets (including the NAc and the dorsal striatum). The aims examine the hypothesis that nicotine-induced changes in the DA system evolve during chronic nicotine exposure and during the withdrawal period. The significance of the study originates from the expectation that these nicotine-induced activities and changes in the DA system contribute to the transition from initial nicotine use to addiction. Tobacco use remains the leading cause of preventable death in the United States, and these studies provide a mechanistic basis for nicotine addiction and for developing therapies to aid smoking cessation.

Cognitive deficits associated with neuronal dysfunction and aging constitute a serious health problem. Dopaminergic systems contribute to a number of cognitive disorders, such as schizophrenia, Alzheimer's dementia, and Parkinson's disease, which cost > $200 billion a year in the USA alone. Successful therapeutic approaches to alleviate cognitive problems should target appropriate neural circuits in the brain. Basic neuroscience research provides that information necessary to identify the networks, neurotransmitters, and mechanisms that underlie proper brain function and are the targets for potential therapies. Proper dopaminergic signaling is essential for cognitive processes such as attention, executive function, learning, and memory. The complex nature of these processes and the paucity of synaptic and cellular data linked to the systems-level behaviors have spurred the proposed studies. Our earlier work showed that induction of in vivo synaptic plasticity associated with a learning task requires local disinhibition of excitatory circuits coupled with an afferent dopamine signal. Recent results from our lab support the view that dopamine signaling in the hippocampus lowers the threshold for synaptic plasticity that underlies learning. Our preliminary results show that local dopaminergic activity is required for in vivo hippocampal long-term synaptic potentiation associated with diverse learning paradigms, such as aversive memory retention and novel object recognition. Presently however, there is a controversy regarding the source, density, and significance of hippocampal dopaminergic innervation and about dopaminergic regulation of synaptic plasticity and memory. In the proposed studies, we will identify the sources of dopaminergic neurotransmission in the hippocampus using multiple independent viral labeling methods. Then, we will examine dopaminergic influences over distinct hippocampal circuits during specific memory tasks. Our working hypothesis is that dopamine acts within critical time windows and controls the magnitude of synaptic plasticity within specific circuits that regulate different types of learning. Dopamine normally contributes to the efficient learning of appropriate behavioral responses motivated by environmental cues. The working hypothesis, however, also helps to explain the cognitive dysfunctions that arise during dopamine signaling imbalances found in diseases where inappropriate sensory gating, attention, and learning produce maladaptive behavior. A multidisciplinary approach that crosses neural levels of integration will be applied to understand the synaptic mechanisms underlying aversive memory retention and novelty detection. We will use an array of anatomical tracing and analytical techniques to determine the origin of dopamine signals that act upon hippocampal circuits. During the performance of behavioral tasks, these endogenous dopaminergic signals will be temporally controlled using optogenetic approaches, and in vivo synaptic plasticity will be measured in real-time. The delineated mechanisms within these critical neural circuits will provide targets for developing future therapeutic strategies that diminish cognitive impairments.

Project Summary. Excessive alcohol use is among the leading causes of preventable death worldwide and costs the USA over $223 billion a year. Stress and nicotine (from tobacco) are well-known risk factors for heavy alcohol consumption and alcohol use disorders. Despite the consistent human epidemiological evidence, in experimental models stress does not always produce increased alcohol consumption. The controversy arises, in part, because the neural mechanisms underlying the interactions among stress, nicotine, and alcohol remain significantly unknown. This proposal arose from our preliminary results showing that pre-exposure to acute nicotine or stress under strictly defined experimental conditions increases alcohol self-administration. We showed in rats that nicotine (like stress) boosts corticosterone levels in rats. Nicotine- or stress-induced glucocorticoid receptor activity is necessary for the subsequent increase in alcohol self-administration that arises owing to altered midbrain GABAergic circuitry. When we inhibited the nicotine/stress-induced glucocorticoid signaling or corrected midbrain GABAergic dysfunction, then alcohol self-administration returned to control levels. In the proposed studies, we will move from the simple acute exposures to nicotine and stress to more biologically realistic experimental situations. Lifetime nicotine and tobacco use almost always begins during adolescence, and adolescent smoking and childhood maltreatment are both high risk factors for increased alcohol consumption and alcohol use disorders in adulthood. Therefore, we will expose adolescent rats to nicotine or stress then allow the animals to age before analyzing their alcohol drinking behavior compared to control rats. Our preliminary results with adolescent nicotine treatments show that later in life, the adolescent- treated rats do drink more alcohol. Furthermore, the increased drinking requires glucocorticoid activity and arises from changes in midbrain GABAergic circuitry. We will measure the consequences of adolescent stress or nicotine exposure in adult rats during initiation, maintenance, extinction, and re-instatement of alcohol self- administration. The experiments will go on to investigate general circuit mechanisms underlying the increase in alcohol self-administration induced by adolescent nicotine or stress. Initially, we will be guided by our recent results indicating that a single, acute exposure to nicotine or stress induces an increase in alcohol self-administration by altering midbrain GABAergic circuitry. Then, guided by our preliminary results we will prevent or reverse the increased drinking caused by adolescent stress or nicotine via molecular and pharmacological manipulations within the midbrain. An aim is to identify a target and test a potential therapeutic drug to aid against increased alcohol consumption.

Project Summary. Cocaine is the most widely abused psychostimulant by a wide margin, and it remains a major public health problem in the US. Cocaine use was slowly declining, but in recent years there has been a resurgence in cocaine abuse accompanied by a sharp increase in cocaine-related hospitalizations and deaths. These facts highlight the need for effective medications for cocaine use disorder (CUD) because there are presently no FDA- approved pharmacologic treatments for CUD. Our exciting preliminary results highlight a novel molecular substrate that could be targeted to attenuate or prevent cocaine taking and seeking. Specifically, we show that cocaine exposure alters the expression of KCC2, a K+-Cl- cotransporter that defines the Cl- gradient in midbrain GABA neurons. Importantly, this cocaine-induced neuroadaptation is associated with circuitry changes in midbrain GABA neurons that promote and elevate further cocaine taking. These findings support the working hypothesis that initial cocaine taking alters midbrain GABAergic circuitry and increases the vulnerability for increased cocaine consumption over time. Thus, KCC2 represents a potential therapeutic target to treat CUD. KCC2 is expressed primarily in the central nervous system, and it is amenable to therapeutic manipulation in humans. KCC2 is highly attractive as a therapeutic target because it is usually constitutively highly active. Therefore, when normal subjects are treated with KCC2 activators, KCC2 activity is already high, such that attempts to increase its activity further do not produce deleterious side effects. Under normal physiological conditions, KCC2 maintains a low intra-neuronal Cl- concentration required for hyperpolarizing, inhibitory GABAergic currents. Our preliminary results indicate that cocaine dose-dependently downregulates KCC2 function in midbrain GABA neurons, thereby altering midbrain GABAergic circuitry. As a consequence of these circuitry changes, downregulation of KCC2 leads to increased cocaine self-administration. That is, cocaine use itself, by downregulating KCC2, perpetuates heavy cocaine self-administration. Our preliminary results indicate that if we prevent KCC2 downregulation or correct KCC2 function, then we decrease cocaine self-administration. The overall goal of this proposal is to characterize the functional state of the midbrain GABAergic circuitry and the disposition of KCC2 function during cocaine self-administration, extinction, and the reinstatement of cocaine seeking (Aims1 & 2). At each phase of the addiction cycle, we will determine the functional state of the midbrain GABAergic circuitry as a causal contributor to cocaine taking or seeking. Finally, we will apply two mechanistically different pharmacotherapies to boost KCC2 function to decrease cocaine self-administration and cocaine-seeking behavior during abstinence (Aim3). These translationally-relevant studies will test potential therapeutic drugs acting to boost KCC2 function to mitigate enhanced cocaine self-administration induced by cocaine itself. The proposed studies of KCC2 as a novel therapeutic target to mitigate CUD are timely, highly significant, and appropriately aimed at the factors underlying the transition to heavy cocaine use.