Martin Sarter - US grants

The proposed research will focus on two different interactions between benzodiazepine receptor ligands and age-related behavioral and neuronal changes. First, although benzodiazepine receptor agonists (BZRa) are the most often used psychotropic drugs in the elderly, and are known to impair cognitive abilities, their interactions with age-related behavioral and neuronal changes have yet to be characterized. The proposed research will test the hypotheses that BZRa and aging act synergistically to compromise attentional abilities. We will determine whether this interaction is based on the GABAergic innervation of functionally declining cholinergic neurons originating in the basal forebrain and innervating cortex. Second, benzodiazepine receptor ligands that exert effects opposite to BZRa (i. e. , benzodiazepine receptor antagonists/ partial inverse agonists) have been demonstrated to attenuate behavioral impairments associated with disruptions in cholinergic systems. The proposed research will extend these findings to the attentional impairments associated with normal aging, and will test the hypothesis that the beneficial behavioral effects of such treatments are mediated via an increase in cortical acetylcholine release. These effects are presumably based on an inhibition of normal GABA-cholinergic interactions in the basal forebrain. These experiments will employ computerized operant conditioning techniques for the testing of attentional abilities in rats of different ages, systemic and intracranial administration of drugs in freely moving animals, and microdialysis for the measurement of cortical acetylcholine release. Thus, the proposed research will contribute to the understanding of the behavioral and neuronal consequences of the use of BZRa in the elderly, and will outline the behavioral and neuronal potential of a novel pharmacological approach for the treatment of age-related cognitive impairments.

Benzodiazepine receptor agonists (BZRa) are well known to induce anterograde amnesia in humans and animals. It has been speculated that changes in stimulus attention and processing represent a major component of this disruption of learning. Indeed, BZRa have been demonstrated to impair the ability to attend to stimuli, particularly in situations which require the subject to detect rarely and unpredictably occurring signals. The proposed research will examine the hypothesis that the effects of BZRa on vigilance represent a specific pharmacological property which is dissociable from effects on locomotor activity and sedation. The major part of the proposed experiments will test the hypothesis that the attentional and locomotor effects of BZRa and partial agonists are mediated via anatomically distinct areas of the brain. The search for these brain areas will be initially guided by findings which suggest that areas preferably enriched with BZ1-receptor subtypes (e.g., globus pallidus) are involved in the locomotor effects, whereas regions characterized by high densities of BZ2-binding sites (e.g., dentate gyrus) mediate the attentional impairments produced by compounds such as chlordiazepoxide (CDP). Rats will be trained in visual signal detection tasks. The effects of systemic administration of the BZ 1-selective zolpidem, the non-se dative partial agonist Rol6-6O28, and the full agonist CDP will be studied in order to characterize the components of the vigilance impairment typically induced by BZRa. Using the technique of intracranial injections in behaving animals, the effects of bilateral infusions of these compounds on task performance will be studied. The regions selected include the globus pallidus, dentate,gyrus, basolateral amygdala, caudate nucleus, substantia innominata, and locus coeruleus. This research will extend our understanding about the behavioral and neuronal mechanisms involved in the effects of BZRa and contribute to the characterization of GABAergic systems involved in these effects.

Attentional processes, such as stimulus selection and stimulus processing, represent critical stages of information processing. Attentional dysfunctions have been considered major components, and even causes, of the cognitive impairments of senile dementia and schizophrenia. Furthermore, normal aging is associated with impairments in attentional abilities. The goal of the proposed research is to determine the role of cortical cholinergic afferents (which originate in the basal forebrain) in sustained and divided attention. The proposed experiments will utilize novel, validated behavioral paradigms for the test of attentional abilities in rats. Results from our previous experiments have suggested that the GABAergic innervation of basal forebrain neurons represents a major anatomical substrate mediating the bidirectional effects of benzodiazepine receptor (BZR) agonists and inverse agonists, respectively, on behavioral measures of learning and memory, and on cortical acetylcholine (ACh) release (measured by in vivo microdialysis). The proposed experiments will test the hypothesis that the basal forebrain GABA-cholinergic link mediates the attentional effects of BZR ligands. Furthermore, it will be determined whether cortical cholinergic deafferentation (achieved by infusions of the immunotoxin 192 IgG-saponin into cortical areas) result in impairments in attentional abilities. A third series of experiments will test the hypotheses that age-related impairments in attentional abilities are due to functional impairments in cortical cholinergic afferents and, thus, can be attenuated by infusions of BZR inverse agonists into the basal forebrain. Taken together, these experiments will determine the role of basal forebrain GABA-cholinergic interactions in general, and of cortical cholinergic projections originating from this area in particular, in attentional abilities. Furthermore, this research will examine the hypothesis that the effects of BZR ligands on vigilance and brain information processing capacity are mediated via cortical cholinergic afferent projections. Finally, hypotheses about a major neuronal substrate of the age-related impairments in attentional abilities will be tested, and a pharmacological approach for the treatment of these impairments will be evaluated. Generally, the findings from these experiments will advance our understanding about the neurobiological mechanisms underlying attentional functions.

This proposal covers three main research areas: (I) the behavioral functions of frontal cortex acetylcholine (ACh), particularly its involvement in attentional abilities; (II) the bidirectional modulation of attentional abilities and of cortical ACh release by compounds that regulate GABAergic transmission (i.e., benzodiazepine receptor [BZR] agonists and selective inverse agonists), and the determination of the neuronal substrates of these GABA-cholinergic interactions and of the cognitive effects of BZR-ligands; (III) the age-related changes in attentional abilities and in the regulation of cortical ACh release, and the BZR-agonist-induced exacerbation and the BZR-selective inverse agonist-induced attenuation of these effects of age. Methodologically, experiments will be conducted using operant behavioral paradigms for the measurement of sustained and divided attention, the technique of bilateral intracranial infusion of compounds in performing animals, and the microdialysis technique for the measurement of ACh release in awake, freely moving animals. Furthermore, as our previous data have indicated that BZR-mediated modulation of cortical ACh release is a function of the activational status of cortical cholinergic afferents, interactions between attentional performance, the effects of BZR-ligands, and cortical ACh release will be studied by dialyzing animals while they perform in attentional tasks (or in behavioral procedures that control for the sensory effects of stimuli and for the motor components of the response requirements). Taken together, the results from this research will add to our understanding of the neuronal mechanisms of attentional abilities and of the behavioral and neuronal components of BZR-agonist-induced impairments of these abilities. Furthermore, this research will test hypotheses about the neuronal mechanisms involved in the increased vulnerability of the elderly to the detrimental cognitive effects of BZR- agonists. Finally, the proposed experiments will examine the hypotheses that the treatment with BZR-selective inverse agonists results in the facilitation of attentional abilities, and that the beneficial behavioral effects of such compounds depend on their potency to enhance the ability of cortical cholinergic afferents to respond to stimulation, particularly in aged subjects.

The present proposal represents a competitive renewal application. The proposed research focuses on the effects of age on the regulation of activity and functions of the cortical cholinergic afferent system. The research conducted during the previous funding period revealed new insights into the effects of age on the regulation of cortical acetylcholine (ACh) release and attentional abilities. In contrast to the rather limited effects of age per se on cortical ACh release and on attentional functions, our recent experiments on the effects of age in animals with an experimentally-induced partial decrease in the density of cortical cholinergic inputs. While the deafferentiation-induced decrease in baseline cortical ACh release did not differ between young and aged animals, and while stimulated cortical ACh release in young- deafferented animals was identical to that from young-sham-deafferented and aged/sham-deafferented animals, aged/deafferented animals exhibited a strongly attenuated increase in cortical ACh release in response to behavioral and neuropharmacological stimuli. These data suggest that age acted as an intervening variable in deafferented animals, resulting in an age-related attenuation of the capacity of residual cholinergic inputs to the cortex to respond to stimulation. The proposed research will test hypotheses about the neuronal mechanisms mediating the interactions between the effects of aging and partial deafferentation on ACh release, and about the significance of such interactions for the attentional abilities of aged rats. As age-related cognitive disorders may derive from interactions between the effects of age and pre-existing neuropathological processes, and as the decline in cortical cholinergic function remains a primary neuropathological candidate in the development of age-related cognitive disorders, this research will reveal important insights in the neuronal processes underlying the effects of age on the functions of a compromised cortical cholinergic input system.

DESCRIPTION: The goal of this research is to determine the cognitive functions of cortical acetylcholine (ACh). Cortical ACh is hypothesized to mediate attentional processes, specifically sustained attention. The proposed experiments will assess the attentional functions of cortical ACh by measuring cortical ACh release and single unit activity in rats while they perform a task designed and validated for the measurement of sustained attention. Our preliminary experiments demonstrated that increased demands on sustained attention, caused by the presentation of distractors and indicated by specific impairments in performance, are associated with increases in ACh release in the medial prefrontal cortex. Additionally, attention-associated increases in neuronal activity were observed and blocked by the removal of cholinergic inputs to the recording area (produced by an infusion of the cholinotoxin 192 IgG-saporin into the recording field). The proposed research will determine the role of cortical ACh in sustained attention by : 1) developing a series of distractors which serve to systematically vary the demands on attention; 2) demonstrating that the attentional performance under taxing conditions depends critically on the integrity of the cortical cholinergic afferent system; 3) demonstrating that increased demands on attention are associated with increases in medial prefrontal ACh release and predictable shifts in single unit activity; 4) demonstrating that the attention-associated increases in neuronal activity are blocked by loss of cholinergic inputs to the recording area; and 5) demonstrating that infusions of a benzodiazepine receptor agonist and an inverse agonist block and augment, respectively, the attentional performance-associated increases in cortical ACh efflux and neuronal activity. Collectively, the presynaptic (i.e., ACh release) and postsynaptic (single unit activity) measures of attention-associated changes in cortical ACh will yield a specific hypothesis about the role of cortical ACh in attention. The determination of the specific cognitive functions of cortical ACh will lead to a better understanding of the disastrous cognitive consequences of deviations in the integrity of cortical cholinergic afferents.

[unreadable] DESCRIPTION (provided by applicant): Cognitive variables are associated with the development of the positive symptoms of schizophrenia. Specifically, persistent impairments in the ability to filter irrelevant stimuli and associations, and in related executive functions such as set switching, planning, and dual-task performance, have been hypothesized to impede the patient's efficacy in using and updating past experiences to interpret and properly respond to current inputs. The proposed research will test the general hypothesis that increases in dopaminergic transmission in the nucleus accumbens (NAC), now widely accepted as a neuronal hallmark of schizophrenia, contribute transynaptically to the increases in (re)activity of cortical cholinergic inputs, and that the increased cortical cholinergic transmission mediates attentional impairments. Preliminary data demonstrate that the repeated administration of amphetamine (AMPH), a procedure that sensitizes the mesolimbic dopaminergic system, results in augmented increases in cortical acetylcholine (ACh) efflux in naive animals. Furthermore, repeated AMPH exposure results in attentional impairments in rats tested in a sustained attention task. Partial loss of cortical cholinergic inputs attenuated the attentional consequences of repeated AMPH administration. Additional preliminary data support the hypotheses that cortical ACh efflux is modulated by the NAC, presumably via projections to the basal forebrain, and that cortical ACh efflux and attentional performance are regulated by basal forebrain GABAergic and glutamatergic mechanisms. The proposed experiments will test hypotheses about the effects of repeated AMPH on attentional performance-associated cortical ACh efflux, the necessity of cortical cholinergic inputs in the manifestation of repeated AMPH-induced attentional impairments, and about basal forebrain afferent neuronal circuits that mediate the effects of repeated AMPH on attentional performance and performance-associated cortical ACh efflux.

DESCRIPTION (provided by applicant): This is a competitive renewal application for a K02 award. During the prior award period, the candidate's research focused on the regulation and function of cortical cholinergic inputs. Results substantiated hypotheses about the mediation of attentional functions by cortical cholinergic inputs, the regulation of this system by basal forebrain afferents, including projections originating in the nucleus accumbens and prefrontal cortex. Furthermore, hypotheses about the contribution of abnormally regulated cortical cholinergic inputs to the cognitive symptoms of major neuropsychiatric disorders were developed and tested. This research utilized a broad range of behavioral neuroscience methods, including operant procedures designed for the assessment of different aspects of attention, and the measurement of acetylcholine (ACh) efflux and neuronal activity in task-performing animals. In addition to continuing research in these areas, three new research avenues are proposed for further development during the next award period. These activities will focus on (1) the validation of a biosensor method for the rapid measurement of choline concentrations as a measure of ACh release with high temporal resolution, and the integration of this method into our main research program, (2) the investigation of interactions between the effects of early-life disruption of trophic factor support of cortical cholinergic inputs, transient ischemic attacks, and aging on the regulation and function of cortical cholinergic inputs, and (3) on the development of a task to be used in a neuropsychological and psychopharmacological research program designed to test hypotheses about the role of the cholinergic system in mediating impairments in the ability to divide attention between competing cognitive demands in humans. The renewal of the KO2 would allow this candidate to devote at least 75% of his time for multidisciplinary research on the neurobiology of attentional functions.

[unreadable] DESCRIPTION (provided by applicant): This Small Grant Application requests support for research involving the development of new research technology and research approaches. The proposed research combines experiments designed to validate further the use of a novel amperometric choline detection method for the assessment of acetylcholine (ACh) release with studies that will explore the usefulness of this methodology for the test of hypotheses about the functions of plastic, long-term changes of the excitability of the cortical cholinergic input system. The initial experiments will examine the relationships between electrical, including tetanic, stimulation of the cholinergic basal forebrain (BF) and evoked choline signals in the auditory cortex. Additional experiments will determine that evoked choline signals reflect choline derived from hydrolysis of ACh, and from ACh released depolarization-dependently. A second series of experiments will employ this methodology to test hypotheses about the role of cortical ACh release in the mediation of the long-term plastic changes of BF stimulation on auditory cortical input processing. Collectively, this research will achieve two major goals. First, it will generate essential evidence concerning the validity and usefulness of the choline biosensor for the assessment of ACh release at high temporal (milliseconds to seconds) and spatial (um) resolution. Second, present techniques for the assessment of ACh release are of limited use for research requiring information about stimulus-evoked changes in ACh release; however, such information is essential for the test of hypotheses about the role of cholinergic transmission in mediating plastic changes in sensory input processing. The proposed research will test the viability of using the choline microelectrode technique to address this central issue. Moreover, the expected results will be useful for future investigations about the role of the cortical cholinergic input system in modulating the attentional processing of stimuli that acquire behavioral, specifically affective, significance. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This R21 application seeks support for the development of a novel screening procedure for candidate therapeutics for the treatment of the cognitive symptoms of neuropsychiatric and neurodegenerative disorders. Productive and informative screening procedures for the detection and characterization of the pro-cognitive and pro-cholinergic properties of putative treatments have remained scarce. The proposed project is designed to investigate the usefulness of a new in vivo screening procedure for the detection and characterization of cognition enhancers acting by, at least in part, increasing prefrontal cholinergic activity. This procedure allows the demonstration of transient cholinergic activity evoked by cues triggering attentional processes. The modulatory effects of candidate drugs on cue-evoked cholinergic activity ("cholinergic footprints") and associated effects on performance are hypothesized to serve as effective screening tools for cognition enhancers. The experiments of Aim 1 will demonstrate that differential ""cholinergic footprints"" are generated by major reference compounds, including amphetamine, nicotine, d-cycloserine and thioperamide. Aim 2 will demonstrate that this screening procedure can be employed to characterize and differentiate the effects of antipsychotic drugs hypothesized to exhibit differential pro-cognitive effects in an animal model of schizophrenia. Future screening of compounds with differential affinity to receptor subtypes, such as subtypes of cholinergic, noradrenergic, or glutamatergic receptors, will demonstrate that differential "cholinergic footprints" are generated by receptor subtype-selective compounds. Furthermore, the characteristics of an "optimal cholinergic footprint" for receptor subtype-selective compounds will be defined for use as a reference for further screening. The long-term goal of this research is to demonstrate that the proposed experimental paradigm serves as an effective screening test for use in programs designed to develop cognition enhancers. Thus, this research will contribute to the development of more effective preclinical strategies for the detection and development of cognition enhancers. Relevance The development of treatments for the cognitive impairments associated with schizophrenia, the dementias and other neuropsychiatric and neurodegenerative disorders has been lacking informative, effective and productive preclinical methods for the detection and characterization of new treatments. As efforts to develop cognition enhancers have focused on drugs modulating forebrain cholinergic neurotransmission, the proposed research is designed to develop and validate a new in vivo screening method for cognition enhancers acting via stimulating prefrontal cholinergic activity. The development of new drug finding [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Administration of nicotine and nicotinic acetylcholine receptor (nAChR) subtype-selective agonists benefit the behavioral and cognitive symptoms of patients with ADHD, schizophrenia, senile dementia and other disorders. However, the neuropharmacological mechanisms underlying the cognitive effects of nAChR agonists have remained unsettled. Cortical nAChRs are situated predominantly on presynaptic terminals and stimulate the release of several neurotransmitters, including acetylcholine (ACh). This research is guided by the general hypothesis that the beneficial attentional effects of nAChR agonists are mediated via stimulation of acetylcholine (ACh) release in the prefrontal cortex (PFC). Preliminary studies utilized enzyme-selective microelectrodes to monitor ACh and glutamate release at a high temporal resolution. Administration of nAChR agonists produced transient increases in ACh and glutamate release. Alpha-4/beta-2 selective nAChR agonists yielded more potent and "sharper" cholinergic signals than nicotine; these signal characteristics are hypothesized to underlie the robust pro-cognitive properties of these compounds. The amplitudes of cholinergic signals depended on ionotropic glutamate receptor activity. In contrast, the slower temporal dynamics of nicotine-evoked cholinergic signals did not seem to be mediated via glutamatergic mechanisms. Preliminary evidence also indicates that in task-performing animals, cues that trigger attentional processes evoke transient increases in cholinergic activity in the PFC and that nicotine administration augmented the amplitude and slowed the decay of cue-evoked cholinergic signals. This research will test hypotheses concerning the neuropharmacological mechanisms mediating the effects of nAChR agonists on cholinergic activity in the PFC, nAChR agonist-induced modulation of attentional cue-evoked cholinergic activity in performing animals, and the cognitive conditions under which beneficial cognitive effects of nAChR agonists are optimally revealed.Narrative/Relevance [unreadable] [unreadable] Alterations in the regulation and expression of nicotinic receptors and abnormal regulation of cholinergic neurotransmission have been suggested to contribute to the cognitive symptoms of several neuropsychiatric and neurodegenerative disorders, including schizophrenia, autism and dementia. The proposed research is expected to demonstrate that the beneficial cognitive effects of nicotine and nicotinic receptor subtype- selective agonists are mediated primarily by modulation of attentional performance-evoked cholinergic activity in the PFC. This research will reveal critical neuronal and cognitive mechanisms underlying the pro-cognitive effects of nicotinic receptor ligands and thereby assist in defining and predicting the clinical potential of this group of compounds. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application proposes interdisciplinary research on the regulation and function of the high-affinity choline transporter (CHT). The CHT imports choline for the synthesis of acetylcholine (ACh) into cholinergic neurons and thereby controls the capacity of cholinergic neurons to sustain increases in cholinergic neurotransmission. Choline uptake is primarily regulated by the density of CHTs in synaptosomal plasma membrane. The rates of CHT internalization and outward trafficking determine the density of CHTs in plasma membrane. Accumulating evidence indicates that these intracellular CHT transport mechanisms are highly regulated by diverse signaling pathways. This research will test the general hypothesis that CHT capacity limitations constrain the ability of cholinergic neurons to mediate heightened demands on cognitive activity, specifically motivated, attentional performance under challenging conditions. We will study mice expressing a reduced level of CHTs and exhibiting an attenuated capacity of cholinergic neurons to sustain increases in ACh release, intact rats following the blockade of CHT-mediated choline uptake in prefrontal cortex, and humans heterozygous for a variant of the CHT that reduces choline transport capacity by 40-50%. This research will employ molecular, neurochemical, neuropsychological and neuroimaging techniques in order to determine the cellular and neuronal mechanisms that limit CHT capacity in situations that tax cholinergic functions and thereby limit cognitive capacity. Results are expected to demonstrate that a reduced capacity for CHT-mediated choline uptake robustly attenuates the recover of attentional performance after performance challenges, and that such impaired performance is mediated via insufficient levels of prefrontal cholinergic neurotransmission (rodents) and insufficient activation of right prefrontal cortex (humans). Collectively, this research will determine the neuronal mechanisms that constrain behavioral and cognitive capacities, reveal neuronal mechanisms that contribute to cognitive decline, and define new targets for the development of preventive and symptomatic treatments for the cognitive symptoms of neuropsychiatric and neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: The abnormal regulation of the cortical cholinergic input system plays a major role in the manifestation of the cognitive impairments of neuropsychiatric and neurodegenerative disorders, specifically schizophrenia, dementia and other age-related cognitive impairments. The high-affinity choline transporter strongly influences the capacity of this neuronal system to sustain elevated levels of activity. This research will utilize a wide range of experimental approaches and conduct research in mice, rats, and humans to determine how the choline transporter is regulated and how this transporter limits cognitive capacity. The results from this research are of direct significance for hypotheses concerning the role of this major neuromodulator system in cognitive disorders and for the development of new treatments for such disorders.

In substance abusers, the propensity to attribute incentive salience to cues and allow these cues to capture attention is thought to be causally related to response inhibition deficits, including cognitive and motor impulsivity, Aim 1 of Project 4 will characterize the attentional capacities of Sign Trackers (STs) and demonstrate that compared to Goal Trackers (GTs), STs exhibit a limited capacity to sustain attention and a propensity for impulsive responses. STs' attentional performance is hypothesized to be due to impaired top- down control mechanisms, including ineffective modification of perfonnance in response to errors and reward loss. Aim 2 concerns the prefrontal cholinergic mechanisms that mediate the relatively poor attentional performance of STs. Preliminary evidence is consistent with the hypothesis that prefrontal giutamatergic-cholinergic interactions are differently regulated in STs and contribute to their comparatively lower attentional performance. Aim 3 will demonstrate that blocking a specific node in this circuit, presynaptic alpha4beta2* nAChRs expressed by thalamic afferents, causes ST-like performance impairments in GTs. Conversely, stimulation of alpha4beta2* nAChRs improves the attentional performance of STs. Aim 4 will extend this research to the impact of cues that were associated with cocaine self- administration and are capable of producing addiction-like behavior and relapse. The presence of such a drug cue is expected to abolish the ability of STs to sustain cognitive performance, in part as a result of cue- evoked, excessively large and lasting prefrontal cholinergic transients that interfere with the detection of task signals. These drug cue-evoked, excessive release events also contribute to the relative inability of STs to re-engage in cognitive task perfonnance. Collectively, this research is guided by the hypothesis that lower levels of cognitive control combined with attentional bias toward drug-related cues are key cognitive traits in individuals vulnerable for addiction. The neurochemical mechanisms underiying the detrimental efTicacy of the drug cue in STs form the basis of therapeutic interventions designed to limit dmg-cue evoked behavior and foster re-engagement in cognitive task performance (Aim 4). RELEV/VNCE (See Instructions): Addiction is a major public health problem in the United States. The goal of this Project is to use a preclinical model to delineate the psychological and neurobiological basis of individual differences in vulnerabilty to develop addiction-like behavior, as this will help identify risk factors that will aid in the development of targeting interventions and treatments.

Project I: Summary/Abstract About two thirds of patients with Parkinson's disease (PD) experience falls. Falls in PD patients are a primary cause of hospitalization and nursing home admission. These debilitating features of PD are resistant to dopamine replacement therapy, emphasizing the critical need for basic research and therapeutic development focused on non-dopaminergic systems that degenerate in PD. The main goals of this research are to determine the role of cholinergic cell loss in the basal forebrain and brain stem pedunculopontine nucleus for the propensity for falls in PD. We developed a novel behavioral apparatus (?MCMCT?) that requires rats to traverse rotating surfaces. This task requires exquisite gait control, including precisely timed placement of weight-shifting steps. Furthermore, we developed a rodent model of the multisystem cholinergic-dopaminergic losses hypothesized to cause falls in PD. The available evidence suggests that dorsal striatal dopaminergic deafferentation impairs the performance of complex behavioral sequences such as gait, resulting in the manifestation of risk factors for falls. This places additional demands on the attentional control of such behaviors. Loss of cholinergic projections impairs the attentional supervision of gait and postural control, thereby contributing to the onset of levodopa-resistant abnormalities of gait and posture, and to falls. PPN cholinergic loss further dysregulates striatal circuitry, thereby increasing the severity of impairments in gait and posture and thus the propensity for falls. The proposed research will determine (1) the cognitive-motor and neuronal mechanisms that underlie falls, (2) the potential usefulness of a rationally-derived treatment approach to prevent falls, and (3) the main neuronal mechanisms mediating fall-related performance and treatment effects. These experimental goals are in accord with the ?highest priority recommendations? of the NINDS PD 2014 Research Report, which include a call for studies aimed at understanding the neural circuit mechanisms of gait and balance disorders in PD, and developing effective treatments for these L-dopa-resistant symptoms (Clinical recommendations 2 & 3; Basic recommendation 3). The proposed experiments are, to our knowledge, the ?rst to explore experimentally the interactions between abnormal basal forebrain and brainstem cholinergic and striatal dopaminergic circuits in the genesis of gait and postural defects, and falls. In addition, this research will establish falls as a useful behavioral endpoint to study cortico-striatal interactions and their modulation by brain stem ascending cholinergic projections.

Risk factors for developing addiction include structural abnormalities in cortical and subcortical regions and associated psychological traits. One such trait concerns the propensity for attribution of incentive salience to drug-associated cues, rendering such cues to be ?attractive? and ?magnetic? and capable of instigating drug- seeking behavior. Sign-tracking rats (STs) approach and contact a Pavlovian conditioned stimulus for food while their counterparts, the goal-trackers (GTs), also learn about predictive nature of such cues but they do not approach them. STs have been extensively demonstrated to be vulnerable for developing addiction-like behaviors and thus have been established as a major animal model of addiction vulnerability. Because impairments in attentional abnormalities are considered an essential component of psychological traits associated with addiction vulnerability, we demonstrated that STs exhibit relatively poor attentional performance that is mediated via low levels of cortical cholinergic neuromodulation. These behavioral and neurochemical characteristics of STs are consistent with the hypothesis that relatively low levels of cholinergic neuromodulation bias the subject away from goal-directed attention and toward cue-driven (or bottom-up) attention. We also showed that, in contrast to GTs, the presence of a Pavlovian cocaine cue fails to increase levels of cholinergic neuromodulation in STs, thereby fostering attention-capture by the cue and cue-directed behavior. The proposed research will first test the hypothesis that a failure of the neuronal choline transporter (CHT) to mobilize in response to stimulation of cholinergic neurons is a cellular mechanism accounting for the attenuated capacity for cholinergic neuromodulation in STs. Second, we will test the hypothesis that by experimentally attenuating CHT function and inhibiting basal forebrain cholinergic activity, sign-tracking behavior manifests and, in GTs, attentional control is diminished, and GTs are more likely to approach a classically conditioned cocaine cue. Third, based on evidence indicating that stimulation of ?4?2* nicotinic acetylcholine receptors (nAChRs) mediates effects of cholinergic neuromodulation on cortical circuitry, we will test the hypothesis that, in STs, such a treatment fosters goal-tracking, improves attentional control and reduces the degree to which a cocaine cue controls behavior. Together, this research will demonstrate that cholinergic-attentional deficits are essential components of addiction vulnerability traits, that a dysregulated CHT is a neuromarker of the trait indexed by sign-tracking, and that sign-tracking and associated vulnerabilities can be reversed by upregulating cholinergic neuromodulation of the cortex.

Project I: Summary/Abstract About two-thirds of patients with Parkinson?s disease (PD) experience falls. Falls in PD patients are a primary cause of hospitalization and nursing home admission. These debilitating features of PD are resistant to dopamine replacement therapy, emphasizing the critical need for basic research and therapeutic development focused on non-dopaminergic systems that degenerate in PD. During the initial funding period, we established a rodent model of PD falls and developed a novel behavioral paradigm that reflect many elements of PD falls. The novel Michigan Complex Motor Control Task (MCMCT) assesses falls resulting from impaired cognitive-motor interactions in rats. We also demonstrated that rats with dual losses of cortical cholinergic and striatal dopamine (DL rats), reflecting PET-based findings in PD fallers, exhibit high rates of falls on the MCMCT. Similar to PD fallers, impairments in attention of DL rats predict fall rates. Importantly, treatment with an ?4?2* nicotinic acetylcholine receptor (nAChR) agonist, or with combination treatments of ACh-esterase inhibitors (donepezil or rivastigmine) and a 5-HT6 receptor antagonist (idalopirdine) reduce fall rates, supporting the translational value of our system. We now propose rigorous systems-neuroscience analyses of the role of basal forebrain cholinergic signaling in falls (Aim 1), of cholinergically-driven cortico-striatal information transfer (Aim 2), and of the role of striatal cholinergic interneurons (Aim 3). The goals of the research of Aim 1 and Aim 3 will directly support the research of Projects III and II, respectively. The proposed research is supported by extensive preliminary evidence demonstrating 1) the impact of optogenetic manipulations of basal forebrain cholinergic signaling on complex movement control; 2) that cues guiding complex movements are ?imported? into the striatum via cortico-striatal glutamatergic activity; 3) that DREADD-based inhibition or stimulation of striatal cholinergic interneuronal activity cause and prevent falls, respectively; 4) and that these interneurons broadly code cues utilized to execute movements. Collectively, the proposed research will develop a cognitive and systems neuroscience approach to understanding falls in PD, establish a valuable preclinical model for therapy development, and substantiate falls as a useful behavioral endpoint for studying the cortico-striatal, cognitive- motor interface.

Addictive urges often have several specific motivation features: the motivational urge is a) highly intense, b) narrowly focused on the addicted target (e.g., drugs), and c) sometimes seemingly `irrational', in the sense of not being explainable by past or expected reinforcement values even for the addicts themselves. For example, relapse can occur even when the addict is no longer in withdrawal, and knows from past experience that the available drug is not very pleasant, and does not expect the future drug to be very pleasant. Here we use optogenetic tools to study brain mechanisms that generate these powerful motivation features. This proposal takes advantage of our recent discovery that optogenetic stimulation of amygdala-related circuitry creates powerful motivational urges for cocaine or sucrose rewards that are a) narrowly focused, b) highly intense, and c) irrational in the sense of exceeding the sum of component reinforcements. This manipulation recreates in the laboratory the same features of motivation that need understanding in addiction. Experiment 1 will identify the crucial anatomical site within amygdala, and identify the particular neuron subpopulations, responsible for generating this intense yet narrow urge for a particular reward, even at the expense of other rewards. Experiment 2 will examine larger brain interactions of the amygdala with mesolimbic circuits, to identify the responsible larger brain circuitry. Experiment 3 will examine how drug-induced mesolimbic sensitization interacts with this circuitry to exacerbate these three features of addictive-like motivation. Altogether, these studies will help clarify how brain mechanisms generate excessive yet narrow, and even irrational, urges (similar to the addictive urges that make drug addicts relapse).

PROJECT II: SUMMARY/ABSTRACT Approximately two thirds of patients with Parkinson?s disease (PD) experience falls; a primary cause of hospitalization and nursing home admission. These debilitating features of PD are resistant to dopamine replacement therapy, emphasizing the urgent need for basic research and therapeutic development focused on non-dopaminergic systems degenerating in PD. We previously established a rodent model of PD falls and developed novel behavioral paradigms that reflect critical elements of PD falls. Our work identified disruptions of the Attentional-Motor Interface (AMI) network as a major pathophysiologic substrate of impaired gait and balance in PD. The novel Michigan Complex Motor Control Task (MCMCT) assesses falls resulting from impaired AMI function in rats. We also demonstrated that rats with dual losses of cortical cholinergic and striatal dopamine (DL rats), reflecting PET-based findings in PD fallers, exhibit high rates of falls on the MCMCT. As in PD fallers, impairments in attention of DL rats predict fall rates. Treatment with an ?4?2* nicotinic acetylcholine receptor agonist, combination treatments of AChase inhibitors and a 5-HT6 receptor antagonist (idalopirdine) reduce fall rates, indicating translational value of our system. We now propose rigorous mechanistic studies identifying critical synaptic dysfunction within key AMI nodes. We will assess the role of basal forebrain cholinergic signaling in falls (Aim 1), of cholinergically-driven cortico-striatal information transfer (Aim 2), and of the role of striatal cholinergic interneurons (Aim 3). This work will directly complement the research of Projects I and III. The proposed research is supported by extensive preliminary evidence demonstrating: 1) the impact of optogenetic manipulations of basal forebrain cholinergic signaling on complex movement control; 2) that cues guiding complex movements are ?imported? into the striatum via cortico-striatal glutamatergic activity; 3) that DREADD- based inhibition or stimulation of striatal cholinergic interneuronal activity cause and prevent falls, respectively; 4) that these interneurons broadly code cues utilized to execute movements. The proposed research will identify mechanisms of nodal and synaptic AMI dysfunctions, identify novel intervention targets, extend a valuable preclinical model for therapy development, and substantiate falls as a useful behavioral endpoint for studying key nodes of the AMI.