Jonathan L. Brigman, Ph.D. - US grants

DESCRIPTION (provided by applicant): Corticostriatal networks and NMDAR mediation of habitual and flexible action in the mouse. The objective of the proposed research is to investigate the neurocircuitry modulating the balance of habitual and flexible action that is thought to be dysfunctionally altered in alcohol dependence. Although studies in other species have provided strong evidence that these behaviors are mediated by networks connecting cortical and striatal subregions, this has not been well established in the mouse. In order for alcohol research to take full advantage of the molecular and genetic techniques that mouse models permit it is essential to first establish the role of these circuits in that species. I propose to elucidate the networks and molecular mechanism underlying habitual and flexible action in the mouse by using an integrative approach that combines traditional techniques (lesion and microinfusion) with emerging genetic and electrophysiological techniques. In order to do this, I propose three specific aims 1) Determine the involvement of corticostriatal networks and NMDAR mediation of well-learned and flexible behavior in the mouse. To accomplish this aim I propose to map endogenous activation patterns and examine the effects of subregion specific lesions in mice performing a task that requires both habitual and flexible action: visual discrimination and reversal. Further, the effects of NMDAR subunit loss in corticostriatal subregions on habitual and flexible performance will be tested using a conditional GluN2B mutant mouse model. 2. Examine in vivo electrophysiological activity of corticostriatal networks and the role of NMDAR during well- learned and flexible behavior. To accomplish this aim I propose to perform in vivo recording of neuronal activity in cortical and striatal subregions during performance of the discrimination-reversal task in both non-mutant mice and the GluN2B conditional mutant model. 3. Examine in vivo electrophysiological activity of corticostriatal networks during higher-order measures of executive control. In order to complete this aim I propose to perform electrophysiological recording in non-mutant and GluN2B mutant mice performing an operant task directly adapted from those used to measure executive control in clinical populations: visual set-shifting. Taken together, the completion of these specific aims will provide strong evidence for the role of corticostriatal networks and NMDAR in the mediation of flexible and habitual actions and provide a strong foundation for future studies investigating how these systems are dysregulated in alcohol abuse and dependence. During my graduate training and fellowship I developed the knowledge of scientific design and conduct necessary to complete the mentored training and development necessary to succeed in the proposed experiments. As a graduate student in the laboratory of Dr. Lawrence Rothblat I was trained in the fundamentals of design and conduct of neuroscience research using operant behavioral tasks with transgenic and mutant mouse models and gained experience in stereotaxic surgical procedures and histological techniques. During my fellowship at NIAAA under the mentorship of Dr. Andrew Holmes I have received extensive hands-on training in a broad range of behavioral measures and a variety of techniques for acute and chronic systemic drug administration as well as tissue micro-dissection for western blotting and high pressure liquid chromatography. In addition I have expanded his surgical techniques to include implantation of indwelling guide cannulae and micro-infusion of bioactive drugs in awake behaving animals. The mentored phase of the proposal will be conducted in the Division of Intramural Clinical and Biological Research at NIAAA under the mentorship of Dr. Andrew Holmes and the co-mentorship of Dr. David Lovinger. Dr. Holmes has extensive expertise in behavioral neuroscience techniques and a well-established research program using behavioral paradigms to investigate genetic and environmental factors underlying addiction and neuropathology using mouse models. Dr. Lovinger is a leader in studying the targets of alcohol and drugs of abuse using in vivo and ex vivo electrophysiology. This environment will provide all the necessary resources necessary to complete the research goals including, but not limited to behavioral apparatus, surgical equipment and animal care, histological resources and multi-channel acquisition systems for in vivo recording. The training and career development resources in the intramural program such as weekly laboratory meetings, seminar series conducted by leading scientists in the field, career development workshops, and training in the responsible conduct of research, make NIAAA an excellent scientific environment for conducting the mentored phase of the proposal. PUBLIC HEALTH RELEVANCE: Alcohol disorders represent a major health issue in the United States as 17.6 million people (H 1 in every 12 adults) abuse alcohol or are alcohol dependent. Current theories of dependence suggest that increases in habitual drug and alcohol seeking during the cycle of dependence may be caused by dysfunction in circuits connecting cortical and striatal brain areas. This project seeks to understand how these systems mediate behavior in the mouse, in order to provide a unique model for investigating the genetic and environmental factors underlying alcohol abuse and dependence.

There is growing evidence that moderate exposure to alcohol during development can lead to behavioral and cognitive deficits that can persist throughout the lifespan. The cognitive impairments associated with Fetal Alcohol Spectrum Disorders (FASDs) include abnormalities in learning and memory, executive control and social behaviors^^ and are often characterized by a hyper-focus on one particular task or aspect of a task, to the detriment of other important behaviors. Measures of cortically-mediated cognition have been shown to be sensitive to high dose ethanol (EtOH) exposure during development in rodents, but little is known regarding the mechanisms responsible for executive function alterations associated with FASD. We propose to investigate the impact of prenatal ethanol exposure on corticostriatal-mediated behavior and learning related cortical and striatal physiology by integrating highly translatable touch-screen behavioral measures previously shown to recruit dorsal striatum and orbitofrontal cortex with in vivo and ex vivo electrophysiology in prenatally exposed and control mice. We hypothesize that moderate prenatal EtOH exposure will decrease activation of neuronal circuits in the orbito-frontal cortex (OFC) impairing executive control behavior and releasing the dorsal striatal (dS) from cortical control, resulting in hyper-focused, unregulated learning. In order to test this hypothesis we propose three specific aims. First, we will investigate whether moderate prenatal ethanol exposure impairs reversal learning by measuring choice learning and shifting in adolescent mice after moderate prenatal alcohol exposure using touch screen paradigm. Next, we will examine whether this ethanol exposure impairs the function of dS neuronal circuits by both performing in vivo multi-electrode array electrophysiological recording to examine dS neuronal firing activity during choice learning and shifting and utilizing in vitro slice electrophysiological techniques to examine synaptic transmission and plasticity in the dS after choice learning and shifting. Finally, we will investigate whether PAE impairs the function of OFC neuronal circuits during reversal learning by performing in vivo recording of OFC neuronal firing activity during choice learning and shifting and performing in vitro slice electrophysiology to measure synaptic transmission and AMPAR/NMDAR ratios in the OFC after choice learning and shifting. Taken together, the completion of these aims will allow us to better understand the mechanisms of cognitive impairment in FASD and provide an important tool for developing more effective therapies for executive dysfunction.

Abstract There is growing evidence that moderate exposure to alcohol during development can lead to behavioral and cognitive deficits that can persist throughout the lifespan. Cognitive impairments associated with Fetal Alcohol Spectrum Disorders (FASD) include abnormalities in learning and memory, executive control and social behaviors and are often characterized by a hyper-focus on one particular task or aspect of a task, to the detriment of other important behaviors. We have recently shown that moderate prenatal alcohol exposure (PAE) can impair executive control in adulthood and that behavioral impairments are accompanied by significant alterations in coherence in the orbitofrontal cortex during choice. Both behavioral deficits and cortical alterations are found when GluN2B subunit containing N-Methyl-D-Aspartate receptors (NMDAR) are lost in the OFC. Given preliminary data that GluN2B is significantly reduced in the OFC after PAE, we propose to investigate whether impairments in behavioral flexibility are driven by alterations in cortical coherence mediated by alterations in NMDAR expression and function and whether these deficits can be rescued. We propose to integrate a well-established voluntary drinking paradigms for moderate PAE with touch-screen behavioral assays, in vivo and ex vivo electrophysiology and optogenetic stimulation. First, we will express channel rhodopsin (ChR2) in the cortex of PAE and SAC mice and examine whether direct stimulation will rescue behavioral flexibility impairments in PAE mice. Next, given evidence that GluN2B is downregulated after PAE and that his subunit contributes to establishing cortical coherence, we will investigate whether behavioral deficits in the model are mediated by alterations in GluN2B subunit expression and recruitment required to induce plasticity and support behavioral flexibility. Finally, we will examine whether changes in connectivity between OFC and dS drives asynchrony and perseveration. We will use retrograde expression to fluorescently tag OFC-dS neurons and characterize function of NMDAR via ex vivo slice physiology. Then we will utilize retrograde expression of ChR2 to test if stimulation restricted to OFC-dS neurons is sufficient to rescue the behavioral deficits. Taken together the completion of these aims will elucidate the mechanisms of cognitive impairment in FASD and provide an important tool for developing more effective therapies for executive dysfunction.

Abstract A common feature in Fetal Alcohol Spectrum Disorders (FASD) is an inability to focus attention appropriately and inhibit responding to stimuli that are similar to, but distinct from, those that reliably lead to positive outcomes. Continuous performance tasks have been used to measure both attention and response inhibition in human subjects and the Five Choice Continuous Performance task (5C-CPT) was developed to examine these processes in an analogous manner in rodents. Together with our collaborators, we have recently validated this task behaviorally in mice and human subjects and our current data shows that the 5C-CPT recruits similar EEG signal from frontal and parietal cortex in rodents and healthy human subjects. Here, we propose to test whether moderate ethanol exposure during the first and second trimester equivalents impairs attention and response inhibition on the touch-screen 5C-CPT. Next, we will perform simultaneous EEG-like dura-resting local field potential (LFP) and depth recording of frontal and parietal cortex to examine whether moderate PAE significantly alters frontal and parietal signaling, and if this signaling is related to alteration in neuronal firing pattern or timing. The combination of these approaches will allow us to look at both individual unit firing and regional activity and compare it to a clinically relevant measure of brain activity. Finally, given evidence that LFP oscillatory signaling is controlled by the activity of fast-spiking parvalbumin positive (FS-PV+) interneurons, we will perform targeted in vivo recording of FS-PV+ neurons, immunohistochemistry and ex vivo electrophysiology to examine whether deficits in cognitive control are mediated by alterations in interneuron number and excitatory tone. Our hypothesis is that PAE leads to inappropriate parietal oscillatory tone via loss of interneuron signaling, with the overall outcome being impaired cognitive control. Taken together, the completion of these aims will allow us to better understand the mechanisms of the long-lasting impairments in cognitive control seen in FASD and provide targets for more effective therapies for executive dysfunction.

Project Summary This is a competitive renewal application for an Alcohol Training Program in Neurosciences at the University of New Mexico (UNM-ARTN). This program is currently ending Year 14 of support. It has had a tremendously positive impact on graduate education at our institution and has increased visibility of alcohol research across campus. The program provides multidisciplinary training that includes molecular biological, biochemical, electrophysiological, imaging, and behavioral techniques. The focus of the UNM-ARTN program is to provide training in alcohol neuroscience research. Our group has strengths in three areas of fetal alcohol spectrum disorder research: 1. Basic mechanisms involved in ethanol neuroteratogenesis; 2. Biomarkers of prenatal alcohol exposure; and 3. Therapeutic interventions to ameliorate FASD-related cognitive deficits. A total of 12 faculty members from the Departments of Neuroscience and Psychology will be directly involved in the program as core faculty. These faculty members are well-funded and productive, and have extensive collaborative interactions in terms of research grants, publications, and mentoring of graduate students. Students will be from the Biomedical Sciences Graduate Program of the Health Sciences Center and the Cognition, Brain and Behavior Program of the Psychology Department. The program will support 4 Ph.D. students per year. The program will be overseen by Dr. Valenzuela, a steering committee composed of members of the training faculty, and an external advisory committee. Our trainees have been very successful in terms of obtaining individual predoctoral fellowships, presenting at scientific meetings, and publishing in well-respected journals. Our goal is to continue to provide high quality graduate students with the necessary training to prepare them for a successful future career in alcohol research.