John A. Wemmie - US grants

[unreadable] DESCRIPTION (provided by applicant): Seizure disorders cause significant morbidity and mortality, and many cases are refractory to current medical management. Thus, improved treatments are needed. Therapeutic advances might be developed from a better understanding of the antiepileptic mechanisms of brain acidosis. It has long been known that low pH effectively inhibits seizures and reduces neuron excitability, but the molecular mechanisms underlying these effects are poorly understood. The recent identification of proton receptors in the brain may provide a molecular link between brain pH and seizures. It was recently found that one of these receptors, the acid sensing ion channel ASIC1a is required for acid-evoked currents in central neurons. And consistent with an inhibitory effect on seizures, preliminary data indicate overexpressing ASIC1a in mice attenuates seizures. In contrast, disrupting ASIC1a makes seizures worse. Together, these observations suggest the hypothesis that ASIC1a mediates the antiepileptic effects of central acidosis and reduces neuron excitability. To test this hypothesis three aims are planned. The first aim will test whether ASIC1a mediates antiepileptic effects of CO2, which rapidly crosses the blood-brain barrier and lowers central pH. Wild-type mice, ASIC1a null mice, and ASIC1a overexpressing transgenic mice will be injected with a chemoconvulsant and the anti-epileptic effects of CO2 relative to air will be compared between genotypes. The results will provide an indication of whether the anti-epileptic properties of ASIC1a and CO2 are related. The second aim will test whether ASIC1a mediates the antiepileptic effects of acid in brain slices where pH can be better controlled. The third aim will test the effects of ASIC1a on acid inhibition of action potentials in cultured hippocampal neurons. Preliminary data suggest ASIC1a may inhibit action potentials, which may help explain how ASIC1a inhibits seizures in vivo. Together these experiments will provide important insight into the historically well established but poorly understood antiepileptic effects of acid. They will also lead to additional mechanistic studies to clarify in more depth how ASIC1a exerts its antiepileptic effects. Importantly, these studies may also suggest ASIC1a as a novel therapeutic target for inhibiting seizures in patients. Identifying novel and broad antiepileptic mechanisms may be especially beneficial to patients with refractory disease. Seizures disorders cause significant morbidity and mortality and are often refractory to medical management. Studying novel features of seizure inhibition may lead to treatments with alternative mechanisms of action. In this application we explore the seizure inhibiting effects of a poorly understood gene, acid-sensing ion channel 1a, and we test whether ASIC1a contributes to the well-established but poorly understood antiepileptic effects of brain acidosis. These studies have the potential to foster the development of Asic1a antagonists as a novel therapeutic approach to seizures. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Anxiety disorders are the most common form of psychiatric illness and exact a huge toll on America's health. Current treatments are often inadequate suggesting more effective, more specific therapies are needed. Clinical studies have firmly established that CO2 inhalation triggers anxiety and panic attacks, and that patients with anxiety disorders are hyper-responsive to CO2. These findings suggest that a better understanding of the molecular mechanisms underlying CO2 sensitivity could lead to novel insight into the causes of anxiety disorders and possibly lead to better treatments. Because CO2 sensitivity has been explored primarily in clinical studies, which are restricted in their ability to identify molecular mechanisms, there is a significant need for animal models to probe the mechanisms underlying CO2 sensitivity. In this proposal we address this need for animal models of CO2-evoked fear, by modeling CO2 behavioral and physiological responses in mice. We investigate the hypothesis that CO2 inhalation lowers brain pH, which activates pH-sensitive receptors in the fear circuit, which in turn increase the behavioral and physiological manifestations of fear, anxiety, and panic. This project may be critical for helping to explain the long recognized, but poorly understood clinical phenomenon of CO2 sensitivity. In addition, these studies are likely to have broader implications. Our preliminary data suggest that CO2 activates novel signaling pathways underlying anxiety disorders, and that these pathways might be therapeutically targeted to prevent anxiety disorders and reduce their symptoms.

DESCRIPTION (provided by applicant): Adequate perfusion is essential for normal brain function and impaired regulation of cerebral blood flow (CBF) may contribute to neurological dysfunction and disease. Despite recent progress, our knowledge of mechanisms that regulate CBF remains inadequate. Two of the most powerful stimuli that affect CBF are hypercapnia and increased cellular activity (cellular metabolism and synaptic activity). Both of these stimuli increase local concentrations of hydrogen ion (reduce extracellular pH). The overall goal of this application is to examine the role of acid-sensing ion channels (ASICs) in control of CBF. We found recently that ASICs are required for acid-evoked effects on synaptic plasticity. Moreover, the ASIC1a subtype functions as a chemosensor in neurons mediating hypercapnia- and acid-evoked behaviors. These findings led to preliminary experiments testing whether ASICs also play a role in regulation of CBF. Although effects of hypercapnia and acidosis have been known for decades, mechanisms that initiate vascular responses to these stimuli remain undefined. Based on this background, we propose two Aims. Aim 1 will examine the hypothesis that ASICs mediate vascular responses to hypercapnia. We will examine vascular effects of hypercapnia and acidosis following manipulation of ASICs using genetic and pharmacological approaches. To define the importance of neuronal ASIC, we will take advantage of mice lacking or overexpressing ASIC1a specifically in neurons. We will also use ASIC inhibitors to pharmacologically probe ASIC function. Aim 2 will use similar approaches to examine the hypothesis that neuronal ASICs contribute to vascular responses in models of neurovascular coupling. In pilot studies, we found that disrupting ASIC1a nearly eliminated hypercapnia-induced vasodilation but also significantly attenuated vasodilator responses in a model of neurovascular coupling. Together these studies will unambiguously determine the importance and site of ASIC action in hypercapnia- and proton-dependent regulation of cerebrovascular responses. The studies may provide new and unprecedented insight into the complex interaction between brain and its vascular supply. Such insight may ultimately lead to improved therapeutic approaches for cerebrovascular disease and brain injury. This project was conceived and will be carried out by an innovative collaboration between investigators with diverse expertise in CBF, neurovascular coupling, pH regulation, and ASICs.

DESCRIPTION (provided by applicant): Drugs of abuse, such as cocaine, produce long-lasting synaptic adaptations that increase the compulsive nature of addiction, undermine self-control, and increase the likelihood of relapse. Identifying and understanding the molecules that regulate these synaptic changes may suggest novel therapies. Recently, we found that acid-sensing ion channels (ASICs) and brain pH play critical roles in the synaptic plasticity thought to underlie addiction. Our findings suggest that ASIC1a is activated during synaptic transmission in medium-spiny neurons (MSNs) of the nucleus accumbens (NAc), a site firmly implicated in addiction-related behavior. Genetically deleting ASIC1a in mice led to a number of synaptic changes paralleling those previously observed following cocaine withdrawal. Consistent with these synaptic effects, disrupting ASIC1a in mice throughout the body or specifically in the NAc increased conditioned place preference (CPP) to cocaine and to morphine, indicating important behavioral consequences that generalize to multiple drugs of abuse. Confirming the NAc as a key site of ASIC1a action in cocaine-dependent behavior, restoring ASIC1a expression to the NAc of ASIC1a-/- mice reversed the synaptic abnormalities and normalized cocaine CPP. We also tested synaptic and behavioral effects of ASIC1a in rats and found results similar to those in mice. In rats, overexpressing ASIC1a in the NAc doubled the ASIC-mediated synaptic current, and significantly reduced cocaine self- administration. Together, these observations indicate that ASIC1a inhibits addiction-related behavior. Furthermore, these results suggest the hypothesis that ASIC1a and brain pH might be targeted to reduce the synaptic changes underlying addiction and relapse. To test this hypothesis, we propose to explore genetic and pharmacological approaches to increase ASIC1a function at synapses and to determine their ability to affect cocaine-related synaptic physiology and behavior in mice and rats. The planned studies capitalize on novel insight into the roles of ASICs and pH in synaptic transmission, and take advantage of state-of-the-art electrophysiological approaches and an innovative collaboration between principal investigators with extensive experience in ASICs, brain pH, and drug-related behavior. Our planned behavioral analyses include models of craving/relapse using long-access cocaine self-administration in rats, widely considered one of the best models of addiction because animals control their own drug intake, thus facilitating assessment of various stages of drug-seeking behavior. Because ASIC1a structure and function in rodents are nearly identical to those in humans, these studies will be highly relevant to the human brain. Moreover, the knowledge gained through these experiments will inform innovative strategies to interrupt addictive behaviors by targeting ASICs and/or brain pH.

Abstract Bipolar disorder (BD) is a frequently devastating psychiatric illness that is challenging to diagnose and treat. Identifying the underlying mechanisms of this illness may provide a foundation for better evidence-based diagnostic and therapeutic techniques. For the first time in the context of psychiatric illness, we recently explored the utility of a magnetic resonance imaging (MRI) strategy called T1 relaxation in the rotating frame (T1?), which is highly sensitive to brain pH. We tested patients with BD in the euthymic state and found prominent T1? differences compared to matched controls. More recently, we also imaged participants with BD in depressed and manic states. These studies suggest that the brain area with the most prominent T1? changes in bipolar disorder is the cerebellum, a structure has been previously suggested to contribute to BD but has received relatively little attention compared to forebrain structures. Neuroanatomical models of BD have largely overlooked the cerebellum despite compelling evidence that the cerebellum is strongly connected to brain regions involved in the emotional control network that has been put forth as a model of the disorder. We hypothesize that cerebellar activity plays a critical role in regulating mood in BD, which will be tested in this proposal using a cross-sectional design and recruiting BD subjects across the mood spectrum as well as matched controls. Participants will undergo psychiatric symptom assessment and brain imaging. Psychiatric symptom assessments will include, the Montgomery-Asberg Depression Rating Scale (MADRS) and Young Mania Rating Scale (YMRS). Brain imaging will include quantitative whole-brain T1? mapping, 31P- and 1H- MRS of the cerebellar vermis, as well as diffusion imaging (DWI) and resting state fMRI. Medications will be assessed and used as covariates in analyses. This data will be used to assess the following aims: Aim 1) Does cerebellar activity play a significant role in mood regulation in BD? We hypothesize that the cerebellum plays a significant role in maintaining a euthymic mood state (i.e., plays a compensatory role). We reason that if cerebellar activity normalizes mood, then its activity should be greatest when BD participants are euthymic and decrease with increasing mood symptom severity. Alternatively, if cerebellar activity drives abnormal moods, then it is likely to be greatest in patients who are manic or depressed. Aim 2) Does connectivity of the cerebellar vermis with the emotional control network differ with mood symptoms in BD? We hypothesize that functional connectivity between the vermis and nodes of the emotional control network will vary with mood state with increased connectivity in the euthymic state and decreased connectivity during exaggerated mood states (depression/mania). We also expect that BD participants in the euthymic state will exhibit increased connectivity relative to healthy controls. These results would provide further evidence that the cerebellum is playing a compensatory role to maintain mood. In addition, this would support a refined model of the neural circuits underlying the pathophysiology of BD.

PROJECT SUMMARY/ABSTRACT Recent work suggests that functional T1? provides a novel imaging technique to study brain function. This signal has been to exist in human and animal studies of the visual cortex. This technique has been shown to be separate from the hemodynamic response typically used to assess brain function using MR imaging. Initial work has shown that this signal responds faster than arterial and venous vascular signals measured using ASL and BOLD imaging respectively. In addition, the T1? signal has been shown to be sensitive to metabolism (glucose, glutamate, and pH) as well as neural currents. Finally, we have shown that the functional T1? signal was sensitive to changes in panic disorder not evident using BOLD imaging as well as shows a decoupling from the BOLD signal in bipolar disorder. These findings highlight the unique capabilities of this new functional imaging technique, which may provide new insight into psychiatric and neurological disorders. However, a number of gaps exist in our current understanding of the functional T1? signal, which this proposal aims to address. The aims of this project include: 1) Determine the relationship between functional T1? responses and local metabolites and neural currents using implanted biosensors in animal models; 2) Enhance spatial coverage and temporal sampling of functional T1? imaging using multi-band and compressed-sensing; 3) Determine the temporal and spatial response functions of T1? relative to BOLD and ASL; and 4) Evaluate the stability, reliability, and accuracy of functional T1? mapping. As part of these specific aims, we will evaluate techniques to eliminate the intravascular signal from the measurements and define a canonical impulse response function that can be used in the analysis of future functional T1? imaging studies. Finally, the reliability analysis proposed in Aim 4 will determine the ability of functional T1? mapping to be adopted by other groups and extend the technique to paradigms outside of the visual cortex. The proposed study will allow us to interpret the functional changes between patient populations by understanding the source of the functional T1? signal. The understanding of the signal source will also highlight many other psychiatric and neurological disorders where this functional imaging technique can be applied.

This revised application requests continuation of funding for a successful and long-standing training program (dating back to 1991). At a critical juncture of change in leadership, the program strives to maintain a strong foundation with the opportunity for growth and restructuring. Rebranded as the Iowa Neuroscience Specialty Program In Research Education (INSPIRE) this program seeks to integrate training in translational neuroscience with an emphasis on a lifespan trajectory perspective. Our approach is to re-shape and reframe our research opportunities based on the NIMH's Research Domain Criteria (RDoC) project by focusing on mechanisms of psychopathology based on functional dimensions rather than the diagnostic criteria that define patient populations. The ultimate goal is to train a group of young investigators who will combine a high level of sophistication about the complexities of the human brain's functional domains, how these systems develop over time, and the mechanisms that may lead to pathologic functioning. . This program is at a pivotal point in time. Nancy Andreasen, a pioneer of brain imaging in the study of schizophrenia, conceptualized and directed this program from 1991 until now. The current Co-PI of the program, Peg Nopoulos, is an alumnus of the program herself and will now be responsible for directing the program. She is joined by John Wemmie, the new Co-PI of the program. This change in leadership has afforded the opportunity to retain the solid foundation, but also to re-shape the program substantially in two broad areas: 1) a focus on the study of neurobiologic mechanisms of psychiatric illness across the lifespan, and 2) implementing a more highly structure training program with emphasis on translational science. In expanding the content of the program beyond the major psychoses, we partner with a new and growing Molecular Psychiatry division, adding six new MD/PhD and PhD scientists as mentors. This helps expand the program to include basic science PhDs, a new phenotype of fellow, and allows for the creation of an interdisciplinary cohort or trainees. This mix of types of trainees adds an addition layer of exposure to translational research with a strong emphasis on team science. Central to the training program is the Master's in Translational Biomedicine (TBM) program which is hosted by our Institute for Clinical and Translational Sciences (ICTS). This program is designed to be individualized and flexible. The INSPIRE program will recruit a total of 4 fellows at the post-doctoral level who are MD, MD/PhD, or PhD trained. Each fellow will be `matched' with an outstanding mentor as well as a mentor team to oversee the primary activity of mentored research activity. In addition, each fellow will develop a program through the TBM that suits their needs while fulfilling requirements (such as Training in Responsible Conduct in Research), utilizing both formal didactics and career development activities. A degree (certificate or Master's) is an option, but not a requirement. The training period is typically 2 years in length but can be expanded to 3 years.

Abstract Survival in a hostile environment requires the ability to assess potential threats and take the most appropriate defensive action. Accumulating evidence suggests that the amygdala is well positioned to integrate information about threats and to guide or shift responses along a spectrum of potential defensive behaviors. In both humans and mice, the amygdala plays an essential role in defensive responses to the threat of suffocation, signaled by rising systemic carbon dioxide (CO2) levels in the body. Lesioning the amygdala, or manipulating it more precisely in other ways, reduces some defensive behaviors evoked by CO2 inhalation (e.g. freezing) while simultaneously increasing others (e.g. fight-or-flight). Because CO2 inhalation evokes a variety of defensive responses in a robust, reproducible, and concentration-dependent manner, CO2 provides a straightforward and translatable approach to studying the amygdala's role in defensive behavior regulation. In this application, we propose to study defensive behaviors evoked by CO2 using state-of-the-art, neuron- specific manipulations and electrophysiological recording to deconstruct roles of select neuron populations in the basolateral amygdala (BLA). We hypothesize that distinct defensive behaviors are differentially regulated by the BLA and that principal neurons and interneurons in the BLA each play unique roles. To test this hypothesis we will use genetic, optogenetic, and electrophysiological approaches to specifically activate and silence specific BLA neuron populations and quantify the effects on neural activity and defensive behaviors. Together these experiments will allow us to discern how these neurons regulate different defensive behaviors. Understanding basic mechanisms that guide or shift defensive behaviors along their spectrum will be essential for identifying abnormalities in these processes and for finding ways to correct them. This knowledge will ultimately impact mental illnesses where defensive behaviors are inappropriately extreme such as panic disorder and post-traumatic stress disorder.

Abstract The US is facing a crisis of opioid overdoses and addiction. Current therapies consist largely of alternative opioids (i.e. maintenance with methadone or buprenorphine) and do not correct neurobiological factors that underlie drug craving and relapse. These factors include the long-lasting changes at glutamatergic synapses in the nucleus accumbens (NAc), which both resemble and differ from changes induced by other highly addictive drugs such as cocaine. Our recent studies suggest these synaptic effects of opioids are opposed by acid- sensing ion channels (ASICs). ASICs conduct inward Na+ and Ca2+ current at post-synaptic dendritic spines where they are activated during synaptic transmission by protons released into the synaptic cleft from neurotransmitter-containing vesicles. Because these protons are removed from the synaptic cleft via the actions of carbonic anhydrase 4 (CA4), genetically disrupting CA4 or pharmacologically inhibiting CA4 with acetazolamide (AZD) dramatically increases synaptic ASIC currents. These observations have led to our hypothesis that AZD will reverse synaptic changes following opioid withdrawal by inhibiting CA4 and increasing ASIC activity, and thereby reduce craving and relapse. In this proposal we plan to test this hypothesis by rigorously assessing effects of opioids on synaptic physiology and behavior. Together the experiments in this proposal will pave the way to a better understanding of the neurobiology underlying opioid addiction and to new molecular targets for treating opioid use disorder (OUD). Knowledge gained from these studies could suggest new ways to treat opioid addiction through non-opioidergic mechanisms, for example by manipulating ASICs, brain pH, or carbonic anhydrase, for which a number of inhibitors are already approved for human use, and might be efficiently repurposed.