Larry Zweifel - US grants

One of the major pathways thought to be important for behavioral responses to natural rewards and drugs of abuse is the mesocorticolimbic dopamine (DA) circuit. Stable alterations within this circuit that lead to drug seeking behavior are thought to involve signaling through the NMDA-type glutamate receptor (NMDAR), however virtually nothing is known about the cell-specific requirements of this signal transduction cascade within DA neurons. To study the cell-specific requirements of NMDAR signaling mice which lack the functional subunit of NMDAR, NR1, within DA neurons will be studied. A new mouse model that will allow for the cell-specific, reversible inactivation of neural activity will also be generated. This approach, based on the chemical dimerization and inactivation of a protein critical for neurotransmitter release, will advance our understanding of the activity-dependence of neurons within reward pathways required for addiction. Attaining an enhanced understanding of the neural mechanisms and circuitry of addiction will hopefully lead to better treatments for individuals who suffer from addiction and lead to the design of Pharmaceuticalsto treat chronic severe pain and other debilitating illnesses without fear of developing dependence.

DESCRIPTION (provided by applicant): Anxiety disorders, such as post-traumatic stress disorder (PTSD) are hypothesized to result from a failure of fear processing centers in the brain to form appropriate associative memories during a traumatic event. Emerging evidence suggests that the dopamine neurotransmitter system is important for associative fear learning, raising the intriguing possibility that disregulation of this system during a fearful experience could be a contributing factor in the development of some anxiety disorders. Consistent with this hypothesis, we recently discovered that genetic disruption of the phasic activation of dopamine neurons impairs Pavlovian fear conditioning in mice, resulting the manifestation of generalized anxiety- like behavior. To date, very little is known about the neural circuitry regulating, or regulated by phasic dopamine signaling. Our hypothesis is that a select excitatory input to dopamine neurons facilitates the phasic activation of a subset of these cells during a fearful experience. Subsequent phasic dopamine release into discrete brain regions engages the dopamine D1 receptor to facilitates the formation of memories related to the fearful event. To test this hypothesis, we will utilize a multidisciplinary approach involving mouse behavior, genetics, molecular biology, viral-mediated gene transfer, and in vivo fiber- optic imaging of dopamine neuron activity in freely behaving mice. We are innovating a technique that will allow for fibered fluorescence microscopy of real-time activity-dependent calcium dynamics within dopamine neurons projecting to specific targets during Pavlovian fear conditioning in mice that will allow us to generate a map of phasic dopamine neuron activation. Additionally, we are establishing a combinatorial viral vector based approach for the conditional inactivation of specific genes in neurons projecting to select targets that will allow us to map the critical inputs to dopamine neurons for fear conditioning. Finally, we have developed a method for conditional gene reconstitution that will allow us to generate a map of the minimal essential brain regions requiring D1R expression for fear conditioning. Together these techniques will help us to establish the precise neural circuitry of dopamine-dependent fear processing and will provide broadly useful tools for the dissection of behaviorally relevant circuits throughout the brain.

DESCRIPTION (provided by applicant): Evidence from humans and animals indicate a critical role for the hippocampus in the formation of long- term episodic memories. The hippocampus consists of anatomically segregated subregions with a diversity of neuronal types hypothesized to encode different aspects of an experience. Although the molecular mechanisms of activity-dependent plasticity in hippocampal neurons have been intensely scrutinized, we have limited understanding of the activity patterns shown by select neuronal populations during memory encoding, consolidation and recall. Therefore, there is a strong demand to develop new imaging approaches that can interrogate the activity of hippocampal circuits in the context of the behavior that they are thought to support. Because neuronal spikes are associated with large intracellular calcium conductances, calcium indicators can be used as a proxy for neuronal activation. In the proposed research, we will implement the use of fiber optic fluorescence endomicroscopy (FFE) to chronically monitor the activation of defined hippocampal neurons in mice subjected to hippocampus-dependent tasks. A genetically-encoded calcium indicator will be conditionally expressed in transgenic mice selectively expressing Cre recombinase in CA1 pyramidal cells, or in dentate granule cells and CA3 pyramidal cells. Our initial proof-of-principl experiments in fully awake, unrestricted mice advocate the feasibility of this approach for large-population imaging of single-neuron activity. In our first aim, we will optimize the parameters of FFE required to achieve stable, long-term imaging of the different neuronal populations. In our second aim, we will use this method to define the neuronal ensembles that encode contextual- and trace-fear conditioning, two hippocampus-dependent variants of associative memory hypothesized to engage different circuits. Time-lapse experiments will allow comparisons of the activation patterns observed during training, consolidation and recall. Through genetic or pharmacological interventions against each one of these memory phases, we will link the activity of select neurons to cognition. Thus, the proposed research will advance the field by introducing FFE as a novel in vivo approach to study memory mechanisms, and will use this technique to begin to address previously inaccessible questions on the cellular basis of memory. PUBLIC HEALTH RELEVANCE: Memory impairments are associated with aging and psychiatric diseases. This project uses advanced calcium imaging in behaving mice to reveal the properties of neuronal activation in different types of hippocampal cells during memory formation and retrieval. Consequently, this research may identify specific cellular targets or activity-dependent network patterns that can be exploited to prevent memory impairments.

Project Summary Serotonergic and dopaminergic systems of the brain are broadly implicated in mental illness including depression and mood-related disorders. How these systems interface to modulate affective state in the presence of stress and recovery from stress remains poorly understood. Dopamine neurons located in the ventral tegmental area (VTA) and serotonin neurons located in the dorsal raphe nucleus (DRN) share common targets. Both project to the prefrontal cortex, nucleus accumbens, hippocampus, and amygdala. Each of these regions has been implicated in depression-related symptom domains and the balance of dopamine and serotonin in these regions is an important regulator homeostatic response to stress. Serotonin neurons of the DRN directly synapse onto dopamine neurons of the VTA, indicating a direct interaction between these systems. To establish the role of this circuit connection in behavioral regulation of stress and behavioral domains relevant to depression, we will perform a detailed circuit analysis of serotonergic inputs from the DRN to dopamine neurons of the VTA and how this connectivity changes in response to social defeat stress in susceptible and resistance mice. To enhance the granularity of our circuit analysis, we will measure excitatory and inhibitory coupling between serotonin and dopamine neurons in dopamine cells that project to specific areas of the brain. To ascertain how reward responding of serotonergic neurons change at the local circuit level of the DRN following social defeat in susceptible and resistant mice, we will monitor in vivo calcium dynamics in all serotonin producing neurons of the DRN and exclusively in those cells that synapse onto dopamine neurons. To establish the functional importance of serotonergic DRN input to the VTA in the mitigation of depression-related symptom domains following social defeat, we will selectively enhance the activity of DRN neurons that synapse onto dopamine cells using designer receptor (DREADD) technology. These studies, combined with the efforts of my collaborators in this center grant will provide an unparalleled assessment the neural circuitry underlying susceptibility and resistance to social defeat stress, elucidate the therapeutic potential of an endogenous opioid system and how it interfaces with these circuits, determine the impact of sex-specific differences in these circuits associated with stress-induced alterations in decision making, and provide detailed assessments of transcriptional and translational changes in treatment resistant females suffering from depression and in rodents susceptible to depression-related symptoms.

? DESCRIPTION (provided by applicant): Activity patterns in the brain establish the manner in which sensory information is perceived and salience is assigned. Disruptions of these patterns through genetic mutations are likely a major cause of mental illness. The midbrain dopamine system plays an essential role in salience assignment and mutations within several ion channels known to regulate action potential firing patterns by dopamine neurons have been identified, yet virtually nothing is known of the impact of these mutations on dopamine physiology, circuit function, and behavior. We have demonstrated that a mutation in the calcium activated, small conductance potassium channel, SK3, identified in a patient with schizophrenia alters dopamine neuron activity pattern regulation. Selective, expression of this dominant-negative human SK3 mutant in dopamine neurons of mice shifts balance between tonic and phasic activity of dopamine neurons towards a more phasic state. The resulting dysregulation of activity patterns in dopamine neurons leads to impairments in sensory and attention gating processes. The major challenge that lies ahead is discovering how alterations in tonic-to-phasic dopamine ratios impact cortical and striatal circuits important for gating sensory information and to further defin behavioral domains impacted by such disruptions. Here, I outline several innovate approaches that we will utilize to determine how imbalances in dopamine activity patterns impact corticostriatal connectivity and function. Utilizing combinatorial viral vector gene delivery, we wll optogentically isolate inputs from the prefrontal cortex to the nucleus accumbens region of the striatum and define how expression of the human SK3 mutation in dopamine neurons impacts the connectivity of these two structures using in vivo fiber-optic confocal microscopy and imaging of a genetically encoded calcium indicator. We will monitor activity- dependent process in the nucleus accumbens using fiber-optic confocal microscopy and define how alterations in tonic-to-phasic dopamine activity influences activity in direct and indirect pathway neurons of the striatum in freely behaving mice during an attention gating task. Finally, we will use viral-mediated circuit dissection to define the minimal network elements in the brain required for dopamine-dependent modulation of sensory and attention gating.

Project Summary Learning to avoid environmental threats is essential for survival. Our knowledge of the neural substrates within the central nervous system responsible for learning to avoid such threats is incomplete. The precise analysis of the function and regulation of specific neurons within neural circuits that mediate threat perception and avoidance is essential for our understanding of these basic neural processes and the etiology of neurological and psychiatric disease. We have identified a population of neurons within the parabrachial nucleus (PBN) that express calcitonin gene-related peptide (CGRP). This specific population of neurons is necessary and sufficient for mediating behavioral responses to foot shock and visceral malaise. Experimental activation of CGRP-neuron terminals in the central nucleus of the amygdala is sufficient to generate fear or taste memories; however, activation of CGRP receptor (CALCRL) neurons in the CeA is sufficient for fear conditioning but not for taste conditioning. This multi-investigator proposal will draw on the expertise of three PIs (Dr. Zweifel, Dr. Palmiter, and Dr. McKnight) to discover how different threats are differentially recognized by the central nucleus of the amygdala (CeA). To address this fundamental question we will integrate cutting-edge techniques in mouse genetics, viral-mediated circuit dissection, behavior, in vivo imaging, electrophysiology, and cell-specific molecular profiling. We will characterize the molecular profile (translated mRNAs) of postsynaptic CGRP receptor (CALCRL)-expressing and non-CALCRL expressing neurons in the CeA to establish the identity of these neurons recruited during conditioned taste aversion and fear. We will elucidate the extent of activation of these circuit components using in vivo calcium imaging of circuit dynamics in freely behaving mice. We will establish how distinct noxious stimuli associated with pain or visceral malaise diverge at the level of CALCRL-and non-CALCRL expressing neurons in the CeA through functional manipulation of these neural circuit connections with light- and drug-activated receptors. Successful completion of this proposal will delineate key circuit components underlying aversive processing in the brain and will serve as a gateway to future investigations into downstream circuit components critical for these processes.

Project Summary Dopamine neurons of the ventral tegmental area (VTA) provide essential modulatory signals to the forebrain limbic system and cortex to facilitate learning and motivational processes. Disruptions in dopamine signaling are broadly implicated in mental illness and likely contribute to a spectrum of behavioral dysfunction through distinct cortico-limbic pathways. The heterogeneity of dopamine neurons in the VTA has long been appreciated, but therapeutic strategies targeting the system are still based on a monolithic perspective. Recent advances in mapping the input/output relationships of distinct dopamine pathways have attempted to resolve the basic organization of the VTA, but have yielded little in the way of establishing how specialized subdivisions might be organized. Unraveling the basic anatomical and functional organization of the VTA is essential if precision therapeutics are to be achieved for treating specific behavioral dysfunctions. We hypothesized that the dopamine system in the VTA can be organized based on peptidergic modulation to gate information through specific output pathways. We have mapped the projections of dopamine neurons in the VTA that express distinct neuropeptide receptors and discovered a remarkable specialization of these outputs. Here we propose to establish the basic electrophysiological, anatomical, and behavioral function of these pathways using novel Cre driver mouse lines in combination with advanced techniques in viral-based circuit dissection. We will also establish novel methods for the genetic characterization of these subpopulations. Successful completion of our proposed aims will provide novel insight into the basic organization of the VTA dopamine system and establish a framework for cracking the dopamine code.

Summary Recent evidence shows that the potent brain stimulatory effects of amphetamine are controlled by the endocannabinoid (eCB) signaling system. Our laboratory established that the enzyme, ?/?-hydrolase domain 6 (ABHD6), represent a novel molecular component of the eCB signaling system. Indeed, this post-synaptic enzyme controls the activity-dependent production of 2-arachidonoylglycerol (2-AG, the most abundant eCB in the brain), and as such controls the levels and efficacy of 2-AG at cannabinoid CB1 receptors (CB1R). We recently evaluated the involvement of ABHD6 in the locomotor responses stimulated by amphetamine in mice and found that its pharmacological inhibition and genetic deletion exerts a profound enhancing effect on the acute amphetamine-stimulated locomotor activity through a CB1R-dependent mechanism. In this R21 grant, we propose to identify brain regions involved in the ABHD6-dependent control of psychostimulants using several recently developed tools, including a brain-penetrant selective inhibitor of ABHD6 (KT-182 and MJN193), a Cre-dependent ABHD6 mouse line (ABHD6lox/lox), a specific antibody for ABHD6, and a CRISPR/Cas9 Slc6a3 (DAT) knockout model of hyperactivity. There are two main questions that will be addressed by this proposal. First, where in the brain is the interaction between ABHD6 and amphetamine occurring? Second, does ABHD6 alone, or in combination with low dose amphetamine, result in paradoxical calming response measured in a mouse model of attention deficit hyperactivity disorder (ADHD)-like phenotypes. Our aims are: 1: Identify brain regions involved in the ABHD6-dependent control of psychostimulants. 2: Establish the dose-dependent effects of ABHD6 inhibition on the paradoxical calming effects of psychostimulants in an animal model of hyperactivity. The completion of these studies will provide foundational results on the molecular mechanism by which ABHD6 regulates psychostimulant behavior in mice. A better understanding of this novel molecular interaction should help optimize the therapeutic use of psychostimulants while reducing their addiction and toxicity profile.