Garret D. Stuber, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The nucleus accumbens has long been implicated as an essential brain region for the manifestation of reward-related behaviors and addiction. Furthermore, it is known that a primary chemical component of 'reward' in the nucleus accumbens comes from dopaminergic input from the ventral tegmental area. Evidence from electrophysiological studies suggests that dopaminergic burst firing (occurring over seconds) may be an important mediator of associative learning between environmental cues and the primary effects of a reinforcer. While previous microdialysis and electrochemical studies have investigated how dopamine levels can change over minutes to hours during cocaine self administration, little is known about how dopamine changes on a second to second timescale. The proposed experiments plan to examine the role of phasic dopamine neurotransmission in the nucleus accumbens during cocaine self-administration in rats. In Experiment 1, we will determine whether differences exist between the two key subregions of the nucleus accumbens (the core and shell) with respect to phasic dopamine during cocaine self-administration. In preliminary experiments transient increases in dopamine were seen immediately after the lever-press for cocaine in the core of the NAc The second proposed experiment will examine how extinction/reinstatement of cocaine self-administration behavior affects phasic dopamine release in the nucleus accumbens. During extinction each lever-press will no longer result in cocaine delivery, and any changes in phasic dopamine will be examined. In reinstatement, cocaine infusions will again be paired with both drug associated cues and lever presses, and we will examine if this subsequent pairing of reinforcer with drug-associated cues alters phasic dopamine. These studies will further the understanding of how dopaminergic signaling is involved in drug reinforcement and conditioning. [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Dopaminergic neurons, originating in the midbrain, and projecting to forebrain areas including the prefrontal cortex, amygdala, and nucleus accumbens are critical for the manifestation of goal directed behavior for both natural rewards and drugs of abuse. While the exact role DAergic transmission plays in goal-directed behavior is still under investigation, one particularly attractive hypothesis is that DAergic neurons act as a learning signal to code relationships between reinforcers and environmental cues that predict them. Although both in vivo electrophysiological and electrochemical studies have demonstrated that DAergic signaling is plastic, and can be modified by manipulating the learned association between cues and reinforcers, little is known about the cellular changes that occur in DAergic neurons following stimulus-reward learning. The proposed experiments plan to examine whether the properties and plasticity of excitatory synapses on DAergic neurons are altered following reward-related learning and extinction. These studies will further the understanding of how dopaminergic signaling is involved in reinforcement and conditioning. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Understanding the neural circuitry that underlies reward-related behavior is essential for developing precise and effective treatments for addictive behavior. While many brain regions are important for the manifestation and expression of reward-related behavior, the nucleus accumbens (NAc) is thought to be a brain nucleus critical for translating the affective state of an organism into a motor action. Because the NAc is heterogeneous;receiving diverse glutamatergic inputs from areas such as the basolateral amygdala (BLA) and medial prefrontal cortex (mPFC) as well as dopaminergic input from the ventral tegmental area, it has been difficult to study the contribution of a specific synaptic connections in controlling behavior. To circumvent this, we propose here to use optogenetic stimulation of channelrhodopsin-2 (ChR2) to specifically control neural transmission from the BLA and mPFC to the NAc. We will first selectively introduce ChR2 into glutamatergic neurons of the BLA and mPFC and characterize their firing properties with optical stimulation. Following expression of ChR2 in specific subsets of glutamate terminals in the NAc, we will characterize afferent-specific neurotransmission by examine synaptic strength and glutamate release probability. Finally, We will assay whether optical stimulation of BLA or mPFC to NAc synapses can reinforce behavioral responding, thus promoting goal-directed behavior. These experiments could provide important and novel information about the ability of particular excitatory inputs into the NAc to sustain reinforcement while also defining a minimal unit within a neural circuit that can drive reward-related behavior.

DESCRIPTION (provided by applicant): Utilizing environmental information to predict future positive and negative outcomes is a behavioral adaptation that is essential for survival. While this process is required for the control of natural motivated behavioral responding to obtain rewards, the neural circuits that encode cue-reward associations are thought to be dysfunctional in neuropsychiatric disorders such as addiction. Therefore, it is essential that further research is conducted to delineate the neural mechanism that underlie responses to reward-predictive cues in an effort to uncover specific neural circuit elements that mediate this phenomena. Signaling by midbrain dopamine neurons is thought to play an important role in controlling the formation and expression of cue-induced reward seeking. In this proposal, we aim to study neural circuit elements within the ventral midbrain that may be important for activating or inhibiting dopaminergic function and therefore influence the acquisition and expression of cue-reward associations. To accomplish this, we will take a multifaceted approach. We will perform in vitro slice electrophysiological experiments to characterize the functional connectivity between specific excitatory inputs to dopaminergic and GABAergic neurons within the midbrain. In addition, we will use in vivo optogenetic stimulation/inhibition experiments to establish or refute causal relationships between genetically and anatomically defined neural circuit elements in the midbrain and the release of dopamine in the nucleus accumbens to reward-predictive cues. The information gained from these studies may greatly advance our understanding of the neural circuits that encode cue-reward associations.

DESCRIPTION (provided by applicant): During abstinence from alcohol, stress in alcoholics can result in negative affect and craving (1), a response which is accompanied by a change in brain functional magnetic resonance imaging (fMRI)-effects of stress not seen in social drinkers (2). To model the negative affect induced by stress during abstinence in alcoholics, an extended period of chronic intermittent alcohol (CIA) exposure was found to cause an enduring adaptation that sensitizes stress-induced negative affect during abstinence. These series of clinical and basic findings are consistent with the kindling/stress hypothesis of alcoholism. Even though the central amygdala (CeA) is known to play an important role in stress-induced negative affect after CIA exposure, unknown is the role input circuits to the CeA have in facilitating this stress-induced anxiety. In addition to CRF, other inputs that synapse on CeA neurons are glutamate (GLU) terminals from the basolateral amygdala (BLA), oxytocin (OXY) terminals from the hypothalamus, and vasopressin (VP ) terminals. Nonetheless, the influence these other inputs have on stress-induced anxiety after CIA exposure has not been identified. Likewise unidentified is whether CeA medial division (CEM) neurons that output terminals to the periaqueductal gray (PAG) and other sites are a critical component of the neural circuit that supports stress-induced anxiety associated with CIA. To guide addressing these unknowns, the hypothesis tested is that stress facilitates anxiety during abstinence from CIA exposure by influencing a neural circuit composed of terminal inputs to the lateral (CEL) and medial (CEM) divisions of the CeA that modulate CEM output. First, to permit optogenetic investigations of the potential role BLA terminals have in facilitating stress-induced anxiety after CIA exposure, an AAV5-eYFP vector with a CAMKII promoter containing the rhodopsin derivatives halorhodopsin (NpHR3.0) or channel- rhodopsin (ChR2) will be placed into the BLA to confirm localization of GLU terminals from the BLA to the lateral (CEL) and the medial (CEM) divisions of the CeA. Subsequently tested with optogenetics will be whether ChR2 activation or NpHR3.0 inhibition of BLA terminal release of GLU on CEL neurons will influence facilitation of stress-induced anxiety after CIA exposure. This latter optogenetic strategy will be complemented by determining if activation of CEL neurons with an OXY receptor agonist will block facilitation of stress-induced anxiety after CIA exposure. Upon completing investigations of CEL neural inputs, optogenetic inhibition or excitation of GLU-containing BLA terminals synapsing on CEM neurons will assess if the stress-induced anxiogenic action after CIA exposure is affected. Because VP-containing terminals synapse on CEM neurons to induce anxiety- like behavior, it is reasoned that activation of CEM neurons with VP may contribute to the anxiety induced by stress following CIA exposure. To confirm a direct involvement of CEM neurons in VP action, an AAV-eYFP vector with the NpHR3 expressed in CEM neurons will test if optogenetic inhibition of CEM neurons will alter both stress- as well as VP-induced anxiety after CIA exposure. To confirm that VP action contributes to stress-induced anxiety after CIA exposure, VP receptor subtypes will be pharmacologically antagonized in the CEM prior to stress to determine if the stress- induced anxiety after CIA exposure is prevented. Upon confirmation of CEM terminal presence in the PAG, involvement of these CEM terminals in the PAG in stress- and VP-induced anxiety observed after CIA exposure will be assessed by optogenetic inhibition or excitation of these CEM terminals- an approach to implicate both the CEM and the PAG in facilitation of the stress-induced anxiety after CIA exposure. Collectively, the proposed pharmacological and optogenetic strategies utilized can be expected to define whether involvement of specific neural inputs to the CEL and CEM, which are accompanied by CEM output to the PAG and other sites, form a circuit that influences facilitation of stress-induced negative affect after CIA exposure. This proposed circuit involving the CeA is felt to be associated with the neuropathology responsible for the facilitated negative affect observed to stress that facilitates craving in alcoholics durin abstinence. New knowledge concerning adaptations in this circuit after CIA exposure could possibly provide clues by which to minimize the barrier to developing new and improved therapeutics to treat the negative symptoms to stress observed in abstinent alcoholics.

Autism spectrum disorder (ASD) is characterized by social impairments, including impaired social cognition, social percepfion, and social attention. Recently, there has been increased interest in examining the impact of motivational systems on social functioning in ASD. The frarinework of the so-called social motivation hypothesis of ASD is that functional disruption in brain circuits that support social motivational may constitute a primary deficit in ASD that may have downstream effects on the development of social cognition. The mesolimbic dopamine system arising in the ventral tegmental area (VTA) and projecfing to the nucleus accumbens (NAc) is an essential substrate for the expression of many forms of motivated behaviors. Human neuroimaging studies have demonstrated reduced mesolimbic activation in ASD to social rewards, suggesting that reduced function of the mesolimbic dopaminergic system may underlie decreased sociial motivation in ASD. Whereas social deficits in ASD may be related to pathological mesolimbic dopamine system activity, it is unknown if precise neural circuit manipulations that can directly control dopamine output in the NAc to promote pro-social behaviors in animal models of ASD. In addition, the neuropeptide oxytocin (OT) is a promising therapeufic to promote social engagement in ASD and is known to regulate VTA activity in response to social rewards specifically. However the functional neural circuitry by which OT neurons regulate VTA dopaminergic activity has not been identified. These are critical gaps in our understanding of the neural cii-cuitry that controls motivated social engagement. We propose a translational project integrating optogenetic circuit manipulafions in a mouse model of ASD with a clinical functional neuroimaging evaluation of the effects of OT on reward circuits in individuals with ASD.

Abstract: Motivated behaviors such as feeding and reward-seeking are critical for an organism's survival. These processes require distributed neuronal networks and supporting cell types in multiple brain regions to be tightly regulated and tuned in order to orchestrate behavioral output. The lateral hypothalamic area (LHA) has long been identified as a critical neuroanatomical substrate for motivated behavior. Despite decades of research, the molecular identity of defined LHA cell types remains poorly understood. Additionally, while LHA neurons have been previously shown to encode appetitive and consummatory behaviors via distinct cellular ensembles, it remains unknown how distinct LHA output neurons contribute to these processes. Here we propose to continue to study the neural circuits of the LHA, and to identify how distinct cell types contribute to feeding and reinforcement. Using an interdisciplinary approach to leverage cutting edge tools such as single cell transcriptional profiling, two photon calcium imaging, and viral we aim to undercover key circuit elements, and novel circuit specific gene expression patterns that can be leveraged for future therapeutic interventions for addiction and other neuropsychiatric disorders.

DESCRIPTION (provided by applicant): The Neurobiology Curriculum (NBIO) at UNC-Chapel Hill (UNC-CH) is one of the oldest neuroscience graduate training programs in the US having granted PhDs continuously for 47 years. NBIO has a reputation for excellence and rigor, and there has been constant recruitment of new faculty which keeps the program innovative. In support of NBIO, we request renewed funding for T32 NS007431, Neuroscience Predoctoral Training at UNC-Chapel Hill. This T32 supports 10 stipends and travel funds for NBIO students in the 1st and 2nd years of training. The Training Grant importantly impacts neuroscience graduate training at UNC-Chapel Hill (UNC-CH) in several ways: 1) T32 support for travel provides access to career-enhancing enrichment activities for the most promising students. 2) T32 support increases student access to the best training labs. 3) T32 support enhances our recruitment and mentoring of URM trainees. 4) T32 support leverages resources provided by the UNC School of Medicine and thereby helps support the seminar series and the annual UNC Neuroscience Symposium. NBIO is a comprehensive neuroscience graduate training program that has strong leadership and standing committees to guide the program, a thoughtfully constructed and rigorous Core course, and a diverse group of electives. There are numerous, well-attended community activities including weekly seminars, a weekly data presentation series for the students, an annual symposium, presentation of the internationally recognized Perl/UNC Neuroscience Prize, and an annual student research day. Mentoring in the program is continuous and there are well-designed activities to educate students about academic and non-academic careers. Faculty and students have outstanding publication records. Multiple trainees (27%) received individual NRSAs during the reporting period. Almost all trainees who received PhDs during the past 10 years are pursuing scientific careers including academic positions, positions in pharma/biotech, other scientific careers, completion of MD/PhD training, and postdoctoral fellowships. There is an outstanding record of recruiting and retaining URM trainees. Renewal would allow UNC-CH to continue training the next generation of neuroscientists including a strong contingent from URM groups.

Project Summary: The neural circuitry that encodes and mediates the establishment of cue-reward associations, an adaptive process that is essential for survival, likely becomes dysfunctional in neuropsychiatric illnesses such as drug addiction. While the full encoding of cue-reward associations require a distributed network of brain nuclei acting in concert to orchestrate behavioral output, neurons in ventral tegmental area and upstream circuits in cortex and hypothalamus are thought to play an important role in reward prediction and assigning incentive salience to environmental cues such as those that become associated with repeated drug use. In this application, we propose to state of the art deep brain two-photon imaging in awake and behaving mice to study how the encoding properties within these circuits emerge and are altered during primary reward exposure as well in associative learning. These experiments will provide important mechanistic information to explain how reward circuits encode and control the development and expression of cue-reward associations relevant to addiction.

Abstract: Opiate addiction extorts a tremendous toll on society, but a mechanistic understanding of how repeated exposure to opioids such as heroin ultimately results in compulsive drug-taking and -seeking behavior in some individuals, but not others, is still not known. A longstanding idea is that enduring changes in neural circuit function occur because of drug-induced gene expression changes in certain brain cells. This facilitates subsequent drug-taking and -seeking behaviors in vulnerable individuals. Unfortunately, identifying cell-type specific alterations following drug use (typically performed in established animal models of addiction), is generally a slow and tedious process as changes in gene expression following in vivo drug exposure are typically assayed in series, within heterogeneous brain regions, in an a-priori hypothesis driven fashion (i.e. previous knowledge predicting a specific gene may be involved). This dramatically limits the throughput of data collection and likely complicates the subsequent interpretation as gene expression patterns data are typically captured from thousands to millions of homogenized cells. Given that the nervous system is composed of highly heterogeneous tissue, re-assessing cell type specific gene expression changes in an unbiased manner from 1000's of individual cells is desperately needed. Here, we propose to combine our expertise in order to generate comprehensive datasets aimed at understanding how single-cell gene expression, circuit connectivity, and neural activity patterns are impacted by previous drug-taking behavior. These data will provide a much-needed cellular atlas and resource for the addiction neuroscience community and will likely lead to the identification of many novel cell type, gene expression changes, and ensembles that can be leveraged for future study.