Dopamine Signaling in Neuropsychiatric Disorders and Addiction

Dopamine (DA) signaling in the brain is requisite for a number of key behaviors, including motivation, reward, motor function, and learning. Multiple neurological and neuropsychiatric disorders exhibit aberrant DA signaling, including addiction, schizophrenia, autism spectrum disorder (ASD), Parkinson's disease, and attention-deficit/hyperactivity disorder (ADHD). Despite the association of these disorders with dopaminergic dysfunction, the molecular mechanisms and neuronal circuits involved in these processes are not well defined. In order to investigate these pressing questions, we leverage a variety of approaches that span from molecules to behavior in mouse models, including in vivo monitoring of neuronal activity and DA signaling using genetically encoded tools. We currently are pursuing multiple lines of investigation:

Regulation of the Cocaine-Sensitive DA Transporter (DAT):  Our laboratory is interested in the circuit- and molecular-specific mechanisms that regulate DA signaling and DA-dependent behaviors. Once released, extracellular DA is temporally and spatially restricted by presynaptic DA reuptake facilitated by the DA transporter (DAT). In addition to its central role in basal synaptic transmission, DAT is the primary target for addictive psychostimulants, cocaine and amphetamine, as well as therapeutic psychoactive drugs, such as methylphenidate (Ritalin) and bupropion (Wellbutrin/Zyban).  These agents block DAT activity and thereby enhance extracellular DA levels and drive dysfunction in DA-depndent behaviors. 

Given DAT’s importance in DAergic neurotransmission and as a psychoactive drug target, cellular mechanisms that impact DAT function are likely to have significant impact on DA signaling and neuropsychiatric disorders.  Multiple DAT coding variants have been identified in ADHD and autism patients, further supporting that altered DAT function is linked to significant behavioral consequences.  Work from our lab investigates the cellular and molecular mechanisms that regulate DAT.  Endocytic trafficking dynamically controls DAT plasma membrane availability, and a variety of cellular signaling pathways and psychostimulant drugs rapid alter DAT trafficking, surface expression and function. We have identified multiple key players that govern DAT trafficking.  Using a variety of cutting edge approaches, such as viral-mediated gene expression, gene silencing (RNAi), optogenetics and chemogenetics, we are investigating how DAT regulation impacts DA neurotransmission and DA-associated behaviors.

Role of modulatory glutamate signaling in motor function, novelty, and reward: Glutamate is the major excitatory neurotransmitter in the brain, but also has a modulatory role by signaling through metabotropic glutamate receptors (mGluRs). Recent work from our laboratory revealed that selective expression of mGluR5 in DA neurons is required for several DA-dependent behaviors and DA signaling. However, the circuit- and mechanistic-specific underpinnings of these processes have not been elucidated. Using a novel conditional knockout model, we are leveraging several intersectional approaches to determine how DAergic mGluR5 impacts DA neuron function and DA-dependent behaviors.

3. Nociceptin: A neuropeptide that impacts cocaine sensitivity

Nociceptin is an endogenous opiate-like neuropeptide that is implicated in pain and reward motivation. Recent studies suggest that nociceptin has the capacity to block cocaine's initial addictive properties, but the mechanisms underlying nociceptin's actions are largely unknown. Work in our lab has discovered that nociceptin blocks cocaine reward, and does so by acting directly on DA axon terminals in the nucleus accumbens, the brain's "reward center". In addition, our results suggest that nociceptin likely shifts cocaine sensitivity, and does so by controlling reward tone differentially in males vs. females. These exciting results may pave the way towards harnesing nociceptin signaling as a potential addiction therapeutic. However, the mechanisms underlying nociceptin's actions are unknown, nor is it known whether nociceptin specificallly impacts cocaine reward, or other drugs of abuse. There are two possible rotation projects:

Project 3a: Impact of nociceptin signaling on other drugs of abuse. Students will conduct addiction behavioral assays while infusing nociceptin directly into the nucleus accumbens in mice. Students will learn mouse handling and stereotaxic surgery, and mouse behavioral assays.

Project 3b: Sex-specific differences in nociceptin signalig. Students will use RT-qPCR to test whether males and female mouse midbrain neurons express differing amounts of the nociceptin receptor, NOPR. Students will learn mouse handling skills, brain harvesting, and RT-qPCR.

Nociceptin infusion into the nucleus accumbens blocks cocaine preference in females. Conditioned Place Preference Assay (CPP). Wildtype female mice received either saline or 10mg/kg cocaine (I.P.) and were trained for 3 days in a 3-chambered CPP aparatus, while being infused with either vehicle or 500nM nociceptin. Preference scores were determined on test days. Control-infused mice exhibited signifiant preference to the cocaine-paired chamber as compared to the saline-paired chamber (****p<0.001), whereas mice infused with nociceptin failed to establish cocaine preference (p=0.74). Two-way ANOVA with Tukey's multiple comparison test, n=4-10.

Potential Rotation Projects

1. Dopamine Transporter (DAT) Trafficking in Cocaine Addiction

DAT is a membrane protein in dopamine (DA) axon teminals whose primary function is to remove released dopamine and thereby terminate synpatic transmission. Addictive psychostimulants, such as cocaine, block DAT function, which increases extracellular DA and is responsible for cocaine's addictive properties. DAT is not static at the plasma membrane, but is dynamically regulated by membrane traffficking. This project is to determine whether DAT membrane trafficking is required for cocaine addiction. Our lab has developed a novel, AAV-mediated, in vivo molecular replacement strategy, that replaces wildytpe DAT with DAT trafficking dysregulated mutants in adult mice. There are 2 possible rotation projects:

Project 1a: Rotation students will assist in behaviorally assessing replacement mice as compared to controls, and will additionally use immumohistochemical approaches to validate mutant protein expression in dopamine neurons in situ. Students will gain experience in mouse behavior, brain dissection, preparation of brain slices,  and immunhistochemical techniques in mouse brain.

Project 1b: Rotation students will develop a CRISPR/Cas9 guide RNA in order to perform gene editing and mutate DAT in vivo, and will test its efficacy in vitro. Students will learn guide RNA design, and mammalian tissue culture, and genomic sequencing.

Cocaine's Synaptic Actions at the Dopamine Transporter. Synaptic models. (A) Dopamine released into the extracellular space is rapidly cleared by the dopamine transporter (purple), thereby limiting the postsynaptic dopamine signal. (B) Cocaine binds to and blocks the dopamine transporter, allowing dopamine to accumulate extracellularly and enhancing the postsynaptic signal.