Neurons in the ventral tegmental area (VTA) and Nucleus Accumbens (NAc) play key roles in reward-seeking and are usurped in addiction. While most work has focused on dopaminergic projections from VTA to NAc, how opioids alter projections from NAc to VTA is largely unknown. The NAc to VTA connection deserves considerable attention since D1-MSN activity drives reward-seeking behavior, D1-MSNs directly modulate VTA neurons, and opioid receptors expressed specifically on D1-MSN to VTA synapses control dopamine release. Opioid-induced transcriptional changes in VTA neurons play an important role in opioid abuse and are likely sensitive to altered D1-MSN activity. However, no studies have examined transcriptional changes based on a specific synaptic input. It is further unknown if molecular adaptations in the VTA caused by D1-MSN activity alter dopamine release. We have adopted a technically innovative approach that combines next-generation single nuclei RNA sequencing with transsynaptic viral tagging to identify transcriptional changes based on synaptic input.  RNAseq is a powerful tool for quantifying thousands of transcripts and can be used to identify changes in expression independent of a priori predictions. 

We are currently profiling transcriptional changes in VTA neurons receiving D1-MSN input after fentanyl self-administration. We will then generate Cre-dependent viruses to manipulate our candidate molecules to block fentanyl self-administration and seeking. With in vivo fast-scan cyclic voltammetry, we are determining how fentanyl and molecular targets in VTA neurons influence NAc dopamine release after self-administration. 

This project is funded by NIH/NIDA R00 DA050575

Opioid use disorder is a life-long burden for many individuals, imposing high personal, financial, and health costs. Even after prolonged abstinence, many individuals in recovery will go on to relapse, including those that received medication-assisted treatments. Repeated opioid exposure usurps normal reward circuit function by producing long-lasting molecular changes that alter physiology and support continued drug use. These cellular adaptations have been implicated in sustained relapse vulnerability, but we lack a clear understanding of what drives their persistence. There is a further lack of information on the precise molecular adaptations underlying altered circuit function, and in which specific circuits they act to promote relapse. Understanding this “who, what, when, and where,” will be key to identifying new therapeutic targets. Here, we will answer these questions using a multi-level approach that allows us to sequence, manipulate, and record from neurons in specific circuits in the context of opioid self-administration and relapse.  

Our preliminary data show that both genetically-distinct and genetically-identical neuron subtypes in the ventral tegmental area (VTA) undergo differential molecular adaptations after fentanyl self-administration, which we hypothesize arises from activity-dependent transcriptional changes in specific circuits. We further hypothesize the transcriptional changes are sustained by methylation and demethylation at the gene promoters. We will first record calcium activity in VTA neurons that project to either the nucleus accumbens (NAc) or amygdala (AMY)—projections known to be important for drug intake and relapse, respectively. Then, in the same neurons from the same animals, we will identify which gene networks are transcriptionally changed after self-administration and persist until relapse testing. Next, we will identify the DNA methylation marks driving sustained differential expression, with an emphasis on genes important for synaptic plasticity. Next, we will use CRISPR/dCas9 fusion constructs to manipulate methylation states at our identified loci in specific circuits. This proposal will allow us to define the specific VTA circuits that support opioid intake and relapse, which gene networks support activity of these circuits, and how DNA methylation cements the transcriptional landscape to alter behavior. This award will allow research into neural mechanisms of opioid use disorder with unprecedented resolution, and has the potential to transform how we approach studying the genetics of substance use disorders. Together, this critical information will help inform new treatment strategies to prevent relapse.

This project is funded by NIH/NIDA DP1DA058661

VTA dopamine neurons mediate behavioral outcomes to chronic social defeat stress (CSDS), an effect driven in part by upstream locus coeruleus (LC) norepinephrine neurons. LC norepinephrine activates adrenergic receptors on VTA dopamine neurons to reverse maladaptive cellular hyperactivity and promote resilient behavioral outcomes. While both LC and VTA neurons are implicated in the pathophysiology of depressive disorders, no work has tested how norepinephrine release and regulation in the VTA are altered by CSDS or the vicarious chronic witness defeat stress (CWDS). There is a further lack of information on how this circuit is altered in stressed females, or how its function might be altered by serotonin-norepinephrine reuptake inhibitor (SNRI) antidepressant treatments. We will examine noradrenergic mechanisms of stress resilience within the LC to VTA circuit with LC-specific optogenetic stimulation, in vivo FSCV, neuropharmacology, and next-generation sequencing.  

The dynamic interplay between stress and drug exposure whereby exposure to one increases the response to the other is usually termed “cross-sensitization”. Historically, most rodent cross-sensitization studies were restricted to rats, excluded females, or introduced confounds (pain, inflammation) that complicate stress and opioid cross-sensitization studies. Prior drug use is associated with increased risk for other psychiatric illness, but most cross-sensitization studies focus on stress’s effect on drug-taking behavior and leave out how drug-taking impacts stress-related behaviors.