Inactivation of ERK1/2 Signaling in Dopaminergic Neurons by Map Kinase Phosphatase MKP3 Regulates Dopamine Signaling and Motivation for Cocaine.

The mesolimbic dopamine system is a crucial component of reward and reinforcement processing, including the psychotropic effects of drugs of abuse such as cocaine. Drugs of abuse can activate intracellular signaling cascades that engender long-term molecular changes to brain reward circuitry, which can promote further drug use. However, gaps remain about how the activity of these signaling pathways, such as ERK1/2 signaling, can affect cocaine-induced neurochemical plasticity and cocaine-associated behaviors specifically within dopaminergic cells. To enable specific modulation of ERK1/2 signaling in dopaminergic neurons of the ventral tegmental area, we utilize a viral construct that Cre dependently expresses Map kinase phosphatase 3 (MKP3) to reduce the activity of ERK1/2, in combination with transgenic rats that express Cre in tyrosine hydroxylase (TH)-positive cells. Following viral transfection, we found an increase in the surface expression of the dopamine transporter (DAT), a protein associated with the regulation of dopamine signaling, dopamine transmission, and cocaine-associated behavior. We found that inactivation of ERK1/2 reduced post-translational phosphorylation of the DAT, attenuated the ability of cocaine to inhibit the DAT, and decreased motivation for cocaine without affecting associative learning as tested by conditioned place preference. Together, these results indicate that ERK1/2 signaling plays a critical role in shaping the dopamine response to cocaine and may provide additional insights into the function of dopaminergic neurons. Further, these findings lay important groundwork toward the assessment of how signaling pathways and their downstream effectors influence dopamine transmission and could ultimately provide therapeutic targets for treating cocaine use disorders.

Matrix metalloproteinase activity during methamphetamine cued relapse.

Relapse to drug seeking involves transient synaptic remodelling that occurs in response to drug-associated cues. This remodelling includes activation of matrix metalloproteinases (MMPs) to initiate catalytic signalling in the extracellular matrix in the nucleus accumbens core (NAcore). We hypothesized that MMP activity would be increased in the NAcore during cue-induced methamphetamine (meth) seeking in a rat model of meth use and relapse. Male and female rats had indwelling jugular catheters and bilateral intracranial cannula targeting the NAcore surgically implanted. Following recovery, rats underwent meth or saline self-administration (6 h/day for 15 days) in which active lever responding was paired with a light + tone stimulus complex, followed by home cage abstinence. Testing occurred after 7 or 30 days of abstinence. On test day, rats were microinjected with a fluorescein isothiocyanate (FITC)-quenched gelatin substrate that fluoresces following cleavage by MMP-2,9, allowing for the quantification of gelatinase activity during cued-relapse testing. MMP-2,9 activity was significantly increased in the NAcore by meth cues presentation after 7 and 30 days of abstinence, indicating that remodelling by MMPs occurs during presentation of meth associated cues. Surprisingly, although cue-induced seeking increased between Days 7 and 30, MMP-2,9 activity did not increase. These findings indicate that although MMP activation is elicited during meth cue-induced seeking, MMP activation did not parallel the meth seeking that occurs during extended drug abstinence.

Chemogenetic Manipulation of Dopamine Neurons Dictates Cocaine Potency at Distal Dopamine Transporters.

The reinforcing efficacy of cocaine is largely determined by its capacity to inhibit the dopamine transporter (DAT), and emerging evidence suggests that differences in cocaine potency are linked to several symptoms of cocaine use disorder. Despite this evidence, the neural processes that govern cocaine potency in vivo remain unclear. In male rats, we used chemogenetics with intra-VTA microinfusions of the agonist clozapine-n-oxide to bidirectionally modulate dopamine neurons. Using ex vivo fast scan cyclic voltammetry, pharmacological probes of the DAT, biochemical assessments of DAT membrane availability and phosphorylation, and cocaine self-administration, we tested the effects of chemogenetic manipulations on cocaine potency at distal DATs in the nucleus accumbens as well as the behavioral economics of cocaine self-administration. We discovered that chemogenetic manipulation of dopamine neurons produced rapid, bidirectional modulation of cocaine potency at DATs in the nucleus accumbens. We then provided evidence that changes in cocaine potency are associated with alterations in DAT affinity for cocaine and demonstrated that this change in affinity coincides with DAT conformation biases and changes in DAT phosphorylation state. Finally, we showed that chemogenetic manipulation of dopamine neurons alters cocaine consumption in a manner consistent with changes in cocaine potency at distal DATs. Based on the spatial and temporal constraints inherent to our experimental design, we posit that changes in cocaine potency are driven by alterations in dopamine neuron activity. When considered together, these observations provide a novel mechanism through which GPCRs regulate cocaine's pharmacological and behavioral effects.SIGNIFICANCE STATEMENT Differences in the pharmacological effects of cocaine are believed to influence the development and progression of cocaine use disorder. However, the biological and physiological processes that determine sensitivity to cocaine remain unclear. In this work, we use a combination of chemogenetics, fast scan cyclic voltammetry, pharmacology, biochemistry, and cocaine self-administration with economic demand analysis to demonstrate a novel mechanism by which cocaine potency is determined in vivo These studies identify a novel process by which the pharmacodynamics of cocaine are derived in vivo, and thus this work has widespread implications for understanding the mechanisms that regulate cocaine consumption across stages of addiction.

Estrogen receptor beta signaling enhances extinction memory recall for heroin-conditioned cues in a sex- and region-specific manner.

Return to use, or relapse, is a major challenge in the treatment of opioid use disorder (OUD). Relapse can be precipitated by several factors, including exposure to drug-conditioned cues. Identifying successful treatments to mitigate cue-induced relapse has been challenging, perhaps due to extinction memory recall (EMR) deficits. Previously, inhibition of estradiol (E2) signaling in the basolateral amygdala (BLA) impaired heroin-cue EMR. This effect was recapitulated by antagonism of BLA estrogen receptors (ER) in a sex-specific manner such that blocking ERα in males, but ERß in females, impaired EMR. However, it is unclear whether increased E2 signaling, in the BLA or systemically, enhances heroin-cue EMR. We hypothesized that ERß agonism would enhance heroin-cue EMR in a sex- and region-specific manner. To determine the capacity of E2 signaling to improve EMR, we pharmacologically manipulated ERß across several translationally designed experiments. First, male and female rats acquired heroin or sucrose self-administration. Next, during a cued extinction session, we administered diarylpropionitrile (DPN, an ERß agonist) and tested anxiety-like behavior on an open field. Subsequently, we assessed EMR in a cue-induced reinstatement test and, finally, measured ERß expression in several brain regions. Across all experiments, females took more heroin and sucrose than males and had greater responses during heroin-cued extinction. Administration of DPN in the BLA enhanced EMR in females only, driven by ERß's impacts on memory consolidation. Interestingly, however, systemic DPN administration improved EMR for heroin cues in both sexes across several different tests, but did not impact sucrose-cue EMR. Immunohistochemical analysis of ERß expression across several different brain regions showed that females only had greater expression of ERß in the basal nucleus of the BLA. Here, in several preclinical experiments, we demonstrated that ERß agonism enhances heroin-cue EMR and has potential utility in combatting cue-induced relapse.

Perirhinal to prefrontal circuit in methamphetamine induced recognition memory deficits.

Return to methamphetamine (meth) use is part of an overarching addictive disorder hallmarked by cognitive sequela and cortical dysfunction in individuals who use meth chronically. In rats, long access meth self-administration produces object recognition memory deficits due to drug-induced plasticity within the perirhinal cortex (PRH). PRH projections are numerous and include the medial prefrontal cortex (mPFC). To evaluate the role of the PRH-mPFC reciprocal circuit in novel object recognition memory, a rgAAV encoding GFP-tagged Cre recombinase was infused into the PRH or the mPFC and rats were tested for recognition memory. On test day, one group explored both familiar and novel objects. A second group explored only familiar objects. GFP and Fos expression were visualized in the mPFC or PRH. During exploration, PRH neurons receiving input from the mPFC were equally activated by exploration of novel and familiar objects. In contrast, PRH neurons that provide input to the mPFC were disproportionately activated by novel objects. Further, the percent of Fos + cells in the PRH positively correlated with recognition memory. As such, the flow of communication appears to be from the PRH to the mPFC. In agreement with this proposed directionality, chemogenetic inhibition of the PRH-mPFC circuit impaired object recognition memory, whereas chemogenetic activation in animals with a history of long access meth self-administration reversed the meth-induced recognition memory deficit. This finding informs future work aimed at understanding the role of the PRH, mPFC, and their connectivity in meth associated memory deficits. These data suggest a more complex circuitry governing recognition memory than previously indicated with anatomical or lesion studies.

Individual differences in dopamine uptake in the dorsomedial striatum prior to cocaine exposure predict motivation for cocaine in male rats.

A major theme of addiction research has focused on the neural substrates of individual differences in the risk for addiction; however, little is known about how vulnerable populations differ from those that are relatively protected. Here, we prospectively measured dopamine (DA) neurotransmission prior to cocaine exposure to predict the onset and course of cocaine use. Using in vivo voltammetry, we first generated baseline profiles of DA release and uptake in the dorsomedial striatum (DMS) and nucleus accumbens of drug-naïve male rats prior to exposing them to cocaine using conditioned place preference (CPP) or operant self-administration. We found that the innate rate of DA uptake in the DMS strongly predicted motivation for cocaine and drug-primed reinstatement, but not CPP, responding when "price" was low, or extinction. We then assessed the impact of baseline variations in DA uptake on cocaine potency in the DMS using ex vivo voltammetry in naïve rats and in rats with DA transporter (DAT) knockdown. DA uptake in the DMS of naïve rats predicted the neurochemical response to cocaine, such that rats with innately faster rates of DA uptake demonstrated higher cocaine potency at the DAT and rats with DAT knockdown displayed reduced potency compared to controls. Together, these data demonstrate that inherent variability in DA uptake in the DMS predicts the behavioral response to cocaine, potentially by altering the apparent potency of cocaine.