The Role of Cocaine and Dopamine D2 Receptors in Conditioned Behaviors

Post by Andrew Vo

What's the science?

Addictive drugs are known to alter brain circuits — specifically the midbrain dopamine system that innervates the dorsal striatum (DSt) and nucleus accumbens (NAc). Dopamine signaling can have different effects in these distinct brain regions depending on the class of dopamine receptor the dopamine reaches: D1 receptors excite medium spiny neurons (MSNs), while D2 receptors have inhibitory effects on behaviour. Although the role of D2 receptors (D2Rs) in motivated behavior is understood and the impact of D2R dysregulation following long-term drug use in addiction is well-established, it remains unknown how initial cocaine exposure regulates D2R signaling in a region-specific manner to alter conditioned behaviors. This week in Neuron, Gong et al. investigated the cellular mechanisms underlying the effect of repeated cocaine exposure on drug-seeking behaviors in mice.

How did they do it?

The authors began by examining a) the effect of repeated cocaine exposure on D2R sensitivity in D2-MSNs and b) whether this effect differed between the DSt and NAc. To measure D2R signaling in D2-MSNs, electrophysiological activity from D2-MSNs was recorded during cocaine exposure while modulating the concentration of dopamine stimulation, generating a dose-response curve. Mice were repeatedly exposed to cocaine for 7 days, followed by a 14-day withdrawal period, before a single injection challenge of cocaine. Response curves were generated after each of these phases.

Next, the authors set out to determine the exact mechanism underlying changes in D2R sensitivity following cocaine exposure. To test whether these changes were caused by alterations in D2R levels, they sampled tissues from the DSt and NAc of mice treated with either cocaine or a saline control and measured the relative density of D2Rs using immunoblotting (a technique for analyzing proteins in a sample using antibody staining). They also generated mice in which D2R levels were either (1) knocked down or (2) overexpressed and observed any changes in the effects of cocaine exposure on D2R sensitivity. To test whether these changes were instead caused by regulation of G proteins, which tightly couple with D2Rs to facilitate dopamine signaling, they sampled tissues from the DSt and NAc of mice following acute, chronic, withdrawn, and cocaine-challenge compared to saline controls and measured G protein levels using western blotting (which detects the type and amount of a specific protein in a mixture). Further, they measured the effects of cocaine exposure on D2R sensitivity in G protein knockdown mice and again after G protein re-expression via a viral rescue procedure.

The authors were also interested in the behavioral effects of cocaine-mediated changes in D2R sensitivity. Using a conditioned place preference paradigm, in which mice learn to associate a previously neutral chamber with a drug, they compared behavior of G protein knockdown mice to controls, as well as following re-expression of G protein levels. In a self-administration task that assessed reinforcement learning, G protein knockdown and control mice were trained to self-administer cocaine in response to a cue, followed by an abstinence period, before a final relapse test.

Finally, the authors explored potential mechanisms that could capture the effect of cocaine exposure on D2R sensitivity changes. They tested the effects of increasing (i.e., administering a dopamine precursor or D2R-specific agonist) or decreasing (i.e., administering D2R or D1R antagonists) dopamine stimulation. They also examined the effects of chemogenetic inactivation of D1Rs in either D1-MSNs or the prefrontal cortex. Last, they tested the role of NMDA plasticity by administering an NMDA-receptor antagonist.

What did they find?

Repeated exposure to cocaine caused a rightward shift in the dose-response curve in the NAc but not DSt, indicating a region-specific reduction in D2R sensitivity. This reduced D2R sensitivity returned to baseline levels after a drug withdrawal period but was immediately reinstated following a single challenge injection of cocaine.

Immunoblotting revealed similar levels of D2R expression in both DSt and NAc in cocaine-treated mice compared to controls. Reducing or enhancing D2R levels, via knockdown or overexpression respectively, did not prevent cocaine-associated decreases in D2R sensitivity in the NAc. Western blotting showed that cocaine exposure reduced G protein levels in the NAc but not DSt. These altered levels returned to baseline after a withdrawal period but were reinstated following a single injection challenge of cocaine. Decreasing G protein levels via knockdown mice successfully reduced D2R sensitivity in the NAc and blocked the effect of chronic cocaine exposure. Viral rescue of G protein levels in these mice recovered the cocaine-associated reduction in D2R sensitivity in NAc. Collectively, these findings indicate that D2R sensitivity changes following cocaine exposure occur independently of changes in D2R levels and are instead related to the regulation of G proteins.

Cocaine conditioned place preference caused a reduction in G protein levels in the NAc. G protein knock-down mice were found to spend more time in the drug-associated chamber. This preference was eliminated after re-expression of G protein levels via viral rescue. In the self-administration task, knock-down mice were unaffected in their ability to self-administer cocaine in response to a cue. Following an abstinence period and subsequent relapse test, these knock-down mice were unaffected in the reinstatement of drug-seeking. Taken together, these results suggest that cocaine-induced changes in G protein expression leading to a reduction in D2R sensitivity in the NAc underlie conditioned drug-seeking behaviors.  

Increasing extracellular dopamine levels did not affect D2R sensitivity, unlike the result of cocaine exposure. D2R antagonism before cocaine exposure did not block reductions in D2R sensitivity in the NAc. In contrast, D1R antagonism could successfully block this cocaine-mediated effect. Further examining the role of D1R regulation of D2R-MSN sensitivity, inactivation of D1Rs in PFC but not D1-MSNs blocked the effect of cocaine on D2R sensitivity in NAc. A similar effect could be achieved by blocking NMDA receptors. These findings illustrate a regulatory role of D1R inputs from the PFC on D2R sensitivity.

What's the impact?

In summary, this study demonstrated that initial chronic exposure to cocaine reduced the sensitivity—but not the level—of D2Rs specifically in the NAc of mice. Reduced D2R sensitivity was caused by decreased expression of G protein in D2-MSNs following cocaine exposure. Together, these changes promoted conditioned drug-seeking behaviors. Uncovering the neural mechanisms through which initial drug exposure regulates drug-seeking behavior has important implications for the treatment of addiction and relapse.

Gong et al. Cocaine shifts dopamine D2 receptor sensitivity to gate conditioned behaviors. Neuron (2021). Access the original scientific publication here.