The opioid epidemic has garnered much of the public’s attention in recent years, but addiction to psychostimulants continues to represent a substantial proportion of the overall addiction crisis the US. Most addictive drugs increase dopamine release in nucleus accumbens [1]. In addition, repeated drug exposure causes long-lasting neural changes that remain in place long after the drug is no longer onboard. This enduring neural plasticity is thought to be mediated in part by epigenetic mechanisms [2], processes that modulate gene expression without altering the DNA sequence. Methylation of DNA at cytosine-phospho-guanine (CpG) dinucleotides can alter the expression of genes and contributes to neuronal changes that play a role in memory and behavioral effects of addictive drugs [3, 4]. A challenging and active research area is to delineate how psychostimulant-induced increased dopamine neurotransmission translates to transcriptional and epigenetic changes that cause long-lasting changes in addiction-related behaviors [5].

In a recent Neuropsychopharmacology publication, Zipperly et al. [6] address this important question using a comprehensive combination of in vivo and in vitro cutting-edge approaches. Gadd45b, a member of the Growth arrest and DNA-damage-inducible gene family is important for synaptic plasticity and memory formation. Gadd45b is also associated with decreased methylation at CpG islands within regulatory DNA regions [3, 4]. Hence, Gadd45b lies at the intersection of experience-dependent neuronal activity and downstream regulation of DNA methylation states [7, 8], making it a promising candidate to link dopamine neurotransmission to cocaine-induced plasticity and epigenetic changes.

Zipperly et al. [6] first showed that acute cocaine injections transiently elicits the transcription of Gadd45b, but not other enzymes involved in DNA methylation such as DNA methyl transferases or ten-eleven translocation enzymes in rat’s nucleus accumbens. Gadd45b was the only component of the DNA methylation machinery tested that behaved like other immediate early genes such as Arc, Fos and Egr1—showing increased expression 1 h but not 24 h after acute cocaine injections. Moreover, cocaine sensitization or re-exposure to a cocaine-associated context was sufficient to increase nucleus accumbens Gadd45b expression, suggesting a potential role of this molecule in mediating cocaine’s behavioral effects. Next, the authors directly tested the functional relevance of Gadd45b in the formation of cocaine-related memories. Constitutive Gadd45b knock out mice did not show cocaine conditioned place preference (CPP), suggesting that Gadd45b expression is necessary for cocaine-context associations. The authors then turned to a targeted gene knockdown approach using a CRISPR cas9 lentiviral construct designed to target exon 2 of Gadd45b. Knockdown of Gadd45b in the nucleus accumbens core of rats also prevented cocaine CPP, demonstrating that local Gadd45b is necessary for cocaine reward memory.

To further delineate the signaling pathways up- and downstream of Gadd45b, Zipperly et al. turned to a highly controllable rat primary striatal neuron culture system. Cultured neurons were first treated with dopamine (1 µM) for 1 h, a regimen that closely mimics the temporal dynamics of dopamine following cocaine administration in vivo. Dopamine increased expression of Gadd45b in striatal cells and this effect was blocked by co-treatment with the D1 dopamine receptor (Drd1) antagonist SCH-23390, suggesting that dopamine-induced expression of Gadd45b is mediated by Drd1 receptors. Consistent with this possibility, treatment with the Drd1 receptor agonist SKF-38393 also increased Gadd45b expression. In contrast, application of the D2/D3 dopamine receptor agonist quiporole did not change Gadd45b expression in cultured neurons. Drd1 receptors are coupled to the GαS G protein, which increases levels of cAMP intracellularly. The adenylyl cyclase activator Forskolin also increased Gadd45b mRNA expression in striatal neurons. Inhibition of the cAMP response element binding protein (CREB) and mitogen-activated protein kinase (MEK) signaling also blunted dopamine-elicited changes in Gadd45b mRNA expression. Together, these experiments demonstrate that dopamine-induced transcription of Gadd45b requires activation of Drd1 receptors and signaling through the cAMP, MAPK and CREB signaling pathways.

To further characterize the role of Gadd45b in regulating transcription and DNA methylation related to Drd1 activation, the authors designed a custom short-hairpin RNA (shRNA) tool to manipulate Gadd45b in cultured striatal neurons. RNA sequencing revealed that Gadd45b knockdown decreased expression of genes related to dopaminergic and glutamatergic synapses. To refine which gene programs were specifically engaged by Drd1 activation, the authors compared the transcriptome of neurons treated with either vehicle or SKF-38393 and expressing either control or Gadd45b shRNA. Interestingly, many of the genes that are most responsive to Drd1 activation in control neurons showed little response following SKF-38393 application when Gadd45b was knocked down, demonstrating that Gadd45b deletion prevents the full activation of dopamine-responsive gene programs in striatal neurons. The authors also examined whether DNA methylation patterns were affected by Gadd45b knockdown using reduced representation bisulfite sequencing. Surprisingly, only a small proportion of CpG sites were affected by Gadd45b knockdown, suggesting that this manipulation leaves baseline DNA methylation landscapes largely intact. In contrast, Gadd45b knockdown prevented the SKF-38393-mediated changes in methylation in striatal cells, suggesting that Gadd45b is critical for both hypermethylation and hypomethylation of loci following Drd1 receptor activation.

Lastly, the authors examined whether Gadd45b knockdown affected the physiological properties of striatal neurons using a high-throughput multi-electrode array system. This approach allows for both baseline measurements and assessments following application of SKF-38393. Given the baseline changes in genes related to glutamate and dopamine signaling, the authors unexpectedly found that neurons transduced with Gadd45b shRNA did not differ from scrambled controls in spontaneous activity, firing rate, mean action potential burst frequency, or number of spikes in each burst. Only burst duration was reduced by Gadd45b deletion. Moreover, SKF-38393 application increased action potential firing rate in both control and Gadd45b shRNA-expressing cells, suggesting that Gadd45b knockdown did not alter the electrophysiological changes associated with Drd1 receptor activation in striatal neurons.

Collectively, these findings highlight the importance of Gadd45b in the nucleus accumbens downstream of Drd1 activation in orchestrating transcriptional and epigenetic alterations that contribute to cocaine physiological and behavioral effects. A better understanding of the neural mechanisms associated with cocaine reward and dopamine signaling lays the groundwork to develop improved therapeutics for substance abuse. Treatments that go beyond treating symptoms associated with drug use and target the underlying long-term brain changes are needed. This elegant and comprehensive study contributes to these efforts and also addresses important aspects of reward-related memory formation. The authors’ findings have broad implications to multiple research disciplines and raise some interesting questions about the molecular underpinnings of reward memories.

Funding and disclosure

The author declares no competing interests.