Substance use disorders (SUD) are long-lasting and perpetuated by associative memories, which can induce motivation to seek drugs. Memories are maintained, in part, by learning-induced structural changes to dendritic spines, which are mediated by actin dynamics (Rudy, 2015). We previously reported that actin remains uniquely dynamic in the basolateral amygdala (BLA) following methamphetamine (METH) treatment. Nonmuscle myosin II (NMII) is a molecular motor ATPase that drives actin polymerization to support this structural plasticity (Rex, Gavin, Rubio, Kramar, Chen, Jia, Huganir, Muzyczka, Gall, Miller, Lynch, and Rumbaugh, 2010). Inhibiting NMII with a single administration of blebbistatin (Blebb) arrests METH-induced actin dynamics and results in dendritic spine loss in the BLA, as well as an immediate, retrieval-independent disruption of established METH-associated memories and METH seeking that persists for at least one month (Rex et al., 2010, Young et al., 2014; Young, Blouin, Briggs, Sillivan, Lin, Cameron, Rumbaugh, and Miller, 2016; Young et al., 2017, Young et al., 2020). The retrieval-independent effect of NMII inhibition is specific to METH and the BLA, as it does not interfere with memories for foot shock, food reward or other commonly abused drugs, including cocaine (COC), and does not disrupt METH memories when infused into dorsal hippocampus (dHPC) or nucleus accumbens (Briggs et al., 2017, Briggs et al., 2018). Moreover, NMII inhibition affects males and females similarly (Young et al., 2017), therefore sex as a biological variable was not considered for these initial exploratory experiments to understand mechanism. We are currently developing a medication for METH use disorder based on these findings. However, the mechanisms underlying this highly specific effect are unknown. The mechanism is not a requirement for FDA approval; however, this work holds the potential to advance our understanding of the neurobiology of stimulant use disorder.

The persistent susceptibility of a METH-associated memory to NMII inhibition was unexpected because actin-myosin dynamics are thought to rapidly stabilize after a learning event (Rudy, 2015). The sustained myosin-dependent actin dynamics in BLA spines (Young et al., 2020) following METH treatment and the ability of Blebb to disrupt a METH-associated memory days to weeks after training (Young et al., 2014, Young et al., 2016) suggests that myosin remains uniquely active following METH exposure. To begin to approach the underlying mechanisms, we focused on the differential susceptibility of METH and COC to NMII inhibition. Given that METH and COC share a number of similarities, such as increased dopamine, serotonin, and norepinephrine release, changes in glutamate receptors, and incubation of craving (Pickens et al., 2011, Zhang et al., 2001), the specificity for METH is somewhat surprising. Very few comparative studies have investigated these illicit drugs, but one well-established difference is their pharmacokinetic properties. METH enters the brain at a higher concentration and has a longer half-life than COC (Benuck et al., 1987, Cho et al., 2001, Nishimura et al., 2017). We explored the hypothesis that longer brain exposure to METH outlasts the signaling cascade that typically inactivates NMII.

We further hypothesized that METH and COC are likely to induce the expression of distinct subsets of transcripts in the BLA and other brain regions that support drug associations (Cooper et al., 2017, Kelley, , 2004). Identification of these differentially expressed genes (DEG) could provide insight into the selective vulnerability of METH-associated memories to NMII inhibition in the BLA. We identified Crhr2, which encodes the corticotropin releasing hormone or factor (CRF) receptor 2 (CRF2), as uniquely upregulated in the BLA following METH treatment. CRF2 plays a key role in the brain’s stress response (Behan et al., 1996, Logrip et al., 2011) and CRF, a ligand for both the CRF1 and CRF2 receptors, is released in the BLA following COC and METH administration (Giardino, Pastor, Anacker, Spangler, Cote, Li, Stenzel-Poore, Phillips, and Ryabinin, 2011; Logrip et al., 2011). In addition, CRF2 has been identified as an upstream regulator of NMII outside the CNS (Karteris, Hillhouse, and Grammatopoulos, 2004). Here we investigated the role of BLA CRF2 in METH and COC-associated learning and memory and its potential to drive differential susceptibility of the memories through NMII.