NR2B selective N-methyl-D-aspartate receptor antagonist(Merck)

Growth arrest and DNA-damage–inducible protein 45β reactivates methylation-silenced neural plasticity-associated genes through DNA demethylation. However, growth arrest and DNA-damage–inducible protein 45β–dependent demethylation contributes to neuropathic allodynia-associated spinal plasticity remains unclear.

Adult male Sprague–Dawley rats (654 out of 659) received a spinal nerve ligation or a sham operation with or without intrathecal application of one of the following: growth arrest and DNA-damage–inducible protein 45β messenger RNA–targeted small interfering RNA, lentiviral vector expressing growth arrest and DNA-damage–inducible protein 45β, Ro 25–6981 (an NR2B-bearing N-methyl-d-aspartate receptor antagonist), or KN-93 (a calmodulin-dependent protein kinase II antagonist) were used for behavioral measurements, Western blotting, immunofluorescence, dot blots, detection of unmodified cytosine enrichment at cytosine-phosphate-guanine site, chromatin immunoprecipitation quantitative polymerase chain reaction analysis, and slice recordings.

Nerve ligation-enhanced growth arrest and DNA-damage–inducible protein 45β expression (n = 6) in ipsilateral dorsal horn neurons accompanied with behavioral allodynia (n = 7). Focal knockdown of growth arrest and DNA-damage–inducible protein 45β expression attenuated ligation-induced allodynia (n = 7) by reducing the binding of growth arrest and DNA-damage–inducible protein 45β to the voltage-dependent T-type calcium channel 3.2 subunit promoter (n = 6) that decreased expression of and current mediated by the voltage-dependent T-type calcium channel 3.2 subunit (both n = 6). In addition, NR2B-bearing N-methyl-d-aspartate receptors and calmodulin-dependent protein kinase II act in an upstream cascade to increase growth arrest and DNA-damage–inducible protein 45β expression, hence enhancing demethylation at the voltage-dependent T-type calcium channel 3.2 subunit promoter and up-regulating voltage-dependent T-type calcium channel 3.2 subunit expression. Intrathecal administration of Ro 25–6981, KN-93, or a growth arrest and DNA-damage–inducible protein 45β–targeting small interfering RNA (n = 6) reversed the ligation-induced enrichment of unmodified cytosine at the voltage-dependent T-type calcium channel 3.2 subunit promoter by increasing the associated 5-formylcytosine and 5-carboxylcytosine levels.

By converting 5-formylcytosine or 5-carboxylcytosine to unmodified cytosine, the NR2B-bearing N-methyl-d-aspartate receptor, calmodulin-dependent protein kinase II, or growth arrest and DNA-damage–inducible protein 45β pathway facilitates voltage-dependent T-type calcium channel 3.2 subunit gene demethylation to mediate neuropathic allodynia.

Cocaine use during adolescence increases vulnerability to drug dependence and decreases the likelihood that individuals will seek treatment as adults. Understanding how early-life cocaine exposure influences decision-making processes in adulthood is thus critically important.

Adolescent or adult mice were exposed to subchronic cocaine, then behavioral sensitivity to changes in the predictive relationship between actions and their consequences was tested. Dendritic spines on the principal pyramidal neurons of the orbitofrontal prefrontal cortex (oPFC) were also imaged and enumerated. To determine whether cytoskeletal regulatory systems in the oPFC influenced decision-making strategies, we then inhibited the activity of Abl family and Rho kinases as well as NR2B-containing N-methyl-D-aspartate receptors. We also attempted to block the reinstatement of cocaine seeking in cocaine self-administering mice.

Adult mice with a history of subchronic cocaine exposure in adolescence engaged habit-based response strategies at the expense of goal-directed decision-making strategies and had fewer dendritic spines in the oPFC. Inhibition of the cytoskeletal regulatory Abl family kinases in the oPFC recapitulated these neurobehavioral deficiencies, whereas Rho kinase inhibition corrected response strategies. Additionally, the NR2B-selective N-methyl-D-aspartate receptor antagonists ifenprodil and CP-101,606 blocked cocaine-induced habits; this was dependent on Abl family signaling in the oPFC. Ifenprodil also mitigated cue-induced reinstatement of cocaine seeking in mice self-administering cocaine.

We suggest that adolescent cocaine exposure confers a bias toward habit-based behavior in adulthood via long-term cellular structural modifications in the oPFC. Treatments aimed at mitigating the durable consequences of early-life cocaine use may benefit from targeting cytoskeletal regulatory systems.