GluN2B inhibition confers resilience against long-term cocaine-induced neurocognitive sequelae

Abstract

Cocaine self-administration can disrupt the capacity of humans and rodents to flexibly modify familiar behavioral routines, even when they become maladaptive or unbeneficial. However, mechanistic factors, particularly those driving long-term behavioral changes, are still being determined. Here, we capitalized on individual differences in oral cocaine self-administration patterns in adolescent mice and revealed that the post-synaptic protein PSD-95 was reduced in the orbitofrontal cortex (OFC) of escalating, but not stable, responders, which corresponded with later deficits in flexible decision-making behavior. Meanwhile, NMDA receptor GluN2B subunit content was lower in the OFC of mice that were resilient to escalatory oral cocaine seeking. This discovery led us to next co-administer the GluN2B-selective antagonist ifenprodil with cocaine, blocking the later emergence of cocaine-induced decision-making abnormalities. GluN2B inhibition also prevented cocaine-induced dysregulation of neuronal structure and function in the OFC, preserving mature, mushroom-shaped dendritic spine densities on deep-layer pyramidal neurons, which were otherwise lower with cocaine, and safeguarding functional BLA→OFC connections necessary for action flexibility. We posit that cocaine potentiates GluN2B-dependent signaling, which triggers a series of durable adaptations that result in the dysregulation of post-synaptic neuronal structure in the OFC and disruption of BLA→OFC connections, ultimately weakening the capacity for flexible choice. And thus, inhibiting GluN2B-NMDARs promotes resilience to long-term cocaine-related sequelae.

Fig. 1. Cocaine persistently disrupts flexible decision-making behavior.

a Response sequencing during oral cocaine self-administration sessions, conducted in adolescence. b Average nose poke (NP) and lever press (LP) responses across last 8 sessions. Stable and escalatory response groups defined by k-means clustering. Inset. Ratio of NP to LP responses. c, d Left panels. NP and LP responses per session. Right panels. Average NP and LP responses across last 8 sessions. e Behavioral procedure assessing flexible decision making later in life. f Responses across training. g, h Choice test responses after one action was unexpectedly not reinforced, first following FR1 training (test 1; impaired in escalating cocaine responders) and then RI training (test 2). Data presented as individual points or mean ± S.E.M. *p < 0.05 (post-hoc). See Table S2for complete statistics and group sizes.

Fig. 2. Quantification of synaptic and plasticity-related protein levels in the OFC following oral cocaine self-administration.

a Synaptic localization of quantified proteins in the OFC. b, c Quantification of synaptic marker protein levels in the OFC: PSD-95 (lower among escalating responders) and synaptophysin. d–g Quantification of glutamate receptor subunit levels in the OFC: GluN1, GluN2A, GluN2B (lower among stable responders), and GluA1. Values normalized to loading controls (HSP-70) and expressed as fold change from sucrose controls. Representative blots show target protein (black arrow) and HSP-70 loading controls (no arrow). h, i Correlation between choice test preference (from Fig. 1g) and PSD-95 or GluN2B levels. 95% confidence intervals (shading). Data presented as individual points. *p < 0.05 (post-hoc), ^#p = 0.07. See Table S3for complete statistics and group sizes.

Fig. 3. GluN2B inhibition by ifenprodil blocks cocaine-induced decision-making deficits.

a Ifenprodil and cocaine administration. b Responses across training. (c) Choice test responses. Cocaine impaired action flexibility, which was protected by ifenprodil. d–f Similar patterns were detected in instrumental reversal learning responses: reversal rate, response acquisition, and perseverative responding. Data presented as individual points or mean ± S.E.M. *p < 0.05 (post-hoc). ^†p < 0.05 (2^-factor ANOVA, cocaine × ifenprodil). See Table S4for complete statistics and group sizes.

Fig. 4. GluN2B inhibition blocks cocaine-induced dendritic spine loss in the OFC.

a Dendritic spine imaging from the OFC. Scale bar = 100 µm. b Representative three-dimensional dendritic spine reconstructions. Scale bar = 2 µm. c Dendritic spine density for all spines, lower following cocaine, and protected by ifenprodil. d Correlation between individual choice behavior and dendritic spine density across all groups. 95% confidence interval (grey shading). e–g Dendritic spine density stratified by mushroom-, thin-, or stubby-type spines. h Mushroom-to-thin spine-type ratio. Data presented as individual points (solid = per animal; transparent = per dendrite). *p < 0.05, ^#p = 0.0.038 (post-hoc). See Table S5for complete statistics and group sizes.

Fig. 5. Preservation of flexible decision making following cocaine by GluN2B inhibition requires functionally intact BLA→OFC projections.

a Combinatorial viral targeting of BLA→OFC projections. b–d eGFP-Cre driving hM4Di-mCherry expression in cell bodies in the BLA and axon terminals in the OFC. Scale bar = 25 µm. e Timing of CNO administration (0.1 or 1.0 mg/kg) occurs following a session when responding is unexpectedly not reinforced, this experimental design allowing us to selectively manipulate projection activity during a period of new memory encoding. f Responses across training. g, h Choice test responses following FR and moderate RI training, which has additive impact with 0.1 mg/kg CNO in obstructing action flexibility. i Timing of ifenprodil, cocaine, and CNO administration. j Responses across training. k Choice test responses following FR training, with effects of ifenprodil obstructed by subthreshold CNO. l Choice test response preference ratios revealing the same pattern. m Choice test responses following moderate RI training. n Choice test response preference ratios. Data presented as individual points or mean ± S.E.M. *p < 0.05 (post-hoc or planned comparison). See Table S6for complete statistics and group sizes.