Reinstatement of nicotine seeking is mediated by glutamatergic plasticity

Abstract

Nicotine abuse and addiction is a major health liability. Nicotine, an active alkaloid in tobacco, is self-administered by animals and produces cellular adaptations in brain regions associated with drug reward, such as the nucleus accumbens. However, it is unknown whether, akin to illicit drugs of abuse such as cocaine or heroin, the adaptations endure and contribute to the propensity to relapse after discontinuing nicotine use. Using a rat model of cue-induced relapse, we made morphological and electrophysiological measures of synaptic plasticity, as well as quantified glutamate overflow, in the accumbens after 2 wk of withdrawal with extinction training. We found an enduring basal increase in dendritic spine head diameter and in the ratio of AMPA to NMDA currents in accumbens spiny neurons compared with yoked saline animals at 2 wk after the last nicotine self-administration session. This synaptic potentiation was associated with an increase in both AMPA (GluA1) and NMDA (GluN2A and GluN2B) receptor subunits, and a reduction in the glutamate transporter-1 (GLT-1). When nicotine seeking was reinstated by presentation of conditioned cues, there were parallel increases in behavioral responding, extracellular glutamate, and further increases in dendritic spine head diameter and ratio of AMPA to NMDA currents within 15 min. These findings suggest that targeting glutamate transmission might inhibit cue-induced nicotine seeking. In support of this hypothesis, we found that pharmacological inhibition of GluN2A with 3-Chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulfonamide (TCN-201) or GluN2B with ifenprodil abolished reinstated nicotine seeking. These results indicate that up-regulated GluN2A, GluN2B, and rapid synaptic potentiation in the accumbens contribute to cue-induced relapse to nicotine use.

Fig. 1.

Nicotine self-administration and reinstatement of nicotine-seeking behavior. (A) Nicotine self-administration and extinction training before reinstatement testing and measurements for either d_h, A/N, microdialysis, or Western blot analysis. (B) Rats acquired lever responding to the active lever to receive i.v. infusions of nicotine (0.02 mg/kg per infusion). During extinction training, active lever presses decreased to inactive lever press rates. (C) Restoration of the conditioned cues to the active lever significantly reinstated lever pressing in animals given 120-min reinstatement of cue-induced nicotine seeking. *P < 0.05, comparing active lever pressing after cues with extinction pressing (average of the last three extinction sessions).

Fig. 2.

Increased extracellular glutamate in the NAcore during cue-induced reinstatement of nicotine seeking. (A) The delivery of conditioned cues induced significant active lever pressing in nicotine-experienced animals, whereas the same cues in yoked saline animals did not alter behavior. (B) Time course of reinstated nicotine seeking revealed increased active lever pressing during the first 40 min after initiating the reinstatement session. (C) Reinstatement of nicotine seeking increased extracellular glutamate levels during the first 40 min after beginning cue-induced reinstatement. Arrowhead refers to initiation of cue-induced reinstatement session. (D) Histological verification of guide cannula placement according to Paxinos and Watson (2007) (41) by an individual unaware of the animal’s treatment group. Numbers refer to mm rostral to Bregma. *P < 0.05 compared with extinction active lever pressing in A, yoked saline in B, or basal glutamate in C. ⁺P < 0.05 comparing nicotine with saline.

Fig. 3.

Nicotine seeking enlarges NAcore spine head diameter. (A) Example of a high-resolution 3D reconstruction of an NAcore MSN. Clear arrow, location of soma; boxed region, segment analyzed in B from a t = 45 animal. (B) Sample dendrites from NAcore spiny cells in yoked saline (d_h = 0.342 µm) or cocaine-trained rats at t = 0 (0.423 µm), t = 15 cue (0.495 µm), and t = 45 cue (0.418 µm) after initiating cue-induced reinstatement. (C) Time course of reinstated active lever pressing in the t = 15 min and t = 45 min groups. (D) Mean d_h was increased after withdrawal from nicotine self-administration (t = 0) and was further increased at t = 15 by contingent conditioned cues. (E) Cumulative d_h frequency plot indicating a rightward shift at t = 0 compared with saline and a further rightward shift at t = 15 min after cue. By t = 45 min after cue the cumulative d_h distribution had returned to t = 0 min. (F) No difference in spine density was found between groups. *P < 0.05 compared with yoked saline animals; ⁺P < 0.05 compared with t = 0.

Fig. 4.

Rapid increase in A/N during cue-induced reinstatement of nicotine seeking. (A) Sample AMPA and NMDA current traces from each group. A/N was significantly elevated in animals withdrawn with extinction training from nicotine self-administration compared with yoked saline animals. The initiation of cue-induced reinstatement of nicotine-seeking further elevated A/N at t = 15. (B) Decay time (time to 37% decay) for NMDA currents in nicotine-extinguished animals was significantly increased in nicotine-extinguished animals (t = 0) as well as animals at t = min compared with yoked saline animals. N in parentheses is number of animals. The number in bars is number of cells recorded in each condition. *P < 0.05 compared with yoked saline animals at t = 0 (white bar); ⁺P < 0.05 compared with t = 0.

Fig. 5.

Increased GluN2A- and GluN2B-containing NMDA receptors mediate cue-induced nicotine reinstatement. (A) Withdrawal from nicotine self-administration (t = 0) significantly elevated membrane-bound GluA1, GluN2A, and GluN2B and reduced GLT1 in the NAcore compared with yoked saline. All data were normalized to calnexin as a loading control. (B) Intra-NAcore injection of the GluN2A antagonist TCN-201 reduced cue-induced nicotine seeking. (C) Systemic injection of the GluN2B antagonist ifenprodil produced a dose-dependent reduction in cue-induced reinstated nicotine seeking. *P < 0.05 compared with either yoked saline (white bar) or extinction active lever presses; ⁺P < 0.05 compared with vehicle active lever presses.