Chronic treatment with N-acetylcysteine decreases extinction responding and reduces cue-induced nicotine-seeking

Abstract

N-acetylcysteine (NAC), a promising glutamatergic therapeutic agent, has shown some clinical efficacy in reducing nicotine use in humans and has been shown to reverse drug-induced changes in glutamatergic neurophysiology. In rats, nicotine-seeking behavior is associated with alterations in glutamatergic plasticity within the nucleus accumbens core (NAcore). Specifically, cue-induced nicotine-seeking is associated with rapid, transient synaptic plasticity (t-SP) in glutamatergic synapses on NAcore medium spiny neurons. The goal of the present study was to determine if NAC reduces nicotine-seeking behavior and reverses reinstatement-associated NAcore glutamatergic alterations. Rats were extinguished from nicotine self-administration, followed by subchronic NAC administration (0 or 100 mg/kg/d) for 4 days prior to cue-induced reinstatement. NAcore synaptic potentiation was measured via dendritic spine morphology and mRNA and protein of relevant glutamatergic genes were quantified. Nicotine-seeking behavior was not reduced by subchronic NAC treatment. Also, NAcore transcript and protein expression of multiple glutamatergic genes, as well as spine morphological measures, were unaffected by subchronic NAC. Finally, chronic NAC treatment (15 days total) during extinction and prior to reinstatement significantly decreased extinction responding and reduced reinstatement of nicotine-seeking compared to vehicle. Together, these results suggest that chronic NAC treatment is necessary for its therapeutic efficacy as a treatment strategy for nicotine addiction and relapse.

Keywords: N-acetylcysteine; Nicotine; Relapse; Synaptic Plasticity.

Figure 1

Subchronic N‐acetylcysteine (NAC) does not attenuate nicotine‐seeking behavior. (A) A timeline of experimental procedures including nicotine self‐administration, extinction training, reinstatement of nicotine‐seeking, NAC administration, and tissue collection (to be used for either RT‐qPCR, western blots, or dendritic spine morphology). (B) Rats acquired lever pressing to the active lever to receive intravenous infusions of nicotine (0.02 mg/kg/infusion) paired with a light+tone compound stimulus during self‐administration. During extinction training, active lever pressing decreased to inactive lever press rates due to no programmed consequence. (C) No difference was found for the total number of infusions received throughout self‐administration, prior to NAC or vehicle treatment. (D) Administration of NAC during extinction had no significant effect on active lever pressing. (E) In a 2‐h cue‐reinstatement session, animals receiving 0 or 100 mg/kg NAC showed no significant effects of treatment on mean active lever pressing. (F) Upon examination of the time‐course of active lever pressing during the 2‐h reinstatement session, NAC treatment did not significantly reduce cumulative active lever pressing compared to vehicle‐treated animals. *P < 0.0001, significant main effect of session (extinction vs. reinstatement). The bar in (E) indicates a significant main effect of session.

Figure 2

The effect of subchronic N‐acetylcysteine (NAC) on glutamatergic genes. (A) Lever presses and infusions during nicotine self‐administration and extinction training of rats tested for transcript expression following administration of 0 or 100 mg/kg NAC prior to the last four sessions of extinction and the reinstatement session. (B) No difference was found for the total number of infusions received throughout self‐administration, prior to NAC or vehicle treatment. (C) High‐dose NAC did not significantly reduce active lever pressing during a 2‐h reinstatement test prior to sacrifice for gene expression analysis. (D) Glutamatergic transcripts of interest (Slc1a2, Gria1, Gria2, Grin2a, and Grin2b) relative to the endogenous control gene GAPDH revealed no differences in mRNA expression due to subchronic 100 mg/kg NAC treatment. Expression is shown as a percentage of control animals. N per group for each gene is listed with each bar. **P < 0.0001 main effect of session (extinction vs. reinstatement). #P < 0.05 main effect of drug (saline vs. nicotine). ‡P < 0.05 main effect of treatment (0 vs. 100 mg/kg NAC). §P < 0.05 interaction between drug and treatment. *P < 0.05 significant difference between 0 and 100 mg/kg NAC in saline animals. The bar in (C) indicates a significant main effect of session.

Figure 3

Subchronic N‐acetylcysteine (NAC) does not alter nucleus accumbens core (NAcore) protein expression. (A) Lever presses and infusions during nicotine self‐administration and extinction training for animals tested for protein expression following treatment with 0 or 100 mg/kg NAC. (B) No difference was found for the total number of infusions received throughout self‐administration, prior to NAC or vehicle treatment. (C) Expression of GLT‐1 protein was not significantly different between groups of nicotine self‐administering and timed saline infusions, as well as between vehicle and 100 mg/kg NAC. Expression is shown as a percentage of animals receiving timed saline infusions and vehicle treatment (0 mg/kg NAC), and calculated using GAPDH as a control protein. (D) NAC did not significantly reduce active lever pressing during a 2‐h reinstatement test prior to sacrifice for protein expression analysis. (E) Treatment with NAC did not significantly alter levels of glutamatergic proteins of interest. Expression is shown as percentage of control animals, and calculated using GAPDH as a control. *P < 0.05 main effect of session (extinction vs. reinstatement). The bar in (D) indicates a significant main effect of session.

Figure 4

Effects of cue‐induced nicotine‐seeking on dendritic spine morphology. (A) A representative medium spiny neuron (MSN) with soma denoted by a white arrow and a sample dendrite used for analysis highlighted within a green box. (B) Sample dendritic segments of MSNs from animals at T = 0 ( top ) or T = 15 ( bottom ). The comparison of mean spine head diameter of animals at T = 0 and T = 15, as well as the cumulative frequency distribution of spine head diameters are shown in (C) and (D), respectively. Additionally the comparison of spine head density (E), spine neck length (F), frequency distribution of spine neck length, (G) and the ratio for spine head diameter to spine neck length (H) between animals undergoing a 15‐min reinstatement session (T = 15) and those that did not (T = 0) are shown. *P < 0.05 compared to animals at T = 0.

Figure 5

Subchronic N‐acetylcysteine (NAC) does not inhibit nicotine reinstatement or t‐SP. (A) Representative micrographs of dendritic spines from animals receiving 0 ( top ) or 100 mg/kg NAC ( bottom ). (B) Self‐administration and extinction of rats receiving either 0 or 100 mg/kg NAC prior to sacrifice for dendritic spine morphology analysis. (C) Total infusions received by group throughout self‐administration. (D) Treatment with 100 mg/kg NAC did not significantly reduce active lever pressing during a 15 min reinstatement session compared to vehicle‐treated animals. (E) High‐dose NAC (100 mg/kg) did not significantly alter mean spine head diameter. No effects of high‐dose NAC (100 mg/kg) were observed on the (F) cumulative frequency distribution of spine neck diameter or spine neck length (I) at T = 15. Comparisons of spine density, mean spine neck length, and head diameter to neck length ratio are shown in (G), (H), and (J), respectively. Dashed line in (G) represents basal (T = 0, vehicle‐treated) levels. *P < 0.05 compared to animals receiving 0 mg/kg NAC in (C). *P < 0.0001 main effect of session (extinction vs. reinstatement) in (D).

Figure 6

Chronic N‐acetylcysteine (NAC) attenuates nicotine‐seeking behavior. Active (A) and inactive lever presses (B) during self‐administration training for animals treated with 0 or 100 mg/kg NAC. No differences were detected due to treatment or session. (C) The number of infusions earned by group during nicotine self‐administration. No significant differences were detected due to treatment, however, a significant effect of session was observed indicating all animals increased infusions earned as training progressed. (D) The total number of infusions earned by treatment group collapsed across session. No significant differences were detected. (E) Active lever pressing during 2‐h extinction sessions significantly decreased across sessions, and was significantly reduced with chronic NAC treatment. (F) Following extinction, rats significantly reinstated to nicotine‐conditioned cues during a 2‐h reinstatement test. Additionally, NAC treatment significantly reduced active lever pressing regardless of session. Bonferroni‐corrected comparisons of vehicle versus treatment lever pressing indicated 100 mg/kg NAC significantly decreased active lever pressing during reinstatement. *P < 0.05, significant main effect of treatment. #P < 0.05, significant main effect of session. +P < 0.05, significant main effect of session.