Long-term effects of juvenile nicotine exposure on abstinence-related social anxiety-like behavior and amygdalar cannabinoid receptor 1 (CB1R) mRNA expression in the novelty-seeking phenotype

Abstract

A rat model of novelty-seeking phenotype predicts vulnerability to nicotine relapse where locomotor reactivity to novelty is used to rank high (HR) versus low (LR) responders. Present study investigates implication of cannabinoid receptor 1 (CB1R) in the basolateral (BLA) and the central (CeA) nuclei of amygdala in behaviorally sensitizing effects of nicotine and accompanying social anxiety following juvenile nicotine training and a 1- or 3-wk injection-free period in the novelty-seeking phenotype. Sprague-Dawley rats were phenotype screened, and received four, saline (1 ml/kg; s.c) or nicotine (0.35 mg/kg; s.c) injections, followed by a 1- or 3-wk injection-free period. Subsequently, animals were challenged with a low dose of nicotine (0.1 mg/kg; s.c.), subjected to the social interaction test and sacrificed. In situ hybridization histochemistry was used to assess CB1R messenger RNA (mRNA) levels in the amygdala. Nicotine pre-trained HRs displayed expression of locomotor sensitization to nicotine challenge along with enhanced social anxiety compared to saline pre-trained controls following a 1- or 3-wk injection-free period. HR-specific behavioral effects were accompanied by decreased CB1R mRNA levels in the CeA and the BLA following a 1-wk injection-free period. Decreased CB1R mRNA levels in both compartments of the amygdala were also observed following nicotine challenge in saline pre-trained HRs after a 3-wk injection-free period compared to HRs after a 1-wk injection-free period. These findings show robust, long-lasting expression of behavioral sensitization to nicotine in HRs associated with changes in amygdalar CB1R mRNA as a potential substrate for abstinence-related anxiety.

Figure 1

Total locomotor reactivity to four intermittent nicotine (0.35 mg/kg, s.c.) or saline (1.0 ml/kg, s.c.) training injections, with 3-d intervals (A), total locomotor reactivity to a low dose nicotine (0.1 mg/kg, s.c.) challenge following 1 wk (1 WK) or 3 wks (3 WKs) of injection-free period (B), and percent time spent interacting with the resident rat in the SI test following 1 wk (1 WK) or 3 wks (3 WKs) of injection-free period (C). Group means ± SEMs are plotted in line (A) and bar (B, C) graphs. In panel A, * represents significant effects in total locomotor reactivity to nicotine in HRs compared to saline-injected controls, whereas # represents significant effects in nicotine-injected LRs compared to saline-injected controls. Significance is set at p = 0.005.

Figure 2

CB1R mRNA expression in the BLA and the CeA of a representative HR rat that is pre-trained with saline and underwent 1 wk of injection-free period (A), HR rat that is pre-trained with nicotine and underwent 1 wk of injection-free period (B), HR rat that is pre-trained with saline and underwent 3 wks of injection-free period (C), HR rat that is pre-trained with nicotine and underwent 3 wks of injection-free period (D). Panels A, B, C and D show images of coronal hemisections of the BLA and the CeA that were radioactively labeled with an antisense cRNA probe against CB1R and exposed on an x-ray film. Means of quantification results for integrated densities ± SEMs are plotted with bar graphs for the BLA (E) and the CeA (F; *: p ≤ 0.05). Scale bar = 250 μm