Low dose nicotine and antagonism of β2 subunit containing nicotinic acetylcholine receptors have similar effects on affective behavior in mice

Abstract

Nicotine leads to both activation and desensitization (inactivation) of nicotinic acetylcholine receptors (nAChRs). This study tested the hypothesis that nicotine and a selective antagonist of β2*nAChRs would have similar effects on affective behavior. Adult C57BL/6J male mice were tested in a conditioned emotional response (CER) assay which evaluates the ability of an aversive stimulus to inhibit goal-directed behavior. Mice lever-pressed for a saccharin reinforcer according to a variable schedule of reinforcement during sessions in which two presentations of a compound light/tone conditioned stimulus (CS) co-terminated with a 0.1 or 0.3 mA, 0.5 s footshock unconditioned stimulus (US). During testing in the absence of the US, mice received doses of i.p. nicotine (0, 0.0032, 0.01, 0.032, 0.1 mg/kg) or a selective β2 subunit containing nAChR (β2*nAChR) antagonist dihydro-beta-erythroidine (0, 0.1, 0.3, 1.0, 3.0 mg/kg DHβE). There was a dose-dependent effect of nicotine revealing that only low doses (0.01, 0.032 mg/kg) increased CER suppression ratios (SR) in these mice. DHβE also dose-dependently increased SR at the 3 mg/kg dose. In ethological measures of fear-/anxiety-like behavior, these doses of nicotine and DHβE significantly reduced digging behavior in a marble burying task and 0.3 mg/kg DHβE promoted open-arm activity in the elevated plus maze. Doses of nicotine and DHβE that altered affective behavior had no effect on locomotor activity. Similar to previous reports with anxiolytic drugs, low dose nicotine and DHβE reversed SR in a CER assay, decreased digging in a marble burying assay and increased open arm activity in the elevated plus maze. This study provides evidence that inactivation of β2*nAChRs reduces fear-like and anxiety-like behavior in rodents and suggests that smokers may be motivated to smoke in part to desensitize their β2*nAChRs. These data further identify β2*nAChR antagonism as a potential therapeutic strategy for relief of negative affect and anxiety.

Figure 1. CER training and drug testing schedule.

To introduce mice to the location of saccharin delivery, magazine training (Phase I) occurred over days 1–3. CER acquisition of lever pressing maintained by 10 mMol saccharin solution took place during overnight sessions with increasingly demanding schedules of reinforcement (Phase II; days 4–9) until mice reached criteria of 70 reinforcers and 100 s of correct magazine entries in a session. This was followed by daily 30 minute sessions (Phase III) where mice lever pressing was maintained by saccharin under a variable schedule of reinforcement. Mice moved onto the next Phase of training when they reached criteria of 40 lever presses and 10 reinforcers during a single 30 minute session (days 10–13). During CER training mice continued operant training but also received 2 explicit pairings of a light and tone conditioned stimulus (CS) which co-terminated with a 0.1 or 0.3 mA footshock unconditioned stimulus (US) (Phase IV; days 14–43). Phase IV continued until all mice showed a stable level of CS and NON-CS responding over 3 days. Drug testing (Phase V) consisted of lever pressing maintained by saccharin in the presence of the CS but in the absence of the US. For these studies that used a within-subject, Latin square design, there were at least 2 CER training days in between drug testing sessions to assure that mice returned to baseline prior to the next injection of drug.

Figure 2. Low dose nicotine and the β2*nAChR antagonist DHβE reverse conditioned suppression.

In mice trained to a 0.3 mA unconditioned stimulus footshock during CER training, A) administration of nicotine resulted in a dose-dependent reversal of conditioned suppression as measured by increased suppression ratios (F _4,9 = 3.101, p<0.05; n = 10). B) The β2*nAChR antagonist DHβE also resulted in a significant increase in suppression ratios in these mice (F _4,6 = 2.934, p<0.05; n = 7), suggesting that inhibition of β2*nAChRs supports anxiolytic-like behavior in the CER assay. C, D) Neither nicotine (F _4,5 = 1.991, p>0.05; n = 6) nor DHβE (F _4,5 = 1.263, p>0.05; n = 6) resulted in significant changes in suppression ratio responding in mice exposed to 0.1 mA US footshock during CER training. Data are presented as means ± SEM. *p<0.05 compared to when mice received saline control injections.

Figure 3. Nicotine and DHβE resulted in fewer marbles buried.

A) The 0.01 and 0.032 mg/kg i.p. nicotine that promoted anxiolytic-like behavior in the CER task also resulted in a significant reduction in digging behavior as measured by fewer marbles buried compared to when mice were administered saline (n = 14). B) Similarly, mice treated with 3 mg/kg i.p. DHβE also buried less marbles compared to when they received saline treatment (n = 15). Data are presented as means ± SEM; *p<0.05 compared to when mice received saline control injections.

Figure 4. Antagonism of β2*nAChRs promoted anxiolysis-like behavior in the elevated plus maze.

A) Mice receiving 0.3 mg/kg i.p. DHβE required less time to explore the end terminus of the open arms of an elevated plus maze, B) made more entries into the open arms of the maze and C) spent more time in the open arms than saline-injected mice. Data are presented as means ± SEM; *p<0.05, #p = 0.067 compared to saline controls.

Figure 5. Nicotine and DHβE did not affect locomotor activity.

Doses of A) nicotine (n = 5−6) and B) DHβE (n = 4−5 per group) that promoted anxiolytic-like behavior in the CER and marble burying tasks did not affect locomotor activity as measured by beam breaks (F _s = 0.072, 1.451, p _s>0.05). Data are presented as means ± SEM.