A Two-Day Continuous Nicotine Infusion Is Sufficient to Demonstrate Nicotine Withdrawal in Rats as Measured Using Intracranial Self-Stimulation

<div><p>Avoidance of the negative affective (emotional) symptoms of nicotine withdrawal (e.g., anhedonia, anxiety) contributes to tobacco addiction. Establishing the minimal nicotine exposure conditions required to demonstrate negative affective withdrawal signs in animals, as well as understanding moderators of these conditions, could inform tobacco addiction-related research, treatment, and policy. The goal of this study was to determine the minimal duration of continuous nicotine infusion required to demonstrate nicotine withdrawal in rats as measured by elevations in intracranial self-stimulation (ICSS) thresholds (anhedonia-like behavior). Administration of the nicotinic acetylcholine receptor antagonist mecamylamine (3.0 mg/kg, s.c.) on alternate test days throughout the course of a 2-week continuous nicotine infusion (3.2 mg/kg/day via osmotic minipump) elicited elevations in ICSS thresholds beginning on the second day of infusion. Magnitude of antagonist-precipitated withdrawal did not change with further nicotine exposure and mecamylamine injections, and was similar to that observed in a positive control group receiving mecamylamine following a 14-day nicotine infusion. Expression of a significant withdrawal effect was delayed in nicotine-infused rats receiving mecamylamine on all test days rather than on alternate test days. In a separate study, rats exhibited a transient increase in ICSS thresholds following cessation of a 2-day continuous nicotine infusion (3.2 mg/kg/day). Magnitude of this spontaneous withdrawal effect was similar to that observed in rats receiving a 9-day nicotine infusion. Our findings demonstrate that rats exhibit antagonist-precipitated and spontaneous nicotine withdrawal following a 2-day continuous nicotine infusion, at least under the experimental conditions studied here. Magnitude of these effects were similar to those observed in traditional models involving more prolonged nicotine exposure. Further development of these models, including evaluation of more clinically relevant nicotine dosing regimens and other measures of nicotine withdrawal (e.g., anxiety-like behavior, somatic signs), may be useful for understanding the development of the nicotine withdrawal syndrome.</p></div

Procedural timeline for Experiment 1.

<p>Mec = 3.0 mg/kg mecamylamine. Sal = Saline. Chronic infusion condition is italicized. See text for further details.</p

Mecamylamine elicits elevations in ICSS latencies in rats receiving a continuous nicotine infusion for 2 or more days.

<p>Mean (±SEM) ICSS latencies (expressed as percent of baseline) during pre- and post-tests in Experiment 1. *^,** Different from Sal + Sal ALL group on that test day, <i>p</i> <0.05 or 0.01.</p

Cessation of a 2-day or 9-day continuous nicotine infusion elicits elevations in ICSS thresholds.

<p>Mean (±SEM) ICSS thresholds (expressed as percent of baseline) following osmotic pump removal in Experiment 2. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144553#sec002" target="_blank">Materials and Methods</a> section for definition of group abbreviations. * 2- or 9-day Nic group different from its respective Sal control group at that time point, p <0.05.</p

Mecamylamine elicits elevations in ICSS thresholds in rats receiving a continuous nicotine infusion for 2 or more days.

<p>Mean (±SEM) ICSS thresholds (expressed as percent of baseline) during pre- and post-tests in Experiment 1. Mecamylamine (+) or saline (-) was administered prior to post-tests. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144553#sec002" target="_blank">Materials and Methods</a> section for definition of group abbreviations. *^,** Different from Sal + Sal ALL group on that test day, p <0.05 or 0.01. # Nic + Mec EVEN group elevated compared to Nic + Mec ALL group on that test day, <i>p</i> < 0.01.</p

MAb7F9 modestly attenuates the ability of acute METH to reverse elevations in ICSS thresholds during METH withdrawal.

<p>ICSS thresholds (A) and response latencies (B) (expressed as percent of baseline, mean ± SEM) in rats administered i.v. PBS or 200 mg/kg mAb immediately following cessation of a chronic METH infusion, followed 20 hr later by s.c. SAL or 0.3 mg/kg METH in Experiment 3. *^, ** Significantly different from SAL-injected animals for that mAb dose, p < 0.05 or 0.01. ^#,mAb 200 + METH significantly different from PBS + METH, p < 0.05.</p

Mean (±SEM) ICSS thresholds (in μA) and response latencies (in sec) in experimental groups during baseline sessions in Experiments 1b, 2, and 3.

<p>Mean (±SEM) ICSS thresholds (in μA) and response latencies (in sec) in experimental groups during baseline sessions in Experiments 1b, 2, and 3.</p

MAb7F9 attenuates the ability of acute METH to reduce baseline ICSS thresholds, ICSS thresholds (A) and response latencies (B) (expressed as percent of baseline, mean ± SEM) following i.v. infusion of vehicle (PBS) or mAb (30, 100, or 200 mg/kg) followed by repeated daily injections of s.c. SAL or 0.3 mg/kg METH in Experiment 2.

<p>** Significantly different from PBS + SAL at that session, p < 0.01. ^{#, ##} 200 mAb + METH significantly different from PBS + METH at that session, p < 0.05 or 0.01. For clarity, significant differences in marginal means between groups are not shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118787#pone.0118787.g003" target="_blank">Fig. 3A or 3B</a>.</p

Acute injection of METH reduces ICSS thresholds.

<p>ICSS thresholds (A) and response latencies (B) (expressed as percent of baseline, mean ± SEM) following s.c. injection of METH (0–0.56 mg/kg) in Experiment 1a. ** Significantly different from 0 mg/kg METH, p < 0.01.</p