Dopamine D1-like receptor activation decreases nicotine intake in rats with short or long access to nicotine

Abstract

The use of nicotine and tobacco products is highly addictive. The dopaminergic system plays a key role in the initiation and maintenance of nicotine intake. Dopamine D1-like receptor blockade diminishes nicotine intake in rats with daily short (1 h) access to nicotine, but little is known about the effects of dopamine receptor antagonists or agonists on nicotine intake in rats with intermittent long (23 h) access. Because of the extended access conditions and high nicotine intake, the intermittent long access procedure might model smoking and vaping better than short access models. We investigated the effects of the dopamine D1-like receptor antagonist SCH 23390 and the D1-like receptor agonist A77636 on nicotine intake in male rats with intermittent short or long access to nicotine. The rats self-administered nicotine for 5 days (1 h/day) and were then given 15 intermittent short (1 h/day) or long (23 h/day) access sessions (3 sessions/week, 0.06 mg/kg/inf). The D1-like receptor antagonist SCH 23390 decreased nicotine intake to a similar degree in rats with short or long access to nicotine. The D1-like receptor agonist A77636 induced a greater decrease in nicotine intake in the rats with long access to nicotine than in rats with short access. Treatment with A77636 induced a prolonged decrease in nicotine intake that lasted throughout the dark and light phase in the long access rats. These findings indicate that blockade and stimulation of D1-like receptors decreases nicotine intake in an intermittent long access animal model that closely models human smoking and vaping.

INTRODUCTION

The use of tobacco products and e-cigarettes is highly addictive and has harmful health effects. Smoking can lead to cancer, cardiovascular disease and accelerates age-related cognitive decline^1–3. Although e-cigarettes deliver fewer harmful chemicals than tobacco products, they are not safe and have adverse health effects as well. E-cigarette aerosol contains compounds that are carcinogenic and can cause respiratory and cardiovascular diseases⁴. Despite that smoking rates are on the decline, smoking remains a major worldwide health burden⁵. In the United States (US), about 40 million people use tobacco products, and worldwide about 1.3 billion people use tobacco products^6,7. In contrast to the decline in smoking, vaping is on the rise^8,9. From 2017 to 2019, the prevalence of vaping doubled among high school students⁸. In the US, about 40% of 12th graders (17–18 years of age) have used e-cigarettes⁸. The COVID-19 pandemic has contributed to a further increase in smoking and vaping¹⁰.

Smoking and vaping often starts during adolescence, and the users of nicotine products gradually become dependent^11,12. Nicotine induces mild euphoria, a feeling of relaxation, and enhances cognitive function and these positive reinforcing properties play a role in the initiation of nicotine intake^13–15. After the development of dependence, negative reinforcement processes play a critical role in maintaining nicotine intake and relapse¹⁶. Rat nicotine self-administration procedures have been developed to study the reinforcing properties of nicotine¹⁷. Rats self-administer nicotine on a short access schedule (1 h/day) after a brief period of food training¹⁸. In nicotine self-administration studies, the delivery of a nicotine infusion is paired with a cue light which facilitates the acquisition of nicotine intake¹⁹. Most nicotine self-administration studies have been done with rats that had short access to nicotine for 5 days per week and were not nicotine dependent^20–22. However, more recent studies have shown that rats with daily long access or intermittent long access have higher levels of nicotine intake than those with short access^22–24. Our prior work showed that rats with long access to nicotine increased their nicotine intake over time and gradually consumed a higher proportion of nicotine during the light phase, which might be a sign of dependence^24,25. Nicotine self-administration facilitates intracranial self-stimulation (ICSS) in rats, which indicates that nicotine enhances brain reward function^24,26. Drugs that facilitate ICSS may have abuse potential in humans²⁷. Nicotine abstinence in dependent animals leads to ICSS inhibition, which reflects a reward deficit or anhedonia^28,29. Interestingly, the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine induces greater ICSS inhibition in rats with intermittent long access to nicotine than in rats with intermittent short access to nicotine²⁴. These findings indicate that intermittent long access to nicotine leads to high levels of nicotine intake and signs of dependence and might, therefore, better model adult smoking and vaping than short access to nicotine.

The neurotransmitter dopamine plays a critical role in the reinforcing properties of nicotine³⁰. In particular, dopaminergic projections from the ventral tegmental area to the nucleus accumbens and prefrontal cortex are activated by nicotine³¹. Dopamine mediates its effect in the brain by stimulating D1-like receptors (D1 and D5 receptors) and D2-like (D2, D3, and D4 receptors) receptors³². Previous work demonstrated that D1-like receptor blockade induces a greater decrease in nicotine intake in rats than D2-like receptor blockade³³. Furthermore, chronic nicotine administration induces adaptations in D1-like, but not D2-like, receptor signaling in the ventral tegmental area of rats³⁴. The D1-like receptors bind dopamine with a high affinity and mediate the rewarding properties of drugs of abuse³⁰. It has been well established that D1-like receptor blockade diminishes nicotine intake in rats with daily short access to nicotine^33,35,36. It is, however, unknown if D1-like receptor blockade also affects nicotine intake in rats with intermittent long access to nicotine. Furthermore, little is known about the effects of drugs that stimulate D1-like receptors on nicotine intake in rats. In the present study, we investigated the effects of D1-like receptor blockade and D1-like receptor stimulation on nicotine self-administration in rats with intermittent short and long access to nicotine. The intermittent self-administration schedule was similar to the one used in previous studies in which rats had 24–48 h off between nicotine self-administration sessions^23,24. In the present study, dopamine receptors were blocked with the selective D1-like receptor antagonist SCH 23390 and activated with the D1-like receptor agonist A77636. This study shows that both blockade and activation of D1-like receptors in rats with short and long access to nicotine leads to reduced nicotine intake.

MATERIALS AND METHODS

Animals

Adult male Wistar rats (200–250 g, 8–9 weeks of age) were purchased from Charles River (Raleigh, NC). The rats were housed with a rat of the same sex in a climate-controlled vivarium on a reversed 12 h light-dark cycle (light off at 7 AM). The rats were gently handled for 2–3 min per day for several days before the food training sessions. During the food training period, the rats were singly housed, and remained singly housed for the rest of the study. Prior to the onset of the studies, food was available ad libitum in the home cage. During the food training and self-administration sessions, the rats were fed 90–95 percent of their ad libitum food intake. Water was available ad libitum throughout the study. The experimental protocols were approved by the University of Florida Institutional Animal Care and Use Committee (IACUC). All experiments were performed in accordance with relevant guidelines and regulations of IACUC and in compliance with the ARRIVE guidelines 2.0 (Animal Research: Reporting of In Vivo Experiments).

Drugs

(−)-Nicotine hydrogen tartrate (Sigma-Aldrich, St. Louis, MO), SCH 23390 hydrochloride (Tocris bioscience, Minneapolis, MN), and A77636 hydrochloride (Tocris bioscience, Minneapolis, MN) were dissolved in sterile saline (0.9 % sodium chloride). SCH 23390 and A77636 were administered subcutaneously (SC) in a volume of 1 ml/kg body weight. Nicotine was dissolved in sterile saline, and the rats self-administered 0.03 or 0.06 mg/kg/inf of nicotine in a volume of 0.1 ml/inf. Nicotine doses are expressed as base, and SCH 23390 and A77636 doses are expressed as salt.

Food training

A schematic overview of the experimental design is depicted in figure 1. Rats were trained to press a lever for food pellets in operant chambers that were placed in sound- and light-attenuated cubicles (Med Associates, St. Albans, VT). Food training was conducted before the catheters were implanted. Responding on the active lever resulted in the delivery of a food pellet (45 mg, F0299, Bio-Serv, Frenchtown, NJ), and responding on the inactive lever was recorded but did not have scheduled consequences. Food delivery was paired with a cue light, which remained illuminated throughout the time-out (TO) period. The food training sessions were conducted for 10 days. Instrumental training started under an FR1-TO1s reinforcement schedule, and the rats remained on this schedule for 5 days (30 min sessions per day). After the fifth food training session, the rats were singly housed and remained so for the rest of the study. On day 6, the time-out period was increased to 10 s. The rats were allowed to respond for food pellets under the FR1-TO10s schedule (20 min sessions) for 5 days. Both levers were retracted during the 10 s time-out period. The rats were fed 90–95 percent of their baseline food intake in the home cage when the food training was ongoing.

Figure 1. Schematic overview of the experiment.

Rats were trained to respond for food pellets and then received intravenous (IV) catheters. The rats were allowed to self-administer nicotine for five daily 1 h baseline sessions (3 days 0.03 mg/kg/inf and 2 days 0.06 mg/kg/inf) and then they self-administered nicotine (0.06 mg/kg/inf) under an intermittent short access (1 h/day) or a long access (23 h/day) schedule for fifteen baseline sessions (3 sessions/week). After the fifteen baseline sessions, the effects of SCH 23390 and A77636 on nicotine self-administration (0.06 mg/kg/inf) in rats with short or long access were investigated.

Intravenous catheter implantation

The catheters were implanted as described before^26,36,37. The rats were anesthetized with an isoflurane-oxygen vapor mixture (1–3%) and prepared with a catheter in the right jugular vein. The catheters consisted of polyurethane tubing (length 10 cm, inner diameter 0.64 mm, outer diameter 1.0 mm, model 3Fr, Instech Laboratories, Plymouth Meeting, PA). The right jugular vein was isolated, and the catheter was inserted 3 cm. The tubing was then tunneled subcutaneously and connected to a vascular access button (Instech Laboratories, Plymouth Meeting, PA). The button was exteriorized through a 1-cm incision between the scapulae. During the 7-day recovery period, the rats received daily infusions of the antibiotic Gentamycin (4 mg/kg, IV, Sigma-Aldrich, St. Louis, MO). A sterile heparin solution (0.1 ml, 50 U/ml) was flushed through the catheter before and after administering the antibiotic or nicotine self-administration. After flushing the catheter or a nicotine self-administration session, 0.05 ml of a sterile heparin/glycerol lock solution (500 U/ml) was infused into the catheter. The animals received carprofen (5 mg/kg, SC) daily for 48 h after the surgery. Two days before the start of the nicotine self-administration sessions, the rats were allowed to respond for food pellets under the FR1-TO10s schedule (one 20-min session).

Experimental design

The rats were allowed to self-administer nicotine for five daily 1 h baseline sessions. During the first three sessions (days 1–3), the rats self-administered 0.03 mg/kg/inf of nicotine under an FR1-TO10s schedule. During the following two sessions (days 4 and 5) the rats self-administered 0.06 mg/kg/inf of nicotine under an FR1-TO60s schedule. The 0.06 mg/kg/inf dose of nicotine leads to higher levels of nicotine intake that the 0.03 mg/kg/inf dose^38,39. High doses of nicotine can cause seizures in rodents^18,40,41. To prevent seizures, the time-out period was increased from 10 to 60 s when the dose of nicotine was increased from 0.03 to 0.06 mg/kg/inf. The time-out period (10 – 60 s) does not affect total nicotine intake over a 1-h nicotine self-administration period¹⁸. During the first day that the rats received the 0.03 or 0.06 mg/kg/inf dose, nicotine intake was limited to prevent aversive effects (i.e., seizures and negative mood state). The maximum number of infusions was set to 20 on the first day that the rats received the 0.03 mg/kg/inf dose and to 10 on the first day that the rats received the 0.06 mg/kg/inf dose. After five daily nicotine self-administration sessions, the rats were allowed to self-administer nicotine (0.06 mg/kg/inf) under an intermittent short access (1 h/day) schedule or intermittent long access (23 h/day) schedule. The short access rats self-administered nicotine during the dark phase. The long access rats self-administered nicotine during the dark (11 h) and the light (12 h) phase. During the light phase, the cubicle was illuminated by a house light (ENV-229M, Med Associates, St. Albans, VT) that was mounted on the ceiling of the cubicle. The light was programmed to turn on automatically and remained on for the entire duration of the light phase. The light intensity in the operant chambers during the light phase was 13 lux. Intermittent short and long access nicotine self-administration sessions were conducted three days per week (Monday, Wednesday, and Friday) for five weeks (15 sessions). Responding on the active lever resulted in the delivery of a nicotine infusion (0.1 ml infused over a 5.6-s period). The initiation of the delivery of an infusion was paired with a cue light, which remained illuminated throughout the time-out period. Responding on the inactive lever was recorded but did not have scheduled consequences. The active and inactive lever were retracted during the time-out period. After 15 baseline sessions, the effects of SCH 23390 (experiment 1: short access n=8; long access n=8) and A77636 (experiment 2: short access n=7; long access n=8) on nicotine self-administration (0.06 mg/kg/inf; FR1-TO60s schedule) in rats were investigated (Fig. 1). Both SCH 23390 and A77636 were administered 15 min before the nicotine self-administration sessions. SCH 23390 (0, 0.003, 0.01, and 0.03 mg/kg, SC) and A77636 (0, 0.1, and 0.3 mg/kg, SC) were administered according to a Latin square design. The highest dose of A77636, 1mg/kg, was not included in the Latin square design and was administered last. There were at least 48 h between injections with SCH 23390 and 72 h between injections with A77636. The doses of SCH 23390 and A77636 were based on a previous study with rats³⁶. During the self-administration period, the rats received 90–95 percent of their normal ad libitum food intake in the home cage. The rats were fed (23 g) immediately after the operant sessions. The long access rats received water and food (23 g) in the operant chamber on self-administration days. A mild level of food restriction facilitates food training and nicotine self-administration in rats^42,43.

Statistics

Baseline nicotine self-administration data were analyzed with a two-way ANOVA with access schedule (short or long access) and phase (dark or light) as a between subjects factor and time as a within subjects factor. The effects of SCH 23390 and A77636 on operant responding for nicotine were analyzed with a two-way ANOVA with access schedule or light phase as a between subjects factor and drug treatment as a within subjects factor. The duration of the effects of SCH 23390 and A77636 on nicotine intake in long access groups were analyzed with a two-way ANOVA with drug treatment and time as within subjects factors. For all statistical analyses, significant effects in the ANOVA were followed by Bonferroni’s posthoc tests to determine which groups differed from each other. P-values that were less or equal to 0.05 were considered significant. Significant main effects, interaction effects, and posthoc comparisons are reported in the results section. Data were analyzed with SPSS Statistics version 28 and GraphPad Prism version 9.3.1. GraphPad Prism was also used to create the figures and heatmaps.

RESULTS

Experiment 1: Effect of the D1-like receptor antagonist SCH 23390 on operant responding for nicotine in rats with intermittent short or long access to nicotine

Baseline nicotine self-administration before SHC 23390 treatment

Before the treatments with SHC 23390 started, the rats self-administered 0.03 mg/kg/inf of nicotine for three days (1 h/day), 0.06 mg/kg/inf of nicotine for 2 days (1 h/day), and 0.06 mg/kg/inf of nicotine for 15 days (1 h or 23 h/day). During the first three nicotine self-administration sessions (0.03 mg/kg/inf), nicotine intake and responding on the active lever decreased and was not affected by the experimental group (short vs. long access) (Fig. S1A, S1B; Nicotine intake, Time F2,28 =7.971, P < 0.01; Access F1,14=3.271, NS; Time x Access F2,28=2.143, NS; Active lever, Time F2,28 =5.936, P < 0.01; Access F1,14=3.676, NS; Time x Access F2,28=1.556, NS). Responding on the inactive lever did not change over time and was not affected by the experimental group (Fig. S1A; Inactive lever; Time F2,28 =0.566, NS; Access F1,14=1.054, NS; Time x Access F2,28=1.016, NS).

During the following two sessions, the rats had access to 0.06 mg/kg/inf of nicotine (1h/day). Nicotine intake, responding on the active lever, and responding on the inactive lever increased and was not affected by the experimental conditions (Fig. S1C, S1D; Nicotine intake, Time F1,14 =8.916, P < 0.05; Access F1,14=2.045, NS; Time x Access F1,14=1.842, NS; Active lever, Time F1,14 =8.494, P < 0.05; Access F1,14=3.892, NS; Time x Access F1,14=2.353, NS; Inactive lever, Time F1,14 =8.61, P < 0.05; Access F1,14=0, NS; Time x Access F1,14=0.859, NS).

During the following 15 sessions (0.06 mg/kg/inf, 1 h or 23 h/day), the long access rats responded more on the active lever than the short access rats (Fig. 2A; Active lever, Access F1,14=71.684, P < 0.001). Responding on the active lever did not change over time in the short access rats, but in the long access rats, responding on the active lever decreased and then stabilized (Fig. 2A; Active lever, Time F14,196 =1.342, NS; Time x Access F14,196=1.877, P < 0.05). The long access rats self-administered more nicotine and responded more on the inactive lever than the short access rats (Fig. 2A, 2B; Nicotine intake, Access F1,14=78.694, P < 0.001; Inactive lever, Access F1,14=56.672, P < 0.001). Nicotine intake and responding on the inactive lever did not change over time (Fig. 2A, 2B; Nicotine Intake, Time F14,196 =1.679, NS; Time x Access F14,196=1.667, NS; Inactive lever, Time F14,196 =1.064, NS; Time x Access F14,196=1.202, NS).

Figure 2. Intermittent short and long access nicotine self-administration.

The figures depicts baseline responding on the active lever and the inactive lever (A, C) and nicotine intake (0.06 mg/kg/inf; B, D) in the short and long access rats before treatment with SCH 23390 and A77636. SCH 23390 baseline group (short access n=8, long access, n=8), A77636 baseline group (short access n=7, long access n=8). Asterisks indicate more active lever responses than inactive lever responses (A, C) and higher nicotine intake in the long access than in the short access sessions (B, D). Plus sign indicate fewer active and inactive lever responses (A, C) and less nicotine intake (D) compared to the first long access session. *, + P<0.05; **, ++ P<0.01; ***, +++ P<0.001. Data are expressed as means ± SEM.

It was also determined if there was a difference in nicotine intake between the dark and light phase in the long access rats during the 15 baseline sessions (0.06 mg/kg/inf). Absolute nicotine intake decreased during the baseline sessions, and the rats self-administered more nicotine during the dark phase than during the light phase (Fig. 3A, S2A; Time F14,196 =2.043, P < 0.05; Phase F1,14=218.714, P < 0.001). Nicotine intake decreased during the dark phase, and nicotine intake during the light phase was relatively stable (Fig S2A; Time x Phase F14,196=3.522, P < 0.001). Nicotine intake during the dark phase as a percentage of total nicotine intake decreased over time and nicotine intake during the light phase increased (Fig. S2B; Time F14,196 =0, NS; Phase F1,14=793.844, P < 0.001; Time x Phase F14,196=3.86, P<0.001).

Figure 3. Heatmap representation of nicotine self-administration in rats with intermittent long access to nicotine.

The figures depict heatmaps of baseline intermittent nicotine intake (0.06 mg/kg/inf) before the onset of the treatments with SCH 23390 (A) and A77636 (B). The rats self-administered a greater amount of nicotine during the dark phase than during the light phase. Asterisks indicate higher nicotine intake during the dark phase than during the light phase across all self-administration sessions. The intensity of the colors is indicative of the level of nicotine intake (dark purple indicates high nicotine intake, light purple indicates low nicotine intake, and white indicates no nicotine intake). Long access, n=8/group. ** P<0.01; *** P<0.001.

Nicotine self-administration after SHC 23390 treatment

Nicotine intake and responding on the active lever was decreased after treatment with the D1-like receptor antagonist SCH 23390 (Fig. 4A, 4C; Nicotine intake, Treatment F3,42 =12.5, P < 0.001; Active lever, Treatment F3,42 =12.779, P < 0.001). The posthoc test showed that 0.01 and 0.03 mg/kg of SCH 23390 decreased active lever responses and nicotine intake in the short access rats (Fig. 4A, 4C). The long access rats had a higher level of nicotine intake and responded more on the active lever than the short access rats (Nicotine intake, Access F1,14=34.998, P < 0.001; Treatment x Access F3,42=1.045, NS; Active lever, Access F1,14=34.679, P < 0.001, Treatment x Access F3,42=1.408, NS). The long access rats also responded more on the inactive lever than the short access rats, and responding on the inactive lever was not affected by treatment with SCH 23390 (Fig. 4B; Inactive lever, Treatment F3,42 =2.343, NS; Access F1,14=28.896, P < 0.001; Treatment x Access F3,42=1.636, NS).

Figure 4. Effect of the D1-like receptor antagonist SCH 23390 on nicotine self-administration in short and long access rats.

Treatment with SCH 23390 decreased responding on the active lever (A) and did not affect responding on the inactive lever (B). Treatment with SCH 23390 decreased nicotine intake (C) in short and long access rats and nicotine intake (D) during the first hour of access in the long access rats. Short access, n=8; long access, n=8. Asterisks indicate a significant difference from rats that received vehicle and were on the same access schedule. IVSA, intravenous self-administration. ** P<0.01; *** P<0.001. Data are expressed as means ± SEM.

An additional time-course analysis was conducted to determine the duration of the effect of SCH 23390 in the long access rats. The D1-like receptor antagonist SCH 23390 only affected nicotine intake at the beginning of the 23-h self-administration period (Fig. 5A, S3A; Nicotine intake, Time F22,154 =18.24, P < 0.001; Treatment F3,21=1.916, NS; Time x Treatment F66,462=1.816, P < 0.01). The post hoc showed that 0.03 mg/kg of SCH 23390 decreased nicotine self-administration during the first hour of the self-administration session but not at later time points (Fig. 5A, S3A). Furthermore, during the first hour of nicotine intake, short access rats had a higher level of nicotine intake than the long access rats, and treatment with SCH 23390 induced a greater decrease in nicotine intake in the short access rats than in the long access rats (Fig. 4C, 4D; Nicotine intake, Treatment F3,42 =34.705, P < 0.001; Access F1,14=21.762, P < 0.001; Treatment x Access F3,42=9.826, P < 0.001). The posthoc test showed that 0.01 and 0.03 mg/kg of SCH 23390 decreased nicotine intake in the short access rats and 0.03 mg/kg of SCH 23390 decreased nicotine intake during the first hour in the long access rats (Fig. 4C, 4D). A separate analysis was conducted to determine the effects of SCH 23390 during the dark and light phase in the long access rats (Fig. 5B). Nicotine intake was higher during the dark phase than during the light phase (Phase F1,14=35.639, P < 0.001). Treatment with SCH 23390 did not affect nicotine intake during the dark or light phase in rats with long access to nicotine (Treatment F3,42 =1.942, NS; Treatment x Phase F3,42=1.279, NS).

Figure 5. Time-course effects of the D1-like receptor antagonist SCH 23390 on nicotine intake in long access rats.

Treatment with 0.03 mg/kg of SCH 23390 decreased nicotine intake during the first hour of the long access session (A). Treatment with SCH 23390 did not affect nicotine intake during the dark (11 h) and light phase (12 h) of long access session (B). Long access, n=8. Asterisks indicate lower nicotine intake in rats treated with 0.03 mg/kg of SCH 23390 than in vehicle treated rats. *** P<0.001. Data are expressed as means ± SEM.

Experiment 2: Effect of the D1-like receptor agonist A77636 on operant responding for nicotine in rats with intermittent short or long access to nicotine

Baseline nicotine self-administration before A77636 treatment

During the first 3 days of nicotine self-administration, nicotine intake and responding on the active lever decreased and these parameters were not affected by the experimental group (short vs. long access) (Fig. S1E, S1F; Nicotine intake, Time F2,26 =16.022, P < 0.001; Access F1,13=3.095, NS; Time x Access F2,26=0.852, NS; Active lever, Time F2,26 =15.95, P < 0.001; Access F1,13=2.902, NS; Time x Access F2,26=0.985, NS). Responding on the inactive lever did not change over time and was not affected by the experimental group (Fig. S1E; Inactive lever, Time F2,26 =2.187, NS; Access F1,13=1.844, NS; Time x Access F2,26=1.624, NS).

After the rats had access to 0.03 mg/kg/inf of nicotine for three days, they were given access to 0.06 mg/kg/inf of nicotine for 2 days. During these two days, nicotine intake and responding on the active lever increased (Fig. S1G, S1H; Nicotine intake, Time F1,13 =12.14, P < 0.01; Access F1,13=3.47, NS, Time x Access F1,13=0.007, NS; Active lever, Time F1,13 =12.14, P < 0.01; Access F1,13=3.47, NS, Time x Access F2,26=0.007, NS). Responding on the inactive lever did not change and was not affected by the experimental group (Fig. S1G; Inactive lever, Time F1,13 = 2.086, NS; Access F1,13=2.68, NS; Time x Access F1,13=3.056, NS).

After the five 1-h sessions, the rats had access to 0.06 mg/kg/inf of nicotine for 15 days. The long access rats had a higher level of nicotine intake and more responses on the active and inactive lever than the short access rats (Fig. 2C, 2D; Nicotine intake, Access F1,13=30.991, P < 0.001; Active lever, Access F1,13=31.661, P < 0.001; Inactive lever, Access F1,13=8.101, P < 0.05). Nicotine intake and responding on the active and inactive lever did not change over time in the short access rats but decreased in the long access rats and then stabilized (Fig. 2C, 2D; Nicotine intake, Time F14,182 =8.088, P < 0.001; Time x Access F14,182=8.1, P < 0.001; Active lever, Time F14,182 =8.245, P < 0.001; Time x Access F14,182=8.394, P < 0.001; Inactive lever, Time F14,182 =3.415, P < 0.001; Time x Access F14,182=4.252, P < 0.001).

An additional analysis was conducted to determine if there was a difference in nicotine intake between the dark and the light phase during the 15 baseline sessions (0.06 mg/kg/inf) in the long access rats. Nicotine intake decreased over time, and nicotine intake was higher during the dark phase than during the light phase (Fig. 3B, S2C; Time F14,196 =11.895, P < 0.001; Phase F1,14=44.77, P < 0.001). Absolute nicotine intake decreased during the dark phase and was relatively stable during the light phase (Fig. S2C; Time x Phase F14,196=8.671, P < 0.001). Nicotine intake as a percentage of total nicotine intake decreased during the dark phase and increased during the light phase (Fig. S2D; Time F14,196 =0, NS; Phase F1,14=221.766, P < 0.001; Time x Phase F14,196=4.956, P < 0.001).

Nicotine self-administration after A77636 treatment

Treatment with the D1-like receptor agonist A77636 decreased nicotine intake and responding on the active lever in the short and long access rats, and the effect of A77636 was greater in the long access rats than in short access rats (Fig. 6A, 6C; Nicotine intake, Treatment F3,39 =9.854, P < 0.001; Treatment x Access F3,39=4.816, P < 0.01; Active lever, Treatment F3,39 =9.302, P < 0.001; Treatment x Access F3,39=4.368, P < 0.05). The posthoc test showed that 1 mg/kg of A77636 decreased active lever responses and nicotine intake in the short access rats, and both 0.1 and 1 mg/kg of A77636 decreased active lever responses and nicotine intake in the long access rats (Fig. 6A, 6C). Treatment with A77636 did not affect responding on the inactive lever (Fig. 6B; Inactive lever, Treatment F3,39 =2.138, NS; Treatment x Access F3,39=2.167, NS). The long access rats had a higher level of nicotine intake and responded more on the active and inactive lever than the short access rats (Nicotine intake, Access F1,13=27.423, P < 0.001; Active lever, Access F1,13=28.019, P < 0.001; Inactive lever, Access F1,13=12.927, P < 0.01).

Figure 6. Effect of the D1-like receptor agonist A77636 on nicotine self-administration in short and long access rats.

Treatment with A77636 decreased responding on the active lever (A) and did not affect responding on the inactive lever (B). Treatment with A77636 also decreased nicotine intake (C) in short and long access rats and nicotine intake (D) during the first hour of access in the long access rats. Short access, n=7; long access, n=8. Asterisks indicate a significant difference from rats that received vehicle and were on the same access schedule. IVSA, intravenous self-administration. * P<0.05; ** P<0.01; ***, +++ P<0.001. Data are expressed as means ± SEM.

A time-course analyses showed that A77636 mainly affected nicotine intake at the beginning of the 23 h self-administration period (Fig. 7A, S3B; Nicotine intake, Time F22,154 =28.199, P < 0.001; Treatment F3,21=8.402, P < 0.001; Time x Treatment F66,462=1.991, P < 0.001). The post hoc analysis showed that 0.1 and 0.3 mg/kg of A77636 decreased nicotine intake during the first hour of the self-administration session (Fig. 7A, S3B). In addition, 1 mg/kg of A77636 decreased nicotine intake during the first four hours of the self-administration session but not at later time points (Fig 7A, S3B). Furthermore, treatment with A77636 decreased nicotine intake in the short and long access rats (Fig. 6C, 6D; Nicotine intake, Treatment F3,39 =13.892, P < 0.001; Access F1,13=14.311, P < 0.01; Treatment x Access F3,39=2.53, NS). The posthoc test showed that 1 mg/kg of A77636 decreased nicotine intake in the short access rats and 0.1, 0.3, and 1 mg/kg of A77636 decreased nicotine intake during first hour in the long access rats (Fig. 6C, 6D). A separate analysis was conducted to determine the effects of A77636 during the dark and light phase in rats with long access to nicotine (Fig. 7B). The rats self-administered more nicotine during the dark phase than during the light phase (Phase F1,14=27.005, P < 0.001). Treatment with A77636 decreased nicotine intake during the dark and light phase (Treatment F3,42 =8.672, P < 0.001; Treatment x Phase F3,42=2.271, NS). The posthoc test showed that 0.1 and 1 mg/kg of A77636 decreased nicotine intake during the dark phase and 1 mg/kg of A77636 decreased nicotine intake during the light phase (Fig. 7B).

Figure 7. Time-course effects of the D1-like receptor agonist on nicotine intake in long access rats.

Treatment with 0.1 and 0.3 mg/kg of A77636 decreased nicotine intake during the first hour of access to nicotine (A). Treatment with 1 mg/kg of A77636 decreased nicotine intake during the first four hours of access to nicotine (A). Treatment with 0.1 mg/kg of A77636 decreased nicotine intake during the dark phase (11 h) and 1 mg/kg of A77636 decreased nicotine intake during the dark (11 h) and light phase (12 h) of the long access sessions (B). Long access, n=8. Plus signs indicate lower nicotine intake in rats treated with 0.1 mg/kg of A77636 than in vehicle treated rats. Pound sign indicate lower nicotine intake in rats treated with 0.3 mg/kg of A77636 than in vehicle treated rats. Asterisks indicate lower nicotine intake in rats treated with 1 mg/kg of A77636 than in vehicle treated rats. *, + P<0.05; ** P<0.01; ***, +++, ### P<0.001. Data are expressed as means ± SEM.

DISCUSSION

In the present study, we investigated the effects of the dopamine D1-like receptor antagonist SCH 23390 and the D1-like receptor agonist A77636 on the self-administration of nicotine in rats with intermittent short or long access to nicotine. The rats with long access to nicotine had a higher level of nicotine intake than the rats with short access to nicotine. The D1-like receptor antagonist SCH 23390 decreased nicotine intake to a similar degree in rats with short or long access to nicotine. Treatment with the D1-like receptor agonist A77636 decreased nicotine intake in the short and long access rats, but A77636 induced a greater decrease in nicotine intake in the long access rats. Treatment with A77636 decreased nicotine intake during the dark and light phase in the long access rats, indicating that this drug has a prolonged effect on nicotine intake. These findings indicate that rats with intermittent long access to nicotine have a high level of nicotine intake compared to rats with intermittent short access. Furthermore, drugs that block or stimulate D1-like receptors decreased nicotine intake in rats with intermittent short or long access to nicotine.

In the present studies, all the rats had daily short access to nicotine for 5 days. Then half the rats were given intermittent short access (1 h) to nicotine, and the other half were given intermittent long access (23 h) to nicotine. Baseline nicotine intake was stable in the rats with intermittent short access to nicotine. In contrast, nicotine intake in the rats with intermittent long access gradually decreased and then stabilized. In previous studies in which Wistar rats had intermittent long access to nicotine, nicotine intake slightly increased and then stabilized^23,24. One major difference between the present studies and those that reported a small increase in nicotine intake is the dose of nicotine. In the present study, the rats self-administered 0.06 mg/kg of nicotine, and in other studies, the rats self-administered 0.03 mg/kg/inf of nicotine^23,24. The 0.06 mg/kg/inf dose of nicotine leads to a higher level of nicotine intake than the 0.03 mg/kg/inf dose of nicotine^44,45. In the present study, the long access rats started out with a very high level of nicotine intake (SCH 23390 group 2.4 mg/kg of nicotine and A77636 group 3.4 mg/kg of nicotine), and during the last session (fifteenth session), they self-administered about 1.8 mg/kg of nicotine. In contrast, in previous studies with the 0.03 mg/kg/inf dose of nicotine, the rats self-administered about 1 mg/kg during the first session and 1.2 mg/kg during the last session^23,24. This indicates that at the onset of the present studies, nicotine intake was 2 to 3 times higher than in previous nicotine self-administration studies with a lower dose of nicotine. Furthermore, these findings indicate that in long access rats with a history of nicotine intake, the average nicotine intake is about 1.8 mg/kg in rats that receive the 0.06 mg/kg dose and 1.2 mg/kg in rats that receive the 0.03 mg/kg dose. In the present study, the rats may have gradually decreased their nicotine intake because high doses of nicotine are aversive. The long access rats self-administered 0.3–0.4 mg/kg of nicotine during the first hour of access (Fig 4D, 6D; vehicle group). Studies in which nicotine was administered noncontingently have shown that these doses are rewarding but that higher doses are aversive. In previous studies, noncontingent administration of low and intermediate doses of nicotine (0.1 – 0.3 mg/kg) facilitated ICSS in rats. The administration of high doses of nicotine (0.6 – 1.25 mg/kg) inhibited ICSS, which suggests that these doses are aversive^29,46,47. Place conditioning studies with rats have also reported that high doses of nicotine are aversive⁴⁸. Whether nicotine intake increases or decreases during the first long access sessions may also be affected by other factors such as prior food training and the history of nicotine intake (e.g., short or long access and daily or intermittent nicotine self-administration) before the rats were given long access to nicotine^22,23,49.

One of the main goals of the present studies was to determine the effects of the D1-like receptor antagonist SCH 23390 and the D1-like receptor agonist A77636 on nicotine intake in rats with intermittent short or long access to nicotine. The intermittent long access model might model smoking better than the short access model. Previous work has shown that abstinence from nicotine leads to increased anxiety-like behavior and allodynia in rats with intermittent long, but not short, access to nicotine⁵⁰. Furthermore, the nAChR antagonist mecamylamine induces a greater inhibition of ICSS in rats with intermittent long access to nicotine than in rats with short access to nicotine²⁴. Blockade of nicotinic receptors also differently affects nicotine intake in rats with a history of short or long access to nicotine. In animals with short access to nicotine, mecamylamine decreases nicotine intake^21,51. In contrast, mecamylamine does not decrease nicotine intake in long access rats because the initial decrease in nicotine intake is followed by a rebound increase in nicotine intake²⁴. In the present study, we found that the D1-like receptor antagonist SCH 23390 decreased nicotine intake in the short and long access rats. The outcome of the posthoc test suggests that SCH 23390 has a larger effect on nicotine intake in the short access rats than in the long access rats. In the short access rats, both the 0.01 and 0.03 dose decreased nicotine intake, but these doses did not affect nicotine intake in the long access rats. The time point analysis showed that in the long access rats, SCH 23390 decreased nicotine intake during the first hour after treatment but not at later time points. We also investigated the effects of the D1-like receptor agonist A77636 on nicotine intake. The D1-like receptor agonist A77636 decreased nicotine intake in the short and long access rats, but the effect of A77636 was greater in the long access rats. The posthoc test showed that only the highest dose of A77636 (1 mg/kg) decreased nicotine intake in the short access rats, but both a low dose (0.1 mg/kg) and a high dose (1 mg/kg) decreased nicotine in the long access rats. All A77636 doses (0.1, 0.3, and 1 mg/kg) decreased nicotine intake during the first hour of nicotine self-administration in the long access rats. This work indicates that A77636 decreases nicotine intake in rats with short and long access to nicotine, but the effect A77636 is greater in the long access rats.

The rewarding effects of nicotine are at least partly mediated via the activation of the mesolimbic dopaminergic system^30,52. Dopamine levels in the nucleus accumbens (NAcc) of rats increase after noncontingent nicotine administration or nicotine self-administration^53,54. Nicotine-induced dopamine release in the NAcc did not change over a six-week self-administration period, which indicates that there is no tolerance to the effects of nicotine self-administration on dopamine release⁵⁴. Dopamine mediates its rewarding effects by stimulating D1- and D2-like receptors. Blockade of D1-like receptors with SCH 23390 decreases nicotine intake in rats^33,36,55. Furthermore, administration of the D1-like receptor antagonist SCH 23390, but not the D2-like receptor antagonist haloperidol, into the NAcc, insular cortex, and the parietal association cortex reduces nicotine self-administration in rats^35,56. In the present study, both blockade (SCH 23390) and stimulation (A77636) of D1-like receptors diminished nicotine self-administration. Similarly, both SCH 23390 and A77636 prevent nicotine-induced conditioned place aversion in mice⁵⁷. In addition, SCH 23390 and A77636 reduce operant responding for food in rats³⁶. These findings suggest that D1-like receptors play a critical role in the rewarding properties of nicotine and natural rewards. Furthermore, systemic administration of SCH 23390 inhibits ICSS (aversive) and blocks the ICSS facilitating effects of nicotine in rats^58,59. In contrast, systemic and intra-NAcc administration of A77636 facilitates ICSS in rats^60,61. In addition, A77636 has a high intrinsic D1 receptor activity (134 % of dopamine) in rat caudate putamen membranes⁶². These studies indicate that blockade of D1 receptors prevents the effects of nicotine on the dopaminergic system, while stimulation of D1 receptors mimics the effects of dopamine. Thus, both blockade and stimulation of D1 receptors blunts the effects of nicotine on the dopaminergic system and decreases nicotine intake. It is also important to note that in the current study A77636 induced a greater reduction in nicotine self-administration than SCH 23390. Previous research suggests that SCH 23390 (Kd = 5.4 nM) and A77636 (Kd = 7.6 nM) have similar affinity for D1 receptors in rat striatal membranes⁶³. However, SCH 23390 caused a 21 % reduction in D1 receptor binding (total number of binding sites (Bmax)= 1.5 pmol/mg protein), whereas A77636 induced a 76 % reduction in D1 receptor binding (Bmax = 0.45 pmol/mg protein) in rat striatal membranes⁶³. Taken together, these studies suggest that A77636 decreases D1 receptor binding sites and thereby diminishes the effects of nicotine on the dopaminergic system.

In the present study, we also determined if intermittent long access to nicotine affects nicotine intake in the dark versus the light phase. Absolute nicotine intake during the dark phase gradually decreased, and nicotine intake during the light phase was relatively stable. However, when we determined nicotine intake during the dark and light phase as a percentage of total nicotine intake, we found that nicotine intake during the dark phase decreased (SCH 23390 group: 91 to 77 percent and A77636 group: 87 to 79 percent) and nicotine intake during the light phase increased (SCH 23390 group: 9 to 23 percent and A77636 group: 13 to 21 percent). This is in line with a previous study in which we found that the percentage of nicotine consumed during the dark phase decreased and during the light phase increased²⁴. Another study that investigated the effects of daily long access to nicotine on nicotine intake during the dark and light phase also found that the difference in nicotine intake between the dark and the light phase decreased over time⁴⁵. In humans, smoking during the night has been associated with a high level of nicotine dependence²⁵. The relative increase in nicotine intake during the light phase in rats could possibly be associated with the gradual development of nicotine dependence. However, this needs to be further explored before firm conclusions can be drawn. We conducted additional analyses to determine the effects of SCH 23390 and A77636 on nicotine intake during the dark and light phase (separate analysis for each phase) in the long access rats. Treatment with the D1-like receptor antagonist SCH 23390 did not affect nicotine intake during the dark or light phase in the rats with long access to nicotine. Interestingly, treatment with the D1-like receptor agonist A77636 decreased nicotine intake during the dark and light phase. This indicates that stimulation of D1-like receptors has a greater and more prolonged effect on nicotine intake than blockade of D1-like receptors.

There are several possible pharmacological mechanisms that might mediate the long-term effects of A77636 on nicotine self-administration. A77636 is a highly potent and selective D1-like receptor agonist (D1 receptors half maximal effective concentration (EC₅₀): rat caudate putamen = 8.97 nM, human D1 receptors cell lines = 3 nM; D1 receptors inhibition constant (Ki) value: rat striatum = 31 nM, monkey striatum = 1 nM, human D1 receptors cell lines: 8 nM; D2 receptors Ki value: rat striatum = 2000 nM, human D2 receptors cell lines: > 4276 nM)^62–64. In both in vitro and in vivo studies, A77636 induced rapid D1-like receptor desensitization and had long-term pharmacological effects. In vitro studies showed that A77636 decreased (76 %) D1 receptor binding in rat striatal membranes and subsequently reduced (60 %) the SKF-81297 (D1-like agonist)-induced increase in cAMP levels in rat striatal neurons^63,64. Similarly, in vivo studies showed that A77636-induced contralateral rotations in 6-hydroxydopamine (6-OHDA) lesioned rats and eye blink rates in cynomolgus monkeys (marker of D1-like receptor activation) rapidly diminished with successive treatments^64,65. Furthermore, A77636 has a high affinity for D1-like receptors (equilibrium dissociation constant (Kd): rat striatum = 7.6 nM) and dissociates slowly from these receptors⁶². This results in prolonged activation of D1-like receptors and pharmacological effects. Systemic administration of A77636 stimulates the release of acetylcholine in the frontal cortex and hippocampus over a 3 h test period in rats⁶⁶. Furthermore, A77636 mediated long-term (> 20 h) anti-Parkinson’s and anorexic effects in rats^62,67. Dopamine agonists increase the spontaneous eye blink rate in monkeys, and this effect is completely blocked with D1-like receptor antagonists but not with D2-like receptor antagonists⁶⁸. Systemic administration of A77636 in cynomolgus monkeys increases the eye blink rate over a 6 h test period⁶⁴. In our study, A77636 decreased nicotine intake during the dark and light phase in the long-access rats and this effect was most pronounced during the first four hours after treatment. Previous research suggests that the pharmacological effects of A77636 are primarily mediated via the activation of D1-like receptors but not D2-like receptors. A77636-induced acetylcholine release in the frontal cortex of rats, rotational behavior in 6-OHDA lesioned rats, and eye blink rates in monkeys are completely blocked by pretreatment with the D1like receptor antagonists (SCH 23390 or SCH39166)^62,64. In contrast, pretreatment with the selective D2-like receptor antagonist haloperidol did not prevent the A77636-induced rotations in 6-OHDA lesioned rats⁶². Taken together, these studies suggest that A77636-mediated D1-like receptor activation and desensitization blunts the rewarding properties of nicotine and leads to a long-term decrease in nicotine intake.

In our previous work, we determined if SCH 23390 and A77636 have sedative effects or induce motor impairments by evaluating their effects on locomotor activity in Wistar rats³⁶. Treatment with 0.01 and 0.03 mg/kg of SCH 23390 decreased locomotor activity in male rats. In contrast, treatment with A77636 did not affect locomotor activity. Other studies have also reported that SCH 23390 dose-dependently (0.01 – 1 mg/kg) decreases locomotor activity, whereas A77636 (0.1 – 1 mg/kg) did not affect locomotor activity in adult male Wistar rats^69,70. Previous studies have found that SCH 23390 has high affinity for serotonin 5-HT1C, 5-HT2, and 5-HT2C receptors and is a potent agonist of these receptors^71–73. Activation of serotonin 5-HT2C receptors with WAY 161503 dose-dependently decreases locomotor activity in rats and this effect is completely blocked with the 5-HT2C antagonist SB 242084⁷⁴. Furthermore, nicotine increases locomotor activity but co-administration of WAY 161503 and nicotine leads to a decrease in locomotor activity⁷⁵. Both SCH 23390 and the potent and selective 5-HT2C agonist CP 809,101 inhibit ICSS (aversive) in rats⁵⁸. Pretreatment with the 5-HT2C antagonist SB 242084 blocks the effects of CP 809,101 but not the effects of SCH 23390 on ICSS responding. This suggests that the aversive effects of SCH 23390 are not mediated via 5-HT2C receptors⁵⁸. Taken together, these findings suggest that doses of SCH 23390 that decreased nicotine intake in the long access and short access rats have sedative effects via off-target mechanisms. In contrast, A77636 has low affinity for α- and β-adrenoreceptors and serotonin 5-HT1A, 5-HT1C, and 5-HT2 receptors⁶², and doses of A77636 that decreased nicotine intake did not have sedative effects. Therefore, the D1-like receptor agonist A77636 could potentially help people quit smoking without causing sedation. In the present study, we only determined the effects of SCH 23390 and A77636 on nicotine intake in male rats. It has been well established that there are sex differences in nicotine intake and other behavioral tests in rodents^76,77. However, in a previous study, we showed that there are no sex differences in the effects of SCH 23390 or A77636 on operant responding for nicotine and food and locomotor activity in rats³⁶.

In conclusion, the present findings show that rats with intermittent long access to nicotine have higher nicotine intake than rats with intermittent short access to nicotine. The D1-like receptor antagonist SCH 23390 decreased nicotine intake in the short and long access rats. The D1-like receptor agonist A77636 induced a greater decrease in nicotine intake in rats with long access to nicotine than in rats with short access to nicotine. Although both SCH 23390 and A77636 decrease nicotine intake, A77636 decreases nicotine intake at doses that do not have sedative effects. This work suggests that the D1-like receptor agonist A77636 could potentially be used to help people quit smoking and vaping.

Data Availability

Data are available from the corresponding author on request.