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. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: Pharmacol Biochem Behav. 2021 Jun 28;207:173224. doi: 10.1016/j.pbb.2021.173224

Adolescent nicotine treatment causes robust locomotor sensitization during adolescence but impedes the spontaneous acquisition of nicotine intake in adult female Wistar rats

Ranjithkumar Chellian 1, Azin Behnood-Rod 1, Ryann Wilson 1, Marcelo Febo 1, Adriaan W Bruijnzeel 1,2
PMCID: PMC8284834  NIHMSID: NIHMS1720011  PMID: 34197844

Abstract

Very few people are able to quit smoking, and therefore it is essential to know which factors contribute to the development of compulsive nicotine use. These studies aimed to investigate if early-adolescent nicotine exposure causes locomotor sensitization and affects anxiety-like behavior and the spontaneous acquisition of intravenous nicotine self-administration. Early-adolescent male and female rats were treated with nicotine from postnatal (P) days 24 to 42, and anxiety-like behavior and locomotor activity were investigated one day after the cessation of nicotine treatment and in adulthood (>P75). The spontaneous acquisition of nicotine self-administration was also investigated in adulthood. The rats self-administered 0.03 mg/kg/infusion of nicotine for six days under a fixed-ratio (FR) 1 schedule and four days under an FR2 schedule (3-h sessions). Repeated nicotine administration increased locomotor activity, rearing, and stereotypies in a small open field in adolescent male and female rats. One day after the last nicotine injection, the percentage of open arm entries in the elevated plus-maze test was decreased in the males and increased in the females. However, locomotor activity in the small open field was unaffected. Adolescent nicotine treatment did not affect anxiety-like behavior and locomotor activity in adulthood. During the 10-day nicotine self-administration period, the females had a higher level of nicotine intake than the males. Adolescent nicotine treatment decreased nicotine intake in the females. In conclusion, these findings indicate that repeated nicotine administration during adolescence causes robust behavioral sensitization and leads to lower nicotine intake in females throughout the acquisition period in adulthood in rats.

Keywords: adolescent, nicotine, sensitization, acquisition, self-administration, sex, rats

1. INTRODUCTION

Despite a worldwide reduction in tobacco use, smoking and smokeless tobacco use remains a significant worldwide health burden in humans (WHO, 2018). There are more than 40 million tobacco users in the US alone, and worldwide there are about 1 billion tobacco users (CDC, 2014). Tobacco use disorder is characterized by compulsive smoking and negative affective withdrawal signs upon smoking cessation (American Psychiatric Association, 2013; Bruijnzeel, 2012). Negative affective withdrawal signs play a role in the continuation of smoking, but nicotine’s positive reinforcing properties play a critical role in smoking initiation. Nicotine induces mild euphoria, enhances cognitive functions, and provides a feeling of relaxation and calmness (Benowitz, 2009; Rezvani and Levin, 2001). The majority of tobacco and e-cigarette users start using nicotine products during adolescence. A recent study showed that about 50 percent of smokers start smoking before eighteen (Ali et al., 2020). Each day more than 3000 adolescents start experimenting with cigarettes, and two-thirds transition to daily cigarette use (HHS, 2014). Tobacco use is on the decline among young people, but e-cigarette use is increasing and may fuel a new wave of nicotine users. The increase in e-cigarette use is supported by data from the Monitoring the Future (MTF) survey. E-cigarette use among high schoolers doubled from 2017 to 2019 (Miech et al., 2019). In the US, about 21 percent of 8th graders (12–13 years of age) and 40 percent of 12th graders (17–18 years of age) have used e-cigarettes (Miech et al., 2019). The present study investigated if adolescent nicotine exposure affects the acquisition of nicotine intake in adulthood. A rapid escalation of nicotine use may increase the risk for tobacco use disorder in humans and causes dependence in animal models (Behrendt et al., 2009; Caille et al., 2012).

Noncontingent exposure to drugs of abuse causes neuronal changes that enhance the response to drugs (Robinson and Berridge, 1993). Repeated injections with psychostimulants enhance the behavioral and neurochemical response to drugs of abuse (Piazza et al., 1989; Piazza et al., 1990; Vanderschuren et al., 1999). Drug sensitization has also been shown to enhance the acquisition of cocaine and amphetamine self-administration in rats (Horger et al., 1992; Piazza et al., 1990; Zhao and Becker, 2010). Several studies have investigated nicotine-induced behavioral and neurochemical sensitization. Repeated nicotine administration leads to an increase in locomotor activity in adult male and female rats (Domino, 2001; Miller et al., 2001). Sensitization processes have also been associated with an increased neurochemical response to nicotine. Repeated nicotine administration leads to increased dopamine transmission and dopamine D3 receptor levels in the nucleus accumbens (Cadoni and Di Chiara, 2000; Le Foll et al., 2003). It has been hypothesized that sensitization processes contribute to the development of drug use disorders, but it is unknown if repeated nicotine administration during adolescence affects the acquisition of nicotine intake in adulthood in males and females. There are conflicting findings regarding the effects of nicotine pretreatment on nicotine intake later in life in rodents. A study with adult male rats showed that nicotine pretreatment (0.4 mg/kg of nicotine base, subcutaneous [SC], 7 days) did not affect the acquisition of nicotine intake in Sprague-Dawley rats and made nicotine intake less likely in Long-Evans rats (Shoaib et al., 1997). A recent study suggests that treating male adolescent Sprague-Dawley rats with high doses of nicotine (1 mg/kg of nicotine base, SC, 14 days) does not affect nicotine self-administration (0.02 mg/kg nicotine base per infusion) in adulthood under an FR1 schedule (Renda et al., 2020). A study with male and female mice showed that chronic noncontingent nicotine administration during adolescence via minipumps (3 mg/kg of nicotine base per day, SC, 14 days) did not affect the oral nicotine intake (2% saccharin solution containing 100 μM nicotine) in operant chambers under a progressive ratio (PR) schedule in adulthood (Cole et al., 2019). However, when the mice were tested under an FR schedule, adolescent nicotine administration increased oral nicotine intake (2% saccharin solution containing 100 μM nicotine) in adult females but not males. The administration of a high (0.5 mg/kg of nicotine base, SC, 4 days / 8 injections), but not a low dose (0.1 mg/kg of nicotine base, SC) of nicotine to early adolescent male mice enhanced the rewarding effect of nicotine (0.5 mg/kg of nicotine base, SC) in adulthood in a conditioned place preference procedure (0.5 mg/kg of nicotine base, SC)(Kota et al., 2009). Sex differences in the long-term effects of adolescent nicotine administration have also been shown to affect reward learning. Adolescent nicotine treatment (0.35 mg/kg of nicotine base, SC, 15 days) improved reward learning in adult males and impaired reward learning in adult females (Quick et al., 2014). Therefore, adolescent nicotine intake could affect drug intake later in life via changes in reward learning.

Several studies have investigated the effects of repeated nicotine injections on locomotor activity in adolescent rats. One of the first studies showed that repeated administration of nicotine (0.4 mg/kg of nicotine salt, SC, equivalent to 0.13 mg/kg nicotine base) sensitizes the locomotor response to nicotine in adolescent male rats. Doses that were lower than 0.13 mg/kg did not induce locomotor sensitization (Schochet et al., 2004). Another study reported that 0.4 mg/kg of nicotine base (SC) induces locomotor sensitization in adolescent male rats (Thompson et al., 2018). A study that compared the effects of nicotine in adolescent males and females showed that median and high doses of nicotine (0.5 and 0.7 mg/kg of nicotine base, intraperitoneal [IP]) induce locomotor sensation in males and females. A lower dose (0.3 mg/kg of nicotine base) only caused behavioral sensitization in the adolescent males (Perna and Brown, 2013)

It is currently unknown if a nicotine treatment regimen that induces robust behavioral sensitization in adolescent rats affects nicotine intake in adulthood. It is also unknown if there are sex differences in the long-term effects of adolescent nicotine treatment on nicotine intake. Based on previous drug sensitization studies, it was hypothesized that a nicotine treatment regimen that leads to behavioral sensitization would also lead to higher levels of nicotine intake during the acquisition phase (Vanderschuren and Pierce, 2010). In the present study, the rats were treated with nicotine from P24 to 42. This period is considered early adolescence in rats, and during this period, rats are more sensitive to the effects of drugs than during late adolescence or adulthood (Badanich et al., 2008; Spear, 2000). The locomotor response to nicotine was investigated from P24 to P29. Locomotor activity and anxiety-like behavior were investigated one day after the last nicotine injection and in adulthood (>P75). The acquisition of intravenous nicotine self-administration was also investigated in adulthood.

2. MATERIALS AND METHODS

2.1. Animals

Male and female Wistar rats (P21, 45–55 g) were purchased from Charles River (Raleigh, NC). The rats arrived in the vivarium at P21 and were kept in standard housing conditions (2 rats of the same sex per cage) in a climate-controlled vivarium on a reversed 12 h light-dark cycle (light off at 7 AM). Food and water were available ad libitum in the home cage. The experimental protocols were approved by the University of Florida Institutional Animal Care and Use Committee.

2.2. Drugs

(−)-Nicotine hydrogen tartrate was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and dissolved in sterile saline (0.9 % sodium chloride). The nicotine solution was administered in a volume of 1 ml/kg body weight (SC), or the rats self-administered 0.1 ml/infusion intravenously (IV) of a nicotine solution. Nicotine doses are expressed as the base.

2.3. Experimental design

The rats were handled for 2–3 min per day, and the body weights were recorded on P22 and P23. The rats were randomly divided into two groups (saline and nicotine injections; n=12/group/sex) and were treated with saline or nicotine from P24 to P42 (See Figure 1 for a schematic overview of the experiment). We determined the effect of nicotine treatment on body weights because a nicotine-induced change in body weight could potentially affect the behavior of the rats in the tests. Nicotine-induced locomotor sensitization was studied from P24 to P29. On the first, second, and sixth treatment day, the rats were placed in a small open field for 30 min and then received an injection with saline or nicotine (0.4 mg/kg, SC). This nicotine dose causes behavioral sensitization and has rewarding effects in adolescent rats (Schochet et al., 2004; Torres et al., 2008). Immediately after the injection, the rats were returned to the small open field, and locomotor activity, rearing, and stereotypies were measured for 60 min. On days 3–5, the rats received an injection and were returned to their home cage immediately afterward. From P30–41, the rats received two saline or nicotine (0.4 mg/kg, SC) injections per day. They received one injection in the morning (10 AM) and one injection in the afternoon (4 PM). The twice-daily injection regimen was based on previous studies that showed that treating adolescent mice with nicotine twice a day increases the rewarding properties of morphine and cocaine in adulthood (Alajaji et al., 2016; Kota et al., 2018). On P42, the rats received an injection in the morning, and 24 h later (P43), they were tested in the elevated plus-maze test and the small open field test to determine if the cessation of nicotine administration affects anxiety-like behavior and locomotor activity. The rats were housed with 2 rats per cage (same-sex) during the first part of the study, and after they were prepared with a catheter, they were singly housed. The rats were handled for 2–3 min per week, and the body weights were recorded on P50, P58, P65, and P71. When the rats reached adulthood (>P75, Figure 1), behavioral tests for locomotor activity (P79), anxiety-like behavior (P80 and 81), and nicotine self-administration sessions (> P90) were performed. The rats were not treated with any drugs immediately before these behavioral tests. We conducted the behavioral tests on three separate days. On the first day, the small open field test was done (10 min), on the second day, the large open field test (10 min), and on the third day, the elevated plus-maze test (5 min). The elevated plus-maze test was conducted twice, both during adolescence and in adulthood. Previous work has shown that there is no habituation in locomotor activity and anxiety-like behavior when the rats are tested in the elevated plus maze twice (Schrader et al., 2018). After the behavioral tests were completed, the rats received a permanent catheter in the right jugular vein, and the acquisition of intravenous nicotine self-administration was investigated for ten days.

Figure 1. Schematic overview of behavioral tests.

Figure 1.

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The rats arrived in the vivarium at postnatal day (P) 21, and they were treated with nicotine from P24 to P42. Twenty four hours after the last nicotine or saline injection, they were tested in the elevated plus-maze test and the small open field. In adulthood (>P75), they were tested in the small open field, large open field, and elevated plus-maze. After completing the behavioral tests, the rats were prepared with catheters and the acquisition of nicotine intake was investigated. Abbreviations: P, postnatal day; INJ, injections; SOF, small open field; LOF, large open field; EPM, elevated plus-maze; IVSA, intravenous self-administration.

2.4. Behavioral tests

2.4.1. Small open field test

The small open field test was done to assess locomotor activity, rearing, and stereotypies (Bruijnzeel et al., 2016; Qi et al., 2016). Horizontal and vertical beam breaks were measured using an automated animal activity cage system (VersaMax Animal Activity Monitoring System, AccuScan Instruments, Columbus, OH, USA). Horizontal beam breaks and total distance traveled reflect locomotor activity, and vertical beam breaks reflect rearing. Repeated interruptions of the same beam is an estimate of stereotypies (Chellian et al., 2020b; Febo et al., 2003; Hayashi et al., 2007). The setup consisted of four animal activity cages made of clear acrylic (40 cm × 40 cm × 30 cm; L x W x H), with 16 equally spaced (2.5 cm) infrared beams across the length and width of the cage. The beams were located 2 cm above the cage floor (horizontal activity beams). An additional set of 16 infrared beams were located 11 cm (adolescent rats) or 14 cm (adult rats) above the cage floor (vertical activity beams). All beams were connected to a VersaMax analyzer, which sent information to a computer that displayed beam data through Windows-based software (VersaDat software). The small open field test was conducted in a dark room, and the cages were cleaned with a Nolvasan solution (chlorhexidine diacetate) between animals. Each rat was placed in the center of the small open field, and activity was measured for 10 or 90 min. The brief, 10-min, small open field session was conducted to determine locomotor activity in animals in a drug-free state. The long 90 min session (30 min baseline and 60 min post nicotine) was conducted to capture the full time-course of effects of nicotine. Nicotine may cause a brief decrease in locomotor activity followed by an increase in locomotor activity; therefore, it is essential to measure activity over a relatively long period (Bruijnzeel et al., 2011).

2.4.2. Elevated plus maze test

The elevated plus-maze test is used to measure anxiety-like behavior in rodents (Walf and Frye, 2007) and was performed as described in our previous work (Chellian et al., 2020b; Qi et al., 2016; Tan et al., 2019). The elevated plus maze (Coulbourn Instruments, Whitehall, PA) consists of two open arms (i.e., without walls; 50 cm × 10 cm; L × W) and two closed arms (i.e., with black walls, 50 cm × 10 cm × 30 cm; L × W × H). The open and closed arms were connected by a central platform, and the open arms had 0.5 cm tall ledges to prevent the rats from falling off. The open arms were placed opposite of each other, and the maze was elevated 55 cm above the floor on acrylic legs. At the beginning of each test, the rats were placed in the central area facing an open arm and were allowed to explore the apparatus for 5 min. The rats were recorded with a camera that was mounted above the maze. The test was conducted in a quiet, dimly lit room (100 lux). The open arm and closed arm duration, number of open and closed arm entries, and total distance traveled were determined automatically (center-point detection) using EthoVision XT 11.5 software (Noldus Information Technology, Leesburg, VA). The percentage of open arm entries (open arm entries/total arm entries) and percentage time on the open arms (open arm time/total time on the arms) were calculated. The apparatus was cleaned with a Nolvasan solution between tests.

2.4.3. Large open field test

The large open field test is another test that is widely used to assess anxiety-like behavior in rodents (Liebsch et al., 1998; Prut and Belzung, 2003). This test was conducted as described in our previous work (Qi et al., 2016; Tan et al., 2019). The open-field arena is 120 × 120 × 60 cm (L x W x H) and is placed in a dimly lit (80 lux) room. The arena is made of black high-density polyethylene panels that are fastened together and are placed on a black plastic bottom plate (Faulkner Plastics, Miami, FL). The rats were recorded with a camera mounted above the arena, and their behavior was analyzed automatically (center-point detection) with EthoVision XT 11.5 software (Noldus Information Technology, Leesburg, VA). The open field was divided into two zones: a border zone (20 cm wide) and a center zone (60 × 60 cm; L x W). The width of the border zone is based on previous studies that used the large open field to study anxiety-like behavior in rodents (Knight et al., 2021; Lamprea et al., 2008; Qi et al., 2016). The 20-cm wide border zone allows the rats to stay close to the border without entering the center zone. The following behaviors were analyzed: duration in the border zone and center zone and distance traveled in the border and center zone. The open field was cleaned with a Nolvasan solution between tests.

2.5. Intravenous catheter implantation and spontaneous operant responding for nicotine

The rats were anesthetized with an isoflurane-oxygen vapor mixture (1–3%) and prepared with a catheter in the right jugular vein. The surgery was conducted as described in our previous studies (Chellian et al., 2020a; Chellian et al., 2020c; Yamada and Bruijnzeel, 2011). The catheters consisted of polyurethane tubing (length 15 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 to a depth of 2.5 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 small, 1-cm incision between the scapulae. After the surgery, the rats were given at least four days to recover. The rats received daily intravenous infusions of the antibiotic Gentamycin (4 mg/kg, Sigma-Aldrich, St. Louis, MO) for seven days. A sterile heparin/glycerol solution (0.1 ml, 500 U/ml, SAI Infusion Technologies, Lake Villa, IL) was infused into the catheter after administering the antibiotic or after nicotine self-administration. The animals received carprofen (5 mg/kg, SC) daily for 48 hours after the surgery. The nicotine self-administration sessions were conducted in Plexiglas operant chambers (56 × 38 × 36 cm; W x H x L) that were placed in melamine sound-attenuating cubicles (Med Associates St. Albans, VT). One of the walls of the operant chamber contained two levers (active lever and inactive lever). The levers were 3 cm wide and placed 3 cm above the metal grid floor of the chamber. All data collection and test sessions were controlled by a Windows computer with Med Associates software. The delivery of the nicotine solution was controlled by a syringe pump (Model A, Razel Scientific Instruments, Stamford, CT). Nicotine was delivered via plastic tubing that was covered with a protective tether (VABR1T/220, Instech Laboratories). The tether was connected to the stainless steel swivel (375/22, Instech Laboratories, Plymouth Meeting, PA) and with a magnet to the vascular access button. On self-administration days, the rats were transported from the vivarium (5-min walk) to the self-administration room. The rats were allowed to self-administer nicotine for three hours at the 0.03 mg/kg/infusion dose for ten days. The rats self-administered nicotine under an fixed-ratio 1, time-out 10 second (FR-1TO10s) schedule for six days and under an fixed-ratio 2, time-out 10 second FR-2TO10s schedule for four days. On the first self-administration day, the maximum number of nicotine infusions was set to 20 to prevent the rats from overdosing on nicotine. Active lever (right lever, RL) responding 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. Inactive lever (left lever, LL) responses were recorded but did not have scheduled consequences. Both levers were retracted during the 10 s time-out period. The self-administration sessions were conducted six days per week. Catheter patency was assessed after the last self-administration session by infusing 0.2 ml of the ultra-short action barbiturate Brevital (1 % methohexital sodium). Rats with patent catheters displayed loss of muscle tone within a few seconds after infusion. If the rats did not respond to Brevital, their self-administration data were excluded from the analysis. Two animals (one female-saline rat and one female-nicotine rat) did not respond to the Brevital, and their nicotine self-administration data were excluded.

2.6. Statistics

Body weights and nicotine sensitization data were analyzed using a three-way ANOVA, with treatment condition (saline and nicotine injection) and sex as between-subject factors and time (days) as a within-subjects factor. A secondary analysis was conducted with the nicotine sensitization data to provide insight into the effects of nicotine and sex on each specific treatment day (Day 1, 2, 6). Nicotine sensitization data for each specific treatment day were analyzed using two-way ANOVAs, with treatment condition and sex as between-subjects factors. Elevated plus maze, small open field, and large open field data were analyzed using two-way ANOVAs, with treatment condition and sex as between-subjects factors. The nicotine self-administration sessions (Day 1–10) were analyzed using a three-way ANOVA, with treatment condition (saline and nicotine injection) and sex as between-subject factors, and session (days) as a within-subjects factor. For all statistical analyses, significant interactions 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 26 and GraphPad Prism version 8.4.

3. RESULTS

3.1. Body weights

Before the onset of the nicotine and saline injections, the females had a higher body weight than the males (P22, Sex F1,44 = 5.048, P<0.05; P23, Sex F1,44 = 4.45, P<0.05, Table S1). Before the start of the injections, there was no difference in body weight between the treatment (nicotine vs. saline) groups (P22, Treatment F1,44 = 0, NS; P23, Treatment F1,44 = 0.014, NS). During the nicotine-treatment period, the males had a higher body weight than the females (Sex F1, 44 = 12.553, P<0.01, Figure 2A). The males gained more weight than the females over time (Time F18, 792 = 3737.713, P<0.0001; Time × Sex F18, 792 = 127.758, P<0.0001), and nicotine treatment reduced body weight gain over time to a similar degree in the males and females (Treatment F1, 44 = 0.444, NS; Time × Treatment F18, 792 = 2.587, P<0.0001; Sex × Treatment F1,44 = 0.073, NS; Time × Sex × Treatment F18, 792 = 0.625, NS).

Figure 2. Nicotine reduces body weight gain in adolescent rats.

Figure 2.

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The rats were treated with nicotine from P24–42, and during (A) and after (B) the nicotine treatment period, the body weights of the rats were recorded. During the nicotine treatment period, the males gained more weight than the females, and nicotine reduced body weight gain. After the nicotine treatment period, the males gained more weight than the females, but nicotine treatment did not affect weight gain. Male-saline n=12, male-nicotine n=12, female-saline n=12, female-nicotine n=12. Data are expressed as means ± SEM.

After the cessation of nicotine treatment (P43–72), the males had a higher body weight than the females and the males gained more weight over time than the females (Sex F1, 44 = 201.799, P<0.0001; Time F4, 176 = 1345.660, P<0.0001; Time × Sex F4, 176 = 218.675, P<0.0001; Figure 2B). Nicotine treatment did not have a long-term effect on weight gain in the males or the females (P43–72; Treatment F1, 44 = 0.306, NS; Sex × Treatment F1,44 = 0.206, NS; Time × Treatment F4, 176 = 0.405, NS; Time × Sex × Treatment F4,176 = 0.38, NS).

During the 10-day nicotine self-administration period, the males had a higher body weight than the females (Sex F1, 42 = 115.635, P<0.0001, Figure S1). The male and female rats did not gain weight during the 10-day nicotine self-administration period and a history of nicotine exposure did not affect the body weights of the rats (Time F9, 378= 0.148, NS; Treatment F1, 42 = 0.001, NS; Time × Sex F9,378 = 1.143, NS; Time × Treatment F9,378= 0.736, NS; Sex × Treatment F1,42= 0.026, NS; Time × Sex × Treatment F9,378= 0.446, NS).

3.2. Nicotine sensitization during adolescence (P24–29)

3.2.1. Total distance traveled

The total distance traveled (Figure 3A) was determined after the first, second, and sixth nicotine injection. Nicotine treatment increased the total distance traveled (Treatment F1, 44 = 79.750, P<0.0001). The effect of nicotine on the total distance traveled increased over time (Time F2, 88 = 42.201, P<0.0001; Time × Treatment F2, 88 = 31.001, P<0.0001). The total distance traveled continued to increase over time in the females but not in the males (Time × Sex F2, 88 = 16.017, P<0.0001). The total distance traveled did not differ between the males and the females, and nicotine treatment had the same effect on the total distance traveled in the males and the females (Sex F1, 44 = 0.303, NS; Sex × Treatment F1, 44 = 0.466, NS; Time × Sex x Treatment F2, 88 = 0.767, NS). The posthoc showed that after the second and sixth nicotine treatment, the distance traveled in the males and females was greater than after the first treatment. In the females, the total distance traveled was also greater after the sixth nicotine treatment than after the second nicotine treatment. For separate analysis for each specific injection day see Figures S2A–C.

Figure 3. Nicotine sensitizes behavioral responses in male and female adolescent rats.

Figure 3.

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The adolescent male and female rats were treated with nicotine from P24–42, and after the first, second, and sixth injection, they were tested in the small open field. The figures show the effect of nicotine on the total distance traveled (A), horizontal beam breaks (B), vertical beam breaks (C), and stereotypies (D). Asterisks indicate a significant difference from rats of the same sex that received the same treatment on Day 1, and plus signs indicate a significant difference from rats of the same sex that received the same treatment on Day 2. Male-saline n=12, male-nicotine n=12, female-saline n=12, female-nicotine n=12. *,+ p<0.05, **,++ p<0.01, *** p<0.001. Data are expressed as means ± SEM.

3.2.2. Horizontal beam breaks

The nicotine-treated rats had more horizontal beam breaks (Figure 3B) than the saline-treated controls, and the effect of nicotine increased over time (Treatment F1, 44 = 72.498, P<0.0001; Time × Treatment F2, 88 = 16.761, P<0.0001). Horizontal activity increased over time in the females but in the males activity slightly decreased on the last day (Sex F1, 44 = 1.464, NS; Sex × Treatment F1, 44 = 0.51, NS; Time F2, 88 = 21.483, P<0.0001; Time × Sex F2, 88 = 14.485, P<0.0001; Time × Sex × Treatment F2, 88 = 1.348, NS). The posthoc showed that after the second and sixth nicotine treatment, the males and females had more horizontal beam breaks than after the first treatment day. The females also had more horizontal beam breaks after the sixth nicotine treatment than after the second nicotine treatment. For separate analysis for each specific injection day see Figures S2D–F.

3.2.3. Rearing

Nicotine increased rearing (Figure 3C) and this effect increased over time (Treatment F1, 44 = 4.952, P<0.05; Time × Treatment F2, 88 = 17.994, P<0.0001). Rearing increased over time in the females, however, in the males rearing initially increased and then decreased (Sex F1, 44 = 2.93, P=0.094 [trend]; Sex × Treatment F1, 44 = 0.584, NS; Time F2, 88 = 16.005, P<0.0001; Time × Sex F2, 88 = 19.407, P<0.0001; Time × Sex × Treatment F2, 88 = 1.887, NS). The posthoc showed that after the second and sixth nicotine treatment, the males and females displayed more rearing than after the first treatment day. The females also displayed more rearing after the sixth nicotine treatment than after the second nicotine treatment. For separate analysis for each specific injection day see Figures S2G–I.

3.2.4. Stereotypies

Nicotine treatment increased stereotypies (Figure 3D) and nicotine-induced stereotypies increased over time (Time F2, 88 = 15.509, P<0.0001; Treatment F1, 44 = 88.938, P<0.0001; Time × Treatment F2, 88 = 16.048, P<0.0001). In females, the number of stereotypies increased over time, but in males, stereotypies first increased and then somewhat decreased (Time × Sex F2, 88 = 8.474, P<0.0001). The males and the females displayed the same number of stereotypies, and nicotine-treatment had the same effect on stereotypies in the males and females (Sex F1, 44 = 2.211, NS; Sex × Treatment F1, 44 = 1.029, NS; Time × Sex x Treatment F2, 88 = 1.11, NS). The posthoc showed that after the second nicotine treatment, the males displayed more stereotypies than after the first nicotine treatment. The females displayed more stereotypies after the second and sixth nicotine treatment than after the first nicotine treatment. For separate analysis for each specific injection day see Figures S2J–L.

3.3. Anxiety-like behavior and locomotor activity in adolescent rats after the cessation of nicotine treatment

3.3.1. Elevated plus-maze test

Anxiety-like behavior was measured in the elevated plus-maze test 24 h after the last nicotine injection. Cessation of nicotine treatment decreased the percentage of open arm entries in the males and increased the percentage of open arm entries in the females (Sex F1, 44 = 1.69, NS; Treatment F1, 44 = 0.467, NS; Sex × Treatment F1, 44 = 4.428, P<0.05; Figure 4A). Cessation of nicotine administration did not affect other behavioral parameters such as open arm duration (Treatment F1, 44 = 0.238, NS; Sex × Treatment F1, 44 = 0.808, NS), percentage of open arm duration (Treatment F1, 44 = 0.225, NS; Sex × Treatment F1, 44 = 0.393, NS), open arm entries (Treatment F1, 44 = 0.111, NS; Sex × Treatment F1, 44 = 0.821, NS), closed arm duration (Treatment F1, 44 = 0.004, NS; Sex × Treatment F1, 44 = 0.001, NS), closed arm entries (Treatment F1, 44 = 0.506, NS; Sex × Treatment F1, 44 = 3.125, NS), and total distance traveled (Treatment F1, 44 = 0.006, NS; Sex × Treatment F1, 44 = 2.18, NS; Figure S3A–E).

Figure 4. Effect of cessation of nicotine administration on anxiety-like behavior and locomotor activity in adolescent rats.

Figure 4.

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The rats were treated with nicotine from P24–42 and tested in the elevated plus-maze and the small open field test at P43, 24 after the last nicotine injection. The figure shows the percentage of open arm entries (A) and the percentage time in the open arms (B) in the elevated plus-maze test. The figure also shows the total distance traveled (C), horizontal bream breaks (D), vertical beam breaks (E), and stereotypies (F) in the small open field test. Male-saline n=12, male-nicotine n=12, female-saline n=12, female-nicotine n=12. Data are expressed as means ± SEM.

There was a main effect of sex for most of the behavioral parameters. The females spent more time on the open arms (Sex F1, 44 = 21.277, P<0.0001) and spent a higher percentage of time on the open arms (Sex F1, 44 = 21.405, P<0.0001, Figure 4B). Compared to the males, the females spent less time in the closed arms (Sex F1, 44 = 12.167, P<0.01), made more open arm entries (Sex F1, 44 = 17.206, P<0.0001), and traveled a greater distance (Sex F1, 44 = 21.67, P<0.0001). There were no sex differences in the number of closed arm entries (Sex F1, 44 = 2.983, NS).

3.3.2. Small open field test

Cessation of nicotine administration did not affect total distance traveled (Treatment F1, 44 = 1.068, NS; Sex × Treatment F1, 44 = 2.098, NS), horizontal beam breaks (Treatment F1, 44 = 0.008, NS; Sex × Treatment F1, 44 = 0.348, NS), rearing (Treatment F1, 44 = 3.378, P=0.073 [trend]; Sex × Treatment F1, 44 = 0.891, NS), and stereotypies (Treatment F1, 44 = 0.102, NS; Sex × Treatment F1, 44 = 0.005, NS) in the small open field test (24h after last nicotine injection, Figure 4C–F). However, the females traveled a greater distance (Sex F1, 44 = 6.439, P<0.05) and displayed more rearing (Sex F1, 44 = 14.067, P<0.01). The males displayed more stereotypies (Sex F1, 44 = 5.179, P<0.05). There was no sex difference in the number of horizontal beam breaks (Sex F1, 44 = 0.11, NS).

3.4. Locomotor activity and anxiety-like behavior in a drug-free state in adulthood

3.4.1. Small open field

Locomotor activity was measured in a drug-free state in the small open field in adulthood (P79). Exposure to nicotine during adolescence did not affect the total distance traveled (Treatment F1, 44 = 0.017, NS; Sex × Treatment F1, 44 = 0.838, NS), horizontal beam breaks (Treatment F1, 44 = 0.116, NS; Sex × Treatment F1, 44 = 0.073, NS), rearing (Treatment F1, 44 = 1.135, NS; Sex × Treatment F1, 44 = 0.431, NS), or stereotypies (Treatment F1, 44 = 0.12, NS; Sex × Treatment F1, 44 = 0.044, NS) in the small open field test in adulthood (Figure 5A–D). Sex differences were found in the behavioral parameters in rats tested in the small open field. The female rats displayed more rearing (Sex F1, 44 = 16.208, P<0.0001) and traveled a greater distance (Sex F1, 44 = 17.376, P<0.0001) compared to the males. The number of stereotypies was higher in the males than in the females (Sex F1, 44 = 4.175, P<0.05). There was no sex difference in horizontal beam breaks (Sex F1, 44 = 0.354, NS).

Figure 5. Long term effects of adolescent nicotine-treatment on locomotor activity.

Figure 5.

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The rats were treated with nicotine from P24–42 and tested in the small open field in adulthood (P79). In the small open field, the total distance traveled (A), horizontal beam breaks (B), vertical beam breaks (C), and stereotypies (D) were determined. N=12/group. Data are expressed as means ± SEM.

3.4.2. Large open field

Anxiety-like behavior was measured in a drug-free state in the large open field in adulthood (P80). Adolescent nicotine exposure had no effect on total distance traveled (Treatment F1, 44 = 0.905, NS; Sex × Treatment F1, 44 = 0.215, NS), distance traveled in the border zone (Treatment F1, 44 = 1.864, NS; Sex × Treatment F1, 44 = 0.099, NS), distance traveled in the center zone (Treatment F1, 44 = 1.323, NS; Sex × Treatment F1, 44 = 2.583, NS), time in the center zone (Treatment F1, 44 = 0.238, NS; Sex × Treatment F1, 44 = 1.748, NS), entries into the center zone (Treatment F1, 44 = 2.025, NS; Sex × Treatment F1, 44 = 2.563, NS), and time in the border zone (Treatment F1, 44 = 0.294, NS; Sex × Treatment F1, 44 = 0.139, NS) in the large open field test in adulthood (Figure 6A–C). However, sex differences were observed for all behavioral parameters in the large open field test. The total distance traveled was greater in the females than the males (Sex F1, 44 = 51.005, P<0.0001), and the females traveled a greater distance in the border zone (Sex F1, 44 = 45.468, P<0.0001) and center zone (Sex F1, 44 = 16.217, P<0.0001). Furthermore, the females spent more time in the center zone (Sex F1, 44 = 12.703, P=0.001) and made more entries into the center zone (Sex F1, 44 = 23.930, P<0.0001). The males spent more time in the border zone than the females (Sex F1, 44 = 29.338, P<0.0001).

Figure 6. Long term effects of adolescent nicotine-treatment on anxiety-like behavior and locomotor activity.

Figure 6.

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The rats were treated with nicotine from P24–42, and the rats were tested in the large open field (P80) and elevated plus-maze (P81) in adulthood. In the larger open field, the total distance traveled, the distance traveled in the border zone, and the center zone was determined (A). The time in the border and center zone (B) and the entries into the center zone (C) were determined. In the elevated plus-maze test, the percentage of open arm entries (D) and the percentage of open arm time (E) were determined. N=12/group. Data are expressed as means ± SEM.

3.4.3. Elevated plus-maze

Anxiety-like behavior was measured in a drug-free state in the elevated plus maze test in adulthood (P81). A history of nicotine exposure did not affect entries into the open arms (Treatment F1, 44 = 1.783, NS; Sex × Treatment F1, 44 = 2.02, NS; Figure S4A) and the percentage of open arm entries (Treatment F1, 44 = 0.886, NS; Sex × Treatment F1, 44 = 1.745, NS; Figure 6D). Exposure to nicotine during adolescence did not affect the amount of time on the open arms (Treatment F1, 44 = 0.921, NS; Sex × Treatment F1, 44 = 0.876, NS; Figure S4B) and the percentage of time on the open arms (Treatment F1, 44 = 1.417, NS; Sex × Treatment F1, 44 = 1.252, NS; Figure 6E) in the elevated plus-maze test in adulthood. Furthermore, nicotine exposure did not affect closed arm entries (Treatment F1, 44 = 0.185, NS; Sex × Treatment F1, 44 = 0.101, NS; Figure S4C), the time in the closed arms (Treatment F1, 44 = 0.83, NS; Sex × Treatment F1, 44 = 0.379, NS; Figure S4D), and total distance traveled (Treatment F1, 44 = 0, NS; Sex × Treatment F1, 44 = 0.324, NS; Figure S4E). There were sex differences for all the behavioral parameters measured. The females had a higher number of open arm entries (Sex F1, 44 = 7.364, P<0.01; Figure S4A) and a higher percentage of open arm entries (Sex F1, 44 = 6.567, P<0.05; Figure 6D). The females also spent more time on the open arms (Sex F1, 44 = 5.479, P<0.05; Figure S4B) and spent a greater percentage of time on the open arms (Sex F1, 44 = 5.931, P<0.05; Figure 6E). The females made more closed arm entries than the males (Sex F1, 44 = 8.42, P<0.01; Figure S4C), but the males spent more time in the closed arms (Sex F1, 44 = 10.614, P<0.01; Figure S4D). The females traveled a greater distance than the males (Sex F1, 44 = 15.974, P<0.0001; Figure S4E).

3.5. Spontaneous acquisition of nicotine self-administration in adulthood

The females had a higher level of nicotine intake than the males during the first hour of nicotine intake from days 1–10 (Sex F1, 42 = 12.593, P<0.01; Figure 7A, S5A). First-hour nicotine intake was diminished by adolescent nicotine treatment, and this was mainly due to the large decrease in nicotine intake in the females (Treatment F1, 42 = 4.079, P<0.05; Sex × Treatment F1, 42 = 4.730, P<0.05; Time × Treatment F9, 378 = 3.422, P<0.0001; Time x Sex x Treatment F9, 378 = 7.447, NS; Figure 7A, S5A). The posthoc showed that nicotine intake in the nicotine-treated females was lower on Days 5, 6, and 8 than in the saline-treated females. Nicotine intake in the males increased under the FR1 schedule and then stabilized under the FR2 schedule. Nicotine intake in females increased under the FR1 schedule and decreased after switching to the FR2 schedule (Time F9, 378 = 12.619, P<0.0001; Time × Sex F9, 378 = 5.936, P<0.0001).

Figure 7. Adolescent nicotine-treatment reduces spontaneous acquisition of nicotine self-administration in adulthood.

Figure 7.

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The rats were treated with nicotine from P24–42, and the acquisition of nicotine self-administration was investigated in adulthood (>P90). The figures depict nicotine intake during the first hour (A) and during the complete 3-h session (B) on days 1–10. The asterisk indicates a significant difference from rats of the same sex that were pretreated saline. Male-saline n=12, male-nicotine n=12, female-saline n=11, female-nicotine n=11. * p<0.05. Data are expressed as means ± SEM.

During the complete 3-h sessions, the female rats self-administered more nicotine than the males, and the rats that had been pre-exposed to nicotine had a lower level of nicotine intake than the saline-pretreated controls (Sex F1, 42 = 13.152, P<0.01; Treatment F1, 42 = 6.724, P<0.05; Figure 7B, S5B). A close look at the figures (Figures 7B and S5) reveals that the main effect of sex was mainly due to the higher nicotine intake of the saline-treated females compared to the saline-treated males under the FR1 schedule. Adolescent nicotine treatment had a greater effect on nicotine intake in females than males (Sex × Treatment F1, 42 = 4.275, P<0.05).

Nicotine intake in the males increased and then stabilized, and nicotine intake in the females increased and then decreased (Time F9, 378 = 9.489, P<0.0001; Time x Sex F9, 378 = 6.981, P<0.0001). Nicotine intake increased more in the saline-pretreated rats than the nicotine-pretreated rats (Time x Treatment F9, 378 = 2.233, P<0.05; Time x Sex x Treatment F9, 378 = 0.737, NS). Nicotine intake in the males increased under the FR1 schedule and stabilized under the FR2 schedule. Nicotine intake in the

females increased under the FR1 schedule and decreased when they were switched to the FR2 schedule (Time F9, 378 = 9.489, P<0.0001). In particular the saline-treated females had a high level of nicotine intake under the FR1 schedule (Time x Sex F9, 378 = 6.981, P<0.0001; Time x Treatment F9, 378 = 2.233, P<0.05; Time x Sex x Treatment F9, 378 = 0.737, NS).

The females had a higher level of responding on the active lever (RL) than the males, and active lever responding was lower in the nicotine-pretreated rats than the saline-pretreated rats (Sex F1, 42 = 10.332, P<0.01; Treatment F1, 42 = 6.186, P<0.05; Figure S6A). Responding on the active lever increased over time, and this effect was more pronounced in the females than in the males and more pronounced in the saline-pretreated rats than in the nicotine-pretreated rats (Time F9, 378 = 29.785, P<0.0001; Time x Sex F9, 378 = 2.566, P<0.01; Time x Treatment F9, 378 = 2.607, P<0.01; Time x Sex x Treatment F9, 378 = 0.292, NS). Pretreatment with nicotine had the same effect on active lever responding in the males and the females (Sex × Treatment F1, 42 = 3.022, P=0.089 [trend]).

There was a non-significant trend toward a lower level of responding on the inactive lever (LL) in the nicotine-pretreated rats compared to the saline-pretreated rats (Treatment F1, 42 = 3.802, P=0.058 [trend]; Figure S6B). The effect of the nicotine pretreatment on inactive lever responding was not significantly affected by the sex of the rats (Sex × Treatment F1, 42 = 3.309, P=0.076 [trend]). The females had a higher level of responding on the inactive lever than the males (Sex F1, 42 = 4.273, P<0.05). Responding on the inactive lever increased over time, and this effect was most pronounced in the saline pretreated rats and was not affected by the sex of the rats (Time F9, 378 = 13.171, P<0.0001; Time x Treatment F9, 378 = 2.376, P<0.05; Time x Sex F9, 378 = 1.33, NS; Time x Sex x Treatment F9, 378 = 0.69, NS)

4. DISCUSSION

The present study investigated the effects of adolescent nicotine treatment on locomotor activity, anxiety-like behavior, and the acquisition of nicotine intake in males and females. Based on previous drug sensitization studies, it was hypothesized that repeated nicotine treatment during adolescence would lead to an increased locomotor response to nicotine during adolescence and increased levels of nicotine intake during the acquisition phase in adulthood. Adolescent nicotine treatment caused robust locomotor sensitization in the adolescent male and female rats. Cessation of nicotine treatment decreased the percentage of open arm entries in the adolescent male rats and increased the percentage of open arm entries in the adolescent female rats (Sex x Nicotine treatment interaction). Pretreatment with nicotine during adolescence did not significantly affect other anxiety and activity parameters in the elevated plus-maze test, small open field, and large open field in adulthood in a drug-free state. After the behavioral tests, the acquisition of nicotine intake was investigated. The females self-administered more nicotine than the males, and treatment with nicotine during adolescence significantly decreased nicotine intake in adulthood in females. These findings indicate that adolescent nicotine treatment causes robust behavioral sensitization, but this does not lead to a higher level of nicotine intake. Exposure to nicotine during adolescence may lead to a lower level of nicotine intake during the acquisition phase in females in adulthood.

In the present study, we determined if repeated nicotine administration during adolescence affects locomotor activity. On the first injection day, the nicotine-treated rats had a significantly higher level of activity than the saline-treated control rats. This is in line with previous studies showing that nicotine (0.1–1 mg/kg of nicotine base) increases locomotor activity in adolescent rats (Elliott et al., 2004). In contrast, in adult animals, nicotine doses above 0.15 mg/kg decrease locomotor activity (Domino, 2001; Stolerman et al., 1973). In both the males and females, locomotor activity (total distance traveled and horizontal beam breaks), rearing, and stereotypies increased over time. The effects of nicotine on locomotor activity, rearing, and stereotypies were not affected by the sex of the rats. This suggests that there are no sex differences in behavioral sensitization in adolescent rats. We are not aware of any other studies that investigated sex differences in nicotine sensitization in adolescent rats; however, many studies have investigated sex differences in nicotine sensitization in adult rats. Several studies did not find sex differences in nicotine sensitization in adult rats (Booze et al., 1999; Ericson et al., 2010; Kanýt et al., 1999). However, greater nicotine-induced behavioral sensitization in females has also been reported (Harrod et al., 2004). Overall, our study suggests that there are no sex differences in behavioral sensitization in early-adolescent rats (P24–29).

The rats were treated with nicotine for 19 days, and one day later, they were tested in the elevated plus-maze test and the small open field. These tests were conducted to determine whether the cessation of nicotine administration affects anxiety-like behavior (elevated plus-maze test) and locomotor activity (small open field) in adolescent rats. Cessation of nicotine administration decreased the percentage of open arm entries in the males and increased the percentage of open arm entries in the females (significant Sex x Nicotine treatment interaction). This indicates that cessation of nicotine treatment increases anxiety-like behavior in adolescent males, and decreases anxiety-like behavior in adolescent females. However, cessation of nicotine administration did not affect other anxiety or activity parameters in the elevated plus-maze test. On a similar note, cessation of nicotine administration did not affect locomotor activity (total distance traveled and horizontal beam breaks), rearing, or stereotypies in the small open field test. To our knowledge, there are no other studies that have investigated anxiety-like behavior and locomotor activity in adolescent female rats after the cessation of nicotine administration. However, one study investigated anxiety-like behavior in adolescent male rats that were treated with nicotine. The adolescent nicotine-treated male rats did not display increased anxiety-like behavior during precipitated withdrawal or after the cessation of nicotine administration (Wilmouth and Spear, 2006). Studies with adult rats suggest that females display more anxiety-like behavior than males during precipitated nicotine withdrawal (Flores et al., 2020). In conclusion, these findings suggest that cessation of nicotine administration in adolescent rats may have a small sex-dependent effect on anxiety-like behavior and does not affect locomotor activity.

Sex differences in anxiety-like behavior and locomotor activity were observed in adolescent rats (P43), and these sex differences were independent of treatment effects. In the elevated plus-maze test, the adolescent females made significantly more entries into the open arms, spent more time in the open arms, and spent a greater percentage of time on the open arms. This indicates that adolescent females display less anxiety-like behavior compared to adolescent males in the elevated plus-maze test. In the small open field test, the females significantly traveled a greater distance and displayed more rearing compared to the males, thus indicating that the females are more active in the small open field test. These findings are in line with a previous study by our group, in which we found that adult female Wistar rats display less anxiety-like behavior in the elevated plus-maze test and are more active in an open field test (Knight et al., 2021). It is interesting to note that there are significant sex differences in anxiety-like behavior in the adolescent rats in the elevated plus-maze test. A previous study with adolescent Wistar rats also reported that adolescent females spent a greater percentage of time in the open arms of the elevated plus maze (Imhof et al., 1993). This suggests that adolescent female Wistar rats, like adult female Wistar rats, exhibit less anxiety-like behavior than male Wistar rats. In contract, other studies reported that there are no sex differences in anxiety-like behavior in the elevated plus-maze test in adolescent Sprague-Dawley and Lister Hooded rats (Doremus-Fitzwater et al., 2009; File and Tucker, 1984; Lynn and Brown, 2010). We used Wistar rats for our study, and the studies that did not find sex differences were done with Sprague-Dawley and Lister Hooded rats. Therefore, it might be possible that the expression of sex differences in anxiety-like behavior in adolescent rats is strain-dependent.

In the present study, we also investigated the effects of adolescent nicotine treatment on anxiety-like behavior and locomotor activity in adulthood. The elevated plus-maze, small open field, and large open field test were conducted 37–39 days after the cessation of nicotine treatment. All these tests provide insight into locomotor activity, and the elevated plus-maze test and large open field test also provide insight into anxiety-like behavior. It is interesting to note that adolescent nicotine treatment had no long-term effects on anxiety-like behavior or locomotor activity. This contrasts with other reports that adolescent nicotine treatment leads to an increase in anxiety-like behavior in adulthood. For example, male Sprague Dawley rats treated with nicotine patches during adolescence spent less time in the center of a large open field and less time on the open arms of the elevated plus-maze in adulthood (Slawecki et al., 2003; Slawecki et al., 2005). The adolescent rats treated with nicotine were also less likely to approach food in the center of an open field (Slawecki et al., 2003). It has been reported that adult male and female Long-Evans rats that received nicotine via minipumps during adolescence spent less time in the center of an open field compared to controls (Smith et al., 2006). In another study, adolescent male Sprague Dawley rats received nicotine injections, and anxiety-like behavior and locomotor activity were investigated in adulthood (Iniguez et al., 2009). Adolescent nicotine treatment did not affect locomotor activity in adulthood in the open field test. However, rats treated with nicotine during adolescence spent less time in the open arms and had a lower percentage of open arms entries in the elevated plus-maze test (Iniguez et al., 2009). A possible explanation for the discrepancy between our findings and those of others is that we used a different strain of rats. We found no long-term effect of adolescent nicotine treatment on anxiety-like behavior in Wistar rats, while the studies that reported long-term effects of adolescent nicotine treatment on anxiety-like behavior used Sprague Dawley or Long Evans rats. Another possibility is that the route of nicotine administration, rate of delivery, doses, or treatment regimen affects the long-term effects of nicotine treatment. In the present study, nicotine was injected twice a day. In contrast, in some studies that found long-term effects nicotine treatment, nicotine was administered via patches or minipumps (nicotine exposure for 24h/day)(Slawecki et al., 2003; Slawecki et al., 2005; Smith et al., 2006).

We also determined sex differences in adult rats in the elevated plus-maze test, small open field test, and the large open field test. We found robust sex differences in all these tests. In the small open field test, the females significantly traveled a greater distance and displayed more rearing. In the large open field test, the females traveled a greater distance and made more entries into the center zone, and spent more time in the center zone. Furthermore, in the elevated plus-maze test, the females spent a greater percentage of time in the open arms, had a higher percentage of open arm entries, and traveled a greater distance. These findings indicate that adult females are more active in open field tests and the elevated plus test. The females display less anxiety-like behavior in the elevated plus-maze test and the large open field test. This sex difference in anxiety-like behavior might be explained by sex differences in androgen levels during development. It has been shown that male rats display less anxiety-like behavior when they are castrated before puberty (Domonkos et al., 2017). Treatment with testosterone does not reverse the long-term effects of hypogonadism in adult males. Therefore, this suggests that the relatively high level of anxiety-like behavior in male rats might be due to exposure to testosterone during development (Domonkos et al., 2017; Domonkos et al., 2018).

It was also investigated if adolescent nicotine exposure and the sex of the rats affected the acquisition of nicotine intake. The females had a higher level of nicotine intake than the males, and prior exposure to nicotine led to lower nicotine intake in females. In one of our previous studies, we did not observe a sex difference in nicotine intake in Wistar rats (Chellian et al., 2020a). A difference between the current study and our previous study is that in the present study, we investigated the acquisition of nicotine intake without food training, and in our previous study, the rats had received food training. Because food training leads to a high level of nicotine intake during the first week of nicotine self-administration, this might mask the minor sex differences that can be observed during the spontaneous acquisition of nicotine intake. Several other studies reported that there is no sex difference in nicotine intake when nicotine intake has been established, and the rats are tested under FR schedules with low response requirements (Chaudhri et al., 2005; Donny et al., 2000; Feltenstein et al., 2012; Grebenstein et al., 2013). However, female rats acquire nicotine self-administer faster and take more nicotine during the acquisition phase (Donny et al., 2000; Park et al., 2007; Wang et al., 2018), which is in line with our finding that females self-administer more nicotine during the acquisition phase.

Prior research indicates that noncontingent drug administration may enhance the acquisition of drug self-administration (Lacy et al., 2018). Adolescent nicotine exposure increases cocaine and fentanyl self-administration in adulthood (Cardenas et al., 2020; Reed and Izenwasser, 2017). Therefore, it was hypothesized that adolescent nicotine treatment would lead to an increase in nicotine self-administration. In contrast to what was predicted, adolescent nicotine treatment led to a significantly lower level of nicotine intake during the acquisition phase, and this effect was most pronounced in the females. There might be several possible explanations for this. One possible explanation is that adolescent nicotine treatment decreases the rewarding effects of nicotine. This is supported by a study that showed that adolescent nicotine treatment diminishes nicotine’s rewarding effects in adulthood in a conditioned place preference procedure (Torres et al., 2008). Furthermore, another study showed that exposure to nicotine during adolescence diminishes nicotine’s locomotor stimulant effects in adult male rats (Brielmaier et al., 2007). Another possibility is that adolescent nicotine treatment affects the motivational state in adulthood. Adolescent nicotine treatment not only decreased nicotine intake but also responding on the inactive lever. Adolescent nicotine treatment increases immobility in the forced swim test and decreases sucrose preference in rats (Iniguez et al., 2009). Based on these findings, it could be suggested that adolescent nicotine treatment causes a motivation deficit that leads to a decrease in nicotine intake.

Overall, these findings indicate that repeated nicotine treatment leads to behavioral sensitization in adolescent male and female rats. Adolescent nicotine treatment did not have a long-term effect on anxiety-like behavior and activity parameters. The adult females self-administered more nicotine than the adult males. Furthermore, adolescent nicotine treatment led to lower nicotine intake in females in adulthood throughout the acquisition phase.

Supplementary Material

Supplement

ACKNOWLEDGMENTS:

This work was supported by a NIDA/NIH and FDA Center for Tobacco Products (CTP) grant (DA042530) and NIDA grant (DA046411) to AB. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Food and Drug Administration.

Footnotes

Conflict of Interest: The authors declare that they have no conflict of interest.

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