Animals

Experiments were conducted on adult male Sprague‒Dawley rats (n = 50; 186–237 g) acquired from a licenced animal breeder (Charles River, Sulzfeld, Germany). Before surgery, a total of 4–5 animals were housed per cage (59 cm × 38 cm × 20 cm), or after surgery, they were housed individually in standard cages (42 cm × 26.5 cm × 18 cm) in a colony room (temperature: 22 ± 2 °C; humidity: 55 ± 10%; 12-h light-dark cycle; lights on at 6:00 h). The animal’s condition was monitored once or twice (after the surgery) daily by the experimenter to ensure their welfare and to detect any signs of distress or illness. The animals had free access to rodent chow throughout the entire experiment. Water was also available ad libitum, except for during the initial training sessions. All experiments were conducted during the early light phase (between 7:00 AM and 2:00 PM) of the light‒dark cycle. The experiments were performed in compliance with the European Community Council Directive 2010/63/EU for Animal Experiments, and were approved by the II Local Ethics Committee at the Maj Institute of Pharmacology, Polish Academy of Sciences (permission numbers: 1240/2015, 1273/2015, 186/2016, 161/2017, 353/2017, 354/2017).

Drugs

The following drugs were used: a thymidine analogue that labels proliferating cells, BrdU (Sigma-Aldrich, USA), and (-)-nicotine hydrogen tartrate salt (Sigma-Aldrich, St. Louis, MO, USA; cat #N5260–25G). The preparation and doses of nicotine (expressed as the free base) used were previously reported (Zaniewska et al. 2010; Higgins et al. 2012; Le Foll et al. 2012; Cousins et al. 2014; Cohen et al. 2015; Briggs et al. 2016; Fletcher et al. 2019). BrdU was dissolved in a filtered (Whatman filters, PP w/GMF 0.2 μm; GE Healthcare, USA) solution of 0.9% saline and 0.007 N NaOH (pH 7.5). The BrdU solution was slowly stirred on a heated magnetic stirrer until dissolved. It was then loaded into a syringe and injected intraperitoneally (ip) at a volume of 2 ml/kg when the syringe was comfortably warm to the touch. Nicotine was dissolved in sterile 0.9% saline, and the pH was adjusted to 7.0 using 20% NaOH. The nicotine solution was stored in the dark at 4 °C, but prior to the experiment, it was allowed to warm to room temperature. For intraveous (iv) self-administration, the nicotine solution (0.03 mg/kg/inf) was filtered through Whatman filters. To induce drug-seeking behaviour in the animals, nicotine (0.4 mg/kg) was administered via subcutaneous injection (sc) at a volume of 1 ml/kg.

The effects of voluntary wheel running on hippocampal neurogenesis during nicotine cessation

Training and intravenous catheter implantation.

Using a previously published protocol (Filip et al. 2007) with small modifications, animals (n = 38; Fig. 1a) were trained to press a lever for 2 h in standard operant chambers (Med-Associates, St. Albans, GA, USA) under a fixed ratio (FR) 1 schedule of water reinforcement for 5 days. Prior to training, the animals underwent 16–18 h of water deprivation. After training, the rats were given free access to food and water, and after 2 days, they underwent surgery. Animals were anaesthetized with a solution containing ketamine (20 mg/kg, intramuscular injection (im); Biowet, Poland) and dexmedetomidine (0.1 mg/kg, im; Orion Corporation, Finland) and were then implanted with a silastic catheter in the external right jugular vein, as previously described (Filip et al. 2007). Following catheter implantation, all animals were allowed to recover for 7–10 days. Animals received 4–5 ml of a 0.9% NaCl/5% glucose solution (sc) for two days after surgery and the anti-inflammatory/analgesic drug meloxicam (0.04 mg/kg, sc; Metacam, Boehringer Ingelheim, Ingelheim/Rhein, Germany) for three days. The catheters were flushed daily with 0.2 ml of sterile 0.9% saline solution containing cefazolin (100 mg/ml; Polpharma, Poland), a broad-spectrum antibiotic, and an anticoagulant heparin (100 IU/ml; Polfa, Poland) to maintain catheter function.

Fig. 1

Effects of wheel-running exercise on hippocampal neurogenesis during long-term nicotine deprivation. Rats were allowed to self-administer nicotine (0.03 mg/kg/inf, NIC SA) or received saline infusions (YSAL) in 2-h sessions for 21 days. Immediately after the last self-administration session, the rats were injected with 5-bromo-2’-deoxyuridine (BrdU, 3 × 50 mg/kg, ip) to label proliferating cells. The rats then entered the deprivation phase for 14 days. On abstinence Day 1, the animals were transferred to cages equipped with running wheels (WR) or locked wheels (LW) for the next 13 abstinence days. The control groups remained in their home cages throughout the entire deprivation period (home). On Day 14 of abstinence, the animals were perfused, and hippocampal neurogenesis was examined. (a) The experimental schedule for NIC SA and WR exposure during nicotine cessation, followed by neurogenesis analyses. (b) The number of lever presses (made under the fixed ratio (FR) schedule, during infusions, and the time-out period) in rats self-administering NIC (n = 20) or receiving YSAL (n = 18) during an increasing schedule of reinforcement (FR(1–5)). The lever presses in session 1 are shown; however, they were not included in the analysis due to the effect of previous water training on lever pressing on this day. (c) NIC infusions in rats (n = 20) throughout 21 self-administration sessions. The infusions in session 1 were not included in the analysis due to the effect of previous water training on infusions on this day. (d) Cumulative NIC intake (mg/kg) during 21 sessions in the three assigned groups (kept in home cages: n = 5; exposed to LW: n = 8; exposed to WR: n = 7, during nicotine cessation). (e) Daily running distance ((km)/d) in the NIC-deprived group (n = 7) and YSAL-treated group (n = 7) with access to WR. (f) Effects of different environmental conditions (home, LW, and WR) on the body weight (g) of rats deprived of NIC (n = 4–8 rats/group). For comparison, the body weights of rats on the last day of self-administration are shown. The data are expressed as the means (± SEM). (b) Post hoc Tukey test: *p < 0.05, ***p < 0.001 vs. NIC–inactive lever presses; #p < 0.05, ###p < 0.001 vs. NIC–active lever presses on session 2; $p < 0.05, $$$p < 0.001 vs. YSAL–active lever presses. (e) p < 0.05: the effect of NIC deprivation vs. YSAL; p < 0.001: the effect of abstinence day: post hoc Tukey: p < 0.01 Day 14 vs. Day 2. (f) p < 0.01: the effect of environment: post hoc Tukey: p < 0.01 WR vs. home; p < 0.001: the effect of abstinence day: p < 0.001 Day 8 vs. 3, Day 11 vs. 3 and 8, Day 14 vs. 3, 8 and 11

Maintenance of self-administration

After recovery from surgery, the animals were trained for one day to press a lever under an FR1 schedule of water reinforcement during a 2-h session. Subsequently, the rats were trained to self-administer nicotine (0.03 mg/kg/inf) using an increasing FR (1–5) schedule of reinforcement (Le Foll et al. 2012). During the maintenance phase, rats were given access to nicotine during 2-h daily sessions (6 days/week; 21 sessions). In the initial sessions, animals had limited access to water for 2 h per day immediately after the session (with free access from Saturday after the session until late Sunday afternoon). Over the subsequent days, water access was gradually increased to ad libitum during the second week of self-administration. The nicotine dose given to each individual rat was adjusted by weighing the animals every second or third day to account for their growth. The ambient light was on throughout each session. Each completion of the FR schedule on the active lever resulted in an infusion of nicotine (0.03 mg/kg per 0.1 ml) and a 5-second presentation of a conditioned stimulus (illumination of a stimulus light + tone). After every infusion, there was a 20-s time-out period during which responses were recorded but not reinforced. Rats were tested simultaneously in two groups, with one group serving as the ‘yoked’ control, which received an injection of saline (YSAL) each time a response-contingent injection of nicotine was self-administered by the paired rat from the second group (NIC). Saline passive injections were accompanied by the presentation of cues (light + tone). Acquisition of the conditioned operant response lasted at least 21 days until the subjects achieved stable self-administration over the last three sessions, with a standard deviation within those days that was < 10% of the average. The results are presented as the number of intravenous infusions per session and the number of lever presses, reported separately for the active and inactive levers. The number of lever presses corresponds to all presses performed under the FR schedules, during infusions, and/or time-out periods.

Nicotine deprivation

After the last self-administration session, the rats were administered three injections of BrdU (50 mg/kg, ip) at 6-h intervals to label proliferating cells during the early deprivation period. The first BrdU injection was administered 15–20 min after the end of the self-administration session, allowing time to remove the animals from the experimental cages and prepare warm BrdU solution. Twenty-four hours after the last self-administration session, the rats were transferred to large polycarbonate cages (41 cm × 51 cm × 21 cm) equipped with either running wheels (diameter: 35 cm; Campden Instruments Ltd., UK; YSAL– WR: n = 7; NIC– WR: n = 7) or locked wheels (YSAL– LW: n = 7; NIC– LW: n = 8) until Day 14 of nicotine abstinence. Animals in the locked wheel condition had access to the wheel but were unable to run, as the wheel was immobilized. The control groups remained in their standard home cages throughout the entire deprivation period (YSAL– HOME: n = 4; NIC– HOME: n = 5).

In the running conditions, a counter attached to the wheel counted the flag number (6 flags per revolution) during the daily 24-h sessions. The total distance run (in km) for the interval experiment was calculated as the revolution count multiplied by 1.1 m (the distance per revolution) divided by 1000.

Immunohistochemistry

At 14 days after cessation, the animals were anaesthetized with a sublethal dose of sodium pentobarbital (90 mg/kg) and pentobarbital (18 mg/kg) (ip; Morbital, PGF Cefarm, Poland) and transcardially perfused with 0.9% NaCl, followed by buffered 4% paraformaldehyde (VWR International, USA). The brains were then isolated and postfixed in a buffered solution of 4% paraformaldehyde for 24 h at 4 °C. After the postfixation period, the brains were cut into 50 μm thick coronal sections at the level of the hippocampus (Bregma = -2.04 to -6.60 mm) according to a stereotaxic atlas of the rat brain (Paxinos and Franklin 2001) using a Leica VT-1000 S vibratome (Leica Microsystems, Heidelberg, Germany). For immunohistochemistry, every ninth section throughout the entire hippocampus was preserved for further processing (7–8 sections from each subject). For immunofluorescence, two sections containing the proximal (Bregma = -2.92 mm) and distal (Bregma = -5.16 mm) parts of the hippocampus were selected. Two methods were used to study neurogenesis: immunoenzymatic labelling and triple immunofluorescence labelling. Immunoenzymatic labelling was used to assess the total number of Ki-67 (marker of cell proliferation)-, BrdU (marker of survival of proliferating cells)-, DCX-, and NeuN-positive cells (Ki-67+, BrdU+, DCX+ or NeuN+, respectively). Triple immunofluorescence labelling was performed to determine changes in the differentiation of the surviving cells. The detailed immunostaining protocols used are described in previous publications (Maćkowiak et al. 2005, 2009; Chocyk et al. 2015; Majcher-Maślanka et al. 2019; Zaniewska et al. 2021; Solarz-Andrzejewska et al. 2023).

Single staining for BrdU

Survival of dividing progenitor cells was assessed by staining for BrdU two weeks after BrdU injections. Free-floating sections were washed with 0.01 M PBS (pH 7.4) and denatured in a solution containing 50% formamide/2x SSC (saline-sodium citrate buffer, pH 7.0; Sigma-Aldrich) for 2 h at 65 °C. The sections were then washed twice in 2x SSC buffer for 5 min and incubated in 2 M HCl for 30 min at 37 °C before being incubated in 0.1 M borate buffer (0.1 M boric acid + NaOH; pH 8.5) for 10 min at room temperature. Subsequently, the brain sections were rinsed with 0.01 M PBS and incubated in blocking buffer (5% normal goat serum; Vector Laboratories, Peterborough, UK and 0.3% Triton X-100 in 0.01 M PBS) for 1 h. Finally, the sections were incubated (48 h at 4 °C) with primary monoclonal anti-BrdU mouse antibodies (1:200; Roche Diagnostics, Indianapolis, IN, USA; cat #11170376001) in 3% normal goat serum supplemented with 0.3% Triton X-100 in 0.01 M PBS. Reactions were visualized using biotinylated goat anti-mouse IgG (1:200, 1 h; Vector Laboratories), followed by incubation with avidin-biotin-horseradish peroxidase complex (1:200, 1 h; Vectastain Elite ABC Kit, Vector Laboratories), 3,3′-diaminobenzidine tetrahydrochloride (DAB)-nickel solution (0.02% DAB + 0.03% NiCl2 in 0.01 M PBS), and 0.01% H2O2. This resulted in the immunoreactive cells appearing as a dark grey colour. Finally, the sections were rinsed in 0.01 M PBS, mounted on SuperFrost Plus slides (Menzel-Gläser, Thermo Scientific, Braunschweig, Germany), air-dried, and coverslipped using Permount (Fisher Scientific) as the mounting medium.

Single staining for K i-67 or DCX

Free-floating brain sections were washed with 0.01 M PBS (pH 7.4) and blocked in blocking buffer containing 5% normal goat serum for Ki-67 or 5% normal rabbit serum for DCX labelling (Vector Laboratories) and 0.3% Triton X-100 in 0.01 M PBS for 1 h. Then, the sections were incubated with either rabbit polyclonal anti-Ki-67 (1:750; Abcam, Cambridge, UK; cat #ab15580) or goat polyclonal anti-DCX (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat #sc-8066) primary antibodies diluted in the appropriate 3% normal serum with 0.3% Triton X-100 in 0.01 M PBS for 48 h at 4 °C. The reaction was visualized using a biotinylated goat anti-rabbit (1:200; Vector Laboratories) or rabbit anti-goat (1:200; Vector Laboratories) IgG and peroxidase complex (1:200, 1 h; Vectastain Elite ABC Kit), followed by incubation with a 0.02% DAB solution and 0.01% H2O2. For DCX, the immunoreactive cells appeared brown in colour. For Ki-67, a DAB-nickel solution (0.02% DAB + 0.03% NiCl2 in 0.01 M PBS) with 0.01% H2O2 was used, resulting in immunoreactive cells with a dark grey colour. Finally, the sections were rinsed in 0.01 M PBS, mounted on SuperFrost Plus slides (Menzel-Gläser), air-dried, and coverslipped using Permount.

Single staining for NeuN

Free-floating sections were washed in 0.01 M PBS (pH 7.4) and incubated in PBS containing 0.3% H2O2 and 0.2% Triton X-100 for 30 min to block endogenous peroxidase activity. Next, the sections were rinsed and blocked in a solution containing 5% normal goat serum and 0.2% Triton X-100 in PBS for 1 h. After blocking, the sections were incubated with a mouse anti-NeuN antibody (1:1000; Millipore) for 48 h at 4 °C. The antibody was diluted in a solution containing 3% normal goat serum and 0.2% Triton X-100 in PBS. The sections were then washed in PBS and incubated with biotinylated goat anti-mouse IgG (1:200; Vector Laboratories) for 1 h, followed by incubation with an avidin-biotin-peroxidase complex (1:100, 1 h; Vectastain Elite ABC Kit) for 1 h. The immunohistochemical reaction was developed using a solution of 0.02% DAB and 0.01% H2O2 in TBS, resulting in brown staining of the immunoreactive cells. Finally, the sections were rinsed in 0.01 M PBS, mounted on SuperFrost Plus slides (Menzel-Gläser), air-dried, and coverslipped using Permount.

Immunofluorescence staining

The differentiation of BrdU+ cells was assessed two weeks after BrdU administration by immunofluorescent triple labelling for BrdU, DCX and NeuN. The sections were first treated to denature DNA (as described above) and then blocked with a buffer containing 5% normal donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA, USA) and 0.3% Triton X-100 in 0.01 M PBS for 1 h. Next, a cocktail of primary antibodies against BrdU (monoclonal rat 1:300; Accurate Chemical and Scientific Corp., Westbury, NY, USA; cat #AB6326), DCX (polyclonal goat 1:500; Santa Cruz Biotechnology) and NeuN (monoclonal mouse 1:1000; EMD Millipore, Temecula, CA, USA; cat #MAB377) in 3% normal donkey serum with 0.3% Triton X-100 in 0.01 M PBS was applied to the sections, which were then allowed to incubate for 48 h at 4 °C. After washing, a mixture of secondary antibodies (Alexa 488-conjugated donkey anti-rat IgG 1:200; Thermo Fisher Scientific, Rockford, IL, USA, Cy3-conjugated donkey anti-goat IgG 1:300; Jackson Immunoresearch Laboratories and Cy5-conjugated donkey anti-mouse IgG 1:300; Jackson Immunoresearch Laboratories) in 3% normal donkey serum with 0.3% Triton X-100 in 0.01 M PBS was applied to the sections, which were then incubated overnight at 4 °C. The sections were then washed, mounted on SuperFrost Plus slides (Menzel-Gläser), and coverslipped with medium containing 0.01 M PBS-buffered glycerol.

Quantitative evaluation of staining

For immunoenzymatic staining, the number of immunoreactive (BrdU+, Ki-67+, DCX+ or NeuN+) cells in the DG of the hippocampus was estimated using unbiased stereological methods (West et al. 1991) following the procedure described by Maćkowiak et al. (Maćkowiak et al. 2007). Every ninth section along the rostrocaudal axis of the hippocampal formation was analysed using optical fractionator sampling, which was performed using a Leica DM 6000B light microscope equipped with a motorized stage (Ludl Electronic Products, Hawthorne, NY, USA) and digital camera (MBF C×9000, Williston, VT, USA). The dorsal DG was outlined under low magnification (2.5×) using Stereo Investigator software v. 8.0 (MBF Bioscience, Williston, VT, USA) according to a stereotaxic atlas of the rat brain (Paxinos and Franklin 2001). Sampling was performed bilaterally under high magnification (63×, oil-immersion objective) using counting frames with areas of 1600 µm2 (the analysis of BrdU+, Ki-67+ or DCX+ cells) or 3600 (the analysis of NeuN+ cells) µm2 and heights of 15 μm. Cells appearing in the upper focal plane were omitted to prevent counting the tops of the cells (− 5 μm of the topmost surface of the section). The mean numerical density of immunoreactive cells was calculated for each animal from the sum of the counts made within the optical dissectors. The final results are presented as the density of immunoreactive cells in the DG of the hippocampus, calculated as the total number of cells estimated by optical fractionator divided by the estimated DG volume in mm3.

For immunofluorescent triple labelling, the colocalization of BrdU, DCX, and/or NeuN immunoreactivity in the sections was visualized using a confocal microscope (Leica TCS SP8, WLL) with excitation wavelengths of 495 nm (Alexa 488), 550 nm (Cy3), and 650 nm (Cy5). The sections were scanned using a 63× objective (HC PL APO CS2 63×/1.40 OIL) along the Z-axis (Z-step size: 1 μm, scan speed: 400 Hz, frame size: 512 × 512, line average: 3). Z-plane stacks of images were collected at every location within the hippocampal DG in which BrdU+ cells were visible. Images were further examined using Leica Application Suite X software (LAS X; Leica Microsystems, Switzerland) to observe the phenotype of the surviving cells. For each rat, the number of single-labelled cells (BrdU+ cells labelled for neither DCX nor NeuN), double-labelled cells (BrdU+ cells labelled for either DCX or NeuN; BrdU+/DCX+ or BrdU+/NeuN+, respectively) and triple-labelled cells (BrdU+ cells expressing both DCX and NeuN; BrdU+/DCX+/NeuN+) were estimated. The results are presented as the percentages of BrdU+ cells labelled for the respective marker(s) for each cell type in the given region.

Preparation of photomicrographs for data presentation

To present examples of cells that exhibited immunoreactivity for the neurogenesis markers used in the analysis, immunoenzymatically stained sections were imaged using a Leica MICA WideFocal Live Cell system (Leica Microsystems), while immunofluorescence staining was displayed in representative confocal microscopy images of Z-plane stacks from a control animal. Final photomicrographs were compiled with ImageJ v. 1.52q (NIH, Bethesda, MA, USA) and CorelDRAW Graphics Suite 2023 (Corel Corporation, Ottawa, Canada) software.

Effects of long-term exercise during nicotine withdrawal on the behaviour of rats during nicotine deprivation

Separate groups of rats (n = 12) were generated through a self-administration procedure. After achieving stable responses on the active lever, the animals were subjected to an abstinence phase. Twenty-four hours after the last self-administration session, the rats were transferred to cages equipped with either running wheels (NIC– WR; n = 6) or locked wheels (NIC– LW; n = 6) until Day 14 of nicotine cessation.

The FST

On Day 13 of nicotine abstinence, the rats were individually placed in a nontransparent cylindrical tank (50-cm high, 23 cm in diameter) filled with water (30-cm deep, 25 ± 1 °C), and they remained there for 15 min (the pretest) (Zaniewska et al. 2021). The rats were then removed, dried, and returned to the appropriate home cages. On Day 14, immediately after the locomotor activity test, the rats underwent the FST for 5 min (the test). The following behavioural parameters were measured by two experimenters: immobility time, swimming and climbing. All the test sessions were recorded by a video camera to allow for repeated measurements. After the FST, all animals were returned to standard home cages without wheels.

Locomotor activity

On Day 14, after the final 24-h exposure to the running wheels (the 13th session), all animals were placed in standard home cages. Their locomotor activity was then assessed without prior habituation to the test environment (spontaneous locomotor activity) (Zaniewska et al. 2021). Activity was recorded in Opto-Varimex cages (Columbus Instruments, USA). Interruptions of the photobeams resulted in the measurement of horizontal locomotor activity, which was defined as the distance travelled (expressed in cm). Locomotor activity was recorded during 5- or 30-min trials. Subsequently, the animals were transferred to their standard home cages.

Nicotine-seeking behaviour

On Day 15, 24 h after the last running wheel exposure, the rats were administered nicotine (0.4 mg/kg, sc, unconditioned stimulus) and immediately introduced to the experimental cages to induce a drug-seeking response. During a 2-h session, pressing the active lever on an FR5 schedule resulted in the intravenous delivery of saline without conditioned cues (light + tone). Similar to the maintenance phase, a 20-s time-out period followed each infusion. The number of lever presses corresponds to presses made under the FR schedules, during infusions, and time-out periods. The strength of nicotine-seeking behaviour, as indicated by the number of active lever presses, was compared between animals that had access to unlocked running wheels and those with access to locked wheels.

Statistical analyses

The data are expressed as means (± SEM). The sample size calculation was based on our preliminary behavioural (self-administration, FST) and neurobiochemical (hippocampal neurogenesis) data showing that to obtain statistically significant differences, a minimum of 6 (behavioural analyses) or 4–5 (neurogenesis analyses) rats per group is necessary. The normality of the data distribution was tested by the Shapiro‒Wilk normality test. After examining all the assumptions (i.e., normal distribution, equality of variance, existence of outliers), appropriate statistical tests were applied. Comparisons between means representing changes from the control values were made using Student’s t test for independent samples (cumulative nicotine intake/FST/locomotor activity). For non-normally distributed data, the Mann‒Whitney U test was used. Two-way analysis of variance (ANOVA) for repeated measures with interaction was used to analyse the lever presses during the maintenance phase across sessions 2 to 21 (factors: lever, treatment (nicotine), session) and body weight gain during nicotine abstinence (neurogenesis experiment). One-way ANOVA for repeated measures with interaction was applied to analyse the running distance (neurogenesis experiment) as well as lever presses during the maintenance phase (factors: lever, session) and body weight gain (factors: environment (locked wheels/running wheels), abstinence day) (behavioural experiment). Repeated measures ANOVA with interaction was used to assess nicotine infusions across sessions 2 to 21 (behavioural and neurogenesis experiments) and running distance (behavioural experiment). The animals’ body weight on the last self-administration day (factors: treatment (nicotine), environment), the immunostaining data (factors: treatment (nicotine deprivation), environment), and the number of lever presses taken during the induction of drug-seeking behaviour (factors: lever, environment) was analysed using two-way ANOVA with interaction. One-way ANOVA was used to analyse the cumulative nicotine intake in the neurogenesis experiment. ANOVA was followed by a post hoc Tukey test. All comparisons were made with an experiment type I error rate (α) set at p < 0.05. Statistics were calculated using GraphPad Prism v.9.3.0 software (GraphPad Software, La Jolla, CA, USA) or Statistica v. 13.3 (TIBCO Statistica).