Chronic Lorcaserin Treatment Reverses the Nicotine Withdrawal-Induced Disruptions to Behavior and Maturation in Developing Neurons in the Hippocampus of Rats

Preclinical data have shown that treatment with serotonin (5-HT)2C receptor agonists inhibits the behavioral effects of nicotine, including self-administration, reinstatement, and locomotor responses to nicotine. Since the data on the effects of 5-HT2C receptor agonism on nicotine withdrawal signs are limited, we aimed to investigate whether 5-HT2C receptor agonism alleviated the behavioral and neurobiochemical (hippocampal neurogenesis) consequences of nicotine withdrawal in Sprague-Dawley rats. Our data indicate that withdrawal from nicotine self-administration induced locomotor hyperactivity, lengthened immobility time (the forced swim test), induced ‘drug-seeking’ behavior and deficits in cognition-like behavior (the novel object recognition task). A two-week exposure to the 5-HT2C receptor agonist lorcaserin attenuated locomotor hyperactivity and induced recovery from depression-like behavior. Analyses of brain slices from nicotine-withdrawn animals revealed that lorcaserin treatment recovered the reduced number of doublecortin (DCX)-positive cells, but it did not affect the number of Ki-67- or 5-bromo-2’-deoxyuridine (BrdU)-positive cells or the maturation of proliferating neurons in drug-weaned rats. To summarize, we show that lorcaserin alleviated locomotor responses and depression-like state during nicotine withdrawal. We propose that the modulatory effect of lorcaserin on the ‘affective’ aspects of nicotine cessation may be linked to the positive changes caused by the compound in hippocampal neurogenesis during nicotine withdrawal.


Drugs
BrdU was dissolved in a filtered (Whatman filters, PP w/GMF 0.2 µm; GE Healthcare, Chicago, IL, USA) solution of 0.9% saline and 0.007N NaOH (pH 7.5). Nicotine was dissolved in sterile 0.9% saline. The pH was adjusted to 7.0 using 20% NaOH. In the case of the iv self-administration paradigm, the nicotine solution (0.03 mg/kg/inf) was filtered using Whatman filters. During the induction of 'drugseeking' behavior, the drug (0.4 mg/kg, sc) was administered at a volume of 1 ml/kg. Lorcaserin (0.1-0.6 mg/kg, sc) was dissolved in sterile 0.9% saline and injected at a volume of 1 ml/kg.

Effects of Lorcaserin in the FST in Naive Rats
On the first day of the FST, rats were individually placed in a nontransparent cylindrical tank (50cm high, 23 cm in diameter) filled with water (30-cm deep, 25 ± 1°C), and they remained there for 15 min (the pretest). The rats were then removed, dried and returned to their home cages. On day 2, the rats were treated with lorcaserin (0.1-0.6 mg/kg, n = 8/group) or its vehicle (n = 8), and 60 min later, the behavior of the rats was tested for 5 min in the FST under identical conditions. The following parameters were measured by two experimenters: immobility time, swimming and climbing. All the test sessions were recorded by a video camera to allow repeating the doubtful measurements.
To verify the specificity of the effects seen in the FST, the effect of the active dose of lorcaserin (0.3 mg/kg; in the FST) on locomotor activity was tested in non-habituated (spontaneous locomotor activity) rats (veh: n = 8; lor(0.3): n = 8). Locomotor activity was recorded individually for each animal 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 traveled (expressed in cm). Measurements of locomotor activity were recorded during 5-or 30-min trials 60 min after injection with lorcaserin (0.3 mg/kg) or its vehicle.

Training and Intravenous Catheter Implantation
After 16-18-h water deprivation, animals were trained for 5 days 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. Two days after lever press training, during which animals had free access to food and water, rats were anesthetized with a solution containing ketamine (20 mg/kg, im; Biowet, Puławy, Poland) and dexmedetomidine (0.1 mg/kg, im; Orion Corporation, Espoo, Finland), and then they were implanted with a silastic catheter in the external right jugular vein. Following catheter implantation, all animals recovered for 7-10 days. For two days after the surgery, animals received 4-5 ml of 0.9% NaCl/5% glucose solution (sc), and for three days, they were given the antiinflammatory/analgesic drug meloxicam (0.04 mg/kg, sc; Metacam, Boehringer Ingelheim, Ingelheim/Rhein, Germany). Catheters were flushed daily with 0.2 ml of a sterile 0.9% saline solution containing cephazolin (100 mg/ml; Polpharma, Warszawa, Poland) and heparin (100 IU/ml; Polfa, Warszawa, Poland) to maintain catheter function.

Maintenance of Nicotine Self-Administration
Rats were given access to nicotine during the daily 2-h session (6 days/week). The ambient light was on throughout each session. Each press on the active lever (i.e., the number of presses was gradually increased from FR1 to FR5) 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). Following each infusion, there was a 20-s time-out period during which responding was recorded but not reinforced. Rats were tested simultaneously in two groups with one of the rat group serving as the 'yoked' control that 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 a minimum of 21 days until the subjects achieved the criteria for stable responding: stable self-administration over the last 3 sessions with a standard deviation within those days that was <10% of the average.

Effects of Nicotine in the LDB
The animals were kept in total darkness for 30 min before the test and throughout the whole experiment. Following habituation, the animals were placed for 10 min in LDB cages (TSE Systems, Bad Homburg, Germany). Each arena, 45 cm × 45 cm × 45 cm, consisted of two compartments: one dark compartment (made of black acrylic, covering 1/3 of the total cage area) and one light compartment (light intensity -60 lx, made of transparent acrylic, covering the remaining 2/3 of the cage), separated from each other by a wall equipped with a central tunnel gate (height x width: 11 cm x 8 cm). The experimental cages were located in soundproof cabinets containing integrated infrared sensors that detected the animals along the X-, Y-(horizontal level) and Z-(vertical level) axes. The sensor frames on the X-, Y-and Z-axes were equipped with 32 sensor pairs mounted 14 mm from each other. The cages were equipped with fans providing background noise (65 dB). The behavior of animals (locomotor activity, the amount of time spent in each compartment, number of transitions between the compartments) was automatically recorded by a camera mounted on the ceiling of the cabinet and was analyzed using Fear Conditioning software (TSE Systems).

Nicotine Withdrawal
Effects of Protracted Nicotine Withdrawal on the Behavior in the NORT 24 h before testing, the rats were habituated to the arena (without any objects) for 5 min. The rats were tested in a dimly lit (25 lx) open field made of dull gray plastic (length × width × height: 66 cm × 56 cm × 30 cm). After each measurement, the floor was cleaned and dried. The following day, the test comprised two 3-min trials separated by an intertrial interval of 1 h. During the first trial (familiarization, T1), two identical objects (A1 and A2) were presented in opposite corners, and they were approximately 10 cm from the walls of the open field. In the second trial (retention, T2), one of the objects was replaced with a novel object (A = familiar and B = novel). The animals were returned to the home cage after T1. The objects used in the test included a glass bulb filled with gravel and a plastic bottle filled with sand. The heights of the objects were comparable (ca. 12 cm), and both objects were heavy enough to not be displaced by the animals. Half of the animals from each group identified the glass bulb as a novel object, and the other half identified the plastic bottle. The location of the novel object in the recognition trial was randomly assigned for each rat. The exploration of an object was defined by looking, licking, sniffing or touching the object while sniffing but not by leaning against, standing or sitting on the object. Any rat spending less than 5 s exploring the two objects within 3 min of T1 or T2 was eliminated from the study. The behavior of the rats was recorded using a camera placed above the arena and connected to an Any-maze® tracking system (Stoelting Co., Wood Dale, IL, USA). An experimenter blinded to the treatment conditions manually assessed the exploration time. Additionally, the distance traveled was automatically measured using an Any-maze® tracking system. Based on the exploration time (E) of the two objects, the discrimination index (DI) was calculated as DI=(EB − EA)/(EA + EB).

Immunohistochemistry
Immediately after the 21st self-administration session, rats were given three injections of BrdU (50 mg/kg, ip) at 6-h intervals during early withdrawal. During the withdrawal period, animals were kept in their home cages and received lorcaserin (0.1 mg/kg) or its vehicle once daily. At 14 days postcessation, animals were deeply anesthetized with a sublethal dose of sodium pentobarbital (90 mg/kg) and pentobarbital (18 mg/kg) (ip; Morbital, PGF Cefarm, Kraków, Poland) and were transcardially perfused with 0.9% NaCl, which was followed by buffered 4% paraformaldehyde (VWR International, Radnor, PA, USA). After perfusion, the brains were isolated from the skull 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 sections (covering the entire hippocampus) using a Leica VT-1000S vibratome (Leica Microsystems, Heidelberg, Germany).

Quantitative Evaluation of Staining
For immunoenzymatic staining, the number of immunoreactive (BrdU + , Ki-67 + or DCX + ) cells in the dentate gyrus (DG) of the hippocampus was estimated under a light microscope (Leica, DM 6000B; Leica Microsystems) equipped with a motorized stage (Ludl Electronic Products, Hawthorne, NY, USA). Briefly, every ninth section along the rostrocaudal axis of the hippocampal formation was analyzed with a 63× planapochromat lens using Stereo Investigator software v.8.0 (MBF Bioscience, Williston, VT, USA). Cells appearing in the upper focal plane were omitted to prevent counting the tops of cells (−5 µm of the topmost surface of the section). For each animal, the mean numerical density of immunoreactive cells was calculated from the sum of the counts made within the optical dissectors. The final results were presented as the number of immunoreactive cells in the DG of the hippocampus, which was calculated automatically by Stereo Investigator software per 1 mm 3 hippocampal area.
For triple-label immunofluorescence, two sections per subject across the studied region were analyzed using a confocal microscope (Leica TCS SP8, WLL, Leica Microsystems) with excitation wavelengths of 499 nm (Alexa 488), 548 nm (Cy3) and 649 nm (Cy5). The sections were scanned using a 63x objective (HC PL APO CS2 63x/1.40 OIL) along the Z-axis (Z-step size: 1-µm, scan speed: 400 Hz, frame size: 1024 x 1024). 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 v.3.5.2.18963 (LAS X; Leica Microsystems) software to observe the phenotype of BrdU + cells. For each rat, the number of single (BrdU + )-, double (BrdU + /DCX + or BrdU + /NeuN + )-and triple (BrdU + /DCX + /NeuN + )-labeled cells was estimated. The results are presented as the mean number of cells per section and the percent of colocalized individual cell type per total BrdU cell population per region.

Preparation of Photomicrographs for Data Presentation
To present examples of cells that exhibited immunoreactivity for the neurogenesis markers used in the hippocampus, immunoenzymatically stained sections were imaged with an Aperio ScanScope slide scanner (Aperio UK, Oxford, UK), while immunofluorescent staining was presented by using a representative Z-plane stack of images from a control ysal animal. Final photomicrographs were compiled with ImageJ (NIH, Bethesda, MA, USA) and CorelDraw v.11.0 (Corel Corporation, Ottawa, Canada) software.

Cumulative Nicotine Intake in Rats within 21 Self-Administration Sessions
Total nicotine intake during 21 sessions did not differ between the three assigned groups that received vehicle or lorcaserin (0.1 or 0.3 mg/kg) during a 14-day withdrawal period (F(2,27) = 0.48, p = 0.63) ( Figure S2).

The Effects of Chronic Lorcaserin Administration on the Behavior of Nicotine-Withdrawn Rats in the NORT
The time spent exploring two identical objects (Trial 1) was not changed following lorcaserin treatment (interaction: F(3,76) = 1.04, p = 0.38) ( Figure S3A). No significant treatment effect was observed in the distance traveled by rats in the familiarization and retention trials (interaction: F(3,38) = 0.56, p = 0.65) ( Figure S3B).