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. Author manuscript; available in PMC: 2024 Jun 1.
Published in final edited form as: Neuropharmacology. 2023 Mar 12;230:109495. doi: 10.1016/j.neuropharm.2023.109495

The involvement of dopamine and D2 receptor-mediated transmission in effects of cotinine in male rats

Xiaoying Tan 1, Elizabeth M Neslund 1, Zheng-Ming Ding 1,2,*
PMCID: PMC10071274  NIHMSID: NIHMS1884017  PMID: 36914092

Abstract

Previous studies indicated that cotinine, the major metabolite of nicotine, supported intravenous self-administration and exhibited relapse-like drug-seeking behaviors in rats. Subsequent studies started to reveal an important role of the mesolimbic dopamine system in cotinine’s effects. Passive administration of cotinine elevated extracellular dopamine levels in the nucleus accumbens (NAC) and the D1 receptor antagonist SCH23390 attenuated cotinine self-administration. The objective of the current study was to further investigate the role of mesolimbic dopamine system in mediating cotinine’s effects in male rats. Conventional microdialysis was conducted to examine NAC dopamine changes during active self-administration. Quantitative microdialysis and Western blot were used to determine cotinine-induced neuroadaptations within the NAC. Behavioral pharmacology was performed to investigate potential involvement of D2-like receptors in cotinine self-administration and relapse-like behaviors. NAC extracellular dopamine levels increased during active self-administration of cotinine and nicotine with less robust increase during cotinine self-administration. Repeated subcutaneous injections of cotinine reduced basal extracellular dopamine concentrations without altering dopamine reuptake in the NAC. Chronic self-administration of cotinine led to reduced protein expression of D2 receptors within the core but not shell subregion of the NAC, but did not change either D1 receptors or tyrosine hydroxylase in either subregion. On the other hand, chronic nicotine self-administration had no significant effect on any of these proteins. Systemic administration of eticlopride, a D2-like receptor antagonist attenuated both cotinine self-administration and cue-induced reinstatement of cotinine seeking. These results further support the hypothesis that the mesolimbic dopamine transmission plays a critical role in mediating reinforcing effects of cotinine.

Keywords: Cotinine, Dopamine, D2 receptor, Eticlopride, Microdialysis, Self-administration

Introduction

Consumption of tobacco products remains a leading public health issue. Nicotine is the main addictive component in tobacco that leads to habitual use (De Biasi and Dani, 2011; Prochaska and Benowitz, 2019). Cotinine is the major metabolite of nicotine, accumulates to higher levels than nicotine, and is better tolerated than nicotine (Benowitz and Jacob, 1994; Hatsukami et al., 1997; Riah et al., 1999). Cotinine has been widely used as a biomarker for tobacco use due to its long half-life (Hukkanen et al., 2005). Both nicotine and cotinine are agonists of nicotinic receptors (nAChRs) with cotinine being several orders of magnitude less potent than nicotine (Abood et al., 1983; Anderson and Arneric, 1994).

Accumulating evidence indicates that cotinine produces its own neurobehavioral effects. It elicited electroencephalogram activation and behavioral arousal (Yamamoto and Domino, 1965), altered food reinforcement (Goldberg et al., 1989; Risner et al., 1985), produced nicotine-like discriminative stimulus effects in drug discrimination tests (Goldberg et al., 1989; Rosecrans and Chance, 1977; Takada et al., 1989), and enhanced attention, learning and memory in animal models of cognitive impairment (Buccafusco and Terry Jr., 2009; Echeverria et al., 2011; Terry Jr et al., 2012). In addition, cotinine altered monoamine neurotransmission and intracellular signal transduction in both brain and periphery (Dwoskin et al., 1999; Fuxe et al., 1979; Schroff et al., 2000; Vainio et al., 2000; Vainio et al., 1998). In humans, cotinine altered abstinence-induced subjective states and withdrawal symptoms in smokers (Benowitz et al., 1983; Hatsukami et al., 1998; Keenan et al., 1994). On the other hand, studies showed that cotinine did not alter the threshold for intracranial self-stimulation in rats (Harris et al., 2015), and failed to substitute for the discriminative stimulus effects of epibatidine (Desai et al., 2016). These results suggest that cotinine’ effects appear to be dependent on model systems.

Recent studies suggest that cotinine may produce its own reinforcing effects. Cotinine supported intravenous self-administration in a schedule- and dose-dependent manner in rats. Cotinine self-administration was less robust than nicotine self-administration. Reliable cotinine self-administration produced blood cotinine levels in the range of ~200-800 ng/ml (Ding et al., 2021). These levels were comparable to those seen in habitual smokers, suggesting that cotinine may be reinforcing at physiologically-relevant concentrations. In addition, relapse-like behaviors were demonstrated in both the reinstatement of drug seeking and incubation of craving models in rats with a history of cotinine self-administration (Tan et al., 2022). Pretreatment with pharmacological agents targeting nAChRs, e.g., mecamylamine or varenicline, attenuated nicotine, but not cotinine self-administration (Ding et al., 2021), suggesting differential involvement of nAChRs in nicotine and cotinine self-administration.

Recent data suggest an involvement of the mesolimbic dopamine (DA) system in mediating effects of cotinine (Tan et al., 2021a). Passive administration of cotinine either centrally into the ventral tegmental area (VTA) or peripherally increased extracellular DA levels in the nucleus accumbens (NAC). Blockade of D1-like receptors (D1Rs) attenuated cotinine self-administration. Given the important role of the mesolimbic DA system in mediating the effects of nicotine (Picciotto and Kenny, 2020), these results suggest that activation of the mesolimbic DA system may be a shared cellular mechanism contributing to both nicotine and cotinine self-administration. However, several questions remain, including (1) whether the mesolimbic DA levels change during active cotinine self-adminsitration, (2) whether chronic exposure to cotinine induces adaptive changes in the DA system, and (3) whether other DA receptors, e.g., D2-like receptors (D2R) are also involved in the reinfocing effects of cotinine.

Nicotine stimulated the mesolimbic DA transmission not only after passive administration, but also during ongoing active self-administration (Benwell and Balfour, 1992; Lecca et al., 2006). Nicotine has been shown to induce neuroadaptions within the mesolimbic DA system. For example, a history of nicotine self-administration reduced the basal extracellular DA concentrations but increased DA reuptake within the NAC (Rahman et al., 2004b). Chronic nicotine exposure altered DA receptor expression and function (Balfour et al., 1998; Reilly et al., 1987). In addtion to the D1R, the D2-like receptor (D2R) has been shown to play an important role in mediating nicotine self-administration and relapse-like behaviors (Corrigall and Coen, 1991; Liu et al., 2010). Given the mesolimbic DA system as a potential shared substrate for nicotine and cotinine self-administration, cotinine may also produce these similar effects.

The objective of the current study was to further explore the involement of the mesolimbic DA transmission in the reiforcing effects of cotinine by investigating remaining questions listed above. Conventional microdialysis was coupled with self-administration to investigate timecourse changes of extracellular DA levels during ongoing active cotinine self-administration. The quantitative no-net-flux microdialysis and Western blot were used to determine adaptive alterations in DA transmission induced by chronic cotinine exposure. In addition, pharmacological studies were employed to determine the role of the D2R in cotinine-related behaviors. The overall hypothesis was that the activation of mesolimbic DA transmission is critical to the reinforcing effects of cotinine.

Materials and methods

Animals

Young adult male Wistar rats (starting at ~ 8 weeks old) were acquired from Envigo (Indianapolis, IN USA) and housed in a vivarium controlled for temperature and humidity. Male rats were tested because our previous study used male rats that generated initial evidence supporting an involvement of the mesolimbic DA system in cotinine’s effects (Tan et al., 2021a). The current study with male rats would allow better comparison between studies. The room was maintained on a reversed 12-h light-dark cycle with light off from 9:30am to 9:30pm. Experimental procedures were performed during the dark phase. Acclimation period was approximately one week. Rats were housed in groups (2-4/cage) upon arrival and individually after surgery. Cages were enriched with a polycarbonate play tunnel and nestlets. Food and water were available ad libitum unless explicitly stated. Protocols used were approved by the Institutional Animal Care and Use Committee at Pennsylvania State University College of Medicine. All experiments were performed in accordance with the principles outlined in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

Intravenous self-administration, extinction, and cue-induced reinstatement

Catheterization surgery followed the procedure previously described (Ding et al., 2021). Briefly, rats were anesthetized with 2-3% isoflurane inhalation and the right jugular vein was exposed surgically. Polyurethane tubing of the catheter [Inner diameter (I.D.) x outer diameter (O.D.) = 0.6 x 1.0 mm; Instech Laboratories, Inc., Plymouth Meeting, PA, USA] was inserted into the vein. The remaining portion of the catheter coursed subcutaneously over the shoulder blade to exit the back of the rat via a 22-gauge back-mount cannula (P1 Technologies, Roanoke, VA, USA). Bupivacaine (Hospira, Inc., Lake Forest, IL, USA) at 0.5% and carprofen (Zoetis Inc., Kalamazoo, MI, USA) at 5 mg/kg were applied as analgesia during surgery. Catheters were flushed daily with ~0.5 ml of 20 IU/ml heparinized saline (Fresenius Kabi, Lake Zurich, IL, USA) containing 0.13 mg/ml gentamicin sulfate (Fresenius Kabi, Lake Zurich, IL, USA). Catheter patency was checked with intravenous administration of ~0.1 ml of 10 mg/ml methohexital sodium (Par Pharmaceutical, Chestnut Ridge, NY, USA) once a week. Rats with failed catheters were excluded from experiments.

Rats were handled on a daily basis following surgery, and self-administration was initiated ~5 days after surgery in standard rat self-administration chambers (Med Associates Inc., St. Albans, VT, USA) as previously described (Ding et al., 2021). Light food restriction remained in place throughout to maintain rats at ~85% body weight. A piece of Froot Loops cereal was placed on the active lever as a bait during the first two sessions to promote exploratory behavior. A fixed-ratio (FR) 1 plus 17 s timeout schedule was employed. Response on the active lever led to an intravenous infusion of either (−)-cotinine (Sigma, St. Louis, MO, USA) at 0.03 mg/kg/infusion or (−)-nicotine hydrogen tartrate (Sigma, St. Louis, MO, USA) with nicotine base dose at 0.03 mg/kg/infusion. These doses of nicotine and cotinine elicited reliable self-administration and clinically-relevant blood nicotine or cotinine concentrations in rats (Corrigall and Coen, 1989; Ding et al., 2021). Infusions were delivered in 55 μl over a 3-s period via a syringe pump (PHM-100, Med Associates Inc., St. Albans, VT, USA). During infusion, house light was turned off and the cue light above the active lever was turned on. The infusion was followed by a 17-s timeout period during which both cue light and house light were off. Lever presses during the infusion and timeout periods were recorded but produced no further infusions. Responses on the inactive lever were recorded, but no programmed consequences ensued. Sessions were 2 hours in duration, and were conducted daily during weekdays for 3-4 weeks.

Following self-administration, rats underwent extinction training to extinguish self-administration behavior as previously described (Tan et al., 2022). Neither the drug nor light cues associated with self-administration was available during extinction sessions. Extinction training lasted for 2 weeks. Then rats were tested for cue-induced reinstatement of drug-seeking behavior during which responses on the level previously associated to drug self-administration led to the delivery of only light cues, but not the drug itself. Responses on the previous inactive lever resulted in no programmed consequence.

Eticlopride treatment during cotinine self-administration and cue-induced reinstatement of cotinine-seeking behavior

To examine effects of inhibiting DA D2Rs on cotinine-related behaviors, S-(−)-eticlopride hydrochloride, a selective D2R antagonist (Sigma, St. Louis, MO, USA) was injected subcutaneously into separated groups of rats self-administering cotinine (n = 8) and undergoing cue-induced reinstatement of cotinine-seeking behavior (n = 7). Eticlopride (0, 5, and 10 μg/kg) was administered approximately 30 min prior to operant sessions using a within-subject design with different doses administered in a random order. Non-treatment sessions were included to allow responses to return to basal levels between treatments. These doses of eticlopride have been shown to attenuate nicotine-related relapse behaviors in rats (Liu et al 2010).

Stereotaxic surgery and probe insertion

Rats underwent stereotaxic surgery for implantation of guide cannulae and probes for microdialysis following procedures previously described (Engleman et al., 2020; Tan et al., 2021a). Briefly, rats were anesthetized with 2-3% isoflurane inhalation, and then implanted with one 18-gauge guide cannula (I.D. x O.D. = 0.82 x 1.27 mm; P1 Technologies, Roanoke, VA, USA) aimed at the NAC (AP +1.7 mm, ML +2.3 mm, DV −8.4 mm). Stylets were inserted into cannula with a 0.5-mm extension beyond the guide cannula. Following surgery, rats were handled on a daily basis and recovered for at least 5 days before probe insertion. For probe insertion, rats were lightly anesthetized with isoflurane, and a loop-style microdialysis probe with 1.5-mm active membrane (I.D. x O.D. = 200 μm x 216 μm, molecular weight cut-off: 13 kDa, Spectrum Laboratories, Inc, Rancho Dominguez, CA, USA) was inserted into the NAC.

No-net-flux microdialysis

The no-net-flux microdialysis is a quantitative technique that has been widely used to measure true basal extracellular DA concentrations and reuptake which are represented by the no-net-flux point and extraction fraction, respectively (Parsons and Justice, 1994). This experiment was performed to examine adaptive changes in extracellular DA transmission within the NAC after a history of cotinine exposure following procedures previously described (Engleman et al., 2020). Passive administration but not self-administration was performed in this study for better control of exposure condition. Our previous study indicates that during self-administration rats elicited more lever responses and obtained greater number of infusions for cotinine than saline. The differences in lever responses and infusion volumes render the saline self-administration a less ideal control group for cotinine self-administration. On the other hand, passive exposure through systemic injections enable exposure to the same volume of cotinine and saline.

Rats were divided into two groups (n = 6-7/group) that received repeated daily subcutaneous injections of either saline or cotinine at 1 mg/kg for 20 days during weekdays. The duration of cotinine exposure was similar to typical periods of cotinine self-administration in previously studies (Ding et al., 2021; Tan et al., 2021a; Tan et al., 2022). This dose of cotinine was in the similar range of cotinine intake during reliable self-administration (Ding et al., 2021) and has been shown to elevate extracellular DA levels in the NAC following acute systemic administration (Tan et al., 2021a). Following the 15th injection, rats underwent stereotaxic surgery for implantation of guide cannula aimed at the NAC. A probe was inserted into the NAC shortly after the 20th injection and no-net-flux microdialysis was conducted ~16-18 h later. During microdialysis, rats were perfused with a ringer solution (147 mM NaCl, 3 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, and 0.2 mM ascorbate) at a flow rate of 0.5 μl/min. After a 90-min equilibration period, three samples were collected for baseline. Then, probes were perfused with three DA solutions (2.5, 10, 20 nM) one after another in a random order. Six samples were collected for each concentration. Following the perfusion with the last DA solution, rats were switch to ringer solution perfusion with two additional samples collected. Samples were collected at 12-min intervals into centrifuge vials containing 3 μl of 100 mM acetic acid, and were immediately stored at −80°C for later analysis.

Microdialysis during ongoing active self-administration

A conventional microdialysis technique was coupled with self-administration to examine time-course changes of extracellular DA levels within the NAC that were associated with ongoing behavior following procedures previously described (Weiss et al., 1993). Rats were trained to self-administer cotinine or nicotine for 14 sessions. Since it was shown that ongoing nicotine self-administration increased extracellular DA levels in the NAC (Lecca et al., 2006), nicotine self-administration was used as a positive control in this study. A guide cannula was implanted after the 10th session and a probe was inserted after the 14th session into the NAC. Immediately following probe insertion, rats underwent a 60-min acclimation period in operant chambers with probes tethered to microdialysis tubing. Levers were made unavailable for responding to avoid potential extinction of self-administration behavior. Rats resumed self-administration with the probe tethered to microdialysis tubing for 1-2 sessions for further habituation to the tether. Microdialysis was conducted ~16-18 h after the final habituation session. On the day of microdialysis, rats were placed into operant chambers with levers hidden from rats. Probes were connected to a perfusion pump that perfused the ringer solution at a flow rate of 1.0 μl/min through the NAC. After a two-hour equilibration period, three baseline samples were collected. Then, levers were made available to rats and the self-administration session started. Rats received the usual drug solution for self-administration. During the 2-h session, twelve samples were collected. After self-administration, rats remained in chambers and six additional samples were collected. Samples were collected at 10-min intervals and stored for future analysis.

DA analysis

Microdialysis samples were analyzed for DA contents with an ALEXYS™ Neurotransmitter Analyzer, a UHPLC system with electrochemical detection (Antec Scientific USA, Boston, MA, USA) as previously described (Tan et al., 2021a). Briefly, samples were loaded via an AS 110 UHPLC autosampler onto an analytical column (ACQUITY UPLC® BEH C18, 1.7 μm, 50 mm × 1.0 mm, Waters Corporation, MA, USA) with a mobile phase containing 100 mM phosphoric acid, 0.1 mM EDTA, 100 mM citric acid, 600 mg/L OSA, and 3.5-4.0% acetonitrile at pH 6.0. DA was detected by a SenCell with a 2-mm glassy-carbon working electrode with a detection limit at 0.1 nM. Oxidation potential was set at + 460 mV and detection range was set at 500 pA/V. The signal was analyzed with DataApex Clarity software.

Western blot

Expression levels of selective protein markers of the DA system were examined in the NAC from rats with a history of self-administration of saline, cotinine at 0.03 mg/kg/infusion, or nicotine at 0.03 mg/kg/infusion. Detailed self-administration procedure and data from these rats have been published elsewhere (Ding et al., 2021). Briefly, these rats were trained for self-administration for 5 weeks, with weeks 1 to 3 under a FR1 schedule, week 4 under a FR2 schedule, and week 5 under a progressive ratio (PR) schedule. Shortly after the last session, rats were euthanized and brains were harvested. Protein levels were determined following procedures previously described (Ding et al., 2017; Farrag et al., 2017). Briefly, brain tissue was micro-punched from the shell and core subregions of the NAC. Total protein was extracted with a NucleoSpin® RNA/Protein purification kit (MACHEREY-NAGEL GmbH & Co., Germany) following the manufacturer’s instruction. Protein content was determined with the Qubit® Protein Assay on a Qubit 2 fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as instructed by the manufacturer. Western blot was carried out on a ProteinSimple Wes automated western blot platform (Bio-techne, Minneapolis, MN, USA). A 12-230 kDa separation microplate kit was used with 0.2-0.5 μg/μl protein loaded onto the plate. Primary antibodies included mouse anti-tyrosine hydroxylase (TH) antibody MAB5280 (1:100; MilliporeSigma, Burlington, MA, USA), rabbit anti-D2R antibody AB5084P (1:100; MilliporeSigma, Burlington, MA, USA), and rat anti-D1R antibody D2944 (1:50; MilliporeSigma, Burlington, MA, USA). Total protein was used as the loading control. Densitometric analysis of bands of interest were performed using the Compass analytical software from ProteinSimple.

Histology

Microdialysis probe placements were verified as previously described (Engleman et al., 2020; Tan et al., 2021a). At the end of microdialysis experiments, rats were euthanized with CO2 overdose and bromophenol blue was perfused through microdialysis probes. Brains were quickly removed and frozen at −80°C. Brain sections (40 μm thick) were sliced on a cryostat microtome and stained with cresyl violet for the determination of placements with the reference to the rat brain atlas of Paxinos & Watson (Paxinos and Watson, 1998).

Statistical analysis

DA data from the conventional microdialysis study and self-administration data were analyze with repeated measures mixed ANOVAs. For no-net-flux data, linear regression analysis was used to extract no-net-flux points (the extracellular DA concentration) and slope (DA extraction fraction) and t-tests were performed to compare differences between groups. For Western blot, densitometric data of the protein of interest were first normalized against the loading control, i.e., total protein. Then, values from the saline group were averaged and were used to normalize other values in the same study. The normalized data were analyzed with one-way ANOVAs. Pharmacological data from eticlopride experiments were analyzed with one-way ANOVA. Post-hoc LSD analyses were performed following the identification of significant main effects. The significant level was set at p < 0.05.

Results

Ongoing self-administration increased extracellular DA levels

Across self-administration training, repeated measures ANOVA revealed significant effects of session (F13, 117 = 16.7, p < 0.001), but not drug (F1, 9 = 1.7, p = 0.2) or drug x session interaction (F13, 117 = 1.0, p = 0.4) on number of infusions across sessions (Fig. 1A). Rats gradually acquired self-administration and increased number of infusions with no significant difference between cotinine and nicotine.

Figure 1.

Figure 1.

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Effects of ongoing self-administration of cotinine (COT; 0.03 mg/kg/infusion) or nicotine (NIC; 0.03 mg/kg/infusion as base) on extracellular dopamine (DA) levels within the nucleus accumbens (NAC). A. Number of infusions per session across sessions of NIC or COT self-administration. B. Time-course of extracellular DA levels within the NAC (lines and left y axis) and corresponding number of infusions (bars and right y axis) in 10-min bins during the ongoing self-administration (SA) session. The insets (1 & 2) show average DA increase during the 1st and 2nd hour of SA. * p < 0.05, significantly greater in the NIC than COT group.

During the session combining microdialysis and self-administration, number of infusions and extracellular DA levels in 10-min bins are summarized in Fig. 1B. For number of infusions, there was significant effect of time (F11, 99 = 2.9, p = 0.003), but not drug (F1, 9 = 0.3, p = 0.6) or time x drug interaction (F11, 99 = 2.9, p = 0.2). Average numbers of infusions were 24 ± 5 for nicotine and 21 ± 2 for cotinine, representing 25 ± 17% and 29 ± 11% reduction, respectively, from the baseline levels averaged from the last three self-administration sessions (34 ± 6 and 35 ± 5 per session for cotinine and nicotine, respectively).

For microdialysis, basal DA levels were 1.9 ± 0.2 nM for nicotine and 1.6 ± 0.2 nM for cotinine (t9 = 1.2, p = 0.3) within the NAC. During the first hour of self-administration, DA levels gradually increased in both nicotine and cotinine group. There was significant effect of time (F6, 54 = 5.0, p < 0.001), but not drug (F1, 9 = 0.2, p = 0.7) or drug x time interaction (F6, 54 = 0.7, p = 0.6). During this period, average DA levels were 132 ± 5% and 129 ± 5% of baseline for nicotine and cotinine, respectively (Fig. 1B inset 1; t9 = 0.3, p = 0.7). During the second hour of self-administration, there was a significant effect of drug (F1, 9 = 7.2, p = 0.03), but not time (F5, 45 = 1.0, p = 0.4) or time x drug interaction (F5, 45 = 1.4, p = 0.3). Overall, nicotine induced greater DA increase than COT did. Indeed, average DA increase during this period was significantly greater in the nicotine group (154 ± 8%) than those in the cotinine group (132 ± 4%) (Fig. 1B inset 2; t9 = 2.7, p = 0.03). After self-administration, DA levels gradually returned toward baseline with no significant difference between groups (F1, 9 = 0.9, p = 0.4).

Chronic self-administration of cotinine altered protein markers of the DA system

Detailed self-administration data were published previously (Ding et al., 2021). Briefly, rats gradually acquired self-administration with more infusions obtained for cotinine or nicotine than for saline toward the end of FR1 schedule, and during both FR2 and PR schedules. Fig. 2A shows average intake of nicotine or cotinine across sessions. At the end of the last FR2 session, cotinine rats attained blood cotinine levels at ~ 450 ng/ml, and nicotine rats exhibited blood nicotine levels at ~25 ng/ml and blood cotinine levels at ~230 ng/ml. Western blot was conducted in protein samples from both NACsh and NACcr subregions of rats collected shortly after the last PR session. There was no significant difference in TH levels (Fig. 2B) among these groups either in NACsh (F2, 24 = 1.4, p = 0.3) or NACcr (F2, 23 = 0.6, p = 0.6). Similarly, no significant difference was found in D1R levels (Fig. 2C) in either NACsh (F2, 19 = 0.8, p=0.5) or NACcr (F2, 22 = 1.4, p = 0.3). On the other hand, significant difference was observed in the NACcr (F2, 21 = 3.5, p < 0.05) for the D2R (Fig. 2D). Cotinine rats displayed significantly lower levels than saline rats (p = 0.02). There was a trend for significantly reduction of D2Rs in nicotine rats than saline rats (p = 0.058).

Figure 2.

Figure 2.

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Effects of chronic self-administration of saline (SAL), cotinine (COT; 0.03 mg/kg/infusion) or nicotine (NIC; 0.03 mg/kg/infusion as base) on protein markers of dopamine system within the shell and core subregions of the nucleus accumbens (NACsh & NACcr). A: NIC and COT intake across self-administration sessions under both fixed-ratio (FR) and progressive-ratio (PR) schedules. B: Protein levels of tyrosine hydroxylase (TH) in both subregions. C: Protein levels of D1 receptors (D1R) in both subregions. D: Protein levels of D2 receptors (D2R) in both subregions. * p < 0.05, significantly lower than the SAL group.

A history of cotinine exposure decreased basal extracellular DA concentrations

Fig. 3 summarizes the no-net-flux microdialysis data showing effects of repeated injections of cotinine or saline on basal extracellular DA concentrations and reuptake within the NAC. Cotinine treatment significantly reduced basal extracellular DA concentrations within the NAC compared to saline treatment (t11 = 2.7, p = 0.02). No difference was observed in DA extraction fraction (t11 = 1.2, p = 0.25), suggesting no difference in DA reuptake.

Figure 3.

Figure 3.

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Effects of repeated daily injections of cotinine (COT; 1.0 mg/kg) or saline (SAL) for 20 days on basal extracellular concentrations and clearance of dopamine (DA) within the nucleus accumbens. A: Linear regression plot of DA concentrations perfused in the brain ([DA]in) against DA concentration difference between those perfused in and those perfused out of the brain. B: Basal extracellular DA concentrations represented by the no-net-flux points shown in the regression plot. * p < 0.05, significantly lower than those in the SAL group. C: Extraction fraction (Ed) of DA represented by the slope of regression lines.

Eticlopride reduced cotinine self-administration

During self-administration training (Fig. 4A), rats gradually increased responses, and responded significantly more on active than inactive lever (session: F14, 98 = 2.6, p = 0.004; lever: F1, 7 = 17.4, p = 0.004; session x lever interaction: F14, 98 = 1.7, p = 0.06). There was a significant effect of eticlopride on number of infusions (Fig. 4B; F2, 21 = 22.2, p < 0.001). Eticlopride at 10 μg/kg significantly reduced infusions (p < 0.05). Eticlopride also reduced active lever responses at 10, but not 5 μg/kg (Fig. 4B; F2, 21 = 7.9, p = 0.003). Eticlopride had no effect on inactive responses (Fig. 4B; F2, 21 = 1.2, p = 0.3).

Figure 4.

Figure 4.

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Effects of the dopamine D2-like receptor antagonist eticlopride (ETIC) on self-administration of cotinine at 0.03 mg/kg/infusion. A: Responses on both active and inactive lever as well as number of infusions per session across self-administration training. * p < 0.05 significant difference between active and inactive lever responses. B: Effects of ETIC on number of infusions, active and inactive responses. * p < 0.05, significantly lower than the saline (SAL) treatment.

Eticlopride reduced cue-induced reinstatement of cotinine seeking

During self-administration (Fig. 5A), rats gradually increased cotinine self-administration, and responded significantly more on active than inactive lever (session: F19, 114 = 2.5, p = 0.002; lever: F1, 6 = 50.1, p < 0.001; session x lever interaction: F19, 114 = 2.4, p = 0.002). During extinction (Fig. 5A), rats gradually reduced responses on active lever over sessions (session: F9, 54 = 3.0, p = 0.006). Although rats did not fully extinguish active lever responses, this was similar to some cohorts of rats in a previous study and might reflect individual differences among animals (Tan et al 2022). This partial extinction did not alter cue-induced reinstatement as rats elicited more active responses during cue exposure than average active responses during the last three extinction sessions (58 ± 17 vs 22 ± 6, p < 0.05). For eticlopride treatment on reinstatement of cotinine seeking (Fig. 5B), there was a significant effect of eticlopride on active responses (F2, 18 = 4.4, p = 0.03), but not on inactive responses (F2, 18 = 0.2, p > = 0.9). Eticlopride at both 5 and 10 μg/kg significantly reduced active responses compared to vehicle treatment (p < 0.05).

Figure 5.

Figure 5.

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Effects of the dopamine D2-like receptor antagonist eticlopride on cue-induced reinstatement of cotinine seeking. A. Number of infusions and responses on both active and inactive levers during cotinine self-administration and extinction. B. Responses on both active and inactive levers during cue-induced reinstatement of cotinine seeking. * p < 0.05, significantly lower than the saline (SAL) treatment.

Discussion

The current study demonstrated that in male rats (1) ongoing active self-administration of cotinine and nicotine both increased extracellular DA levels within the NAC with more robust increase in nicotine than cotinine self-administration, (2) repeated injections of cotinine decreased basal extracellular DA concentrations in the NAC without altering DA reuptake, (3) chronic self-administration of cotinine reduced protein levels of D2Rs within the NACcr, but not NACsh, without altering expression of either TH and D1Rs, (4) chronic self-administration of nicotine had no significant effect on protein levels of TH, D1Rs or D2Rs, and (5) systemic administration of the D2R antagonist eticlopride attenuated both cotinine self-administration and cue-induced reinstatement of cotinine seeking. These results extended findings from a previous study which demonstrated that passive administration of cotinine increased extracellular DA levels in the NAC, and that inhibition of D1Rs with SCH23390 reduced cotinine self-administration (Tan et al., 2021a). Together, these findings further support the hypothesis that activation of the mesolimbic DA transmission plays a critical role in the reinforcing effects of cotinine.

Activation of the mesolimbic DA system is associated with reinforcing effects of drugs of abuse (Koob and Volkow, 2016). Drugs supporting self-administration in rodents, e.g., cocaine, morphine, and nicotine, have all been shown to elevate extracellular DA levels in the NAC following passive exposure and during self-administration (Di Chiara and Imperato, 1988; Willuhn et al., 2010). In our study, both cotinine and nicotine self-administration increased extracellular DA levels in the NAC, suggesting that activation of NAC DA transmission may contribute to the reinforcing effects of nicotine and cotinine. These findings agree with a pervious study showing that nicotine self-administration increased DA levels in both subregions of the NAC across a six-week self-administration period (Lecca et al., 2006). The peak DA increase in the Lecca et al study was ~145% of baseline occurring around 3-4 weeks of nicotine self-administration. This is similar to the highest DA levels at ~150% of baseline observed at the end of 3 weeks of nicotine self-administration in the current study. In addition, DA elevation was less robust in rats self-administering cotinine than nicotine, which suggests that cotinine may be less reinforcing than nicotine. Consistently, a previous study found that nicotine induced greater breakpoint than cotinine under a PR schedule at the dose of 0.03 mg/kg/infusion (Ding et al., 2021), suggesting greater reinforcing efficacy of nicotine than that of cotinine. Taken together, these results further support that the magnitude of DA increase during self-administration may be associated with the reinforcing efficacy of a drug.

The mechanism underlying differential DA increase during nicotine and cotinine self-administration remains unknown. The number of infusions were similar between nicotine and cotinine during both self-administration training and the self-administration-microdialysis session. In addition, temporal patterns of infusions were not different between cotinine and nicotine self-administration during microdialysis. Therefore, the difference in DA increase may not be explained by total drug intake or timing of these infusions. Cotinine has been shown to be a much less potent agonist of nAChRs than nicotine (Tan et al., 2021b), which may also contribute to the difference in DA increase between cotinine and nicotine self-administration. DA levels sampled with microdialysis technique mainly reflect tonic release of DA (Willuhn et al 2010). Therefore, the current study suggests that nicotine self-administration may induce greater tonic release of DA than cotinine self-administration. While nicotine has been shown to increase phasic DA release (Willuhn et al 2010), there is lack of information on effect of cotinine on phasic DA release. Therefore, it remains undetermined whether the different DA increase observed in the current study may be due to difference in phasic DA release between nicotine and cotinine. In addition, cotinine has been shown to be a relatively more potent desensitization agent to ganglion nicotinic receptors than nicotine (Buccafusco et al 2007, 2009). If this is also the case for nicotinic receptors within the mesolimbic DA system, the difference in desensitization effect may contribute to different DA increase during nicotine and cotinine self-administration.

The current study is consistent with previous findings that passive systemic administration of cotinine increased extracellular DA levels within the NAC (Tan et al., 2021a). Cotinine was shown to be self-administered into the VTA, and local injection of cotinine into the VTA increased DA release within the NAC (Tan et al., 2021a). Therefore, DA increase during cotinine self-administration could be mediated, at least in part, by cotinine activation of VTA DA neurons projecting to the NAC. The peak DA increase in the current study was ~130% of baseline, which is comparable to DA levels at ~125% of baseline following passive injection (Tan et al., 2021a). Average number of cotinine infusions was 21 during microdialysis. The unit dose of cotinine during self-administration was 0.03 mg/kg/infusion, which would result in total cotinine intake at ~0.63 mg/kg. This intake level is slightly lower than the 1.0 mg/kg dose used in the passive administration study. These results suggest that there may be other factors contributing to the comparable DA increase between these two studies. One possibility is that the motivational process may contribute to DA increase during active self-administration in addition to cotinine. Intravenous drug infusion during self-administration may be another factor. Direct infusion of cotinine into the vein would result in rapid distribution of cotinine into the brain before it is diluted. On the other hand, subcutaneous injection would lead to slow absorption and distribution of cotinine into the brain after cotinine is diluted during circulation.

Repeated injections of cotinine reduced basal extracellular DA concentrations in the NAC revealed by the quantitative no-net-flux microdialysis study. On the other hand, DA reuptake indexed by extraction fraction was not altered. These results suggest that lower basal extracellular DA concentrations may be due mainly to decreased presynaptic DA release, or increased DA metabolism within the NAC, or both. Interestingly, a previous study using the same no-net-flux technique reported that chronic exposure to nicotine, through either active self-administration or yoked passive administration, decreased basal extracellular DA concentrations within the NAC (Rahman et al., 2004b). Taken together, these findings suggest that cotinine, as the major metabolite of nicotine, may contribute to NIC-induced reduction of basal extracellular DA concentrations in the NAC. Chronic nicotine exposure decreased DA reuptake in the Rahman et al study, whereas cotinine had no effect on DA reuptake (Fig. 3). These results suggest that nicotine and cotinine may produce differential effects on DA reuptake. On the other hand, this difference may also be due to different strains of rats employed in these two studies, i.e., Wistar vs Long-Evans rats. It should be noted that extraction fraction derived from no-net-flux microdialysis is only an indirect measure of DA reuptake. Therefore, it will be valuable in the future to directly investigate DA reuptake kinetics or protein expression of DA transporters following chronic cotinine exposure. Several studies indicated that passive administration of nicotine was shown to lower tissue DA content in the NAC (Fung et al., 1996; Lapin et al., 1989), which may contribute to nicotine-induced decrease of extracellular DA concentrations. It will be interesting to investigate in the future whether cotinine may also reduce tissue DA content.

Cotinine appeared to produce subregion- and target-specific effects on protein levels of DA system markers in the NAC. Specifically, chronic cotinine self-administration reduced D2Rs levels only in the NACcr but not NACsh subregions. Cotinine did not alter levels of D1Rs or TH in either subregion. In contrast, chronic nicotine self-administration failed to alter expression of any target protein in any subregion, although there was a trend of reduction in D2Rs in the NACcr subregion. The effects of nicotine on D1Rs and TH are consistent with previous findings showing no effect of nicotine on D1R binding, D1R gene expression, or TH activity within the NAC (Carr et al., 1989; Fung et al., 1996; Le Foll et al., 2003). On the other hand, findings are inconsistent regarding the effects of chronic nicotine exposure on D2R expression and function. Several studies found that NIC did not alter D2R binding (Carr et al., 1989; Fung and Lau, 1991; Janson et al., 1992) or mRNA levels (Le Foll et al., 2003) in NAC. Some studies reported that nicotine exposure reduced D2R binding (Fung et al., 1996; Janson et al., 1992). One study found increased D2R binding following nicotine exposure (Reilly et al., 1987). Experimental procedures may contribute to these differences, e.g., nicotine dose, administration routes, exposure period, and D2R ligands used in binding studies. Consistent with NIC-induced reduction of D2R binding sites, several studies indicated that nicotine exposure downregulated the function of D2 auto-receptors within the NAC (Balfour et al., 1998; Rahman et al., 2004a). Using microdialysis technique, these studies showed that nicotine exposure attenuated D2 auto-receptor regulation of extracellular DA levels. Taken together, our study suggest that cotinine may contribute to NIC-induced downregulation of D2Rs in the NAC. Our study didn’t identify the relative contribution of presynaptic auto-receptors and post-synaptic D2Rs to cotinine-induced D2R reduction, which will be of interest for future investigation.

Given the extensive effects of cotinine on mesolimbic DA transmission, the current study also examined effects of pharmacological inhibition of DA transmission on reinforcement-related behavioral effects of cotinine. Systemic administration of the D2R antagonist eticlopride dose-dependently reduced number of cotinine infusions and active responses during cotinine self-administration (Fig. 4), suggesting that activation of D2R-mediated DA transmission may contribute to the reinforcing effects of cotinine. These findings are consistent with previous reports that D2R antagonists, e.g., haloperidol and spiperone, attenuated nicotine self-administration (Abela et al., 2019; Corrigall and Coen, 1991). In addition, eticlopride blocked cue-induced reinstatement of cotinine seeking in rats with a history of cotinine self-administration (Fig. 5), which suggests that D2R-mediated DA transmission may play a critical role in cotinine-related relapse-like behaviors. These results agree with previous findings that D2R antagonists including eticlopride and haloperidol reduced cue- or nicotine-induced reinstatement of nicotine seeking behaviors in rats (Abela et al., 2019; Liu et al., 2010). The previous study indicated that eticlopride in the range of 5-10 μg/kg did not alter food self-administration (Liu et al., 2010), suggesting that the effects of eticlopride in the current study may not be due to inhibition of general operant activity. In addition, eticlopride did not alter responses on inactive lever during either cotinine self-administration (Fig. 4) or cue-induced reinstatement of cotinine seeking (Fig. 5). These results further suggest lack of effect of eticlopride on general activity, although caution should be taken with this interpretation due to a potential floor effect as a result of low basal inactive responses.

One weakness is that the current study was performed only in male rats. This design allowed comparison of microdialysis data between the current study and a previous study as discussed above (Tan et al 2021a). In addition, our previous dose-effect study indicates that male and female rats did not differ in lever responses, cotinine infusions, and breakpoints for cotinine during self-administration of cotinine (Ding et al 2021). Given the critical role of the mesolimbic DA system in cotinine reinforcement, these results suggest that cotinine may produce similar effects on the mesolimbic DA system between male and female rats. This hypothesis will be further tested in future studies.

In summary, our study reveals that cotinine self-administration activated the mesolimbic DA system and increased extracellular DA levels in the NAC. As a result, blockade of D2R-mediated DA transmission attenuated cotinine self-administration and relapse-like cotinine seeking behaviors. In addition, chronic cotinine exposure induced adaptive changes in the NAC represented by lower extracellular DA concentrations and reduced protein levels of selective DA receptors. These neuroadaptations may lead to a hypodopaminergic state, which may contribute to maintaining drug self-administration behavior. The extensive effects of cotinine on the mesolimbic DA system suggest that DA transmission may be a shared cellular mechanism involved in effects of both nicotine and cotinine, and that cotinine, as a metabolite of nicotine, may contribute to behavioral and pharmacological effects of nicotine.

Highlights.

  1. Extracellular dopamine levels in the nucleus accumbens increased during active self-administration of cotinine and nicotine

  2. Repeated injections of cotinine decreased basal extracellular concentrations of dopamine in the nucleus accumbens

  3. Chronic cotinine self-administration led to lower protein levels of dopamine D2 receptors in the core subregion of the nucleus accumbens

  4. The dopamine D2 receptor antagonist eticlopride attenuated cotinine self-administration and cue-induced reinstatement of cotinine seeking

Acknowledgements

This study was supported in part by the US National Institutes of Health grant DA044242. The authors declare no conflict of interest. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of National Institutes of Health.

Footnotes

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