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. Author manuscript; available in PMC: 2022 Jun 15.
Published in final edited form as: J Psychopharmacol. 2019 Nov 7;34(1):137–144. doi: 10.1177/0269881119885917

Effects of an adenosine A2A agonist on the rewarding associative properties of nicotine and neural plasticity in a rodent model of schizophrenia

Wesley Drew Gill 1, Heath W Shelton 1, Katherine C Burgess 1, Russell W Brown 1
PMCID: PMC9199013  NIHMSID: NIHMS1810269  PMID: 31694445

Abstract

Background:

Adenosine A2a receptors form a mutually inhibitory heteromeric complex with dopamine D2 receptors such that each receptor exhibits lower sensitivity to its agonist after the opposing receptor agonist is bound. This study analyzed the effects of CGS 21680, an adenosine A2A agonist, on nicotine conditioned place preference (CPP) in adolescence using a rodent model of schizophrenia (SZ).

Methods:

Rats were treated from postnatal day (P) 1 to P21 with saline or the dopamine D2/D3 agonist quinpirole (NQ treatment) and raised to P41. After an initial preference test, rats were conditioned with saline or nicotine (0.6 mg/kg base) from P43 to P51. CGS 21680 (0.03 or 0.09 mg/kg) was given 15 minutes before nicotine was administered. The post-conditioning test was administered on P52. On P53, the nucleus accumbens (NAcc) was analyzed for brain-derived neurotrophic factor (BDNF) and glial cell-lined neurotrophic factor (GDNF).

Results:

Results revealed that NQ treatment enhanced nicotine CPP, and both doses of CGS 21680 alleviated this enhancement. Nicotine also resulted in a CPP in controls, which was alleviated by both doses of CGS 21680. BDNF closely followed the behavioral results: CGS 21680 alleviated the enhancement in NAcc BDNF in NQ-treated animals, and eliminated the increase in NAcc BDNF produced by nicotine in controls. NQ-treated animals conditioned to nicotine resulted in an increase of NAcc GDNF, but this was eliminated by CGS 21680. Both BDNF and GDNF correlated with CPP performance.

Conclusions:

Results revealed that an adenosine A2A agonist decreased the rewarding aspects of nicotine and its accompanying neural plasticity changes in a model of SZ.

Keywords: Adenosine A2A receptors, dopamine D2 receptor, nicotine, schizophrenia, BDNF, GDNF, adolescence

Introduction

Schizophrenia (SZ) is a neurological disorder found in approximately 1% of the population (Kahn et al., 2015). The symptoms of this disorder include anhedonia, delusions, hallucinations, movement disorders, and cognitive deficits. A neurobiological hallmark of SZ is increased sensitivity of dopamine D2 receptors (Abi-Dargham et al., 2000). Therefore, antipsychotic treatment of SZ has largely focused on the antagonism of D2 receptors. However, antipsychotic medications have demonstrated limited efficacy and, in many cases, harmful side effects (Atti et al., 2014; Caroff, 2012).

An emerging target in the treatment of SZ is the adenosine system, which may contribute to both the pathophysiology of psychosis and substance abuse comorbidity known to be present in the disorder (Jastrzębska et al., 2014; Lopes et al., 2011; Rial et al., 2014). It has been shown that the nucleoside adenosine modulates both dopamine and glutamate neurosignaling, both of which are abnormal in SZ but are also central in psychostimulant drug addiction (Boison and Aronica, 2015). Adenosine A2A receptors form a heteromeric complex with dopamine D2 receptors in several brain regions, including the striatum (Rial et al., 2014). This interaction between receptors is mutually inhibitory, with each receptor exhibiting lower sensitivity after the agonist for the opposing receptor is bound (Filip et al., 2012). There have been equivocal data on whether adenosine A2A receptor expression is changed in SZ, with studies showing either no change (Urigüen et al., 2009; Zhang et al., 2012) or upregulation in the hippocampus (Hwang et al., 2013). Further, adenosine kinase is the enzyme that breaks down adenosine, and inhibition of adenosine kinase has been shown to have antipsychotic properties (Boison et al., 2012; Shen et al., 2012). Therefore, an adenosine A2A agonist could decrease dopamine D2 receptor sensitivity, potentially having not only antipsychotic properties, but also decreasing the dopaminergic response to commonly abused drugs.

Individuals diagnosed with SZ smoke tobacco at a rate that is four times higher than the total population (Featherstone et al., 2015). Compared with other diagnostic groups, patients with SZ are more likely to be heavy smokers, defined as those who smoke more than one and a half packs a day (Salokangas et al., 2006). Tobacco smoking leads to a diminishment of the quality of life, as well as an increased risk of lung and related cancers, cardiovascular disease, and respiratory illnesses (Board on Population Health and Public Health Practice, 2015). In addition, tobacco smoking may reduce blood levels of certain antipsychotic agents (Carillo et al., 2003; Jann et al., 1986; Perry et al., 1993), and due to this decrease, individuals with SZ who smoke may require a higher daily dose of neuroleptic (Vinarova et al., 1984). Evidence suggests that the reason for increased nicotine abuse in individuals diagnosed with SZ may be that it provides relief from cognitive and affective symptoms (LeDuc and Mittleman, 1995), although this has not been fully delineated. Interestingly, adenosine A2A receptor agonists have been shown to reduce behavioral sensitization to nicotine in rats (Jastrzębska et al., 2014), as well as reduce methamphetamine behavioral sensitization (Shimazoe et al., 2000) and conditioned place preference (CPP; Kavanagh et al., 2015). Cocaine self-administration has also been reduced through pretreatment with an adenosine A2A agonist (Wydra et al., 2015). Therefore, there is a strong literature indicating that the adenosine A2A receptor as a potential target for the treatment of SZ symptoms and comorbid psychostimulant substance abuse.

The present study focused on analyzing changes in the associative properties of nicotine, as well as neural plastic changes produced by nicotine in a rodent model of SZ and the effects of an adenosine A2A agonist on these effects. The rodent model of SZ used is the neonatal quinpirole (NQ) model, which we developed in our laboratory. In this model, rats are neonatally administered quinpirole, a dopamine D2/D3 agonist, from postnatal day (P) 1 to P21. This treatment results in increased D2 receptor sensitivity for the duration of the animal’s lifetime without an increase in the number of D2 receptors (Kostrzewa, 1995). We have shown that NQ treatment results in enhanced behavioral responses to nicotine, as well as increased dopamine release in response to nicotine in the nucleus accumbens (NAcc), a brain area that mediates the rewarding aspects of drugs in the brain (Perna et al., 2013). Further, we have shown that NQ treatment enhanced brain-derived neurotrophic factor (BDNF; Peterson et al., 2017) and glial cell-line derived neurotrophic factor (GDNF) protein in response to nicotine in the NAcc (Brown et al., 2018a, 2018b). BDNF is expressed ubiquitously in the brain and plays an important role in neuroplasticity and has been implicated in the development of addiction (Kowiański et al., 2018). GDNF is associated with dopamine terminal areas in the brain that play an important role in dopaminergic neuronal plasticity. The overall hypothesis is that NQ-induced enhancement of the behavioral response to nicotine can be mitigated by adenosine A2A activation and changes in BDNF and GDNF signaling in the NAcc.

Methods

Subjects

A total of 131 Sprague Dawley rats (71 females, 60 males) that were the offspring of 15 litters served as subjects in this study. The day of birth was treated as P0. One male and one female per litter were assigned to each drug condition to control for within-litter variance, and the litter was with the female dam until weaning at P21. From P21 until the end of the study (P52), animals were socially housed two to four per cage. Animals were housed on a 12-hour/12-hour light/dark cycle, and food was available ad libitum throughout the experiment. All procedures were approved by the East Tennessee State University Animal Care and Use Committee, which is congruent with the National Institutes of Health Guide for the Care and Use of Animals.

Drugs

The drugs used in this study include quinpirole HCl (product # Q102), the adenosine A2A agonist CGS 21680 (product #C141), and nicotine hydrogen tartarate (product # SML 1236), which were all from Sigma–Aldrich (St. Louis, MO).

Neonatal drug treatment

Beginning on P1, animals were administered either saline (0.9% NaCl) or quinpirole HCl (1 mg/kg) intraperitoneally (i.p.) daily through to P21. We have shown that neonatal NQ treatment using this dose and developmental time period results in an increase of dopamine D2 receptor sensitivity throughout the animal’s lifetime (Kostrzewa, 1995). We have verified this effect through both a behavioral yawning test (Thacker et al., 2006) and decreased expression of regulator of G-protein signaling 9 (RGS9) in the dopamine terminal areas medial frontal cortex, dorsal striatum, and NAcc (Maple et al., 2007). RGS9 is a regulator of G-protein signaling at the dopamine D2 receptor, and decreased expression of RGS9 is consistent with both increased dopamine D2 signaling and postmortem data in individuals diagnosed with SZ (Celver et al., 2010; Seeman et al., 2006).

Adolescent drug treatment and group coding

The dose of nicotine (0.6 mg/kg free base) and number of days of conditioning were chosen based on past work demonstrating this dose produced nicotine CPP in adolescent rats (Brown et al., 2018a). The doses of CGS 21680 were chosen based on pilot data that revealed that these two doses minimally reduced locomotor activity, and we aimed to administer a dose that had minimal side effects. In addition, all animals administered CGS 21680 were also conditioned with nicotine because the focus of the study was to analyze the effects of this adenosine A2A agonist on the behavioral effects of nicotine, and not the effects of CGS 21680 alone. In all figures below, the group codes are neonatal drug treatment with neonatal saline treatment (NS) or neonatal NQ treatment. Neonatal drug treatment was followed by the drug condition in adolescence: saline (S), nicotine (N), CGS 21680 0.03 mg/kg followed by nicotine (0.03CGS-N), or CGS 21680 0.09 mg/kg followed by nicotine (0.09CGS-N).

CPP

A three-chambered Plexiglas CPP apparatus was employed that measured 81 cm in length, 33 cm in width and 61 cm in depth. All three chambers were equal in size (27×33 cm) and separated by removable doors with distinct visual and tactile stimuli. The middle chamber was painted gray, whereas the end chambers were painted with either black or white vertical or horizontal stripes.

Animals were given two preconditioning preference tests on both P42 and P43. Conditioning began the day after the initial preference tests on P44, and removable dividers were placed into the apparatus. In the groups to be conditioned with nicotine, the choice of the paired context was balanced across animals and was based on an analysis of the initial preference test such that there were no significant differences in the amount of time spent in the initial context preference across groups. Animals were always given saline in the morning session and placed into the “unpaired” context. In the afternoon session (four hours later), animals were administered saline or an i.p. injection of CGS 21680 (0.03 or 0.09 mg/kg) followed 15 minutes later by an injection of saline or nicotine (0.6 mg/kg base). Approximately 15 minutes after the final injection, rats were placed into their assigned “paired” context for a 10-minute conditioning trial. Conditioning occurred every consecutive day for eight days from P44 to P51.

A post-conditioning preference test was conducted 24 hours later on P52. This test was identical to the initial preference test, with all animals administered two saline i.p. injections spaced apart in time identical to the injections all animals received during conditioning. The dependent measure was the mean percentage time spent in the paired context on the preconditioning preference test subtracted from the percentage time spent in the paired context on the post-conditioning preference test. Brain tissue was harvested on P53, and the NAcc was dissected away from the rest of the brain for BDNF and GDNF enzyme-linked immunosorbent assay (ELISA).

BDNF ELISA

The NAcc was removed from each hemisphere and placed into a 1.5 mL vial and weighed. Tissue was homogenized using a Fisher sonic dismembrator model 500 in a RIPA lysis buffer with a phosphatase/protease inhibitor cocktail (Sigma Aldrich; #P8340, #P0044, #P5726, phenylmethylsulfonyl fluoride (PMSF)). Homogenates were then centrifuged at 14,000 g for 20 minutes at 4°C. The resulting supernatants were refrigerated until the following day when the ELISA was performed. To analyze BDNF, we used the BDNF Emax immunoassay kit from Promega (Madison, WI) and followed the protocol instructions. In brief, anti-BDNF monoclonal antibody (mAb) was added to a carbonate coating buffer (pH 9.7), and 100 μL of the coating buffer was added to each well of a 96-well polystyrene ELISA plate (VWR #62402-942) and incubated overnight at 4°C. All wells were washed using a Tris-buffered saline/Tween-20 wash buffer and incubated at room temperature for one hour. The BDNF standard curve was prepared using the BDNF standard supplied by the manufacturer (1 μg/mL). The standard was diluted in Block & Sample 1× buffer (provided) to achieve a concentration range of 0–1000 pg/mL. The standards and samples were incubated with shaking at room temperature for two hours. Anti-human BDNF pAB was then added to each well plate and incubated at room temperature (two hours), which was followed by incubation (one hour) with anti-IgY horseradish peroxidase (HRP) conjugate. Visualization was achieved by adding TMB (3,3’ 5,5 tetramethylbenzidine) solution to each well followed by an incubation period of 10–12 minutes at room temperature. This reaction was stopped by adding 1N hydrochloric acid to each well (100 μL), and plates were read within five minutes of stopping the reaction. Optical density was measured using a Bio-Tek ELx 800 microplate reader (Winooski, VT) using a 450 nm filter.

GDNF ELISA

The same procedures were followed from the BDNF ELISA, and the protocol provided with the kit (Promega) was followed. However, there are a few differences in the protocol. Anti-GDNF mAb was added to a carbonate coating buffer (pH 7.4), and 100 μL of the coating buffer was added to each well of a 96-well polystyrene ELISA plate (VWR #62402-942) and incubated overnight at 4°C. The incubation time after the standard and samples were added to the plate was six hours, and the incubation time after the anti-human polyclonal antibody (pAb) was overnight at 4°C. All other steps were similar to the BDNF ELISA protocol, and the plate was read using a 450 nm filter.

Statistical analyses

The primary statistic used was the analysis of the variance (ANOVA), and Newman–Keuls was used as the post hoc test to analyze all significant interactions (p=0.05). Between-subjects factors included sex (male, female), neonatal drug treatment (saline, quinpirole), and adolescent drug treatment (saline–saline, saline–nicotine, CGS 0.03 mg/kg–nicotine; CGS 0.09 mg/kg–nicotine). The number of subjects per group for the CPP behavioral test ranged from seven to nine per group. It should be noted that a subset of subjects was used for both the BDNF and GDNF ELISAs, and the number of subjects per drug condition ranged from six to eight.

Results

CPP

The difference in percentage time spent in the paired context on the post-conditioning test versus the mean preconditioning test is presented as a function of neonatal and adolescent drug treatment in Figure 1(a) (females) and (b) (males). As can be observed, neonatal quinpirole treatment enhanced the rewarding associative properties of nicotine, and although the higher dose of CGS 21680 reduced these rewarding properties, the lower dose was less effective, particularly in males. A three-way ANOVA confirmed these observations and revealed significant main effects of neonatal drug treatment (F(1, 130)=17.46, p<0.001) and adolescent drug treatment (F(3, 130)=23.93, p<0.001), a significant two-way interactions of sex×neonatal drug treatment (F(1, 130)=5.09, p<0.02) and neonatal drug treatment×adolescent drug treatment (F(3, 130)=3.81, p<0.01), as well as a significant three-way interaction of sex×neonatal drug treatment×adolescent drug treatment (F(3, 130)=3.32, p<0.02). Based on these statistically significant results, we will analyze males and females separately, followed by a description of sex differences.

Figure 1.

Figure 1.

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Difference in percentage time spent in the paired context on the pre- and post-conditioning test is presented as a function of neonatal and adolescent drug treatment for females (a) and males (b). (a) Female group NQ-SN demonstrated significantly greater CPP than female group NS-SN (**p<0.05). Group NS-SN was significantly greater than NS-SS (*p<0.05). Female groups NS–0.03 CGS-N and NS–0.09CGS-N and groups NQ–0.03CGS-N and NQ–0.09CGS-N were all equivalent and were significantly greater than group NS-SS (*p<0.05). (b) Male group NQ-SN demonstrated significantly greater CPP than male group NS-SN (**p<0.05). Male groups NS-SN, NQ–0.03CGS-N, and NQ–0.09CGS-N were all equivalent and greater than NS-SS controls (*p<0.05). Further, male NQ–0.03CGS-N was not significantly different from male group NQ-SN (#p>0.05). NQ: neonatal quinpirole; CPP: conditioned place preference; NS: neonatal saline; SN: saline–nicotine (0.6 mg/kg base); SS: saline–saline; 0.03CGS-N: 0.03 mg/kg CGS-21680 followed by nicotine (0.6 mg/kg base); 0.09CGS-N: 0.09 mg/kg CGS-21680 followed by nicotine (0.6 mg/kg base).

In Figure 1(a), Newman–Keuls post hoc tests revealed that the female group NQ-N (n=8) was significantly greater than the female group NS-SN (n=8, p<0.05). Therefore, NQ treatment enhanced nicotine CPP. Both female NQ-treated groups administered CGS 21680 (n=9 for both groups) were significantly greater than NS-SS controls (n=9), and equivalent to female group NS-SN (n=8). Therefore, this result indicates that CGS 21680 was effective in reducing the enhanced nicotine CPP produced by NQ treatment, but did not eliminate nicotine CPP. Regarding NS-treated females in Figure 1(a), group NS-SN (n=8) was significantly greater than controls (NS-SS; p<0.05), revealing nicotine-produced CPP in females. However, both female NS-treated groups treated with CGS 21680 (n=8 for both groups) were equivalent to female group NS-SN and significantly greater than group NS-SS (n=9), indicating that neither dose of CGS 21680 blocked nicotine CPP in controls.

Males are represented in Figure 1(b). Male group NQ-N (n=8) was significantly greater than male (n=6) group NS-N (p<0.05), replicating previous work that NQ treatment enhanced the rewarding associative properties of nicotine. Male groups NQ–0.03CGS-N (n=9), NQ–0.09CGS-N (n=7), and NS-SN were all equivalent and significantly greater than male group NS-SS (n=7; p<0.05). Therefore, as with females, CGS 21680 was effective in reducing enhanced nicotine CPP produced by NQ treatment, but did not eliminate nicotine CPP. Both male groups NS–0.03CGS-21680-N (n=8) and NS–0.09CGS-26180-N (n=6) resulted in a lack of nicotine CPP and were equivalent to NSS controls. Therefore, unlike females, CGS 21680 blocked nicotine CPP in NS-treated males compared to NS-treated females

In terms of sex comparisons, both male and female groups NQ-N were equivalent to male group NQ–0.03CGS-N (p>0.05), but were significantly greater than female group NQ–0.03CGS-N (n=9). Therefore, the 0.03 mg/kg dose of CGS 21680 was ineffective in blocking the enhanced effects of NQ on nicotine CPP in males, but this was not the case in females. However, both male and female groups NQ–0.09CGS were equivalent but significantly lower than male and female groups NQ-N, demonstrating that the 0.09 mg/kg dose of CGS 21680 was effective in blocking the enhancing effects of NQ on nicotine CPP in both males and females. However, it is also important to point out that across both males and females, NQ-treated animals administered CGS 21680 demonstrated nicotine CPP, and were significantly above saline-treated controls (p<0.05). In controls, males demonstrated a significantly higher sensitivity to CGS 21680 because both doses of CGS 21680 were effective in blocking nicotine CPP and were equivalent to saline-treated controls.

BDNF ELISA

BDNF (pg/mg) is presented as a function of neonatal and adolescent drug treatment in Figure 2. Through observation, it is clear that male and female groups NQ-N demonstrated an increase in accumbal BDNF compared to all other groups, and CGS 21680 reduced BDNF regardless of dose and neonatal drug treatment. A three-way ANOVA confirmed these observations, and revealed a significant main effects of sex (F(1, 111)=8.54, p<0.004), neonatal drug treatment (F(1, 111)=13.2, p<0.001), and adolescent drug treatment (F(1, 111)=20.26), as well as significant two-way interactions of sex×adolescent drug treatment (F(3, 111)=6.74, p<0.001) and neonatal drug treatment×adolescent drug treatment (F(3, 111)=11.58, p<0.001). Newman–Keuls post hoc analyses revealed male (n=6) and female (n=7) groups NQ-N were equivalent and demonstrated significantly increased levels of BDNF in the NAcc than all other groups (p<0.05). Further, male (n=6) and female (n=8) groups NS-N were also equivalent, and significantly greater than male (n=6) and female (n=6) controls (group NS-S). These effects replicate past work (Peterson et al., 2017) revealing that NQ enhanced the increased BDNF protein response to nicotine, but also that nicotine resulted in a significant increase of BDNF protein in the NAcc. Female groups NS–0.03 CGS (n=8) and NS–0.09CGS (n=6) were equivalent to female group NS-N, and significantly greater than female group NS-SS (p<0.05). This result demonstrated that CGS 21680 was only effective in blocking the enhancement of BDNF produced by nicotine administered to NQ-treated female rats, but CGS 21680 did not block the effects of nicotine on BDNF in NS-treated females. Interestingly, female groups NQ–0.03CGS-N, NS–0.03CGS-N, and NS–0.09CGS-N (p<0.05) demonstrated significantly greater BDNF protein than all male groups given CGS 21680, regardless of neonatal drug treatment, revealing a sex difference in the sensitivity to the adenosine A2A agonist. Finally, we investigated whether BDNF correlated with nicotine CPP using Pearson’s correlation coefficient, and indeed there was a significant correlation (p=0.002, one-tailed).

Figure 2.

Figure 2.

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BDNF (pg/mg weight of tissue) is presented as a function of neonatal and adolescent drug treatment for females (a) and males (b). Both male and female group NQ-SN demonstrated significantly greater BDNF protein than all other groups (**p<0.05). Male and female group NS-SN demonstrated a significant increase of BDNF compared to NS-SS controls (*p<0.05). Female groups NS–0.03CGS-N and NS–0.09CGS-N were equivalent to female group NS-SN and demonstrated significantly greater BDNF protein levels than group NS-SS (*p<0.05). In addition, female groups NQ–0.03CGS-N, NS–0.03CGS-N, and NS–0.09CGS-N demonstrated significantly greater BDNF protein than all male groups given CGS-21680, regardless of neonatal drug treatment (^p<0.05).

GDNF ELISA

GDNF (pg/mg) is presented as a function of neonatal and adolescent drug treatment in Figure 3. Through observation, it appears that both female and male groups NQ-N demonstrated significantly greater GDNF protein levels than all other groups. A three-way ANOVA initially supported this observation. However, there were no effects involving sex as a factor. Therefore, we dropped sex as a factor from this analysis and used a two-way ANOVA, with neonatal drug treatment and adolescent drug treatment as the two factors. In Figure 3, females and males are presented separately in order to be consistent with Figures 1 and 2, but a third panel (Figure 3(c)) has been added that does not include sex as a factor. The two-way ANOVA revealed significant main effects of both neonatal (F(1, 110)=10.17, p<0.002) and adolescent drug treatment (F(3, 110)=3.28, p<0.02) and a significant interaction of neonatal drug treatment×adolescent drug treatment (F(3, 110)=3.60, p<0.01). As indicated in Figure 3(c), both male (n=6) and female (n=6) groups NQ-N demonstrated significantly greater levels of GDNF protein than all other groups (p<0.05). In addition, both NS and NQ-treated groups that received 0.09 mg/kg CGS 21680 (n=7 in all groups except for male NQ–0.09CGS21680-N, n=9) combined with nicotine demonstrated significantly higher GDNF levels than controls (p<0.05). There were no other significant differences between groups. Thus, replicating previous work, nicotine administered to controls did not produce a significant increase in GDNF, although NQ combined with adolescent nicotine treatment did result in a significant increase of accumbal GDNF. However, both the 0.03 and 0.09 mg/kg dose of CGS 21680 were effective in decreasing the increase in GDNF observed in NQ animals conditioned to nicotine. As with BDNF, we investigated whether GDNF correlated with nicotine CPP, and indeed there was a significant correlation between these two dependent measures (p=0.016, one-tailed).

Figure 3.

Figure 3.

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GDNF (pg/mg weight of tissue) is presented as a function of neonatal and adolescent drug treatment for females (a) and males (b) and females and males combined (c). The rationale for panel (c) is that there were no sex differences; males and females were combined in the analysis, and all statistical differences are indicated in this graph. Both male and female group NQ-SN demonstrated significantly greater GDNF protein than all other groups (**p<0.05), and NS–0.09CGS-N was significantly greater than controls (*p<0.05).

Discussion

This study demonstrates that NQ treatment enhanced the associated properties of nicotine, consistent with previous work (Brown et al., 2018a; Peterson et al., 2017). Most importantly, enhanced nicotine CPP produced by NQ was alleviated by the adenosine A2A agonist CGS 21680, regardless of the dose (0.3 or 0.9 mg/kg). As can be observed in Figure 1, CPP was reduced to statistically equivalent levels in animals treated neonatally with saline conditioned with nicotine. This result demonstrates that an adenosine A2A agonist is effective in reducing the enhanced rewarding properties of nicotine in NQ rats, but not effective at completely reducing the rewarding aspects of nicotine at the doses used. In rats neonatally treated with saline (NS), it was observed that CGS 21680 was effective in alleviating nicotine CPP, but only in males. In females treated neonatally with saline, CGS 21680 was not effective in reducing nicotine CPP. These results demonstrate a sex difference in the behavioral response to CGS 21680 and suggest a sex difference in the adenosine system. There have not been any studies to demonstrate sex differences in the response to CGS 21680 or any adenosine A2A agonist and its interaction with psychostimulants. This work is consistent with past work that has shown that CGS 21680 was effective in reducing the behavioral response to nicotine in adult male rats, although doses from this past study were higher than those used here (0.1–0.4 mg/kg) (Jastrzębska et al., 2014). In addition, past work has shown CGS 21680 reduces nicotine-induced dopamine release in striatal nerve terminals (Garção et al., 2013).

The analyses of BDNF largely echoed the behavioral results. NQ treatment also enhanced the accumbal BDNF protein response to nicotine treatment in both males and females, which was reduced in groups treated with either dose of CGS 21680. However, this reduction was more extreme in NQ-treated males, where both doses of CGS 21680 were effective in lowering BDNF expression to levels comparable to levels of NS-treated controls. In female NQ-treated rats, the 0.09 mg/kg dose of CGS 21680 reduced BDNF expression in NQ rats to control levels, but the 0.03 mg/kg dose of CGS 21680 only reduced the expression to levels comparable to NS rats treated with nicotine at the time of testing. Regarding BDNF, males appear to demonstrate an increased sensitivity to the adenosine A2A agonist. In NS-treated females, nicotine increased accumbal BDNF compared to NS-treated controls, replicating previous work (Peterson et al., 2017). However, both doses of CGS 21680 failed to block this effect. Therefore, it appears that in both CPP and in BDNF protein, there is a sex difference in the response to CGS 21680, with males demonstrating an increased sensitivity both behaviorally and in the BDNF response compared to females. There are not any previous studies that have analyzed sex differences in the interaction of CGS 21680 or other adenosine A2A agonists and psychostimulants.

Several studies have shown that adenosine A2A activation is involved in increasing BDNF expression in cultured neurons (Jeon et al., 2011; Komaki et al., 2012). Further, the A2A receptor is involved in inducing long-term potentiation related to BDNF activation in the hippocampus (Fontinha et al., 2008; Rodrigues et al., 2014). Nevertheless, there have not been any studies to analyze adenosine A2A activation and its influence on BDNF in vivo. Based on the current data and past work, it appears that adenosine A2A activation may affect plasticity differently in the NAcc versus the hippocampus. This would be sensible based on the fact that A2A receptors form a mutually inhibitory heteromer with the dopamine D2 receptor, which are in high density in the NAcc (~70 fmol/mg) but in lower density in the hippocampus (~40–45 fmol/mg; Tarazi and Baldessarini, 2000). In addition, the interaction of CGS 21680 with nicotine could have a different interaction with BDNF compared to these past studies, which analyzed the adenosine A2A agonist without the influence of a psychostimulant. Regardless, the interaction of the adenosine system with BDNF appears intriguing and likely complex.

Regarding GDNF, NQ treatment enhanced the accumbal GDNF response to nicotine in both males and females, but this was not observed in NS-treated rats conditioned with nicotine. Rats treated neonatally with saline did not show significant differences in GDNF expression when treated with saline or nicotine at the time of testing (Figure 3). However, NQ animals conditioned with nicotine demonstrated significant increases in accumbal GDNF, an effect that was alleviated by both doses of CGS 21680 across both sexes. The increase in GDNF observed in the NQ-treated rats conditioned with nicotine replicates previous data from our laboratory showing a similar effect (Brown et al., 2018a), as well as a lack of an increase NS-treated rats conditioned with nicotine. Past work has shown that nicotine increased GDNF in the NAcc up to eight hours after nicotine treatment (Takarada et al., 2012), but not 24 hours later, suggesting that GDNF may increase as dopaminergic tone is increased after drug treatment, and decreases over time much more rapidly than BDNF. There is much less information on the relationship between adenosine A2A receptors and GDNF. In striatal synaptosomes, two past studies have shown that GDNF stimulation of dopamine and glutamate release is dependent or can be potentiated through activation of adenosine A2A receptors with CGS 21680 (Gomes et al., 2006, 2009). These past studies help to explain the increase in accumbal GDNF observed in the NS-treated group administered the higher 0.09 mg/kg dose of CGS 21680 in combination with nicotine. Interestingly, this same potentiation did not occur in NQ-treated rats administered either dose of CGS 21690 in combination with nicotine. Thus, increased dopamine D2 receptor sensitivity may influence the relationship between nicotine and GDNF.

The neurotrophic factor data here suggest that BDNF is reliably increased by nicotine 48 hours after nicotine treatment, whereas GDNF is only increased in NQ-treated rats administered nicotine at this same time point. Based on these effects, we hypothesize that BDNF may play a larger role in the long-term synaptic changes and synaptic maintenance that underlies nicotine addiction. GDNF may be important in the relatively immediate changes in dopamine synaptic plasticity once nicotine is administered, but clearly does not remain increased unless there is an increase in dopaminergic tone, as is produced by NQ treatment. Several past studies have analyzed the roles of BDNF and/or GDNF in drug self-administration through injection of BDNF directly into the NAcc, or increased expression of GDNF via viral vector into the ventral tegmental area. In both cases, infusion or increased expression of these neurotrophic factors enhanced cocaine (Graham et al., 2007) or heroin self-administration (Lu et al., 2009; Pickens et al., 2011), respectively. However, the current study analyzed endogenous BDNF and GDNF. Importantly, we have yet to analyze GDNF at a time point shorter than 48 hours after nicotine injection, and this conjecture is based on the past work by Takada et al. (2012).

Overall, this study demonstrated that NQ treatment in rats enhanced the effects of nicotine in regard to its associative rewarding properties, as well as its effect on two different neurotrophic factors (BDNF and GDNF), which have been implicated in drug addiction. This is the first study to demonstrate that the enhanced effects of NQ on the rewarding aspects of nicotine can be alleviated by an adenosine A2A receptor agonist. This finding suggests that the adenosine A2A receptor is a potential target to reduce the rewarding effects of nicotine and possibly be applied as a treatment for nicotine abuse comorbidity in SZ. Findings in this study also suggest that targeting the A2A receptor as a treatment for nicotine addiction in otherwise healthy individuals could provide alternate treatment options, as nicotine CPP in male control rats and elevated BDNF expression following nicotine treatment in male control rats were both alleviated by CGS 21680.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: a portion of this work was supported by a grant from the East Tennessee State University School of Graduate Studies to W.D.G.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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