Heroin dependency has become a global problem and has caused significant clinical and socioeconomic burdens along with devastating medical consequences. Chronic drug exposure alters the expression and functional activity of 5-hydroxytryptamine (serotonin) 2A receptors (5-HT2ARs) in the brain. Furthermore, pharmacological blockade of 5-HT2ARs reduces cue-induced cocaine craving behaviors. In this study, we explored the influence of 5-HT2ARs on heroin-withdrawal behaviors in mice. Black C57BL/6J mice were given gradually increasing (10–50 mg/kg over 4.5 days) doses of heroin to induce heroin dependency, after which naloxone was given to precipitate withdrawal symptoms. MDL100907, a selective and potent 5-HT2AR antagonist, attenuated naloxone-precipitated withdrawal symptoms in these mice. In addition, 5-HT2AR protein levels increased significantly in the medial prefrontal cortex (mPFC), while phosphorylation of extracellular signal-regulated kinase (p-ERK) decreased in the mPFC after heroin exposure. In conclusion, these results suggest that 5-HT2ARs might be involved in the development of opioid dependency and that pharmacological blocking of 5-HT2ARs might be a new therapeutic strategy for heroin dependency.
Diacetylmorphine (heroin) is one of the most addictive drugs. Heroin use disorders have become a global problem and have caused significant clinical and socioeconomic burdens along with devastating medical consequences. As a morphine derivative, heroin has a high-addictive potency. Once it enters the brain, heroin is rapidly hydrolyzed to 6-acetylmorphine and morphine by serum cholinesterase, which then binds with opioid receptors to activate dopaminergic neurons. Heroin derivatives have a high affinity for opioid receptors in the brain. The opioid receptor superfamily consists of δ (delta), κ (kappa), and μ (mu) opioid receptors (DOR, KOR, and MOR, respectively). MOR is hyperactivated after drug binding and modulates neurotransmitter efflux to reinforce addictive behaviors.
Heroin addiction is increasing, especially in adolescents and young adults. Rehabilitation following heroin addiction takes a long time, and antidrug treatment can cause serious side effects in patients. Furthermore, deaths related to heroin overdose have been increasing at an alarming rate in the United States and Canada. Heroin use can also cause long-term health complications, such as viral infections, chronic obstructive pulmonary disease, and cerebellar dysfunctions.
Heroin disrupts the physiological neurotransmitter signaling cascades, especially those involving 5-hydroxytryptamine (5-HT) neurotransmitters. The 5-HT receptors modulate drug-addictive behaviors. For example, activation of 5-HT 2C receptors (5-HT2CRs) inhibits behavioral sensitization and drug dependency in heroin-treated mice. 5-Hydroxytryptamine (serotonin) 2A receptors (5-HT2ARs) also participate in opiate addiction in rodents and humans. Recently, it was reported that long-term exposure to heroin leads to adaptive changes in 5-HT2ARs in the human brain. 5-HT2ARs activate phospholipase C through Gα q/11, which contributes to inositol 1,4,5-triphosphate and 1,2-diacylglycerol accumulation, intracellular Ca 2+ release, and protein kinase C activation.
5-Hydroxytryptamine (serotonin) 2A receptors regulate neuropsychological functions and have been shown to play a role in a number of neurological disorders, including Alzheimer’s disease, Parkinson’s disease, obsessive–compulsive disorder, schizophrenia, autism spectrum disorder, depression, anxiety, insomnia, and obesity. The 5-HT2AR antagonist volinanserin or MDL100907 (Aventis Pharmaceuticals) has been used to treat neuropsychological dysfunctions in drug-addicted patients, and a synergism between the 5-HT2AR and 5-HT2CR affects the severity of addictive behaviors. 5-HT2ARs are ubiquitously expressed in all brain regions, with the highest receptor densities in the frontal and motor cortices. The dorsomedial prefrontal cortex (PFC), specifically the prelimbic cortical region, plays a pivotal role in drug addiction reinforcement behaviors in rodents. Overexpression of 5-HT2ARs has been associated with chronic dependency on opioids, worsening withdrawal symptoms, and relapse behavior after heroin exposure. These effects may have been mediated by downregulation of ERK/mitogen-activated protein kinase signaling in the PFC.
In this study, we aimed to investigate the impacts of 5-HT2AR antagonists on heroin-withdrawal symptoms in mice. We showed that the selective 5-HT2AR antagonist, MDL100907 (volinanserin), significantly inhibits heroin-induced abnormal motor activities and withdrawal behavior in male mice.
Male C57BL/6J adult mice, aged 8–12 weeks old [license # SCXY (Su) 2011-0003] and weighing 20 ± 2 g, were purchased from Cavion (Cavion Experimental Animal Co., Changzhou, China). Mice were housed in groups of four in 29 cm × 18 cm × 12 cm polycarbonate cages with ad libitum access to water and food under a controlled temperature (23 ± 1°C) and 12-h light/dark cycle (dark phase 7:00 p.m. to 7:00 a.m.). Only male mice were used in this study to avoid the effects of the female estrous cycle on behavioral parameters. All animal procedures followed the National Institutes of Health guidelines for the care and use of research rodents, and the Institutional Animal Care and Use Committee reviewed and approved the study protocol.
Heroin was provided by the Anhui provincial public security department (Hefei, China). MDL100907 was purchased from Sigma-Aldrich (Sigma-Aldrich, United States) and was dissolved in dimethyl sulfoxide (DMSO) to prepare different doses (i.e., 0.01, 0.03, 0.1, and 0.3 mg/kg) for intraperitoneal (IP) injection. The final concentrations of DMSO of these four doses were 0.6, 0.2, 0.06, and 0.02%, respectively.
Naloxone and saline were purchased from The First Affiliated Hospital of Anhui University of Science and Technology (Huainan, China). The time points of MDL100907 treatment were determined based on a previously published report, and doses were optimized in our laboratory. Naloxone (5 mg/kg) was administered by IP injection. To minimize background interference of heroin on naloxone binding to various opioid receptors and spontaneous withdrawal precipitation, naloxone was injected 2 h after heroin was administered. Naloxone was dissolved in 0.9% saline solution and administered at a dose of 5 mg/kg. Saline solution was used as a placebo.
For immunoblotting, we used anti-5-HT2AR (ab16028, Abcam, United States) and anti-phospho-ERK1/2 (Cell Signaling Technology, United States) primary antibodies and a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (SA00001-2, Proteintech, United States). Protein was quantified using a bicinchoninic acid (BCA) assay kit provided by the Beyotime Institute of Biotechnology (Haimen, China).
The open-field test was conducted as described elsewhere. Briefly, mice were placed in the center of a white open field with dimensions of 30 cm × 30 cm × 37.5 cm. The movement and activities of the mice were tracked by a video camera equipped with EthoVision-XT-5.1 behavioral tracking software (Noldus Information Technology, Netherlands). The behavioral parameters tested in this study were distance traveled and duration of immobility at a certain coordinate.
Naïve mice were randomly distributed into three groups (n = 8–18 per group), which received either placebo (saline), M100907 (0.03 mg/kg), or naloxone (5 mg/kg) at designated time points. Behaviors were monitored for 60 min to evaluate drug-induced behavioral changes. The Noldus PhenoTyper system could accurately calculate the horizontal distance traveled, but not fine behaviors. For example, the system could not distinguish between the head and tail when detecting the direction of movement. Behaviors were monitored with video monitoring.
