Methamphetamine (MA) is an amphetamine-based psychostimulant, which is one of the most common drugs abused by illicit drug users. In humans, chronic drug abuse leads to flaws in executive functioning, information processing, and memory (Scott
Several studies have focused on the role of glutamate in relation to MA addiction (Nash and Yamamoto, 1992; Abekawa
Histone deacetylases (HDACs) are classified into Class I (HDAC1, HDAC2, HDAC3 and HDAC8), Class II (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9 and HDAC10), Class III (sirtuins, SIRT 1-7) and Class IV (HDAC11) (Seto and Yoshida, 2014). HDAC has been extensively studied for synaptic plasticity and long-term memory as the core of drug addiction (Vecsey
Accordingly, HDACs inhibitors represent important agents for the development of pharmacological therapies combating the negative effects of psychostimulant exposure. MA-induced deacetylation of α-tubulin, an indicator of cellular structural loss in endothelial cells was also prevented by treatment with HDAC inhibitors (Fernandes
MeBib is a HDAC6 inhibitor derived from the benzimidazole scaffold. Benzimidazole is a heterocyclic aromatic compound, consisting of a phenyl ring fused with an imidazole ring. Numerous benzimidazole-containing compounds have attracted the attention of drug discovery efforts due to a wide spectrum of pharmacological activities (Lazer
In this study, we investigated whether the HDAC6 inhibitor MeBib reduced the self-administration of MA in rats. We also investigated the effect of MeBib on the molecular mechanism of neurotransmission by glutamate receptors, which are induced by MA.
In this study, male Sprague-Dawley rats (200-250 g, 8 weeks of age, Hyochang Company, Daegu, Korea) were used. Rats were individually housed for free access to food and water. In an animal room with a constant light and dark cycle, the temperature and the humidity were kept constantly at 21-23°C and 55-65% each. The MA self-administration study was conducted during their dark cycle. Experimental procedures and animal care were in accordance with the Institutional Animal Care and Use Committee at the Keimyung University (Daegu, Korea; EXP-IRB number: KM_2018-007; the date of approval: 12 June 2018) The experiments were carried out in accordance with the Keimyung university’s scientific research guidelines and regulations.
Methamphetamine (MA) was purchased from the Ministry of Food and Drug Safety (Daejeon, Korea). MeBib was purified and received from Dr. Seo Young-ho, a professor of the college of Pharmacy, Keimyung University. MA was dissolved in sterile saline. The purity of MeBib is >95%. MeBib was dissolved in 10% DMSO. MeBib (10 mg/kg/mL) and its vehicle (10% DMSO in saline) were injected intraperitoneally in rats.
Rats were tested in operant conditioning chambers (Med Associates, St. Albans, VT, USA). Each chamber was equipped with two response levers (4.8×1.9 cm), a cue light (3 W, 28 V), and a house light (3 W, 28 V). A cue light was located above each response lever. The floor of the chamber was lined with wood chip bedding and covered with a metal grid. The chambers were placed within a sound-attenuating wooden enclosure, equipped with a ventilation fan. A syringe in the infusion pump (Razel Scientific Instruments, Stamford, CT, USA) was connected to a fluid swivel (Med Associates) with Tygon tubing. A Tygon tubing shield with a metal spring connected the swivel to the animal’s catheter cannula assembly.
45 mg food pellets (Bio-Sub, French Town, NJ, USA) were used for lever press acquisition. The rats were fasted for about 1 day and then trained to press the lever to obtain 45 mg food pellets for 1 h to facilitate the acquisition of self-administration under food restriction. Rats which consumed all 100 food pellets during the 3 consecutive sessions had passed the lever press training and had a preparation period for intravenous catheterization surgery. Rats were fed an unlimited amount of food and water in the home cage for at least 2 days prior to intravenous catheterization surgery.
Rats which had undergone lever press training were anesthetized with a pentobarbital anesthesia (50 mg/kg, i.p.) and the chronic indwelling jugular catheter (Instech Solomon, Plymouth Meeting, PA, USA) was implanted into the right jugular vein. Catheters were secured to the jugular vein with Mersilene surgical mesh (Ethicon Inc., Somerville, NJ, USA) and exteriorized via a skin incision in the animal’s back through a 22-gauge stainless-steel cannula (Plastics One, Roanoke, VA, USA), fixed to the head assembly with dental cement, and secured with a Prolene surgical mesh (Ethicon Inc.). Daily washing was performed with 0.2 mL saline containing heparin (20 U/mL) and gentamicin sulfate (0.33 mg/mL) to prevent clogging of the catheter due to blood flow.
Rats undergoing intravenous catheterization surgery were subjected to a lever press response to MA (0.1 mg/kg per infusion over 5 s in a 0.1 mL volume) at a fixed rate of 1 (FR1) after a 1 week recovery period. When the active lever was pressed, the cue indicator of the active lever was turned on for 5 s and the house light was turned off. After saline or MA was injected, the house light was turned off for 10 s timeout. Presses on the inactive lever were recorded but had no programmed consequence. Typically, this required 14 days following the initiation of MA self-administration. First, we tried to find out whether MeBib is a drug that helps suppress the stability of MA induction. Thus, rats were pretreated with MeBib (10 mg/kg/mL) or vehicle by intraperitoneal injection 30 min before starting the self-administration test from the start date. As a criterion for MA dependency, self-administration experiments were performed until a stable response pattern was established at less than 10% variance in the total number of responses for three consecutive sessions. Subsequently, the rats were sacrificed and samples were collected 1 h after completion of the MA self-administration.
We performed a food reinforcement experiments based on the previous study (Jang
The brain was cut using a cryostat microtome and both the dorsal and ventral hippocampal tissues were taken and used for data analysis. These tissues were homogenized in protein extraction buffer containing protease and phosphatase inhibitors. Protein concentrations were measured using a PierceTM BCA Protein assay kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of total protein (30 µg) were separated on an 8% or 12% SDS-PAGE gel and transferred to a PVDF membrane (Merck Millipore, MA, USA). After being blocked in 5% milk for 1 h at room temperature, membranes were incubated overnight at 4°C with the following primary antibodies against AMPA receptor 2 (GluA2; 1:1000, 13607, Cell Signaling, Beverly, MA, USA), BDNF (1:1000, ab108319, Abcam, Cambridge, UK), phospho-CREB (ser133,1:1000, 9198, Cell Signaling, Beverly, MA, USA), CREB (1:1000, 4820, Cell Signaling) phospho-p44/42 MAPK (ERK1/2; 1:1000, 9101, Cell Signaling), p44/42 MAPK (ERK1/2; 1:1000, 9102, Cell Signaling), NMDA receptor2B (GluN2B; 1:1000, 4212, Cell Signaling), and β-Actin (1:1,000, 4970, Cell Signaling). Membranes were washed in TBST for 20 min and probed with Horseradish Peroxidase (HRP) conjugated secondary antibodies: Anti-Mouse IgG (1:5,000, 7076, Cell Signaling) and Goat Anti-Rabbit IgG (1:5,000, 7076, Cell Signaling); Rabbit Anti-Goat IgG H&L (HRP) (ab6741, Abcam). After chemiluminescent substrate (Amersham Bioscience, Buckinghamshire, UK) was distributed on the membrane, images were captured using a Fusion SOLO S system (Vilber Lourmat, Eberhardzell, Germany). Protein bands were quantified with ImageJ software (National Institute of Mental Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij). The values were normalized to β-actin intensity levels.
Statistical significances between groups were determined using the two-way analysis of variance (ANOVA) with repeated measures (RM), followed by Bonferroni’s post hoc test, respectively. Analysis was conducted using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). Data were presented as mean ± SEM for each group (n=6 per group). Statistical significance was accepted for
Rats were divided into 4 groups (n=6). On each of 14 consecutive days, rats were allowed to acquire a lever press response for MA (0.1 mg/kg per infusion over 5 s in a 0.1 mL volume) under a fixed-ratio 1 (FR1) schedule. When the active lever is pressed, the cue light above the active lever turns on for 5 s, the house light turns off. After each infusion, an additional timeout period of 10 s was added with the house light off. Pressing of the inactive lever is recorded, but no programmed result. The daily averages for active lever presses, inactive lever presses, and infusions for MA self-administering rats over a 14-day period are shown in Fig. 1. The MA self-administered rat group (MA group) showed a higher MA seeking behavior with a significant number of infusion lever presses compared to the control group (
To test whether MeBib affects generalized behavioral responses, food reinforcement training was conducted using sucrose pellets. MeBib (10 mg/kg) and its vehicle (10% DMSO in saline) were injected intraperitoneally 30 min before the food reinforcement test. As shown in Fig. 2, MeBib did not affect food reinforcement training (
Glutamate release by MA is known to activate N-methyld-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors that induce excitotoxicity (Simoes
According to previous studies, the hippocampus participates in the formation and consolidation of new memories mediated via MAPK and ERK (Cammarota
CREB signaling pathway, including the NMDA receptor, plays an important role in MA-induced behavioral sensitization and rewarding effects (Nestler, 2001; Brami-Cherrier
Drug-induced changes in serum brain-derived neurotrophic factor (BDNF) in the cortex and hippocampus have been observed in MA users (Kim
MA is one of the most commonly abused drugs by illicit drug users worldwide. Many studies reported that MA abuse leads to cognitive impairment, and memory deficits. Besides, MA induces neuronal degeneration of the brain area linked to cognitive function. Therefore, understanding the synaptic changes and neuronal mechanisms that are affected by MA exposure has become imperative. In this study, we elucidated the effects of HDAC6 inhibitor MeBib on MA self-administration. Our results demonstrated that the number of active lever presses in MA self-administered rats was reduced by pretreatment with MeBib, which also suppressed the expression of the NMDA receptor GluN2B and inhibited the ERK/CREB/BDNF signaling pathway.
We found that the expression of GluA2 and GluN2B was increased in the hippocampus of MA self-administered rats. These data are consistent with previous reports suggesting that the expression of NMDA receptor was regulated after MA exposure and modulated the excitotoxic effect (Bowyer and Ali, 2006; Kalivas and Volkow, 2011). Another study also reported that MA self-administration increased the NMDA receptor currents and surface expression of the GluN2B subunit (Mishra
Previously, researchers reported that ERK activation occurs as a calcium-dependent NMDAR-mediated response, which transmits extracellular stimuli to the nucleus and regulates synaptic plasticity and learning (Huo
Next, we found that CREB, one of the most important transcription factors mediating various neuronal connections (Sakamoto
BDNF release in the hippocampus increases tyrosine phosphorylation of the GluN2B subunit at Tyr-1472, which is a critical step in the stimulation of synaptic transmission (Lin
Collectively, we found that MA self-administration induced GluN2B expression, associated with the activation of sequential ERK/CREB/BDNF pathway in an animal model; however, it was suppressed by HDAC6 inhibitor MeBib (Fig. 5). Thus, we suggest that MeBib prevents MA seeking response induced by MA and therefore, represents a potent candidate as an MA addiction inhibitor.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1A6A1A03011325), and was supported by the Keimyung University Research Grant of 2018 (B.D.P).