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Cannabidiol Counteracts Amphetamine-Induced Neuronal and Behavioral Sensitization of the Mesolimbic Dopamine Pathway through a Novel mTOR/p70S6 Kinase Signaling Pathway

Justine Renard

1 Addiction Research Group,

2 Department of Anatomy and Cell Biology, and

Michael Loureiro

1 Addiction Research Group,

2 Department of Anatomy and Cell Biology, and

Laura G. Rosen

1 Addiction Research Group,

2 Department of Anatomy and Cell Biology, and

Jordan Zunder

1 Addiction Research Group,

2 Department of Anatomy and Cell Biology, and

Cleusa de Oliveira

2 Department of Anatomy and Cell Biology, and

Susanne Schmid

2 Department of Anatomy and Cell Biology, and

Walter J. Rushlow

1 Addiction Research Group,

2 Department of Anatomy and Cell Biology, and

3 Department of Psychiatry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada

Steven R. Laviolette

1 Addiction Research Group,

2 Department of Anatomy and Cell Biology, and

3 Department of Psychiatry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada

Author contributions: J.R., W.J.R., and S.R.L. designed research; J.R., M.L., L.G.R., J.Z., S.S., and C.d.O. performed research; J.R., M.L., W.J.R., and S.R.L. analyzed data; J.R., W.J.R., and S.R.L. wrote the paper.

Abstract

Schizophrenia-related psychosis is associated with disturbances in mesolimbic dopamine (DA) transmission, characterized by hyperdopaminergic activity in the mesolimbic pathway. Currently, the only clinically effective treatment for schizophrenia involves the use of antipsychotic medications that block DA receptor transmission. However, these medications produce serious side effects leading to poor compliance and treatment outcomes. Emerging evidence points to the involvement of a specific phytochemical component of marijuana called cannabidiol (CBD), which possesses promising therapeutic properties for the treatment of schizophrenia-related psychoses. However, the neuronal and molecular mechanisms through which CBD may exert these effects are entirely unknown. We used amphetamine (AMPH)-induced sensitization and sensorimotor gating in rats, two preclinical procedures relevant to schizophrenia-related psychopathology, combined with in vivo single-unit neuronal electrophysiology recordings in the ventral tegmental area, and molecular analyses to characterize the actions of CBD directly in the nucleus accumbens shell (NASh), a brain region that is the current target of most effective antipsychotics. We demonstrate that Intra-NASh CBD attenuates AMPH-induced sensitization, both in terms of DAergic neuronal activity measured in the ventral tegmental area and psychotomimetic behavioral analyses. We further report that CBD controls downstream phosphorylation of the mTOR/p70S6 kinase signaling pathways directly within the NASh. Our findings demonstrate a novel mechanism for the putative antipsychotic-like properties of CBD in the mesolimbic circuitry. We identify the molecular signaling pathways through which CBD may functionally reduce schizophrenia-like neuropsychopathology.

SIGNIFICANCE STATEMENT The cannabis-derived phytochemical, cannabidiol (CBD), has been shown to have pharmacotherapeutic efficacy for the treatment of schizophrenia. However, the mechanisms by which CBD may produce antipsychotic effects are entirely unknown. Using preclinical behavioral procedures combined with molecular analyses and in vivo neuronal electrophysiology, our findings identify a functional role for the nucleus accumbens as a critical brain region whereby CBD can produce effects similar to antipsychotic medications by triggering molecular signaling pathways associated with the effects of classic antipsychotic medications. Specifically, we report that CBD can attenuate both behavioral and dopaminergic neuronal correlates of mesolimbic dopaminergic sensitization, via a direct interaction with mTOR/p70S6 kinase signaling within the mesolimbic pathway.

Introduction

Schizophrenia is a devastating psychiatric disorder characterized by delusions, hallucinations, and cognitive filtering disturbances (McGrath et al., 2008). For decades, schizophrenia has been treated using antipsychotic drugs targeting dopamine (DA) receptors. However, there are significant side effects associated with currently available antipsychotics (Awad and Voruganti, 2004), and no mechanistically novel treatment has emerged to replace them. Disturbances in the brains endocannabinoid system are increasingly recognized as etiological factors underlying schizophrenia-related symptoms (Tan et al., 2014). Exposure to extrinsic cannabinoids, such as marijuana (MJ), can induce psychotomimetic effects acutely, or following chronic neurodevelopmental exposure (D’Souza et al., 2004; Renard et al., 2014). Nevertheless, MJ contains a complex mixture of phytochemicals, the two largest being Δ-9-tetra-hydrocannabinol (THC) and cannabidiol (CBD). THC and CBD possess highly distinct pharmacological and psychotropic profiles. Whereas THC exposure is associated with psychotomimetic effects, recent evidence suggests that CBD, a nonpsychoactive component of MJ, has promising potential as an antipsychotic treatment.

In preclinical models of schizophrenia, CBD reduces schizophrenia-like behaviors induced by psychotomimetic drugs and has a neuropharmacological profile similar to atypical antipsychotics. For example, CBD is more effective than haloperidol and similar to clozapine, in attenuating ketamine-induced hyperlocomotion (Moreira and Guimarães, 2005). CBD has been shown also to reverse MK-801-induced sensorimotor gating deficits in mice (Long et al., 2006) and MK-801-induced social withdrawal in rats (Gururajan et al., 2011). CBD is comparable with haloperidol in terms of reducing apomorphine-induced hyperlocomotion, but in contrast to haloperidol, is devoid of extrapyramidal side effects, even at high doses (Zuardi et al., 1991). A recent clinical trial has confirmed that CBD possesses properties similar to antipsychotic medications and effectively reduces psychotic symptoms with equal efficacy to traditional medications, but with significantly fewer side effects (Leweke et al., 2012). However, the neuronal and molecular mechanisms through which CBD may exert these effects are entirely unknown.

At the molecular level, considerable evidence links schizophrenia with disturbances in signaling pathways associated with DA receptor function. These include the wingless (Wnt) signal transduction pathway, protein kinase B (Akt), glycogen synthase kinase-3 (GSK-3), and β-catenin. Importantly, both typical and atypical antipsychotic medications can activate these pathways (Alimohamad et al., 2005b; Sutton et al., 2007; Freyberg et al., 2010). In addition, increasing evidence identifies the mammalian target of rapamycin (mTOR) pathway, which regulates downstream activity of p70S6 kinase (p70S6K), as a crucial molecular substrate underlying schizophrenia-related psychopathology and antipsychotic efficacy (Gururajan and van den Buuse, 2014; Liu et al., 2015).

In the present study, we used amphetamine (AMPH)-induced sensitization and sensorimotor gating in rats, two preclinical behavioral procedures relevant to schizophrenia-related psychopathology, combined with molecular analyses and in vivo neuronal electrophysiology to characterize the potential antipsychotic-like properties of CBD within the mesolimbic system. We report that CBD attenuates AMPH-induced psychomotor sensitization and AMPH-induced sensorimotor gating deficits. Furthermore, we report that CBD produces its effects through modulation of the phosphorylation states of the mTOR/p70S6K signaling pathways in the nucleus accumbens shell (NASh). Finally, we demonstrate that CBD within the NASh can normalize AMPH-induced dysregulation of mesolimbic DA neuron activity states.

Materials and Methods

Animals.

Male Sprague Dawley rats (300–350 g) were obtained from Charles River Laboratories. At arrival, rats were housed under controlled conditions (12 h light/dark cycle, constant temperature, and humidity) with access to food and water ad libitum. All procedures were performed in accordance with Governmental and Institutional guidelines for appropriate animal care and experimentation.

Surgical procedures.

Rats were anesthetized with an intraperitoneal (i.p.) injection of ketamine (80 mg/ml)-xylazine (6 mg/kg) mixture. Meloxicam (1 mg/kg; s.c.) was administered postoperatively to reduce pain and inflammation. Rats were placed in a Kopf stereotaxic device and stainless steel guide cannulae (22-gauge) were implanted bilaterally into the NASh using flat skull stereotaxic coordinates as follows (12° angle, in mm from bregma): anteroposterior 1.8 mm, lateral ±2.6 mm, dorsoventral −7.4 mm from the dural surface. Guide cannulae were held in place using jeweler’s screws and dental acrylic cement. Rats were single-housed after surgeries.

Drug preparation and administration

One week after surgery, rats received Intra-NASh bilateral infusions of CBD (Tocris Bioscience, 100 ng in 20% DMSO and 80% NaCl (0.9%); 0.50 μl per side), vehicle (VEH, 20% DMSO and 80% NaCl (0.9%); 0.50 μl per side), coadministration of Torin2 (Tocris Bioscience, 40 ng in 20% DMSO and 80% NaCl (0.9%); 0.50 μl per side) and CBD (Torin2+CBD) and coadministration of PF 4708671 (PF, Tocris Bioscience, 100 ng in 50% DMSO and 50% NaCl (0.9%); 0.50 μl per side) and CBD (PF+CBD) over 5 consecutive days using an injection cannulae connected to a Hamilton syringe with Teflon tubing and a microinfusion pump. A total volume of 0.5 μl per side was delivered over a period of 1 min. Microinjectors were left in place for an additional 1 min following drug infusion to ensure adequate diffusion from the tip. Immediately following the microinfusions, the rats received an intraperitoneal injection of d -AMPH sulfate (AMPH; Sigma-Aldrich; 5 mg/kg in 0.9% NaCl) or VEH (0.9% NaCl). Following the final AMPH or VEH treatment injection (on day 5), rats were left undisturbed in home cages until test day (locomotor activity or prepulse inhibition [PPI]) on sensitization day 16, when rats received the VEH or AMPH challenge (1 mg/kg; i.p.).

AMPH-induced hyperlocomotor activity

Locomotor activity, stereotypy, and rearing counts were recorded for 60 min in an automated open-field activity chamber (Med Associates). The final number of rats in each group was as follows: VEH/Intra-NASh VEH group (VEH/VEH), n = 9; VEH/Intra-NASh CBD group (VEH/CBD), n = 10; AMPH/Intra-NASh VEH group (AMPH/VEH), n = 8; AMPH/Intra-NASh CBD group (AMPH/CBD), n = 10; AMPH/Intra-NASh Torin2+CBD, n = 8; and AMPH/Intra-NASh PF+CBD, n = 8.

Protein extraction and Western blotting

After completion of locomotor sensitization tests, rats received an overdose of sodium pentobarbital (240 mg/kg, i.p., Euthanyl). Under deep anesthesia, rats were decapitated and brains removed and frozen. Coronal sections (60 μm) containing the nucleus accumbens (NAc) were cut on a cryostat and slide mounted. Some sections were stained with cresyl violet for microinfusion site verification with light microscopy. For remaining sections, bilateral micropunches of the NAc, adjacent to, but not including injection sites, were obtained for protein isolation. The Western blotting procedure was performed as described previously (Lyons et al., 2013). Primary antibody dilutions were as follows: α-tubulin (1:120,000; Sigma-Aldrich), phosphorylated GSK-3α/β ser21/9 (p-GSK-3α/β; 1:1000; Cell Signaling Technology), total GSK-3α/β ser21/9 (t-GSK-3α/β; 1:1000; Cell Signaling Technology), phosphorylated Akt Ser473 (p-Akt; 1:1000; Cell Signaling Technology), total Akt (t-Akt; 1:1000; Cell Signaling Technology), β-catenin (1:10,000; Sigma-Aldrich), phosphorylated mTOR ser2448 (p-mTOR;1:2000; Cell Signaling Technology), total mTOR (t-mTOR; 1:2000, Cell Signaling Technology), phosphorylated p70S6K thr389 (p-p70S6K; 1:1000; Cell Signaling Technology), and total p70S6K (t-p70S6K; 1:1000; Cell Signaling Technology). Secondary antibodies (Thermo Scientific) were all used at a dilution of 1:20,000.

PPI of startle reflex

Rats were acclimated to the startle chambers (Med Associates) for 5 min over 3 d. On the last day of acclimation, rats were tested in an input/output (I/O) function consisting of 12 increasing startle pulses (from 65 to 120 dB, 5 dB increments) to determine the appropriate gain setting for each individual rat. The testing procedure consisted of the following phases: the acclimation phase, a habituation phase (Block 1), and PPI measurement (Block 2). During acclimation, rats were exposed to the chambers and white background noise (68 dB) for 5 min. During Block 1, 10 pulse alone trials (110 dB white noise, 20 ms duration) were delivered at 15–20 s intertrial intervals. Block 2 consisted of 9 different trials presented 10 times in a pseudo-randomized order at 15–20 s intervals: 10 pulse-alone trials, and 10 of each of the three different prepulse-pulse trial types (72, 76, 80) with interstimulus intervals of 30 and 100 ms. Pulse-alone trials consisted of a startle stimulus-only presentation, whereas prepulse-pulse trials consisted of the presentation of a weaker nonstartling prepulse (white noise, 20 ms duration) before the startling stimulus. PPI was calculated for each animal and each trial condition as PPI (%) = (1 − average startle amplitude to pulse with prepulse/average startle amplitude to pulse only) × 100. The final number of rats in each group was as follows: VEH/Intra-NASh VEH group (VEH/VEH), n = 8; VEH/Intra-NASh CBD group (VEH/CBD), n = 9; AMPH/Intra-NASh VEH group (AMPH/VEH), n = 9; AMPH/Intra-NASh CBD group (AMPH/CBD), n = 9; AMPH/Intra-NASh Torin2+CBD, n = 10; and AMPH/Intra-NASh PF +CBD, n = 10.

In vivo ventral tegmental area (VTA) neuronal recordings

Effects of Intra-NASh VEH, -CBD, -CBD+Torin2 or – CBD+PF 4708671 (PF) pretreatment on AMPH-induced PPI deficit. Exposure to AMPH (5 d, 5 mg/kg) followed by an 11 day sensitization period caused PPI deficit in Intra-NASh VEH-pretreated rats. Intra-NASh CBD pretreatment significantly decreases the AMPH-induced PPI deficit observed in Intra-NASh VEH-pretreated rats. Intra-NASh CBD cotreatment with either Torin2 or PF significantly reverses CBD-induced increases in PPI. VEH/Intra-NASh VEH (VEH/VEH), n = 8; VEH/Intra-NASh CBD (VEH/CBD), n = 9; AMPH/Intra-NASh VEH (AMPH/VEH), n = 9; AMPH/Intra-NASh CBD (AMPH/CBD), n = 9; AMPH/Intra-NASh Torin2+CBD (AMPH/Torin2+CBD), n = 10; and AMPH/Intra-NASh PF +CBD (AMPH/PF+CBD), n = 10. **p Figure 5 A. A microphotograph of a representative VTA neuronal recording placement is shown in Figure 5 B. Two-way ANOVA comparing firing frequency rates relative to preinfusion baseline levels showed a significant interaction between treatment (VEH vs CBD) and recording epoch time (F(5,113) = 4.17, p Fig. 5 C). Conversely, Intra-NASh CBD-treated rats displayed significantly decreased VTA DA neuronal firing frequency (p Fig. 5 C). Furthermore, frequency rates in CBD versus VEH-treated DA neurons were significantly lower at the 18–24 min epoch (p Fig. 5 C). Comparing DA neuron spikes firing in bursting mode, two-way ANOVA showed a significant interaction between treatment (VEH vs CBD) and recording epoch time (F(5,107) = 4.12, p Fig. 5 D). Post hoc comparisons demonstrated that, whereas VEH-treated neurons showed significantly increased bursting levels relative to baseline beginning 30 min after AMPH (p Fig. 5 D). In addition, DA neuron bursting levels were significantly lower in CBD versus VEH-treated neurons at the 6–12 min epoch (p Fig. 5 D). Sample VTA DA neuronal recording traces from VEH or CBD-treated neurons after AMPH exposure are presented in Figure 5 E, F. Thus, Intra-NASh CBD effectively attenuated AMPH-induced VTA DA neuronal sensitization effects both in terms of firing frequency and bursting levels.

Cannabidiol Counteracts Amphetamine-Induced Neuronal and Behavioral Sensitization of the Mesolimbic Dopamine Pathway through a Novel mTOR/p70S6 Kinase Signaling Pathway Justine Renard 1 ]]>