Self-Administration of Entactogen Psychostimulants Dysregulates Gamma-Aminobutyric Acid (GABA) and Kappa Opioid Receptor Signaling in the Central Nucleus of the Amygdala of Female Wistar Rats

This rodent study (2021) assessed whether if female rats escalate self-administration of the entactogens MDMA, methylone and pentylone, and investigated the impact this has on GABAA receptor and kappa-opioid receptor (KOR) signalling in the amygdala. It was found that GABA transmission increased in pentylone and MDMA rats compared to those administered saline while pentylone and MDMA disrupted KOR signalling. These findings suggest that GABA and KOR mechanisms play a critical role in entactogen self-administration like those observed with escalation of alcohol or cocaine self-administration.

Abstract

“Male rats escalate intravenous self-administration of entactogen psychostimulants, 3,4-methylenedioxymethcathinone (methylone) and 3,4-methylenedioxymethamphetamine (MDMA) under extended access conditions, as with typical psychostimulants. Here, we investigated whether female rats escalate self-administration of methylone, 3,4-methylenedioxypentedrone (pentylone), and MDMA and then studied consequences of MDMA and pentylone self-administration on GABA_A receptor and kappa opioid receptor (KOR) signaling in the central nucleus of the amygdala (CeA), a brain area critically dysregulated by extended access self-administration of alcohol or cocaine. Adult female Wistar rats were trained to self-administer methylone, pentylone, MDMA (0.5 mg/kg/infusion), or saline-vehicle using a fixed-ratio 1 response contingency in 6-h sessions (long-access: LgA) followed by progressive ratio (PR) dose-response testing. The effects of pentylone-LgA, MDMA-LgA and saline on basal GABAergic transmission (miniature post-synaptic inhibitory currents, mIPSCs) and the modulatory role of KOR at CeA GABAergic synapses were determined in acute brain slices using whole-cell patch-clamp. Methylone-LgA and pentylone-LgA rats similarly escalated their drug intake (both obtained more infusions compared to MDMA-LgA rats), however, pentylone-LgA rats reached higher breakpoints in PR tests. At the cellular level, baseline CeA GABA transmission was markedly elevated in pentylone-LgA and MDMA-LgA rats compared to saline-vehicle. Specifically, pentylone-LgA was associated with increased CeA mIPSC frequency (GABA release) and amplitude (post-synaptic GABAA receptor function), while mIPSC amplitudes (but not frequency) was larger in MDMA-LgA rats compared to saline rats. In addition, pentylone-LgA and MDMA-LgA profoundly disrupted CeA KOR signaling such as both KOR agonism (1 mM U50488) and KOR antagonism (200 nM nor-binaltorphimine) decreased mIPSC frequency suggesting recruitment of non-canonical KOR signaling pathways. This study confirms escalated self-administration of entactogen psychostimulants under LgA conditions in female rats which is accompanied by increased CeA GABAergic inhibition and altered KOR signaling. Collectively, our study suggests that CeA GABA and KOR mechanisms play a critical role in entactogen self-administration like those observed with escalation of alcohol or cocaine self-administration.”

Authors: Sophia Khom, Jacques D. Nguyen, Sophia A. Vandewater, Yanabel Grant, Marisa Roberto & Michael A. Taffe

Summary

INTRODUCTION

MDMA, Methylone and Pentylone are commonly abused substances that are monoamine transporter inhibitors and substrates with increased selectivity for serotonin over dopamine or norepinephrine transporters. MDMA exhibits low efficacy as a reinforcer in male rats, but is more effective when trained to self-administer mephedrone or under higher ambient temperature conditions. Additionally, 4-methylmethcathinone is a robust reinforcer in rat IVSA models.

Within the class of entactogen stimulants, Pentylone appears to be more efficacious as a reinforcer than Methylone in a dose-substitution comparison in male and female rats originally trained to self-administer methamphetamine and -pyrrolidinopentiophenone, respectively.

Methylone, a psychomotor stimulant, appears to be more effective than MDMA under 6 h daily access conditions, but has not been well characterized apart from two above-mentioned dose substitution studies in animals trained on other drugs.

In this study, female rats received long-access IVSA of three entactogens. The study tested the hypothesis that extended access sessions would lead to increased IVSA of methylone relative to MDMA. We aimed to investigate neuroadaptations in synaptic transmission in the central nucleus of the amygdala (CeA) associated with chronic administration of drugs of abuse including ethanol, cocaine, methamphetamine, or opioids.

Administration of drugs of abuse recruits these CeA stress systems, and MDMA-LgA and Pentylone-LgA affect CeA neuronal activity including GABAergic transmission and its regulation by the dynorphin/KOR system.

Here we used long-access (6-h) self-administration of MDMA and Pentylone to assess potential differences in behavioral patterns in entactogen self-administration. We performed ex vivo slice electrophysiology to assess changes in CeA GABA transmission and its regulation by the dynorphin/KOR system.

Animals

Wistar rats were housed in humidity and temperature-controlled vivaria on 12:12 h light:dark cycles. All experimental procedures were conducted in scotophase and were approved by the Institutional Care and Use Committees of The Scripps Research Institute.

Drugs

PentyloneHCl and MethyloneHCl were obtained from Cayman Chemical, 3,4-methylenedioxymethamphetamine (MDMA) HCl was obtained from NIDA Drug Supply, and the MDMA analog was obtained from Fox Chase Chemical Diversity Center.

INTRAVENOUS CATHETERIZATION

Rats were anesthetized with isoflurane/oxygen vapor mixture and prepared with chronic intravenous catheters as described previously. The catheters consisted of a 14-cm length polyurethane-based tubing fitted to a guide cannula. A minimum of 4 days were allowed for surgical recovery prior to starting an experiment. Catheter patency was assessed once a week, beginning in the third week of training, via administration through the catheter of 0.2 ml (10 mg/ml) of the ultra-short-acting barbiturate anesthetic, Brevital sodium.

Experiment 1 Acquisition

Rats were trained to self-administer MDMA, methylone, pentylone or saline vehicle using a fixed-ratio 1 response contingency. One individual in the Pentylone group was lost due to the catheter being chewed off by the cage mate. Operant conditioning chambers were used for self-administration studies. Rats received a priming infusion non-contingently if no response was made in the first 30 min of the session, and acquisition training was conducted for 14 – 15 sessions depending on the group.

Experiment 1 Progressive-Ratio Dose-Response Testing

Rats were subjected to dose substitution with the respective training drug, followed by dose substitution with methamphetamine, in a randomized order under a Progressive Ratio (PR) response contingency. One individual in the Pentylone group was lost due to the catheter being chewed off by the cage mate.

Experiment 2 Acquisition

Rats were trained to self-administer MDMA, pentylone, or saline vehicle following catheter implantation. Rats were trained for 11 – 14 sessions depending on the group, and were euthanized after 11 sessions.

Animals for Electrophysiology

Electrophysiological recordings were performed from 27 rats: 8 from the saline-control group, 11 from the MDMA-LgA group, and 8 from the Pentylone-LgA group. 2 MDMA rats, 0 Pentylone rats and 4 saline rats received priming infusions and were subjected to electrophysiological experiments. The estrous cycle was determined upon sacrifice and a potential impact of the estrous cycle on CeA GABA transmission as well as KOR function could not be excluded.

Electrophysiological Recordings

We prepared acute brain slices containing the central nucleus of the amygdala (CeA) and recorded from 135 neurons using whole-cell patch technique. The slices were incubated in 37 C warm, oxygenated artificial cerebrospinal fluid (aCSF) for 30 min.

Data Analysis and Statistics

IVSA experiments were performed on rats with Pentylone training. The number of infusions obtained was analyzed by repeated measures rmANOVA with Sessions or Dose as within-subjects factors.

mIPSCs were recorded using MiniAnalysis software and analyzed for frequency, amplitude, and current kinetics. The effects of the KOR agonist U-50488 and the KOR antagonist nor-binaltorphimine were then determined by one-way ANOVA and Dunnett post hoc analyses.

Female Wistar Rats Escalate Self-Administration of Entactogen Psychostimulants Under Extended Access (6-h) Conditions

Rats trained on vehicle saline had lower mean drug-intake than rats trained on pentylone, methylone, or MDMA. Analysis of the data showed that infusions were significantly increased in the Methylone, Pentylone, and MDMA groups compared to the first session, and that the Pentylone group was significantly different from the Vehicle group during Sessions 5 and 10 – 14 and from the MDMA group during Sessions 5 – 6, 13 – 14.

Dose Substitution in Female Wistar Rats Following Escalation of Self-Administration of Entactogen Psychostimulants

Analysis confirmed that rats trained on Methylone and Pentylone increased their intake to an approximately similar extent and received higher number of infusions compared to rats trained on MDMA. Pentylone-trained rats reached higher breakpoints in PR tests than Methylone and MDMA-trained groups.

The trained group obtained more infusions of Pentylone and MDMA than the vehicle group, regardless of the training drug. Within each drug, the groups did not differ, and exhibited similar dose-effect functions.

Methylenedioxymethamphetamine or Pentylone Heightens Central Nucleus of the Amygdala Inhibitory Signaling

We assessed whether intravenous self-administration of MDMA (LgA) or Pentylone (LgA) impacts CeA GABA transmission by recording pharmacologically isolated action-potential independent miniature inhibitory postsynaptic currents (mIPSCs) in 42 neurons from saline-control animals, 33 neurons from MDMA-LgA and 52 neurons from Pentylone-LgA rats.

Pentylone-LgA and MDMA-LgA increase mIPSC amplitudes and rise times, and MDMA-LgA and Pentylone-LgA induce similar mIPSC rise times and decay times, which indicate that these drugs induce similar neuroadaptations to increase CeA GABA signaling.

Methylenedioxymethamphetamine and Pentylone Self-Administration Disrupt Endogenous Kappa Opioid Receptor Signaling

We tested whether MDMA-LgA or Pentylone-LgA would also alter KOR-mediated regulation of vesicular CeA GABA release. Both drugs increased mIPSC frequency, indicating that dynorphin/KOR signaling regulates GABA signaling under physiological conditions also in female rats.

U-50488 decreased mIPSC frequency in MDMA-LgA and Pentylone-LgA rats, but did not alter mIPSC amplitudes or current kinetics. This indicates that KOR-activation after Pentylone-self-administration also decreases post-synaptic GABAA receptor function presumably leading to reduced neuronal inhibition. MDMA and Pentylone self-administration under long-access conditions induce significant neuroadaptations of KOR receptor signaling, which is confirmed by the fact that the KOR-antagonist norBNI decreased CeA vesicular GABA release in MDMA-LgA and Pentylone-LgA rats compared to saline-controls.

DISCUSSION

Female rats readily acquire the self-administration of methylone, pentylone, and MDMA under 6-h long-access (LgA) daily training conditions. Moreover, the study shows that the self-administration of entactogen cathinones and MDMA in female rats causes a profound dysregulation of CeA neuronal activity.

Motivated drug-seeking behavior was observed in the self-administration of the MDMA analog compound, but the potency was reduced and the efficacy was similar to MDMA.

MDMA-LgA and Pentylone-LgA increased CeA GABA transmission by increasing pre-synaptic GABA release and/or enhancing post-synaptic GABAA receptor function, respectively, in a dose-substitution procedure. This resulted in enhanced local inhibition and a presumably stronger CeA neuronal inhibition.

Our study revealed that activation of the dynorphin/KOR system in female rats decreases CeA GABA release, while KOR antagonism increases CeA GABA release, supporting a tonic role of KOR in the basal CeA GABA activity. Furthermore, both MDMA and Pentylone self-administration under long-access conditions disrupted CeA regulation by the KOR system.

Similar paradoxical effects of norBNI on CeA GABA signaling have been previously reported after cocaine-LgA, but the KOR agonist U-50488 decreased CeA GABA release in our study. The neuroadaptations at GABAergic synapses in response to cocaine, MDMA and pentylone self-administration may be explained by the different mechanisms of action of these drugs, or by the fact that norBNI is not just a KOR antagonist but can also activate alternative signaling cascades.

The paradoxical effect of norNI might be explained by changes in KOR signaling rather than a loss of CeA dynorphin, however, future studies utilizing different KOR antagonists will facilitate more insights into this phenomenon.

This study replicates the extended-access IVSA intake of entactogen cathinones and MDMA in female rats, and reveals similar profound neuroadaptations of CeA GABA transmission and its regulation by the dynorphin/KOR system in both groups.

FUNDING

This research was supported by NIH/NIDA grants, NIAAA grants, the Austrian Science Fund, and the Pearson Center for Alcoholism and Addiction Research.