The role of extracellular serotonin and MDMA in the sensitizing effects of MDMA

This rodent study (2022) assessed the impact repeated high-dose exposure to MDMA has on markers of serotonin neurotransmission and if this is related to the sensitizing effects of MDMA. The results suggest that the sensitizing effects of MDMA are not due to changes in MDMA-produced synaptic overflow of serotonin but are more likely related to alterations in serotonin receptor mechanisms and/or dopamine neurotransmission.

Abstract

Introduction: MDMA is a non-selective monoamine releasing stimulant with potent serotonergic effects – a pharmacological effect not typically associated with drugs of misuse or efficacious reinforcers. Nonetheless, MDMA is misused by humans and self-administered by laboratory animals. We have previously shown that repeated exposure to MDMA sensitized both the locomotor activating and reinforcing effects of MDMA in rats. Because repeated MDMA exposure often results in decreased markers of serotonin neurotransmission, it is possible that this might underlie the sensitizing effects of MDMA. This was examined in the current study.

Methods: Male Sprague-Dawley rats were stereotaxically implanted with guide cannula in the medial striatum. They were then pre-treated with saline (n = 11) or MDMA (10 mg/kg, i.p.; n = 10), once daily for five days. Two-days later, all rats received ascending doses of MDMA (0.0, 5.0, 10.0, mg/kg, i.p.) administered at 2 hr intervals, during which locomotor activity was measured and microdialysis samples were collected. Microdialysates were analyzed using liquid chromatography-mass spectrometry and the concentrations of serotonin and MDMA were quantified.

Results: Acute MDMA administration produced dose-dependent increases in locomotor activity, which was significantly enhanced by MDMA pre-treatment. Acute MDMA also produced dose-dependent increases in medial-striatal serotonin and MDMA, but this was not impacted by MDMA pre-treatment.

Conclusion: These results suggest that the sensitizing effects of MDMA are not due to changes in MDMA-produced synaptic overflow of serotonin in the medial striatum or the absorption/elimination of systemically administered MDMA. More likely candidates are alterations in serotonin receptor mechanisms and/or dopamine neurotransmission following repeated exposure.

Authors: Ross van de Wetering, Jan A. Vorster, Sophie Geyrhofer, Joanne E. Harvey, Robert A. Keyzers & Susan Schenk

Summary

Male Sprague-Dawley rats were implanted with a guide cannula in the medial striatum and then received ascending doses of MDMA (0.0, 5.0, 10.0 mg/kg, i.p.) at 2 hr intervals. Their locomotor activity was measured and their microdialysates were analyzed using liquid chromatography-mass spectrometry.

Acute MDMA administration produced dose-dependent increases in locomotor activity, which were enhanced by MDMA pre-treatment. MDMA also produced dose-dependent increases in medial-striatal serotonin and MDMA, but this effect was not impacted by MDMA pre-treatment.

MDMA is an amphetamine analogue and popular recreational drug of misuse. Some users develop symptoms consistent with a substance use disorder, including cravings, urges to use, tolerance, and withdrawal symptoms.

MDMA is self-administered by laboratory animals and has a response profile similar to other psychostimulant drugs of abuse. The response is dose-dependent and escalates with continued testing.

MDMA is a potent releasing stimulant and re-uptake inhibitor of the monoamine neurotransmitters serotonin (5-HT), dopamine (DA), and norepinephrine. Its long half-life and non-linear pharmacokinetics make it difficult to dose accurately and may lead to higher-than-expected brain concentrations of the drug.

After 17-20 days of MDMA self-administration, 5-HT levels were lower, and 5-HT production was decreased in the nucleus accumbens.

We suggested that the development of 5-HTergic deficits as a result of repeated exposure to MDMA may disinhibit the reinforcing effects of MDMA and increase self-administration. We also measured MDMA concentrations in the medial-striatal 5-HT system.

24 male Sprague-Dawley rats were used in the experiment. They were housed individually in a vivarium at 21 °C and 55% humidity, and were fed ad libitum except during the experiment.

()-MDMAHCl was dissolved in a sterile 0.9% NaCl solution for i.p. injections and in artificial cerebrospinal fluid (aCSF) for dialysis perfusate and standard dilutions. All chemicals were purchased from Sigma Aldrich except for D2-5-HTC2O4H2, which was synthesized in-house.

Serotonin was synthesized from 5-benzyloxyindole by the procedure of Saari . It was characterized by 1H NMR, 13C NMR, HRMS, and ESI m/z.

MDMA pre-treatment and locomotor sensitization were carried out as previously described . The medial striatum was chosen as the site for microdialysis because of its role in the sensitization of other drugs of abuse and its increased expression of the transcription factor, FosB, following repeated MDMA exposure.

Locomotor activity was measured in clear plexiglass chambers set in sound-attenuating boxes. Rats were placed into the chambers for 30 min prior to receiving saline or 10 mg/kg, i.p. MDMA and remained in the chambers for an additional 60 min thereafter.

Following two drug-free days, microdialysis probes were implanted at 0800 and testing began 3 hours later. Locomotor activity was measured at 30 min intervals.

Microdialysis probes with a membrane surface area of 0.5 mm x 3 mm were used to analyze aCSF. The samples were stored at -80 °C until analyzing, and then brains were rapidly removed and placed in 4% paraformaldehyde fixative overnight before being frozen at -80 °C until sectioning.

Analysis of microdialysate samples was carried out using an Agilent Technologies 6530 Q-TOF LC-MS equipped with a Jet-Stream electrospray ionization (ESI) source. The flow rate was 0.45 mL/min until 10.7 min, increased to 0.5 mL/min from 10.7 – 11.7 min, and reduced back to 0.45 mL/min at 11.7 min.

5-HT and MDMA concentrations were determined by analyzing external standards diluted in aCSF at 0.1, 0.5, 1, 5, and 10 nM and 0.1, 5, and 10 mM, respectively. A weighted (1/SD2) linear model was used to calculate the concentrations of the targeted analytes.

The time course of locomotor data and 5-HT/MDMA concentration data were analyzed using separate 2 (pre-treatment: Saline vs MDMA 10.0) x 16 (time: 30 – 480 min) mixed-measures ANOVAs. The area under the curve (AUC) was also calculated for each 2-hr time period associated with each MDMA dose.

Locomotor and dialysate data were obtained from 21 of the 24 rats. Saline and MDMA pre-treatments produced significant increases in locomotor activity, with MDMA producing greater increases in locomotor activity at 300, 330, 390, 420, and 450 min. Figure 2B shows that there was a significant two-way interaction between pre-treatment and MDMA dose, and that there were significant simple main effects of MDMA dose for both the saline and MDMA pre-treatment groups. Figure 3A shows mean MDMA concentrations as a function of MDMA pre-treatment and time/acute MDMA administration. There was no significant interaction between pre-treatment and time/acute MDMA administration, and no significant effect of MDMA dose on overall MDMA concentrations.

We developed a procedure for the quantification of extracellular concentrations of 5-HT and MDMA in the medial striatum. We then applied this procedure to determine the role of medial-striatal, extracellular 5-HT and MDMA in sensitized MDMA-produced behavior following repeated, intermittent exposure.

Acute MDMA administration produced dose-dependent increases in locomotor activity and extracellular concentrations of MDMA in the medial striatum. MDMA pre-treatment had no effect on extracellular concentrations of MDMA following acute administration.

Acute MDMA exposure increased extracellular concentrations of 5-HT in the medial striatum, but MDMA pre-treatment did not significantly impact this increase. This suggests that a certain amount of exposure is required to produce significant 5-HTergic deficits.

MDMA exposure did not decrease 5-HT overflow in the medial striatum, suggesting that other mechanisms, such as changes in receptor densities, may be more important than 5-HT in sensitizing MDMA-produced locomotor activity.

DA is a likely candidate for the development of MDMA behavioral sensitization, as DA levels are elevated and MDMA-produced DA release is enhanced following repeated exposure. DA D1 and DA D2 antagonists prevent the development of MDMA behavioral sensitization.

The dose-dependent increase in 5-HT concentrations was non-linear, and the second dose produced minimal increases. This may be because the 5-HTergic effects of MDMA have become saturated. MDMA-induced 5-HT release and transporter binding peaked at approximately 6 and 14 M, respectively, in rat brain tissue slices, suggesting that saturation of MDMA-produced 5-HT release/transporter binding would have occurred. MDMA doses used in the current study are common for behavioral studies, and rats self-administer similar or greater amounts of MDMA during a single session. However, the DAergic effects of MDMA do not saturate at these doses, and higher doses may have more potent DAergic effects.

There is a dependence syndrome for ecstasy, according to several studies.

An ecological momentary assessment study of MDMA use in adolescents found that the use of MDMA leads to dependence and psychopathology. MDMA use develops a profile of dependence with extended testing and abstinence, and the use of MDMA leads to psychopathology in adolescents. MDMA (ecstasy) reinforces cocaine seeking in drug-naive and cocaine-trained rats. The reinforcing effects of MDMA are predicted by regional changes in fosB expression in rat brain following MDMA selfadministration.

M. Slane, K.T. Ball, S. Schenk, D. Gittings, J. Colussi-Mas, E. Daniela, Effects of priming injections of MDMA and cocaine on reinstatement of MDMA- and cocaine-seeking in rats. MDMA (“ecstasy”) abuse as an example of dopamine neuroplasticity. MDMA pharmacokinetics in humans and rats are non-linear, and its pharmacodynamic consequences are influenced by dose and route of administration.

A number of studies have been done on the relationship between serotonergic activity and reinforcing effects of a series of amphetamine analogs, including the effects of fluoxetine on amphetamine self-administration in rats and the effects of fenfluramine on amphetamine self-administration in rhesus monkeys. Self-administered MDMA produces dose- and time-dependent serotonin deficits in the rat brain. Current and former ecstasy (MDMA) users have decreased serotonin transporter availability. Researchers have found that ecstasy users have reduced cognitive performance and serotonergic function, and that these effects are related to the use of ecstasy. Multiple studies have shown that repeated MDMA administration increases MDMA-produced locomotor activity and facilitates the acquisition of MDMA self-administration. These studies have also shown that benzoyl chloride derivatization and liquid chromatography – Mass Spectrometry can be used to monitor in vivo neurochemical monitoring. MDMA elicits behavioral and neurochemical sensitization in rats. A linear regression for calibration lines revisited: weighting schemes for bioanalytical methods, Journal of Chromatography A. 1446 (2016) 78 – 90, https://doi.org/10.1016/j.chroma.2016.04.006.

Behavioral sensitization to 3,4-methylenedioxymethamphetamine is long-lasting and modulated by the context of drug administration. In addition, dopamine cell bodies and terminals are reduced in adult rats exposed to a low dose regimen of MDMA during adolescence. Researchers have found that 3,4-methylenedioxymethamphetamine (MDMA) induces serotonin release at serotonin transporters in the brain, which may be the mechanism by which MDMA induces behavioral sensitization in rats.

The study of the effects of methylenedioxymethamphetamine on the striatal monoaminergic systems in the rat brain was conducted by C.J. Schmidt, J.A. Levin, W. Lovenberg, D. Crespi, T. Mennini, M. Gobbi, S. Koch, M.P. Galloway, and G.A. Gudelsky.

Figure 2 shows that pre-treatment with Saline or MDMA 10.0 significantly reduced locomotor activity, while MDMA 0.0, 5.0, and 10.0 administered at different times led to greater AUC.

MDMA concentrations increased with time following administration of increasing doses. The area under the curve (AUC) was highest at 10.0 mg/kg, i.p., administered 360 min after pre-treatment with MDMA 10.0.

Figure 4 shows that 5-HT concentrations increased with time/acute MDMA administration, with the highest concentration occurring 360 min after injection.