This animal study (n=36) investigated the effects of ibogaine (0, 20, 40 mg/kg) in rats and found that higher doses promoted the expression of Glial cell Derived Neurotrophic Factor (GDNF) and that both doses promoted proBDNF expression in the Nucleus Accumbens, which may be underlying mediators of its long-lasting effect on reducing drug dependence.

Abstract

“Introduction: Ibogaine is an atypical psychedelic alkaloid, which has been subject of research due to its reported ability to attenuate drug-seeking behavior. Recent work has suggested that ibogaine effects on alcohol self-administration in rats are related to the release of Glial cell Derived Neurotrophic Factor (GDNF) in the Ventral Tegmental Area (VTA), a mesencephalic region which hosts the soma of dopaminergic neurons. Although previous reports have shown ibogaine’s ability to induce GDNF expression in rat midbrain, there are no studies addressing its effect on the expression of GDNF and other neurotrophic factors (NFs) such as Brain Derived Neurotrophic Factor (BDNF) or Nerve Growth Factor (NGF) in distinct brain regions containing dopaminergic neurons.

Methods: In this work, we examined the effect of ibogaine acute administration on the expression of these NFs in the VTA, Prefrontal Cortex (PFC), Nucleus Accumbens (NAcc) and the Substantia Nigra (SN). Rats were i.p. treated with ibogaine 20 mg/kg (I₂₀), 40 mg/kg (I₄₀) or vehicle, and NFs expression was analyzed after 3 and 24 h.

Results: At 24 h an increase of the expression of the NFs transcripts was observed in a site and dose dependent manner. Only for I₄₀, GDNF was selectively upregulated in the VTA and SN. Both doses elicited a large increase in the expression of BDNF transcripts in the NAcc, SN and PFC, while in the VTA a significant effect was found only for I₄₀. Finally, NGF mRNA was upregulated in all regions after I₄₀, while I₂₀ showed a selective upregulation in PFC and VTA. Regarding protein levels, an increase of GDNF was observed in the VTA only for I₄₀ but no significant increase for BDNF was found in all the studied areas. Interestingly, an increase of proBDNF was detected in the NAcc for both doses. These results show for the first time a selective increase of GDNF specifically in the VTA for I₄₀ but not for I₂₀ after 24 h of administration, which agrees with the effective dose found in previous self-administration studies in rodents. Further research is needed to understand the contribution of these changes to ibogaine’s ability to attenuate drug-seeking behavior.”

Authors: Soledad Marton, Bruno González, Sebastián Rodríguez-Bottero, Ernesto Miquel, Laura Martínez-Palma, Mariana Pazos, José Pedro Prieto, Paola Rodríguez, Dalibor Sames, Gustavo Seoane, Cecilia Scorza, Patricia Cassina & Ignacio Carrera

Summary

INTRODUCTION

Ibogaine, an indole alkaloid isolated from the root bark of the African shrub Tabernanthe iboga, has been reported to reduce craving and self-administration of several drugs of abuse in humans and animal models, with long-lasting effects that persist beyond pharmacokinetic elimination of the drug.

Although ibogaine binds to numerous central nervous system (CNS) targets at the micromolar range, the mechanism of action of its ability to attenuate drug-seeking behavior remains unresolved. Its longer-lived active metabolite, noribogaine, may be responsible for the enduring effects elicited by ibogaine.

Ibogaine and noribogaine were shown to increase the expression of Glial Cell Derived Neurotrophic Factor (GDNF) in the midbrain of rats and mice for up to 24 h, and to reduce ethanol self-administration. This mechanism may reverse the biochemical adaptations to chronic exposure to drugs of abuse in the reward system.

NFs are proteins that promote the growth, differentiation, synaptogenesis, and survival of neurons. NFs are also important in the adult brain and may mediate neuronal remodeling processes that occur during the development of substance use disorders. The administration of BDNF or GDNF can either promote or inhibit drug-taking behaviors depending mainly on the brain site of administration, along with other several factors such as the drug type, the addiction phase, and the time interval between site-specific NFs injections and the related behavioral assessments.

We analyzed the effect of a single administration of ibogaine on the expression of GDNF and BDNF in the VTA, PFC and NAcc.

A behavioral study was performed on rats to examine the impact of ibogaine administration on the expression of other relevant NFs and to determine if the effects observed at 24 h were due to long lasting mechanisms elicited by the drug.

Ibogaine HCl

Ibogaine was chemically synthesized using voacangine, which was extracted from the root bark of Voacanga africana. It was purified using column chromatography and used in this study.

Voacangine was decarboxylated by adding KOH to EtOH and heating to reflux for 5 min. The residue was dissolved in a 6% HCl solution and then analyzed by TLC. Ibogaine was obtained by treating a solution of starting material with 50% NaOH (pH 10 – 11), precipitating ibogaine as a white solid, and extracting three times with EtOAc. Ibogaine was purified using column chromatography purification and analyzed by 1 H and 13 C NMR.

Experimental Animals

Thirty-six male Wistar adult rats were used in this study and were assigned to one of the following groups: vehicle group, ibogaine 20- treated group, and ibogaine 40- treated group. They were housed four to five per cage and maintained on a 12-h light/dark cycle.

Behavioral Analysis

The open field apparatus (OF) was placed in a quiet experimental room with controlled temperature (22 -2 C), and rats were randomly assigned to different experimental groups and were used only once. The total distance traveled in meters (m) during 30 min was measured using a camera connected to a computer equipped with the Ethovision XT 12.0 software.

Ex vivo Studies Brain Dissection

Three or twenty-four hours after I20 , I40 or vehicle injection, animals were sacrificed by decapitation, and the brains were carefully removed and chilled in ice cold saline.

Semiquantitative qPCR

Total RNA was extracted from different brain regions and reverse-transcribed to cDNA using 200 U M-MLV-reverse transcriptase. The resulting cDNA was diluted in Biotools Quantimix Easy master mix and used for quantitative PCR analysis of GAPDH, BDNF, GDNF and NGF. We used PCR amplification over 40 cycles to calculate the relative amounts of our gene of interest, and analyzed the data using Rotor Gene 6000 software.

Western Blot Analysis

The selected brain regions were lysed in a lysis buffer containing 50 mM NaCl, 50 mM HEPES, 2 mM sodium orthovanadate, 1% Triton X-100, and SigmaFAST Protease inhibitor cocktail, and then transferred into a nitrocellulose membrane. The bands were detected using the Odyssey system.

Data Analysis

GraphPad Prism software 5 was used to design figure graphs and data analysis. Six animals per group were assessed for behavioral and PCR studies, and 4 animals per group were assessed for western blot analysis.

RESULTS

We investigated the behavioral effect of ibogaine treatment on novelty-induced locomotion in rats at 3 and 24 h after injection. Ibogaine did not alter animal locomotion at either time point, and animals were qualitatively indistinguishable from the vehicle group animals.

qPCR results showed that ibogaine acute administration differentially regulated GDNF mRNA expression levels in the selected brain regions in a dose and time-dependent manner.

Ibogaine treatment produced an appreciable downregulation of BDNF in the PFC at 3 h after injection, while no response was seen for the other brain areas at this time point. At 24 h, ibogaine treatment increased the mRNA expression of BDNF in all the brain regions studied in a dose-dependent manner.

NGF mRNA content was increased in PFC, NAcc, VTA, and SN after I20 treatment. However, the increase was not as high as that for BDNF.

GDNF, BDNF and proBDNF Protein Content by Western Blot

After 24 h of ibogaine administration, we found no significant change in the mature protein content of BDNF or GDNF in any of the studied brain regions. However, the content of proBDNF was increased in the NAcc by 2.7 and 2.8 fold, respectively, compared to the control group.

DISCUSSION

We have demonstrated that ibogaine administration alters the transcripts levels of GDNF and BDNF, and that ibogaine administration increases the protein content of these NFs. These effects may be related to ibogaine’s ability to attenuate drug-seeking behavior.

The novelty-related motor activity was decreased 24 h after I40, and this decrease was not observed 3 h after the same treatment. This decrease may be related to the neurochemical imbalance in the basal ganglia output, eliciting a decrease in the animal overall motivation.

At 3 h after I20 and I40 treatments, no alteration of GDNF transcript content was found in all the studied brain areas, whereas after 24 h, GDNF expression and mature protein content increased in the NAcc.

GDNF is upregulated in the VTA of the rat after 24 h of ibogaine administration, which is important because the VTA is the brain region that mediates ethanol self-administration. We show that I20 administration does not increase GDNF expression in any of the studied brain areas, and that I40 administration increases GDNF expression in the VTA, which is consistent with the reports indicating that GDNF mediates negative regulatory effects on chronic morphine-induced neuroadaptations in VTA of rodents.

Ibogaine was able to attenuate the cell loss in the SN and the biochemical changes at the striatum throughout the NFs expression in an experimental model of Parkinson’s Disease.

The first hours after ibogaine administration were marked by a downregulation of BDNF expression in the PFC, while no changes in other areas were observed. At 24 h, BDNF expression was highly increased, but this increase was not reflected in an increase in BDNF mature protein levels.

We found that the proBDNF content was increased in the NAcc for both ibogaine doses. This could be explained by the opposite effect of ibogaine on cocaine-seeking behavior and vulnerability to substance abuse.

Despite implicit assumption that differentially expressed mRNAs are reflected in protein content, numerous previous studies have concluded that the correlation is poor. It is possible that the time frame of protein synthesis is different for both neurotrophic factors, or that post-transcriptional regulation is involved.

Ibogaine administration increases serotonin transmission, which leads to an increase in BDNF and GDNF expression. Ibogaine and its long-lasting metabolite noribogaine are serotonin-reuptake inhibitors, which could account for the observed effect on BDNF and GDNF expression after 24 h of ibogaine administration.

Ibogaine modulated the expression of GDNF and BDNF, as well as of NGF, 24 h after treatment, while no changes were found at 3 h. NGF is likely involved in mediating important responses related to chronic intake of drugs of abuse.

The modifications of NFs levels induced by ibogaine/noribogaine may underlie neuroplasticity processes in the discrete brain regions analyzed, and could explain, at least in part, the ability of ibogaine to attenuate drug-seeking behavior in rodents.

CONCLUSION AND FUTURE PERSPECTIVES

Ibogaine administration alters the expression of GDNF, BDNF, and NGF transcripts in rat brain regions related to the dopamine neurotransmission in a dose- and time-dependent manner. This study adds relevant information concerning specific brain areas involved in the increment of GDNF levels as a putative mechanism of action underlying the anti-addictive effect of ibogaine.

AUTHOR CONTRIBUTIONS

SM, BG, EM, SR-B, MP, LM-P, BG, IC, PC, GS, and CS performed the qPCR experiments, EM, SR-B, and MP performed the Western Blot experiments, and MP, PC, GS, and CS wrote the manuscript.

ACKNOWLEDGMENTS

We thank the ANII and CSIC-UdelaR for financial support, and Dr. Kenneth Alper for important discussions regarding ibogaine and noribogaine pharmacology.