Ibogaine modifies GDNF BDNF and NGF

Ibogaine Modifies GDNF, BDNF, and NGF Expression in Brain Regions Involved in Mesocorticolimbic and Nigral Dopaminergic Circuits
Soledad Marton¹†, Bruno González²†, Sebastián Rodríguez¹, Ernesto Miquel¹, Laura Martínez Palma¹, Mariana Pazos², José Pedro Prieto³, Paola Rodríguez², Dalibor Sames⁴, Gustavo Seoane², Cecilia Scorza³*, Patricia Cassina¹*, Ignacio Carrera²*
¹Departamento de Histología y Embriología, Facultad de Medicina – Universidad de la República, Montevideo – Uruguay ²Laboratorio de Síntesis Orgánica, Departamento de Química Orgánica, Facultad de Química – Universidad de la República, Montevideo – Uruguay ³Departamento de Neurofarmacología Experimental, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo – Uruguay ⁴Department of Chemistry, Columbia University, New York, NY – USA
†These authors contributed equally to this work
*Correspondence: Cecilia Scorza (cscorza@iibce.edu.uy), Patricia Cassina (pcassina@fmed.edu.uy), Ignacio Carrera (icarrera@fq.edu.uy)

Abstract
Ibogaine is a psychedelic alkaloid which has been subject of intense scientific research due to its reported ability to attenuate drug-seeking behavior. Recent work suggested that ibogaine effects on alcohol self-administration in rats was related to the release of Glial Cell Derived Neurotrophic Factor (GDNF) in the Ventral Tegmental Area (VTA), a mesencephalic region which hosts soma of dopamine neurons. It is well known that neurotrophic factors (NFs) mediate the neuroadaptations induced in the mesocorticolimbic dopaminergic system by repeated exposure to drugs.
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, Brain Derived Neurotrophic Factor (BDNF) or Nerve Growth Factor (NGF) in distinct regions containing dopaminergic neurons. 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 hours. Only at 24 h an increase of the expression for the three NFs were observed in a site and dose dependent manner. Results for GDNF showed that only I40 selectively upregulated its expression in the VTA and SN. Both doses of ibogaine elicited a large increase in the expression of BDNF in the NAcc, SN and PFC, while a significant effect was found in the VTA only for I40.
Finally, NGF was found to be upregulated in all regions after I40, while a selective upregulation was found in PFC and VTA for the I20 treatment. An increase in the content of mature GDNF was observed in the VTA but no significant increase in the mature BDNF protein content was found in all the studied areas. Interestingly, an increase in the content of proBDNF was detected in the NAcc for both treatments. Further research is needed to understand the neurochemical bases of these changes, and to confirm their contribution to the anti-addictive properties of ibogaine.
1. Introduction
Background on Ibogaine
Ibogaine is the main indole alkaloid isolated from the root bark of the African shrub Tabernanthe iboga. Traditionally used in African religious ceremonies as a psychedelic, ibogaine became the subject of interest of the scientific community due to its reported ability to reduce craving and self-administration of several drugs of abuse in humans. These effects found mainly in uncontrolled clinical trials and observational studies, have been reported to be long-lasting enduring weeks to months after a single administration of large doses of ibogaine.
In animal models for drug dependence, ibogaine also reduces the self-administration of morphine and heroin, cocaine, and alcohol, with long-lasting effects that persists beyond pharmacokinetic elimination of the drug. In addition, ibogaine administration to animals also reduces naloxone or naltrexone precipitated-withdrawal signs.
Challenges and Controversies
Several ibogaine effects observed in humans, such as the dissociative or psychedelic effects, tremor induction, and prolongation of the QTC interval in the EKG (which has been associated with sudden death cases after ibogaine intake), have made it a controversial treatment option and resulted in reluctance to pursue rigorous controlled clinical trials. In this regard, unveiling the mechanism of action for ibogaine’s anti-addictive property is critical for the development of safer derivatives for clinical use, and for a deeper understanding of the neurobiological basis of substance dependence and its possible treatment.
Mechanism of Action
Although a vast amount of research has been done regarding the pharmacology of ibogaine, the mechanism of action of its anti-addictive effects remains unresolved. Ibogaine binds to numerous central nervous system (CNS) targets at the micromolar range such as: nicotinic acetylcholine receptors (nAChR α3β4 and α2β4), N-methyl-D-aspartate (NMDA), kappa and mu opioid, 5HT2A and 5HT3 receptors and the dopamine and serotonin transporters.
However, these ibogaine-receptor interactions do not seem to account for the long-lasting effects of ibogaine found in rodents which are described to last for 48 to 72 hours after ibogaine administration. In rodents, ibogaine has a short half-life of 1-2 hours raising the hypothesis that its longer-lived active metabolite, noribogaine, should be responsible for the enduring effects elicited by ibogaine. However, no appreciable amounts of noribogaine have been found in rodents’ brain tissue 19 hours after ibogaine intraperitoneal (i.p.) administration, and only approximately 5% of the noribogaine Cmax was detected in serum 24 hours after the same treatment.
The GDNF Hypothesis
A few years ago, a novel hypothesis linking ibogaine’s attenuation of alcohol self-administration in rodents to its ability to modulate the expression of Glial Cell Derived Neurotrophic Factor (GDNF) in the brain was proposed. It was shown that a single ibogaine i.p. administration (40 mg/kg) increased the expression of GDNF in the midbrain of rats and mice for up to 24 hours. In addition, microinjection of ibogaine into the Ventral Tegmental Area (VTA), produced a long-lasting reduction of ethanol self-administration, a response that was attenuated by the intra-VTA delivery of anti-GDNF neutralizing antibodies.
These results suggested that ibogaine mediates its effects against ethanol consumption by increasing GDNF content in the VTA. Accordingly, another study from the same research group showed that the intra-VTA infusion of noribogaine induced a long-lasting decrease in ethanol self-administration. Further, ibogaine-derived synthetic derivatives were recently shown to induce the release of GDNF in vitro, in established cell line systems.
These observations formed the basis for a new rationale to explain the long-lasting effects of ibogaine. The induction of GDNF by ibogaine/noribogaine may activate an autocrine loop, leading a long-term synthesis and release of GDNF that persists beyond elimination of both substances. This mechanism may reverse the biochemical adaptations to chronic exposure to drugs of abuse in the reward system.
Role of Neurotrophic Factors
Neurotrophic Factors (NFs), such as GDNF, BDNF (Brain Derived Neurotrophic Factor) or NGF (Nerve Growth Factor) are small proteins that promote the growth, differentiation, synaptogenesis, and survival of neurons. Their expression in the nervous tissue is relatively high during the development of the CNS, where substantial growth, differentiation and remodeling of the nervous system occur. More recently, it has been discovered that NFs play important roles in the adult brain where they modulate maintenance, protection, repair and plasticity of the nervous tissue.
Furthermore, accumulating evidence has suggested that NFs mediate neuronal remodeling processes that occur during the development of substance use disorders (SUDs). Particularly, the role of GDNF and BDNF in the neuroadaptations in the mesocorticolimbic dopamine system (Prefrontal Cortex, PFC-VTA-Nucleus Accumbens, NAcc pathway) induced by repeated exposure to drugs of abuse has been extensively studied, including the impact of manipulating NFs levels on drug-seeking behavior in animal models.
It has been shown that 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 (initiation, maintenance, abstinence or relapse), the time interval between site-specific NFs injections and the related behavioral assessments. For example, BDNF infusion into the NAcc increases cocaine-seeking behavior, while BDNF infusion into the medial prefrontal cortex (mPFC) suppresses it.
The effect of administrating other NFs (as NGF or Fibroblast Growth Factor, FGF) on drug-seeking behavior and modulation of neuroadaptations produced by chronic administration of drugs of abuse has been less studied. In the case of NGF, for example, it was found that NGF content decreases in the hippocampus and hypothalamus of alcohol-treated mice, and that its administration into the central nucleus of the amygdala mimicked the morphine reward sensitization. In contrast to BDNF, a single infusion of NGF into the VTA produced no changes in cocaine-seeking behavior.
Study Objectives
Given the importance and the site-specificity of the elicited responses, we decided to analyze the effect of a single administration of ibogaine on the expression of GNDF, BDNF and NGF at two time points, 3 and 24 h after i.p. injection, in those brain areas which define the mesocorticolimbic dopamine system such as VTA, PFC and NAcc. As the Substantia Nigra (SN) is a major nucleus of dopaminergic neurons important in the basal ganglia functioning, the expression of these NFs in this region was also studied. In addition, locomotion of control and drug treated animals was recorded using an open field test.
2. Materials and Methods
2.1 Ibogaine·HCl Preparation
The ibogaine used in this study was chemically synthesized using voacangine as starting material, which was extracted from the root bark of Voacanga africana using a modification of a previously described procedure. Briefly, 100g of grounded root bark of Voacanga africana was extracted with a 1% aqueous solution of HCl (6 x 500 mL). The combined aqueous extracts were basified by adding concentrated NH4OH until pH 10-11. A brown precipitate was separated by centrifugation and dried at 60°C for 24 h.
This solid was taken in acetone and filtered to discard root impurities. The solvent was evaporated in vacuo to afford a total alkaloid extract of 3.5-4.0 g. Column chromatography allowed to obtain 1g of pure voacangine which was analyzed by ¹H and ¹³C NMR. Voacangine was then decarboxylated through a multi-step process involving reflux with KOH in ethanol, followed by acidification and basification to yield ibogaine free base with an 86% yield.
Crystallization from EtOH afforded a crystalline solid which was converted to the corresponding hydrochloride by treatment with diethyl ether saturated with HCl(g). Purity of ibogaine·HCl was determined by GC-MS analysis as 98.3%. Dissolution of ibogaine-HCl to prepare the samples for i.p. injection was carried out using warm saline that was previously degassed by nitrogen bubbling.
2.2 Experimental Animals
Thirty-six Wistar adult rats (270-300 g) were used in this study and assigned to one of the following groups: Vehicle group at 3 and 24 h (n=6 per each group); Ibogaine 20 mg/kg (I20) treated group at 3 h and 24 h (n=6 per each group) and Ibogaine 40 mg/kg (I40) treated group at 3 and 24 h (n=6 per each group). Animals were housed four to five per cage and maintained on a 12-h light/dark cycle (lights on at 07.00 h) with food and water freely available before and after i.p. injection of vehicle or ibogaine until behavioral testing and sacrifice.
All experimental procedures were conducted in agreement with the National Animal Care Law (#18611) and with the “Guide to the care and use of laboratory animals” (8th edition, National Academy Press, Washington D. C., 2010). Furthermore, the Institutional Animal Care Committee approved the experimental procedures. Adequate measures were taken to minimize pain, discomfort or stress of the animals, and all efforts were made to use the minimal number of animals necessary to obtain reliable scientific data.
2.2.1 Behavioral Analysis
Animals were brought to the experimental room in their home cages, identified and weighed prior to the behavioral test. An open field (OF) apparatus consisting of a square area (45 cm wide×45 cm long×40 cm high) with transparent plastic walls indirectly illuminated (35 luxes) to avoid reflection and shadows were employed. The OF was placed in a quiet experimental room with controlled temperature (22 ± 2 ºC).
As rats were not habituated to the OF before drug or vehicle administration, novelty-induced motor activity was automatically recorded by a camera connected to a computer equipped with the Ethovision XT 12.0 software. Using this video tracking software, we specifically measured the total distance traveled in meters during 30 min, starting 3 and 24 h after ibogaine or vehicle administration. Animals were randomly assigned to different experimental groups and were used only once.
Taking into account that immediately after i.p. administration ibogaine can produce a dose-dependent unusual motor profile and some prototypical serotonergic syndrome-related behaviors (e.g., tremor, flat body posture, forepaw treading), these specific behaviors were assessed by a trained investigator every 5 min (for a total of 30 min) starting 3 and 24 h after ibogaine administration. During all experiments, the OF was cleaned with 30% alcohol before placing the following rat. All experiments were done between 9 AM and 3 PM.
2.3 Ex Vivo Studies
2.3.1 Brain Dissection
Three or twenty-four hours after I20, I40 or vehicle (i.p.) injection, animals were sacrificed by decapitation and the brains were carefully removed and chilled in ice cold saline. According to Paxinos and Watson, the whole NAcc (shell and core), PFC (including mPFC), Substantia Nigra (SN, pars compacta-SNpc and pars reticulata-SNpr) and VTA were dissected out on ice and the tissue obtained was immediately frozen and rapidly stored at -80°C until the processing day.
2.3.2 Semiquantitative qPCR
For RT-PCR analysis total RNA was extracted from the different brain regions using Trizol reagent followed by chloroform extraction and isopropanol precipitation. Possible DNA contaminations were eliminated with DNase treatment using DNase free Kit. RNA quality was evaluated by agarose gel electrophoresis followed by ethidium bromide staining and quantified using a NanoDrop 1000 Spectrophotometer.
500 ng of this total RNA was reverse-transcribed using 200 U M-MLV-reverse transcriptase following manufacturer instructions. 25 ng of the resulting cDNA was diluted in Biotools Quantimix Easy master mix in 10 μl volume. All reactions were performed in triplicates in strip tubes, using specific forward and reverse primers. PCR amplification was done over 40 cycles using a Rotor-Gene 6000 System and data were analyzed using Rotor Gene 6000 software. Quantification was performed with ΔΔCt method using rats treated with vehicle as a negative control, and GAPDH mRNA as reference.
2.3.3 Western Blot Analysis
The selected brain regions were sonicated in a lysis buffer containing 50 mM NaCl, 50 mM HEPES, 2 mM sodium orthovanadate, 1% Triton X-100, and SigmaFAST Protease inhibitor cocktail. After quantification and denaturation, the samples were loaded and separated by 12% SDS-PAGE gels and then transferred into a nitrocellulose membrane.
The membranes were incubated for 1 h in blocking solution and incubated overnight at 4°C with primary antibodies to GDNF (1:500 in BS; Abcam ab119473), BDNF (1:400 in BS; Promega G1641), or proBDNF (1:500 in BS; Invitrogen PA1-18360), together with anti-alpha-tubulin (1:3000 in BS; Abcam ab184613) as loading control. Afterwards, the membranes were washed and incubated for 1 hour at room temperature with IRDye conjugated secondary antibodies.
The Odyssey system (LI-COR Biosciences) was used to detect the bands. Quantification of band intensity was performed using Image Studio software version 5.2.5.
2.4 Data Analysis
GraphPad Prism software 5 was used to design figure graphs and data analysis. Data are presented as mean ± S.E.M values. Six animals per group were assessed for comportamental and PCR studies. In some cases, some data was excluded from the analysis due to insufficient sample or high deviation from the mean of the group, rendering a lower n, but never smaller than 4.
For western blot analysis, samples from 4 animals per group were assessed. Data from qPCR and western blot were analyzed and compared by one-way ANOVA followed by post hoc Tukey’s Multiple Comparison Test. In all cases, statistical significance was set at p < 0.05. Data from motor activity were analyzed by two-way (treatment, time, and interaction between factors) ANOVA for repeated measures followed by Newman-Keuls multiple comparison post hoc test and Unpaired-t-test.
3. Results
Behavioral Effects of Ibogaine
In a previous study, we reported a very high impact of the I40 treatment on novelty-induced locomotion after two hours of ibogaine administration and the concomitant induction of some of the behavioral signs related to the serotonergic syndrome. Thus, we decided to analyze the behavioral effect of ibogaine treatment in the time points used in the present study.
Compared to the control group, novelty-induced locomotion was not altered by I20 at any evaluated time. Whereas I40 was not effective to induce any behavioral alterations 3 h after i.p. administration, it elicited a significantly reduction of the animal locomotion 24 h after injection. No abnormal behaviors were present for both time points and animals were qualitatively indistinguishable from the vehicle group animals. Immediately after each behavioral test, animals were sacrificed to pursue brain dissection for the qPCR and Western Blot studies.
3.1 qPCR Quantification of Neurotrophic Factors mRNA
GDNF Expression
qPCR results for GDNF showed that ibogaine acute administration differentially regulated GDNF mRNA expression levels in the selected brain regions in a dose and time-dependent manner. At 3 hours, no changes in the GDNF mRNA expression was found for both doses of ibogaine in all the studied areas.
In contrast, after 24 hours of treatment, changes in the expression of GDNF were found in a dose and site-specific manner. While the I20 dose did not affect the GDNF expression in any of the studied areas, the I40 dose selectively increased GDNF mRNA content in the midbrain regions: VTA (12-fold increase compared to the control group) and SN (6-fold increase vs the control group) with no appreciable effects in the PFC and NAcc.
BDNF Expression
For BDNF, ibogaine treatment produced an appreciable downregulation of its expression in the PFC at 3 hours after injection (1.7 and 2-fold decrease for I20 and I40 respectively, compared to control), while no response was seen for the other brain areas at this time point.
At 24 hours, ibogaine administration upregulated the mRNA expression of BDNF in all the brain regions studied in a dose-dependent manner. A large effect was found in the NAcc for both doses of ibogaine (220-fold increase compared to the control for I20, and 340-fold increase for I40). The I20 dose increased BDNF expression in PFC (55-fold increase compared to the control) but not in the VTA or SN.
On the other hand, in addition to the NAcc, the I40 dose also upregulated BDNF expression in PFC (107-fold increase compared to the control), VTA (43-fold increase compared to the control) and SN (21-fold increase compared to the control).
NGF Expression
For NGF, no difference in the content of mRNA was found 3 hours after ibogaine treatments. At 24 hours, an upregulation of NGF mRNA content was found in: PFC (14-fold increase compared to the control), NAcc (15-fold increase compared to the control), VTA (11-fold increase compared to the control), and SN (4-fold increase compared to the control).
For the I20 dose a significant effect was only found in the PFC (7-fold increase compared to the control) and VTA (5-fold increase compared to the control). However, the levels of increase in the NGF mRNA were not as high as those for BDNF.
3.2 GDNF, BDNF and proBDNF Protein Content
Considering the changes found for the expression of NFs after 24 hours of ibogaine administration, we decided to analyze the content of mature proteins BDNF and GDNF for all the studied brain regions, because of their involvement in the addictive behavior. Precursor of BDNF, proBDNF was also considered since it is well described that it shows opposite effects to the mature protein because of a higher affinity to the p75 receptor.
GDNF Protein Content
For GDNF, a single dose of ibogaine affected mature protein content in a region- and dose-dependent manner. While no changes in GDNF content were observed for I20 in any of the studied regions, GDNF content was increased in VTA for the I40 dose (2-fold increase compared to the control group). No effect was observed in the GDNF content at the NAcc, SN and PFC in comparison to the control group.
BDNF and proBDNF Protein Content
For BDNF no significant change in the mature protein content was detected for all the studied regions for both doses of ibogaine. Nevertheless, in the case of proBDNF we found a selective increase in the protein content for I20 and I40 in the NAcc (2.7 and 2.8-fold increase for I20 and I40 doses respectively, compared to control), while no significant change was detected in the other brain areas.
4. Discussion
Overview of Findings
In the present study, we have demonstrated that ibogaine administration simultaneously alters the expression of the two main trophic factors involved in addictive behavior: GDNF and BDNF, but also NGF in rat brain regions related to the dopamine neurotransmission in a dose and time dependent manner. In addition, we showed that after 24 h of treatment, I40 selectively increased the content of mature GDNF in the VTA, while proBDNF content was increased selectively in NAcc by both doses.
Considering that dopamine neurotransmission, specifically in the mesocorticolimbic pathway, is related to rewarding/reinforcing and motivational actions of most drugs of abuse, our findings contribute to shed light on a mechanism underlying the anti-addictive action of ibogaine.
Pharmacokinetics and Time Course
According to previous pharmacokinetics reports in rats using i.p. administration, ibogaine concentration in blood rapidly decreases in the first hour while noribogaine concentration is at maximum at 2.4 h and lasts up to 24 h. Regarding concentrations in the brain, no appreciable amounts of noribogaine have been found in the brain tissue of rodents 19 h after ibogaine i.p. administration.
Given these previous reports, we chose to study NFs expression/content in the selected brain areas at 3 h, where the parent drug and its metabolite are present in relevant concentrations, and at 24 h where both drugs are no longer detectable in the brain. In this manner, the observed effects found at 24 h involving NFs expression and the outcome of the locomotion study, would be due to signaling mechanisms elicited by the drug which remain after it has been cleared from the brain, and not from the acute effects of ibogaine/noribogaine.
These results may provide the rationale for the previously reported long-lasting anti-addictive effects of ibogaine.
Motor Activity Findings
Regarding the motor function, a decrease in the novelty-related motor activity was observed 24 h after I40 (while 3 h after the same treatment, animals displayed a similar activity than the control). There is no evidence at this point to establish a potential connection between this intriguing behavior and the observed changes in NFs expression.
In this regard, considering the changes in the expression of NFs at 24 h in the SN, it is plausible that a neurochemical imbalance in the basal ganglia output may underlie the changes in the motor activity. Also, since the induced increase of GDNF content in the VTA by ibogaine has been proposed previously as a putative mechanism to reduce motivational behaviors in alcohol self-administration paradigms, we cannot rule out that this acute motor impairment is related to this neurochemical effect eliciting a decrease in the animal overall motivation.
GDNF Expression Findings
At 3 h after I20 and I40 treatments, no alteration of GDNF expression was found in all the studied brain areas. This is in contrast with previous work reported by He et al., where a significant GDNF upregulation was found 3 h after I40 treatment in the midbrain of rats. Differences between both reports may rely in the analyzed brain regions. We studied the effect of ibogaine administration on GDNF expression in specific brain areas (PFC, VTA, NAcc and SN), while the whole midbrain was used in the study by He et al.
On the other hand, after 24 h, we found that the I40 dose increases GDNF expression and mature protein content specifically in the rat VTA, which was also found in the whole midbrain at this time point in the mentioned previous report. In this manner our study identifies the VTA as the key brain region of the mesocorticolimbic system where GDNF is upregulated after 24 h of ibogaine administration.
This finding is important since the ability of ibogaine to attenuate ethanol self-administration had previously been proposed to be mediated, at least in part, by the increase in GDNF content in the VTA. Furthermore, we show that I20 administration does not increase GDNF expression in any of the studied brain areas, which is in accordance with the observation that this dose was not effective in reducing drug self-administration in the majority of previous studies in rodents.
In addition, our results are in line with the reports indicating that GDNF infusions into the VTA has been effective in reducing drug self-administration or conditioned place preference in rodents (for cocaine and alcohol), and with the proposal that upregulation of the GDNF pathway represents a potential strategy to treating SUDs. Lastly, I40 administration increases GDNF expression in the SN, which was not accompanied with a significant increase of the GDNF protein content at this time point.
Cellular Sources of GDNF
Since in this study the mRNA content was analyzed in whole tissue from the different regions, the precise cell source of GDNF was not identified. Literature indicates that GDNF may be produced either by neuronal or glial cells. Ibogaine treatment upregulated GDNF secretion in dopaminergic neuron-like SHSY5Y cells in culture, however since astrocytes are a major source of NFs the glial origin cannot be excluded.
This observation deserves further attention, since an increase in GDNF in different cell types in regions containing dopaminergic neurons by ibogaine/noribogaine administration could be important for future development of therapeutics for neurodegenerative disorders.
BDNF Expression Findings
With regard to BDNF, a selective downregulation of its expression in the PFC for both doses of ibogaine was found after 3 h of administration, while no changes in other areas were observed. Ibogaine and noribogaine administration in rats stimulate the secretion of corticosterone, being ibogaine a more potent releaser. Since corticosterone decreases BDNF expression in the frontal cortex, ibogaine induced corticosterone secretion during the first hours after treatment (where ibogaine concentrations in blood are high), could be the reason behind this result.
In contrast, at 24 h, an impressive upregulation of BDNF expression was found, which was much more pronounced compared to the effect on GDNF and NGF expression in all the studied brain areas at this time point. Nevertheless, this high effect on BDNF expression was not reflected on an increase in the content of BDNF mature protein, since no significant differences were found between both treatments and the control group at this time point, although trending toward increased BDNF protein levels in NAcc and VTA for both doses.
proBDNF Findings and Implications
Since BDNF is synthesized in a precursor form, we included proBDNF in our experimental design. A selective increase in the proBDNF content was selectively found for NAcc for both ibogaine doses. It is known that the mature BDNF protein and its precursor proBDNF have opposite effects on neuronal protection, axonal growth, maturation of dendrites and synaptic plasticity, owing to different affinities of each form to the TrkB and p75 receptors.
These opposite effects have been hypothesized specifically in the NAcc in the context of animal models of depression as learned helplessness and social defeat stress, where BDNF content is increased while proBDNF content is decreased compared to control animals in this brain area. In this regard, since it is well-documented that an increase in BDNF content in the NAcc increases cocaine-seeking behavior and vulnerability to substance abuse, an increase in proBDNF in this brain area could have an opposite impact.
In this line of reasoning, the increase in proBDNF content in NAcc generated by ibogaine after 24 h of administration in rats, could also be implicated in ibogaine’s effect in drug self-administration paradigms. Further experiments are required to address this hypothesis.
mRNA-Protein Correlation
Despite implicit assumption that differentially expressed mRNAs are reflected in protein content, numerous previous studies comparing mRNA and protein levels concluded that the correlation is poor. While the increase in GDNF mRNA expression was linked to augmented mature protein content, our data showing an impressive increase in BDNF mRNA expression and no changes in mature protein are intriguing.
The possibility exists that the time frame of protein synthesis is different for both NFs, however many other factors should be considered to explain this incongruousness. These include post-transcriptional regulation, for example miRNA-based translation repression or alternative splicing, or translational and post-translational modifications.
Indeed, it has been previously described that sortilin, an intracellular chaperon, acts as a regulatory switch for delivery of BDNF to the regulatory secretory pathway or to degradation in the lysosome, modulating in this way the neurotrophic factor availability. Interestingly, BDNF levels have been shown to be modified in PFC after chronic ethanol exposure.
Mechanisms of NF Upregulation
How does ibogaine administration produce this long-term upregulation of GDNF and BDNF? It is well established that an increase in serotonin transmission leads to an increase in BDNF expression/signaling both in vitro and in vivo. In addition, serotonin and SSRIs (Selective Serotonin Re-uptake Inhibitors) induce GDNF expression in vitro, and recently it has been shown that chronic treatment in mice using SSRIs induce GDNF content in SN and Striatum.
It is well-established that ibogaine and noribogaine increase serotonin transmission. Both substances are serotonin-reuptake inhibitors, and noribogaine is more potent at increasing serotonin levels in the NAcc than ibogaine, which correlates with the ability of both compounds to inhibit SERT in vitro (IC50 of 3.85 and 0.18 μM for ibogaine and noribogaine, respectively).
In this manner, a sustained enhancement on serotonin transmission due to ibogaine and its long-lasting metabolite noribogaine could account, at least in part, for the observed effect on BDNF and GDNF expression after 24 h of ibogaine administration.
NGF Findings
Finally, in addition to GDNF and BDNF, ibogaine also modulated the expression of NGF 24 h after treatment, while no changes were found at 3 h. The effect of NGF administration in specific brain areas on drug-seeking behavior has been less studied in comparison to GDNF and BDNF, and scarce data is available on the effects of NGF in brain regions related to the dopaminergic mesocorticolimbic circuitry.
Nevertheless, NGF (as other neurotrophins) is likely involved in mediating important responses related to chronic intake of drugs of abuse, as illustrated by a previous study that points to a role NGF plays in the central amygdala in the development of increased sensitivity to opioid reward.
Neuroplasticity Implications
The modifications in NFs levels induced by ibogaine/noribogaine, may underlie neuroplasticity processes in the discrete brain regions analyzed as has been described by several drugs used in clinical practice including drugs of abuse. Most of these drugs regulate the expression of NFs, reactivating a process defined as induced plasticity (iPlasticity), which allows networks reorganization in the adult brain.
5. Conclusions and Future Perspectives
This study demonstrates for the first time that ibogaine administration simultaneously alters the expression of GDNF, BDNF and NGF in rat brain regions related to the dopamine neurotransmission in a dose and time dependent manner. Our results add relevant information concerning specific brain areas involved in the increment of GDNF levels (VTA) as a putative mechanism of action underlying the anti-addictive effect of ibogaine.
In addition, we showed that only I40 promoted this increase in GDNF content, which is in accordance with previous reports where the I20 treatment was not effective in reducing drug self-administration in rodents. Also, we found that both doses of ibogaine produced an increase in the proBDNF content in NAcc after 24 h of treatment, which could be an important factor mediating ibogaine anti-addictive long-lasting effects, in addition to the already highlighted increase in GDNF.
Future experiments are needed in order to clarify these important implications.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be considered as a potential conflict of interest.
Author Contributions
SM, BG and SR did the qPCR experiments. EM and SR performed the Western Blot experiments. SM, BG, LMP and MP contributed in the experiments and in the analysis of the data. BG prepared the ibogaine HCl used in this study. JPP, PR and CS, did the experiments with animals and the brain dissection. IC, PC, GS and CS provided the funding for the experiments. MP, PC, GS, CS, DS and IC planned the experiments, and wrote the manuscript. All the authors participated in critical revision the manuscript, added important intellectual content, and approved the definitive version.
Funding
Agencia Nacional de Investigación e Innovación (ANII, Montevideo – Uruguay) Project Fondo María Viñas 103488, Comisión Sectorial de Investigación Científica (UdelaR) – Projects Grupos I+D 981 and 1104, and Programa de Desarrollo de Ciencia Básicas (PEDECIBA).
Acknowledgments
We would like to thank Agencia Nacional de Investigación e Innovación (ANII) and Comisión Sectorial de Investigación Científica (CSIC-UdelaR) for financial support. We thank Inés Carrera, Analia Richeri and Juan Pablo Rodríguez for their help in designing the manuscript figures. We also would like to thank Dr. Kenneth Alper for important discussions regarding ibogaine and noribogaine pharmacology.