|Written by George Battaglia|
|Thursday, 06 November 2008 18:29|
Parameters of Degeneration and
Recovery of Brain, Serotonin Neurons
GEORGE BATTAGLIA, S. Y. YEH AND ERROL B. DE SOUZA'
Neuroscience Branch, Addiction Research Center
National Institute on Drug Abuse, Baltimore, MD 21224
BATTAGLIA, G., S. Y. YEH AND E. B. DE SOUZA. MDMA-induced neurotoxicity : Parameters of degeneration and recovery of brain serotonin neurons. PHARMACOL BIOCHEM BEHAV 29(2) 269-274, 1988-This study investigates a number of parameters that influence the neurotoxic effects of 3.4-methylenedioxymethamphetamine (MDMA) on serotonin (5-HT) neurons in brain. Both the dose and number of injections of MDMA affect the degree of neurotoxicity on 5-HT axons and terminals as assessed by decreases in the content of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) and the density of 5-HT uptake sites. Repeated systemic administration of various doses of MDMA (5-20 mg/kg twice daily for 4 consecutive days) results in dose-dependent decreases in 5-HT, 5-HIAA and 5-HT uptake sites. Increasing the number of injections of MDMA resulted in progressively greater reductions in 5-HT and 5-HIAA which occurred prior to decreases in 5-HT uptake sites. In contrast, no significant changes were observed in the density of norepinephrine uptake sites following single or repeated injections of 20 mg/kg MDMA. With respect to neuronal regeneration, following an initial 90,70 loss of 5-HT uptake sites after treatment with MDMA, the recovery of these sites occurred over a protracted period of time; a marked 25% reduction was seen at 6 months and the concentration of 5-HT uptake sites returned to control levels at 12 months following treatment with MDMA. Pretreatment with the selective 5-HT uptake blocker, citalopram, prior to each injection of MDMA prevented the neurotoxic effocts of MDMA on the 5-HT parameters described above suggesting that active uptake of MDMA or a MDMA-related subAance into brain 5-HT neurons was involved in the neurotoxic actions of the drug. In addition, the neurodegenerative effects of MDMA on 5-HT neurons exhibited some species specificity as comparable decreases in cerebral cortical 5-HT, 5-HIAA and 5-HT uptake sites were observed in rat and guinea pig while no significant changes in any of these serotonergic parameters were seen in mouse brain.
Neurotoxicity 5-HT uptake sites Regeneration 5-HT 5-HIAA
Since 5-HT uptake sites are highly concentrated on 5-HT containing nerve terminals [ 17], we have taken the approach of using specific radioligands such as IHparoxetine to directly label 5-HT uptake sites in brain and to assess the neurodegeneration of 5-HT neurons by measuring changes in the density of 5-HT uptake sites. We have recently reported the feasibility of using the measurement of 3H-paroxetinelabeled 5-HT uptake sites to quantify the neurotoxic effects of MDMA on 5-HT neurons in various regions of rat brain [2.32). Visualization of MDMA-induced destruction of 5-HT axons and terminals using antibodies directed against serotonin [22.231 and preliminary autoradiographic studies demonstrating corresponding decreases in ~H-paroxetinelabeled 5-HT uptake sites in slide-mounted brain sections of MDMA-treated rats [31 further validate the use of changes in the density of 5-HT uptake sites as an appropriate index of 5-HT neurodegeneration.
The present study was designed to elucidate some of the parameters that may contribute to the marked neurotoxic effects of MDMA on brain serotonin neurons. By measuring changes in 5-HT uptake sites as well as---traditional-markers used to assess such as 5-HT and 5-HIAA content, we have assessed the neurodegenerative effects of MDMA on serotonin neurons with respect to: (1) the effects of dose and dose regimen (i.e., number of injections), (2) the time course of neuronal recovery, (3) the role of the 5-HT transporter complex and (4) the effects in various species.
FIG. 1. The effect of repeated systemic administration of various doses of MDMA on the content of serotonin (5-HT) and 5-hydroxyindoleacetic (5-HIAA) and on the density of 5-HT uptake sites in frontal cortex. Rats were administered saline or MDMA I" ice a day for 4 consecutive days and sacrificed 18 hours after the last injection. One group of rats was pretreated with 10 mg/kg citalopram 30 minutes prior to each administration of 10 mg/kg MDMA. Data represent the meantS.E.M. from 3-5 rats and are expressed as a percent of values in control, saline-injected rats. Control values for 5-HT and 5-HIAA levels were 387:L61 and 15 1 -20 pg/mg tissue, respectively. The density of 5-HT uptake sites in the frontal cortex in controls was 396- 15 fmol/mg protein. Data were analyzed by oneway ANOVA and Duncan's multiple range test. *, ** and *** indicate significant differences atp<0.05,p<0.0I and p<0.001, respectively, from control saline-treated rats. 't and i ', t indicate significant differences at p<0.01 and p<0.001, respectively. from all other groups.
FIG. 2. The effects of single and multiple injections of MDIXIA on the content of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5HIAA) and on the density of 5-HT uptake sites in rat frontal cortex. Rats were injected the specified number of times with either saline or 10 mg/kg MDMA and sacrificed 18 hours after the last injection. Data which represent the mean and S.E.M from three to five animals are plotted as a percent of respective values for each of the parameters in control, saline-injected rats. Control levels of 5-HT and 5-HIAA were 475±24and 332--t24 pmol/mg tissue. respectiNely. The density of 5-HT uptake sites was 349±24 fmol/mg protein in controls. Data were analyzed by one-way ANOVA and Duncan's multiple range test. * indicates a significant difference at p<0.05 from corresponding control saline-injected rats: -I" and - 1 -1" indicate significant differences at p<0.01 andp<0.001, respectively, from all other groups.
FIG. 3. Time course of recovery of serotonin (5-HT) uptake sites in rat frontal cortex following repeated systemic administration of XIDMA. Rats were treated with either saline or 20 mg/kg MDMA twice a day for 4 consecutive days and then sacrificed at various times following the last injection up to 12 months. Salineinjected control rats were killed at each of the time points, and the data Ahich represent the mcan±S.E.M. of five rats per group are plotted as a percent of the value of control animals at the respective time.
single versus Mulliple Injections
Previous studies have demonstrated that the repeated systemic administration of MDMA is neurotoxic to 5-HT neurons in rat brain [2, 3, 7, 22, 23, 27, 28, 30-321. The present study was designed to investigate the influence of certain treatment parameters on the neurotoxic effects of MIJIMA and to determine if these changes are reversible. We report that the neurotoxic actions of MDMA on serotonergic neurons appear to be a function of both the dose administered as well as the number of times the drug is given. Furthermore, the dose and/or number of injections of MDMA can differentially affect the various serotonergic markers used to assess neu rode generation. With respect to the dose of MIJIMA, 5-HT levels appear to be more readily decreased (45% reduction at 5 mg/kg) while 5-HIAA levels are only significantly decreased (-60,7c) following four or eight doses of either 10 or 20 mglkg MDMA. We have previously demonstrated that in either homogenates [2,321 or slidemounted brain sections [31, the loss of 5-HT uptake sites provides an appropriate index of the degeneration of serotonergic neurons in that this measure corresponds to the degree of loss of neuronal axons and terminals detected using 5-HT immunocytochemical techniques [22,231. By using decreases in the density of 5-HT uptake sites as an index of neurodegeneration, it is apparent that MDMA causes the dose-dependent destruction of cortical 5-HT axons and terminals with a significant decrease observed even at the lowest dose of MDMA employed (5 mg/kg). These results demonstrate the dose-dependence of 5-HT neurotoxicity following a repetitive injection regimen of MDMA.
FIG. 4. The effects of repeated systemic administration of MDMA on the content of serotonin (5-HT) (A), 5-hydroxyindoleacetic acid (5-HIAA) (13) and the density of 5-14T uptake sites (C) in rat, guinea pig and mouse frontal cortex. Animals were treated with saline or 20 mg/kg MDMA twice a day for 4 consecutive days then sacrificed 7 days after the last injection. Data represent the meant S.E.M. of five animals per group and are expressed as a percent of safine-injected control values in the respective species. In rat, guinea pig and mouse, control values of 5-HT were 275:L41, 296tI4 and 449t36 pg/mg tissue, respectively; control values of 5-HIAA were 345t4O, 92t4 and 319t34 pg/mg tissue, respectively, control values of 5-HT uptake sites were 397tI0, 216t6 and 233tI2 fmoUmg protein, respectively. Data were analyzed by Students t-test. *** indicates a significant difference at p <0.001 from respective control values.
Since MDMA has been reported to increase the release of serotonin [15,21] and cause suppression of tryptophan hydroxylase activity following systemic administration [30,311, it is important to differentiate between the suppressive effects of MDMA on 5-HT metabolic processes in structurally intact neurons and the neurodegenerative effects of MDMA on the structural integrity of 5-HT axons and terminals. We have addressed this question by comparing the effects of single versus multiple injections of MDMA on each of the serotonergic parameters described. The correspondence between changes in 5-HT or 5-HIAA content and decreases in the density of 5-HT uptake sites, which is indicative of 5-HT neurodegeneration, can be used to assess the relationship between changes in metabolic activity and actual destruction of neurons following treatment With M13MA. These studies (Fig. 2) demonstrating changes in 5-HT and 5-HIAA levels following two and four injections of MDMA but no significant decrease in 5-HT uptake sites until eight repetitive injections of the drug suggest that M13MAinduced changes in 5-HT metabolic activity can precede and may occur independent of the processes determining destruction of 5-HT axons and terminals.
To date, no investigations have reported whether 5-HT neurons can recover from the neurotoxic effects of MDMA and what the time course of neuronal recovery might be. We report here a recovery time course covering a 6-month period following administration of MDMA which indicates that cortical 5-HT axons and terminals can undergo a regeneration process subsequent to an initial 9Wo reduction. The time course of recovery in the frontal cortex, however, occurs over an extremely long period of time as there is a 25% deficit in 5-HT uptake sites up to 6 months and a return to control values at 12 months after MDMA treatment. The persistent neurotoxic effects on 5-HT neurons following MDMA is similar to that observed following parachloroamphetamine (PCA) in which marked reductions in 5-HT have been observed up to 4 months after a single injection [8, 9, 16, 261. As we have used the recovery of 5-HT uptake sites as an index of neuronal regeneration, it is unclear from the present data whether there is an actual regeneration of neurons that have previously undergone axon or terminal degeneration or whether the increased density of uptake sites is a consequence of increased collateral sprouting of neurons unaffected by the drug treatment. It has previously been reported that following 5,6-dihydroxytryptamineinduced axotomy, axonal sprouting occurs within 4-5 days and the appearance of new axonal sprouts correlates with the recovery of 3 H-5-HT uptake [51. Evidence from both immunocytochemical data [22,231 and autoradiographic studies of 5-HT uptake sites [31 indicating that 5-HT cell bodies appear to be insensitive to the neurotoxic effects of repeated systemic administration of MDMA provide a mechanism by which terminal regeneration of MDMA-affected neurons may occur.
The data of the present study demonstrate that destruction of 5-HT axons by MDMA involves the 5-HT active uptake carrier as blockade of this carrier by administration of citalopram, a selective 5-HT uptake blocker [131, prior to administration of MDMA can prevent the decreases in 5-HT markers elicited by MDMA alone (see Fig. 1). These data are consistent with previous reports for other potent 5-HT neurotoxins demonstrating that pretreatment with 5-HT uptake blockers can prevent the neurotoxic effects of PCA [25,26]. It has recently been shown that MDMA-induced neurotoxicity can be prevented or reversed if a 5-HT uptake blocker is administered no later than 12 hours after MDMA treatment .
High doses of MDMA (20 mg/kg) which markedly decrease all parameters of cortical 5-HT neurons in the rat and guinea pig produce no significant changes in the content of 5-HT and 5-HIAA and in the density of 5-HT uptake sites in the mouse. While differences in sensitivity to the neurotoxic effects of PCA on 5-HT neurons have been observed in mouse when compared to its effects in rat and guinea pig [8, 9, 16, 26], the differential sensitivity may be due, in part, to speciesdependent differences in the half-life of PCA . Active neurotoxic metabolites or metabolic intermediates of PCA have previously been postulated as being responsible for its neurotoxic effects on 5-HT neurons [10,26], although to date no such active species has been identified. While there has been no direct demonstration of a neurotoxic metabolite of MDMA, some preliminary data suggest that an active metabolite of MDMA may be responsible for elicitng its neurotoxic effects. We have previously reported that in contrast to the marked 5-HT neurodegenerative effects following systemic administration of MDMA or MDA, direct intracerebral injections of MDMA or MDA are without effect on cortical 5-HT neurons as visualized directly using an antibody directed against serotonin . Our observation of marked species differences in sensitivity to MDMA-induced 5-HT neurotoxicity would be consistent with the hypothesis of a peripherally produced neurotoxic metabolite of MDMA. While these findings suggest that a peripheral metabolite of MDMA may be the active neurotoxic species, in preliminary studies, we have found that in rats pretreated with the cytochrome P-450 enzyme inhibitor, SKF 525A, there was no potentiation or attenuation of the neu rodege ne ration found following repeated administration of 10 mg/kg of MDMA (unpublished observation). Additional studies are required to identify the neurotoxic species of MDMA. In summary, our findings indicate that in rat, MDMA-induced neurotoxicity of brain 5-HT terminals is dependent on the dose of drug used as well as the number of times the drug is administered. Following extensive destruction of 5-HT neurons, regeneration can occur but requires a substantial amount of time. The ability of 5-HT uptake blockers to prevent the neurotoxic effects of MDMA suggests that the active uptake process is involved in the mechanism of neurotoxicity of MDMA.
We thank Terrie Pierce and Mary Flutka for manuscript preparation. This work was supported in part from funds provided by the U.S. Food and Drug Administration.
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