Anatomic evidence for a neurotoxic effect of (±)-fenfluramine upon serotonergic projections in the rat

Brain Research, 511 (1990) 165-168

165

Elsevier

BRES 24013

Anatomic evidence for a neurotoxic effect of (+_)-fenfluramine upon serotonergic projections in the rat Derek C. Molliver and Mark E. Molliver The Johns Hopkins University, School of Medicine, Departments of Neuroscience and Neurology, Baltimore, MD 21205 (U.S.A.)

(Accepted 5 December 1989) Key words: Serotonin; Fenfluramine; Neurotoxicity;Degeneration; Cerebral cortex; Indoleamine; Amphetamine; Raphe

Immunocytochemistrywas used to determine whether (+)-fenfluramine causes structural damage to serotonergic (5-HT) neurons. Sections from rat forebrain were examined 4 h, 36 h and 2 weeks after various dose regimens of fenfluramine. At all time points there was a reduction of fine 5-HT axon terminals in the forebrain, while beaded axons were spared. The presence of markedly swollen, fragmented 5-HT axons 36 h after injection is indicative of axonal degeneration, and provides morphologicevidence for a neurotoxic effect of (+-)-fenfluramineupon 5-HT axon terminals. Fenfluramine is a halogenated amphetamine derivative that is used as an appetite suppressant in the clinical management of obesity 16'21, and is currently being investigated for the treatment of autism 2°. The anorectic effect of fenfluramine appears to result from the release of serotonin (5-HT) 4'5'26, which is dependent upon an effect of fenfiuramine at the 5-HT uptake carrier 4. Fenfluramine causes large, acute decreases in brain levels of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) and in tryptophan hydroxylase activity2,15,25. A single 100 /tmol/kg dose causes significant depletion of 5-HT that lasts more than 30 days6, while multiple doses cause 5-HT depletion lasting for at least 4 months 1'23,25. In contrast, the half-lives of fenfluramine and of its primary active metabolite, norfenfluramine, in rats are approximately 4 h and 24 h, respectively21. These observations raise the possibility that the reduction in 5-HT markers may result from a neurotoxic effect of fenfluramine upon serotonin neurons. However, since fenfluramine may conceivably cause transient changes in 5-HT metabolism, direct structural evidence is required to determine whether the prolonged reductions in biochemical levels are due to neuronal degeneration. The present experiment utilized immunocytochemical staining for 5-HT neurons with three goals: to assess anatomic evidence of an acute effect of fenfluramine upon serotonergic neurons, to determine whether the drug has lasting neurotoxic effects, and to identify specific neurons and neuronal processes that may be damaged. The effects of fenfluramine upon 5-HT neurons were

assessed at three survival times. Animals were sacrificed at different intervals after drug treatment in order to examine short and long-term effects of fenfluramine and to determine whether neuronal degeneration occurs. Experimental animals were injected with (+)-fenfluramine and controls were given saline vehicle injections. In these experiments, at least 3 treated animals and 3 controls were studied for each dosage and treatment regimen. To study aEute drug effects, rats were administered a single subcutaneous injection of 5 or 10 mg/kg of (+)-fenflurarnine (free base) and were perfused 4 h later. To detect morphologic evidence of neuronal degeneration, rats were injected with (+)-fenfluramine (5 mg/kg) at 12 h intervals (3 doses total), and peffused 36 h after the third injection. (In pilot studies, we previously determined that a large number of degenerating axons were seen at 36-48 h surffivals.) For the study of persistent neurotoxic effects, rats were given single daily injections of (+)-fenfluramine (5 or 10 mg/kg) for 5 days and were peffused two weeks after the last injection. In all experiments, male Sprague-Dawley rats (145170 g) were administered various doses of raeemic fenfluramine (Sigma, St. Louis) by subcutaneous injection (see regimen above) and sacrificed by transcardial perfusion with 4% paraformaldehyde in a 0.15 M phosphate buffer, pH 7.4. The brains were removed and 30 gm parasagittal sections were prepared either for staining with Cresyl violet or for immunocytochemistry using an antibody to serotonin previously characterized

Correspondence.. M.E. Molliver, Department of Neuroscience, The Johns Hopkins University, School of Medicine, 725 N. Wolfe Street,

Baltimore, MD 21205, U.S.A.

0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

166 by Lidov and coworkers ~°. Immunocytochemical methods used are identical to those described elsewhere TM. Four hours after a single dose of (+)-fenfluramine (5 or 10 mg/kg) there was a marked decrease in the number of 5-HT-immunoreactive axons throughout the forebrain. The decrease in 5-HT axon staining had a consistent regional distribution, with the most profound depletion seen in neocortex, striatum, hippocampus, and medial hypothalamus. There was considerable sparing of 5-HT axons in brainstem, lateral hypothalamus, dentate gyrus and in the olfactory glomeruli. Even in severely affected areas, some 5-HT-immunoreactive axons remained. The loss of staining was restricted to fine 5-HT axon terminals, while a second class of 5-HT axons s, those with large, spherical varicosities was consistently spared. Preterminal axons (in the median forebrain bundle) and the cell bodies of 5-HT neurons in the raphe nuclei did not show changes after fenfluramine treatment. In the animals sacrificed two weeks after drug administration there was a profound loss of 5-HT axon staining, with the same regional distribution that was found at 4 h. As in the short survivals, fine axons were lost while beaded axons were spared. The decreased density of fine axons was clearly evident (although incomplete) at the 5 mg/kg dose and more pronounced at the 10 mg/kg dose (Fig. 1). The decrease in axon staining was highly

Contrnl

consistent among treated animals, with little or no variability between rats in each dosage group. In all cases, the 5 mg/kg dose produced a noticeable decrease in the density of stained 5-HT axons that unequivocally distinguished treated from control animals. At 36 h survival times the decrease in immunocytochemical staining of 5-HT axons was similar to that seen after 4 h, and exhibited the same regional and morphological distribution. However, in all areas of cortex and basal forebrain, intensely stained 5-HT fibers exhibited cytopathologic features. Two kinds of structural changes were observed. Many 5-HT axons were irregularly shaped, with extremely large, abnormal varicosities (Fig. 2). These enormously swollen axons were seen within cortical gray matter, where 5-HT axon terminals are normally found. In many cases interruption or fragmentation of fibers was evident. These damaged 5-HT axons, approximately 5-10 times larger than normal fibers, were not seen in controls and were clearly different from the intact fine or beaded axons in untreated animals. A second type of degenerative change was seen in subcortical white matter, anterior to the genu of the corpus caliosum, and in lateral hypothalamus. The latter changes consist of swollen axon stumps that reflect degeneration of the distal segment of preterminal axons. The present immunocytochemical study confirms that

Fenfluramine 5 mQ/kg

Fenfluramine 10 mg/kg

Fig. 1. The effects of fenfluramine on 5-HT axons in parietal cortex of the rat demonstrated by 5-HT immunocytochemistry 2 weeks after treatment. Animals received a single daily dose of (_+)-fenfluramine (s.c.) for 5 consecutive days, and were sacrificed 2 weeks later. There is an unequivocal loss of immunoreactive axons after multiple doses of 5 mg/kg, with a more striking decrease at 10 mg/kg. Dark-field photomicrograph. Bar = 100/~m.

167 (+)-fenfluramine initially causes depletion of transmitter from serotonergic axons in forebrain. The presence of structural damage to 5-HT axons observed at 36 h, together with the persistent loss of stained 5-HT axons after 2 weeks survival, furnishes morphologic evidence which supports the interpretation that (+)-fenfluramine causes degeneration of 5-HT axon terminals. These results provide an anatomic explanation for the previously reported biochemical findings of long-lasting reductions in levels of 5-HT, 5-HIAA, tryptophan hydroxylase activity and synaptosomal uptake 2,5,8,15,25. The effects of (+)-fenfluramine on 5-HT axon terminals are similar to those of the neurotoxic amphetamine derivatives p-chloroamphetamine (PCA) and 3,4-methylenedioxyamphetamine (MDA). All 3 drugs cause acute release of 5-HT 2'3'5'1s followed by a long-lasting reduction in brain levels of this transmitter 3,8,~7,22,25 and a persistent loss of 5-HT axon terminals 11,12,14,17. These effects are characterized by selective vulnerability of fine, but not beaded, 5-HT axon terminals ~1'~3'14. These two 5-HT axon types have overlapping but different distributions, with predominantly fine axons in neocortex but

beaded axons in brainstem and restricted areas of forebrain 9-~'27. The differential regional distribution of the two 5-HT axon types 11'14'27 explains the regional differences in reduction of 5-HT levels after administration of these drugs. Harvey and McMaster 6 ascribed the fenfluramineinduced loss of 5-HT to the degeneration of cell bodies in the B-9 group. Their evidence for persistent decreases in 5-HT levels has been repeatedly verified, but the interpretation that the losses are due to cell body damage has not been confirmed 24. The present experiment shows that axons of passage and cell bodies of origin are spared after fenfluramine administration. These findings indicate that, as shown for M D A and PCA TM, fenfluramine has a restricted neurotoxic effect upon specific structural compartments of 5-HT neurons. The demonstration that large doses of (_+)-fenfluramine are neurotoxic in rats indicates the importance of further study in primates in order to evaluate the safety of clinical use. The usual dose for human clinical administration of (+)-fenfluramine (approx. 1 mg/kg/ day, p.o. 21) is much less than that used in this study.

eO 4 i,

~Q

A I

#

q

I

Q

B i

t

Fig. 2. Bright-field photomicrographs of 5-HT axons in forebrain of the rat. A: vehicle-treated controls. B: 36 h after fenfluramine (5 mg/kg x 3). In fenfluramine-treated animals (B), intense immunoreactive staining, huge abnormal varicosities, swollen intervaricose segments and fragmentation of fibers indicate degeneration of axon terminals. These damaged axons are distinct from beaded axons normally found in controls (A). All panels are printed at the same final magnification. Bar = 10/~m.

168 Differences in rates of drug metabolism between species and in methods of administration make it difficult to generalize from this study to h u m a n effects of fenfluramine. Yet, vulnerability of 5-HT neurons is also present in monkeys since m e t h y l e n e d i o x y m e t h a m p h e t a m i n e ( M D M A ) , a related a m p h e t a m i n e derivative, produces significant neurotoxic effects in primates ~8'27. Moreover,

1 Clineschmidt, B.V., Zacchei, A.G., Totaro, J.A., Pflueger, A.B., McGuffin, J.C. and Wishousky, T.I., Fenfluramine and brain serotonin, Ann. N Y Acad. Sci., 305 (1978) 222-241. 2 Duhault, J. and Verdavainne, C., Modification du taux de serotonine cerebrale chez le rat par le trifluoromethyl-phenyl2-ethyl aminopropane (fenfluramine 768 S.), Arch. Int. Pharrnacodyn. Ther., 170 (1967) 276-286. 3 Fuller, R.W., Perry, K.W. and Molloy, B.B., Reversible and irreversible phases of serotonin depletion by 4-chloroamphetamine, Eur. J. Pharmacol., 33 (1975) 119-124. 4 Fuller, R.W., Shoddy, H.D. and Robertson, D.W., Mechanisms of effects of d-fenfluramine on brain serotonin metabolism in rats: uptake inhibition versus release, Pharmacol. Biochem. Behav., 30 (1988) 715-721. 5 Fuxe, K., Larnebo, L.-O., Hamberger, B. and Ogren, S.-O., On the in vivo and in vitro actions of fenfluramine and its derivatives on central monoamine neurons, especially 5-hydroxytryptamine neurons, and their relation to the anorectic activity of fenfluramine, Postgrad. Med. J., 51 (Suppl. 1) (1975) 35-45. 6 Harvey, J.A. and McMaster, S.E., Fenfluramine: evidence for a neurotoxic action on midbrain and a long-term depletion of serotonin, Psychopharmacol. Commun., 1 (1975) 217-228. 7 Johnson, M.P., Hoffman, A.J. and Nichols, D.E., Effects of the enantiomers of MDA, MDMA and related analogues on [3H]serotonin and [3H]dopamine release from superfused rat brain slices, Eur. J. Pharmacol., 132 (1986) 269-276. 8 Kleven, M.S., Schuster, C.R. and Seiden, L.S., Effect of depletion of brain serotonin by repeated fenfluramine on neurochemical and anorectic effects of acute fenfluramine, J. Pharrnacol. Exp. Ther., 246 (1988) 822-828. 9 Kosofsky, B.E. and Molliver, M.E., The serotoninergic innervation of cerebral cortex: different classes of axon terminals arise from dorsal and median raphe nuclei, Synapse, 1 (1987) 153-168. 10 Lidov, H.G.W., Grzanna, R. and Molliver, M.E., The serotonin innervation of the cerebral cortex in the rat - An immunohistochemical analysis, Neuroscience, 5 (1980) 207-227. 11 Mamounas, L.A., Mullen, C. and Molliver, M.E., Morphologically dissimilar serotonergic axon types in rat cerebral cortex are differentially vulnerable to the neurotoxin p-chloroamphetamine (PCA), Soc. Neurosci. Abstr., 14 (1988) 210. 12 Molliver, D.C. and Molliver, M.E., Selective neurotoxic effects of (+)-fenfluramine upon 5-HT axons in rat brain: immunocytochemical evidence, Soc. Neurosci. Abstr., 14 (1988) 210. 13 Molliver, M.E., Mamounas, L.A. and Carr. P., Reinnervation of cerebral cortex by 5-HT axons after denervation by psychotropic amphetamine derivatives, Soc. Neurosci. Abstr., 15 (1989) 417. 14 O'Hearn, E., Battaglia, G., De Souza, E.B., Kuhar, M.J. and Molliver, M.E., Methylenedioxyamphetamine (MDA) and

repeated large injections of d-fenfluramine have recently been shown to cause lasting neurotoxic effects upon 5-HT projections to forebrain in the primate 19'23.

We wish to thank Patrice Carr for technical assistance, and Patricia O'Neill for help in preparing the manuscript. This work was supported by USPHS research Grant DA 04431.

methylenedioxymethamphetamine (MDMA) cause selective ablation of serotonergic axon terminals in forebrain: immunocytochemical evidence for neurotoxicity, J. Neurosci., 8 (1988) 2788-2803. 15 Opitz, K., Anorexigene Phenylalkylamine und Serotoninstoffwechsel, Naunyn-Schmiedeberg's Arch. Pharmacol. Exp. Pathol., 259 (1967) 56-65. 16 Pinder, R.M., Brogden, R.N., Sawyer, P.R., Speight, T.M. and Avery, G.S., Fenfluramine: a review of its pharmacological properties and therapeutic efficacy in obesity, Drugs, 10 (1975) 241-323. 17 Ricaurte, G.A., Bryan, G., Strauss, L., Seiden, L. and Schuster, C., Hallucinogenic amphetamine selectively destroys brain serotonin nerve terminals, Science, 229 (1985) 986-988. 18 Ricaurte, G.A., Forno, L.S., Wilson, M.A., DeLanney, L.E., Irwin, I., Molliver, M.E. and Langston, J.W., (+)3,4-methylenedioxymethamphetamine (MDMA) selectively damages central serotonergic neurons in nonhuman primates, J. Am. Med. Assoc., 260 (1988) 51-55. 19 Ricaurte, G.A., Molliver, M.E., Witkin, J.M., Molliver, D.C., Wilson, M.A. and Katz, J.L., d-Fenfluramine produces longterm effects on central serotonin neurons in nonhuman primates, Soc. Neurosci. Abstr., 15 (1989) 419. 20 Ritvo, E.R., Freeman, B.J., Yuwiler, A., Geller, E., Schroth, P., Yokota, A., Mason-Brothers, A., August, G.J., Klykylo, W., Leventhal, B., Lewis, K., Piggott, L., Realmuto, G., Stubbs, E.G. and Umansky, R., Fenfluramine therapy for autism: promise and precaution, Psychopharmacol. Bull., 22 (1986) 133-140. 21 Rowland, N.E. and Carlton, J., Neurobiology of an anorectic drug: fenfluramine, Prog. Neurobiol., 27 (1986) 13-62. 22 Sanders-Bush, E. and Steranka, L.R., Immediate and long-term effects of p-chloroamphetamine on brain amines, Ann. N. E Acad. Sci., 305 (1978) 208-221. 23 Schuster, C.R., Lewis, M. and Seiden, L.S., Fenfluramine: neurotoxicity, Psychopharmacol. Bull., 22 (1986) 148-151. 24 Sotelo, C. and Zamora, A., Lack of morphological changes in the neurons of the B-9 group in rats treated with fenfluramine, Curt. Med. Res. Opin., 6 (1978) 55-62. 25 Steranka, L.R. and Sanders-Bush, E., Long-term effects of fenfluramine on central serotonergic mechanisms, Neuropharmacology, 18 (1979) 895-903. 26 Trulson, M.E. and Jacobs, B.L., Behavioral evidence for the rapid release of CNS serotonin by PCA and fenfluramine, Eur. J. Pharmacol., 36 (1976) 149-154. 27 Wilson, M.A., Ricaurte, G.A. and Molliver, M.E., Distinct morphologic classes of serotonergic axons in primates exhibit differential vulnerability to the psychotropic drug 3,4-methylenedioxymethamphetamine, Neuroscience, 28 (1989) 121-137.