REVERSIBLE, SHORT-LASTING, AND DOSE-DEPENDENT EFFECT OF (+)-FENFLURAMINE ON NEOCORTICAL SEROTONERGIC AXONS

Dextrofenfluramine ((+)-fenfluramine) is the dextro-optical isomer of the racemic compound (+/-)-fenfluramine. This compound stimulates the release of serotonin (5-HT) and blocks its re-uptake in serotonergic nerve terminals. (+)-Fenfluramine and its nor metabolite which have been localized in significant amounts in the rat brain are useful anorectic agents in animals. In humans, (+)-fenfluramine is used as an anti-obesity agent when administered orally in doses of 0.25 mg/kg/twice a day. Studies in some animal species (such as the rat and monkey, but not mice) using high doses of (+)-fenfluramine (administered subcutaneously) have shown long-term neurochemical and immunocytochemical effects in selected brain regions. In the present study we used the rat to determine the mechanism underlying the anorectic effect of orally administered (+)-fenfluramine. The rat was selected because long-term effects of (+)-fenfluramine have been previously described in this species. In addition, a variety of other aspects of orally administered (+)-fenfluramine have been addressed in this study. For example, how long does the depletion of 5-HT in the nerve terminals last following cessation of the drug treatment? i.e. is the effect reversible? Is this depletion of 5-HT and the resultant abnormal morphology of 5-HT-immunoreactive nerve terminals seen at high doses dose-dependent? Since some of these questions relate to morphological evaluation of this drug in brain 5-HT systems, we have examined this system as part of our ongoing effort to examine brain monoaminergic systems under perturbed conditions. We have used a morphological (immunocytochemical) approach to answer these questions. The primary function of this study was to evaluate the effects of short-term exposure (4 days) to varying doses of orally administered (+)-fenfluramine on 5-HT-immunoreactive nerve terminals in the frontal cortex of the rat. The frontal cortex was selected because it contains a homogeneous population of nerve fibers and terminals unlike other cortical regions, the hippocampus, striatum and the hypothalamus where a mixed population of coarse and fine fibers has been described. Since the previously reported effect of fenfluramine on 5-HT nerve terminals was the appearance of coarse fibers, the region of cortex selected for this study showed no coarse fibers in the pair-fed control. This essential feature of control regions has not been used in previous studies on this subject. The present study demonstrates that (+)-fenfluramine produces a dose-dependent reduction in 5-HT immunoreactivity of 5-HT nerve terminals in the neocortex of adult rats. Repeated oral administration of a (+)-fenfluramine 'loading dose' for 4 days resulted in a progressive, dose-dependent decrease in 5-HT immunoreactivity in the frontal cortex as well as in other cortical areas. This phenomenon is completely reversible, with complete return to normal of 5-HT immunoreactivity at 15 days post-treatment. Furthermore, repeated administration of this drug produced no behavioral responses in the animals. In addition to reduction in 5-HT immunoreactivity, earlier reports using massive doses of (+/-)-fenfluramine administered parenterally into rats, describe thick 5-HT-immunoreactive axons in the neocortex. At the outset of our study we repeated this study with (+/-)- and (+)-fenfluramine (24 and 48 mg/kg/day) administered subcutaneously for 4 days in the rat ((+)-fenfluramine is twice as potent as (+/-)-fenfluramine in order to examine these thick 5-HT-immunoreactive profiles. In most of the experiments in this study we used oral doses ranging from 2 mg/kg/day to 48 mg/kg/day. The lowest dose at which the thick profiles were first observed with oral administration was 16 mg/kg/day (which is far in excess of the anorectic dose of 0.06-1.25 mg/kg/day). Although it is not possible to compare the doses between rats and humans because of marked differences in the metabolism in these two species, it is unlikely that the human therapeutic dose of 0.2-0.4 mg/kg of (+)-fenfluramine would produce these thick profiles. Furthermore, the rapid and complete return of 5-HT immunoreactivity in the frontal cortex of the rats within 15 days after treatment indicates that the effect of this drug is merely to deplete the nerve terminals of 5-HT resulting in their inability to be 5-HT-immunoreactive, and once the drug has been discontinued the mechanisms for synthesizing 5-HT are able to re-accumulate this putative neurotransmitter (5-HT) in the nerve terminals resulting in the return of 5-HT immunoreactivity. Loading with (+)-fenfluramine and its response in 5-HT-immunoreactive terminals in the brain represents an example of a robust, chemically, specific change in the immunoreactivity of a specific population of nerve terminals that is labile and reversible.