Characterization of d-fenfluramine-induced hypothermia: evidence for multiple sites of action

Abstract

The effects of d-fenfluramine on core body temperature has been largely investigated under conditions of either high or low ambient temperature, whereas little research has focused on this response under normal environmental conditions. Moreover, there has been neglect in research on the mechanisms underlying changes in body temperature. In this study, we demonstrate that d-fenfluramine (5 and 10 mg/kg) induces a sustained decrease in body temperature in the rat under normal ambient temperatures. Pre-treatment with the selective serotonin reuptake inhibitor sertraline (5 mg/kg), the full 5-HT_1A receptor antagonist 4-fluoro-N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-2-pyridinyl benzamide], WAY 100635 (0.15 mg/kg) and the 5-HT_2C receptor antagonist benzofuran-2-carboxamidine, RO 43-0440 (2.5 mg/kg) blocked d-fenfluramine-induced hypothermia. Depletion of 5-hydroxytryptamine (5-HT) stores following treatment with the serotonergic neurotoxin parachlorophenylalanine reversed the initial hypothermic effects of d-fenfluramine but not the later effects, as d-fenfluramine produced a delayed hypothermia (>120 min post-challenge) in animals pre-treated with parachlorophenylalanine. Such findings are consistent with a requirement for d-fenfluramine uptake into 5-HT neurons followed by release of 5-HT from intracellular stores and stimulation of post-synaptic 5-HT receptors to reduce body temperature. The hypothermic response to d-fenfluramine was potentiated by ketanserin pre-treatment 30 min post-challenge but then antagonized at later time intervals. Pre-treatment with the dopamine, D₂ antagonist, haloperidol (1 mg/kg) and sulpiride (30 mg/kg) had a similar effect in blocking the hypothermia as WAY 100635, suggesting a role for dopamine D₂ receptors in the response. Pre-treatment with the α₂-adrenoceptor antagonist yohimbine failed to block the hypothermic response. These results suggest multiple sites of action mediating d-fenfluramine-induced hypothermia and may be the result of a combined effect of d-fenfluramine and its active metabolite norfenfluramine affecting not only the release of 5-HT but also stimulation of post-synaptic receptors.

Introduction

d-Fenfluramine is the potent isomer of the substituted amphetamine fenfluramine, which stimulates 5-hydroxytryptamine (5-HT) release and increases extracellular 5-HT levels in many brain regions Auerbach et al., 1989, Schwartz et al., 1989, Kreiss et al., 1993, Series et al., 1994. The d-isomer of fenfluramine is a more specific 5-HT probe, being free of the catecholamine effects of the racemic d,l-fenfluramine (Garattini et al., 1987). A plethora of 5-HT-mediated behavioural and physiological responses (including feeding and thermoregulation) have also been assessed using d-fenfluramine or its racemate Rose et al., 1997, Baumann et al., 1998. It has been used extensively as a neuroendocrine probe of serotonergic function in both psychiatric patients and healthy individuals O'Keane and Dinan, 1991, O'Keane et al., 1992, Feeney et al., 1993, Cleare et al., 1996.

Rodents that are exposed to d- or d,l-fenfluramine have been shown to have impaired thermoregulatory ability and alterations in core body temperature Jespersen et al., 1969, Preston et al., 1990, Malberg and Seiden, 1997, Stewart et al., 1997. Under elevated environmental temperature conditions (>25°C) d- and d,l-fenfluramine induce a hyperthermic response in the rat Frey, 1975, Sulpizio et al., 1978, Sugrue, 1984 in a similar fashion as other substituted amphetamines, such as 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine. In contrast, under lower ambient temperature conditions (<10°C) a distinct hypothermic response to fenfluramine has been observed (Malberg and Seiden, 1997). While many studies have focused on the effects of fenfluramine-induced temperature changes under extreme environmental conditions, there has been a relative neglect in the characterization of its effects on core body temperature under normal laboratory conditions. In addition, the receptor subtypes involved in mediating these temperature responses at any environmental temperature remain uncertain. Recently, it has been demonstrated that d- or d,l-fenfluramine, induces a reduction in core body temperature in both rats and mice, kept under normal laboratory temperature (20–24°C) O'Callaghan and Miller, 1994, Miller and O'Callaghan, 1995, Malberg and Seiden, 1997. This is in contrast to the traditional view that d-, or d,l-fenfluramine evokes little change in core temperature under these conditions Sugrue, 1984, Stewart et al., 1997.

Both noradrenergic and dopaminergic neurotransmitter systems have been implicated in the mediation of the hypothermic response in many species, with both α₂-adrenoceptors Myers et al., 1987, Minor et al., 1989, Menon et al., 1990 and dopamine receptors Nunes et al., 1991, Zarrindast and Tabataba, 1992, Millan et al., 1994, Parada et al., 1995 thought to play a major role. In addition, the relationship between the serotonergic system and hypothermic mechanism has long been associated Lin et al., 1983, Lin et al., 1998, Won and Lin, 1988 with hypothermia as a consequence of 5-HT_1A receptor activation being the best characterized (see Millan et al., 1993, De Vry, 1995, Cryan et al., 1999a). Agonists at this receptor subtype such as 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), flesinoxan and ipsapirone show a dose-dependent hypothermic response following administration and this response has been used as an in vivo marker of 5-HT_1A receptor activation in rodents and man Goodwin et al., 1985, Hjorth, 1985, Lesch et al., 1990, De Vry, 1995, Pitchot et al., 1995, Cryan et al., 1999b.

The aim of the present study was to characterize the effects of d-fenfluramine on core body temperature under normal laboratory conditions in rats. The contribution of the 5-HT reuptake site, 5-HT_1A, 5-HT_2A and 5-HT_2C receptors in conjunction with the role of intracellular 5-HT stores were assessed in the d-fenfluramine-induced response. In addition, we further investigated the selectivity of the responses by assessing the interaction of other ligands with affinity for other monoamine receptors implicated in altering core body temperature. The results suggest that d-fenfluramine-induced hypothermia is the result of d-fenfluramine uptake, induction of 5-HT release and stimulation of post-synaptically located receptors.