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Administration of fenfluramine at different ambient temperatures produces different core temperature and 5-HT neurotoxicity profiles
Brain Research 765 Ž1997. 101–107
Research report
Administration of fenfluramine at different ambient temperatures produces different core temperature and 5-HT neurotoxicity profiles Jessica E. Malberg, Lewis S. Seiden
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Department of Pharmacological and Physiological Sciences, UniÕersity of Chicago, 947 East 58th Street, Chicago, IL 60637, USA Accepted 8 April 1997
Abstract This study investigated the effect of two different ambient temperatures on fenfluramine-induced 5-HT neurotoxicity. Fenfluramine ŽFEN. Ž12.5 mgrkg= 4; injections made hourly. or saline ŽSAL. was administered to rats in either a normal laboratory temperature of 248C or a warm environment of 308C. Animals were kept at that ambient temperature for 20 h after FEN administration. Ambient temperature was controlled to "0.58C and rat core temperature was continually measured using a non-invasive apparatus. FEN-treated rats at 248C displayed a core temperature hypothermia with a peak low of 33.88C, and this core temperature hypothermia lasted for 20 h after FEN administration. Rats treated with FEN at 308C displayed a significant core temperature hyperthermia for 4 h after the first drug injection compared to SAL-treated groups, with a peak core temperature of 38.68C. 2 weeks after FEN injections, brain regions were analyzed by HPLC. Both groups of FEN-treated rats showed decreases in 5-HT and 5-HIAA in the hippocampus, frontal cortex, somatosensory cortex, striatum, hypothalamus and septum. However, FEN rats treated at 308C had significantly greater decreases Ž26–35%. in 5-HT compared to FEN-treated rats at 248C in the frontal cortex, hippocampus, striatum and somatosensory cortex and significantly greater decreases Ž26–50%. in 5-HIAA in the frontal cortex, hippocampus and somatosensory cortex. This study indicates fenfluramine can produce neurotoxicity in rats that display either a core temperature hypothermia or hyperthermia, although hyperthermic rats have greater 5-HT and 5-HIAA depletions than the hypothermic rats. q 1997 Elsevier Science B.V. Keywords: Fenfluramine; Amphetamine; Neurotoxicity; Temperature; Thermoregulation; 5-HT; 5-HIAA
1. Introduction D,L-Fenfluramine
ŽFEN. is a substituted amphetamine which is used clinically for its anorectic effects w1,20x. Similar to other amphetamine analogs, such as methamphetamine ŽMETH. and methylenedioxymethamphetamine ŽMDMA., FEN has been shown to be neurotoxic to the 5-HT system. This neurotoxicity has been demonstrated experimentally by meeting a number of criteria. FEN administration produces a decrease in 5-HT and 5-HIAA levels in rats w15,30x, guinea pigs w27x and monkeys w27x, a decrease in tryptophan hydroxylase w30x, a decrease in the number of 5-HT uptake sites and morphological evidence that is consistent with FEN-induced degeneration of 5-HT
Abbreviations: FEN, fenfluramine; SAL, saline; MDMA, methylenedioxymethamphetamine; METH, methamphetamine; 5-HT, serotonin; 5HIAA, 5-hydroxyindole acetic acid. ) Corresponding author. Fax: q1 Ž773. 702-3003; E-mail: [email protected] 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 5 1 7 - 9
nerve terminals w25,33x that lasts beyond the period of FEN administration. These effects have been found 2 weeks after FEN administration w15,25,27,30,33x, and the decrease in 5-HT levels have been reported at 8 w15,27x and 16 w27x weeks. Zaczek et al. w35x have shown that the number of 5-HT transporters was still reduced at 8 months. FEN has been shown to acutely release 5-HT w21x and this 5-HT release is blocked by fluoxetine w2x, indicating that this release is mediated by the uptake carrier though the mechanism of exchange diffusion w29x. The FEN-induced neurotoxicity has also been blocked by fluoxetine pretreatment w10,30x. These studies indicate that FEN uptake via the 5-HT uptake transporter or 5-HT release is necessary for FEN-induced 5-HT neurotoxicity. In addition to its neurotoxic effects on the 5-HT system, FEN has been shown to affect core temperature w8,13,24x and thermoregulatory ability w17,26x. Many studies have been performed looking at the effects of doses of FEN on core body temperature. These studies are varied in their results; both hypothermia w8,14x and hyperthermia w9,32x have been reported after FEN administration. This appar-
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ent contradiction has been explained by the ambient temperature at which the animal was dosed. Yehuda and Wurtman w34x have administered FEN at 4, 20 and 378C; at the high environmental temperature a core temperature hyperthermia was seen, and at the low environmental temperatures a core temperature hypothermia was seen. Additionally, Preston et al. w26x and Ma and Preston w17x have reported that at a dosing ambient temperature of 288C FEN produced hyperthermia, increased metabolic rate, increased blood flow and increased whole-body oxygen consumption, but that at an ambient temperature of 208C core temperature was reduced and the thermogenesis that was seen in control rats was also reduced. Based on these studies, Preston et al. w26x and Ma and Preston w17x concluded that FEN can affect thermogenesis and thermoregulatory parameters, such as blood flow and metabolic rate, but these effects are dependent on the ambient temperature at which the drug is administered. More recently, studies by Farfel and Seiden w8x and Miller et al. w22x have looked at the interaction of rat core temperature and neurotoxicity when FEN was administered in a normal laboratory temperature of 228–248C. Both of these studies reported a FEN-induced hypothermia in rats and also reported FEN-induced 5-HT and 5-HIAA depletions. The finding that FEN produces 5-HT neurotoxicity with an accompanying hypothermia is a striking difference from the other substituted amphetamines, such as METH and MDMA. In these drugs, animals that become hypothermic during METH or MDMA administration show protection against METH- or MDMA-induced neurotoxicity w3,19x. Conversely, in these drugs, neurotoxicity has been associated with an increase in body temperature w5,8,18x. Although FEN, METH and MDMA are all 5-HT-releasing agents, there seems to be a large difference in their core temperature and neurotoxicity profiles in the rat, where hypothermia accompanies FEN-induced neurotoxicity, but protects against MDMA- or METH-induced neurotoxicity. Although it is known that FEN produces an inability to thermoregulate and is also neurotoxic to the 5-HT system, it is not known whether the lack of thermoregulation is related to the neurotoxicity. This study aimed to investigate if rats treated with FEN in a high ambient temperature Ž308C. would show a different core temperature and neurotoxicity profile than rats treated at a normal laboratory temperature of 248C. To pursue this line of study, our laboratory has developed a core temperature measurement apparatus which controls ambient temperature to "0.58C and measures rat core temperature once per minute using a non-invasive apparatus. We now report that rats treated with FEN at an ambient temperature of 248C show a core temperature hypothermia compared to SAL-treated rats during the entire 20-h testing period. Rats treated with FEN at an ambient temperature of 308C show a core temperature hyperthermia compared to SAL-treated rats for the first 4 h after the drug injection.
Both FEN-treated groups of rats had decreases in 5-HT and 5-HIAA levels, but rats treated at the high ambient temperature of 308C had greater 5-HT and 5-HIAA depletions than the rats treated at 248C.
2. Materials and methods 2.1. Subjects Sprague–Dawley rats ŽHoltzman, Madison, WI. weighing between 250 and 300 g at time of surgery were used. Throughout the experiment, rats had access to food Ž4% Tek Lab rat chow. and water ad libitum. Rats were maintained on a 12:12 h lightrdark cycle with a room temperature of 22–248C, except on the injection day as indicated in the Section 2.5. Rats were housed 4–5 per cage except for the injection day, when they were housed individually in the temperature chambers. 2.2. Drugs Ž".-Fenfluramine ŽFEN. was obtained from Sigma Chemical Co. ŽSt. Louis, MO. and dosages are expressed in terms of the weight of the salt. Ketamine and Nembutal were obtained from Abbott Laboratories ŽChicago, IL.. 2.3. Surgery For rat core temperature measurements, temperaturesensitive radio transmitters ŽMinimitter Co., Sunriver, OR. were implanted in the peritoneum of the rat. Rats were anesthetized with Nembutal Ž40 mgrkg., and given supplemental 1-ml injections of ketamine as needed. A midline cut was made into the peritoneum and a sterilized transmitter inserted into the peritoneal cavity. Rats were given a minimum of 3 days to recover from the surgery. 2.4. Temperature measurement apparatus The temperature measurement chambers are modified compact refrigerators Ž7 ft 3 ; Absocold Corp, FL. with a Plexiglass window in the door to allow observation of the rats and to keep the lightrdark cycle in synchrony. A strip heater, fan and thermistor to record ambient temperature are mounted in the back of the chamber and a cage inside the chamber contains the rat. Four AM radios placed directly outside this cage receive frequency signals, corresponding to core temperature, from a temperature-sensitive transmitter implanted in the peritoneum of the rats. The radios and thermistor are interfaced to a microcomputer Ž33 MHA 386 DX.. To monitor core temperature, the computer receives the frequency signals from the radios and through an A-to-D converter, converts the frequency signals into core temperature values for each rat. To control for ambient temperature, the computer has digital
J.E. Malberg, L.S. Seidenr Brain Research 765 (1997) 101–107
output lines which turn the refrigerator compressor and strip heater off and on as needed to maintain the desired ambient temperature. The computer receives ambient and core temperature values every second, and these are averaged together to produce an ambient and a core temperature value for each minute of the experiment. 2.5. Procedure After arrival in the colony, rats were implanted with core temperature-sensitive radiotransmitters. One week after implantation, rats were placed in the temperature chambers for 1 h at an ambient temperature of 248C Žnormal laboratory temperature. for a baseline measure. After the baseline hour, the ambient temperature either remained at 248C or was increased to 308C. Rats were then injected with FEN Ž12.5 mgrml, 1 mlrkg. or SAL Ž1 mlrkg. once an hour for four hours and remained in the temperature chamber at 24 or 308C for 20 h. This dosage and injection schedule has been previously used by Farfel and Seiden w8x. Two weeks after the injections, rats were killed by decapitation and their brains were dissected as described by Heffner et al. w12x. Tissue samples were stored in liquid nitrogen. 5-HT and 5-HIAA levels were determined for the frontal cortex, somatosensory Žoccipital–parietal. cortex, hippocampus, striatum, hypothalamus and septum using high-performance liquid chromatography ŽHPLC. as described in Kotake et al. w16x.
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2.6. Statistics The core temperature data were quantified by using an area-under-the-curve ŽAUC. analysis. For each rat, a core temperature vs. time graph was generated, and the AUC was determined ŽIgor, Wavemetrics.. This AUC value was denoted as the ‘total core temperature’ response for each rat. The core temperature responses for all rats in each experimental group were summed together. Differences were determined by ANOVA followed by a Tukey post-hoc test ŽInstat for MacIntosh, GraphPad.. Single-point comparisons were determined using a Tukey post-hoc test. For the neurotoxicity data, differences were determined by ANOVA followed by Student’s t-test comparisons.
3. Results 3.1. Core temperature data Analysis of the total core temperature change Žtotal AUC., showed that the four experimental groups, FEN given at 248C ŽFEN-248C., FEN given at 308C ŽFEN-308C., SAL given at 248C ŽSAL-248C. and SAL given at 308C ŽSAL-308C., showed a significant treatment effect Ž F s 49.53, P - 0.0001. throughout the testing period ŽFig. 1.. The average core temperatures of the SAL-248C and SAL308C groups during the entire testing period were 37.66 "
Fig. 1. Core temperature vs. time graph for the 4 experimental groups ŽFEN-308C, FEN-248C, SAL-308C and FEN-248C.. Rats were treated with FEN Ž12.5 mgrkg= 4, n s 8. or SAL Ž1 mlrkg= 4, n s 6. at 0, 60, 120 and 180 min at either 24 or 308C. Values represent means" S.E.M.
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0.028C ŽS.E.M.. and 37.59 " 0.028C and their total core temperature responses were not significantly different from each other. After the first injection of FEN, the FEN-248C group became hypothermic compared to the FEN-308C, SAL248C and SAL-308C groups. From that time until the end of the testing period Ž20 h., the FEN-248C group had total core temperature responses that were significantly below the FEN-308C Ž F s 49.5, P - 0.001., SAL-248C Ž P 0.001. and SAL-308C groups Ž P - 0.001.. The FEN-248C group had a peak low of 33.88C, which is 3.78C below baseline. This peak low occurred at 297 min after the first injection and 117 min after the fourth and final injection of FEN. FEN-308C animals showed an increase in core temperature for 242 min Ž4 h. after the first injection of FEN. The total core temperature response for that time period was higher than the SAL-308C Ž F s 28.3, P - 0.0001., SAL248C Ž P - 0.05. and FEN-248C groups Ž P - 0.001., indicating a significant hyperthermic response. The average temperature for this time period was 38.4 " 0.038C, with a peak of 38.68C at 222.5 min after the first injection, which was 42.5 min after the fourth injection. After 242 min until the end of the testing period, the average core temperature
was for the FEN-308C group was 37.8 " 0.018C and the total core temperature response did not differ from the SAL-248C and SAL-308C groups. These data indicate that although a 248C and 308C dosing environment has no effect on SAL-treated rats, FEN-248C rats display a significant hypothermia throughout the 20-h testing period, while FEN-308C rats display a hyperthermia for the first 4 h after FEN administration. 3.2. Neurotoxicity data For HPLC analysis, SAL-treated animals have been placed in one group for comparison with the FEN-248C and FEN-308C treated animals. There is a significant effect of treatment on 5-HT levels in hippocampus Ž F s 113.8, P - 0.0001., frontal cortex Ž F s 72.64, P - 0.0001., somatosensory cortex Ž F s 169.14, P - 0.0001., striatum Ž F s 19.406, P - 0.0001., hypothalamus Ž F s 38.36, P 0.0001. and septum Ž F s 13.15, P - 0.001.. There is also a significant effect of treatment on 5-HIAA levels in the hippocampus Ž F s 84.83, P - 0.0001., frontal cortex Ž F s 136.46, P - 0.0001., somatosensory cortex Ž F s 151.24, P - 0.0001., striatum Ž F s 9.15, P - 0.01., hypothalamus Ž F s 17.87, P - 0.0001. and septum Ž F s 6.66, P - 0.01..
Fig. 2. 5-HT levels from rats treated with FEN Ž12.5 mgrkg= 4, n s 8. or SAL Ž1 mlrkg= 4, n s 6. at ambient temperatures of 24 or 308C. All SAL-treated animals were grouped together Ž n s 12.. All animals were killed 14 days after FEN or SAL injection. Values represent means " SEM expressed in ngrmg of wet tissue weight. ) P - 0.01 compared to SAL; ) ) ) P - 0.05 compared to FEN-248C.
J.E. Malberg, L.S. Seidenr Brain Research 765 (1997) 101–107
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Fig. 3. 5-HIAA levels from rats treated with FEN Ž12.5 mgrkg= 4, n s 8. or SAL Ž1 mlrkg= 4, n s 6. at ambient temperatures of 24 or 308C. All SAL-treated animals were grouped together Ž n s 12.. All animals were killed 14 days after FEN or SAL injection. Values represent means " SEM expressed in ngrmg of wet tissue weight. ) P - 0.01 compared to SAL; ) ) ) P - 0.05 compared to FEN-248C.
Post-hoc tests indicate that both FEN-248C and FEN308C rats had significantly Ž P - 0.05. lower levels of 5-HT and 5-HIAA in all regions examined compared to SAL-treated animals. The FEN-308C animals had greater depletions of 5-HT compared to the FEN-248C rats in the frontal cortex Ž30% greater 5-HT depletion., striatum Ž35%., hippocampus Ž26%. and somatosensory cortex Ž28%.. FEN-308C animals also had greater depletions of 5-HIAA compared to FEN-248C animals in the frontal cortex Ž50% greater 5-HIAA depletion., hippocampus Ž26%. and somatosensory cortex Ž46%.. Although both FEN-248C and FEN-308C groups had very large depletions compared to control values, in all regions studied the FEN-308C animals had greater depletions of 5-HT and 5-HIAA than the FEN-248C animals. This was true even for regions where the differences in the FEN-248C and FEN-308C groups did not reach statistical significance.
4. Discussion This experiment confirms previous findings that FEN is neurotoxic to the 5-HT system w15,27x and demonstrates that FEN administration causes a hypothermia in rats when
administered at a normal laboratory ambient temperature Ž24 " 0.58C. w8x, but causes a hyperthermia in rats when administered in a warm environment Ž30 " 0.58C., indicating a lack of thermoregulatory ability. In addition, these FEN-treated hyperthermic animals have greater 5-HT and 5-HIAA depletions than the FEN-treated animals that have become hypothermic. This study indicates that there is an interaction between the changes in core temperature and the 5-HT neurotoxicity engendered by FEN administration. It can be seen that FEN-treated rats that are either hypothermic ŽFEN-248C. or hyperthermic ŽFEN-308C. both show large depletions in 5-HT and 5-HIAA. This is surprising in the face of the large amount of data gathered on the protective effect of cold on METH- and MDMA-induced neurotoxicity w4,5,18,19,23,24x. Animals that are administered drugs that produce hypothermia or placed in a cold environment are protected against the MDMA- or METH-induced neurotoxicity w4,5,8,19x. However, the present study indicates that in rats, there is a dissociation between the neurotoxic profiles of FEN and other drugs, such as MDMA and METH, since hypothermia protects against MDMA- or METH-induced 5-HT depletions, but not FEN-induced 5-HT depletions. Although the FEN-308C group had significantly more
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5-HT and 5-HIAA depletions than the FEN-248C group in the frontal cortex, hippocampus and somatosensory cortex, it should be noted that both groups ŽFEN-248C and FEN308C. had extremely large FEN-induced 5-HT and 5-HIAA depletions ŽFigs. 2 and 3.. Although it is clear that the high ambient temperature engendered more FEN-induced neurotoxicity, the large doses of FEN used in this study may have created a ceiling effect and masked a more robust difference between the two different environmental temperature groups. With smaller doses of FEN, the neurotoxic differences between the two groups may become more pronounced. Further studies using other doses are warranted. This study was undertaken to see if the administration of FEN at different temperatures would lead to different neurotoxic profiles. Our hypothesis was that the high ambient temperature would affect the rats’ thermoregulatory ability, and this, in turn, would affect the neurotoxicity. However, FEN-248C rats also seem to have reduced thermoregulatory ability since they become hypothermic at normal laboratory temperature compared to SAL-treated animals. Preston et al. w26x report that rats treated at 238C had a decrease in core temperature and a decrease in metabolic rate, indicating a deficit in thermoregulation compared to SAL-treated rats. It may be that rats treated at both ambient temperatures ŽFEN-248C and FEN-308C. have thermoregulatory deficits which may lead to 5-HT neurotoxicity. However, this interpretation is confounded by the fact that at similar laboratory temperatures Ž248C. METHand MDMA-treated rats, which also show impairments in their thermoregulatory abilities w7,11x, do not become hypothermic w19x. In addition, it is surprising that the FENinduced hypothermia has the long Ž20 h. time course that is seen in this study. The half-life of FEN is approximately 2.6 h in the rat w6x. Therefore, the hypothermia cannot be attributed to the acute pharmacological effects of FEN. However, FEN-induced acute 5-HT release may be responsible for the shorter hyperthermia that is seen in the 308C group. It is not known at what point in the mechanism of FEN-induced neurotoxicity the different ambient temperatures exert their effects. Stewart et al. w31x have administered FEN at 22 and 288C and reported that the pharmacokinetics of fenfluramine and norfenfluramine were not altered by the different ambient temperatures, although the core temperatures of these animals were different. Therefore, the ambient temperatures may be affecting other reactions leading to neurotoxicity. For example, the ambient temperature effect on FEN-induced neurotoxicity may be related to the resulting core temperature affecting the velocity of the 5-HT uptake transporter and subsequent 5-HT release. This transporter-mediated 5-HT release is hypothesized to be related to the neurotoxicity w28x, since blockade of serotonin release by fluoxetine or other uptake inhibitors prevents the 5-HT neurotoxicity w10,30x. Although it is clear that the transporter is active at 248C,
since neurotoxicity is seen at that ambient temperature, the velocity of the 5-HT transporter may be increased at 308C ambient temperature. This increase in the rate of 5-HT transporter-mediated exchange diffusion would lead to additional 5-HT release and additional neurotoxicity. Hyperthermia at specific ambient temperatures along with increased neurotoxicity would place FEN more in line with the previously studied amphetamines such as MDMA or METH which show increased neurotoxicity in a warm environment w3,19x. Aside from the action on the 5-HT transporter, different ambient temperatures could also affect other reactions leading to FEN-induced neurotoxicity. This paper provides evidence that rats treated with FEN display a hypothermia in a normal laboratory temperature of 248C, but display a hyperthermia when FEN is administered at 308C. Although both the FEN-248C and FEN-308C groups have large 5-HT and 5-HIAA depletions, the FEN308C groups have more depletions in certain areas. This is a departure from the temperature and neurotoxicity profiles seen with other amphetamines, such as MDMA and METH, where hypothermia affords protection against the amphetamine-induced neurotoxicity. This study suggests that the FEN-induced neurotoxic reactions which occur in the face of hypothermia may be increased at a high ambient temperatures.
Acknowledgements The authors wish to thank Georgetta Vosmer for her technical assistance and advice.
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Research report
Administration of fenfluramine at different ambient temperatures produces different core temperature and 5-HT neurotoxicity profiles Jessica E. Malberg, Lewis S. Seiden
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Department of Pharmacological and Physiological Sciences, UniÕersity of Chicago, 947 East 58th Street, Chicago, IL 60637, USA Accepted 8 April 1997
Abstract This study investigated the effect of two different ambient temperatures on fenfluramine-induced 5-HT neurotoxicity. Fenfluramine ŽFEN. Ž12.5 mgrkg= 4; injections made hourly. or saline ŽSAL. was administered to rats in either a normal laboratory temperature of 248C or a warm environment of 308C. Animals were kept at that ambient temperature for 20 h after FEN administration. Ambient temperature was controlled to "0.58C and rat core temperature was continually measured using a non-invasive apparatus. FEN-treated rats at 248C displayed a core temperature hypothermia with a peak low of 33.88C, and this core temperature hypothermia lasted for 20 h after FEN administration. Rats treated with FEN at 308C displayed a significant core temperature hyperthermia for 4 h after the first drug injection compared to SAL-treated groups, with a peak core temperature of 38.68C. 2 weeks after FEN injections, brain regions were analyzed by HPLC. Both groups of FEN-treated rats showed decreases in 5-HT and 5-HIAA in the hippocampus, frontal cortex, somatosensory cortex, striatum, hypothalamus and septum. However, FEN rats treated at 308C had significantly greater decreases Ž26–35%. in 5-HT compared to FEN-treated rats at 248C in the frontal cortex, hippocampus, striatum and somatosensory cortex and significantly greater decreases Ž26–50%. in 5-HIAA in the frontal cortex, hippocampus and somatosensory cortex. This study indicates fenfluramine can produce neurotoxicity in rats that display either a core temperature hypothermia or hyperthermia, although hyperthermic rats have greater 5-HT and 5-HIAA depletions than the hypothermic rats. q 1997 Elsevier Science B.V. Keywords: Fenfluramine; Amphetamine; Neurotoxicity; Temperature; Thermoregulation; 5-HT; 5-HIAA
1. Introduction D,L-Fenfluramine
ŽFEN. is a substituted amphetamine which is used clinically for its anorectic effects w1,20x. Similar to other amphetamine analogs, such as methamphetamine ŽMETH. and methylenedioxymethamphetamine ŽMDMA., FEN has been shown to be neurotoxic to the 5-HT system. This neurotoxicity has been demonstrated experimentally by meeting a number of criteria. FEN administration produces a decrease in 5-HT and 5-HIAA levels in rats w15,30x, guinea pigs w27x and monkeys w27x, a decrease in tryptophan hydroxylase w30x, a decrease in the number of 5-HT uptake sites and morphological evidence that is consistent with FEN-induced degeneration of 5-HT
Abbreviations: FEN, fenfluramine; SAL, saline; MDMA, methylenedioxymethamphetamine; METH, methamphetamine; 5-HT, serotonin; 5HIAA, 5-hydroxyindole acetic acid. ) Corresponding author. Fax: q1 Ž773. 702-3003; E-mail: [email protected] 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 5 1 7 - 9
nerve terminals w25,33x that lasts beyond the period of FEN administration. These effects have been found 2 weeks after FEN administration w15,25,27,30,33x, and the decrease in 5-HT levels have been reported at 8 w15,27x and 16 w27x weeks. Zaczek et al. w35x have shown that the number of 5-HT transporters was still reduced at 8 months. FEN has been shown to acutely release 5-HT w21x and this 5-HT release is blocked by fluoxetine w2x, indicating that this release is mediated by the uptake carrier though the mechanism of exchange diffusion w29x. The FEN-induced neurotoxicity has also been blocked by fluoxetine pretreatment w10,30x. These studies indicate that FEN uptake via the 5-HT uptake transporter or 5-HT release is necessary for FEN-induced 5-HT neurotoxicity. In addition to its neurotoxic effects on the 5-HT system, FEN has been shown to affect core temperature w8,13,24x and thermoregulatory ability w17,26x. Many studies have been performed looking at the effects of doses of FEN on core body temperature. These studies are varied in their results; both hypothermia w8,14x and hyperthermia w9,32x have been reported after FEN administration. This appar-
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ent contradiction has been explained by the ambient temperature at which the animal was dosed. Yehuda and Wurtman w34x have administered FEN at 4, 20 and 378C; at the high environmental temperature a core temperature hyperthermia was seen, and at the low environmental temperatures a core temperature hypothermia was seen. Additionally, Preston et al. w26x and Ma and Preston w17x have reported that at a dosing ambient temperature of 288C FEN produced hyperthermia, increased metabolic rate, increased blood flow and increased whole-body oxygen consumption, but that at an ambient temperature of 208C core temperature was reduced and the thermogenesis that was seen in control rats was also reduced. Based on these studies, Preston et al. w26x and Ma and Preston w17x concluded that FEN can affect thermogenesis and thermoregulatory parameters, such as blood flow and metabolic rate, but these effects are dependent on the ambient temperature at which the drug is administered. More recently, studies by Farfel and Seiden w8x and Miller et al. w22x have looked at the interaction of rat core temperature and neurotoxicity when FEN was administered in a normal laboratory temperature of 228–248C. Both of these studies reported a FEN-induced hypothermia in rats and also reported FEN-induced 5-HT and 5-HIAA depletions. The finding that FEN produces 5-HT neurotoxicity with an accompanying hypothermia is a striking difference from the other substituted amphetamines, such as METH and MDMA. In these drugs, animals that become hypothermic during METH or MDMA administration show protection against METH- or MDMA-induced neurotoxicity w3,19x. Conversely, in these drugs, neurotoxicity has been associated with an increase in body temperature w5,8,18x. Although FEN, METH and MDMA are all 5-HT-releasing agents, there seems to be a large difference in their core temperature and neurotoxicity profiles in the rat, where hypothermia accompanies FEN-induced neurotoxicity, but protects against MDMA- or METH-induced neurotoxicity. Although it is known that FEN produces an inability to thermoregulate and is also neurotoxic to the 5-HT system, it is not known whether the lack of thermoregulation is related to the neurotoxicity. This study aimed to investigate if rats treated with FEN in a high ambient temperature Ž308C. would show a different core temperature and neurotoxicity profile than rats treated at a normal laboratory temperature of 248C. To pursue this line of study, our laboratory has developed a core temperature measurement apparatus which controls ambient temperature to "0.58C and measures rat core temperature once per minute using a non-invasive apparatus. We now report that rats treated with FEN at an ambient temperature of 248C show a core temperature hypothermia compared to SAL-treated rats during the entire 20-h testing period. Rats treated with FEN at an ambient temperature of 308C show a core temperature hyperthermia compared to SAL-treated rats for the first 4 h after the drug injection.
Both FEN-treated groups of rats had decreases in 5-HT and 5-HIAA levels, but rats treated at the high ambient temperature of 308C had greater 5-HT and 5-HIAA depletions than the rats treated at 248C.
2. Materials and methods 2.1. Subjects Sprague–Dawley rats ŽHoltzman, Madison, WI. weighing between 250 and 300 g at time of surgery were used. Throughout the experiment, rats had access to food Ž4% Tek Lab rat chow. and water ad libitum. Rats were maintained on a 12:12 h lightrdark cycle with a room temperature of 22–248C, except on the injection day as indicated in the Section 2.5. Rats were housed 4–5 per cage except for the injection day, when they were housed individually in the temperature chambers. 2.2. Drugs Ž".-Fenfluramine ŽFEN. was obtained from Sigma Chemical Co. ŽSt. Louis, MO. and dosages are expressed in terms of the weight of the salt. Ketamine and Nembutal were obtained from Abbott Laboratories ŽChicago, IL.. 2.3. Surgery For rat core temperature measurements, temperaturesensitive radio transmitters ŽMinimitter Co., Sunriver, OR. were implanted in the peritoneum of the rat. Rats were anesthetized with Nembutal Ž40 mgrkg., and given supplemental 1-ml injections of ketamine as needed. A midline cut was made into the peritoneum and a sterilized transmitter inserted into the peritoneal cavity. Rats were given a minimum of 3 days to recover from the surgery. 2.4. Temperature measurement apparatus The temperature measurement chambers are modified compact refrigerators Ž7 ft 3 ; Absocold Corp, FL. with a Plexiglass window in the door to allow observation of the rats and to keep the lightrdark cycle in synchrony. A strip heater, fan and thermistor to record ambient temperature are mounted in the back of the chamber and a cage inside the chamber contains the rat. Four AM radios placed directly outside this cage receive frequency signals, corresponding to core temperature, from a temperature-sensitive transmitter implanted in the peritoneum of the rats. The radios and thermistor are interfaced to a microcomputer Ž33 MHA 386 DX.. To monitor core temperature, the computer receives the frequency signals from the radios and through an A-to-D converter, converts the frequency signals into core temperature values for each rat. To control for ambient temperature, the computer has digital
J.E. Malberg, L.S. Seidenr Brain Research 765 (1997) 101–107
output lines which turn the refrigerator compressor and strip heater off and on as needed to maintain the desired ambient temperature. The computer receives ambient and core temperature values every second, and these are averaged together to produce an ambient and a core temperature value for each minute of the experiment. 2.5. Procedure After arrival in the colony, rats were implanted with core temperature-sensitive radiotransmitters. One week after implantation, rats were placed in the temperature chambers for 1 h at an ambient temperature of 248C Žnormal laboratory temperature. for a baseline measure. After the baseline hour, the ambient temperature either remained at 248C or was increased to 308C. Rats were then injected with FEN Ž12.5 mgrml, 1 mlrkg. or SAL Ž1 mlrkg. once an hour for four hours and remained in the temperature chamber at 24 or 308C for 20 h. This dosage and injection schedule has been previously used by Farfel and Seiden w8x. Two weeks after the injections, rats were killed by decapitation and their brains were dissected as described by Heffner et al. w12x. Tissue samples were stored in liquid nitrogen. 5-HT and 5-HIAA levels were determined for the frontal cortex, somatosensory Žoccipital–parietal. cortex, hippocampus, striatum, hypothalamus and septum using high-performance liquid chromatography ŽHPLC. as described in Kotake et al. w16x.
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2.6. Statistics The core temperature data were quantified by using an area-under-the-curve ŽAUC. analysis. For each rat, a core temperature vs. time graph was generated, and the AUC was determined ŽIgor, Wavemetrics.. This AUC value was denoted as the ‘total core temperature’ response for each rat. The core temperature responses for all rats in each experimental group were summed together. Differences were determined by ANOVA followed by a Tukey post-hoc test ŽInstat for MacIntosh, GraphPad.. Single-point comparisons were determined using a Tukey post-hoc test. For the neurotoxicity data, differences were determined by ANOVA followed by Student’s t-test comparisons.
3. Results 3.1. Core temperature data Analysis of the total core temperature change Žtotal AUC., showed that the four experimental groups, FEN given at 248C ŽFEN-248C., FEN given at 308C ŽFEN-308C., SAL given at 248C ŽSAL-248C. and SAL given at 308C ŽSAL-308C., showed a significant treatment effect Ž F s 49.53, P - 0.0001. throughout the testing period ŽFig. 1.. The average core temperatures of the SAL-248C and SAL308C groups during the entire testing period were 37.66 "
Fig. 1. Core temperature vs. time graph for the 4 experimental groups ŽFEN-308C, FEN-248C, SAL-308C and FEN-248C.. Rats were treated with FEN Ž12.5 mgrkg= 4, n s 8. or SAL Ž1 mlrkg= 4, n s 6. at 0, 60, 120 and 180 min at either 24 or 308C. Values represent means" S.E.M.
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0.028C ŽS.E.M.. and 37.59 " 0.028C and their total core temperature responses were not significantly different from each other. After the first injection of FEN, the FEN-248C group became hypothermic compared to the FEN-308C, SAL248C and SAL-308C groups. From that time until the end of the testing period Ž20 h., the FEN-248C group had total core temperature responses that were significantly below the FEN-308C Ž F s 49.5, P - 0.001., SAL-248C Ž P 0.001. and SAL-308C groups Ž P - 0.001.. The FEN-248C group had a peak low of 33.88C, which is 3.78C below baseline. This peak low occurred at 297 min after the first injection and 117 min after the fourth and final injection of FEN. FEN-308C animals showed an increase in core temperature for 242 min Ž4 h. after the first injection of FEN. The total core temperature response for that time period was higher than the SAL-308C Ž F s 28.3, P - 0.0001., SAL248C Ž P - 0.05. and FEN-248C groups Ž P - 0.001., indicating a significant hyperthermic response. The average temperature for this time period was 38.4 " 0.038C, with a peak of 38.68C at 222.5 min after the first injection, which was 42.5 min after the fourth injection. After 242 min until the end of the testing period, the average core temperature
was for the FEN-308C group was 37.8 " 0.018C and the total core temperature response did not differ from the SAL-248C and SAL-308C groups. These data indicate that although a 248C and 308C dosing environment has no effect on SAL-treated rats, FEN-248C rats display a significant hypothermia throughout the 20-h testing period, while FEN-308C rats display a hyperthermia for the first 4 h after FEN administration. 3.2. Neurotoxicity data For HPLC analysis, SAL-treated animals have been placed in one group for comparison with the FEN-248C and FEN-308C treated animals. There is a significant effect of treatment on 5-HT levels in hippocampus Ž F s 113.8, P - 0.0001., frontal cortex Ž F s 72.64, P - 0.0001., somatosensory cortex Ž F s 169.14, P - 0.0001., striatum Ž F s 19.406, P - 0.0001., hypothalamus Ž F s 38.36, P 0.0001. and septum Ž F s 13.15, P - 0.001.. There is also a significant effect of treatment on 5-HIAA levels in the hippocampus Ž F s 84.83, P - 0.0001., frontal cortex Ž F s 136.46, P - 0.0001., somatosensory cortex Ž F s 151.24, P - 0.0001., striatum Ž F s 9.15, P - 0.01., hypothalamus Ž F s 17.87, P - 0.0001. and septum Ž F s 6.66, P - 0.01..
Fig. 2. 5-HT levels from rats treated with FEN Ž12.5 mgrkg= 4, n s 8. or SAL Ž1 mlrkg= 4, n s 6. at ambient temperatures of 24 or 308C. All SAL-treated animals were grouped together Ž n s 12.. All animals were killed 14 days after FEN or SAL injection. Values represent means " SEM expressed in ngrmg of wet tissue weight. ) P - 0.01 compared to SAL; ) ) ) P - 0.05 compared to FEN-248C.
J.E. Malberg, L.S. Seidenr Brain Research 765 (1997) 101–107
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Fig. 3. 5-HIAA levels from rats treated with FEN Ž12.5 mgrkg= 4, n s 8. or SAL Ž1 mlrkg= 4, n s 6. at ambient temperatures of 24 or 308C. All SAL-treated animals were grouped together Ž n s 12.. All animals were killed 14 days after FEN or SAL injection. Values represent means " SEM expressed in ngrmg of wet tissue weight. ) P - 0.01 compared to SAL; ) ) ) P - 0.05 compared to FEN-248C.
Post-hoc tests indicate that both FEN-248C and FEN308C rats had significantly Ž P - 0.05. lower levels of 5-HT and 5-HIAA in all regions examined compared to SAL-treated animals. The FEN-308C animals had greater depletions of 5-HT compared to the FEN-248C rats in the frontal cortex Ž30% greater 5-HT depletion., striatum Ž35%., hippocampus Ž26%. and somatosensory cortex Ž28%.. FEN-308C animals also had greater depletions of 5-HIAA compared to FEN-248C animals in the frontal cortex Ž50% greater 5-HIAA depletion., hippocampus Ž26%. and somatosensory cortex Ž46%.. Although both FEN-248C and FEN-308C groups had very large depletions compared to control values, in all regions studied the FEN-308C animals had greater depletions of 5-HT and 5-HIAA than the FEN-248C animals. This was true even for regions where the differences in the FEN-248C and FEN-308C groups did not reach statistical significance.
4. Discussion This experiment confirms previous findings that FEN is neurotoxic to the 5-HT system w15,27x and demonstrates that FEN administration causes a hypothermia in rats when
administered at a normal laboratory ambient temperature Ž24 " 0.58C. w8x, but causes a hyperthermia in rats when administered in a warm environment Ž30 " 0.58C., indicating a lack of thermoregulatory ability. In addition, these FEN-treated hyperthermic animals have greater 5-HT and 5-HIAA depletions than the FEN-treated animals that have become hypothermic. This study indicates that there is an interaction between the changes in core temperature and the 5-HT neurotoxicity engendered by FEN administration. It can be seen that FEN-treated rats that are either hypothermic ŽFEN-248C. or hyperthermic ŽFEN-308C. both show large depletions in 5-HT and 5-HIAA. This is surprising in the face of the large amount of data gathered on the protective effect of cold on METH- and MDMA-induced neurotoxicity w4,5,18,19,23,24x. Animals that are administered drugs that produce hypothermia or placed in a cold environment are protected against the MDMA- or METH-induced neurotoxicity w4,5,8,19x. However, the present study indicates that in rats, there is a dissociation between the neurotoxic profiles of FEN and other drugs, such as MDMA and METH, since hypothermia protects against MDMA- or METH-induced 5-HT depletions, but not FEN-induced 5-HT depletions. Although the FEN-308C group had significantly more
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5-HT and 5-HIAA depletions than the FEN-248C group in the frontal cortex, hippocampus and somatosensory cortex, it should be noted that both groups ŽFEN-248C and FEN308C. had extremely large FEN-induced 5-HT and 5-HIAA depletions ŽFigs. 2 and 3.. Although it is clear that the high ambient temperature engendered more FEN-induced neurotoxicity, the large doses of FEN used in this study may have created a ceiling effect and masked a more robust difference between the two different environmental temperature groups. With smaller doses of FEN, the neurotoxic differences between the two groups may become more pronounced. Further studies using other doses are warranted. This study was undertaken to see if the administration of FEN at different temperatures would lead to different neurotoxic profiles. Our hypothesis was that the high ambient temperature would affect the rats’ thermoregulatory ability, and this, in turn, would affect the neurotoxicity. However, FEN-248C rats also seem to have reduced thermoregulatory ability since they become hypothermic at normal laboratory temperature compared to SAL-treated animals. Preston et al. w26x report that rats treated at 238C had a decrease in core temperature and a decrease in metabolic rate, indicating a deficit in thermoregulation compared to SAL-treated rats. It may be that rats treated at both ambient temperatures ŽFEN-248C and FEN-308C. have thermoregulatory deficits which may lead to 5-HT neurotoxicity. However, this interpretation is confounded by the fact that at similar laboratory temperatures Ž248C. METHand MDMA-treated rats, which also show impairments in their thermoregulatory abilities w7,11x, do not become hypothermic w19x. In addition, it is surprising that the FENinduced hypothermia has the long Ž20 h. time course that is seen in this study. The half-life of FEN is approximately 2.6 h in the rat w6x. Therefore, the hypothermia cannot be attributed to the acute pharmacological effects of FEN. However, FEN-induced acute 5-HT release may be responsible for the shorter hyperthermia that is seen in the 308C group. It is not known at what point in the mechanism of FEN-induced neurotoxicity the different ambient temperatures exert their effects. Stewart et al. w31x have administered FEN at 22 and 288C and reported that the pharmacokinetics of fenfluramine and norfenfluramine were not altered by the different ambient temperatures, although the core temperatures of these animals were different. Therefore, the ambient temperatures may be affecting other reactions leading to neurotoxicity. For example, the ambient temperature effect on FEN-induced neurotoxicity may be related to the resulting core temperature affecting the velocity of the 5-HT uptake transporter and subsequent 5-HT release. This transporter-mediated 5-HT release is hypothesized to be related to the neurotoxicity w28x, since blockade of serotonin release by fluoxetine or other uptake inhibitors prevents the 5-HT neurotoxicity w10,30x. Although it is clear that the transporter is active at 248C,
since neurotoxicity is seen at that ambient temperature, the velocity of the 5-HT transporter may be increased at 308C ambient temperature. This increase in the rate of 5-HT transporter-mediated exchange diffusion would lead to additional 5-HT release and additional neurotoxicity. Hyperthermia at specific ambient temperatures along with increased neurotoxicity would place FEN more in line with the previously studied amphetamines such as MDMA or METH which show increased neurotoxicity in a warm environment w3,19x. Aside from the action on the 5-HT transporter, different ambient temperatures could also affect other reactions leading to FEN-induced neurotoxicity. This paper provides evidence that rats treated with FEN display a hypothermia in a normal laboratory temperature of 248C, but display a hyperthermia when FEN is administered at 308C. Although both the FEN-248C and FEN-308C groups have large 5-HT and 5-HIAA depletions, the FEN308C groups have more depletions in certain areas. This is a departure from the temperature and neurotoxicity profiles seen with other amphetamines, such as MDMA and METH, where hypothermia affords protection against the amphetamine-induced neurotoxicity. This study suggests that the FEN-induced neurotoxic reactions which occur in the face of hypothermia may be increased at a high ambient temperatures.
Acknowledgements The authors wish to thank Georgetta Vosmer for her technical assistance and advice.
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