- Email: [email protected]
Fluoxetine and desipramine selectively attenuate 2′-NH2-MPTP-induced depletions in serotonin and norepinephrine
European Journal of Pharmacology, 250 (1993) 215-221
Elsevier Science Publishers B.V.
EJP 53405
Fluoxetine and desipramine selectively attenuate 2'-NH2-MPTP-induced depletions in serotonin and norepinephrine A n n e M. A n d r e w s
,,a,b
a n d D e n n i s L. M u r p h y
a
a Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD, USA, and 9 Department of Chemistry, American University, Washington, DC, USA
Received 5 July 1993, accepted 17 September 1993
We recently reported that the novel MPTP analog 1-methyl-4-(2'-aminophenyl)-l,2,3,6-tetrahydropyridine (2'-NHz-MPTP) administered to C57BL/6 mice produced substantial decreases in forebrain serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine, with negligible effects on brain dopamine or dopamine metabolites. In the present report, we confirm and extend our original results to include dose-response data and the effect of selective uptake inhibition on the levels of monoamine neurotransmitters in various regions of the mouse brain following treatment with 2'-NHz-MPTP. In a dose-ranging study, 2'-NH2-MPTP (10 mg/kg x 4) produced a 25-30% reduction in frontal cortex 5-HT, 5-HIAA, and norepinephrine. When 4 x 20 mg/kg 2'-NHz-MPTP was administered, 70-75% reductions in 5-HT, 5-HIAA, and norepinephrine in both frontal cortex and hippocampus were seen 1 week after treatment. No changes in dopamine were found in striatum or in any of the other brain regions examined at either dose. Doses of 40 and 60 mg/kg were lethal shortly after a single injection. In mice receiving either fluoxetine or desipramine (10 mg/kg) prior to 2'-NHz-MPTP (20 m g / k g × 4), decreases in 5-HT and norepinephrine, respectively, were significantly attenuated by ~ 30-40%. These data suggest that 2'-NHz-MPTP acts in a dose-dependent manner and that the serotonergic and noradrenergic uptake systems are involved in the mechanism by which 2'-NHz-MPTP causes selective deficits in cortical and hippocampal 5-HT and norepinephrine. MPTP (1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine); MPTP analog; MPTP derivative; 5-HT (5-hydroxytryptamine, serotonin); Norepinephrine; Fluoxetine; Desipramine
1. Introduction
The dopaminergic neurotoxicity of 1-methyl-4phenyl-l,2,3,6-tetrahydropyridine (MPTP; fig. 1) is mediated via uptake of its primary metabolite, 1-methyl4-phenylpyridinium (MPP+), by the dopamine transporter (Javitch et al., 1985). Evidence includes the fact that M P T P toxicity can be attenuated by pharmacologic inhibition of the dopamine transporter with either mazindol or amfolenic acid (Javitch et al., 1985; Mayer et al., 1986; Melamed et al., 1985; Sundstr6m and Jonsson, 1985). 1-Methyl-4-(2'-aminophenyl)-l,2,3,6-tetrahydropyridine (2'-NH2-MPTP; fig. 1) is an analog of M P T P that produces marked decreases in cortical and hippocampal serotonin (5-hydroxytryptamine; 5-HT) and n o r e p i n e p h r i n e without decreasing striatal dopamine in mouse brain when administered in a standard dosing paradigm for M P T P (20 m g / k g x 4)
* Corresponding author. Address: National Institute of Mental Health, Building 10, Room 3D41, 9000 Rockville Pike, Bethesda, MD 20892, USA. Tel. (301)496-3421, fax (301)402-0188.
(Andrews and Murphy, 1993). In this study, we investigated the effects of varying the concentration of 2'N H z - M P T P administered to mice. In addition, because the dopamine transporter is involved in the events leading to M P T P toxicity, we also studied whether the serotonin and norepinephrine transporters were of similar importance in the mechanism of action of 2'NH2-MPTP.
2. Materials and methods 2.1. Materials
2 ' - N H 2 - M P T P was synthesized by Ecochem Research (Chaska, MN). The identity of 2 ' - N H 2 - M P T P was verified by GCMS and [IH]NMR. It was stored desiccated at - 7 0 ° C and checked periodically for stability by HPLC. Fluoxetine was a gift from Eli Lilly and Company (Indianapolis, IN), and desipramine was purchased from Aldrich Chemical Company (Milwaukee, WI). All other drugs and chemicals were obtained from Sigma Chemical Company (St. Louis, MO).
216
2.2. Animal treatments C57BL/6 mice (Charles River Laboratories) weighing 25 g at the beginning of the study were housed under standard conditions with food and water ad libitum. In the dose-ranging study, 0, 10, 20, 40, or 60 mg/kg 2'-NH2-MPTP (calculated as the free base) was administered in four injections at 2 h intervals. In a second experiment designed to study the effect of uptake inhibitors, three groups of mice were injected with 20 mg/kg 2'-NH2-MPTP every 2 h for a total of four injections. Ninety minutes prior to the first dose of 2'-NH2-MPTP, mice were injected with either 10 mg/kg fluoxetine, 10 m g / k g desipramine, or saline. In a separate experiment, three groups of mice were injected with either 10 mg/kg fluoxetine, 10 mg/kg desipramine, or saline in order to determine the effects of the uptake inhibitors alone. All injections were administered i.p. in a volume of 0.1 ml. In the third experiment, control animals and those receiving 4 × 20 mg/kg 2'-NH2-MPTP were monitored for changes in body temperature. Mice were acclimated to the testing room temperature of 19.3 + 1.0°C for 1 h prior to the experiment. Colonic temperature was measured with a rectal probe (Harvard Apparatus) and digital thermometer (Sensortek Inc., Clifton, N J). The thermocouple was inserted 2 cm into the colon of mice which were gently restrained by the tail prior to drug administration to establish a baseline temperature. Thirty minutes after each of the four injections of either saline or 2'-NH2-MPTP, rectal temperature was measured again.
2.3. HPLC analysis Seven days post-treatment, the mice were killed by cervical dislocation and their brains were rapidly removed and dissected on ice. Brain regions including frontal cortex, hippocampus, striatum, hypothalamus, and brainstem were dissected according to The Atlas of the Mouse Brain and Spinal Cord (Sidman et al., 1971). These tissues were individually analyzed for monoamine neurotransmitters and metabolites by high perCH3 t
CH3 I
2'-NH2-MPTP
MPTP
Fig. 1. 1-Methyl-4-(2'-aminophenyl)-l,2,3,6-tetrahydropyridine (2'NH2-MPTP) and its parent compound, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP).
formance liquid chromatography coupled with electrochemical detection (HPLC-ECD) at +0.85 V using established methods (Mefford, 1981). Briefly, individual samples were sonicated in 200250/zl 0.1 M H f l O 4 and centrifuged at 7200 g (12000 rpm) for 10 min. Each supernatant (50/zl) was injected onto a 100 mm by 4.6 mm Axxiom 3/zm octadecylsulfate (ODS) reversed-phase chromatography column in a mobile phase containing 0.25 M citric acid, 5-9% acetonitrile, 450 m g / l octanesulfonic acid, 0.3-0.4% triethylamine, and 100 mg/l EDTA. 5-Hydroxy-Nmethyltryptamine (N-Me-5-HT) was used as the internal standard and calculations were performed by comparing the relative peak areas of sample peaks with those of external standards. 5-Hydroxytryptamine, 5hydroxyindoleacetic acid (5-HIAA), norepinephrine, dopamine, and the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were all able to be measured in a single chromatogram. The order of elution and representative retention times are as follows: norepinephrine (4.4), DOPAC (6.5), dopamine (7.7), 5-HIAA (10.4), HVA (12.7), 5-HT (17.9), and N-Me-5-HT (21.2) min, respectively. Protein was quantitated by the method of Lowry et al. (Lowry et al., 1951). Concentrations are expressed in ng/mg protein (mean + S.E.M.).
2.4. Statistics One-way analysis of variance (ANOVA) was used to detect overall statistically significant differences among treatment groups. Individual t-tests compared least squares means between groups. Probability < 0.05 was considered significant following multiplication by a Bonferroni correction factor (g) where g = n * ( n - l ) / 2 , n being the number of treatment groups.
3. Results
3.1. Dose-ranging study with 2'-NH2-MPTP In mice injected with 2'-NH2-MPTP, statistically significant reductions (25-30%) in cortical 5-HT, 5HIAA, and norepinephrine were evident at the lowest, 4 × 10 mg/kg dose (P < 0.01, 0.001, and 0.001, respectively; table 1). At 4 × 20 mg/kg, more marked changes in 5-HT, 5-HIAA, and norepinephrine were observed, especially in cortex and hippocampus where 2'-NH 2MPTP caused decrements on the order of 70% (P < 0.001; table 1). Smaller reductions in striatal 5-HT and 5-HIAA were also seen at this dose, as well as a decrease in norepinephrine in brainstem (P
217 TABLE 1 Dose-ranging study with 2 ' - N H 2-MPTP. Two groups of mice (n = 8) were injected 4 times i.p. with either 10 or 20 m g / k g 2 ' - N H 2 - M P T P at 2 h intervals. In two additional groups injected with 40 or 60 m g / k g 2 ' - N H 2 - M P T P (n = 8), > 80% of the animals died shortly after the first injection. Control animals received four injections of saline (n = 8). Seven days post-treatment, the mice were killed and their brains were analyzed for neurotransmitters and metabolites as described in Materials and methods. T h e results are expressed as percentages of the respective control group m e a n s _+S.E.M. which are similar in magnitude to the absolute concentrations shown in figs. 2 and 4. There were no statistically significant changes in hypothalamus (data not shown). Cortex
Hippocampus
Striatum
cant attenuation (P < 0.01 vs. 2'-NH2-MPTP-treated mice) of the cortical and hippocampal 5-HT decrements caused by 2'-NH2-MPTP (fig. 2A). The levels ot
5-HT
Ao 10" 9" 8"
rT
7'
Brainstem
5-HT 10 m g / k g 20 m g / k g 5-H/A4 10 m g / k g 20 m g / k g
×4 ×4
7 6 . 8 5 : 4 . 5 b 1 0 6 _+12 99.0_+4.3 98.65:6.7 32.3+ 3.2 c 31.7+ 4.2 c 80.9+2.1 b 7 9 . 3 5 : 4 . 8
4.
×4 ×4
69.1_+ 3.3 c 8 9 . 5 5 : 7 . 1 93.4_+3.1 91.5_+10 31.1_+ 3.0 c 3 1 . 1 5 : 2 . 3 c 77.0+2.5 c 65.4_+ 8.8
2'
68.9_+ 3.0 c 78.7 + 7.2 ND 32.1+ 3.8 c 26.1_+ 3.0 c N D
72.9 5 : 4 . 5 60.35:8.6 b
0
96.8-+ 7.6 93.5_+10
80.2-+ 6.9 78.4_+12
I'
Norepinephrine 10 m g / k g × 4 20 m g / k g × 4
Cortex
Hippocampus
Striatum
Hippocampus
Striatum
Dopamine 10 m g / k g × 4 20 m g / k g × 4
105 -+12 84.2+ 3.9
112 +4.3 103 _+3.2
B.
DOPAC 10 m g / k g 20 m g / k g HVA 10 m g / k g 20 m g / k g
×4 ×4
86.0-+14 102 + 2 6
×4 ×4
107 + 8.0 86.3+11
ND ND 116 + 7.1 112 + 7.8
83.8-+5.1 91.5+7.4 91.4+2.7 85.0+6.8
73.9:t: 8.5 64.6+ 8.7 105 _+19 81.2-+11
5-HIAA 8"
7" 6"
.i
5"
b p < 0.01, c p < 0.001. Entries marked ' N D ' were not detectable.
4"
ment with 2'-NH2-MPTP. The two higher doses of 2'-NH2-MPTP (40 and 60 mg/kg) led to lethality in > 80% of the animals following a single injection.
/r',A
2"
1'
3.2. Pretreatment with up take inhibitors 0 Cortex
Mice pretreated with fluoxetine, a selective inhibitor of 5-HT uptake (Wong et al., 1975) showed a signifiNE
Co 7' Fig. 2. Effects of pretreatment with fiuoxetine on the changes in 5-HT, 5-HIAA, and norepinephrine 1 week after 2'-NH2-MPTP. Two groups of mice were injected i.p. 4 times with 20 m g / k g 2 ' - N H 2 - M P T P at 2 h intervals. O n e group received saline 90 min prior to 2 ' - N H 2 - M P T P (n = 7; diagonally hatched bars) and the second group received a single injection of 10 m g / k g fluoxetine (n = 7; shaded bars). Control animals (n = 7; u n s h a d e d bars) received four injections of saline every 2 h. After 1 week, 5-HT, 5-HIAA, and norepinephrine were m e a s u r e d by H P L C - E C D as described in Materials and Methods. Control group m e a n s + S.E.M. in cortex, hippocampus, and striatum, respectively are as follows: 5-HT, 7.28+0.46, 8.14+0.38, and 3.92+0.32; 5-HIAA, 3.43+0.30, 6.92 + 0.70, and 2.92 + 0.39; norepinephrine, 4.15 + 0.45, 5.70 + 0.39, and none detected ( n g / m g protein). Probabilities indicated in the figure are as follows: * P < 0.05 * * P < 0.01, and * * * P < 0.001 different from control, and * p < 0.01 different from 2'-NH2-MPTP.
6" 5'
,T
I'
not detectable
0 Cortex
Hlppocampus
Strlatxma
218
5-HIAA in mice pretreated with fluoxetine were not statistically different from the levels in animals that received 2'-NH2-MPTP alone (fig. 2B), however, the general trend toward attenuation paralleled that seen for 5-HT (fig. 2A). Fluoxetine pretreatment had no significant effect on 2'-NH2-MPTP-induced decreases in norepinephrine (fig. 2C). In a separate experiment, mice receiving 10 mg/kg fluoxetine by itself (n = 7) had levels of 5-HT, 5-HIAA, and NE 1 week posttreatment which were not significantly different from animals injected with saline (n = 7; data not shown). On the other hand, the norepinephrine-selective uptake inhibitor desipramine (Carlsson et al., 1966; Lidbrink et al., 1971) significantly diminished 2'-NH 2MPTP's effects on cortical and hippocampal norepinephrine (P < 0.01 vs. 2'-NH2-MPTP-treated mice: fig. 3C) without affecting the magnitude of the 2'NH2-MPTP-induced decreases in 5-HT (fig. 3A) or 5-HIAA (fig. 3B). Desipramine treatment by itself (n = 7) had no effect on 5-HT, 5-HIAA, or norepinephrine levels measured 1 week post-treatment (data not shown). In striatum, the levels of 5-HT and 5-HIAA in all treatment groups were not statistically different from control in this experiment (figs. 2A, 2B, 3A, and 3B). Levels of dopamine were also unchanged in the three brain regions examined (fig. 4A). Minimal effects on dopamine metabolites included small decreases in hippocampal DOPAC following all three drug treatments (fig. 4B) and a small increase in hippocampal HVA after 2'-NH2-MPTP (fig. 4C).
min after each dose, subsided before the administration of the next dose, and became less marked aftel each subsequent dose.
A.
5-HT 10~ 9" 8-
•
z-I,7-
7-
3
:
:
I o
Cortex
Hippocampus
Striatum
5-HIAA
B. 8
,T
:e 4"
3.3. Behavior and body temperature Mice that received 4 × 20 mg/kg 2'-NH2-MPTP exhibited behavior which appeared to be similar to the serotonin syndrome (Modigh and Svensson, 1972) and consisted of moderately paced walking in circles around the cage, Straub tails and occasional rearing. In addition, some animals exhibited piloerection and excessive salivation. This behavior began approximately 15-20
2" I" 0 Cortex
C.
Hippocampus
Striatum
NE
,T Fig. 3. Effect of pretreatment with desiprimine on changes in 5-HT, 5-HIAA, and norepinephrine 1 week after 2'-NH2-MPTP. Two groups of mice were injected i.p. 4 times with 20 m g / k g 2 ' - N H 2M P T P at 2 h intervals. O n e group receive d saline 90 rain prior to 2 ' - N H 2 - M P T P (n = 7; diagonally hatched bars), the second group received a single injection of 10 m g / k g desiprimine (n = 6; shaded bars). Control animals (n = 7; u n s h a d e d bars) received four-injections of saline every 2 h. After 1 week, 5-HT, 5-HIAA, and norepinephrine were m e a s u r e d by H P L C - E C D as described in Materials and methods. Control group m e a n s + S . E . M , are the same as those reported in the legend of fig. 2. Probabilities indicated in the figure are as follows: * P < 0.05, * * P < 0.01, and * * * P < 0.001 different from control, and t p < 0.01 different from 2'-NH2-MPTP.
:s
4" 3 2 not detectable
I 0
Cortex
Hippocampus
Striatum
219
Colonic temperature was recorded after each of four injections of 2'-NH2-MPTP or saline in a representative sample of animals. In the case of both 2'NH2-MPTP-treated (n -- 8) and control groups (n = 6),
mean body temperature after each injection was unchanged from baseline temperatures of 38.4 + 0.18 and 38.4 + 0.12°C, respectively (data not shown).
4. Discussion
DA
Ao
120
12
100
10
80
I'i "60
6
~
4
0
2
0
Cortex
B.
Hippocampus
Striatum
DOPAC 20 18' 16 14' 12" I0' 8" 6" 4"
0
Cortex
C.
H/ppocampus
Striatum
ttVA 12"
10"
6'
4"
2"
o Cortex
Hippocampus
2'-NH2-MPTP differs considerably from other previously described MPTP analogs (Bradbury et al., 1985; Fuller et al., 1985; Youngster et al., 1989) in the fact that it causes selective decreases in serotonin and norepinephrine in mouse forebrain without affecting striatal dopamine. Because most of the analogs which have proven to be more potent dopaminergic neurotoxins than MPTP bear a substituent at the 2'-position (i.e., 2'-CH3-MPTP, 2'-Et-MPTP, 2'-C1-MPTP, 2'-FMPTP, and 2'-CF3-MPTP) (Youngster et al., 1989), the -NH 2 substituent itself and not simply substitution at the 2'-position appear to be important for the differential actions of 2'-NH2-MPTP. This study confirms the 2'-NH2-MPTP-induced decreases of 60-70% in cortical and hippocampal 5-HT and norepinephrine which we reported in an independent study (Andrews and Murphy, 1993) and extends the findings to include data on the dose-dependent effects of 2'-NH2-MPTP. From the latter results, the LD50 of 2'-NH2-MPTP can be estimated to be between 20 and 40 mg/kg when given i.p. to mice. Modest decreases ( ~ 20%) in striatal 5-HT and 5-HIAA were observed in this report and in separate experiments from our previous work (Andrews and Murphy, 1993); however, these smaller decrements are more variable and not always statistically significant. 2'-NHa-MPTP is similar to MPTP and many of its analogs in that it seems to require active uptake by the neurotransmitter-specific transporters located on the monoaminergic neurons that are vulnerable to its toxic effects. As the results of this study demonstrate, sero-
Striatum
Fig. 4. The effects of pretreatment with uptake inhibitors on changes in dopamine, D O P A C , and H V A 1 week after 2'-NH2-MPTP. Three groups of mice were injected i.p. 4 times with 20 m g / k g 2 ' - N H 2M P T P at 2 h intervals. O n e group received saline 90 min prior to 2'-NHE-MPTP ( n = 7 ; diagonally hatched bars), a second group received a single injection of 10 m g / k g fluoxetine (n = 7; shaded bars), and the third was pretreated with 10 m g / k g desipramine (n = 6; cross-hatched bars). Control animals (n = 7; u n s h a d e d bars) received four injections of saline every 2 h. O n e week later, dopamine and its metabolites D O P A C and H V A were m e a s u r e d by H P L C - E C D as described in Materials and methods. Control group m e a n s + S.E.M. in cortex, hippocampus and striatum, respectively are as follows: dopamine, 0.499+0.10, 0.392_+ 0.089, and 107.8_+ 7.8; D O P A C , 2.57 _+0.40, 1.95_+0.16, and 17.2_+2.5; H V A , 1.03_+0.13, 0.386_+0.074, and 9.69 _+ 1.02 ( n g / m g protein). Probabilities indicated in the figure are as follows: * P < 0.05, * * P < 0.01, and * * * P < 0.001 different from control, and t p < 0.01 different from 2'-NHE-MPTP.
220
tonin and norepinephrine uptake inhibitors selectively attenuated 2'-NH2-MPTP-induced decreases in hippocampal and cortical 5-HT and norepinephrine, respectively. These data suggest that 2'-NH2-MPTP , or possibly a pyridinium metabolite, as is the case for MPTP (Javitch et al., 1985; Markey et al., 1984), is actively transported via the 5-HT transporter into serotonergic nerve terminals and the norepinephrine transporter into noradrenergic nerve terminals before causing decreases in each of these neurotransmitters. Complete protection by the selective uptake inhibitors from the effects of 2'-NH2-MPTP was not observed in this study. Among several possible considerations, pharmacokinetic factors may provide one explanation. For example, 2'-NH2-MPTP or a metabolite may have a much longer half-life in brain compared to fluoxetine or desipramine. The MPTP metabolite MPP + is retained on the order of days to weeks in primate and dog brain (Herkenham et al., 1991; Johannessen et al., 1991). A second explanation involves the possibility that the 10 mg/kg dose of uptake inhibitor was not sufficient to completely block uptake in mice. We have performed additional experiments wherein a 10 mg/kg dose of fluoxetine or desipramine was given before, during, and after the 4 x 20 mg/kg 2'-NH 2MPTP regimen; however, only 30% of the mice survived this particular treatment regimen. Further investigation of different dosing paradigms could prove worthwhile. Finally, 2'-NH2-MPTP or its metabolite(s) may gain access to serotonergic and noradrenergic neurons via other mechanisms not exclusively involving active transport. A nonspecific passive diffusion mechanism seems unlikely though, due to the fact that the -NH 2 substituent is nonlipophilic. It is unclear why the 5-HT uptake inhibitor fluoxetine failed to produce a statistically significant effect on the serotonin metabolite 5-HIAA; however decreased formation of 5-HIAA due to inactivation of monoamine oxidase (MAO) by 2'-NH2-MPTP may be involved. It has been demonstrated that MPTP and some other 2'-substituted MPTP analogs are capable of mechanism-based inactivation of MAO (Krueger et al., 1990). Irreversible inhibition of MAO by 2'-NH 2-MPTP or a metabolite would decrease the formation of 5-HIAA and partially cancel any increase expected due to the reduced toxicity associated with fluoxetine pretreatment. The ability of other serotonergic and noradrenergic neurotoxins to be actively transported by the 5-HT and norepinephrine reuptake systems has already proven to be important in determining their toxic selectivity (Battaglia et al., 1988; Fuller et al., 1975; Jonsson, 1980). 2'-NH2-MPTP appears to be no exception. However, beyond uptake by the 5-HT and norepinephrine transporters, 2'-NH2-MPTP may be acting by a mechanism which is different from these other neurotoxins. For
example, 5-HT is believed to play a role in mammalian thermoregulation (Jacob and Girault, 1979; Lin et al., 1983) and serotonergic neurotoxins such as fenfluramine, p-chloroamphetamine and 3,4-methylenedioxymethamphetamine are reported to cause dose-dependent hyperthermia when administered to rats (Frey, 1975; Nash et al., 1988; Pawlowski, 1981; Schmidt et al., 1990). The fact that 2'-NH2-MPTP did not cause an increase in body temperature in our study may be an indication that its mechanism of action is somewhat different from the substituted amphetamines. However, a few studies have reported that different ambient temperatures can modify the magnitude and direction of the observed change in body temperature (hyperthermia verses hypothermia) following administration of substituted amphetamines (Frey, 1975; Nash et al., 1988; Pawlowski, 1981; Schmidt et al., 1990); therefore, experiments to determine 2'-NH2-MPTP's effects on colonic temperature at varying ambient temperatures may be warranted. Finally, because the effects of 5-HT on thermoregulation are different in rats and mice (Jacob and Girault, 1979), data obtained with substituted amphetamines in rats may not be directly applicable to studies in mice. While the acute behavioral effects observed following 2'-NHz-MPTP bear some resemblance to the serotonin syndrome, specific aspects of this syndrome are not well-documented in mice. The behaviors elicited by 2'-NH2-MPTP may also be attributed, in part, to centrally or peripherally released norepinephrine as well as 5-HT, or to other undetermined factors. Further investigation will be able to provide additional evidence of the similarities between 2'-NH2-MPTP's mechanism of action and those of MPTP analogs versus other classes of serotonergic and noradrenergic neurotoxins.
Acknowledgments The authors wish to thank Dr. R. Carlson for his help with 2'-NH2-MPTP synthesis and Dr. J.L. Hill for his statistical advice. A.M. Andrews was partially supported by a fellowship from the U.S. Department of Education.
References Andrews, A.M. and D.L. Murphy, 1993, Sustained depletion of cortical and hippocampal serotonin and norepinephrine but not striatal dopamine by 1-methyl-4-(2'-aminophenyl)-l,2,3,6-tetrahydropyridine (2'-NH2-MPTP): a comparative study with 2'-CH 3MPTP and MPTP, J. Neurochem. 60, 1167. Battaglia, G., S.Y. Yeh and E.B. De Souza, 1988, MDMA-induced neurotoxicity: parameters of degeneration and recovery of brain serotonin neurons, Pharmacol. Biochem. Behav. 29, 269. Bradbury, A.J., B. Costall, A.M. Domeney, B. Testa, P.G. Jenner,
221 C.D. Marsden and R.J. Naylor, 1985, The toxic actions of MPTP and its metabolite MPP ÷ are not mimicked by analogues of MPTP lacking an N-methyl moiety, Neurosci. Lett. 61, 121. Carlsson, A., K. Fuxe, B. Hamberger and M. Lindqvist, 1966, Biochemical and histochemical studies on the effects of imipraminelike drugs and (+)-amphetamine on central and peripheral catecholamine neurons, Acta Physiol. Scand. 67, 481. Frey, H.-H., 1975, Hyperthermia induced by amphetamine, p-chloroamphetamine, and fenfluramine in the rat, Pharmacology 13, 163. Fuller, R.W., K.W. Perry and B.B. Molloy, 1975, Effect of 3-(p-trifluoromethylphenoxy)-N-methyl-3-phenylpropylamine on the depletion of brain serotonin by 4-chloroamphetamine, J. Pharmacol. Exp. Ther. 193, 796. Fuller, R.W., S.K. Hemrick-Luecke and D.W. Robertson, 1985, Comparison of 1-methyl-4-(p-chlorophenyl)-l,2,3,6-tetrahydropyridine, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) and p-chloroamphetamine as monoamine depletors, Res. Commun. Chem. Pathol. Pharmacol. 50, 57. Herkenham, M., M.D. Little, K. Bankiewicz, S.-c. Yang, S.P. Markey and J.N. Johannessen, 1991, Selective retention of MPP ÷ within the monoaminergic systems of the primate brain following MPTP administration: an in vivo autoradiographic study, Neuroscience 40, 133. Jacob, J.J. and J.-M.T. Girault, 1979, 5-Hydroxytryptamine, in: Body Temperature: Regulation, Drug Effects, and Therapeutic Implications, ed. Lomax, P. and E. Sch6nbaum (Marcel Dekker, Inc., New York) p. 183. Javitch, J.A., R.J. D'Amato, S.M. Strittmatter and S.H. Snyder, 1985, Parkinsonism-inducing neurotoxin N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine: uptake of the metabolite N-methyl-4-phenylpyridinium by dopamine neurons explains selective toxicity, Proc. Natl. Acad. Sci. USA 82, 2173. Johannessen, J.N., T.J. Sobotka, V.K. Weise and S.P. Markey, 1991, Prolonged alterations in canine striatal dopamine metabolism following subtoxic doses of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) and 4'-amino-MPTP are linked to the persistence of pyridinium metabolites, J. Neurochem. 57, 981. Jonsson, G., 1980, Chemical neurotoxins as denervation tools in neurobiology, Annu. Rev. Neurosci, 3, 169. Krueger, M.J., K. McKeown, R.R. Ramsay, S.K. Youngster and T.P. Singer, 1990, Mechanism-based inactivation of monoamine oxidases A and B by tetrahydropyridines and dihydropyridines, Biochem. J. 268, 219. Lidbrink, P., G. Jonsson and K. Fuxe, 1971, The effect of imipramine-like drugs and antihistamine drugs on uptake mechanisms in the central noradrenaline and 5-hydroxytryptamine neurons, Neuropharmacology 10, 521. Lin, M.T., J.J. Wu and B.L. Tsay, 1983, Serotonergic mechanisms in the hypothalamus mediate thermoregulatory responses in rats, Naunyn-Schmied. Arch. Pharmacol. 322, 271.
Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193, 265. Markey, S.P., J.N. Johannessen, C.C. Chiueh, R.S. Burns and M.A. Herkenham, 1984, Intraneuronal generation of a pyridinium metabolite may cause drug-induced parkinsonism, Nature 311, 464. Mayer, R.A., M.V. Kindt and R.E. Heikkila, 1986, Prevention of the nigrostriatal toxicity of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine by inhibitors of 3,4-dihydroxyphenylethylamine transport, J. Neurochem. 47, 1073. Mefford, I.N., 1981, Application of high performance liquid chromatography with electrochemical detection to neurochemical analysis: measurement of catecholamines, serotonin and metabolites in rat brain, J. Neurosci. Methods 3, 207. Melamed, E., J. Rosenthal, O. Cohen, M. Globus and A. Uzzan, 1985, Dopamine but not norepinephrine or serotonin uptake inhibitors protect mice against neurotoxicity of MPTP, Eur. J. Pharmacol. 116, 179. Modigh, K. and T.H. Svensson, 1972, On the role of central nervous system catecholamines and 5-hydroxytryptamine in the nialamide-induced behavioural syndrome, Br. J. Pharmacol. 46, 32. Nash, J.F., H.Y. Meltzer and G.A. Gudelsky, 1988, Elevation of serum prolactin and corticosterone concentrations in the rat after the administration of 3,4-methylenedioxymethamphetamine, J. Pharmacol. Exp. Ther. 245, 873. Pawlowski, L., 1981, Different action of 5-hydroxytryptamine (5-HT) uptake inhibitors on fenfluramine- but not p-chloramphetamineinduced hyperthermia in rats, J. Pharm. Pharmacol. 33, 538. Schmidt, C.J., C.K. Black, G.M. Abbate and V.L. Taylor, 1990, Methlylenedioxymethamphetamine-induced hypertherrnia and neurotoxicity are independently mediated by 5-HT 2 receptors, Brain Res. 529, 85. Sidman, R.L., J.B. Angevine and E.T. Pierce, 1971, Atlas of the Mouse Brain and Spinal Cord (Harvard University Press, Cambridge, MA). Sundstr6m, E. and G. Jonsson, 1985, Pharmacological interference with the neurotoxic action of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) on central catecholamine neurons in the mouse, Eur. J. Pharmacol. 110, 293. Wong, D.T., F.P. Bymaster, J.S. Horng and B.B. Molloy, 1975, A new selective inhibitor for uptake of serotonin into synaptosomes of rat brain: 3-(p-trifluoromethylphenoxy)-N-methyl-3-phenylpropylamine, J. Pharmacol. Exp. Ther. 193, 804. Youngster, S.K., P.K. Sonsalla, B.-A. Sieber and R.E. Heikkila, 1989, Structure-activity study of the mechanism of l-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. I. Evaluation of the biological activity of MPTP analogs, J. Pharmacol. Exp. Ther. 249, 820.
Elsevier Science Publishers B.V.
EJP 53405
Fluoxetine and desipramine selectively attenuate 2'-NH2-MPTP-induced depletions in serotonin and norepinephrine A n n e M. A n d r e w s
,,a,b
a n d D e n n i s L. M u r p h y
a
a Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD, USA, and 9 Department of Chemistry, American University, Washington, DC, USA
Received 5 July 1993, accepted 17 September 1993
We recently reported that the novel MPTP analog 1-methyl-4-(2'-aminophenyl)-l,2,3,6-tetrahydropyridine (2'-NHz-MPTP) administered to C57BL/6 mice produced substantial decreases in forebrain serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine, with negligible effects on brain dopamine or dopamine metabolites. In the present report, we confirm and extend our original results to include dose-response data and the effect of selective uptake inhibition on the levels of monoamine neurotransmitters in various regions of the mouse brain following treatment with 2'-NHz-MPTP. In a dose-ranging study, 2'-NH2-MPTP (10 mg/kg x 4) produced a 25-30% reduction in frontal cortex 5-HT, 5-HIAA, and norepinephrine. When 4 x 20 mg/kg 2'-NHz-MPTP was administered, 70-75% reductions in 5-HT, 5-HIAA, and norepinephrine in both frontal cortex and hippocampus were seen 1 week after treatment. No changes in dopamine were found in striatum or in any of the other brain regions examined at either dose. Doses of 40 and 60 mg/kg were lethal shortly after a single injection. In mice receiving either fluoxetine or desipramine (10 mg/kg) prior to 2'-NHz-MPTP (20 m g / k g × 4), decreases in 5-HT and norepinephrine, respectively, were significantly attenuated by ~ 30-40%. These data suggest that 2'-NHz-MPTP acts in a dose-dependent manner and that the serotonergic and noradrenergic uptake systems are involved in the mechanism by which 2'-NHz-MPTP causes selective deficits in cortical and hippocampal 5-HT and norepinephrine. MPTP (1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine); MPTP analog; MPTP derivative; 5-HT (5-hydroxytryptamine, serotonin); Norepinephrine; Fluoxetine; Desipramine
1. Introduction
The dopaminergic neurotoxicity of 1-methyl-4phenyl-l,2,3,6-tetrahydropyridine (MPTP; fig. 1) is mediated via uptake of its primary metabolite, 1-methyl4-phenylpyridinium (MPP+), by the dopamine transporter (Javitch et al., 1985). Evidence includes the fact that M P T P toxicity can be attenuated by pharmacologic inhibition of the dopamine transporter with either mazindol or amfolenic acid (Javitch et al., 1985; Mayer et al., 1986; Melamed et al., 1985; Sundstr6m and Jonsson, 1985). 1-Methyl-4-(2'-aminophenyl)-l,2,3,6-tetrahydropyridine (2'-NH2-MPTP; fig. 1) is an analog of M P T P that produces marked decreases in cortical and hippocampal serotonin (5-hydroxytryptamine; 5-HT) and n o r e p i n e p h r i n e without decreasing striatal dopamine in mouse brain when administered in a standard dosing paradigm for M P T P (20 m g / k g x 4)
* Corresponding author. Address: National Institute of Mental Health, Building 10, Room 3D41, 9000 Rockville Pike, Bethesda, MD 20892, USA. Tel. (301)496-3421, fax (301)402-0188.
(Andrews and Murphy, 1993). In this study, we investigated the effects of varying the concentration of 2'N H z - M P T P administered to mice. In addition, because the dopamine transporter is involved in the events leading to M P T P toxicity, we also studied whether the serotonin and norepinephrine transporters were of similar importance in the mechanism of action of 2'NH2-MPTP.
2. Materials and methods 2.1. Materials
2 ' - N H 2 - M P T P was synthesized by Ecochem Research (Chaska, MN). The identity of 2 ' - N H 2 - M P T P was verified by GCMS and [IH]NMR. It was stored desiccated at - 7 0 ° C and checked periodically for stability by HPLC. Fluoxetine was a gift from Eli Lilly and Company (Indianapolis, IN), and desipramine was purchased from Aldrich Chemical Company (Milwaukee, WI). All other drugs and chemicals were obtained from Sigma Chemical Company (St. Louis, MO).
216
2.2. Animal treatments C57BL/6 mice (Charles River Laboratories) weighing 25 g at the beginning of the study were housed under standard conditions with food and water ad libitum. In the dose-ranging study, 0, 10, 20, 40, or 60 mg/kg 2'-NH2-MPTP (calculated as the free base) was administered in four injections at 2 h intervals. In a second experiment designed to study the effect of uptake inhibitors, three groups of mice were injected with 20 mg/kg 2'-NH2-MPTP every 2 h for a total of four injections. Ninety minutes prior to the first dose of 2'-NH2-MPTP, mice were injected with either 10 mg/kg fluoxetine, 10 m g / k g desipramine, or saline. In a separate experiment, three groups of mice were injected with either 10 mg/kg fluoxetine, 10 mg/kg desipramine, or saline in order to determine the effects of the uptake inhibitors alone. All injections were administered i.p. in a volume of 0.1 ml. In the third experiment, control animals and those receiving 4 × 20 mg/kg 2'-NH2-MPTP were monitored for changes in body temperature. Mice were acclimated to the testing room temperature of 19.3 + 1.0°C for 1 h prior to the experiment. Colonic temperature was measured with a rectal probe (Harvard Apparatus) and digital thermometer (Sensortek Inc., Clifton, N J). The thermocouple was inserted 2 cm into the colon of mice which were gently restrained by the tail prior to drug administration to establish a baseline temperature. Thirty minutes after each of the four injections of either saline or 2'-NH2-MPTP, rectal temperature was measured again.
2.3. HPLC analysis Seven days post-treatment, the mice were killed by cervical dislocation and their brains were rapidly removed and dissected on ice. Brain regions including frontal cortex, hippocampus, striatum, hypothalamus, and brainstem were dissected according to The Atlas of the Mouse Brain and Spinal Cord (Sidman et al., 1971). These tissues were individually analyzed for monoamine neurotransmitters and metabolites by high perCH3 t
CH3 I
2'-NH2-MPTP
MPTP
Fig. 1. 1-Methyl-4-(2'-aminophenyl)-l,2,3,6-tetrahydropyridine (2'NH2-MPTP) and its parent compound, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP).
formance liquid chromatography coupled with electrochemical detection (HPLC-ECD) at +0.85 V using established methods (Mefford, 1981). Briefly, individual samples were sonicated in 200250/zl 0.1 M H f l O 4 and centrifuged at 7200 g (12000 rpm) for 10 min. Each supernatant (50/zl) was injected onto a 100 mm by 4.6 mm Axxiom 3/zm octadecylsulfate (ODS) reversed-phase chromatography column in a mobile phase containing 0.25 M citric acid, 5-9% acetonitrile, 450 m g / l octanesulfonic acid, 0.3-0.4% triethylamine, and 100 mg/l EDTA. 5-Hydroxy-Nmethyltryptamine (N-Me-5-HT) was used as the internal standard and calculations were performed by comparing the relative peak areas of sample peaks with those of external standards. 5-Hydroxytryptamine, 5hydroxyindoleacetic acid (5-HIAA), norepinephrine, dopamine, and the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were all able to be measured in a single chromatogram. The order of elution and representative retention times are as follows: norepinephrine (4.4), DOPAC (6.5), dopamine (7.7), 5-HIAA (10.4), HVA (12.7), 5-HT (17.9), and N-Me-5-HT (21.2) min, respectively. Protein was quantitated by the method of Lowry et al. (Lowry et al., 1951). Concentrations are expressed in ng/mg protein (mean + S.E.M.).
2.4. Statistics One-way analysis of variance (ANOVA) was used to detect overall statistically significant differences among treatment groups. Individual t-tests compared least squares means between groups. Probability < 0.05 was considered significant following multiplication by a Bonferroni correction factor (g) where g = n * ( n - l ) / 2 , n being the number of treatment groups.
3. Results
3.1. Dose-ranging study with 2'-NH2-MPTP In mice injected with 2'-NH2-MPTP, statistically significant reductions (25-30%) in cortical 5-HT, 5HIAA, and norepinephrine were evident at the lowest, 4 × 10 mg/kg dose (P < 0.01, 0.001, and 0.001, respectively; table 1). At 4 × 20 mg/kg, more marked changes in 5-HT, 5-HIAA, and norepinephrine were observed, especially in cortex and hippocampus where 2'-NH 2MPTP caused decrements on the order of 70% (P < 0.001; table 1). Smaller reductions in striatal 5-HT and 5-HIAA were also seen at this dose, as well as a decrease in norepinephrine in brainstem (P
217 TABLE 1 Dose-ranging study with 2 ' - N H 2-MPTP. Two groups of mice (n = 8) were injected 4 times i.p. with either 10 or 20 m g / k g 2 ' - N H 2 - M P T P at 2 h intervals. In two additional groups injected with 40 or 60 m g / k g 2 ' - N H 2 - M P T P (n = 8), > 80% of the animals died shortly after the first injection. Control animals received four injections of saline (n = 8). Seven days post-treatment, the mice were killed and their brains were analyzed for neurotransmitters and metabolites as described in Materials and methods. T h e results are expressed as percentages of the respective control group m e a n s _+S.E.M. which are similar in magnitude to the absolute concentrations shown in figs. 2 and 4. There were no statistically significant changes in hypothalamus (data not shown). Cortex
Hippocampus
Striatum
cant attenuation (P < 0.01 vs. 2'-NH2-MPTP-treated mice) of the cortical and hippocampal 5-HT decrements caused by 2'-NH2-MPTP (fig. 2A). The levels ot
5-HT
Ao 10" 9" 8"
rT
7'
Brainstem
5-HT 10 m g / k g 20 m g / k g 5-H/A4 10 m g / k g 20 m g / k g
×4 ×4
7 6 . 8 5 : 4 . 5 b 1 0 6 _+12 99.0_+4.3 98.65:6.7 32.3+ 3.2 c 31.7+ 4.2 c 80.9+2.1 b 7 9 . 3 5 : 4 . 8
4.
×4 ×4
69.1_+ 3.3 c 8 9 . 5 5 : 7 . 1 93.4_+3.1 91.5_+10 31.1_+ 3.0 c 3 1 . 1 5 : 2 . 3 c 77.0+2.5 c 65.4_+ 8.8
2'
68.9_+ 3.0 c 78.7 + 7.2 ND 32.1+ 3.8 c 26.1_+ 3.0 c N D
72.9 5 : 4 . 5 60.35:8.6 b
0
96.8-+ 7.6 93.5_+10
80.2-+ 6.9 78.4_+12
I'
Norepinephrine 10 m g / k g × 4 20 m g / k g × 4
Cortex
Hippocampus
Striatum
Hippocampus
Striatum
Dopamine 10 m g / k g × 4 20 m g / k g × 4
105 -+12 84.2+ 3.9
112 +4.3 103 _+3.2
B.
DOPAC 10 m g / k g 20 m g / k g HVA 10 m g / k g 20 m g / k g
×4 ×4
86.0-+14 102 + 2 6
×4 ×4
107 + 8.0 86.3+11
ND ND 116 + 7.1 112 + 7.8
83.8-+5.1 91.5+7.4 91.4+2.7 85.0+6.8
73.9:t: 8.5 64.6+ 8.7 105 _+19 81.2-+11
5-HIAA 8"
7" 6"
.i
5"
b p < 0.01, c p < 0.001. Entries marked ' N D ' were not detectable.
4"
ment with 2'-NH2-MPTP. The two higher doses of 2'-NH2-MPTP (40 and 60 mg/kg) led to lethality in > 80% of the animals following a single injection.
/r',A
2"
1'
3.2. Pretreatment with up take inhibitors 0 Cortex
Mice pretreated with fluoxetine, a selective inhibitor of 5-HT uptake (Wong et al., 1975) showed a signifiNE
Co 7' Fig. 2. Effects of pretreatment with fiuoxetine on the changes in 5-HT, 5-HIAA, and norepinephrine 1 week after 2'-NH2-MPTP. Two groups of mice were injected i.p. 4 times with 20 m g / k g 2 ' - N H 2 - M P T P at 2 h intervals. O n e group received saline 90 min prior to 2 ' - N H 2 - M P T P (n = 7; diagonally hatched bars) and the second group received a single injection of 10 m g / k g fluoxetine (n = 7; shaded bars). Control animals (n = 7; u n s h a d e d bars) received four injections of saline every 2 h. After 1 week, 5-HT, 5-HIAA, and norepinephrine were m e a s u r e d by H P L C - E C D as described in Materials and Methods. Control group m e a n s + S.E.M. in cortex, hippocampus, and striatum, respectively are as follows: 5-HT, 7.28+0.46, 8.14+0.38, and 3.92+0.32; 5-HIAA, 3.43+0.30, 6.92 + 0.70, and 2.92 + 0.39; norepinephrine, 4.15 + 0.45, 5.70 + 0.39, and none detected ( n g / m g protein). Probabilities indicated in the figure are as follows: * P < 0.05 * * P < 0.01, and * * * P < 0.001 different from control, and * p < 0.01 different from 2'-NH2-MPTP.
6" 5'
,T
I'
not detectable
0 Cortex
Hlppocampus
Strlatxma
218
5-HIAA in mice pretreated with fluoxetine were not statistically different from the levels in animals that received 2'-NH2-MPTP alone (fig. 2B), however, the general trend toward attenuation paralleled that seen for 5-HT (fig. 2A). Fluoxetine pretreatment had no significant effect on 2'-NH2-MPTP-induced decreases in norepinephrine (fig. 2C). In a separate experiment, mice receiving 10 mg/kg fluoxetine by itself (n = 7) had levels of 5-HT, 5-HIAA, and NE 1 week posttreatment which were not significantly different from animals injected with saline (n = 7; data not shown). On the other hand, the norepinephrine-selective uptake inhibitor desipramine (Carlsson et al., 1966; Lidbrink et al., 1971) significantly diminished 2'-NH 2MPTP's effects on cortical and hippocampal norepinephrine (P < 0.01 vs. 2'-NH2-MPTP-treated mice: fig. 3C) without affecting the magnitude of the 2'NH2-MPTP-induced decreases in 5-HT (fig. 3A) or 5-HIAA (fig. 3B). Desipramine treatment by itself (n = 7) had no effect on 5-HT, 5-HIAA, or norepinephrine levels measured 1 week post-treatment (data not shown). In striatum, the levels of 5-HT and 5-HIAA in all treatment groups were not statistically different from control in this experiment (figs. 2A, 2B, 3A, and 3B). Levels of dopamine were also unchanged in the three brain regions examined (fig. 4A). Minimal effects on dopamine metabolites included small decreases in hippocampal DOPAC following all three drug treatments (fig. 4B) and a small increase in hippocampal HVA after 2'-NH2-MPTP (fig. 4C).
min after each dose, subsided before the administration of the next dose, and became less marked aftel each subsequent dose.
A.
5-HT 10~ 9" 8-
•
z-I,7-
7-
3
:
:
I o
Cortex
Hippocampus
Striatum
5-HIAA
B. 8
,T
:e 4"
3.3. Behavior and body temperature Mice that received 4 × 20 mg/kg 2'-NH2-MPTP exhibited behavior which appeared to be similar to the serotonin syndrome (Modigh and Svensson, 1972) and consisted of moderately paced walking in circles around the cage, Straub tails and occasional rearing. In addition, some animals exhibited piloerection and excessive salivation. This behavior began approximately 15-20
2" I" 0 Cortex
C.
Hippocampus
Striatum
NE
,T Fig. 3. Effect of pretreatment with desiprimine on changes in 5-HT, 5-HIAA, and norepinephrine 1 week after 2'-NH2-MPTP. Two groups of mice were injected i.p. 4 times with 20 m g / k g 2 ' - N H 2M P T P at 2 h intervals. O n e group receive d saline 90 rain prior to 2 ' - N H 2 - M P T P (n = 7; diagonally hatched bars), the second group received a single injection of 10 m g / k g desiprimine (n = 6; shaded bars). Control animals (n = 7; u n s h a d e d bars) received four-injections of saline every 2 h. After 1 week, 5-HT, 5-HIAA, and norepinephrine were m e a s u r e d by H P L C - E C D as described in Materials and methods. Control group m e a n s + S . E . M , are the same as those reported in the legend of fig. 2. Probabilities indicated in the figure are as follows: * P < 0.05, * * P < 0.01, and * * * P < 0.001 different from control, and t p < 0.01 different from 2'-NH2-MPTP.
:s
4" 3 2 not detectable
I 0
Cortex
Hippocampus
Striatum
219
Colonic temperature was recorded after each of four injections of 2'-NH2-MPTP or saline in a representative sample of animals. In the case of both 2'NH2-MPTP-treated (n -- 8) and control groups (n = 6),
mean body temperature after each injection was unchanged from baseline temperatures of 38.4 + 0.18 and 38.4 + 0.12°C, respectively (data not shown).
4. Discussion
DA
Ao
120
12
100
10
80
I'i "60
6
~
4
0
2
0
Cortex
B.
Hippocampus
Striatum
DOPAC 20 18' 16 14' 12" I0' 8" 6" 4"
0
Cortex
C.
H/ppocampus
Striatum
ttVA 12"
10"
6'
4"
2"
o Cortex
Hippocampus
2'-NH2-MPTP differs considerably from other previously described MPTP analogs (Bradbury et al., 1985; Fuller et al., 1985; Youngster et al., 1989) in the fact that it causes selective decreases in serotonin and norepinephrine in mouse forebrain without affecting striatal dopamine. Because most of the analogs which have proven to be more potent dopaminergic neurotoxins than MPTP bear a substituent at the 2'-position (i.e., 2'-CH3-MPTP, 2'-Et-MPTP, 2'-C1-MPTP, 2'-FMPTP, and 2'-CF3-MPTP) (Youngster et al., 1989), the -NH 2 substituent itself and not simply substitution at the 2'-position appear to be important for the differential actions of 2'-NH2-MPTP. This study confirms the 2'-NH2-MPTP-induced decreases of 60-70% in cortical and hippocampal 5-HT and norepinephrine which we reported in an independent study (Andrews and Murphy, 1993) and extends the findings to include data on the dose-dependent effects of 2'-NH2-MPTP. From the latter results, the LD50 of 2'-NH2-MPTP can be estimated to be between 20 and 40 mg/kg when given i.p. to mice. Modest decreases ( ~ 20%) in striatal 5-HT and 5-HIAA were observed in this report and in separate experiments from our previous work (Andrews and Murphy, 1993); however, these smaller decrements are more variable and not always statistically significant. 2'-NHa-MPTP is similar to MPTP and many of its analogs in that it seems to require active uptake by the neurotransmitter-specific transporters located on the monoaminergic neurons that are vulnerable to its toxic effects. As the results of this study demonstrate, sero-
Striatum
Fig. 4. The effects of pretreatment with uptake inhibitors on changes in dopamine, D O P A C , and H V A 1 week after 2'-NH2-MPTP. Three groups of mice were injected i.p. 4 times with 20 m g / k g 2 ' - N H 2M P T P at 2 h intervals. O n e group received saline 90 min prior to 2'-NHE-MPTP ( n = 7 ; diagonally hatched bars), a second group received a single injection of 10 m g / k g fluoxetine (n = 7; shaded bars), and the third was pretreated with 10 m g / k g desipramine (n = 6; cross-hatched bars). Control animals (n = 7; u n s h a d e d bars) received four injections of saline every 2 h. O n e week later, dopamine and its metabolites D O P A C and H V A were m e a s u r e d by H P L C - E C D as described in Materials and methods. Control group m e a n s + S.E.M. in cortex, hippocampus and striatum, respectively are as follows: dopamine, 0.499+0.10, 0.392_+ 0.089, and 107.8_+ 7.8; D O P A C , 2.57 _+0.40, 1.95_+0.16, and 17.2_+2.5; H V A , 1.03_+0.13, 0.386_+0.074, and 9.69 _+ 1.02 ( n g / m g protein). Probabilities indicated in the figure are as follows: * P < 0.05, * * P < 0.01, and * * * P < 0.001 different from control, and t p < 0.01 different from 2'-NHE-MPTP.
220
tonin and norepinephrine uptake inhibitors selectively attenuated 2'-NH2-MPTP-induced decreases in hippocampal and cortical 5-HT and norepinephrine, respectively. These data suggest that 2'-NH2-MPTP , or possibly a pyridinium metabolite, as is the case for MPTP (Javitch et al., 1985; Markey et al., 1984), is actively transported via the 5-HT transporter into serotonergic nerve terminals and the norepinephrine transporter into noradrenergic nerve terminals before causing decreases in each of these neurotransmitters. Complete protection by the selective uptake inhibitors from the effects of 2'-NH2-MPTP was not observed in this study. Among several possible considerations, pharmacokinetic factors may provide one explanation. For example, 2'-NH2-MPTP or a metabolite may have a much longer half-life in brain compared to fluoxetine or desipramine. The MPTP metabolite MPP + is retained on the order of days to weeks in primate and dog brain (Herkenham et al., 1991; Johannessen et al., 1991). A second explanation involves the possibility that the 10 mg/kg dose of uptake inhibitor was not sufficient to completely block uptake in mice. We have performed additional experiments wherein a 10 mg/kg dose of fluoxetine or desipramine was given before, during, and after the 4 x 20 mg/kg 2'-NH 2MPTP regimen; however, only 30% of the mice survived this particular treatment regimen. Further investigation of different dosing paradigms could prove worthwhile. Finally, 2'-NH2-MPTP or its metabolite(s) may gain access to serotonergic and noradrenergic neurons via other mechanisms not exclusively involving active transport. A nonspecific passive diffusion mechanism seems unlikely though, due to the fact that the -NH 2 substituent is nonlipophilic. It is unclear why the 5-HT uptake inhibitor fluoxetine failed to produce a statistically significant effect on the serotonin metabolite 5-HIAA; however decreased formation of 5-HIAA due to inactivation of monoamine oxidase (MAO) by 2'-NH2-MPTP may be involved. It has been demonstrated that MPTP and some other 2'-substituted MPTP analogs are capable of mechanism-based inactivation of MAO (Krueger et al., 1990). Irreversible inhibition of MAO by 2'-NH 2-MPTP or a metabolite would decrease the formation of 5-HIAA and partially cancel any increase expected due to the reduced toxicity associated with fluoxetine pretreatment. The ability of other serotonergic and noradrenergic neurotoxins to be actively transported by the 5-HT and norepinephrine reuptake systems has already proven to be important in determining their toxic selectivity (Battaglia et al., 1988; Fuller et al., 1975; Jonsson, 1980). 2'-NH2-MPTP appears to be no exception. However, beyond uptake by the 5-HT and norepinephrine transporters, 2'-NH2-MPTP may be acting by a mechanism which is different from these other neurotoxins. For
example, 5-HT is believed to play a role in mammalian thermoregulation (Jacob and Girault, 1979; Lin et al., 1983) and serotonergic neurotoxins such as fenfluramine, p-chloroamphetamine and 3,4-methylenedioxymethamphetamine are reported to cause dose-dependent hyperthermia when administered to rats (Frey, 1975; Nash et al., 1988; Pawlowski, 1981; Schmidt et al., 1990). The fact that 2'-NH2-MPTP did not cause an increase in body temperature in our study may be an indication that its mechanism of action is somewhat different from the substituted amphetamines. However, a few studies have reported that different ambient temperatures can modify the magnitude and direction of the observed change in body temperature (hyperthermia verses hypothermia) following administration of substituted amphetamines (Frey, 1975; Nash et al., 1988; Pawlowski, 1981; Schmidt et al., 1990); therefore, experiments to determine 2'-NH2-MPTP's effects on colonic temperature at varying ambient temperatures may be warranted. Finally, because the effects of 5-HT on thermoregulation are different in rats and mice (Jacob and Girault, 1979), data obtained with substituted amphetamines in rats may not be directly applicable to studies in mice. While the acute behavioral effects observed following 2'-NHz-MPTP bear some resemblance to the serotonin syndrome, specific aspects of this syndrome are not well-documented in mice. The behaviors elicited by 2'-NH2-MPTP may also be attributed, in part, to centrally or peripherally released norepinephrine as well as 5-HT, or to other undetermined factors. Further investigation will be able to provide additional evidence of the similarities between 2'-NH2-MPTP's mechanism of action and those of MPTP analogs versus other classes of serotonergic and noradrenergic neurotoxins.
Acknowledgments The authors wish to thank Dr. R. Carlson for his help with 2'-NH2-MPTP synthesis and Dr. J.L. Hill for his statistical advice. A.M. Andrews was partially supported by a fellowship from the U.S. Department of Education.
References Andrews, A.M. and D.L. Murphy, 1993, Sustained depletion of cortical and hippocampal serotonin and norepinephrine but not striatal dopamine by 1-methyl-4-(2'-aminophenyl)-l,2,3,6-tetrahydropyridine (2'-NH2-MPTP): a comparative study with 2'-CH 3MPTP and MPTP, J. Neurochem. 60, 1167. Battaglia, G., S.Y. Yeh and E.B. De Souza, 1988, MDMA-induced neurotoxicity: parameters of degeneration and recovery of brain serotonin neurons, Pharmacol. Biochem. Behav. 29, 269. Bradbury, A.J., B. Costall, A.M. Domeney, B. Testa, P.G. Jenner,
221 C.D. Marsden and R.J. Naylor, 1985, The toxic actions of MPTP and its metabolite MPP ÷ are not mimicked by analogues of MPTP lacking an N-methyl moiety, Neurosci. Lett. 61, 121. Carlsson, A., K. Fuxe, B. Hamberger and M. Lindqvist, 1966, Biochemical and histochemical studies on the effects of imipraminelike drugs and (+)-amphetamine on central and peripheral catecholamine neurons, Acta Physiol. Scand. 67, 481. Frey, H.-H., 1975, Hyperthermia induced by amphetamine, p-chloroamphetamine, and fenfluramine in the rat, Pharmacology 13, 163. Fuller, R.W., K.W. Perry and B.B. Molloy, 1975, Effect of 3-(p-trifluoromethylphenoxy)-N-methyl-3-phenylpropylamine on the depletion of brain serotonin by 4-chloroamphetamine, J. Pharmacol. Exp. Ther. 193, 796. Fuller, R.W., S.K. Hemrick-Luecke and D.W. Robertson, 1985, Comparison of 1-methyl-4-(p-chlorophenyl)-l,2,3,6-tetrahydropyridine, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) and p-chloroamphetamine as monoamine depletors, Res. Commun. Chem. Pathol. Pharmacol. 50, 57. Herkenham, M., M.D. Little, K. Bankiewicz, S.-c. Yang, S.P. Markey and J.N. Johannessen, 1991, Selective retention of MPP ÷ within the monoaminergic systems of the primate brain following MPTP administration: an in vivo autoradiographic study, Neuroscience 40, 133. Jacob, J.J. and J.-M.T. Girault, 1979, 5-Hydroxytryptamine, in: Body Temperature: Regulation, Drug Effects, and Therapeutic Implications, ed. Lomax, P. and E. Sch6nbaum (Marcel Dekker, Inc., New York) p. 183. Javitch, J.A., R.J. D'Amato, S.M. Strittmatter and S.H. Snyder, 1985, Parkinsonism-inducing neurotoxin N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine: uptake of the metabolite N-methyl-4-phenylpyridinium by dopamine neurons explains selective toxicity, Proc. Natl. Acad. Sci. USA 82, 2173. Johannessen, J.N., T.J. Sobotka, V.K. Weise and S.P. Markey, 1991, Prolonged alterations in canine striatal dopamine metabolism following subtoxic doses of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) and 4'-amino-MPTP are linked to the persistence of pyridinium metabolites, J. Neurochem. 57, 981. Jonsson, G., 1980, Chemical neurotoxins as denervation tools in neurobiology, Annu. Rev. Neurosci, 3, 169. Krueger, M.J., K. McKeown, R.R. Ramsay, S.K. Youngster and T.P. Singer, 1990, Mechanism-based inactivation of monoamine oxidases A and B by tetrahydropyridines and dihydropyridines, Biochem. J. 268, 219. Lidbrink, P., G. Jonsson and K. Fuxe, 1971, The effect of imipramine-like drugs and antihistamine drugs on uptake mechanisms in the central noradrenaline and 5-hydroxytryptamine neurons, Neuropharmacology 10, 521. Lin, M.T., J.J. Wu and B.L. Tsay, 1983, Serotonergic mechanisms in the hypothalamus mediate thermoregulatory responses in rats, Naunyn-Schmied. Arch. Pharmacol. 322, 271.
Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193, 265. Markey, S.P., J.N. Johannessen, C.C. Chiueh, R.S. Burns and M.A. Herkenham, 1984, Intraneuronal generation of a pyridinium metabolite may cause drug-induced parkinsonism, Nature 311, 464. Mayer, R.A., M.V. Kindt and R.E. Heikkila, 1986, Prevention of the nigrostriatal toxicity of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine by inhibitors of 3,4-dihydroxyphenylethylamine transport, J. Neurochem. 47, 1073. Mefford, I.N., 1981, Application of high performance liquid chromatography with electrochemical detection to neurochemical analysis: measurement of catecholamines, serotonin and metabolites in rat brain, J. Neurosci. Methods 3, 207. Melamed, E., J. Rosenthal, O. Cohen, M. Globus and A. Uzzan, 1985, Dopamine but not norepinephrine or serotonin uptake inhibitors protect mice against neurotoxicity of MPTP, Eur. J. Pharmacol. 116, 179. Modigh, K. and T.H. Svensson, 1972, On the role of central nervous system catecholamines and 5-hydroxytryptamine in the nialamide-induced behavioural syndrome, Br. J. Pharmacol. 46, 32. Nash, J.F., H.Y. Meltzer and G.A. Gudelsky, 1988, Elevation of serum prolactin and corticosterone concentrations in the rat after the administration of 3,4-methylenedioxymethamphetamine, J. Pharmacol. Exp. Ther. 245, 873. Pawlowski, L., 1981, Different action of 5-hydroxytryptamine (5-HT) uptake inhibitors on fenfluramine- but not p-chloramphetamineinduced hyperthermia in rats, J. Pharm. Pharmacol. 33, 538. Schmidt, C.J., C.K. Black, G.M. Abbate and V.L. Taylor, 1990, Methlylenedioxymethamphetamine-induced hypertherrnia and neurotoxicity are independently mediated by 5-HT 2 receptors, Brain Res. 529, 85. Sidman, R.L., J.B. Angevine and E.T. Pierce, 1971, Atlas of the Mouse Brain and Spinal Cord (Harvard University Press, Cambridge, MA). Sundstr6m, E. and G. Jonsson, 1985, Pharmacological interference with the neurotoxic action of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) on central catecholamine neurons in the mouse, Eur. J. Pharmacol. 110, 293. Wong, D.T., F.P. Bymaster, J.S. Horng and B.B. Molloy, 1975, A new selective inhibitor for uptake of serotonin into synaptosomes of rat brain: 3-(p-trifluoromethylphenoxy)-N-methyl-3-phenylpropylamine, J. Pharmacol. Exp. Ther. 193, 804. Youngster, S.K., P.K. Sonsalla, B.-A. Sieber and R.E. Heikkila, 1989, Structure-activity study of the mechanism of l-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. I. Evaluation of the biological activity of MPTP analogs, J. Pharmacol. Exp. Ther. 249, 820.