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The Journal of Neuroscience, November 1, 1998, 18(21):9069-9077
Functional Consequences of Central Serotonin Depletion Produced
by Repeated Fenfluramine Administration in Rats
Michael H.
Baumann,
Mario A.
Ayestas, and
Richard B.
Rothman
Clinical Psychopharmacology Section, Intramural Research Program,
National Institute on Drug Abuse, National Institutes of Health,
Baltimore, Maryland 21224
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ABSTRACT |
Repeated administration of D,L-fenfluramine (FEN) is
known to cause prolonged depletion of forebrain serotonin (5-HT) in
animals. Ironically, few studies have evaluated functional consequences of such FEN-induced 5-HT loss. In the present work, we examined neuroendocrine and behavioral responses evoked by acute FEN injection in rats that had previously received a 4 d FEN-dosing regimen known to deplete forebrain 5-HT (D,L-FEN, 20 mg/kg, s.c.,
b.i.d.). Rats were fitted with indwelling jugular catheters before the study to allow for repeated intravenous challenge injections and stress-free blood sampling. At 1 and 2 weeks after the 4 d dosing regimen, acute FEN (1.5 or 3.0 mg/kg, i.v.) produced dose-related elevations in plasma corticosterone and prolactin; these hormonal responses were markedly attenuated in FEN-pretreated rats. Behavioral effects of acute FEN, namely flat body posture and forepaw treading, were also blunted in FEN-pretreated rats. Interestingly, rats exposed
to repeated FEN did not display overt abnormalities in hormonal or
behavioral parameters under basal (i.e., unprovoked) conditions,
despite dramatic decreases in postmortem tissue levels of 5-HT in
numerous brain areas. Our results suggest that FEN-induced 5-HT
depletion is accompanied by multiple impairments in 5-HT function.
Although the clinical relevance of our data are debatable, the findings
clearly show the utility of the FEN challenge test for uncovering
in vivo functional deficits that might otherwise go
undetected. FEN should remain an important pharmacological tool for
determining the role of 5-HT neurons in mediating diverse physiological
and behavioral processes.
Key words:
fenfluramine; serotonin depletion; neuroendocrine; corticosterone; prolactin; behavior; neurotoxicity; serotonin function; rat
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INTRODUCTION |
Fenfluramine
(N-ethyl- -methyl-m-[trifluoromethyl]-phenethylamine)
is an appetite suppressant with proven efficacy (Silverstone, 1992 ).
Two forms of the drug that exhibit similar pharmacology have been
prescribed: the D,L-racemic mixture (FEN) and the more potent D-isomer (D-FEN) (McTavish and Heel,
1992). Despite structural similarities to amphetamine, FEN is not a
locomotor stimulant and is seldom abused. The unique properties of FEN
are related to its preferential activation of central serotonin (5-HT)
neurons (for review, see Rowland and Carlton, 1986). For example, FEN stimulates 5-HT release and inhibits 5-HT reuptake in brain tissue preparations (Fuxe et al., 1975; Garattini et al., 1975). In
vivo microdialysis studies in rat brain demonstrate that systemic
or local administration of FEN elevates extracellular levels of 5-HT (Auerbach et al., 1989; Schwartz et al., 1989); this effect of FEN is
blocked by 5-HT reuptake inhibitors like fluoxetine, suggesting involvement of 5-HT transporters (Berger et al., 1992; Sabol et al.,
1992). Although FEN influences both 5-HT release and reuptake, the
5-HT-releasing capability of the drug appears to predominate in
vivo (Fuller et al., 1988; Carboni et al., 1989).
A primary concern with the clinical use of FEN relates to its potential
adverse effects on 5-HT neurons (for review, see McCann et al., 1997).
It is well known that administration of FEN at sufficient doses can
cause long-lasting (>2 weeks) depletion of 5-HT in rodent brain
(Clineschmidt et al., 1976; Harvey and McMaster, 1975; Sanders-Bush et
al., 1975). Such depletions are associated with a loss of
5-HT-immunoreactive nerve fibers (Appel et al., 1989; Molliver and
Molliver, 1990) and a reduction in 5-HT reuptake sites (Appel et al.,
1990; Zaczek et al., 1990). The deleterious effects of FEN on 5-HT
systems have been reported in every animal species examined to date
(McCann et al., 1997), prompting speculation that FEN is a 5-HT
neurotoxin that may be hazardous to human patients (Ricaurte et al.,
1991; McCann et al., 1994).
Interestingly, few preclinical studies have found impairments in 5-HT
function after FEN-induced 5-HT depletion (for review, see Ricaurte et
al., 1994). This observation could be regarded as evidence for a lack
of such deficits; alternatively, more sensitive testing methods may be
required to reveal subtle changes in 5-HT neuronal dynamics. In the
present study, we used an in vivo pharmacological approach
to assess the functional integrity of 5-HT neurons in FEN-treated rats.
More specifically, we evaluated neuroendocrine and behavioral responses
elicited by acute FEN challenge in rats that had previously received a
4 d FEN-dosing regimen known to deplete central 5-HT (20 mg/kg,
s.c., b.i.d., 4 d). The stimulatory effect of acute FEN on
circulating adrenocorticotropin (ACTH), corticosteroids, and prolactin
is well documented in both animals and humans (Van de Kar, 1991; Yatham
and Steiner, 1993). Because these FEN-evoked hormonal changes are
mediated, at least in part, by activation of 5-HT pathways in the
hypothalamus (Levy et al., 1994a), they can be used as sensitive
measures of brain 5-HT function. It is noteworthy that FEN challenge
tests have been widely used by clinical investigators to demonstrate
changes in 5-HT sensitivity associated with depression and other
psychiatric disorders (Siever et al., 1991).
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MATERIALS AND METHODS |
Animals and surgery. Male Sprague Dawley rats
weighing 250-300 gm were housed in standard vivarium conditions
(lights on, 7:00 A.M.-7:00 P.M.) with ad libitum
access to food and water. Animals were maintained in facilities fully
accredited by the American Association of the Accreditation of
Laboratory Animal Care, and experiments were performed in accordance
with the Animal Care and Use Committee of the National Institute on
Drug Abuse (NIDA) Intramural Research Program (IRP). All of the rats in
our studies were anesthetized with methoxyflurane (Pittman-Moore, Phillipsburg, NJ), and indwelling jugular catheters were surgically implanted as described (Baumann et al., 1995). Briefly, catheters (0.5 mm inner diameter, 1.0 mm outer diameter) were made by joining 8 cm of
vinyl tubing to 3 cm of SILASTIC (Dow Corning, Midland, MI) tubing with
a 1 cm 23 gauge needle tube connector. During surgery, the SILASTIC end
of the catheter was inserted into the jugular vein and advanced to the
atrium, whereas the vinyl end was exteriorized on the nape of the neck.
Rats were singly housed postoperatively and allowed 1 week to recover;
during this period catheters were flushed daily with 0.3 ml of 50 IU/ml
heparin saline.
Intravenous challenge test procedures. On the day of a
challenge test, rats were moved to the laboratory in their home cages at 10:00-11:00 A.M. One hour later, polyethylene extension tubes (PE
50, Clay Adams, Parsippany, NJ) were connected to intravenous catheters and threaded outside of the cages. This arrangement enabled
the investigator to perform intravenous injections and serial blood
sampling without disturbing the rats. Furthermore, the rats were able
to move freely about the cage during the experimental sessions.
Catheters were flushed with 0.3 ml of 50 IU/ml heparin saline, and
acute intravenous challenge injections were administered between 12:00
and 3:00 P.M. In all cases, blood samples (0.5 ml) were withdrawn via
the catheters immediately before and at 15, 30, and 60 min after
intravenous injections. Blood was collected into 1 ml syringes,
transferred to 1.5 ml tubes, and spun for 10 min at 5000 rpm. Plasma
was decanted and stored at  80°C until the time of assay.
Acute fluoxetine pretreatment study. Because there is
disagreement in the literature concerning the role of 5-HT neurons in mediating FEN-induced corticosterone secretion (McElroy et al., 1984;
Van de Kar et al., 1985), we first assessed the ability of fluoxetine
pretreatment to affect FEN-induced neuroendocrine secretion. Groups of
rats were pretreated with intraperitoneal fluoxetine (5 mg/kg) or its
water vehicle (1 ml/kg), 2 hr before receiving intravenous challenge
injection of FEN (D,L-racemic mixture given at 3 mg/kg) or
its saline vehicle (1 ml/kg). Fluoxetine HCl was generously provided by
Eli Lilly and Company (Indianapolis, IN), whereas FEN was obtained from
the NIDA IRP Pharmacy (Baltimore, MD). Drugs were dissolved in their
respective vehicles immediately before use. Serial blood samples were
withdrawn, and plasma was collected as described above.
Repeated FEN administration study. Rats were randomly
assigned to groups receiving a 4 d injection regimen of either FEN
(D,L-racemic mixture given at 20 mg/kg, s.c., b.i.d.) or
saline (1 ml/kg, s.c., b.i.d.). Similar repeated FEN-dosing procedures
have been shown to cause long-lasting depletion of forebrain 5-HT in
rats (Appel et al., 1989; Zaczek et al., 1990). One week after the
final injection of the 4 d regimen, rats received acute
intravenous challenge injections of FEN (1.5 or 3.0 mg/kg) or saline (1 ml/kg). Serial blood samples were withdrawn, and plasma was collected
as described above. The intravenous doses of FEN used herein have been
shown to produce significant increases in circulating corticosterone in
rats (Baumann et al., 1995). The challenge test procedure was repeated
1 week later (i.e., 2 weeks after cessation of repeated treatment) so
that all rats were tested twice with the identical acute treatment.
The occurrence of specific 5-HT-mediated behaviors was evaluated in the
same subjects during the test sessions. At 2, 10, 20, and 30 min after
intravenous injections, rats were observed, and the presence of forepaw
treading (FPT), flat body posture (FBP), and penile erections (PE) were
scored using the graded scale: 0 = absent, 1 = equivocal,
2 = present, and 3 = intense (Tricklebank et al., 1985). Rats
were given a single score for each behavior that consisted of the
summed scores across all time points.
Prolactin and corticosterone radioimmunoassays. Plasma
hormone levels were quantified using double-antibody radioimmunoassay (RIA) procedures. Samples from the fluoxetine experiments and the
repeated FEN-dosing experiments were analyzed in separate assays.
Corticosterone levels were determined using commercially available
[125I]-corticosterone RIA kits (ICN Biomedicals,
Irvine, CA). Plasma samples (10 µl) were aliquoted in duplicate, and
the average intra-assay coefficient of variability was 6.5%. Prolactin
levels were determined using materials generously provided by the
National Hormone and Pituitary Program (Rockville, MD). Antiserum
directed against rat prolactin (rPRL-S-9) was diluted 1:437,500, and
rPRL-RP-3 was the reference standard.
[125I]-Prolactin was obtained from Hazleton
Laboratories (Vienna, VA). Plasma samples (50 µl) were aliquoted in
duplicate, and the intra-assay coefficient of variability was
7.8%.
Microdissection and neurotransmitter analyses. Rats from the
repeated FEN-dosing study that received acute saline challenge (i.e.,
repeated saline/acute saline and repeated FEN/acute saline) were
killed by decapitation shortly after the 2 week test. Brains were rapidly removed, frozen on dry ice, and stored at 80°C. The
postmortem tissue levels of 5-HT and its metabolite,
5-hydroxyindoleacetic acid (5-HIAA), were determined in various brain
regions to verify the extent of 5-HT depletion produced by the 4 d
FEN-dosing regimen. Coronal sections 300 µm in thickness were cut
from frozen brains, and discrete regions were microdissected using
stainless steel needle tubing as described (Palkovits and Brownstein,
1988). Specific brain regions and their respective locations relative
to bregma are summarized in Table 1.
Tissue punches of a given region from individual rats were homogenized
in 100 µl of cold 0.1 N perchloric acid and centrifuged at 15,000 rpm
for 15 min. The concentrations of 5-HT and 5-HIAA were quantified in
the supernatant using high-pressure liquid chromatography coupled to
electrochemical detection (HPLC-EC) according to published methods
(Baumann et al., 1993). Briefly, 20 µl aliquots of supernatant were
injected onto a C-18 reversed-phase column that was linked to a
coulometric electrochemical detection system (Environmental Sciences
Associates, Bedford, MA). Mobile phase consisting of 50 mM
sodium phosphate monobasic (pH 2.75), 250 µM
Na2 EDTA, 0.025% sodium octane sulfonic acid, and 25%
methanol was recirculated at a flow rate of 0.7 ml/min. Chromatographic data were exported to a MAXIMA 820 software system (Waters Associates, Milford, MA) for peak amplification, integration, and analysis. Peak
heights of unknowns were compared with peak heights of 5-HT and 5-HIAA
standards. The lower limit of detectability (3× baseline noise level)
was 10 pg for both analytes. Tissue pellets were resolubilized in 1.0 N
NaOH and assayed for protein (Bradford, 1976).
Data analysis. All data are expressed as mean ± SEM
for n = 7 or 8 rats per group. Hormone data were
evaluated using a two-way (pretreatment × acute treatment)
ANOVA with repeated measures, whereas behavioral data were
evaluated using two-way ANOVA (pretreatment × acute treatment).
Neurochemical data were evaluated using one-way (pretreatment) ANOVA.
When significant F values were obtained, Duncan's Multiple
Range test was performed to determine differences (p < 0.05) between group means.
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RESULTS |
Fluoxetine pretreatment study
Tables 2 and
3 summarize the effect of fluoxetine
pretreatment on FEN-induced secretion of corticosterone and prolactin. Fluoxetine did not influence baseline (time 0; t = 0 min) levels of either hormone. As shown in Table 2, FEN produced a
sustained rise in plasma corticosterone (F(1,28) = 55.49; p < 0.0001), and this response was diminished
in rats pretreated with fluoxetine (pretreatment × acute
treatment interaction: F(1,28) = 10.52; p < 0.003). Similarly, Table 3 shows that FEN elevated
plasma prolactin (F(1,28) = 60.54;
p < 0.0001), and this response was reduced by
fluoxetine pretreatment (pretreatment × acute treatment interaction: F(1,28) = 15.55; p < 0.001). Post hoc tests revealed that fluoxetine
significantly attenuated FEN-induced corticosterone and prolactin
secretion at all time points after intravenous challenge injection. It
is noteworthy, however, that fluoxetine pretreatment did not completely
block the hormonal effects elicited by FEN.
Repeated FEN-dosing study: neuroendocrine findings
Figures 1 and
2 illustrate the corticosterone responses
evoked by intravenous FEN challenge administered 1 and 2 weeks after cessation of the 4 d saline or FEN treatment regimen. Baseline (time 0) hormone levels were not altered by the repeated FEN-dosing regimen on either challenge day. As depicted in Figure 1, acute FEN
increased corticosterone in a dose-related manner at 1 week (F(2,42) = 37.99; p < 0.00001).
There was a significant main effect of pretreatment
(F(1,42) = 39.8; p < 0.00001)
and a significant pretreatment × acute treatment interaction
(F(2,42) = 5.6; p < 0.01).
Post hoc tests demonstrated that corticosterone
responses in FEN-pretreated rats were significantly diminished after
all intravenous challenge conditions, i.e., saline (zero dose), 1.5 mg/kg FEN, and 3.0 mg/kg FEN. Analogous to the 1 week findings, Figure
2 shows that acute FEN increased corticosterone at 2 weeks (F(2,42) = 31.7; p < 0.00001),
and this effect was modified by pretreatment condition
(F(1,42) = 9.75; p < 0.01).
Post hoc tests revealed that corticosterone responses
were blunted in FEN-pretreated rats at 1.5 and 3.0 mg/kg doses of acute
FEN.
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Figure 1.
FEN-induced corticosterone release determined 1 week after cessation of the 4 d saline (1 ml/kg, s.c., b.i.d.) or
FEN (20 mg/kg, s.c., b.i.d.) dosing regimen. Rats from each
pretreatment group were challenged with intravenous saline or FEN (1.5 or 3.0 mg/kg). Serial blood samples were withdrawn immediately before
(time 0) and at 15, 30, and 60 min after intravenous injection.
Corticosterone values are mean ± SEM for n = 8 rats per group. *p < 0.05 with respect to
corresponding saline pretreatment group at the specified time
point.
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Figure 2.
FEN-induced corticosterone release assessed 2 weeks after the 4 d saline or FEN-dosing regimen. The data
depicted here are obtained from the same subjects described in Figure
1. Refer to the legend of Figure 1 for further details.
n = 7 or 8 rats per group. *p < 0.05 with respect to corresponding saline pretreatment group.
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Figures 3 and
4 illustrate the FEN-induced prolactin
responses 1 and 2 weeks after the 4 d saline or FEN treatment
regimen. Baseline (time 0) prolactin levels were not affected by the
repeated FEN-dosing regimen on either challenge day. As depicted in
Figure 3, acute FEN elevated plasma prolactin in a dose-related manner at 1 week (F(2,42) = 27.85; p < 0.00001). Although there was no main effect of pretreatment
(F(1,42) = 3.16; p < 0.08), a
significant pretreatment × acute treatment interaction was noted
(F(2,42) = 4.47; p < 0.02).
Post hoc evaluation demonstrated that FEN-pretreated rats had attenuated prolactin responses after the high (3.0 mg/kg) challenge dose of FEN. The data presented in Figure 4 show that acute
FEN elevated plasma prolactin at 2 weeks
(F(2,42) = 36.18; p < 0.0001).
There was a main effect of pretreatment (F(1,42) = 9.27; p < 0.01) and a significant acute × pretreatment interaction (F(2,42) = 11.61;
p < 0.0001). Post hoc tests revealed
that the FEN-induced prolactin response was again blunted only at the
3.0 mg/kg challenge dose of FEN.
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Figure 3.
FEN-induced prolactin secretion determined 1 week
after cessation of the 4 d saline (1 ml/kg, s.c., b.i.d.) or FEN
(20 mg/kg, s.c., b.i.d.) dosing regimen. Rats from each pretreatment
group were challenged with intravenous saline or FEN (1.5 or 3.0 mg/kg). Serial blood samples were withdrawn immediately before (time 0)
and at 15, 30, and 60 min after intravenous injection. Prolactin levels
are mean ± SEM expressed as nanogram per milliliter equivalents
of rPRL-RP-3 for n = 8 rats per group.
*p < 0.05 with respect to corresponding saline
pretreatment group at the specified time point.
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Figure 4.
FEN-induced prolactin secretion assessed 2 weeks
after the 4 d saline or FEN-dosing regimen. The data depicted here
are obtained from the same subjects described in Figure 3. Refer to the
legend of Figure 3 for further details. n = 7 or 8 rats per group. *p < 0.05 with respect to
corresponding saline pretreatment group.
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Repeated FEN-dosing study: behavioral findings
Table 4 shows the effects of the
4 d FEN-dosing regimen on body weight for all rats used in this
study. FEN exposure significantly reduced the body weight gain of rats
when assessed at 1 week (F(1,42) = 49.91;
p < 0.0001) and 2 weeks
(F(1,42) = 15.61; p < 0.0004) after the last repeated dose. Figures 5
and 6 depict the effect of acute FEN
challenge on 5-HT-mediated behaviors 1 and 2 weeks after the 4 d
saline or FEN pretreatment. As shown in Figure 5, acute FEN
increased flat body posture at 1 week (F(2,42) = 5.77; p < 0.006). There was a main effect of
pretreatment (F(1,42) = 18.47; p < 0.0001) and a significant pretreatment × acute treatment interaction (F(2,42) = 6.44; p < 0.004). Post hoc tests revealed the occurrence of
FEN-induced flat body posture was virtually absent in FEN-pretreated
rats. Acute FEN also increased the incidence of forepaw treading
(F(1,42) = 16.60; p < 0.0001),
and this behavioral effect was influenced by previous FEN exposure
(F(1,42) = 4.76; p < 0.04). In
this case, post hoc tests showed that forepaw treading was attenuated in FEN-pretreated rats only at the highest challenge dose of FEN (3.0 mg/kg). FEN challenge stimulated penile erections at 1 week (F(2,42) = 9.93; p < 0.0003), but this effect was not modified by the 4 d FEN-dosing
regimen (F(1,42) = 0.38; p < 0.54). The data illustrated in Figure 6 show that the behavioral
results from the 2 week FEN challenge tests were very similar to those from the 1 week challenge sessions.
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Figure 5.
FEN-induced behavioral responses determined 1 week
after cessation of the 4 d saline (1 ml/kg, s.c., b.i.d.) or FEN
(20 mg/kg, s.c., b.i.d.) dosing regimen. Rats from each pretreatment
group were challenged with intravenous saline or FEN (1.5 or 3.0 mg/kg). The occurrence of flat body posture (FBP),
forepaw treading (FPT), and penile erections
(PE) was assessed at 2, 10, 20, and 30 min after
injection. Numerical scores were recorded for each behavior at each
time point according to a graded rating scale (see Materials and
Methods). Rats received a final "behavioral score" for each
behavior, which consisted of the summed scores for that behavior over
all time points. Data are expressed as mean ± SEM for
n = 8 rats per group. *p < 0.05 with respect to corresponding saline pretreatment group.
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Figure 6.
FEN-induced behavioral responses evaluated 2 weeks
after the 4 d saline or FEN-dosing regimen. The data depicted here
are obtained from the same subjects described in Figure 5. Refer to the
legend of Figure 5 for further details. n = 8 rats
per group. *p < 0.05 with respect to corresponding
saline pretreatment group.
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Repeated FEN-dosing study: neurochemical findings
Figures 7 and
8 summarize the postmortem tissue levels
of 5-HT and 5-HIAA from the brains of acute saline-treated rats killed 2 weeks after the 4 d FEN regimen. Figure 7 shows that repeated FEN significantly reduced 5-HT in forebrain areas such as the caudate
putamen (45% decrease) (F(1,14) = 16.32;
p < 0.001), olfactory tubercle (50% decrease)
(F(1,14) = 15.19; p < 0.01),
hippocampus (55% decrease) (F(1,14) = 19.14;
p < 0.001), and amygdala (45% decrease)
(F(1,14) = 17.38; p < 0.001).
In hypothalamus, FEN-pretreated rats displayed significantly diminished
5-HT in the ventromedial region (46% decrease)
(F(1,14) = 18.2; p < 0.001) but
not in the lateral region (6% decrease)
(F(1,14) = 0.33; p < 0.58). In
general, the 5-HIAA data in Figure 8 parallel the findings noted for
5-HT.
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Figure 7.
Postmortem tissue levels of 5-HT in microdissected
brain regions from rats challenged with acute saline. Rats had
previously received repeated saline or FEN for 4 d and were
decapitated 2 weeks later after the final challenge test session.
Micropunches of tissue were assayed for 5-HT by HPLC-EC techniques (see
Materials and Methods). Regions examined were caudate putamen
(CP), nucleus accumbens (NAC), olfactory
tubercle (OT), hippocampus CA3
(HIP), lateral hypothalamus (LH),
ventromedial hypothalamus (VMH), and basolateral
amygdala (AMY). Data are mean ± SEM
expressed as nanograms per milligrams of protein for
n = 8 rats per group. *p < 0.05 compared with saline pretreatment group.
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Figure 8.
Postmortem tissue levels of 5-HIAA in
microdissected brain regions from rats challenged with acute saline.
Rats had previously received repeated saline or FEN for 4 d and
were decapitated 2 weeks later after the final challenge test session.
Micropunches of tissue were assayed for 5-HIAA by HPLC-EC techniques
(see Materials and Methods). Data are mean ± SEM expressed as
nanograms per milligram of protein for n = 8 rats
per group. *p < 0.05 compared with saline
pretreatment group.
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DISCUSSION |
In the current study, FEN-induced depletion of forebrain 5-HT was
accompanied by a reduction in neuroendocrine and behavioral responsiveness to subsequent FEN challenge. The simplest explanation for these results is that diminished sensitivity to FEN reflects presynaptic 5-HT dysfunction in rats previously exposed to the drug.
Our data are reminiscent of the findings of Kleven et al. (1988) who
demonstrated tolerance to the anorectic effect of FEN in rats
pretreated with 5-HT-depleting doses of FEN. It is noteworthy that no
overt hormonal or behavioral abnormalities were observed under basal
conditions in the FEN-pretreated rats from our study. Only after
perturbation of the 5-HT system via acute FEN injection were the
functional changes unmasked.
An accurate interpretation of our data relies on the assumption that
the FEN-induced responses we chose to examine involve central 5-HT
mechanisms. The neural substrates responsible for 5-HT-mediated
neuroendocrine and behavioral effects are presumably localized in the
hypothalamus and brainstem, respectively (Levy et al., 1994a; Wilkinson
and Dourish, 1991). Although the FEN-evoked prolactin response is known
to involve central 5-HT systems, the role of 5-HT in the accompanying
corticosterone response is controversial (McElroy et al., 1984; Levy et
al., 1994b; Van de Kar et al., 1985). We used the criterion of
fluoxetine reversibility to evaluate the 5-HT specificity of hormonal
effects of FEN; fluoxetine antagonizes in vivo actions of
FEN by blocking 5-HT reuptake sites and thereby preventing 5-HT-release
(Levy et al., 1994a). Our data showed that fluoxetine significantly
attenuates FEN-induced corticosterone and prolactin secretion. However,
fluoxetine did not totally abolish the hormonal effects of FEN, and
there are several possibilities that might explain this finding. For
example, the dose of fluoxetine that we used (i.e., 5 mg/kg, i.p.) may
not be sufficient to completely block all 5-HT transporter sites
(Fuller et al., 1977; Levy et al., 1994b). Alternatively, FEN may
possess direct postsynaptic receptor actions that are not antagonized
by fluoxetine pretreatment (Oluyomi et al., 1994; Raiteri et al.,
1995). Irrespective of the such arguments, our results indicate that
FEN-induced corticosterone and prolactin responses are at least
partially mediated by 5-HT mechanisms. Trulson and Jacobs (1976)
reported that the 5-HT syndrome elicited by FEN is prevented after
depletion of 5-HT with the synthesis inhibitor parachlorophenylalanine.
Thus, the available evidence supports the notion that FEN-induced
endpoints assessed in the present study represent valid indices of
central 5-HT function.
Prior exposure to repeated FEN administration markedly reduced
subsequent FEN-evoked corticosterone secretion, and this effect was
observed at both challenge doses for at least 2 weeks. Our data agree
with those of Appel et al. (1991) who found corticosterone responses to
FEN are blunted on the final day of a 4 d FEN-dosing regimen.
Similarly, Pinto et al. (1996) demonstrated that
D-FEN-stimulated ACTH secretion is impaired 2 weeks after
exposure to repeated doses of D-FEN >2.0 mg/kg. In their
study, deficits in ACTH secretion were positively correlated with the
degree of central 5-HT depletion. The collective data indicate that
repeated FEN administration causes functional impairments in 5-HT
neurons modulating the hypothalamic-pituitary-adrenal (HPA) axis. A
related finding from our work was that stress-induced corticosterone
secretion appeared compromised in rats with 5-HT depletion. Our
challenge test procedure represents a minor stressor that causes a
reliable, albeit modest, elevation of plasma corticosterone (Baumann et
al., 1995). The typical transient increase in corticosterone produced
by saline injection was completely absent in rats pretreated with FEN.
Whether the sensitivity to other more potent stressors is altered after
FEN pretreatment is unknown, but our findings suggest 5-HT plays a role
in the stress-induced activation of the HPA axis.
Prior exposure to FEN also decreased the FEN-induced secretion of
prolactin, but this effect was qualitatively different from the
corticosterone results. More precisely, prolactin responses to FEN were
blunted only at the high challenge dose in rats exhibiting 5-HT
depletion. Our results are at odds with those of Pinto et al. (1996)
who found repeated D-FEN pretreatment did not alter subsequent prolactin responses to D-FEN. It is feasible
that methodological differences between our work and theirs can account
for this discrepancy. In any event, the data from both studies imply
that 5-HT control of prolactin secretion remains relatively stable
despite reductions in brain 5-HT. On the other hand, FEN-induced 5-HT
depletion may disrupt physiologically relevant prolactin secretion when
large surges of the hormone are common, such as during lactation.
Rowland et al. (1978) reported that pretreatment with
parachloroamphetamine, which depletes brain 5-HT similar to FEN,
reduces the suckling-induced prolactin response in lactating rats.
Distinct components of the 5-HT behavioral syndrome, namely flat body
posture and forepaw treading, are mediated predominately by 5-HT
receptors (Tricklebank et al., 1985; Wilkinson and Dourish, 1991). We
found that FEN-induced flat body posture was nearly eliminated in
FEN-pretreated rats, whereas forepaw treading was less affected. These
results are comparable to the findings of Trulson et al. (1976) who
found a marked reduction in the ability of FEN to elicit the 5-HT
behavioral syndrome after depletion of 5-HT with the neurotoxin,
5,7-dihydroxytryptamine (5,7-DHT). Likewise, Kleven et al. (1988)
reported tolerance to the anorectic effect of FEN in FEN-pretreated
rats, and this effect lasted up to 8 weeks. In contrast, FEN-induced
penile erections were not altered by previous FEN exposure. One
possible explanation for this finding is that FEN evokes penile
erections by direct 5-HT receptor activation. As eluded to previously,
accumulating evidence suggests that some effects of FEN are mediated
independent of presynaptic 5-HT actions (Oluyomi et al., 1994; Raiteri
et al., 1995).
The present neurochemical data showed that FEN-pretreated rats display
a significant loss of 5-HT and 5-HIAA in the ventromedial hypothalamus
(VMH), whereas the lateral hypothalamus (LH) is unaffected. The degree
of 5-HT depletion in the VMH was similar to other vulnerable areas such
as the caudate putamen and amygdala. It is noteworthy that the
microdissected brain region we refer to as the VMH actually included
the medial and lateral subdivisions of the ventromedial hypothalamic
nucleus, as well as portions of the arcuate nucleus (Palkovits and
Brownstein, 1988); these nuclei are known to contain high densities of
5-HT nerve terminals (Steinbusch, 1981; Molliver, 1987). The
neighboring region we designate as the LH contained predominately axon
tracts of the median forebrain bundle. Based on our results from the
hypothalamus, it appears that 5-HT nerve terminals are more susceptible
to FEN-induced 5-HT depletion when compared with fibers of passage.
Some studies have found the hypothalamus to be resistant to the
5-HT-depleting effects of FEN (Kleven et al., 1988; Zaczek et al.,
1990). This apparent incongruity can be resolved on the basis of the
dissection method used to excise the hypothalamus: a dissection which
includes the LH would tend to mask the severity of 5-HT depletion
occurring in the VMH. The present findings show the utility of the
micropunch technique for sampling subdivisions of heterogenous brain
areas like the hypothalamus.
Similar to the findings of others (for review, see Ricaurte et al.,
1994), we found no overt changes in basal neuroendocrine or behavioral
parameters in FEN-pretreated rats. Stated more simply, it was
impossible to distinguish which rats exhibited 5-HT depletion without
testing them in a very specific manner. This observation is surprising
based on the alleged role of 5-HT in the integration of diverse motor
and vegetative functions (Jacobs and Fornal, 1995). One feasible
explanation for this apparent paradox is that neuroadaptive mechanisms
are recruited to keep synaptic 5-HT levels constant despite FEN-induced
depletion of brain 5-HT. Recent in vivo microdialysis
findings from rats support this proposal. Series et al. (1994) depleted
brain 5-HT using various amphetamine derivatives, including FEN, and
then monitored dialysate 5-HT in the cortex 2 weeks later. These
investigators found that 5-HT depletion did not affect basal dialysate
levels of 5-HT but severely impaired the ability of FEN to elicit 5-HT
release. Virtually identical results were reported by Kirby et al.
(1995) who lesioned brain 5-HT neurons with 5,7-DHT. Thus, it appears
that compensatory mechanisms maintain optimal levels of synaptic 5-HT
when tissue levels of 5-HT are depleted. Only on acute perturbation of
the 5-HT system by pharmacological challenge can the functional
deficits in 5-HT transmission be revealed.
The potential clinical relevance of our data deserves comment.
Recently, racemic FEN and D-FEN have been withdrawn from
clinical use because of the occurrence of cardiac valve abnormalities
in some patients (Connolly et al., 1997). Despite the fact that FEN is
no longer prescribed, lingering doubts about long-term neurotoxicity might still be a concern for people who have taken the drug. With respect to this issue, several points are worth mentioning. First, whether FEN-induced 5-HT depletion in animals actually reflects true
neurotoxicity is a debatable issue, and the mechanisms underlying such
5-HT loss are not known (Baumann and Rothman, 1998; McCann et al.,
1997). Second, because of profound species differences in vulnerability
to FEN-induced 5-HT depletion (McCann et al., 1994; Fracasso et al.,
1995), direct extrapolation of animal data to humans is unwarranted.
Finally, it should be noted that the dose of FEN used in the present
study to decrease brain 5-HT levels (i.e., 20 mg/kg, s.c.) is much
greater than the typical doses used to suppress feeding (i.e., 1-3
mg/kg, i.p.) in rats (Cox and Maickle, 1972; Kleven et al., 1988).
Thus, the present findings per se do not provide support
for, or against, the occurrence of 5-HT neurotoxicity in humans. Our
results do suggest that the FEN challenge paradigm is a plausible
method for identifying potential 5-HT dysfunction in patients who have
taken FEN as an appetite suppressant. Indeed, the FEN challenge test
has been widely used in psychiatry as a safe and reliable way to assess
5-HT neurotransmission (Siever et al., 1991).
In summary, the data presented herein confirm that repeated FEN
administration decreases forebrain 5-HT in rats, and the degree of 5-HT
depletion in the VMH was similar to other susceptible brain regions.
More importantly, FEN-induced 5-HT loss was associated with reduced
neuroendocrine and behavioral responsiveness to acute FEN challenge.
These effects were sustained, lasting for at least 2 weeks. In
conjunction with in vivo microdialysis studies, our results
indicate that blunted sensitivity to FEN reflects deficits in
presynaptic 5-HT function in the rats with central 5-HT depletion. As
discussed above, the clinical relevance of our data are not known.
However, the present findings clearly illustrate the usefulness of the
FEN challenge test for uncovering changes in 5-HT neurotransmission that might otherwise go undetected (Baumann et al., 1995). Thus, although FEN is no longer available as an appetite suppressant, this
drug should remain an important tool for examining the involvement of
5-HT neurons in diverse physiological and behavioral processes.
| |
FOOTNOTES |
Received April 20, 1998; revised July 10, 1998; accepted Aug. 13, 1998.
This work was generously supported by the Intramural Research Program
of the National Institute on Drug Abuse. We thank Turaya Bryant and
Brian Reddick for their excellent technical assistance.
Correspondence should be addressed to Dr. Michael H. Baumann, Clinical
Psychopharmacology Section, Intramural Research Program, National
Institute on Drug Abuse, National Institutes of Health, P.O. Box 5180, 5500 Nathan Shock Drive, Baltimore, MD 21224. E-mail: mbaumann{at}intra.nida.nih.gov
| |
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Abstract
Introduction
Compound via MeSH
