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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 283:581–591, 1997
Vol. 283, No. 2
Printed in U.S.A.
In Vivo Criteria To Differentiate Monoamine Reuptake Inhibitors
from Releasing Agents: Sibutramine Is a Reuptake Inhibitor1
C. GUNDLAH,2 K. F. MARTIN,3 D. J. HEAL3 and S. B. AUERBACH2
Department of Biological Sciences, Rutgers University, Piscataway, New Jersey
Accepted for publication July 8, 1997
Many compounds have high affinity, without being substrates, for the 5-HT reuptake carrier and thereby increase
extracellular levels of 5-HT in the CNS by blocking reuptake.
The increase in extracellular 5-HT after inhibition of reuptake is dependent on impulse-mediated release (Fuller and
Wong, 1990; Fuller, 1993). In contrast, other compounds can
evoke 5-HT release by a mechanism that is mainly independent of neuronal activity (Kuhn et al., 1985; Sharp et al.,
1986; Carboni and DiChiara, 1989; Raiteri et al., 1995).
These are termed “releasing agents.” Some releasing agents
exert their effects by entering the nerve terminal via the
reuptake carrier where they displace 5-HT from its storage
pool (Mennini et al., 1981; Rudnick and Wall, 1992; Berger et
al., 1992). Because releasing agents such as fenfluramine
and PCA act as substrates for the 5-HT transporter, they also
appear to block reuptake at low concentrations (Garattini et
al., 1986; Berger et al., 1992).
Received for publication February 24, 1997.
1
This work was supported by Grant MH51080A from National Institutes of
Health to S.B.A., a grant from Knoll Pharmaceuticals and a grant from the
John & Anne B. Leathem Scholarship to C.G.
2
Current address: Department of Biological Sciences, PO Box 1059, Rutgers University, Piscataway, NJ 08855.
3
Current address: Knoll Pharmaceuticals, Nottingham, U.K.
However, systemic injection of fenfluramine during its local
infusion does not attenuate this increase. 4) Reuptake inhibitor
pretreatment attenuates fenfluramine-induced increases in 5HT. According to these criteria, the in vivo effects of the novel
antiobesity drug sibutramine are consistent with its characterization as a 5-HT reuptake inhibitor and not a 5-HT releaser.
Thus, sibutramine produced increases in hypothalamic 5-HT
similar in magnitude to the effects of the known reuptake inhibitors, and the increase was attenuated by 8-OH-DPAT. Also,
sibutramine attenuated fenfluramine-induced 5-HT release.
Systemic administration of sibutramine failed to attenuate the
increase in 5-HT produced by its local infusion, suggesting that
this criterion is not applicable to compounds with low affinity for
the 5-HT transporter.
Monoamine reuptake inhibitors and releasing agents both
produce increases in extracellular neurotransmitter levels.
Thus, it is difficult to establish experimental criteria for
classification of these compounds in vivo (Fuller et al., 1988).
However, previous observations suggest four distinguishing
criteria: 1) The increase in extracellular 5-HT in response to
systemic administration of reuptake inhibitors (Perry and
Fuller, 1992; Rutter and Auerbach, 1993) is relatively small
compared with the effect of releasing agents (Kalén et al.,
1988; Sabol et al., 1992b). This is because the discharge of
5-HT neurons is largely inhibited by acute systemic administration of a reuptake inhibitor and the increase in extracellular 5-HT is dependent on depolarization-induced release
(Blier et al., 1987; Adell and Artigas, 1991; Rutter and Auerbach, 1993). These autoinhibitory mechanisms should not
modify the actions of 5-HT-releasing agents. 2) The effect of
reuptake inhibitors (Carboni and DiChiara, 1989; Perry and
Fuller, 1992; Rutter and Auerbach, 1993), but not releasing
agents (Carboni and DiChiara, 1989; Gobbi et al., 1993), is
attenuated by agents that inhibit 5-HT neuronal discharge,
e.g., 8-OH-DPAT or TTX. 3) The large increase in extracellular 5-HT occurring during local infusion of these drugs into
a terminal field is attenuated by their acute peripheral administration (Rutter and Auerbach, 1993; Hjorth and Auer-
ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; NE, norepinephrine; CNS, central
nervous system; TTX, tetrodotoxin; MDMA, 3,4-methylenedioxymethamphetamine; PCA, p-chloroamphetamine; DRN, dorsal raphe nucleus.
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ABSTRACT
Because monoamine reuptake inhibitors and releasing agents
both increase extracellular neurotransmitter levels, establishing
in vivo experimental criteria for their classification has been
difficult. Using microdialysis in the hypothalamus of unanesthetized rats, we provide evidence that serotonin- (5-HT) selective
and nonselective reuptake inhibitors can be distinguished from
the 5-HT-releasing agent fenfluramine by four criteria: 1) Systemic fenfluramine produces a much greater increase in 5-HT
than the reuptake inhibitors. 2) The 5-HT somatodendritic autoreceptor agonist, (6)-8-hydroxy-(dipropylamino)tetralin (8OH-DPAT), attenuates the increase in 5-HT produced by reuptake inhibitors, but not by fenfluramine. 3) The large increase
in 5-HT produced by infusion of reuptake inhibitors into the
hypothalamus is attenuated by their systemic administration.
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Methods
Animals and guide cannula implantation. Male SpragueDawley rats (Harlan Sprague Dawley, Indianapolis, IN) (250-400 g)
were housed singly on a reversed 12:12 hr light-dark cycle (lights off
at 9:30 A.M.) from the day of arrival, at least 10 days before experimentation, with free access to food and water. All experiments were
performed during the dark cycle. All animal use procedures were in
strict accordance with National Institutes of Health guidelines for
the care and use of animals and were approved by the Rutgers
University Institutional Review Board. Rats were anesthetized with
xylazine (Rompun; 4 mg/kg i.p., Mobay Corp., Shawnee, KS) and
ketamine (Ketaset; 100 mg/kg i.p., Fort Dodge Laboratories, Fort
Dodge, IA) and guide cannulae were implanted as described previously (Auerbach et al., 1989). After surgery, the guide cannulae were
plugged with stylets and the rats were allowed a minimum recovery
period of 5 days.
Dialysis probe implantation and perfusion. The dialysis
probe was an I-shaped concentric design. The working surface was a
3.5 mm length of permeable nitrocellulose dialysis tubing of 0.25 mm
outer diameter and 6000 MW cutoff (Spectrum Medical Industries,
Los Angeles, CA). Fluid entered through an outer tube of 26-gauge
stainless-steel and exited through an inner tube of hollow glass silica
fiber (Polymicro Technologies, Phoenix, AZ). The probe length was
adjusted to place the tip 0.2 mm above the base of the hypothalamus
[flat skull position: 6.2 mm anterior and 0.9 mm lateral relative to
intra-aural 0; and 9.2 mm below dura (Paxinos and Watson, 1982)].
The evening before an experiment, rats were briefly immobilized
with a volatile anesthetic, methoxyflurane, and a dialysis probe was
lowered slowly through the guide cannula and cemented in place.
Rats were then placed in a cylindrical enclosure 12 inches in diameter, and attached to a counterweighted cable and fluid swivel that
allowed the animals to move freely and have free access to food and
water.
The dialysis solution (in millimolar: NaCl, 147; KCl, 4; and CaCl2,
1.8, unadjusted pH 6.3) was perfused through the probe at a rate of
1.1 ml/min by a syringe pump. The perfusate was collected at 30-min
intervals and analyzed immediately for 5-HT content, as previously
described in detail (Auerbach et al., 1989). Extracellular 5-HT was
very high immediately after probe implantation, but fell to steady
levels within 8 h (Auerbach et al., 1989). These initially high levels
were most probably derived from plasma (Kalén et al., 1988). Therefore, all experiments were started no sooner than 8 hr after probe
implantation.
Analysis of 5-HT. High-performance liquid chromatography with
electrochemical detection was used for analysis of dialysis samples.
Separation of 5-HT from other electroactive compounds was achieved
on a 10 cm 3 3.2 mm column with ODS 3 mm packing (BAS Inc., W.
Lafayette, IN) and a mobile phase of 0.15 M chloroacetic acid, 0.12 M
sodium hydroxide, 0.18 mM EDTA, 60 ml/liter of acetonitrile and 1.0
mM sodium octane sulfonic acid. Monoamines were measured by
using a dual potentiostat electrochemical detector (EG&G PARC,
Princeton, NJ) and dual glassy carbon working electrodes in the
parallel configuration. Applied potentials, relative to an Ag/AgCl
reference electrode, were set at approximately maximal and halfmaximal for oxidation of 5-HT. These values were checked frequently and were usually about 590 and 530 mV. The detection limit
for 5-HT was approximately 300 fg, based on a signal-to-noise ratio
of 3:1.
Procedures and data analysis. The experimental protocol involved either systemic injection or local delivery of drugs by reverse
dialysis into the hypothalamus. Mean baseline 5-HT levels were
calculated as the average of four consecutive 30-min samples immediately before drug administration. In calculating and presenting the
results, levels (predrug baseline and postdrug samples) were expressed as a percentage of the mean baseline value 6 S.E. This
preserves variability associated with random changes in extracellular levels across time although normalizing between animal differences in absolute baseline levels. Within dose, data were analyzed by
repeated measures analysis of variance (general linear model),
whereas between dose data were analyzed by repeated measures
multivariate analysis of variance (general linear model). Factorial
analysis, using Scheffé’s F test was applied for post hoc determination of significant differences (P , .05).
Histology. After probe sites were used, the rats were anaesthetized deeply with pentobarbital and perfused intracardially with
physiological saline followed by 10% formalin. The brains were removed and 80-mm sections were mounted on slides and stained with
cresyl violet. Probe location was determined with the aid of a microscope.
Materials. All chemicals and solvents were of at least analytical
grade. Drugs were obtained from the following sources: d,l-fenfluramine and imipramine (Sigma Chemical Co., St. Louis, MO); fluoxetine and nisoxetine (Lilly Research Laboratories, Indianapolis, IN);
sibutramine and BTS 54 505 (Knoll Pharmaceuticals Research and
Development, Nottingham UK); paroxetine (SmithKline Beecham
Pharmaceuticals, Surrey, UK); 8-OH-DPAT (Research Biochemicals
Inc., Natick, MA). All drugs were weighed as the salt and administered in a volume of 2.0 ml/kg. For systemic administration, all drugs
were dissolved in water and heated and sonicated until fully dissolved. For systemic administration, fenfluramine, fluoxetine, nisoxetine, imipramine, sibutramine, BTS 54 505 and paroxetine were
injected i.p., and 8-OH-DPAT was injected s.c. For local administration into the hypothalamus, the drugs were dissolved in, and diluted
with, the dialysis solution.
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bach, 1994). This attenuation is due to the indirect activation
of somatodendritic autoreceptors after systemic administration of a reuptake inhibitor (Auerbach et al., 1995; Rutter et
al., 1995). It is unlikely that this would be a property of
releasing agents, assuming that they produce depolarizationindependent release of 5-HT. 4) Because both reuptake inhibitors and releasing agents such as fenfluramine bind to
the 5-HT carrier, pretreatment with reuptake inhibitors can
attenuate the increase in extracellular 5-HT produced by
fenfluramine (Sabol et al., 1992a; Gobbi et al., 1992).
The primary aim of our research was to determine the
validity of these four criteria for in vivo differentiation of
monoamine reuptake inhibitors from releasing drugs. Although there are in vitro tests available, because the pharmacological profile of compounds can be altered by metabolism, it is important to perform in vivo tests. In order to
achieve this aim, microdialysis in the hypothalamus of unanesthetized rats was used to compare fenfluramine, which
is a 5-HT releasing agent (Berger et al., 1992), with the
reuptake inhibitors fluoxetine, paroxetine and imipramine.
The second aim of this study was to then use these criteria to
determine whether sibutramine (BTS 54 524; N-1-(1-[4-chlorophenyl]cyclobutyl)-3-methyl-butyl-3-N, N-dimethylamine
hydrochloride monohydrate) and its primary amine metabolite, BTS 54 505, act as reuptake inhibitors or releasing
agents in vivo. Previous studies have established that sibutramine weakly inhibits NE and 5-HT reuptake in vitro
(Buckett et al., 1988). However, the primary and secondary
amine metabolites of sibutramine are potent NE and 5-HT
reuptake inhibitors in vitro (Luscombe et al., 1989). Thus, the
in vivo effects of sibutramine and BTS 54 505 were compared
with those of fenfluramine and known reuptake inhibitors.
Some of these results were previously presented in abstract
form (Gundlah et al., 1996).
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Uptake Inhibitors vs. Releasers
583
Results
Fig. 1. Effect of paroxetine (A) and imipramine (B) on extracellular 5-HT
in the hypothalamus. Values (mean 6 S.E.) are expressed as a percent
of the average of four baseline samples. The mean baseline levels for
the five paroxetine treatment groups were not significantly different and
the combined value was 1.1 6 0.07 pg/30-min sample (n 5 40).
Similarly, the mean baseline levels for the four imipramine groups were
not significantly different and the combined value was 2.0 6 0.11
pg/30-min sample (n 5 23). Drugs or vehicle were administered at time
0 (arrow). Paroxetine (0.1, 1, 10, 30 mg/kg i.p.) produced a significant
dose-dependent increase in 5-HT [F(4,35) 5 12.9, P , .0001) (n 5 4-11
for each dose]. Imipramine (5, 10, 20 mg/kg i. p.) also produced a
significant dose-dependent increase in 5-HT [F(3,19) 5 10.19, P ,
.0003] (n 5 5-6 for each dose). *Significantly different from vehicle
(Scheffé’s post hoc test, P. , .05).
Effect of reuptake inhibitors, fenfluramine, sibutramine, and BTS 54 505 on extracellular 5-HT. The 5-HT
selective reuptake inhibitor paroxetine produced an increase
in extracellular 5-HT in the hypothalamus after systemic
(i.p.) administration (fig. 1A). This effect was dose dependent,
with injections of 0.1, 1, 10 and 30 mg/kg producing maximum increases of ;60, 150, 200 and 200% above baseline,
respectively. At the highest doses, there was a stable elevation of 5-HT between 60 min and the end of sampling at 4 hr
after injecting paroxetine.
The NE and 5-HT reuptake inhibitor imipramine induced
an increase in hypothalamic extracellular 5-HT in the first 30
min after systemic (i.p.) injection (fig. 1B). This was a dosedependent effect, with injections of 5, 10 and 20 mg/kg producing maximum increases of ;20, 80 and 100% above baseline, respectively. At the two higher doses, 5-HT increased
rapidly, then gradually declined over the 4 hr time course.
Systemic administration of d,l-fenfluramine (1, 3, 10 and
20 mg/kg i.p.) produced a dose-dependent increase in extracellular 5-HT in the hypothalamus. The 1, 3, 10 and 20 mg/kg
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Fig. 2. Effect of fenfluramine on extracellular 5-HT in the hypothalamus. Values (mean 6 S.E.) are expressed as a percent of the average
of four baseline samples. The mean baseline levels for the three treatment groups were not significantly different and the combined value
was 1.12 6 0.08 pg/30-min sample (n 5 30). d,l-Fenfluramine (1, 3, 10,
20 mg/kg i. p.) or vehicle was administered at time 0 (arrow) and
extracellular 5-HT was measured for 2 hr. Fenfluramine produced a
dose-dependent increase in 5-HT [F(4,25) 5 73.65, P , .0001] (n 5 5-7
for each dose). *Significantly different from vehicle (Scheffé’s post hoc
test, P , .05). N.B. Figure 2 is plotted on a different scale from figures
1A and 1B.
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Fig. 3. Effect of sibutramine (A) and BTS 54 505 (B) on hypothalamic
5-HT. Values (mean 6 S.E.) are expressed as a percent of the average
of four baseline samples. The mean baseline levels for the four sibutramine treatment groups were not significantly different and the combined value was 1.09 6 0.12 pg/30-min sample (n 5 26). The mean
baseline levels for the three BTS 54 505 treatment groups were not
significantly different and the combined value was 1.0 6 0.04 pg/30 min
sample (n 5 19). Drug was administered at time 0 (arrow) and extra
cellular 5-HT was measured every 30 min for 4 hr after sibutramine, and
every 2 hr for 6 hr after BTS 54 505. Sibutramine (1, 3, 10, 30 mg/kg i.p.)
produced a significant dose-dependent increase in 5-HT [F(3,22) 5
12.34, P , .0001] (n 5 5-7 for each dose). BTS 54 505 (1, 3, 10 mg/kg
i.p.) also produced a significant dose-dependent increase in extracellular 5-HT [F(2,16) 5 40.32, P , .0001] (n 5 6-7 for each dose).
*Significantly different from the low dose (Scheffé’s post hoc test, P ,
.05).
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doses increased 5-HT to a maximum of ;180, 580, 1000 and
2600% above baseline, respectively (fig. 2). Peak levels were
observed within 60 min after injection.
Sibutramine produced a dose-dependent increase in hypothalamic 5-HT (fig. 3A) similar in magnitude to the effect of
the reuptake inhibitors (fig. 1) but not fenfluramine (fig. 2).
Systemic injection of 1, 3, 10 and 30 mg/kg i.p. increased
5-HT to a maximum of ;40, 60, 100 and 200% above baseline, within 1 hr after injection. The sibutramine metabolite,
BTS 54 505, produced a similar dose-dependent increase in
hypothalamic 5-HT (fig. 3B). Systemic administration of 1, 3
and 10 mg/kg i.p. increased extracellular 5-HT to a maximum
of ;20, 220 and 280% above baseline, within 2 hr after
administration.
Effect of 8-OH-DPAT on the increase in extracellular
5-HT produced by reuptake inhibitors, fenfluramine
or sibutramine. The 5-HT1A receptor agonist, 8-OH-DPAT,
was used to determine if increases in 5-HT were dependent
on 5-HT neuronal discharge. At a low dose, 8-OH-DPAT (0.1
mg/kg s.c.) maximally stimulates somatodendritic autoreceptors (Sharp et al., 1989) without having significant effects on
postsynaptic 5-HT1A receptors (Goodwin et al., 1987). In the
absence of reuptake inhibitors, this results in decreases in
extracellular 5-HT to ;30 to 40% of baseline levels. Because
8-OH-DPAT is rapidly metabolized, its effect on 5-HT is
transient, lasting about 1 hr (Sharp et al., 1989; Auerbach et
al., 1989).
Administration of 8-OH-DPAT (0.1 mg/kg s.c.) 2.5 hr after
injection of the reuptake inhibitors paroxetine (10 mg/kg i.p.)
or 2 hr after fluoxetine (10 mg/kg i.p.) produced a significant
but transient attenuation of the increase in hypothalamic
5-HT (fig. 4A, 4B). Administration of 8-OH-DPAT (0.1 mg/kg
s.c.) 2.5 hr after sibutramine (10 mg/kg i.p.) also produced a
significant, transient attenuation (fig. 4C).
The peak increase in extracellular 5-HT after systemic
fenfluramine was short-lasting. Therefore, 8-OH-DPAT (0.1
mg/kg s.c.) was administered either simultaneously (fig. 5A),
or 30 min after d,l-fenfluramine (10 mg/kg i.p.) (fig. 5B) to
determine if the increase in 5-HT was dependent on neuronal
discharge. In both cases, 8-OH-DPAT had no significant effect on the magnitude or time course of fenfluramine-induced
changes in 5-HT.
Effect of peripheral injection of reuptake inhibitors,
fenfluramine, sibutramine and BTS 54 505 on extracellular 5-HT during their local infusion. The increase in
extracellular 5-HT during local infusion of a 5-HT reuptake
inhibitor into a forebrain site is attenuated by subsequent
systemic administration of fluoxetine, citalopram or sertraline (Rutter and Auerbach, 1993; Auerbach et al., 1995).
Antagonists of 5-HT1A receptors block the decrease induced
by systemic administration of these high affinity, selective
5-HT reuptake inhibitors. This suggests that the decrease is
mediated by somatodendritic autoreceptors and consequent
inhibition of 5-HT release (Rutter et al., 1995; Auerbach et
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Uptake Inhibitors vs. Releasers
585
Fig. 4. Effect of 8-OH-DPAT on paroxetine-, fluoxetine- or sibutramine-induced increases in hypothalamic 5-HT. Values (mean 6 S.E.)
are expressed as a percent of the average of four baseline samples.
The mean baseline levels for the paroxetine (A), fluoxetine (B), sibutramine (C) and vehicle treatment groups were not significantly different
and the combined value was 1.7 6 0.1 pg/30-min sample (n 5 20).
Paroxetine (10 mg/kg i.p., n 5 5), fluoxetine (10 mg/kg i.p., n 5 5),
sibutramine (10 mg/kg i.p., n 5 5) or vehicle (data replotted in A, B and
C) was administered at time 0 (first arrow). 1Significant increases
compared with vehicle after paroxetine [F(1,8) 5 12.79 P , .007],
fluoxetine [F(1,8) 5 11.65, P , .009] and sibutramine[F(1,8) 5 17.75,
P , .003]. The increase in 5-HT observed after 10 mg/kg i.p. paroxetine
in this experiment was somewhat, but not significantly smaller than in
figure 1 ]F(1,14) 5 1.36, P , .264]. 8-OH-DPAT (0.1 mg/kg s.c.) administered at the time indicated by the second arrow reversed the druginduced increases in hypothalamic 5-HT: Paroxetine pretreatment
[F(7,28) 5 6.31, P , .0002]; fluoxetine [F(7,28) 5 6.64, P , .0001];
sibutramine [F(7,28) 5 24.68, P , .0001]. *Significantly decreased from
extracellular 5-HT during the 2-hr period just before 8-OH-DPAT treatment (Scheffé’s post hoc test, P , .05).
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al., 1995). However, the nonselective reuptake inhibitors imipramine, clomipramine and amitriptyline had low efficacy in
this “local-peripheral” experimental paradigm (Auerbach et
al., 1995). The correlation between low selectivity and low
efficacy suggested the possibility that an enhancement of
extracellular NE uptake inhibition may offset autoreceptormediated inhibition of 5-HT release. The following experiments were designed to test this hypothesis using drugs that
alone, or in combination block the reuptake of 5-HT and NE
to compare the efficacy of known reuptake inhibitors to fenfluramine and sibutramine in the local-peripheral experimental paradigm. Doses of these drugs were chosen according to their ability to produce similar maximal increases in
5-HT.
As shown in figures 6 and 7, reverse dialysis infusion of
either paroxetine (10 mM), imipramine (80 mM), fluoxetine
plus the NE reuptake inhibitor nisoxetine (each 10 mM) or
d,l-fenfluramine (100 mM) into the hypothalamus produced a
maximum increase in 5-HT to ;400 to 600% above baseline.
During the period of local infusion of paroxetine or nisoxetine
plus fluoxetine, systemic injection of the corresponding
drug(s) (each 10 mg/kg i.p.) resulted in a significant decrease
in 5-HT to ;150% above baseline (fig. 6A, C). Systemic injection of imipramine (10 mg/kg i.p.; fig. 6B) during its local
infusion had no effect on extracellular 5-HT. Systemic d,lfenfluramine (10 mg/kg i.p.; fig. 7) during its local infusion
slightly increased 5-HT levels.
Local infusion of sibutramine (2 mM) or its metabolite BTS
54 505 (10 m) increased extracellular 5-HT to a maximum of
;400% above baseline. Although there was a tendency toward a decrease, systemic sibutramine (10 mg/kg i.p.) had no
significant effect on hypothalamic 5-HT concentrations during its local infusion (fig. 8A). In contrast, systemic injection
of BTS 54 505 (10 mg/kg i.p.) during its local infusion significantly decreased extracellular 5-HT in the hypothalamus to
;120% above baseline (fig. 8B).
Effect of paroxetine, fluoxetine or sibutramine on
fenfluramine-induced 5-HT release. Animals were
treated with fluoxetine (10 mg/kg i.p.), paroxetine (10 mg/kg
i.p.), sibutramine (10 mg/kg i.p.) or saline 2 hr before d,lfenfluramine challenge (10 mg/kg i.p.). As shown in figure 9,
fenfluramine alone produced a 1400% increase above baseline 5-HT. Pretreatment with fluoxetine or paroxetine significantly attenuated the effect of fenfluramine on 5-HT. The
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Vol. 283
largest attenuation was observed with paroxetine, which almost completely blocked the increase in 5-HT, whereas fluoxetine produced an attenuation to ;100% above baseline.
Pretreatment with sibutramine significantly attenuated the
fenfluramine-induced increase to 320% above baseline 5-HT
(fig. 9).
Discussion
DPAT [F(11,33) 5 12.60, P , .0001]. *Significantly increased from
baseline (Scheffé’s post hoc test, P , .05). Treatment with 8-OH-DPAT
had no significant influence on the fenfluramine-induced increase in
5-HT: In comparison to fenfluramine alone: Fenfluramine with 8-OHDPAT [F(1,12) 5 3.43, P , .09]; fenfluramine before 8-OH-DPAT [F(1,9)
5 2.30, P , .17].
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Fig. 5. Effect of 8-OH-DPAT on fenfluramine-induced increases in hypothalamic 5-HT. Values (mean 6 S.E.) are expressed as a percent of
the average of four baseline samples. The mean baseline levels for the
three treatment groups were not significantly different, and the combined value was 1.9 6 0.2 pg (n 5 18). d,l-Fenfluramine (10 mg/kg i.p.)
was administered either alone (n 5 7, same data replotted in A and B)
or administered simultaneously with 8-OH-DPAT (0.1 mg/kg s.c., n 5 7,
A) or 30 min before 8-OH-DPAT (0.1 mg/kg s.c., n 5 4, B). Extracellular
5-HT was elevated from baseline: Fenfluramine alone [F(7,42) 5 56.98,
P , .0001]; fenfluramine administered with 8-OH-DPAT [F(7,42) 5
84.94, P , .0001]; fenfluramine administered 30 min before 8-OH-
Unequivocal classification of 5-HT reuptake inhibitors and
releasing agents is difficult because both drug types produce
increases in extracellular 5-HT. Four criteria for in vivo
differentiation of the effects of 5-HT reuptake inhibitors and
releasing agents were evaluated using the extensively characterized 5-HT reuptake inhibitors paroxetine and fluoxetine, the NE and 5-HT reuptake inhibitor imipramine, and
the 5-HT releasing agent fenfluramine. Using these criteria,
experiments were then performed to determine whether sibutramine and its primary amine metabolite, BTS 54 505, act
similarly to 5-HT reuptake inhibitors or releasing agents in
vivo.
Magnitude of increase in extracellular 5-HT. The first
criterion was the difference in the magnitude of the maximal
increase in extracellular 5-HT after acute systemic administration of reuptake inhibitors compared with releasing
agents. Thus, the 2- to 4-fold maximal increase in hypothalamic 5-HT in response to acute systemic administration of
paroxetine or imipramine is in agreement with the effect of
other reuptake inhibitors such as fluoxetine and citalopram
(Rutter and Auerbach, 1993; Perry and Fuller, 1992; Hjorth
and Sharp, 1993). Presumably, the small increase in 5-HT
represents a balance between blocking reuptake and the
strong but incomplete inhibition of release that is a consequence of autoreceptor stimulation (Rutter et al., 1995). The
sustained effect of paroxetine and BTS 54 505 is probably due
to the fact that they are not rapidly metabolized to inactive
compounds (Preskorn, 1994; K. F. Martin and D. J. Heal,
unpublished data). Similarly, the prolonged effect of sibutramine is due to the sustained presence of its primary amine
metabolite BTS 54 505 (K. F. Martin and D. J. Heal, unpublished data). The decrease from peak levels of extracellular
5-HT relatively soon after imipramine administration could
be a reflection of its rapid metabolism to the selective NE
reuptake inhibitor desmethyl imipramine (Sato et al., 1994).
In contrast, moderate systemic doses of 5-HT releasing
agents, e.g., fenfluramine, MDMA and PCA, produce much
larger increases in extracellular 5-HT concentrations. In our
study, systemic injection of fenfluramine increased hypothalamic 5-HT to a transient peak, ;20-fold above baseline
levels. This is consistent with the previous observation that
fenfluramine produced an ;30-fold increase in hippocampal
5-HT (Sabol et al., 1992a). Similarly, after systemic administration of MDMA or PCA, extracellular 5-HT was increased
more than 10-fold in rat striatum (Kalén et al., 1988; Gudelsky and Nash, 1996). The relatively large increase in extracellular 5-HT is probably because the effect of fenfluramine
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Uptake Inhibitors vs. Releasers
587
at high doses is mainly independent of 5-HT neuronal activity and calcium influx (Fuller et al., 1988; Berger et al., 1992;
Levi and Raiteri, 1993). As compared to the reuptake inhibitors, there was a rapid decline from peak 5-HT levels after
fenfluramine administration. This may be due to partial depletion of the releasable pool combined with inhibition of
synthesis which would reduce the replenishment of 5-HT
(K. F. Martin, S. Aspley and D. J. Heal, unpublished data).
Fig. 6. Effect of systemic administration of paroxetine, imipramine or
nisoxetine combined with fluoxetine on the increase in extracellular
5-HT produced by local infusion of these drugs into the hypothalamus.
Values (mean 6 S.E.) are expressed as a percent of the average of four
baseline samples. The mean baseline levels for the treatment groups
were not significantly different, and the combined value was 2.9 6 0.1
pg/30-min sample (n 5 29). Beginning at time 0, paroxetine (A), imipramine (B) or nisoxetine with fluoxetine (C) was infused by reverse
dialysis into the hypothalamus (horizontal bars). Infusion of paroxetine
(10 mM), imipramine (80 mM) or nisoxetine plus fluoxetine (each 10 mM)
caused a significant increase in extracellular 5-HT from baseline levels:
paroxetine [F(7,84) 5 73.71, P , .0001]; imipramine [F(7,70) 5 32.43,
P , .0001]; nisoxetine plus fluoxetine [F(7,28) 5 26.72, P , .0001]. At
the time indicated by the arrow, either the corresponding drug (10
mg/kg i.p.) or the vehicle was administered systemically. This resulted
in a significant decrease in extracellular 5-HT after systemic injection of
paroxetine [F(6,30) 5 33.71, P , .0001] and nisoxetine plus fluoxetine
[F(5,20) 5 2.33,P , .0001] but not imipramine [F(5,25) 5 1.24, P , .319]
(n 5 5-7 for each group). *Significant decrease from the level before
peripheral paroxetine or nisoxetine and fluoxetine administration
(Scheffé’s post hoc test, P , .05).
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Fig. 7. Effect of systemic administration of fenfluramine on the increase in extracellular 5-HT produced by its local infusion into the
hypothalamus. Values (mean 6 S.E.) are expressed as a percent of the
average of four baseline samples. The mean baseline levels for the
treatment groups were not significantly different, and the combined
value was 2.6 6 0.2 pg/30 min sample (n 5 10). Beginning at time 0,
d,l-fenfluramine (100 mM) was infused by reverse dialysis into the
hypothalamus (horizontal bars). This treatment caused a significant
increase in extracellular 5-HT from baseline levels [F(7,63) 5 25.65, P ,
.0001]. At the time indicated by the arrow, d,l-fenfluramine (10 mg/kg
i.p.) or the vehicle was administered systemically. This did not result in
a decrease in extracellular 5-HT after systemic fenfluramine [F(1,8) 5
0.58, P , .5] (n 5 5 for each group).
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Vol. 283
Fig. 8. Effect of systemic administration of sibutramine (A) or BTS 54
505 (B) on the increase in extracellular 5-HT produced by local infusion
of these drugs into the hypothalamus. Values (mean 6 S.E.) are expressed as a percent of the average of four baseline samples. The
mean baseline levels for the treatment groups were not significantly
different, and the combined value was 1.7 6 0.1 pg/30-min sample (n 5
25). Beginning at time 0, drug was infused by reverse dialysis into the
hypothalamus (horizontal bars). Infusion of sibutramine (2 mM) or BTS
54 505 (10 mM) caused a significant increase in extracellular 5-HT from
baseline levels: sibutramine F(8,96) 5 17.69 P , .0001; BTS 54 505
F(7,77) 5 35.79, P , .0001. At the time indicated by the arrow, either
the corresponding drug (10 mg/kg i.p.) or the vehicle was administered
systemically. This resulted in a significant decrease in extracellular
5-HT after systemic injection of BTS 54 505 [F(7,35) 5 3.89 P , .003)
(n 5 6 for each group)] but not sibutramine [F(6,36) 5 1.54, P , .195]
(n 5 6-7 for each group). *Significant decrease from the level before
peripheral BTS 54 505 administration (Scheffé’s post hoc test, P , .05).
In our experiments, doses of the reuptake inhibitors and
fenfluramine were in the range of the ED50 for effects on
feeding. For example, ;1, 3 and 10 mg/kg are the ;ED50
values for the hypophagic effect of d,l-fenfluramine (Francis
et al., 1995), sibutramine (Fantino and Souquet, 1995; Jackson et al., 1997) and paroxetine (Caccia et al., 1993), respectively. Thus, at three times the ED50 for inhibiting feeding,
the effect of the reuptake inhibitors was maximal at ;3-fold
above baseline 5-HT. In contrast, fenfluramine at three times
its ED50 produced a 6-fold increase in extracellular 5-HT
with increasingly larger effects at higher doses. Together
with previously published results, our data confirm that the
magnitude of the response to peripheral administration can
be used as a distinguishing criterion between reuptake inhibitors and releasing agents.
Dependence on 5-HT neuronal activity. The somatodendritic autoreceptor agonist, 8-OH-DPAT, has previously
been shown to reverse the increase in extracellular 5-HT
produced by systemic administration of fluoxetine to unanesthetized rats (Perry and Fuller, 1992; Rutter and Auerbach,
1993). This suggests that 5-HT neuronal activity is not com-
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Fig. 9. Effect of pretreatment with reuptake inhibitors or sibutramine
on fenfluramine-induced increases in hypothalamic 5-HT. Average
baseline levels of 5-HT (pg) for the 2-hr pretreatment period were: 1.4 6
0.13 for paroxetine (n 5 5), 0.60 6 0.05 for fluoxetine (n 5 4), 2.4 6 0.24
for sibutramine (n 5 6) and 1.3 6 0.12 for the vehicle (n 5 7). After
stable baseline levels were obtained, paroxetine, fluoxetine, sibutramine, d,l-fenfluramine (10 mg/kg i.p.) or the saline vehicle was administered. Values (mean 6 S.E.) are expressed as a percent of the level at
time 0, the fourth sample (2 hr) after systemic administration of pretreatment compound. Mean absolute values 2 hr after systemic injection were 5.7 6 1.3 pg for paroxetine, 3.1 6 0.6 pg for fluoxetine, 4.2 6
0.4 pg for sibutramine and 1.0 6 0.2 pg for the vehicle. *Significantly
different from pretreatment with paroxetine, fluoxetine or sibutramine
(Scheffé’s post hoc test, P , .05).
1997
589
Efficacy in the local-peripheral experimental paradigm
was significantly correlated with selectivity for blocking
5-HT reuptake (Auerbach et al., 1995). This suggested that
enhancement of extracellular NE might attenuate autoreceptor-mediated inhibition of 5-HT release. However, results
indicate that efficacy in this paradigm is also correlated with
affinity (fig. 10) and suggest that blocking NE reuptake is not
the factor responsible for the weak effect of some NE and
5-HT reuptake blockers. Paroxetine and fluoxetine, drugs
with high affinity and selectivity for the 5-HT transporter
had high efficacy. Combining the NE reuptake inhibitor
nisoxetine with fluoxetine did not alter the ability of peripheral fluoxetine to modulate the effect of local infusion. Duloxetine and BTS 54 505 have similar profiles to the combination of nisoxetine and fluoxetine, being high affinity, NE
and 5-HT reuptake inhibitors, and were similarly efficacious.
Thus, the drugs that displayed good efficacy all have high
affinity for the 5-HT reuptake site, but several show little
selectivity between 5-HT and NE (fig. 10). The drugs with the
lowest affinity for the 5-HT reuptake site, imipramine and
sibutramine, had the lowest efficacy. In summary, our results suggest that low affinity for the 5-HT reuptake carrier,
not ability to block NE reuptake, is the factor more likely
responsible for low efficacy in these “local-peripheral” experiments. However, because most nonselective monoamine reuptake inhibitors also have relatively low affinity for the
5-HT reuptake site, both factors appear to be correlated with
efficacy, and further experiments will be necessary to determine the explanation for these variable results.
Although extracellular levels were increased 7-fold during
local infusion of fenfluramine, 5-HT release was not decreased but was slightly increased, by subsequent systemic
administration of fenfluramine. This further supports the
conclusion that the enhancement in extracellular 5-HT after
administration of fenfluramine is not dependent on 5-HT
neuronal discharge. In summary this criterion can differentiate high affinity reuptake inhibitors from 5-HT releasing
agents.
Ability to attenuate fenfluramine-induced 5-HT release. The releasing effect of fenfluramine involves its binding to the 5-HT reuptake carrier. Thus, because reuptake
inhibitors block the binding of fenfluramine to the transporter (Garattini et al., 1989), they attenuate fenfluramineinduced 5-HT release in vitro and in vivo (Raiteri et al., 1995;
Sabol et al., 1992a; Gobbi et al., 1992; Kreiss et al., 1993).
Furthermore, fluoxetine markedly attenuates the 5-HT releasing effects of MDMA (Bel and Artigas, 1992). Consistent
with these data, pretreatment with fluoxetine or paroxetine
greatly reduced the large increase in extracellular 5-HT produced by fenfluramine. This confirms the validity of this
criterion, i.e., reuptake inhibitors attenuate the releasing
effect of fenfluramine.
Similar to the reuptake inhibitors paroxetine and fluoxetine, sibutramine also attenuated the large increase in 5-HT
produced by fenfluramine. Sibutramine is a drug with low
affinity for the 5-HT reuptake site, and produced a smaller
attenuation than the higher affinity drug, fluoxetine. Paroxetine has even higher affinity than fluoxetine, and produced
the greatest attenuation. Thus, affinity for the reuptake
transporter was correlated with the efficacy of these drugs in
preventing fenfluramine-induced 5-HT release.
D-Fenfluramine may act as a pure reuptake inhibitor at
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pletely suppressed as might be inferred from the strong inhibitory effect of reuptake inhibitors on single unit activity in
the DRN of anesthetized rats (Sheard et al., 1972; Aghajanian, 1972). Our results are consistent with the conclusion
that the increase in extracellular 5-HT produced by reuptake
inhibitors is dependent on residual activity of 5-HT neurons
in freely behaving animals. Thus, we have shown that 8-OHDPAT reversed the increase produced by systemic paroxetine
and fluoxetine. This effect was also demonstrated with the
putative reuptake inhibitor, sibutramine. In contrast, the
relatively large effect of fenfluramine, MDMA and PCA on
extracellular 5-HT may be due to a mechanism of release
that is independent of 5-HT neuronal activity (Fuller et al.,
1988; Gudelsky and Nash, 1996). Thus, in vitro and in vivo
studies indicate that fenfluramine, MDMA- and PCA-induced release of 5-HT is not attenuated by TTX or removal of
calcium from the perfusion medium (Kuhn et al., 1985; Sharp
et al., 1986; Carboni and DiChiara, 1989; Bonnano et al.,
1994). Our results are consistent with this conclusion, as the
fenfluramine-induced increase in hypothalamic 5-HT was
not attenuated by 8-OH-DPAT. These data, in combination
with published results, therefore provide evidence to support
the validity of the second criterion to differentiate 5-HT reuptake inhibitors from releasing agents.
Systemic drug administration during local infusion.
In comparison to the effects of reuptake inhibitors after systemic administration, their perfusion by reverse dialysis into
forebrain sites produces larger effects on extracellular 5-HT.
For example, fluoxetine infusion into the diencephalon resulted in 6-fold increases in extracellular 5-HT (Rutter and
Auerbach, 1993). Local administration into a forebrain terminal site can completely block reuptake while avoiding activation of somatodendritic autoreceptors. Subsequent peripheral injection of these reuptake blockers then produces a
net decrease in forebrain extracellular 5-HT as a consequence of inhibition of 5-HT neuronal discharge and release
(Rutter et al., 1995).
In our experiments, systemic administration of reuptake
blockers during their local infusion had inconsistent effects.
Several 5-HT reuptake inhibitors produced the expected response. Thus, during local paroxetine or fluoxetine infusion,
extracellular 5-HT was increased to ;600% above baseline
levels, and systemic administration of the corresponding
high affinity, 5-HT selective drug resulted in a decrease in
extracellular 5-HT to ;150-300% above baseline levels.
Our results are also in agreement with previous observations that some NE and 5-HT monoamine reuptake inhibitors including imipramine and amitriptyline have low efficacy in local-peripheral tests (Auerbach et al., 1995). In
contrast, duloxetine, a NE and 5-HT monoamine reuptake
inhibitor with high affinity for the 5-HT transporter produced a large attenuation in 5-HT when tested in the localperipheral experimental paradigm (S. B. Auerbach and
S. Hjorth, unpublished observations). In addition, our results
indicate that nisoxetine, a high affinity NE selective reuptake inhibitor (Tejani-Butt et al., 1990), in combination
with fluoxetine, produced a decrease in 5-HT similar to that
seen with fluoxetine alone. Systemic administration of sibutramine during its local infusion did not produce a decrease
in hypothalamic 5-HT. In contrast, systemic BTS 54 505, as
with duloxetine, and the combination of fluoxetine with
nisoxetine, did attenuate its locally induced increase in 5-HT.
Uptake Inhibitors vs. Releasers
590
Gundlah et al.
Vol. 283
membrane (Rudnick and Wall, 1992). However, the attenuation produced by pretreatment with reuptake inhibitors
indicates that this is unlikely to be the predominant mechanism for fenfluramine-induced 5-HT release.
In conclusion, we have identified and tested four criteria,
which, when applied together, differentiated 5-HT reuptake
inhibitors from 5-HT releasing agents in vivo. Using these
criteria, we have also determined that the novel antiobesity
drug sibutramine acts as a 5-HT reuptake inhibitor in vivo,
and not as a 5-HT releasing agent. This is consistent with in
vitro data indicating that sibutramine is a 5-HT and NE
reuptake inhibitor (Buckett et al., 1988; Cheetham et al.,
1993, Cheetham et al., 1996).
Acknowledgments
References
Fig. 10. Correlation of efficacy of uptake blockers for producing decreases in extracellular 5-HT with affinity (A) for the 5-HT transporter
and relative selectivity (B) for blocking 5-HT in comparison to NE
uptake. For each compound tested, the maximal decreases in extracellular 5-HT levels after systemic administration (efficacy) are plotted
as a function of the (A) Ki values (nM) for blocking 5-HT uptake and (B)
ratio of Ki values for blocking 5-HT and NE uptake (Ki NE/Ki 5-HT). The
values for affinity and relative selectivity were obtained from the literature (Cheetham et al., 1993, 1996; Hyttel, 1994; Wong et al., 1993;
Tejani-Butt et al., 1990). Some values are replotted from Auerbach et al.
(1995). There is a similar correlation between efficacy in producing
decreased extracellular 5-HT and affinity for the 5-HT transporter (r2 5
0.772), and selectivity for blocking 5-HT uptake relative to NE (r2 5
0.726).
doses between 0.2 to 0.5 mg/kg i.p. and a releaser at doses
higher than 1 mg/kg i.p. in the rat (Fattaccini et al., 1991).
The releasing action of some amphetamine derivatives at
higher doses may be enhanced by diffusion across the plasma
ADELL A. AND ARTIGAS F.: Differential effects of clomipramine given locally or
systemically on extracellular 5-hydroxytryptamine in raphe nuclei and frontal cortex. Naunyn-Schmiedeberg’s Arch. Pharm. 343: 237–244, 1991.
AGHAJANIAN, G. K.: Chemical feedback regulation of serotonin-containing neurons in brain. Annals N.Y. Acad. Sci. 193: 86–94, 1972.
AUERBACH, S. B., LUNDBERG, J. F. AND HJORTH S.: Differential inhibition of
serotonin release by 5-HT and NA reuptake blockers after systemic administration. Neuropharmacology 34: 89–96, 1995.
AUERBACH, S. B., MINZENBERG, M. J. AND WILKINSON, L. O.: Extracellular serotonin and 5-hydroxyindoleacetic acid in hypothalamus of the unanesthetized
rat measured by in vivo dialysis coupled to high-performance liquid chromatography with electrochemical detection: dialysate serotonin reflects neuronal release. Brain Res. 499: 281–290, 1989.
BEL, N. AND ARTIGAS, F.: Fluvoxamine preferentially increases extracellular
5-hydroxytryptamine in the raphe nuclei: an in vivo microdialysis study.
Eur. J. Pharmacol. 229: 101–103, 1992. [published erratum appears in Eur.
J. Pharmacol. 232: 326, 1993].
BERGER, U. V., GU, X. F. AND AZMITIA, E. C.: The substituted amphetamines
3,4-methylenedioxymethamphetamine, methamphetamine, p-chloroamphetamine and fenfluramine induce 5-hydroxytryptamine release via a
common mechanism blocked by fluoxetine and cocaine. Eur. J. Pharmacol.
215: 153–160, 1992.
BLIER P., DE MONTIGNY C. AND CHAPUT Y.: Modifications of the serotonin system
by antidepressant treatments: Implications for the therapeutic response in
major depression. J. Clin. Psychopharm. 17: 24s–35s, 1987.
BONNANO, G., FASSIO, A., SEVERI, P., RUELLE, A. AND RAITERI, M.: Fenfluramine
releases serotonin from human brain nerve endings by a dual mechanism.
J. Neurochem. 63: 1163–1166, 1994.
BUCKETT, W. R., THOMAS, P. C. AND LUSCOMBE, G. P.: The pharmacology of
sibutramine hydrochloride (BTS 54 524), a new antidepressant which induces rapid noradrenergic down-regulation. Prog. Neuro-psychopharmacol.
Biol. Psychiatry 12: 575–584, 1988.
CACCIA, S., ANELLI, M., CODEGONI, A. M., FRACASSO, C. AND GARRATTINI, S.: The
effects of single and repeated anorectic doses of 5-hydroxytryptamine uptake
inhibitors on indole levels in rat brain. Br. J. Pharmacol. 110: 355–359, 1993.
CARBONI, E. AND DICHIARA, G.: Serotonin release estimated by transcortical
dialysis in freely-moving rats. Neuroscience 32: 637–645, 1989.
CHEETHAM, S. C., VIGGERS, J. A., BUTLER, S. A., PROW, M. R. AND HEAL, D. J.:
[3H]Nisoxetine-a radioligand for noradrenaline reuptake sites: correlation
with inhibition of [3H]noradrenaline uptake and effect of DSP-4 lesioning
and antidepressant treatments. Neuropharmacology 35: 63–70, 1996.
CHEETHAM, S. C., VIGGERS, J. A., SLATER, N. A., HEAL, D. J. AND BUCKETT, W. R.:
[3H]Paroxetine binding in rat frontal cortex strongly correlates with [3H]5HT uptake: Effect of administration of various antidepressant treatments.
Neuropharmacology 32: 737–743, 1993.
FANTINO, M. AND SOUQUET, A. M.: Effects of metabolites 1 and 2 of sibutramine
on the short-term control of food intake in the rat. Int. J. Obesity 19: 145,
1995.
FATTACCINI, C. M., GOZLAN, H. AND HAMON, M.: Differential effects of dfenfluramine and p-chloroamphetamine on H75/12-induced depletion of 5hydroxytryptamine and dopamine in the rat brain. Neuropharmacology 30:
15–23, 1991.
FRANCIS, J., CRITCHLEY, D., DOURISH, C. T. AND COOPER, S. J.: Comparison
between the effects of 5-HT and dl-fenfluramine on food intake and gastric
emptying in the rat. Pharmacol. Biochem. and Behav. 50: 581–585, 1995.
FULLER, R. W.: Microdialysis studies with 5-HT reuptake inhibitors. Trends
Pharmacol. Sci. 14: 397–398, 1993.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fluoxetine and nisoxetine were generous gifts from Eli Lilly Co.
Paroxetine was generously provided by SmithKline Beecham Pharmaceuticals. Sibutramine and BTS 54 505 were provided by Knoll
Pharmaceuticals Research and Development.
1997
591
contribution of metabolites to the rapid and potent down-regulation of rat
cortical b-adrenoceptors by the putative antidepressant sibutramine hydrochloride. Neuropharmacology. 28: 129–134, 1989.
MENNINI, T., BORRONI, E., SAMANIN, R. AND GARATTINI, S.: Evidence of the existence of two different intraneuronal pools from which pharmacological
agents can release serotonin. Neurochem. Int. 3: 289–294, 1981.
PAXINOS, G. AND WATSON, C.: The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney, Australia, 1982.
PERRY, K. W. AND FULLER, R. W.: Effect of fluoxetine on serotonin and dopamine
concentration in microdialysis fluid from rat striatum. Life Sci. 50: 1683–
1690, 1992.
PRESKORN, S.: Targeted pharmacotherapy in depression management: comparative pharmacokinetics of fluoxetine, paroxetine and sertraline. Int. Clin.
Psychopharm. 9: (Suppl. 3:) 13–19, 1994.
RAITERI, M., BONANNO, G. AND VALLEBUONA, F.: In vitro and in vivo effects of
d-fenfluramine: no apparent relation between 5-hydroxytryptamine release
and hypophagia. J. Pharmacol. Exp. Ther. 273: 643–649, 1995.
RUDNICK, G. AND WALL, S. C.: p-Chloroamphetamine induces serotonin release
through serotonin transporters. Biochemistry 31: 6710–6718, 1992.
RUTTER, J. J. AND AUERBACH, S. B.: Acute uptake inhibition increases extracellular serotonin in the rat forebrain. J. Pharmacol. Exp. Ther. 265: 1319–
1324, 1993.
RUTTER, J. J., GUNDLAH, C. AND AUERBACH, S. B.: Systemic uptake inhibition
decreases serotonin release via somatodendritic autoreceptor activation.
Synapse 20: 225–233, 1995.
SABOL, K. E., RICHARDS, J. B. AND SEIDEN, L. S.: Fluoxetine attenuates the d,
l-fenfluramine-induced increase in extracellular serotonin as measured by
in vivo dialysis. Brain Res. 585: 421–424, 1992a.
SABOL, K. E., RICHARDS, J. B. AND SEIDEN, L. S.: Fenfluramine-induced increases
in extracellular hippocampal serotonin are progressively attenuated in vivo
during a four-day fenfluramine regimen in rats. Brain Res. 571: 64–72,
1992b.
SATO, Y., SHIBANOKI, S., SUGAHARA, M. AND ISHIKAWA, K.: Measurement and
pharmacokinetic analysis of imipramine and its metabolite by brain microdialysis. Br. J. Pharmacol. 112: 625–629, 1994.
SHARP, T., BRAMWELL, S. R. AND GRAHAME-SMITH, D. G.: 5-HT1 agonists reduce
5-hydroxytryptamine release in rat hippocampus in vivo as determined by
brain microdialysis. Br. J. Pharmacol. 96: 283–290, 1989.
S HARP , T., Z ETTERSTROM , T., C HRISTMANSON , L. AND U NGERSTEDT , U.: pChloroamphetamine releases both serotonin and dopamine into rat brain
dialysates in vivo. Neurosci. Lett. 72: 320–324, 1986.
SHEARD, M. H., ZOLOVICK, A. AND AGHAJANIAN, G. K.: Raphe neurons: Effect of
tricyclic antidepressant drugs. Brain Res. 43: 690–694, 1972.
TEJANI-BUTT, S. M., BRUNSWICK, D. J. AND FRAZER, A.: [3H]Nisoxetine: A new
radioligand for norepinephrine uptake sites in brain. Eur. J. Pharmacol.
191: 239–243, 1990.
WONG, D. T., BYMASTER, F. P., MAYLE, D. A., REID, L. R., KRUSHINSKI, J. H. AND
ROBERTSON, D. W.: LY248686, a new inhibitor of serotonin and norepinephrine uptake. Neuropharmacology 8: 23–33, 1993.
Send reprint requests to: Dr. Sidney Auerbach, Department of Biological
Sciences, P.O. Box 1059, Rutgers University, Piscataway, NJ 08855.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
FULLER, R. W., SNODDY, H. D. AND ROBERTSON, D. W.: Mechanisms of effects of
d-fenfluramine on brain serotonin metabolism in rats: uptake inhibition
versus release. Pharmacol. Biochem. Behav. 30: 715–721, 1988.
FULLER, R. W. AND WONG, D. T.: Serotonin uptake and serotonin uptake inhibition. Ann. N.Y. Acad. Sci. 600: 68–72, 1990.
GARATTINI, S., MENNINI, T., BENDOTTI, C., INVERNIZZI, R. AND SAMANIN, R.: Neurochemical mechanism of action of drugs which modify feeding via the
serotoninergic system. Appetite 7: 15–38, 1986.
GARATTINI, S., MENNINNI, T. AND SAMANIN, R.: Reduction of food intake by
manipulation of central serotonin. Br. J. Psychiatry 155: 41–51, 1989.
GOBBI, M., FRITTOLI, E., MENNINNI, T. AND GARATTINI, S.: Releasing activities of
d-fenfluramine and fluoxetine on rat hippocampal synaptosomes preloaded
with [3H] serotonin. Naunyn-Schmiedeberg Arch. Pharmacol. 345: 1–6,
1992.
GOBBI, M., FRITTOLI, E., USLENGHI, A. AND MENNINNI, T.: Evidence of an exocytotic-like release of [3H]5-hydroxytryptamine induced by d-fenfluramine in
rat hippocampal synaptosomes. Eur. J. Pharmacol. 238: 9–17, 1993.
GOODWIN, G. M., DE SOUZA, R. J., GREEN, A. R. AND HEAL, D. J.: The pharmacology of the behavioural and hypothermic responses of rats to 8-hydroxy2-(di-n-propylamino)tetralin (8-OH-DPAT). Psychopharmacology. 91: 506–
511, 1987.
GUDELSKY, G. A. AND NASH, J. F.: Carrier-mediated release of serotonin by
3,4-methylenedioxymethamphetamine: implications for serotonin-dopamine
interactions. J. Neurochem. 66: 243–249, 1996.
GUNDLAH, C., MARTIN, K. F., HEAL, D. J., SCHJOTT, J. AND AUERBACH, S. B.: In vivo
criteria to differentiate monoamine uptake inhibitors (maris) from serotonin
releasing drugs: sibutramine is a mari. Soc. Neurosci. Abstract 244: 13,
1996.
HJORTH, S. AND AUERBACH, S. B.: Further evidence for the importance of 5-HT1A
autoreceptors in the action of selective serotonin reuptake inhibitors. Eur.
J. Pharmacol. 260: 251–255, 1994.
HJORTH, S. AND SHARP, T.: In vivo microdialysis evidence for central serotonin1A
and serotonin1B autoreceptor blocking properties of the beta adrenoceptor
antagonist (-)penbutolol. J. Pharmacol. Exp. Ther. 265: 707–712, 1993.
HYTTEL, J.: Pharmacological characterization of selective serotonin reuptake
inhibitors (SSRIs). Int. Clin. Psychopharm. 9: (Suppl. 1) 19–26, 1994.
JACKSON, H. C., NEEDHAM, A. M., HUTCHINS, L. J., MAZURKIEWICZ, S. E. AND HEAL,
D. J.: Comparison of the effects of sibutramine and other monoamine reuptake inhibitors on food intake in the rat. Br. J. Pharmacol. 121:1758–
1762, 1997.
KALÉN, P., STRECKER, R. E., ROSENGREN, E. AND BJORKLUND, A.: Endogenous
release of neuronal serotonin and 5-hydroxyindoleacetic acid in the caudateputamen of the rat as revealed by intracerebral dialysis coupled to highperformance liquid chromatography with fluorimetric detection. J. Neurochem. 51: 1422–1435, 1988.
KREISS, D. S., WIELAND, S. AND LUCKI, I.: The presence of a serotonin uptake
inhibitor alters pharmacological manipulations of serotonin release. Neuroscience 52: 295–301, 1993.
KUHN, D. M., WOLF, W. A. AND YOUDIM, M. B. H.: 5-Hydroxytryptamine-release
in vivo from a cytoplasmic pool: studies on the 5-HT behavioral syndrome in
reserpinized rats. Br. J. Pharmacol. 84: 121–129, 1985.
LEVI, G. AND RAITERI, M.: Carrier-mediated release of neurotransmitters.
Trends Neurosci. 16: 415–419, 1993.
LUSCOMBE, G. P., HOPCROFT, R. H., THOMAS, P. C. AND BUCKETT, W. R.: The
Uptake Inhibitors vs. Releasers