Ftibanserin, a Mixed S-HT~AAgonist / 5-HTzAAntagonist, Decreases Palatable Food lntake
in Operant and Non-Operant Feeding Paradigrns in Rats.
Susan Elizabeth Gilbert Evans
A thesis submitted in confomity with the requirements
for the degree of Master of Science,
Graduate Department of the Institute of Medical Science,
University of Toronto
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Flibanserin, a Mixed 5-HllA Agonist / 5-HT2AAntagonist, Decreases Palatable Food lntake
in Operant and Non-Operant Feeding Paradigms in Rats.
Master of Science, 2000
Susan Elizabeth Gilbert Evans
lnstitute of Medical Science, University of Toronto
The present research examined the effeds of flibanserin. a serotonin (5-Hl)l~agonist and 5-
HTX antagonist, in operant and non-operant models of sucrose feeding in rats. Five days of intrapentoneal @p.) injections of flibanserin 5 mglkg producd a significant reduction in the free-intake of sucrose. In operant experiments where rats were required to lever press for sucrose on a progressive ratio schedule, acute flibanserin 15 makg, as well as acute and repeated (1 3-day) fluoxetine 1O mg/kg, produced a significant reduction in the break point for sucrose reinforcement.
Neither flibanserin 5 rnglkg nor fluoxetine 10 mgkg produced profound effects on locomotor adivity.
The finding of decreased sucrose intake with flibanserin is consistent with the widely accepted view that enhanced serotonin neurotransmission causes a reduction in food intake and, given fiibanserin's mechanism of action, provides preliminary evidence that post-synaptic 5-HT1~receptors are involved in mediating this hypophagic effed. Acknowlednements
I wish to extend my wamest thanks to my supervisor, Dr. franco Vaccanno, for his valuable guidance and support throughout my Master's program, and to my program advisory cornmittee members, Dr. Paul Fletcher and Dr. Sidney Kennedy, for their most helpful cornments and suggestions. 1 would also like to thank my fellow graduate students and Research Associates, Bert
DeSousa, Glen Wundedich and David Bush, Andrew Arifunaman. Toni DeCristofaro, Camela
Presta and Franca Placenza, for welcoming me into the lab, providing support and expert technical assistance, and fostering a creative atmosphere both within and outside of the University. Thanks must also be extended to Boehringer lngelheim (Canada) Ltd. for the generous gift of flibanserin.
Finally, 1 am indebted to my husband, Ken Evans, who always provides me with an unending source of inspiration. Table of Contents
INTRODUCTION...... 1 BACKGROUND...... 1
5-HTIA RECEPTORS ...... 2
5-HTzARECEPTORS ...... 4
POST-SYNAPTIC5-HTiA AGONISM1 5-HTZAANTAGONISM ...... 5
5-HT AND FEEDING...... 7
EXPERIMENTALPURPOSE AND HYPOTHESIS...... 10
METHODS ...... 12 SUBJECTSAND APPARATUS...... 12
Experimenl 1...... 14
Experiment 2 ...... 15
Expriment 3 ...... 17
Experiment 4 ...... 18
TESTCOMPOUNDS ...... 19
STATIST~CALANALYSIS ...... 19
RESULTS ...... 20 EXPERIMENT1 ...... 20
DISCUSSION AND CONCLUSIONS...... 48 FREE-FEEDINGOF SUCROSE...... 48
SUCROSEVERSUS CHOWFEEDING ...... 49 PROGRESSIVERATIO EXPERIMENTS ...... 51
FEEDINGAND REWARD...... 54
FEEDINGAND ACTIVITY...... 55
THE ROLEOF 5-HT RECEPTORSIN FLIBANSERIN'SFEEDING EFFECTS ...... 57
CONCLUSIONS...... 60
IMPLICATIONS. NEW RESEARCH QUESTIONS AND FUTURE DIRECTIONS... 61 REFERENCES...... 65 APPENDIX 1: PROGRESSIVE RATIO SCHEDULE OF REINFORCEMENT...... 75 List of Tables
Table 1: Phamacological and Behaviouraf Characteristics of Flibansenn ...... 6
Table 2: Acute and Chronic Drug Administration and Test Regimen for Expriment 1 ...... 15
Table 3: Dosing / testing schedule for Expenment 3 ...... 18
Table 4: Mean (S.E.M.) Body Weight (grarns) Measured During Expeflrnent 1 ...... 21
Table 5: Mean (S.E.M.) Baseline Responding for Sucrose in Experiment 2 ...... 26
Table 6: Mean (S.E.M.) Percent Accuracy for the Active Lever in Experiment 2 ...... 28 List of Figures
Figure 1: The effed of acute and repeated flibanserin on sugar and chow intake (Experiment 1). .. 22
Figure 2: The effed of acute and repeated flibanserin on sugar intake in HlGH or LOW baseline
sugar feeders (Experiment 1) ...... 24
Figure 3: The effed of a single i.p. injection of vehicle, flibanserin 5 mglkg or fluoxetine 1O mglkg on
the break point for sucrose reinforcement (Experiment 2)...... 29
Figure 4: The number of reinforcers obtained dunng the 0-30, 30-60, 60-90 and 90-120 minute time
intervals of the 2-hour progressive-ratio test session (Experiment 2). ..-...... -..--...-.-.--..----31
Figure 5: The effeds of repeated i.p. administration of vehicle, flibanserin 5 mglkg or fluoxetine 10
mg/kg on the break point for sucrose reinforcernent (Experiment 2)...... 33
Figure 6 The effed of repeated i.p. administration of vehicle, flibanserin 5 mglkg or fiuoxetine 10
mgkg on the absolute change from baseline body weight (Experiment 2)- ...... 35
Figure 7: The effed of a single i.p. injection of flibanserin (0, 1.67, 5 or 15 mglkg) on the break point
for sucrose reinforcement (Experiment 3)...... 39
Figure 8 The number of reinforcers obtained during the 0-30, 30-60, 60-90 and 90-1 20 minute time
intervals of the 2-hour progressive-ratio test session (Experiment 3)...... 41
Figure 9: The effed of a single i-p. injection of vehicle, flibanserin 5 mg/kg or fluoxetine 10 mglkg on
total exploratory locomotor activity (Experiment 4)...... 44
Figure 10:The effed of a single i.p. injection of vehicle, flibanserin 5 mg/kg or fiuoxetine 10 mg/kg on
exploratory locomotor activity in the 0-30, 30-60, 60-90 and 90-120 minute tirne intewals of
the two-hour test session (Experiment 4)...... 46
vii List of Abbreviations
5,7-DHT 5,7-dihydroxytryptamine
5-Hl Serotonin
5-HTlA Serotonin 1A receptor
S-HT2~ Serotonin 2A receptor
5-HT2= Serotonin 2C receptor
8-OH-DPAT 8-h ydroxy-2-(di-n-propy1arnino)-tetralin
ANOVA Analysis of Variance
001 1-(2,S-dimethyloxy-4-iodophenyl)-2-aminopropane
ELA Exploratory locomotor adivity
FR Fixed Ratio
i.p. Intraperitoneal
I-Tso Charge (Current (1) x Time (7') ) required to inhibit 50% of neurons
MAO1 Monoamine oxidase inhibitor
mCPP m chlorophenylpiperaUne
PCPA Parachiorophenylalanine
PR Progressive ratio
S.E.M. Standard error of the mean
SNRl Serotonin and norepinephrine reuptake inhibitor
SSRl Selective serotonin reuptake inhibitor
ti~z Half-life of elimination
viii Introduction
Background
Since the introduction of the tBcydic antidepressants (TCAs) in the 1960's. the therapeutic
efficacy of serotonergic antidepressant drugs has been well established and provides a major Iine of
evidence to support the widely accepted view that serotonin (S-HT) deficiency underlies depressive
disorders (Blier et al., 1987). Dnigs within the three main dasses of serotonergic antidepressants,
the selective serotonin reuptake inhibitors (SSRls), TCAs and monoamine oxidase inhibitors (MAOls),
share the cornmon property of enhancing serotonergic neurotransmission, an action which is thought
to mediate their antidepressant action. Though these drugs provide invaluable therapeutic benefit to
depressed patients, the understanding of how they exert their ultimate antidepressant action is far
from complete. Further, certain drawbacks associated with these drugs, such as a delay in onset of
clinical efficacy (Baldessarini, 1990; Artigas et al., 1996), and their respective side effect profiles,
demonstrate the deficiencies in antidepressant treatments and highlight the need for a better
understanding of the neurobiology of depression-
Serotonergic antidepressants are thought to exert their therapeutic action either directly.
through pharrnacological interaction with 5-HT receptors, or indiredly, by causing an increase in the
synaptic 5-HT that is available for binding with receptors. The identification of multiple 5-Hl receptor
subtypes (for a review, see Saxena 1995) has facilitated the characterization of many antidepressants
according to their 5-HT receptor binding profiles and as a result. antidepressant drugs now serve as
indispensable tools for the study of serotonergic receptor mechanisms controlling various aspects of mammalian behaviour. Understanding the interadions between serotonergic dmgs and their receptors has enabled researchers to begin to more clearly define the role of the various 5-HT systems. and has promoted the developrnent of newer antidepressants with specific receptor adivity.
Two particular 5-HT receptor subtypes, the S-HTI* and 5-HTU\ receptors, have been the subject of recent antidepressant research (Stahl, 1994; Borsini, 1994), and new treatment strategies involving 5- HTIA and/or 5-HT- receptor adivity are currently being investigated to address one important
therapeutic need, a faster onset of antidepressant efficacy (Borsini, 1994).
SHT~Areceptors
5-HTIA receptors located in the CNS may be organized into two main populations. pre- and
post-synaptic, according to their anatomical distribution. Pre-synaptic 5-HTIA receptors are found on
the cell bodies and dendrites of serotonergic neurons in the dorsal and median raphe nuclei, where
they act as somatodendritic autoreceptors, while the post-synaptic population is located diffusely in
many serotonin projection areas, including the frontal cortex, hippocampus and amygdala (Welner et
al., 1989). Stimulation of 5-HTIA receptors leads to membrane hyperpolarization, which induces a
decrease in neuronal firing. However. because of their anatomical organization. stimulation of pre-
versus post-synaptic 5-HTIA receptors produces opposite net effeds on the serotonin system.
Whereas post-synaptic stimulation mediates a net inhibitory effect on cells in 5-HT projection regions,
pre-synaptic stimulation inhibits the 5-HT cell itself. resulting in a decrease in 5-HT output which
lessens the inhibitory influence of 5-HT in projection regions. Further, it is thought that pre-synaptic
brain regions contain larger 5-HT1~recepfor reserves than post-synaptic projedion areas, such that
lower doses of 5-HTIA agonists will typically stimulate pre-synaptic receptors, whereas higher doses are necessary to elicit post-synaptic effeds (for a review, see De Vry. 1995).
Evidence from both clinical and preclinical studies has implicated 5-HTIA receptors in depression and antidepressant response. For example, normal 5-HTIA-mediated physicrlogical responses, such as hypothermia and cortisol secretion, may be blunted in depressed patients (Stahl,
1994). Further, preclinical research indicates that S-HT1~agonists are active in the Porsolt model of depression in rats and mice. even following destruction of pre-synaptic 5-HT neurons with the 5-HT neurotoxin 5, 7-dihydroxytryptamine (5.7-DHT) or the 5-Hf synthesis inhibitor parachlorophenylalanine (PCPA) (Wieland and Lucki, 1990; Matsuda et al., 1995), indicating that post-synaptic 5-HTIA rnechanims are sufficient to support these antidepressant-like effects. Such findings have led some researchers to propose that pst-synaptic 5-HTIA receptors are an important site of action for the therapeutic adion of serotonergic antidepressant dmgs (Welner et al.. 1989;
Stahl, 1994; Blier and de Montigny, 1994; Goodwin, 1996; Blier and de Montigny, 1998). In contrast,
because stimulation of somatodendritic 5-kiTfAautoreceptors results in a decrease in 5-Hl output, which attenuates the pst-synaptic inhibitory actions of 5-HT, it has also kenproposed that
somatodendritic 5-HTIA receptors hinder antidepressant response and are involved in the delay in onset of clinical efficacy (Blier and de Montigny. 1998).
Typically, antidepressant dnigs must be administered for at least two weeks before clinical efficacy emerges (Baldessarini, 1990; Soares and Gershon, 1996). Since most serotonergic antidepressants share the property of increasing extracellular 5-HT (typically, through the blockade of
5-HT reuptake), the initial effed of antidepressant administration would be to increase the 5-HT available for binding at pre- and pst-synaptic 5-HTIA receptors. However. since binding of 5-Hf at pre-synaptic 5-HTIA autoreceptors teads to an inhibition of 5-HT cell firing, it has been proposed that the result of short term antidepressant administration is to decrease 5-HT output, which effedively attenuates the net antidepressant effect - post-synaptic 5-HTiA receptor stimulation. Studies of acute and chronic antidepressant treatment have suggested that over time, somatodendritic 5-Hf ,A receptors functionally desensitize in the presence of repeated antidepressant treatment, thus attenuating 5-HT cell body inhibition and allowing newly released 5-HT to bind with post-synaptic 5-
HT,A receptors and produce antidepressant effeds. In support of this hypothesis are findings that short terni antidepressant treatment inhibits firing adivity in the dorsal raphe nucleus, which recovers to baseline levels following chronic treatment. suggesting a fundional desensitization of the somatodendritic autoreceptors following repeated antidepressant treatment (Blier and deMontigny,
1985; Blier et al., 1987). Further, repeated, but not acute fluoxetine administration decreases the number of spontaneously active neurons in the frontal cortex (Ceci et al., 1994). while repeated administration of the SSRl fluvoxamine increases extracellular 5-HT in the frontal cortex, but not the midbrain raphé nuclei (Bel and Artigas, 1993), indicating that post-synaptic serotonergic inhibitory effects only emerge with repeated treatment. It has been hypothesized that the time required for the presynaptic 5-HflA auforeceptors to desensitize is associated with the delay in the onset of clinical antidepressant action (Blier and de Montigny, 1994) and accordingly. new antidepressant treatment strategies have been direded at averting stimulation of pre-synaptic 5-HT1, receptors.
Both animal and clinical studies support the notion of a faster onset of adion through blockade of pre-synaptic 5HTlA receptors. In rats, the seledive 5-HTl~antagonist WAY 100,635 has been shown to reverse the firing-inhibitory effect of an acute dose of the SSRl paroxetine in the dorsal raphé nucleus, and to augment extracellular 5-HT in the frontal cortex (Gartside et al., 1995). Such an effect suggests that blockade of the presynaptic 5-HTtA receptor prevents the ability of newly released 5-HT to inhibit 5-HT cells, and facilitates an increase in 5-HT neurotransmission at post- synaptic sites. This strategy is also king explored in clinical trials with pindolol, a dnig which is thought to selectively inhibit pre-synaptic 5HTlA receptors (Romero et al., 1996). Pindolol has ken shown in certain studies to accelerate the onset of eftkacy of SSRl treatment, presurnabiy through a blockade of the pre-synaptic 5-i+TjA mechanisrns associated with a delayed onset of clinical effi~acy
(Artigas et al., 1996; Blier and Bergeron, 1995).
SHTu Receptors
In addition to 5-HTiA receptors, 5-HT2Areceptors have also been studied for their role in depression. 5-HTzAreceptors appear to be distributed pst-synaptically on non-serotonergic cells of many brain regions, with high density in the frontal cortex, anterior cingulate cortex, olfactory tuberde and claustmm (Pazos et al-. 1985). Functionaily, the 5-HlzAreceptor, coupled to phosphotidyl inositide turnover, mediates cell depolarization and neuronal stimulation (Saxena, 1995), an effed which is opposite to the physiological role of 5-HTIA receptors. It has kensuggested that 5-HTa receptors are important in depression, with evidence to support this notion arising from investigations of !he post mortem brains of depressed suicide vidims, which revealed an abnomally high number of
5-HTU\ (fomierly 5-HT2,see Saxena 1995) brain receptors in the frontal cortex (Hrdina et al. 1993). and from dinical studies which demonstrate the efficacy of a 5-HTa antagonist in depressive illness
(Bersani et al., 1991; Bakish et al., 1993). Studies of the rat brain have indicated that 5-HTz~receptors can be found CO-localizedwith 5-
HT,, receptors on the same cell, particularly in the frontal cortex region (Araneda and Andrade.
1991). The overlap of 5-HTIA and 5-HTzAreceptors, which mediate opposing effects on neuronal
firing, is relevant in that it raises the possibility that selective antagonisrn at the S-HT2Areceptor could
enhance post-synaptic 5-HTqA-mediateci effects. In support of this notion. studies have shown such
a functional interaction between 5-HTzAantagonists and 5-HTIA agonists. For exampie, S-HT~A
antagonists have been found to potentiate the firing-suppressant effects of 5-HT (Lakoski and
Aghajanian, 1985), and the S-HTIA agonist 8-hydroxy-2-(di-n-propylamino) tetraline (8-OH-DPAT)
(Ashby et al., 1994) in the prefrontal cortex. Based on the hypothesized fundional interadion
between 5-HTIA and 5-HT2Areceptors, and the fad that both receptor subtypes have been irnpiicated
in depression, several researchers have proposed that a phamacological agent which averts stimulation of sornatodendritic 5m1Areceptors and also acts as an antagonist at S-HTa receptors,
may produce a more robust antidepressant response (Borsini, 1994; Blier and de Montigny, 1998).
Flibanserin is a novel putative antidepressant (Borsini et al., 1998). which is purported to possess the phamacological properties of a cornbined S-HT,, agonist / 5-HTa antagonist (Borsini et al., 1995a), and is currently in clinicat development as a fast-acting antidepressant. Aspects of flibanserin's receptor binding and behavioural profiles are summarized in Table 1: Table 1: Phamiacological and Behavioural Characteristics of Flibanserin
Binding afftnity at 1 Ki (nM) 1 Reference various 54T receptors
5-HTIA 19+2 Borsini et al., 1998
1 1 5-HTW 1 133127 ( Borsini et al.. 1998
5-HTzc 4,431 2 1580 Borsini et al., 1998
I 5-HT uptake site 3.354 2 499 Borsini et al., 1998
Behavioural Models of Active Dose(s) Reference Depression I Leamed helplessness 48 mgkg (oral) Borsini et al., 1997 (rats) I I
Forcedswimtest(mice) 16mg/kg(i.p.) Cesana et al., 1995 I 1 I Mild chronic unpredidable 1 2.5 - 5 mglkg @p.) 1 D'Aquila et al., 1997 stress (m ice)
Olfactory bu1bectomy 1 O mg/kg (i-p.) Borsini et al., 1997 (rats) I I 'for cornpiete list see Borsini et al., 1998
Unlike typical selective S-HT1~agonists, flibanserin appears to be the first serotonergic compound that selectively activates pst-synaptic 5-HTIA receptors, a highly important phannacological property which should confer the advantage of avoiding the somatodendntic SHTIA desensitization period associated with the delay in onset of clinical efficacy.
Evidence to indicate flibanserin's preferential pst-synaptic activity has arisen mainly from in vivo electrophysiological studies in anaesthetized rats. lntravenous administration of flibanserin has been shown to inhibit fronto-cortical neurons, an effed that was blocked by the selective 5-HTTA antagonist WAY 100.1 35. This fronto-cortical inhibition was not affeded by 5,7-DHT lesions (which
destroy 5-HT neurons), indicating that the effect was not dependent on pre-synaptic serotonergic
mechanisms (Borsini et al., 1995b). Additionally, Rueter et al. (1 998) demonstrated that while
flibanserin behaved as a 5-HflA full agonist in both pre-synaptic (dorsal raphe nucleus) and post-
synaptic (media1 prefrontal cortex) regions, microiontophoretically applied flibanserin demonstrated a
higher degree of potency as a 5-HllA receptor agonist (according to I-TS0values) in the post-synaptic
brain regions, hippocampus and medial prefrontal cortex, than in the dorsal raphé nucleus (1.T-
values of 260, 1260 and 1365 nanocoulombs, respectively). Although these studies provide some
evidence of preferential stimulation of post-synaptic receptors in efectrophysiological preparations,
additional support for this concept could be gained through behavioural studies that distinguish
between pre- and post-synaptic 5-HTIA effects.
5-HT and Feeding
In an effort to further characterize flibansenn's behavioural profile in animais. and to explore the
concept of preferential adivity at pre- versus post-synaptic 5-HTrAreceptors, the present studies were
undertaken to explore the effects of the dnig in a behavioural model which may be sensitive to such
distinctions. Feeding behaviour in rats has been found to respond with a good degree of reliability to
manipulations with 5-HT,A agonists and a variety of serotonergic antidepressants, and might serve as
a model which will allow discrimination between pre- and post-synaptic 5-HTIA receptor mechanisms.
It is generally accepted that increases in extracellular 5-HT can lead to an inhibition of feeding
in rats (Samanin, 1989). This effect has been demonstrated through a variety of phamacological
manipulations which enhance extracellular 5-HT, for example with the 5-HT releaser d-fenfluramine
(McGuirk et al., 1992a) as well as the selective serotonin reuptake inhibitors (SSRls) fluoxetine and sertraline, and the serotonin and norepinephnne reuptake inhibitors (SNRls) venlafaxine, duloxetine
and sibutramine (Wong et al., 1988; Lucki et al., 1988; Jackson et al.. 1997). Although the receptor
mechanisms underlying the hypophagic effeds of SSRls and other 5-HT-enhancing manipulations have not been clearly defined, the 5-HTla receptor represents one possible locus mediating such
effects.
The rote of the 5-HT1~receptor in mammalian feeding has been the subject of much
investigation, and evidence suggests that this receptor mediates a biphasic effect on feeding - at relatively high doses, feeding is inhibited, however, at low doses feeding is stimulated (Montgomery et
al., 1991). For example, the prototypic S-HTqAagonist. 8-OH-DPAT has been shown to reliably
induce feeding in rats when administered at fow doses (Dourish et al., 1985a, b; Lu0 et al., 1990;
Fletcher et al, 1991). Such hyperphagic effeds can also be elicited by direct injection of 8-OH-DPAT
into the cell body regions (dorsal, median raphé) (Hutson et al.. 19û6; Bendotti and Samanin, 1986).
and can be blocked by pretreatment with $iiïlA selective antagonists, providing eviâence for the
involvement of presynaptic 5-HTIA receptors (Hutson et al., 1988; Hartley and Fletcher, 1994).
Sirnilarly, other S-HTIA agonists, such as buspirone, gepirone, ipsapirone and MDL 72832 (Gilbert
and Dourish, 1987; Neill and Cooper, 1988; Fletcher et al, 1991). have also been shown to produce
hyperphagia in rats. Such findings have led researchers to propose that the pre-synaptic S'HllA
receptor mediates hyperphagia by causing a decrease in 5-HT synthesis and reduction in overall S-
HT neurotransrnission (Hutson et al., 1986; Hutson et al., 1988; Simansky, 1996). At higher doses,
8-OH-DPAT has been found to decrease feeding (Vickers et al., 1996; Dourish et al., 1985a). an effect which is blocked by pretreatment with the pre- and post-synaptic 5-HTIA antagonist WAY
100635 (Vickers et al., 1996), indicating the involvement of 5-HTIA receptors.
The biphasic effect of typical 5-Hf,A agonists on feeding is consistent with the opposing effects on overall 5-HT neurotransrnission mediated by pre- venus pst-synaptic 5-HTAAreceptors (post- synaptic receptors mediate a net neuronal inhibitory effect in 5-HT projection regions, while pre- synaptic receptors act as autoreceptors and shut down the 5-HT system). Further, it is consistent with the concept that lower doses of 5-HTIA agonists elicit pre-synaptic effeds, whereas higher doses are required to elicit post-synaptic effeds (DeVry. 1995). fhus, since stimulation of somatodendntic
5-HTIA receptors causes an increase in feeding, it is plausible that the direct stimulation of post- synaptic S-HTIA receptors mediates the opposite effect - a decrease in feeding. Further, since post- synaptic 5-HTtn receptors have been identified as an important target for serotonergic antidepressant
effects (Blier et al., 1997). these receptors may also be involved in mediating the hypophagic effects
associated with certain antidepressant drugs such as the SSRls.
Thus far, at least two fadors have hindered the study of ~-HT~Arecepfors as mediators of
hypophagia. First, the drugs that typically induce such effeds are agents that non-selectively
increase 5-HT levels, such as the 5-Hl releaser fenfluramine, and the SSRl fluoxetine. Such a non-
selective increase in 5-HT would increase binding not only at the 5-HTln subtype, but also at other 5-
HT receptor subtypes. Indeed, attempts to block the hypophagic effeds of these drugs with selective
5-HT,A antagonists have not been successful Wckers et al., 1996; Ciccocioppo et al., 1997).
However, the failure to reverse SSRI-induced hypophagia with a $FITlA antagonist cannot entirely
exclude the post-synaptic S-HT,A receptor as a mediator of hypophagia, since in the presence of 5-
HTIA receptor Mockade, dher receptor subtypes, such as 5-HT,B or 5-HTzc, may also have
contributed to the hypophagic efiect (Bendotti and Samanin, 1987; Sirnansky and Vaidya, 1990).
Second, using selective 5-HTIA agonists such as 8-OH-DPAT to elicit hypophagic effeds is
complicated by the fact that post-synaptic 5-mIA receptor stimulation requires high doses which also
induce the "serotonin syndrome", a cluster of behaviours which include flat body posture, reciprocal
forepaw treading. headweaving, tremor and hindlirnb abduction (Tricklebank et al.. 1984: Dourish et
al., 1985a; Smith and Peroutka, 1986). Therefore, it remains unclear whether the reduction in feeding
is an effed that is specifically mediated by post-synaptic %HllA receptors, or one that is influenced by
a generai disnrption in behaviour.
Flibansenn differs from other typical SHTIA agonists in that it appears to selectively activate
post-synaptic 5-HTIA receptors and, therefore, may provide a useful tooi to study selective pst-
synaptic 5-HT,A effects without requiring the high doses to overcorne pre-synaptic receptor stimulation. Further, flibansenn elicits sorne evidence of the serotonin syndrome only at doses of 64
mgfkg and above (Borsini et al., 1998), doses which are considerably higher than those active in behavioural models of depression. Therefore, it may also be possible to demonstrate feeding effeds
in dose ranges that do not induce gros behavioural changes. Based on the phannacology of
flibanserin, and the proposal that these receptors may be involved in the control of feeding, it was
predicted that flibanserin would produce a decrease in feeding, which is consistent with the effeds
produced by typical serotonergic antidepressants.
Expenmental Purpose and Hypothesis
The present experiments were mnducted to explore the hypothesis that flibanserin would
suppress feeding in rats. Experiment 1 sought to detemine the effeds of flibanserin on the intake of
sugar and chow in a non-operant feeding paradigm in non-food deprived rats. This free-feeding
paradigm sewed as the first means of detemining whether flibanserin would have any general effects
on feeding. A choice between sucrose and chow was implemented to investigate whether any
change in feeding was specific to palatable food or to general food intake. The effects of both acute
and repeated flibanserin administrations were investigated, to determine whether any feeding effects
were dependent on the duration of drug treatment. An additional independent variable, individual
differences in sugar feeding, was also incorporated into this experiment. Previous research indicates
that animals categonzed as either 'highn or "lownsugar feeders, according to baseline sucrose intake,
demonstrate differential baseline responsivity in behavioural paradigms of anxiety. (DeSousa et al.,
1998), and different responses to the effects of various psychostimulants (Sills and Vaccarino, 1994;
Sills and Vaccarino, 1998). Such findings suggest that 'lown versus 'highn sugar feeders may be
differentiated on the basis of fleurotransmitter tone (e.g. dopaminergic or opioidergic tone), which may
also provide an indication of differential sensitivity to vanous behaviours. This variable was
incorporated in Experiment 1 to detennine whether individual differences in response to flibanserin
could be detected.
Once the effects of flibanserin were established in the free feeding paradigm, flibanserin's effects were assessed in a feeding paradigm that required operant responding on a progressive ratio schedule of reinforcernent for 45 mg sucrose pellet rewards (Experiments 2 and 3). In such a paradigm, rats are required to produce an increasing number of responses (lever presses) to obtain successive rewards (Richardson and Roberts. 1996). The dependent measure, break point, represents the point at which an animal will no longer respond for the next reward, and is thought to provide a sensitive indication of the animal's motivation to obtain the reward. Operant testing was conducted in two settings, in Experirnents 2 and 3. In Expriment 2. the effeds of a single dose of flibanserin and an active control drug, the SSRl antidepressant fluoxetine, on break points were examined. Fluoxetine was selected as the control drug on the basis that it has been shown to reliaMy induce hypophagia (as discussed above), and would, therefore, provide an indication that the operant
PR paradigm was sensitive to detemining feeding effects of serotonergic agents. ln Experiment 3, the dose-response relationship of flibanserin's effects on progressive ratio responding was examined.
Experiment 4 assessed the effeds of a single dose of flibanserin and fiuoxetine on exploratory locornotor activity (as measured by photocell beam breaks in a locomotor activity test chamber), to deterrnine whether these drugs produced any non-specific effects on activily that could have influenced food intake. Methods
Subjects and Apparatus
Male Wistar rats (Charles River, Montreal. Canada) sewed as subjects in each of the four
experiments. In Experiment 1, 48 rats weighing approximately 225-300 g at the start of the
experiment were used. In Experiment 2 and 4, 24 rats weighing approximately 400-500 g at the start
of the experiment were used. The same rats were used for both experiments. In Experiment 3.24
rats weighing approximately 350-450 g at the start of the experiment were used. Animals were
individually housed in polycarbonate cages (Allantown Caging equipment, NJ, U.S.A.) and given free
access to water and chow (Laboratory Rodent Chow 5001, PMI Feeds, MO, U.S.A-) at al1 times
outside the experirnental test sessions, except during the initial stages of operant training in
Expenments 2 and 3 (see below). The vivarium was maintained at a constant temperature (21 î 1
OC) and on a 12 hour IighUdark cycle (lights on at 7:00 a.m. E.S.T.). Animals were allowed a
minimum of four days in which to habituate to this environment. All testing was conducted during the
light cycle.
In Experiment 1, sugar and chow intake was measured in the animals' home cages using two
identical hanging stainless steel feeding cups, 8 cm diameter, 4.5 cm height. topped by a stainless steel lid with a 3 cm opening which was observed to minimize food spillage during feeding sessions
(no further attempt was made to measure spillage from the cups). One cup was filfed with granulated table sugar (Redpath Sugar), the other with standard rat chow (Laboratory Rodent Chow 5001, PMI
Feeds, MO, U.S.A.), pulverized to a powdered consistency to resembie the sugar texture. Sugar cups were placed to the right in relation to the front of the cage, and the chow cups were placed on the left. Feeding cups were rnanuaily weighed on a Mettler PJ 160 eledronic balance to the nearest
0.1 g, before and after each one-hour feeding session. In Experiments 2 and 3, eight operant chambers, individually enclosed in sound-attenuating
cabinets equipped with a ventilation fan (Med Associates Inc., St. Albans, VT, USA) served as the
test chambers. Chambers consisted of a cage 22 cm in height x 32 cm width x 24 cm depth with a
Plexiglas front, back and ceiling, stainless steel side walls and a stainless steel wire floor. Chambers
were equipped with two non-retractable levers with a white stimulus IigM directly above each lever,
and a central pellet delivery chamber located between the levers. Responding on the lever designated "active" resulted in the delivery of one 45 mg sucrose pellet (Noyes Precision Pellets, PJ
Noyes Company Inc., Lancaster, NH) according to the designated schedule of reinforcement. The stimulus fight above the active lever was illuminated for 1 second imrnediately following completion of the designated schedule of reinforcement. The lever designated "inactive" produced no consequence, though responses on this lever were recorded. In Experiment 2. the right lever was designated active and the leït inactive. In Experiment 4, the levers were counterbalanced such lhat half the animals were trained and tested with the left lever active and right lever inadive, with the order being reversed for the other half of the animals. A white house light and the ventilation fan remained on dunng the entire test session. Operant chambers were automatically controlled from a personal computer within the tesfing room, using Schedule Manager PC-software (Med Associates
Inc., St. Albans, VT, USA). The software wntrofled the duration of the test session as well as schedules of reinforcement, and recorded the number of active and inactive lever responses and sucrose reinforcers delivered.
In Experiment 4. sixteen adivity boxes (32.5 cm x 32.5 cm x 32.5 cm) were used to measufe locomotion dunng test days. Locomotor activity was measured by two sets of horizontal photocell beams mounted approximately 5 cm from the floor, in the front 1/3 and rear 113 of the cage. Data collection was accomplished using custom-designed DOS-based software loaded on a personat computer in an adjacent room, which recorded the total number of consecutive photocell beam interruptions over the two-hour test session. Experiment 1 the Effects of Flibanserin on Sucrose and Chow lntake in the Rat
This was a parallel group study investigating the effeds of acute and repeated flibanserin (O,
0.5 and 5.0 mgkg) on sugar and chow intake in rats. On fwe consecutive habituation test days, two stainless steel feeding cups, one containing granulated sugar and the other containing powdered chow, were hung in the home cage for a one hour feeding period to habituate animals to the feeding cups and food. During the test sessions. al1 regular chow was rernoved from the home cage feeding hopper, though animals continued to have free access to water. On the sixth habituation day animals were injected with 0.9% saline immediately pnor to the introduction of the feeding cups, and after a one-hour feeding session, sugar and chow intake was detemined, animals were ranked in order of sucrose intake and categorized as either a 'high" or a 'low" baseline sugar feeder. Animals from each of these two categofles were assigned in a counterbalanced fashion to the three dose groups
(flibanserin 0, 0.5 or 5.0 mgfkg; n = 16 per group).
On Study Test Day 7. one day afier the saline injection, a test regimen was initiated to study the effeds of acute (single injection) and repeated (five days of injections) flibanserin on sugar and chow intake. This regimen is descnbed in Table 2 (below): Table 2: Acute and Chronic Orug Administration and Test Regimen for Experiment 1
Study Test Day
Acute RePq.id DW iiw - " Procedure 7" - 8 -9 . . O fi . 12 13 -- 14 15 16 - -. Drug Injection ...... J J J J J ...... A ... - . . . .m.. -- ... Feeding Test - J J J d 4 J J 4 J J './" indicates days on which the piromdure was conducted. Days 1-6 (not shown) were trainhg / habituation days. Days 8-1 1 represent drug-free washout days between the acute and repeated drug administration.
On "drug injectionwtest days animals were injected with the appropriate dose of flibanserin 20 minutes before placement of the feeding cups in the cage. Sugar and chow intake over the one hour session was assessed by subtracting the post-session feeding cup weight (grams) from the pre- session weight. Body weight was measured during the repeated injedion phase, on the morning of each test day (prior to testing).
Experiment 2
The Effects of Flibanserin and Fluoxetine on Responding for Sucrose on a Progressive Ratio of Reinforcement
This was a parallel group study examining the effects of flibanserin 5 mg/kg and f uoxetine 1O mg/kg on operant responding for sucrose on a progressive ratio schedule of reinforcement in rats.
The expenment consisted of two phases, training and testing.
Training:
a) Fixed Ratio (FR): On each of six FR training days, animals were placed in the operant chamber for a one to two hour test session. Animals were food restncted to 20-25 g of ch0w from days 24 of fixed-ratio training, to encourage feeding dunng the operant training sessions. During the
first three training sessions animals responded for sucrose reinforcement on a FR-1 schedule, where
a single lever-press delivered one sucrose pellet. In the following three sessions animals responded
under a FR-3 schedule. Once stable responding was achieved across the test groups, the schedule
of reinforcement was switched to a progressive ratio.
b) Proaressive Ratio: In a progressive ratio (PR) scheduie, the delivery of a single reinforcer
requires a successively greater number of lever presses. The point at which an animal stops working to receive a reinforcer is known as the 'break point", and is thought to provide a sensitive indication of the efftcacy of the reinforcer. In this experiment, the PR schedule followed a pre-determined series of fixed-ratio scheâules (rnodified from Hubner and Koob, 1990), as describecl in Appendix 1. The PR training period consisted of six days, with the rate at which the fured-ratio schedutes increased (Le. the amount of work required to obtain sucrose pellets) being increased every two days. The final training day serued as the baseline for acute dnig testing. The total duration of each session was two hours. The "break point" was defined as the total number of sucrose pellets obtained during the ho- hour test session, which is consistent with DePoortere et al. (1993). who described that the number of reinforcers obtained in a PR session serves as a useful indicator of breaking point.
Testing:
Animais were assigned to one of three treatment groups (vehicie, flibanserin 5 mg/kg or fluoxetine 10 mg/kg) in a randornized fashion. During the test phase, animals were placed in the operant chambers for a two-hour test period. A baseline (drug-free) testing day preceded the start of each of the test phases (acute and repeated administration, described below). Body weights were measured daily pnor to the start of esch test session. The drug administration / test session schedule is descn bed as follows: a) Acute Dnia Administration: A single injection of vehicle, flibanserin 5 mglkg or fiuoxetine
10 mg/kg was administered 20 minutes prior to the staR of the progressive ratio test session. Animals
were then placed in the operant chambers and tested for two hours.
b) Re~eatedDrua Administration: Single injections of flibanserin, fluoxetine or vehicle were
administered on each of thirteen consecutive test days. Animals were tested for two hour sessions in
the operant chamber on days 1.2.7 md 13 of dnig treatment. Injections were given 20 minutes prior
to the test session on testing days, while on non-testing days dnig was administered at approximately
the same time as it was on the test days.
All testing occurred approximately between the hours 1Oh30 and 16h30. The number of adive
and inactive lever presses, as well as the number of sucrose pellets delivered, was recorded at 10-
minute intervals over the two-hour test session.
Experiment 3
The Effects of Flibanserin 1.67, 5 and 15 mglkg on Operant Responding for Sucrose on a
Progressive Ratio Schedule of Reinforcement
This was a within-subjects crossover design study investigating the effects of flibansenn 0,
1.67, 5 and 15 mg/kg on operant responding for sucrose on a progressive ratio schedule of reinforcement. The expriment consisted of two phases. training and testing. The FR and PR training phases were identical to those camed out in Experiment 2.
Prior to the drug testing phase, the 24 animals were assigned to one of four groups in a counterbalanced fashion according to baseline body weight. Eâch group consisted of six animals, receiving each of the four test doses throughout the expenrnent according to the scherne described in
Table 3: Table 3: Dosing / testing schedule for Experiment 3
Test Day 1
Group 1
Group 2
Group 3 5 15 O 1.67
Group 4 15 O 1.67 5
Orug was administered 20 minutes prior to the start of each test session. Anirnals were placed in the operant chambers and tested for a period of two hours. Each dnig test day was separated by two drug-free, no testing days. The flumber of active and inactive lever presses, as well as the number of sucrose pellets delivered, was recorded at 10-minute intewals over the two-hour test session. "Break point" was defined as the total number of sucrose pellets obtained during the two- hour test session.
Experiment 4
The Effects of Acute Flibanserin and Fluoxetine Exploratory Locomotor Activity in the Rat
This wîs a parallel group study examining the effects of acute administration of fibanserin 5 mg/kg and fluoxetine 10 mg/kg on exploratory locomotor activity in the rat. The same subjects from
Experiment 2 were used in this study, with al1 animals retaining their previous treatment group assignments.
On locomotor testing days anirnals were removed from their home cage and placed in one of the sixteen activity boxes, where locomotor activity was recorded for two hours. f esthg of ail 24 animals was accomplished in two separate test runs of 12 anirnals each, with equal nurnbers of animals from each dose group assigned to each test run. Anirnals were tested between 1200h and 1700h. The first two consecutive test days were conducted without treatrnent, in order to habituate
animals to the activity boxes. On the third consecutive test day. rats were challenged with a single
i.p. injection of flibanserin, fluoxetine or vehide (according to the assigned dose group) and placed
back in their home cages. Twenty minutes later, animals were placed in the locornotor adivity cage
where exploratory locomotor activity was recorded over the subsequent ho-hour period.
Exploratory locomotor adivity was assessed using customized data collection soffware, which counted the number of crossovers (the nurnber of times both the front and back photocell
beams were intempted consecutively).
Test Compounds
Flibanserin (BIMT-17, Boehringer lngelheim ltalia s.p.a., Milano, Italy) was dissolved in a vehicle of 25% polyethylene glycol 400, 75% distilled water (adjustecl with HCI / NaOH to a final pH of approximately 3). Fluoxetine HCI (Research Biochemicals Inc., Natick, Massachusetts, U.S.A.) was dissolved in an identical vehide (final pH approximately 3). AI1 test drugs were injected intraperitoneally in a volume of 1 mUkg body weight.
Statisücal analysis
In Experiments 1, 2 and 4, data were analysed using between-subjects analysis of variance
(ANOVA). One repeated-measures factor, time, was included where appropriate to account for testing over successive days or over successive time intewals within a test session. In Experiment 1, the effeds of flibanserin on 'high* and 'fow" sugar feeders were analyzed with separate ANOVAs. In
Experiment 3, data were analysed using within-subjects ANOVA. Post-hoc test were conducted, where appropriate, using Tukey's HSD tests. All tests assumed a p < 0.05 level of significance.
Statistical analyses were conduded with the assistance of the amputer software package statisticam. version 5.0. Erperimenf 1
The Effects of Flibanserin on Sucrpse and Chow lntake in the Rat
1. Suaar lntake
Sugar intake for the acute and chronic phases of the study is displayed in Figure la. All
values are reported as the mean ISEM. sucrose intake (g). Day 6 sugar intake was similar for al1
three groups (1.S9 i 0.23 g (vehicle), 1.56 -c 0.21 g (flibanserin 0.5 mg/kg), 1-56 i 0.21 g (flibanserin
5.0 mglkg)), indicating stable baseline feeding. On the acute dmg test day (Day 7). a dose-
dependent, though non-signifiant trend toward a reduction in sucrose intake was apparent with
flibanserin treatment. In the four-âay period following acute injection sugar intake was stable across
the three groups, with a slight increase over time (likely associated with normal body weight gain).
During the five-day repeated injection phase flibansenn 5.0 mglkg significantly attenuated sugar
intake, as indicated by a main effed of dose, F (2, 42) = 6.44, p c 0,004, followed by a significant
Tukey's post-hoc test (p < 0.004 for flibanserin 5 mg/kg vs. vehicle). There was no significant effed of time or the interaction of dose x time.
2. Chow lntake
The average baseline consumption of chow (0.09 t 0.04 g) was much lesthan consumption of sugar (1.39 2 0.1 3 g), indicating a high preference for sugar. ANOVA revealed that fiibanserin treatment produced no significant effeds on chow intake during the acute or repeated administration phases of the study (Figure 1b).
3. Hiah vs. Low Feeder Differences On the sixth day of habituation (Day 6 - 'baseline") animals were given an i.p. injection of
0.9% saline immediately @or to sucrose and chow intake tests. Following this session, animals were classed as either a high ('HIGH") or low ('LOW) baseline sugar feeder based on a median split of the sugar intake data. This classification produced a HlGH group that consumed an average of 2-25 î
0.1 0 g, and a LOW group that consumed an average of 0.89 î 0.10 g. Acute flibanserin had no
significant effect on sugar intake in either group. aithough visual inspection of the data indicated that
the dose-dependent trend toward a reduction in intake appeared to be more pronounceci in the HIGH
group versus the LOW group (Figure 2). There was a significant main effed of dose in both HlGH
and LOW groups following repeated nibanserin (HIGHS: dose, F (2, 20) = 5.61, p < 0.02, LOWS:
dose, F (2, 19) = 4.39, p c 0.03). Further, there was a significant effed of time in the HlGH group (F
(4, 80) = 3.901, p < 0.01), but no dose x tirne interaction (F(8, 80) = 0.958, p > 0.05). while in the low
group thert? was no main effect of time (F(4, 76) = 1.421. p > 0.05) but a significant dose x time
interaction (F(8, 76) = 2.764, p < 0.01).
4. Bodv Weiaht
Table 4 displays the mean body weights for each dose group. Body weights increased
naturally in al1 groups over the course of the experirnent- ANOVA reveakd no significant differences
in body weight between any of the three test groups, either during the acute test phase or on any of the five repeated phase test days.
Table 4: Mean (SEM.) Body Weight (grams) Measured During Expriment 1
Test Day
Day 6 Day 7 Day12 Day13 Day14 Day15 Day16
0 mgfkO 290.1 9 295.1 3 331.25 336.31 339.25 345.06 347.1 3
(2.91) (2.86) (3.01) (2.74) (3.45) (3.44) (3 -77)
0.5 mgfkg 285.69 297.75 33134 337.1 3 336.69 345.69 352.00 Figure 1: The effect of acute and repeated flibanserin on sugar and chow intake
(Experiment 4).
Individual points represent the mean (+ S.E.M.) sugar (Panel A) or chow (Panel 8) intake, in grams,
for each dose group during the one-hour feeding test session. Arrows indicate the acute and
repeated flibanserin administration pefiods- Tukey's pst-hoc test following a significant main effect of dose revealed a significant reduction in sucrose intake in the flibanserin 5 mg/kg test group during the 5-day repeated administration phase @ < 0.004). - - - - 6 7 8 9 10 11 12 13 14 15 16 Test Day
6 7 8 9 10 11 12 13 14 15 16 Test Day Figure 2: The effect of acute and repeated flibanserin on sugar intake in HIGH or LOW
baseline sugar feeders (Experiment 1).
Animals were classified as a HlGH (upper panel) or LOW (lower panel) sugar feeder. according to
their level of sugar intake on the baseline test day. Individual points represent the rnean (k SEM.)
sugar intake in grams for each group during the one-hour test session. Arrows indicate the acute and
repeated flibanserin administration periods. Tukey's post-hoc tests revealed a significant reduction in sucrose intake in the flibanserin 5 mg/kg test group during the Mayrepeated administration in both
HlGH (p < 0.05) and LOW @ c 0.05) groups. I I 0.00 : 6 7 8 9 10 11 12 13 14 15 16 Test Day
0.00 1 r 6 7 8 9 10 11 12 13 14 15 16 Test Day Experiment 2
The Effects of Flibanserin and FIuoxetine on Responding îbr Sucrose on a Progressive Ratio
of Reinforcement
1. Res~ondinafor Sucrose
Responding for sucrose reinforcement during the baseline tests that immediately preceded
acute and chronic drug administration is displayed in Table 5. Values indicate stable responding
across al1 test groups.
Table 5: Mean (S.E.M.) Baseline Responding for Sucrose in Expriment 2
Baseline Preceding Acute Testing Baseline Preceding Chronic Testing
C I # Lever # Sucrose # Lever # Sucrose
Presses Pellets Obtained Presses Pellets Obtained
Vehicie 208.8 35.0 223.4 35.8
(41 -1) (2.1) (49.4) (2.2)
Flibansefin 135.1 31.3 212.6 34.4
(1 9.0) (1-3) (59.0) (2-5)
Fluoxetine 182.6 34.0 227.6 35.9
(31.6) (1 -9) (48.6) (2-1 I 1 I 1 I
Acute dnig administration produced a significant effect of treatment (F(2, 21) = 8.903, p <
0.002),with Tukey's post-hoc test revealing a significant reduction in break point for sucrose reinforcement in the fluoxetine 10 rng/kg group wrnpared to vehicle @ < 0.003) (Figure 3). but no effect on break point in the flibanserin 5 mg/kg group @ > 0.05). In order to charaderize the nature of responding within the ho-hour test session, the acute test session was divided into four 30-minute intervals and the number of sucrose pellets obtained in each intewal was examined (Figure 4). ANOVA revealed significant effects of treatment (F(2. 21) = 8.90, p < 0.002), time (F(3'63) = 204.7, p
c 0.0000) and treatment x time (F(6, 63) = 8.13, p < 0.0000). In the first 30 minutes, animals in the
fluoxetine group obtained significantly fewer sucrose pellets than control animals @ < 0.0002).
whereas responding during the second, third and final 30-minute time periods did not differ across
treatment groups.
There was decrease in break point following repeated administration of fluoxetine, but not
flibanserin (Figure 5). ANOVA revealed an overall significant main effect of treatment (F(2. 21) =
7.807, p < 0.003; with Tukey's pst-hoc test revealing a significant difference frorn vehicle, p < 0.01,
for the fluoxetine group, but not for flibanserin @ > 0.05). Further. there was a main effect of time
(F(3, 63) = 3.724, p < 0.02), but no significant treatment x tirne interaction (F (6, 63) = 1.706. p >
O.OS), although the effec. of fluoxetine did appear to increase with time. The effed of fluoxetine on
break points appeared to be reversible, with ANOVA revealing that break points rneasured one and
two weeks after the cessation of dnig administration were not significantly different from the control
group.
2. Percent Accuracv for Active Lever
The percent accuracy for the active lever was detemined in order to compare the activity
directed at obtaining sucrose reward against generalized, non-specific lever pressing activity-
Percent accuracy was calculated by dividing the number of responses directed at the active lever by the total number of lever responses direded at both levers (active + inadive)- There was a high
degree of accuracy for the adive lever that did no! significantly differ across the three groups on any of the test days (Table 6). 28
Table 6: Mean (S.E.M.) Percent Accuracy for the Active Lever in Experiment 2
Baseline ' Acute Chronic Chronic Chronic Chronic
Test Day 1 Test Day 2 Test Day 7 Test Day 13
Vehicle 96.0 96.9 97.9 97.8 97.5 97.7
(0.6) (1.1) (0.7) (0.9)
Flibanserin 96.9 98.0 99.0 98.7 99.2 98.4
(0.5) (0.5) (0.4) (0- 5) 11 Ftuoxetine 97.0 99.3 98.2 99.4 (1 -5) (1 -0) (1-7) (0.4) (1 -4) (0-3) representative baseline test, one day prior 1 acute testing
3. Bodv Weiaht n Body weights were assessed during the repeated drug administration phase in ternis of the absolute change from pre-drug baseline (rneasured one day prior to the repeated dnig phase).
During this phase, there was a slight increase in body weight relative to baseline in the fiibanserin and vehicle groups, an expeded result of body weight gain in nomal anirnals (Figure 6). In contrast, body weights in the fiuoxetine group decreased over time relative to baseline (main effect of treatment
(F(2, 21) = 72.65, p < 0.0000). time (F(12, 252) = 10.08, p < 0.0000, and the interaction of treatment x time (F(12,252) = 53.35, p < 0.0000). By the final day of drug administration, the mean body weight in the fluoxetine group had diminished to 91 -5% of the baseline value. One and two weeks following cessation of drug administration. an upward trend in body weight gain was apparent in the fluoxetine group, suggesting a return to control levels. Figure 3: The effect of a single i.9. injection of vehicle, flibanserin 5 mglkg or fluoxetine
1O mglkg on the break point for sucrose reinforcement (Expriment 2).
Bars represent the mean (+ S.E.M.) break point (the final number of sucrose pellets obtained in the 2- hour session) for each group during the baseline and acute test day. 'Denotes statistical significance from vehicie @ c 0.003).as deterrnined by Tukey's post-hoc test.)
Figure 4: The number of reinforcers obtained dunng the 0-30, 30-60, 60-90 and 90-120 minute time intervals of the 24our progressive-ratio test session (Expriment 2).
Individual points represent the mean (+ S.E.M.) number of reinforcers obtained in each 30-minute time bin, following a single i.p. injection of vehide. flibanserin 5 mgkg or fluoxetine 10 rngfkg. '
Oenotes statistical significance from vehicle @ < 0.0002) as detemined by Tukey's pst-hoc test. 30-60 60-90 Time lntewal (minutes)
-t- Fluoxetine Figure 5: The effects of repeated i.p. administration of vehicle, flibanserin 5 mgtkg or fluoxetine 10 mgikg on the break point for sucrose reinforcement (Expriment 2).
Rats were treated for a total of 13 days (indicated with arrows), with operant testing conducted at baseline (Day O), Days 1, 2, 7 and 13. Bars represent the mean (I SEM.) break points (the final number of sucrose pellets obtained in the 2-hour session) for each group on the test days indicated.
Days 20 and 27 represent drug-free test days conducted one and two weeks following the cessation of dnig administration. Tukey's pst-hoc test revealed a significant redudion in break points in the fluoxetine group, cornpared to controls, during the drug administration period (p < 0.02). Test Day
[g Vehicle Flibanserin 5 mglkg Fluoxetine 10 mglkg Figure 6 The effect of repeated i.p. administration of vehicle, flibanserin 5 mglkg or fluoxetine 10 mg/kg on the absolute change from badine body weight (Experiment 2).
Individual points represent the mean (k S.E.M.) difference from baseline body weight (in grams), measured on each day of drug administration (days 1 through 13). and one and two weeks (days 20 and 27) foltowing cessation of drug treatment.
Gperiment 3
The Effects of FIibanserin 1.67,s and iSmg/kg on Operant Responding for Sucrose on a
Progressive Ratio Schedk~leof Reinlbrcement
1 Res~ondinafor Sucrose
Acute flibanserin 15 mglkg significantly attenuated the break point for sucrose reinforcement
(F(3,66) = 10.36, p < 0.00001 1) (Figure 7). In order to characterize the nature of responding within the two-hour test session, the session was divided into four 30-minute intervals and the nurnber of sucrose pellets obtained in each interval was examined. Wdhin the test session, there appeared to be a biphasic time effed of flibanserin 15 mg/kg on the number of sucrose pellets obtained (Figure
8). ANOVA revealed a marked redudion in the number of sucrose pellets obtained in the 0-30 minutes of the session in the flibanserin 15 rngtkg group (F(3,66) = 36.576. p < 0.0000). However, the number of pellets obtained was increased relative to control in the 30-60 and 60-90 minute intervals (30-60 minutes: F(3, 66) = 4.297, p < 0.008, Tukey's post-hoc p < 0.03; 60-90 minutes: F(3,
66) = 10.942, p < 0.0000, Tukey's post-hoc, p < 0.0002). The number of sucrose pellets obtained in the final interval (90-120 minutes) was marginal, with no significant differences between any dose group.
2. Percent Accuracv for Active Lever
The percent accuracy for the adive lever was detemined in order to compare the activity directed at obtaining sucrose reward against generalized. non-specific lever pressing activity.
Percent accuracy was calculated by dividing the number of responses directed at the active lever by the total number of lever responses direded at both levers (active + inadive). The mean (* S.E.M.) percent accuracy for the active lever was similar for al1 four treatment doses: 95.3 +: 0.7% (vehicle),
95.7 + 0.7% (1.67 mg/kg), 96.5 + 0.6% (5.0 rngikg), 96.7 t 0.9% (15 mg/kg). 3. Lever Preference
In order to control for possible preference of the right lever versus the left lever, half the rats
were trained and tested with the left lever designated active 0.e. resulting in sucrose delivery). while
the other half were tested with the right lever designated active. ANOVA revealed no significant
differences in responding when the active lever was located on the right side of the chamber, versus when the lever was located on the left (main effed of lever. F(l, 21) = 0.028, p > 0.05; dose x lever
interaction, F (3, 63) = 0.096, p > 0.05). Figure 7: The effect of a single i.p. injection of flibanserin (0, 1.67,s or 15 mg/kg) on the break point for sucrose reinforcement (Expriment 3).
Bars represent the mean (+ SEM.) break points for each treatrnent dose. ' Denotes statistical significance from vehicle (p < 0.00001 1). as detemined by Tukey's post-hoc test.
Figure 8 The number of reinforcers obtained during the 0-30, 30-60, 60-90 and 90-120 minute time intenrals of the 2hour progressive-ratio test session (Experiment 3). lndividual points represent the mean (f S.E.M.) number of reinforcers obtained during each time interval following a single i.p. injedion of flibanserin (0, 1.67. 5 or 15 mg/kg). Statistical significance frorn vehicie (detemined by Tukey's post-hoc test) is denoted by 'p< 0.01 30 - 60 60 - 90 90 - 120
MinuteTime Pedod The Effecfs of Acufe Flihnserin and Fluoxetine Exploratory Locomotor AwvifV in the Rat
There appeared to be no overall significant effed of either fluoxetine or nibanserin on exploratory locomotor activity (ELA) for the total two hour session (F(2, 21 ) = 0.6945. p > 0.05)
(Figure 9). However, examination of activity over the first, second, third and final 30-minute time intervak revealed a pattern of activity in the fluoxetine group that differed from mritrol anirnals. In the first 30 minutes (Figure 1O), there was a signifiant redudion in ELA in the fluoxetine group cornpared to the vehicle group (F(2, 21) = 4.52, p < 0.03, followed by Tukey's test, p < 0.03). This effect diminished from 30 - 90 minutes, where no significant differences between groups were detected.
However in the final 30 minutes, the fluoxetine group demonstrated a significantly greater ELA cornpared to vehicle (F(2, 21) = 5.73, p < 0.02, foflowed by Tukey's test, p c 0.02). Figure 9: The effect of a single i.p. injection of vehicle, fiibanserin 5 mgIlcg or fluoxetine
10 mglkg on total exploratory locomotor activity (Expriment 4).
Bars represent the mean (2 SEM.) total number of crossovers for each dose group for the two-hour test penod. Vehicle Fli banserin Fluoxetine Figure IO: The effect of a single i.p. injection of vehicle, nibanserin 6 mg/kg or fluoxetine
10 mg/kg on exploratory locomotor activity in the 0-30, 30-60,60-90 and 90-1 20 minute time
intervals of the two-hour test session (Experirnent 4).
Individual points represent the mean (I SEM.) number of crossovers for each group at the time interval indicated, ' Denotes statistical significance from vehicle @ < 0.05), as detemined by Tukey's post- hoc test. 4Ve hicle 4Flibanserin 5 mglkg +Fluoxetine 10 mg/kg
30-60 60-90 Time Interval Discussion and Conclusions - - ppp - -- - -
The present research indicates that in non-deprived rats, the putative antidepressant flibansenn
produced a decrease in sucrose intake in a free-feeding paradigm, and decreased the break point for sucrose reinforcement (the final number of sucrose pellets obtaineû) in an operant progressive ratio
paradigm. These findings support the initial hypothesis that fiibanserin would produce a decrease in sucrose feeding in non-deprived rats. The decrease in sucrose consumption obsewed with flibanserin contrasts with the hyperphagic effects elicited by typical 5-wlA âg0nistS SUC~as 8-OH-
DPAT, effeds that have been atthbuted to activation of somatodendritic S-HTIA receptors. This is relevant because, unlike typical 5-HTIA agonists, flibanserin is purported to act preferentially at post- synaptic 5-HTIAreceptors. a phamacological property which may also confer a more rapid onset of antidepressant response (Borsini, 1994; Blier and de Montigny, 1998).
A dose-dependent reduction in the free-feeding of granulated sucrose was evident following acute administration of flibanserin, though statistically significant reductions were 0nly seen with repeated (five day) administration of the 5 mg/kg dose. This difference between acute and chronic dosing may refiect the phamacokinetics of flibanserin, such that while a single dose may have been sufficient to produce mild hypophagic effects, repeated administration may have been required ta produce adequate drug levels before more substantial, significant effects were obsewed. A potentiation of effeds with repeated drug administration has also been found with clinical dosing of antidepressants, where antidepressant efficacy emerges only after several weeks of repeated administration in depressed patients (Baldessarini, 1990). It has been hypothesized that the delayed onset of eftïcacy obsewed with typical serotonergic antidepressants is associated with the time required for inhi bitory pre-synaptic 5-HTlA autoreceptors to desensitize before p~~t-synapticeffects emerge (Blier and de Montigny, 1994; Blier et al., 1997). Fiibanserin, however, is purported to avoid the penod of pre-synaptic 5-WIAautoreceptor desensitization by virtue of a direct preferential stimulation of post-synaptic receptors (Borsini et al., 1995b), which should confer a more immediate onset of antidepressant efficacy (Borsini, 1994). The observed difference in feeding effeds between
acute and repeated flibanserin administration may appear to argue against a fast-onset of action of
flibanserin, however, this notion cannot be substantiated presently, since it is not dear whether the
sugar feeding model addresses behavioural or pharmacological issues relevant to the onset of
antidepressant effÏcacy. Indeed, there is some preliminary evidence for a fast onset of action with
fiibansetin, since in the 'leamed helplessnessRand 'chronic mild unpredidable stress" animal models
of depression. acute flibanserin has been shown to produce antidepressant-like effects, whereas
reference antidepressants induce such effects only following repeated administration (Borsini et al.,
1997; DTAqui1aet al., 1997). Only clinical tests will confirm whether flibanserin truly produces a more
rapid onset of antidepressant efficacy.
Sucrose versus Chow Feeding
Under baseline conditions in the free-feeding experiment, rats consumed more granulated sucrose than powdered chow, indicating a preference for sugar wtiich is consistent with previous studies (Sills and Vaccarino, 1998; DeSousa et al., 1998). Flibanserin administration caused a reduction in feeding of sugar, but not chow, which rnay sirnply reflect the fact that baseline chow intake was too low for any subsequent reductions in feeding to be observed. However, the fact that only sugar feeding appeared to be affected by fiibansenn also raises the possibility that the drug's hypophagic effects may be selective for carbohydrate-nch foods. Though the macronutnent content was not specifically controlled in the present experirnents. inferences could be made from the sugar- chow choice paradigm, which, qualitatively, represents a high versus low carbohydrate choice. The selective redudion in sugar intake with fiibanserin 5 mgkg is in the opposite direction to the reported effects of the 5-HTIA agonists 8-OH-DPAT and buspirone, which have been shown to selectively increase carbohydrate intake while having no effed on protein intake (Luo et al., 1990)- Because flibanserin may ad preferentially at post-synaptic 5-HTjAreceptors, whereas buspirone and 8-OH-
DPAT are thought to act preferentially at somatodendritic 5-HTIA receptors, the opposing effects on carbohydrate intake provide another Iine of evidence to support the concept that pre- and pst- synaptic 5-HTiA receptors mediate opposite effects on the 5-HT system. Consistent with flibanserin's decrease in sugar intake, the SSRl antidepressant fluoxetine has also kenshown to selectively decrease carbohydrate intake (Luo et al., 1990). a finding which further charaderizes flibanserin's
similanties to the antidepressant class of drugs.
Flibanserin did not produce any effects on body weight during the repeated administration phase of Experiment 1, which may indicate that rats were consuming nomal amounts of regular ch0w outside the experimental test session. The presumption that chow feeding was unaffeded by flibanserin treatment could provide further evidence to indicate a selective effed on sugar intake, though such a conclusion would require substantiation in more detailed feeding experiments examining macronutrient consumption.
Individual DHerences in Free Feeding of Sucrose
Previous research has suggested that individual differences in baseline sugar intake can predict differential responsivity to various behavioural and pharrnacological challenges (DeSousa et al., 1998; Sills and Vaccanno, 1994; Sills et al., 1998). To extend such findings, Experiment 1 incorporated a measure of 'highn versus 'low" baseline sugar feeding animals to detemine whether individual differences in feeding response could be detected following flibanserin challenge. Results indicated that with acute flibanserin administration, a dosedependent trend toward a reduction of sugar intake was more apparent in the 'highn versus "lown animals, though the trend was non- significant. Repeated flibanserin significantly reduced sugar intake in both 'highn and 'lown animals, while chow intake was unaffeded in both 'highsn and 'lows" with acute and repeated flibanserin.
These results suggest that while there may a slight selectivity for a reduction in sugar intake in 'high" versus "lown animals with acute treatment, overall, rats do not demonstrate substantial individual differences in their feeding response to flibanserin. Progressive atio experimen ts
Following the observation that flibanserin 5 mg/kg decreased sugar feeding in Experirnent 1,
this effed was further expiored in an operant feeding paradigm where rats were required to lever-
press under a progressive ratio (PR) schedule of reinforcement to obtain successive rewards. The
PR paradigm has been used extensively to study the rewarding efficacy of dmgs of abuse (for a
review, see Richardson and Roberts. 1996). and has also been used to study the effeds of dmgs on other general reinforcers such as food (Roberts et al., 1994) or sweet solutions (Barr and Philiips,
1998). The PR paradigm differs from fixed-ratio operant paradigms in that it is used to assess not only whether a particular stimulus (in the present research, a sucrose pellet) can adas a reinforcer, but also to assess the degree of reinforcing efficacy. Typically, an increase in break point indicates an increase in reinforcing efficacy (and is observed with typical drugs of abuse), while a decrease in the break point may indicate a decrease in reinforcing effcacy (Richardson and Roberts, 1996). In the present experiments, the PR paradigm was employed to determine whether flibanserin and fluoxetine affected the rats' motivation to obtain sucrose rewards.
The results of the present PR experiments indicate that acute flibanserin 15 mgkg, as well as acute and repeated fluoxetine 10 mglkg induced a significant decrease in break points. The finding that fluoxetine reduced the number of sucrose pellets obtained suggests a hypophagic effed of the dmg which is consistent with previous studies of fluoxetine and other SSRls (Wong et al., 1988; Lucki et al., 1988; Jackson et al.. 1997). and supports the validity of the progressive ratio operant paradigm for detecting the hypophagic effects of SSRIs. The dose of fluoxetine used in this experiment, 10 rnglkg, is considered to be sufficient to block 5-HT reuptake (Fuller et al., 1994). indicating that the behavioural effects observed in the present experiment are indeed within the active dose range of fluoxetine. Although the intake of regular chow was not measured outside the experimental test sessions, the signifiant decline in body weight throughout repeated fluoxetine treatment rnay suggest that regular food intake was also attenuated, which is consistent with previous findings showing a reduction in 24-hour food intake following SSRl treatment (Heisler et al., 1997). Interestingly, the pattern of operant responding within the two hour session differed for acute
flibansenn 15 mg/kg (Expriment 3) and acute fluoxetine 10 rnglkg (acute test day in Expenrnent 2).
Fluoxetine produced a significant redudion in the nurnber of sucrose pellets obtained in the first 30
minutes of the test session, with no subsequent effects the other intervals, suggesting that
ffuoxetine's feeding effeds were lirnited to the first 30 minutes. Flibanserin also produced a
significant decrease in the nurnber of reinforcers obtained in the first 30 minutes, but later produced a
significant increase in the nurnber of reinforcers obtained during the 30-60 and 60-90 minute intervals
relative to control animals.
This difference in pattern of responding for flibanserin and fluoxetine may be explained by the
different phamawkinetics of these two drugs. Fluoxetine is metabolized more slowly than
flibanserin: in rats, the mean half-life of elimination (t~~)for a single oral dose of fiibanserin 5 rng/kg
(1 5 mglkg has not been tested) has ken reported as approxirnately 1.9 hours (Boehtinger lngelheirn
GmbH, data on file), compared with a mean value of approximately 7.7 hours for a single oral dose of
fluoxetine 10 mg/kg (Caccia et al., 1990). Further, fluoxetine is metabolized to the active metabolite
norfiuoxetine, which is eliminated even more slowly frorn plasma than the parent drug. It is, therefore,
Iikely that fluoxetine, with its slower elimination time, produced its influence on responding throughout the entire 2-hour test session, whereas flibanserin, with a faster elirnination, produced shorter lasting
effects which may have diminished during the session. At the point where flibanserin's effects began to diminish, the rats would not have obtained as rnany sucrose pellets as those in the vehicfe group, and would require fewer lever presses to obtain subsequent ones. Wth this easier work requirernent, the fl ibanserin-treated animals would then be more likeiy to demonstrate increases in responding relative to vehicle animals, which may explain the observed increase in responding in the flibanserin group at 30-90 minutes of the test session compared to controls. The appearance of such biphasic time effects highlights the imporiance of timing the start of a test session with respect to drug administration, since certain effects may be missed depending on the phamacokinetic profile of the test compound and the timing of the test, The fact that fli banserin was able to reduce sugar feeding in both free-feeding and an operant
progressive ratio models suggests that the drug affects feeding mechanisms which are common to
both types of paradigms. However, there may exist a difFerence in the sensitiv-w to flibanserin within these two behavioural paradigrns, since 5 mgkg produced no effects in break points for sucrose
reinforcement either following acute or repeated administration, whereas the same dose did produce a decrease in sucrose intake in a free-feeding paradigrn. One possible explanation for this discrepancy is that the well-trained operant response is more resistant than free-feeding to disruption by phamacological means, such that a higher dose of flibanserin was necessary to alter progressive- ratio responding than free feeding. Different dose sensitivity to operant versus free-feeding paradigms has also previously been reported by Ebenezer (1996). though other methodological considerations (male versus fernale subjects, different timing of drug administration) rnay have conttibuted to the differences in that study. The possibifity of differential sensitivity to the two feeding paradigms warrants confirmation in future experiments.
It may be of note that the PR schedule emptoyed in the present experiments was somewhat less demanding than other schedules that have been used with food (Roberts et al., 1994) or sucrose solution (Barr and Phillips, 1998) as reinforcers Therefore, although effects were observed in the present experiments with both fluoxetine and flibanserin, the magnitude of effed may have been different had the dernand characteristics been greater. It should also be noted. however, that in the present experiments. under training conditions with the least demanding PR schedule, rats would work to obtain approximately 90 sucrose pellets (data not shown), whereas under the more difficult test schedule rats would obtain only approximately 30-35 sucrose pellets at baseline (Experiments 2 and 3). Therefore, under the test schedule rats did not consume enough sucrose pellets to reach satiety. This indicates that the break point was not sirnpiy a rneasure of satiety, but was in fact a function of the work required to obtain sucrose reinforcement. Feeding and Reward
The relevance of a redudion in break point for sucrose reward extends beyond a general discussion of hypophagic effects. The fact that this effed was demonstrated in the progressive ratio operant paradigm, which is purporteâ to measure reinforcing effÏcacy (Richardson and Roberts, lW6), indicates that flibanserin and fluoxetine may produce a decrease in the motivation to respond for sucrose, and raises the possibility that serotonergic antidepressants produce a general decrease in rewarded behaviours. Evidence to support this notion can be found in previous research studying the effeds of serotonergic manipulations on different types of reward. For exampie, fluoxetine has been shown to decrease the intravenous self-administration of cocaine (Richardson and Roberts,
1991; Carroll et al., 1990), and the intake of a sweet solution (Carroll et al., 1990), and to devate reward thresholds for intracranial self-stimulation of the ventral tegmental area or median forebrain bundle (Lee and Kometsky, 1998). al1 of which are effeds consistent with a decrease in rewarded behaviour. Further, Higgins et al. (1993) found that the indirect 5-HT agonist dexfenfluramine caused a decrease in heroin self-administration, an effed which was attenuated by the non-selective 5-HTl~ antagonist metergoline, suggesting the involvement of 5-HTIR receptors in reinforced behaviour.
Complementary findings of Roberts et al. (1994) indicate that 5.7-DHT lesioning of the 5-HT system, which produces a decrease in extracellular 5-HT, elicits an iiicrease in reward as evidenced by an increase in break points for cocaïne or food reward. These findings suggest that increasing 5-HT neurotransmission can effect a decrease in motivated behaviours (Amit et al., 1991), while decreases in 5-HT may produce the opposite effect (Richardson and Roberts, 1991). The present results are consistent with this notion, since both flibanserin and fluoxetine (direct and indirect 5-HT agonists. respectively) were found to decrease the rewarding efficacy of palataMe sucrose.
ft is notable that in contrast to the evidence suggesting a decrease in reward with antidepressants in freely behaving animals, several researchers have found antidepressant treatment to increase rewarded behaviours in a stress-related model of depression. In such studies, rats or mice that have been subjected to mild chronic stress demonstrate a reduction in their consumption of a palatable sucrose solution compared with non-stressed controls. This 'stress-induced" reduction in sucrose consumption has been described as an anhedonic effect. and is thought to provide face validity for the model, since 'anhedonia", or a reduction of pleasure in adivities that once were pleasurable, is one of the cardinal features of dinical depression mtlner, 1997). The reduction in sucrose consumption, or 'anhedonia", is attenuated following treatment with various typical antidepressants (Muscat et al., 1992; Cheeta et al., 1994; Monleon et al., 1995), as well as flibansenn
(D'Aquila et al., 1997), an effect which has been purported to model antidepressant efficacy. Based on these observations, it appears that 'stressed" animals can actually demonstrate an Mcrease in rewarded behaviours following antidepressant treatment. This potential difference in responsiviîy to antidepressant treatment between 'stressed" and *non-stressed" animals raises interesting questions about the baseline fundioning of the serotonin system and the effects of antidepressants on reward- relevant behaviours in depressed and non-depressed individuals. Such issues should be expiored in future studies.
Feeding and Activï2y
Acute fluoxetine 10 rngtkg. in addition to decreasing break points for sucrose reinforcement, also altered locomotor activity. Such alterations were not apparent in the total locomotor activity score for the two hour test session, however, examination of 30-minute time intervals revealed a biphasic time effect of the dnig. There was a significant reduction in adivity in the first 30-minutes of the session, which also corresponded to the time in which the maximum reduction in sucrose pellets obtained was observed with operant responding. In the final 30-minute time interval (90-120 minutes), locomotor activity was slightly increased relative to control animals, which may account for the fact that the total locomotor adivity score for the fluoxetine treated anirnals did not significantly differ from control animals.
The observed reduction in locomotor adivity in the fluoxetine group within the first 30-minutes of the test session could indicate a reduced motivation to explore an environment, an interpretation which, together with the reduction in sucrose break points observed during this time period in the PR experiments, could suggest a general reduction in motivated behaviour following acute fluoxetine administration. This notion rnay be viewed as consistent with the previously discussed effects of
fluoxetine to attenuate behaviours directeci at vanous rewards, such as cocaïne self-administration
and intracranial self-stimulation.
It is also possible that the reduction in locomotor adivity represents a general sedative effect of
fluoxetine that could also have caused deficits in responding for sucrose in the PR experirnent.
Indeed, fluoxetine and other serotonergic antidepressants have kenshown to produce decreases in
locomotor activity in animais, and sedation is a common dinical side effed of these drugs (Tucker
and File, 1986). Nonetheless, sedation rnay not entirely account for the obsetved reduction in
sucrose intake, since fluoxetine and other antidepressants have also been shown to alter specific components of feeding which rnay be independent of sedative effeds. Studies of animal feeding
behaviour have led to the charactenzation of a senes of typical behaviours that comprise a
"behavioural satiety sequence". The sequence begins with feeding, followeâ by various active behaviours including exploration and grooming, then resting (see Simansky, 1996 for a review).
Evidence suggests that enhancing 5-HT neurotransmission can alter this pattern of satiety. For example, the SSRls fluoxetine, paroxetine, femoxetine and sertraline, and the 5-HTlAIlBagonist eltoprazine have been shown to accelerate the onset of satiety, as evidenced by decreased feeding and increased resting, without significantly altering other components of feeding (McGuirk et al.,
1992a; McGuirk et al., 1992b; Simansky and Vaidya, 1990). The fact that the adive khaviours were not reduced in these experiments rnay suggest that drug-induced sedation was not a primary factor in the hypophagic effects of these drugs. Thus, in the present experiments the reductions in locomotor activity and sucrose intake rnay reflect completely different aspects of fluoxetine's behavioural effects.
Acute flibanserin 5 mg/kg produced no significant effeds on locomotor activity, even though in the free feeding paradigm this dose of flibanserin did reduce sucrose intake in a dose-dependent trend. This finding rnay indicate that the mild hypophagic effects of acute flibanserin in this paradigm were not related to sedative effeds. In the operant feeding model, acute flibansenn 15 mglkg significantly attenuated break points for sucrose reinforcement; however, this dose of flibanserin was not tested for its effects on locomotor adivity. so it is unknown whether sedation contributed to the PR effects obsewed with this dose. The effects of flibanserin on locomotor activity and the behavioural satiety sequence should be determined in future experiments.
The Role of &HT Receptots in Flibanserin's Feeding EffectS
The reduction in sucrose feeding following flibansenn is consistent with the hypophagic effects associated with many serotonergic antidepressant drugs. and provides further evidence that enhanced serotonergic activity causes a redudion in feeding (Samanin, 1989; Simansky, 1996).
However, while these typical antidepressants act to increase overall levels of 5-HT through blockade of the 5-HT reuptake mechanism, flibanserin acts diredly at post-Synaptic S'HllA and 5-HT2~ receptors without having appreciabie binding affinity for other major receptor subtypes (Borsini et al.,
1998). This specific pharmacology suggests that 5-HTIA andlor 5-HTx receptors may mediate flibansenn's hypophagic effects.
Although the present experiments were not designed to specifically address the question of which receptor subtypes mediate flibanserin's feeding effeds, there is converging evidence to suggest that the post-synaptic 5-HTlAreceptor may be involved. As previously discussed, pre- and post-synaptic S-HT,* receptors cm mediate opposite effects on 5-HT neurotransmission: both receptors cause neuronal hyperpolarization, though while stimulation of post-synaptic 5-HTIA receptors results in the net inhibitory effect of 5-HT in brain regions under serotonergic control, the net effect of pre-synaptic 5-HTIA receptor stimulation is to shut down the 5-HT cell and decrease 5-HT output (see De Vry, 1995). Based on the notion that pre- and post-synaptic 5-HTIA receptors mediate opposing physiological roles, it was hypothesized that flibanserin would produce hypophagia, a reduction in feeding, which is opposite to the hyperphagia associated with pre-synaptic 5-HT1, receptor activation.
In the present experiments, flibanserin produced hypophagic effects in two models of sucrose feeding, but did not induce feeding at any test dose lower than the anorectic doses. For example, in Experiment 3, 15 rnglkg reduced responding for sucrose, however, a 9-fold lower dose, 1.67 mg/kg, produced no effed. Though further low doses would need to be tested, the lack of a biphasic dose effect on feeding with the dose range tested in the present expeflments may suggest that the drug does not produce feeding effects through pre-synaptic 5-HTlk receptorç, distinguishing flibanserin from other typical 5-HTIA agonists. This finding , demonstrated in a behavioural paradigm, extends the eledrophysiological evidence that flibansen'n selectively activates post-synaptic 5-HTln receptors
(Borsini et al., 1995b. Rueter et al., 1998). Additionally. although the present experiments did not specifically test for evidence of the serotonin syndrome, previous studies of flibanserin have shown that the syndrome is only elicited with doses stafting at 64 mg/kg (Borsini et al., 1998). much higher than the highest test dose of 15 mg/kg in the present experiments; on the other hand, the 5-HTln agonist 8-OH-DPAT induces hypophagia at high doses which also elicit the serotonin syndrome
(Simansky and Vaidya, 1990). This observation may provide preliminary evidence that the hypophagia induced by flibanserîn is not due to a general behavioural disruption from the serotonin syndrome. Taken together, these findings provide some supportive evidence that flibanserin produces hypophagia through preferential adivation of post-synaptic 5-HTIA receptors, at doses which do not significantly disrupt other behaviours.
In support of the concept that post-synaptic S-HTTAreceptors mediate hypophagic effeds,
Vickers et al. (1 996) have shown that a high dose of 8-OH-DPAT, which probably stimulates post- synaptic 5-HTIA receptors, elicited a decrease in wet mash feeding which was completely reversed by the 5-HT,, antagonist WAY100635. On the other hand, some investigators have argued against a role for the 5-HTln receptor in mediating the hypophagia associated with serotonergic agents. based on evidence that blockade of 5-HTIA recepfors with pre- and post-synaptic 5-HTi~antagonists such as WAY 100635 or WAY 100135 do not modify the hypophagic effects of the 5-HT enhancing drugs d-fenfluramine or fluoxetine, or the 5-HT precursor 5-hydroxytryptophan (Vickers et al., 1996;
Ciccocioppo et al., 1997). However, such findings would not refute the possibility of involvement of the 5-HTIA receptor in mediating nibanserin's hypophagic effeds, since it is possible that in the aforementioned experiments, hypophagic effects were supported by more than one receptor subtype. 5-HT precursors, releasers and reuptake blockers act to increase overall levels of endogenous 5-HT,
so that bloc~ingone receptor subtype (e-g. the 5-Hf,, receptor) with a selective antagonist would
enhance the synaptic availability of 5-HT for binding with other 5-HT receptors that could rnediate
hypophagia. Indeed, other 5-HT receptors have been implicated as important in producing
hypophagic effects. For exampie, stimulation of 5-Hf,B and / or 5-HTzc receptors using agents with
varying degrees of selectivity, such as RU 24969, mCPP, and 001, can also reduce feeding in rats
(Bendotti and Samanin. 1987; Simansky and Vaidya, 1990). The relevance of the present findings is
that flibanserin's inhibitory effect on sucrose feeding cannot be attributed to other 5-HT,, 5-HTzc, 5-
HT3.4. 5.6 or 7 receptors, or the 5-HT reuptake site since the drug does not possess appreciable affinity
for these other receptors (Borsini et al., 1998). The involvement of the post-synaptic 5-HTIA receptors
in flibanserin's feeding effects warrants further exploration in controlled experiments that are
specifically designed to address this question.
In addition to stimulating the post-synaptic 5-HT,, receptor, flibanserin also acts as an antagonist at 5-HT2~receptors, though prediding the relative contribution of 5-HlzAantagonism to flibanserin's hypophagic effects is cornplex, since previous literature has not provided a clear understanding of the role of these receptors in feeding. Based on electrophysiological and behavioural evidence, which suggests a fundional interaction between 5-HT,, and 5-HT2Areceptors
(Lakoski and Aghajanian, 1985; Ashby et al., 1994; Berensden 1995), it would be predided that a 5-
HT2Aantagonist could enhance the fundional activity of the post-synaptic 5-HT,, receptor. Indeed, it is possible that flibanserin's purported preferential adivity at post-synaptic S'iiTlA receptors is influenced by the concurrent antagonism at post-synaptic 5-HTU\ receptors. In feeding rnodels, if post-synaptic 5-HTi~and 5-HTzAreceptors have sorne role in controlling feeding, a 5-HTa antagonist might be expected to decrease feeding when adrninistered alone, or to enhance the hypophagic effects of post-synaptic 5-HTIA agonism- However, previous literature does not provide consistent support of this notion, since antagonists that block 5-HTtA receptors have been found to increase
(Fletcher, 1988), decrease (Wong et al., 1988) or have no effea (Massi and Marini, 1987) on food intake in rats. Further, they may reverse (Grignaschi et al., 1992; Halford and Blundell, 1996). or have no effect (Lucki et al., 1988; Wong et al., 1988; Lightowler et al., 1996) on the hypophagic effects of SSRls. The results of the present expenments provide no indication of how 5-HT, antagonism contributes to flibanserin's effed, since no attempt was made to control for this variable.
Further studies are required to confimi if, and how. 5-HTZAantagonism moderates the hypophagic effects of flibanserin.
Conclusions
ln conclusion, the resutts of these expenments indicate that the 5-HTIA agonist f ~H&A antagonist flibanserin decreased the intake of sucrose in nondeprived rats. This finding was demonstrated in two different feeding paradigms, one investigating the free-feeding of granulated sucrose and powdered chow, and the other, an operant paradigm which required responding on a progressive ratio schedule for sucrose. The overall effect of fibansefln to decrease the break point for sucrose reinforcement on progressive ratio schedule was also obsefved with the comparator antidepressant fluoxetine. However, the rats treated with fiuoxetine and flibanserin differed in their time course of responding for sucrose within the test session, which may refled the different pharmacokinetic profiles of the two dnigs. The finding of decreased sucrose intake with flibansetin is consistent with the widely accepted view that enhanced serotonin neurotransmission causes a reduction in food intake, and further, based on flibanserin's mechanism of action, provides preliminary evidence that post-synaptic 5-HTIA receptors are involved in mediating this hypophagic effect. Implications, New Research Questions and Future Directions
The present research provided evidence that flibanserin decreases free-feeding and operant feeding of sucrose in non-depn'ved rats. This research has imrnediate implications for the study of serotonergic antidepressants and feeding. It represents the first known examination of the effeds of a putative post-synaptic selective 5HTlAagonist on feeding in rats. The fact that nibanserin decreased sucrose feeding is consistent with the notion that pre- and post-synaptic 5-HT1* receptors mediate opposite physiological effects, and provides an indication that post-synaptic 5-rn1~receptors support hypophagic effects.
These results also have broader implications, and may contribute to the understanding of the serotonin SyStem and its role in depression and eating disorders. The post-synaptic S-HT,A receptor has been impiicated as an important locus for antidepressant effects, and knowing that this receptor may also mediate hypophagic effects will help to further understand the rnechanisrn of action of antidepressant dnigs and their side effects. Additionally, vanous serotonergic agents. such as fenfluramine and sibutramine, have already been explored for their potential anorectic properties in obese patients. Knowing which 5-HT receptor subtype(s) mediate the effects of these drugs will help to further elucidate the role of the 5-Hf system in the control of feeding.
Due to the lirnited scope of this projed, several important unanswered questions remain to be addressed in future studies. These questions are outlined in, but are not limited to, the following points:
1. Receptor Subtype(s) Mediating Flibanserin's Effects on Feeding Flibansetin has been described as an agonist at pst-synaptic 5-Hf,, receptors and an antagonist at 5-HT2~receptors, with no appreciable affinity for other 5-HT or other major CNS receptors (Borsini et al., 1998). As discussed above, it is plausiMe that post-synaptic 5-HTln receptor activation mediates the hypophagic effeds of this drug, though this hypothesis would need to be confirmed with funher experiments. Thus, one future direction arising from this research would be to systematically explore the involvement of 5-HT receptors on flibansenn-induced feeding effeds.
Such a systematic investigation could involve convergent approaches to study receptor mechanisms and feeding. inciuding investigations of a) peripheral versus central receptors (using
5-HT agonists and antagonists selective for central or peripheral receptors, and by peripheral vs. intracerebroventricular injedion of flibanserin), b) pre- versus post-synaptic receptors (using central injections of flibanserin, alone or in combination with selective antagonists, in pre- versus post- synaptic brain regions), and c) diwerent S-HT receptor subtypes (using antagonists selective for 5-
HT receptor subtypes).
2. Effect of Flibansenn on Feeding Patterns and Microstructure
The present experiments determined that flibansenn is capable of inducing an overall decrease in sucrose feeding in free-feeding and operant progressive ratio feeding paradigms. However, these findings do not provide an indication of how flibansefln alters feeding. Investigations of rneal patterning would provide valuable information on whether flibanserin aiters parameters such as meal number, duration and frequency. Further, several investigators have adopted the approach of studying the 'behavioural satiety sequence" as an indication of which components of feeding, within a meal, are affeded by dmg manipulations. Therefore, another important new direction aflsing from the present research would be to explore the effeds of flibansefin on the feeding patterns and the behavioural satiety sequence in freely feeding rats. This investigation would provide an indication of how flibanserin alters feeding behaviours, and particulady, whether or not flibansefln decreases feeding in a manner similar to other antidepressants.
3. Effect of Flibanserin on Macronutrient Consumption
The present research also provided some indication that flibansefin aitered sucrose intake, but not chow intake, during free-feeding sessions, raising the possibility that flibanserin selectively aners specific food types. Therefore, an additional issue to be addressed in feeding studies would be to determine whether flibanserin produces differential effeds on controlled carbohydrate, protein or fat diets.
4. Effects of Flibanserin on Motor Performance and Feeding
Expriment 4 indicated that acute fluoxetine produced a decrease in locomotor activity in the first 30-minutes of a locomotor test session. which corresponds to the time period in which the reduction in operant responding for food was most apparent. Expriment 4 also indicated that flibansenn 5 mglkg, the dose that mildly suppressed the free feeding of sucrose, did not atter locomotor activity. However, since these expenments were not designed to examine locomotor activity in detail, the results do not provide a complete indication of how flibanserin and fluoxetine affect locomotor activity, and whether alterations in motor activity influence feeding behaviour. Thus, future expenments should be camed out to detemine in a more controlled fashion the effeds of a broad range of flibanserin doses on feeding and exploratory locomotor activity, in cornparison with other reference antidepressants such as fluoxetine, and other typical 5-HTtaagonists such as 8-OH-
DPAT.
5. The Effects of Flibanserin on Other Reward-Relevant Behavioun
The present research indicates that both flibanserin and the comparator antidepressant fluoxetine decrease the break point for sucrose reinforcement in rats working under a progressive ratio schedule. This paradigm is particularly sensitive to examining alterations in reinforcing efficacy, and one interpretation of the present finding is that flibanserin and fluoxetine produce a decrease in the rewarding efficacy of sucrose. To extend this finding, investigations could be cctmed out to detemine whether this reward alteration was specific to sucrose, or could be generalized to regular chow, and even to non-food reward. Such a question could be addressed by studying fiibanserin's effects on operant responding for regular chow pellets, and on non-ingestive rewards such as intracranial self-stimulation or psychostimulant reward and, further, in non-operant paradigms such as the conditioned place preference paradigm. Such investigations could further the understanding of the interactions between the serotonergic system and reward mechanisms, which is particularly relevant given that diminished reward or 'pleasure" is considered to be a primary feature of clinical depression.
6. The Effects of Flibanserin on Eating 8ehaviour in Human Subjects
The use of different serotonergic agents. such as dexfenfluramine and sibutramine, in weight loss programs in humans highlights the dinical utility of studying the rote of serotonin in feeding.
Since flibanserin appears to produce anorectic effeds in animals, the question of its utility in human weight loss also anses. Thus, future studies could be direded at assessing flibanserin's effects on feeding behaviour in clinical trials in humans. References
Amit Z, Smith BR, Gill K (1 991) Serotonin uptake inhibitors: effects on rnotivated corisumrnatory
behaviors. Journal of Clinical Psychiatry 52: 55-60
Araneda R, Andrade R (1 991) 5-Hydro~ytryptamine~and 5-hydro~ytryptarnine,~receptors mediate
opposing responses on membrane excitability in rat association cortex. Neuroscience 40:
339-412
Artigas F, Romero L. De Montigny C, Blier P (1996) Acceleration of the effed of selected
antidepressant drugs in major depression by 5-HTIA antagonists. Trends in Neuroscience 19: 378-383
Ashby CR Jr., Edwards E, Wang RY (1994) Electrophysiological evidence for a fundional interaction
between 5-HTiA and SHT2Areceptors in the rat medial prefrontal cortex: an iontophoretic
study. Synapse 17: 173-181
Bakish O, Lapierre YD, Weinstein R, Klein J, Mens A, Jones B, Hom El Browne M, Bourget D,
Blanchard A, Thibaudeau C, Waddell C, Raine O (1993) Ritanserin. imipramine and placebo
in the treatment of dysthymic disorder. Journal of Clinical Psychophamacology 13: 409-414
Baldessarini RJ (1990) Dmgs and the treatment of psychiatrie disorders. In: Goodman Gilman A, Rall
TW, Nies AS, Taylor P (eds) Goodman and Gilman's The Phannacological Basis of
Therapeutics. Eighth edition. Pergamon Press, New York, pp 383-435
Barr AM, Phillips AG (1 998) Chronic mild stress has no effect on responding by rats for sucrose under
a progressive ratio schedule. Physiology and Behavior 64: 591-597
Bel N, ARigas F (1993) Chronic treatment with fluvoxamine increases extracellular serotonin in frontal
cortex but not in raphé nuclei. Synapse 15: 243-245 Bendotti C, Samanin R (1986) 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) elicits eating in
free-feeding rats by acting on central serotonin neurons. European Journal of Pharmacology
121 : 147-150
Bendotti C, Samanin R (1987) The role of putative 5-HT ,A and 5-HTls receptors in the wntrol of
feeding in rats. Life Sciences 41 : 635-642
Berensden HHG (1995) Interactions between Shydroxytryptarnine receptor subtypes: is a disturbed
receptor balance contnbuting to the symptomatology of depression in humans?
Pharmacology and Therapeutics 66: 17-37
Bersani G, Poui F, Marini S, Grispini A, Pasini A, Ciani N (1991) 5-HTz receptor antagonism in
dysthymic disorder. a double-blind pîacebo-controlled study with ritanserin. Acta Psychiatn'ca
Scandinavica 83: 244-248
Blier P, De Montigny C (1985) Serotoninergic but not noradrenergic neurons in rat central nervous
system adapt to long-terni treatment with monoamine oxidase inhibitors. Neuroscience 16:
949-955
Blier P. De Montigny C, Chaput Y (1 987) Modifications of the serotonin system by antidepressant
treatments: implications for the therapeutic response in major depression. Journal of Clinical
Psychopharrnacology 7: 24S-35s
Blier P. De Montigny C (1994) Current advances and trends in the treatment of depression. Trends in
Pharrnacological Science 15: 220-226
Blier P. Bergeron R (1 995) Effectiveness of pindolol with selected antidepressant dmgs in the
treatment of major depression. Journal of Clinical Psychopharrnacology 15: 21 7-222
Blier P, Bergeron R, De Montigny C (1 997) Selective activation of postsynaptic S-HTIA receptors
induces rapid antidepressant response. Neuropsychopharrnacology 16: 333-338 Blier P. De Montigny C (1 998) Possible serotonergic mechanisms underlying the antidepressant and
anti-obsessive-compuIsive disoder responses. Biological Psychiatry 44: 31 3-323
Borsini F (1 994) Balance between cortical 5'Wl~and 5-Hf2 receptor function: hypothesis for a faster
antidepressant action. Phamacological Research 30: 1-1 1
Borsini F, Giraldo E, Monferini E, Antonini G, Parenti M, Bietti G, Donetti A (1 995a) BIMT-17, a 5-
HTU\ receptor antagonist and 5-HT,A receptor full agonist in rat cerebral cortex. Naunyn-
Schmiedeberg's Archives of Phannacology 352: 276-282
Borsini F, Ceci A, Bietti G, Donetti A (1 995b) BIMT-17, a 5-HTIA receptor agonist15-HTzAreceptor
antagonist, diredly activates postsynaptic 5-HT inhibitory responses in the rat cerebral cortex.
Naunyn-Schmiedeberg's Archives of Pharmacology 352: 283-290
Borsini F, Cesana R, Kelly J, Leonard BE, McNamara M, Richards J, Seiden LS (1997) BIMT-17: a
putative antidepressant with a fast onset of action? Psychophannacology 134: 378-386
Borsini F, Brambilla A, Ceci A, Cesana R, Giraldo E, Ladinsky H. Monferini E, Turconi M (1998)
Flibanserin - Antidepressant, 5-HT1* receptor agonist, 5-HTU\ receptor antagonist. Drugs of
the Future 23: 9-16
Caccia S. Cappi M. Fracasso C, Garattini S (1990) Influence of dose and route of administration on
the kinetics of fluoxetine and its metabolite norfluoxetine in the rat. Psychophamacology
100: 509-5 14
Carroll ME, Lac ST, Asencio M, Kragh R (1990) Fluoxetine reduces intravenous cocaine self-
administration in rats. Phamacology, Biochemistry and Behavior 35: 237-244
Ceci A, Fodritto F, Borsini F (1994) Repeated treatment with fluoxetine decreases the number of
spontaneously active cells per track in frontal cortex. European Journal of Pharmacology
271 : 231-234 Cesana R, Ciprandi C, Borsini F (1995) The effects of BlMT 17, a new potential antidepressant, in the
forced swimming test in mice. Behavioral Phamacology 6: 688-694.
Cheeta S. Broekkamp C. Willner P (1994) Stereospecific reversal of stress-induced anhedonia by
mianserin and its (+)-enantiomer. Psychopharmacology 116: 523-528
Ciccocioppo R, Panocka 1, Polidori C, Dourish CT, Massi M (1 997) Blockade of pre- and post-
synaptic S-HT,Areceptors does not rnodify the effect of fluoxetine or 5-hydroxytryptophan on
ethanol and food intake in rats. Psychophamacology 134: 55-63
D'Aquila P, Monleon S. Borsini F, Brain P, Willner P (1 997) Anti-anhedonic actions of the novel
serotonergic agent flibanserin, a potentially rapidly-acting antidepressant. European Journal
of Phamacology 340: 121-1 32
De Vry J (1995) 5-HT1~receptor agonists: recent developments and wntroversial issues.
Psychopharmacology 121: 1-26
Depoortere RY. Li OH. Lane JD, Emmett-Oglesby MW (1993) Parameters of self-administration of
cocaïne in rats under a progressive-ratio schedule. Phamacology, Biochemistry and
Behavior 45: 539-548
DeSousa NJ, Wunderiich GR, De Cabo C. Vaccarino FJ (1998) Individual differences in sucrose
intake predict behavioral reactivity in rodent models of anxiety. Phannacology, Biochemistry
and Behavior 60: 841-846
Dourish CT, Curzon G (1 985a) Characteristics of feeding induced by the serotonin agonist 8-hydroxy-
2-(di-n-propylamino) tetralin (8-OH-DPAT). Brain Research Bulletin 15: 377-384
Dourish CT. Hutson PH, Curzon G (1985b) Low doses of the putative serotonin agonist û-hydroxy-2-
(di-n-propylamino) tetralin (8-OH-DPAT) elicit feeding in the rat. Psychophannacology 86:
197-204 Ebenezer IS (1 996) Effeds of buspirone on operant and nonoperant food intake in food deprived rats.
Methods and Findings in Experimental Clinical Pharmacology 18: 475-480
Fletcher PJ (1 988) lncreased food intake in satiated rats induced by the 5-HT antagonists
rnethysergide, rnetergoline and ritanserin. Psychopharmacology 96: 237-242
Fletcher PJ, Zack MH, Coscina DV (1991) Influence of taste and food texture on the feeding
responses induced by 8-OH-DPAT and gepirone. Psychopharmacology 104: 302-306
Fuller RW, Hernrick-Luecke SK. Snoddy HD (1994) Fluoxetine at a~orecticdoses does not have
properties of a dopamine uptake inhibitor. Journal of Neural Transmission [General Section]
6: 165-177
Gartside SE. Umbers V, Hajos M. Sharp T (1 995) Interaction between a selective 5-HTIA receptor
antagonist and an SSRl in vivo: effects on 5-HT cell firing and extracellular 5-HT. British
Journal of Pharmacology 115: 1064-1070
Gilbert F, Dounsh CT (1987) Effects of the novel anxiolytics gepirone, buspirone and ipsapirone on
free feeding and on feeding induced by 8-OH-OPAT. Psychopharmacology 93: 349-352
Goodwin GM (1 996) How do antidepressants affed serotonin receptors? The role of serotonin
receptors in the therapeutic and side effed profile of the SSRls. Journal of Clinical Psychiatry
57: 9-13
Grignaschi G, Samanin R (1 992) Role of serotonin and catecholamines in brain in the feeding
suppressant effed of fiuoxetine. Neuropharrnacotogy 31 : 445449
Halford JCG, Blundell JE (1 996) Metergoline antagonizes fiuoxetine-induced suppression of food
intake but not changes in the behavioural satiety sequence. Pharmacology, Bioctiemistry
and Behavior 54: 745-751
Hartley JE, Fletcher A (1994) The effects of WAY-100135 and 8-hydroxy-2-(di-n-propylamino) tetralin
on feeding in the rat. European Journal of Phamacology 252: 329-332 Heisler LK, Kanarek RB, Gerstein A (1997) Fluoxetine decreases fat and protein intakes but not
carbohydrate intake in male rats. Phamacology. Biochemistry and Behavior 58: 767-773
Higgins GA, Wang Y, Sellers EM (1993) Preliminary findings with the indirect 5-HT agonist
dexfenfluramine on heroin discrimination and self-administration in rats. Pharmacology.
Biochemistry and Behavior 45: 963-966
Hrdina PD, Demeter E, Vu TB, S6t6nyi P. Palkovits M (1993) 5-HT uptake sites and 5-HTz receptors
in brain of antidepressant-free suicide victims 1 depressives: increase in 5-HT2 sites in cortex
and arnygdala. Brain Research 614: 37-44
Hubner CB, Koob GF (1990) The ventral pallidum plays a role in mediating cocaine and heroin self-
administration in the rat. Brain Research 508: 20-29
Hutson PH, Dourish CT, Curzon G (1986) Neurochemical and behavioural evidence for mediation of
the hyperphagic action of 8-OH-DPAT by 5-HT cell body autoreceptors. European Journal of
Pharmacology 129: 347-352
Hutson PH, Dourish CT, Curzon G (1988) Evidence that the hyperphagic response to 8-OH-DPAT is
mediated by 5-HTt~receptors. European Journal of Phamacology 150: 361-366
Jackson HC, Needharn AM, Hutchins W. Mazurkiewicz SE, Heal DJ (1997) Cornpanson of the effeds
of sibutramine and other monoamine reuptake inhibitors on food intake in the rat. British
Journal of Phamacology 121: 1758-1762
Lakoski JM, Aghajanian GK (1 985) Effects of ketanserin on neuronal responses to serotonin in the
prefrontal cortex, lateral geniculate and dorsal raphé nucleus. Neurophamiacology 24: 265-
273
Lee K, Kometsky C (1 998) Acute and chronic fluoxetine treatment decreases the sensitivity of rats to
rewarding brain stimulation. Pharmacology, Biochemistry and Behavior GO: 539-544 Lightowfer S, Wood M, Brown T, Glen A, Blackburn 1,Tulloch 1, Kennett G (1996) An investigation of
the mechanism respcmsible for fluoxetine-induced hypophagia in rats. European Journal of
Phannacology 296: 137-143
Lucki 1, Kreider MS, Simansky KJ (1988) Reduction of feeding behavior by the serotonin uptake
inhibitor sertraline. Psychophamacobgy 96: 289-295
Luo S. Ransom T, Li ETS (1990) Selective increase in carbohydrate intake by rats treated with 8-
hydroxy-2-(di-n-propy1amino)-tetraline or buspirone. Life Sciences 46: 1643-1648
Massi M, Marini S (1987) Effed of the 5HT2 antagonist ritanserin on food intake and on SHT induced
anorexia in the rat. Phamacology, Biochernistry and Behavior 26: 333-340
Matsuda T, Sornboonthum P, Suzuki M. Asono S, Baba A (1995) Antidepressant-like effect by
postsynaptic 5-HT,, receptor activation in mice. European Journal of Phamacology 280:
235-238
McGuirk J. Muscat R. Willner P (1992a) Effects of chronically administered fluoxetine and
fenfluramine on food intake, body weight and the behavioural satiety sequence.
Psychophamacology 106: 401 -407
McGuirk J, Muscat R, Willner P (1992b) Effects of the 5-HT uptake inhibitors, femoxetine and
paroxetine, and a 5-HTIm agonist, eltoprazine, on the behavioural satiety sequence.
Phamacology, Biochemistry and Behavior 41: 801-805
Monleon S. D'Aquila P, Parra A, Simon VM, Brain PF, Wllner P (1995) Attenuation of sucrose
consumption in mice by chronic mild stress and its restoration by imipramine.
Psychophamiacology 117: 453457
Montgomery AMJ, Rose IC, Herberg LJ (1991) 5-HTlA agonis& and dopamine: the effects of 8-OH-
DPAT and buspirone on brain-stimulation reward. Journal of Neural Transmission [General
Section] 83: 139-148 Muscat R, Papp M, Willner P (1992) Reversal of stress-induced anhedonia by the atypical
antidepressants, fluoxetine and maprotiline. Psychopharrnacology 109: 433-438
Neill JC, Cooper S3 (1 988) MDL 72832, a selective 5-HT 1A receptor ligand. stereospecifically
increases food intake. European Journal of Phamacology 151: 329-332
Pazos A, Cortés R, Palacios JM (1985) Quantitative autoradiographic mapping of serotonin receptors
in the rat brain. II. Serotonin-2 receptors. Brain Research 346: 231-249
Richardson NR, Roberts DCS (1991) Fluoxetine pretreatment reduces breaking points on a
progressive ratio schedule reinforced by intravenous cocaine self-administration in the rat.
Life Sciences 49: 833-840
Richardson NR, Roberts DCS (1 996) Progressive ratio schedules in drug self-administration studies
in rats: a method to evaluate reinforcing efficacy. Journal of Neuroscience Methods 66: 1-1 1
Roberts DCS, Loh €A, Baker GB, Vickers G (1994) Lesions of central serotonin systems affect
responding on a progressive ratio schedule reinforced either by intravenous cocaine or by
food. Phamacology, Biochemistry and Behavior 49: 177-182
Romero L, Bel N, ARigas F, De Montigny C. Blier P (1996) Effed of pindolol on the fundion of pre-
and postsynaptic 5-HTIA receptors: in vivo microdialysis and electrophysiological studies in
the rat brain. Neuropsychophamacology 15: 349-360
Rueter LE, De Montigny C, Blier P (1998) In vivo electrophysiological assessrnent of the agonistic
properties of flibansenn at pre- and postsynaptic 5-HTIAreceptors in the rat brain. Synapse
29: 392-405
Samanin R (1 989) Serotonin and feeding. In: Bevan P, Cook AR, Archer T (eds) Behavioral
Phamacology of 5-HT. Lawrence Erlbaurn Associates, Hillsdale. pp 259-281
Saxena P (1 995) Serotonin receptors: subtypes, functional responses and therapeutic relevance.
Phamacology and Therapeutics 66: 339-368 Sills TL, Vaccarino FJ (1994) Individual differences in sugar intake predict the locomotor response to
acute and repeated amphetamine administration. Psychopharmacology 116: 1-8
Sills TL, Vaccarino FJ (1 998) Individual differences in the feeding and locomotor stimulatory effects of
acute and repeated morphine treatments. Phamacology, Biochemistry and Behavior 60:
293-303
Simansky KJ, Vaidya AH (1990) Behavioral mechanisms for the anorectic action of the serotonin (5-
HT) uptake inhibitor sertraline in rats: compatison with directly acting 5-HT agonists. Brain
Research Bulletin 25: 953-960
Simansky KJ (1996) Serotonergic control of the organization of feeding and satiety. Behavioural
Brain Research 73: 37-42
Smith LM. Peroutka S (1986) Differential effects of 5-hydr~xytryptarnine,~selective drugs on the 5-HT
behavioral syndrome. Phamacology, Biochemistry and Behavior 24: 1513-1 519
Soares JC, Gershon S (1 996) Prospects for the development of new treatments with a rapid onset of
action in affective disorders. Drugs 52: 477-482
Stahl S (1 994) S-HTIA receptors and phannacotherapy: is serotonin receptor down-regulation linked
to the mechanisrn of adion of antidepressant dnigs? Psychopharmacology Bulletin 30: 39-43
Tricklebank MD, Forler C, Fozard JR (1984) The involvement of subtypes of the 5-HT, receptor and
of catecholaminergic systems in the behavioural response to 8-hydroxy-2-(di-n-
propy1amino)tetralin in the rat. European Journal of Phamacology 1û6: 271-282
Tucker JC, File SE (1986) The effects of trycyclic and 'atypical' antidepressants on spontaneous
locomotor activity in rodents. Neuroscience and Biobehavioral Reviews 10: 115-1 21
Vickers SP, Clifton PG, Dourish CT (1996) Behavioural evidence that cl-fenfiuramine-induced
anorexia in the rat is not mediated by the S-HT~Areceptor subtype. Psychophamacoiogy
125: 168-175 Welner SA, De Montigny C, Desroches J, Desjardins P. Suranyi-Cadotte BE (1989) Autoradiographic
quantification of serotoniniA receptors in rat brain following antidepressant drug treatment.
Synapse 4: 347-352
Wieland S, Lucki 1 (1 990) Antidepressant-like adivity of 5-HTTAagonists measured with the forced
swim test. Psychopharmacology 101: 497-504
Willner P (1997) The chronic mild stress procedure as an animal model of depression: valid,
reasonably reliable, and useful. Psychopharmacology 134: 371-377
Wong DT, Reid LR, Threlkeld PG (1988) Suppression of food intake in rats by fluoxetine: cornparison
of enantiorners and effeds of serotonin antagonists. Pharmacology, Biochemistry and
Behavior 31 : 475479 Appendix 1: Pronressive Ratio Schedule of Reinforcement
The following taMe describes the series of successive fmed-ratio schedules which comprised
the progressive-ratio schedule of reinforcement, and indicates the cumulative number of responses
made and cumulative nurnber of sucrose reinforcers received corresponding to each point in the
schedule. This series was derived by applying a multiplication factor of 0.1 {with the resulting ratio
rounded to the nearest integer) to the succession of FR responses described in a schedule from
Hubner and Koob (1990). During the first two and second two PR training days a multiplication factor of 0.001 and 0.01, respectively, was instead applied, thereby gradually training the animals to the increasing difficuity of the reward schedule.
For example: An animal that has just completed the FR-12 ratio has made f28 responses and has received 32 reinforcers, and wjll now be requjred to make 14 responses to receive the next (3p) reinforcer. I Fixed Ratio / Cumulative Number of 1 Cumulative Nurnber of I Responses Made Reinforcers Obtained I Fixed Ratio Cumulative Number of Cumulative Number of Responses Made 1 Reinforcers Obtained Fixed Ratio Cumulative Number of Cumulative Nurnber of
Responses Made Reinforcers Obtained
18 192 36 Fixed ~ati6 Cumulative ~umberof Cumulative Number of
Responses Made Reinforcers Obtained