Serotonergic Manipulations Both Potentiate and Reduce Brain Stimulation Reward in Rats: Involvement of Serotonin-1A Receptors

  1. Amanda A. Harrison1 and
  2. Athina Markou
  1. Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California
     
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    Abstract

    A discrete-trial current-threshold self-stimulation procedure was used to assess the effects of increased and decreased serotonergic neurotransmission, and 5-HT1A receptor activation on brain stimulation reward. Systemic administration of the 5-HT1Areceptor agonist 8-OH-DPAT had a biphasic effect on brain reward thresholds, without affecting the latency to respond, a measure of performance. The low dose of 8-OH-DPAT (0.03 mg/kg) lowered reward thresholds, whereas higher doses (0.1 and 0.3 mg/kg) elevated thresholds. The 5-HT1A receptor antagonistp-MPPI had no effect on brain stimulation behavior, but reversed both the 8-OH-DPAT-induced lowering and elevation of thresholds, indicating that both of these effects of 8-OH-DPAT are mediated through the 5-HT1A receptor. Injections of 8-OH-DPAT into the median raphé nucleus also lowered brain reward thresholds, without affecting measures of performance, whereas injections of 8-OH-DPAT into the dorsal raphé nucleus had no effect. A high dose of the selective serotonin reuptake inhibitor fluoxetine (10 mg/kg) elevated reward thresholds and responses latencies, whereas lower doses (2.5 and 5.0 mg/kg) increased response latencies without affecting thresholds. Furthermore, the coadministration of a 5-HT1A antagonist,p-MPPI, and a previously ineffective dose of fluoxetine, a drug combination that increases serotonin levels, significantly elevated thresholds. Thus, it is suggested here that the reward-potentiating effects of systemically administered low doses of 8-OH-DPAT may be the result of reduced serotonergic neurotransmission, mediated by activation of 5-HT1A somatodendritic autoreceptors in the median, but not the dorsal, raphé nucleus. In conclusion, the present data support the hypothesis that serotonin exerts an inhibitory influence on reward processes.

    Serotonergic neurotransmission is hypothesized to be involved in motivational processes and reward-related behaviors (Miliaressis, 1977; Bendotti and Samanin, 1986; Hillegaart et al., 1991). Nevertheless, the exact role of serotonin (5-HT) on reward processes remains unclear. That is, increases in serotonin either increase or decrease reward, and decreases in serotonin also have produced inconsistent results (Poschel et al., 1974; Redgrave 1978; Ahlenius et al., 1981; McClelland et al., 1989; Hillegaart et al., 1991; Fletcher et al., 1995). A method used to study reward is the brain stimulation reward (BSR) procedure (Markou and Koob, 1992). In this procedure, the reinforcing efficacy of electrical stimulation of brain sites is indicated by the fact that subjects perform an operant response to receive the brief electrical stimuli (Liebman, 1983).

    One hypothesis is that serotonin exerts an inhibitory influence on reward processes. This hypothesis is supported by reports that reductions in serotonergic neurotransmission induced by either intraventricular 5,6-dihydroxytryptamine or systemicpara-chlorophenylalanine (PCPA) increased BSR reflected in increased response rates (Poschel and Ninteman, 1971; Poschel et al., 1974). However, the hypothesized serotonergic mediation of BSR remained controversial due to lack of correlation between the time course of serotonin depletion and the behavioral effects (Katz and Baldrighi, 1979; Gratton, 1982). Furthermore, (±)-8-hydroxy-2-(di-n-propyl-amino)tetralin hydrobromide (8-OH-DPAT), a 5-HT1A receptor agonist (Hall et al., 1985), injected into the median raphé nucleus lowered BSR thresholds reflecting also increased reward (Fletcher et al., 1995). This effect is probably due to activation of 5-HT1A somatodendritic autoreceptors in the raphé nuclei leading to reduced firing of serotonin neurons and reduced serotonin release (Aghajanian et al., 1987; Sinton and Fallon, 1988). Consistent with the above-mentioned effects are the observations that increasing serotonin by systemic administration ofd-fenfluramine, 5-hydroxytrytophan, fluoxetine, or serotonin injections into the nucleus accumbens or the caudate-putamen decreased BSR (Bose et al., 1974; Redgrave, 1978; McClelland et al., 1989; Lee and Kornetsky, 1998).

    The above-mentioned studies used rats with stimulating electrodes in the medial forebrain bundle, including the area of the lateral hypothalamus. However, studies in subjects with electrodes in the raphé nuclei demonstrated a facilitatory role of serotonin on BSR. Electrodes in either the median (Miliaressis, 1977; van der Kooy et al., 1978) or the dorsal raphé (Simon et al. 1976; van der Kooy et al., 1978) maintained self-stimulation behavior that was reduced by PCPA, suggesting that self-stimulation of the raphénuclei is serotonergically mediated. Response rates for self-stimulation of the hippocampus also were decreased by PCPA administration (van der Kooy et al., 1977). Taken together, these findings indicate that the role of serotonin in BSR may depend on the stimulation site.

    A limitation of many of the above-mentioned studies is that changes in reward were measured as increases or decreases in response rates. Response rates are more affected by changes in behavioral activation induced by manipulations than threshold measures of reward (Liebman, 1983), which is relevant because serotonin manipulations affect both response inhibition and locomotor behavior (Soubrié, 1986).

    The 5-HT1A receptor has received considerable research interest due to its hypothesized link to disorders such as depression and anxiety (Maes and Meltzer, 1995; File et al., 2000). High densities of 5-HT1A recognition sites are found in the hippocampus, amygdala, raphé nuclei, lateral septum, entorhinal cortex, and certain hypothalamic nuclei (Pazos and Palacios, 1985). The 5-HT1A receptor exists in two forms in the brain, as somatodendritic autoreceptors and postsynaptic receptors in terminal regions. 5-HT1A receptor-mediated serotonin neurotransmission is involved in the control of various motivated behaviors, including locomotor activity, feeding, sexual behavior, and BSR (Ahlenius et al., 1981; Bendotti and Samanin, 1986;Hutson et al., 1986; Hillegaart et al., 1989, 1991; Hillegaart, 1990;Montgomery et al., 1991; Fletcher et al., 1995).

    The purpose of the present experiments was to systematically assess the role of serotonin, and more specifically of 5-HT1A receptors, in BSR. A response-rate-independent discrete-trial procedure that provides current intensity thresholds as a measure of reward and response latencies as a measure of performance was used. The effects of systemically administered 8-OH-DPAT, a 5-HT1Areceptor agonist, were first examined. The selectivity of the 8-OH-DPAT systemic effects was then assessed using the 5-HT1A receptor antagonist p-MPPI (Kung et al., 1994). Discrete median and dorsal raphé nucleus infusions of 8-OH-DPAT assessed 1) the effects of reduced serotonin induced by activation of 5HT1A autoreceptors, and 2) the differential involvement of the two raphé nuclei on BSR behavior. Finally, the effects of enhancing serotonin neurotransmission by the coadministration of fluoxetine, a selective serotonin reuptake inhibitor, and a 5-HT1A receptor antagonist on BSR also were examined.

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    Materials and Methods

    Subjects

    Male Wistar rats (Charles River, Hollister, CA) were housed in pairs in a temperature- and humidity-controlled environment with a 12-h light/dark cycle (lights on 6:00 AM). The subjects were tested during the light phase of their light/dark cycle. Food and water were available ad libitum in the home cages. All subjects were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the animal facilities and experimental protocols were in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care.

    Apparatus

    The experimental apparatus consisted of 16 Plexiglas chambers (30.5 × 30 × 17 cm) encased in sound-attenuating boxes. Each operant chamber consisted of a stainless steel grid floor and a metal wheel manipulandum located on the front wall, which required a 0.2-N force to rotate it a quarter turn. Gold-contact swivel commutators and bipolar leads (Plastics One, Roanoke, VA) connected the animals to constant current stimulators (Stimtek 1200; San Diego Instruments, San Diego, CA). The stimulation parameters, data collection, and all programming functions were controlled by a microcomputer.

    Surgical Procedure

    When the rats reached 320 g in body weight they underwent surgical implantation of stainless steel bipolar intracranial self-stimulation electrodes (diameter = 0.25 mm, length = 11 mm; Plastics One). The subjects were anesthetized with a halothane/oxygen vapor mixture (1.0–1.5%), placed in a Kopf stereotaxic frame (David Kopf Instruments, Tujunga, CA) with the incisor bar set at −3.3 mm below the interaural line (i.e., flat skull; Paxinos and Watson, 1998). An electrode was implanted into the posterior lateral hypothalamus according to the coordinates AP, −2.8 mm from bregma; L, ±1.7 mm; DV, −8.8 mm from skull surface. The electrode was implanted in the right brain hemisphere in half of the subjects and the left hemisphere for the others. In experiments 5 and 6, subjects were prepared with both electrodes in the lateral hypothalamus and guide cannulae (23-gauge stainless steel tubing) 2 mm away from either the median raphé (22o from vertical; AP, −7.8 mm from bregma; L, +3.39 mm; DV, −7.06 mm from skull surface, guide cannulae were 13 mm in length) or the dorsal raphé (32o from vertical; AP, −7.8 mm from bregma; L, +40.0 mm; DV, −5.55 mm from skull surface, guide cannulae were 11 mm in length). The cannulae were implanted in the right hemisphere and the electrodes in the left hemisphere in all subjects. The subjects were given a 7-day postoperative recovery period before behavioral training.

    Drugs

    8-OH-DPAT (Sigma, St. Louis, MO) was dissolved in 0.9% saline and administered subcutaneously in a volume of 1 ml/kg of body weight. 4-Iodo-N-[2-[4-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide hydrochloride (p-MPPI) (Research Biochemicals International, Natick, MA) was dissolved in sterile water and sonicated for 10 to 20 min, and then brought to a pH of approximately 5.2 with 0.1 M NaOH.p-MPPI was administered subcutaneously in a volume of 4 ml/kg of body weight. Infusions of 8-OH-DPAT into either the median or the dorsal raphé nucleus were administered in a volume of 0.5 μl over 1 min, with the injection needle left in place for 1 min after the infusion to minimize efflux. (+)-N-Methyl-γ-[4-(trifluoromethyl)phenoxy]-benzenepropanamine hydrochloride (fluoxetine) (Research Biochemicals International) was dissolved in saline and administered intraperitoneally in a volume of 1 ml/kg of body weight.

    Intracranial Self-Stimulation Behavioral Procedure

    The intracranial self-stimulation discrete-trial current-threshold procedure is a modified version (Markou and Koob, 1992) of a procedure initially developed by Kornetsky and coworkers (Lee and Kornetsky, 1998). The subjects were initially trained to turn the wheel manipulandum on a fixed ratio 1 schedule of reinforcement during which each quarter turn of the wheel resulted in the delivery of a contingent electrical reinforcer. The electrical reinforcer had a train duration of 500 ms and consisted of 0.1-ms rectangular cathodal pulses that were delivered with 100-Hz frequency. The current intensity delivered was adjusted for each animal and typically ranged from 100 to 250 μA. After successful familiarization with this procedure (two sessions of 100 reinforcers in less than 20 min), the rats were gradually trained on the discrete-trial, current-threshold procedure.

    At the start of each trial, rats received a noncontingent electrical stimulus. During the following 7.5 s, the limited hold, if the subjects responded by turning the wheel manipulandum a quarter turn (positive response) they received a second contingent stimulus identical to the previous noncontingent stimulus. During a 2-s period immediately after a positive response, further responses had no reinforcement or task consequences. If no response occurred during the 7.5-s limited hold period, a negative response was recorded. The intertrial interval (ITI), which followed the limited hold period, had an average duration of 10 s (range 7.5–12.5 s). Responses that occurred during the ITI resulted in a further 12.5-s delay of the onset of the next trial. During training, the duration of the ITI and delay periods imposed by inappropriate ITI responding were gradually increased until the standard task parameters were reached. Stimulation intensities were varied according to the classical psychophysical method of limits. Thus, the subjects received four alternating series of ascending and descending current intensities starting with a descending series. Within each series the stimulus intensity was altered by 5-μA steps between each set of trials (three trials per set). The initial stimulus intensity was set at 40 μA above the baseline current threshold for each animal. A series was terminated after either 15 stimulus increments (or decrements) had occurred, or after the determination of the threshold for the series (see below). Each test session typically lasted 30 min and provided two dependent variables.

    Thresholds.

    The current threshold for each descending series was defined as the stimulus intensity between the successful completion of a set of trials (positive responses during two or more of the three trials) and the stimulus intensity for the first set of trials, of two consecutive sets, during which the animal failed to respond positively on two or more of the three trials. During the ascending series, the threshold was defined as the stimulus intensity between the unsuccessful completion of a set of trials (negative responses during two or more of the three trials) and the stimulus intensity for the first set of trials, of two consecutive sets, during which the animal responded positively on two or more of the trials. Thus, during each test session, four thresholds were determined and the mean of these values was taken as the threshold for each subject.

    Response Latency.

    The latency between the onset of the noncontingent stimulus at the start of each trial and a positive response was recorded as the response latency. The response latency for each test session was defined as the mean response latency of all trials during which a positive response occurred.

    After training in the above-mentioned brain stimulation reward procedure, rats were tested until stable baseline thresholds had been achieved (±10% over a 5-day period). Drug testing was initiated only after performance had stabilized, which typically occurred after 2 to 3 weeks of baseline testing. Return to baseline threshold levels was required between drug injections. Unless otherwise stated (experiments 2 and 7) experimentally naive subjects were used in each experiment.

    Experiment 1: Effects of Systemically Administered 8-OH-DPAT, a 5-HT1A Agonist, on Brain Stimulation Reward.

    After establishment of stable baseline thresholds, 8-OH-DPAT (0, 0.03, 0.1, 0.3 mg/kg s.c., n = 11) was administered (30-min pretreatment) according to a within-subjects Latin square design, with a minimum of 3 days between consecutive drug treatments.

    Experiment 2: Effects of p-MPPI, a 5-HT1A Antagonist, on Brain Stimulation Reward.

    p-MPPI (0, 1, 3, 10 mg/kg s.c., 135-min pretreatment,n = 8) was administered according to a within-subjects Latin square design, with a minimum of 4 days between consecutive drug treatments.

    Experiment 3: Effects of p-MPPI, a 5-HT1A Antagonist, on the 8-OH-DPAT-Induced Lowering of Brain Reward Thresholds.

    Eight combination treatments ofp-MPPI and 8-OH-DPAT were administered according to a within-subjects Latin square design, with a minimum of 3 days between consecutive combination drug treatments (n = 10). The treatments involved one of four doses of p-MPPI (0, 0.03, 0.3, 1 mg/kg s.c., 135-min pretreatment) followed by one of two doses of 8-OH-DPAT (0, 0.03 mg/kg s.c., 30-min pretreatment).

    Experiment 4: Effects of p-MPPI on the 8-OH-DPAT-Induced Elevation of Brain Reward Thresholds.

    Six combination treatments of p-MPPI and 8-OH-DPAT were administered according to a within-subjects Latin square design, with a minimum of 3 days between consecutive combination drug treatments (n = 10). The treatments involved one of three doses ofp-MPPI (0, 0.3, 1 mg/kg s.c., 135-min pretreatment) followed by one of two doses of 8-OH-DPAT (0, 0.3 mg/kg s.c., 30-min pretreatment).

    Experiment 5: Effects of 8-OH-DPAT Infusions into the Median Raphé Nucleus on Brain Stimulation Reward.

    8-OH-DPAT (0, 1.0, 2.5, 5.0 μg/0.5 μl/1 min, 5-min pretreatment,n = 11) was infused directly into the median raphé nucleus through 15-mm 30-gauge stainless steel tubing injectors using a Harvard pump (Harvard Apparatus Inc., Holliston, MA) according to a within-subjects Latin square design. Each microinfusion was followed by a 1-min period in which the infusion needle was left in place to minimize efflux.

    Experiment 6: Effects of 8-OH-DPAT Infusions into the Dorsal Raphé Nucleus on Brain Stimulation Reward.

    8-OH-DPAT (0, 1.0, 2.5, 5.0 μg/0.5 μl/1 min, 5-min pretreatment,n = 11) was infused directly into the dorsal raphé nucleus in a similar manner to that described above in the median raphé experiment (experiment 5), using 13-mm, 30-gauge stainless steel tubing injectors.

    Experiment 7: Effects of Fluoxetine, a Selective Serotonin Reuptake Inhibitor, on Brain Stimulation Reward.

    Fluoxetine (0, 2.5, 5, 10 mg/kg i.p., 120-min pretreatment, n = 10) was administered according to a within-subjects Latin square design, with a minimum of 4 days between consecutive drug treatments. Eight of the subjects in this experiment were also subjects in experiment 2, half completed experiment 2 first, while the other half completed experiment 7 first (i.e., crossover design). Stable baseline thresholds were established between drug treatments and between experiments.

    Experiment 8: Effects of p-MPPI and Fluoxetine on Brain Stimulation Reward.

    Four combination treatments ofp-MPPI and fluoxetine were administered according to a within-subjects Latin square design, with a minimum of 7 days between consecutive combination drug treatments (n = 10). The treatments involved one of four doses of p-MPPI (0, 1, 3, 10 mg/kg s.c., 135-min pretreatment) followed by fluoxetine (5 mg/kg s.c., 120-min pretreatment).

    Histology

    After completion of behavioral testing in experiments 5 and 6 all subjects were sacrificed with an overdose of sodium pentobarbital (Abbott Laboratories, Abbott Park, IL) and perfused intracardially with phosphate-buffered saline and then 4% formaldehyde solution (Fisons, Rochester, NY). The brains were removed from the skull, fixed, frozen, and sliced into 50-μm sections. Mounted sections were examined under a light microscope to determine the accuracy of cannula placement.

    Data Analyses

    All reward threshold and response latency data were expressed as a percentage of the previous baseline day's data. The percentage scores then were analyzed using the one-way repeated-measures ANOVA. The data from experiments 3 and 4 were treated in an identical manner to those of the other experiments except that the data were analyzed using two-way repeated-measures ANOVAs with agonist and antagonist doses being the two factors. Statistically significant results were followed by post hoc comparisons of group means using the Newman-Keuls analysis. All statistical analyses were performed using the SuperAnova statistical package (Abacus Concepts Inc., Berkeley, CA). The level of significance reported is p < 0.05.

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    Results

    Baseline Thresholds and Response Latencies.

    Baseline data were analyzed using one-way ANOVAs to assess potential drifts of baseline performance. No significant differences in thresholds or response latencies were found in any of the experiments (see Table1 for ranges of thresholds and responses latencies for each experiment).

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    Table 1

    Range of mean baseline thresholds and response latencies for all experiments

    Experiment 1: Effects of Systemically Administered 8-OH-DPAT, a 5-HT1A Agonist, on Brain Stimulation Reward.

    The 5-HT1A receptor agonist 8-OH-DPAT had a biphasic effect on brain stimulation reward thresholds [F(3,30) = 16.028, p < 0.001]. The lowest dose administered, 0.03 mg/kg, significantly lowered thresholds compared with thresholds after saline treatment, whereas the two highest doses, 0.1 and 0.3 mg/kg, significantly elevated thresholds compared with thresholds observed after administration of either saline or 0.03 mg/kg 8-OH-DPAT (Fig. 1A). 8-OH-DPAT had no effect on response latencies, a measure of general motoric performance [F(3,30) = 1.725, p > 0.1] (Fig.1B).

    Figure 1
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    Figure 1

    Effects of systemically administered 8-OH-DPAT, a 5-HT1A agonist, on brain stimulation reward thresholds (A) and response latencies (B), (mean ± S.E.M.). Asterisks (∗) denote statistically significant differences from thresholds after vehicle treatment. *p < 0.05, **p < 0.01. Crosses (†) denote statistically significant differences compared with thresholds after administration of 0.03 mg/kg 8-OH-DPAT, indicating dose-dependent effects of 8-OH-DPAT, ††p < 0.01.

    Experiment 2: Effects of p-MPPI, a 5-HT1A Antagonist, on Brain Stimulation Reward.

    Systemic administration of p-MPPI, had no statistically significant effect on either reward thresholds [F(3,21) = 1.783, p > 0.1] (Fig.2A), or the latency to respond [F(3,21) = 1.688, p > 0.1] (Fig.2B).

    Figure 2
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    Figure 2

    Effects of p-MPPI, a 5-HT1A antagonist, on brain reward thresholds (A) and response latencies (B) (mean ± S.E.M.). p-MPPI had no effect on brain stimulation reward thresholds or response latencies.

    Experiment 3: Effects of p-MPPI, a 5-HT1A Antagonist, on the 8-OH-DPAT-Induced Lowering of Brain Reward Thresholds.

    Main effects of both 8-OH-DPAT [F(1,9) = 38.553, p < 0.001] andp-MPPI [F(3,27) = 19.282, p< 0.001] were observed. Post hoc Newman-Keuls analysis of an 8-OH-DPAT × p-MPPI interaction [F(3,27) = 16.076, p < 0.001] demonstrated that, as seen in experiment 1, the administration of vehicle + 0.03 mg/kg 8-OH-DPAT significantly lowered reward thresholds compared with thresholds after vehicle + vehicle treatment.p-MPPI (0.03, 0.3, or 1.0 mg/kg + vehicle) had no effect on reward thresholds compared with thresholds after vehicle + vehicle treatment, but did reverse the 8-OH-DPAT-induced lowering of thresholds in a dose-dependent manner (Fig. 3A). None of the drug combinations had any effect on response latencies [F(3,27) = 1.538, p > 0.1] (Fig.3B).

    Figure 3
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    Figure 3

    Reversal of 8-OH-DPAT-induced lowering of reward thresholds by p-MPPI (A) with no treatment effects on response latencies (B), (mean ± S.E.M.). Asterisks (∗) denote statistically significant differences from thresholds after the corresponding p-MPPI + vehicle treatment, **p < 0.01. Crosses (†) denote statistically significant differences from thresholds after administration of vehicle + 0.03 mg/kg 8-OH-DPAT, ††p < 0.01. Hashes (#) denote a statistically significant difference from thresholds after 0.03 mg/kg p-MPPI + 0.03 mg/kg 8-OH-DPAT, indicating a p-MPPI-induced dose-dependent reversal of the effects of 8-OH-DPAT,##p < 0.01.

    Experiment 4: Effects of p-MPPI on the 8-OH-DPAT-Induced Elevation of Brain Reward Thresholds.

    Main effects of both 8-OH-DPAT [F(1,9) = 27.948,p < 0.001] and p-MPPI [F(2,27) = 9.4, p < 0.01] were observed. Post hoc Newman-Keuls analysis of an 8-OH-DPAT ×p-MPPI interaction [F(2,18) = 12.286,p < 0.001] demonstrated that, as seen in experiment 1, the administration of vehicle + 0.3 mg/kg 8-OH-DPAT significantly elevated reward thresholds compared with thresholds after vehicle + vehicle treatment. p-MPPI (0.3 or 1.0 mg/kg + vehicle) had no effect on reward thresholds compared with thresholds after vehicle + vehicle treatment, but dose dependently reversed the threshold elevations induced by 0.3 mg/kg 8-OH-DPAT (Fig.4A). The drug combinations had no effect on response latencies [F(2,18) = 0.792,p > 0.1] (Fig. 4B).

    Figure 4
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    Figure 4

    Reversal of 8-OH-DPAT-induced elevations of reward thresholds by p-MPPI (A) with no treatment effects on response latencies (B), (mean ± S.E.M.). Asterisks (∗) denote statistically significant differences from thresholds after the corresponding p-MPPI + vehicle treatment, *p < 0.05, **p < 0.01. Crosses (†) denote statistically significant differences from thresholds after administration of vehicle + 0.3 mg/kg 8-OH-DPAT,†p < 0.05,††p < 0.01. Hashes (#) denote a statistically significant difference from thresholds after 0.3 mg/kgp-MPPI + 0.3 mg/kg 8-OH-DPAT treatment, indicating ap-MPPI-induced dose-dependent reversal of the effects of 8-OH-DPAT, #p < 0.05,##p < 0.01.

    Experiment 5: Effects of 8-OH-DPAT Infusions into the Median Raphé Nucleus on Brain Stimulation Reward.

    Histological analysis of brain tissue indicated that cannula placements in all subjects were accurate (Fig. 5A). Central infusions of 8-OH-DPAT (1.0 and 2.5 μg/0.5 μl) into the median raphé nucleus significantly lowered brain reward thresholds compared with thresholds after vehicle administration [F(3,30) = 7.674, p < 0.001] (Fig.5B), without affecting response latencies [F(3,30) = 0.307, p > 0.1] (Fig. 5C).

    Figure 5
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    Figure 5

    Effects of median raphé infusions (histologies,Paxinos and Watson 1998) (A) of 8-OH-DPAT on brain stimulation reward thresholds (B) and response latencies (C) (mean ± S.E.M.). Infusions of 8-OH-DPAT into the median raphé nucleus lowered brain stimulation reward thresholds without affecting response latencies. Asterisks (∗) denote statistically significant differences from thresholds after vehicle treatment. *p < 0.05, **p < 0.01.

    Experiment 6: Effects of 8-OH-DPAT Infusions into the Dorsal Raphé Nucleus on Brain Stimulation Reward.

    Histological analysis of brain tissue indicated that the cannula placements in four subjects were inaccurate (Fig.6A). Thus, the behavioral data of these subjects were eliminated from all analyses. Central infusions of 8-OH-DPAT into the dorsal raphé nucleus (in the remaining subjects, n = 7) had no statistically significant effect on either brain stimulation reward thresholds [F(3,18) = 0.578, p> 0.1] (Fig. 6B) or response latencies [F(3,18) = 0.52, p > 0.1] (Fig. 6C).

    Figure 6
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    Figure 6

    Effects of dorsal raphé infusions (histologies,Paxinos and Watson, 1998) (A) of 8-OH-DPAT on brain stimulation reward thresholds (B) and response latencies (C) (mean ± S.E.M.). Infusions of 8-OH-DPAT into the dorsal raphé nucleus had no effect on reward thresholds or response latencies.

    Experiment 7: Effects of Fluoxetine, a Selective Serotonin Reuptake Inhibitor, on Brain Stimulation Reward.

    Fluoxetine (10 mg/kg) significantly elevated reward thresholds compared to thresholds after vehicle treatment, and 2.5 or 5.0 mg/kg fluoxetine [F(3,30) = 6.489, p < 0.01] (Fig.7A). All doses of fluoxetine administered increased the latency to respond [F(3,30) = 3.988,p < 0.05] compared with the response latency after vehicle treatment (Fig. 7B).

    Figure 7
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    Figure 7

    Effects of fluoxetine on brain stimulation reward thresholds (A) and response latencies (B) (mean ± S.E.M.). Fluoxetine (10 mg/kg) elevated brain stimulation reward thresholds. Fluoxetine (2.5, 5.0, or 10 mg/kg) increased the latency to respond. Asterisks (∗) denote statistically significant differences from thresholds or latencies after vehicle treatment. *p< 0.05, **p < 0.01.

    Experiment 8: Effects of Combined p-MPPI and Fluoxetine Treatment on Brain Stimulation Reward.

    The coadministration of 10 mg/kg p-MPPI and a previously ineffective dose of fluoxetine (5 mg/kg) significantly elevated reward thresholds compared with thresholds after vehicle + 5 mg/kg fluoxetine treatment [F(3,27) = 3.349, p < 0.05] (Fig. 8A). Neither 10 mg/kgp-MPPI (under Results of experiment 2) nor 5 mg/kg fluoxetine (present experiment) had an effect on brain reward thresholds when administered alone. None of the drug combination treatments had an effect on response latencies [F(3,27) = 1.939, p > 0.1] (Fig.8B).

    Figure 8
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    Figure 8

    .. Effects of p-MPPI + fluoxetine on brain stimulation reward thresholds (A) and response latencies (B) (mean ± S.E.M.). p-MPPI (10 mg/kg) + fluoxetine (5 mg/kg) elevated reward thresholds without affecting response latencies. Asterisks (∗) denote statistically significant differences from thresholds after vehicle treatment and 5 mg/kg fluoxetine. *p < 0.05.

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    Discussion

    Systemic administration of the 5-HT1Areceptor agonist 8-OH-DPAT, depending on the dose, increased or decreased BSR reflected in changes in reward thresholds. 8-OH-DPAT did not affect response latencies, an independent measure assessing performance effects. These results indicate that the effects of 8-OH-DPAT on reward thresholds were not confounded by performance effects and are consistent with previous reports of biphasic effects of 8-OH-DPAT on BSR (Montgomery et al., 1991).

    Although the systemic administration of the 5-HT1A antagonist p-MPPI had no effect on BSR behavior, p-MPPI blocked both the reward potentiating and the reward reducing effects of systemically administered 8-OH-DPAT, without affecting response latencies. p-MPPI has high affinity and selectivity as a 5-HT1A receptor antagonist with Kd = 0.36 nM, whereas the Ki values for α1, α2, β, D2, and 5-HT2 receptors were 35, 181, 740, 19, and 270 nM, respectively (Kung et al., 1994). Taken together, these results suggest that both the reward-enhancing and the reward-reducing effects of 8-OH-DPAT are primarily mediated through 5-HT1A receptor activation. The biphasic effects of systemically administered 8-OH-DPAT may be due to a dose-related selective stimulation of the two types of 5-HT1A receptors, the somatodendritic autoreceptors and the postsynaptic receptors. 8-OH-DPAT has affinity for both subtypes of 5-HT1A receptors (Hall et al., 1985). Systemic administration of 8-OH-DPAT reduced serotonin release in the striatum and ventral hippocampus, effects indicative of autoreceptor activation (Kreiss and Lucki, 1997). Thus, the reward-enhancing effect may be attributable to decreased serotonin release resulting from activation of 5-HT1Asomatodendritic autoreceptors in the raphé nuclei, whereas the reward-reducing effect may be attributable to activation of 5-HT1A postsynaptic receptors.

    To assess the effects of 8-OH-DPAT on the 5-HT1Asomatodendritic autoreceptors, 8-OH-DPAT was injected into either the median or the dorsal raphé nucleus. Dissociable effects on BSR were observed. Injections into the median raphé nucleus potentiated BSR, an effect consistent with previous reports (Fletcher et al., 1995), whereas injections into the dorsal raphé nucleus had no effect. The similarity of the reward-potentiating effects of 8-OH-DPAT administered either systemically at low doses or directly into the median raphé nucleus suggests that reduced serotonergic neurotransmission induced by activation of 5-HT1Aautoreceptors in the median raphé nucleus may be responsible for the systemic effect. The present results are reminiscent of reports that 8-OH-DPAT injections into the median raphé nucleus increased, whereas injections into the dorsal hippocampus that is rich in 5-HT1A receptors (Pazos and Palacios, 1985) decreased social interactions in rats (File et al., 2000). Based on the above-mentioned findings, it is predicted that activation of postsynaptic 5-HT1A receptors through microinjections in terminal serotonergic regions rich in 5-HT1A receptors, such as the hippocampus and the amygdala, would decrease BSR.

    The lack of effect of 8-OH-DPAT injections into the dorsal raphénucleus is surprising for two reasons. First, because the number of 5-HT1A receptors in the dorsal raphé is approximately 5-fold greater than those in the median raphé(Weissmann-Nanopoulos et al., 1985). Second, because the serotonin cells of the dorsal raphé respond more sensitively than those of the median raphé to 8-OH-DPAT in terms of both decreases in neuronal discharge rates (Sinton and Fallon, 1988), and decreases in forebrain serotonin synthesis (Invernizzi et al., 1991). Nevertheless, the present results are consistent with similar behavioral dissociations between the effects of dorsal and median raphéinfusions of 8-OH-DPAT. 8-OH-DPAT injections into the dorsal raphé reduced locomotor activity and had no effect on male sexual behavior, whereas 8-OH-DPAT injections into the median raphéincreased both locomotor activity and sexual behavior (Hillegaart, 1990; Hillegaart et al., 1991). These behavioral dissociations are probably related to the different targets of the ascending projections from the dorsal and median raphé nuclei. The median raphéprojects primarily to limbic forebrain areas such as the septum, amygdala, and hippocampus, whereas the dorsal raphé projects to striatal areas (Azmitia and Segal, 1978). Infusions of 8-OH-DPAT into the dorsal raphé reduce serotonin synthesis in the striatum, nucleus accumbens, and cortex but not in the hippocampus and hypothalamus, whereas median raphé infusions reduced serotonin synthesis in all of the above-mentioned areas (Invernizzi et al., 1991). Thus, the median raphé effects may be mediated through reduced serotonergic neurotransmission in the hypothalamus that was the stimulation site in the present study. This hypothesis is consistent with data summarized above indicating an inhibitory role for serotonin on hypothalamic BSR (see the Introduction).

    Consistent with the present data and previous reports (Lee and Kornetsky, 1998), enhancing serotonergic neurotransmission by the acute administration of fluoxetine, a selective serotonin reuptake inhibitor, elevated reward thresholds at the highest (10-mg/kg) dose administered. Fluoxetine also increased response latencies at all doses administered. Thus, the threshold-elevating effect of acute fluoxetine may not be a reward-related effect but instead may be due to alterations of motor performance. However, previous reports indicated elevated BSR thresholds after fluoxetine administration without effects on response latencies (Lee and Kornetsky, 1998). Thus, the effects of fluoxetine on response latencies are not consistent, and response latencies, as assessed by the discrete-trial BSR procedure, may not necessarily be the best measure of nonspecific effects of manipulations. In conclusion, the fluoxetine data also support the hypothesis that enhancement of serotonergic neurotransmission decreases BSR. Other than the 5-HT1A receptor, other receptors that also may be involved in serotonin-induced inhibition of reward include the 5-HT1B receptor (Harrison et al., 1999).

    The coadministration of p-MPPI, a 5-HT1A antagonist, and a previously ineffective dose of fluoxetine (5 mg/kg) elevated reward thresholds without affecting response latencies. Although there was a tendency for dose dependence of the effects of p-MPPI on augmenting the effects of a previously ineffective dose of fluoxetine (5 mg/kg) on threshold, this dose dependence was not statistically reliable in the present study. Nevertheless, previous work with p-MPPI and fluoxetine demonstrated that 3 mg/kg p-MPPI combined with 5 mg/kg fluoxetine was sufficient to induce threshold elevations in two independent replications (Harrison et al., 2001). Thus, thep-MPPI-induced potentiation of fluoxetine's effect on thresholds is probably mediated by 5-HT1Areceptor blockade in the raphé nuclei, presumably by potentiating the effects of fluoxetine on serotonin levels. The present behavioral data are consistent with neurochemical data indicating that fluoxetine-induced enhancement of serotonergic neurotransmission is potentiated by pretreatment with a 5-HT1Aantagonist (Hjorth, 1993). These results are again consistent with the hypothesis that serotonin plays an inhibitory role in lateral hypothalamic BSR.

    Nevertheless, it should be noted that the dose of p-MPPI required to block the reward-decreasing effects of 8-OH-DPAT (0.3 mg/kgp-MPPI) was significantly lower than that demonstrated to augment the reward-reducing effects of fluoxetine previously (Harrison et al. 2001) and in the present study (3 and 10 mg/kg, respectively). Thus, the possibility remains that p-MPPI-induced augmentation of fluoxetine's effect on BSR may be mediated by antagonism at other serotonin, and potentially nonserotonin, receptors for which p-MPPI may have affinity at high concentrations (Kung et al., 1994).

    As discussed above, fluoxetine reliably decreases BSR (Lee and Kornetsky, 1998; present study). Fluoxetine, however, is a clinically effective antidepressant treatment that reversesreward deficits in depressed individuals (Wong et al., 1995). Furthermore, the coadministration of pindolol, a 5-HT1A, 5-HT1B, and β-adrenergic receptor antagonist, together with a selective serotonin reuptake inhibitor either accelerates the onset or augments the antidepressant actions of selective serotonin reuptake inhibitors in humans (for review, see McAskill et al., 1998). The present demonstration of increased serotonin neurotransmission decreasing reward appears contradictory to the clinical observation that drugs that enhance serotonin neurotransmission are effective antidepressants that restore sensitivity to rewarding stimuli in humans. Recent data may explain this apparent discrepancy. It has been demonstrated that the effects of increased serotonergic neurotransmission on BSR depend on the “hedonic” state of the subject at the time of treatment. That is, coadministration of fluoxetine and p-MPPI reversed reward deficits associated with nicotine or amphetamine withdrawal (Harrison et al., 2001), whereas the same drug combination decreased reward in unperturbed subjects such as the ones used in the present and previous studies (Harrison et al., 2001).

    In conclusion, the present data indicate that systemic administration of the 5-HT1A receptor agonist 8-OH-DPAT increased BSR at low doses and decreased reward at higher doses. Furthermore, both the reward-potentiating and reward-reducing effects of systemically administered 8-OH-DPAT were shown to be mediated through activation of 5-HT1A receptors, as indicated by blockade of both effects by the 5-HT1A receptor antagonist p-MPPI. Local administration of 8-OH-DPAT into the median, but not the dorsal, raphé nucleus potentiated lateral hypothalamic BSR. Thus, it is suggested here that the reward-potentiating effects of systemically administered low doses of 8-OH-DPAT may be the result of reduced serotonergic neurotransmission, mediated by activation of 5-HT1A somatodendritic autoreceptors in the median raphé nucleus. Finally, enhancing serotonergic neurotransmission by the acute administration of fluoxetine or the coadministration of fluoxetine together with the 5-HT1A receptor antagonist reduced lateral hypothalamic BSR. The present data support the hypothesis that serotonin exerts an inhibitory influence on reward processes. Taken together with previous findings (Harrison et al., 2001), it appears that the reward-modulating effects of serotonin depend on the hedonic state of the subjects.

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    Acknowledgments

    We thank Mike Arends for computer and library searches and editorial assistance, Robert Lintz and Robyn Bianco for technical support, and Richard Schroeder for help with histologies.

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    Footnotes

    • Send reprint requests to: Athina Markou, Ph.D., Department of Neuropharmacology, CVN-7, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. E-mail:amarkou{at}scripps.edu

    • ↵1 Current address: School of Psychology, University of Leeds, Leeds, LS2 9JT, England, UK.

    • This study was supported by National Institute on Drug Abuse Grant DA11946 and a Novartis Research Grant to A.M. This is publication 13580-NP from The Scripps Research Institute.

    • Abbreviations:
      BSR
      brain stimulation reward
      PCPA
      para-chlorophenylalanine
      8-OH-DPAT
      8-hydroxy-2-(di-n-propylamino)tetralin
      5-HT
      5-hydroxytryptamine
      p-MPPI
      4-iodo-N-[2-[4-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide hydrochloride
      ITI
      intertrial interval
      • Received September 27, 2000.
      • Accepted January 5, 2001.
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