Abstract

Central serotonin is implicated in a variety of emotional and behavioral control processes. Serotonin depletion can lead to exaggerated aversive processing and deficient response inhibition, effects that have been linked to serotonin's actions in the amygdala and orbitofrontal cortex (OFC), respectively. However, a direct comparison of serotonin manipulations within the OFC and amygdala in the same experimental context has not been undertaken. This study compared the effects of infusing the serotonin neurotoxin, 5,7-dihydroxytryptamine into the OFC and amygdala of marmosets performing an appetitive test of response inhibition. Marmosets had to learn to inhibit a prepotent response tendency to choose a box containing high-incentive food and instead choose a box containing low-incentive food, to obtain reward. OFC infusions caused long-lasting reductions in serotonin tissue levels, as revealed at postmortem, and exaggerated prepotent responses. In contrast, the significantly reduced prepotent responses following amygdala infusions occurred at a time when serotonin tissue levels had undergone considerable recovery, but there remained residual reductions in extracellular serotonin, in vivo. These opposing behavioral effects of serotonin manipulations in the same experimental context may be understood in terms of the top-down regulatory control of the amygdala by the OFC.

Introduction

Serotonin (5-hydroxytryptamine, 5-HT) plays an important role in a wide variety of emotional, cognitive, and behavioral control processes. Disturbances in serotonin function are associated not only with affective disorders such as depression and anxiety (Deakin 1988; Meltzer 1989) but also with impulse control disorders such as mania, alcohol-induced aggression (Cocarro 1989; Linnoila and Virkkunen 1992; Thakore et al. 1996), and obsessive compulsive disorder (OCD, Blier and de Montigny 1998). Changes in aversive processing have been linked to the effect of serotonin at the level of the amygdala in both humans and animals (Stutzmann and LeDoux 1999; Burghardt et al. 2004; Harmer et al. 2006), whereas behavioral disinhibition is associated with serotonergic dysfunction in the medial prefrontal cortex (PFC) and orbitofrontal cortex (OFC) (Clarke et al. 2004; Winstanley, Theobald, Cardinal, and Robbins 2004; Winstanley, Theobald, Dalley, et al. 2004; Clarke et al. 2005; Walker et al. 2006; Winstanley et al. 2006). In particular, reductions of serotonin within OFC produces marked perseverative responding on a range of appetitive visual discrimination tasks (Clarke et al. 2004; 2005; Walker et al. 2006, 2009).

However, the OFC and amygdala are reciprocally interconnected (Carmichael and Price 1995; Ghashghaei et al. 2007), and both regions have been implicated in emotional regulation (Banks et al. 2007) and behavioral flexibility (Jones and Mishkin 1972; Dias et al. 1996; Izquierdo and Murray 2005; Saddoris et al. 2005; Stalnaker et al. 2007). Moreover, dysregulation in both regions has been reported in affective disorders (Drevets 2001; Dannlowski et al. 2007) as well as OCD (Szeszko et al. 1999; Saxena and Rauch 2000; Cannistraro et al. 2004). Thus, in order to understand, more fully, the role of serotonin in emotional and behavioral control processes and the contribution of serotonergic dysregulation to the various symptoms of disorders such as depression and OCD, it is necessary to compare the effects of serotonin manipulations at the level of the OFC and amygdala in animals performing the same behavioral test.

The present study compared the effects of 5,7-dihydroxytryptamine (5,7-DHT)-induced depletions of 5-HT in the OFC and amygdala of marmosets on performance of a test requiring the inhibition of a prepotent response. Two clear Perspex boxes, half filled with either high- or low-incentive food, were presented, and animals had to learn to inhibit their initial preference to respond to the high-incentive food box and to learn instead to respond to the low-incentive food box in order to obtain food reward. Successful performance on this test has already been shown to depend upon an intact OFC in marmosets (Man et al. 2009).

Materials and Methods

Subjects

Eleven common marmosets (Callithrix jacchus, 4 females, 7 males) were subjects in the present study. Animals were housed in pairs in temperature- and humidity-controlled conditions on a 12-h light/dark cycle. Monkeys were maintained on a daily diet of sandwiches (bread, egg, and marmoset jelly [Special Diet Services, Essex, United Kingdom]), and 2 pieces of carrot or a slice of orange, with additional weekend supplements of fruit, Farley's rusk, and peanuts. Water was available ad libitum.

Prior to entering this study, all animals had received a telemetric implant into the descending aorta, undergone behavioral testing on an appetitive pavlovian conditioning paradigm identical to that described in Reekie et al. (2008), and received a 5,7-DHT or sham lesion of the OFC or amygdala (5,7-DHT OFC, n = 4; 5,7-DHT Amygdala, n = 3; and sham controls, n = 2[OFC] and n = 2[Amygdala]). All procedures were conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986.

Surgery

Lesions of the serotonergic innervation of the OFC and amygdala were made following a procedure adapted from Clarke et al. (2005) using 5,7-DHT (9.92 mM: creatinine sulfate salt; Sigma–Aldrich) in 0.1% L-ascorbic acid in 0.9% saline. The noradrenaline (NA) uptake blocker nisoxetine (25 mM; Sigma–Aldrich) and the dopamine (DA) uptake blocker 1-(2-(bis-(4-fluorophenyl) methoxy)ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (GBR 12909, 1.0 mM; Sigma–Aldrich) were administered concomitantly to protect NA and DA innervation, respectively.

Marmosets were premedicated with ketamine hydrochloride (0.1 mL of a 100 mg/mL solution, i.m.; Pfizer, Kent, United Kingdom) and a prophylactic analgesic (Norocarp; 0.03 mL of 50 mg/mL carprofen, s.c.; Pfizer), before being intubated and maintained on isoflurane gas anesthetic (flow rate: 2.5% isoflurane in 0.2 L/min O2; Novartis Animal Health UK, Herts, United Kingdom). Animals were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) with incisor and zygoma bars specially adapted for the marmoset. Injections were made at a rate of 0.05 μL/20 s through a glass cannula attached to a 2-μL Hamilton syringe (Precision Sampling Co, Baton Rouge, LA). The coordinates and volumes of toxin administered are shown in Table 1. Sham controls underwent identical surgical procedures with the toxin omitted from the infusate. Dexamethasone phosphate (0.2 mL i.m.; Fauling Pharmaceuticals plc, Warwicks, United Kingdom) was administered on completion of surgery to prevent tissue inflammation. The analgesic Metacam (meloxicam, 0.1 mL of a 1.5 mg/mL oral suspension; Boehringer Ingelheim, Germany) was given every 24 h for 3 days postoperatively for further pain relief. Animals were returned to their home cage and had ad libitum access to water and supplementary diet during a recovery period of at least 12 days.

Table 1

Stereotaxic coordinates for lesions

Lesion area Coordinates (mm)
 
Volume injected (μL) 
AP LM 
OFC 16.75 ± 2.5 0.7* 0.50 
 ± 4.8 0.7* 0.50 
17.75 ± 2.0 0.7* 0.50 
 ± 4.8 0.7* 0.50 
18.5 ± 2.0 0.7* 0.60 
 ± 4.0 0.7* 0.50 
Amygdala 9.3 ± 5.6 0.35 
  0.35 
Lesion area Coordinates (mm)
 
Volume injected (μL) 
AP LM 
OFC 16.75 ± 2.5 0.7* 0.50 
 ± 4.8 0.7* 0.50 
17.75 ± 2.0 0.7* 0.50 
 ± 4.8 0.7* 0.50 
18.5 ± 2.0 0.7* 0.60 
 ± 4.0 0.7* 0.50 
Amygdala 9.3 ± 5.6 0.35 
  0.35 

Note: Coordinates (based on the interaural plane) and volume for each injection. OFC, orbitofrontal cortex; * 0.7 mm above the base of the brain.

Apparatus

The Wisconsin General Test Apparatus (WGTA) and training within the apparatus has previously been described in detail (see Man et al. 2009). Briefly, animals sat in a Perspex carry box behind the apparatus. When an opaque screen was lifted, they could see into a test compartment and could reach through the bars of the carry box, toward a test tray containing 2 food wells (20-mm diameter and 6-mm depth, spaced 115 mm apart). The experimenter could view the test compartment through a 1-way mirror and control the screen and objects without the animals being able to see the experimenter.

Behavioral Testing

Preliminary training consisted of simple 2-choice object discriminations of 2 3-dimensional objects of differing color and shape, placed directly over the prebaited food wells. Animals had to touch the object to choose it, whereby the object was retracted, to reveal either “syrup bread” reward (0.5-cm cube of wholemeal bread soaked in glucose BP solution; William Ranson & Son Plc, United Kingdom) in the underlying food well (rewarded object) or an empty food well (unrewarded object). The position of the rewarded object was pseudorandomly allocated to the left or right food wells across trials. Subjects received up to 30 trials per daily session. Criterion for all tasks was set at 90% correct responses within a 30-trial session (27/30).

After completing 2 pairs of object discriminations, the objects were substituted with clear Perspex boxes (5 cm × 5 cm × 5 cm), one of which was half filled with multicolored marshmallows (high-incentive object), whereas the other was half filled with dry laboratory chow food pellets (low-incentive object). The food types were clearly visible within the sealed boxes, which the animals could not access. In this discrimination task with incongruent incentive objects, animals were required to inhibit their prepotent response of reaching toward the high-incentive food object and instead choose the low-incentive food object in order to receive a syrup bread reward (Fig. 1A–C). Choosing the incorrect high-incentive object received no reward. Thus, the incentive values of the food objects were incongruent with the reward contingencies. The response (correct or incorrect) was recorded. High- and low-incentive objects were pseudorandomly allocated to the left and right food wells and balanced across each block of 10 trials. A correction procedure was implemented if a subject displayed a significant side bias of 6 consecutive responses to 1 side.

Figure 1.

A schematic diagram of the test apparatus (A) where a marmoset is presented with a choice of 2 food objects. Choosing the incorrect high-incentive object results in that object being retracted revealing an empty food well, and thus, no reward (B), whereas when the correct, low-incentive food object is chosen and retracted, the animal gets access to food reward (C).

Figure 1.

A schematic diagram of the test apparatus (A) where a marmoset is presented with a choice of 2 food objects. Choosing the incorrect high-incentive object results in that object being retracted revealing an empty food well, and thus, no reward (B), whereas when the correct, low-incentive food object is chosen and retracted, the animal gets access to food reward (C).

Behavioral Measures

The main measures of performance were the total number of incorrect responses (errors) and overall trials made before reaching criterion of 90% correct across thirty trials. In addition, using signal detection theory (Macmillan and Creelman 1991), a measure of discrimination (d' value) was calculated for blocks of 10 trials and averaged within groups per block of trials. This was compared with criterion values of a 2-tailed z-test (each tail P = 0.05) to determine the classification of each 10-trial block into perseverative (d' < 0.05; prepotent responding to the incorrect stimulus significantly above chance) and nonperseverative error types (d' > 0.05; responding to the correct stimulus at or above chance). For a detailed explanation of these calculations, see Clarke et al. (2004). This analysis of the pattern of errors on a discrimination task has proven particularly sensitive to detecting the effects of serotonergic lesions in previous studies, revealing differences between the experimental and control groups that were not evident from the analysis of overall errors to criterion (Clarke et al. 2007; Walker et al. 2009).

In Vivo Assessment of Extracellular 5-HT Using Microdialysis

Given the length of time since 5,7-DHT amygdala lesion surgery (8–10 months), immediately following completion of WGTA testing, extracellular levels of 5-HT were assessed in the amygdala of 2 lesioned and 2 sham-operated control animals using in vivo microdialysis.

Following isoflurane anesthesia, probes were implanted acutely in the following stereotaxic coordinates based on the interaural plane: anterior–posterior +9.3 mm; lateral −5.6 mm; dorsal–ventral +4.0 mm. A concentric-design microdialysis probe was constructed using an outer 24-gage thin-wall stainless-steel tubing (length 24 mm), an inner polyamide-coated silica glass capillary tubing (SGE, United Kingdom, internal diameter 80 μm, outer diameter 110 μm) and an exposed active 1.0-mm length of dialysis membrane (Fitral 16; Hospal, United Kingdom, outer diameter 220 μm). Harvard microsyringe pumps with 2.5-mL gas-tight syringes perfused artificial cerebrospinal fluid (aCSF) through the dialysis probe at a flow rate of 1.0 μL/min. The aCSF had the following composition (mM): NaCl 147, KCl 3.0, CaCl2 1.3, MgCl2 1.0, sodium phosphate buffer 1.5 (pH 7.4), and the selective 5-HT reuptake inhibitor citalopram (1 μL) to augment extracellular levels of 5-HT.

After approximately 3 h to allow the implanted probes to equilibrate, dialyzate fractions were collected every 20 min into 2 μL 0.01M perchloric acid. Samples consisted of 3 baseline fractions, followed by a 10 min potassium (75 mM, K+) challenge to cause depolarization and neurotransmitter release and then a further 3 fractions. Samples were stored on dry ice and then at −80 °C before being analyzed.

Dialyzate samples were analyzed for 5-HT and the metabolite 5-hydroxyindolacetic acid (5-HIAA) content using reversed-phase high-performance liquid chromatography (HPLC) and electrochemical detection following the methods outlined in Dalley et al. (2002). The signal was integrated using Chromeleon software (version 6.2, Dionex, United Kingdom).

Postmortem Lesion Assessment

Postmortem neurochemical analysis was used to determine the specificity and extent of the 5,7-DHT lesion in the OFC and amygdala. On completion of the study (ca. 8–12 months postsurgery) animals were euthanized with Dolethal (1 mL of pentobarbital sodium ph.Eur.200 mL/mL solution i.p; Merial Animal Health, Ltd, Essex, United Kingdom), and tissue samples were taken from cortical and subcortical regions. Samples were homogenized in 200 μL of 0.2 M perchloric acid, centrifuged at 6000 rpm for 20 min at 4 °C, and the supernatant was analyzed by reversed-phase HPLC and electrochemical detection.

Chilled 15-μL samples were separated on a C18 silica-based analytical column (100 × 4.6 mm 3 μm octadecylsilane) using a mobile phase (13.6 g/L KH2PO4.H2O, 185 mg/L octane sulfonic acid, and 18% methanol, pH 2.75) delivered at 0.8 mL/min. Tissue levels of 5-HT and 5-hydroxyindoleacetic acid were quantified using a dual-electrode analytical cell and electrochemical detector (Coulochem II; ESA, Chelmsford, MA) with electrode 1 set at −150 mV and electrode 2 set at 180 mV (5014b analytical cell; ESA) with reference to a palladium electrode. The resultant signal was integrated using Chromeleon software (version 6.2, Dionex, United Kingdom). The HPLC system was calibrated using standards containing known amounts of 5-HT, NA, and DA.

Statistical Analysis

Data were analyzed using SPSS for Windows (version 16.0, SPSS Inc, Chicago, IL, United States). Statistical significance was set at P ≤ 0.05, and data were square root transformed as necessary if variances deviated significantly (as measured by Levene′s test of homogeneity). Analysis of neurochemical data involved multiple independent 1-way analysis of variance (ANOVA) with Sidak correction for multiple comparisons with uncorrected posthoc t-tests. Behavioral measures of total number of trials and errors to criterion were compared across the 3 groups using 1-way ANOVA with posthoc Tukey's analysis. Error types were examined using repeated measures ANOVA (within-group factor: error type (2), perseverative, nonperseverative; between-group factor: lesion group (3), 5,7-DHT OFC-treated, 5,7-DHT amygdala-treated, control). Simple main and interaction effects underwent posthoc analysis using independent samples t-tests. All data are presented as mean ± standard error of the mean (SEM).

Results

Lesion Assessment

Postmortem Levels of Serotonin in the OFC and Amygdala

Injections of 5,7-DHT into the OFC caused substantial depletions of 5-HT in the OFC at 8–12 months postsurgery. There was an additional depletion in the neighboring lateral PFC. In contrast, amygdala 5-HT depletion following 5,7-DHT injections into the amygdala was less extensive at 8–12 months postsurgery (Fig. 2A and Table 2). Two of the 3, 5,7-DHT amygdala-treated animals (A1, A2) had depletions of 27% and 38%, whereas the third (A3) had only 1%. All 3 did however have reduced 5-HIAA levels, 32%, 35%, and 56%, respectively. Pretreatment with the NA and DA reuptake blockers nisoxetine and GBR 12909 successfully protected NA and DA, respectively, within the OFC of 5,7-DHT OFC-treated animals and the amygdala of 5,7-DHT amygdala-treated animals, compared with controls. No significant depletions of 5-HT, DA, or NA were seen in any other cortical or striatal regions. The failure to find significant postmortem amygdala 5-HT depletions may be due to recovery over time. Pilot data measuring the extent of 5-HT depletion following 5,7-DHT infusions into the amygdala at 2 weeks (n = 1) and 4 months (n = 1) in postmortem tissue revealed depletions of 80% and 50%, respectively (Fig. 2B).

Table 2

Postmortem 5-HT and 5-HIAA tissue levels

Brain region Serotonin levels
 
5-HIAA levels
 
 % Depletion
 
 % Depletion
 
Control 5,7-DHT OFC 5,7-DHT Amygdala Control 5,7-DHT OFC 5,7-DHT Amygdala 
OFC* 0.8 ± 0.04 67.1 ± 4.5** (58.5–63.8) −34.4 ± 7.4 (−48.4–23.2) 1.1 ± 0.07 56.4 ± 2.1 (52.7–59.8) 16.8 ± 11.5 (−10.2–39.5) 
Amygdala 1.7 ± 0.3 12.5 ± 8.2 (−3.4–23.6) 26.1 ± 10.9 (1.2–37.8) 6.6 ± 0.7 13.8 ± 15.0 (−15.6–33.6) 43.6 ± 5.8 (32.2–56.3) 
LPFC 0.9 ± 0.06 66.8 ± 4.8* −11.2 ± 13.2 0.6 ± 0.07 58.4 ± 1.0 21.2 ± 14.8 
DPFC 0.7 ± 0.1 31.0 ± 6.9 7.4 ± 7.0 0.7 ± 0.09 33.9 ± 9.0 28.6 ± 11.3 
MPFC 1.0 ± 0.06 41.1 ± 4.2 −23.1 ± 13.1 1.5 ± 0.06 38.1 ± 6.4 34.2 ± 10.6 
PM/M 0.7 ± 0.07 21.3 ± 2.3 −13.3 ± 6.1 0.8 ± 0.05 22.1 ± 11.4 22.4 ± 11.7 
Peri-amygdala 1.6 ± 0.4 20.9 ± 13.1 28.6 ± 23.9 4.3 ± 0.7 22.7 ± 17.3 46.9 ± 12.2 
Hippocampus 0.9 ± 0.1 −0.2 ± 11.8 −3.9 ± 47.3 3.8 ± 0.8 18.2 ± 16.3 18.6 ± 14.0 
C1 0.7 ± 0.07 9.0 ± 6.2 −35.9 ± 17.2 1.3 ± 0.15 15.4 ± 6.6 21.3 ± 10.5 
C2 0.8 ± 0.03 8.5 ± 5.3 −33.9 ± 7.3 1.0 ± 0.07 11.4 ± 17.2 8.4 ± 12.5 
Caudate1 1.1 ± 0.3 −4.1 ± 9.4 −40.8 ± 15.5 3.9 ± 0.4 11.4 ± 14.0 −53.7 ± 31.5 
Caudate2 1.7 ± 0.1 18.4 ± 3.5 −3.1 ± 23.1 5.2 ± 0.4 34.8 ± 16.0 −9.6 ± 33.2 
Putamen1 1.6 ± 0.2 12.0 ± 11.5 7.6 ± 3.9 6.8 ± 0.7 28.9 ± 22.9 −0.7 ± 23.5 
Putamen2 1.8 ± 0.2 14.8 ± 3.6 −3.8 ± 16.5 9.1 ± 1.1 43.1 ± 9.4 −30.3 ± 25.4 
NAcc 1.9 ± 0.3 −15.7 ± 1.7 −20.2 ± 16.5 5.9 ± 0.4 7.0 ± 17.1 −11.6 ± 27.9 
Hypothalamus 2.8 ± 0.5 19.1 ± 2.6 −43.4 ± 15.7 7.2 ± 1.3 23.1 ± 13.9 −26.0 ± 33.1 
Brain region Serotonin levels
 
5-HIAA levels
 
 % Depletion
 
 % Depletion
 
Control 5,7-DHT OFC 5,7-DHT Amygdala Control 5,7-DHT OFC 5,7-DHT Amygdala 
OFC* 0.8 ± 0.04 67.1 ± 4.5** (58.5–63.8) −34.4 ± 7.4 (−48.4–23.2) 1.1 ± 0.07 56.4 ± 2.1 (52.7–59.8) 16.8 ± 11.5 (−10.2–39.5) 
Amygdala 1.7 ± 0.3 12.5 ± 8.2 (−3.4–23.6) 26.1 ± 10.9 (1.2–37.8) 6.6 ± 0.7 13.8 ± 15.0 (−15.6–33.6) 43.6 ± 5.8 (32.2–56.3) 
LPFC 0.9 ± 0.06 66.8 ± 4.8* −11.2 ± 13.2 0.6 ± 0.07 58.4 ± 1.0 21.2 ± 14.8 
DPFC 0.7 ± 0.1 31.0 ± 6.9 7.4 ± 7.0 0.7 ± 0.09 33.9 ± 9.0 28.6 ± 11.3 
MPFC 1.0 ± 0.06 41.1 ± 4.2 −23.1 ± 13.1 1.5 ± 0.06 38.1 ± 6.4 34.2 ± 10.6 
PM/M 0.7 ± 0.07 21.3 ± 2.3 −13.3 ± 6.1 0.8 ± 0.05 22.1 ± 11.4 22.4 ± 11.7 
Peri-amygdala 1.6 ± 0.4 20.9 ± 13.1 28.6 ± 23.9 4.3 ± 0.7 22.7 ± 17.3 46.9 ± 12.2 
Hippocampus 0.9 ± 0.1 −0.2 ± 11.8 −3.9 ± 47.3 3.8 ± 0.8 18.2 ± 16.3 18.6 ± 14.0 
C1 0.7 ± 0.07 9.0 ± 6.2 −35.9 ± 17.2 1.3 ± 0.15 15.4 ± 6.6 21.3 ± 10.5 
C2 0.8 ± 0.03 8.5 ± 5.3 −33.9 ± 7.3 1.0 ± 0.07 11.4 ± 17.2 8.4 ± 12.5 
Caudate1 1.1 ± 0.3 −4.1 ± 9.4 −40.8 ± 15.5 3.9 ± 0.4 11.4 ± 14.0 −53.7 ± 31.5 
Caudate2 1.7 ± 0.1 18.4 ± 3.5 −3.1 ± 23.1 5.2 ± 0.4 34.8 ± 16.0 −9.6 ± 33.2 
Putamen1 1.6 ± 0.2 12.0 ± 11.5 7.6 ± 3.9 6.8 ± 0.7 28.9 ± 22.9 −0.7 ± 23.5 
Putamen2 1.8 ± 0.2 14.8 ± 3.6 −3.8 ± 16.5 9.1 ± 1.1 43.1 ± 9.4 −30.3 ± 25.4 
NAcc 1.9 ± 0.3 −15.7 ± 1.7 −20.2 ± 16.5 5.9 ± 0.4 7.0 ± 17.1 −11.6 ± 27.9 
Hypothalamus 2.8 ± 0.5 19.1 ± 2.6 −43.4 ± 15.7 7.2 ± 1.3 23.1 ± 13.9 −26.0 ± 33.1 

Note: Mean levels of 5-HT and 5-HIAA (pmol/mg tissue weight ± SEM) in the control group and depletions of 5-HT and 5-HIAA calculated as a percentage change from mean control levels (± SEM) in marmosets with 5,7-DHT lesions of the OFC and amygdala. Value ranges (in parentheses) are provided for OFC and amygdala regions of interest. LPFC, lateral prefrontal cortex; DPFC, dorsal prefrontal cortex; MPFC, medial prefrontal cortex; PM/M, premotor/motor cortex; C1, anterior cingulate cortex; C2, midcingulate cortex; Caudate1, anterior caudate; Caudate2. midcaudate; Putamen1, anterior putamen; putamen2, midputamen; NAcc, nucleus accumbens. *P < 0.05; **P < 0.01

Figure 2.

Depletion of 5-HT in postmortem tissue of 5,7-DHT–treated animals calculated as a percentage change from the mean of control levels (n = 4). (A) 5-HT depletion in OFC and in amygdala of animals that received either a 5,7-DHT infusion into the OFC (5,7-DHT OFC, n = 3) or amygdala (5,7-DHT Amygdala, n = 3). ** Within OFC, 5-HT OFC-lesioned group significantly different from 5-HT amygdala-lesioned group, P < 0.01. (B) 5-HT depletion in amygdala at a range of time points (2 weeks, 4, 9, and 11 months) following 5,7-DHT infusions into the amygdala. The gray bar indicates the range of time postlesion during which behavioral testing in the current study occurred; between 8 and 11.5 months postlesion. A1-3 are individual 5,7-DHT amygdala-lesioned animals.

Figure 2.

Depletion of 5-HT in postmortem tissue of 5,7-DHT–treated animals calculated as a percentage change from the mean of control levels (n = 4). (A) 5-HT depletion in OFC and in amygdala of animals that received either a 5,7-DHT infusion into the OFC (5,7-DHT OFC, n = 3) or amygdala (5,7-DHT Amygdala, n = 3). ** Within OFC, 5-HT OFC-lesioned group significantly different from 5-HT amygdala-lesioned group, P < 0.01. (B) 5-HT depletion in amygdala at a range of time points (2 weeks, 4, 9, and 11 months) following 5,7-DHT infusions into the amygdala. The gray bar indicates the range of time postlesion during which behavioral testing in the current study occurred; between 8 and 11.5 months postlesion. A1-3 are individual 5,7-DHT amygdala-lesioned animals.

Comparison of 5-HT levels across groups in 16 brain regions using ANOVA (with Sidak correction for multiple comparisons) revealed a significant main effect of group for 5-HT levels in OFC (F(2,10) = 67.49, P = 0.0002) but no significant effect in amygdala (F(2,10) < 1). Uncorrected posthoc t-tests demonstrated that the 5,7-DHT OFC-treated group had significantly lower 5-HT levels within the OFC compared with both controls (t6 = 8.9, P < 0.001) and 5,7-DHT amygdala-treated animals (t4 = −11.8, P = 0.001). There was also a significant reduction in 5-HT levels in the lateral PFC (F(2,10) = 22.5, P = 0.02) due to 5,7-DHT OFC-treated animals showing significant 5-HT depletions compared with controls (t6 = 7.6, P = 0.001). We have previously reported significant depletions in this area using this lesion method (Clarke et al. 2004, 2005; Walker et al. 2006, 2009). Analysis of 5-HIAA levels across regions revealed significant reductions in OFC but these did not survive correction for multiple comparisons (F(2,10) = 11.07, P = 0.077; % depletions, 56.4 ± 2.0).

Analysis for DA and NA revealed that 5,7-DHT infusions into the OFC did not affect these other monoamines within the OFC or neighboring frontal brain regions (lateral, dorsal, and medial PFC; all F's<1). Similarly DA and NA levels were not affected consistently in the amygdala or neighboring brain regions (hippocampus, peri-amygdala cortex, nucleus accumbens, putamen, and hypothalamus) of 5,7-DHT amygdala-treated animals (all F's < 1).

It is unclear why 5-HT depletion, as measured by postmortem tissue levels of extracellular 5-HT, was greater in the OFC compared with the amygdala, 8–12 months postsurgery. However, 5-HT tissue levels were 2-fold greater in the amygdala than in the OFC of control animals (OFC: 0.8 pmol/mg tissue weight vs. Amygdala: 1.7 pmol) which is likely to reflect a greater density/number of terminals in the amygdala, and this may underlie the greater propensity to recover from 5,7-DHT lesions in the amygdala.

Extracellular Measurement of Serotonin in the Amygdala Using In Vivo Microdialysis

Although postmortem tissue levels of amygdala showed a significant degree of recovery 8–12 months postsurgery, amygdala 5-HT function, assessed using in vivo microdialysis, was still significantly compromised at approximately 10 months postsurgery. Figure 3A shows that extracellular 5-HT levels were below the level of detection (2.0 fmol/20 mins) at both baseline and following a K+ challenge, in the 2 5,7-DHT amygdala-treated animals that underwent microdialysis, including the animal with no discernable 5-HT depletion in postmortem tissue (see animal A3 in Fig. 2B). In contrast, in the 2 control animals that underwent dialysis, 5-HT levels were well above threshold (ca. 9.5 fmol) and K+ caused an almost 2-fold increase in 5-HT levels. In addition, concentrations of the 5-HT metabolite, 5-HIAA, in 5,7-DHT amygdala-treated animals were approximately half that observed in controls (Fig. 3B). Taken together, these results suggest a functional depletion of serotonin at 8–12 months postsurgery.

Figure 3.

Extracellular concentrations of 5-HT (A) and 5-HIAA (B) in the amygdala of 2 controls (C1,C2) and 2 animals with 5,7-DHT lesions of the amygdala (A2,A3). Mean concentrations of 3 20-min baseline dialyzate fractions (basal, open bar, ±SEM) and the subsequent 20-min dialyzate fraction including the 10-min 75 mM potassium infusion (K+, striped bar). The dashed lines indicate the detection thresholds for 5-HT and 5-HIAA.

Figure 3.

Extracellular concentrations of 5-HT (A) and 5-HIAA (B) in the amygdala of 2 controls (C1,C2) and 2 animals with 5,7-DHT lesions of the amygdala (A2,A3). Mean concentrations of 3 20-min baseline dialyzate fractions (basal, open bar, ±SEM) and the subsequent 20-min dialyzate fraction including the 10-min 75 mM potassium infusion (K+, striped bar). The dashed lines indicate the detection thresholds for 5-HT and 5-HIAA.

Behavioral Assessment

Two simple object discriminations were successfully learned by all animals during preliminary training (mean of the summed errors across the 2 discriminations to reach criterion ± SEM, control: 13.0 ± 3.4; 5-HT OFC lesion: 26.8 ± 12.8; 5-HT amygdala lesion: 16 ± 2.3). One-way ANOVA showed no effect of group on the total number of trials to criterion or total errors, for either discrimination (all F < 1). The large number of errors for the 5,7-DHT OFC-treated group was due to 1 of the 4 animals making 4 times more errors, than the mean errors of the remaining 3, on 1 of the discriminations. There were no perseverative errors made during training, and there was no significant effect of group on nonperseverative errors (F < 1 for both discriminations). Therefore, depletion of 5-HT in the OFC or amygdala had no effect on learning object–reward associations in a 2-choice discrimination task.

Discrimination Task with Incongruent Incentive Objects

All monkeys showed an initial prepotent tendency to choose the unrewarded, high-incentive object, but over time, were able to learn to inhibit this response and choose the low-incentive object (Fig. 4A and B). However, although 5,7-DHT amygdala-treated monkeys made significantly fewer prepotent or perseverative responses to the high-incentive unrewarded object compared with controls, 5,7-DHT OFC-treated monkeys made significantly more responses (see Fig. 4C).

Figure 4.

Behavioral assessment of discrimination task with incongruent incentive objects. (A) Learning curves of 5,7-DHT Amygdala group (n = 3) compared with controls (n = 4) and (B) 5,7-DHT OFC group (n = 4) compared with same control data. “d′ value” calculated using signal-detection theory on blocks of 10 trials and averaged within groups per block of trials. Italicized numbers adjacent to data points represent the number of animals contributing to the mean d′ value. Points within the upper and lower dotted lines reflect chance performance. Shaded area represents the number of trials required for controls to reach chance performance, which were (A) greater compared with 5,7-DHT Amygdala-treated animals but (B) fewer than 5,7-DHT OFC-treated animals. (C) Mean numbers of perseverative and nonperseverative errors (square root ± SEM) to reach criterion in control, 5,7-DHT OFC and 5,7-DHT Amygdala groups. *, 5,7-DHT Amygdala group made significantly fewer perseverative errors than controls; **, 5,7-DHT OFC group made significantly greater perseverative errors than controls;‡, 5,7-DHT OFC group made more nonperseverative errors than controls.

Figure 4.

Behavioral assessment of discrimination task with incongruent incentive objects. (A) Learning curves of 5,7-DHT Amygdala group (n = 3) compared with controls (n = 4) and (B) 5,7-DHT OFC group (n = 4) compared with same control data. “d′ value” calculated using signal-detection theory on blocks of 10 trials and averaged within groups per block of trials. Italicized numbers adjacent to data points represent the number of animals contributing to the mean d′ value. Points within the upper and lower dotted lines reflect chance performance. Shaded area represents the number of trials required for controls to reach chance performance, which were (A) greater compared with 5,7-DHT Amygdala-treated animals but (B) fewer than 5,7-DHT OFC-treated animals. (C) Mean numbers of perseverative and nonperseverative errors (square root ± SEM) to reach criterion in control, 5,7-DHT OFC and 5,7-DHT Amygdala groups. *, 5,7-DHT Amygdala group made significantly fewer perseverative errors than controls; **, 5,7-DHT OFC group made significantly greater perseverative errors than controls;‡, 5,7-DHT OFC group made more nonperseverative errors than controls.

An initial 1-way ANOVA on the total number of errors to reach criterion revealed a significant Group effect (F(2,10) = 20.79, P = 0.001), with the 5,7-DHT OFC group making significantly more errors compared with the 5,7-DHT amygdala-treated (P = 0.003) and control groups (P = 0.001), although there was no difference between the latter 2 groups on this measure. An equivalent pattern was seen when analyzing the total number of trials to criterion (Group effect: F(2,10) = 18.86, P = 0.001).

The errors were then analyzed further using signal-detection theory to distinguish between perseverative and nonperseverative components (Fig. 4C). A 2-way ANOVA confirmed a main effect of Group (F(2,8) = 21.9, P = 0.001), Error type (F(1,8) = 38.39, P > 0.001) and a Group by Error type interaction (F(2,8) = 11.24, P = 0.005). Simple main effects revealed that 5,7-DHT OFC-treated animals made significantly more perseverative errors compared with 5,7-DHT amygdala-treated (P < 0.001) and control groups (P = 0.003). In contrast, the 5,7-DHT amygdala-treated animals made significantly less perseverative errors compared with controls (P = 0.042). With respect to the nonperseverative errors, 5,7-DHT OFC-treated animals made more such errors than controls (P = 0.005) and 5,7-DHT amygdala-treated animals (P = 0.01), whereas 5,7-DHT amygdala-treated animals were no different to controls.

Discussion

5,7-DHT infusions into the OFC of marmosets resulted in selective reductions of postmortem tissue levels of 5-HT within the OFC and adjacent lateral PFC and impaired the performance of marmosets on the discrimination task with incongruent incentive objects. Early in learning, marmosets with OFC 5,7-DHT infusions made significantly more responses to the high-incentive object compared with controls, despite receiving no reward for doing so. They also took more trials overall to reach criterion, compared with controls. 5,7-DHT infusions into the amygdala also affected performance on the task but produced the converse behavioral effect to that of 5,7-DHT infusions into the OFC. Marmosets switched away from the high-incentive object in significantly fewer trials than controls. Although postmortem tissue levels of serotonin in the amygdala were not significantly reduced compared with controls, evidence for a functional lesion was provided by in vivo microdialysis performed immediately after task completion. The latter revealed a marked reduction in extracellular levels of 5-HT within the amygdala of 5,7-DHT amygdala-treated monkeys and a significantly blunted response to potassium stimulation.

The impaired ability of marmosets with 5,7-DHT infusions within the OFC to perform the incongruent incentive discrimination task is consistent with our previous studies demonstrating that this same lesion impairs performance on a visual discrimination reversal task (Clarke et al. 2005) and a detour-reaching task (Walker et al. 2006). In all cases, the 5-HT–depleted animals persist in performing a previously rewarded or prepotent response, despite that response no longer resulting in reward.

The present study extends these previous results in showing that the deficit is present regardless of whether the animal has to 1) inhibit responding to an arbitrary stimulus that has become associated with reward (discrimination reversal) and instead select the previously unrewarded stimulus, 2) inhibit a direct reach for food reward and instead learn an alternative response (detour-reaching task), or 3) inhibit a prepotent response to reach for a high-incentive food object and instead learn to select a low-incentive food object (present study). Such response biases, however, are not only driven by prior reward associations, but they also occur in a context in which animals have to select against an initial stimulus preference in a visual discrimination task using conditioned reinforcers (Walker et al. 2009). In this case, the response bias was most likely the result of the increased perceptual saliency of one of the stimuli. Additional evidence that the deficit is independent of reward-related processing per se comes from the finding that 5,7-DHT infusions into the OFC do not prolong responding during extinction of a visual discrimination, when responding to the previously rewarded stimulus no longer results in reward (Walker et al. 2009). 5-HT–depleted animals show a similar rate of extinction as controls, but unlike controls, persist in responding to the previously rewarded stimulus during extinction and make very few responses to the previously unrewarded stimulus.

These findings highlight how a disruption of serotonin function within the PFC results in attentional/response biases developing toward intrinsically or reward-related salient stimuli, resulting in perseverative-like responding, likely due to a loss of top-down regulation. However, in contrast to our previous studies, the 5,7-DHT OFC-treated monkeys in the present study not only displayed perseverative responding to the high-incentive, unrewarded stimulus, but were also significantly slower to learn to approach the low-incentive, rewarded stimulus. One possible explanation for this difference may lie in the level of active withdrawal induced by the alternative stimulus to which the animal must respond, in order to get reward. This is likely to be far stronger for low-incentive laboratory chow (present study) than an abstract visual pattern previously associated with no reward (discrimination-reversal study); the issue does not arise at all in the detour-reaching task as the animal is learning an alternative response still directed at the same food reward. Thus, the impaired ability to learn to approach the low-incentive food box may also reflect a loss of top-down regulation, this time though, with respect to withdrawal responses. Such withdrawal responses, if left unchecked, as a consequence of PFC dysfunction, will make it less likely that the animal will approach the low-incentive laboratory chow.

These effects are most likely due to loss of 5-HT within the OFC, rather than the lateral PFC as excitotoxic lesions of the OFC cause similar stimulus-bound responding on all 3 tests that have been shown to be sensitive to selective 5-HT depletions, namely, discrimination reversal, detour reaching, and, as shown here, incongruent discrimination. Moreover, although lesions of the lateral PFC have not been studied in the context of the incongruent discrimination task, such lesions do not affect discrimination reversal or detour reaching, making it unlikely that the deficit seen here was due to 5-HT depletion in the lateral PFC.

Serotonin depletions in the amygdala also affected performance on the incongruency-discrimination task but, in contrast to serotonin depletions within the OFC, caused monkeys to switch away from the high-incentive food reward more rapidly than controls; although their overall speed of learning was no different to controls. Before considering the underlying psychological mechanism of this effect, it is important to first consider the nature of the functional lesion caused by 5,7-DHT infusions into the amygdala. Postmortem tissue levels of 5-HT in the amygdala are reduced to approximately 20% of control levels (i.e., 80% depletion) just 2 weeks after the lesion. However, recovery does occur, such that the depletion is approximately 50% at 4 months and 20% at 11 months, postsurgery. Because the animals in this study were tested between 8 and 11 months postsurgery, then considerable recovery had taken place. Despite this, in vivo microdialysis revealed that at the time of behavioral testing, basal extracellular 5-HT levels were below the detection threshold (2.0 fMol), and levels did not exceed this threshold upon potassium stimulation. Hence, extracellular basal levels and stimulated release were markedly reduced in the amygdala.

Although there appeared to be increases in 5-HT and 5-HIAA postmortem tissue levels in the striatum and hypothalamus following 5,7-DHT infusions into the amygdala, these were not significant (F < 1) and were due to large increases in just 1 animal (see very large “SEM” values for caudate and hypothalamus in Table 2). Thus, they are unlikely to contribute to the reductions in perseverative responding that were seen in all 3 5,7-DHT amygdala-lesioned animals.

Whether the improved ability to switch away from the high-incentive food reward that accompanied reduced 5-HT release was due to 1) an enhanced sensitivity to reward loss, 2) a decline in the strength of the approach response to high-incentive food, or 3) a reduced ability to represent the relative reward value of the high- and low-incentive foods remains to be determined. The latter seems least likely because all 5,7-DHT amygdala-treated animals still showed an initial preference for the high-incentive food box, making, on average, 2.6 ± 1.2 responses to the latter before selecting the low-incentive food box for the first time. In contrast, given the contribution of the amygdala to pavlovian appetitive, conditioned approach responses, reduced 5-HT release in the amygdala may have resulted in a decline in the strength of the pavlovian approach response to the sight of the high-incentive food reward (Cardinal et al. 2002). An equally plausible hypothesis is that 5-HT reductions led to enhanced sensitivity to reward loss. Certainly, acute central 5-HT depletion in humans, induced by dietary depletion of the 5-HT precursor tryptophan, has been shown to enhance prediction of the “nonrewarded” outcome on a pavlovian probabilistic discrimination-reversal task (Cools et al. 2008). Such an effect could underlie the improved ability to switch away from the “nonrewarded” high-incentive food reward in the present study.

An important consideration is whether the reduced perseverative responding following 5,7-DHT infusions in the amygdala is a direct consequence of reduced 5-HT functioning within the amygdala or is the result of an exaggerated serotonin response as a consequence of postsynaptic compensatory changes. Although such changes are possible following all neurochemical lesions in which there is recovery over time, it is particularly relevant to the amygdala results of the present study given the more rapid recovery of postmortem serotonin tissue levels in the amydala than in the OFC. Certainly, exaggerated phasic levels of 5-HT following dietary depletion of the 5-HT precursor tryptophan in humans have been proposed to underlie the enhanced prediction of nonreward (Cools et al. 2008). Whether this is the case in the present study cannot be ascertained. In addition, changes in postsynaptic-receptor expression, including changes in the balance of the different receptor subtypes, may have contributed to the behavioral effect. This is an important issue that requires further investigation especially because compensatory changes in 5-HT–receptor expression following serotonin-transporter blockade may underlie the therapeutic effects of antidepressants (Deakin 1988) and drug treatments for OCD (De Leeuw and Westenberg 2008; Joel et al. 2008).

It should be highlighted that, unlike 5,7-DHT induced, 5-HT depletions, excitotoxic-induced lesions of the intrinsic cell bodies of the amygdala in marmosets do not affect learning of a discrimination with incongruent incentive objects (Man et al. 2009). This is consistent with the finding that excitotoxic lesions also do not affect discrimination-reversal learning (Izquierdo and Murray 2007; Clarke et al. 2008). However, combined excitotoxic lesions of the amygdala and OFC in rats do block the reversal deficit observed following excitotoxic OFC lesions alone (Stalnaker et al. 2007). One explanation for this effect has been that although the amygdala is not critical for reversal learning, it has a modulatory role. Because reversal-related neuronal activity in the amygdala is disrupted following an OFC lesion (Saddoris et al. 2005), it is proposed that the resulting aberrant signal from the amygdala is responsible for the deficit in reversal learning. The amygdala may play a similar modulatory but not critical role in this appetitive test of inhibitory control in the present study, and so effects are seen with aberrant amygdala signaling (as caused by 5,7-DHT infusions) but not an absence of amygdala signaling altogether (as caused by excitotoxic cell-body lesions).

The finding that 5-HT manipulations have contrasting effects on behavioral flexibility, at the level of the OFC and amygdala, may be understood in terms of the top-down regulatory control of the amygdala by the OFC. Indeed, contrasting effects of amygdala and OFC excitotoxic lesions have been reported previously in extinction of responding for reward (Izquierdo and Murray 2005; Clarke et al. 2008) and delay of reinforcement using a delay-discounting procedure (Winstanley, Theobald, Cardinal, and Robbins 2004; Winstanley, Theobald, Dalley, et al. 2004). There are both excitatory and inhibitory projections from the OFC into the amygdala (Ghashghaei and Barbas 2002), and a recent study has shown that the flexibility of neuronal activity patterns in the amygdala, in response to changes in reward contingencies, is dependent on the OFC (Saddoris et al. 2005). The present results suggest that the behavioral flexibility provided by such OFC–amygdala interactions is regulated by 5-HT both at the level of the OFC and amygdala. A role for 5-HT in modulating prefrontal–amygdala interactions has already been proposed with respect to medial prefrontal cortex. Thus, in humans, short allele carriers of a functional 5 promotor polymorphism of the serotonin transporter gene show relative uncoupling of a perigenual cingulate–amygdala feedback circuit implicated in the extinction of negative affect (Pezawas et al. 2005). In addition, 5-HT2A density in the medial PFC has been shown to be inversely correlated with threat-related amygdala reactivity and positively correlated with amygdala habituation (Fisher et al. 2009). Moreover, although rapid increases in amygdala 5-HT release are associated with the initiation of fear-related behaviors in rats, relatively delayed 5-HT release in medial PFC is associated with their decline (Forster et al. 2006). A similar relationship may exist between OFC–amygdala interactions and 5-HT but whether the actions of 5-HT serve a common behavioral function across these different circuits remains to be determined. Two related but somewhat competing hypotheses propose that 5-HT is involved in either punishment processing (Dayan and Huys 2009) or the drive to withdraw from aversive or high-stimulation environments (Tops et al. 2009). According to either view, the changes in behavior observed following manipulation of 5-HT in the amygdala and OFC in the present study could be seen as an alteration in the balance between “withdrawal from” and “engagement with” environmental cues, thereby slowing down or speeding up, respectively, learning about the altered contingencies. Whatever the mechanism, these findings provide direct evidence that 5-HT manipulations can cause opposing effects in cortical and subcortical structures and may account for some of the apparently opposing behavioral symptoms that can co-occur in affective disorders.

Funding

Medical Research Council (MRC) Programme Grant (G0401411) to A.C.R.; Conducted within the University of Cambridge Behavioural and Clinical Neuroscience Institute, supported by Medical Research Council and the Wellcome Trust (joint award).

We would like to thank David Theobald for the neurochemical analysis. Conflict of Interest: None declared.

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