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European Journal of Pharmacology 590 (2008) 136–149

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European Journal of Pharmacology

j ourna l homepage: www.e lsev ie r.com/ locate /e jphar

Evidence for serotonin synthesis-dependent regulation of in vitro neuronal firingrates in the midbrain raphe complex

Andrew K. Evans a,c,⁎, Niels Reinders a, Katie A. Ashford a,b, Isabel N. Christie a,Jonathan B. Wakerley b, Christopher A. Lowry a,c

a Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Dorothy Hodgkin Building, Bristol BS1 3NY, United Kingdomb Department of Anatomy, University of Bristol, Bristol, United Kingdomc Department of Integrative Physiology, University of Colorado at Boulder, Boulder, CO 80309-0354, United States

⁎ Corresponding author. Department of Integrative Phyat Boulder, Boulder, CO 80309-0354, United States. Tel.:492 0811.

E-mail address: [email protected] (A.K.

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.06.014

A B S T R A C T
A R T I C L E I N F O
Article history:
Evidence suggests that 5-hy Received 19 February 2008Received in revised form 16 May 2008Accepted 2 June 2008Available online 7 June 2008
Keywords:TryptophanSerotoninTryptophan hydroxylase5-HT1A autoreceptorDorsal raphe nucleusMDMA

droxytryptamine 1A (5-HT1A) receptor-mediated autoregulation of serotonergicneuronal firing rates is impaired in stress-related neuropsychiatric disorders. In vitro models may provideinsight into neural mechanisms underlying regulation of serotonergic systems. However, serotonin synthesisand tonic autoregulation of serotonergic neuronal firing rates are impaired in in vitro preparations lackingtryptophan. We describe the effects of perfusion of living rat brain slices with tryptophan on both 1) tissueconcentrations of serotonin metabolites and 2) neuronal firing rates within the dorsal raphe nucleus. Brainslices were perfused with artificial cerebrospinal fluid lacking tryptophan for 4 h, followed by exposure to 1)40 μM tryptophan (0–60 min) or 2) 0–400 μM tryptophan (23 min) and microdissected for analysis of indoleconcentrations. Parallel studies examined effects of tryptophan on neuronal firing rates and interactions withdrugs expected to alter synaptic concentrations of serotonin. Tryptophan resulted in time-dependent andconcentration-dependent increases in serotonin and serotonin metabolites, effects that were correlated withrestoration of tonic autoinhibition of dorsal raphe nucleus neuronal firing rates. Inhibition of serotoninsynthesis resulted in time-dependent and concentration-dependent increases in 5-hydroxtryptophan thatcorrelated with reversal of the tryptophan-mediated autoinhibition of neuronal firing rates. Tryptophanmodulated effects of several drugs on neuronal firing rates, including a selective 5-HT1A receptor antagonist(WAY-100635), a monoamine oxidase inhibitor (pargyline), a selective serotonin reuptake inhibitor(fluoxetine), and a serotonin-releasing agent (methylenedioxymethamphetamine). These studies supportthe hypothesis that tonic autoregulation of serotonergic neuronal firing rates is dependent on tryptophanavailability and characterise conditions necessary to study this process in vitro.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Within the central nervous system, serotonin exerts a modulatoryinfluence over a diverse array of physiological and behaviouralprocesses ranging from general motor activity and sleep–wake-arousal cycles to temperature control, appetite, aggression, and sexualbehaviour (Jacobs and Azmitia, 1992; Lucki, 1998). In addition,serotonergic systems regulate physiological and behaviouralresponses to stress-related stimuli (Carrasco and Van de Kar, 2003;Chaouloff, 1993; Lowry, 2002; Maier and Watkins, 2005) and areimplicated in themodulation of anxiety states (Millan, 2003; Gray andMcNaughton, 2000; Lowry et al., 2005; Graeff et al., 1997) anddepression (Barnes and Sharp, 1999; Millan, 2006).

siology, University of Colorado+1 303 492 8154; fax: +1 303

Evans).

l rights reserved.

Clinical evidence implicates dysregulation of serotonergic systemsin the aetiology of stress-related neuropsychiatric disorders as indicatedby serotonin-related gene expression and 5-hydroxytryptamine 1A (5-HT1A) autoreceptor binding and function (mechanisms responsible fornegative feedback regulation of serotonergic neuronal firing rates).Human depressed suicide patients have increased expression of therate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase(TPH), in subregions of the midbrain raphe complex (Bach-Mizrachiet al., 2008; Boldrini et al., 2005; Bonkale et al., 2006; Underwood et al.,1999). Allelic variation of TPH has been linked to abnormal amygdalarreactivity in processing of emotional stimuli (Brown et al., 2005; Canliet al., 2005) and can be a genetic predictor of 1) depression (Zill et al.,2004; Haghighi et al., 2008), 2) suicide risk among depressed patients(Lopez et al., 2007), and 3) responses to antidepressant treatment(Peters et al., 2004). 5-HT1A autoreceptor function has also beenassociated with amygdalar reactivity to emotional stimuli (Fisher et al.,2006), and patients with bipolar or unipolar depression, or panicdisorder have reduced 5-HT1A receptor binding in the midbrain raphe

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137A.K. Evans et al. / European Journal of Pharmacology 590 (2008) 136–149

complex (Drevets et al., 2000; Neumeister et al., 2004). Detailed studiesof mechanisms regulating TPH activity and autoreceptor-mediatedregulation of neuronal firing rates in vitro in animal models are animportant step toward understanding the relationships among stress-related physiology, increased TPH expression, autoreceptor-mediatedregulation of serotonergic systems and stress-related neuropsychiatricdisorders.

However, it has been suggested that serotonin synthesis, animportant element of 5-HT1A autoreceptor-mediated regulation ofserotonergic neuronal activity, is impaired in an in vitro slicepreparation in the absence of the synthetic precursor, tryptophan(Liu et al., 2005). Following 2–4 h of perfusion of living brain sliceswith artificial cerebrospinal fluid (aCSF) lacking exogenous trypto-phan, a tonic 5-HT1A receptor-mediated inhibition of neuronal firingrate, commonly observed in vivo (Fornal et al., 1996; Mundey et al.,1996; Hajos et al., 2001), is not present in the dorsal raphe nucleus invitro (Mlinar et al., 2005; Liu et al., 2005; Johnson et al., 2002).Moreover, serotonin concentrations are depleted as measured by theintensity of serotonin-immunostaining (Liu et al., 2005) or analysis ofserotonin concentrations measured in homogenates of whole brainslices using high performance liquid chromatography (HPLC) withelectrochemical detection (Mlinar et al., 2005; Trulson and Freder-ickson, 1987). Both tissue concentrations of serotonin and a tonic 5-HT1A receptor-mediated inhibition of neuronal firing rate can bepartially restored with continuous bath application of tryptophan (Liuet al., 2005; Mlinar et al., 2005), although the time course andconcentration-dependency of these responses have not been exten-sively characterised. It is also unclear to what degree tryptophan-induced changes in serotonin synthesis andmetabolism are correlatedwith changes in neuronal firing rates. Previous in vivo experimentshave demonstrated that neuronal firing rates in the midbrain raphecomplex are sensitive to manipulations designed to alter extracellularserotonin concentrations via dysregulation of serotonin catabolism(Sharp et al., 1997), clearance (Gartside et al., 1997b), or vesicularstorage (Gartside et al., 1997a). Hence it would seem likely that theeffects of these manipulations in vitro are dependent on exogenoustryptophan.

The aims of this study were to characterise the effects of perfusionof living brain slices with aCSF containing exogenous tryptophan ontissue concentrations of tryptophan, serotonin and 5-hydroxyindo-leacetic acid (5-HIAA) in the midbrain raphe complex and todetermine how these effects correlate with the effects of tryptophanon serotonergic neuronal firing rates. Additionally, we examined therole of tryptophan availability on regulatory mechanisms thought toinfluence serotonergic neuronal firing rates via changes in the tone onsomatodendritic autoreceptors. We predicted that serotonin synth-esis, and therefore autoreceptor-mediated regulation of neuronalfiring rates, would be dependent on exogenous tryptophan in an invitro slice preparation. A thorough analysis of the time course andconcentration-dependent characteristics of the response to trypto-phan would seem important as a basis for future in vitro studiesinvestigating molecular and cellular responses that might beinfluenced by changes in tryptophan availability, serotonin synthesis,or somatodendritic autoreceptor signalling.

2. Materials and methods

2.1. Rats

Male Wistar rats (100–200 g) were obtained from a colonymaintained at the University of Bristol (derived from Harlan, Oxon,UK) under standard environmental conditions (21±2 °C; 55±10%humidity; illumination of approximately 300 lx at 1 m). Rats werehoused in groups of 4–6 in Techniplast cages (48 cm length×38 cmwidth×21 cm height; Techniplast, Kettering, UK) on a 14:10 h light–dark cycle with lights on at 5 AM. For some electrophysiological

experiments, rats were housed in RC1 cages (50 cm length×33 cmwidth×23 cm height; North Kent Plastics, Dartford, UK) either ingroups (2–5) or individually (animals arrived in groups of 5 and weresacrificed 1 per day; hence a fully randomised population of ~20%were single housed for at least 24 h). Food (B&K CRM rat chow; B&K,Hull, UK) and tap water were provided ad libitum. All procedures wereapproved by the Ethical Review Group at the University of Bristol andwere conducted in accordance with UK Home Office guidelines andthe Scientific (Animal Procedures) Act 1986.

2.2. Drugs

Phenylephrine hydrochloride, 5-hydroxytryptamine hydrochloride,L-tryptophan, NSD-1015 (m-hydroxybenzylhydrazine), WAY-100635(N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride), fluoxetine hydrochloride,pargyline, and (±)-3,4-methylenedioxymethamphetamine hydrochlor-ide (MDMA) were obtained from Sigma-Aldrich (Poole, UK). All drugswere dissolved in aCSF for application. The concentrations of NSD-1015and WAY-100635 were chosen based on previous work demonstratingtheir efficacies in inhibiting aromatic amino acid decarboxylase (AADC)and selectively antagonizing 5-HT1A receptors respectively in vitro(Hashimoto et al., 2003; Johnson et al., 2002). The concentrations ofpargyline (Huang et al., 1997), fluoxetine (Johnson et al., 2002) andMDMA (Sprouse et al., 1989; Sprouse et al., 1990) used in electro-physiology studies were based on previous in vitro brain slice studies.

2.3. In vitro slice preparation for neurochemistry and electrophysiologystudies

Coronal brain slices were prepared between 8 and 12 AM on themorning of each experiment. Following an overdose of sodiumpentobarbital (Euthatal; Vericore, Dundee, UK; 200 mg/kg bodyweight, i.p.) and rapid decapitation, the brain was rapidly removed,blocked in a coronal plane rostral to the raphe complex, and mountedon a vibratome block (DSK Microslicer; DTK-1000) using cyanoacry-late adhesive. Within 3 min from the time of decapitation, the brainwas immersed in aCSF (4 °C) consisting of (in mM): 124 NaCl, 3.25 KCl,2.4 MgSO4, 1 CaCl2, 1.25 KH2PO4, 10 D-glucose, and 26 NaHCO3

equilibrated with 95% O2/5% CO2 for sectioning. Coronal sections(450 μm) containing the dorsal raphe nucleus were obtained startingcaudally around −8.85 mmBregma andmoving rostrally for 4 sectionsending rostral to the thickening of the decussation of the superiorcerebellar peduncle, approximately −7.50 mm Bregma (Paxinos andWatson, 2005). Following removal of cortical tissues, 4 sections (levels(mm Bregma): −8.85 (−9.08 to −8.63); −8.40, (−8.63 to −8.18); −7.95,(−8.18 to −7.73); and −7.50, (−7.73 to −7.28)) from each brain weretransferred to a sloping interface perfusion chamber (either a holdingor recording chamber) on a bed of double-layered lens tissueunderneath an additional single layer of lens tissue to prevent thetop of the section from drying out. Sections were maintained at 35 °Cand perfused with warmed, oxygenated aCSF at a flow rate of 750 μLpermin. For electrophysiological experiments, sliceswere individuallymoved from a holding chamber to a recording chamber; conditionswere identical between holding and recording chambers. Sectionswere allowed to acclimate to the recording chamber for at least 1 hprior to electrophysiological recordings. Due to the absence ofnoradrenergic input to serotonergic neurones in vitro, 3 μM pheny-lephrine hydrochloride, a specificα1-adrenoceptor agonist, was addedto the aCSF at least 10 min prior to the beginning of recordings torestore spontaneous firing rates of serotonergic neurones to thoseobserved in vivo during active waking states (Vandermaelen andAghajanian, 1983). This tonic adrenergic stimulatory input is essentialfor studying tonic autoregulatory properties of serotonergic neuronesin vitro. In a majority of recordings, when testing for inhibitory effectsof drugs on neuronal firing rates, elevated baseline firing rates were

138 A.K. Evans et al. / European Journal of Pharmacology 590 (2008) 136–149

induced by using aCSF containing 1 mM Ca2+ instead of 2 mM Ca2+

(Lowry et al., 2000) which reliably and reversibly doubles the firingrate of putative serotonergic neurones (data for 2mM Ca2+ not shown;also see (Trulson and Crisp, 1985)).

2.4. Neurochemistry

An in vitro slice perfusion model was designed to replicate conditionsfrom electrophysiology experiments (described below) and to evaluate 1)time-dependent and concentration-dependent effects of exogenoustryptophan on tissue concentrations of tryptophan, serotonin, and 5-HIAA in the midbrain raphe complex, and 2) effects of an inhibitor ofaromatic amino acid decarboxylase (AADC), NSD-1015, on tissue concen-trations of 5-hydroxytryptophan (5-HTP) in the presence of exogenoustryptophan as an index of TPH activity and de novo serotonin synthesis.Brain slices from up to eight rats per day were prepared and placed inparallel chambers of an eight lane sloping interface perfusion chambercustom designed with respect to the chamber used for electrophysiologybut enabling multiple parallel drug applications. The following methodsdescribe 4 specific neurochemistry experiments in which the followinginitial conditions were identical: brain slices were first acclimated to theperfusion chamber for 1 h after sectioning and in vivo-like tonic firing ratesfor serotonergic neurones were restored with the addition of 3 μMphenylephrine to the perfusion medium 3 h prior to indicated drugapplications. Tryptophanand/orNSD-1015wereadministered, asdescribedin detail below, by bath application in standard aCSF containing 3 μMphenylephrine. After each experiment, sections were rinsed for 2 s intryptophan-free aCSF (35 °C), mounted on clean slides, rapidly frozen ondry ice and stored at −80 °C for latermicrodissection andhighperformanceliquid chromatography analysis of indole tissue concentrations. Alltreatments were randomly assigned to each rat and treatment order wasrandomised and balanced acrossmultiple days to control for variation thatmay occur with time of day as well as daily variation.

2.4.1. Tryptophan time courseRat brain slices were exposed by bath application to 40 μM

tryptophan (0, 3, 9, 15, 30 and 60 min; n=6).

2.4.2. Tryptophan concentration-responseAfter determination of an appropriate time point from the

tryptophan time course study at which the effects of 40 μM trypto-phan were half-maximal, the time point was used to determine aconcentration-response to tryptophan application. Brain sliceswere exposed to tryptophan (0, 4, 10, 40, 100, and 400 μM; n=6) for23 min.

2.4.3. NSD-1015 concentration-responseThe rate-limiting enzyme for serotonin synthesis, TPH, converts

tryptophan to 5-HTP. As 5-HTP is rapidly converted to serotonin andtissue concentrations of serotonin reflect both newly synthesised aswell as previously stored serotonin, 5-HTP accumulation in thepresence of an inhibitor or AADC serves as an index of TPH activity.Indeed, inhibition of AADC with NSD-1015 in vivo leads to increases intissue concentrations of 5-HTP that reflect TPH activity (Carlsson andLindqvist, 1970). As 5-HTP accumulation is dependent on bothtryptophan availability and TPH activity, the in vitro slice preparationis particularly suited for measuring TPH activity as tryptophanavailability is controlled by the experimenter. Brain slices wereexposed by bath application to 40 μM tryptophan for 40 min with asimultaneous exposure to NSD-1015 (0, 0.05, 0.5, 5, 50, and 500 μM;n=6) during the last 30 min.

2.4.4. NSD-1015 time courseRat brain slices were exposed to 40 μM tryptophan for 40 minwith

a simultaneous exposure to 5 μM NSD-1015 for the final 0, 10, 20 or30 min (n=6) of that 40 min application.

2.5. Brain microdissection

Brain microdissection combined with high pressure liquid chro-matography with electrochemical detection of tryptophan, serotonin,5-HIAA, and 5-HTP was based on a previously described procedure(Renner and Luine, 1984). Individual brain regions were microdis-sected at −10 °C using the Palkovits punch technique (Palkovits andBrownstein, 1988) using a stainless steel microdissecting needle (690or 410 μm diameter, Neuropunch #18036-19 and #18036-22, FineScience Tools, Foster City, CA, USA). Microdissected tissues fromindividual brain regions from individual rats were expelled intoseparate tubes containing 75 µl acetate buffer (pH 5.0), and thenstored at −80 °C until they were analysed for tissue concentrations oftryptophan, serotonin, and 5-HIAA.

2.6. Selection of raphe regions

In order to investigate potential regional variation in treatmenteffects of perfusion with 40 µM tryptophan on serotonin metabolismin the raphe complex, fifteen regions were selected from therostrocaudal extent of the midbrain raphe complex (−8.85 to−7.50 mm Bregma; Supplementary data online, Fig. S1). Microdis-sected regions included the dorsal raphe nucleus, caudal part (DRC),interfascicular part (DRI), dorsal part (DRD), ventral part (DRV), andventrolateral part (DRVL), the median raphe nucleus (MnR), and thecaudal linear nucleus of the raphe (CLi). Some regions were sampledseparately at two or three different rostrocaudal levels. Anterior–posterior levels of sections selected for specific microdissections wereidentified by comparisons with a standard rat brain stereotaxic atlas(Paxinos andWatson, 2005). All samples represent individual puncheswith the exception of the MnR and DRVL, which were each sampledmultiple timeswithin a single rostrocaudal level, then pooled for HPLCanalysis. Although data were collected for these multiple regions, thisreport will focus on data from the DRV in a 450 μm brain slice centredaround −7.95 mm Bregma, a region corresponding to the location of amajority of the electrophysiological recordings and a region contain-ing the most serotonergic neurones (see Supplementary data onlinefor complete regional analysis).

2.7. HPLC analysis

Microdissected samples in acetate buffer were thawed at 4 °C andcentrifuged at 13,000 RPM (~12,000 g) for 3 min. The supernatant wasdrawn off and the pellet was reconstituted with 150 or 200 μl of 0.2 MNaOH for later assay of protein content (Pierce Protein MicroassayProtocol, Perbio Science UK Ltd., Cramlington, UK). Thirty-five µl of thesupernatant from each sample was then placed in an ESA 542autosampler (ESA Analytical, Ltd., Huntington, UK) maintained at 4 °Cwith the column oven temperature set to 29 °C. Ten µl of supernatantfrom each sample was then injected onto the chromatographicsystem. Chromatographic separation was accomplished using anintegrated precolumn/column system consisting of a guard cartridge(4.6 × 5 mm) attached to an Ultrasphere XL-ODS cartridge(4.6×70 mm; Beckman Coulter, Fullerton, CA, USA). The mobilephase consisted of 9.53 g/l KH2PO4, 200 mg/l 1-octanesulfonic acid,and 35mg/l ethylenediaminetetraacetic acid in 13%methanol; pHwasadjusted to 3.45 using orthophosphoric acid. Electrochemical detec-tion was accomplished using an ESA Coulochem II multi-electrodedetector with an ESA conditioning cell 5021 and an ESA analytical cell5011 with electrodes set at −0.10 and +0.55 V. The mean peak heights(pg/cm) of known concentrations of tryptophan, serotonin and 5-HIAA standards were determined from the peak heights of twochromatographs run before and after each set of samples. Concentra-tions of tryptophan, serotonin, and 5-HIAA in the microdissectedsamples were determined based on peak heights measured using acomputerised analysis system (EZChrom Elite for Windows, ver 2.8;


Scientific Software, Inc., Pleasanton, California, US) while the analystwas blind to the nature of the treatment groups. Indole concentrationswere expressed as pg/µg protein.

2.8. Electrophysiological recording

Recording procedures were essentially as described earlier (Lowryet al., 2000) with some exceptions. Extracellular single unit recordingswere made using glass microelectrodes filled with aCSF and coupledto an alternating current differential preamplifier (1000×). Single unitswere discriminated using an amplitude window, and data wererecorded using Spike 2 software (version 3.03, Cambridge ElectronicsDesign, Cambridge, UK). Units were screened for properties consistentwith a serotonergic phenotype (Vandermaelen and Aghajanian, 1983;Beck et al., 2004;Marinelli et al., 2004). All recordingswere performedfrom the dorsal and ventral subregions of the dorsal raphe nucleusbetween −8.85 and −7.50 mm Bregma. After isolation of a single unit,the neuronal firing rate was recorded for approximately 5 min toobtain baseline data. All brain slices were then perfused with aCSFcontaining 50 μM 5-hydroxytryptamine hydrochloride (serotonin; 5-HT) for 2 min. Single units with long duration biphasic or triphasicaction potentials that were reversibly inhibited by serotonin anddemonstrated the highly regular and relatively slow firing pattern(0.5–2.8 Hz in the presence of 2mMCa2+; 1.0–5.6 Hz in the presence of1 mM Ca2+) characteristic of serotonergic neurones in vivo (Jacobs andFornal, 1991) were accepted as putative serotonergic neurones.Approximately 10 min following serotonin application, allowing forat least 6 min of a stable firing rate after recovery from serotonin,sections were perfused with aCSF containing 40 μM tryptophan andactivity was recorded for 15–30 min. The tryptophan concentrationused in these studies was based on the Km of neuronal tryptophanhydroxylase (TPH2) for its substrate, tryptophan [20–50 μM; (Fried-man et al., 1972; McKinney et al., 2005)], and the reportedphysiological concentrations of extracellular tryptophan in the brain[10–30 μM (Boadle-Biber, 1993)]. Effects of additional drugs (NSD-1015, WAY-1000635, fluoxetine, pargyline, and MDMA) in thepresence of tryptophan were tested at least 6 min (typically 6 to9 min) after establishment of a new stable baseline firing rate in thepresence of tryptophan (which normally occurred after 15–30 min oftryptophan exposure).

2.9. Analysis of drug-induced changes in neuronal firing rates

The response of each neurone to serotonin application wascalculated as a mean change in firing rate during the first 2 min ofserotonin application expressed as a percentage of the mean firingrate during the period 3 min prior to serotonin application. Thedynamic effects of bath application of aCSF containing 40 μMtryptophan on neuronal firing rates over a period of up to 30 minwere calculated as mean firing rates during consecutive 3 min blocksbeginning 3 min prior to tryptophan application and expressed as apercentage of the mean firing rate over a 3 min block beginning 6 minprior to tryptophan application. This time course allowed for both thedetermination of a stable firing rate prior to drug application aswell asan inhibition of firing rate during drug application. A majority ofneurones reached a new lower and stable baseline firing rate within15min following the onset of exposure to tryptophan; however, not allputative serotonergic neurones were responsive to tryptophan andtherefore each neurone was classified as tryptophan-responsive ortryptophan non-responsive based on the magnitude of the change inneuronal firing rate 15 min after tryptophan application. Neuronesshowing a 5% or greater inhibition of neuronal firing rate after 15 minin the presence of tryptophan were classified as responsive whileneurones showing less than 5% inhibition of neuronal firing rate wereclassified as non-responsive for further analyses. The responses ofneurones to application of drugs predicted to either reverse (NSD-

1015 and WAY-100635) or potentiate (fluoxetine, pargyline andMDMA) the effects of tryptophan on neuronal firing rates wereevaluated based on the mean firing rate during consecutive 3 minblocks beginning 6–9 min prior to drug application and expressed as apercentage of the pre-tryptophan baseline firing rate.

2.10. Statistics

All statistical analyses used Statistical Package for the SocialSciences (SPSS) version 14.0 (SPSS, Woking, UK). The Greenhouse–Geisser correction was used to correct for differences in varianceacross repeated measures (Vasey and Thayer, 1987). NonlinearRegression analyses used GraphPad Prism version 4.0 (GraphPadSoftware, San Diego, California, US). All reported values are meanvalues and standard errors of the means (S.E.M.). In all cases, two-tailed significance was accepted at Pb0.05.

2.10.1. NeurochemistryEffects of Tryptophan or NSD-1015 TREATMENT (TIME or CON-

CENTRATION) and REGION on tissue indole concentrations wereanalysed using a single multifactor analysis of variance (ANOVA) withrepeated measures using TREATMENT as the between-subjects factorand REGION as the within-subjects factor for repeated measuresanalysis. A Grubb's test was used to eliminate outliers from the dataset. Outliers (0.9%) and missing values (0.7%) were replaced by themethod of Petersen (Petersen, 1985) prior to the multifactor ANOVAwith repeated measures analysis, but the original data were used forpost hoc analysis and for graphic representation of the data. Whenappropriate, Fisher's Protected LSD tests were used for post hoccomparisons. Adjustments for multiple comparisons were made,when indicated, using either Bonferroni's correction or Dunnett's testfor multiple comparisons with a single control.

2.10.2. ElectrophysiologyTime-dependent effects of tryptophan, NSD-1015, WAY-100635,

pargyline, fluoxetine, and MDMA application on neuronal firing ratewere analysed with univariate ANOVA with repeated measures, withTIME as the within-subjects factor for repeated measures analysis.Main effects of TIME on tryptophan-induced changes in neuronalfiring rate were further analysed with post hoc pairwise comparisonsusing Bonferroni's test for multiple comparisons to determine thetime point at which a change in firing rate occurred as well as when anew, stable plateau in firing rate occurred. Main effects of TIME onNSD-1015-induced and WAY-100635-induced changes in neuronalfiring rate were further analysed using Dunnett's test for multiplecomparisons with a single control to determine the time point atwhich the firing rate was no longer significantly different from pre-tryptophan baseline conditions. Main effects of TIME on pargyline-,fluoxetine-, and MDMA-induced changes in neuronal firing rate werefurther analysed using Dunnett's test to determine the time point atwhich the firing rate was significantly different from pre-drugbaseline conditions.

3. Results

3.1. Neurochemistry

3.1.1. Exogenous tryptophan resulted in time-dependent increases intissue concentrations of tryptophan, serotonin, and 5-HIAA

In order to determine the time-dependent effects of tryptophan ontissue concentrations of tryptophan, serotonin, and 5-HIAA, brain sliceswere perfusedwith40 μMtryptophanacross a range of 0–60min. 40 μMtryptophan resulted in time-dependent increases in tryptophan,serotonin, and 5-HIAA concentrations in the midbrain raphe complex.The multifactor repeated measures ANOVA revealed a significantREGION×TIME interaction for tryptophan (F(5,30)=6.644; Pb0.001),

Fig. 1. Exposure of rat brain slices to aCSF containing exogenous tryptophan resulted in A) time-dependent (with 40 μM tryptophan) and B) concentration-dependent (23 min)increases in tissue concentrations of tryptophan, serotonin and 5-HIAA in the dorsal raphe nucleus. Data from panel A) and B) represent separate time course and concentration-response experiments (n=6). Tissue concentrations of indoles are indicated in pg/μg protein. The time at which there was a half-maximal accumulation of 5-HIAA (t1/2) and thetryptophan concentration at which there is a half-maximal accumulation of 5-HIAA (EC50) are indicated in the respective graphs. The microdissected ventral part of the dorsal raphenucleus (DRV) at −7.95 mm Bregma from a 450 μm thick brain slice is indicated in the diagram on the left; scale bar, 1 mm (Paxinos and Watson, 2005). Values represent means±S.E.M.Asterisks indicate values significantly different than the control condition (0 min or 0 μM TRP; Pb0.05; Dunnett's).

Fig. 2. Exposure of rat brain slices to the aromatic amino acid decarboxylase (AADC)inhibitor, NSD-1015, resulted in A) time-dependent and B) concentration-dependentincreases in tissue concentrations of 5-HTP in microdissected tissue punches containingthe DRV at −7.95 mm Bregma (Paxinos and Watson, 2005). Graphs indicate increases intissue concentrations of 5-HTP (pg/μg protein) following A) 5 μM NSD-1015 application(0, 10, 20 and 30 min; n=6), or B) NSD-1015 application (0, 0.05, 0.5, 5, 50 and 500 μM;n= 6) for 30 min. 5-HTP concentrations represent means±S.E.M. Asterisks indicategroups that are different than the control condition (Pb0.05, Dunnett's).


serotonin (F(5,30)=2.809; Pb0.001) and 5-HIAA ((F(5,30)=6.976; Pb0.001)concentrations indicating regional variation in time course effects. Theresults reported here, however, will focus on the DRV in a 450 μm brainslice centred around −7.95 mm Bregma, a region that contains largenumbers of serotonergic neurones (Steinbusch, 1981) and was repre-sentative of the overall trends across regions. For a complete report ofdata for all regions, see Supplementary data online, Tables S1 and S2 andFig. S2. One-way ANOVAwithin the DRV at −7.95 mm Bregma revealedan effect of TIME on tissue concentrations of tryptophan (F(5,34)=17.972;Pb0.001), serotonin (F(5,34)=5.866; P=0.001), and 5-HIAA (F(5,34)=16.262; Pb0.001; Fig. 1A). Further post hoc pairwise comparisons ofmean tryptophan, serotonin, and 5-HIAA concentrations in the DRVrevealed time-dependent changes in tissue concentrations of all threeindoles (Fig. 1A). Time-dependent increases, relative to time 0, in tissueconcentrations of both tryptophan (P=0.002) and 5-HIAA (P=0.025)were seen at 15 min. In contrast, increases in tissue concentrations ofserotonin only approached significance at 30 min (P=0.077) but weresignificant at 60 min (P=0.007). Data for time-dependent increases intissue concentrations of 5-HIAA following tryptophan application werefit with a non-logarithmic sigmoidal regression curve (Prism GraphPad4; Y=Bottom+[Top−Bottom][xn /(Ka

n+xn)]), resulting in an approxima-tion of a time point (Ka, T1/2) at which a half-maximal 5-HIAAconcentration was reached following application of aCSF containing40 μMtryptophan. The T1/2 for tissue accumulation of 5-HIAA in theDRVat −7.95 was 18.6 min.

3.1.2. Exogenous tryptophan resulted in concentration-dependentincreases in tissue concentrations of tryptophan, serotonin and 5-HIAA

In order to determine the concentration-dependent effects oftryptophan on tissue concentrations of tryptophan, serotonin, and 5-HIAA, brain slices were perfused with varying concentrations oftryptophan for 23 min. This time point was selected based on theaverage T1/2 for the midline dorsal raphe nucleus (DRC, DRD, DRV)pooled across sections (22.95±5.77min). Perfusion of brain slices withtryptophan for 23 min resulted in concentration-dependent increasesin tryptophan, serotonin, and 5-HIAA concentrations in the midbrainraphe complex. As with time-dependent changes, multifactorrepeated measures ANOVA revealed significant REGION×CONCEN-TRATION interactions for tissue concentrations of tryptophan (F(5,30)=3.505; Pb0.001), serotonin (F(5,30)=3.538; Pb0.001), and 5-HIAA

((F(5,30)=7.002; Pb0.001) indicating regional variation in concentra-tion-dependent effects. As indicated above, the results reported herewill focus on the DRV at the level of −7.95 mm Bregma which wasrepresentative of the overall trend across regions. For a completereport of concentration-dependent results for all regions see Supple-mentary data online (Tables S3 and S4 and Fig. S3). One-way ANOVAwithin the DRV revealed an effect of CONCENTRATION on tissueconcentrations of tryptophan (F(5,35)=22.391; Pb0.001), serotonin(F(5,34)=27.573; Pb0.001), and 5-HIAA (F(5,35)=30.678; Pb0.001; Fig.1B).Further post hoc pairwise comparison of means for tryptophan,


serotonin, and 5-HIAA concentrations revealed concentration-depen-dent changes in tissue concentrations of all three indoleswithin theDRV(Fig. 1B). Exogenous tryptophan application (100–400 μM) resulted inincreased tissue concentrations of tryptophan (Pb0.001). Increases intissue concentrations of serotoninwere observed following exposure toexogenous tryptophan concentrations of at least 40 μM (Pb0.001) withno further increase at 400 μM. A robust concentration-dependentincrease in tissue concentrations of 5-HIAA was observed in the DRVfollowing exposure to exogenous tryptophan (40–400 μM Pb0.001). Inorder to estimate the tryptophan concentration required for half-maximal accumulationof 5-HIAA (EC50;medianeffective concentration)following 23 min of tryptophan application, data for tissue concentra-tions of 5-HIAA were fit with a sigmoidal regression curve (PrismGraphPad 4). The EC50 value for tissue accumulation of 5-HIAA in theDRV was 37.4 μM.

3.1.3. NSD-1015 application resulted in time-dependent and concentration-dependent increases in tissue concentration of 5-HTP

In order to obtain an index of de novo serotonin synthesis (TPHactivity) in the in vitro slice preparation, brain slices were first exposedto aCSF lacking the essential amino acid tryptophan for 4 h followed

Fig. 3. Application of artificial cerebrospinal fluid (aCSF) containing 40 μM exogenous trypneurones. A) Spike frequency histogram from an extracellular single unit recording depicts thillustrating the time course of the effects of tryptophan application on neuronal firing rasquare). C) Diagrams of coronal sections from a standard rat brain atlas (Paxinos andWatson,non-responsive (gray square) recordings. In tryptophan-responsive neurones, maximal inhibfiring rate within 15min. The neuronal firing rate at each time point is normalised as a percento tryptophan application. Each symbol represents the mean firing rate over a period of 3 minare different than the first time point prior to tryptophan application (Pb0.05, Dunnett's).

by exposure to aCSF containing 40 μM tryptophan for 40 min witheither 1) co-application of 5 μM NSD-1015 for the last 0, 10, 20 or30 min, or 2) co-application of NSD-1015 (0, 0.05, 0.5, 5, 50 and500 μM) for the last 30 min. NSD-1015 application resulted in a time-dependent increase in 5-HTP accumulation in the DRV. One-wayANOVA revealed an effect of TIME on tissue concentrations of 5-HTP(F(3,21)=13.702; Pb0.001; Fig. 2A). Post hoc pairwise comparisons of 5-HTP concentrations revealed a time-dependent increase that reachedsignificance at 20 min (P=0.001). NSD-1015 application resulted in aconcentration-dependent increase in tissue concentrations of 5-HTPin the DRV. One-way ANOVA revealed an effect of CONCENTRATIONon tissue accumulation of 5-HTP (F(5,28)=31.288, Pb0.001; Fig. 2B).Further post hoc analyses revealed an increase in tissue concentra-tions of 5-HTP with 5, 50 and 500 μM NSD-1015 (Pb0.001).

3.2. Electrophysiology

3.2.1. Tryptophan application resulted in inhibition of neuronal firingrate in a subset of putative serotonergic neurones

In parallel extracellular electrophysiological studies designed tounderstand the role of exogenous tryptophan in autoregulatory

tophan inhibited the neuronal firing rate of a subpopulation of dorsal raphe nucleuse effects of serotonin (50 μM), and tryptophan (40 μM) on neuronal firing rate. B) Graphte (tryptophan-responsive, n=95, open circle; tryptophan non-responsive, n=11, gray1998) indicate anatomical sites for tryptophan-responsive (open circle) and tryptophanition of firing rate was evident within 6 min and established a new lower stable baselinetage of the baseline firing rate represented by the first point on the graph at 4.5 min priorbetween each time point indicated on the x-axis±S.E.M. Asterisks indicate groups that


mechanisms controlling neuronal firing rates in the dorsal raphenucleus, 106 putative serotonergic neurones were studied with respectto a response to sequential bath application of 50 μM serotonin and40 μM tryptophan (for example see Fig. 3A). The mean decrease inneuronal firing rate in response to bath application of aCSF containing50 μM serotonin, expressed as a percentage of baseline firing rate, was65.4±2.5% (S.E.M.). The mean decrease in neuronal firing rate following15 min of bath application of aCSF containing 40 μM tryptophan,expressed as a percentage of baseline firing rate,was 75.5±2.0% (S.E.M.).However, not all neurones that responded to serotonin with aninhibition of neuronal firing rate also responded to tryptophanwith aninhibition of neuronalfiring rate. Eighty-nine% of neurones in the dorsalraphenucleus that responded to serotonin applicationwith inhibition ofneuronal firing rate also responded to application of aCSF containing40 μM tryptophan with ≥5% inhibition of neuronal firing rate within15 min (tryptophan-responsive; n=95; Fig. 3B). The remainder ofneurones (tryptophan non-responsive; n=11) had b5% inhibition ofneuronal firing rate within 15 min of tryptophan application. Locationsof tryptophan-responsive and tryptophan non-responsive neurones areindicated in Fig. 3C. Separate analysis of firing rates of neuronesclassified as responsive and non-responsive using univariate repeatedmeasures ANOVA, revealed a main effect of TIME on firing rate in boththe tryptophan-responsive group (F(7,658)=149.063; epsilon=0.241;Pb0.001; Fig. 3B) and in the tryptophan non-responsive group (F(7,70)=14.908; epsilon=0.340; Pb0.001). Post hoc comparison of the change inneuronal firing rate compared to the pre-tryptophan baseline firing raterevealed that, whereas an inhibition of neuronal firing rate in the non-responsive neurones eventually occurred after 18 min (P=0.027),tryptophan-responsive neurones had an inhibition of neuronal firingratewithin 6min (Pb0.001) and reached anew, stable baselineneuronalfiring rate within 9 min (Fig. 3B). This time course for inhibition closelymatches the time course of increasing tissue concentrations of 5-HIAAfrom the DRV at −7.95 mm Bregma, the region inwhich the majority ofthe electrophysiology recordings were made (Fig. 4).

Fig. 4. There was a temporal relationship between increases in tissue concentrationsof 5-HIAA and inhibition of neuronal firing rates in the DRV following perfusion of livingbrain slices with aCSF containing 40 μM exogenous tryptophan. A) Graph illustrating theeffects of 40 μM exogenous tryptophan on tissue concentrations of 5-HIAA in the DRV at−7.95 mm Bregma (n=6). 5-HIAA concentrations represent means±S.E.M. B) Graphillustrating the time course of the effects of 40 μM exogenous tryptophan on inhibition ofneuronal firing rate (0 through 7.5 min, n=99; 10.5 min, n=98; 13.5 min, n=95; 16.5 min,n=85; 19.5 min, n=72; 22.5 min, n=55; 25.5 min, n=54; 28.5 min, n=41). Dotted linerepresents beginning of tryptophan application at time=0 min across both studies. Dataare the same as those depicted in Figs. 2 and 3 but with an additional 3 time points for theneuronal firing rate data. The neuronal firing rate at each time point is normalised as apercentage of the baseline firing rate represented by the first point on the graph at 4.5minprior to tryptophan application. Each symbol represents themean firing rate over a periodof 3 min between each time point indicated on the x-axis±S.E.M.

In order to determine if the tryptophan-responsive and tryptophannon-responsive groups of neurones could be differentiated based onneuronal properties, we compared baseline firing rate and magnitudeof serotonin-induced inhibition of neuronal firing rates in the twogroups. The mean baseline firing rate of all neurones tested was 3.5±0.2 Hz. The mean neuronal firing rate for the tryptophan-responsivegroup (3.3±0.2 Hz; n=95) was significantly less than that of thetryptophan non-responsive group (4.9±0.4 Hz; n=11) (t(101)=3.253;P=0.002 ). Note that these firing rates are greater than reported inprevious studies (0.5–2.5 Hz; (Jacobs and Fornal, 1991; Lowry et al.,2000)) due to the use of aCSF containing 1mM instead of 2mMCa2+ asnoted in the methodology. The magnitude of serotonin-inducedinhibition of neuronal firing rates expressed as a percentage ofbaseline was not different between tryptophan-responsive and non-responsive groups (t(101)=1.025; P=0.308). Post hoc comparison ofslice history, revealed that while the mean time at which sectionswere prepared in the morning was equivalent between groups (10:46AM) the tryptophan non-responsive neurones were recorded later inthe day (longer incubation time) with respect to responsive neurones(324±24 min after slice preparation compared to 258±13 min, t(101)=2.413, P=0.028). Incubation timeswere not correlatedwithmagnitudeof inhibition following tryptophan application.

3.2.2. Tryptophan modulation of neuronal firing rate was dependent onde novo serotonin synthesis, 5-HT1A receptor activation, and clearance,storage, and metabolism of serotonin

We designed a series of studies in order to determine if thetryptophan-induced decreases in neuronal firing rates of dorsal raphenucleus neurones were dependent on de novo serotonin synthesis,activation of 5-HT1A receptors, and serotonin catabolism, clearance andvesicular storage (instead of direct effects of tryptophan on 5-HT1Areceptors, or effects on kynurenine metabolites (Ruddick et al., 2006)).We exposed dorsal raphe nucleus neurones to aCSF containing 40 μMtryptophan for between 15 and 30 min, a time when most neuronesdisplay new, lower, but stable baseline firing rates.We then exposed theneurones to aCSF containing tryptophan in combination with 1) NSD-1015, an inhibitor of AADC; 2)WAY-100635, a selective 5-HT1A receptorantagonist; 3) pargyline, an inhibitor of monoamine oxidase; 4)fluoxetine, a selective serotonin reuptake inhibitor; or 5) MDMA, aserotonin-releasing agent (targeting vesicular monoamine transporter2 (VMAT2) and the serotonin transporter).

3.2.3. NSD-1015NSD-1015 application was effective in reversing the tryptophan-

induced inhibition of cell firing rates to pre-tryptophan baseline firingrates (Fig. 5, n=7), demonstrating that the tryptophan-mediatedinhibition of neuronal firing rates in the dorsal raphe nucleus ismediated via conversion of tryptophan to serotonin (via theintermediate 5-HTP). Analysis of the mean firing rates of neuronesacross a time course following NSD-1015 application in the presenceof a tryptophan-induced inhibition of neuronal firing, using univariaterepeated measures ANOVA, revealed a main effect of TIME (F(8,48)=43.217; epsilon=0.219; Pb0.001) on neuronal firing rate. Further posthoc analysis revealed that firing rates were initially significantlydifferent than pre-tryptophan firing rates (due to the tryptophan-induced inhibition of neuronal firing rate), remained significantlydifferent through 6 min following application of NSD-1015, andreturned to pre-tryptophan firing rates by 9 min. A concentration-response examining electrophysiological effects of NSD-1015 (Fig. 5A)corroborated the neurochemical evidence that 5 μMNSD-1015 was aneffective concentration for inhibition of AADC resulting in tissueaccumulation of 5-HTP occurring between 10 and 20 min (Fig. 2).

3.2.4. WAY-100635WAY-100635 application was effective in reversing the trypto-

phan-induced inhibition of neuronal firing rates to pre-tryptophan

Fig. 5. Exposure of rat brain slices to the AADC inhibitor, NSD-1015, reversed the inhibition of neuronal firing rate following application of aCSF containing 40 μM tryptophanwithin atime course and at concentrations that blocked the conversion of 5-HTP to serotonin and resulted in increases in tissue concentrations of 5-HTP in parallel neurochemical studies (seeFig. 2). A), B) Spike frequency histograms from extracellular single unit recordings depict the effects of serotonin (50 μM), tryptophan (40 μM), and NSD-1015 (0.05–5 μM) on neuronalfiring rate. B) Graph illustrating the time course of the effects of 5 μM NSD-1015 application on neuronal firing rate (n=7). The firing rate returns to pre-tryptophan baseline rateswithin 12 min following NSD-1015 application. The neuronal firing rate at each time point is normalised as a percentage of pre-tryptophan baseline firing rate (indicated by thedotted line) to depict when the firing rate returns to a pre-tryptophan rate and represents means±S.E.M.

Fig. 6. Bath application of WAY-100635, a selective antagonist at the 5-HT1A receptor, reverses the inhibition of neuronal firing rate following application of aCSF containing 40 μMtryptophan. A) Spike frequency histogram from an extracellular single unit recording depicting the effects of tryptophan (40 μM), and WAY-100635 (20 nM) on neuronal firing rate.B) Graph illustrating the time course of the effect of WAY-100635 on neuronal firing rate (n=9). The neuronal firing rate at each time point is normalised as a percentage of pre-tryptophan baseline firing rate (indicated by the dotted line) to depict when the firing rate returns to a pre-tryptophan rate and represents means±S.E.M.



baseline firing rates (Fig. 6, n=8), linking the tryptophan-mediatedinhibition of neuronal firing rates in the dorsal raphe nucleus to 5-HT1A receptor activation. Univariate repeated measures ANOVA of thenormalised neuronal firing rate across a time course following 20 nMWAY-100635 application, in the presence of a tryptophan-inducedinhibition of neuronal firing rate, revealed a main effect of TIME(F(13,91)=26.609; epsilon=0.126; Pb0.001). Further post hoc analysisrevealed that firing rates were initially significantly different than pre-tryptophan firing rates, remained significantly different through15 min following application of WAY-100635, and returned to pre-tryptophan firing rates by 18 min.

3.2.5. PargylineWe predicted that an inhibitor of monoamine oxidase would

increase synaptic concentrations of serotonin in the presence oftryptophan and potentiate the effects of tryptophan on inhibition ofneuronal firing rates. In the presence of a stable firing rate following40 μM tryptophan application, 20 μMpargyline application resulted ina further inhibition of neuronal firing rates in the midbrain raphecomplex (5/6 recordings) but had no effect in the absence ofexogenous tryptophan (5/5 recordings; Fig. 7). Analysis of time courseeffects of pargyline on neuronal firing rates using univariate repeatedmeasures ANOVA, revealed a main effect of TIME in the presence oftryptophan (F(7,28)=41.822; epsilon=0.167; P=0.001; n=5) but not inthe absence of tryptophan ((F(7,28)=4.391; epsilon=0.207; P=0.076;n=5). Further post hoc analyses of recordings in the presence oftryptophan revealed an inhibition in neuronal firing rate within 9 min(P=0.008) of pargyline application. Importantly, in cells in whichpargyline exposure was examined in the absence of tryptophan,pargyline exposure was followed by exposure to tryptophan, resultingin a robust inhibition of neuronal firing rate (Fig. 7B). Pargylineinhibition of monoamine oxidase is long-lasting (Taylor et al., 1960)accounting for the tryptophan result subsequent to pargylineexposure.

Fig. 7. In the presence of 40 μM tryptophan, bath application of 20 μM pargyline, a monoamineffect on neuronal firing rates in the absence of tryptophan. A), B) Spike frequency histograneuronal firing rates (A) in the presence of tryptophan (40 μM), and (B) in the absence of trypthe effect of 20 μM pargyline on neuronal firing rates (open circles: no tryptophan, 5 out of 6rate at each time point is normalised as a percentage of the baseline firing rate as representedstable firing rate prior to pargyline application. Each symbol represents the mean firing rat

3.2.6. FluoxetineWe predicted that a selective serotonin reuptake inhibitor would

increase synaptic concentrations of serotonin by inhibiting itsclearance from the synaptic space in the presence of tryptophan andpotentiate the effects of tryptophan on inhibition of neuronal firingrates. In the presence of a stable firing rate during exogenous 40 μMtryptophan exposure, 1 μM fluoxetine application resulted in aninhibition of neuronal firing rate in approximately 6/10 recordings inthe midbrain raphe complex but had no effect in 4/5 neurones in theabsence of exogenous tryptophan (Fig. 8A). However, a higher dose offluoxetine (10 μM) resulted in inhibition of neuronal firing rates in 6/7neurones in the presence of exogenous tryptophan and in 5/9neurones in the absence of exogenous tryptophan (Fig. 8B). Thedistribution of individual responses to each treatment was notsuitable for statistical analysis and is displayed as a scatter plot ofindividual responses across a time course (Fig. 8) depicting individualvariation and the interaction between tryptophan availability anddose of fluoxetine.

3.2.7. MDMAWe predicted that a monoamine releasing agent would potentiate

the effects of tryptophan on inhibition of neuronal firing rates. MDMA(5 μM) resulted in an inhibition of neuronal firing rate in both thepresence and absence of 40 μM tryptophan. Analysis of time courseeffects of 5 μM MDMA on neuronal firing rates in 20 recordings(tryptophan=11, no tryptophan=9), using univariate repeated mea-sures ANOVA, revealed a main effect of TIME in the presence oftryptophan (F(10, 100)=201.253; epsilon=0.; Pb0.001; Fig. 9) as well asin the absence of tryptophan (F(10, 80)=67.574; epsilon=0.; Pb0.001;Fig. 9). Post hoc analysis of changes in firing rates across a time coursein the presence or absence of tryptophan revealed an inhibition ofneuronal firing rate by 180 s following MDMA application in thepresence of tryptophan (Pb0.001) and by 540 s in the absence oftryptophan (Pb0.001). To test whether the effects of MDMA in the

e oxidase inhibitor, inhibits neuronal firing rates in the dorsal raphe nucleus, but has noms from extracellular single unit recordings depict the effects of pargyline (20 μM) ontophanwith subsequent tryptophan application. C) Graph illustrating the time course ofrecordings; closed circles: with tryptophan, 5 out of 5 recordings). The neuronal firingby the first point on the graph, at 7.5min prior to pargyline application, to demonstrate ae over a period of 3 min between each time point indicated on the x-axis±S.E.M.

Fig. 8. In the presence of 40 μM tryptophan, bath application of 1 or 10 μM fluoxetine, a selective serotonin reuptake inhibitor, inhibits neuronal firing rates in the dorsal raphenucleus. Scatter plot diagrams for A) 1 and B) 10 μM fluoxetine applications indicate that whereas in the presence of tryptophan (right column), both 1 and 10 μM fluoxetineapplication result in inhibition of neuronal firing rate across a time course of 3–15 min, in the absence of tryptophan (left column) 1 μM fluoxetine has a minimal effect on neuronalfiring rates across the time course while 10 μM fluoxetine results in an inhibition of neuronal firing rates in approximately half of the recordings. The neuronal firing rate at each timepoint is normalised as a percentage of the baseline firing rate at 7.5 min prior to fluoxetine application as indicated by the dashed line. Each data point represents the meannormalised firing rate from an individual recording over a period of 1.5 min before and after each time point indicated on the x-axis. Solid bars represent the mean response for eachtimepoint.

Fig. 9. MDMA, a serotonin-releasing agent, inhibits neuronal firing rates in the dorsal raphe nucleus both in the presence and absence of exogenous tryptophan in a brain slicepreparation. Spike frequency histograms from extracellular single unit recordings depict the effects of MDMA (5 μM) on neuronal firing rates in the absence (A) and presence (B) oftryptophan (40 µM). C) Graph illustrating the time course of the effect of 5 μMMDMA on neuronal firing rates (open circles: in the absence of exogenous 40 μM tryptophan, n=9/10recordings; closed circles: in the presence of exogenous 40 μM tryptophan, n=11/12 recordings). Each symbol represents the mean firing rate over a period of 3 min between eachtime point indicated on the x-axis normalised as a percentage of the baseline firing rate as represented by the first point on the graph at −7.5 min prior to MDMA application±S.E.M.



absence of tryptophan were due to release of previously storedserotonin versus de novo serotonin synthesis, NSD-1015 was appliedto 2 neurones following an MDMA-induced inhibition of neuronalfiring rate in the absence of tryptophan; NSD-1015 failed to reversethe MDMA-induced inhibition of neuronal firing rate (data notshown).

4. Discussion

In this study we demonstrated that continuous perfusion of brainslices with aCSF containing exogenous tryptophan resulted in time-and concentration-dependent increases in tissue concentrations oftryptophan, serotonin, and 5-HIAA in the dorsal raphe nucleus. Theseneurochemical effects of exogenous tryptophan application weretemporally correlated with the effects of tryptophan on neuronalfiring rates. In addition, only a subpopulation of the neurones thatresponded to exogenous serotonin application with an inhibition ofneuronal firing rate established new, lower, stable baseline firing ratesfollowing tryptophan application, suggesting that, as opposed to awidespread tonic serotonin release and activation of serotoninreceptors in the dorsal raphe nucleus, tonic somatodendritic releaseof de novo serotonin may be restricted to specific synaptic zones. Ourdata indicated that tryptophan-mediated tonic autoregulation ofneuronal firing rates in the dorsal raphe nucleus is dependent on denovo serotonin synthesis and activation of 5HT1A receptors. Inaddition, we provided the first evidence that the ability of themonoamine oxidase inhibitor, pargyline, and the serotonin reuptakeinhibitor, fluoxetine, to inhibit neuronal firing rates in the dorsal raphenucleus is dependent on tryptophan availability in vitro, and weprovided evidence that higher doses of fluoxetine are capable ofinhibiting neuronal firing rates in the absence of exogenoustryptophan, potentially via non-serotonergic mechanisms. Our datasuggest that MDMA is capable of inhibiting neuronal firing rates viarelease of previously stored as well as newly synthesised serotonin,and the magnitude of the MDMA inhibition is influenced bytryptophan availability. Our data indicate that 5HT1A receptor-mediated tonic inhibition of neuronal firing rate is dependent ontryptophan availability and influenced by serotonin synthesis, storage,reuptake and catabolism.

4.1. Tryptophan application increased tissue concentrations oftryptophan, serotonin, and 5-HIAA

Tryptophan application resulted in concentration- and time-dependent increases in tissue concentrations of tryptophan, serotoninand 5-HIAA within the dorsal raphe nucleus. Tissue concentrations oftryptophan, serotonin and 5-HIAA were detectable following 4 h oftryptophan depletion, but demonstrated robust increases followingtryptophan loading. While robust increases in tryptophan concentra-tions are consistent with widespread tryptophan uptake by bothserotonergic and non-serotonergic neurones alike (Kuhar et al., 1972),temporally dynamic changes in tissue concentrations of serotonin and5-HIAA reveal important dynamics regarding tryptophan utilizationspecifically in serotonergic neurones.

Interpretation of tissue concentrations of 5-HIAA is widelydebated; they may reflect intraneuronal metabolism of unreleasedserotonin (Lookingland et al., 1986), metabolism of serotonin follow-ing reuptake (Reinhard andWurtman,1977), or a combination of both.A ratio of 5-HIAA/serotonin is sometimes used to reflect serotoninmetabolism, although this ratio is not interpretable under situationswhen rapid synthesis of serotonin coincides with widespread releaseand catabolism (Ten Eyck et al., 2005) as in the present paradigm. Wehave therefore not reported metabolite ratios as these data did notprovide any additional information beyond that available from theindividual metabolites. The present study suggests that tissueconcentrations of 5-HIAA in this paradigm 1) may reflect metabolism

of newly synthesised serotonin as tissue concentrations of 5-HIAAwereclosely linked to tryptophan availability, and 2) may be proportional toconcentrations of released serotonin as tissue concentrations of 5-HIAAwere temporally correlated with a tryptophan-mediated inhibition ofneuronal firing rate, which is likely mediated via actions of releasedserotonin at 5-HT1A inhibitory autoreceptors (Fig. 4). These conclusionsare consistent with a study using radiolabelled tryptophan to tracknewly synthesised serotonin in which it was found that the specificactivity of released serotonin increases over time at a greater rate thanthe specific activity of serotonin remaining in the tissue, suggesting thatnewly synthesised serotonin is preferentially packaged for release asopposed to intraneuronal storage (Elks et al., 1979). Tissue concentra-tion of 5-HIAA in this model may serve as a marker for vesicularserotonin available for tonic release or as a reflection of de novoserotonin synthesis. Indeed, EC50 values derived from the present 5-HIAA accumulation data (~40 μM) corroborate previous EC50 valuesderived from tryptophan-mediated inhibition of neuronal firing rates(Liu et al., 2005) aswell asKmvalues of neuronal tryptophanhydroxylase2 (TPH2) (McKinney et al., 2005).

In contrast to dynamic changes in tissue concentrations of 5-HIAAfollowing exogenous tryptophan application, concentrations of serotoninwere more stable, demonstrating a temporally delayed increase in theDRVat−7.95mmBregma. The stabilityof serotonin concentrations in thispreparation is consistent with previous in vitro slice perfusion studiesfollowing 2–8 h of incubation in tryptophan-free medium examiningeither whole slice tissue concentrations of serotonin or serotonin-immunohistochemistry (Liu et al., 2005; Mlinar et al., 2005; Trulson andFrederickson, 1987) suggesting that there are persistent stores ofserotonin resistant to tryptophan depletion over time. The relativelyminor increase in tissue concentrations of serotonin as compared to 5-HIAA is further evidence that newly synthesised serotonin may bepreferentially packaged for release followed by rapid metabolism to 5-HIAA.

Following 4 h of exposure of rat brain slices to aCSF lackingtryptophan, absence of 5-HT1A receptor-mediated inhibition ofneuronal firing rate in the presence of a persistent store of serotoninprovided evidence that the persistent store of serotonin may beindependent of serotonin normally available for release. Pools ofserotonin available for tonic release versus long-term storage may bedifferentially sensitive to tryptophan depletion. Indeed, Sprouse et al.(1989) demonstrate that tonic serotonin release is undetectable bymicrodialysis in slices perfused without exogenous tryptophan for atleast 2 h, a time when there are still detectable tissue concentrationsof serotonin, and demonstrate that serotonin is then detectablefollowing MDMA exposure and this detection correlates with anMDMA-induced inhibition of neuronal firing rate. These data areconsistent with a persistent store of serotonin that is not tonicallyreleased following tryptophan depletion; it is possible that MDMA isable to release these stores of serotonin through its actions at theserotonin transporter and VMAT2 (Schuldiner et al., 1993). Based onthis evidence we predict that a persistent store of serotonin observedfollowing tryptophan depletion could be more thoroughly exhaustedwith a concurrent MDMA application.

4.2. NSD-1015 had time- and concentration-dependent effects on tissueconcentrations of 5-HTP

We observed concentration-dependent (5–500 μM NSD-1015) andtime-dependent (10–30min) increases in tissue concentrations of 5-HTPin the DRV following inhibition of AADC reflecting de novo conversion oftryptophan to 5-HTP by TPH. The effective concentration of 5 μM NSD-1015 was consistent with evidence from single unit extracellularrecordings demonstrating that 5 μM but not 0.05 or 0.5 μM NSD-1015was effective in blocking serotonin synthesis and reversing a tryptophan-induced inhibition of neuronal firing rate. These data provide afoundation for future studies examining manipulations thought to


modulate TPH activity, including stress-related stimuli or stress-relatedneuropeptides. These data corroborate in vivo studies inwhich inhibitionof AADC was used to block serotonin synthesis and 5-HTP accumulationwas used as a measure of TPH activity (Carlsson et al., 1972; Dilts andBoadle-Biber, 1995).

4.3. Tryptophan application inhibited neuronal firing rates in asubpopulation of neurones in the dorsal raphe nucleus

Along with an increase in neurochemical indices of serotoninmetabolism following exposure to exogenous tryptophan, weobserved tonic 5-HT1A receptor-mediated inhibition of neuronal firingrate resembling in vivo tonic autoregulation of neuronal firing rates(Fornal et al., 1996, 1994). Tryptophan availability can explain thediscrepancy between the existence of tonic 5-HT1A receptor-mediatedinhibition of neuronal firing rate in vivo versus in vitro (Johnson et al.,2002; Craven et al., 1994). Our data support findings from whole cellpatch clamp and single unit extracellular recordings performed invitro demonstrating that a tonic 5-HT1A receptor-mediated autoinhi-bition of serotonergic neurones in the dorsal raphe nucleus isdependent on tryptophan availability (Liu et al., 2005; Mlinar et al.,2005) and are consistent with previous studies showing thatincreasing serotonin precursor availability in vivo results in inhibitionof neuronal firing rates (Gallager and Aghajanian, 1976; Trulson andJacobs,1976). The finding thatwe can block these effects of tryptophanon neuronal firing rates with an inhibitor of AADC, NSD-1015,corroborates previous studies using a similar inhibitor of serotoninsynthesis, benserazide (Ro-4-4602; 30 μM) in the perfusate in vitroand systemically in vivo (Gallager and Aghajanian, 1976; Liu et al.,2005).

We demonstrated that only a subpopulation of dorsal raphenucleus neurones that responded to exogenous serotonin applicationwith an inhibition of neuronal firing rate was also inhibited byexogenous tryptophan (95/106). This ratio of tryptophan-responsiveto serotonin-responsive neurones in the dorsal raphe nucleus issimilar to that reported by Liu and colleagues in vitro (2005; 20/25). 5-HT1A receptor-mediated inhibition of neuronal firing rate has beendemonstrated for both serotonergic and non-serotonergic neurones inthe raphe nucleus (Kirby et al., 2003). Liu et al. (2005) havedemonstrated with co-labelling studies that 95% of serotonin-immunopositive neurones are inhibited by tryptophan whereas 10%of immunonegative neurones are inhibited by tryptophan. Theyfurther note that in situ hybridization studies (Day et al., 2004)show that 5HT1A receptors are expressed on nearly all serotonergicneurones but only 10% of GABAergic neurones in the raphe nuclei. Asubpopulation of neurones that we have identified with a serotonin-mediated inhibition of neuronal firing rate but no response totryptophan application may represent a population of non-serotoner-gic neurones (e.g. GABAergic) or a population of serotonergicneurones that possess autoreceptors, but does not receive serotoner-gic innervation, or whose innervation has been severed in the slicepreparation. A different plane of sectioning may preserve more axonalprocesses and functional somatodendritic contacts. It is possible thattryptophan non-responsive neurones that demonstrate an inhibitionof neuronal firing rate following exposure to exogenous serotoninmayrepresent a population of neurones in which somatodendritic releaseof serotonin is extrinsically mediated via a mechanism absent fromour preparation. A recent study has demonstrated that serotoninrelease in the raphe nucleus can be mediated via NMDA receptoractivation in the absence of serotonergic neuronal firing (de Kocket al., 2006). It is also possible that a serotonin-responsive buttryptophan non-responsive recording is from a serotonergic neuronein which there was a loss of TPH activity over time as TPH has beenshown to be highly sensitive to oxidative deactivation following30 min incubation at room temperature (Kuhn et al., 1980). Post hocanalyses revealed that the average incubation time for the tryptophan

non-responsive neurones was 60 min longer than the averageincubation time for tryptophan-responsive neurones. It remainsunclear whether the tryptophan-induced inhibition of neuronal firingrate is truly an autoinhibition in which responsive neurones areutilizing the exogenous tryptophan to produce serotonin and subse-quently release that serotonin onto their own somatodendriticautoreceptors. However, intracellular recording studies suggest thatthis might be the case as inhibitors of tryptophan hydroxylase activityapplied directly through the pipette, which would only affect therecorded neurone, can reverse the tryptophan-mediated inhibition ofneuronal firing rate (Liu et al., 2005).

4.4. Modulation of serotonergic neuronal firing rates via dysregulation ofnormal serotonin catabolism, clearance, and release is dependent ontryptophan availability in vitro

Previously reported effects of pargyline on neuronal firing rates invivo (Sharp et al., 1997) can be replicated in vitro only in the presenceof exogenous tryptophan suggesting that these effects are dependenton tryptophan availability and de novo serotonin synthesis. Pargylinehas been demonstrated to interfere with phenylephrine metabolismin in vitro preparations resulting in increased noradrenergic tone(Raffel and Wieland, 1999). This effect of pargyline may contribute towhat appears to be a slight (but not significant) increase in neuronalfiring rate following pargyline application in the absence of exogenoustryptophan (Fig. 7C).

This is the first study to clearly demonstrate a dynamic, tryptophan-sensitive inhibition of neuronal firing rate with fluoxetine in vitro.Serotonin reuptake inhibitors (fluoxetine, 0.8 mg/kg i.v. and paroxetine,0.8mg/kg i.v.) have been demonstrated to increase extracellular serotoninconcentrations and to inhibit neuronal firing rates in the dorsal raphenucleus in vivo (Gartside et al.,1995; Romero et al., 2003). Previous in vitrostudies have reported no effect of fluoxetine on baseline serotonergicneuronal firing rates ((Liu et al., 2005; Sprouse et al., 1989) although see(TrulsonandFrederickson,1987)). In contrast to theseprevious studies,wereport concentration-dependent effects of fluoxetine in the absence oftryptophan, and it is possible that the inhibition of neuronal firing ratesthat we have observed with high doses of fluoxetine are dependent onother mechanisms, independent of tryptophan availability and serotoninrelease. For example it has been well documented that fluoxetine hasactions at nicotinic acetylcholine receptors (Garcia-Colunga et al., 1997),5HT3 receptors (Eisensamer et al., 2003), calcium channels (Kim et al.,2005; Kecskemeti et al., 2005), as well as indirect and direct effects onGABAergic receptors, the latter two of which are capable of directlysuppressing serotonergic neuronal firing rates.

MDMA is capable of inhibiting neuronal firing rates in the presenceor absence of exogenous tryptophan in vitro. This is consistent withprevious evidence in which MDMA has been shown to increaseextracellular serotonin concentrations and to inhibit neuronal firingrates in the raphe complex both in vitro and in vivo (Sprouse et al.,1989, 1990; Gartside et al., 1997a). This inhibition of neuronal firingrate in vitro has previously been shown to be correlated withmicrodialysis measurements of serotonin release even in the absenceof exogenous tryptophan (Sprouse et al., 1989). As discussed earlier, ifMDMA is capable of releasing previously stored pools of serotonin,this would explain its efficacy in the absence of exogenous tryptophan.We confirm previous studies showing that MDMA has increasedefficacy in inhibiting neuronal firing rates in the presence ofexogenous tryptophan (Sprouse et al., 1990). It is likely that thegreater effect of MDMA in the presence of exogenous tryptophan is acumulative effect of release of previously stored serotonin in additionto release of newly synthesised serotonin, due to the actions of MDMAon both the serotonin transporter and VMAT2 (Partilla et al., 2006).

Paradoxically,microdialysismeasurements of extracellular serotoninare undetectable in brain slices both in the presence and absence ofexogenous tryptophan, yet MDMA application results in detectable


serotonin release that correlates with inhibition of neuronal firing rate(Sprouse et al., 1989, 1990). Although it is unclear why tonic serotoninrelease remainsundetectable in thepresence of tryptophan, it is possiblethat tonic serotonin synthesis and release in the presence of tryptophanis discrete and localised to synapses, undetectable with a microdialysisprobe in vitro, whereas MDMA application results in indiscriminatemore widespread release of vesicular serotonin. Based on this hypoth-esis, wewould predict that any cell demonstrating a change in neuronalfiring rate following exogenous serotonin application would respondsimilarly to MDMA application. In our studies we came across severalrecordings in which serotonin application resulted in an excitation ofneuronal firing rate. These recordings were not included in this studyalthough theyhave some interesting implications in that these neuroneswere likely non-serotonergic and therefore incapable of utilizingexogenous tryptophan for conversion to serotonin; indeed they didnot respond to exogenous tryptophan with a similar increase inneuronal firing rate. On the other hand, in the few instances in whichthose excitatory serotonin responses were followed with an MDMAapplication, the result was an increase in neuronal firing rate in supportof a non-specific MDMA release of serotonin as compared to what maybe amore anatomically-selective tryptophan-dependent tonic release ofserotonin.

The present model can be useful for 1) studies of mechanismsregulating dendritic/somatic release of serotonin, as already demon-strated by de Kock et al. (2006); 2) studies of effects of neurotrans-mitters or neuropeptides that are thought to act via effects on TPHactivity, such as corticotropin-releasing factor, or other presynapticmechanisms; or 3) studies of changes in neuronal activity in models ofaltered presynaptic gene expression e.g. increases in TPH expressionfollowing adverse early life experiences (Gardner et al., 2005) orchronic mild stress (McEuen and Bale, 2006). The latter is particularlyintriguing because human depressed suicides have increased TPHmRNA and protein expression in the dorsal raphe nucleus (Bach-Mizrachi et al., 2006, 2008; Boldrini et al., 2005; Underwood et al.,1999; Bonkale et al., 2006), but we know virtually nothing about howthis altered TPH expression might affect function of autoregulatoryfeedback systems in serotonergic neurones. This model system couldprovide a foundation to begin to investigate these questions.

4.5. Conclusions

Perfusion of living brain slices with aCSF containing tryptophanresulted in time-dependent and concentration-dependent increasesin indices of serotonin synthesis and metabolism in the midbrainraphe complex that were temporally correlated with the restoration ofan in vivo-like inhibition of neuronal firing rate. Tryptophan-inducedinhibition of neuronal firing rate occurred in only a subpopulation ofserotonin-sensitive neurones suggesting that tryptophan-dependentserotonin release results in an anatomically selective, as opposed towidespread, activation of serotonin receptors in the dorsal raphenucleus in vitro, providing support for potential context- or region-specific regulation of neuronal firing rates in the dorsal raphe nucleus.Previously reported effects of pargyline and fluoxetine on neuronalfiring rates in vivo were observed in vitro only in the presence ofexogenous tryptophan, although higher concentrations of fluoxetinealtered neuronal firing rates in the absence of tryptophan, potentiallyvia non-serotonergic mechanisms. These studies support the hypoth-esis that tryptophan availability is an important determinant ofautoregulatory mechanisms controlling neuronal firing rates withinthe midbrain raphe complex.

Acknowledgements

C. A. Lowry was supported by a Wellcome Trust Research CareerDevelopment Fellowship (RCDF 068558/Z/02/Z) and is a recipient of a2007 Young Investigator Award from NARSAD: The Mental Health

Research Association; A. K. Evans was a recipient of an OverseasResearch Scholarship Award. We thank Dr. Matthew W. Hale forcritical comments on the manuscript. We wish to express ourgratitude to Dr. Daniel R. Staub for the design of the 8-chamberparallel perfusion system that made this work feasible.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ejphar.2008.06.014.

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