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Research Report

Inhibition of uptake 2 (or extraneuronal monoaminetransporter) by normetanephrine potentiates theneurochemical effects of venlafaxine

Zia Rahman⁎, Robert H. Ring, Kimberly Young, Brian Platt, Qian Lin, Lee E. Schechter,Sharon Rosenzweig-Lipson, Chad E. BeyerWyeth Research, Discovery Neuroscience, Depression and Anxiety Disorders, CN 8000, Princeton, NJ 08543-8000, USA

A R T I C L E I N F O

⁎ Corresponding author. Fax: +1 732 274 4020.E-mail address: rahmanz@wyeth.com (Z.

0006-8993/$ – see front matter © 2008 Elsevidoi:10.1016/j.brainres.2008.01.062

A B S T R A C T

Article history:Accepted 21 January 2008Available online 5 February 2008

Two distinct norepinephrine (NE) transporter mechanisms (uptake 1 and uptake 2) regulateextracellular NE concentrations. An association has been observed between the gradualimprovement in patients treated with antidepressants that inhibit the NE transporter (NET/uptake 1) and increases inurinarynormetanephrine, theO-methylatedNEmetabolite andpotentinhibitor of uptake 2. These observations led to the hypothesis that increased levels ofnormetanephrine, and consequently inhibition of uptake 2, may partly be responsible forthe clinical efficacy of some antidepressants. To investigate this hypothesis, we employedmicrodialysis techniques in the rat frontal cortex to monitor extracellular changes innormetanephrine following chronic administration of the clinically effective antidepressant,venlafaxine (a serotonin (5-HT) and NE reuptake inhibitor). We evaluated the neurochemicaleffects of inhibiting uptake 2 alone, or in conjunction with venlafaxine, on extracellular levels ofNE and 5-HT. Chronic venlafaxine administration (14 days, 10 mg/kg, s.c.) elicited significantincreases in cortical NE and 5-HT while producing a non-significant trend to increase corticallevels of normetanephrine. Additional studies revealed that combining normetanephrine withvenlafaxine (10 mg/kg, i.p.), at a dose of normetanephrine (10 mg/kg, i.p.) that did not producechanges in extracellular levels of NE on its own, potentiated antidepressant-induced increases inextracellular NE. We also report mouse behavioral data involving the tail suspension test thatcomplement theneurochemical observations. These preclinical findings, taken together, suggestthat inhibiting both uptake 1 and uptake 2 via venlafaxine and normetanephrine, respectively,elicits a greater increase in cortical levels of NE than inhibiting either transporter alone.

© 2008 Elsevier B.V. All rights reserved.

Keywords:AntidepressantsVenlafaxineNorepinephrineNormetanephrineMicrodialysisOCT3

1. Introduction

Centrally released monoamine neurotransmitters such as nor-epinephrine (NE), dopamine (DA) and serotonin (5-HT) are ac-tively cleared fromtheextracellular environmentby theactionofspecialized transport mechanisms. By regulating monoamine

Rahman).

er B.V. All rights reserved

concentrations in the synapse, these transport mechanismscontrol the magnitude and duration of monoaminergic neuro-transmission, and consequently, play a critical role in the func-tion of a variety of physiological systems. Two distinct transportmechanisms mediating the reuptake of released monoamineshave been described (Eisenhofer, 2001). The predominant

.

Fig. 1 – Detection of cortical levels of normetanephrine usingmicrodialysis. A). In vitro standards: HPLC was done using invitro standards for normetanephrine, norepinephrine (NE),serotonin (5-HT), dopamine (DA) and 3-methoxy-4-hydroxyphenylglycol (MHPG). Normetanephrine was detected anddistinguished from the other neurotransmitters of interest.B). In vivo microdialysis: Cortical normetanephrine levelswere measured following systemic normetanephrineadministration. Male adult SD rats were administered either1, 3 or 10 mg/kg (i.p.) normetanephrine and extracellularnormetanephrine levels in the frontal cortex were measuredusingmicrodialysis. Therewas a dose-dependent increase innormetanephrine levels observed in the cortex, with a 1000%increase over basal levels at the highest dose tested. Data areexpressed as mean+SEM (n=4–6 per treatment group).

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mechanism, referred to as uptake 1, includes the NE transporter(NET), the DA transporter (DAT) and the 5-HT transporter (SERT),(Eisenhofer, 2001; Amara and Kuhar, 1993). In addition to uptake1, extracellular monoamines may also be inactivated by the ac-tions of an extraneuronal transport mechanism, referred to asuptake 2 (Trendelenburg, 1991). By comparison, uptake 1 is asodium- and calcium-dependent, high-affinity and low-capacitytransport system that is primarily located on presynaptic nerveendings (Eisenhofer, 2001), while uptake 2 is a sodium- andcalcium-independent, low-affinity and high-capacity transportsystem found inextraneuronal cells such asmyocardial, smoothmuscle or glandular cells and in glial cells in the central nervoussystem (CNS) (Trendelenburg, 1991). Therefore, uptake 2 is dis-tinguishable from uptake 1 not only with respect to transportmechanisms, but also in drug sensitivity, substrate affinity andselectivity (Eisenhofer, 2001; Koepsell, 2004).

Uptake 2 was originally described over 40 years ago in periph-eral tissues (Iversen, 1965) and has been subsequently implicatedtomediatemonoamine reuptake in the brain (Hendley et al., 1970;Wilson et al., 1988; Russ et al., 1996). Molecular correlates of thismonoamine transport mechanism have been recently identified(Grundemann et al., 1998; Kekuda et al., 1998; Wu et al., 1998) (forreview see Eisenhofer (2001)). Based on functional characteriza-tion, three polyspecific organic cation transporters (OCTs), OCT1,OCT2 and OCT3, have been described as the molecular correlatesofuptake2 (Koepsell, 2004).Although there is considerableoverlapin the substrate and inhibitor specificities of the 3 OCTs, there aredistinct differences in affinity and maximal transport ratesbetween them (Koepsell et al., 2003; Amphoux et al., 2006; Martelet al., 1999). In rats, expression of OCT1 and OCT2 is restrictedmainly to peripheral tissues including kidney, liver and intestinewhereas OCT3 is expressed in skeletal muscle, smooth muscle,placenta and in the CNS (Martel and Azevedo, 2003). Among the 3organic cation transporters, OCT3 expression is highest in the ratbrain (Wu et al., 1998; Hayer-Zillgen et al., 2002; Haag et al., 2004),which, based on its distribution pattern and pharmacologicalprofile, is implicated to be most closely related to the uptake 2mechanism (Jonker and Schinkel, 2004). Correspondingly, thehuman orthologue of OCT3 is referred to as the extraneuronalmonoamine transporter (EMT) (Grundemann et al., 1998).

Alterations in the concentrations of NE in theCNShave beenimplicated in the etiology and pathophysiology of psychiatricillnesses such as depression (Blier and de Montigny, 1994;Heninger et al., 1996). Asdescribedabove, regulationofNE levelsin the synapse are predominatelymediated by the action of theNET (Glowinski et al., 1966; Graefe and Henseling, 1983; Axelrod,1971), a protein that is inhibited by several classes of antidepres-sants such as selective NE reuptake inhibitors and dual 5-HT/NE reuptake inhibitors (SNRIs; e.g., venlafaxine) (Trendelenburg,1991; Frazer, 1997). These antidepressant drugs exert their psy-chotropic action, in part, by inhibiting the activity of the NET,resulting in a rise in extracellular levels of NE in the synapse(Frazer, 1997). In addition to the actions of the NET, extracellularNE can also be taken up into adjacent glial cells by uptake 2.Within glial cells, NE is metabolized to normetanephrine by theaction of the enzyme catechol-O-methyltransferase (COMT)(Kopin, 1985). The pharmacological properties of normetanephr-ine have been described with the most pronounced role beingthat of a potent inhibitor of uptake 2 (Schanberg et al., 1968;Goldstein et al., 2003; Callingham and Burgen, 1966). This

inhibition of uptake 2 by normetanephrine may prevent anysubsequent uptake of extracellular NE into glial cells resulting inincreased levels of NE in the synapse. Therefore, antidepressantselevating extracellular levels of NE may result in an indirectaccumulation of normetanephrine, which in turn may inhibituptake 2, and consequently, result in greater increases in theextracellular concentrations of NE. Based on this mechanism, ithas been hypothesized that some effective antidepressants may

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produce their clinical activity, in part, by directly and indirectlyinhibiting NET and uptake 2, respectively (Schildkraut andMooney, 2004).

Support for this hypothesis comes from several clinicalstudies reporting altered levels of NE and normetanephrine inthe CSF, plasma, and urine of depressed patients (Schildkrautet al., 1966, 1969; Potter and Manji, 1994; Maas et al., 1987).Additionally, in studies of depressed patients treated withnoradrenergic-preferring antidepressants, a gradual increase inurinary normetanephrine was observed during the period oftherapeutic improvement (Schildkraut et al., 1966). Therefore,this association between clinical efficacy and increases in uri-nary normetanephrine, which is consistent with the delay inonset of antidepressant activity, was only observed in patientsfollowing long-term, chronic treatment (i.e., 3 weeks) but notshort-term (<1 week) antidepressant administration (Schildk-raut andMooney, 2004). Overall, these collective data imply thatantidepressant drugs may, in addition to increasing NE levels,elevate levels of normetanephrine. The downstream effects ofelevatingnormetanephrine includeeffectively inhibitinguptake

Fig. 2 – Chronic antidepressant treatment produced a trend to incfor chronic antidepressant treatment studies: 4 groups of adult S10mg/kg, s.c.) or vehicle (water) for 14 days followed by a challengon day 15 and subsequent microdialysis/HPLC analysis. B) Chroncortical levels of extracellular normetanephrine following a chal(29%, P=0.199). C, D, E) Chronic treatment with an SNRI (venlafaxextracellular levels ofNE (C) and5-HT (D) butnoeffect onextracellulvenlafaxine (10 mg/kg, s.c.). Asterisks represent significant (P<0.0

2 in the CNS, which in turn may play a role in regulating therelease and reuptake of NE. Given that normetanephrine pos-sesses high affinity and selectivity for uptake 2 (Martel andAzevedo, 2003; Burgen Asv, 1965; Callingham and Burgen, 1966),it could be argued that the extraneuronal NE transporter mayindirectly be involved in mediating the therapeutic effects ofantidepressant drugs that work via increasing noradrenergicneurotransmission.

The present studies employed in vivo microdialysis techni-ques in rats and behavioral tests inmice to explore this hypoth-esis from a preclinical perspective, and to evaluate whetherinhibiting uptake 2 via normetanephrine could influence CNSlevels of NE. Initial studies evaluated if chronic (2 weeks) treat-ment with venlafaxine, a clinically effective antidepressant thatinhibits the activity of theNET (or uptake 1),would elicit changesin extracellular levels of normetanephrine in the rat frontalcortex. Subsequent studies evaluated the acute effects ofnormetanephrine alone, and in combination with, venlafaxine,in the same brain region. The primary results observed in thesestudies showed that acute normetanephrine pretreatment

rease cortical levels of normetanephrine. A) Treatment groupsD rats were treated daily either with an SNRI (venlafaxine;e with the drug (venlafaxine; 10mg/kg, s.c.) or vehicle (water)ic antidepressant treatment produced a trend to increase

lenge with venlafaxine (10 mg/kg, s.c.) compared to controline; 10 mg/kg, s.c.) for 14 days produced robust increases inar levels ofDA (E) in the frontal cortex followinga challengewith5) treatment effects compared to vehicle-treated animals.

Fig. 2 (continued).

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potentiated the increase in extracellular levels of NE followingacute treatmentwith venlafaxine. Additional studiesmeasuringantidepressant-like behavior in mice using the tail suspensiontest (TST) demonstrated that pretreatment with normetanephr-ine potentiated the antidepressant-like effects of desipramine, atricyclic antidepressant that inhibits the activity of the NET.

These preclinical studies are important insomuch that theyelucidate a role for uptake 2 inhibition in the neurochemicaleffects of antidepressants, and as such, reveal a potential novelmolecular target for the treatment of psychiatric illnesses.

2. Results

2.1. Detection of normetanephrine levels in the rat frontalcortex using microdialysis techniques

In vivomicrodialysis andHPLC techniqueswere used tomeasurecortical levels of normetanephrine in rats following systemicadministration of normetanephrine. Preliminary experimentsusing HPLC and an external standard curve demonstrated ourability to electrochemically detect normetanephrine and distin-guish it from other neurotransmitters of interest. The HPLC chro-matogram for normetanephrine was found to be distinct fromother neurotransmitters including NE, DA and 5-HT (Fig. 1 A)validating the use of this method to quantify cortical levels ofnormetanephrine. To confirm that we could also measure nor-metanephrine levels in vivo, we performedmicrodialysis experi-ments in the rat frontal cortex following systemic administrationof normetanephrine. A single injection of normetanephrine (1, 3or 10 mg/kg, i.p.) dose-dependently increased extracellularnormetanephrine levels in the rat frontal cortex (Fig. 1 B), withthe highest dose tested producing a 1000% increase in corticalnormetanephrine levels compared to baseline levels. This in-crease in cortical normetanephrine levels was observed within40 min of a systemic injection of normetanephrine (with 3 and10 mg/kg, i.p.). Collectively, these data suggest that we can sepa-rate and reliably quantify levels of normetanephrine in vivo.

2.2. Chronic antidepressant treatment produced a trend toincrease cortical levels of normetanephrine

To evaluate the effects of chronic antidepressant treatment oncentral normetanephrine levels, rats were injected in their homecages once daily with an SNRI (venlafaxine, 10 mg/kg, s.c.) or ve-hicle for 14 days. The various treatment groups of animals testedare outlined in Fig. 2 A. Chronic antidepressant treatmentproduced a 29% increase in extracellular levels of normetanephr-ine in the rat frontal cortex followingachallengewithvenlafaxinecompared to controls (Fig. 2 B). This trend, however, was notfound to be statistically different from controls (P=0.199). It isimportant to note that in these same animals, chronic venlafax-ine treatment produced significant increases in both cortical NE(F(20,200)=11.46, P=0.0001) and 5-HT (F(19,190)=7.56, P=0.0016)(Fig. 2 C and D) but not in cortical DA (F(19,190)=0.64, P=0.5961)(Fig. 2 E). These latter neurochemical effects of venlafaxine areentirely consistent with its reported activity as an SNRI withantidepressant-like activity in behavioral models (Redrobe et al.,1998; Koch et al., 2003).

2.3. Acute normetanephrine pretreatment potentiates theincreases in extracellular levels of NE following administrationof venlafaxine

Consistent with previous neurochemical data (Beyer et al.,2002) and its in vitro activity as an SNRI (Stahl et al., 2005),

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acute administration of venlafaxine (10 mg/kg, i.p.) elicited asignificant (F(21,210)=7.78, P=0.0011) increase in the extra-cellular levels of NE in the rat frontal cortex (Fig. 3 A). Themaximal increasewas∼150% above baseline levels at 120-minpost-injection. Acute administration of normetanephrine(10 mg/kg, i.p.) alone did not produce any significant(P=0.9407) change in extracellular NE levels in the rat frontalcortex (Fig. 3 A). However, when administered in combination, a20-min pretreatment with normetanephrine (10 mg/kg, i.p.),followed by venlafaxine (10 mg/kg, i.p.) produced a significantly

Fig. 3 – Acute normetanephrine treatment potentiates the neuroextracellular NE measured in animals that received a single injesignificantly altered. Acute administration of venlafaxine (10mg/NE as compared to the basal levels in the frontal cortex. To deterconjunction with venlafaxine, animals received an initial injectioadministration of the SNRI given 20min later, (venlafaxine; 10mgproduced a significantly greater increase in the extracellular levecortex in response to administration of venlafaxine (10mg/kg, i.p(P<0.05) treatment effects compared to vehicle- and venlafaxine-t5-HT measured in the same animals (as in A) that received a sinarrow)were not significantly altered. Acute administration of venextracellular levels of 5-HT as compared to the basal levels in thekg, i.p.; first arrow) did not produce greater increases in the extrathat received administration of venlafaxine (10 mg/kg, i.p.) alonegroup).

(P=0.0012) greater increase in the extracellular levels of NEcompared to either drug alone (Fig. 3 A). In the combinationstudy using venlafaxine and normetanephrine, the maximalincrease in NE was ∼300% above baseline and observed to besignificantly different from the venlafaxine alone treatmentgroup (P=0.0058). These neurochemical effects were specificfor NE since no significant differences in effects were observedon cortical 5-HT (F(18,180)=2.67, P=0.0787) (Fig. 3 B) or DA (datanot shown) following this treatment combination. Takentogether, these data demonstrate that acute normetanephrine

chemical effects of an antidepressant. A). Cortical levels ofction of normetanephrine alone (10 mg/kg, i.p.) were notkg, i.p.) produced an increase (~150%) in extracellular levels ofmine the effects of acute normetanephrine administered inn of normetanephrine (10 mg/kg, i.p.; first arrow) followed by/kg, i.p.; second arrow). Acute normetanephrine pretreatmentls of NE (~300%) over extracellular NE levels in the rat frontal.) alone. Asterisk and number symbol (#) represent significantreated animals, respectively. B). Cortical levels of extracellulargle injection of normetanephrine alone (10 mg/kg, i.p. firstlafaxine (10mg/kg, i.p. second arrow) produced an increase infrontal cortex. Acute normetanephrine pretreatment (10 mg/cellular levels of 5-HT in the rat frontal cortex in the animals. Data are expressed as mean+SEM (n=4–6 per treatment

Fig. 4 – Acute normetanephrine potentiates the antidepressant-like behavioral effects of an antidepressant in the tailsuspension test in SwissWebstermice. A). Acute normetanephrine treatment (17 and 56mg/kg, i.p.) does not affect immobilitytime relative to vehicle (Veh) treatment. B). Acute desipramine treatment (10mg/kg, i.p.) does not effect immobility time relativeto vehicle treatment. C). Acute normetanephrine (Nm) treatment potentiates the antidepressant-like effects of desipramine(DMI). Co-treatment of 56 mg/kg (i.p.) of normetanephrine with 10 mg/kg (i.p.) of desipramine produced a significant (P<0.05)decrease of immobility time relative to vehicle treatment.

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potentiates the effects of venlafaxine on cortical levels of NE inthe rat frontal cortex.

2.4. Acute normetanephrine pretreatment potentiates theantidepressant-like behavioral effects of desipramine in thetail suspension test in mice

To evaluate if normetanephrine influences the behavioral effectsof an antidepressant drug that inhibits the NET, we tested theeffects of normetanephrine alone or in combination with de-sipramine in the mouse tail suspension test (TST). When admin-isteredalone,neithernormetanephrine (17mg/kgor56mg/kg, i.p.)(Fig. 4 A) nor desipramine (10 mg/kg, i.p.) (Fig. 4 B) produced sig-nificant changes in immobility time compared to vehicle treat-ment. These results suggest a lack of antidepressant-like effectsof these drugs at the doses tested. However, when administeredin combination, a 20-min pretreatment with 56 mg/kg (i.p.) of

normetanephrine followed with 10 mg/kg (i.p.) of desipramine,produced a significant (P=0.0147 relative to vehicle+vehicle con-trols) reduction (36%)of immobility time (Fig. 4C).This response isconsistent of an antidepressant-like activity of the combinationtreatment.

3. Discussion

Several clinical reports indicate that levels of normetanephrine,the O-methylated metabolite of NE and a potent inhibitor ofuptake 2, are elevated in the CSF, plasmaandurine of depressedpatients undergoing antidepressant therapy (Schildkraut et al.,1966, 1971; Potter and Manji, 1994; Maas et al., 1987). Moreover,an association has been observed between the gradual clinicalimprovement in patients treated with antidepressants and theincrease in urinary normetanephrine leading to the hypothesis

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that blockade of uptake 2 by normetanephrine contributes to orhastens the action of antidepressant drugs (Schildkraut andMooney, 2004). The present preclinical experiments investi-gated this hypothesis by using microdialysis techniques in therat frontal cortex to study the effects of normetanephrine on theneurochemical actions of the antidepressant venlafaxine. Theneurochemical findings reported here suggest that inhibition ofuptake 2 by normetanephrine potentiates the increase in cor-tical levels of NE induced by the action of venlafaxine.

The intrinsic biological properties of normetanephrine werefirst described in the context of its presence in peripheral tissues(BurgenAsv, 1965; Schanberget al., 1968), but itspharmacologicaland neurochemical effects have not been extensively explored(Schildkraut and Mooney, 2004). Using in vivo microdialysis andHPLC techniques, we have been successful in measuring dose-dependent increases incortical levelsofnormetanephrine inratsfollowing systemic administration of normetanephrine (Fig. 1).One potential limitation of this initial study is that it was notdesigned to evaluate whether the normetanephrine monitoredin the frontal cortex was the same normetanephrine injectedsystemically. Therefore, these results may either be due to theability of normetanephrine to directly cross into the CNS, or bethe result of a series of events initiating in the periphery thatculminate in the large, dose-dependent increases in normeta-nephrine observed centrally. Despite not having this informa-tion, to our knowledge, these initial microdialysis results are thefirst demonstration that levels of normetanephrine in the ratfrontal cortex can be quantified via in vivomicrodialysis coupledto HPLC techniques. Overall, these results provide the initialcharacterization of the analytical conditions required for thesuccessful detection of normetanephrine in vivo (see below).

Chronic administration of venlafaxine produced significantincreases in levels of both cortical NE and 5-HT (Fig. 2), which isconsistent with published results for this antidepressant (Beyeret al., 2002; Koch et al., 2003). Quantification of cortical normeta-nephrine levels in these rats revealed a trend towards increasedcortical levels of normetanephrine over baseline values (29%)(Fig. 2). Although the observed increases in cortical normeta-nephrine levels were not statistically significant (P=0.199), it isconceivable that increasing the duration of antidepressant treat-ment from 2 weeks to perhaps 3 or 4 weeks, or increasing thedose of antidepressant used, may result in a more robust andstaistically significant increase incortical levelsofnormetanephr-ine. It is noteworthy to mention that these preclinical observa-tions reported here are consistent with clinical reports showingthat there is a gradual increase in the release of urinary norme-tanephrine levels during chronic administration of tricyclicantidepressant drugs (Schildkraut et al., 1966). Since, increasesin levels of normetanephrine levels were observed in patientsonly during long-term (starting ∼ 3 weeks) but not after short-term (<1 week) administration of antidepressant drugs (Schildk-raut et al., 1966; Schildkraut andMooney, 2004), future studieswillbe designed to evaluate the neurochemical effects with otherantidepressants and after longer time intervals.

Themost significant observation of this studywas that acutenormetanephrine treatment potentiates the neurochemicaleffects of venlafaxine on cortical levels of NE. Results from themicrodialysis experiments described here indicate that acutenormetanephrine pretreatment potentiates the effects of ven-lafaxine on cortical levels of NE (Fig. 3). Although acute intra-

peritoneal administration of 10 mg/kg normetanephrine alonefailed toproducesignificant changes inextracellularNE levels inthe rat frontal cortex, acute venlafaxine (10 mg/kg, i.p.) admin-istration produced an increase of ∼150% in extracellular levelsof NE over basal levels in the frontal cortex. This latter neuro-chemical response is consistent in both magnitude and dura-tion to the effects of venlafaxine previously reported (Beyeret al., 2002). When acute normetanephrine was given as a pre-treatment tovenlafaxine, a significantlygreater (∼300%) increasein the extracellular levels of NEwas observed. The cortical levelsof extracellular 5-HT measured in the same animals were notsignificantly altered (Fig. 3 B). These data suggest that acutenormetanephrine treatment potentiates the neurochemicaleffects of the SNRI venlafaxine on cortical levels of NE but noton 5-HT. Although 5-HT levels were unaffected when normeta-nephrinewas given alone or when administered in combinationwith venlafaxine, other studies have shown that inhibition ofuptake 2 modulates 5-HT uptake in other brain regions. Forexample, local infusion of the uptake 2 inhibitor, decynium22, inthe medial hypothalamus in rats via microdialysis resulted in areversible dose-dependent increase of extracellular 5-HT con-centrations (Feng et al., 2005). The reason for these seeminglydisparate findings isnot obvious, however, is likely a reflectionofregional differences in the role of uptake 2 in the frontal cortexversus the hypothalamus and/or pharmacological differencesbetween the uptake 2 inhibitor used in each study.

To extend the neurochemical findings further, we evaluatedthe effects of inhibiting NET and uptake 2 in a behavioral modelof antidepressant-like activity. The tail suspension test (TST) is abehavioral test widely used for measuring antidepressant drug-like activity inmice (Cryanet al., 2005; O'Learyet al., 2007). In thistest, acute administration of antidepressant drugs typicallyreduces the time spent immobile and promotes the occurrenceof escape-relatedbehaviorwhich is indicativeof antidepressant-like activity (Steru et al., 1985). We have utilized the TST inmiceto evaluate if normetanephrine influences the behavioral effectsof desipramine, an antidepressant drug that inhibits the activityof the NET. Mice pretreated with 56 mg/kg (i.p.) of normeta-nephrine, followed by a dose of desipramine (10mg/kg, i.p.) thatdid not elicit any antidepressant-like activity when adminis-tered alone, produced a significant reduction of immobility time(36%; P=0.0147 relative to vehicle+vehicle controls) (Fig. 4 C)indicating antidepressant-like activity. These behavioral resultssuggest that acute normetanephrine pretreatment, which pre-sumably inhibits uptake 2, potentiates the antidepressant-likebehavioral effects of the NET inhibitor desipramine. Thesebehavioral data in mice complement the neurochemical obser-vations in rat suggesting that inhibiting both uptake 2 and NETproduces a greater antidepressant-like effect than inhibitingeither mechanism or transporter alone.

Biological effects ofNE in the synapseare regulated, inpart, bythe action of efficient clearancemechanisms for signal termina-tion (Blakely et al., 1994). The NET is the principle transporterresponsible for the inactivation of centrally released NE (Tren-delenburg, 1991) and is correspondingly amajormolecular targetfor the actions of several antidepressant drugs (Stahl et al.,2005; Barker and Blakely, 1995). In addition to the action of theNET, the extraneuronalmonoamine transporter or uptake 2 alsocontributes to inactivate levels of NE in the synapse (Iversen,1965;Grundemannet al., 1998). Uptake 2mayact asanadditional

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mechanism that inactivates monoamines that have escapedneuronal reuptake, and thus prevents uncontrolled spreading ofthe signal. The neurochemical data presented here suggest thatin rats inhibition of uptake 2 via normetanephrine alone is notsufficient to elevate cortical levels of NE. Indeed these data areconsistent with the notion that uptake 2 is not the primaryuptakemechanismfor removingNE fromtheextracellular space.Conversely, acute administration of an SNRI like venlafaxine,inhibitsNEuptakeviaNET (oruptake1), resulted inan increase inthe extracellular levels of NE. However, when acute normeta-nephrine is administered in conjunction with the SNRI, theactions of both, NET and uptake 2 are inhibited, resulting in asignificantly greater increase in the extracellular levels of NEcompared to the increase achieved by inhibiting NET alone withvenlafaxine. These neurochemical results suggest that althoughinhibition of uptake 2 via normetanephrine is not sufficient toelevate cortical levels of NE, a combination approach to inhibitboth uptake 1 and uptake 2may produce a greater increase inNEthan antidepressant treatment alone. This is also supported bythemouse TST data described in this report. Therefore, a similarcombination approach may result in antidepressant drugs withfaster onset of antidepressant action, a feature that is an im-portant unmet clinical need for all marketed antidepressants(Rosenzweig-Lipson et al., 2007).

Recent studies involving genetic manipulation of OCT3,which correlatesmost closely to uptake 2, further demonstratedthe functional involvement of uptake 2 in CNS functions. Datafromstudies in involving geneticmanipulationofOCT3 (Slc22a3)in mice implicate its role in salt-intake regulation (Vialou et al.,2004). In another study, down regulation of OCT3 inmice by i.c.v.infusion of antisense oligonucleotides results in reduced immo-bility time in the forced swim test, a response consistent withantidepressant-like activity in this model (Kitaichi et al., 2005).The results of the current studies in rats suggest that inhibitionof bothNET and uptake 2 via venlafaxine and normetanephrine,respectively, elicits a greater increase in cortical levels ofNE thanby inhibition of either NET or uptake 2 alone. This finding sup-ports the hypothesis that inhibition of uptake 2 in the brainmayincrease the efficacy of norepinephrine reuptake inhibitor anti-depressant drugs. To our knowledge, these findings constitutethe first neurochemical demonstration of potentiation of neuro-chemical effects of venlafaxine by systemic administration ofnormetanephrine in rats.

Due to its physiological profile and the expression patternwithin the CNS, EMT, the correlate of uptake 2 mechanism inhumans, is suggested to be a candidate gene for various neuro-psychiatric disorders (Grundemannet al., 1998; Eisenhofer, 2001).For example, single-nucleotide polymorphisms (SNPs) have beendetectedwithin the promoter region of the EMT gene in humans(Lazar et al., 2003) providing a basis for association studies andcandidate gene approaches. An association between 2 SNPs(rs 509707 and rs 4709426) in the EMT gene and development ofpolysubstance use among patients with methamphetamine(MAP) dependence has been reported suggesting a role for EMTinMAPdependence (Aoyamaetal., 2006). Ina recent report,novelmutations of the EMT gene have been reported in children andadolescents with obsessive–compulsive disorder (OCD) (Lazar etal., 2007). In vitro functional analysis of an EMT mutant allele(Met370Ile) in a luciferase reporter assay indicated impairedtransport activity for NE providing functional significance of the

mutation in NE uptake (Lazar et al., 2003). While this emergingbody of evidence suggesting a direct association of the EMT genein a variety of neuropsychiatric disorders is intriguing, additionalstudies interrogating the physiological significance of thismech-anism and its precise relationship with catecholamine signalingare still required.

In summary,wehaveevaluated thehypothesis that increasedlevels of normetanephrine, and consequently inhibition of up-take 2, may partly be responsible for the clinical efficacy ofantidepressant drugs that increase extracellular levels of NE.Using microdialysis studies in rats, we have demonstrated thatnormetanephrine potentiates the neurochemical effects ofvenlafaxine as evidenced by increased cortical levels of NE.Similarly, using the TST in mice, we have demonstrated thatnormetanephrine potentiates the antidepressant-like behavioraleffects of desipramine. The findings presented in this reportsuggest that inhibiting both NET and uptake 2 produces a greaterincrease in NE than antidepressant treatments that block NETalone. Based on this observation, it can be suggested that com-pounds that increase levels of normetanephrineor inhibit uptake2 on glial cells, would accelerate the onset of clinical action ofantidepressants that increase NE levels in the synapse by in-hibiting the NET. Therefore, development of a single moleculethat blocksbothuptakesites, or alternatively, apotent inhibitor ofuptake 2 could be used as adjunctive therapy to current anti-depressants toelicit superior antidepressant activity indepressedpatients.

4. Experimental procedures

4.1. Animal housing, dosing and surgery

All experimentswereconductedaccording to thespecificationsofboth the National Institutes of Health guide for the Care and Useof Laboratory Animals (Pub. 85-23, rev 1996) andWyeth's InternalAnimal Care and Use Committee (IACUC). For all microdialysisexperiments, adult male Sprague-Dawley rats (Charles River,Wilmington,MA)weighing between 280–350 gwere used. For thetail suspension test, male Swiss Webster mice (Charles River)weighing 25–35 g were used. All animals were group housed,independent of their treatment, in anAAALAC-accredited facilityand maintained on a 12-h light/dark cycle. All experimentationwas conducted during the light period (lights on at 0600 h).

Normetanephrinewasdissolved inwaterandadministeredat1, 3 or 10 mg/kg (i.p.). For chronic studies, animals received asingle injection of vehicle (water) or venlafaxine (10 mg/kg, s.c.)once a day for 14 consecutive days followed by a challenge withthe drug (venlafaxine; 10mg/kg, s.c.) or vehicle (water) on day 15.These injections were administered in a volume of 1 ml/kg andgiven to the animal in their home cage. For acute treatmentmicrodialysis studies animals received a single injection ofnormetanephrine (10 mg/kg, i.p.) and/or venlafaxine (10 mg/kg,i.p.). For theTST,animals receivednormetanephrine (17or56mg/kg i.p.) and/or desipramine (10 mg/kg i.p.)

For the microdialysis experiments, on day 1 of the acutestudies or on day 14 of the chronic treatment study, stereotaxicsurgery was performed. Under 2–3% halothane anesthesia(Fluothane; Zeneca, Cheshire, UK), animals were secured in astereotaxic frame with ear and incisor bars (David Kopf,

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Tujunga, CA), while a microdialysis guide cannula (CMA/12;CMA Microdialysis, Stockholm, Sweden) was directed at thedorsal lateral frontal cortex using the following coordinates:+3.2 mm anterior to bregma, −3.5 mm lateral from the midlineand −1.3 mm ventral to dura with a flat skull (Paxinos andWatson, 1986). Guide cannulae were fixed to the skull with twostainless steel screws (Small Parts, Roanoke, VA) and dentalacrylic (Plastics One, Roanoke, VA). Following surgery, animalswere individually housed in Plexiglas cages (45 cm2) for ap-proximately 24 h and had access to food and water ad libitum.

4.2. In vivo microdialysis in rats

4.2.1. Testing proceduresMicrodialysis procedures were conducted either on day 2 (acutestudies) or on day 15 (chronic studies) as described previously(Beyer et al., 2002). Briefly, microdialysis probes (CMA/12; 20 kDcut-off) were purchased from CMA Microdialysis (Sweden) andconsistedofa2-mmactivemembrane (OD0.5mm)witha14-cmstainless steel shaft (OD 0.64 mm). Probes were perfused withartificial CSF (aCSF; 125mMNaCl, 3mMKCl, 0.75mMMgSO4 and1.2 mM CaCl2, pH 7.4) in a glass beaker for at least 18 h prior toexperimentationaccording tomanufacturer's specifications.Onthe day of experiments, microdialysis probes were inserted, viathe guide cannula, into the frontal cortex and perfused withaCSF at a flow rate of 1 μl/min. A 3-h stabilization period wasallowed following probe insertion after which time dialysatewas collected every 20min. Initially, five sampleswere collectedto demonstrate a steady baseline. Next, animals received aninjection of either vehicle (water) or venlafaxine (10 mg/kg, i.p.)and samples were collected every 20 min for at least the fol-lowing 3 h. At the end of the experiment, animals were eutha-nized and probe placement was verified histologically. Datafrom animals with incorrect probe placement were discardedfrom the final analysis.

4.2.2. HPLC conditionsDialysate (20μl) fromthe rat frontal cortexwasanalyzedbyhigh-pressure liquid chromatography (HPLC) with electrochemicaldetection. Samples containing normetanephrine, NE, 5-HT andDA were separated by HPLC (C18 ODS3 column, 150×3.0 mm,Metachem, Torrance, CA) and detected using an ANTEC electro-chemical detector (ANTEC, Netherlands) set at a potential of0.65 V vs. a Ag/AgCl reference electrode. Mobile phase (0.15 MNaH2PO4, 0.25 mM EDTA, 1.75 mM 1-octane sulphonic acid, 2%isopropanol and 4%methanol, pH=4.8) was delivered by a JascoPU1580HPLCpump (Jasco Ltd, Essex, U.K) at a flow rate of 0.5ml/min. Neurochemical data were compared to an external stan-dard curve and all data was acquired using the Atlas softwarepackage (Thermo Labsystems, Beverley, MA) for the PC.

4.2.3. Statistical analysisThe fmol concentrations of all neurotransmitters during thebaseline samples were averaged and this value was denoted as100%. Subsequent sample valueswere expressed as a percentageof this preinjection baseline value (% of baseline). Neurochemicaldata, excluding preinjection values, were analyzed by a two-wayanalysis of variance (ANOVA) with repeated measures (time).Post-hoc analyses were made using the Bonferroni/Dunns ad-justment for multiple comparisons. All statistical calculations

were performed using the Statview software application (AbacusConcepts Inc., Berkeley, CA) for the PC.

4.3. The tail suspension test (TST)

The procedure followed in this study was a variant of the oneoriginally described by Steru et al. (1985). Normetanephrinewas administered 20 min prior to desipramine. 60 min fol-lowing desipramine treatment, the mice were suspendedupside down by the tail using adhesive laboratory tape (VWRInternational), to a flat metal bar connected to a strain gaugewithin a tail suspension chamber (Med Associates). The timespent immobile during a 6-min test session was automaticallyrecorded. 8 mice were simultaneously tested within separatechambers. Data collected were expressed as a mean of im-mobility time and statistical analysis was performed using aone-way ANOVA with least significant difference (LSD) post-hoc test.

4.4. Chemicals

Venlafaxine was synthesized by Wyeth's Chemical andScreening Sciences group (Princeton, NJ) and its purityverified by standard analytical methods. All other chemicalsused for microdialysis and HPLC experiments were ofanalytical grade and purchased from Sigma-Aldrich chemi-cals (Milwaukee, WI).

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