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Research Report

Tyrosine depletion lowers dopamine synthesis anddesipramine-induced prefrontal cortex catecholamine levels

Rodolfo Bongiovannia, Erica Newboulda, George E. Jaskiwa,b,⁎aPsychiatry Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Brecksville, OH, USAbDepartment of Psychiatry, Case Western Reserve University, Cleveland, OH, USA

A R T I C L E I N F O

⁎ Corresponding author. Psychiatry Service 11E-mail address: gxj5@case.edu (G.E. Jaskiw

0006-8993/$ – see front matter. Published bydoi:10.1016/j.brainres.2007.10.079

A B S T R A C T

Article history:Accepted 28 October 2007Available online 4 November 2007

The relationship between limited tyrosine availability, DA (dopamine) synthesis and DAlevels in themedial prefrontal cortex (MPFC) of the ratwas examinedby invivomicrodialysis.We administered a tyrosine- and phenylalanine-free mixture of large neutral amino acids(LNAA(−)) IP to lower brain tyrosine, and the norepinephrine transporter inhibitordesipramine (DMI) 10 mg/kg IP to raise MPFC DA levels without affecting DA synthesis. Forexamination of DOPA levels, NSD-1015 20 μM was included in perfusate. Neither NSD-1015nor DMI affected tyrosine levels. LNAA(−) lowered tyrosine levels by 45%, and lowered DOPAlevels as well; this was not additionally affected by concurrent DMI 10 mg/kg IP. In parallelstudies DMI markedly increased extracellular levels of DA (420% baseline) andnorepinephrine (NE) (864% baseline). LNAA(−) had no effect on baseline levels of DA or NEbut robustly lowered DMI-induced DA (176% baseline) as well as NE (237% baseline) levels.Even when DMI (20 μM) was administered in perfusate, LNAA(−) still lowered DMI-inducedDA and NE levels. We conclude that while baseline mesocortical DA synthesis is indeeddependent on tyrosine availability, the MPFC maintains normal extracellular DA and NAlevels in the face of moderately lower DA synthesis. During other than baseline conditions,however, tyrosine depletion can lower ECF DA and NE levels in MPFC. These data offer apotential mechanism linking dysregulation of tyrosine transport and cognitive deficits inschizophrenia.

Published by Elsevier B.V.

Keywords:Prefrontal cortexDopamineNorepinephrineDOPANSD-1015Schizophrenia

1. Introduction

Tyrosine depletion studies in man suggest that prefrontalcortex catecholamine-mediated processes depend on ade-quate tyrosine availability (Gijsman et al., 2002; Harmer et al.,2001; Harrison et al., 2004). Studies in the rat show that alimited supply of precursor can constrain both catecholaminesynthesis and release under appropriate conditions (MilnerandWurtman, 1986; Sved and Fernstrom, 1981). The ratmedialprefrontal cortex (MPFC) is thought to meet such conditions; it

6A(B), LSC VAMC, 10000 B).

Elsevier B.V.

is characterized by a high proportion of activated TH (IuvoneandDunn, 1986), rapidDA turnover (Bannon et al., 1981), a highdegree of coupling between DA synthesis and release (Gallo-way et al., 1986) and a high rate of DA release per DA terminal(Cass and Gerhardt, 1995; Garris and Wightman, 1994). Thisprofile predicts a marked sensitivity to tyrosine availability(Bradberry et al., 1989; Tam et al., 1990). Yet, while tyrosinedepletion has been reported to lower the rate of DA synthesisinMPFC (Bradberry et al., 1989;McTavish et al., 1999; Tamet al.,1990), it does not appear to lower basal extracellular fluid (ECF)

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DA levels (Jaskiw et al., 2005; Jaskiw et al., 2006). One possibilityis that compensatory mechanisms in MPFC maintain steadyECF DA levels in the face of moderately lower DA synthesis.However, the approaches used to lower tyrosine availability,measure DA synthesis (Bradberry et al., 1989; McTavish et al.,1999; Tam et al., 1990) and quantify ECF DA levels (Jaskiw et al.,2005; Jaskiw et al., 2006) have varied considerably acrossstudies. Thus, how limited tyrosine levels affect MPFC DAsynthesis and ECF DA under similar experimental conditionsremains to be determined.

In view of this, we measured MPFC DOPA levels using thesame tyrosine depletion paradigm (McTavish et al., 1999) andin vivo approach used to examine DA and NE levels previously( Jaskiw et al., 2005, 2006). First we determined that introduc-tion of the DOPA-decarboxylase inhibitor NSD-1015 in perfu-sate did not affect tyrosine levels. Subsequently, with NSD-1015 in perfusate, we examined how tyrosine depletion affectsDOPA levels. Our primary hypothesis was that tyrosinedepletion would modestly lower basal MPFC DOPA accumula-tion in vivo. This would suggest that the MPFC could maintainstable basal DA levels in the face of moderately loweredtyrosine levels and DA synthesis ( Jaskiw et al., 2005, 2006).

The relationship between tyrosine availability, DA levels andDA synthesis under non-physiological conditions is a differentmatter. The longstanding assumption has been that DA systemsare tyrosine-dependent if and only if tyrosine hydroxylase (TH)activity is enhanced (Milner and Wurtman, 1986; Sved andFernstrom, 1981). Accordingly, drugs that do not per se increaseDA synthesis should not influence the effects of tyrosinedepletion on either DA levels or DA synthesis. Desipramine(DMI) is a highly selective inhibitor of the norepinephrinetransporter (NET) (Roth, 2006). DMI robustly elevates both MPFCDA and norepinephrine (NE) levels (Di Chiara et al., 1992; Greschet al., 1995) without affecting MPFC DA synthesis (Bongiovanniet al., 2005). Hence, our secondary hypothesis was that DMIwould not affect the ability of tyrosine depletion to lower MPFCDOPA accumulation. Furthermore, tyrosine depletion would notaffect the DMI-induced elevation of DA and NE levels.

2. Results

2.1. Basal levels

Basal MPFC concentrations were as follows: tyrosine 1.88±0.23 μM, DOPA 0.90±0.30 nM, DOPAC 31.2±7.8 nM, NA 0.744±0.059nM (mean±SEM). DAandNE levelswere examined in twoseparate experiments conducted severalmonths apart. Acrossindividual groups DA ranged 0.33–0.55 nM. However, theaverage basal level in the first study (0.39±0.05 nM) (Fig. 4A)was not significantly different (t=0.37, p=0.71) from that in thesecond (0.43±0.04 nM) (Fig. 5A). Tyrosine and NE levels did notvary between studies.

2.2. Route of NSD-1015 administration affectstyrosine levels

Typical doses of systemically administered NSD-1015 elevatetissue tyrosine levels (Carlssonet al., 1972;Westerink andWirix,1983). To determine how NSD-1015 affects ECF tyrosine levels

we administered NSD-1015 either IP (100mg/kg) or in perfusate(20 μM) and compared MPFC tyrosine levels by a two-wayANOVA with route of administration as the primary factor andtime as the repeated factor. There were significant effects ofroute [F(1,80)=31.20, p<0.0001], time [F(9,80)=3.30, p<0.002] andtime×route [F(9,80)=2.59, p<0.01]. Post hoc tests showed thatthe two groups were significantly different at t=2 h and t=2.5 h(p<0.01) (Fig. 2). Bonferroni-corrected multiple comparisonsshowed that when NSD-1015 was administered in perfusate,therewere no significant changes in tyrosine levels at tN1 com-pared to t=0. In contrast, when NSD-1015 was administered IP,tyrosine levels rose to a maximum of 240% baseline and weresignificantly greater at t=2 h and t=2.5 h than at t=0 h (p<0.05).

2.3. DMI does not affect tyrosine levels

To determine whether DMI affects tyrosine levels, these weremeasured after different doses of DMI with concomitantadministration of NSD-1015 in perfusate (20 μM). An ANOVAwith DMI dose (0, 10, or 20 mg/kg IP) as the primary factor andtime as the repeated factor showed no significant effects ofDMI dose [F(2,77)=2.72, pN0.07], time [F(10,77)=0.64, pN0.78] orDMI dose×time interaction [F(20,77)=0.56, pN0.93] (data notshown). Tyrosine levels after administration of DMI 10 mg/kgIP alonewere further examined using ANOVAwith time as therepeated factor (data not shown). There was no effect of time[F(10,30)=0.65, pN0.76].

2.4. Tyrosine depletion lowers MPFC DOPA levels. DMI hasno additional effect

To determine the effect of tyrosine depletion on DA synthesis,NSD-1015 (20 μM)was added to perfusate and either VEHAA or amixture of large neutral amino acids (LNAA) that did notcontain tyrosine or phenylalanine (LNAA(−)) was administeredIP. This was followed by either VEHD or DMI IP. The ANOVA fortyrosine showed significant treatment [F(3,245) = 25.89,p<0.0001], time [F(11,245)=4.23, p<0.0001] and time×treat-ment [F(33,245)=2.06, p<0.001] effects. In both LNAA(−)-treatedgroups, MPFC tyrosine levels were significantly lower withrespect to their VEHAA controls (Fig. 3A). There were nosignificant differences in tyrosine levels between the groupsVEHAA+VEHD and VEHAA+DMI. After the introduction of NSD-1015 in perfusate, DOPA levels rose and then stabilized byt=2.5 h (Fig. 3B). The ANOVA for DOPA showed significanttreatment [F(3,196)=14.96, p<0.001], time [F(12,196)=23.58,p<0.0001] but not time×treatment [F(36,196)=0.99, pN0.5]effects. Post-hoc tests showed that by tN4 h, DOPA levels inthe LNAA(−)+VEHD group were significantly lower than thosein VEHAA+VEHD animals (Fig. 3B). Similarly, DOPA levels in theLNAA(−)+DMI groupwere lower than those in LNAA(−)+VEHAA

animals (Fig. 3B). There were no significant differences inDOPA levels for VEHAA+VEHD versus VEHAA+DMI, nor forLNAA(−)+VEHD versus LNAA(−)+DMI. The ANOVA for DOPACshowed significant treatment [F(3,263)=5.55, p<0.001], time [F(11,263)=1.96, p=0.03] but not time×treatment [F(33,263)=0.44,p=0.997] effects. DOPAC levels in VEHAA+VEHD as well as inVEHAA+DMI groups were not significantly different from oneanother (Fig. 3C). DOPAC levels in the LNAA(−)+DMI groupwere significantly below those in the VEHAA+DMI group. By

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t=4.5 h, DOPAC levels in the LNAA(−) groups were at 54%baseline (Fig. 3C).

2.5. Tyrosine depletion lowers DMI-induced DA andNE levels

To determine how tyrosine depletion affects DMI-inducedcatecholamine levels, rats receivedVEHAA or LNAA(−) and thenDMI 10 mg/kg IP. NSD-1015 was not included. The overallANOVA for DA showed significant treatment [F(1,64)=121,p<0.0001], time [F(12,64)=32.1, p<0.0001] and treatment×time[F(12,64)=7.7, p<0.0001] effects (Fig. 4A). There were nosignificant differences between DA levels in the groupsreceiving VEHAA and LNAA(−) during the 60-min intervalbetween administration of LNAA(−) and subsequent adminis-tration of DMI or VEH. After administration of DMI, DA levelsincreased rapidly to a maximum of 420% and 176% baseline inanimals pretreated with VEHAA and LNAA(−), respectively(Fig. 4A). For the entire period after administration of DMI, DAlevels in LNAA(−)-pretreated animals were significantly low-er than those in VEHAA-treated animals. In the same groupof rats, the overall ANOVA forNE showed significant treatment[F(1,64)=121, p<0.0001], time [F(12,64)=32.1, p<0.0001] andtreatment×time [F(12,64)=7.68, p<0.0001] effects (Fig. 4B).There were no significant differences between NE levels inthe groups receiving VEHAA and LNAA(−) during the 60-mininterval between administration of LNAA(−) and subsequentadministration of DMI or VEH. After administration of DMI, NElevels increased rapidly to a maximum of 864% and 237%baseline in animals pretreated with VEHAA and LNAA(−),respectively. For the entire period after administration ofDMI, NE levels in LNAA(−)-pretreated animals were signifi-cantly lower than those in VEHAA controls (Fig. 4B).

We were surprised that LNAA(−) lowered DMI-induced DAand NE levels (Figs. 4A, B) and repeated the precedingexperiment but this time with DMI (20 μM) in perfusate. Ratswere randomly assigned to receive either VEH or LNAA(−) aftercollection of the first dialysate sample (Figs. 5A, B). There werenodifferences inDAandNE levels between theVEH- and LNAA(−)-treated groups for the subsequent two samples, and so thefirst three samples were considered to be the baseline. TheANOVA for DA showed significant treatment [F(1,90)=32.0,p<0.0001], time [F(9,90)=26.6, p<0.0001] and treatment×time[F(9,90)=2.9, p<0.005] effects. There were no significantdifferences between DA levels in the groups receiving VEHAA

and LNAA(−) during the 60-min interval between administra-tion of LNAA(−) and subsequent administration of DMI or VEH(Fig. 5A). DA levels in the VEHAA+DMI group (peak 368%) weresignificantly higher than those in the LNAA(−)+DMI group(peak 207%) formost of the period tN1.5 h, p<0.001) (Fig. 5A). Inthe same group of rats, the ANOVA for NE showed significanttreatment [F(1,89) =6.95, p<0.01] and time [F(9,89)=35.1,p<0.0001] but not treatment×time [F(9,89)=1.01, p=0.4] effects.There were no significant differences between NE levels in thegroups receiving VEHAA and LNAA(−) during the 60-mininterval between administration of LNAA(−) and subsequentadministration of DMI or VEH. NE levels in the VEHAA+DMIgroup (maximum 550% baseline) were significantly higherthan those in the LNAA(−)+DMI group (maximum 404%baseline) at t=2.0 h (p<0.05) (Fig. 5B).

3. Discussion

3.1. Main findings

Our data show that MPFC DOPA accumulation in vivo islowered by tyrosine depletion. While there is no apparentinteraction of tyrosine depletion and DMI as far as DOPAaccumulation is concerned, tyrosine depletion consistentlylowered DMI-induced DA and NE levels in microdialysate.These data must be critically evaluated and understood inlight of both methodological and theoretical considerations.

3.2. Lowering brain tyrosine

Brain levels of tyrosine are determined mainly by transportacross the blood–brain barrier (Pardridge, 1998). Since theblood–brain barrier transporter also carries other large neutralamino acids (LNAAs) (Kanai et al., 1998; Pardridge, 1977), braintyrosine levels are lowered when serum levels of one or morecompeting LNAAs become elevated (Fernstrom and Faller,1978; Fernstrom and Fernstrom, 1995; McTavish et al., 1999).This can be done for instance, by administering a largesystemic dose of a single LNAA such as valine (Tam et al.,1990) or by inducing diabetes mellitus to elevate branchedchain amino acids (Bradberry et al., 1989). Both these para-digms lowerMPFC tissue DOPA accumulation (Bradberry et al.,1989; Tam et al., 1990). However, high dose valine also lowerscortical tryptophan, serotonin turnover and release (Gartsideet al., 1992; Kennett and Joseph, 1981); such changes also affectDOPAaccumulation (LeMasurier et al., 2006). Diabetesmellitusper se lowers central TH levels and TH activity (Bitar et al., 1986;Figlewicz et al., 1996). In contrast, a tyrosine- and phenylala-nine-free mixture of large neutral amino acids (LNAA(−))acutely lowers brain tyrosine without affecting serotonergicindices (McTavish et al., 1999). LNAA(−) and variants thereoflowered NSD-1015-induced DOPA accumulation in a tissuesample that included frontal and orbital cortex in addition tothe MPFC (Le Masurier et al., 2006; McTavish et al., 1999). Themesocortical DA innervation across these regions, however,showsbothanatomic and functional heterogeneity (Descarrieset al., 1987; Herman et al., 1982; Lindvall and Bjorklund, 1987).For these reasons, we considered the inferential value of theseearlier data uncertain and adopted the LNAA(−) paradigm inconjunction with microdialysate collected from the MPFC.

3.3. Measuring DOPA levels

Basal brain tissue levels of DOPA are low and unstable(Westerink and Spaan, 1982); basal DOPA levels in brain ECFare even lower (Carboni et al., 1992). There is a single report inwhich basal DOPA levels were measured by in vivo micro-dialysis in striatum (Carboni et al., 1992); there are no suchstudies in the MPFC. In our own laboratory, we have beenunable to measure meaningful basal DOPA levels in micro-dialysate (Bongiovanni et al., 2006). Accordingly, the usualapproach is to administer a DOPA-decarboxylase inhibitor andthen measure DOPA accumulation in tissue (Carlsson et al.,1972) or microdialysate (Nakahara and Nakamura, 1999;Westerink et al., 1990). The implicit assumption is that the

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selected inhibitor does not affect other experimentally rele-vant processes. However, DOPA-decarboxylase inhibitors alsoinhibit transamination, the main catabolic pathway fortyrosine (Moran and Sourkes, 1965). Since catabolism of theother LNAAs is less dependent on transamination, systemi-cally administered DOPA-decarboxylase inhibitors elevateserum levels of tyrosine more than that of other LNAAs andthus increase net tyrosine influx into the brain (Hilton et al.,1998). A standard dose of NSD-1015 (100 mg/kg IP) elevatesbrain tissue tyrosine levels to 140% baseline within 30 min(Carlsson et al., 1972) and 230% baseline within 60 min(Westerink and Wirix, 1983); tyrosine levels in ECF show thesame degree of elevation (Fig. 2). We recently demonstratedthat drug-induced DA indices in the striatum and MPFC arepotentiated when brain tyrosine levels are elevated by around50–100% of baseline (Jaskiw et al., 2005). This is precisely therange of elevation induced by systemic NSD-1015 (Carlssonet al., 1972; Westerink and Wirix, 1983) (Fig. 2) and must beconsidered as a potential confound.

In vivo microdialysis permits NSD-1015 to be introducedvia perfusate (Westerink et al., 1990). Since NSD-1015 is 2000times more potent as an inhibitor of rat brain DOPAdecarboxylase (IC50 0.015 μM) than of tyrosine aminotransfer-ase (IC50 30 μM) (Dyck, 1987), we expected that at lowconcentrations NSD-1015 would not significantly affect trans-amination. Indeed, brain ECF tyrosine levels were not affectedby NSD-1015 (20 μM) (Figs. 2 and 3A). Furthermore, with NSD-1015 in perfusate, LNAA(−) lowered ECF tyrosine levels in ECFby 45% (Fig. 3A), the same degree of depletion achievedwithoutNSD-1015 (Jaskiw et al., 2005, 2006). Thus,we concludethat NSD-1015 (20 μM) in perfusate does not significantly affectbrain tyrosine aminotransferase.

3.4. Tyrosine depletion lowers MPFC DA synthesis

LNAA(−) lowered DOPA and DOPAC levels (Figs. 3B, C). Thissupports our primary hypothesis, namely that tyrosinedepletion lowers the rate of MPFC DA synthesis in vivo. Theusual range of intracellular tyrosine levels (110–150 μM) andthe affinity of TH for tyrosine (10–20 μM) suggest that underbaseline conditions rat brain TH is 50–80% saturated withtyrosine (Kaufman and Kaufman, 1985; Kaufman, 1995). Theimplicit assumption of course, is that tyrosine levels withinDA terminals match those in brain tissue. That is open toquestion. First, brain tissue offers no direct information abouttyrosine levels in DA terminals (Milner et al., 1987). Second,tyrosine hydroxylation can lower intracellular tyrosine levels,even in the face of adequate tyrosine levels in the surroundingmedium (Vaccaro et al., 1980) or brain ECF (Bongiovanni et al.,2006). Thus, tyrosine levels within highly active catechola-mine terminals are likely to be lower than in the surroundingtissue, and terminal TH less than 50–80% saturated bytyrosine. A further lowering of tyrosine levels by LNAA(−)would be expected to lower the rate of tyrosine hydroxylation.

Tyrosine hydroxylation takes place both in DA and in NEterminals. In rodent MPFC, however, most TH protein, THactivity (Emson and Koob, 1978; Miner et al., 2003; Schmidt andBhatnagar, 1979) and most DOPA accumulation are attributedto DA terminals (Wolf et al., 1986). In agreement, we confirmour earlier report (Bongiovanni et al., 2005) that a dose of

systemically administered DMI that depresses electrophysio-logical activity of the locus coeruleus (Haskins et al., 1985;Mateo et al., 1998) and lowers the rate of NE synthesis in wholebrain (Nielsen, 1975) has no effect on DOPA levels in the MPFC(Fig. 3B). Since NE terminals appear to make a minorcontribution to net MPFC DOPA accumulation, LNAA(−) mustlower the rate of tyrosine hydroxylation within MPFC DAterminals.

3.5. Tyrosine depletion lowers DMI-induced but not basalDA and NE levels

DA or NE levels do not change after LNAA(−) for the 60-minperiod examined currently (Figs. 4A and 5A), nor for the 4-hperiod examined earlier (Jaskiw et al., 2005, 2006). These levelsare regulated by several overlapping mechanisms. Impulse-dependent DA release, for instance, is determined mainly bythe rate of neuronal stimulation (Cooper et al., 2002) but is alsomodulated by terminal DA release-modulating autoreceptors(Cooper et al., 2002) as well as by adrenergic alpha-2autoreceptors (Valentini et al., 2006). DA terminals preferen-tially release newly synthesized rather than stored DA (Chenet al., 2003; Yavich and MacDonald, 2000). Even though theMPFC is characterized by a relatively high degree of couplingbetween DA synthesis and ECF DA levels (Cass and Gerhardt,1995; Galloway et al., 1986; Garris and Wightman, 1994), ECFDA levels remain stable (Figs. 4A and 5A) (Jaskiw et al., 2005,2006) during a treatment that lowers the rate of tyrosinehydroxylation (Fig. 3B). One possibility is that tyrosinedepletion indeed lowers DA efflux from MPFC terminals, butthat terminal release-modulating autoreceptors (Cooper et al.,2002) respond so effectively that, within the temporal resolu-tion of in vivo microdialysis, ECF DA levels appear stable.

Tyrosine depletion robustly attenuated DMI-induced DAand NE levels (Figs. 4A, B). This was unexpected. Most datasuggest that tyrosine influences on catecholamine levelsemerge only when the rate of tyrosine hydroxylation isincreased and coupled to elevated catecholamine levels(Fernstrom, 1983; Jaskiw et al., 2001; Milner and Wurtman,1986; Tam et al., 1990). DMI does not affect tyrosine hydro-xylation in MPFC (Fig. 3B) (Bongiovanni et al., 2005), thecoupling between catecholamine synthesis and release, orenzymes which catabolize catecholamines. DMI 10 mg/kg IPattains brain levels of about 10 nM (van Wijk et al., 1977). Thisdoes not appreciably affect the adrenergic-alpha2 autorecep-tor (Ki 10 μm) but potently inhibit cortical NET (Ki 0.4 nM) (Roth,2006). NET has a higher affinity for DA than for NE (Gu et al.,1994) and is a major determinant of ECF DA levels (Carboniet al., 2006; Di Chiara et al., 1992; Yamamoto and Novotney,1998). Indeed, the DMI-induced elevation of MPFC ECF DA andNE levels is largely attributed to local NET blockade (Di Chiaraet al., 1992; Tanda et al., 1997; Yamamoto and Novotney, 1998).The magnitude of these elevations, however, still depends onthe continuing terminal efflux of catecholamines. A fall in ECFDA below basal levels would normally trigger a compensatoryresponse mediated by DA release-modulating autoreceptors.We posit that such a compensatory response would not betriggered if elevated levels of DA were lowered but stillremained above basal levels. Stated otherwise, we speculatethat the LNAA(−)-mediated lowering of DMI-induced DA levels

Fig. 1 – Typical probe placement in the MPFC. Thesuperimposed brain region boundaries are from the 3.2 mmlevel (Paxinos and Watson, 1982).

Fig. 2 – Effect of NSD-1015 on tyrosine (TYR) levels inmicrodialysate from MPFC. NSD-1015 was administeredeither once as a single IP injection (arrow) or by perfusion.The black line below “in perfusate 20μM” denotes the periodof perfusion. Each point is the mean±SEM (shown onlyunidirectionally). *p<0.05, **p<0.01, ***p<0.001 from baseline(□ n=6; ▼ n=6).

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from 420% to 176% baseline (Fig. 5A) would not lead to aphysiologically significant lowering of occupancy of the DArelease-mediating autoreceptor. Such a mechanism couldexplain why LNAA(−) lowers DMI-induced DA levels (Figs. 4Aand 5A) without affecting basal DA levels ( Jaskiw et al., 2005,2006).

Tyrosine depletion also lowered DMI-induced NE levels(Figs. 4B and 5B). There are competing theories as to the sourceof most of the NE in the ECF of the MPFC. Anatomic data offerthe possibility that mesocortical NE terminals take up ECF DAfor conversion to NE (Miner et al., 2003). Alternatively, since NEsystems even in DA poor regions can be tyrosine sensitive,LNAA(−) could limit synthesis of NE from tyrosine within NEterminals (Fernstrom, 1983; Milner and Wurtman, 1986). Ourdata from the MPFC do not distinguish between thesepossibilities. It would be informative to examine LNAA(−)effects on DMI-induced levels in DA poor cortical regions suchas the occipital or parietal cortices where most of the NE ispresumably synthesized directly from tyrosine (Valentiniet al., 2004;Valentini et al., 2006). In such regions, an LNAA(−)-induced lowering of ECF NE levels would likely reflect aprimary lowering of the NE synthesized from tyrosine withinNE terminals.

3.6. Summary and conclusions

Our first conclusion is a methodological one. For in vivomeasurements of DOPA accumulation, it would be advisableto administer NSD-1015 locally rather than systemically.Second, we confirm in vivo, what had been suggested earlier(Bradberry et al., 1989; McTavish et al., 1999; Tam et al., 1990);under physiological conditions, MPFC DA synthesis is indeeddependent on tyrosine availability. Third, compensatorymechanisms within the MPFC can maintain normal DA andNE levels in ECF even when tyrosine depletion modestlylowers DA synthesis. Fourth, elevated MPFC DA and NE levelscan be tyrosine-dependent under conditions which on theirown do not affect DA synthesis.

These findings may begin to explain why normal functionof prefrontal cortex appears to be contingent on adequate

tyrosine availability (Gijsman et al., 2002; Harmer et al., 2001;Harrison et al., 2004). Cognitive tasks specifically mediated bythe mesocortical catecholamine innervation (Phillips et al.,2004; Rossetti and Carboni, 2005) are associated with anelevation of ECF catecholamine levels in prefrontal cortex ofnon-human primates (Watanabe et al., 1997) as well as in therat (Rossetti and Carboni, 2005). We posit that tyrosinedepletion would not affect catecholamine levels in the restingstate but would lower cognitive demand-driven or pharma-cologically induced elevations of ECF DA and NE levels inprefrontal cortex.

These considerations are of particular relevance to thepathophysiology of schizophrenia and related disorders.Schizophrenia is associated with an abnormally lowmesocor-tical DA innervation (Akil et al., 1999, 2000) and an increasedrate of cortical DA turnover (Lindstrom et al., 1999). These datapredict that prefrontal cortex DA-mediated processes inschizophrenia would be unusually sensitive to tyrosine avail-ability. It is intriguing then that schizophrenia is alsoassociated with a generalized abnormality of tyrosine trans-port (Flyckt et al., 2001; Hagenfeldt et al., 1987; Ramchand et al.,1996;Wiesel et al., 1991, 1994, 1999) linked tomesocortical DA-mediated cognitive dysfunction (Wiesel et al., 2005). Thisdysregulation of tyrosine transport into the brain (Wieselet al., 1999) is thought to limit tyrosine availability (Bjerken-stedt et al., 2006), which as our data show, can lead to lowerstate-dependent prefrontal cortex catecholamine levels.Future studieswill determinehow to exploit this dysregulationfor research and treatment purposes.

4. Experimental procedures

4.1. Animals, surgery and microdialysis

Male Sprague-Dawley Rats (Zivic–Miller Zelienople, PA) (225–250 g) were housed 2 to a plastic cage (30×30×36 cm3) and

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maintained on a standard 12-h on/off light cycle with foodand water ad libitum in an AAALAC-accredited facility.Procedures were approved by Animal Care Committee andconducted in strict accordance with the NIH Guide for theCare and Use of Laboratory Animals. Each rat was used onlyonce, either for a tissue study or for microdialysis.

In preparation for microdialysis, rats were anaesthetizedwith a mixture of ketamine (70 mg/kg) and xylazine (6 mg/kg) IP and placed in a stereotaxic frame with the incisor bar

set at 7 mm. A stable level of anesthesia was maintained byadministering an additional 0.1 ml of ketamine/xylene whennecessary. Using a Dremel drill (bit FG 3; Roboz SurgicalInstrument, Inc. Washington, DC), a hole for the cannula wasdrilled into the skull. Three additional holes 9 mm from oneanother were drilled around the cannula hole. Three screws(0.8×1/8 in. were placed in the holes so that they protrudedapproximately 3 mm above the skull. A stainless steel guidecannula (ID 0.56 mm, length 11 mm, 21 GA) was lowered ontothe skull surface above the MPFC (AP +3.2, ML ±0.7) (Paxinosand Watson, 1982). Cranioplastic cement (Plastic One,Roanoke, VA) was used to build up a mound encasing theprotruding screws and ending about 5 mm below the top ofthe cannula. In addition, a male spade was placed 9 mm tothe rear of the last screw (nearest to bregma) in the moundthat was later use for the tether. After the cannula wasplaced, a stylus was inserted to maintain patency. One guidecannula was implanted in each rat and the side of cannula-tion (R vs. L) was balanced between rats. Animals wereallowed to recover for 24–48 h. On the evening before thestart of microdialysis, the rats were gently restrained, thestylus was removed from the guide cannula and the probe(exposed membrane tip 4.5×0.2 mm; MWCO 13,000; Spec-trum, CA, USA) implanted. The probe was connected topolyethylene tubing (0.965 mm OD, 0.58 mm ID, length 2 m)to a swivel and perfusion pump. The following morning, thedialysis probe was perfused at 1.0 μl/min for routine sampleanalysis. The perfusate consisted of Dulbecco's phosphate-buffered saline containing (in mM) 137 NaCl, 2.7 KCl,0.5 MgCl2, 1.5 KH2PO4, 8.1 Na2HPO4, 1.2 CaCl2, 5 glucose(final pH 7.4).

DOPA levels in microdialysate were assessed either aftersystemic administration of 100 mg/kg IP NSD-1015 (Sigma)or during perfusion of the microdialysis probe with 20 μMNSD-1015 (Sigma) (Nakahara and Nakamura, 1999). After asteady baseline was reached (3 consecutive basal levelswith a <10% CV) the test drug was administered. Sampleswere collected every 30 min and analyzed for DA, dihy-droxyphenylacetic acid (DOPAC), DOPA and NE. Thesesamples were analyzed immediately for the catechols andthe remainder of the microdialysate stored at −80 °C for theanalysis of amino acids. The brain was removed and storedat −40 °C. Serial sections were cut at 50-μm intervals andthe probe placement verified using a digitized image (Fig. 1)(Bert et al., 2004; Paxinos and Watson, 1982). If the probeextended outside of the targeted region, the data werediscarded.

Fig. 3 – Effects of LNAA(−) and DMI on (A) tyrosine (TYR),(B) DOPA, and (C) DOPAC levels in MPFC microdialysate froma single cohort of rats. The black line below “NSD-101520μM” indicates the duration of perfusion. LNAA(−) or VEHAA

was administered at two time points and DMI administeredonce as indicated by the arrows. Each point is themean±SEM(shown only unidirectionally). Asterisks above the graphshow significant differences betweenVEHAA/VEHD and LNAA(−)/VEHD. Asterisks below the graph show significantdifferences between VEHAA/DMI and LNAA(−)/DMI. *p<0.05,**p<0.01 (□ n=8; • n=8; ▪ n=6; ○ n=6).

Fig. 4 – Effect of LNAA(−) on DMI (10 mg/kg IP)-induced levelsof (A) dopamine (DA)and (B) norepinephrine (NE) in MPFCmicrodialysate from a single cohort of rats. LNAA(−) or VEHAA

was administered at two time points and DMI administeredonce as indicated by the arrows. Each point is themean±SEM(shown only unidirectionally). *p<0.05, **p<0.01, ***p<0.001.The same level of statistically significant difference applies todata points within the span of the bracket below (***) (• n=4;○ n=4).

Fig. 5 – Effect of LNAA(−) onDMI (20μMinperfusate)-inducedlevels of (A) dopamine (DA) and (B) norepinephrine (NE) inMPFC microdialysate from a single cohort of rats. LNAA(−) orVEHAA was administered at the two time points indicated bythe arrows. The black line below “DMI 20 μM” denotes theperiod of DMI perfusion. Each point is the mean±SEM(shown only unidirectionally). *p<0.05, **p<0.01, ***p<0.001(• n=6; ○ n=6).

45B R A I N R E S E A R C H 1 1 9 0 ( 2 0 0 8 ) 3 9 – 4 8

4.2. Assay of tyrosine and catechols

For assay of catechols (DA, DOPA, DOPAC, and NE), sampleswere separated on a 100×4.6 mm C18 column 3 μm particles.The mobile phase consisted of 10 mM citrate, 10 mM acetate,10 mM octylsulfonic acid, 0.1 mM EDTA, with 10% (v/v)methanol adjusted to pH 3.9 with o-phosphoric acid. It wasdelivered at a flow rate of 0.6 ml/min. An electrochemicaldetector was used with a glassy carbon electrode andmaintained at a relative potential of 0.70 V to an Ag/AgClreference electrode (Model LC-4; Bioanalytical Systems, IN,USA). The detection limit for the assay was 50 fg/10 μl at a 3:1signal to noise ratio.

For amino acid assay, the HPLC system consisted of areverse phase C18 column (15×4.6 cm., 3 μm particle size), anelectrochemical detector (ECD) operated at a relative potentialof 0.75 V to an Ag/AgCl reference electrode. The mobile phaseconsisted of 0.133MNa2HPO4 and 25%methanol (v/v) adjustedto pH 6.8 with o-phosphoric acid. To detect the amino acids, a

derivatizing agent was used (OPA-S; 10 mg o-phthaldehydeand 30 mg sodium sulfite diluted to 5.0 ml with 0.1 M sodiumcarbonate pH 10.4) for the reaction media. To a series of 0.3 mltubes were added 10 μl of sample, standards (0.1 to 2.5 μg/ml),and blanks, followed by 10 μl of internal standard (0.5 μg/mlnorvaline) and 10 μl OPA-S, reacted for 5 min and brought to afinal volume of 75 μl with HPLC mobile phase. A 10-μl samplewas injected on-column (Bongiovanni et al., 2001).

4.3. Administered drugs and amino acids mix

The tyrosine (and phenylalanine)-free amino acid mix (LNAA(−)) was prepared as follows: to a 10-ml vial were added 100mgof methionine, 200 mg threonine, 50 mg tryptophan, 350 mglysine, and 350 mg valine, followed by 5 ml of 1 N NaOH (stir5 min). Afterwards, 415.5 mg isoleucine methyl ester·HCl and623.2 mg leucine methyl ester·HCl were added and thesolution brought to a final volume of 6.5 ml (∼pH 6.8). Thisformulation (McTavish et al., 1999) differs from earlierdepleting solutions (Biggio et al., 1976; Fernstrom and Fern-strom, 1995) in that it is administered IP rather than orally and

46 B R A I N R E S E A R C H 1 1 9 0 ( 2 0 0 8 ) 3 9 – 4 8

does not contain histidine, the precursor for histamine. Whilehistamine–catecholamine interactions are known (Subrama-nian and Mulder, 1977), there are no studies suggesting thatbrain histamine levels are lowered by peripheral administra-tion of LNAAs. Furthermore, in previous studies, we haddemonstrated that LNAA(−) effects on MPFC DA levels wasmediated by tyrosine depletion rather than by non-specificeffects of the LNAA solution (Jaskiw et al., 2005).

LNAA(−) (total 1 g/kg IP) or a saline vehicle saline vehicle(VEHAA) was administered in two equal volumes 1 hr apart. Forsystemic administration, DMI-hydrochloride (Sigma) andNSD-1015 hydrochloride were dissolved in distilled water,adjusted to pH 6.5 with 1 N NaOH and delivered IP; controlsreceived saline (VEHD). For local administration, DMI (20 μM)was administered in perfusate (Bongiovanni et al., 2005). Alldoses refer to the free base.

4.4. Statistics

For the study of tyrosine levels (Fig. 2), DOPA levels (Figs. 3A,B, C) and DA levels with DMI administered IP (Figs. 4A, B), theaverage of the three consecutive 30-min dialysis samplespreceding treatment was considered to be the baseline. In thecase of DMI administered in perfusate, rats were randomlyassigned to receive eitherVEHor LNAA(−) after collectionof thefirst dialysate sample (Figs. 5A, B). Statistical analyses wereconducted on the percent change from baseline values. Two-way analysis of variance (ANOVA) with time as the repeatedfactor was used to analyze treatment and time effects. If theoverall ANOVAwas significant, it was followed by Bonferroni'spost-hoc t-tests. Data are expressed as mean±SEM.

Acknowledgments

This material is based on work supported by the Office ofResearch and Development Medical Research Service, Depart-ment of Veterans Affairs.

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