*Division of Pharmacology and Toxicology, Faculty of Pharmacy, University of Helsinki, Finland

�Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland

�In Vivo Voltammetry Contract Research Laboratory, Department of Pharmacology and Toxicology, University of Kuopio, Finland

The Ret gene was originally discovered as an oncogene andsubsequently was shown that it encodes a transmembranereceptor tyrosine kinase (RTK) (Takahashi and Cooper1987). Germline mutations in the Ret proto-oncogene areresponsible for dominantly inherited cancer syndrome calledmultiple endocrine neoplasia 2 (MEN2) characterized bymedullary thyroid carcinoma, pheochromocytoma and hyper-parathyroidism. In MEN2A, missense mutations of Ret geneaffect the extracellular domain of the receptor and lead to itsactivation by covalent Ret dimerization, while MEN2Bsubtype is associated primarily with a single activating,missense mutation of codon 918 (Met918Thr) affecting thecatalytic tyrosine kinase domain (Eng 1999; Takahashi2001). Mouse model for MEN2B was generated byintroducing a single point mutation Met918Thr into themouse endogenous Ret gene which leads to constitutiveactivation of Ret receptor (Smith-Hicks et al. 2000). Both

heterozygous (M/+) and homozygous (M/M) mice displaymajor features of human MEN2B syndrome.

Ret receptor tyrosine kinase is a common signalingreceptor for glial cell line-derived neurotrophic factor

Received December 5, 2007; revised manuscript received January 20,2008; accepted January 23, 2008.Address correspondence and reprint requests to Jelena Mijatovic,

Division of Pharmacology and Toxicology, Faculty of Pharmacy, POBox 56, FIN 00014, University of Helsinki, Finland.E-mail: jelena.mijatovic@helsinki.fiAbbreviations used: CPA, constant potential amperometry; DA,

dopamine; DAT, dopamine transporter; DOPA, 3,4-dyhydroxylphenyl-alanine; DOPAC, dihydroxyphenylacetic acid; GDNF, glial cell line-derived neurotrophic factor; HVA, homovanilic acid; M/+, heterozygousMEN2B mice; M/M, homozygous MEN2B mice; MAO, monoamineoxidase; MEN2B, multiple endocrine neoplasia type 2B; MFB, medialforebrain bundle; RTK, receptor tyrosine kinase; TH, tyrosine hydr-oxylase; VMAT2, vesicular monoamine transporter 2; Wt, wild-type.

Abstract

The Ret receptor tyrosine kinase is the common signaling

receptor for the glial cell line-derived neurotrophic factor

(GDNF) family ligands. The Met918Thr mutation leads to

constitutive activation of Ret and is responsible for dominantly

inherited cancer syndrome MEN2B. Previously, we found that

the mice carrying the mutation (MEN2B mice) have profoundly

increased tissue dopamine (DA) concentrations in the striatum

as well as increased striatal levels of tyrosine hydroxylase

(TH) and dopamine transporter. The aim of this study was to

characterize the striatal dopaminergic neurotransmission in

MEN2B mice and to clarify the mechanisms by which they

compensate their over-production of DA. We found that tyro-

sine hydroxylase activity and DA synthesis are increased in

MEN2B mice. Augmented effects of a-methyl-para-tyrosine

(aMT, an inhibitor of TH) and tetrabenazine (VMAT2 blocker)

on DA levels suggest that also storage of DA is increased in

MEN2B mice. There was no difference in the basal extracel-

lular DA concentrations or potassium-evoked DA release

between the genotypes. The effects of cocaine and haloper-

idol were also similar between the genotypes as assessed by

in vivo microdialysis. However, with in vivo voltammetry we

found increase in stimulated DA release in MEN2B mice and

detailed analysis of DA overflow showed that uptake of DA

was also enhanced in MEN2B mice. Thus, our data show that

enhanced synthesis of DA leading to increased storage and

releasable pools in pre-synaptic terminals in MEN2B mice

apparently also leads to increased DA release, which in turn is

compensated by higher dopamine transporter activity.

Keywords: brain dopamine, dopamine transporter, glial cell

line-derived neurotrophic factor, in vivo voltammetry, micro-

dialysis, Ret receptor, tyrosine hydroxylase.

J. Neurochem. (2008) 105, 1716–1725.

JOURNAL OF NEUROCHEMISTRY | 2008 | 105 | 1716–1725 doi: 10.1111/j.1471-4159.2008.05265.x

1716 Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1716–1725� 2008 The Authors

(GDNF) and other ligands of GDNF family (Durbec et al.1996; Treanor et al. 1996; Trupp et al. 1996). GDNF haspotent effects on dopaminergic system, such as survival-promotion of midbrain dopamine (DA) neurons in vitro andin vivo in animal models of Parkinson disease (Lin et al.1993; Hoffer et al. 1994; Tomac et al. 1995; Gash et al.1996; Rosenblad et al. 2000), and stimulation of DA neuronfunction (Hudson et al. 1995; Beck et al. 1996; Pothos et al.1998; Salvatore et al. 2004). Thus, the Ret-GDNF signalingrepresents a highly interesting target for novel therapies forParkinson’s disease.

Supposing that constitutive Ret signaling in MEN2Bmice would correspond to continuous activation of Ret byGDNF, we previously studied the dopaminergic system inknock-in MEN2B mouse model (Mijatovic et al. 2007)and found robustly increased tissue concentrations of DAand its metabolites in the striatum, cortex, and hypothal-amus. The concentrations of brain serotonin were notaffected and those of norepinephrine were slightlyincreased only in the lower brain stem. Tyrosine hydrox-ylase (TH) protein levels were also increased in thestriatum and substantia nigra/ventral tegmental area (SN/VTA) and TH mRNA levels were increased in SN/VTA ofMEN2B mice suggesting that constitutive Ret activityincreases DA tissue content by increasing its synthesis.Also, the striatal dopamine transporter protein (DAT) levelswere increased in the MEN2B mice, which agree withhigher sensitivity of these mice to the stimulatory effectsof cocaine we found. All this makes MEN2B mice uniquesince to our knowledge no other treatment or altered genefunctioning has led to selective and long-term elevation ofthe tissue DA in this magnitude (Huotari et al. 2002; Eells2003; Chen et al. 2004; Popova et al. 2004). Also, abetter understanding of regulatory mechanisms of braindopaminergic neurotransmission is of a considerableinterest as its disruption is underlying several humanconditions.

MEN2B mice are one of the most interesting tools forstudying how the brain dopaminergic neurons handleexcessive amounts of DA. We hypothesized that complexchanges in synthesis, storage, release, and re-uptake of DAoccur in dopaminergic neurons of MEN2B mice. Therefore,in the present work, we examined the function of TH, DAT,vesicular monoamine transporter (VMAT2) and D2-dopa-mine autoreceptors in MEN2B mice using classical pharma-cological tools. For that purpose, we measured and comparedtissue concentrations of DA and its metabolites using HPLCas well as their extracellular concentrations by in vivomicrodialysis after treatments with drugs that block TH,DAT, VMAT2, and D2 receptors between MEN2B andwild-type mice. We also used in vivo voltammetry that allowsreal-time measurements of neurotransmitter release and re-uptake and reveals possible differences in the DA neuro-transmission between the genotypes.

Materials and methods

AnimalsThe generation and genotyping of MEN2B knock-in mice is

described elsewhere (Smith-Hicks et al. 2000; Mijatovic et al.2007). Mice were bred locally in the Laboratory Animal Center,

University of Helsinki on C57BL/6 X 129Sv hybrid background.

Male mice were used at 8–16 weeks of age. The chief veterinarian

of the county administrative board approved the experimental set-

up. The experiments were conducted according to the ‘European

Convention for the Protection of Vertebrate Animals used for

Experimental and other Scientific purposes’. The mice were housed

in groups of 2–8 to a cage and had free access to mouse chow and

water. They were maintained under 12 : 12 h light/dark cycle

with lights on from 6 AM to 6 PM at an ambient temperature of

20–22�C.

Brain dissectionThe mice were killed by decapitation and their brains were rapidly

removed from the skull and placed on an ice-cooled brain matrix

(Stoelting, Wood Dale, IL, USA). Two coronal cuts were made by

razor blades at about 1.5 and )0.3 mm from the bregma according

to mouse brain atlas of Franklin and Paxinos (1997). From the

obtained section the dorsal striatum was punched below the corpus

callosum by using a sample corer (inner diameter of 2 mm).

Dissected tissue pieces were immediately placed into frozen

microcentrifuge tubes and after weighing they were stored at

)80�C until assayed.

Estimation of monoamines and their metabolitesConcentrations of DA, DOPA, L-dihydroxyphenylacetic acid

(DOPAC) and homovanillic acid (HVA), from brain samples were

analyzed using high-performance liquid chromatography with

electrochemical detection as described by (Airavaara et al. 2006).The values of monoamines and their metabolites are presented as

nanograms per gram (ng/g) wet weight of tissue.

In vivo microdialysis

SurgeryThe mice were under general isoflurane (3.5% induction, 2%

maintenance) and local lidocaine anesthesia as microdialysis guide

cannulae (AgnTho’s, Lidingo, Sweden) were implanted using the

stereotaxic device (Stoelting, Wood Dale, IL, USA). Mice were

given buprenorphine (0.1 mg/kg s.c.) to relieve pain. The coordi-

nates for guide cannulae were calculated relative from bregma, and

guide cannulae were aimed in the striatum above the point (A/

P = + 0.6, L/M = +1.8 and D/V = )2.3) according to mouse brain

atlas (Franklin and Paxinos 1997). The cannula was fastened to the

skull with dental cement (Aqualox; Voco, Cuxhaven, Germany) and

two stainless steel screws. After surgery, mice were placed into

individual test cages and allowed to recover in the cages for at least

7 days before the experiment.

ExperimentAt about 4 PM on the day before experiment, a microdialysis probe

(AgnTho’s, Lidingo, Sweden, 1 mm membrane, outer diameter

0.24 mm) was inserted into the guide cannula, and the probe was

infused with Ringer solution (147 mM NaCl, 1.2 mM CaCl2,

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1716–1725

Striatal DAergic transmission in MEN2B mice | 1717

2.7 mM KCl, 1.0 mM MgCl2, and 0.04 mM ascorbic acid) at the

flow rate of 0.5 lL/min. In the morning of the experimental day

(about 8 AM), the flow rate was increased to 2 lL/min, and after 2 h

of stabilization period, the collection of actual microdialysis samples

(every 20 min, 40 lL/sample) was started. The concentrations of

DA, DOPAC, and HVA were determined by HPLC with an

electrochemical detector (Coulochem II; ESA, Inc., Chelmsford,

MA, USA) equipped with a model 5014B microdialysis cell, a

HPLC pump (Jasco PU2080, Tokyo, Japan) and a pulse damper

(SSI LP-21; Scientific Systems, State College, PA, USA). The

column (Spherisorb ODS2, 3 lm, 4.6 · 100 mm; Waters, Milford,

MA, USA) was kept at 40�C, with a column heater (Croco-Cil;

Bordeaux, France). The mobile phase consisted of 0.1 M NaH2PO4

buffer, pH 4.0, 0.8–1.2 mM octanesulphonic acid, 10% methanol

and 1.0 mM EDTA. The flow rate of the mobile phase was 1.0 mL/

min. Twenty microliters of the dialysate sample was injected into the

column with a CMA/200 autoinjector (CMA, Stokholm, Sweden).

Dopamine was reduced with an amperometric detector (potential

)120 mV) after being oxidized with a coulometric detector

(+300 mV); DOPAC and HVA were oxidized with the coulometric

detector. The chromatograms were processed and integrated with

Azur version 4.0 Chromatography Software and Data Acquisition

System (version 4.0, Datalys, Theix, France). Basal extracellular

concentrations of neurotransmitters (not corrected with in vitro-recovery) were determined as mean of concentrations of four stable

pre-drug samples collected during the first 80 min of sampling

(variation < 20%). Post-drug neurotransmitter levels were converted

to a percentage of this baseline value, which was defined as 100%.

After completion of the experiments, the positions of the probes

were verified histologically by fixing coronal brain sections on

gelatin/chrome-coated slides and checking the respective placements

of the implanted probes in the striatum. Potassium stimulation was

performed by manually switching the perfusion medium, Ringer, to

the one containing 100 mM KCl and 27.5 mM NaCl (remaining

composition identical to that described above).

In vivo voltammetry

Preparation of animalsWild-type and homozygous MEN2B mice were anaesthetized with

chloral hydrate (450 mg/kg, i.p.) and fixed to a stereotaxic frame.

Additional injections of anesthetics were made at 45–60 min

intervals. Rectal temperature was kept at 37�C with a heating lamp.

The carbon fiber working electrode was inserted through an opening

in the skull to the caudate nucleus (AP: 1.18 mm, L: 1.5 mm, H:

3.4 mm vs. bregma) and a bipolar stimulating electrode was

implanted in the medial forebrain bundle (AP: )2.1 mm, L:

1.1 mm, H: 5.0–5.2 mm) according to mouse brain atlas (Paxinos

and Franklin 2000). The exact placement of the stimulating

electrode in the dorsoventral coordinate was adjusted for maximal

dopamine release. A small leak-free Ag/AgCl reference electrode

(AH 69-0023, Harvard Apparatus, Holliston, MA, USA) in a saline

bridge was placed on the skull. A stainless steel screw was fixed

onto the skull as the auxiliary electrode.

Electrochemical techniqueStimulated release of dopamine was measured using constant

potential amperometry (CPA). A single Nafion-coated carbon fiber,

30 lm in diameter (WPI, Sarasota, FL, USA) was protruded in

pulled capillary glass and insulated with epoxy glue. The exposed

tip of the fiber was 300 lm. A custom-built three-electrode

potentiostat held the working electrode at 0.4 V against an Ag/

AgCl reference electrode. The current at the working electrode was

converted to voltage at a headstage converter located near the

working electrode. The data were digitized at 10 KHz and recorded

with a personal computer for further off-line analysis. The

electrochemical signals in response to stimulations were stabilized

in about 90 min after implantation of the working electrodes. After

the experiments, the working electrodes were rinsed with deionized

water and calibrated for dopamine in phosphate-buffered saline

(pH = 7.4).

Electrical stimulation and experimental protocolA battery-operated constant current unit (A365, WPI) run by a

personal computer was used for the stimulation. Constant current

pulses (1 ms in duration) of 180–200 lA were delivered to the

stimulation electrode at 10–60 Hz in 2 s bursts. The intervals

between the bursts were 1–6 min. Longer intervals corresponded

with higher stimulation frequencies.

Another stimulation protocol was used to obtain peaks of DA

overflow of different amplitudes, from which peaks of similar

amplitudes can be directly compared. We used 50 Hz stimulations

applied in the bursts of 0.2, 0.4, 0.8, and 1.6 s lengths at 1–4 min

intervals.

The right slope of the dopamine overflow curve reflects

dynamics of dopamine elimination from the extracellular space

(Wightman et al., 1988). This curve can be approximated with a

good fit by a standard exponential function f(t) = Ae)t/s + C.

Curve fitting was made with Clampfit v.8 (Axon Instruments/

Molecular Devices Corporation, Sunnyvale, CA, USA) with peaks

of similar amplitudes. We analysed and depicted dopamine decay

from the extracellular space using parameter tau (s) which is time

constant of dopamine decay curve. The effect of cocaine was

analyzed 25–45 min after the administration at different stimu-

lation frequencies (10–60 Hz, 2 s burst length) and lengths

(0.2–1.6 s, at 50 Hz).

Drug treatmentsA blocker of L-amino acid decarboxylase (AADC; EC 4.1.1.28)

NSD-1015 (BioChemika, Sigma, China) was dissolved in saline

(0.9% NaCl solution) and given to mice i.p.; 150 mg/kg. Half an

hour after NSD-1015 treatment the mice were decapitated, and

striata dissected out for estimation of L-dopa levels. Alpha-

methyl-paratyrosine (a-MT; Labkemi AB, Stockholm, Sweden), a

blocker of tyrosine hydroxylase, was dissolved in saline and given

i.p.; 250 mg/kg. The mice were decapitated 100 min after the

injection. A VMAT2 blocker, tetrabenazine (Tocris, Avonmouth,

UK) was first dissolved in small amount of acidic saline,

after which the pH was adjusted to four with NaOH, rest

of the saline was added and the mice were injected i.p.;

5 mg/kg. The mice were decapitated 60 min after the drug

administration. Cocaine HCl (University Pharmacy, Helsinki,

Finland, 10 or 20 mg/kg) and haloperidol (Janssen Pharmaceutica

N.V., Beerse, Belgium, 0.5 mg/kg) were given i.p. The

drugs were administered in a volume of 0.1 mL/10 g of body

weight.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1716–1725� 2008 The Authors

1718 | J. Mijatovic et al.

Data presentation and statistical analysisData obtained from monoamine tissue measurements were analy-

zed with one-way or two-way ANOVA, followed by Tukey/Kramer

post hoc comparisons. One-way ANOVA and Tukey/Kramer post hoctest were used to analyze statistically significant differences in basal

extracellular concentrations of DA, DOPAC, and HVA between

genotypes. One-way ANOVA for repeated measures was used to

analyze the drug effects on output of extracellular levels of DA and

DOPAC in MEN2B mice and their wild-type littermates.

In voltammetry experiments DA overflow was expressed in

molar concentration (lM/L) on the basis of post-calibration data.

The effects of cocaine were calculated as the peak dopamine

response before and after the treatment. The effects of cocaine and

peak dopamine release after stimulations at increasing frequencies

were statistically analyzed with multivariate analysis of variance for

repeated measures (MANOVA, Pillai’s Trace was calculated with SPSS

statistical package, SPSS Inc., Chicago, IL, USA). In this analysis,

the effects of cocaine (treatment, two levels) and the stimulation

frequencies (frequency, six levels) were used as the within-subject

factors and genotype (two levels) as the between-subject factors.

Time constants of uptake in two genotypes (between-subject factors,

two levels) and after cocaine (within-subjects factors, two levels)

were compared using MANOVA as above and t-test was used for

comparison of individual pairs. Data are presented as mean ± SEM.

Results

Synthesis and storage of dopamine are increased in MEN2BmiceOur previous data (Mijatovic et al. 2007) suggested thatincreased synthesis of DA might be behind elevated tissuelevels of DAwe found in MEN2B mice. Quantification of L-dopa accumulation after inhibition of AADC (EC 4.1.1.28)by NSD-1015 provides an index of TH activity (Carlsson andLindqvist 1973). Thus, the mice were injected with NSD-1015 150 mg/kg i.p. 30 min prior killing, neurochemicalmeasurement of L-dopa levels were performed and we found41% and 68% increase in striatal L-dopa levels in heterozy-gous (M/+) and homozygous (M/M) mice, respectively, ascompared with their wild-type (Wt) littermates (genotypeeffect F2, 26 = 20.552, p < 0.001; Fig. 1). This shows thatTH activity and DA synthesis are increased in MEN2B mice.

As shown in Fig. 2, DA depletion induced by an inhibitorof TH, a-methyl-para-tyrosine (aMT) was significantlyaugmented in heterozygous and homozygous MEN2B mice(33% and 40%, respectively) as compared with the wild-typemice (27%; genotype · treatment interaction F2,.32 = 3.594,p = 0.0391). aMT depletes so-called releasable pool ofintracellular DA that is dependent on synthesis rate of DA(Moore and Dominic 1971; Yavich 1996). Therefore,especially the levels of newly synthesized DA are decliningmore in MEN2B mice than in the Wt mice, which agreeswith enhanced activity of their TH and its increasedsensitivity to the blockade by aMT. However, we cannotexclude the possibility that also increased DA release and its

subsequent metabolism at least to some extent account forthe increased aMT-induced depletion of DA after synthesisand supply of new DA are blocked.

As DA synthesis and tissue DA levels are increased inMEN2B mice, this suggests that storage of DA is increased.To examine this, we used tetrabenazine (5 mg/kg i.p., 1 hprior decapitation) which blocks VMAT2 and leads todepletion of tissue DA content. Tetrabenazine redistributesDA from vesicles to cytoplasm rendering it vulnerable tometabolism by monoamine oxidase (MAO) (Colzi et al.1993; Fumagalli et al. 1999) which subsequently leads toincreased production of DOPAC. We found that tetrabenzine-induced depletion of DA was significantly higher inheterozygous and homozygous MEN2B (75% and 81%,respectively) as compared with Wt mice (69%; geno-type · treatment interaction F2, 22 = 14.311, p = 0.0001;Fig. 3a). Accordingly, levels of DOPAC were increased

Fig. 1 Accumulation of L-DOPA is augmented in heterozygous (M/+)

and homozygous (M/M) MEN2B mice as compared with wild-type (Wt)

mice 30 min after NSD-1015 administration (150 mg/kg, i.p.). The

columns give the concentrations (ng/g) of L-DOPA (means ± SEM).

Statistical results were obtained with one-way ANOVA and Tukey/

Kramer post hoc analysis. **p < 0.01 when compared with wild-type

mice; n = 7–15/genotype.

Fig. 2 Depletion of dopamine (DA) induced by alpha-methyl-para-

tyrosine (aMT; 250 mg/kg, i.p., 100 min after administration) was

increased in heterozygous M/+ and homozygous (M/M) MEN2B mice

as compared with wild-type (Wt) mice. The columns give the

concentrations (ng/g) of DA in the dorsal striatum (mean ± SEM).

Statistical results were obtained with two-way ANOVA. n = 4–8/group.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1716–1725

Striatal DAergic transmission in MEN2B mice | 1719

significantly more in the heterozygous and homozygousMEN2B mice (341% and 373%, respectively) as comparedwith Wt mice (279%; genotype · treatment interactionF2, 23 = 3.901, p = 0.0348; Fig. 3b). Thus, also VMAT2activity appeared to be increased in MEN2B mice suggestingthat dopaminergic neurons in MEN2B mice are capable ofstoring the larger amount of DA that they produce.

Extracellular levels of DA and its metabolitesTo study whether also the extracellular levels of DA areincreased in MEN2B mice we measured DA concentrationsin the dorsal striatum by microdialysis. Unlike tissue DA, wefound no differences in the basal extracellular concentrationsof DA between the genotypes (genotype effect F2, 43 = 0.02,p = 0.98; Fig. 4).

On the other hand, basal extracellular levels of the DAmetabolite, DOPAC were significantly higher in bothheterozygous and homozygous MEN2B mice (58% and71%, respectively) as compared with those of their Wtlittermates (genotype effect F2, 43 = 6.334, p = 0.039;Fig. 4). However, levels of HVA in both MEN2B genotypes

were elevated only by about 30% and not significantly(genotype effect F2, 43 = 1.702, p = 0.1944; Fig. 4).

Also, we examined the release of DA in the striatum afterhigh K+ stimulus. High K+ stimulation via the microdialysisprobe (see Materials and methods) results in widespread and

Fig. 3 The effects of tetrabenazine (TBN; 150 mg/kg, i.p., 60 min

after administration) on the concentrations (ng/g) of dopamine (DA, a)

and 3,4-dyhydroxylphenylacetic acid (DOPAC, b) in the dorsal stria-

tum of the wild-type (Wt), heterozygous (M/+) and homozygous (M/M)

MEN2B mice. Tetrabenazine induced-depletion of DA and tetraben-

azine-induced accumulation of DOPAC were augmented in knock-in

MEN2B mice as compared with Wt mice. The columns give means ±

SEM. Statistics were obtained with two-way ANOVA. n = 3–7/group.

Fig. 4 The basal extracellular concentrations (not corrected by in vitro

recovery) of dopamine (DA) and its metabolites 3,4-dyhydroxylphe-

nylacetic acid (DOPAC) and HVA in the striatal dialysates of the wild-

type (Wt), heterozygous (M/+) and homozygous (M/M) MEN2B mice.

Four consecutive 40 lL dialysate samples were collected at 20 min

intervals from each animal. The concentrations as nM were averaged.

The columns give means ± SEM from data obtained from 13–18 mice/

group. Statistical results were obtained with two-way ANOVA followed

by Tukey/Kramer post hoc test; **p < 0.01 when compared with Wt

mice.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1716–1725� 2008 The Authors

1720 | J. Mijatovic et al.

clamped depolarizations that induce massive neurotransmit-ter release. We found no difference in the K+-evoked DArelease between the genotypes as potassium increased outputof DA similarly in all three genotypes, by about 20-fold(genotype effect F2, 25 = 0.843, p = 0.4421; Fig. 5b).

Previously, we found increased striatal DAT levels inMEN2B mice and enhanced behavioral response to cocaineas compared with Wt mice (Mijatovic et al. 2007). Thissuggested that DAT function is enhanced and might be acompensatory mechanism that maintains the extracellularDA levels within the normal range. Therefore, we studied theeffects of cocaine (10 and 20 mg/kg) on extracellular DAlevels in MEN2B mice. Unexpectedly, we found no differ-ences in the extracellular DA concentrations in response toeither doses of cocaine (genotype effects: F2, 21 = 0.390,p = 0.6821 and F2, 24 = 0.012, p = 0.9884, for cocaine 10and 20 mg/kg, respectively; Fig. 5a and b).

D2-type DA receptors control the neuronal activity ofDAergic neurons as well as synthesis and release of DA. Westudied whether there are differences in the response to theblockade of D2 receptors by haloperidol. Haloperidol(0.5 mg/kg, i.p.) similarly increased the extracellular levels

of DA in all genotypes (genotype effect F2, 24 = 0.708,p = 0.5027; Fig. 6a) suggesting no difference in the func-tioning or sensitivity of D2 type autoreceptors controlling therelease of DA. Nevertheless, haloperidol increased extracel-lular concentrations of DOPAC significantly more in homo-zygous MEN2B mice (genotype effect F2, 19 = 5.600,p = 0.0122; p < 0.01 Tukey/Kramer post hoc; Fig. 6b) thanin heterozygous and wild-type mice in which the increasewas the same. As DOPAC is mostly an index of DAsynthesis this suggests that D2 autoreceptors controlling thesynthesis of DA might be functioning more actively inMEN2B mice and thus being more sensitive to the blockadeby an antagonist.

In vivo voltammetryStimulation of the MFB at increasing frequencies producednonlinear increase in the peak DA overflow (Fig. 7a and b).This increase described initially as a result of elevated release(Gonon, 1988) has been later explained on the basis ofMichaelis-Menten kinetics as a dominance of the release overthe re-uptake at high (more than 30 Hz) frequencies ofstimulation (Kawagoe et al., 1992). Knock-in MEN2B miceshowed much larger increase in DA overflow in compa-rison with wild-type control mice (frequency · genotype,

Fig. 5 Effects of cocaine (a and b) and high potassium stimulation (b,

Ringer solution containing 100 mM KCl perfused for 40 min) on the

extracellular concentrations of DA did not differ between the wild-type

(Wt), heterozygous (M/+) and homozygous (M/M) MEN2B mice. Co-

caine (10 or 20 mg/kg, i.p) was given or potassium perfusion was

started at times indicated by arrows. Values are given as percentages

of the baseline (± SEM). n = 6–12 mice/group. Note that the scales in

the figures differ.

Fig. 6 Effects of haloperidol (HAL, 0.5 mg/kg, i.p.) on the extracellular

concentrations of DA (a) and DOPAC (b) of the wild-type (Wt), het-

erozygous (M/+) and homozygous (M/M) MEN2B mice. The arrows

indicate the time of the drug injection. Data are presented as mean

percentage change values (n = 7–10 mice/group) of the baseline

values (= 100%) and analyzed with repeated measures ANOVA.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1716–1725

Striatal DAergic transmission in MEN2B mice | 1721

F(5) = 5.4, p < 0.05). This is a sign of the larger DA releaseper stimulation pulse or/and lower re-uptake of DA inMEN2B mice. However, the levels of DAT in MEN2B micewere increased (Mijatovic et al. 2007), which indicates thatmost probably they have elevated DA release.

Cocaine increased peak DA overflow in a frequency-depended manner (treatment · frequency, F(5) = 6,6, p <0.05, MANOVA), which can be entirely explained in terms of

release and re-uptake, but the effects of cocaine did not differbetween genotypes (treatment · genotype, F(1) = 0.2,p = 0.69, MANOVA). However, the lack of the overallstatistical significance arises as a result of the frequency-specific effect of cocaine. The drug produced the largestincrease in DA concentrations at 20 and 30 Hz stimulations,but had practically no effects on the peak DA overflow at thehigher frequencies of stimulations. We calculated thepercentage of increase of DA overflow after cocaine at eachstimulation frequency in the two genotypes (Fig. 7c) andfound, testing within-subject contrasts, that at 30 Hz stim-ulation cocaine was significantly more effective in increasingpeak extracellular DA concentrations in MEN2B mice thanin the control, wild-type mice (frequency · genotype,F(1) = 6.47, p = 0.029). The effect was even larger at20 Hz stimulation, but because of the high variations in theeffect of cocaine in MEN2B mice it did not reach statisticalsignificance.

Comparisons of the rate of DA elimination from theextracellular space after the end of stimulation (right slope ofthe DA overflow curve (Fig. 8a) allows testing the hypoth-esis that DA release is increased in MEN2B mice which is innon-stimulated conditions compensated by the higher re-uptake. Figure 8(a) shows examples of DA overflow in awild-type and knock-in mouse in response to 0.8 and 0.2 sstimulation before (small peaks) and after (larger peaks)cocaine treatment. Different lengths of stimulations werechosen to obtain peaks of similar amplitudes, which permitdirect comparisons of time constants of DA uptake. Thedifferences between the peak amplitudes and time constantsof DA decay after cocaine are apparent in both genotypes(see statistics in the legend). It is also important to note thatDA decay from the extracellular space was three times fasterin MEN2B mice without any treatment (Fig. 8b). The effectof cocaine on DA elimination was significantly larger inMEN2B mice (Fig. 8c). These results nicely correspond withour previous study (Mijatovic et al. 2007), where wereported more DAT protein in striatum in MEN2B micethan in the wild-types and higher sensitivity of MEN2B miceto cocaine.

Discussion

Previously, we reported that MEN2B mice with constitu-tively active Ret receptor tyrosine kinase have robustincreases in DA tissue content; highest increase (about100%) was found in the dorsal striatum. Also, we foundincreased TH levels in the striatum and SN/VTA, increasedstriatal DAT levels and enhanced locomotion after cocaine inMEN2B mice (Mijatovic et al. 2007). As dopaminergicsystem is known to be incredibly flexible in regulation of itsneurotransmission in this study we examined synthesis,storage, uptake and release of DA and their regulation inMEN2B mice as compared with their wild-type controls. We

Fig. 7 The effects of cocaine (10 mg/kg) on peak dopamine overflow

at different frequency of stimulation of the MFB in MEN2B transgenic

(M/M) and wild-type (Wt) mice. Wild-type (a) and MEN2B mice (b)

mice were stimulated with bursts of increasing frequencies (10–60 Hz,

2 s in length) before and 25–45 min after cocaine treatment. Each

point on the figure represents mean ± SEM of the peak dopamine

overflow (lM) obtained in six mice/genotype. (c) Percentage of in-

crease of peak dopamine overflow after cocaine treatment for both

genotypes was compared for each frequency of stimulation. *p < 0.05

for the effects of cocaine between genotypes at 30 Hz stimulation.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1716–1725� 2008 The Authors

1722 | J. Mijatovic et al.

found that in spite of increased synthesis, storage and tissuelevels of DA in the MEN2B mice there was no difference inthe basal extracellular concentration of DA. Our data suggestthat higher re-uptake of DA could be the compensatory factorfor the increased DA release in MEN2B mice keeping theextracellular concentration of DA similar to those of thewild-type mice, while the D2 type autoreceptors controllingthe release appeared to function normally in the MEN2Bmice.

We previously reported that striatal tissue DA content,DOPAC/DA ratio, an indicator of DA synthesis as well asTH levels are increased in MEN2B mice which suggest thatconstitutively active Ret leads to increased DA tissue levelsvia enhanced synthesis of DA. However, in the present work,we wanted to directly verify this assumption by using aninhibitor of AADC, NSD-1015, and found increased accu-mulation of L-dopa in the MEN2B mice showing that theactivity of TH is indeed increased in MEN2B mice. This issupported by our finding that the effect of an inhibitor of TH,aMTwas higher in MEN2B mice as compared with their Wt.The augmented a-MT response also suggests that releasablepool of DA, which is known to be aMT –sensitive, is largerin the MEN2B mice (Moore and Dominic 1971; Yavich1996). On the other hand, since the pre-synaptic storage pool(reserpine/tetrabenazine-sensitive) accounts for most of thetissue neurotransmitter (Rizzoli and Betz 2005) we supposedthat MEN2B mice would have larger storage pool toaccommodate the larger amount of DA they synthesize. Wedid find greater response to tetrabenazine (a VMAT2 blocker)in MEN2B mice than in the Wt mice showing that indeedMEN2B mice have larger storage pool of DA.

It has been shown that increased DA synthesis andVMAT2 activity increase DA quantal size of release [definedas the number of neurotransmitter molecules released by asingle synaptic vesicle during exocytosis (Pothos 2002)].Therefore, we examined whether increases in the synthesis,storage and tissue DA levels would translate to the increasein the extracellular levels of DA in MEN2B mice bymeasuring the extracellular concentrations of DA in striatalmicrodialysates. We found no differences in basal concen-tration of extracellular DA, neither we found difference in theK+-evoked DA release between the genotypes. The extra-cellular DA levels measured by in vivo microdialysis reflect abalance between the neurotransmitter release and reuptake.As the striatal DAT levels and the behavioral response tococaine are increased in MEN2B mice (Mijatovic et al.2007), we supposed that enhanced activity of DAT isunderlying the seemingly unchanged DA release in MEN2Bmice. Thus, to test this, we studied the cocaine-inducedchanges in the extracellular DA concentrations in thestriatum by microdialysis. Unexpectedly, there was noincrease in the response to cocaine in MEN2B mice ascocaine increased striatal extracellular DA similarly in allthree genotypes.

Because microdialysis did not reveal alterations in theDAT function, we used in vivo voltammetry to study evokedDA release at different frequencies of stimulations ofascending pathways and the effects of cocaine on thestimulated DA release. MEN2B mice showed much largerincrease in stimulated DA overflow in comparison with Wtmice (Fig. 7). At the same time elimination of DA fromextracellular space was significantly faster in these mice,which is in line with our previous finding that striatal DAT

Fig. 8 Analysis of re-uptake of dopamine in MEN2B mice and the

effects of cocaine on dopamine re-uptake. (a) Examples of recordings

of evoked dopamine overflow in the caudate nucleus in wild-type (left

panel) and MEN2B mice (right panel) before (small peaks) and after

(larger peaks) treatment with cocaine (10 mg/kg). Time of stimulation

of the MFB producing similar peak amplitudes is marked with hori-

zontal bars under the curves in (0.8 and 0.2 s in this example in wild-

type and M/M mice, respectively). Re-uptake was analyzed for peaks

of similar amplitudes using parameter s, which is time constant (in

milliseconds) of dopamine decay. The smaller is the time constant the

more effective is re-uptake. (b) Summary of the re-uptake data as

mean ± SEM (six mice/genotype). Note that MEN2B mice (M/M) had

significantly smaller time constant of re-uptake before treatment than

wild-type control (Wt). The effects of cocaine on time constant were

also significant in both genotypes (the effects of treatment,

F(1) = 54.20, p = 0.0001), but we did not find statistically significant

genotype · treatment interactions. (c) When the effects of cocaine

were expressed in percentage to pre-drug levels for each individual

animal it was twice larger in MEN2B mice then in wild-type mice.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1716–1725

Striatal DAergic transmission in MEN2B mice | 1723

levels are increased in MEN2B mice (Mijatovic et al. 2007).Thus, the data suggest that in MEN2B mice release of DAper pulse of stimulation is increased that is counteracted byenhanced re-uptake of DA. Therefore, the elevated re-uptakeof DA is most probably a compensatory mechanismbalancing net DA overflow in MEN2B mice, which mayexplain the absence of differences in DA levels betweengenotypes in microdialysis experiments. On the other hand,more effective re-uptake of DA in MEN2B mice makes themmore vulnerable to cocaine as its effects on DA clearancewere enhanced in the MEN2B mice. Interestingly, there wasno statistical difference in the overall effects of cocainebetween the genotypes when analysis was made across allstimulation frequencies, and significant difference was foundonly within one distinct frequency of stimulation (30 Hz).Thus, we speculate that differences in cocaine’s effectsbetween MEN2B and Wt mice can only be seen atfrequencies of stimulations comparable with frequencies ofneuronal bursting (for a review see Grace et al. 2007). Thiswould provide basis for behavioral differences in the effectsof cocaine that we have previously found between genotypes.Moreover, the frequency/firing mode specificity of the effectof cocaine might explain why we did not see genotype-related differences in the effect of cocaine on extracellularDA by microdialysis, as DA in microdialysate is supposed tomostly reflect the tonic, low frequency activity of DAneurons (Wightman and Robinson 2002).

Besides by DAT, the extracellular DA levels are tightlyregulated by D2-type DA autoreceptors. The D2 DA autore-ceptors are involved in the control of the synthesis and releaseof DA as well as the regulation of the neuronal activity ofDAergic neurons. With well known D2 receptor antagonist,haloperidol we tested whether there are differences betweenthe genotypes in the activity of D2 receptors by usingmicrodialysis. We did not find any difference in the effects ofhaloperidol on the extracellular DA concentrations betweenthe genotypes. However, the extracellular concentrations ofDOPAC, which mostly indicate DA synthesis rate, wereincreased significantly more in the homozygousMEN2Bmiceas compared with the Wt mice. This suggests that function ofD2 receptors controlling of synthesis of DA is enhanced inMEN2B mice whereas function of D2 autoreceptors control-ling the release/neuronal activity of DA is not altered.

Well in line with increased DA production, also extracel-lular concentration of mainly intracellularly produced DAmetabolite, DOPAC (Roffler-Tarlov et al. 1971) was in-creased in MEN2B mice as compared with Wt mice.However, the levels of HVA were not significantly increasedin MEN2B mice. As HVA is formed only outside of DAneurons (Wood and Altar 1988), modest but not significantincrease in extracellular HVA levels in MEN2B mice couldbe because of prior conversion of elevated extracellularDOPAC to HVA. Also, this correlates well with negligibledifferences we found in extracellular DA levels.

In conclusion, our data shows that constitutive Ret activityin DA system in MEN2B mice leads to increased synthesisand storage of DA in MEN2B mice. Although there was nodifference in the extracellular concentration of DA our datasuggest that release of DA is enhanced which is compensatedby increased DAT function.

Acknowledgements

This work was supported by Paivikki and Sakari Sohlberg

Foundation and Finnish Parkinson Foundation. We thank Paolo

Alberton for help and Ritva Ala-Kulju and Marjo Vaha for excellent

technical assistance.

References

Airavaara M., Mijatovic J., Vihavainen T., Piepponen T. P., Saarma M.and Ahtee L. (2006) In heterozygous GDNF knockout mice theresponse of striatal dopaminergic system to acute morphine isaltered. Synapse 59, 321–329.

Beck K. D., Irwin I., Valverde J., Brennan T. J., Langston J. W. and HeftiF. (1996) GDNF induces a dystonia-like state in neonatal rats andstimulates dopamine and serotonin synthesis. Neuron 16, 665–673.

Carlsson A. and Lindqvist M. (1973) In-vivo measurements of trypto-phan and tyrosine hydroxylase activities in mouse brain. J NeuralTransm. 34, 79–91.

Chen K., Holschneider D. P., Wu W., Rebrin I. and Shih J. C. (2004) Aspontaneous point mutation produces monoamine oxidase A/Bknock-out mice with greatly elevated monoamines and anxiety-likebehavior. J. Biol. Chem. 279, 39645–39652.

Colzi A., D’Agostini F., Cesura A. M., Borroni E. and Da Prada M.(1993) Monoamine oxidase-A inhibitors and dopamine metabolismin rat caudatus: evidence that an increased cytosolic level ofdopamine displaces reversible monoamine oxidase-A inhibitors invivo. J. Pharmacol. Exp. Ther. 265, 103–111.

Durbec P., Marcos-Gutierrez C. V., Kilkenny C. et al. (1996) GDNFsignalling through the Ret receptor tyrosine kinase. Nature 381,789–793.

Eells J. B. (2003) The control of dopamine neuron development, func-tion and survival: insights from transgenic mice and the relevanceto human disease. Curr. Med. Chem. 10, 857–870.

Eng C.(1999) RET proto-oncogene in the development of human cancer.J. Clin. Oncol. 17, 380–393.

Franklin K. and Paxinos G. (1997) The Mouse Brain in StereotaxicCoordinates. Academic Press INC, San Diego.

Fumagalli F., Gainetdinov R. R., Wang Y. M., Valenzano K. J., MillerG. W. and Caron M. G. (1999) Increased methamphetamine neu-rotoxicity in heterozygous vesicular monoamine transporter 2knock-out mice. J. Neurosci. 19, 2424–2431.

Gash D. M., Zhang Z., Ovadia A. et al. (1996) Functional recovery inparkinsonian monkeys treated with GDNF. Nature 380, 252–255.

Gonon F. G. (1988) Nonlinear relationship between impulse flow anddopamine released by rat midbrain dopaminergic neurons asstudied by in vivo electrochemistry. Neurosci. 24, 19–28.

Grace A. A., Floresco S. B., Goto Y. and Lodge D. J. (2007) Regulationof firing of dopaminergic neurons and control of goal-directedbehaviours. Trends Neurosci. 30, 220–227.

Hoffer B. J., Hoffman A., Bowenkamp K., Huettl P., Hudson J., MartinD., Lin L. F. and Gerhardt G. A. (1994) Glial cell line-derivedneurotrophic factor reverses toxin-induced injury to midbraindopaminergic neurons in vivo. Neurosci. Lett. 182, 107–111.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1716–1725� 2008 The Authors

1724 | J. Mijatovic et al.

Hudson J., Granholm A. C., Gerhardt G. A. et al. (1995) Glial cell line-derived neurotrophic factor augments midbrain dopaminergiccircuits in vivo. Brain Res. Bull. 36, 425–432.

Huotari M., Gogos J. A., Karayiorgou M., Koponen O., Forsberg M.,Raasmaja A., Hyttinen J. and Mannisto P. T. (2002) Brain cate-cholamine metabolism in catechol-O-methyltransferase (COMT)-deficient mice. Eur. J. Neurosci. 15, 246–256.

Kawagoe K. T., Garris P. A., Wiedemann D. J. and Wightmann R. M.(1992) Regulation of transient dopamine concentration gradients inthe microenvironment surrounding nerve terminals in the ratstriatum. Neurosci. 51, 55–64.

Lin L. F., Doherty D. H., Lile J. D., Bektesh S. and Collins F. (1993)GDNF: a glial cell line-derived neurotrophic factor for midbraindopaminergic neurons. Science 260, 1130–1132.

Mijatovic J., Airavaara M., Planken A., Auvinen P., Raasmaja A.,Piepponen T. P., Costantini F., Ahtee L. and Saarma M. (2007)Constitutive Ret activity in knock-in multiple endocrineneoplasia type B mice induces profound elevation of braindopamine concentration via enhanced synthesis and increases thenumber of TH-positive cells in the substantia nigra. J. Neurosci.27, 4799–4809.

Moore K. E. and Dominic J. A. (1971) Tyrosine hydroxylase inhibitors.Fed. Proc. 30, 859–870.

Paxinos G. and Franklin K. (2000) The Mouse Brain in StereotaxicCoordinates, 2nd edn. Academic Press, San Diego.

Popova N. K., Gilinskii M. A. and Amstislavskaya T. G. (2004) Effect ofmonoamine oxidase gene knockout on dopamine metabolism inmouse brain structures. Bull. Exp. Biol. Med. 137, 382–384.

Pothos E. N. (2002) Regulation of dopamine quantal size in midbrainand hippocampal neurons. Behav. Brain Res. 130, 203–207.

Pothos E. N., Davila V. and Sulzer D. (1998) Presynaptic recording ofquanta from midbrain dopamine neurons and modulation of thequantal size. J. Neurosci. 18, 4106–4118.

Rizzoli S. O. and Betz W. J. (2005) Synaptic vesicle pools. Nat. Rev.Neurosci. 6, 57–69.

Roffler-Tarlov S., Sharman D. F. and Tegerdine P. (1971) 3,4-dihydr-oxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetic acidin the mouse striatum: a reflection of intra- and extra-neuronalmetabolism of dopamine? Br. J. Pharmacol. 42, 343–351.

Rosenblad C., Kirik D. and Bjorklund A. (2000) Sequential adminis-tration of GDNF into the substantia nigra and striatum promotesdopamine neuron survival and axonal sprouting but not striatalreinnervation or functional recovery in the partial 6-OHDA lesionmodel. Exp. Neurol. 161, 503–516.

Salvatore M. F., Zhang J. L., Large D. M. et al. (2004) Striatal GDNFadministration increases tyrosine hydroxylase phosphorylation inthe rat striatum and substantia nigra. J. Neurochem. 90, 245–254.

Smith-Hicks C. L., Sizer K. C., Powers J. F., Tischler A. S. andCostantini F. (2000) C-cell hyperplasia, pheochromocytoma andsympathoadrenal malformation in a mouse model of multipleendocrine neoplasia type 2B. EMBO J. 19, 612–622.

Takahashi M. (2001) The GDNF/RET signaling pathway and humandiseases. Cytokine Growth Factor Rev. 12, 361–373.

Takahashi M. and Cooper G. M. (1987) ret transforming gene encodes afusion protein homologous to tyrosine kinases. Mol. Cell. Biol. 7,1378–1385.

Tomac A., Lindqvist E., Lin L. F., Ogren S. O., Young D., Hoffer B. J.and Olson L. (1995) Protection and repair of the nigrostriataldopaminergic system by GDNF in vivo. Nature 373, 335–339.

Treanor J. J., Goodman L., de Sauvage F. et al. (1996) Characterizationof a multicomponent receptor for GDNF. Nature 382, 80–83.

Trupp M., Arenas E., Fainzilber M., Nilsson A. S., Sieber B. A., Gri-goriou M., Kilkenny C., Salazar-Grueso E., Pachnis V. and ArumaeU. (1996) Functional receptor for GDNF encoded by the c-retproto-oncogene. Nature 381, 785–789.

Wightman R. M., Amatore C., Engstrom R. C., Hale P. D., KristensenE. W., Kuhr W. G. and May L. J. (1988) Real-time characterizationof dopamine overflow and uptake in the rat striatum. Neurosci. 25,513–523.

Wightman R. M. and Robinson D. L. (2002) Transient changes inmesolimbic dopamine and their association with ‘reward’.J. Neurochem. 82, 721–735.

Wood P. L. and Altar C. A. (1988) Dopamine release in vivo fromnigrostriatal, mesolimbic, and mesocortical neurons: utility of 3-methoxytyramine measurements. Pharmacol. Rev. 40, 163–187.

Yavich L. (1996) Two simultaneously working storage pools of dopa-mine in mouse caudate and nucleus accumbens. Br. J. Pharmacol.119, 869–876.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1716–1725

Striatal DAergic transmission in MEN2B mice | 1725