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ORIGINAL ARTICLE

Quantification of [11C]yohimbine binding to α2 adrenoceptorsin rat brain in vivoJenny-Ann Phan1,2, Anne M Landau1,3, Dean F Wong4,5,6, Steen Jakobsen1, Adjmal Nahimi1,6, Doris J Doudet1,7 and Albert Gjedde3,4,6,8

We quantified the binding potentials (BPND) of [11C]yohimbine binding in rat brain to alpha-2 adrenoceptors to evaluate

[11C]yohimbine as an in vivo marker of noradrenergic neurotransmission and to examine its sensitivity to the level of noradrenaline.Dual [11C]yohimbine dynamic positron emission tomography (PET) recordings were applied to five Sprague Dawley rats at baseline,followed by acute amphetamine administration (2 mg/kg) to induce elevation of the endogenous level of noradrenaline. Thevolume of distribution (VT) of [

11C]yohimbine was obtained using Logan plot with arterial plasma input. Because alpha-2adrenoceptors are distributed throughout the brain, the estimation of the BPND is complicated by the absence of an anatomicregion of no displaceable binding. We used the Inhibition plot to acquire the reference volume, VND, from which we calculated theBPND. Acute pharmacological challenge with amphetamine induced a significant decline of [11C]yohimbine BPND of ~ 38% in allvolumes of interest. The BPND was greatest in the thalamus and striatum, followed in descending order by, frontal cortex, pons, andcerebellum. The experimental data demonstrate that [11C]yohimbine binding is sensitive to a challenge known to increase theextracellular level of noradrenaline, which can benefit future PET investigations of pathologic conditions related to disruptednoradrenergic neurotransmission.

Journal of Cerebral Blood Flow & Metabolism advance online publication, 7 January 2015; doi:10.1038/jcbfm.2014.225

Keywords: [11C]yohimbine; alpha-2 adrenoceptors; amphetamine challenge; Inhibition plot; reference region

INTRODUCTIONDysfunction of noradrenergic neurotransmission is implicated in arange of brain disorders, such as major depression and neuro-degeneration.1,2 Full understanding of the regulation of noradre-nergic neurotransmission has been hindered by the lack ofselective tracers of noradrenaline’s receptors in vivo. Yohimbine isan alkaloid herbal compound, which can be extracted from theWest African plants, Pausinystalia Yohimbine tree and the roots ofRauvolfia Serpentina.3 In vitro, yohimbine binds to the alpha-2adrenoceptors (α2R) with high affinity, whereas it binds to the α1adrenoceptors and the 5-HT1A, 5-HT1B and 5-HT1D receptors withlower affinity. Yohimbine is an antagonist of the α2R and a partialagonist of the 5-HT1A receptors.4 However, at the low concentra-tion used in positron emission tomography (PET), the radioligand[11C]yohimbine binds with high selectivity to the α2R.

5 Recent PETstudies of pig brain showed that [11C]yohimbine binding is asensitive marker of noradrenaline release, as shown by thesignificant decrease of the volume of distribution (VT) in responseto acute amphetamine treatment.5,6 These findings suggest that[11C]yohimbine can be used to advantage in studies of the normalfunction and pathophysiologic alterations of noradrenergicneurotransmission.In the present study, we determined the [11C]yohimbine bind-

ing potential (BPND) in the absence of a region devoid of specific

binding sites, i.e., a ‘reference’ region. The BPND reflects the ratio atsteady state of specifically bound to non-displaceable tracerquantities in the tissue.To determine the BPND, it is necessary to know the steady-

state volume of distribution of the non-displaceable partitionvolume (VND), as well as the total volume of distribution ina given brain region (VT). There are two ways to obtain the VNDvalue, either by the identification of a brain region withoutbinding sites,7 or by complete blocking of the binding sites in thebrain tissue as a whole. For tracers that bind to differentiallydistributed receptors, such as radioligands of the dopaminereceptors, the cerebellum is such a reference tissue with littlespecific binding. In contrast, the α2R are distributed throughoutthe brain with no well-defined region(s) of non-displaceable (i.e.,non-specifically bound) accumulation. In this case, the alternativeapproach is to block the displaceable binding with a suitableunlabeled ligand. Such an approach is presented here in the formof the Inhibition plot,8,9 derived to yield the volume of non-displaceable distribution (VND) from the linear relationship of thevalues of VT in the challenge condition to those of the baselinecondition.To examine the sensitivity of [11C]yohimbine binding to mono-

aminergic competition, we challenged the binding with acuteadministration of amphetamine, a drug known to induce the

1Department of Nuclear Medicine and PET Centre, Aarhus University Hospital, Aarhus, Denmark; 2Department of Biomedicine, Aarhus University, Aarhus, Denmark; 3Center ofFunctionally Integrative Neuroscience, Aarhus University, Aarhus, Denmark; 4Department of Radiology and Radiological Science, Division of Nuclear Medicine, Johns HopkinsUniversity School of Medicine, Baltimore, Maryland, USA; 5Department of Psychiatry, Neuroscience and Environmental Health Sciences, Johns Hopkins Medical Institutions,Baltimore, Maryland, USA; 6Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark; 7Department of Medicine/Neurology, Universityof British Columbia, Vancouver, British Columbia, Canada and 8Department of Neurology, McGill University, Montreal, Quebec, Canada. Correspondence: Professor A Gjedde,Department of Neuroscience and Pharmacology, Panum Institute, University of Copenhagen, Blegdamsvej 3, Copenhagen 2200 N, Denmark.E-mail: gjedde@sund.ku.dkThis study received financial support from the Department of Nuclear Medicine and PET Centre, Aarhus University Hospital, and Aarhus University, and the Danish Council forIndependent Research.Received 13 August 2014; revised 21 October 2014; accepted 19 November 2014

Journal of Cerebral Blood Flow & Metabolism (2015), 1–11© 2015 ISCBFM All rights reserved 0271-678X/15

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release of endogenous monoamines, particularly noradrenalineand dopamine. Amphetamine is a psychostimulant thatacts as a substrate for the monoamine re-uptake and vesiculartransporters (VMAT-2), leading to reversal of the membranetransport and depletion of synaptic vesicles, and ultimateelevation of the extracellular concentration of endogenousmonoamines.10

Here, we apply the Inhibition plot to obtain a steady-stateestimate of the VND in the absence of a reference region, toexamine the specificity of [11C]yohimbine as a marker of α2Ravailability and noradrenaline release in rat brain in vivo.

MATERIALS AND METHODSTheoryKinetic analysis of [11C]yohimbine uptake and distribution. Conventionalkinetic analysis of PET radioligand distribution is based on compartmentalmodels described by linear differential equations with transfer coefficients.When the bound and free quantities of tracers are in steady state, thetracer distribution in the brain compartment is dominated by theexchange between the circulation and the tissue compartment acrossthe blood–brain barrier (BBB). The tracer exchange across BBB is governedby the concentration of tracer in plasma (kbq/ml) and the quantity in braintissue (kbq/cm3). We applied the one-tissue compartment model, wherethe distribution of tracer is described by two first order differentialequations as follows,

dm1

dt¼ V0

dCa

dtð1Þ

and

dm2

dt¼ K1Ca - k’2m2 ð2Þ

where m1 is the tracer quantity in the vascular compartment with thevolume V0 and the concentration Ca as function of time. The term m2

denotes the tracer quantity in the brain tissue, which depends on theclearance K1 from plasma to brain and k2’ the rate constant of the reversedirection from the tissue compartment. Here, at steady state, K1 is definedas,

K1 ¼ F 1 - e - PS=F� �

ð3Þwhere F is blood flow, PS is the permeability-surface area product of thetracer. In addition, k2’ equals the ratio of K1 to total volume of distribution,VT, at steady state,

k’2 ¼ k21þ BPND

¼ K1

VND 1þ BPNDð Þ ¼F 1 - e - PS=F� �

VND 1þ BPNDð Þ; ð4Þ

where BPND denotes the binding potential, and VND is the partitionvolume of non-displaceable tracer in the tissue compartment. Rearrange-ment and combination of equations (3) and (4) yield the transfercoefficient k2,

k2 ¼ k’2 1þ BPNDð Þ ¼ K1

VNDð5Þ

Combination of equations (1) and (2) shows that the change of mass of[11C]yohimbine in the brain as a whole obeys the equation,

dmdt

¼ dm1

dtþ dm2

dt¼ V0

dCa

dtþ K1Ca - k’2m2 ð6Þ

The total mass of radiotracer, m, is obtained by the integration ofequation (6),

m ¼ m1 þm2 ¼ V0Ca þ K1

Z T

0Cadt - k’2

Z T

0m2dt; ð7Þ

where the unknown quantity, m2, is solved by substitution of m2 =m−m1

m ¼ V0Ca þ K1

Z T

0Cadt - k’2

Z T

0m 1 -

m1

m

� �dt ð8Þ

According to equation (8), the kinetics obey a linear relationship whensteady state is approximated, which strictly occurs at times when the m1/mratios are of negligible magnitude. When the ratio m1/m becomes

negligible, equation (8) can be simplified to the basic equation for theone-compartment analysis,

m ¼ K1

Z T

0Cadt - k’2

Z T

0mdt; ð9Þ

According to equation (9), the mass exchange can be described by a linearequation at the time when m1/m becomes negligible. Here, m1/m ratio of0.01 was chosen as the threshold of negligible vascular radioactivity,implying that m1 is negligible when 1% or less of radiotracer remains inplasma relative to brain tissue.The dynamic parameters in equation (9) can be estimated after

rearrangement, allowing linear regression combinations of any two ofthe three kinetic variables, including the apparent volume of distribution,Vapp(T),

Vapp Tð Þ ¼R T0 mdtR T0 Cadt

; ð10Þ

the apparent clearance, Kapp(T), of tracer from the vascular compartment tobrain tissue,

Kapp Tð Þ ¼ m Tð ÞR T0 Cadt

; ð11Þ

and the apparent residence time of tracer in tissue, Θ(T),

Θ Tð Þ ¼R T0 mdt

m Tð Þ ð12Þ

In the following, we applied two linear plots derived from equation (9) toestimate the time of onset of steady state, and we subsequently appliedthe Logan plot11 to estimate VT. To determine the onset of steady state, wefirst applied the linearized model described by Reith et al,12 where the K1 isdirectly obtained from the slope, and k2’ from the y-intercept,

1Θ Tð Þ ¼ K1

1Vapp Tð Þ - k’2 ð13Þ

We assumed that steady state is present at the time interval, when the bestlinear fit is obtained with equation (13). To confirm this assumption, weapplied a second linearized solution as presented by Gjedde et al.13

Vapp Tð Þ ¼ -1k’2

Kapp Tð Þ þ VT; ð14Þ

where k2’ is determined from the reciprocal value of the slope. Finally, weused the range of times consistent with steady state to estimate the VTfrom the slope of the Logan plot,

Θ Tð Þ ¼ VT1

Kapp Tð Þ -1k’2

ð15Þ

Binding potential relative to non-displaceable uptake. The bindingpotential relative to non-displaceable uptake, BPND, can be calculated interms of VT when the non-displaceable partition volume, VND is known. Tocompute the VND of [11C]yohimbine in rat brain, we applied the linearizedbinding equation known as the Inhibition plot, which is derived from twomeasures of receptor occupancy at baseline and upon pharmacologicalchallenge. The BPND is a parameter that represents the ratio of bound tofree quantities of tracer in the free solution in brain tissue.14 The relativereceptor availability equals the ratio of the binding potentials at inhibition(BPND(i)) and baseline (BPND(b)),

1 - s ¼ BPND ið ÞBPND bð Þ

; ð16Þ

where 1-s expresses the fraction of receptors available for binding in thepresence of a competitor, and s is the fraction of receptors actuallyoccupied by the competitor. The BPND can also be expressed in terms ofthe distribution volumes, such that,

BPND bð Þ ¼VT bð Þ - VND bð Þ

VND bð Þð17Þ

and

BPND ið Þ ¼VT ið Þ - VND ið Þ

VND ið Þ; ð18Þ

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where VT is the total volume of distribution, of which VND is thenon-displaceable volume of distribution, and the subscripts (b) and (i)denote the baseline and challenge conditions. When the VND valueremains the same at an amphetamine challenge, compared with baseline(VND(i)= VND(b) = VND), combination of equations (16 to 18) can be reducedto yield the relative receptor availability in terms of the volumes,

1 - s ¼ VT ið Þ - VND

VT bð Þ - VND; ð19Þ

from which the VND value can be obtained to yield the binding potential,

BPND ¼ VT

VND- 1 ð20Þ

Three linearized versions of the relative receptor availability (equation (19))previously were derived to obtain the VND value by linear regression,including the Inhibition plot,8,9 the Saturation (or Lassen15) plot, and theOccupancy plot.16 Gjedde et al 8,9 linearized equation (19) in the form ofthe Inhibition plot that yields the inhibited binding volume, VT(i), as afunction of the baseline volume, VT(b)

VT ið Þ ¼ 1 - sð ÞVT bð Þ þ sVND; ð21Þwhere the estimate of VND is the intercept of the linear regression line andthe line of identity. Lassen et al15 linearized equation (19) in the form of theSaturation plot that yields the baseline volume of distribution, VT(b),

VT bð Þ ¼ 1sVT þ VND; ð22Þ

from which VND can be obtained as the ordinate intercept of the linearregression. Cunningham et al16 derived the third linearization, commonlyknown as the Occupancy plot, to yield the difference between the volumesof distribution at baseline relative to the challenge condition, ΔVT, and VNDcan be obtained from the x-intercept,

ΔVT ¼ sVT bð Þ - sVND; ð23Þwhere VND is obtained as the abscissa intercept. To use any of thelinearized plots, at least two consecutive PET recordings with two differentlevels of receptor occupancy are required. For the Inhibition plot, unlikethe Saturation and Occupancy plots, the dependent (VT(i)) and indepen-dent (VT(b)) variables are not intermixed, such that the fractional receptoravailability and VND are obtained directly from the volumes of distribution.As the three linearizations are derived from the same original relativereceptor availability formulation (equation (19)), they are all based on thecommon fundamental assumptions that there are different brain regionswith different receptor densities (Bmax), that the Bmax values remainunchanged in the challenge condition, and that receptor affinity and non-displaceable binding volumes have the same values in all the regions andremain constant upon challenge.

Plasma-free fraction, fp. A further essential prerequisite for the linearizationsof relative receptor availability (equation (19)) is that the value of VNDmust remain the same at baseline and inhibition, which entails thatthe free available concentration of radiotracer in the plasma, fp, atequilibrium must remain constant at baseline to challenge condition. This isbecause a change in fp will consequently introduce changes in VND.Amphetamine is known to not alter the binding of yohimbine to peripheralα2R on platelets, as reported for humans.17 Therefore, we performed plasmaultrafiltration to test the hypothesis that amphetamine does not affect plasmaprotein binding as reflected in measures of the plasma-free fraction, in supportof the assumption underlying the Inhibition plot that the values of VND areunchanged.The concentrations of tracer free in plasma, CFP, or freely dissolved in

tissue water, CFT, can be expressed in terms of total concentration of thetracer in plasma, Ca, and non-displaceable concentration in tissue, CND, andthe free fractions in plasma, fp, and in brain tissue, fFT,

CFP ¼ f pCa ð24Þ

CFT ¼ f FTCND ð25ÞThe volume of non-displaceable tracer is defined as ratio of the concen-tration of non-displaceable tracer in tissue, CND, and the total plasmaconcentration, Ca,

VND ¼ VdCND

Ca; ð26Þ

where Vd is the physical volume of distribution of the tracer in the solventin which CND is measured. The combination of equations (24 to 26) gives,

VND ¼ Vdf pfND

CFT

CFPð27Þ

Hence, VND is defined as the product of a physical volume of distributionand two unitless ratios, including the ratio fp/fND, which is determined bynonspecific binding in plasma and brain, and the second ratio CFT/CFP thatdepends on the properties of the BBB. When it is assumed that aradioligand penetrates the BBB by passive diffusion, the concentrationsof radioligand will assume a steady state depending on the relativesolubilities on the two sides of the BBB. In this case, equation (27) can bereduced to,

VND ¼ Vdf pfND

ð28Þ

It follows that the linearizations derived from the relative receptoravailability can only be applied with validity when fp of the radioligandis unchanged from the baseline to the challenge condition. Therefore,plasma ultrafiltration was performed in this work to determine [11C]-yohimbine fp to confirm that it remains constant across the two conditionsto consolidate the basic assumptions applicable for Inhibition plot.

Animal experimentsAll the animal experiments were conducted under humane conditions withan approval from the Danish Animal Experiments Inspectorate and inaccordance with the guidelines set by the Danish Ministry of Food,Agriculture, and Fisheries in agreement with European Legislation for theprotection of vertebrate animals used for experimental and other scientificpurposes. Animals were housed two per cage under a 12/12 hours light/dark cycles, and with ad libitum access to food and water.Initially, six Sprague Dawley rats (250 to 300 g) were subjected to dual

PET sessions with [11C]yohimbine at baseline and after amphetaminechallenge. One animal was excluded because of difficulties with theintravenous catheter, which led to amphetamine administration throughthe femoral artery and resulted in a different kinetic profile, which was notcomparable with the animals that received intravenous amphetamine.A second subset of experiments (four Sprague Dawley rats) was

designed to measure the free available fraction of [11C]yohimbine inplasma at the baseline and post-amphetamine condition. Anesthesia wasinduced in a chamber filled with 2% isoflurane and maintained throughoutthe studies with a mask for delivery of isoflurane (1.8 to 2.0%). Physiologicparameters were monitored during all dynamic recordings. We catheter-ized the femoral artery for blood sampling and the tail vein for tracerinjection and amphetamine administration. At the end of the experiments,the animals were anesthetized with intravenous pentobarbital (100mg/kg)before decapitation.

Positron emission tomography data acquisitionEach animal underwent two consecutive PET acquisitions with [11C]-yohimbine of 90 minutes on the same day, first at baseline and thenfollowed by amphetamine challenge (2 mg/kg intravenously). Approxi-mately 20 minutes after baseline recording, we administered 2mg/kgamphetamine as a bolus intravenous injection. The second [11C]yohimbinePET acquisition began 5 to 10 minutes after this administration. Time–activity curves of [11C]yohimbine uptake were normalized to the plasmaintegral to enable the comparison of dynamic data among individualacquisitions and among animals, as normalization to the plasma integralcorrects for the effects of bioavailability factors, such as the body weight,peripheral washout, and radioactivity dose.Initial studies in rats demonstrated that yohimbine is a p-glycoprotein

substrate at the BBB, which restricts the transfer of [11C]yohimbine to thebrain. Thus, cyclosporine (50 mg/kg), a nonspecific p-glycoprotein inhi-bitor, was administered through the tail vein 30 minutes before traceradministration to facilitate the delivery of [11C]yohimbine into the brain.Cyclosporine pretreatment was only done once before the first imagingsession, as cyclosporine has a long half-life of more than 10 hours in bothrat brain and peripheral tissue, when treated with doses higher than30mg/kg.18

Animals were placed prone in the aperture of the small animal tomo-graph (microPET R4, CTI Concorde, Knoxville, TN, USA) and the head wasimmobilized with a custom-built Plexiglas head holder, allowing repro-ducible and comparable positioning of all animals. The spatial resolution

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(FWHM) of the R4 tomograph is 2 mm at the center of the field of view,indicating a volume of resolution of 8 mm3. We obtained a transmissionscan with a rotating Ge-68 point source for attenuation correction.Dynamic emission recording began upon injection of the radiotracer. Theinjected activity averaged 28± 4Mbq at baseline and 24± 9Mbq afteramphetamine (± s.d., based on injected activity of five animals). The totaltomography length of 90 minutes consisted of multiple frames increasingin duration from 15 seconds to 10 minutes. Arterial plasma input wasprocessed from a total of nine arterial blood samples per session. Plasmasamples of 200 μL were collected in heparinized tubes at 1,5,10,20, 30, 45,60, 75, and 85 minutes after radiotracer injection. To generate plasma, wecentrifuged the whole blood in 10 minutes at 4 °C at 1,000 g, and activitieswere counted with a gamma counter (Packard Cobra Gamma Counter,Model D5003) and decay corrected.

Image processingDynamic PET acquisitions were processed using Montreal NeurologicalInstitute MINC software. We reconstructed the photon attenuation correc-tion of dynamic recordings by three-dimensional-filtered back projection,resulting in a 128×128× 63 matrix. Summed emission recordings weremanually registered to a digital magnetic resonance imaging (MRI) atlas ofthe rat brain, using the program Register (Montreal Neurological Institute)with nine degrees of freedom. The dynamic emission recordings wereresampled to the stereotaxic space. Using the program Display, masks ofvolumes of interest, including frontal cortex, striatum, thalamus, pons, andcerebellum were drawn on the MRI atlas according to the stereotaxic atlasof the rat brain. The volume of interest masks were then used to extracttime–activity curves from the resampled dynamic data sets. We obtainedthe total volumes of distribution VT(b) and VT(i) with the Logan analysis ofthe time–activity curves for each region, and the corresponding plasmaactivity curve as input.11 Using radio-HPLC, no radioactive metaboliteswere detected in plasma samples (data from two animals), similar to thepreviously reported absence of yohimbine metabolites in pig plasma.5

Thus, we assumed that plasma input was not influenced by plasma meta-bolites. Parametric maps of VT were processed by voxel-wise normalizationusing Logan plot with arterial plasma input. We used the open-sourcesoftware, three-dimensional Slicer (4.3.1 r22599), to create surface brainmodels processed from the MRI atlas of the rat brain to illustrate theanatomic location of the coronal, horizontal, and sagittal slices of theparametric maps.

Determination of plasma-free fraction, fp[11C]yohimbine fp had to be determined because the VND value estimatedwith the Inhibition plot is based on the assumption that the value remainsconstant from baseline to inhibition, implying that [11C]yohimbine fpis the same in the two conditions. The amount of blood required foradequate determination would have been too large to be obtained in thesame animals that underwent PET in addition to the sampling of theplasma input curve, as blood loss may affect the estimates of kineticparameters. Thus, we used a separate group of four rats, treated inthe same manner as the rats that underwent PET acquisition. Administra-tion of cyclosporine (50 mg/kg) was done through an intravenous catheterin the tail vein, and we catheterized the femoral artery for blood sampling.Blood from the baseline condition was collected 30 minutes aftercyclosporine treatment, and then, to obtain blood from the challengecondition, we administered amphetamine (2 mg/kg intravenously) andsampled after 30 minutes. To generate plasma, the blood was centrifuged20 minutes at 4 °C. With slight modifications, the ultrafiltration wasconducted as previously described by Gandelman et al.19 We usedCentrifree Centrifugal filter device (Millipore, Bedford, MA, USA) with amolecular cutoff of 30 kDa for all the samples. Each sample unit of 1,000 μLrat plasma was spiked with 50 μL [11C]yohimbine tracer. After 10-minuteincubation at room temperature, aliquots of 50 μL of this solution wereused to measure the total activity (unfiltered plasma). For triplicatemeasurement of the filtered plasma, the remaining volume was dividedinto three ultrafiltration devices and centrifuged for 20 minutes usinga centrifuge with a fixed angle rotor at 1,000 g. After filtration, weremoved aliquots of 50 μL to assess the activity in protein-free plasma. Theactivity of filtered plasma was determined in triplicates. All the sampleswere counted in a gamma device (Packard Cobra Gamma Counter, ModelD5003). Nonspecific binding of the tracer to the ultrafiltration device wasrecovered by performing the same measurements with a phosphate-buffered solution. The correction factor, γ, was determined as the ratio

of the activity in the unfiltered phosphate buffer (APB, U) to the filteredbuffer (APB, F),

γ ¼ APB;UAPB;F

ð29Þ

The plasma-free fraction, fp was calculated as the ratio of the activity of thefiltered plasma (APlasma,F) to the unfiltered plasma (APlasma,U), multiplied bythe correction factor, γ, for recovery,

f p ¼ Aplasma;F

Aplasma;Uγ ð30Þ

StatisticsWe used repeated measures two-way ANOVA at Po0.05 to determinewhether pharmacological challenge with amphetamine significantlyaltered the regional BPND of [11C]yohimbine. For the plasma-free fraction,nonparametric test and paired t-test were applied to test whether the freefraction after amphetamine challenge significantly differed from baselinecondition. Paired t-test was used to determine whether VT estimatesobtained from the Logan plot were significantly different when dynamicdata were fitted at 6 to 28minutes compared with 28 to 85minutes.

RESULTS[11C]yohimbine clearance declined robustly in response to amphe-tamine in all examined regions, compared with the baselinecondition, presented as regional time–activity curves, normalizedto the plasma integral in Figures 1A–1E. The reduced clearancereveals the sensitivity of [11C]yohimbine binding to the release ofendogenous monoamines. Time–activity curves were normalizedto the corresponding plasma integral to enable accurate compar-ability among animals, as the plasma integral directly reflectsthe administered quantity of radiotracer, which depends on theindividually injected radioactive dose, the body weight, and theextent of peripheral washout. As shown in Figure 1F, the expo-nentially time-interpolated plasma activity curves were similar inshape and magnitude, implying that the difference of binding inbrain tissue post amphetamine was not due to differences of inputfunction, tracer delivery, or other bioavailability factors.The steady-state period was determined by identifying the

interval, in which the transfer coefficient estimates, K1 and k2’,remained constant. Figure 2A displays the linear solution pre-sented by Reith et al,12 in which K1 was obtained from the slopeand k2’ from the ordinate intercept, indicating steady-stateapproximation at the time interval from 6 to 28minutes, whereK1 is constant. A second linear solution13 yielded k2’ as thereciprocal value of the slope, as presented in Figure 2B. Together,the two linear plots confirmed that steady state is present in thetime interval of 6 to 28 minutes. We estimated the regional VTfrom Logan plots with arterial plasma input, fitted in the timeinterval of 6 to 28 minutes, as exemplified in Figure 2C. Allestimates of the steady-state parameters are listed in Table 1A.The clearance K1 decreased in response to amphetamine, indicat-ing a decrease in tracer uptake in brain tissue in the challengecondition relative to baseline. Likewise, the rate constant k2calculated from equation (5) declined post amphetamine,consistent with [11C]yohimbine being displaced by release ofendogenous monoamines. In contrast, the k2’ estimates wereunchanged in two regions, including frontal cortex and striatum.The m1/m ratio was negligible ffi1% in this period (listed inTable 1B), as verification that the negligible magnitude of the m1/m ratio coincides with the linearity of equation (9). Notably, k2’values estimated from the linearization of Reith et al12 are similarto those estimated from the solution presented by Gjedde et al,13

and the VT values obtained from this plot are equal to theestimates from the Logan plot.11

Figure 3 displays the parametric maps of [11C]yohimbine VTsuperimposed on an MRI atlas (Figure 3A), showing an abundant

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Figure 1. (A–F) Regional time–activity curves of [11C]yohimbine uptake. Time–activity curves of [11C]yohimbine from each volume of interestare normalized to the respective plasma input integrals (A–E). The displacement by amphetamine was uniform in all VOIs, as shown by thereduction of ~ 34% of the area under the curves of amphetamine (green curve), relative to baseline (black curve). The error bands representthe s.e.m. of five animals. The time-interpolated plasma activity curves (F) at baseline (black curve) and post-amphetamine (green curve) aresimilar in profile and magnitude, which excludes the possibility that the observed effect is because of differences in the dose of administeredradiotracer. CEREB, cerebellum; FC, frontal cortex; STR, striatum; THA, thalamus; VOI, volume of interest.

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distribution throughout the brain at baseline condition (Figure 3B)and a marked decrease upon amphetamine challenge (Figure 3C)because of competition with released monoamines.The VT values from the solution presented by Logan et al11 were

used in the Inhibition plot, shown in Figure 4A. The VT values afteramphetamine were plotted as a function of baseline, which

produced a VND value of 0.286 ± 0.136 mL/cm3. The slope of theInhibition plot represents the fraction of unoccupied receptors(1− s), which declined with the amphetamine challenge comparedwith baseline, as expected after the release of endogenousligands. A reduction of receptor availability of 23 to 56% occurredin response to amphetamine, supporting yohimbine’s sensitivityto endogenous transmitters. The absolute values of receptoroccupancy, s, exerted by endogenous monoamines are shown inFigure 4A for individual animals.Figure 4B shows the binding potentials calculated in terms of

the known VND value. At baseline, the BPND was greatest in thethalamus (5.84 ± 0.95) and striatum (5.82 ± 1.09), followed indescending order by frontal cortex (5.22±0.99), pons (5.10±0.85),and cerebellum (4.02 ± 0.66).We determined values of fp by plasma ultrafiltration in a

separate group of animals, treated in the same manner as thosethat underwent dynamic imaging. We found a baseline fp of0.23 ± 0.03 that did not significantly differ from fp at post-amphetamine of 0.23 ± 0.02 (P40.87). Individual values of fp arelisted in Table 2. The result of the statistical test of plasma-freefractions was not at variance with the null hypothesis of nodifference, as shown with a nonparametric as well as a t-test. Theunchanged plasma-free fraction supports constant VND valuesfrom baseline to amphetamine challenge and confirms theassumption of no change underlying the Inhibition plot.

DISCUSSIONIn this work, we used the Inhibition plot to evaluate [11C]-yohimbine as a marker of monoaminergic competition at the α2Rin brain. First, we determined the steady-state interval by usingtwo linear plots to show that the transfer coefficients K1 and k2’ areconstant at 6 to 28 minutes. Correspondingly, the m1/m ratio wasnegligible ffi1% in this period, confirming that the tracerexchange across BBB followed the linear relationship predictedby equation (9). Interestingly, the magnitude of k2’ estimatesobtained from the linearized plot presented by Reith et al12 weresimilar to the estimates from Gjedde et al,13 suggesting that theestimates obtained from different linear solutions approximate thesame parameter values when estimated at steady state. Themagnitude of the blood–brain clearance K1 declined in all theexamined regions in response to amphetamine, whereas thevalues of k ’2 remained constant in particular regions (frontal cortexand striatum), indicating the opposite effects of reduced clearanceand increased binding on the magnitude of this rate constant.Calculations of k2 using equation (5) verified that the magnitude ofthis rate constant k2 declined with the magnitude of the clearanceK1 post amphetamine. The suggestion of reduced blood flow instriatum and frontal cortex in response to amphetamine in thisstudy is consistent with prior reports of low striatal blood flow inmonkeys and rats in response to amphetamine administration,20

and reduced cortical blood flow in response to noradrenalinerelease after locus coeruleus stimulation.21

Figure 2. (A–C) Determination of time of steady-state onset. Typicalexamples of regression to linearizations of the one-compartmentmodel to determine the steady-state interval of acquisition data.First, the times when K1 was constant were identified by applyingthe linear model described by Reith et al12 (A), in which K1 wasdirectly estimated as the slope. The equilibrium period was 6 to28minutes, which was further confirmed with the linear plotdescribed by Gjedde et al13 (B), where k2’ is obtained as thereciprocal of the slope. Ultimately, the steady-state interval wasapplied also to the Logan plot11 to estimate the equilibrium volumeof distribution, VT, (C). CEREB, cerebellum; FC, frontal cortex; STR,striatum; THA, thalamus.

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Although no radioactive metabolites were detected in plasma,the two linear plots used to estimate K1 and k2’ revealed that theresults may have been influenced by metabolites at times laterthan 28 minutes, as exemplified by the curvatures seen in theplots of Figures 5A and 5B after 28 minutes of circulation. Thecurvature was not immediately apparent in the Logan plots, asshown in Figure 5C, but the slopes are significantly different whenobtained for the two intervals before and after 28 minutes ofcirculation (Po0.05, data from five animals). Here, the globalbaseline VT estimate at 6 to 28 minutes averaged 1.71 ± 0.10 mL/cm3, compared with the global estimate of 1.58 ± 0.07 mL/cm3 at28 to 85 minutes. The curvatures suggest that the steady state isaffected by plasma metabolites at times later than 28 minutes,perhaps explaining the well-known underestimation by the Loganplot of the value of VT, as reported previously.22 The comparison ofthe early and late Logan slopes demonstrates that departuresfrom steady state may be difficult to detect with this plot towardsthe end of circulation times with the increased spacing of the lastpoints. The curvatures and the decline of the VT estimates,associated with a possible end of steady state, may indicate thepresence of low concentrations of plasma metabolites below thelimit of detection by HPLC, possibly related to the negligibleamounts of tracer in small animals, the limited plasma volume inrodents, and a low level itself of the plasma metabolites. However,the effect was circumvented by identification of times ofpreserved steady state before the effective appearance of plasmametabolites.We have demonstrated that [11C]yohimbine binding across

the brain varied as expected from previous binding assays inrat brain in vitro with [3H]yohimbine, in which a high level ofTa

ble1A.

Reg

ional

kinetic

param

etersat

baselinean

dwitham

phetam

ine

VOIs

Reith

etal12

Gjedd

eet

al13

Loga

net

al11

Inhibitio

nplot

8,9

%Ch

ange

K1(ml/m

inute)

k 2'(per

minute)

k 2(per

minute)

k 2'(perminute)

k 2(per

minute)

VT(ml/cm

3 )VT(ml/cm

3 )BP N

D(ratio)

Mean

±s.e.m.

Mean

±s.e.m.

Mean

±s.e.m.

Mean

±s.e.m.

Mean

±s.e.m.

Mean

±s.e.m.

Mean

±s.e.m.

Mean

±s.e.m.

Mean

±s.e.m.

Baseline

FC0.28

0.06

0.18

0.05

5.85

0.99

0.18

0.05

5.89

0.98

1.72

0.28

1.74

0.28

5.09

0.97

STR

0.29

0.08

0.18

0.06

6.49

1.08

0.17

0.06

6.53

1.06

1.88

0.31

1.91

0.30

5.67

1.06

THA

0.43

0.15

0.25

0.10

6.94

0.94

0.25

0.10

6.94

0.92

1.90

0.28

1.91

0.27

5.69

0.93

PONS

0.68

0.32

0.45

0.23

7.96

1.91

0.49

0.28

7.03

1.11

1.70

0.24

1.71

0.24

4.97

0.83

CER

EB0.61

0.26

0.50

0.24

7.40

2.32

0.59

0.33

5.77

0.83

1.40

0.19

1.41

0.18

3.92

0.65

Post-amph

etam

ine

FC0.18

0.02

0.19

0.04

3.11

0.47

0.19

0.04

3.14

0.47

1.03

0.13

1.05

0.13

2.68

0.45

−37

.63

2.15

STR

0.19

0.02

0.18

0.03

3.42

0.43

0.18

0.03

3.46

0.44

1.10

0.11

1.14

0.12

2.97

0.43

−38

.18

5.46

THA

0.21

0.02

0.20

0.04

3.49

0.47

0.20

0.04

3.54

0.47

1.11

0.12

1.14

0.13

2.99

0.45

−39

.01

5.07

PONS

0.26

0.02

0.28

0.05

3.23

0.37

0.28

0.05

3.28

0.38

1.01

0.10

1.03

0.11

2.62

0.37

−37

.49

6.09

CER

EB0.25

0.03

0.31

0.06

2.65

0.29

0.31

0.06

2.68

0.29

0.87

0.08

0.88

0.09

2.09

0.31

−35

.25

5.95

CER

EB,cerebellum;FC

,frontalco

rtex;ST

R,striatum;TH

A,thalam

us;VOI,vo

lumeofinterest.Steady-stateestimates

ofkinetic

param

eterswereobtained

from

theinterval

of6to

28minuteswiththethree

linearizedplots

usedhere.

Theestimates

areco

mparab

leacross

thethreeplots.Th

emag

nitudeofthetran

sfer

coefficien

tk 2

was

calculatedfrom

k’ 2acco

rdingto

equation(5).

Thebold

entriesaremad

eto

improve

thedeliveryofinform

ationso

that

themeanvalues

caneasily

beseparated

from

thestan

darderrorvalues.

Table 1B. m1/m ratios

m1/m ratio

Midframe time Typical example Baseline AMPH

(minute) Baseline AMPH Mean ± s.d. Mean ± s.d.

1.13 0.017 0.025 0.014 0.006 0.021 0.0031.38 0.016 0.023 0.014 0.005 0.020 0.0031.63 0.015 0.021 0.013 0.005 0.018 0.0021.88 0.014 0.020 0.012 0.004 0.018 0.0022.25 0.012 0.018 0.011 0.004 0.016 0.0022.75 0.011 0.016 0.010 0.003 0.015 0.0023.25 0.010 0.014 0.009 0.003 0.014 0.0023.75 0.009 0.013 0.009 0.002 0.013 0.0014.5 0.008 0.012 0.008 0.002 0.012 0.0025.5 0.007 0.011 0.007 0.002 0.011 0.0027 0.006 0.010 0.006 0.002 0.010 0.0029 0.005 0.009 0.006 0.002 0.009 0.002

12.5 0.004 0.008 0.005 0.001 0.008 0.00217.5 0.004 0.008 0.005 0.001 0.008 0.00222.5 0.004 0.008 0.005 0.001 0.008 0.00227.5 0.004 0.008 0.005 0.001 0.007 0.00235 0.004 0.008 0.005 0.001 0.007 0.00245 0.005 0.008 0.005 0.001 0.007 0.00255 0.005 0.008 0.006 0.001 0.007 0.00265 0.005 0.008 0.006 0.001 0.007 0.00275 0.006 0.008 0.006 0.001 0.007 0.00285 0.006 0.008 0.007 0.002 0.007 0.002

Typical examples of m1/m ratios of the data, presented in Figures 2 and 5,are listed here with the average m1/m ratio± s.d. from five animals,demonstrating the onset of steady state for ratios o1% for baseline andwith amphetamine (AMPH) acquisitions.The bold entries are made to improve the delivery of information so thatthe mean values can easily be separated from the standard error values.

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binding was found throughout the brain. Regions with thegreatest [3H]yohimbine binding included striatum, cerebral cortex,hypothalamus, hippocampus, cerebellum, and pons.23 Thesefindings agree with the present in vivo data with the same rankorder of magnitudes of [11C]yohimbine binding.In the rat central nervous system, the expression of α2R has

three subtypes, including α2A, α2B, and α2C, which are classified onthe basis of the pharmacological characteristics and molecularcloning.24,25 In rats, the central α2R are regionally heterogeneous.It is known from in situ hybridization of α2R gene expression thatthe cerebral cortex, hippocampus, thalamic nuclei, and cerebellumpredominantly express both α2A and α2C subtypes, while the basalganglia express α2C, and the α2B subtype is expressed exclusivelybut weakly in the thalamus.25 Further binding assays in humanbrains have shown that α2AR co-exist with α2CR in the caudatestriatum, based on [3H]yohimbine binding that was displaced byoxymetazoline and prazosine, both drugs known to bind withhigh affinity for α2AR and α2CR, respectively.

26 Consistent withthese findings, in vitro autoradiography of rat brain with the two

α2R antagonists, [3H]rauwolscine and [3H]idazoxan, revealedbinding to sites in striatum that was displaced by noradrenergicbut not dopaminergic ligands, which further supports theexistence of striatal α2C and α2A sites in rat brain.27

Figure 4. (A–B) The Inhibition plot and [11C]yohimbine bindingpotentials, BPND. Steady-state volumes of distribution, VT, obtainedfrom the Logan plot,11 applied to the inhibition8,9 plot to yield thereference volume, VND (A). The regional volumes of distribution inresponse to pharmacological challenge, VT(i), of each animal wereplotted as a function of the volume of distribution at baseline, VT(b).The dashed line, representing the linear regression of the averagedbaseline data (n= 5), also represents the line of identity. The solidlines are the linear regressions of amphetamine challenge againstbaseline in individual animals (n= 5). Each point represents thevolume of distribution in a VOI, and the linear regressions are basedon five VOIs, including frontal cortex (FC), striatum (STR), thalamus(THA), pons (PO), and cerebellum (CEREB). The slope of the linearregressions of amphetamine challenge, (1− s), is the receptoravailability, shown here as the result of competition with endogen-ous transmitters released by amphetamine. The estimate of VND wasobtained from the shared intercept of the linear reggressions withthe baseline curve, with a value of 0.286± 0.136mL/cm3. The VNDestimated from the Inhibition plot was used to calculate the regionalbinding potentials, BPND, presented in B. Values of BPND werehighest in the thalamus and striatum, followed in descending orderby, frontal cortex, pons, and cerebellum. BPND declined significantlyin response to acute amphetamine (black bars) (Po0.05), comparedwith baseline (white bars) across all brain regions. Regional BPNDfrom individual animals are presented with a line that connects thevalues at baseline and post-amphetamine to indicate the pharma-cological effect in each animal. VOI, volume of interest.

Figure 3. (A–C) Parametric maps of [11C]yohimbine VT. Representa-tive parametric maps of [11C]yohimbine VT were superimposed onan average MRI rat atlas (A), shown in horizontal (left column),coronal (middle column), and sagittal planes (right column). Themagnitude of VT was obtained from the Logan plot with voxel-wisenormalization to plasma input and linear regression in the steady-state interval of 6 to 28minutes. VT remained uniformly highthroughout the brain at baseline (B) and declined markedly withacute amphetamine challenge (C), suggesting that [11C]yohimbinebinding is sensitive to the elevated level of monoamines. MRI,magnetic resonance imaging.

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In contrast, [11C]yohimbine is not specific to any of thesesubtypes and thus labels α2R without discriminating amongsubtypes. The combination of known α2R subtype expressionin vitro and [11C]yohimbine PET in vivo is a valuable tool ofprediction of which α2R subtype possibly may be implicated in aparticular condition. Especially noteworthy is the finding thatin vivo striatal [11C]yohimbine binding significantly exceeds that ofthe frontal cortex, because striatum is known to receive generousdopaminergic innervation via nigrostriatal afferents but very littlenoradrenergic input.28 The discrepancy between α2R distributionand noradrenergic innervation is in agreement with previousautoradiography of the rat brain, for example, demonstratingmore numerous binding sites of the α2R antagonist [3H]-rauwolscine in striatum than in cerebral cortex.29 In light ofthe known regional distribution of α2R subtypes in rat brain, theobserved striatal [11C]yohimbine binding may be ascribed to thelabeling of both α2C and α2A sites.Of course, the functional role of α2R in a region that has limited

noradrenergic input28 is not immediately evident. However,interaction between other monoamines and these receptorspossibly means that α2R in striatum has a role in, and respondsto, dopaminergic neurotransmission. This interpretation is sup-ported by evidence from work in zebra finches, in whichdopamine binds to α2R, although with considerably lower affinitythan for noradrenaline.30 Further studies have shown that theaffinity of α2CR for dopamine was higher than that of α2AR fornoradrenaline in rat striatum, suggesting that dopamine may bethe preferred neurotransmitter of the striatal α2CR.

31 In the light ofthis evidence in support of homology between noradrenergic anddopaminergic receptors, it is likely that the displacement of [11C]-yohimbine binding in striatum with amphetamine is because ofcompetition with dopamine released from dopaminergic term-inals in the presence of minimal noradrenergic input. We observedsignificantly displaced [11C]yohimbine binding after amphetaminein regions of minimal dopaminergic input, including frontalcortex, thalamus, pons, and cerebellum, suggesting that thisdisplacement may be ascribed to competition with endogenousnoradrenaline in these brain areas, where the distribution ofnoradrenergic projections and α2R are known to coincide.28,29

The dense binding in both frontal cortex and striatum could beascribed, in part, to an effect of partial volume, attributable to theproximity of these regions, together with the limited resolution ofa microPET tomograph. However, partial volume effects cannotfully explain the [11C]yohimbine binding in striatum, becauserecent in vivo studies in pigs with much larger brains show [11C]-yohimbine binding in caudate nucleus and putamen that is similarto the binding in cortical regions.5,6

Table 2. Plasma-free fractions at baseline and with amphetamine

Animal Baseline Post-amphetamine

fp fp

1 0.30 0.202 0.21 0.273 0.16 0.214 0.23 0.25Mean± s.e.m. 0.23± 0.03 0.23± 0.02

No significant differences of plasma-free fractions at baseline and withamphetamine challenge, as measured with plasma ultrafiltration (n= 4,P= 0.87). This confirms that the same VND estimate can be used for baselineand in response to amphetamine, as required by the Inhibition plot.The bold entries are made to improve the delivery of information so thatthe mean values can easily be separated from the standard error values.

Figure 5. (A–C) Linear fitting of acquisition data from 28 to85 minutes in comparison with 6 to 28 minutes. The comparisonof the regression results of the periods of 6 to 28 minutes (dashedcurves) and the later time points of 28 to 85 minutes (solid curves),to illustrate the differential properties of the three linearizedversions of one-compartment model. The linearizations describedby Reith et al (A) and by Gjedde et al (B), respectively, show that dataat times later than 28 minutes are nonlinear, indicating probableinfluence from radioactive metabolites, which is less visible on theLogan plot (C).

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This study successfully yielded estimates of receptor availabilityor BPND of [11C]yohimbine in rat brain in the absence of areference region. When the BPND declined significantly in responseto the amphetamine challenge, the Inhibition plot yielded the VNDvalue that must be known to calculate the BPND of radiotracerswith displaceable accumulation in all parts of the brain. Similarfindings were reported in recent PET studies in pigs, where [11C]-yohimbine binding was significantly displaced by competitionwith endogenous transmitters, as shown by a decline of the VT inresponse to acute amphetamine treatment,6 although the bindingpotential was not calculated because the magnitude of VND variedamong the few subjects studied.In contrast to the significant decline of [11C]yohimbine BPND

found in present study, an ex vivo study in rat brain showed thatthe binding of [3H]RX821002, a highly selective α2R antagonist,declined only in three cortical regions (frontopolar, anterior cingu-late, and frontal parietal cortices) in response to amphetamine,with no changes observed in other cortical or subcortical struc-tures.32 On the basis of previous characterizations of RX821002selectivity with equal affinity by α2AR and α2CR and a threefoldlower affinity by the α2B subtype,

33 in parallel with the presence ofα2A and α2C subtypes in both cerebral cortex and cerebellum,25

and the rich noradrenergic input from locus coeruleus,28 we arguethat the choice of cerebellum as reference would be at variancewith the presence of displaceable binding sites in cerebellum,which would lead to the underestimation of BPND.Since cyclosporine administration was used in this study to

block p-glycoprotein action at the BBB, the influence of potentialmetabolic interactions of cyclosporine, yohimbine, and ampheta-mine must be weighed. It has been reported that the half-life ofcyclosporine is dose-dependent, with a duration above 10 hourswhen treated with doses higher than 30mg/kg in rats.18 The doseused in this study (50 mg/kg) may saturate metabolic enzymesand compromise the metabolic degradation of yohimbine andamphetamine. Although direct pharmacological interaction betweencyclosporine and yohimbine has not been described, interactionof enzymatic metabolism is unlikely because degradation iscatalyzed by different rate-limiting enzymes. Metabolism of cyclo-sporine is primarily catalyzed by CYP3A enzyme,34 whileamphetamine primarily is metabolized by CYP2D6,35 such thatthe pharmacological action of amphetamine is not influenced byinteraction with cyclosporine metabolism. However, cyclosporineessentially is lipophilic, and reports in humans have indicated that98.5% of cyclosporine is bound to plasma proteins,36 which mayreduce the protein-binding capacity for yohimbine and amphe-tamine. Although it cannot be excluded that cyclosporine maypotentiate the bioavailability of yohimbine and amphetamine, thispotential interference would not compromise the binding proper-ties of yohimbine to α2R, nor the sensitivity to (endogenous)monoamines in this work. The decline of yohimbine binding withthe amphetamine challenge can be attributed to factors otherthan monoamine release, including a decrease of the pharmaco-logical action of cyclosporine, which was administered as a singledose (50 mg/kg). However, the long half-life of cyclosporine madeit reasonable for us to assume constant pharmacological actionthroughout the study. In contrast, yohimbine is peripherallymetabolized by oxidation to 11-hydroxy-yohimbine and 10-hydroxy-yohimbine, as catalyzed by the CYP2D6 and CYP3A4enzymes37 that degrade amphetamine and cyclosporine. It ispossible that the metabolism of yohimbine is competitivelysuppressed to a greater extent with amphetamine administrationthan at baseline. However, this potential difference of yohimbinemetabolism in the two conditions has minimal influence on theresults because the dynamic data from both conditions wereanalyzed at times of apparent steady state before as discussedabove. Central metabolism of yohimbine may also have an impacton the binding profile, but the expression of CYP450 enzymes inbrain tissue is only 1% of the expression in liver,38 in support of

the claim that peripheral metabolism is more dominant thancentral metabolism.As PET in humans generally is conducted without anesthesia, the

effects of anesthetics in animals must be considered carefully whenwe use the present findings to predict the results that would beobtained in future human PET studies of noradrenergic neuro-transmission. The use of isoflurane in the present study is apotential confound for two reasons. First, compared with the awakecondition, isoflurane evokes a substantial three- to fourfold increaseof noradrenaline in the rat preoptic area.39 However, as the durationof the microdialysis was only 50 minutes, it is not a simple matter topredict whether noradrenaline release remains stable or declineswith the prolonged anesthesia used in the present study. When weconsider the possibility that the effect of isoflurane on the level ofnoradrenaline could be constant, we would expect an under-estimation of the BPND in both the baseline and inhibition states,compared with an awake state. Isoflurane has been shown toenhance the pharmacological actions of amphetamine and poten-tiate the release of monoamines, which has affected the bindingof certain radioligands of dopamine receptors.40 Thus, althoughthe possibility that isoflurane may influence the magnitude ofnoradrenaline release and interfere with [11C]yohimbine bindingcannot be excluded, we argue that the binding properties of [11C]-yohimbine and its sensitivity to endogenous noradrenaline is notcompromised by persistently elevated noradrenaline.Altered α2R expression has been reported in a spectrum of brain

disorders, including postmortem analysis that demonstrated amarked increase of α2R in the brains of suicide victims.1 In futureinvestigations, [11C]yohimbine can be used to determine α2Rdensity in vivo. We suggest at least two consecutive PET radio-tracer acquisitions at high, followed by low, specific activity, withEadie–Hoffstee or Scatchard plot analysis of binding to quantifyreceptor density and affinity.In summary, [11C]yohimbine can be used to visualize binding to

α2R, and the binding is sensitive to noradrenaline and othermonoamine release. The Inhibition plot8,9 yields an estimate of theVND of [11C]yohimbine that overcomes the absence of a referenceregion of non-displaceable uptake. Unlike the Logan plot,11 thelinearized plots of Reith et al12 and Gjedde et al13 assist in theidentification of the onset and termination of true steady state ofthe dynamic acquisition data when plasma metabolism may inter-fere with the maintenance of steady state. In the future, the use of[11C]yohimbine with PET can be beneficial to in vivo investigationof α2R density and affinity, and noradrenaline and other mono-amine release in physiologic and pathologic states of the brain.

DISCLOSURE/CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGMENTSThe authors thank Mette Simonsen for excellent technical assistance.

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