RESEARCH ARTICLE

Small-Animal Single-Photon Emission Computed

Tomographic Imaging of the Brain Serotoninergic Systems

in Wild-Type and Mdr1a Knockout Rats

Noe Dumas, Marcelle Moulin-Sallanon, Nathalie Ginovart, Benjamin B. Tournier, Peggy Suzanne, Thomas Cailly,Frederic Fabis, Sylvain Rault, Yves Charnay, and Philippe Millet

Abstract

The pharmacokinetic properties of radiotracers are crucial for successful in vivo single-photon emission computed tomographic

(SPECT) imaging. Our goal was to determine if MDR1A-deficient animals could allow better SPECT imaging outcomes than wild-type

(WT) animals for a selection of serotoninergic radioligands. Thus, we compared the performances of 123I-p-MPPI, 123I-R91150, 123I-

SB207710, and 123I-ADAM radioligands, for imaging of their respective targets (5-hydroxytryptamine [5-HT]1A, 5-HT2A, 5-HT4, and

serotonin transporter [SERT]), in WT and Mdr1a knockout (KO) rats. With 123I-SB207710, virtually no SPECT signal was recorded in

the brain of WT or KO animals. For 123I-p-MPPI, low nondisplaceable binding potentials (BPND, mean 6 SD) were observed in WT (0.49

6 0.25) and KO (0.89 6 0.52) animals. For 123I-ADAM, modest imaging contrast was observed in WT (1.27 6 0.02) and KO (1.31 6 0.09)

animals. For 123I-R91150, the BPND were significantly higher in Mdr1a KO (3.98 6 0.65) animals compared to WT animals (1.22 6 0.26).

The pharmacokinetics of 123I-SB207710 and 123I-p-MPPI do not make them ideal tracers for preclinical SPECT neuroimaging. 123I-

ADAM showed adequate brain uptake regardless of Mdr1a expression and appeared suitable for preclinical SPECT neuroimaging in

both animal strains. The use of Mdr1a KO animals significantly improved the brain penetration of 123I-R91150, making this animal

strain an interesting option when considering SPECT neuroimaging of 5-HT2A receptors in rat.

I N THE PAST DECADE, intense research efforts have

been dedicated to the development of imaging systems

that meet the requirements of small-animal imaging in

terms of spatial resolution and detection sensitivity. These

efforts have led to the invention of multipinhole collimation

and advanced image reconstruction algorithms, enabling

the recording of submillimeter resolution single-photon

emission computed tomographic (SPECT) images in rats

and mice (see Nuyts and colleagues1 for a review on

multipinhole SPECT).

In the field of neuropsychiatry, conventional SPECT

has been used in humans to perform quantitative

measurement of brain neuroreceptor availability in vivo,

making this technique valuable for the study of neuro-

psychiatric disorders such as depression,2 schizophrenia,3

and feeding disorders.4 However, despite this demon-

strated potential in humans, the use of SPECT in

combination with animal models of neuropsychiatric

diseases has barely been investigated. The rarity of efficient

SPECT tracers for neuroreceptors is partly explaining this

situation, and this paucity in tracers is in turn partly due to

the stringency of the requirements on tracer pharmacoki-

netics for successful SPECT imaging.

The MDR1A transporters (also called P-glycoprotein or

Pgp) are members of the ABC transporter superfamily5 and

are known to influence the kinetics of many pharmaceu-

ticals,6 notably by preventing them from crossing the blood-

brain barrier, making them a potentially interesting target

for the modulation of the pharmacokinetics of SPECT

From the Clinical Neurophysiology and Neuroimaging Unit, Division of

Neuropsychiatry, Department of Psychiatry, University Hospitals of

Geneva, Switzerland; INSERM, Unit 1039, J. Fourier University, La

Tronche, France; Department of Psychiatry, University of Geneva,

Switzerland; Normandie Universite, France; Universite de Caen Basse-

Normandie, CERMN (EA 4258 - FR CNRS 3038 INC3M - SF 4206

ICORE) UFR des Sciences Pharmaceutiques, Bd Becquerel, F-14032

Caen, France; Morphology Unit, Division of Neuropsychiatry, Division of

Neuropsychiatry, Department of Psychiatry, University Hospitals of

Geneva, Switzerland.

Address reprint requests to: Philippe Millet, PhD, Service de Neuropsychiatrie

Unite de Neurophysiologie Clinique et Neuro-imagerie, Hopitaux

Universitaires de Geneve, Belle-Idee Chemin du Petit-Bel-Air, 2 Chene-

Bourg 1225, Geneva, Switzerland; e-mail: Philippe.Millet@hcuge.ch.

DOI 10.2310/7290.2013.00072

# 2014 Decker Publishing

Molecular Imaging, Vol 13, 2014. 1

tracers. A variety of pharmacologic inhibitors have been

developed to achieve the blockade of MDR1A transporters7;

however, in preclinical research, the use of knockout (KO)

animals is also an interesting option because it offers the

advantage of being a stable modification of the system,

which is more convenient than a transient blockade of

transporters by competitive inhibitors, when willing to

study the pharmacokinetics of tracers.

Existing neuroreceptor SPECT tracers of the serotonin-

ergic systems allow imaging in small animals with varying

degrees of success (see Paterson and colleagues for a

review8). Our efforts have been focused on the tracers

described hereafter. 123I-p-MPPI has shown some poten-

tial as a 5-hydroxytryptamine1A receptor (5-HT1AR)

tracer in in vitro and ex vivo studies in rodents9 as well

as in vivo in monkeys.10 The 5-HT1AR has, for example,

been shown to participate in the regulation of feeding

behavior11 or in depression.12 123I-R91150 is a selective 5-

HT2AR ligand and has been successfully used for SPECT

in the clinic.13 The 5-HT2AR is notably thought to play a

role in schizophrenia.14 123I-SB207710 has shown some

potential as a 5-HT4R tracer in vitro and ex vivo in

rodents as well as in vivo in nonhuman primates.15 The 5-

HT4R has been involved, for example, in cognitive

functions such as learning and memory.16 123I-ADAM is

a selective serotonin transporter (SERT) ligand17 and has

been successfully used in human SPECT studies.2 The

SERT is most notably known for its involvement in

depression.18

The main goal of this study was to compare the

performances of this selection of serotoninergic radio-

tracers in both wild-type (WT) and Mdr1a KO rats, to

determine if the use of this genetically modified animal

strain, with a more permissive blood-brain barrier, could

result in improved SPECT imaging outcomes for certain

tracers. Preliminary evaluations by ex vivo autoradiogra-

phy were performed to obtain reference images for the in

vivo SPECT images. The SPECT data obtained in WT

and KO animals were compared by means of binding

potentials. Additionally, modelization of the pharmacoki-

netics of 123I-R91150 using the Simplified Reference Tissue

Model (SRTM)19 was performed to provide insight into

the effects of MDR1A on this tracer’s kinetics.

Material and Methods

We followed the guidelines proposed by Stout and

colleagues20 to provide a detailed description of the

methods used in this work.

Animals

Male Sprague Dawley Mdr1a KO rats (SD-Abcba1tm1sage)21

and WT controls of the same background weighing 300 to

350 g were obtained from Sigma Advance Genetic

Engineering Labs (St. Louis, MO). Animals were housed

in groups, with a 12 hours light/12 hours dark cycle, with

water and food ad libitum. All animal experiments were

approved by the Swiss Cantonal Veterinary Office. The size

of the cohorts for the SPECT experiments can be seen in

Table 1. The ex vivo autoradiographic experiments were

performed on one subject per tracer. During SPECT scans,

the body temperature and respiration rate were monitored.

The body temperature was maintained at 37 6 1uC with a

thermostatically controlled heating blanket.

Chemicals

123I radioiodide was purchased from GE Healthcare

(Eindhoven, the Netherlands). SB207710 trimethyltin pre-

cursor and p-MPPI tributyltin precursor were obtained from

Eras Labo (Grenoble, France). ADAM tributyltin precursor

was obtained from Syntheval (Caen, France). R91150

precursor was synthesized by introducing 1-[3-(4-fluorophe-

noxy)propyl]-4-methylpiperidin-4-amine (0.40 g, 15 mmol),22

dichloromethane (8 mL), 4-amino-2-methoxybenzoic acid

(0.40 g, 18 mmol), 1-hydroxybenzotriazole (0.24 g, 15 mmol),

and 3-(ethyliminomethyleneamino)-N,N-dimethylpropan-1-

amine hydrochloride (0.34 g, 15 mmol) in a round-bottomed

flask. The mixture was cooled to 0uC, and triethylamine

(0.76 mL, 54 mmol) was added dropwise. The mixture was

Table 1. BPND at Equilibrium in Wild-Type and Knockout

Animals*

WT BPND Mdr1a KO BPND

Mean 6 SD n Mean 6 SD n

p

Value

123I-p-MPPI 0.493 6 0.253 4 0.887 6 0.524 4 .224123I-R91150 1.221 6 0.261 4 3.981 6 0.648 4 , .002123I-SB207710 0.087 6 0.032 3 0.111 6 0.055 2 .568123I-ADAM 1.271 6 0.018 4 1.311 6 0.085 4 .393

BPND 5 nondisplaceable binding potential; KO 5 knockout; WT 5 wild

type.

*Determined as the ratio of mean activity in a tracer-relevant, receptor-rich

region over the mean activity in the tracer-poor cerebellum region, minus 1.

The target-rich regions, tracer and target wise, were 123I-p-MPPI/5-HT1AR,

hippocampus; 123I-R91150/5-HT2AR, orbitofrontal cortex; 123I-SB207710/

5-HT4R, olfactory tubercles; 123I-ADAM/SERT, midbrain; p value from an

unpaired t-test.

2 Dumas et al

allowed to reach room temperature, stirred overnight, washed

with water (3 3 20 mL), washed with brine (3 3 20 mL), dried

over MgSO4, and evaporated. The crude was then purified by

column chromatography using AcOEt/MeOH/NEt3 (99/1/1)

as the eluant to afford 4-amino-N-{1-[3-(4-fluorophenoxy)

propyl]piperidin-4-yl}-2-methoxybenzamide22 (R91150 pre-

cursor) with 40% yield and to conform to analytical standards.

All other chemicals were purchased from Sigma-Aldrich

(Buchs, Switzerland), with the highest purity available, and

were used without any further purification.

Radiotracer Preparation

All radiotracers were obtained by incubation of the

specified reaction mixtures (see below) for 20 minutes at

room temperature. Purification was conducted directly

from the reaction mixture on a high-performance liquid

chromatography (HPLC) system (Knauer GmbH, Berlin,

Germany) with a reversed-phase column (Bondclone C18

10 mm 300 3 7.8 mm, Phenomenex, Schlieren,

Switzerland) and eluted with the specified mobile phase

(see below) at a flow rate of 3 mL/min. Fractions

containing the radiolabeled compound of interest were

collected and concentrated using a rotary evaporator, and

the final product was diluted in saline prior to animal

administration. During the HPLC run, ultraviolet absor-

bance and radioactivity were monitored, allowing for the

measurement of specific activity thanks to calibration

curves established with the cold reference compound.

Specific activities of preparations were always above 20%

of 123I carrier-free specific activity (ie, above 1,760 MBq/nmol).

Radiochemical purities, assessed by HPLC, were above 98%.

123I-p-MPPI

Fifty micrograms of p-MPPI precursor in 50 mL ethanol was

mixed with 50 mL of HCl 1 M, 15 mL of carrier-free 123I

sodium iodide (10 mCi) in 0.05 M NaOH, and 50 mL of 3%

H2O2. Purification was achieved by HPLC with a 10-minute

gradient of 30 to 60% acetonitrile (ACN) in water with

7 mM H3PO4. Radiotracer retention time was 8.8 minutes.

123I-R91150

Three hundred micrograms of R91150 precursor in 3 mL

ethanol was mixed with 3 mL of glacial acetic acid, 15 mL of

carrier-free 123I sodium iodide (10 mCi) in 0.05 M NaOH,

and 3 mL of 30% H2O2. 123I-R91150 was isolated by an

isocratic HPLC run (ACN/water 50/50, 10 mM acetic acid

buffer pH 5). The radiotracer retention time was 11.0

minutes.

123I-SB207710

Fifty micrograms of trimethyl stannyl precursor in 50 mL

ethanol was mixed with 50 mL of 1 M HCl, 15 mL of

carrier-free 123I sodium iodide (10 mCi) in 0.05 M NaOH,

and 50 mL of 3% H2O2. 123I-SB207710 was isolated by

isocratic HPLC (ACN/water 30/70, 7 mM H3PO4). The

retention time of 123I-SB207710 was 9.3 minutes.

123I-ADAM

Fifty micrograms of ADAM precursor in 5 mL ethanol was

mixed with 5 mL of 1 M HCl, 15 mL of carrier-free 123I

sodium iodide (10 mCi) in 0.05 M NaOH, and 5 mL of

30% H2O2. 123I-ADAM was isolated by an isocratic HPLC

run (ACN/water 60/40, 10 mM acetic acid buffer pH 5).

The retention time was 12 minutes.

Dynamic SPECT Imaging

While maintained under isoflurane anesthesia (1.8–2.5%)

with pure oxygen and installed in a U-SPECT-II imaging

system (MILabs, Utrecht, the Netherlands), the animals

received a bolus injection in the tail vein with 55 MBq of123I-labeled radiotracer in 0.6 mL saline. The imaging

system was fitted with a general purpose rat and mouse

collimator (GP-RM, 75 pinholes of 1.5 mm diameter). The

scan volume was kept to the minimum around the skull,

and image frames of a length of 1 minute were acquired.

Additionally, a phantom consisting of a 2 mL plastic tube

containing a known activity concentration was scanned

under the same conditions for the purpose of activity

calibration. SPECT tomograms were reconstructed with a

pixel ordered subsets expectation maximization algo-

rithm23 (P-OSEM, 0.4 mm voxels, 4 iterations, 6 subsets)

using MILabs proprietary software and finally smoothed

with a gaussian filter of 0.8 mm full width half maximum.

SPECT data were corrected for radioactive decay; no

correction of attenuation and scatter was applied.

Data Coregistration and Analysis

SPECT data were analyzed with PMOD software (PMOD

Technologies, Zurich, Switzerland). SPECT data were

manually coregistered to a region of interest (ROI)

template for the rat brain (atlas provided in PMOD: ‘‘Px

Rat (W. Schiffer)’’24) (Figure 1). The mean activity in each

Mdr1a KO Rats for 5-HT Receptor SPECT 3

ROI was calculated for each frame to obtain time-activity

curves. Left and right ROI of paired structures were

considered as a whole, and the subregions of hippocampus

and cerebellum were pooled: hereafter, hippocampus ROI

refers to the combination of the template’s anterior and

posterior hippocampus ROI and cerebellum ROI refers to

the combination of the gray and white matter cerebellum

ROI. As a means of evaluating the imaging ability of tracers

and to compare their performance in WT and KO animals,

an estimate of the nondisplaceable binding potential

(BPND)25 was obtained by calculating the value of the

specific uptake ratio (SUR) during pseudoequilibrium. SUR

is the ratio between the mean activity in a tracer-relevant

receptor-rich region and in the tracer-poor cerebellum

region, minus 1. The pseudoequilibrium interval was

defined as the period during which SUR values remained

stable, as described by Catafau and colleagues.26 The target-

rich regions, tracer and target wise, were 123I-p-MPPI/5-

HT1AR, hippocampus; 123I-R91150/5-HT2AR, orbitofrontal

cortex; 123I-SB207710/5-HT4R, olfactory tubercles; and 123I-

ADAM/SERT, midbrain.

123I-R91150 Pharmacokinetics Analysis

In the case of 123I-R91150, pharmacokinetic parameters

were calculated from time-activity curves using the

SRTM19 in PMOD software, with cerebellum as a re-

ference region. The SRTM relies on three parameters: R1

is the relative delivery to ROI compartments versus

reference region (R1 5 K1/K19), k2 is the efflux constant

from brain to plasma, and BPND SRTM is the non-

displaceable binding potential derived from the SRTM

(BPND SRTM 5 k3/k4).

Ex Vivo Autoradiography

WT rats were anesthetized with isoflurane, injected in the

tail vein with < 10 MBq of radiotracer and sacrificed

by decapitation after 30 minutes for 123I-SB207710 and123I-p-MPPI and after 60 minutes for 123I-R91150 and

123I-ADAM. The choice of the time points was based on

preliminary experiments conducted at 30 and 60 minutes

postinjection. The brains were quickly removed and frozen

in precooled isopentane at 220uC. Twenty micrometer

thick coronal sections were collected with a cryomicro-

tome (Leica) at 220uC. The sections were mounted on

glass slides, air-dried at room temperature, and exposed to

phosphor imaging plates overnight (Fuji Photo Film Co.,

Tokyo, Japan). Finally, the plates were scanned with a Fuji

Bio-Imaging Analyzer BAS 1800II scanner (Fuji Photo

Film Co.), at 50 mm resolution, to obtain the ex vivo

autoradiograms. Image quantification was performed

using ImageJ software (National Institutes of Health,

Bethesda, MD) to obtain ex vivo SURs, which were

calculated as the ratio between a target region and the

tracer-poor cerebellum region, minus 1.

Results

When evaluated by ex vivo autoradiography in WT rats

(Figure 2D), 123I-p-MPPI showed marked uptake (ex

vivo SUR at 30 minutes postinjection) in hippocampus

(0.43) and septum (0.37) and more modest uptake in

amygdala (0.29), cortex (0.14), and hypothalamus (0.17).

Meanwhile, cerebellum was showing a low level of tracer

uptake. When evaluated in vivo, the time-averaged SPECT

images obtained with 123I-p-MPPI showed a slightly

superior imaging contrast in Mdr1a KO animals (Figure

2C) in comparison with WT animals (Figure 2B), as

evidenced by the BPND calculated in hippocampus versus

cerebellum (see Table 1), which was very small in WT

animals (0.49 6 0.25, mean 6 SD) and was higher but not

significantly different in KO animals (0.89 6 0.52, mean 6

SD, p 5 .224). The regional time-activity curves (Figure

2E) showed that the tracer brain uptake was approximately

doubled in KO rats; however, the clearance from the brain

remained fast in KO, and, as a result, activity levels in the

5-HT1AR-rich hippocampus region reached the levels

observed in the 5-HT1AR-poor cerebellum region within

an hour.

Figure 1. Sectional views of Schifferand colleagues’ rat brain region ofinterest atlas24 with a reference MRItemplate.

4 Dumas et al

When evaluated by ex vivo autoradiography in WT rats

(Figure 3D), 123I-R91150 showed highest uptake (ex vivo

SUR at time 60 minutes postinjection) in frontal regions

such as orbitofrontal cortex (0.70) and more moderate

uptake in the rest of the cortex (0.52), midbrain (0.44),

and hypothalamus (0.36). Meanwhile, cerebellum was

showing a very low level of tracer uptake. When evaluated

by in vivo imaging in WT rats, SPECT images showed low

uptake of 123I-R91150 in brain and relatively higher uptake

in the pituitary gland and harderian glands (Figure 3B). In

KO animals, however, SPECT images showed a marked

uptake in frontal regions and the rest of the cortex and

relatively lower uptake in cerebellum (Figure 3C). The

time-activity curves showed marked differences in tracer

kinetics between KO and WT animals, with an approxi-

mately threefold increase in tracer uptake in the orbito-

frontal cortex region of KO rats (Figure 3E). The BPND

calculated for 123I-R91150 in the orbitofrontal cortex

region versus cerebellum (see Table 1) was modest in WT

animals (1.22 6 0.26, mean 6 SD) and was significantly

higher in Mdr1a KO animals (3.98 6 0.65, mean 6 SD,

p , .002) animals. The effect of MDR1A proteins on 123I-

R91150 kinetics was also apparent in the variation of

SRTM parameters derived for the orbitofrontal cortex

region with cerebellum as reference, as Table 2 also shows

an increase in BPND SRTM by a factor of around 3 in KO

compared to WT animals. However, no significant

variations were observed between the two animal strains

in brain efflux rates (k2) or target versus reference delivery

rates (R1). Fitting other ROIs using SRTM also revealed

markedly higher BPND SRTM in other frontal regions of

KO rats, such as medial prefrontal cortex and frontal

cortex (Figure 4). However, the BPND SRTM observed in

the hypothalamus and entorhinal cortex regions was higher

in WT rats compared to KO animals. Overall, the in vivo123I-R91150 SPECT data from KO rats (see Figure 3C)

visually correlated well with ex vivo autoradiograms (see

Figure 3D), whereas SPECT images in WT rats were

dwarfed by activity accumulation in the harderian glands

and in the pituitary gland (see Figure 3B).

Figure 2. 123I-p-MPPI brain distribution study. Anatomic atlas templates adapted from the Paxinos and Watson atlas41 (A), time-averaged imagesof representative SPECT scans between 15 and 35 minutes after tracer injection in wild-type (WT) (B) and Mdr1a knockout (KO) rats (C), ex vivoautoradiograms obtained 30 minutes after tracer injection in WT rats (D), and regional time-activity curves measured by SPECT after tracerinjection (E). Color scales are relative and relevant to individual experiments. Acb 5 accumbens nucleus; Am 5 amgydala; BS 5 brainstem;Cb 5 cerebellum; Cx 5 cortex; HG 5 harderian gland; Hip 5 hippocampus; Hyp 5 hypothalamus; MB 5 midbrain; OFC 5 orbitofrontal cortex;Pit 5 pituitary gland; Spt 5 septum; Thal 5 thalamus.

Mdr1a KO Rats for 5-HT Receptor SPECT 5

When evaluated by ex vivo autoradiography in WT

rats (Figure 5D), 123I-SB207710 accumulated markedly

(ex vivo SUR at time 30 minutes postinjection) in the

olfactory tubercles (1.48) and globus pallidus (1.13) and

to a lesser extent in the hypothalamus (0.75), accumbens

nucleus (1.14), caudate putamen (0.78), and hippocam-

pus (0.58). Meanwhile, cerebellum showed a very low

level of tracer uptake. After injection of 123I-SB207710,

virtually no specific SPECT signal was detected in vivo

in the brain of either WT (Figure 5B) or Mdr1a KO

animals (Figure 5C). In Table 1, the BPND calculated

from mean activity in the olfactory tubercle region versus

cerebellum was negligible in WT animals (0.09 6 0.03,

mean 6 SD) and not significantly different from that

observed in Mdr1a KO animals (0.11 6 0.06, mean 6 SD,

p 5 .568). Moreover, the time-activity curves of any

ROI were practically indistinguishable from each other

(Figure 5E).

When evaluated by ex vivo autoradiography in WT

rats (Figure 6D), 123I-ADAM showed marked uptake

(ex vivo SUR at 60 minutes postinjection) in hypothala-

mus (0.88) and midbrain (0.93) and a slightly dimmer

uptake in numerous other regions, including olfactory

tubercles (0.76), septum (0.78), thalamus (0.72), hippo-

campus (0.52), globus pallidus (0.81), and pons (0.71).

Meanwhile, cerebellum was showing a very low level of

tracer uptake. When evaluated by in vivo imaging, time-

averaged 123I-ADAM SPECT images showed significant

Figure 3. 123I-R91150 brain distribution study. Anatomic atlas templates adapted from the Paxinos and Watson atlas41 (A), average SPECT imagesbetween 100 and 120 minutes after tracer injection in wild-type (WT) (B) and Mdr1a knockout (KO) rats (C), ex vivo autoradiograms obtained 60minutes after tracer injection in WT rats (D), and regional time-activity curves measured by SPECT after tracer injection (E). Color scales arerelative and relevant to individual experiments. Acb 5 accumbens nucleus; Am 5 amgydala; BS 5 brainstem; Cb 5 cerebellum; Cx 5 cortex;HG 5 harderian glands; Hip 5 hippocampus; Hyp 5 hypothalamus; MB 5 midbrain; OFC 5 orbitofrontal cortex; Pit 5 pituitary gland;Spt 5 septum; Thal 5 thalamus.

Table 2. 123I-R91150 SRTM Parameters Obtained in

Orbitofrontal Cortex with Cerebellum as a Reference Region

WT (n 4) Mdr1a KO (n 5 4)

Mean 6 SD Mean 6 SD p Value

R1 0.487 6 0.033 0.525 6 0.064 .332

k2 (min–1) 0.046 6 0.003 0.042 6 0.003 .108

BPND SRTM 0.959 6 0.039 2.656 6 0.234 , .001

BPND 5 nondisplaceable binding potential; KO 5 knockout; SRTM 5

Simplified Reference Tissue Model; WT 5 wild type.

p Value from an unpaired t-test.

6 Dumas et al

brain uptake with similar levels in both WT (Figure 6B)

and KO animals (Figure 6C) and exhibited preferential

uptakes in hypothalamus and midbrain and lower uptake

in cerebellum, which is in accordance with the ex vivo

autoradiograms (see Figure 6D). The BPND calculated

from mean activity in the midbrain ROI versus cerebellum

(see Table 1) was moderate in WT animals (1.27 6 0.02,

mean 6 SD) and not significantly different from that

observed in Mdr1a KO animals (1.31 6 0.09, mean 6 SD,

p 5 .393). The regional time-activity curves (Figure 6E)

showed no clear difference in the kinetics of 123I-ADAM

between the two animal strains.

Figure 5. 123I-SB207710 brain distribution study. Anatomic atlas templates adapted from the Paxinos and Watson atlas41 (A), average SPECTimages between 30 and 60 minutes after tracer injection in wild-type (WT) (B) and Mdr1a knockout (KO) (C) rats, ex vivo autoradiogramsobtained 30 minutes after tracer injection in WT rats (D), and regional time-activity curves measured by SPECT after tracer injection (E). Colorscales are relative and relevant to individual experiments. Acb 5 accumbens nucleus; BS 5 brainstem; Cb 5 cerebellum; CPu 5 caudate putamen;Cx 5 cortex; GP 5 globus pallidus; HG 5 harderian gland; Hip 5 hippocampus; Hyp 5 hypothalamus; MB 5 midbrain; OFC 5 orbitofrontalcortex; Pit 5 pituitary gland; Thal 5 thalamus; Tu 5 olfactory tubercles.

Figure 4. 123I-R91150 nondisplace-able BP (mean 6 SD) observed byfitting the Simplified Reference TissueModel (SRTM) to time-activity curvesextracted from WT (n 5 4) and KO (n5 4) regions of interest adapted fromSchiffer and colleagues’ ROI tem-plate.24 Acb 5 accumbens nucleus;BPND 5 nondisplaceable bindingpotential; CgC 5 cingulate cortex;CPu 5 caudate putamen; EC 5

entorhinal cortex; FC 5 frontal cor-tex; Hip 5 hippocampus; Hyp 5

hypothalamus; MC 5 motor cortex;MPFC 5 medial prefrontal cortex;OFC 5 orbitofrontal cortex; SC 5

somatosensory cortex; Spt 5 septum;Thal 5 thalamus; Tu 5 olfactorytubercles.

Mdr1a KO Rats for 5-HT Receptor SPECT 7

Discussion

The 5-HT1AR tracer 123I-p-MPPI gave ex vivo autoradio-

gram results in agreement with the work of others9;

however, the quality of the SPECT images obtained in

vivo was hampered by the tracer short residence time in

target-rich regions. Overall, the use of this tracer for small-

animal SPECT neuroimaging will be hindered by its fast

washout from target brain regions, in both WT and Mdr1a

KO rats. The short retention time of tracer in the target-

rich regions could be explained by a fast dissociation rate

from the target receptor. Nevertheless, the regional time-

activity curves showed a pattern of distribution that

coincided with the tracer distribution observed ex vivo,

with higher uptake in hippocampus compared to cere-

bellum. The 18F-MPPF, a positron emission tomographic

(PET) analogue of 123I-p-MPPI, has been successfully used

for 5-HT1AR imaging in small animals,27 and other

investigators have reported a two- to threefold increase

in its brain uptake, using ex vivo autoradiography, when

administered to Mdr1a KO mice.28 Our in vivo SPECT

data show a similar increase in the iodinated 123I-p-MPPI

ligand brain uptake in Mdr1a KO animals, indicating that

this ligand is subject to efflux transport by MDR1A

proteins, just like its fluoride analogue. However, this

enhanced brain penetration did not significantly improve

the imaging contrast observed by SPECT in KO animals.

Our data for the 5-HT2A receptor ligand 123I-R91150

show that the in vivo quantification of 5-HT2AR in WT

animals is compromised by the low brain uptake of tracer

and by the strong accumulation of activity that occurs in

harderian glands and the pituitary gland, making neigh-

boring brain regions prone to spillover and falsely elevated

binding potentials. In contrast, SPECT data from Mdr1a

KO rats corresponded remarkably well with ex vivo

autoradiograms, allowing a more trustworthy quantifica-

tion of 5-HT2ARs.

The ex vivo distribution was in agreement with the

literature,29 with a pronounced binding in cortex,

especially in frontal cortical regions. However, the brain

uptake in WT animals was relatively low compared to that

in KO animals, in which the tracer kinetics was radically

Figure 6. 123I-ADAM brain distribution study. Anatomic atlas templates adapted from the Paxinos and Watson atlas41 (A), average SPECT imagesbetween 70 and 90 minutes after tracer injection in wild-type (WT) (B) and Pgp knockout (KO) rats (C), ex vivo autoradiograms obtained60 minutes after tracer injection in WT rats (D), and regional time-activity curves measured by SPECT after tracer injection (E). Color scales arerelative and relevant to individual experiments. Acb 5 accumbens nucleus; BS 5 brainstem; Cb 5 cerebellum; Cx 5 cortex; HG 5 harderiangland; Hip 5 hippocampus; Hyp 5 hypothalamus; MB 5 midbrain; OFC 5 orbitofrontal cortex; Pit 5 pituitary gland; Thal 5 thalamus.

8 Dumas et al

modified with an approximately threefold increase in brain

uptake. As a result, the correlation between the tracer

distributions observed by SPECT versus ex vivo auto-

radiography was higher in KO animals compared to WT

animals (determination coefficient R2 5 .9 in KO versus

R2 5 .2 in WT animals).

However, the comparison of pharmacokinetic para-

meters derived for 123I-R91150 using SRTM in both WT

and Mdr1a KO rats surprisingly showed no significant

variation in the tracer efflux rate from brain tissue (k2):

indeed, given the function of the MDR1A proteins,

consisting in transporting substrates outside from brain

tissue, a decrease in k2 value in KO animals could have

been expected. Other studies have suggested that MDR1A

proteins have the ability to extrude substrates directly from

the plasma membrane,30 thus effectively interfering

simultaneously with both tissue uptake rate (K1) and

tissue efflux rate (k2). Moreover, the time scale on which

the efflux transport occurs at the molecular level is much

more rapid than the time resolution of the SPECT

acquisition,31 making it impossible to completely distin-

guish between a decrease in k2 and an increase in K1, due,

for instance, to MDR1A inhibition or loss of function.

Thus, it is plausible that MDR1A absence predominantly

induces an increase in K1 and would explain why no

significant alteration in k2 is detected when comparing KO

and WT animals. However, the SRTM only allows access

to the ratio of delivery rates in target ROI over reference

region (R1). In our case, we observed no change in the

value of R1, indicating that delivery rates in both target

and reference regions were affected in a proportional way

by MDR1A. This observation is in agreement with the

work of Seegers and colleagues,32 which showed that

MDR1A is expressed at comparable levels in frontal cortex

and cerebellum. Nevertheless, the time-activity curves

clearly showed higher tracer uptake in both cerebellum

and orbitofrontal cortex of KO animals, thus strongly

suggesting an increased delivery rate in the KO condition.

Further pharmacokinetic modeling experiments with

blood sampling will be needed to precisely quantify the

impact of MDR1A protein expression on tissue brain

delivery (K1). Finally, the BPND SRTM values were higher

in most ROI of Mdr1a KO compared to WT animals,

except in ROI adjacent to the harderian glands or the

pituitary gland. Indeed, in the case of WT animals, these

extracerebral organs took up a significant amount of

activity; thus, activity spillover from these glands could

explain why superior BPND SRTM were recorded in the

entorhinal cortex or hypothalamus of WT animals.

Nevertheless, these regions set apart, our data clearly show

that 123I-R91150 brain uptake is delivery limited: regional

uptake depends mostly on the crossing of the blood-brain

barrier (K1).

One other spectacular effect of the absence of MDR1A

proteins is the disappearance of signal from the harderian

glands in KO animal 123I-R91150 SPECT scans. These

glands, however, are indeed anatomically present in the

KO strain; thus, our results suggest that MDR1A proteins

are expressed in the harderian glands, where they are

responsible for a nonspecific accumulation of the tracer

and/or its radiometabolites. The presence of MDR1A

proteins in the harderian glands would not be surprising as

the Mdr1a gene is known for its widespread expression,33

and it has been reported that at least one other transporter

of the same ABC superfamily is expressed in the harderian

glands,34 where it plays a role in the excretion of

protoporphyrin metabolites.

The very clear difference in the distribution pattern of123I-R91150 between Mdr1a KO and WT animals also

suggests that this tracer might be an interesting tool to

quantify the activity of MDR1A transporters in vivo.

Indeed, 123I-R91150 has already been successfully used in

humans in a number of studies,8 and it has been reported

to undergo only little metabolization in humans26; more-

over, we reported here a significant threefold increase in

brain signal in Mdr1a KO animals, and others even

reported a fivefold increase in brain uptake with the use of

50 mg/kg cyclosporine as an MDR1A proteins inhibitor.35

According to the criteria defined by Kannan and

colleagues,30 these properties suggest that 123I-R91150

would be a suitable SPECT tracer to quantify efflux

proteins function in vivo in humans. Nowadays, the

assessment of efflux protein function is attracting growing

attention as a means to predict the response to certain

pharmacologic treatments, notably anticancer therapies,

and to study the influence of blood-brain barrier integrity

on brain physiology.36 Thus, the assessment of MDR1A

transporters function could one day be useful for clinical

practice. Currently, such studies are usually performed

with PET tracers, most frequently using [11C]-verapamil,37

but in the future, the use of a SPECT imaging alternative

might be more convenient as SPECT is a more widespread,

cheaper, and slightly less cumbersome technique than PET.

The 5-HT4R tracer 123I-SB207710 gave ex vivo auto-

radiograms in agreement with the results found in the

literature,38 showing a marked labeling of 5-HT4R-rich

regions; however, this tracer did not yield enough cerebral

signal in either WT or Mdr1a KO rats to be useful for in

vivo SPECT scans. Other evidence suggests that this

tracer brain uptake is limited by different causes than

Mdr1a KO Rats for 5-HT Receptor SPECT 9

MDR1A-mediated efflux: indeed, it has been reported by

Marner and colleagues that the 123I-SB207710 PET

analogue 11C-SB207145 is quickly metabolized in human

plasma and that this degradation is prevented by plasmatic

esterase inhibitors.39 Both 123I-SB207710 and its PET

analogue are esters, and in an ex vivo experiment, we

observed a threefold increase in the rat brain uptake after

pretreatment with plasmatic esterase inhibitors (data not

shown). Although the SPECT data showed similar brain

uptake in both animal strains, it cannot be concluded from

this experiment whether this tracer is a substrate of

MDR1A proteins or not as the recorded signals were not

significantly above noise. Taken together, these data

indicate that, whether or not 123I-SB207710 undergoes

efflux by MDR1A, this is not the main reason why its use

in small-animal SPECT neuroimaging is intractable. It

shows, however, that 123I-SB207710 in vivo brain penetra-

tion is significantly reduced, indirectly, owing to its fast

metabolism by plasmatic esterase.

The SERT tracer 123I-ADAM gave ex vivo autoradiogram

results in accordance with the literature,17 with intense

binding notably in thalamus and hypothalamus among

other brain areas known to be enriched in 5-HT neuronal

elements.40 The SPECT scans showed sufficient brain

uptake, allowing for the recording of meaningful images in

both WT and Mdr1a KO animals. No significant difference

in 123I-ADAM pharmacokinetics was observed in Mdr1a KO

rats, indicating that it is not a substrate for this transporter.

Overall, this tracer showed favorable kinetics for SPECT

neuroimaging in both WT and Mdr1a KO animals.

Conclusion

The advent of small-animal SPECT scanners has offered

unprecedented opportunities for the noninvasive study of

animal models of disease. However, application of this

technology to the field of preclinical neuroimaging is

limited by the availability of tracers with suitable

pharmacokinetics. We reported here on the evaluation of

a selection of serotoninergic tracers of potential interest for

small-animal SPECT neuroimaging. Tracers such as 123I-

SB207710 and 123I-p-MPPI are efficient tools for ex vivo or

in vitro studies, but their pharmacokinetics do not make

them ideal tracers for preclinical SPECT neuroimaging.

The SERT tracer 123I-ADAM showed decent brain uptake

regardless of Mdr1a expression and appeared suitable for

preclinical SPECT neuroimaging in WT animals. Although123I-R91150 uptake was readily detectable in SPECT scans

of WT animals, tracers that are MDR1A substrates, such as123I-R91150, might benefit from the use of Mdr1a KO

animals and see a significant increase in their brain

penetration, making Mdr1a KO strains an interesting

option when considering SPECT neuroimaging studies in

rats. This study also suggests that 123I-R91150 could be a

convenient tool for assessing the function of MDR1A

proteins at the blood-brain barrier.

Acknowledgment

Financial disclosure of the authors: This work was supported

by the Swiss National Science Foundation (grant no.

310030_120369), the Geneva Neuroscience Centre, and the

Ernst and Lucie Schmidheiny Foundation. The authors are

grateful for the contributions of the BioPark Platform in

Archamps, the Fondation Caisse d’Epargne Rhone-Alpes, the

ABC laboratory of the European Scientific Institute (ESI) and

the Association IFRAD Suisse, which was created in 2009 at the

initiative of the Fondation pour la Recherche sur Alzheimer

(formerly IFRAD France). We also thank the Conseil Regional

de Basse Normandie for its financial support.

Financial disclosure of reviewers: None reported.

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