Live imaging of neuronal connections by magnetic resonance: Robust transport in the...

Reward circuitry is perturbed in the absence of the serotonin transporter

Elaine L. Bearer a,b, Xiaowei Zhang a, Davit Janvelyan a, Benoit Boulat a, Russell E. Jacobs a,!a Biological Imaging Center, Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USAb Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02906, USA

a b s t r a c ta r t i c l e i n f o

Article history:Received 13 October 2008Revised 10 March 2009Accepted 11 March 2009Available online xxxx

Keywords:Serotonin transporterKnock-outManganese enhanced MRISynaptic transmissionTransportMorphometryStatistical parametric mappingDTIMRS

The serotonin transporter (SERT) modulates the entire serotonergic system in the brain and in!uences boththe dopaminergic and norepinephrinergic systems. These three systems are intimately involved in normalphysiological functioning of the brain and implicated in numerous pathological conditions. Here we use high-resolution magnetic resonance imaging (MRI) and spectroscopy to elucidate the effects of disruption of theserotonin transporter in an animal model system: the SERT knock-out mouse. Employing manganese-enhanced MRI, we injected Mn2+ into the prefrontal cortex and obtained 3D MR images at speci"c timepoints in cohorts of SERT and normal mice. Statistical analysis of co-registered datasets demonstrated thatactive circuitry originating in the prefrontal cortex in the SERT knock-out is dramatically altered, with a biastowards more posterior areas (substantia nigra, ventral tegmental area, and Raphé nuclei) directly involvedin the reward circuit. Injection site and tracing were con"rmed with traditional track tracers by opticalmicroscopy. In contrast, metabolite levels were essentially normal in the SERT knock-out by in vivo magneticresonance spectroscopy and little or no anatomical differences between SERT knock-out and normal micewere detected by MRI. These "ndings point to modulation of the limbic cortical–ventral striatopallidal bydisruption of SERT function. Thus, molecular disruptions of SERT that produce behavioral changes also alterthe functional anatomy of the reward circuitry in which all the monoamine systems are involved.

© 2009 Elsevier Inc. All rights reserved.

Introduction

The serotonin transporter (SERT) regulates serotonin levels in thesynaptic cleft through active uptake from the extracellular space (Li,2006) and is encoded by a single gene in mouse (Bengel et al., 1997).SERT is the target of a large class of psychoactive drugs used in anumber of anxiety disorders, as well as drugs of abuse such as cocaineand methylenedioxymethamphetamine (MDMA). Moreover, SERT isthe principal regulator of the entire serotonergic system (Murphyet al., 2004) and dysregulation of SERT gene expression is implicatedas a risk factor for a number of affective disorders (Gainetdinov andCaron, 2003; Murphy et al., 2004; Murphy et al., 2001).

Mouse knock-outs for SERT and the other two monoaminetransporters, dopamine transporter (DAT) and norepinephrine trans-porter (NET), have been used extensively to study the pharmacolo-gical, behavioral, and anatomic consequences of disruption (Caron,1999; Dykstra et al., 2003; Gainetdinov and Caron, 2003; Gainetdinovet al., 2002; Hall et al., 2004; Kita et al., 2003; Numachi et al., 2007;Reith, 2005; Rocha, 2003; Torres and Caron, 2005; Uhl, 2003; Uhl etal., 2002; Xu et al., 2000; Yamashita et al., 2006). Single and multipleknock-outs of the monoamine transporters have been especiallyuseful in investigations aimed at linking the molecular actions andbehavioral consequences of drugs of abuse (Sora et al., 2001; Uhl and

Lin, 2003). These studies have generated a wealth of informationabout speci"c aspects of these model systems at the molecular level(e.g. up/down regulation of monoamine receptors in response touptake inhibition, altered concentrations of monoamine metabolitesand related molecules), and at the behavioral level (e.g. conditionedplace preference, locomotor response, drug induced response)(Homberg et al., 2007; Li et al., 2003; Numachi et al., 2007; Rocha,2003; Shen et al., 2004).

Here we explore brain circuitry in SERT knock-out mice to linkmolecular alterations to anatomical and behavioral observations. SERTknock-out mice exhibit avoidance and hyperarousal and are morevulnerable to stress than wild-type mice (Adamec et al., 2008). Inaddition, SERT mice display an initial impairment of food- andcocaine-self-administration (Thomsen et al., 2009). A number ofbehaviors, including addiction, anxiety, aggression, and affectivedisorders such as depression, have been linked to anatomical brainregions, speci"cally the limbic cortical–ventral striatopallidal circuitry(Berton and Nestler, 2006; Everitt and Robbins, 2005; Murphy andLesch, 2008; Nelson and Trainor, 2007; Robbins and Everitt, 2002).The prefrontal cortex (PFC) is believed to perform executive functionsin this circuit (Berton and Nestler, 2006; Robbins and Everitt, 2002)where it has been implicated in working memory, affect, tempera-ment, attention, response initiation and management of autonomiccontrol and emotion (Canli et al., 2001; Groenewegen and Uylings,2000; Groenewegen et al., 1997; Hagen et al., 2002; Zald et al., 2002).The PFC is also densely innervated by serotonergic neurons arising in

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! Corresponding author. Fax: +1 626 449 5163.E-mail address: [email protected] (R.E. Jacobs).

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Please cite this article as: Bearer, E.L., et al., Reward circuitry is perturbed in the absence of the serotonin transporter, NeuroImage (2009),doi:10.1016/j.neuroimage.2009.03.026

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the median Raphé nuclei of the brain stem (Puig et al., 2004). Recentwork in monkey demonstrates that injection of Mn2+ into the PFCtraces expected pathways deeper into the brain (Simmons et al.,2008). How these pathways might be altered by a loss of SERT activityremains an open question.

Due to the importance of the PFC and its connections, and theexpected involvement of serotonin in this circuit, we chose to exploitthe SERT knock-out mouse to probe circuitry originating in the PFCwith and without SERT activity. After stereotaxic injection of nanolitervolumes of MnCl2 into the PFC of knock-out and normal control mice,we followed the time course of Mn2+ uptake, transport, andaccumulation over the "rst 24 h post-injection by sequential high-resolution MRI. We also employed MRS to compare metabolite levelsin living brains of normal versus SERT knock-out mice. After in vivoMR imaging, we "xed the brains and used diffusion tensor imaging toobtain additional structural information and then processed them forhistology and analysis by microscopy. Co-injection of !uorescenttracer with the Mn2+ allowed de"nitive identi"cation of the injectionsite, con"rming its location and lack of damage at the cellular level.Detection of this conventional !uorescent tracer at distant sites wasexamined to verify the MEMRI results.

Finally, we adopted a non-biased comprehensive approach toidentify all connections traced byMn2+ throughout the brain after PFCinjection. Whole brain MRI data sets from both genotypes at all timepoints were co-registered into the same 3D space (Kovacevic et al.,2005; Lee et al., 2005) using a straightforward linear and nonlinearalignment (Bearer et al., 2007b; Tyszka et al., 2006). Image alignmentallows an automated voxel-wise comparison of 3D MR images(Hammers et al., 2003; Kassubek et al., 2004; Lee et al., 2005;Mechelli et al., 2005; Toga and Mazziotta, 2002). This allowsidenti"cation of those voxels with statistically signi"cant intensitychanges across time and between cohorts (Bearer et al., 2007b; Crosset al., 2004). By comparing the intensities between one time point andthe next, we detected the pathway of the Mn2+ as it progressed alongneuronal circuits in each genotype. This allowed us to probe changesin the reward/addiction circuitry (limbic cortical–ventral striatopalli-dal) due to loss of SERT activity.

Materials and methods

Animals

Mice were obtained from Taconic Farms, Inc (Hudson, NY). Tenserotonin transporter (SERT) knock-out mice (Taconic: B6.129-Slc6a4tm1Kpl N10) and ten normal mice (C57Bl/6NTac) were used inthis study. Mice were female between the ages of 19 and 23 weeks. Allexperiments were performed in accordance with protocols approvedby the Institutional Animal Care and Use Committee of the CaliforniaInstitute of Technology.

Stereotaxic injections

Stereotaxic injection procedure was similar to that employed byBearer et al. (2007b). Mice were anesthetized by spontaneousinhalation of 1% iso!urane and placed in a stereotaxic frame (KopfInstruments, Tujunga, CA). 5 nl of 600 mM MnCl2 with 0.5 mg/ml 3krhodamine dextran-amine (RDA) (Molecular Probes/Invitrogen,Eugene, OR) was injected unilaterally into the right prefrontal cortex(coordinates x !0.5 mm (lateral), y 1.0 mm (anterior–posterior A–Pwith Bregma=0), z 1.0 mm (dorso–ventral D–V with brain sur-face=0) (Paxinos and Franklin, 2001)) over 5 min using a quartzmicropipette (1 mm OD quartz capillary pulled to approximately80 μm OD tip). The animal then received 0.2 ml glycopyrrolate(0.02 mg/kg) and 0.1 ml dextrose (5%) subcutaneously; with 0.2 mllactated Ringer's solution (10 ml/kg) IP. It was then immediatelyplaced in the MR scanner under 0.8% iso!urane anesthetic.

Preparation for ex vivo imaging

From 1 to 10 days after in vivo imaging, animals were sacri"ced andbrains "xed via transcardiac perfusion with 4% paraformaldehyde(PFA) in phosphate buffered saline, pH 7.2–7.4, as previously described(Tyszka et al., 2006). After overnight rocking in 4% PFA at 4 °C themouse headwas cleaned of skin, lower jaw, ears and cartilaginous nosetip and then rocked in 50 ml 0.01% sodium azide in PBS for 7 days at4 °C. The head was then transferred to a 5 mM solution of gadoteridol(Prohance®, Bracco Diagnostics Inc, Princeton NJ) and 0.01% sodiumazide in PBS and rocked for 7 days at 4 °C prior to MR imaging.

Magnetic resonance imaging and spectroscopy

Each animal was scanned before the stereotaxic injection; andbeginning at 0:38±0:14,1:20±0:14, 2:00±0:16, 2:44±0:14, 4:08±0:14, and 22:51±1:02 h post-injection. Times are averages over allanimals±standard deviation. We use the midpoint of each 40 minutescan as the “scan time” and for convenience call these the 1 h, 1 h40 m, 2 h 20 m, 3 h, 4 h 20 m and 24 h time points. An 11.7T 89 mmvertical bore Bruker BioSpin Avance DRX500 scanner (Bruker BioSpinInc, Billerica, MA) equipped with a Micro2.5 gradient systemwas usedto acquire all mouse brain images and spectroscopic data with a35 mm linear birdcage RF coil. For in vivo imaging the animal's headwas secured in a Te!on stereotaxic unit within the RF coil to minimizemovement and aid in reproducible placement. Temperature andrespiration were continuously monitored during data acquisition andremained within normal ranges. We employed a 3D RARE imagingsequence (Hennig et al., 1986) with RARE factor of 4, 4 averages,TR/TEeff=250 ms/24 ms; matrix size of 160!128!78; FOV16 mm!12.8 mm!7.8 mm; yielding 100 μm isotropic voxels with40minute scan time. The short TR provides T1 weighting to emphasizethe location of the paramagnetic Mn2+, while the relatively longeffective TE (24 ms) provides T2 weighting that aids in providingcontrast between different anatomical features.

All in vivo mouse brain magnetic resonance spectroscopy (MRS)experiments were conducted using Point Resolved Spectroscopy(PRESS) (Bottomley, 1987) with a short echo time TE of 7.267 ms,recycle time of 2.3 s, a spectral width of 7 kHz, 4000 data points ineach free induction decay signal (FID), and 128 averages. Thesequence was preceded by a VAPOR water suppression module(Tkáč et al., 1999) interleaved with outer volume saturation.Optimized second order shimming was done with the Fastmaproutine (Gruetter, 1993) in a 5 mm cube centered in the striatum.The PRESS spectrawere then recorded inside a 2mm3 volume (8 μl) atthe center of the volume used for shimming.

For ex vivo imaging, two intact "xed heads were secured in aTe!on® holder and submerged in a per!uoropolyether (Fomblin®,Solvay Solexis, Inc, Thorofare, NJ) within a 50 ml vial and imaged. Theambient bore temperature was maintained at 4 °C by thermostaticallycontrolled air!ow. Diffusion weighted images were acquired using aconventional pulsed-gradient spin echo (PGSE) sequence (TR/TE=300 ms/11.9 ms, 256!150!120 matrix, 25.6 mm!15 mm!12 mm FOV, 100 μm isotropic voxel size, 1 average, δ=3 ms,Δ=5.2 ms, Gd=1125 mT/m, nominal b-factor=3370 s/mm2). Anoptimized six point icosahedral encoding scheme (Hasan et al., 2001)was used for diffusion weighted acquisitions with a single un-weighted reference image for a total imaging time of 14.5 h.

Histology

Brains were "xed in pairs (SERT/normal) at different time points(1, 2, 4, 6, 8,11,12,13,16 and 21 days) after injection to allowhistologicdetermination of transport differences. After "xation and ex vivo MRimaging, brains were dissected from the calvarium and sent toNeuroscience Associates (NSA, Knoxville, TN) for gelatin embedding

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and serially sectioned coronally at 30–35 μm. All 20 brains wereembedded in register into the same gelatin block, allowing review ofthe same location in the entire set on one microscope slide. Sectionswere collected into 24 cups sequentially, such that each cup containedevery 24th section spanning the whole brain of all 20 individuals.Alternate cups were selected for staining with either Thionine/Nisslfor cellular morphology, or for mounting unstained in anti-quenchwith DAPI (Vector Lab, Burlingame, CA) to image the RDA !uores-cence. Sections were imaged on a Zeiss AxioImager Z1 equipped withboth hallogen andmercury illumination, He43 "lter cube, and 5!, 10!,20!, 40!, 63! and 100! neo!uor objectives. Images were captured byan Axiocam HRC or MRM using AxioVision 4.5 software.

Image alignment and statistical parametric mapping

Pre and post Mn2+ injection MR images were skull-stripped usingthe Brain Surface Extractor (BSE) within BrainSuite 2 (Shattuck andLeahy, 2001) to remove all non-brain material. Inaccuracies werecorrected bymanually editing themasks using BrainSuite 2. After skullstripping, "eld inhomogeneities were corrected using the N3 method(Sled et al., 1998) and each was scaled to the mode of its intensityhistogram (Bearer et al., 2007b; Kovacevic et al., 2005). A minimumdeformation target (MDT) was produced as described (Kochunovet al., 2001). Brie!y, all pre-injection images were aligned to arepresentative individual with a 12-parameter full-af"ne transforma-tion using Alignlinear (AIR 5.2.5) (Woods et al., 1998a,1998b)with theleast squares with intensity rescaling cost function. The resultingtransformations were averaged and all pre-injection images werealigned to the average with a 12-parameter full-af"ne model. Theresulting transformations for each cohort were averaged and the pre-injection images were then warped into this MDT common spacebeginning with a 2nd order 30 parameter model and ending with a5th order 168 parameter model using Align_warp (AIR 5.2.5). For eachsample, the post-injection images were linearly aligned (12 parametermodel) to the pre-injection image using Alignlinear, followed by thepolynomial warp "eld used to transform that sample's pre-injectionimage into the MDT. All automated image processing was performedin the LONI Pipeline Processing Environment (Rex et al., 2003) usingeither a 32-processor Onyx 200 or 64-processor Origin 3000 super-computer (SGI). This process placed all the images in the same spaceso that voxel-wise comparisons could be made among the differentdata sets. Final images were blurred with a 0.3 mm Gaussian kerneland a paired Student's t-test, as implemented in SPM5 (WellcomeTrust Centre for Neuroimaging, University College London), was usedto determinewhich voxels increased in intensity when comparing onetime point to the next. Similar processing was used to comparerotationally invariant indices derived fromDTI datasets, except that anunpaired Student's t-test was used to compare knock-out and normalcohorts. Parametric maps of voxels with statistically signi"cantchanges in intensity were created to display the results and tocorrelate increases with underlying anatomy (Bearer et al., 2007b).Anatomy was determined with reference to Hof et al. (2000) andthe Allen Brain Atlas (Dong, 2008) (http://www.brain-map.org/welcome.do). Sagittal sections were typically more helpful thancoronal in identifying and comparing anatomical structures in the invivo MR images.

Tensor-based morphometry

A deformation "eld analysis implemented by Thompson andcoworkers (Lepore et al., 2008) was used to analyze whether MRIscans of the SERT knock-out differ anatomically from normal mousebrain scans. This approach has been used previously in studies of bothmouse (Kovacevic et al., 2005; Ma et al., 2005; Spring et al., 2007;Verma et al., 2005) and human (Lepore et al., 2008; May and Gaser,2006; Perani et al., 2005; Reading et al., 2005). Brie!y, all scans of a

particular contrast (e.g. pre-injection, iDWI) were mapped into anMDT. The resulting displacement "eld (JTJ, with J the deformationJacobian) for each scan was employed in the actual analysis. Maps ofthe determinant of JTJ were used in a voxel-wise Hotteling's T2 testyielding p-value maps to gauge statistical differences between thetwo cohorts at each voxel in the image.

Determination of relative metabolite concentrations

For each mouse brain the spectrum of the relative amount ofmetabolites inside the experimental PRESS volume was quanti"edusing the QUEST (quantitation based on quantum estimation)module (Ratiney et al., 2005) available inside the Java MagneticResonance User Interface (JMRUI) package (Naressi et al., 2001). Abasis set comprising nine metabolites was used to perform the "t. Tothis end PRESS spectra of creatine, choline, N-acetyl aspartate (NAA),taurine, myo-inositol, lactate, glutamine, glutamate and gamma-amino-butyric acid (GABA) were simulated using the NMR-SCOPE(Graveron-Demilly et al., 1993) simulation package within JMRUI. Thespin parameters (number of spins, chemical shifts, J-couplings) wereobtained from Govindaraju (Govindaraju et al., 2000). The choice touse a simulated basis set rather than a measured one was based onrecent results showing no statistically signi"cant differences betweenestimates obtained using either basis set (Cudalbu et al., 2007).Metabolite amounts obtained by the QUEST were normalized tocreatine.

Diffusion tensor image construction

Reconstruction of the apparent diffusionweighted images includedspatial radial Gaussian "ltering (0.25 voxel width) to smooth the co-registration cost function and improve the SNr of all subsequentcalculations. The apparent diffusion tensor was calculated convention-ally by inversion of the encoding b-matrix. The b-matrix for eachdiffusion encoding was determined by numerical simulation of thepulse sequence k-space trajectory in order to account for gradientcross-terms (Mattiello et al., 1997). Eigenvalues, eigenvectors, tensortrace and fractional anisotropy were calculated conventionally usingbuilt-in and customMatlab functions (TheMathworks Inc., NatickMA).The six diffusion weighted images were averaged to generate a highSNr isotropic diffusionweighted image (iDWI). Diffusion tensor images(and associated images of eigenvalue, λi; trace, Tr(D); and fractionalanisotropy, FA) were placed into the same space for voxel-wisecomparisons using the methods outlined above for in vivo MR images.

Rendering

Visualization of the MR images and statistical parametric mapswas performed with ResolveRT4 (Mercury Computer Systems, Inc.,Hudson, NH) and MRIcro (Rorden and Brett, 2000).

Results

Injection site location and condition

The injection sites were within a 0.3 mm radius for all 20 animals,as demonstrated in MR images recorded 1 h after Mn2+ injectioninto the PFC (Fig. 1). The average injection site for all 20 animalswas: x (lateral) +0.45±0.16 mm, y (A–P), +0.92±0.28 mm, z(D–V)!0.86±0.27mm; for the SERT knock-outs: x+0.43±0.16mm,y +0.74±0.25 mm, z !0.83±0.26 mm; and for the normals:x +0.47±0.17 mm, y +1.1±0.18 mm, z !0.88±0.29 mm. Co-injection of !uorescent dextran allowed identi"cation of the injectionsite by histology. We therefore performed a comprehensive histolo-gical analysis of all 20 brains to verify the location of the injection site,detect any morphologic evidence of injury, and validate that the

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http://www.brain-map.org/welcome.do

http://www.brain-map.org/welcome.do


injectate was delivered into the pathway previously identi"ed bytraditional tracers (Gabbott et al., 2005; Sesack and Pickel, 1992). Theinjection site could only be identi"ed in 8 of 20 brains (4 normals and4 SERT) that were "xed at 1–8 days after injection. At later time points,the injection site could not be identi"ed. In 4 of the 20 brains, theinjection site was only identi"able by the presence of !uorescentdextran as no other sign could be found in our serial histologicsections, whether stained for morphology with DAPI or with Nissl/Thionine (Fig. 2). Nor could any gliosis, an indicator of brain injury, befound at later time points (Fig. 2). In those brains where the injectionsite was identi"ed by the presence of rhodamine-dextran, thesurrounding tissue showed no increased cellularity, evidence ofbleeding, or other damage/injury response, although the injectatedid appear as a small droplet (50–100 μm diameter) displacing braintissue at 1 day post-injection. The location of identi"ed injection sitesin the PFC matched the injection position in MR images.

In both SERT knock-out and normal mice microscopic examinationof locations distant from the injection site showed a signi"cantnumber of rhodamine-positive cells. These areas were restricted to

regions representing predicted targets in the pathway from PFC, suchas the globus pallidus (Fig. 3).

Metabolite concentrations are similar in SERT and wild-type mice

In vivomagnetic resonance spectroscopy offers a means tomonitornon-invasively low molecular weight metabolite levels within theintact brain (Choi et al., 2003; Jansen et al., 2006; Maudsley, 2002;Tkac et al., 2004). In this study we determined concentrations ofmetabolites relative to creatine, which is assumed to remain constantamong mice and between the two cohorts (Fig. 4). All spectra wererecorded from a 2 mm3 volume centered in the striatum (Fig. 4B).Although Student's t-test statistical comparisons between the meta-bolite levels from the SERT knock-out and normal cohorts indicatethat none of the metabolites are statistically different at a pb0.05level, the choline and taurine levels are different at pb0.053; bothbeing larger in the SERT knock-out versus normals.

Voxelwise analysis detects no anatomical differences in SERT comparedto normal mice

We aligned 3D MR images of 20 living mice, 10 for each genotype,into a single 3D atlas and used statistical parametric mapping tocompare brain structure between genotypes. Two separate statisticalanalyses were performed. In the "rst, the extent of anatomicalmorphometric alterations undergone by the SERT knock-out wasdetermined by tensor-based morphometry (TBM) of both in vivo and"xed MRI data sets. Secondly, the impact of SERT knock-out on tissuestructure manifest in altered water diffusional characteristic wasdetermined by image alignment and statistical parametric mapping ofimages of FA and Tr(D) (Verma et al., 2005).

The TBM analysis revealed that both the pre-injection in vivo scansand iDWI ex vivo scans had an extremely small number (less than 0.1%of the brain volume) of statistically different (pb0.01) voxelsrandomly scattered about the brain (colored areas in Fig. 5). For theTBM analysis all the pre-injection data sets were mapped to the sameMDT, the Jacobian of the deformation "eld calculated, and p-valuemaps noting statistical signi"cance of differences in the deformationsof the two cohorts determined. A similar calculation was donebeginning with the iDWI data sets measured on "xed brains. In Fig. 5green areas denote voxels that differ signi"cantly between the SERTknock-out and normal mouse brain scans at pb0.01.

After warping into the same space, an unpaired Student's t-testwas used to gauge whether the normal and SERT knock-out havedifferent patterns of FA or Tr(D). No patterns of differences wereobserved with either FA or Tr(D) measurements with very fewsigni"cantly different voxels between the brains of SERT knock-outand normal mice: 0.4% for FA and 0.2% for Tr(D) (Fig. 6). Coloredvoxels show the differences between the SERT knock-out and normalmouse at a signi"cance level of pb0.01.

Visualization of Mn2+ transport in the limbic system of living mice

In contrast to the results of spectroscopy and DTI imaging, wherelittle to no differences between SERT knock-out and normalmicewerefound,Mn2+ accumulation over timewas profoundly different in SERTknock-outs compared to normals. Alignment and a consistentintensity scaling allowed us to visually and computationally followthe changes due to the appearance of Mn2+ induced hyperintensity instructures distant from the injection site in averaged co-alignedimages, such as the slices shown in Fig. 7. We aligned all of the SERTknock-out in vivo MR images, then at each time point averaged theimages from the 10 individual SERT knock-out mice. The averagedimages preserve the "delity of the original images — anatomicalstructures are clear and distinct (e.g. ventricles, corpus callosum,hippocampus, internal and external capsule are all easily identi"able)

Fig. 1. Injection sites are closely grouped within the prefrontal cortex. Location of theinjection site is measured as the center of the hypointense region in the MR imagerecorded 1 h post-injection. In the three projections, injection sites for individualanimals are shown as orange spheres for SERT knock-outs and gray spheres for normalmice. Scale bar=1 mm.


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(Fig. 7). Signal intensities in the globus pallidus (GP) and substantianigra (SNr) regions are qualitatively unchanged from pre-injection tothe 3-hour time point, then increase at 4 h and increase again at 24 h(Fig. 7A). Intensity measurements in a region of interest (ROI) withinthe GP ipsilateral to the injection site as a function of time afterinjection in averaged images from each time point and genotypecon"rm the visual assessment (Fig. 7B)

Statistical mapping of Mn2+ tracing from PFC injection: profounddifferences between SERT knock-out and normal mice

For an unbiased assessment of the whole brain, we performedautomated voxel-wise statistical parametric mapping to determinethe spatial extent of Mn2+ accumulation as a function of time afterinjection (Fig. 8 and Supplemental data). For clarity, normal and SERTknock-out cohorts are shown separately, and volumes with signi"cantincreases (pb0.025) are color coded. Comparisons between theearliest two time periods are shown in pure green for both the SERTknock-out and normal animals, while later times are displayed withincreasing amounts of blue for the SERT knock-out and red for thenormal animal.

The injection site was readily detected in the 1 h compared to pre-injection statistical map, as shown in green in Fig. 8. The pure greencolored regions are voxels that are signi"cantly more intenseimmediately post-injection than before injection of a small amountof Mn2+ into the prefrontal cortex. As the green areas (1 hNpre) showin slice 1 of the coronal view (Figs. 8A and B) and slices 3–5 of thesagittal view (Figs. 8C and D), after the injection a spheroid of

hyperintensity in the MR image appears, likely due to Mn2+ diffusion.Comparison of the 1 h 40m image data sets with those recorded at 1 hpost-injection reveals no signi"cant changes in either the SERT knock-outs or normal animals.

By 2 h 20 m post-injection, Mn2+-induced intensity changes hadalready progressed from the injection point to more distal locations.Wide spread changes were already apparent at 2 h 20 m post-injection between the two genotypes. (Fig. 8, 2 h 20 mN1 h 40 m). Forthe SERT knock-outs, at the most dorsal level of the injection site onthe ipsilateral side only, the hyperintensity increase expands outwardthrough layers 3 and 4 of the cortex (blue-green regions in Figs. 8B,slices 1 and 2, D, slices 1–3). At 1.5 mm off midline, the hyperintensityincrease dips into the dorsal anterior portion of the caudate putamen(CP), courses across the CP and down into the globus pallidus (GP),bed nucleus of the stria terminalis (BST), and most rostral portion ofthe thalamus (TH). These changes are especially apparent in theipsilateral sagittal slices (Fig. 8D, slices 1–5). For the normal animals,we see relatively small isolated volumes of hyperintensity increase inthe CP ipsilateral to the injection site and just outside the injectionvolume on the contralateral side (Figs. 8A, slice 1; C, slices 2, 7, 8).

From 2 h 20 m to 3 h post-injection, the SERT knock-outs showadditional hyperintensity increase in the ipsilateral CP; spreadingipsilateral to contralateral in a discontinuous arc through severalnuclei of the anterior TH (Fig. 8B, slices 2–4). For the normal cohort,we note hyperintensity increase dorsal to ventral across the anteriorthird of the ipsilateral CP, connecting to the medial edge of the CP anddown through the BST with a small amount in the paraventricular TH;and hyperintensity increase contralateral in the dorsal and mid level

Fig. 2. Injection site shows minimal injury. All 20 brains were examined by histology. Examples of a section through the PFC of brains imaged by MR and then "xed for histology,sectioned coronally in the same block in register, and stained in parallel for Nissl/Thionine are shown. The brain in (A)was "xed at 2 days and (B) 16 days after injection. At 2 days (A)an apparent needle track is found in the Nissl/Thionine stained section and con"rmed as the injection site in a parallel section imaged by !uorescence (C). The !uorescent tracer hasalready traveled along neural processes into deeper regions of the brain and along the corpus callosum. Highermagni"cation (D) reveals no hemorrhage, or evidence of in!ammationat the injection site or along the track. At 16 days (B) no histologic evidence of the injection can be found nor is any evidence of residual injury detected. Bar=500 μm.


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CP, medial portion of the GP, and more ventrally in the BST with smallamounts in the TH and the nucleus accumbens (ACB). See Figs. 8A,slices 3–5 and C, slices 1–4.

From 3 h to 4 h 20 m post-injection, the SERT knock-outs haveadditional hyperintensity increase with more volume occupied in thesame locations as 3 hN2 h 20 m statistical parametric map (SPM). Thearc through the TH is now broader and continuous to the contralateralGP. For the normal animals, hyperintensity increases are displayed inmajor portions of the ipsilateral CP and medial portions of the GP,leading down to the BST, then into the reticular nucleus (RT) andcentral portions of the TH with additional intensity increases in dorsalregions of the hypothalamus (HY). No changes are seen contralateralto the injection site in this comparison (Fig. 8, 4 h 20 mN3 h).

Major differences in the accumulation of Mn2+ signal evolvedthroughout the time course of the experiment in both cohorts. Theextent and location of these changes become increasingly differentbetween the two cohorts over time. Between 4 h 20 m and 24 h,widespread changes were seen in images from both knock-out andnormal cohorts (pure red and blue in Fig. 8).

In the normal animals, three signi"cant threads of hyperintensitycan be identi"ed, with none extending further posterior than the TH at!2.5 mm Bregma. One thread runs from Layer 5 cortex at 0.5 mm

Bregma tracing diagonally back to the midline at !1.6 mm Bregma inthe retrospinal cortex (Fig. 8A, slices 2–4). The second thread isipsilateral to the injection site "lling much of the CP, and largeportions of the GP, ACB, and substantia innominata (SI); plus theclaustrum (CLA) and nearby cortex. This thread further expands to "llhalf the TH farthest from themidline (Figs. 8A, slices 4 and 5, C slices 1and 2). The third thread is contralateral to the injection site andsomewhat smaller than the second thread but occupies substantiallythe same anatomy (Figs. 8A slices 4 and 5, C, slices 8 and 9).

In the SERT knock-outs by 24 h, three distinct differences withnormals become obvious. First, instead of distinct threads progressingfrom the injection site, in the knock-outs we note amore general spreadof the hyperintensity posterior to themid level THwith a larger volumeoccupied contiguously across the entire TH, i.e. compare the 24 hN4 h30 m SPM with the 4 h 20 mN3 h SPM (Fig. 8B, slice 5). Second, theenhanced region extends more ventrally to occupy much of thesubstantia nigra (SNr) both ipsilateral and contralateral to the injectionsite (Figs. 8B, slices 6 and 7, D, slices 2 and 3), as well as the ipsilateralventral tegmental area (VTA) (Figs. 8B, slice 8, D, slice 4). Finally,

Fig. 3. Neuronal transport of rhodamine-dextran and Mn2+ overlap in the globuspallidus (GP). Panel A shows !uorescence microscopy of a coronal section throughBregma !0.3 mm in the GP lateral to the internal capsule. It reveals a cluster ofrhodamine-positive neurons ipsilateral to the injection in the same normal mouse asshown in Fig. 2A that was co-injected with Mn2+ and rhodamine-dextran, imaged byMR and "xed by perfusion 48 h later. Rhodamine dextran (red) appears as !uorescentparticles in the neuronal cell bodies (insert at 2!). Nuclei are stained blue with DAPI.Note the normal neuronal morphology of the dextran-positive cells. Panel B shows thecorresponding MR image slice from the 3D data set of the average 24-hour post-injection normal mouse that includes the images from the mouse shown in (A). Noteenhanced intensity in the GP. The rectangle in the MR image corresponds to the locationand size of !uorescence image in (A).

Fig. 4. MR spectroscopy detects little difference between SERT knock-out and normalbrains. (A) Typical in vivo proton spectra from a small volume in the striatum revealgood signal-to-noise ratio. Metabolite relative concentrations shown in B aredetermined from these spectra. (B) Metabolite concentrations relative to creatinedetermined from analysis of in vivo spectroscopy data. Only choline and taurine vergeon being different in the SERT knock-out (knock-out — shaded bars) versus the normal(normal — unshaded bars) mouse. Student's t-test comparison reveals pb0.054 (!) forthese two metabolites. Only the major peak(s) for each metabolite are noted. Lac:lactate, NAA: N-acetyl aspartate, Glu: glutamine, Gln: glutamate, Cr: creatine, Cho:choline, Tau: taurine, GABA: γ-aminobutyric acid, M-Ins: myo-inositol.


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signi"cant intensity increases are observed far posterior into the dorsalnucleus Raphé (DNR), red nucleus (RN) and pontine reticular nucleus(PRN) (Figs. 8B, slices 9–12, D, slices 3–5). This accumulation ofMn2+ inthe posterior regions of the brain was not detected in normals.

Discussion

Here we show profound differences in the neuronal circuitrycaused by a genetic disruption of the serotonin transporter in a mousemodel system using a panel of magnetic resonance methods. Bothmetabolite detection by MRS and anatomy by TBM and DTI

demonstrated no detectible differences between SERT knock-outand normal mice. In contrast, widespread differences were revealedby neuronal transport of the MR contrast agent, Mn2+. After injectionof Mn2+ into the prefrontal cortex of living mice, the classic pathwayto the limbic system became highlighted over 24 h in normal mice,whereas in SERT knock-out, Mn2+-induced increased intensityoccurred more caudally, progressing into the dorsal nuclei Raphé ofthe brain stem. Thus by 24 h little overlap in the highlighted anatomybetween the two types of mice was found (Table 1). This dramaticdifference in Mn2+ accumulation cannot be explained by alterationsin metabolite levels or anatomy of the mutants.

Fig. 5. Tensor-based morphometry from in vivo 3D-RARE and isotropic diffusionweightedMRI data of SERT knock-out and normal mice reveal no meaningful anatomic differences. Aand B show para-sagittal and coronal sections, respectively, from the average of all pre-injection 3D RARE in vivoMR scans from both cohorts. C and D show para-sagittal and coronalsections, respectively, from the average of all iDWI ex vivoMR scans from both cohorts. Green areas denote those voxels that differ (pb0.01) between cohorts in their transformationto the same canonical mouse brain. Scale bar=1 mm.

Fig. 6. Statistical parametricmapping comparisons of Tr(D) and FA data of SERT knock-out and normal mice exhibit nomeaningful differences. A and B show para-sagittal and coronalsections, respectively, from the average of all Tr(D) images derived from ex vivo DTI scans from both cohorts. C and D show para-sagittal and coronal sections, respectively, from theaverage of all FA images derived from ex vivoDTI scans from both cohorts. Colored areas show Student's t-test comparisons of the FA and Tr(D) values between the two cohorts wherered implies SERT knock-out values are greater than normal animal values and blue less. Only pb0.01 values are shown and they comprise less than 0.5% of the brain volume. Scalebar=1 mm. (For interpretation of the references to colour in this "gure legend, the reader is referred to the web version of this article.)


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MRS detectable metabolite levels in the caudate putamen ofknock-out mice are similar to normals. Thus neuronal viability(indicated by NAA/Cr), excitatory and inhibitory system integrity(indicated by Glu/Cr and GABA/Cr), membrane turnover (indicatedby Cho/Cr), and glial volume (indicated by Tau/Cr) are notsigni"cantly different between the SERT knock-out and normal animal(Brian Ross, 2001; Dedeoglu et al., 2004; Hetherington et al., 1997;Lyoo and Renshaw, 2002; Marjanska et al., 2005; Morris, 1999;Rothman et al., 2003; Rudin et al., 1995).

Histology also demonstrated no detectible toxic effect of theinjectate containing 3 nmol of Mn2+ on the brain parenchyma ineither genotype. This agrees with work of Canals and coworkers whofound a threshold for neuronal cell death of 16 nmol and forastrogliosis of 8 nmol Mn2+ (Canals et al., 2008). Thus differences inMEMRI delineated pathways are not due to selective anatomicaldamage by the injection in either genotype.

Tensor-based morphometry (TBM) also failed to show any grossanatomical differences between the two cohorts, i.e. no differences inthe size or shape of structures, no missing structures, no cerebellar orhippocampal dysmorphism. We used TBM to compare the SERTknock-out and the normal mouse brain based on in vivo pre-injectionMRI and ex vivo iDWI data sets. Thus, the lack of change must beviewed within the context of the limited spatial resolution (100 μm)and contrast of the images. Histological examinations reveal that SERTknock-out mice have altered barrel "eld morphology (Persico et al.,2001) and other cellular level changes in the cortex and other parts ofthe brain (Altamura et al., 2007; Li, 2006; Persico et al., 2000a;Wellman et al., 2007). The TBM methodology as implemented here isinsensitive to changes at the higher resolution used to detect thesemicroscopic anatomical differences.

Fractional anisotropy (FA) and the trace of the water diffusiontensor (Tr(D)) have been used to gain insight into changes in whitematter tracts and other ordered brain structures (Drobyshevsky et al.,2005; Tyszka et al., 2006; Wieshmann et al., 1999). FA and Tr(D) aretwo rotationally invariant indices derived from the diffusion tensorthat provide information about underlying tissue characteristics(Alexander et al., 2001; Basser,1995; LeBihan,1995;Mori et al., 2001;Papadakis et al., 1999; Pfefferbaum et al., 2000; Song et al., 2002; vanDoorn et al., 1996). Fractional anisotropy is a scalar measure of thediffusion anisotropy (i.e. propensity of water to diffuse along, ratherthan perpendicular to, nerve bundles). Tr(D) is a measure of theorientation-independent mean diffusivity of water in the tissue.Statistical parametric maps of our data failed to detect any mean-ingful differences between FA and Tr(D) datasets from SERT knock-out and normal mice. Hence the tracts and ordered structuresdetected by FA and Tr(D) are essentially normal in the SERT knock-out. Thus, anatomical differences at the 100 μm resolution of this dataare unlikely to contribute to the large differences in Mn2+

distribution.The dramatic differences in Mn2+ enhanced MR images in the

SERT knock-out mice compared to normal animals were found both inthe anatomical distribution of Mn2+-induced intensity increases andin the time course of their appearance. After introduction of nanoliteramounts of Mn2+ into the prefrontal cortex, both knock-out andnormal mice displayed Mn2+ accumulation in the CP, GP, andthalamus; while only the knock-out showed additional accumulationfurther basal and posterior in the hypothalamus, SNr, VTA, RN, andPRNr over time.

Mn2+ is an anterograde trans-synaptic tracer thought to beselectively taken up through Ca2+ or other divalent cation channelsand transported via fast axonal transport within neurons toaccumulate at distant sites in suf"cient amounts to producesigni"cant intensity changes detected by T1-weighted MRI (Mur-ayama et al., 2006; Silva et al., 2004; Van der Linden et al., 2007).Moreover, Mn2+ is an activity dependent tracer, as it is taken up byactive neurons via voltage gated Ca2+ channels (Aoki et al., 2002;Drapeau and Nachshen, 1984; Narita et al., 1990) and crosses activesynapses (Bearer et al., 2007a; Pautler et al., 2003; Saleem et al.,2002). There is no known speci"city of Mn2+ for any particular type ofneuron. Thus, the projections observed in this work likely arise fromall neurons at the forebrain injection site, regardless of theirneurotransmitter speci"city. In this regard, several cellular leveldifferences in the prefrontal cortex have been observed betweenSERT knock-out and normal mice, including increased neuronaldensity, changes in layer thickness, and increased branch length inpyramidal neuron dendrites (Altamura et al., 2007; Persico et al.,2003; Persico et al., 2001; Wellman et al., 2007). These cellulardifferences must modulate to some extent the initial uptake and, thus,could modulate the temporal distribution of Mn2+ originating in theforebrain. Cellular differences due to SERT knock-out have also beennoted in locations such as the somatosensory cortex, amygdala,hypothalamus, striatum, and dorsal Raphé (Kim et al., 2005; Li, 2006;Li et al., 2003; Lira et al., 2003; Persico, 2004; Persico et al., 2000b;Wellman et al., 2007). These structural, cellular, and moleculardifferences all contribute to the functional differences in neuronalcircuits in the two cohorts of mice that affect the transport/relocationof Mn2+. This interpretation is consistent with other studies of SERTknock-out mice showing that they exhibit signi"cant adaptivechanges at the molecular/synaptic/cellular level; including regionspeci"c changes in various serotonin receptor densities and alteredresponses in other neurotransmitter systems (Fox et al., 2007; Kim etal., 2005; Li, 2006; Murphy et al., 2004; Murphy and Lesch, 2008; Shenet al., 2004; Wellman et al., 2007).

Mn2+ transport in the normal animal delineates some, but not all,of the expected connections between the neocortex and the basalganglia of the rodent brain (Carr and Sesack, 2000; Gabbott et al.,2005; Paxinos, 2004). Over the course of 3 h, Mn2+ is transportedfrom the prefrontal cortex to the caudate putamen (CP) and then ontothe globus pallidus (GP), nucleus accumbens (ACB), and thalamus(TH). Similar connections were observed in the rat after stereotaxicinjection onMn2+ into the CP (Soria et al., 2008). By 4 h post-injectionhyperintensity is seen in the bed nucleus of the stria terminalis (BST)and dorsal regions of the hypothalamus (HY). We interpret the largevolume of the striatum occupied in the 24 hN4 h 20 m statisticalparametric map (SPM) of the normal mice to be a consequence ofthree factors: 1) continued input from the prefrontal cortex; 2)transfer among striatal interneurons; and 3) feedback along thethalmo-striatal pathway. Likewise, feedback along the thalmo-corticalpathway accounts for highlighted voxels in the retrospinal cortex. Wedo not observe the pathways (direct or indirect) to the substantianigra (SNr) or projections to the ventral tegmental area (VTA) in theseexperiments, implying relatively lower activity of neurons projectingto these locations in the normal mouse.

Both temporal and spatial patterns of Mn2+ accumulation in theserotonin transporter knock-out mouse were profoundly differentthan those observed in the normal mouse. As noted by others, lifelong

Fig. 7. Visualization of MEMRI shows Mn2+ accumulation over time far from the injection site. (A) Coronal slices show Mn2+ induced hyperintensity near and far from the originalinjection site. The left and right columns show slices at 0.4 mm and 3.5 mm posterior to Bregma, respectively. Slices are derived from the averaged aligned in vivo 3DMR images of 10SERT knock-out mice. Hyperintensity in the cortex (arrowhead — left column 2nd row) is due to Mn2+ passive diffusion from the injection site (Bregma +0.92 mm, 0.5 mm offmidline, 0.9 mm from surface), while hyperintensity far from the injection site (e.g. GP and SNr) is due to active transport and accumulation. A sagittal slice from the pre-injectionimage at the bottom notes the locations of the slices. Red circle in the GP notes the location of the center of volume of interest (VOI) used in B. Scale bar=1 mm. GP: globus pallidus;SNr: substantia nigra. (B) Average intensity in a 0.065 μl volume of interest centered in the globus pallidus (red circle in A) increases signi"cantly as a function of time after injection.Squares: SERT knock-out mice; diamonds: normals. Error bars are one standard deviation. Scale bar=1 mm.


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lack of SERT causes rearrangement of the relative concentrations anddistributions of the various serotonin receptors (Gobbi et al., 2001;Holmes et al., 2003; Li et al., 2003; Mathews et al., 2004; Montanez etal., 2003; Murphy and Lesch, 2008) that are thought to lead to a loss offunctional integration and inhibitory regulation in neural circuitry

related to emotion and effects of drugs of abuse (Hariri and Holmes,2006; Uhl et al., 2002). Moreover, SERT knock-out mice show changesin dendritic morphology in the PFC (longer apical dendritic branchesin the knock-out) and the basolateral amygdala (greater neuronaldensity) (Wellman et al., 2007); as well as signi"cant increases in

Fig. 8. Color-coded statistical parametric maps of the time dependence of Mn2+ accumulation after injection in the prefrontal cortex show widespread differences between normaland SERT knock-out mice. (A and B) show coronal slices from normal and SERT knock-out mice, respectively. In both panels coronal slices proceed anterior to posterior from upperleft to lower right with placement as shown in the sagittal section. (C and D) show sagittal slices from normal and SERT knock-out mice, respectively. In both panels sagittal slicesproceed ipsilateral to contralateral of the injection site with placement as shown in the coronal section. Colored areas denote those regions with signi"cant (pb0.025) intensitychange between consecutive time points indicating Mn2+ tracing to those locations. The 1 h versus pre-injection comparison is shown in pure green for both the SERT knock-out andnormal animals, while later times are displayed with increasing amounts of blue for the SERT knock-out and red for the normal animal. The average pre-injection MR image is shownas grayscale background.


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neuronal cell density in the neocortex (Altamura et al., 2007).Although neurons in the PFC are known to project to the basolateralamygdala (BLA) (Gabbott et al., 2005), no Mn2+ transport to the BLAfrom the injection site in the PFC is seen in the knock-out mouse andonly very minor amounts in the normal mouse at 24 h after injection.We conclude from this that the PFC-BLA circuit is less robust thanconnections to the more medial structures (CP, ACB, and TH) in boththe normal and SERT knock-out mice.

If activity dependent uptake and release are dominant factorsdetermining the Mn2+ distribution, then differences in the statisticalparametric maps of the normal and SERT knock-out mice likely re!ectdifferences in neuronal circuit activity. At early times, we observedMn2+ accumulation in the CP of SERT knock-outs well before normalmice; indicative of increased activity in the PFC (thus increased Mn2+

uptake) and/ormore functionally dense connections between the PFCand CP in SERT knock-outs as compared to normal mice. Increaseduptake of Mn2+ in the PFC of the SERT knock-out is consistent withchanges in dendrite morphology and cortical neuronal cell densitiesobserved by others (Altamura et al., 2007; Wellman et al., 2007).

Subsequent connections from the CP to the GP and TH were alsofound to be more active (and/or denser) in the SERT knock-out versusnormal, as evidenced by the occurrence of Mn2+ induced intensityincreases in GP and TH of the SERT knock-out well before the normal(appearing 2–3 h post-injection in SERT knock-out versus 3–4 h post-injection in the normal mice). Although the normal animals areknown to have important circuits involving the SNr, VTA, and DNR; nohint of Mn2+ transport to these locations was observed in the normalanimal. In contrast, at 24 h post-injection we do note Mn2+

accumulation in these same structures in the SERT knock-out. Thus,one or more of three conditions apply in the SERT knock-out whencompared with the normal mouse: 1) increased uptake because ofincreased activity in neurons projecting from CP/GP to SNr/VTA/DNRand subsequent delivery of Mn2+; 2) more neurons projecting tothese areas; and/or 3) increased dendritic uptake by neurons in theseareas.

Mn2+ labels a well de"ned subset of the known projections fromthe PFC. Labeled projections are qualitatively different in normal ascompared to SERT knock-out animal. The schematic in Fig. 9 shows thethree most striking general differences observed between the SERTknock-out and normal mouse: 1) connections from the PFC to the ACBare observed in normal, but not the SERT knock-out; 2) connectionsfrom the PFC to the VTA and SNr are observed in the SERT knock-out,but not the normal mouse; 3) connections in the SERT knock-outprogress as far posterior as the dorsal nucleus Raphé and PRN, but stopmid thalamus in the normal mouse. Although connections from theVTA to the ACB are well known in the rodent (Carr and Sesack, 2000;

Gabbott et al., 2005; Gerfen, 2004; Sesack and Pickel, 1992), they arenot observed here in either the normal or knock-out mice. These"ndings imply that in the SERT knock-out the mesocortical andnigrostriatal pathways are emphasized at the expense of themesolimbic pathway. Morphological changes noted by others (e.g.changes in barrel "eld density, neuronal density, and dendriticmorphology) likely in!uence the functional responses reported byMn2+ enhanced MRI. Thus, we predict that PFC projections terminat-ing in the SNr, VTA and PRNr will have different morphology in the

Fig. 9. Overview of difference in Mn2+ accumulation between SERT knock-out andnormal mice. (A) Side view of increase in Mn2+ hyperintensity at 24 h compared to 4 h20 m post-injection with normal animals shown in red, SERT knock-out shown in blue,injections site is shown as a brown sphere, and gray semi-transparent background is theaverage normal animal brain. B) Schematic of circuitry delineated byMn2+ tract tracingshows signi"cant differences between the normal animal and the SERT knock-out. In(B) the red arrows denote pathways highlighted in the normal animal, which includethe nucleus accumbens (ACB) and extend only as far posterior as the mid thalamus(TH). Blue arrows denote pathways highlighted in the SERT knock-out, which includethe ventral tegmental area (VTA) and extend as far posterior as the pontine reticularnucleus (PRNr). Basil nuclei of the stria terminalis: BST; caudate putamen: CP;hypothalamus: HY; prefrontal cortex: PFC; red nucleus: RN; substantia nigra: SNr;substantia innominata: SI.

Table 1Anatomical features highlighted by Mn2+ MRI as a function of time after injection into the prefrontal cortex.

Anatomical feature Time after injection when change observed

1 h 1 h 40 m 3 h 4 h 20 m 24 h

PFC: prefrontal cortex KO and Ncortex KO and NCP: caudate putamen KO and N KO and Nic KO and N Nic

GP: globus pallidus KO KOic and N KOic KO and Nic

TH: thalamus KO KOic KO and N KOic and Nic

ACB: nucleus accumbens N Nic

Hypothalamus LHA KOHypothalamus DMH N KOSI: substantia innominata Nic

SNr: substantia nigra KOic

VTA: ventral tegmental area KORN: red nucleus KOPRN: pontine reticular nucleus KODNR: dorsal nucleus Raphé KO

Anatomical features were identi"ed by comparing images like those shown in Fig. 8 with standard mouse brain atlases ((Hof et al., 2000) and the Allen Brain Atlas (Dong, 2008)).Sagittal sections were typically more helpful than coronal in identifying anatomical structures in the in vivo MR images. KO: SERT knock-out; N: normal control; no subscript:ipsilateral; IC subscript: ipsilateral and contralateral.


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SERT knock-out compare to the normalmouse: likely greater neuronaland or dendritic spine density.

The limbic cortical–ventral striatopallidal circuitry is intimatelyinvolved in addiction, aggression, and affective disorders (Berton andNestler, 2006; Everitt and Robbins, 2005; Murphy and Lesch, 2008;Nelson and Trainor, 2007; Robbins and Everitt, 2002). The SERTknock-out animal model used here re!ects many human traitsassociated with particular human SLC6A4 variants (Murphy et al.,2004; Murphy et al., 2003; Uhl, 2006). We have employed Mn2+

enhanced MRI to investigate descending neuronal projectionsoriginating in the most anterior part of this circuit: the prefrontalcortex. Using MRS, TBM, and DTI we observe similar metabolitepro"les, gross anatomy, and structural characteristics in both normaland SERT knock-outmice. MRI provides a view of the brain bridging itscellular–molecular and the behavioral aspects. From the mesoscopicview afforded by MRI, we observe that active circuitry originating inthe prefrontal cortex in the SERT knock-out is biased towards moreposterior areas (substantia nigra, ventral tegmental area, and dorsalnucleus Raphé) directly involved in the reward circuit pointing tospeci"c modulation of this circuit by the altered lifelong serotonintone. As illustrated in this work, MRI can be used to link the microlevel and macro level aspects of animal models of disease; in this caseproviding important insights into how a speci"c molecular alteration(SERT knock-out) is manifested in the complicated and criticallyimportant cortico-limbic circuit.

Acknowledgments

We thank Mike Tyszka at Caltech for creation and implementationof the DTI routines; and Cornelius Hojatkashani, Ilya Eckstein, BorisGutman, Natasha Lepore, Igor Yanovsky and Mubeena Mirza at theLaboratory for NeuroImaging at UCLA for invaluable assistance withLONI pipeline and TBM analysis. The project was funded in part by theBeckman Institute, NIH NIGMS GM47368, NINDS NS046810, P20RR018757 (E.L.B.), NIDA R01DA18184, and NCRRU24 RR021760MouseBIRN (R.E.J.).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.neuroimage.2009.03.026.

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