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Journal of Neuroscience Methods 204 (2012) 9– 18

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Journal of Neuroscience Methods

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Basic neuroscience

A robust experimental protocol for pharmacological fMRI in rats and mice

Livia Ferrari a,1, Giuliano Turrinia,1, Valerio Crestana,1, Simone Bertania,1, Patrizia Cristoforib,Angelo Bifonec, Alessandro Gozzic,∗

a GlaxoSmithKline Medicine Research Centre, Via Fleming 4, 37135 Verona, Italyb GlaxoSmithKline Pathology Department, SA Ware, Hertford, UKc Centre for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy

a r t i c l e i n f o

Article history:Received 29 July 2011Received in revised form 20 October 2011Accepted 22 October 2011

Keywords:fMRIAnaesthesiaRatMousephMRISurgery

a b s t r a c t

Pharmacological Magnetic Resonance Imaging (phMRI) methods have significantly expanded the stim-ulation repertoire available to preclinical fMRI research, by allowing to selectively probe the activity ofspecific brain circuitries and neurotransmitter systems. However, the application of phMRI to animalmodels is constrained by a number of experimental factors. Firstly, in order to prevent motion artefactsand reduce restraint-induced stress, phMRI studies are typically performed under anaesthesia. Moreover,several psychoactive drugs produce blood pressure changes and alterations in respiratory frequencythat may perturb central haemodynamic readouts of brain function. Hence, the quality and outcomeof phMRI studies is critically dependent on the ability to monitor and control peripheral physiologicalparameters (i.e. blood pressure, arterial blood gases) that could alter phMRI readouts. Here we provide athorough methodological description of a robust protocol to measure drug-induced cerebral blood vol-ume changes in anaesthetised rats and mice. We show that the protocol ensures stable physiologicalparameters and robust phMRI response to the psychostimulant drug d-amphetamine in three differentrat strains. We also document the successful application of the protocol to map the central effects pro-duced by d-amphetamine in C57Bl/6J mice, a strain commonly used as background for the generation oftransgenic lines, thus paving the way to the implementation of phMRI in genetically engineered animals.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Functional Magnetic Resonance Imaging (fMRI) has been exten-sively applied to study the functional organization of the humanbrain in healthy and disease states. A strong rationale exists toexpand the application of these methods to laboratory animalspecies. However, the vast repertoire of paradigms used to inves-tigate cognitive or emotional brain function in humans cannot bestraightforwardly applied to rodent species under the constrainedexperimental conditions of an fMRI experiment. As a result, despitethe considerable translational potential of fMRI for both basic andapplied brain research, the application of this technique to labora-tory species has failed to keep pace with the clinic.

An interesting approach to overcome some of the constraintsrelated to the implementation of fMRI in small animals relies onpharmacological manipulation, and has been dubbed “pharmaco-logical MRI” (phMRI) (Leslie and James, 2000), i.e. fMRI applied to

∗ Corresponding author. Tel.: +39 050 509032.E-mail address: alessandro.gozzi@iit.it (A. Gozzi).

1 Present address: Aptuit, Via Fleming 4, 37135 Verona, Italy.

map spatio-temporal patterns of brain activity induced by phar-macological challenges. Originally developed to study the circuitalbasis of pharmacological activity, the approach has demonstratedthe ability to elicit reliable fMRI signals and to probe multiple facetsof brain function through the selective stimulation of different neu-rotransmitter systems (reviewed by Bifone and Gozzi, in press).However, rodent functional and pharmacological MRI studies aretypically performed under general anaesthesia, a practice that isrequired to reduce motion artefacts (Peeters et al., 2001; Sicardet al., 2003), and to minimise the stress produced by restraint andMRI gradient noise (for recent reviews see Jenkins et al., 2003; Silvaet al., 2011; Steward et al., 2005; Van der Linden et al., 2007). More-over, the use of psychoactive drugs and psychostimulants in phMRIis often associated with peripheral cardiovascular and respiratoryeffects that may confound central haemodynamic measurementsof brain function (Kalisch et al., 2001; Tuor et al., 2002). Hencethe success of phMRI studies critically relies on the implemen-tation of experimental procedures to monitor vital functions andto prevent the onset of confounding supra-physiological states. Bymeans of example, artificial ventilation can be employed to pre-vent drug-induced alterations in the respiratory rate which couldalter blood CO2 levels and produce stimulus-unrelated vasodilatory

0165-0270/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.jneumeth.2011.10.020

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10 L. Ferrari et al. / Journal of Neuroscience Methods 204 (2012) 9– 18

responses (Jones et al., 2005; Steward et al., 2005). Likewise, periph-eral blood pressure monitoring is often employed to make sure thatthe mapped phMRI responses are not contaminated by peripherallydriven cardiovascular changes that could overcome the autoregu-latory cerebral blood flow range (Gozzi et al., 2006, 2007). As aresult, animal preparation and physiological monitoring in rodentphMRI studies imposes the application of multiple complex proce-dures of critical importance for the successful implementation ofthe technique.

Here we provide a detailed methodological description of arobust phMRI protocol that we have developed and successfullyapplied in the rat and the mouse to study the brain functionalresponses to a number of psychoactive drugs (Gozzi et al., 2010b,2011a). Moreover, we show that this protocol ensures control ofphysiological parameters (arterial blood gases and blood pressure)under different anaesthetic regimens (halothane, and isoflurane),and elicits robust phMRI responses in different rat strains byusing the psychostimulant drug d-amphetamine. Importantly, wedescribe the implementation of the protocol in C57/Bl6J mice, astrain commonly used as background for the generation of trans-genic lines, and we document its successful application to map thecentral effects produced by the d-amphetamine.

This methodological account is expected to promote the dif-fusion of pre-clinical phMRI methods across different areas ofneuroscience and psychopharmacology.

2. Materials and methods

All the experiments were carried out in accordance with Ital-ian regulation governing animal welfare and protection (whichacknowledges the European Directive 86/609/EEC).

2.1. Anaesthetics and chemicals

General anaesthesia was induced Animals were anaesthetisedwith halothane (HAL) (Merial Bristol, UK) or isoflurane (IF) (AbbottLtd., Italy). Neuromuscular blockade was induced with d(+)-tubocurarine chloride hydrate (Tocris, Bristol, UK; 0.25 mg/mL insaline solution) or pancuronium bromide (Sigma–Aldrich, Milano,Italy; 0.25 mg/mL or 0.0375 mg/mL saline solution for adminis-tration to rats or mice, respectively). Sodium heparin (25 UI/mL)(Eparina Vister, Pfizer, Italy) was added to these solutions to ensurearterial catheter patency throughout the MRI experiment. Sen-sitisation of MRI times-series to cerebral blood volume (CBV)was achieved by using blood-pool super-paramagnetic iron oxidenanoparticles (Endorem, Guebert, France). As Endorem produc-tion has now recently discontinued by Guerbert, the contrastagent Ferex (Biopal Inc., Worcester, MA, USA) can be used asan effective replacement. The two agents have comparable ironoxide concentrations (11.2 mg/mL vs. 10.0 mg/mL, respectively)and mean particle size (80–150 nm vs. 50–150 nm, respectively).The psychostimulant d-amphetamine sulphate was purchasedfrom Sigma–Aldrich (Milano, Italy).

2.2. Animals

Adult male Sprague Dawley rats (268–364 g) were providedby Charles River Italy. Male Lister Hooded rats (305–357 g) wereprovided by Charles River UK. Male Wistar rats (385–437 g) andC57BL6/J mice (24–33 g) were provided by Charles River France.Animals were housed in stable groups of five individuals in polycar-bonate cages with sawdust litter, and fed ad libitum with standardrodent chow. Animals were maintained on a light:dark cycle of12:12 h (lights on at 6 a.m.), room temperature within the range

Fig. 1. Schematic illustration of the main experimental steps employed for animalpreparation according to the protocols described in Section 2.

20–22 ◦C, and relative humidity 45–65%. After arrival, animals wereacclimatized for at least 5 days before experimental use.

2.3. Animal preparation

Partial accounts of the surgical protocol described in the presentmanuscript have been given elsewhere (Gozzi et al., 2006, 2008a,b,2010a, 2011a,b; Schwarz et al., 2008). A schematic illustration ofthe main experimental procedures is illustrated in Fig. 1. Table 1summarises the anaesthetic levels used during the different exper-imental phases in rats and mice.

2.3.1. Induction of anaesthesiaAnaesthesia was induced with 3% HAL or 5% IF in rats and with

4% IF in mice. The use of HAL for surgical preparation in mice is

Table 1Gas anaesthesia levels employed at different stages of animal preparation for phMRIstudy rat and mouse. Anaesthetics were delivered through an intra-tracheal cannula.

Study phases Rat anaesthesia Mouse anaesthesia

HAL (%) IF (%) HAL (%) IF (%)

Induction 3 5 3 4Surgery 1.5 3 1.5 3Maintenance 0.8 1.1 0.8 1.2

Abbreviations: HAL: halothane; IF: isoflurane.

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L. Ferrari et al. / Journal of Neuroscience Methods 204 (2012) 9– 18 11

not recommended as this can produce fatal hypotension. Anaes-thetic gases were delivered in a 1:1 mixture (0.9 L/min + 0.9 L/min)of O2/N2 in an induction chamber connected to a vaporizer (BurtonsMedical Equipment Ltd., UK). The animal was then placed in supineposition on a homeothermic (37 ◦C ± 1) heating blanket connectedto a rectal probe (Harvard Apparatus, UK), and gaseous anaesthesiawas continuously delivered through a face mask.

2.3.2. TracheostomyPrior to surgical incision, each rat received a subcutaneous

(SC) infiltration of tetracaine solution 0.1% at each surgical site(neck and femoral area) at a volume of 0.1 mL/point (0.2 mL/rat).Mice received a SC infiltration of tetracaine solution 0.05% at eachsurgical site (neck and femoral area) at volume of 0.02 mL/point(0.04 mL/mouse). This practice is introduced to provide supple-mentary analgesia and prevent sub-threshold activation of painpathways that may confound central measurements of brain func-tion (Ferrari et al., 2010). The neck and femoral areas were thenshaved with an electrical shaver and the skin disinfected. A gauzeroll was placed under the neck to slightly extend the trachea andfacilitate its subsequent exposure. A midline skin incision was madealong the length of the neck, the muscular planes separated toexpose the trachea. A small incision in the sub laryngeal regionof the trachea was performed, and a G14 (rat) or a G22 (mouse)bevelled butterfly cannula (Vygon, France) was inserted into thetrachea as previously described (Rigalli and Di Loreto, 2009). Tofacilitate connection with the ventilator, the cannula was short-ened to a length of approximately 1 and 0.7 cm for rats and mice,respectively. The cannula was then secured with a 3–0 silk suturetread (Ethicon, Johnson-Johnson Intl., Belgium) passed through theholes of its butterfly. A modified intra-tracheal cannula (depictedin Fig. S1) was used in mouse studies as an alternative to the wholebutterfly cannula, with the aim to reduce its dimension, thus allow-ing a better positioning of the animal on the imaging cradle. To thispurpose, the distal 0.7 cm of a G22 butterfly cannula were cut off,and joined to a Y tubing junction (OD 2 mm, 2Biological Instru-ments, Italy) connected to a closed tubing system connected tothe ventilation pump. After tracheostomy, anaesthetic delivery wasswitched from the facial mask to the ventilation pump (Inspira ASV,Harvard Apparatus, UK). A ventilation frequency of 70 bpm wasused both in rat and mouse studies. Tidal ventilation volumes wereadjusted on the basis of the result of arterial blood gas analysis.In order to keep the anaesthesia and animal physiology apparatusoutside the magnet stray field, the ventilator was fitted with 4-m-long tubing. As a result, nominal tidal volumes had to be adjustedto compensate for the increased flow resistance associated to theextended length of the tubing. This resulted in the use of typically1.6–3.5 cc for rat studies, and 0.8–1 cc for mouse experiments. Asarterial blood gas analysis in the mouse was performed only atthe end of the phMRI study, the initial tidal volume was set basedon pilot experiments where a range of initial tidal volumes wereexplored and their corresponding blood gas levels were recorded.As a result of this activity, we noted that, while 5–20% tidal volumeadjustments were often necessary in rat studies to account for dif-ferences in animal weight, this was not the case with mice, wherethe use of constant tidal volumes ensured stable blood gases acrosssubjects of comparable age and genetic background.

2.3.3. Femoral vessels cannulationAfter tracheostomy, HAL and IF anaesthesia levels were

decreased to 1.5 and 3%, respectively (Table 1). This practiceaccounts for the more effective delivery of anaesthetic agentsachieved by the use of a tracheotube, and prevents the onset ofhypotensive states. These levels were maintained throughout thecannulation procedure.

Rat. The left leg was extended, taped on to the surgical sup-port, and a 2-cm skin incision was made with a scalpel in theventro-femoral area as previously described (Rigalli and Di Loreto,2009). The left femoral artery was isolated and cannulated with apolyethylene catheter (PE50, OD 0.97 mm, ID 0.58 mm, Clay Adams,USA) filled with heparinized physiologic solution (25 UI/mL) con-taining 0.25 g/mL of d(+)-tubocurarine. The catheter was connectedto a CRITIFLO TA4004 device (flow/flush device; Becton Dickinson,NJ, USA) coupled to an arterial blood pressure transducer (TSD04,Biopac Systems Corp., Goweta, USA). This device was in turn con-nected via a PE50 catheter to an infusion pump (KDS200, KDScientific, Massachusetts, USA) that was used for continuous infu-sion of a solution of heparin and paralyzing agent (described above).An intra-arterial bolus of 1 mL/kg of 0.25 mg/mL d(+)-tubocurarinewas administered immediately after femoral artery cannulation toinduce rapid neuromuscular blockade. Tubocurarine was infused ata rate of 1 mL/kg/h throughout the experiment. Pancuronium bro-mide was used as neuromuscular blocker in Lister Hooded rats.In this case, an intra-arterial bolus of 1 mL/kg of pancuroniumbromide 0.25 mg/mL was administered immediately after femoralartery cannulation, followed by a continuous infusion of 1 mL/kg/hthroughout the imaging experiment. Arterial blood pressure wasrecorded with an invasive blood pressure transducer connectedto a multi-channel MP150 Biopac system (Biopac Systems Corp.,Goweta, USA).

The left femoral vein was cannulated with a PE50 catheter aspreviously described (Rigalli and Di Loreto, 2009). The catheterwas connected to a 1 mL syringe filled with saline to permit theadministration of MRI contrast agent and test compounds (e.g. d-amphetamine).

Mouse. The right femoral artery was cannulated to allow forcontinuous monitoring of blood pressure and blood sampling forarterial gas analysis. The arterial access was also used for intravas-cular delivery of substances (paralyzing agent, contrast agent,drug or vehicle challenge), as the frail nature of femoral veinsin this strain made the cannulation procedure lengthy and unre-liable. The femoral artery was cannulated with a PE10 catheter(OD 0.61 mm, ID 0.28 mm) as described above and connected to acustom-made three-way plexiglass cube that was used as “switch”for intravascular access (Fig. S2). Upon cannulation, an intra-arterialbolus of heparinized physiologic solution (25 UI/mL) containing0.0375 mg/mL of pancuronium bromide was administered withan infusion volume of 0.335 mL/kg, which resulted in an almostimmediate neuromuscular blockade. Pancuronium bromide wasthen infused intra-arterially at rate of 6.7 mL/kg/h throughout theexperiment. The PE10 catheter was connected to the plexiglass(Fig. S2) through a 2-cm of PVC40 (OD 0.90 mm, ID 0.50 mm) junc-tion inserted into a piece of Silicone tubing (French size 3, e.g. outerdiameter approx. 1 mm). This three-way system allowed simulta-neous recording of MABP and infusion of paralyzing agent (way1), as well as the injection of compounds (way 2), upon temporaryclamping of the line to the infusion pump (way 1) to prevent refluxof injection solution.

In studies requiring intraperitoneal (IP) or subcutaneous (SC) ofcompounds (Gozzi et al., 2008a,b; e.g. Gozzi et al., 2011b), a G23infusion cannula was directly inserted in the dorsal skin of the ani-mal, or in the abdominal peritoneum. The cannula was coupled toa PE50 catheter through a short silastic junction and secured to theskin with a few drops of tissue glue (Vetbond 3M®). The admin-istration of dense or viscous suspensions was performed via G20cannulas connected to a PVC60 catheter (OD 1.22 mm, ID 0.76 mm).

2.3.4. Maintenance phaseAt the end of surgical phase (25–35 min of duration) the ani-

mals were placed in prone position onto a customized stereotacticholder (Bruker, Ettlingen, Germany) and anaesthetic levels were

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12 L. Ferrari et al. / Journal of Neuroscience Methods 204 (2012) 9– 18

decreased to maintenance level. Rat studies were performed with0.8% HAL or 1.1% IF (Table 1). Mouse studies were performed eitherwith 1.2% IF, or with 0.8% HAL (e.g. with d-amphetamine). In the lat-ter case, the IF vaporizer used for surgery was replaced with a HALone prior to the maintenance phase. An MRI-compatible thermo-couple probe was used to measure rectal temperature in all animals.The body temperature of all subjects was maintained within phys-iological range (37 ± 1.8 ◦C) throughout the experiment by using awater heating system incorporated in the stereotactic holder.

2.3.5. Arterial blood gas measurementsA blood gas analyzer (AVL OPTI CCA Critical Care Analyzer,

Roswell, USA) was used to measure arterial oxygen (paO2) and car-bon dioxide (paCO2) concentration. An insulin syringe connectedwith a Miraject blunt needle (0.9 mm × 22 mm, Hager Werken,Germany) was used to sample arterial blood (ca. 150 �L) fromthe femoral cannula. In rats, measures performed at the begin-ning of the maintenance phase were used to adjust ventilationvolumes as appropriate. In mouse studies, arterial sampling (ca.100 �L) was only performed at the end of the experiment, withthe aim to avoid haemodynamic interferences related to thesampling of a significant portion (ca. 5%) of the animal’s bloodpool. Terminal measurements performed at the end of the phMRIacquisition in rats and mice were used as a subject exclusion cri-terium when supra-physiological parameters were found (paO2levels < 90 mmHg and paCO2 > 50 mmHg). Terminal blood samplingwas performed by removing the three-way arterial cube and lettingthe arterial catheter to spill blood into an insulin syringe (200 �L)which was then connected to the blood gas analyzer. At the end ofthe experiment the animals were euthanized with an overdose ofanaesthetic followed by cervical dislocation.

2.4. Experimental groups and protocols

The composition of the experimental groups and treatments issummarised below:

(1) SD-HAL. Sprague Dawley (SD) rats surgically prepared andimaged under HAL anaesthesia. Paralyzing agent tubocurarine.During the imaging session, the rats received an IV challengewith d-amphetamine (0.5 mg/rat, 1 mL/kg in saline, n = 10).

(2) LH-HAL. Lister Hooded (LH) rats surgically prepared and imagedunder HAL anaesthesia. Paralyzing agent tubocurarine. Duringthe imaging session, the rats received an IV challenge with d-amphetamine (0.5 mg/rat, n = 10).

(3) Wistar-HAL. Wistar rats surgically prepared and imaged underHAL anaesthesia. Paralyzing agent pancuronium bromide. Dur-ing the imaging session, the rats received an IV challenge withd-amphetamine (0.5 mg/rat, n = 14).

(4) SD-IF. SD rats surgically prepared and imaged under IF anaes-thesia. Paralyzing agent pancuronium bromide (n = 15). Theseanimals did not receive any stimulation, but were used to verifyphysiological stability under isoflurane anaesthesia.

(5) C57-HAL. C57BL6/J mice surgically prepared with IF and imagedunder HAL anaesthesia. Paralyzing agent pancuronium bro-mide. The mice received an intra-arterial administration ofsaline (5 �L/g) followed by an intra-arterial challenge with d-amphetamine (3 mg/kg) 20 min later (n = 11).

(6) C57-IF. C57BL6/J mice surgically prepared and maintainedwith IF. Paralyzing agent pancuronium bromide (n = 8). Theseanimals did not receive any stimulation, but were used toverify physiological stability under isoflurane anaesthesia. Thesame protocol has been recently used to assess the functionalresponse produced by the serotonin antagonist 8-OH-DPAT intransgenic mouse lines (Gozzi et al., 2010b).

(7) SD rats surgically prepared and imaged under HAL anaesthesia.The subjects received an IV vehicle injection (saline, 1 mL/kg)and were used as baseline phMRI reference for the statisticalanalysis across all rat cohorts.

Differences in the number of animals between groups are notdue to outliers or aborted experiments, but reflect the differentindependent experiments. The dose of d-amphetamine used inrat studies has been selected based on previous in vivo works(Gozzi et al., 2011b; Micheli et al., 2007; Schwarz et al., 2007a).The dose used in mice has been selected based on the results ofpilot dose–response phMRI studies.

2.5. Relative cerebral volume (rCBV) measurement

Rat. MRI data were acquired using a Bruker 4.7 Tesla system. TherCBV measurement protocol used has been previously described ingreater detail elsewhere (Gozzi et al., 2010a, 2011b). Briefly, theMR acquisition for each subject comprised T2-weighted anatom-ical images using the RARE sequence followed by a time seriesacquisition with the same spatial coverage and similar parame-ters, but with a lower in-plane spatial resolution (128 × 128). TotalMRI time-series acquisition time was 60–80 min for all groups.Following six reference images, 2.67 mL/kg of the blood pool con-trast agent Endorem (Guerbet, France) was injected through thefemoral vein of rat to sensitise images to rCBV (Mandeville et al.,1998; Schwarz et al., 2003). Intravenous administration of d-amphetamine (0.5 mg/kg) was performed after an equilibrationperiod of 10–15 min and MRI data were acquired over a period of30 min following the administration of the amphetamine challenge.

Mouse. The rCBV measurement protocol used in the phMRI andfMRI study has been described in greater detail elsewhere (Gozziet al., 2010b). Co-centred anatomical images and fMRI time serieswere acquired using a RARE sequence (anatomical: TReff = 5597 ms,TEeff = 76 ms, RARE factor 8, FOV 40 mm, 256 × 256 × 24, 16 contigu-ous 0.75 mm slices; fMRI time series: TReff = 5436 ms, TEeff = 112 ms,RARE factor 32, 128 × 128; NA = 2, dt = 20 s, Nr = 100, correspond-ing to 70 min total acquisition time). Images were sensitised toreflect alterations in rCBV using 3.75 �L/g of blood-pool contrastagent (Endorem, Guerbet; Mandeville et al., 1998). Fifteen minuteslater each subject received vehicle injection (saline, 5 �L/g, i.a.) fol-lowed 28 min later by a challenge with amphetamine (3 mg/kg i.a.;Sigma–Aldrich).

2.6. Data analysis

Rat. fMRI and phMRI time series were analyzed as previouslydescribed (Gozzi et al., 2008a, 2011b). Signal intensity changesin the time series were converted into fractional relative cerebralblood flow (rCBV) (Schwarz et al., 2003). Individual subjectswere spatially normalized mapping their T2-weighted anatomicalimages to a stereotaxic rat brain MRI template set (Schwarz et al.,2006a) and applying the resulting transformation matrix to theaccompanying rCBV time series. rCBV time series for amphetamineor vehicle challenge were normalized to a common injection timepoint. Image based analysis was carried out using FEAT, part of FSL(FMRIB’s Software Library, www.fmrib.ox.ac.uk/fsl), with 0.8 mmspatial smoothing and using a boxcar function depicting the pres-ence of d-amphetamine (Gozzi et al., 2011b). Higher-level groupcomparisons (d-amphetamine vs. vehicle) were carried out usingFLAME (FMRIB’s Local Analysis of Mixed Effects). Z (GaussianisedT/F) statistic images were thresholded using clusters determinedby Z > 1.6 and a corrected cluster significance threshold of P = 0.01(Worsley et al., 1992). VOI timecourses for d-amphetamine wereextracted from unsmoothed rCBV time series data using a 3D

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L. Ferrari et al. / Journal of Neuroscience Methods 204 (2012) 9– 18 13

digital reconstruction of a rat brain atlas co-registered with theMRI template (Schwarz et al., 2006a).

Mouse. d-Amphetamine phMRI data were analyzed as previ-ously described (Gozzi et al., 2010b). A mouse brain templatewas created by co-registering and overlaying all the anatomi-cal scans to a representative subject using FSL/FLIRT. Subjectswere co-registered by rigid body alignment to the template usingFLIRT. Signal intensity changes were converted into fractional rCBVchanges, and rCBV time series before and after i.a. injections ofvehicle or d-amphetamine were calculated. Activation maps wereanalyzed using FEAT with 0.8 mm spatial smoothing and a box-car function covering the presence of d-amphetamine. Voxelwisestatistics was carried out using FLAME, and Z statistic images werethresholded using clusters determined by Z > 1.6 and a correctedcluster significance threshold of P = 0.01. rCBV time series for d-amphetamine and vehicle injections were extracted bilaterally forspecific regions of interest (ROIs). Mean arterial blood pressure datawere re-binned in 10 sample sub-divisions and plotted using 40 sbins.

3. Results

Arterial blood gas levels (paCO2 and paO2) for all the experimen-tal groups and anaesthetic conditions (IF and HAL) are summarisedin Table 2. Pre- and post-acquisition mean paCO2 values werewithin 30–45 mmHg and mean paO2 was >100 mmHg (correspond-ing to a haemoglobin saturation >97%) in all the rats strains andunder all the anaesthetic conditions examined. Post-MRI bloodgas level measurements in mice under isoflurane or halothaneanaesthesia yielded mean paCO2 of ≈30 mmHg and paO2 levels>90 mmHg.

Baseline (pre-injection) mean arterial blood pressure (MABP)values in Sprague-Dawley (SD), Wistar and Lister Hooded (LH) ratsunder different anaesthetic regimens are reported in Fig. 2. Underhalothane anaesthesia, SD and LH showed comparable MABP values(≈80–90 mmHg), whereas Wistar rats exhibited a slightly higherbaseline MABP (≈100–120 mmHg). Isoflurane-anaesthetised SDsubjects showed substantially higher MBAP values than thoserecorded under halothane anaesthesia (≈120–130 mmHg). Acuted-amphetamine administration was accompanied by a transient(5–8 min) MABP increase in all the strain examined (Fig. 2). Despitedifferences in the pre-injection levels, both the absolute and rela-tive MABP increases were comparable across all the strains (30, 24and 21 mmHg and 33%, 23% and 27% in SD, Wistar and LH, respec-tively). Vehicle injection produced a slight (ca. 5%) and transient(ca. 6 min) increase in MABP (data not shown).d-Amphetamine administration produced robust rCBV

increases in all the three rat strains (Fig. 3), an effect thatwas prominent in several cortical and subcortical areas. The rCBVtimecourse of d-amphetamine-induced rCBV changes in LH andSD rats was comparable and characterised by a sharp increasefollowed by a sustained plateau of activation. In Wistar rats, the

Fig. 2. Temporal profile of MABP as a function of rat strain and maintenance anaes-thesia (SD: Sprague-Dawley, LH: Lister Hooded, IF: isoflurane; HAL: halothane). Top:baseline MABP data prior to pharmacological challenge. Bottom: MABP responseproduced by acute d-amphetamine (amph) administration (0.5 mg/kg i.v.). Thearrow indicates time of injection (SD-IF n = 15, SD-HAL n = 10, LH-HAL n = 14, Wistar-HAL n = 14). Data are reported as mean ± SEM. Corresponding functional activationmaps are presented in Fig. 3.

effect was characterised by a peak response followed by a slowbut constant decline over the 30-min time-window examined. Nocorrelation between the temporal dynamics of d-amphetamine-induced MBAP and the corresponding rCBV changes was observed(cf. Fig. 2 with Fig. 3). All the subjects imaged exhibited robustamphetamine responses, with the exception of a subject in theWistar-HAL group, which showed no response to the challenge.The subject was found to have hypercapnic blood levels (pCO2 = 86)due to partial tracheotube occlusion and was thus removed fromthe cohort. Vehicle injection was accompanied by a small and tran-sient decrease in rCBV, an effect that probably reflects temporarydilution of intravascular contrast agent.

Baseline MBAP data in C57BL6/J mice under halothane andisoflurane anaesthesia are reported in Fig. 4. Vehicle injectionproduced a slight transient MABP increase in both experimentalcohorts. Consistent with rat data, basal MABP in halothane-anaesthetised subjects was lower than that measured in the isoflu-rane cohort (≈80–90 mmHg vs. ≈65–75 mmHg). d-Amphetamine

Table 2Arterial blood gas levels and animal weight.

Group N Weight (g) paCO2 (mmHg) paO2 (mmHg)

Pre Post Pre Post

SD-HALa 10 268–331 31.5 ± 3.6 37.9 ± 5.8 208.7 ± 37.1 181.9 ± 29.2LH-HALa 10 305–357 30.6 ± 5.3 37.1 ± 8.5 167.9 ± 74.1 198.1 ± 78.2Wistar-HALa 14 385–437 29.1 ± 5.6 36.1 ± 7.3 251.4 ± 18.6 180.2 ± 56.9SD-IF 15 295–364 31.5 ± 4.1 31.9 ± 4.1 237.1 ± 29.7 208.7 ± 32.5C57-HALa 11 24.7–33.2 – 29.4 ± 8.7 – 203.2 ± 19.4C57-IF 8 24.1–26.5 – 31.0 ± 8.4 – 332.3 ± 51.6

Abbreviations: paCO2: partial pressure of arterial CO2; paO2: partial pressure of arterial O2. Both parameters were measured at the beginning (pre) and at the end (post) ofthe functional MRI time series. Arterial blood gas values are expressed in mmHg (mean ± SEM).

a Groups challenged with d-amphetamine.

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14 L. Ferrari et al. / Journal of Neuroscience Methods 204 (2012) 9– 18

Fig. 3. phMRI activation (Z statistical maps) produced by acute d-amphetamine (amph) administration vs. vehicle as a function of rat strain. All the subjects were imagedunder halothane anaesthesia. For each strain, mean rCBV profile in a representative anatomical volume (somatosensory cortex) is reported on the right panel, together withthe rCBV timecourse produced by vehicle administration in a group of control rats (Sprague Dawley, n = 6). (A) Lister Hooded n = 14, (B) Sprague-Dawley n = 10, and (C) Wistarn = 14. Corresponding MABP timecourses are presented in Fig. 2 (LH-HAL, SD-HAL and Wistar-HAL, respectively).

administration in halothane-anaesthetised mice produced a tran-sient (≈10-min) increase in MABP (absolute MABP increase≈30 mmHg, corresponding to a relative MABP increase of 46%).

The drug elicited widespread and sustained rCBV responses inthe brain of all halothane-anaesthetised C57Bl/6J subjects imaged.Voxelwise group statistics (d-amphetamine vs. vehicle) revealed aclear delineation of ventrostriatal and thalamic areas as regions ofstronger and most reproducible response (Fig. 5). A trend towardsa linear increase in phMRI baseline signal was observed in bothvehicle and amphetamine-treated mice. The trend reflects the

constant elimination of the contrast agent from the blood pool. Asthe effect was constant and gently sloped, no baseline detrendingwas introduced prior to MR signal analysis. As with rat studies, thetemporal profiles of rCBV and MABP response to d-amphetaminedid not appear to be correlated.

4. Discussion

Here we provide a detailed methodological description of arobust protocol for pharmacological fMRI in rats and mice. We

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L. Ferrari et al. / Journal of Neuroscience Methods 204 (2012) 9– 18 15

Fig. 4. Temporal profile of MABP recorded in C57BL6/J mice under HAL or IF main-tenance anaesthesia. Top: baseline MABP data prior to pharmacological challenge.The subjects received a vehicle injection at the time indicated by the arrow. Bot-tom: MABP response produced by acute d-amphetamine (amph) administration(0.5 mg/kg i.v.). The arrow indicates time of d-amphetamine injection. Data arereported as mean ± SEM. Corresponding functional activation maps are presentedin Fig. 5.

show that the protocol ensures controlled arterial blood gas levelsand blood pressure with two different gaseous anaesthetics anddemonstrate its use to reliably map the functional response elicitedby a robust dose of psycho-stimulant drug (d-amphetamine) bothin rats and C57Bl/6J mice.

Previous accounts on the implementation of phMRI methods inrodents have been recently reported from us (Gozzi et al., 2010a,b,

2011b) and other groups (Perles-Barbacaru et al., 2011; Silva et al.,2011; Steward et al., 2005; Van der Linden et al., 2007). The presentmanuscript provides for the first time a step-by-step highly detaileddescription of the materials and critical procedures needed tosuccessfully and robustly implement phMRI in rats and mice. More-over, we provide evidence of the feasibility of the protocol underdifferent anaesthetic conditions in two different rat strains and in amouse line (C57Bl/6J) commonly used as background for the gen-eration of genetically modified animals. Finally, to the best of ourknowledge this is the first description of a protocol that enablesconcurrent continuous blood pressure measurements and func-tional imaging in the mouse. This option is of critical importancefor phMRI studies where the vasopressive effects of drugs can giverise to confound that cannot be straightforwardly separated fromthe central effect of the drug in absence of reliable inter-subjectmeasurements.

The protocol relies on the use of artificial ventilation intracheostomised rodents under neuromuscular blockade. Thisexperimental option has proven very effective in ensuring an accu-rate control of blood partial pressure of carbon dioxide (paCO2),a potent vasodilatory agent (Yang and Krasney, 1995) that couldconfound haemodynamic-based measurements of neuronal func-tion. The onset of hypercapnia and acidosis is frequently observedin spontaneously breathing anaesthetised animals (Steward et al.,2005; Zausinger et al., 2002), a condition that may give rise tostimulus-unrelated haemodynamic responses which could maskthe neural effect of interest (Jones et al., 2005). A tight control ofpaCO2 is of particular importance in phMRI, where the responsefunction produced by pharmacological agents is unknown a priori(Schwarz et al., 2006b), and may take the form of gradual signalchanges that could be easily mistaken with (or even neutralisedby) vasodilatory responses associated to hypercapnic conditions(Sicard and Duong, 2005). In the present study, arterial paCO2measurements before and after the phMRI time series highlightedthe presence of normocapnic values consistent with the valuesrecorded in conscious freely breathing rats (Pepelko and Dixon,1975) and mice (Kline et al., 1998). For rat studies, this was achievedunder two different anaesthetic regimens and in three different ratstrains, a testimony of the versatility of the protocol. Importantly,normocapnic values were also observed in animals challengedwith d-amphetamine, a psychostimulant that alters respiratory fre-quency and thus may lead to blood gas alterations (Mathew andWilson, 1985).

Fig. 5. phMRI activation (Z statistical maps) produced by acute d-amphetamine (amph) administration vs. vehicle (saline) in C57BL6/J mice under halothane maintenanceanaesthesia. The panel on the right illustrates mean rCBV profile in a representative anatomical volume (somatosensory cortex) together with the rCBV timecourse producedby vehicle administration in the same subjects. Corresponding MABP timecourses are presented in Fig. 4 (C57-HAL).

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16 L. Ferrari et al. / Journal of Neuroscience Methods 204 (2012) 9– 18

The protocol described in the present work entails the use of anenriched oxygen atmosphere (50%). In our experience this optionallows for the detection of robust and reproducible rCBV responsesto a variety of drugs in rats and mice, while minimising the riskof adverse hypoxic conditions. The use of such enriched oxygenatmosphere is not expected to significantly alter the magnitudeand quality of drug-induced haemodynamic responses. Consistentwith this, a recent systematic evaluation of the role of inspiredoxygen on blood-oxygen level dependent (BOLD) signals, cerebralblood flow (CBF) and cerebral metabolic rate of oxygen in freelybreathing animals, did not reveal major differences in any of theseparameters between hyperoxic (100% O2) and normal air (21%O2) (Sicard and Duong, 2005). It should also be noted that arte-rial pO2 levels measured before and after the imaging session inour studies were comparable to those recorded in conscious ani-mals under similar oxygen-enriched atmosphere (Kline et al., 1998;Sicard and Duong, 2005).

The combined use of artificial ventilation and neurovascu-lar blockade has also the important merit of virtually abolishingrespiration-induced motion artefacts, a finding reported also byother groups (Ma et al., 2008). As a result, the pre-processing offunctional data could be performed without the need to intro-duce motion correction algorithms, a procedure that result in afaster and more reliable assessment of inter-subject functionalresponses (Huettel et al., 2004). This is of crucial importance inphMRI, where drug effects are best assessed by comparison witha separate group of vehicle- or placebo-treated control subjects(Schwarz et al., 2006b).

The potentially serious confounding contribution of drug-induced blood pressure (MABP) changes on central haemodynamicparameters (Kalisch et al., 2001; Tuor et al., 2002) justifies theimplementation of invasive procedure to measure cardiovascularparameters in phMRI studies. In our protocol, the catheterisation ofa femoral artery was introduced to permit continuous monitoringof MABP and thus obtain individual correlations between phMRIresponses and drug-induced vasoactive changes throughout theimaging experiment. When the temporal dynamics of these twoprocesses are uncorrelated, this information can be employed todisprove confounding peripheral cardiovascular contributions tocentral measurements of neuronal function. However, when theperipheral and central responses elicited by a drug are temporallycorrelated, measurements of CBF autoregulation, i.e. the abilityof reactive blood vessels to homeostatically compensate periph-eral MABP changes to maintain constant perfusion (Edvinssonand Krause, 2002), can be employed to assess the contributionof MABP to the haemodynamic readout of choice. In the case ofthe rat halothane protocol described here, we previously mappedwith great accuracy the MABP range of CBF autoregulation, anddemonstrated accordingly that within this range (60–1200 mmHg)abrupt positive (Gozzi et al., 2007) or negative MABP (Gozzi et al.,2006) changes do not result in central microvascular rCBV alter-ations. These findings define an operational window that canbe used to discard subjects with exceeding MABP responses oridentify doses of drugs that are not expected to produce centralhaemodynamic perturbations in CBV-based measurements. Anal-ogous investigations performed by other authors with IF showedthat the maintenance concentrations used in the IF protocolsdescribed here only marginally attenuate autoregulation responses(Lee et al., 1994).

In the mouse protocol, the arterial route was also exploited asan intravascular access for systemic injection of contrast agent anddrugs. Although femoral veins can be cannulated in mice, we did notsucceed in standardising this procedure, and thus opted for the useof a custom-made three-way piece that can be used to switch flowbetween the MABP transducer and injection line. This catheterisa-tion strategy proved robust, easy to standardise and amenable to

rapid implementation (ca. 15 min with trained operators) on mul-tiple mouse strains and weight ranges. All surgical wounds, bothin rats and mice, were pre-infiltrated with tetracaine to providesupplementary analgesia and prevent sub-threshold activation ofpain pathways that may confound central measurements of brainfunction. The compound has been previously shown to producenegligible central nervous system exposure (Ferrari et al., 2010).

The phMRI experiments reported in this manuscript wereperformed using intravascular superparamagnetic iron oxidenanoparticles (SPION) to sensitise images to CBV (Mandeville et al.,1998). Coupled to the use of spin-echo sequences, the method hasthe advantages of increasing the functional contrast at our field (i.e.4.7 T, Chen et al., 2001) and improving the parenchymal specificityof the measurements by reducing the extra-vascular effects fromlarge vessels (Boxerman et al., 1995; Kim and Ugurbil, 2003). More-over, by minimising the contribution of susceptibility artefacts atair–tissue interfaces, the use of spin-echo sequences greatly facili-tates the implementation of whole-brain functional imaging of therodent brain. The possibility to cover entire brain volumes withnegligible artefactual contributions is of pivotal importance for thesuccessful implementation of phMRI because the precise locationof the substrates modulated by psychopharmacological agents istypically unknown and cannot be predicted a priori.

The psychostimulant d-amphetamine was used as a tool com-pound to demonstrate the feasibility of the approach both inrats and mice. The drug stimulates dopamine release and pro-duces a composite effect that involves mesolimbic and mesocorticaldopamine pathways, resulting in a composite pattern of increasedbrain activity (Gozzi et al., 2011b; Schwarz et al., 2007a,b).The widespread activation observed in all the three rat strainsexamined and in the mouse experiment is consistent with themechanism of action of the drug and previous imaging stud-ies (Chen et al., 1997; Preece et al., 2007; Rausch et al., 2002;Schwarz et al., 2004a, 2009). Interestingly, differences in themagnitude (LH > Wistar > SD) and time-profile of the response to d-amphetamine across rat strains were observed. Whether this effectreflects different pharmacokinetics or haemodynamic response tothe drug remains to be determined.

The performance of phMRI studies in anaesthetised animalsrequires a careful assessment of potential interactions between theanaesthetic and the drug of interests, especially when phMRI dataare used to draw conclusion to non anaesthetised conditions (Gozziet al., 2008c; Steward et al., 2005). For example, functional stud-ies with cocaine (a dopamine-transporter inhibitor) consistentlyproduced brain activation and increased metabolism in HAL- andalpha-chloralose-anaesthetised rats (Ceolin et al., 2004; Marotaet al., 2000; Schmidt et al., 2006; Schwarz et al., 2004b) (Du et al.,2009) as well as in awake animals (Febo et al., 2005), but showednegative functional responses in isoflurane-anaesthetised rodents(A. Gozzi, unpublished results, Du et al., 2009), probably a result ofdifferential interaction of the anaesthetics with striatal dopamin-ergic function (Salord et al., 1997). In the light of the knowninteraction between IF and dopaminergic function, we did not studythe effect of d-amphetamine in IF-anaesthetised rats, and we intro-duced a post-surgery switch to HAL as maintenance anaesthesiain the mouse d-amphetamine study. However, in our experiencethese interactions are typically neurotransmitter-specific. Consis-tent with this, recent studies performed in our lab showed thatIF can be reliably used for phMRI studies of non-dopaminergicmechanism. Specifically, serotonergic and adrenergic drugs pro-duced robust and plausible functional responses in mice andrats consistent with receptor distribution patterns and comple-mentary functional readouts performed in conscious subjects(Gozzi et al., 2010b).

A limitation of the protocol described here is the use of invasiveprocedures such as tracheostomy, or femoral vessel cannulation

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L. Ferrari et al. / Journal of Neuroscience Methods 204 (2012) 9– 18 17

which prevent its application to recovery experiments. However,experimenters interested in adapting this procedure to longi-tudinal studies should be able to substitute tracheostomy withendotracheal intubation (Stark et al., 1981), achieve physiolog-ical control of pCO2 by using a transcutaneous pCO2 monitors(Mueggler et al., 2001; Ramos-Cabrer et al., 2005), and obtainintravascular access using lateral tail vein cannulation (Weber et al.,2006). The use of non invasive tail-cuff systems for non invasiveblood pressure measurement (Krege et al., 1995) could in principlebe used to replace the use of intra-arterial probes. However, prelim-inary experiments performed in our laboratory using a commercialsystem (Columbus Instr, USA) highlighted a strict dependence onbody temperature and poor sensitivity of the method, particularlywhen hypotensive states occur, which may complicate the imple-mentation of this method within the confined environments of anMR scanner.

In conclusion, we provide a detailed methodological descrip-tion of a robust phMRI protocol which ensures stable physiologicalparameters and reproducible functional responses to psychoac-tive drugs in rats and mice. The tight control of physiologicalparameter achievable through artificial ventilation and continuousmonitoring of blood pressure is ideally suited for phMRI studieswhere the use of psychoactive agents may alter peripheral respi-ratory and cardiovascular parameters that could confound centralhaemodynamic readouts. We expect this methodological accountto encourage the diffusion of pre-clinical phMRI methods acrossdifferent areas of neuroscience and psychopharmacology.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jneumeth.2011.10.020.

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