Mapping of brain activation in response to pharmacological agents using fMRI in the rat

Magnetic Resonance Imaging 19 (2001) 905–919

Mapping of brain activation in response to pharmacological agents using fMRI in the rat Gavin C. Houstona, Nikolas G. Papadakisa,b, T. Adrian Carpentera,1, Laurance D. Halla, Bhashkar Mukherjeeb, Michael F. Jamesc, Christopher L-H. Huangb,* a

Herchel Smith Laboratory for Medicinal Chemistry, University of Cambridge Clinical School, University Forvie Site, Robinson Way, Cambridge, CB2 2PZ, UK b Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK c Neuroscience Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (North), Third Avenue, Harlow, Essex, CM19 5AW, UK

Abstract Functional MRI (fMRI) was used to investigate the effects of psychotropic compound activity in the rat brain in vivo. The effects of dizocilpine (MK-801) an N-methyl-D-aspartate receptor antagonist and m-chlorophenylpiperazine (mCPP), a 5-HT2b/2c-receptor agonist on rat brain activity were investigated over a time interval of about 1 h and the results were compared to published glucose utilisation and cerebral blood flow data. Signal magnitude increases were observed predominantly in limbic regions following MK-801 administration (0.5 mg/kg i.v) whereas signal decreases were restricted to neocortical areas; a characteristic, time dependent pattern of regional changes evolved from the thalamic nuclei to cortical regions. In contrast, mCPP (25 mg/kg i.p) produced gradual signal intensity increases in limbic and motor regions with signal decreases restricted to the visual, parietal and motor cortices. The results from both compounds show remarkable similarity with autoradiographic measurements of cerebral blood flow and glucose uptake. These experiments suggest that the spatiotemporal capabilities of fMRI may be applied to the in vivo investigation of psychoactive compound activity with potential for clinical applications. © 2001 Elsevier Science Inc. All rights reserved. Keywords: fMRI; MK-801; mCPP; Autoradiography fMRI

1. Introduction Autoradiographic radionucleotide tracer techniques have been used extensively to study changes of cerebral function in neuropharmacological investigations [1,2,3,4]. Such methods combine a comprehensive anatomic coverage of localised functional changes with high spatial resolution required for such studies in the small laboratory animal. Thus, Nehls et al. [2] utilised both [14C]-2-deoxyglucose [5] and iodo [14C]-antipyrine [6] in conscious rats to quantify regional glucose uptake and blood flow responses following MK-801 administration. However, such techniques inherently provide limited information because each experimental group of animals only provides data at a single time point postmortem; consequently, temporal changes in cerebral * Corresponding author. Tel.: ⫹44-1223-333822; fax: ⫹44-1223333840. E-mail address: [email protected] (C.L-H. Huang). 1 Present address: Wolfson Brain Imaging Centre, University of Cambridge, Box 65, Addenbrookes Hospital, Cambridge, CB2 2QQ, UK.

function induced by the compound under investigation require large numbers of experimental animals. Functional magnetic resonance imaging (fMRI), based on blood oxygenation level dependent (BOLD) contrast [7], offers an approach by which one can both localise functional changes and map these sequentially over time in the same animal. Signal changes observed in BOLD imaging result from a change in the ratio of oxygenated to deoxygenated haemoglobin. Thus, haemodynamic responses to increased neuronal activity such as increased cerebral blood flow and volume, result in an increase in the ratio (hyperaemia) [8,9,10,5], observed as an increased signal intensity. The ability of ultra-fast imaging sequences [11,12,13] to acquire time resolved multiple slice datasets in the same subject enables dynamic processes to be imaged. Thus, fMRI studies have demonstrated measurable signal changes following somatosensory stimuli [14,15,16,17] and the administration of psychoactive compounds [18,19,20] in anaesthetised rats. This study explores the use of such MRI methods in the investigation of the effects of two compounds. Of these, the

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non-competitive NMDA receptor antagonist MK-801 is a neuroprotective compound with well documented effects on cerebral blood flow and metabolism [2,3,4] and is therefore ideal for establishing the fMRI methods for tracking functional changes following the administration of psychoactive compounds in physiological preparations. The resultant techniques were then applied to characterise the spatial and temporal function of the 5HT2b/2c receptor partial agonist mCPP; this agent produces differential cerebral effects at distinct (but close) anatomic regions [21]. Our results indicate that fMRI can characterise the time course of the actions of psychoactive compounds in the rat brain in vivo. The findings also suggest the possibility of such applications in man.

2. Materials and methods 2.1. Animal procedures All physiological procedures were performed in accordance with the UK Home Office Animals (Scientific Procedures) Act, 1986. Sprague Dawley male rats (210 –310 g) (Harlan UK Ltd, UK) were anaesthetised with 5% halothane and a 70:30% mixture of O2:N2O. The animals were randomly allocated to groups as follows: saline only (n ⫽ 2); MK-801 (n ⫽ 4); mCPP (n ⫽ 4 animals), where one individual from each drug study was first test-imaged following both saline and the drug treatment. Surgical anaesthesia, assessed by pinch and blink reflexes, was maintained with 1–2% halothane throughout all surgical procedures. The left femoral artery was cannulated for blood pressure monitoring and to sample blood for pCO2, pO2 & pH measurements. Similarly the left femoral vein was cannulated for administration of anaesthetic and test substances. All animals were tracheotomised and mechanically ventilated with a Harvard Miniature ventilator (87 breaths/min, tidal volume 1.8 –2.5 ml). Core body temperature was maintained (37 ⫾ 0.2°C) using a homeothermic blanket regulated by a rectal temperature probe (Harvard Instruments, UK). After surgery, anaesthesia was maintained with an i.v. administration of 80 mg/kg ␣-chloralose (E.Merk, Darmstadt, Germany), dissolved in saline (0.9% w/v) with an equal mass of Borax (sodium tetraborate decahydrate, Aldrich, UK). Chloralose was supplemented with i.v. doses of 40 mg/kg every 90 min thereafter. At least 20 min elapsed between any given supplementary dose and the start of the functional imaging period. The animal, together with blood pressure transducer, rectal probe and heating blanket was then moved to the cylindrical (birdcage) transmit resonator and positioned in a supine position above the (receive only) surface coil. The animal’s head was held securely in the centre of the transmit resonator with foam wedges. The probe together with the rat was placed on a self-sufficient physiological monitoring and support trolley consisting of a small animal ventilator (Har-

vard Instruments, UK), gas cylinders, homeothermic blanket regulated via a rectal probe (Harvard Instruments Ltd, UK), blood pressure and heart rate monitor all continually powered by an uninterruptable power supply (Emerson computer power supply SL 1000, RS Components, UK) [22]. Hence, uninterrupted physiological monitoring and maintenance during the surgical procedures, transport of the animal to the magnet and the imaging protocol was achieved. Image acquisition commenced after the animal had achieved a steady heart rate and blood pressure following the surgery and change of anaesthetic. 2.2. MR hardware The MR imaging was performed using a 2T (Oxford Instruments, Oxford, UK) 31 cm horizontal bore superconducting magnet. The magnet was equipped with a custom built 11 cm internal diameter, Maxwell-Golay gradient set with each axis driven by a pair of Techron 7570 power supplies (Crown International Inc., Indiana, USA) giving a maximum field strength of 198 mT m⫺1. The data acquisition, gradient and radiofrequency waveform generation were performed by a Bruker Analystische Messtechnik MSL 100 console operating Tomikon imaging software (Bruker Medical GmbH, Ettlingen, Germany). Separate transmit and receive coils were used to maximise the signal to noise ratio (SNR), radio-frequency (r.f.) homogeneity and to minimise space constraints. A sine-spaced birdcage resonator [23] was used for homogeneous r.f. excitation and signal was received through an approximately 1 cm diameter surface coil made from PTFE coated silver wire and mounted within the transmit resonator. 2.3. Image quality assurance Observed fMRI signal changes typically produce deviations from baseline of the order of only a few percent; additionally the echo planar imaging (EPI) family of pulse sequences are sensitive to hardware instability. Complete quality assurance (QA) testing of the hardware was therefore necessary in order to distinguish experimental signal change resulting from physiological effects from any possible equipment instabilities. The QA tests established the performance of the system hardware by measuring the signal to noise ratio (SNR) and the stability of the MRI hardware prior to each experiment by collecting multiple replicate images for a period equivalent to the experiment’s duration. The SNR was measured using a standard protocol (multi-spin echo (SE): repetition time (TR) ⫽ 2360 ms; echo time (TE) ⫽ 40 ms; number of echos ⫽ 4; image matrix ⫽ 256; field of view (FoV) ⫽ 6 cm; slice thickness (SlTh) ⫽ 1.4 mm) using a phantom of doped water (1.25 g/liter CuSO4, 2.4 g/liter NaCl). All fMRI experiments were performed at an SNR greater than 120 (The ratio mean signal intensity/standard deviation of the noise was 142 ⫾ 10.1 and 137 ⫾ 9.6 for the MK-801

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and the mCPP experimental series respectively). Replicate images of the phantom were acquired with the ASE-EPI imaging protocol used for acquisition of experimental data, i.e. Tr ⫽ 3000 and 7500 ms, Tr ⫽ 71 and 67 ms for the MK-801 & mCPP experiments respectively, FoV ⫽ 3 cm, SlTh ⫽ 2 mm, TE offset ⫽ 20 ms. The stability of the signal was assessed over a period of an hour by plotting signal intensity against time and calculating the percentage deviation of the signal from the mean of the time series (1 cm diameter region of interest; signal variability ⬍1.7% overall). Image motion was assessed by viewing subtraction images. 2.4. Imaging protocols Pilot scans (gradient echo (GE), Tr ⫽ 100 ms, TE ⫽ 10 ms, flip angle ␣ ⫽ 60°, matrix ⫽ 128 ⫻ 64, SlTh ⫽ 2 mm, FoV ⫽ 5 cm) checked the position and orientation of the animal’s head. Subsequent to any repositioning of the animal, the magnet was shimmed and further pilot scans of brain anatomy in the sagittal and coronal planes were obtained in order to position the functional imaging slice. High resolution T1- weighted anatomic images (in-plane resolution of 117 ␮m) of the selected slice(s) were acquired using a fast refocused GE pulse sequence with image acquisition parameters of Tr ⫽ 200 ms, TE ⫽ 10 ms, ␣ ⫽ 60°, matrix ⫽ 256 ⫻ 256, FoV ⫽ 3 cm, SlTh ⫽ 2 mm. The acquisition of multiple images over extended periods of time before and following drug administration was limited by the data storage capacity on the spectrometer’s local disk. Accordingly, the imaging protocols entailed a compromise between temporal resolution, brain coverage determined by the number of slices through the brain acquired at each time point, and the duration of the imaging period. Thus, the total number of images, simply calculated as (no. slices acquired x the duration of imaging protocol (s))/TR (s), where TR is the repetition time or delay between successive scans, were optimised specifically for each of the two compounds studied. 2.5. MK-801 The first series of experiments used 0.5 mg/kg MK-801 administered i.v. (i/v) to induce BOLD signal changes. Image acquisition was optimised to maximise the sampling rate by acquiring a single slice in the horizontal plane which encompassed most of the regions of interest that were previously identified as sites of MK-801 activity [2,3,4], including the frontal, temporal and entorhinal cortices, hippocampus, dentate and medial geniculate and mediodorsal thalamic nuclei [24]. Data were acquired using an asymmetric spin-echo echo planar imaging (ASE-EPI) sequence [12,13] with the parameters Tr ⫽ 3000 ms, TE ⫽ 71 ms, TE offset ⫽ 20 ms, FoV ⫽ 3 cm, SlTh ⫽ 2 mm, matrix ⫽ 64 ⫻ 64. A single shot image was acquired every 3 s for 1 h (total number of images ⫽ 1200). Typically 200 images (10 mins)

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were acquired prior to 0.5 mg/kg MK-801 i/v administration and the remaining 1000 images were obtained following drug induced signal changes over the remaining 50 min. 2.6. mCPP The studies that explored the effects of mCPP attempted a more extensive coverage of brain anatomy in order more fully to characterise its fMRI effect [1]. The imaging plane was altered to the conventional coronal plane and four contiguous slices anterior to the cerebellum were acquired. Preliminary analysis of the MK-801 data established that temporal resolution could be reduced without compromising data analysis. In addition, the spectrometer’s disk was repartitioned, to increase the maximum local storage by about 750 images to 1960. Thus it was possible to acquire 4 slices through the brain at time points every 7500 ms, to give 480 frames over a time course of 1hr. Images were acquired for 10 min prior to the administration of mCPP 25 mg/kg i/p or vehicle alone (saline 0.9%) and 50 min following test substance administration using asymmetric SE-EPI (Tr ⫽ 7500 ms, TE ⫽ 67 ms, TE offset ⫽ 20 ms, FoV ⫽ 3 cm, SlTh ⫽ 2 mm, matrix ⫽ 64 ⫻ 64). Acquisition of data were uninterrupted by the administration of the test substance. 2.7. Data analysis The anatomic and ASE-EPI images were transferred from the spectrometer to the network via ASL (Aspect to SCSI Link, Cambridge Magnetic Resonance Systems (CaMReS), U.K.). EPI data were reconstructed to images (128 ⫻ 128 matrix) using a template scan for phase correction of the image [25]. Motion artefacts were initially assessed by visual inspection of a movie of the time series in CMRVIEW (CaMReS) and subsequently assessing subtraction images for pixel level motion. Any detectable motion was corrected using Woods Automated Image Registration (AIR) motion correction algorithm [26,27]. Statistical analysis of the resulting aligned image stacks was performed in IDL (Interactive Data Language, Colorado, USA) using t statistics. BOLD changes were measured at different times following drug administration by comparing the 50 baseline images acquired just prior to drug administration with a running average of 20 test images that was temporally shifted by one image for each test. Values from each pixel in the resulting average were tested against the corresponding pixel in the baseline average using a pixel by pixel t test that incorporated a Bonferroni correction factor equal to the number of pixels in a frame (i.e. 1282). Pixels with statistically significant changes of P ⬍ 0.001 were false coloured and overlaid onto corresponding T1 weighted anatomic images. The resulting images were concatenated into a single stack for viewing as a movie. Regions of interest (ROI’s) were selected from these probability maps based on clusters

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of statistically significant signal change representing volumes lying within the anatomic structure of interest. Such structures were selected with reference to a standard stereotaxic atlas [24] with the position of the centre of the imaging slice estimated from the pilot scans and compared to the corresponding page in the stereotaxic atlas. These were used to plot the time course of the percentage signal change normalised to the pre-treatment baseline calculated from the mean signal intensity of frames 10 –59. The first ten frames were excluded to allow the hardware and T1 relaxation to reach steady state. The present approach to signal analysis accordingly treated each pixel as representing an independent time series. It was consequently computationally more intensive than previous analyses that detected signal change from averaged values derived from standardized regions of interest [2]. It is possible that analyses that involved such averaging of pixel values over large regions of interest would offer a more sensitive detection of the signal changes being sought. However, such an approach would give signal to noise ratios and therefore detection sensitivities that varied with the size of the region of interest. In contrast, the analysis adopted here independently tested each individual value within each pixel for statistical differences from the baseline signal from the same pixel prior to stimulus application. Its results were thus independent both of values in surrounding pixels, of any variations that would take place in the selection of the regions of interest, and of the precise anatomic identifications inherent in such a selection. They therefore offered a more objective albeit conservative detection and analysis of signal change. Data are presented both as a plot of the signal change for five selected ROI’s and as a time course of corrected pvalues (see figure legends) calculated as if multiplied by a Bonferroni correction factor of 1282: therefore a p-value of 0.05 on the plots actually represents the Bonferroni corrected p-value of 0.05/(128 ⫻ 128). All quoted p-values were derived using the multiple Bonferroni comparisons method. The analysis was replicated for the control studies using vehicle alone and employing equivalent imaging planes and route of administration. All graphs incorporate a corresponding on/off flicker plot in which periods of significant signal change are denoted as the raised regions of the graphs indicating the duration of the significant change (P ⬍ 0.001).

throughout the 60 min data acquisition period (1 cm diameter region of interest). Examination of subtraction images from the individual QA data sets revealed no obvious motion artefact. Control i.v. or intraperitoneal (i.p.) saline (0.9%) injections administered during image acquisition in the horizontal (MK-801) or coronal plane (mCPP) produced no areas of statistically significant signal change in cerebral tissue. Time course data based on analysis of regions of interest are presented alongside equivalent analysis from each drug study. No deviation in blood pressure was observed following saline administration for either the i.v. or i.p. route. In contrast to the variation in serial images of the phantom, rat images exhibited greater high frequency fluctuation about the mean signal intensity (i.e. noise), typically 5% from the baseline for the regions of interest studied. This was greater than the variability calculated in the QA tests (1.5% of the mean) and can be attributed to averaging across smaller ROI’s and the contribution of physiological variations to the MR signal. However, this high frequency variation would not be expected to influence the slow onset and offset of drug induced changes in BOLD images, occurring over more than 20 –30 s. Discrete regions of hyperintensity after saline administration were observed in regions with a preponderance of cerebrospinal fluid (CSF), such as the ventricular spaces, and similarly the cerebrospinal fluid ‘pocket’ formed between the entorhinal cortex and cerebellum. Thresholding identified these areas and excluded them from further analysis. 3.2. MK-801 A single horizontal slice was acquired using anatomic pilot scans (Fig. 1) which encompassed most regions of interest identified from the cerebral metabolism and blood flow literature as being sites of MK801 action [28,2,29]. Administration of MK-801 (0.5 mg/kg) caused a gradual and prolonged decrease of 10 –22% in mean arterial blood pressure from pre-injection levels which became statistically significant 25– 40 min following injection (P ⬍ 0.05, paired t test). No significant changes in the blood gases collected before and at the end of the imaging period were observed: pO2 94 ⫾ 6.5 mmHg, pCO2 34 ⫾ 2.7 mmHg pH 7.37 ⫾ 0.02 (mean ⫾ standard deviation, n ⫽ 4). 3.3. MK-801 stimulated BOLD signal changes

3. Results 3.1. Quality assurance The quality assurance protocols performed prior to the acquisition of MK-801 and mCPP functional data gave signal to noise ratios (SNR) of 142 ⫾ 10.1 (3 data sets) and 137 ⫾ 9.6 (4 data sets) respectively (mean SNR ⫾ standard deviation). Signal variability (excluding the first ten frames) remained less than 1.7% of the mean signal intensity

Figs. 2 and 3 show time course data from five regions of interest highlighted in the left hand column of Fig. 2. Mean percent signal changes for each ROI are shown (left graphs) with corresponding probability plots of changes to the mean as determined by the t test (right hand graphs) in equivalent regions of interest (a)–(e) in typical experiments following administration of MK-801 or saline (Figs. 2 and 3 respectively). Fig. 3 illustrates negative results following control injection of vehicle (0.9% saline).

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Fig. 1. Coronal pilot scan (left) marked with the position of the target scan for MK-801 fMRI, shown as T1-weighted anatomic (middle) and equivalent ASE-EPI (right) images.

Fig. 2 demonstrates the irregular nature of the signal changes by plotting the time course of the mean percentage signal change time course for five regions of interest. A varying but characteristic temporal pattern of functional maps was observed that was observed in all four individual subjects studied. The immediate and initial (⫹0 min) regions of activation centred around the medial geniculate nucleus and mesencephalic trigeminal nucleus representing synaptic areas of specific sensory pathways (auditory and somatosensory). Parallel to the subsequent decrease in spatial extent of these activations, typically 20 min after MK801 administration, cortical regions began to exhibit significant signal change (Fig. 2d). Furthermore the significant changes in the cortical regions initially were unilateral in nature (n ⫽ 4) at earlier time points (20 –30 min post administration) and only became bilateral 30⫹ minutes following MK-801 administration. The time plots in Fig. 2

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show that regional activations were ‘clustered’ in time. For example the mesencephalic trigeminal nucleus (Fig. 2c) showed significant signal changes within a minute of i/v injection of MK-801 and toward the end of the imaging period (40⫹mins) with a relatively quiet period in between. The corresponding probability plots show that the BOLD changes in the medial geniculate nucleus were oscillatory in comparison to the mesencephalic trigeminal nucleus during the quiet period. The mesencephalic trigeminal nucleus also showed an inversion of signal change from the early increase in BOLD signal through to a decrease in BOLD signal. Fig. 3 shows the percentage signal intensity change from five regions of interest (left panels) and the corresponding probability plot (right panels) for a representative control animal receiving i.v. saline (0.9%). No statistically significant signal intensity change was observed in any region analysed, although a transient signal attenuation was observed in discrete regions such as the mesencephalic trigeminal nucleus (Fig. 3c). Similarly, false coloured functional probability maps derived from images acquired following i/v saline administration showed no region of significant signal change. Both sets of graphs (Figs. 2 and 3) show a corresponding flicker plot which denote periods of significant signal change as raised regions of the graphs. Such on/off (significant/non-significant) flicker plots indicate that the duration of significant change at a threshold of P ⬍ 0.001 can be as short as a minute, close to the time course examined by flow autoradiography [2]. As mentioned previously if the stringency of the statistics applied to the data are relaxed, e.g. to P ⬍ 0.05 or P ⬍ 0.01 the spatial extent of the activated region increases. Correspondingly the duration of signal change at the probability threshold also increases. It should be noted that the on/off flicker traces represent at least two contiguous pixels of significant change within the region of interest (0.1 mm3 of tissue). Fig. 4 provides a visual representation of the spatial extent and time course of regions with statistically significant change (P ⬍ 0.001). Thresholding at a less stringent p-value resulted in an increase of the spatial and temporal extent of significant signal deviation observed in the coloured functional maps (data not shown) without significantly altering the anatomic loci. Equivalent probability maps showed no area of significant signal change for vehicle alone treated animals (data not shown). In summary, statistically significant fMRI signal increases (P ⬍ 0.001) following MK-801 administration were observed in limbic structures including the hippocampus, dentate gyrus, entorhinal cortex, in the auditory region of the medial geniculate nucleus, the parietal cortex (neocortical areas) and discrete regions within the cerebellum. In contrast significant signal decreases were restricted to the neocortical regions of the frontal and auditory cortex. No significant signal changes were observed in the sensory and

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Fig. 2. Representative time courses of fMRI signal change following administration of MK-801 in five selected regions of interest (far left). Graphs in the middle column show the percentage signal change within the coloured regions delineated as red areas on the images (left column). Corresponding p-value plots from a single pixel within the region of interest are shown on the right. Periods when the signal intensity and probability change achieved significance at the 0.1% level are indicated by the flicker plots on the abscissa of the graphs. (a) medial geniculate nucleus (b) mediodorsal thalamic nucleus (c) mesencephalic trigeminal nucleus (d) parietal cortex (e) caudate putamen (a non-activated region).

associated sensory-motor areas such as the caudate putamen (basal ganglia). Table 1 compares results from the autoradiography literature and this fMRI study representing all of the regions of interest investigated in this study [2]. Significant changes of MR signal (or blood flow and glucose metabolism) are thresholded at three p-values of 0.05, 0.01 and 0.001. In contrast to the time resolved fMRI data, such tabulations give no indication of the time course or spatial extent of discrete regions of BOLD signal change. The autoradiography data were taken from Nehls et al. (1990), where the authors attempted to match the tracer kinetics of the cerebral

blood flow (data integrated over 1 min) and glucose uptake experiments (data integrated over 45 min) to a single time point of 15 min following MK-801 administration [2]. An overall similarity between the results exists between the anatomic regions covered in the fMRI experiment and the corresponding ones in the autoradiographic studies with the exceptions of the caudate putamen (striatum) and the medial geniculate nucleus. It seems reasonable to suggest that the caudate putamen, involved in sensory-motor co-ordination, was strongly influenced by the anaesthesia required during the fMRI experiment, highlighting the difficulties comparing data obtained in conscious and anaesthetised subjects. Similarly, the

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Fig. 3. Control results. i/v saline (0.9%) injection showed no notable areas of signal change. The graphs on the left are percentage signal change plots over time (saline administration at t ⫽ 0) with the horizontal bars indicating the mean ⫾ the standard deviation. The graphs on the right are inferred p-values with the horizontal lines representing the P ⫽ 0.05 and P ⫽ 0.001 threshold levels. Regions of interest (a-e) are the same size (number of pixels) and equivalent in anatomic location to those shown in the images in Fig. 3 (a) medial geniculate nucleus (b) mediodorsal thalamic nucleus (c) mesencephalic trigeminal nucleus (d) parietal cortex (e) caudate putamen.

detection of changes in the medial geniculate nucleus highlights the advantage of temporal resolution and duration that fMRI affords. Fig. 2(a) demonstrates the transient nature of functional changes in this region and the lack of statistically

significant (P ⬍ 0.05) change approximately 5–18 min after MK-801 administration when the autoradiographic measurements were obtained [2], emphasising the limitation of techniques that measure functional changes at a single time point.

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Fig. 4. Colour maps representing regions of statistically significant increased BOLD signal evoked by 0.5 mg/kg MK-801 i/v at the P ⬍ 0.001 level. The horizontal images are orientated with the olfactory bulb to the left of the frame and the cerebellum at the right. The images show every 50th frame of the activation series and the corresponding time point in minutes is shown at the bottom left of each frame.

3.4. Results: mCPP 3.4.1. Quality assurance & general physiological changes The QA protocols gave an SNR of 137 ⫾ 9.6 (signal magnitude ⫾ standard deviation) and the signal variability remained less than 1.7% of the mean throughout the time course of the protocol. These figures are comparable to the quality analysis findings from the protocols used in the study of MK-801. There was no evidence of image motion in subtraction images in any of the data sets (n ⫽ 4). mCPP (25 mg/kg i.p.) administration caused an immediate and prolonged (typically 1 h) increase in mean arterial blood pressure from pre-injection levels 36 ⫾ 12% (P ⬍ 0.01, paired t test). Injection of vehicle (saline) was without significant effect for any individual animal (n ⫽ 3). No significant change of the blood gases sampled prior to and after the imaging period was observed: mean pO2 89 ⫾ 7.1 mmHg, pCO2 38 ⫾ 2.8 mmHg, pH 7.43 ⫾ 0.03 (mean of all samples ⫾ standard deviation). 3.5. mCPP stimulated BOLD changes The experiments with mCPP acquired a series of contiguous coronal slices anterior to the cerebellum (Fig. 5). The increased amount of data collected at each time point was at

the expense of some temporal resolution. Administration of mCPP caused gradual signal changes from baseline (pretreatment) levels that extended beyond 50 min and occurring predominantly in the motor and limbic regions. Signal increases (P ⬍ 0.001) were seen in the motor regions of the substantia nigra reticulata, occulomotor complex (motor), subthalamic nucleus (motor, basal ganglia), lateral habenular nucleus (subthalamic), lateral hypothalamic area, paraventricular nucleus (limbic), thalamic nuclei (anteroventral, anteromedial (both association nuclei, limbic) and parafasicular) and the hippocampus (limbic) as well as white fibre tracts (fornix and corpus callosum). Significant decreases in signal (P ⬍ 0.001) occurred in cortical regions such as the visual, auditory, parietal somatosensory and motor cortex. Fig. 6 shows the position of the four slices acquired with colour overlays of significant areas of signal increase and decrease (red pixels) at P ⬍ 0.001. In contrast to the responses to MK-801, the time course profile of signal change evolved in an almost uniform fashion for the duration of the imaging period. Fig. 7 and 8 plot typical time courses of signal changes within three regions of interest for mCPP (25 mg/kg i/p) alone and saline (0.9%) administration respectively. The monotonic change in signal from pre-injection baseline levels in Fig. 7 is typical of all the regions and animals

G. Houston et al / Magnetic Resonance Imaging 19 (2001) 905–919 Table 1 Effects of MK-801 (0.5 mg kg⫺1) upon cerebral blood flow, local glucose utilisation and fMRI (data pooled n ⫽ 4). 0.000 § no significant change; increase (⫹)0.050 § p ⬍ 0.05, (⫹)0.010 § p ⬍ 0.01, (⫹)0.001 § p ⬍ 0.001; decrease (⫺)0.050 § p ⬍ 0.05, (⫺)0.010 § p 0.01, (⫺)0.001 § p ⬍ 0.001, nip ⫽ not in images. Where a slash (/) appears there are differences/ambiguities in nomenclature of brain regions between authors and the regions have been pooled. (see reference Nehls et al. 1990) Region CEREBELLUM Medulla/pons MESENCEPHALON Inferior colliculus Substantia nigra reticulata DIENCEPHALON Medial geniculate Lateral geniculate Lateral habenular nucleus Ventroposterior thalamic nucleus Mediodorsal thalamic nucleus Anterior thalamic nucleus TELENCEPHALON Hippocampus Dentate gyrus Globus Pallidus Caudate Putamen Visual cortex Entorhinal cortex Auditory cortex Cingulate cortex Parietal cortex Sensory motor cortex Frontal cortex MYELINATED FIBRE TRACTS Internal capsule Corpus callosum Genu of corpus collosum

Cerebral Glucose blood flow utilisation ƒMRI 0.000 0.000

0.000 0.000

(⫹)0.001 nip

(⫺)0.010 0.000

(⫺)0.001 0.000

(⫺)0.010† 0.000

0.000 0.000 (⫹)0.050 0.000 (⫹)0.010 0.000

0.000 0.000 (⫺)0.010 0.000 0.000 (⫹)0.010

(⫹)0.001 0.000 nip 0.000 (⫹)0.001 (⫹)0.001†

(⫹)0.010 (⫹)0.001 0.000 (⫹)0.010 (⫹)0.010 (⫹)0.001 0.000 (⫹)0.050 (⫹)0.050 0.000 0.000

(⫹)0.001 (⫹)0.050 0.000 (⫹)0.050 0.000 (⫹)0.001 (⫺)0.050 (⫹)0.001 (⫺)0.050 (⫺)0.001 (⫺)0.010

(⫹)0.050 (⫹)0.001† 0.000 0.000 nip (⫹)0.001† (⫺)0.010 nip (⫺)0.001 0.000 (⫺)0.001

0.000 (⫹)0.010 (⫹)0.001

0.000 0.000 0.000

0.000 nip nip

† The p-value represents the median of the p-values (i.e. most but not all animals exhibited significant change at this probability threshold).

that were examined (n ⫽ 4). The notable exception was the visual cortex, which showed immediate (less than 1 min) and prolonged statistically significant increase in signal intensity (P ⬍ 0.001, 0 –50 mins). The graphs in Fig. 7 indicate the stringency of the statistics employed for this study. The plots of percentage signal change show a clear trend away from pre-treatment levels that was only observed as statistically significant in the false coloured maps (P ⬍ 0.001) when the trend of signal change was clearly observable. The corresponding probability plots show that statistical significance at the P ⬍ 0.05 level occurs at approximately 10 mins post administration in the thalamus and parietal cortex, as opposed to 20 & 30 mins respectively for P ⬍ 0.001 (Fig. 7a & b). As with the MK-801 increasing the probability threshold level (e.g. from P ⬍ 0.001 to P ⬍ 0.05) increased the temporal duration and the spatial extent of significant regions. Fig. 8 shows the time course data of signal change following i.p. saline administration obtained using equiva-

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lent regions of interest (see Fig. 7). As with the i.v. saline injection, small, short-lived deviations from the baseline (pre-administration) level were observed in the probability plots (e.g. Fig. 8b, right hand column) following the saline injection. However, the signal change was not significant at the P ⬍ 0.05 level. Table 2 presents comparable anatomic regions studied with deoxyglucose autoradiography [1]. The results are presented with three probability thresholds of P ⬍ 0.05, 0.01 & 0.001. Regions exhibiting fMRI signal change have corresponding changes in glucose utilisation as reported in the literature, with the two exceptions being the corpus callosum exhibiting an increase in BOLD signal and the parietal cortex showing a decrease in BOLD with no corresponding change reported in the glucose utilisation literature.

4. Discussion The present study first applied functional MRI techniques to examine the effects of MK-801, a compound whose cerebral metabolism and blood flow effects are well characterised [28,2,29,3,4]. These techniques were then applied to examine the effects of mCPP the major metabolite of the trazodone antidepressant family [30,31,32] widely employed for pharmacological studies in animals and humans with glucose utilisation data available in the literature [1,21,33]. Although intrinsic signal change observed with fMRI is based on a different physiological phenomenon than that assessed with autoradiography, fMRI gave a pattern of functional change that closely resembles results from the established autoradiographic techniques (Tables 1 and 2). The experiments required development of specific MR acquisition protocols, physiological preparation and maintenance capabilities, and data analysis procedures. Significantly, in all four animals characteristic time dependent regional signal changes were observed (n ⫽ 4, Figs. 2 and 4). Significant signal increases, lasting approximately 5–10 min, immediately followed administration of MK-801 in thalamic regions including the medial geniculate nucleus and mesencephalic trigeminal nucleus. Concurrent with the subsidence of these significant changes, neocortical areas began to exhibit significant signal changes that continued until imaging terminated. During the concluding 10 min of imaging, sizeable thalamic regions again exhibited significant signal change contemporaneous with the cortical regions. In contrast, the i.p. administration of mCPP elicited a gradual augmentation of signal changes in limbic and motor regions lasting the duration of the imaging protocol (Fig. 6). Only the visual cortex responded with an immediate and significant change in signal. Anaesthesia was required to minimise animal movement and permit physiological monitoring and mechanical ventilation of the animal, ensuring reproducibility of the physiological conditions during imaging. Importantly, blood gases were maintained within normal physiological values

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Fig. 5. Sagittal pilot scan (top) marked with the positions of the target scan slices for mCPP fMRI. The 4 contiguous coronal slices are shown as T1-weighted anatomic (upper row) and equivalent ASE-EPI images (bottom row). Reference to this pilot image shows that the coronal slices were positioned from approximately bregma – 8 mm to 0 mm. Further reference to a standard stereotaxic atlas [24] confirmed that these slices contained the following structures; their approximate co-ordinates [24] are given in brackets: subthalamic nucleus (Bregma –3.6 to – 4.3 mm), lateral hypothalamic areas (Bregma – 0.92 to – 4.8 mm), thalamus (Bregma -1.8 to – 4.8 mm) and hypothalamus (Bregma –1.3 to – 4.4 mm).

limiting reflex changes in cerebral blood flow, and hence the fMRI signal [34], coupled with variations in arterial pCO2 and pO2 [35,36]. Nonetheless, general anaesthetics depress cerebral metabolic activity and can adversely influence the

local circulatory and metabolic responses to the compound being investigated [37,38,39,2,29,40], rendering a blunted fMRI response [41,42]. Accordingly ␣-chloralose, reported to preserve stable cerebral blood flow that increases with

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Fig. 6. Mapping of fMRI signals obtained in response to i.p. administration of mCPP 25 mg/kg. The top panel of figure 5 shows the position of the four contiguous experimental slices anterior to the cerebellum. All four slices are shown at three time points (-5, 20 and 45 min with respect to mCPP injection) and the corresponding overlay is at the P ⬍ 0.001 level. Movies of the activation maps shows the signal changes are prolonged in contrast to transient effects with MK-801.

neuronal activation, unlike several other anaesthetics commonly used in MRI investigations, was used to maintain anaesthesia [43,15,44,45]. While patterns of altered function in the present study and the published autoradiographic data are broadly similar despite appreciable differences between the methods and physiological preparations, Tables 1 and 2 show that specific anatomic regions exhibited a disparity of results in the two techniques. Comparisons between data collected from anaesthetised and conscious animals are not ideal and possibly account for the differences noted in sensory and sensory-motor co-ordination regions including the caudate putamen (MK-801), sensory motor cortex (MK-801) and the parietal cortex (MK-801 & mCPP). Furthermore, such regions exhibited either a constant (globus pallidus and caudate putamen, Figs. 2e and 7c) or gradual decrease (ventral posterolateral nucleus, ventral posteromedial thalamic nucleus and parietal cortex) of signal magnitude over the time course of the experiment, consistent with the modulation

and/or inhibition of these regions by general anaesthetics including ␣-chloralose. The ability to acquire multiple time points from a single animal is a major advantage of fMRI over autoradiography. By contrast with time resolved fMRI conducted in vivo, autoradiography techniques return a single measurement of cerebral blood or glucose utilisation integrated over 1 min and 45 min respectively. Thus, transient responses to compound administration are captured only if either the timing of the blood flow experiment following drug administration coincided with any transient change, or the transient metabolic response to the compound significantly influenced the deoxyglucose uptake averaged over the 45 min. Although the present study was limited to an imaging period of 1 h, determined by the interval between supplementary doses of ␣-chloralose, the additional time points revealed that the cerebellum and medial geniculate nucleus from the MK-801 study and the corpus callosum in the mCPP study exhibited statistically significant signal increases with no correspond-

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Fig. 7. mCPP 25 mg/kg. (a) thalamus (b) parietal cortex (c) caudate putamen (a non-activated region). Graphs to the left show mean signal change over the ROI through time with an on/off activation box indicating where significant signal changes occur (P ⬍ 0.001). An apparent mismatch between the signal change and the on/off trace is apparent. This is due to the temporal averaging imposed by the t-test. Using 20 active frames effectively averages the signal across 2.5 mins in time. Strong deviations in the signal magnitude are observed in the probability and on/off traces at an earlier time point. Smaller significant signal changes appear later.

ing change reported in the autoradiography literature (Tables 1 and 2). Specifically, Nehls et al. (1990) observed no significant change in cerebral blood flow and glucose utilisation of the medial geniculate nucleus uptake ten minutes after administration of MK-801. In agreement, the fMRI time courses in Figs. 2(a) & 4 show no significant signal change (P ⬍ 0.05) around the 10 min mark. However the temporal capabilities of fMRI do reveal that transient and significant increases of signal magnitude occurred at other times during the time course of the imaging experiment. Modification of the anaesthetic regime to a constant infusion method would enable the compound’s effects to be monitored over longer periods [44,46]. Figs. 4 and 6 show that many of the activations occur at the edges of the brain, particularly in the mCPP study (Fig. 6). These might potentially be associated with contributions from image motion and/or signal change from large vessels [47]. Such sources of signal were minimised by the careful and consistent implementation of the developed experimental protocols. Thus, for each experiment, image motion resulting from animal movement and hardware drift was minimised using anaesthesia plus head restraint and exten-

sive quality assurance (QA) respectively. The success of such precautions were monitored both through analysis of the QA data prior to imaging and through post-acquisition image registration [26,27]. Additionally, theoretical, numerical and phantom studies comparing SE, ASE and GE sequences have shown that the family of asymmetric spin echo sequences is both relatively insensitive to magnetic susceptibility changes occurring in larger vessels and preferentially sensitive to BOLD changes at the capillary level [48,49]. Thus, the appropriately calibrated [50] ASE-EPI used in these studies would localise the signal changes to sites where changes in neuronal metabolism were taking place. It is unlikely that the blood pressure changes (36 ⫾ 12%) observed following mCPP administration were responsible for the concurrent signal changes measured in proximity to large surface vessels. Zaharchuck [51] investigated the effect of cerebral blood flow and volume on the BOLD signal; no signal changes were observed following blood pressure changes between 50 and 140 mmHg [51]. The blood pressures in all the control and subjects in the present study remained well within that upper limit of 140 mmHg. It is

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Fig. 8. Mapping of fMRI signals produced with 0.9% saline i/p. (a) thalamus (b) parietal cortex (c) caudate putamen. The regions of interest (ROI’s) are anatomically equivalent to those in Fig. 7. These data show that saline has no significant effect on the BOLD signal although a small deviation can be seen in the probability traces on the right when the saline is administered. Horizontal bars represent mean ⫾ standard deviation (left panels) and P ⫽ 0.05, P ⫽ 0.001 (right panels).

therefore likely that with the present use of motion minimisation, quality assurance testing and ASE-EPI the observed signal changes primarily result from neuronal metabolism rather than such alternative influences. Differences in the route of administration and drug action are likely to account for the marked temporal and spatial differences observed in the actions of the two compounds. i.v. MK-801 injection would be expected rapidly to produce widespread BOLD changes in the brain. Their varied temporal characteristics in different regions could well reflect the complex relationship between the functional consequences of NMDA receptor blockade and the noncompetitive nature of this antagonism which glutaminergic neurones attempt to surmount with increased firing [28]. In contrast, in addition to competition with endogenous 5HT for its binding site, the i.p. administration that would result in slower absorption of mCPP, could contribute to a slower onset of significant signal change (Fig. 7). The gradual and sustained BOLD changes are likely to be a consequence of the partial agonist activity of this compound stimulating long-term modulatory effects.

The 5HT2 receptors, with which mCPP interacts, are expressed in many regions of the mammalian brain [52]. The great number of 5HT2C receptors in the cortex [52] may mediate the widespread mCPP induced signal decrease in these areas. However, such an association is not observed across all regions. Thus, high receptor numbers are also expressed in basal ganglia regions including the substantia nigra, which exhibits significant signal change (fMRI & glucose uptake), and the globus pallidus, where no significant changes are observed. The influence of general anaesthesia in producing such discrepancies can not be excluded, especially in regions such as the globus pallidus. However, disparities between receptor distribution and the elicited functional response topography are frequently observed and may be caused by other mechanisms such as drug action at other sites, regional variability between receptor density and function and ‘downstream’ effects arbitrated by neural projections and networks. Data acquisition and data analysis strategies can enhance the sensitivity of the fMRI experiment. The present study used a simple rolling t test statistic with derived p-values

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Table 2 fMRI compared to 2DG autoradiography following administration of mCPP (data pooled n ⫽ 4) Region MESENCEPHALON Inferior colliculus Superior colliculus Substantia nigra reticulata Oculomotor complex DIENCEPHALON Subthalamic nucleus Lateral habenular nucleus Lateral hypothalamic area Paraventricular nucleus Periventricular nucleus Arcuate THALAMUS Anteroventral Anteromedial Parafasicular TELENCEPHALON Hippocampus Globus Pallidus Caudate Putamen Visual cortex Parietal cortex Motor cortex Somatosensory cortex Prefrontal cortex WHITE MATTER Corpus callosum Fornix

Glucose utilisation

ƒMRI

(⫺)0.010 (⫺)0.010 (⫹)0.010 (⫹)0.010

(⫺)0.001 (⫺)0.001 (⫹)0.001 (⫹)0.001

(⫹)0.010 (⫹)0.010 (⫹)0.010 (⫹)0.010 0.000 (⫹)0.010

(⫹)0.001 (⫹)0.001 (⫹)0.050† (⫹)0.050† 0.000 (⫹)0.001†

(⫹)0.010 (⫹)0.010 (⫹)0.010

(⫹)0.001 (⫹)0.001 (⫹)0.001

(⫹)0.010 0.000 (⫹)0.050 (⫺)0.010 0.000 (⫺)0.010 0.000 0.000

(⫹)0.001 0.000 0.000 (⫺)0.001 (⫺)0.001 (⫺)0.001 0.000 0.000

0.000 (⫹)0.050

(⫹)0.001 (⫹)0.001

† Exact anatomical identification of the ROI in fMRI images is ambiguous due to the small size of anatomical region and the resolving power of fMRI. Significant signal increase thresholds: (⫹)0.050 § p ⬍ 0.05, (⫹)0.010 § p ⬍ 0.01, (⫹)0.001 § p ⱕ 0.001; signal decrease thresholds: (⫺)0.050 § p ⬍ 0.05, (⫺)0.010 § p ⬍ 0.01, (⫺)0.001 § p ⬍ 0.001. It should be noted that regions of significant change may lie within the anatomical region of interest and not represent significant change across the whole structure.

using the Bonferroni multiple comparison method and a significance threshold of P ⬍ 0.001 resulting in conservative probability maps that would therefore only detect prominent signal changes. A modest increase in sensitivity can be achieved by reducing the stringency of the threshold to P ⬍ 0.05. This results in both a modest increase in area of brain (⬃4%) exhibiting significant signal change and an earlier onset of significant change (Figs. 4 and 7). Further refinement of data analysis techniques should render more information about the spatio-temporal pattern of functional change elicited by the compound being studied. In summary, the fMRI signal changes closely mirror a spatial pattern of functional change established by autoradiographic methods. In addition, high temporal resolution time course information of the drug’s activity can be acquired. Combined with the potential for serial study this makes fMRI an exciting prospect for the study of novel therapeutic agents in both experimental and human systems.

Acknowledgments Support for this research was obtained from the Biotechnology and Biological Sciences Research Council (BBSRC) and SmithKline Beecham Pharmaceuticals plc. This work was performed at the Herchel Smith Laboratory for Medicinal Chemistry and it is a pleasure to thank Herchel Smith for providing the facilities used. Dr C. L-H. Huang also thanks the Medical Research Council (MRC) and the Royal Society (UK) for generous support.

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