SYNAPSE 69:203–212 (2015)
Pharmacological MRI Response to a Selective Dopamine Transporter Inhibitor, GBR12909, in Awake and Anesthetized Rats YUTO KASHIWAGI,1,2* TAKEMI ROKUGAWA,1 TOMOMI YAMADA,1 ATSUSHI OBATA,1 HIROSHI WATABE,3 YOSHICHIKA YOSHIOKA,2,4 AND KOHJI ABE1 1 Department of Drug Metabolism and Pharmacokinetics, Research Laboratory for Development, Shionogi and Co., Ltd., Osaka, Japan 2 Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan 3 Division of Radiation Protection & Safety Control, Cyclotron and Radioisotope Center, Tohoku University, Sendai, Japan 4 Laboratory of Biofunctional Imaging, Immunology Frontier Research Center, Osaka University, Osaka, Japan
KEY
WORDS
pharmacological isoflurane
MRI;
GBR12909;
DAT
inhibitor;
dopamine;
ABSTRACT Pharmacological magnetic resonance imaging (phMRI) is a powerful tool for imaging the effects of drugs on brain activity. In preclinical phMRI studies, general anesthesia used for minimizing head movements is thought to influence the phMRI responses to drugs. In this study we investigated the phMRI responses to a selective dopamine transporter (DAT) inhibitor, GBR12909, and a dopamine (DA) releaser, D-amphetamine (AMPH), in the isoflurane anesthetized and awake rats using a relative cerebral blood volume (rCBV) method. AMPH (1 mg/kg i.p.) caused an increase in rCBV in the dopaminergic circuitry in the both anesthetized and awake rats. The striatal rCBV change was correlated with the change of the striatal DA concentration induced by AMPH in the both anesthetized and awake rats. GBR12909 (10 mg/kg i.p.) caused a positive rCBV response and showed a similar regional pattern of rCBV response to AMPH in the awake rats, and the correlation between the change of the striatal rCBV and the striatal DA concentration was observed. However, in the anesthetized rats, GBR12909 induced a widespread negative rCBV response, whereas an increase in striatal DA concentration was observed. These findings indicate that phMRI responses to activation of DA neurotransmission by GBR12909 or AMPH are overall identical in the awake state, while the phMRI response to a DAT inhibitor, GBR12909 but not to AMPH was changed by isoflurane anesthesia. For the evaluation of neuroactive drugs using phMRI, isoflurane anesthesia might be complicated the interpretation of pharmacodynamic effects of drugs in preclinical studies. Synapse 69:203–212, 2015. VC 2015 Wiley Periodicals, Inc INTRODUCTION Imaging technologies, such as magnetic resonance imaging (MRI) and positron emission tomography (PET), have enabled noninvasive investigation of the pharmacological effects of psychoactive drugs in the brain. Pharmacological MRI (phMRI) can detect signal changes that reflect cerebrovascular responses to acute drug challenges, which is considered as a surrogate for changes in the underlying neuronal activity (Wise and Tracey, 2006). In recent years, this method has been applied to studying the neuronal effects of central acting drugs in both human and animal models. In preclinical studies, phMRI techniques have Ó 2015 WILEY PERIODICALS, INC.
been successfully applied to mapping the specific activation patterns of psychoactive drugs such as amphetamine (AMPH) (Chen et al., 1997) or cocaine (Marota et al., 2000). Furthermore, phMRI studies have revealed central effects produced by drugs acting on different molecular targets such as GABA
*Correspondence to: Yuto Kashiwagi, Department of Drug Metabolism and Pharmacokinetics, Research Laboratory for Development, Shionogi & Co., Ltd., Osaka, Japan. E-mail: [email protected] Received 8 September 2014; Accepted 6 January 2015 DOI: 10.1002/syn.21803 Published online 22 (wileyonlinelibrary.com).
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(Reese et al., 2000), cannabinoid (Chin et al., 2008), nicotine (Skoubis et al., 2006), glutamate (Gozzi et al., 2008), and serotonin (Scanley et al., 2001). For most preclinical brain imaging experiments, it is usually necessary to maintain animals under general anesthesia to minimize head movements and the stress induced by restraint. Under these conditions, phMRI responses to the drug of interest can vary considerably compared with the awake state (Luo et al., 2007; Skoubis et al., 2006; Zhang et al., 2000), because anesthetic agents are likely to alter the properties of neuronal activity and neurovascular coupling, due to their action on different receptors (Alkire et al., 2008). However, for many anesthetics, the target receptors or neurotransmitters remain unknown, making it difficult to predict the influence of anesthetics on underlying neural circuits following pharmacological stimulation (Steward et al., 2005). Dopamine (DA) receptor stimulation often leads to neuronal activation that involves oxygen consumption and the support of an adequate hemodynamic supply (Attwell and Laughlin, 2001; Hoge et al., 1999). For the dopaminergic system, neuronal activity along the dopaminergic circuitry can be probed using phMRI when challenged with a DA ligand, such as AMPH, a DA releaser, or cocaine, a DA transporter (DAT) inhibitor. At the dose range from 0.5 to 3 mg/kg, AMPH leads to positive phMRI responses in DA-rich brain areas, such as the striatum and nucleus accumbens where hemodynamic changes are correlated with extracellular DA concentrations measured by microdialysis (Chen et al., 1999; Choi et al., 2006; Ren et al., 2009), and in downstream structures, such as the thalamus and cortex in rat brain. Positive phMRI responses to AMPH challenge are observed under the conditions with anesthetic agents, such as halotane (Chen et al., 1997, 1999, 2005; Choi et al., 2006; Ren et al., 2009), a-chloralose (Shih et al., 2007), or isoflurane (Easton et al., 2007). Similarly, most of the phMRI studies assessing the effects of cocaine on rat brain have reported that cocaine induced positive phMRI responses in regions with rich dopaminergic innervation under halothane (Marota et al., 2000; Schwarz et al., 2004; Chen et al., 2011) or urethane (Luo et al., 2009) anesthesia. However, some studies conducted under isoflurane anesthesia showed negative cerebrovascular responses to cocaine challenge. Using optical techniques, Du et al. (2009) have reported that cocaine induced positive hemodynamic responses under a-chloralose anesthesia whereas negative responses were observed under isoflurane anesthesia in rat brain. Taken together, these data suggest that effects of anesthetic condition on phMRI responses might be different between a DA releaser, such as AMPH and a DAT inhibitor, such as cocaine. While there have been some reports investigating the Synapse
phMRI response to cocaine in several anesthetic conditions, the interpretation of phMRI response induced by cocaine might be complicated because cocaine can block not only DAT but also norepinephrine and serotonin transporters. Therefore, it is considered to be important to know the phMRI response associated with activations of dopaminergic neuron by using a selective DAT inhibitor in the anesthetized and awake animals. In this study, we conducted the relative cerebral blood volume (rCBV) weighted phMRI in the isoflurane anesthetized and awake rats to investigate phMRI responses to challenge with GBR12909 (1-[2[bis (4-fluorophenyl) methoxy] ethyl]-4-[3-phenylpropyl]-piperazine), which selectively binds to DAT or AMPH. The extracellular DA concentration in the striatum was measured using microdialysis in a separate group of animals to compare the rCBV response and the increase in DA concentration induced by GBR12909 or AMPH challenge. MATERIALS AND METHODS Animal procedures and treatment Adult male Wistar rats [729 weeks old, weight (mean 6 SEM) 326 6 4.0 g; Charles River Japan] were allowed free access to solid chow and tap water and were housed in a temperature-controlled room maintained on a 12:12 light:dark cycle with lights on at 8:00 am. The animals were maintained until the end of experiment with free access to water and rat chow. The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Shionogi Research Laboratories. The animals were placed in an induction chamber and anesthetized with 5% isoflurane and anesthesia was maintained by inhalation of 2% isoflurane through a face mask during surgery. The left femoral artery was cannulated for monitoring mean arterial blood pressure (MABP) and heart rate (HR) and for blood sampling to monitor arterial blood gases. The tail veins were catheterized for administration of contrast agent and muscular relaxant. A polyethylene tube (SP31, Natsume Seisakusho, Japan) was also inserted intraperitoneally for drug treatment. All wounds were infiltrated with lidocaine before suture. Immediately after tracheotomy, the rat was secured into a customized stereotactic holder and artificially ventilated with a mechanical respirator (Model 683, Harvard Apparatus, MA). The isoflurane level was set at 1.5% for a 1:2 O2:N2O gas mixture in anesthetized animals or at 0% for a 2:3 O2:N2 gas mixture in awake animals. The animals were paralyzed with a 0.25 mg/kg i.v. bolus of D-tubocurarine (Wako Pure Chemical, Japan) followed by continuous infusion of 0.25 (in anesthetized animals) or 0.5 (in awake animals) mg/kg/h i.v. Ventilation volume was adjusted in order to keep the arterial blood gases values within
PHMRI RESPONSE TO GBR12909 IN AWAKE AND ANESTHETIZED RATS
the physiological range (30 < pCO2 < 40 mmHg; pO2 > 100 mmHg). A rectal fiber-optic probe (SA Instruments, NY) was used to monitor the core body temperature, which was maintained at 36.5 6 0.5 C by blowing warm air into the magnet. At the end of the experiment, the animals were euthanized with an overdose of anesthetic. phMRI experiment All MRI experiments were performed on a Varian MRI System 7T/210 (Agilent Technologies, CA) using an actively decoupled volume transmission coil and a surface receiver coil (RAPID Biomedical, Germany). Spin-echo sequence (TR 5 4000 ms, TE 5 50 ms, field of view 5 35 3 35 mm2, imaging matrix 5 128 3 128, number of averages 5 1, number of slices 5 20, slice thickness 5 1 mm) was used to acquire T2-weighted anatomical images. Serial T2*-weighted images for rCBV-phMRI were acquired using a multi-slice gradient-echo sequence with imaging parameters: TR 5 468.75 ms, TE 5 20 ms, imaging matrix 5 128 3 64 (zero filled to 128 3 128), number of averages 5 1, flip angle 5 20 , and temporal resolution of 30 s. After 10 reference images had been recorded, 10 mg Fe/kg of the blood pool contrast agent Molday ION (Bio PAL, MA) was injected so that subsequent signal changes would reflect alterations in relative rCBV. Following an equilibration period of 20 min, D-amphetamine (1 mg/kg) or GBR12909 (10 mg/kg, Sigma) was intraperitoneally injected. The MRI data were acquired over a period of at least 40 min following administration of the drug. Data analysis The T2-weighted anatomical images for each subject were spatially normalized by a nine-degree-offreedom affine transformation mapping to a standard rat brain template and applying the resulting transformation matrix to the accompanying fMRI timeseries (FSL/FLIRT). The MR signal intensity changes in the time series data were converted according to Mandeville et al. (1998) to percent change in rCBV on a pixel-by-pixel basis using the transform:
rCBVðtÞ5ln½SðtÞ=S0 =ln S0 =Spre where S(t) is the signal intensity after the drug infusion, S0 is the baseline signal before the drug injection, and Spre is the mean signal intensity before the administration of contrast agent. The regions of interest (ROIs) were drawn on the rCBV time series maps according to the rat brain atlas (Paxinos and Watson, 2007) and the time courses of rCBV for ROIs were obtained from individual animals. To determine systemic vascular changes associated with the pharmacological stimulus, we also analyzed the signal change in an extra-cerebral ROIs located in the tem-
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poralis muscle as systemic circulatory change (PerlesBarbacaru et al., 2011). For the statistical analysis, an individual rCBV time series map was averaged from 0 min to 40 min after drug administration. Using the averaged rCBV maps for each treatment group, Student’s t-test was performed on a pixel-bypixel basis to test the significance of the rCBV response to the drugs compared with the vehicle. The averaged rCBV maps were masked by the statistical significance of the t-test (P < 0.05) and overlaid onto the standard rat brain template. Microdialysis Microdialysis was conducted for separate groups of rats to measure the DA concentrations upon AMPH and GBR12909 challenge in the striatum. Rats were anesthetized with isoflurane (5% for induction, 2% for maintenance) and placed in a stereotaxic apparatus for small animals. A guide cannula (Eicom, Japan) was inserted through a hole drilled on the skull. The coordinates with respect to the bregma were as follows (Paxinos and Watson, 2007) anterior 10.48 mm; ventral 24.0 mm; lateral 23.0 mm. The guide cannula was secured with dental cement and two screws were anchored onto the skull at two additional holes. Rats were then housed with food and water available ad libitum. After 227 days for recovery, the animals were used for microdialysis measurements. To compare the results of microdialysis with those of phMRI, the experimental conditions such as anesthetic level, ventilation parameter, and drug dosage were made to conform to those in the phMRI studies. The cannula stylet was removed and a microdialysis probe was inserted into the guide cannula. Continuous infusion of artificial cerebrospinal fluid (NaCl 147 mM, KCl 4 mM, CaCl2 2.3 mM) was delivered at 1 ll/min for at least 1 h to stabilize the brain tissue against probe-induced trauma. Dialysates were assayed at 6-min time intervals. Dopamine concentration was measured using high-performance liquid chromatography (HPLC) with electrochemical detection: six baseline dialysates were collected before the administration of drugs, followed by 10 post drug dialysates (60 min). RESULTS We firstly investigated the temporal changes in signal intensity after injection of contrast agent for vehicle treatment. As shown in Figure 1, vehicle had no effect on rCBV in all brain regions in the both anesthetized and awake rats. Additionally, the basal rCBV had also been kept at constant level without attenuation within measured time. Figure 2 shows the group average rCBV maps from animals treated with AMPH under isoflurane anesthesia and in the awake state. Significant increases in rCBV were observed from several cortical (e.g., Synapse
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Fig. 1. Temporal profile of rCBV in representative brain regions for the animals treated with vehicle under isoflurane anesthesia (n 5 6) or in awake state (n 5 5). Data are plotted as mean 6 SEM within each group. Str, striatum; PFC, prefrontal cortex; SSCx: somatosensory cortex.
Fig. 2. Maps show the degree of the rCBV changes induced by AMPH between 0 and 40 min after AMPH administration in the isoflurane anesthetized rats (A, n 5 5) or the awake rats (B, n 5 5). Colored pixels represent brain areas that showed group averaged rCBV values significantly different from vehicle (P < 0.05, scale bar
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prefrontal, motor, cingulate, and somatosensory cortex) and subcortical (e.g. nucleus accumbens, striatum, and thalamus) brain regions. As can be seen from Figure 3, which shows averaged time courses of rCBV in the respective brain regions, positive rCBV responses induced by AMPH were observed in the both anesthetized and awake rats. Interestingly, the positive rCBV response in the prefrontal cortex (PFC) was significantly higher in the awake state compared to under isoflurane anesthesia (P < 0.01, repeated measures ANOVA). The increase in the striatal rCBV seems to be higher in the isoflurane anesthetized rats compared to the awake rats. The change of the striatal DA concentration in response to AMPH and the time course of striatal rCBV in the anesthetized and awake rats are shown in Figure 4. The striatal DA concentration was increased by AMPH and closely correlated with the striatal rCBV changes in the both anesthetized and awake rats. The increase in the striatal DA concentration in the anesthetized rats was significantly higher than that in the awake rats (P < 0.01, repeated measures ANOVA). AMPH had no significant effect on blood gas parameters in the anesthetized rats (Table I). In the awake rats, a significant decrease in pO2 was observed by AMPH,
hue indicates averaged rCBV value). Statistical significance was determined using pixel-by-pixel t-test analysis (FSL) comparing the rCBV values for the drug injection group to those for the vehicle group.
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Fig. 4. Effects of AMPH on the striatal rCBV (0.5 min temporal resolution) and the striatal DA concentration in the isoflurane anesthetized rats (A) or the awake rats (B). The extracellular DA concentration in the striatum was measured using microdialysis (in 5 min time bins) for a separate group of animals under isoflurane anesthesia (n 5 4) or in the awake state (n 5 5). There is a tight correlation in time and amplitude between the rCBV and the extracellular DA concentration in the both anesthetized and awake rats. Data are plotted as mean 6 SEM within each group.
TABLE I. Blood gas parameters before and after drug administration pO2 (mmHg) Fig. 3. Temporal profile of AMPH-induced rCBV response in rep-
resentative brain regions of interest in the isoflurane anesthetized rats (n 5 5) or the awake rats (n 5 5). Data are plotted as mean6 SEM within each group. Str, striatum; PFC, prefrontal cortex; NAc, nucleus accumbens; SSCx, somatosensory cortex; Thal, Thalamus.
while the pO2 value 20 min after AMPH administration (101 6 6.6 mmHg) was within the physiological range, and no significant change of pCO2 was observed. Figures 5 and 6 show the group average rCBV maps and rCBV time courses, respectively, from animals treated with GBR12909 under isoflurane anesthesia and in the awake state. Significant decreases in rCBV were observed in extensive brain regions in the anesthetized rats. On the other hand, significant increases in rCBV were observed in the awake rats in some brain regions (striatum, PFC, somatosensory cortex, and thalamus). The change of the striatal DA concentration in response to GBR12909 and the time course of rCBV in the striatum in the anesthetized and awake rats are shown in Figure 7. In the awake rats, the striatal DA concentration was increased and closely correlated with the rCBV change in the striatum. However, an increase in
Baseline D-Amphetamine Isoflurane 123 Awake 146 GBR12909 Isoflurane 157 Awake 159
Baseline
Post drug (20 min)
126 6 10.4 101 6 6.6**
32.7 34.1
6 0.7 32.0 6 1.8 6 0.8 40.7 6 2.2
6 18.3 150 6 22.1 6 14.7 140 6 6.6
31.9 33.6
6 2.1 36.8 6 0.9 6 2.5 33.4 6 3.7
6 6
7.7 6.9
Post drug (20 min)
pCO2 (mmHg)
**P < 0.01 compared with baseline (determined by paired Student’s t-test).
striatal DA concentration induced by GBR12909 was also observed in the anesthetized animals, a result contradictory to the rCBV changes. Similar to the result of amphetamine, the increase in DA concentration by GBR12909 was significantly higher in the isoflurane anesthetized rats than the awake rats (P < 0.01, repeated measures ANOVA). GBR12909 had no significant effect on blood gas parameters in the both anesthetized and awake rats (Table I). The baseline values of MABP were 126 6 4.6 and 159 6 3.6 mmHg, and those of HR were 370 6 8.4 and 422 6 17.8 bpm in the anesthetized and awake rats, respectively. Figure 8 shows the temporal profile of relative MABP, HR, and blood volume in the muscle tissue before and after drug administration in the anesthetized and awake rats. In the anesthetized Synapse
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Fig. 5. Maps show the degree of the rCBV changes induced by GBR12909 between 0 and 40 min after GBR12909 administration in the isoflurane anesthetized rats (A, n 5 6) or the awake rats (B, n 5 5). Colored pixels represent brain areas that showed group averaged rCBV values significantly different from the vehicle (P < 0.05,
scale bar hue indicates averaged rCBV value). Statistical significance was determined using pixel-by-pixel t-test analysis (FSL) comparing the rCBV values for the drug injection group to those for the vehicle group.
rats, AMPH caused an increase in HR and a decrease in MABP. However, no effect of AMPH on blood volume in the muscle tissue was observed. On the other hand, GBR12909 caused a gradual decrease in blood volume in the muscle tissue as well as HR and MABP. In the awake rats, AMPH had little effect on HR, MABP, and blood volume in muscle tissue, and GBR12909 caused slight increases in HR, MABP, and blood volume in the muscle tissue.
1999; Choi et al., 2006; Ren et al., 2009). In this study, GBR12909 and AMPH induced the positive rCBV responses in the awake rats, and the responses closely correlated with the increased extracellular DA concentration in the striatum. The close parallelism between the time course of the extracellular DA concentration and that of rCBV changes suggests that the extracellular DA drives the cerebrovascular responses. In the isoflurane anesthetized rats, the increase in the striatal rCBV induced by AMPH seems to be higher compared with that in the awake rats. Similarly, the increase in striatal DA concentration by AMPH was significantly higher in the anesthetized rats compared with the awake rats. These results are in agreement with previous reports which show the increase in extracellular DA concentration by DAT inhibitors (nomifensine or GBR12909) or DA releasers (methamphetamine or amphetamine) is higher under isoflurane or halothane anesthesia compared with in the awake state (Opacka-Juffry et al., 1992, Fink-Jensen et al., 1994, Adachi et al., 2001). Furthermore, the correlation between the change of the striatal DA concentration and the striatal rCBV response induced by AMPH was also observed in the isoflurane anesthetized rats. These results indicate that isoflurane anesthesia may not affect the cerebrovascular responses to changes of extracellular DA level.
DISCUSSION In this study, we demonstrated that GBR12909, a selective DAT inhibitor, induced positive rCBV responses in areas rich in DA receptors and with high DA utilization in the awake rats and the regional pattern of the rCBV change was similar to that of AMPH. However, in the anesthetized rats, negative rCBV responses were detected on GBR12909 treatment, while positive rCBV responses were observed for AMPH. Microdialysis study showed an increase in striatal DA concentration induced by GBR12909 or AMPH challenge in the both anesthetized and awake rats. Previous studies showed that the rCBV changes induced by AMPH or 2b-carbomethoxy-3b-(4-fluorophenyl) tropane (CFT; a DAT inhibitor) challenge are linearly correlated with the DA concentrations in the extracellular space using microdialysis (Chen et al., Synapse
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Fig. 7. Effects of GBR12909 on the striatal rCBV (0.5 min temporal resolution) and the striatal DA concentration in the isoflurane anesthetized rats (A) or the awake rats (B). The extracellular DA concentration in the striatum was measured using microdialysis (in 5 min time bins) for a separate group of animals under isoflurane anesthesia (n 5 4) or in the awake state (n 5 4). There is a tight correlation in time and amplitude between the rCBV and the extracellular DA concentration in the awake rats but not in the anesthetized rats. Data are plotted as mean 6 SEM within each group. Fig. 6. Temporal profile of GBR12909-induced rCBV response in representative brain regions of interest in the isoflurane anesthetized rats (n 5 6) or the awake rats (n 5 5). Data are plotted as mean 6 SEM within each group. Str, striatum; PFC, prefrontal cortex; NAc, nucleus accumbens; SSCx, somatosensory cortex; Thal, Thalamus.
Meanwhile, the increase in rCBV in the PFC was significantly attenuated in the isoflurane anesthetized rats compared to the awake rats. The PFC mediating higher brain function (Fuster, 2008) has been reported to be affected by general anesthesia (Sellers et al., 2013). These findings suggest that rCBV or DA response in the PFC might be affected by isoflurane anesthesia. However, it is uncertain whether the increase in the DA concentration in the PFC induced by AMPH was different between in the anesthetized and awake rats, because the DA concentration in the PFC could not be measured in this study. Thus, it is considered to be important to know effects of isoflurane on the DA release induced by AMPH in the PFC to clear the cause of difference in the rCBV response to AMPH in the PFC between in the anesthetized and awake rats. On the other hand, GBR12909 induced a negative rCBV response over a widespread brain region in the
anesthetized rats, although the increase in extracellular DA concentration in the striatum was also observed. Based upon previous studies using D1 and D2 agonists, negative rCBV responses can be interpreted as reflecting the agonism of D2/D3 receptors (Chen et al., 2005; Choi et al., 2006), whereas positive rCBV changes are associated with agonism of the D1/ D5 receptors (Choi et al., 2006). From these reports, we speculated that the negative rCBV responses to GBR12909 challenge in the anesthetized rats might be due to D2/D3 receptor activation. Ren et al. (2009) reported that negative rCBV responses are observed with a lower dose (0.25 mg/kg) of AMPH challenge, which becomes a positive response at higher doses (1–3 mg/kg). They suggested that at a lower extracellular DA concentration induced by low-dose AMPH challenge, DA primarily binds to D2 and D3 receptor rather than to the D1 receptor because of higher affinity of the D2 and D3 receptors, which leads to a decrease in rCBV. However, in the present study, GBR12909 at 10 mg/kg challenge induced a significant higher increase in the striatal DA concentration in the anesthetized rats rather than in the awake rats. In addition the increase in striatal DA Synapse
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Fig. 8. Effects of AMPH and GBR12909 on MABP, HR, and blood volume in muscle tissue in the isoflurane anesthetized rats (A) or the awake rats (B). Data for MABP and HR for 20225 min after drug administration were absent due to arterial blood sampling for blood gas measurement. Data are plotted as mean 6 SEM within each group.
concentration induced by 10 mg/kg of GBR12909 in the anesthetized rats seems to be higher than that induced by 1 mg/kg AMPH. Therefore, the negative rCBV responses to GBR12909 observed in the anesthetized rats are unlikely to be induced by D2/D3 receptor activation due to lower extracellular DA concentration. Totally, 10 mg/kg of GBR12909 is effective dose reducing impulsive choice in the delayed reward paradigm evaluating the impulsive behavior such as attention deficit/hyperactivity disorder (Baarendse and Vanderschuren, 2011). In addition, the dose of AMPH inducing comparable effects to 10 mg/kg of GBR12909 is 1 mg/kg in the delayed reword paradigm. These previous findings suggest that 10 mg/kg of GBR12909 is considered to be appropriate dose for studying the CNS effects of GBR12909 using phMRI in rats. Synapse
Many drugs for the central nervous system can also induce significant peripheral effects, including severe alterations of cardiovascular parameters. Under physiological conditions, the cerebral autoregulation maintains relatively constant cerebral circulation in the presence of changes in systemic circulation. However, large and rapid changes in MABP may cause a breakdown in the cerebral autoregulation system, thus introducing potential confounding factors in the interpretation of phMRI (Gozzi et al., 2007). In this study, although changes of cardiovascular parameters (MABP and HR) and blood volume in muscle tissue were observed in response to GBR12909 and AMPH in the awake rats, there were regions with no rCBV response such as the cerebellum. These results suggest that cerebral circulation is kept constant by the cerebral autoregulation system in the awake state, and therefore the
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essential rCBV response associated with the increase in extracellular DA caused by GBR12909 and AMPH could be detected. However, in the anesthetized rats, GBR12909 induced negative rCBV responses in nonspecific brain regions as well as decreases in cardiovascular parameters and blood volume in muscle tissue. In addition the temporal profile of the negative rCBV response was similar to that of blood volume in muscle tissue. Previous papers have reported that isoflurane anesthesia impairs the cerebral autoregulation system (Summors et al., 1999, McPherson et al., 1988). Therefore, due to breakdown in the cerebral autoregulation system under isoflurane anesthesia, the decrease in systemic circulation by GBR12909 might induce a decrease in cerebral circulation. In response to AMPH, a decrease in MABP and an increase in HR, which might be due to a baroreceptors function, were observed in the anesthetized rats. As a result, systemic and cerebral circulations were kept constant, and therefore the regional rCBV response associated with the increase in extracellular DA caused by AMPH could be detected even under isoflurane anesthesia. In conclusion, we investigated the phMRI responses to GBR12909, a selective DAT inhibitor and AMPH, a DA releaser in the isoflurane anesthetized and awake rats and demonstrated that GBR12909 induced positive rCBV responses in the awake rats and the regional pattern of the rCBV response was similar to that induced by AMPH. In addition we found that the rCBV responses to GBR12909 but not to AMPH were changed by isoflurane anesthesia. Although the mechanisms for the negative rCBV response to GBR12909 challenge under isoflurane anesthesia remains uncertain, one possible explanation is due to breakdown in the cerebral autoregulation system by isoflurane anesthesia and decrease in systemic circulation by GBR12909. Further study will be needed to clear whether the negative rCBV responses to GBR12909 under isoflurane anesthesia are due to the inherent property of GBR12909 or the common effect of DAT inhibitors. REFERENCES Adachi YU, Watanabe K, Satoh T, Vizi ES. 2001. Halothane potentiates the effect of methamphetamine and nomifensine on extracellular dopamine levels in rat striatum: A microdialysis study. Br J Anaesth 86:837–845. Alkire MT, Hudetz AG, Tononi G. 2008. Consciousness and anesthesia. Science 322:876–880. Attwell D, Laughlin SB. 2001. An energy budget for signaling in the grey matter of the brain. J Cereb Blood Flow Metab 21:1133– 1145. Baarendse PJ, Vanderschuren LJ. 2012. Dissociable effects of monoamine reuptake inhibitors on distinct forms of impulsive behavior in rats. Psychopharmacology (Berl) 219:313–326. Chen YI, Galpern WR, Brownell AL, Matthews RT, Bogdanov M, Isacson O, Keltner JR, Beal MF, Rosen BR, Jenkins BG. 1997. Detection of dopaminergic neurotransmitter activity using pharmacologic MRI: Correlation with PET, microdialysis, and behavioral data. Magn Reson Med 38:389–398.
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Chen YI, Brownell AL, Galpern W, Isacson O, Bogdanov M, Beal MF, Livni E, Rosen BR, Jenkins BG. 1999. Detection of dopaminergic cell loss and neural transplantation using pharmacological MRI, PET and behavioral assessment. Neuroreport 10:2881–2886. Chen YI, Choi JK, Andersen SL, Rosen BR, Jenkins BG. 2005. Mapping dopamine D2/D3 receptor function using pharmacological magnetic resonance imaging. Psychopharmacology (Berl) 180: 705–715. Chen YI, Famous K, Xu H, Choi JK, Mandeville JB, Schmidt HD, Pierce RC, Jenkins BG. 2011. Cocaine self-administration leads to alterations in temporal responses to cocaine challenge in limbic and motor circuitry. Eur J Neurosci 34:800–815. Chin CL, Tovcimak AE, Hradil VP, Seifert TR, Hollingsworth PR, Chandran P, Zhu CZ, Gauvin D, Pai M, Wetter J, Hsieh GC, Honore P, Frost JM, Dart MJ, Meyer MD, Yao BB, Cox BF, Fox GB. 2008. Differential effects of cannabinoid receptor agonists on regional brain activity using pharmacological MRI. Br J Pharmacol 153:367–379. Choi JK, Chen YI, Hamel E, Jenkins BG. 2006. Brain hemodynamic changes mediated by dopamine receptors: Role of the cerebral microvasculature in dopamine-mediated neurovascular coupling. Neuroimage 30:700–712. Du C, Tully M, Volkow ND, Schiffer WK, Yu M, Luo Z, Koretsky AP, Benveniste H. 2009. Differential effects of anesthetics on cocaine’s pharmacokinetic and pharmacodynamic effects in brain. Eur J Neurosci 30:1565–1575. Easton N, Marshall F, Fone KC, Marsden CA. 2007. Differential effects of the D- and L- isomers of amphetamine on pharmacological MRI BOLD contrast in the rat. Psychopharmacology (Berl) 193:11–30. Fink-Jensen A, Ingwersen SH, Nielsen PG, Hansen L, Nielsen EB, Hansen AJ. 1994. Halothane anesthesia enhances the effect of dopamine uptake inhibition on interstitial levels of striatal dopamine. Naunyn Schmiedebergs Arch Pharmacol 350:239–244. Fuster JM. 2008. The Prefrontal Cortex. San Diego: Elsevier, p. vii410. Gozzi A, Ceolin L, Schwarz A, Reese T, Bertani S, Crestan V, Bifone A. 2007. A multimodality investigation of cerebral hemodynamics and autoregulation in pharmacological MRI. Magn Reson Imaging 25:826–833. Gozzi A, Herdon H, Schwarz A, Bertani S, Crestan V, Turrini G, Bifone A. 2008. Pharmacological stimulation of NMDA receptors via co-agonist site suppresses fMRI response to phencyclidine in the rat. Psychopharmacology (Berl) 201:273–284. Hoge RD, Atkinson J, Gill B, Crelier GR, Marrett S, Pike GB. 1999. Linear coupling between cerebral blood flow and oxygen consumption in activated human cortex. Proc Natl Acad Sci USA 96: 9403–9408. Luo F, Li Z, Treistman SN, Kim YR, King JA, Fox GB, Ferris CF. 2007. Confounding effects of volatile anesthesia on CBV assessment in rodent forebrain following ethanol challenge. J Magn Reson Imaging 26:557–563. Luo F, Schmidt KF, Fox GB, Ferris CF. 2009. Differential responses in CBF and CBV to cocaine as measured by fMRI: Implications for pharmacological MRI signals derived oxygen metabolism assessment. J Psychiatr Res 43:1018–1024. Marota JJ, Mandeville JB, Weisskoff RM, Moskowitz MA, Rosen BR, Kosofsky BE. 2000. Cocaine activation discriminates dopaminergic projections by temporal response: an fMRI study in rat. Neuroimage 11:13–23. McPherson RW, Traystman RJ. 1988. Effects of isoflurane on cerebral autoregulation in dogs. Anesthesiology 69:493–499. Opacka-Juffry J, Ahier RG, Cremer JE. 1991. Nomifensine-induced increase in extracellular striatal dopamine is enhanced by isoflurane anaesthesia. Synapse 7:169–171. Paxinos G, Watson C. 2007. The rat brain in stereotaxic coordinates, 6th ed. San Diego: Academic Press. Perles-Barbacaru TA, Procissi D, Demyanenko AV, Hall FS, Uhl GR, Jacobs RE. 2011. Quantitative pharmacologic MRI: Mapping the cerebral blood volume response to cocaine in dopamine transporter knockout mice. Neuroimage 55:622–628. Reese T, Bjelke B, Porszasz R, Baumann D, Bochelen D, Sauter A, Rudin M. 2000. Regional brain activation by bicuculline visualized by functional magnetic resonance imaging. Time-resolved assessment of bicuculline-induced changes in local cerebral blood volume using an intravascular contrast agent. NMR Biomed 13: 361–370. Ren J, Xu H, Choi JK, Jenkins BG, Chen YI. 2009. Dopaminergic response to graded dopamine concentration elicited by four amphetamine doses. Synapse 63:764–772.
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Pharmacological MRI Response to a Selective Dopamine Transporter Inhibitor, GBR12909, in Awake and Anesthetized Rats YUTO KASHIWAGI,1,2* TAKEMI ROKUGAWA,1 TOMOMI YAMADA,1 ATSUSHI OBATA,1 HIROSHI WATABE,3 YOSHICHIKA YOSHIOKA,2,4 AND KOHJI ABE1 1 Department of Drug Metabolism and Pharmacokinetics, Research Laboratory for Development, Shionogi and Co., Ltd., Osaka, Japan 2 Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan 3 Division of Radiation Protection & Safety Control, Cyclotron and Radioisotope Center, Tohoku University, Sendai, Japan 4 Laboratory of Biofunctional Imaging, Immunology Frontier Research Center, Osaka University, Osaka, Japan
KEY
WORDS
pharmacological isoflurane
MRI;
GBR12909;
DAT
inhibitor;
dopamine;
ABSTRACT Pharmacological magnetic resonance imaging (phMRI) is a powerful tool for imaging the effects of drugs on brain activity. In preclinical phMRI studies, general anesthesia used for minimizing head movements is thought to influence the phMRI responses to drugs. In this study we investigated the phMRI responses to a selective dopamine transporter (DAT) inhibitor, GBR12909, and a dopamine (DA) releaser, D-amphetamine (AMPH), in the isoflurane anesthetized and awake rats using a relative cerebral blood volume (rCBV) method. AMPH (1 mg/kg i.p.) caused an increase in rCBV in the dopaminergic circuitry in the both anesthetized and awake rats. The striatal rCBV change was correlated with the change of the striatal DA concentration induced by AMPH in the both anesthetized and awake rats. GBR12909 (10 mg/kg i.p.) caused a positive rCBV response and showed a similar regional pattern of rCBV response to AMPH in the awake rats, and the correlation between the change of the striatal rCBV and the striatal DA concentration was observed. However, in the anesthetized rats, GBR12909 induced a widespread negative rCBV response, whereas an increase in striatal DA concentration was observed. These findings indicate that phMRI responses to activation of DA neurotransmission by GBR12909 or AMPH are overall identical in the awake state, while the phMRI response to a DAT inhibitor, GBR12909 but not to AMPH was changed by isoflurane anesthesia. For the evaluation of neuroactive drugs using phMRI, isoflurane anesthesia might be complicated the interpretation of pharmacodynamic effects of drugs in preclinical studies. Synapse 69:203–212, 2015. VC 2015 Wiley Periodicals, Inc INTRODUCTION Imaging technologies, such as magnetic resonance imaging (MRI) and positron emission tomography (PET), have enabled noninvasive investigation of the pharmacological effects of psychoactive drugs in the brain. Pharmacological MRI (phMRI) can detect signal changes that reflect cerebrovascular responses to acute drug challenges, which is considered as a surrogate for changes in the underlying neuronal activity (Wise and Tracey, 2006). In recent years, this method has been applied to studying the neuronal effects of central acting drugs in both human and animal models. In preclinical studies, phMRI techniques have Ó 2015 WILEY PERIODICALS, INC.
been successfully applied to mapping the specific activation patterns of psychoactive drugs such as amphetamine (AMPH) (Chen et al., 1997) or cocaine (Marota et al., 2000). Furthermore, phMRI studies have revealed central effects produced by drugs acting on different molecular targets such as GABA
*Correspondence to: Yuto Kashiwagi, Department of Drug Metabolism and Pharmacokinetics, Research Laboratory for Development, Shionogi & Co., Ltd., Osaka, Japan. E-mail: [email protected] Received 8 September 2014; Accepted 6 January 2015 DOI: 10.1002/syn.21803 Published online 22 (wileyonlinelibrary.com).
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(Reese et al., 2000), cannabinoid (Chin et al., 2008), nicotine (Skoubis et al., 2006), glutamate (Gozzi et al., 2008), and serotonin (Scanley et al., 2001). For most preclinical brain imaging experiments, it is usually necessary to maintain animals under general anesthesia to minimize head movements and the stress induced by restraint. Under these conditions, phMRI responses to the drug of interest can vary considerably compared with the awake state (Luo et al., 2007; Skoubis et al., 2006; Zhang et al., 2000), because anesthetic agents are likely to alter the properties of neuronal activity and neurovascular coupling, due to their action on different receptors (Alkire et al., 2008). However, for many anesthetics, the target receptors or neurotransmitters remain unknown, making it difficult to predict the influence of anesthetics on underlying neural circuits following pharmacological stimulation (Steward et al., 2005). Dopamine (DA) receptor stimulation often leads to neuronal activation that involves oxygen consumption and the support of an adequate hemodynamic supply (Attwell and Laughlin, 2001; Hoge et al., 1999). For the dopaminergic system, neuronal activity along the dopaminergic circuitry can be probed using phMRI when challenged with a DA ligand, such as AMPH, a DA releaser, or cocaine, a DA transporter (DAT) inhibitor. At the dose range from 0.5 to 3 mg/kg, AMPH leads to positive phMRI responses in DA-rich brain areas, such as the striatum and nucleus accumbens where hemodynamic changes are correlated with extracellular DA concentrations measured by microdialysis (Chen et al., 1999; Choi et al., 2006; Ren et al., 2009), and in downstream structures, such as the thalamus and cortex in rat brain. Positive phMRI responses to AMPH challenge are observed under the conditions with anesthetic agents, such as halotane (Chen et al., 1997, 1999, 2005; Choi et al., 2006; Ren et al., 2009), a-chloralose (Shih et al., 2007), or isoflurane (Easton et al., 2007). Similarly, most of the phMRI studies assessing the effects of cocaine on rat brain have reported that cocaine induced positive phMRI responses in regions with rich dopaminergic innervation under halothane (Marota et al., 2000; Schwarz et al., 2004; Chen et al., 2011) or urethane (Luo et al., 2009) anesthesia. However, some studies conducted under isoflurane anesthesia showed negative cerebrovascular responses to cocaine challenge. Using optical techniques, Du et al. (2009) have reported that cocaine induced positive hemodynamic responses under a-chloralose anesthesia whereas negative responses were observed under isoflurane anesthesia in rat brain. Taken together, these data suggest that effects of anesthetic condition on phMRI responses might be different between a DA releaser, such as AMPH and a DAT inhibitor, such as cocaine. While there have been some reports investigating the Synapse
phMRI response to cocaine in several anesthetic conditions, the interpretation of phMRI response induced by cocaine might be complicated because cocaine can block not only DAT but also norepinephrine and serotonin transporters. Therefore, it is considered to be important to know the phMRI response associated with activations of dopaminergic neuron by using a selective DAT inhibitor in the anesthetized and awake animals. In this study, we conducted the relative cerebral blood volume (rCBV) weighted phMRI in the isoflurane anesthetized and awake rats to investigate phMRI responses to challenge with GBR12909 (1-[2[bis (4-fluorophenyl) methoxy] ethyl]-4-[3-phenylpropyl]-piperazine), which selectively binds to DAT or AMPH. The extracellular DA concentration in the striatum was measured using microdialysis in a separate group of animals to compare the rCBV response and the increase in DA concentration induced by GBR12909 or AMPH challenge. MATERIALS AND METHODS Animal procedures and treatment Adult male Wistar rats [729 weeks old, weight (mean 6 SEM) 326 6 4.0 g; Charles River Japan] were allowed free access to solid chow and tap water and were housed in a temperature-controlled room maintained on a 12:12 light:dark cycle with lights on at 8:00 am. The animals were maintained until the end of experiment with free access to water and rat chow. The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Shionogi Research Laboratories. The animals were placed in an induction chamber and anesthetized with 5% isoflurane and anesthesia was maintained by inhalation of 2% isoflurane through a face mask during surgery. The left femoral artery was cannulated for monitoring mean arterial blood pressure (MABP) and heart rate (HR) and for blood sampling to monitor arterial blood gases. The tail veins were catheterized for administration of contrast agent and muscular relaxant. A polyethylene tube (SP31, Natsume Seisakusho, Japan) was also inserted intraperitoneally for drug treatment. All wounds were infiltrated with lidocaine before suture. Immediately after tracheotomy, the rat was secured into a customized stereotactic holder and artificially ventilated with a mechanical respirator (Model 683, Harvard Apparatus, MA). The isoflurane level was set at 1.5% for a 1:2 O2:N2O gas mixture in anesthetized animals or at 0% for a 2:3 O2:N2 gas mixture in awake animals. The animals were paralyzed with a 0.25 mg/kg i.v. bolus of D-tubocurarine (Wako Pure Chemical, Japan) followed by continuous infusion of 0.25 (in anesthetized animals) or 0.5 (in awake animals) mg/kg/h i.v. Ventilation volume was adjusted in order to keep the arterial blood gases values within
PHMRI RESPONSE TO GBR12909 IN AWAKE AND ANESTHETIZED RATS
the physiological range (30 < pCO2 < 40 mmHg; pO2 > 100 mmHg). A rectal fiber-optic probe (SA Instruments, NY) was used to monitor the core body temperature, which was maintained at 36.5 6 0.5 C by blowing warm air into the magnet. At the end of the experiment, the animals were euthanized with an overdose of anesthetic. phMRI experiment All MRI experiments were performed on a Varian MRI System 7T/210 (Agilent Technologies, CA) using an actively decoupled volume transmission coil and a surface receiver coil (RAPID Biomedical, Germany). Spin-echo sequence (TR 5 4000 ms, TE 5 50 ms, field of view 5 35 3 35 mm2, imaging matrix 5 128 3 128, number of averages 5 1, number of slices 5 20, slice thickness 5 1 mm) was used to acquire T2-weighted anatomical images. Serial T2*-weighted images for rCBV-phMRI were acquired using a multi-slice gradient-echo sequence with imaging parameters: TR 5 468.75 ms, TE 5 20 ms, imaging matrix 5 128 3 64 (zero filled to 128 3 128), number of averages 5 1, flip angle 5 20 , and temporal resolution of 30 s. After 10 reference images had been recorded, 10 mg Fe/kg of the blood pool contrast agent Molday ION (Bio PAL, MA) was injected so that subsequent signal changes would reflect alterations in relative rCBV. Following an equilibration period of 20 min, D-amphetamine (1 mg/kg) or GBR12909 (10 mg/kg, Sigma) was intraperitoneally injected. The MRI data were acquired over a period of at least 40 min following administration of the drug. Data analysis The T2-weighted anatomical images for each subject were spatially normalized by a nine-degree-offreedom affine transformation mapping to a standard rat brain template and applying the resulting transformation matrix to the accompanying fMRI timeseries (FSL/FLIRT). The MR signal intensity changes in the time series data were converted according to Mandeville et al. (1998) to percent change in rCBV on a pixel-by-pixel basis using the transform:
rCBVðtÞ5ln½SðtÞ=S0 =ln S0 =Spre where S(t) is the signal intensity after the drug infusion, S0 is the baseline signal before the drug injection, and Spre is the mean signal intensity before the administration of contrast agent. The regions of interest (ROIs) were drawn on the rCBV time series maps according to the rat brain atlas (Paxinos and Watson, 2007) and the time courses of rCBV for ROIs were obtained from individual animals. To determine systemic vascular changes associated with the pharmacological stimulus, we also analyzed the signal change in an extra-cerebral ROIs located in the tem-
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poralis muscle as systemic circulatory change (PerlesBarbacaru et al., 2011). For the statistical analysis, an individual rCBV time series map was averaged from 0 min to 40 min after drug administration. Using the averaged rCBV maps for each treatment group, Student’s t-test was performed on a pixel-bypixel basis to test the significance of the rCBV response to the drugs compared with the vehicle. The averaged rCBV maps were masked by the statistical significance of the t-test (P < 0.05) and overlaid onto the standard rat brain template. Microdialysis Microdialysis was conducted for separate groups of rats to measure the DA concentrations upon AMPH and GBR12909 challenge in the striatum. Rats were anesthetized with isoflurane (5% for induction, 2% for maintenance) and placed in a stereotaxic apparatus for small animals. A guide cannula (Eicom, Japan) was inserted through a hole drilled on the skull. The coordinates with respect to the bregma were as follows (Paxinos and Watson, 2007) anterior 10.48 mm; ventral 24.0 mm; lateral 23.0 mm. The guide cannula was secured with dental cement and two screws were anchored onto the skull at two additional holes. Rats were then housed with food and water available ad libitum. After 227 days for recovery, the animals were used for microdialysis measurements. To compare the results of microdialysis with those of phMRI, the experimental conditions such as anesthetic level, ventilation parameter, and drug dosage were made to conform to those in the phMRI studies. The cannula stylet was removed and a microdialysis probe was inserted into the guide cannula. Continuous infusion of artificial cerebrospinal fluid (NaCl 147 mM, KCl 4 mM, CaCl2 2.3 mM) was delivered at 1 ll/min for at least 1 h to stabilize the brain tissue against probe-induced trauma. Dialysates were assayed at 6-min time intervals. Dopamine concentration was measured using high-performance liquid chromatography (HPLC) with electrochemical detection: six baseline dialysates were collected before the administration of drugs, followed by 10 post drug dialysates (60 min). RESULTS We firstly investigated the temporal changes in signal intensity after injection of contrast agent for vehicle treatment. As shown in Figure 1, vehicle had no effect on rCBV in all brain regions in the both anesthetized and awake rats. Additionally, the basal rCBV had also been kept at constant level without attenuation within measured time. Figure 2 shows the group average rCBV maps from animals treated with AMPH under isoflurane anesthesia and in the awake state. Significant increases in rCBV were observed from several cortical (e.g., Synapse
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Fig. 1. Temporal profile of rCBV in representative brain regions for the animals treated with vehicle under isoflurane anesthesia (n 5 6) or in awake state (n 5 5). Data are plotted as mean 6 SEM within each group. Str, striatum; PFC, prefrontal cortex; SSCx: somatosensory cortex.
Fig. 2. Maps show the degree of the rCBV changes induced by AMPH between 0 and 40 min after AMPH administration in the isoflurane anesthetized rats (A, n 5 5) or the awake rats (B, n 5 5). Colored pixels represent brain areas that showed group averaged rCBV values significantly different from vehicle (P < 0.05, scale bar
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prefrontal, motor, cingulate, and somatosensory cortex) and subcortical (e.g. nucleus accumbens, striatum, and thalamus) brain regions. As can be seen from Figure 3, which shows averaged time courses of rCBV in the respective brain regions, positive rCBV responses induced by AMPH were observed in the both anesthetized and awake rats. Interestingly, the positive rCBV response in the prefrontal cortex (PFC) was significantly higher in the awake state compared to under isoflurane anesthesia (P < 0.01, repeated measures ANOVA). The increase in the striatal rCBV seems to be higher in the isoflurane anesthetized rats compared to the awake rats. The change of the striatal DA concentration in response to AMPH and the time course of striatal rCBV in the anesthetized and awake rats are shown in Figure 4. The striatal DA concentration was increased by AMPH and closely correlated with the striatal rCBV changes in the both anesthetized and awake rats. The increase in the striatal DA concentration in the anesthetized rats was significantly higher than that in the awake rats (P < 0.01, repeated measures ANOVA). AMPH had no significant effect on blood gas parameters in the anesthetized rats (Table I). In the awake rats, a significant decrease in pO2 was observed by AMPH,
hue indicates averaged rCBV value). Statistical significance was determined using pixel-by-pixel t-test analysis (FSL) comparing the rCBV values for the drug injection group to those for the vehicle group.
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PHMRI RESPONSE TO GBR12909 IN AWAKE AND ANESTHETIZED RATS
Fig. 4. Effects of AMPH on the striatal rCBV (0.5 min temporal resolution) and the striatal DA concentration in the isoflurane anesthetized rats (A) or the awake rats (B). The extracellular DA concentration in the striatum was measured using microdialysis (in 5 min time bins) for a separate group of animals under isoflurane anesthesia (n 5 4) or in the awake state (n 5 5). There is a tight correlation in time and amplitude between the rCBV and the extracellular DA concentration in the both anesthetized and awake rats. Data are plotted as mean 6 SEM within each group.
TABLE I. Blood gas parameters before and after drug administration pO2 (mmHg) Fig. 3. Temporal profile of AMPH-induced rCBV response in rep-
resentative brain regions of interest in the isoflurane anesthetized rats (n 5 5) or the awake rats (n 5 5). Data are plotted as mean6 SEM within each group. Str, striatum; PFC, prefrontal cortex; NAc, nucleus accumbens; SSCx, somatosensory cortex; Thal, Thalamus.
while the pO2 value 20 min after AMPH administration (101 6 6.6 mmHg) was within the physiological range, and no significant change of pCO2 was observed. Figures 5 and 6 show the group average rCBV maps and rCBV time courses, respectively, from animals treated with GBR12909 under isoflurane anesthesia and in the awake state. Significant decreases in rCBV were observed in extensive brain regions in the anesthetized rats. On the other hand, significant increases in rCBV were observed in the awake rats in some brain regions (striatum, PFC, somatosensory cortex, and thalamus). The change of the striatal DA concentration in response to GBR12909 and the time course of rCBV in the striatum in the anesthetized and awake rats are shown in Figure 7. In the awake rats, the striatal DA concentration was increased and closely correlated with the rCBV change in the striatum. However, an increase in
Baseline D-Amphetamine Isoflurane 123 Awake 146 GBR12909 Isoflurane 157 Awake 159
Baseline
Post drug (20 min)
126 6 10.4 101 6 6.6**
32.7 34.1
6 0.7 32.0 6 1.8 6 0.8 40.7 6 2.2
6 18.3 150 6 22.1 6 14.7 140 6 6.6
31.9 33.6
6 2.1 36.8 6 0.9 6 2.5 33.4 6 3.7
6 6
7.7 6.9
Post drug (20 min)
pCO2 (mmHg)
**P < 0.01 compared with baseline (determined by paired Student’s t-test).
striatal DA concentration induced by GBR12909 was also observed in the anesthetized animals, a result contradictory to the rCBV changes. Similar to the result of amphetamine, the increase in DA concentration by GBR12909 was significantly higher in the isoflurane anesthetized rats than the awake rats (P < 0.01, repeated measures ANOVA). GBR12909 had no significant effect on blood gas parameters in the both anesthetized and awake rats (Table I). The baseline values of MABP were 126 6 4.6 and 159 6 3.6 mmHg, and those of HR were 370 6 8.4 and 422 6 17.8 bpm in the anesthetized and awake rats, respectively. Figure 8 shows the temporal profile of relative MABP, HR, and blood volume in the muscle tissue before and after drug administration in the anesthetized and awake rats. In the anesthetized Synapse
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Fig. 5. Maps show the degree of the rCBV changes induced by GBR12909 between 0 and 40 min after GBR12909 administration in the isoflurane anesthetized rats (A, n 5 6) or the awake rats (B, n 5 5). Colored pixels represent brain areas that showed group averaged rCBV values significantly different from the vehicle (P < 0.05,
scale bar hue indicates averaged rCBV value). Statistical significance was determined using pixel-by-pixel t-test analysis (FSL) comparing the rCBV values for the drug injection group to those for the vehicle group.
rats, AMPH caused an increase in HR and a decrease in MABP. However, no effect of AMPH on blood volume in the muscle tissue was observed. On the other hand, GBR12909 caused a gradual decrease in blood volume in the muscle tissue as well as HR and MABP. In the awake rats, AMPH had little effect on HR, MABP, and blood volume in muscle tissue, and GBR12909 caused slight increases in HR, MABP, and blood volume in the muscle tissue.
1999; Choi et al., 2006; Ren et al., 2009). In this study, GBR12909 and AMPH induced the positive rCBV responses in the awake rats, and the responses closely correlated with the increased extracellular DA concentration in the striatum. The close parallelism between the time course of the extracellular DA concentration and that of rCBV changes suggests that the extracellular DA drives the cerebrovascular responses. In the isoflurane anesthetized rats, the increase in the striatal rCBV induced by AMPH seems to be higher compared with that in the awake rats. Similarly, the increase in striatal DA concentration by AMPH was significantly higher in the anesthetized rats compared with the awake rats. These results are in agreement with previous reports which show the increase in extracellular DA concentration by DAT inhibitors (nomifensine or GBR12909) or DA releasers (methamphetamine or amphetamine) is higher under isoflurane or halothane anesthesia compared with in the awake state (Opacka-Juffry et al., 1992, Fink-Jensen et al., 1994, Adachi et al., 2001). Furthermore, the correlation between the change of the striatal DA concentration and the striatal rCBV response induced by AMPH was also observed in the isoflurane anesthetized rats. These results indicate that isoflurane anesthesia may not affect the cerebrovascular responses to changes of extracellular DA level.
DISCUSSION In this study, we demonstrated that GBR12909, a selective DAT inhibitor, induced positive rCBV responses in areas rich in DA receptors and with high DA utilization in the awake rats and the regional pattern of the rCBV change was similar to that of AMPH. However, in the anesthetized rats, negative rCBV responses were detected on GBR12909 treatment, while positive rCBV responses were observed for AMPH. Microdialysis study showed an increase in striatal DA concentration induced by GBR12909 or AMPH challenge in the both anesthetized and awake rats. Previous studies showed that the rCBV changes induced by AMPH or 2b-carbomethoxy-3b-(4-fluorophenyl) tropane (CFT; a DAT inhibitor) challenge are linearly correlated with the DA concentrations in the extracellular space using microdialysis (Chen et al., Synapse
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Fig. 7. Effects of GBR12909 on the striatal rCBV (0.5 min temporal resolution) and the striatal DA concentration in the isoflurane anesthetized rats (A) or the awake rats (B). The extracellular DA concentration in the striatum was measured using microdialysis (in 5 min time bins) for a separate group of animals under isoflurane anesthesia (n 5 4) or in the awake state (n 5 4). There is a tight correlation in time and amplitude between the rCBV and the extracellular DA concentration in the awake rats but not in the anesthetized rats. Data are plotted as mean 6 SEM within each group. Fig. 6. Temporal profile of GBR12909-induced rCBV response in representative brain regions of interest in the isoflurane anesthetized rats (n 5 6) or the awake rats (n 5 5). Data are plotted as mean 6 SEM within each group. Str, striatum; PFC, prefrontal cortex; NAc, nucleus accumbens; SSCx, somatosensory cortex; Thal, Thalamus.
Meanwhile, the increase in rCBV in the PFC was significantly attenuated in the isoflurane anesthetized rats compared to the awake rats. The PFC mediating higher brain function (Fuster, 2008) has been reported to be affected by general anesthesia (Sellers et al., 2013). These findings suggest that rCBV or DA response in the PFC might be affected by isoflurane anesthesia. However, it is uncertain whether the increase in the DA concentration in the PFC induced by AMPH was different between in the anesthetized and awake rats, because the DA concentration in the PFC could not be measured in this study. Thus, it is considered to be important to know effects of isoflurane on the DA release induced by AMPH in the PFC to clear the cause of difference in the rCBV response to AMPH in the PFC between in the anesthetized and awake rats. On the other hand, GBR12909 induced a negative rCBV response over a widespread brain region in the
anesthetized rats, although the increase in extracellular DA concentration in the striatum was also observed. Based upon previous studies using D1 and D2 agonists, negative rCBV responses can be interpreted as reflecting the agonism of D2/D3 receptors (Chen et al., 2005; Choi et al., 2006), whereas positive rCBV changes are associated with agonism of the D1/ D5 receptors (Choi et al., 2006). From these reports, we speculated that the negative rCBV responses to GBR12909 challenge in the anesthetized rats might be due to D2/D3 receptor activation. Ren et al. (2009) reported that negative rCBV responses are observed with a lower dose (0.25 mg/kg) of AMPH challenge, which becomes a positive response at higher doses (1–3 mg/kg). They suggested that at a lower extracellular DA concentration induced by low-dose AMPH challenge, DA primarily binds to D2 and D3 receptor rather than to the D1 receptor because of higher affinity of the D2 and D3 receptors, which leads to a decrease in rCBV. However, in the present study, GBR12909 at 10 mg/kg challenge induced a significant higher increase in the striatal DA concentration in the anesthetized rats rather than in the awake rats. In addition the increase in striatal DA Synapse
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Fig. 8. Effects of AMPH and GBR12909 on MABP, HR, and blood volume in muscle tissue in the isoflurane anesthetized rats (A) or the awake rats (B). Data for MABP and HR for 20225 min after drug administration were absent due to arterial blood sampling for blood gas measurement. Data are plotted as mean 6 SEM within each group.
concentration induced by 10 mg/kg of GBR12909 in the anesthetized rats seems to be higher than that induced by 1 mg/kg AMPH. Therefore, the negative rCBV responses to GBR12909 observed in the anesthetized rats are unlikely to be induced by D2/D3 receptor activation due to lower extracellular DA concentration. Totally, 10 mg/kg of GBR12909 is effective dose reducing impulsive choice in the delayed reward paradigm evaluating the impulsive behavior such as attention deficit/hyperactivity disorder (Baarendse and Vanderschuren, 2011). In addition, the dose of AMPH inducing comparable effects to 10 mg/kg of GBR12909 is 1 mg/kg in the delayed reword paradigm. These previous findings suggest that 10 mg/kg of GBR12909 is considered to be appropriate dose for studying the CNS effects of GBR12909 using phMRI in rats. Synapse
Many drugs for the central nervous system can also induce significant peripheral effects, including severe alterations of cardiovascular parameters. Under physiological conditions, the cerebral autoregulation maintains relatively constant cerebral circulation in the presence of changes in systemic circulation. However, large and rapid changes in MABP may cause a breakdown in the cerebral autoregulation system, thus introducing potential confounding factors in the interpretation of phMRI (Gozzi et al., 2007). In this study, although changes of cardiovascular parameters (MABP and HR) and blood volume in muscle tissue were observed in response to GBR12909 and AMPH in the awake rats, there were regions with no rCBV response such as the cerebellum. These results suggest that cerebral circulation is kept constant by the cerebral autoregulation system in the awake state, and therefore the
PHMRI RESPONSE TO GBR12909 IN AWAKE AND ANESTHETIZED RATS
essential rCBV response associated with the increase in extracellular DA caused by GBR12909 and AMPH could be detected. However, in the anesthetized rats, GBR12909 induced negative rCBV responses in nonspecific brain regions as well as decreases in cardiovascular parameters and blood volume in muscle tissue. In addition the temporal profile of the negative rCBV response was similar to that of blood volume in muscle tissue. Previous papers have reported that isoflurane anesthesia impairs the cerebral autoregulation system (Summors et al., 1999, McPherson et al., 1988). Therefore, due to breakdown in the cerebral autoregulation system under isoflurane anesthesia, the decrease in systemic circulation by GBR12909 might induce a decrease in cerebral circulation. In response to AMPH, a decrease in MABP and an increase in HR, which might be due to a baroreceptors function, were observed in the anesthetized rats. As a result, systemic and cerebral circulations were kept constant, and therefore the regional rCBV response associated with the increase in extracellular DA caused by AMPH could be detected even under isoflurane anesthesia. In conclusion, we investigated the phMRI responses to GBR12909, a selective DAT inhibitor and AMPH, a DA releaser in the isoflurane anesthetized and awake rats and demonstrated that GBR12909 induced positive rCBV responses in the awake rats and the regional pattern of the rCBV response was similar to that induced by AMPH. In addition we found that the rCBV responses to GBR12909 but not to AMPH were changed by isoflurane anesthesia. Although the mechanisms for the negative rCBV response to GBR12909 challenge under isoflurane anesthesia remains uncertain, one possible explanation is due to breakdown in the cerebral autoregulation system by isoflurane anesthesia and decrease in systemic circulation by GBR12909. Further study will be needed to clear whether the negative rCBV responses to GBR12909 under isoflurane anesthesia are due to the inherent property of GBR12909 or the common effect of DAT inhibitors. REFERENCES Adachi YU, Watanabe K, Satoh T, Vizi ES. 2001. Halothane potentiates the effect of methamphetamine and nomifensine on extracellular dopamine levels in rat striatum: A microdialysis study. Br J Anaesth 86:837–845. Alkire MT, Hudetz AG, Tononi G. 2008. Consciousness and anesthesia. Science 322:876–880. Attwell D, Laughlin SB. 2001. An energy budget for signaling in the grey matter of the brain. J Cereb Blood Flow Metab 21:1133– 1145. Baarendse PJ, Vanderschuren LJ. 2012. Dissociable effects of monoamine reuptake inhibitors on distinct forms of impulsive behavior in rats. Psychopharmacology (Berl) 219:313–326. Chen YI, Galpern WR, Brownell AL, Matthews RT, Bogdanov M, Isacson O, Keltner JR, Beal MF, Rosen BR, Jenkins BG. 1997. Detection of dopaminergic neurotransmitter activity using pharmacologic MRI: Correlation with PET, microdialysis, and behavioral data. Magn Reson Med 38:389–398.
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