1
Molecular Imaging and Biology 2005; 7(4): 262-272.
VISUALIZATION AND QUANTIFICATION OF NEUROKININ-1 ( NK1) RECEPTORS IN THE HUMAN BRAIN
Jarmo Hietala, M.D., Ph.D. 1,2, Mikko J. Nyman, M.D.1, Olli Eskola, M.Sc.3, Aki Laakso, M.D., Ph.D. 1,4, Tove Grönroos, M.Sc.3, Vesa Oikonen, M.Sc.1, Jörgen Bergman, Ph.D.3, Merja Haaparanta, M.Sc.5, Sarita Forsback, M.Sc.5, Päivi Marjamäki, M.Sc.5, Pertti Lehikoinen, Ph.D.3, Michael Goldberg, M.D. 6, Donald Burns, Ph.D.6, Terence Hamill, Ph.D. 6, Wai-Si Eng, Ph.D. 6, Alexandre Coimbra, Ph.D. 6, Richard Hargreaves, Ph.D.6 and Olof Solin, Ph.D.3 1) 2) 3) 4)
Turku PET Centre, Neuropsychiatric Imaging, 20520 Turku, Finland Department of Psychiatry, Turku University Central Hospital, 20520 Turku, Finland Turku PET Centre, Accelerator laboratory, 20520 Turku, Finland Department of Pharmacology and Clinical Pharmacology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland 5) Turku PET Centre, Medicity PET, 20520 Turku, Finland 6) Merck Research Laboratories, Rahway NJ, USA
Correspondence: Prof. Jarmo Hietala, MD, Ph.D. Neuropsychiatric Imaging Turku PET Centre, Turku University Central Hospital Kiinamyllynkatu 4-8 20520-Turku, FINLAND tel: +358-2-313 2891 fax: +358-2-231 8191 E-mail:
[email protected]
Running Header: PET visualization of human brain NK1 receptors in vivo Key words: Substance P, NK1 receptor, Positron emission tomography, human, in vivo
2 ABSTRACT
PURPOSE: To develop a new positron emission tomography (PET) method to visualize NK1 receptor systems in the human brain in vivo to examine their neuroanatomical distribution and facilitate investigations of the role of substance P, NK1 receptors and NK1 receptor antagonists in CNS function and dysfunction. PROCEDURES: PET studies were conducted in 10 healthy male volunteers using a novel selective high affinity NK1 receptor antagonist labeled with fluorine-18 to very high specific radioactivity (up to 2000 GBq/µmol) [18F]SPA-RQ. Data were collected in 3D mode for greatest sensitivity. Different modeling methods were compared and regional receptor distributions determined for comparison with in vitro autoradiographic studies using postmortem human brain slices with [18F]SPA-RQ. RESULTS:The studies showed that the highest uptake of [18F]SPA-RQ was observed in the caudate and putamen. Lower binding was found in globus pallidus and substantia nigra. [18F]SPA-RQ uptake was also widespread throughout the neocortex and limbic cortex including amygdala and hippocampus. There was very low specific uptake of the tracer in the cerebellar cortex. The distribution pattern was confirmed using in vitro receptor autoradiography with [18F]SPA-RQ on post-mortem human brain slices. Kinetic modeling of the [18F]SPA-RQ uptake data indicated a binding potential between 4 and 5 in the basal ganglia and between 1.5 and 2.5 in the cortical regions. CONCLUSIONS:[18F]SPA-RQ is a novel tool for exploration of the functions of NK1 receptors in man. [18F]SPA-RQ can be used to define receptor pharmacodynamics and focus dose selection of novel NK1 receptor antagonists in clinical trials thereby ensuring adequate proof of concept testing particularly in therapeutic applications related to CNS dysfunction. Running Header; PET visualization of human brain NK1 receptors in vivo Key words: Substance P, NK1 receptor, Positron emission tomography, human, in vivo
3 INTRODUCTION
Tachykinins are a family of 10-12 amino acid peptides that were originally grouped together according to their ability to produce fast smooth muscle contraction [1]. Three main mammalian tachykinins are currently known: substance P (SP), neurokinin A (NKA) and neurokinin B (NKB); these all share a common carboxy-terminal sequence ‘Phe-X-Gly-LeuMet-amide’ in their structure. SP was found over 70 years ago and is by far the bestcharacterized tachykinin family neuropeptide [2,3,4]. SP can be classified as a neurotransmitter and brain neuromodulator which coexists with other neurotransmitters, in particular monoamine neurotransmitters but also acetylcholine and glutamate in the nerve terminals [5,6,7,8]. SP also serves an important transmitter role in the primary afferent sensory neurons. Previous immunohistochemical studies [4,9,10] indicate that in the brain SP is especially enriched in the basal ganglia and substantia nigra. SP is also found throughout the neocortex, in limbic areas (amygdala and hypothalamus), habenula, periaqueductal gray and the midbrain nuclei (nucleus of the solitary tract, locus coeruleus and raphe)
The tachykinin peptides mediate their effects via specific G-protein coupled receptors using predominantly the phosphoinositol system as a second messenger pathway. The tachykinin receptors are currently divided into three subtypes, neurokinin 1 (NK1; formerly called the SP receptor), neurokinin 2 (NK2: formerly the substance K/substance E receptor/NKA receptor) and neurokinin 3 (NK3: formerly the NK B receptor) receptors. The effects of SP in the brain are mediated primarily via the NK1 receptor subtype that was cloned and characterized in 1991 [11]. The distribution of SP and NK1 receptors in the nervous system [12], and the targeted disruption of the NK1 receptor in mice have provided hypotheses for the possible
4 function of SP in the central nervous system. It is has been suggested, mainly on the basis of diverse preclinical studies, that SP may be involved in the regulation of movement, emotions, reward, stress responses, neurogenesis and vigilance [13,14,15,16,17,18,19]. The dense SP innervation of substantia gelatinosa of the spinal cord and the nucleus of solitary tract suggests a role for SP in modulation of pain and emesis, respectively [13,20]. SP is also believed to play a transmitter role in neurogenic inflammation, vasomotor control and many gastrointestinal functions. Recent clinical trials with the NK1 receptor antagonist aprepitant have shown that central blockade of substance P is a highly effective strategy for the prevention of acute and delayed chemotherapy-induced nausea and vomiting [21,22,23]. Aprepitant was recently registered worldwide and is an important improvement to anti-emetic control during chemotherapy. Early clinical studies also suggested that aprepitant may have anti-depressant activity implicating SP in the modulation of mood and anxiety in man [24,25]. However recent results from phase III clinical trials indicate that aprepitant is not effective for the treatment of depression (Merck and Co Inc., November 12th 2003: public announcement: http;//pubaff.merck.com/media/111203.html: Merck and Co Data on file: Keller et al: submitted)
Relatively little is known about the functions mediated by NK1 receptors in man as methodology for direct assessment of central nervous system activity of the tachykinins has not been available. Measurement of cerebrospinal fluid SP-like immunoreactivity has been previously reported [26] this approach does not provide anatomical resolution. Due to major species differences in the distribution of SP and NK1 receptors it is particularly important to explore these functions in man. During the last ten years specific and highly selective nonpeptide-structure NK1 receptor antagonists penetrating into the central nervous system have
5 been discovered and explored for their clinical effects [20,21,22,23,24,25,27,28,29,30,31]. These compounds can potentially be labeled with short-lived radioactive isotopes and used in positron emission tomography (PET) as direct probes of receptor function. PET enables direct visualization and also absolute quantification of specific neurotransmitter receptor binding. The experimental compound SPA-RQ is a selective NK1 receptor antagonist with a very high affinity for this receptor (IC50 = 67 pM, Merck, data on file). We have labeled SPA-RQ with fluorine-18 to a very high specific activity, and preclinical studies with this tracer gave high signal/noise ratios suggesting it may have clinical utility as a high affinity imaging agent for positron emission tomography in man [32]. We now report autoradiographic studies in human post-mortem brain and PET studies in vivo that characterize the pharmacology and kinetics of [18F]SPA-RQ binding in human brain.
6 SUBJECTS AND METHODS
Subjects
The study was approved by the ethics committee of the Turku University/University Hospital (Turku, Finland) and was performed in accordance with the ethical standards of the World Medical Association Declaration of Helsinki (Recommendations Guiding Medical Physicians in biomedical Research involving human subjects). Ten healthy male volunteers with no history of psychiatric disorders, substance abuse, or somatic illnesses were recruited. No individual had clinically significant abnormalities in their physical examination, laboratory values (blood and urine) and all had negative urine drug screens. MRI scans (1.5 T Siemens Magnetom, Iselin, NJ USA,) were performed to ensure that there were no structural abnormalities in the brain of the participants. All were non-smokers with ages ranging from 19-33 years (25 ± 4 years, given as mean ± SD) and weights from 61-81 kg (71 ± 7 kg). Written informed consent was obtained prior to inclusion in the study.
PET methods
Radiosynthesis of [18F]SPA-RQ The NK1 receptor antagonist, SPA-RQ (MW = 450 g/mol) was labeled with the positron emitter fluorine-18 (18F; T½=109.8 min). Details of the precursor (Merck, USA), the radiosynthesis and quality control have been described previously [32]. Briefly, [18F]SPA-RQ, (S,S)-[2-[18F]Fluoromethoxy-5-(5-Trifluoromethyltetrazol-1-yl)benzyl]-(2-phenylpiperidin-3-yl)-
7 amine, (described previously as [18F] L-829, 165), was prepared by [18F]fluoroalkylation of a deprotonated phenolic hydroxyl group with [18F]FCH2Br in dimethyl formamide followed by removal of the BOC protecting group. [18F]FCH2Br was prepared from [18F]F- and dibromomethane and purified via preparative gas chromatography [33]. After fluoroalkylation, dimethyl formamide was removed in ~ 7 min by applying a stream of helium over the surface of the solution in the reaction vessel which was simultaneously heated at 110 °C. The t-BOC protecting group was removed by treatment with trifluoroacetic acid at room temperature for 1-2 min, and the trifluoroacetic acid was evaporated by passing a stream of helium through the reaction vessel. The resulting [18F]SPA-RQ was purified via gradient preparative HPLC. The final product was formulated in ethanolic D-glucose solution buffered to pH 7. The specific radioactivity was very high and at least 400 GBq/µmol at the time of injection.
PET scanning
The PET experiments were performed using a whole-body PET scanner (GE advance, Milwaukee, WI, USA) with 35 slices of 4.25 mm thickness covering the whole brain. The basic performance characteristics tests on this camera indicate transaxial and axial spatial resolutions (FWHM) of 4.3 mm and 4.3 mm, respectively. The camera was used in the 3D mode to increase sensitivity. Head fixation was achieved by using a commercial head holder (GE) supporting the head from the top, back and sides. Two beams of laser light were used in the head positioning according to canthomeatal and sagittal lines. Several points were marked on the skin of the subject and followed during the scan to control for any movement.
8 Transmission scan was acquired before first dynamic scan to correct for tissue attenuation using two solid rod sources of Ge-68 with 400 MBq of radioactivity.
The right antecubital vein and left radial artery were cannulated for injection of [18F]SPA-RQ and blood sampling, respectively. [18F]SPA-RQ (125-135 MBq) was given as rapid bolus injection and flushed with 10 cc of saline. The specific radioactivity of [18F]SPA-RQ was on average 1995 GBq/µmol (range 160 – 4150) at the time of the injection (corresponding mass of injected tracer 114 ng (range 14-360 ng). Brain radioactivity was measured for 87 min (3 x 1 min, 6 x 3 min frames and thereafter using 6 min frames) followed by additional dynamic scans consisting of six 10 min frames from 120-180 min, from 210 – 270 min and 300 – 360 min for the first 6 subjects (group 1). Three dynamic scans were conducted for another group of 4 subjects (group 2). The first scan, 87 min in duration, was acquired immediately after injection followed by a 40 minute scan from 120 min after injection and a third, 60 min scan, from 180 min after injection. To obtain arterial input function, an automated blood sampling system was used for the first 3.5 min after which arterial blood samples were taken manually up to 90 min.
[18F]SPA-RQ and radiolabeled metabolite analysis in plasma
Unchanged [18F]SPA-RQ in arterial plasma was determined with planar chromatography and digital autoradiography only in group 2 (n=4) studied with dynamic scans. Deproteinized plasma samples were applied to a high performance thin layer chromatography (TLC) plate with an automatic TLC sampler. After the migration, the TLC plates were exposed to a
9 phosphorimaging plate (Fuji BAS-TR2025, Fuji Photo Film Co., Ltd., Japan) for 4±0.5 hours and analyzed for photostimulated luminescence (Tina 2.1, Raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany). The arterial plasma input function for [18F]SPA-RQ was generated by curve fitting to measurements of the unchanged tracer fraction using a Hill function of the general form Y(L)/Y(0) = 1- Lh / (Kh + Lh) where L is the independent variable and routines described previously by [34].
Region-of-interest definition and determination of receptor binding parameters
The magnetic resonance images for each individual were resliced according to PET tomography [35]. The anatomical regions of interest (see Table 1) were defined on the MR images using ImadeusR software (version 1.0, Forima Inc., Finland) before being transferred to the PET images for tracer analyses. Regions of interest were delineated on three to four consecutive slices. Regional time-radioactivity curves were generated using the Imadeus software and analyzed using a standard three compartment model as a starting point [36,37]. For group 2 where arterial plasma sampling had been performed, the data was analyzed with kinetic modeling [38] using a reference region and metabolite corrected arterial plasma as inputs (for details see next section below) to obtain an estimate of binding potential for [18F]SPA-RQ. In order to account for variations in the sampling rate over time, time activity curve data was weighted, in the manner described previously [34], to give each frame equal weight according the length and total counts in the frame.
Binding potential estimates with kinetic analysis and arterial input.
10 Rate constants (k1, k2, k3 and k4) were estimated in various brain regions with a nonlinear least squares global optimization procedure using tissue and metabolite-corrected plasma time-radioactivity curves. Total blood activity was used to account for the vascular radioactivity and tissue time-radioactivity curves were corrected assuming a fixed 4% vascular volume. After 90 min the radioactivity in blood was relatively low and therefore the analysis was constrained to the first 90 min. In the present analysis we have defined regional binding potential (BP) as k3/k4. Regional binding potentials were estimated using an indirect approach via calculation of [18F]SPA-RQ regional distribution volumes from the rate constants k1, k2, k3 and k4 [39]. Constants k1 and k2 and K1/k2 were not assumed to be same in all regions. In this unconstrained indirect approach regional binding potential BP = (volume distribution in region of interest/Volume of distribution in reference region)-1 = k3/k4 =f2 (free fraction of ligand in brain) Bmax/Kd. Regional BP (k3/k4) was also estimated from a 3 compartment fit with regional K1/k2 fixed to that of the cerebellum
Kinetic analysis with cerebellum input was performed with the reference tissue model [40,41]. This method provides parameters RI, k2 and BP (k3/k4) relative to f2.
Post-mortem human brain receptor autoradiography
The post-mortem human brain material was obtained from the University of Kuopio, Finland (three males, age 35-50 years, post-mortem delay 9.5 –18.5 h, with no known history of neurological or psychiatric diseases). 100 µm-thick horizontal slices from the left cerebral hemisphere were incubated for 60 min at room temperature with 50 pM [18F]SPA-RQ in Tris-
11 HCl-buffer (pH 7.5) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Nonspecific binding was estimated by competing the tracer binding with 1 µM excess of a cold specific high affinity NK1 receptor antagonist GR203040 (2-methoxy-5-tetrazol-1-yl-benzyl-(2phenyl-piperidin-3-yl)-a mine). After washing and drying, the slices were apposed to an imaging plate (Fuji Imaging Plate BAS-TR2025, Fuji Photo Film Co., Ltd., Japan) for digital autoradiography. The imaging plates were scanned after four hours exposure with the Fuji Analyzer BAS-5000.
RESULTS
Plasma analysis
The radioactivity in blood decreased rapidly and could not be measured reliably during the later dynamic PET scans after 90 min (for full details of scan times see above in PET scanning methods section). The amount of unchanged [18F]SPA-RQ as a function of time is shown in Figure 1. At 80 min about 30-40% of the tracer was unchanged. The radio-TLC analysis also showed an as yet unidentified hydrophilic metabolite and free fluoride.
Brain uptake of [18F]SPA-RQ
The tracer readily penetrated the blood-brain barrier into human brain in vivo. Highest uptake was observed in the putamen and in the caudate nucleus whereas lowest uptake was observed in cerebellum. In neocortex and thalamus uptake levels were between striatal and cerebellar regions (Figure 2). Highest cortical binding was noted in the primary visual cortex
12 whereas lowest uptake was in the medial prefrontal cortex. There was also moderate uptake in the midbrain, medulla and also in the pons (Figure 7). When the time-radioactivity curves from six subjects were combined, it was observed that total radioactivity of putamen peaked on average at 215 min and in caudate at 225 min with putamen/cerebellum and caudate/cerebellum ratios between 5 and 6. The specific binding (cerebellum subtracted) peaked at 323 ± 33 min, 327 ± 44 min and 132 ± 17 min in caudate, putamen and occipital cortex, respectively (means ± SD) (Figure 3).
Plasma input models Cerebellum. Cerebellar time activity curves (corrected for the vascular contribution) were analyzed with 2- and 3-compartmental modeling (Figure 4) . Even the two-compartment model described adequately the kinetics of [18F]SPA-RQ in the cerebellum although the 3compartment model fit was statistically better according to Akaike Information and Schwarz criteria (data not shown).
Target regions. Regional distribution volumes derived indirectly from the unconstrained kinetic analysis indicated high total distribution volume values ranging from 5.6 in cerebellum to 26.6 in putamen. The results are summarized in Table 1 for caudate, putamen and occipital cortex. The methods provided reasonable estimates of the binding potential but with relatively high coefficients of variation and the binding potentials were unexpectedly low compared to the binding potential estimates derived from the simple ratio data (Table 1). In many of the smaller regions the full kinetic model proved to be unstable and not suitable for quantification. Fixing the k1/k2 to that of cerebellum improved the stability of the method but the binding potential underestimation was still apparent.
13
Reference tissue input model The reference tissue model described the data adequately and was used to assess the optimum data acquisition times for [18F]SPA-RQ. At the earliest time intervals, the binding potential estimates were highly variable and underestimated but stabilized with longer acquisition time (see Figure 5). The 240 minute scanning time seemed to be sufficient for most of the structures but the binding potential was still slightly underestimated in putamen with a 240 min data acquisition. In, general the binding potential estimates with the simplified reference tissue model and longer data acquisition were clearly higher than those derived from plasma input models and 90 min data acquisition. The reference tissue model was robust enough for estimation of NK1 receptor binding potential in discrete brain regions. This analysis showed that the highest binding potentials were in the basal ganglia but the binding potential in substantia nigra was relatively low (Table 2). There was also relatively high binding potential in the midbrain/pons probably representing groups of nuclei such as the dorsal raphe, locus coeruleus and periaqueductal gray.
Uptake in skull bone Radioactivity in the skull was noted in some subjects during the later scanning times. This uptake had accumulation-type kinetics and most likely represents free 18F from the in vivo metabolism of the tracer.
14 In vitro receptor autoradiography studies in basal ganglia The distribution of [18F]SPA-RQ binding in vitro followed the in vivo pattern with highest binding in striatum > cortical regions= thalamus > cerebellum. The striatum vs. cortex binding contrast varied and was slightly less than that seen in young healthy male volunteers in vivo. The addition of an excess high concentration (1 µM) of cold NK1 receptor antagonist (GR203040) did not affect cerebellar binding (57 ± 8 vs. 60 ± 7 PSL/mm2, means ± SD, n=3). In contrast the presence of GR203040 prevented caudate, putamen and occipital cortical specific binding of [18F]SPA-RQ almost completely (percentage blockade were 98± 2, 94 ± 2 and 95 ± 4, means ± SD, n= 3 respectively) (Figure 6).
DISCUSSION
The NK1 receptor has proved to be an enigmatic target for neuroscience drug discovery and development drug development. Despite the widespread distribution of SP and NK1 receptors in the CNS and the potential application of NK1 receptor antagonists in diverse therapeutic indications, NK1 receptor antagonists have to date proven efficacy only in the prevention of acute and delayed chemotherapy induced nausea and vomiting. The primary aim of the present studies was to develop and characterize a radiotracer method for direct measurement of NK1 receptor binding characteristics in man using positron emission tomography. The availability of a PET tracer will facilitate further clinical exploration and proof of concept testing of NK1 receptor antagonists in potential CNS therapeutic applications.
15 Evaluation of [18F]SPA-RQ as a PET tracer
[18F]SPA-RQ can be labeled to a very high specific radioactivity. This is an important goal in the development of very high (picomolar) affinity receptor ligands, such as SPA-RQ, in order to avoid significant receptor occupancy by the tracer [42]. The injected mass of SPA-RQ per subject was well below 500 ng in this study and the possibility of significant NK1 receptor occupancy (> 1%) with [18F]SPA-RQ is practically non-existent.
Previous in vivo experiments in guinea pigs indicated a good signal/noise-ratio for [18F]SPARQ with striatum/cerebellum ratio of about 20 at 3 hours post-injection. These studies also provided early evidence for appropriate pharmacology of the signal in striatum as the nonspecific cerebellar signal could not be blocked by high doses of a competing cold NK1 receptor antagonist [32]. The autoradiography studies with [18F]SPA-RQ binding to postmortem human brain slices showed clear striatal and cortical binding and that the signal in the cerebellar cortex could not be competed away by a high concentration of a selective NK1 receptor antagonist. Taken together these findings indicated that 1) the density of NK1 receptors is negligible in human cerebellar cortex; 2) the cerebellar cortex can be potentially used as a reference region and as an estimate of free and nonspecific binding in PET studies with [18F]SPA-RQ in vivo. This interpretation is supported by the findings of Bergstrom et al [43] who showed that there is no change in the cerebellar signal in humans before and after high blocking doses of the NK1 receptor antagonist aprepitant in vivo [43].
After injection of [18F]SPA-RQ in healthy volunteers, the brain radioactivity had to be followed up to 6 hours due to the slow tracer kinetics. The total uptake was highest in the caudate-
16 putamen followed by cortical regions (especially visual cortex), thalamus, midbrain, medulla, and pons whereas low uptake was noted in the cerebellar cortex. The total binding peaked at about 210 min in the striatum and earlier in other regions indicating the reversibility of the ligand-receptor recognition process. The radioactivity in the blood was relatively low and could not be measured reliably during the scans >90 min duration. At 90 min, about 40 % of the radioactivity in plasma was unchanged [18F]SPA-RQ. We noted free fluoride and one polar radiolabeled metabolite in the blood. The metabolite was unidentified by our TLC methodology but is hydrophilic in nature and so is unlikely to enter the brain significantly in comparison with the parent [18F]SPA-RQ. Due to the slow clearance/kinetics of the tracer, the first 90 min is clearly non-optimal for detailed quantification of NK1 receptor binding. The different analysis approaches with full kinetic modeling resulted in unexpectedly low binding potential estimates and relatively high percentage of failures in the estimation procedure in smaller brain nuclei. In the cerebellum, the tissue data could be described with a 2- or 3-compartment model although the latter described the data statistically better. This has been observed for many valid tracers such as [11C]raclopride and is likely explained by rapid and slow components of the non-specific binding in the cerebellum [44] although other possibilities still cannot entirely be excluded, such as a radioactive metabolite(s) of [18F]SPARQ. Specific binding can be excluded as there was no binding of [18F]SPA-RQ in the cerebellar cortex in the presence of a cold excess concentration of GR203040 a high affinity selective NK1 receptor antagonist in human post-mortem brain slices in vitro and data from NK1 antagonist studies in vivo shows no change in cerebellar signal before and after blockade of >95% striatal NK1 receptors [43].
17 The reference tissue model using cerebellar input was robust giving data similar to the ratio method and thus seems to be a promising option for kinetic modeling and quantification of the NK1 receptors in baseline situations. The time required for stable determination of binding potential was generally around 240 min but, even at this time, the specific binding in high density areas (e.g. putamen) may be slightly underestimated in some subjects. For practical imaging purposes such as occupancy studies, even simpler ratio methods may be useful [39].
During later scans radioactivity was noted in the skull bone. SP receptors have been reported to exist in bone [45] but the radioactivity is most likely due to uptake of free fluoride by the bone since the uptake kinetics showed clear accumulation for up to 6 hours after tracer injection.
Human brain NK1 receptor distribution measured with [18F]SPA-RQ in vivo and in vitro
Before the completion of our studies there was limited data on the distribution of NK1 receptors in human brain. Several publications have highlighted potential species differences in NK1 receptor distribution that flag caution when extrapolating rodent data to man [4,12,46]. Moreover, another complicating factor is a mismatch, in some brain regions, between the distribution of SP containing neurons and the NK1 receptor in the brain [4,47]. The NK1 receptor distribution in the rat and in man has been predominantly studied with radiolabeled SP. NK1 receptors are widely found in the central and peripheral nervous systems. In the rat CNS, SP and NK1 receptors are most abundant in striatum and spinal cord but can also be found in solitary nucleus, hippocampus, habenula, interpeduncular nucleus, hypothalamus, raphe nuclei and medulla oblongata [48]. The substantia nigra has high amounts of SP but a
18 low number of SP binding sites. Relatively low amounts of SP and SP binding sites are found in the cortex and cerebellum of the rat [4] whereas cortical SP-like immunoreactivity and NK1 receptor mRNA is fairly abundant in man [10,12].
The present in vivo and in vitro studies of NK1 receptor distribution with [18F]SPA-RQ showed the highest density of NK1 receptors to be found in striatum but, unlike in rat, the NK1 receptor density was relatively high throughout the human cortex. Recent studies indicate high NK1like immunoreactivity and NK1 receptor mRNA in human basal ganglia [12,49,50]. Immunohistochemical studies in human basal ganglia have shown that NK1 receptor-positive neurons are preferentially localized in the striosomal compartment in striatum. This cannot be detected with PET due to its limited (4-5mm) anatomical resolution. Rodent in situ hybridization and immunohistochemical studies suggest that the large NK1 receptorimmunoreactivity positive striatal neurons express choline acetyl transferase, calretinin, somatostatin and nitric oxide synthase [51,52,53] but human tissue has not been similarly studied.
There appear to be species differences in the distribution of midbrain NK1 receptors. In the midbrain dopamine cells there is a high abundance of NK3 receptor mRNA in the rat, whereas in human midbrain dopamine cells NK1 predominates [54]. The specific NK1 binding detected by PET in substantia nigra in the present studies was somewhat lower than expected but the BP is probably somewhat underestimated due to partial volume effects. Other studies [55,56] have provided indirect supportive evidence for the likely presence of NK1 receptors in substantia nigra as a result of SP innervation within the direct GABAergic pathway that innervates the pars reticulata as well as the internal segment of globus pallidus
19
The NK1 receptor distribution in the cortical regions was relatively uniform in both in vivo and in vitro studies with [18F]SPA-RQ. The NK1 receptors probably have a modulatory role in the cortical neural pathways and have been suggested to participate at least in the control of cortical dopamine and acetylcholine release [57,58]. The striatum vs. cortex NK1 receptor binding contrast was less marked in two of the three post-mortem brains analyzed compared to that seen in vivo with PET. Possible reasons for this may be aging or factors related to post-mortem delay. One of the cortical regions with highest uptake was the occipital cortex, including the cuneus/posterior cingulate and in the striate cortex including the primary visual cortex. SP has been suggested to be involved in processing of direction specific visual stimuli based on experimental studies and SP receptor binding pattern in human visual cortex [59]. However, practically nothing is known about the modulation of vision by SP in man. It is important to note however that although dense populations of NK1 receptors were visualized in human brain in nigrostriatal pathways and visual cortex using our PET ligand that no motor or visual system adverse events have been associated with the acute or long term use of the NK1 receptor antagonist aprepitant even though these systems were effectively blocked around the clock [43] (Keller et al submitted). The functional role of these receptors thus remains unknown.
Equally high cortical NK1 binding in vivo was observed in the limbic cortex, such as hippocampus and amygdala. It has been hypothesized that SP and NK1 receptors in these nuclei may be involved in neurogenesis and modulation of emotions, anxiety and stress [17,60]. Relatively high uptake of [18F]SPA-RQ was also noted in the midbrain including the nucleus of the solitary tract, periaqueductal gray, raphe nuclei and also locus coeruleus
20 [6,8,12,61].The latter two nuclei were implicated as possible antidepressant sites of action for NK1 receptor antagonists [62,63,64] but this was not supported by extensive clinical trials with the substance P receptor antagonist aprepitant (Keller et al submitted). In contrast the PET tracer binding observed at the base of the IVth ventricle in the vicinity of the, area postrema, nucleus of the solitary tract and motor nuclei involved in the vomiting reflex is consistent with central anti-emetic site of action defined pre-clinically for NK1 receptor antagonists [30] and auto-radiographic studies of the binding of [125I]-SP and its displacement by the anti-emetic NK1 receptor antagonist CP99,994 in ferret brain [65]. Indeed the central site of action for NK1 receptor antagonists is thought to underpin their broad spectrum anti-emetic activity and their synergy with 5HT3 receptor antagonists (e.g. granisetron) that act mainly in the periphery against chemotherapy induced nausea and vomiting [21]. The resolution of PET does not allow the full visualization of these nuclei, but in vitro human brain autoradiography studies and newer high resolution pre-clinical microPET and clinical PET cameras may help to reliably quantify NK1 binding, even in these smaller brain stem nuclei. The NK1 receptor binding in spinal cord or the peripheral nervous system was not explored in this study but will be of interest in the future.
Conclusion
This is the first time that the distribution of SP NK1 receptors has been visualized in vivo in the human brain. Our study demonstrates that NK1 receptors are widely distributed throughout the brain with highest density in the striatum and visual cortex. Their function therein, and whether they provide targets for future CNS therapeutics, is currently unknown. [18F]SPA-RQ has suitable properties for an in vivo imaging agent and can yield accurate estimates of NK1
21 receptor density in brain regions. The tracer could be a valuable tool to help increase our understanding of the involvement of SP in human physiological functions and potential clinical uses for SP NK1 receptor antagonists particularly in CNS driven disorders.
Acknowledgements: This study was supported by Merck Research Laboratories, USA. The help of the staff in the Turku PET laboratory and in MRI unit is appreciated. We thank Drs Jari Tiihonen, Erkki Tupala (Kuopio University) and Terttu Särkioja (Oulu University) for the help with post-mortem human brain slices. The authors would also like to thank Dr. David Sciberras for oversight of the studies during his tenure at Merck. References 1. Stout S.C.; Owens M.J., and Nemeroff C.B.; Neurokinin(1) receptor antagonists as potential antidepressants. Annual Review of Pharmacology and Toxicology 41:877-906, 2001. 2. Von Euler U.S. and Gaddum J.H.; An unidentified depressor substance in certain tissue extracts. J. Physiol. 72:74-87, 1931. 3. Pernow B.; Substance-P. Pharmacological Reviews 35:85-141, 1983. 4. Otsuka M. and Yoshioka K.; Neurotransmitter Functions of Mammalian Tachykinins. Physiological Reviews 73:229-308, 1993. 5. Vincent S.R.; Satoh K.; Armstrong D.M.; Panula P.; Vale W., and Fibiger H.C.; Neuropeptides and Nadph-Diaphorase Activity in the Ascending Cholinergic Reticular System of the Rat. Neuroscience 17:167-&, 1986.
22 6. Baker K.G.; Halliday G.M.; Hornung J.P.; Geffen L.B.; Cotton R.G.H., and Tork I.; Distribution, Morphology and Number of Monoamine-Synthesizing and Substance PContaining Neurons in the Human Dorsal Raphe Nucleus. Neuroscience 42:757-775, 1991. 7. Nicholas A.P.; Pieribone V.A.; Arvidsson U., and Hokfelt T.; Serotonin-Like, Substance PLike and Glutamate Aspartate-Like Immunoreactivities in Medullo-Spinal Pathways of Rat and Primate. Neuroscience 48:545-559, 1992. 8. Sergeyev V.; Hokfelt T., and Hurd Y.; Serotonin and substance P co-exist in dorsal raphe neurons of the human brain. Neuroreport 10:3967-3970, 1999. 9. Ljungdahl A.; Hokfelt T., and Nilsson G.; Distribution of Substance P-Like Immunoreactivity in Central Nervous-System of Rat .1. Cell Bodies and Nerve-Terminals. Neuroscience 3:861-&, 1978. 10.
Mai J.K.; Stephens P.H.; Hopf A., and Cuello A.C.; Substance-P in the Human-Brain. Neuroscience 17:709-739, 1986.
11.
Takeda Y.; Chou K.B.; Takeda J.; Sachais B.S., and Krause J.E.; Molecular-Cloning, Structural Characterization and Functional Expression of the Human Substance-P Receptor. Biochemical and Biophysical Research Communications 179:1232-1240, 1991.
12.
Caberlotto L.; Hurd Y.L.; Murdock P. et al.; Neurokinin 1 receptor and relative abundance of the short and long isoforms in the human brain. Eur. J. Neurosci. 17:17361746, 2003.
23 13.
De Felipe C.; Herrero J.F.; O'Brien J.A. et al.; Altered nociception, analgesia and aggression in mice lacking the receptor for substance P. Nature 392:394-397, 1998.
14.
Murtra P.; Sheasby A.M.; Hunt S.P., and De Felipe C.; Rewarding effects of opiates are absent in mice lacking the receptor for substance P. Nature 405:180-183, 2000.
15.
Laird J.M.A.; Olivar T.; Roza C.; De Felipe C.; Hunt S.P., and Cervero F.; Deficits in visceral pain and hyperalgesia of mice with a disruption of the tachykinin NK1 receptor gene. Neuroscience 98:345-352, 2000.
16.
Rupniak N.M.J.; Carlson E.C.; Harrison T. et al.; Pharmacological blockade or genetic deletion of substance P (NK1) receptors attenuates neonatal vocalisation in guinea-pigs and mice. Neuropharmacology 39:1413-1421, 2000.
17.
Morcuende S.; Gadd C.A.; Peters M. et al.; Increased neurogenesis and brain-derived neurotrophic factor in neurokinin-1 receptor gene knockout mice. Eur. J. Neurosci. 18:1828-1836, 2003.
18.
van der Hart M.G.C.; Czeh B.; de Biurrun G. et al.; Substance P receptor antagonist and clomipramine prevent stress-induced alterations in cerebral metabolites, cytogenesis in the dentate gyrus and hippocampal volume. Molecular Psychiatry 7:933-941, 2002.
19.
Bondy B.; Baghai T.C.; Minov C. et al.; Substance P serum levels are increased in major depression: Preliminary results. Biol. Psychiatry 53:538-542, 2003.
20.
Navari R.M.; Reinhardt R.R.; Gralla R.J. et al.; Reduction of cisplatin-induced emesis by a selective neurokinin-1-receptor antagonist. L-754,030 Antiemetic Trials Group. N. Engl. J. Med. 340:190-195, 1999.
24 21.
Hesketh P.J.; Van Belle S.; Aapro M. et al.; Differential involvement of neurotransmitters through the time course of cisplatin-induced emesis as revealed by therapy with specific receptor antagonists. European Journal of Cancer 39:1074-1080, 2003.
22.
Hesketh P.J.; Grunberg S.M.; Gralla R.J. et al.; The oral neurokinin-1 antagonist aprepitant for the prevention of chemotherapy-induced nausea and vomiting: A multinational, randomized, double-blind, placebo-controlled trial in patients receiving highdose cisplatin - The Aprepitant Protocol 052 Study Group. Journal of Clinical Oncology 21:4112-4119, 2003.
23.
De Wit R.; Herrstedt J.; Rapoport B. et al.; Addition of the oral NK1 antagonist aprepitant to standard antiemetics provides protection against nausea and vomiting during multiple cycles of cisplatin-based chemotherapy. Journal of Clinical Oncology 21:41054111, 2003.
24.
Kramer M.S.; Cutler N.; Feighner J. et al.; Distinct mechanism for antidepressant activity by blockade of central substance P receptors. Science 281:1640-1645, 1998.
25.
Kramer M.S.; Winokur A.; Kelsey J. et al.; Demonstration of the efficacy and safety of a novel substance P (NK1) receptor antagonist in major depression. Neuropsychopharmacology 29:385-392, 2004.
26.
Rimon R.; Legreves P.; Nyberg F.; Heikkila L.; Salmela L., and Terenius L.; Elevation of Substance P-Like Peptides in the Csf of Psychiatric-Patients. Biol. Psychiatry 19:509516, 1984.
25 27.
Snider R.M.; Constantine J.W.; Lowe J.A. et al.; A Potent Nonpeptide Antagonist of the Substance-P (Nk1) Receptor. Science 251:435-437, 1991.
28.
McLean S.; Ganong A.; Seymour P.A. et al.; Characterization of CP-122,721; A nonpeptide antagonist of the neurokinin NK1 receptor. Journal of Pharmacology and Experimental Therapeutics 277:900-908, 1996.
29.
Rupniak N.M.J. and Kramer M.S.; Discovery of the anti-depressant and anti-emetic efficacy of substance P receptor (NK1) antagonists. Trends in Pharmacological Sciences 20:485-490, 1999.
30.
Tattersall F.D.; Rycroft W.; Francis B. et al.; Tachykinin NK1 receptor antagonists act centrally to inhibit emesis induced by the chemotherapeutic agent cisplatin in ferrets. Neuropharmacology 35:1121-1129, 1996.
31.
Tattersall F.D.; Rycroft W.; Cumberbatch M. et al.; The novel NK1 receptor antagonist MK-0869 (L-754,030) and its water soluble phosphoryl prodrug, L758,298, inhibit acute and delayed cisplatin-induced emesis in ferrets. Neuropharmacology 39:652-663, 2000.
32.
Solin O.; Eskola O.; Hamill T.G. et al.; Synthesis and characterization of a potent, selective, radiolabeled substance-P antagonist for NK(1) receptor quantitation: ([(18)F]SPA-RQ). Mol. Imaging Biol. 6:373-384, 2004.
33.
Bergman J.; Eskola O.; Lehikoinen P., and Solin O.; Automated synthesis and purification of [18F]bromofluoromethane at high specific radioactivity. Appl. Radiat. Isot. 54:927-933, 2001.
26 34.
Gunn R.N.; Sargent P.A.; Bench C.J. et al.; Tracer kinetic modeling of the 5-HT1A receptor ligand [carbonyl-11C]WAY-100635 for PET. NeuroImage 8:426-440, 1998.
35.
Hietala J.; Syvalahti E.; Vuorio K. et al.; Presynaptic dopamine function in striatum of neuroleptic-naive schizophrenic patients. Lancet 346:1130-1131, 1995.
36.
Mintun M.A.; Raichle M.E.; Kilbourn M.R.; Wooten G.F., and Welch M.J.; A Quantitative Model for the Invivo Assessment of Drug-Binding Sites with Positron Emission Tomography. Annals of Neurology 15:217-227, 1984.
37.
Slifstein M. and Laruelle M.; Models and methods for derivation of in vivo neuroreceptor parameters with PET and SPECT reversible radiotracers. Nuclear Medicine and Biology 28:595-608, 2001.
38.
Parsey R.V.; Slifstein M.; Hwang D.R. et al.; Validation and reproducibility of measurement of 5-HT1A receptor parameters with [carbonyl-C-11]WAY-100635 in humans: Comparison of arterial and reference tissue input functions. Journal of Cerebral Blood Flow and Metabolism 20:1111-1133, 2000.
39.
Ito H.; Hietala J.; Blomqvist G.; Halldin C., and Farde L.; Comparison of the transient equilibrium and continuous infusion method for quantitative PET analysis of [C11]raclopride binding. Journal of Cerebral Blood Flow and Metabolism 18:941-950, 1998.
40.
Cunningham V.J.; Hume S.P.; Price G.R.; Ahier R.G.; Cremer J.E., and Jones A.K.P.; Compartmental Analysis of Diprenorphine Binding to Opiate Receptors in the Rat Invivo and Its Comparison with Equilibrium Data Invitro. Journal of Cerebral Blood Flow and Metabolism 11:1-9, 1991.
27 41.
Lammertsma A.A. and Hume S.P.; Simplified reference tissue model for PET receptor studies. NeuroImage 4:153-158, 1996.
42.
Sudo Y.; Suhara T.; Inoue M. et al.; Reproducibility of [C-11]FLB 457 binding in extrastriatal regions. Nuclear Medicine Communications 22:1215-1221, 2001.
43.
Bergstrom M.; Hargreaves R.J.; Burns H.D. et al.; Human positron emission tomography studies of brain neurokinin 1 receptor occupancy by aprepitant. Biol. Psychiatry 55:1007-1012, 2004.
44.
Oikonen V.; Allonen T.; Nagren K.; Kajander J., and Hietala J.; Quantification of [carbonyl-C-11]WAY-100635 binding: Considerations on the cerebellum. Nuclear Medicine and Biology 27:483-486, 2000.
45.
Goto T.; Kido M.A.; Yamaza T., and Tanaka T.; Substance P and substance P receptors in bone and gingival tissues. Med Electron Microsc. 34:77-85, 2001.
46.
Maeno H.; Kiyama H., and Tohyama M.; Distribution of the substance P receptor (NK-1 receptor) in the central nervous system. Brain Res. Mol. Brain Res. 18:43-58, 1993.
47.
Hokfelt T.; Broberger C.; Xu Z.Q.D.; Sergeyev V.; Ubink R., and Diez M.; Neuropeptides - an overview. Neuropharmacology 39:1337-1356, 2000.
48.
Mantyh P.W.; Hunt S.P., and Maggio J.E.; Substance-P Receptors - Localization by Light Microscopic Autoradiography in Rat-Brain Using [H-3]Sp As the Radioligand. Brain Res. 307:147-165, 1984.
28 49.
Parent A.; Cicchetti F., and Beach T.G.; Striatal neurones displaying substance P (NK1) receptor immunoreactivity in human and non-human primates. Neuroreport 6:721724, 1995.
50.
Mounir S. and Parent A.; The expression of neurokinin-1 receptor at striatal and pallidal levels in normal human brain. Neuroscience Research 44:71-81, 2002.
51.
Gerfen C.R.; Substance P (neurokinin-1) receptor mRNA is selectively expressed in cholinergic neurons in the striatum and basal forebrain. Brain Res. 556:165-170, 1991.
52.
Chen L.W.; Wei L.C.; Liu H.L.; Qiu Y., and Chan Y.S.; Cholinergic neurons expressing substance P receptor (NK1) in the basal forebrain of the rat: a double immunocytochemical study. Brain Res. 904:161-166, 2001.
53.
Kaneko T.; Shigemoto R.; Nakanishi S., and Mizuno N.; Substance P receptorimmunoreactive neurons in the rat neostriatum are segregated into somatostatinergic and cholinergic aspiny neurons. Brain Res 631:297-303, 1993.
54.
Whitty C.J.; Paul M.A., and Bannon M.J.; Neurokinin receptor mRNA localization in human midbrain dopamine neurons. J. Comp. Neurol. 382:394-400, 1997.
55.
Whitty C.J.; Walker P.D.; Goebel D.J.; Poosch M.S., and Bannon M.J.; Quantitation, Cellular-Localization and Regulation of Neurokinin Receptor Gene-Expression Within the Rat Substantia-Nigra. Neuroscience 64:419-425, 1995.
56.
Futami T.; Hatanaka Y.; Matsushita K., and Furuya S.; Expression of substance P receptor in the substantia nigra. Molecular Brain Research 54:183-198, 1998.
29 57.
Feuerstein T.J.; Gleichauf O., and Landwehrmeyer G.B.; Modulation of cortical acetylcholine release by serotonin: The role of substance P interneurons. NaunynSchmiedebergs Archives of Pharmacology 354:618-626, 1996.
58.
Lejeune F.; Gobert A., and Millan M.J.; The selective neurokinin (NK)(1) antagonist, GR205,171, stereo specifically enhances mesocortical dopaminergic transmission in the rat: a combined dialysis and electrophysiological study. Brain Res. 935:134-139, 2002.
59.
Kus L.; Mazzone S.B.; Paxinos G., and Geraghty D.P.; Autoradiographic localisation of substance P (NK1) receptors in human primary visual cortex. Brain Res. 794:309-312, 1998.
60.
Smith D.W.; Hewson L.; Fuller P.; Williams A.R.; Wheeldon A., and Rupniak N.M.; The substance P antagonist L-760,735 inhibits stress-induced NK(1) receptor internalisation in the basolateral amygdala. Brain Res 848:90-95, 1999.
61.
Nomura H.; Shiosaka S., and Tohyama M.; Distribution of Substance P-Like Immunoreactive Structures in the Brain-Stem of the Adult Human-Brain - An Immunocytochemical Study. Brain Res. 404:365-370, 1987.
62.
Conley R.K.; Cumberbatch M.J.; Mason G.S. et al.; Substance P (neurokinin 1) receptor antagonists enhance dorsal raphe neuronal activity. J. Neurosci. 22:7730-7736, 2002.
63.
Maubach K.A.; Martin K.; Chicchi G. et al.; Chronic substance P (NK1) receptor antagonist and conventional antidepressant treatment increases burst firing of monoamine neurones in the locus coeruleus. Neuroscience 109:609-617, 2002.
30 64.
Froger N.; Gardier A.M.; Moratalla R. et al.; 5-hydroxytryptamine (5-HT)1A autoreceptor adaptive changes in substance P (neurokinin 1) receptor knock-out mice mimic antidepressant-induced desensitization. J. Neurosci. 21:8188-8197, 2001.
65.
Watson J.W.; Gonsalves S.F.; Fossa A.A. et al.; The Antiemetic Effects of Cp-99,994 in the Ferret and the Dog - Role of the Nk1 Receptor. British Journal of Pharmacology 115:84-94, 1995.
Table 1. Distribution volumes (DV) and binding potential (BP) estimates in striatum and occipital (primary visual) cortex with different models. * arterial input ** Indirect approach (unconstrained with derivation of distribution volumes from k-values); ***k1/k2 fixed to that of cerebellum. SRTM = Simplified reference tissue model with cerebellar input. Data acquisition 0-90 min for other methods but 0-240 min for the SRTM method. Means SD are shown; n=4.
Caudatus Putamen Occipital cortex Cerebellum
DV*
BP (kinetic, indirect)**
BP (kinetic, k1/k2 fixed)***
BP (SRTM)
23.0 ± 3.6 26.6 ± 6.6 16.5 ± 1.7 5.6 ± 0.6
3.08 ± 0.48 3.71 ± 1.00 1.93 ± 0.10 -
3.36 ± 0.49 4.23 ± 1.29 2.20 ± 0.16 -
4.90 ± 0.26 5.53 ± 0.32 2.44 ± 0.45 -
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Table 2. Neurokinin NK1 receptor binding potentials as measured with [18F]SPA-RQ in the human brain; simplified reference tissue model (means ± SD; n= 4)
Caudate (head) Putamen Substantia Nigra Ventral midbrain Thalamus
5.24 5.72 0.72 2.60 1.59
± 0.40 ± 0.33 ± 0.30 ± 0.60 ± 0.28
Occipital lobe Primary visual cortex Posterior cingulate cortex
2.44 ± 0.41 2.60 ± 0.54
Parietal lobe Gyrus supramarginalis Gyrus angularis
2.46 ± 0.45 2.40 ± 0.41
Temporal lobe Superior temporal gyrus Medial temporal gyrus Inferior temporal gyrus
2,47 ± 0.42 2.33 ± 0.33 2.16 ± 0.22
Amygdala Hippocampus/ Parahippocampal gyrus
2.60 ± 0.13 1.57 ± 0.21
Frontal lobe Medial frontal cortex Dorsolateral prefrontal cortex Anterior cingulate cortex
1.77 ± 0.40 2.36 ± 0.54 1.91 ± 0.62
32
Figure legends:
Figure 1. Fraction of unchanged [18F]SPA-RQ in arterial plasma of four healthy volunteers (mean ± SD; n= 4). Figure 2. Distribution of [18F]SPA-RQ uptake in the brain of a healthy male volunteer. Consecutive axial slices of an integrated image from 120-180 min is shown. Figure 3. [18F]SPA-RQ: time- radioactivity curves in the striatum, occipital cortex and cerebellum. Total uptake is depicted in the Fig. 3a and specific binding (total binding – cerebellum) in the Fig. 3b (n=6, mean curves). Figure 4. Kinetic modelling of [18F]SPA-RQ in the cerebellum using arterial plasma input. Two and three compartment fits and original data points are shown (healthy volunteers, n=4).
Figure 5. Estimation of regional binding potential with the simplified reference tissue model and [18F]SPA-RQ. Dependency of time for data acquisition (n=6, means ± SD are shown). Figure 6. Distribution of human NK1 receptors in vitro using whole hemisphere receptor autoradiography and [18F]SPA-RQ. Axial slices at the caudate-putamen (upper panel) and cerebellar level (lower panel). Total binding and binding after displacement with 1 µM GR203040 on consecutive slices are shown. High NK1 receptor binding is observed in the head of caudate and putamen and also cerebral cortex. Note that cerebellar binding is unaffected by a high concentration of a NK1 receptor antagonist. Figure 7. Distribution of [18F]SPA-RQ uptake in the midbrain-pons-medulla of a healthy male volunteer. Sagittal slice of an integrated image from 190-240 min is aligned with the structural MR-image from the same subject. There is moderate uptake in the superior and inferior colliculi of the midbrain as well as in the nuclei group in medulla (e.g. nucleus solitaris, n.
33 ambigus and other nuclei of vagus, trigeminal nucleus, inferior salivary nucleus and area postrema). In addition, there is some uptake in the pons representing tegmentum and possibly raphe nuclei. Due to limitations in resolution these locations are tentative. Note also high [18F]SPA-RQ binding in ventral striatum and cortex, moderate binding in thalamic nuclei and very low cerebellar uptake in this slice.
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