Anders Ettrup

Blood BDNF concentrations reflect brain-tissue BDNF levels across species.

Brain-derived neurotrophic factor (BDNF) is involved in synaptic plasticity, neuronal differentiation and survival of neurons. Observations of decreased serum BDNF levels in patients with neuropsychiatric disorders have highlighted the potential of BDNF as a biomarker, but so far there have been no studies directly comparing blood BDNF levels to brain BDNF levels in different species. We examined blood, serum, plasma and brain-tissue BDNF levels in three different mammalian species: rat, pig, and mouse, using an ELISA method. As a control, we included an analysis of blood and brain tissue from conditional BDNF knockout mice and their wild-type littermates. Whereas BDNF could readily be measured in rat blood, plasma and brain tissue, it was undetectable in mouse blood. In pigs, whole-blood levels of BDNF could not be measured with a commercially available ELISA kit, but pig plasma BDNF levels (mean 994±186 pg/ml) were comparable to previously reported values in humans. We demonstrated positive correlations between whole-blood BDNF levels and hippocampal BDNF levels in rats (r2=0.44, p=0.025) and between plasma BDNF and hippocampal BDNF in pigs (r2=0.41, p=0.025). Moreover, we found a significant positive correlation between frontal cortex and hippocampal BDNF levels in mice (r2=0.81, p=0.0139). Our data support the view that measures of blood and plasma BDNF levels reflect brain-tissue BDNF levels.

11C-NS14492 as a Novel PET Radioligand for Imaging Cerebral α7 Nicotinic Acetylcholine Receptors: In Vivo Evaluation and Drug Occupancy Measurements

Small-molecule α7 nicotinic acetylcholine receptor (α7nAChR) agonists are currently validated for use as treatment for cognitive disturbances in schizophrenia and in Alzheimer disease. A suitable radiolabeled α7nAChR PET tracer would be important for in vivo quantification of α7nAChR binding in humans and to measure α7nAChR occupancy of α7nAChR drug candidates. Here, we present the radiosynthesis and in vivo evaluation of 11C-NS14492 as a selective α7nAChR PET radioligand. Methods: The high-affinity α7nAChR-selective partial agonist NS14492 was radiolabeled by methylation of its desmethyl precursor using 11C-methyl triflate. Female Danish Landrace pigs were studied at baseline and after intravenous administration of blocking doses of either the α7nAChR partial agonist SSR180711 or the unlabeled NS14492. 11C-NS14492 was given as an intravenous bolus injection, and the pigs were scanned for 90 min both at baseline and in the blocked conditions. Arterial blood was collected during the scanning, plasma was counted, and parent compound fraction was determined with radio–high-performance liquid chromatography. PET data were quantified with a graphical analysis with arterial input; 11C-NS14492 regional distribution volumes were calculated, and α7nAChR occupancy was determined using an occupancy plot. Results: 11C-NS14492 had a high uptake in the pig brain, with the highest binding in the cerebral cortex and thalamus in accordance with α7nAChR distribution. Pretreatment with NS14492 and SSR180711 consistently decreased distribution volumes of 11C-NS14492 in all examined regions, in a dose-dependent manner, supporting the finding that the radioligand binds selectively to α7nAChR in vivo. Conclusion: We report here that 11C-NS14492 is the first, to our knowledge, PET radioligand for α7nAChR showing a dose-dependent decline in cerebral binding after receptor blockade. This compound is considered a promising PET tracer for in vivo measurements of α7nAChR binding in the human brain.

Serotonin 2A receptor agonist binding in the human brain with [11C]Cimbi-36

[ 11 C]Cimbi-36 was recently developed as a selective serotonin 2A (5-HT2A) receptor agonist radioligand for positron emission tomography (PET) brain imaging. Such an agonist PET radioligand may provide a novel, and more functional, measure of the serotonergic system and agonist binding is more likely than antagonist binding to reflect 5-HT levels in vivo. Here, we show data from a first-in-human clinical trial with [ 11 C]Cimbi-36. In 29 healthy volunteers, we found high brain uptake and distribution according to 5-HT2A receptors with [ 11 C]Cimbi-36 PET. The two-tissue compartment model using arterial input measurements provided the most optimal quantification of cerebral [ 11 C]Cimbi-36 binding. Reference tissue modeling was feasible as it induced a negative but predictable bias in [ 11 C]Cimbi-36 PET outcome measures. In five subjects, pretreatment with the 5-HT2A receptor antagonist ketanserin before a second PET scan significantly decreased [ 11 C]Cimbi-36 binding in all cortical regions with no effects in cerebellum. These results confirm that [ 11 C]Cimbi-36 binding is selective for 5-HT2A receptors in the cerebral cortex and that cerebellum is an appropriate reference tissue for quantification of 5-HT2A receptors in the human brain. Thus, we here describe [ 11 C]Cimbi-36 as the first agonist PET radioligand to successfully image and quantify 5-HT2A receptors in the human brain.

Radiosynthesis and Evaluation of 11C-CIMBI-5 as a 5-HT2A Receptor Agonist Radioligand for PET

PET brain imaging of the serotonin 2A (5-hydroxytryptamine 2A, or 5-HT2A) receptor has been widely used in clinical studies, and currently, several well-validated radiolabeled antagonist tracers are used for in vivo imaging of the cerebral 5-HT2A receptor. Access to 5-HT2A receptor agonist PET tracers would, however, enable imaging of the active, high-affinity state of receptors, which may provide a more meaningful assessment of membrane-bound receptors. In this study, we radiolabel the high-affinity 5-HT2A receptor agonist 2-(4-iodo-2,5-dimethoxyphenyl)-N-(2-[11C-OCH3]methoxybenzyl)ethanamine (11C-CIMBI-5) and investigate its potential as a PET tracer. Methods: The in vitro binding and activation at 5-HT2A receptors by CIMBI-5 was measured with binding and phosphoinositide hydrolysis assays. Ex vivo brain distribution of 11C-CIMBI-5 was investigated in rats, and PET with 11C-CIMBI-5 was conducted in pigs. Results: In vitro assays showed that CIMBI-5 was a high-affinity agonist at the 5-HT2A receptor. After intravenous injections of 11C-CIMBI-5, ex vivo rat studies showed a specific binding ratio of 0.77 ± 0.07 in the frontal cortex, which was reduced to cerebellar levels after ketanserin treatment, thus indicating that 11C-CIMBI-5 binds selectively to the 5-HT2A receptor in the rat brain. The PET studies showed that the binding pattern of 11C-CIMBI-5 in the pig brain was in accordance with the expected 5-HT2A receptor distribution. 11C-CIMBI-5 gave rise to a cortical binding potential of 0.46 ± 0.12, and the target-to-background ratio was similar to that of the widely used 5-HT2A receptor antagonist PET tracer 18F-altanserin. Ketanserin treatment reduced the cortical binding potentials to cerebellar levels, indicating that in vivo 11C-CIMBI-5 binds selectively to the 5-HT2A receptor in the pig brain. Conclusion: 11C-CIMBI-5 showed a cortex-to-cerebellum binding ratio equal to the widely used 5-HT2A antagonist PET tracer 18F-altanserin, indicating that 11C-CIMBI-5 has a sufficient target-to-background ratio for future clinical use and is displaceable by ketanserin in both rats and pigs. Thus, 11C-CIMBI-5 is a promising tool for investigation of 5-HT2A agonist binding in the living human brain.

Characterization of [11C]Cimbi-36 as an agonist PET radioligand for the 5-HT2A and 5-HT2C receptors in the nonhuman primate brain

Only recently the first successful serotonin 2A (5-HT2A) receptor agonist PET radioligands have been described, with [(11)C]Cimbi-36 reported as the most promising in the pig brain so far. Agonist radioligands may target specifically the G protein-coupled state of the receptors and thereby provide a more meaningful assessment of available receptors than antagonist radioligands. In the current study we characterized [(11)C]Cimbi-36 receptor binding in the primate brain. On five experimental days, a total of 14 PET measurements were conducted in three female rhesus monkeys. On each day, PET measurements were conducted after intravenous injection of [(11)C]Cimbi-36 during baseline conditions and after intravenous infusion of the 5-HT2 receptor antagonist ketanserin (n=3) or the 5-HT2C receptor antagonist SB 242084 (n=2). On four of the experimental days an additional baseline PET measurement was conducted after injection of [(11)C]MDL 100907. All PET measurements were performed for 2h in a HRRT PET system and arterial blood was obtained for measurement of the [(11)C]Cimbi-36 input function. Quantification of [(11)C]Cimbi-36 receptor binding was performed using kinetic and graphical analysis. After injection of [(11)C]Cimbi-36 the regional distribution of radioactivity in brain was in accordance with the known 5-HT2 receptor distribution. The two-tissue compartment model was superior for the description of the time-radioactivity curves of all examined brain regions. BPND values obtained with reference tissue models correlated with corresponding values obtained with kinetic modeling. Administration of ketanserin decreased the binding in all brain regions but did not affect the cerebellar distribution volume. The BPND of [(11)C]Cimbi-36 was 56±8% of [(11)C]MDL 100907 across cortical regions, but higher in other brain regions including choroid plexus. After administration of SB 242084, [(11)C]Cimbi-36 binding was nearly completely inhibited in the choroid plexus, partly reduced in several subcortical regions (e.g. hippocampus), but not affected in the cortical regions. In conclusion, the receptor binding of [(11)C]Cimbi-36 can be quantified using kinetic modeling and the cerebellum was found to be a suitable reference region. The difference between [(11)C]Cimbi-36 and [(11)C]MDL 100907 binding in the choroid plexus is related to 5-HT2C receptor binding of [(11)C]Cimbi-36. [(11)C]Cimbi-36 is the first agonist radioligand suitable for examination of 5-HT2A receptors in the cortical regions and of 5-HT2C receptors in the choroid plexus of the primate brain.

No change in [11C]CUMI-101 binding to 5-HT1A receptors after intravenous citalopram in human

The main objective of this study was to determine the sensitivity of [11C]CUMI‐101 to citalopram challenge aiming at increasing extracellular 5‐HT. CUMI‐101 has agonistic properties in human embryonic kidney 293 cells transfected with human recombinant 5‐HT1A receptors (Hendry et al. [2011] Nucl Med Biol 38:273–277; Kumar et al. [2006] J Med Chem 49:125–134) and has previously been demonstrated to be sensitive to bolus citalopram in monkeys (Milak et al. [2011] J Cereb Blood Flow Metab 31:243–249). We studied six healthy individuals. Two PET‐scans were performed on the same day in each individual before and after constant infusion of citalopram (0.15 mg/kg). The imaging data were analyzed using two tissue compartment kinetic modeling with metabolite corrected arterial input and Simplified Reference Tissue Modeling using cerebellum as a reference region. There was no significant difference in regional distribution volume or non‐displaceable binding potential values before and after citalopram infusion. The mean receptor occupancy was 0.03 (range −0.14 to 0.17). Our data imply that [11C]CUMI‐101 binding is not sensitive to citalopram infusion in humans. Synapse, 2012.

cerebral Markers of the Serotonergic System in Rat Models of Obesity and After Roux-en-Y Gastric Bypass

Food intake and body weight are regulated by a complex system of neural and hormonal signals, of which the anorexigenic neurotransmitter serotonin (5‐hydroxytryptamine or 5‐HT) is central. In this study, rat models of obesity and weight loss intervention were compared with regard to several 5‐HT markers. Using receptor autoradiography, brain regional‐densities of the serotonin transporter (SERT) and the 5‐HT2A and 5‐HT4 receptors were measured in (i) selectively bred polygenic diet‐induced obese (pgDIO) rats, (ii) outbred DIO rats, and (iii) Roux‐en‐Y gastric bypass (RYGB)–operated rats. pgDIO rats had higher 5‐HT4 and 5‐HT2A receptor binding and lower SERT binding when compared to polygenic diet‐resistant (pgDR) rats. The most pronounced difference between pgDIO and pgDR rats was observed in the nucleus accumbens shell (NAcS), a brain region regulating reward aspects of feeding. No differences were found in the 5‐HT markers between DIO rats, chow‐fed control rats, and DIO rats experiencing a weight loss. The 5‐HT markers were also similar in RYGB and sham‐operated rats except for a downregulation of 5‐HT2A receptors in the NAcS. The higher receptor and lower SERT binding in pgDIO as compared to pgDR rats corresponds to what is reported in overweight humans and suggests that the dysfunctions of the 5‐HT system associated with overeating or propensity to become overweight are polygenically determined. Our results support that the obesity‐prone rat model has high translational value and suggests that susceptibility to develop obesity is associated with changed 5‐HT tone in the brain that may also regulate hedonic aspects of feeding.

Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain.

Viral vector-mediated gene transfer has emerged as a powerful means to target transgene expression in the central nervous system. Here we characterized the efficacy of serotypes 1 and 5 recombinant adeno-associated virus (rAAV) vectors encoding green fluorescent protein (GFP) after stereotaxic delivery to the neonatal rat and minipig striatum. The efficiency of GFP expression and the phenotype of GFP-positive cells were assessed within the forebrain at different time points up to 12 months after surgery. Both rAAV1-GFP and rAAV5-GFP delivery resulted in transduction of the striatum as well as striatal input and output areas, including large parts of the cortex. In both species, rAAV5 resulted in a more widespread transgene expression compared to rAAV1. In neonatal rats, rAAV5 also transduced several other areas such as the olfactory bulbs, hippocampus, and septum. Phenotypic analysis of the GFP-positive cells, performed using immunohistochemistry and confocal microscopy, showed that most of the GFP-positive cells by either serotype were NeuN-positive neuronal profiles. The rAAV5 vector further displayed the ability to transduce non-neuronal cell types in both rats and pigs, albeit at a low frequency. Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity to study effects of genetic manipulation in this non-primate large animal species. Finally, we generated an atlas of the Göttingen minipig brain for guiding future studies in this large animal species.

An approach for serotonin depletion in pigs: Effects on serotonin receptor binding

Depletion of central serotonin (5‐HT) levels and dysfunction in serotonergic transmission are implicated in a variety of human CNS disorders. The mechanisms behind these serotonergic deficits have been widely studied using rodent models, but only to a limited extent in larger animal models. The pig is increasingly used as an experimental animal model especially in neuroscience research. Here, we present an approach for serotonin depletion in the pig brain. Central serotonin depletion in Danish Landrace pigs was achieved following 4 days treatment with para‐chlorophenylalanine (pCPA). On day 5, tissue concentrations of 5‐HT in seven distinct brain structures from one hemisphere: frontal and occipital cortex, striatum, hippocampus, cerebellum, rostral, and caudal brain stem, were determined. The other hemisphere was processed for receptor autoradiography. Treatments with 50 mg/kg and 100 mg/kg pCPA caused average decreases in 5‐HT concentrations of 61% ± 14% and 66% ± 16%, respectively, and a substantial loss of 5‐HT immunostaining was seen throughout the brain. The serotonin depletion significantly increased 5‐HT4 receptor binding in nucleus accumbens, but did not alter 5‐HT1A and 5‐HT2A receptor or serotonin transporter binding in any brain region. In conclusion, 4 days treatment with pCPA effectively reduces 5‐HT levels in the pig brain. Further, whereas several 5‐HT markers did not change after the pCPA treatment, 5‐HT4 receptors were consistently upregulated, indicating a greater susceptibility of this receptor to altered 5‐HT levels. This porcine model of serotonin depletion will be useful in future studies of cerebral serotonergic dysfunction. Synapse 2011.

The Center for Integrated Molecular Brain Imaging (Cimbi) database

We here describe a multimodality neuroimaging containing data from healthy volunteers and patients, acquired within the Lundbeck Foundation Center for Integrated Molecular Brain Imaging (Cimbi) in Copenhagen, Denmark. The data is of particular relevance for neurobiological research questions related to the serotonergic transmitter system with its normative data on the serotonergic subtype receptors 5-HT1A, 5-HT1B, 5-HT2A, and 5-HT4 and the 5-HT transporter (5-HTT), but can easily serve other purposes. The Cimbi database and Cimbi biobank were formally established in 2008 with the purpose to store the wealth of Cimbi-acquired data in a highly structured and standardized manner in accordance with the regulations issued by the Danish Data Protection Agency as well as to provide a quality-controlled resource for future hypothesis-generating and hypothesis-driven studies. The Cimbi database currently comprises a total of 1100 PET and 1000 structural and functional MRI scans and it holds a multitude of additional data, such as genetic and biochemical data, and scores from 17 self-reported questionnaires and from 11 neuropsychological paper/computer tests. The database associated Cimbi biobank currently contains blood and in some instances saliva samples from about 500 healthy volunteers and 300 patients with e.g., major depression, dementia, substance abuse, obesity, and impulsive aggression. Data continue to be added to the Cimbi database and biobank.