Quantitative imaging of 33P in plant materials using 14C polymer references.

Phosphorus (P) research still lacks techniques for rapid imaging of P use and allocation in different soil, sediment, and biological systems in a quantitative manner. In this study, we describe a time-saving and cost-efficient digital autoradiographic method for in situ quantitative imaging of 33P radioisotopes in plant materials. Our method combines autoradiography of the radiotracer applications with additions of commercially available 14C polymer references to obtain 33P activities in a quantitative manner up to 2000 Bq cm-2. Our data show that linear standard regressions for both radioisotopes are obtained, allowing the establishment of photostimulated luminescence equivalence between both radioisotopes with a factor of 9.73. Validating experiments revealed a good agreement between the calculated and applied 33P activity (R2 = 0.96). This finding was also valid for the co-exposure of 14C polymer references and 33P radioisotope specific activities in excised plant leaves for both maize (R2 = 0.99) and wheat (R2 = 0.99). The outlined autoradiographic quantification procedure retrieved 100% ± 12% of the 33P activity in the plant leaves, irrespective of plant tissue density. The simplicity of this methodology opens up new perspectives for fast quantitative imaging of 33P in biological systems and likely, thus, also for other environmental compartments.

Preclinical evaluation of [18 F]MA3: a CB2 receptor agonist radiotracer for PET.

BACKGROUND AND PURPOSE: Non-invasive in vivo imaging of cannabinoid CB2 receptors using PET is pursued to study neuroinflammation. The purpose of this study is to evaluate the in vivo binding specificity of [18 F]MA3, a CB2 receptor agonist, in a rat model with local overexpression of human (h) CB2 receptors. METHODS: [18 F]MA3 was produced with good radiochemical yield and radiochemical purity. The radiotracer was evaluated in rats with local overexpression of hCB2 receptors and in a healthy non-human primate using PET. KEY RESULTS: Ex vivo autoradiography demonstrated CB2 -specific binding of [18 F]MA3 in rat hCB2 receptor vector injected striatum. In a PET study, increased tracer binding in the hCB2 receptor vector-injected striatum compared to the contralateral control vector-injected striatum was observed. Binding in hCB2 receptor vector-injected striatum was blocked with a structurally non-related CB2 receptor inverse agonist, and a displacement study confirmed the reversibility of tracer binding. This study identified the utility of mutated inactive vector model for evaluation of CB2 receptor agonist PET tracers. [18 F]MA3 PET scans in the non-human primate showed good uptake and fast washout from brain, but no CB2 receptor-specific binding was observed. CONCLUSION AND IMPLICATIONS: Evaluation of [18 F]MA3 in a rat model with local overexpression of hCB2 receptors showed CB2 receptor-specific and reversible tracer binding. [18 F]MA3 showed good brain uptake and subsequent washout in a healthy non-human primate, but no specific binding was observed. Further clinical evaluation of [18 F]MA3 in patients with neuroinflammation is warranted. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Preclinical anaylses of [18F]cEFQ as a PET tracer for imaging metabotropic glutamate receptor type 1 (mGluR1).

Metabotropic glutamate receptor type 1 (mGluR1) is related with various neurological and psychiatric diseases, such as anxiety, depression, epilepsy, Parkinson's disease, and neuropathic pain. Hence, mGluR1 is an important target for drug development and imaging. We synthesized [18F]cEFQ (3-ethyl-2-[18F]fluoroquinolin-6-yl cis-(4-methoxycyclohexyl)methanone) as a PET tracer for selective mGluR1 imaging and evaluated its properties in rodents. A chloroquinoline precursor was labeled by a nucleophilic substitution reaction, and the resulting [18F]cEFQ was obtained with high radiochemical purity (>99%) and specific activity (63-246 GBq/µmol). The log D value was 3.24, and the initial brain uptake at 10 min was over 4% of injected dose per gram in BALB/c mice. According to PET/CT and autoradiography in SD rats, [18F]cEFQ showed wide distribution in the whole brain and the highest uptake in the cerebellum. Pre-treatment with unlabeled cEFQ or the mGluR1-specific antagonist JNJ16259685 blocked the uptake of [18F]cEFQ. However, the uptake was not blocked by pre-treatment with the mGluR5-specific antagonist ABP688. The trans isomer [18F]tEFQ did not show high uptake in the mGluR1-rich region. [18F]cEFQ was straightforwardly prepared using a chloro-derivative precursor. Its feasibility as a specific and selective PET agent for imaging mGluR1 was proved by in vitro and in vivo experiments using rodents.

Unique Distribution of Benzodiazepine Receptors in the Brain during the First Two Years of Life.

BACKGROUND: 123I-iomazenil (IMZ) single-photon emission computed tomography (SPECT) is a tool for evaluating epileptic foci and brain damage. To apply the method to children, information regarding the age-specific expression of benzodiazepine receptors (BDZ-Rs) is required. Unfortunately, there is no information currently available for children <2 years of age. METHODS: We used IMZ SPECT once in infants aged 3-8 months and again at 2 years of age in order to describe the maturational changes in BDZ-R distribution. RESULTS: No neurological deficits were found in any of the infants at the first examination. The BDZ-Rs were more dominantly distributed in the occipital lobe than in the frontal lobe before the age of 2 years. The frontal-occipital gradients of the distribution were obvious in children <8 months of age. Magnetic resonance imaging showed a spreading of myelination toward the frontal lobes simultaneously with BDZ-R expression. CONCLUSION: Information regarding the alteration in the BDZ-R distribution pattern is useful when assessing infantile epilepsy and brain injury. The age-related pattern of BDZ-R distribution could correspond with myelination, cerebral blood flow, metabolism and behavioral development.

Isolation of Catharanthus roseus (L.) G. Don Nuclei and Measurement of Rate of Tryptophan decarboxylase Gene Transcription Using Nuclear Run-On Transcription Assay.

BACKGROUND: An accurate assessment of transcription 'rate' is often desired to describe the promoter activity. In plants, isolation of transcriptionally active nuclei and their subsequent use in nuclear run-on assays has been challenging and therefore limit an accurate measurement of gene transcription 'rate'. Catharanthus roseus has emerged as a model medicinal plant as it exhibits an unsurpassed spectrum of chemodiversity, producing over 130 alkaloids through the terpenoid indole alkaloid (TIA) pathway and therefore serves as a 'molecular hub' to understand gene expression profiles. RESULTS: The protocols presented here streamline, adapt and optimize the existing methods of nuclear run-on assay for use in C. roseus. Here, we fully describe all the steps to isolate transcriptionally active nuclei from C. roseus leaves and utilize them to perform nuclear run-on transcription assay. Nuclei isolated by this method transcribed at a level consistent with their response to external stimuli, as transcription rate of TDC gene was found to be higher in response to external stimuli i.e. when seedlings were subjected to UV-B light or to methyl jasmonate (MeJA). However, the relative transcript abundance measured parallel through qRT-PCR was found to be inconsistent with the synthesis rate indicating that some post transcriptional events might have a role in transcript stability in response to stimuli. CONCLUSIONS: Our study provides an optimized, efficient and inexpensive method of isolation of intact nuclei and nuclear 'run-on' transcription assay to carry out in-situ measurement of gene transcription rate in Catharanthus roseus. This would be valuable in investigating the transcriptional and post transcriptional response of other TIA pathway genes in C. roseus. Isolated nuclei may also provide a resource that could be used for performing the chip assay as well as serve as the source of nuclear proteins for in-vitro EMSA studies. Moreover, nascent nuclear run-on transcript could be further subjected to RNA-Seq for global nuclear run-on assay (GNRO-Seq) for genome wide in-situ measurement of transcription rate of plant genes.

An alternative approach to histopathological validation of PET imaging for radiation therapy image-guidance: a proof of concept.

PURPOSE: In radiotherapy, PET images can be used to guide the delivery of selectively escalated doses to biologically relevant tumour subvolumes. Validation of PET for such applications requires demonstration of spatial coincidence between PET tracer uptake pattern and the histopathologically confirmed target. This study introduces a novel approach to histopathological validation of PET image segmentation for radiotherapy guidance. METHODS AND MATERIALS: Sequential tissue sections from surgically excised whole-tumour specimens were used to acquire full 3D-sets of both histopathological images (microscopy) and PET tracer distribution images (autoradiography). After these datasets were accurately registered, a full 3D autoradiographic distribution of PET tracer was reconstructed and used to obtain synthetic PET images (sPET) by simulating the image deterioration induced by processes involved in PET image formation. To illustrate the method, sPET images were used in this study to investigate spatial coincidence between high FDG uptake areas and the distribution of viable tissue in two small animal tumour models. RESULTS: The reconstructed 3D autoradiographic distribution of the PET tracer was spatially coherent, as indicated by the high average value of the normalised pixel-by-pixel correlation of intensities between successive slices (0.84 ± 0.05 and 0.94 ± 0.02). The loss of detail in the sPET images versus the 3D autoradiography was significant as indicated by Dice coefficient values corresponding to the two tumours (0 and 0.1 at 70% threshold). The maximum overlap between the FDG segmented volumes and the extent of the viable tissue as indicated by Dice coefficient values, was 0.8 for one tumour (for the image thresholded at 22% of max intensity) and 0.88 for the other (threshold of 14% of max intensity). CONCLUSION: It was demonstrated that the use of synthetic PET images for histopathological validation allows for bypassing a technically challenging and error-prone step of registering non-invasive PET images with histopathology.

Detection of remote neuronal reactions in the Thalamus and Hippocampus induced by rat glioma using the PET tracer cis-4-[¹8F]fluoro-D-proline.

After cerebral ischemia or trauma, secondary neurodegeneration may occur in brain regions remote from the lesion. Little is known about the capacity of cerebral gliomas to induce secondary neurodegeneration. A previous study showed that cis-4-[(18)F]fluoro-D-proline (D-cis-[(18)F]FPro) detects secondary reactions of thalamic nuclei after cortical infarction with high sensitivity. Here we investigated the potential of D-cis-[(18)F]FPro to detect neuronal reactions in remote brain areas in the F98 rat glioma model using ex vivo autoradiography. Although the tumor tissue of F98 gliomas showed no significant D-cis-[(18)F]FPro uptake, we observed prominent tracer uptake in 7 of 10 animals in the nuclei of the ipsilateral thalamus, which varied with the specific connectivity with the cortical areas affected by the tumor. In addition, strong D-cis-[(18)F]FPro accumulation was noted in the hippocampal area CA1 in two animals with ipsilateral F98 gliomas involving hippocampal subarea CA3 rostral to that area. Furthermore, focal D-cis-[(18)F]FPro uptake was present in the necrotic center of the tumors. Cis-4-[(18)F]fluoro-D-proline uptake was accompanied by microglial activation in the thalamus, in the hippocampus, and in the necrotic center of the tumors. The data suggest that brain tumors induce secondary neuronal reactions in remote brain areas, which may be detected by positron emission tomography (PET) using D-cis-[(18)F]FPro.

Adenoviral-mediated Cre expression effectively suppresses GlyT1 binding in the thalamic area of GlyT1 conditional knock-out mice.

To properly understand the function of genes of neurological interest, in vivo manipulation in the adult is essential, particularly when the target gene is involved in brain development. Moreover, since the physiological effects of target protein may be region-specific, targeting a distinct brain region could be required to dissect these effects in specific brain locations. Infection of somatic tissues of transgenic mice bearing loxP-flanked gene sequences with a viral vector expressing Cre recombinase provides a means of allowing flexible spatio-temporal control of target gene expression. Viral vector-mediated Cre expression could be used to mediate localized gene modulation in a specific brain region. In the present study this technology was applied to the glycine transporter type-1 (GlyT1) protein which is responsible for the uptake of synaptic glycine in the forebrain and has been implicated as a therapeutic target for the treatment of schizophrenia. Since GlyT1 is widely expressed in glial cells, we employed an adenoviral-based vector (Ad5) to deliver Cre protein, due to the preferentially transduction of glial cells by adenoviral vectors in rodent brain. We show significant reduced GlyT1 binding specifically in the thalamic area of conditional GlyT1 (GlyT1c) transgenic mice injected with Ad5-Cre virus, as measured by GlyT1 autoradiography. In conclusion, we demonstrated the validity of viral vector-mediated delivery of Cre to loxP targeted transgenic mice as a novel strategy to investigate target gene function in selected subregions of the adult brain, which provides a valuable technique to investigate gene function both in normal physiology and in disease models.

Isolation of antioxidants from Psoralea corylifolia fruits using high-speed counter-current chromatography guided by thin layer chromatography-antioxidant autographic assay.

A combinative method using high-speed counter-current chromatography (HSCCC) and thin layer chromatography (TLC) as an antioxidant autographic assay was developed to separate antioxidant components from the fruits of Psoralea corylifolia. Under the guidance of TLC bioautography, eight compounds including five flavonoids and three coumarins were successfully separated from the fruits of P. corylifolia by HSCCC with an optimized two-phase solvent system, n-hexane-ethyl acetate-methanol-water (1:1.1:1.3:1, v/v/v/v). The separation produced 5.91mg psoralen, 6.26mg isopsoralen, 3.19mg psoralidin, 0.92mg corylifol A, and 2.43mg bavachinin with corresponding purities of 99.5, 99.8, 99.4, 96.4, and 99.0%, as well as three sub-fractions, in a single run from 250mg ethyl acetate fraction of P. corylifolia extract. Following an additional clean-up step by preparative TLC, 0.4mg 8-prenyldaidzein (purity 91.7%), 4.18mg neobavaisoflavone (purity 97.4%) and 4.36mg isobavachalcone (purity 96.8%) were separated from the three individual sub-fractions. The structures of the isolated compounds were identified by (1)H NMR and (13)C NMR. The results of antioxidant activity estimation by electron spin resonance (ESR) method showed that psoralidin was the most active antioxidant with an IC50 value of 44.7microM. This is the first report on simultaneous separation of eight compounds from P. corylifolia by HSCCC.

In vivo target recognition with high-resolution imaging: significance for drug development.

In vivo target identification is basic for understanding mechanisms of drug action. Target identification requires cellular resolution. Extrapolation from blood bioavailability, low-resolution scans, radioassays, or in vitro tests regularly produce false-negatives and false-positives. Common ADME procedures disrealize organ complexities. While low-specificity high-capacity sites of deposition are easily recognized, high-specificity low-capacity receptor sites remain hidden. Serious limitations of target recognition are revealed in comparative studies with three methods: high-resolution microscopic autoradiography, radioassay, and whole-body autoradiography. With radioassays and whole-body autoradiography, many targets are simply undetectable. For example, high-resolution microscopic target information for vitamin D, gained 20-30 years ago, was widely ignored. The narrow calcium focus for this multi-target and multi-function hormone was perpetuated until recently through deficient results from conventional assays together with related expert bias. Thus, follow-up has been delayed on discoveries from the use of unconventional histopharmacology methods, pointing at important actions and therapies beyond systemic calcium regulation. High-resolution 'in vivo' target identification with associated functional characterization is useful not only for understanding mechanisms of action, but also for providing leads for innovative and successful drug development and prediction.

Psychotropic drug-induced locomotor hyperactivity and prepulse inhibition regulation in male and female aromatase knockout (ArKO) mice: role of dopamine D1 and D2 receptors and dopamine transporters.

RATIONALE AND OBJECTIVES: The aim of the present study was to investigate the possible role of oestrogen in schizophrenia by comparing aromatase knockout (ArKO) mice, which are unable to produce oestrogen, with wild-type controls using two behavioural animal models with relevance to the illness, psychotropic drug-induced locomotor hyperactivity and prepulse inhibition (PPI). RESULTS: Baseline PPI was not different between ArKO and controls. Treatment with apomorphine, MK-801 and amphetamine caused disruption of PPI in all groups. However, in female but not male ArKO mice, the effect of both apomorphine and amphetamine was reduced. In female ArKO mice, amphetamine-induced hyperlocomotion was markedly reduced, but in male mice, the genotype difference was far smaller. Female but not male ArKO mice also showed a reduction of phencyclidine-induced locomotor hyperactivity. The density of dopamine transporters, but not D1 and D2 receptors, was significantly increased in the caudate putamen of male but not female ArKO mice compared to wild-type mice. This could represent a compensatory dopaminergic upregulation in male ArKO mice. CONCLUSION: Because of their lack of oestrogen production, it was anticipated that ArKO mice would display enhanced effects of amphetamine on locomotor activity and PPI. Instead, in these animals, aromatase knockout appeared to be 'protective'. This may represent limitations in the ability to model a complex illness such as schizophrenia in a constitutive knockout model, such as ArKO mice. Moreover, the current results may point at the involvement of other sex steroids, which are also altered in ArKO mice, in dopaminergic control of behaviour.

The brain selenoproteome: priorities in the hierarchy and different levels of selenium homeostasis in the brain of selenium-deficient rats.

The application of radionuclides for the localization of essential trace elements in vivo and the characterization of their binding proteins is a story of intermittently made improvements of the techniques used for their detection. In this study we present the use of neutron activation analysis and different autoradiographic imaging methods including real-time digital autoradiography to reveal new insights in the hierarchy of selenium homeostasis. Selenoproteins containing the essential trace element selenium play important roles in the CNS. Although the CNS does not show the highest selenium concentration in the case of selenium-sufficient supply in comparison with other organs, it shows a high priority for selenium uptake and retention in the case of dietary selenium deficiency. To characterize the hierarchy of selenium supply in the brain, in vivo radiotracer labeling with (75)Se in rats with different selenium status was combined with autoradiographic detection of (75)Se in brain tissue sections and (75)Se-labeled selenoproteins after protein separation by two-dimensional gel electrophoresis. This study demonstrates significant differences in the uptake of (75)Se into the brain of rats with different selenium status. A brain region-specific uptake pattern of the radiotracer (75)Se in selenium-deficient rats could be revealed and the CSF was identified as a key part of the brain selenium homeostasis.

Increased novelty-induced motor activity and reduced depression-like behavior in neuropeptide Y (NPY)-Y4 receptor knockout mice.

There is growing evidence that neuropeptide Y (NPY) acting through Y1 and Y2 receptors has a prominent role in modulating anxiety- and depression-like behavior in rodents. However, a role of other Y-receptors like that of Y4 receptors in this process is poorly understood. We now investigated male Y2, Y4 single and Y2/Y4 double knockout mice in behavioral paradigms for changes in motor activity, anxiety and depression-like behavior. Motor activity was increased in Y2, Y4 and Y2/Y4 knockout mice under changing and stressful conditions, but not altered in a familiar environment. Y4 and Y2 knockout mice revealed an anxiolytic phenotype in the light/dark test, marble burying test and in stress-induced hyperthermia, and reduced depression-like behavior in the forced swim and tail suspension tests. In Y2/Y4 double knockout mice, the response in the light/dark test and in the forced swim test was further enhanced compared with Y4 and Y2 knockout mice, respectively. High levels of Y4 binding sites were observed in brain stem nuclei including nucleus of solitary tract and area postrema. Lower levels were found in the medial amygdala and hypothalamus. Peripheral administration of pancreatic polypeptide (PP) induced Y4 receptor-dependent c-Fos expression in brain stem, hypothalamus and amygdala. PP released peripherally from the pancreas in response to food intake, may act not only as a satiety signal but also modulate anxiety-related locomotion.

Sizing particles of natural uranium and nuclear fuels using poly-allyl-diglycol carbonate autoradiography.

Theoretical and experimental methods were developed to assess the size distribution of alpha-emitting particles captured on air-sampler filters. The particle size of oxides of low enriched, depleted and natural uranium and also aged plutonium in mixed oxide reactor fuels of known composition was determined using poly-allyl-diglycol carbonate (PADC) autoradiography, the commercial product TASTRAK((R)), solid-state nuclear track detectors. The exposed PADC was chemically etched to reveal clusters of tracks, radially dispersing from central points. A theoretical model was developed which converted the number of tracks in a track cluster to the hot particle diameter. The diameters of 26 particles of natural uranium oxide were measured (4-130 microm) using an optical microscope. There was a good agreement between these particle size measurements and a theoretical assessment based on the track cluster count.

Muscarinic receptors in the thalamus in progressive supranuclear palsy and other neurodegenerative disorders.

Progressive supranuclear palsy (PSP) is a neurodegenerative disease with motor, cognitive, and behavioral symptomatology. Cholinergic dysfunction is thought to underpin several key symptoms. There is known pathologic involvement of the corticobasal ganglia-thalamocortical loops in PSP, but little attention has been focused on potential thalamic dysfunction. Using autoradiography, we measured muscarinic M2 and M4 receptors in specific thalamic nuclei involved in the limbic and motor loops in patients with PSP (n = 11) and compared results from brain tissue of subjects with Lewy body dementias (including dementia with Lewy bodies and Parkinson disease with dementia, n = 31), Alzheimer disease (n = 22) and normal elderly control subjects (n = 27). In the thalamus M2 receptors were more abundant than M4 receptors and were most densely concentrated in the anteroprincipal (AP) and mediodorsal (MD) nuclei, which connect to limbic cortices. M2 receptor binding was reduced in the AP nucleus in PSP compared with control subjects and those with Lewy body dementias. M4 receptors were markedly reduced in the MD nucleus in those with PSP compared with control subjects. M4 receptors were also reduced in the subthalamic nucleus in patients with PSP. M4 receptor binding was reduced in the MD nucleus in the Lewy body dementia and Alzheimer disease groups compared with control subjects. There were no significant changes in the ventrolateral nucleus (motor). Cholinergic dysfunction within the AP and MD nuclei of the thalamus may contribute to behavioral and cognitive disturbances associated with PSP.

Biological characterization of rodent and human vasopressin V1b receptors using SSR-149415, a nonpeptide V1b receptor ligand.

[(3)H]SSR-149415 is the first tritiated nonpeptide vasopressin V(1b) receptor (V(1b)R) antagonist ligand. It was used for studying rodent (mouse, rat, hamster) and human V(1b)R from native or recombinant origin. Moreover, a close comparison between the human and the mouse V(1b)R was performed using SSR-149415/[(3)H]SSR-149415 in binding and functional studies in vitro. [(3)H]SSR-149415 binding was time-dependent, reversible, and saturable. Scatchard plot analysis gave a single class of high-affinity binding sites with apparent equilibrium dissociation constant (K(d)) approximately 1 nM and maximum binding density (B(max)) values from 7,000 to 300,000 sites/cell according to the cell line. In competition experiments, [(3)H]SSR-149415 binding was stereospecific and dose-dependently displaced by reference peptide and nonpeptide arginine vasopressin (AVP)/OT ligands following a V(1b) rank order of affinity: SSR-149415 = AVP > dCha > dPen > dPal > dDavp > SSR-126768A > SR-49059 > SSR-149424 > OT > SR-121463B. Species differences between human, rat, mouse, and hamster V(1b)R were observed. Autoradiography studies with [(3)H]SSR-149415 on rat and human pituitary showed intense specific labeling confined to corticotroph cells and absence of labeling in the other tissues examined. SSR-149415 potently and stereospecifically antagonized the AVP-induced inositol phosphate production and intracellular Ca(2+) increase (EC(50) from 1.83 to 3.05 nM) in recombinant cell lines expressing either the mouse or the human V(1b)R. AVP (10(-7) M) exposure of AtT20 cells expressing mouse or human EGFP-tagged V(1b)R induced their rapid internalization. Preincubation with 10(-6) M SSR-149415 counteracted the internalization process. Moreover, recycling of internalized receptors was observed upon 10(-6) M SSR-149415 treatment. Thus SSR-149415/[(3)H]SSR-149415 are unique tools for studying animal and human V(1b)R.

Quantitative evaluation of 11C-ABP688 as PET ligand for the measurement of the metabotropic glutamate receptor subtype 5 using autoradiographic studies and a beta-scintillator.

In this study we assessed the new glutamatergic ligand (11)C-ABP688 with regard to the following characteristics: (A) brain distribution, (B) first pass extraction fraction, (C) suitable model to describe tracer kinetics and (D) specificity for the mGlu5 receptor. These parameters were assessed using autoradiography and a beta-scintillator positioned in the striatum. The study included 13 male rats. In 2 animals cerebral blood flow was measured using H(2)(15)O. The (11)C-ABP688 data were analyzed using compartmental modeling. A two-tissue compartment model turned out to fit the data more adequately (parameters: K(1), k(2)('), k(3)('), k(4), total distribution volume DV(tot)=K(1)/k(2)(') (1+k(3)(')/k(4)) than a one-tissue compartment model. The autoradiographic studies revealed high uptake in hippocampus, striatum and cortex and low accumulation in thalamus and cerebellum. The uptake was markedly reduced following blockade with the mGlu5 antagonist M-MPEP. The first pass extraction fraction exceeded 85%. Baseline DV(tot) was 15.16+/-2.67 ml plasma/ml tissue and decreased by 56, 67 and 72% following blockade with 1, 2 and 6 mg/kg M-MPEP, respectively. These results show that (11)C-ABP688 is a promising PET ligand for the quantification of mGlu5 receptors in humans and animals. It readily crosses the blood-brain barrier and binds with high specificity to the mGlu5 receptor. The study furthermore demonstrates the usefulness of a beta-scintillator, if necessary in connection with autoradiography, to evaluate new receptor tracers.

Angiotensin II AT1 receptor blockade prevents the hypothalamic corticotropin-releasing factor response to isolation stress.

Sustained pretreatment with angiotensin II AT(1) receptor antagonists prevents the sympathoadrenal and hormonal responses to 24 h isolation stress. To elucidate the mechanism of the anti-stress effects of AT(1) receptor antagonism, we examined the effect of subcutaneous infusion of candesartan, a non-competitive AT(1) receptor antagonist, 0.5 mg/kg/day for 14 days, to Wistar rats on the hypothalamic pituitary adrenal (HPA) axis after 24 h isolation stress. In the morning of day 15, we measured AT(1) receptors corticotropin-releasing factor (CRF) mRNA and immunoreactive CRF in the paraventricular nucleus (PVN), the pituitary adrenocorticotropin hormone (ACTH) and adrenal corticosterone content, and the urinary corticosterone excretion. In rats not treated with candesartan, 24 h isolation stress increased pituitary ACTH, adrenal corticosterone content and AT(1) receptor binding in the PVN but decreased CRF mRNA and CRF content in the PVN. This indicates enhanced CRF utilization not compensated by CRF gene transcription and effective glucocorticoid feedback inhibition in spite of the increase in AT(1) receptor expression. The effects of stress on HPA axis activation and CRF mRNA and content in the PVN were prevented by candesartan pretreatment, suggesting that activation of AT(1) receptors is required for the HPA axis response to isolation. Our results support the hypothesis that the activity of PVN AT(1) receptors is part of the mechanism necessary for development of a full stress-induced HPA axis activation. Inhibition of central AT(1) receptors limits the CRF response to stress and should be considered as a therapeutic tool to preserve homeostasis under chronic stress conditions.

Molecular imaging of vascular cell adhesion molecule-1 expression in experimental atherosclerotic plaques with radiolabelled B2702-p.

PURPOSE: VCAM-1 plays a major role in the chronic inflammatory processes present in vulnerable atherosclerotic plaques. The residues 75-84 (B2702-p) and 84-75/75-84 (B2702-rp) of the major histocompatibility complex-1 (MHC-1) molecule B2702 were previously shown to bind specifically to VCAM-1. We hypothesised that radiolabelled B2702-p and B2702-rp might have potential for the molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) expression in atherosclerotic plaques. METHODS: Preliminary biodistribution studies indicated that 125I-B2702-rp was unsuitable for in vivo imaging owing to extremely high lung uptake. 123I- or 99mTc-labelled B2702-p was injected intravenously to Watanabe heritable hyperlipidaemic rabbits (WHHL, n=6) and control animals (n=6). After 180 min, aortas were harvested for ex vivo autoradiographic imaging, gamma-well counting, VCAM-1 immunohistology and Sudan IV lipid staining. RESULTS: Robust VCAM-1 immunostaining was observed in Sudan IV-positive and to a lesser extent in Sudan IV-negative areas of WHHL animals, whereas no expression was detected in control animals. Significant 2.9-fold and 1.9-fold increases in 123I-B2702-p and 99mTc-B2702-p aortic-to-blood ratios, respectively, were observed between WHHL and control animals (p<0.05). Tracer uptake on ex vivo images co-localised with atherosclerotic plaques. Image quantification indicated a graded increase in 123I-B2702-p and 99mTc-B2702-p activities from control to Sudan IV-negative and to Sudan IV-positive areas, consistent with the observed pattern of VCAM-1 expression. Sudan IV-positive to control area tracer activity ratios were 17.0+/-9.0 and 5.9+/-1.8 for 123I-B2702-p and 99mTc-B2702-p, respectively. CONCLUSION: Radiolabelled B2702-p is a potentially useful radiotracer for the molecular imaging of VCAM-1 in atherosclerosis.

Region specific galanin receptor/neuropeptide Y Y1 receptor interactions in the tel- and diencephalon of the rat. Relevance for food consumption.

The aim of this work was to determine the interactions between NPY and GAL receptor (GALR) subtypes in the hypothalamus and the amygdala using quantitative receptor autoradiography to analyze the binding characteristics of NPY-Y1 and Y2 receptor subtypes in the presence and absence of GAL. Food intake in satiated animals was evaluated after intraventricular co-injections of GAL and NPY-Y1 or Y2 agonists. The expression of c-Fos IR in both regions was also investigated. GAL decreases NPY-Y1 agonist binding in the arcuate nucleus by about 15% (p<0.01), but increases NPY-Y1 agonist binding in amygdala (18%) (p<0.01). These effects were blocked with the GAL antagonist M35. Y2-agonist binding was not modified by GAL. GAL blocked the food intake induced by the Y1 agonist (p<0.01). Co-injections of Y1 agonist and GAL also reduced the c-Fos expression induced by the Y1 agonist in the arcuate nucleus and the dorsomedial hypothalamic nucleus but increased c-Fos expression in amygdala. These results indicate the existence of antagonistic interactions between GALR and NPY-Y1 receptors in the hypothalamus and their functional relevance for food intake. In contrast, a facilitatory interaction between GALR and Y1 receptors exists in the amygdala which may be of relevance for fear related behaviour.