Search

Background: Myocardial infarction (MI) evokes an organized remodeling process characterized by the activation and transdifferentiation of quiescent cardiac fibroblasts to generate a stable collagen rich scar. Early fibroblast activation may be amenable to targeted therapy, but is challenging to identify in vivo. We aimed to non-invasively image active fibrosis by targeting the fibroblast activation protein (FAP) expressed by activated (myo)fibroblasts, using a novel positron emission tomography (PET) radioligand [68Ga]MHLL1 after acute MI. Methods: One-step chemical synthesis and manual as well as module-based radiolabeling yielded [68Ga]MHLL1. Binding characteristics were evaluated in murine and human FAP-transfected cells, and stability tested in human serum. Biodistribution in healthy animals was interrogated by dynamic PET imaging, and metabolites were measured in blood and urine. The temporal pattern of FAP expression was determined by serial PET imaging at 7 d and 21 d after coronary artery ligation in mice as percent injected dose per gram (%ID/g). PET measurements were validated by ex vivo autoradiography and immunostaining for FAP and inflammatory macrophages. Results: [68Ga]MHLL1 displayed specific uptake in murine and human FAP-positive cells (p = 0.0208). In healthy mice the tracer exhibited favorable imaging characteristics, with low blood pool retention and dominantly renal clearance. At 7 d after coronary artery ligation, [68Ga]MHLL1 uptake was elevated in the infarct relative to the non-infarcted remote myocardium (1.3 ± 0.3 vs. 1.0 ± 0.2 %ID/g, p < 0.001) which persisted to 21 d after MI (1.3 ± 0.4 vs. 1.1 ± 0.4 %ID/g, p = 0.013). Excess unlabeled compound blocked tracer accumulation in both infarct and non-infarct remote myocardium regions (p < 0.001). Autoradiography and histology confirmed the regional uptake of [68Ga]MHLL1 in the infarct and especially border zone regions, as identified by Masson trichrome collagen staining. Immunostaining further delineated persistent FAP expression at 7 d and 21 d post-MI in the border zone, consistent with tracer distribution in vivo. Conclusion: The simplified synthesis of [68Ga]MHLL1 bears promise for non-invasive characterization of fibroblast activation protein early in remodeling after MI.

Monoamine oxidase B (MAO-B) is an important enzyme regulating the levels of monoaminergic neurotransmitters. Selective MAO-B inhibitors have been labeled with carbon-11 or fluorine-18 to visualize the localization of MAO-B in vivo by positron emission tomography (PET) and thereby have been useful for studying neurodegenerative diseases. The aim of this study was to develop promising fluorine-18 labeled reversible MAO-B PET radioligands and their biological evaluation in vitro by autoradiography. Radiolabeling was achieved by classical one-step fluorine-18 nucleophilic substitution reaction. The stability and radiochemical yield was analyzed with HPLC. All five fluorine-18 labeled compounds were tested in human whole hemisphere autoradiography experiments. Five compounds (GEH200439, GEH200448, GEH200449, GEH200431A, and GEH200431B) were successfully radiolabeled with fluorine-18, and the incorporation yield of the fluorination reactions varied from 10 to 45% depending on the compound. The radiochemical purity was higher than 99% for all at the end of synthesis. Radioligands were found to be stable, with a radiochemical purity of >99% in a sterile phosphate buffered saline (pH = 7.4) over the duration of the study. The ARG binding density of only 18F-GEH200449 was consistent with known MAO-B expression in the human brain. Radiolabeling of five new fluorine-18 MAO-B reversible inhibitors was successfully accomplished. Compound 18F-GEH200449 binds specifically to MAO-B in vitro postmortem brain and could be a potential candidate for in vivo PET investigation.

Introduction The deposition of islet amyloid composed of amylin aggregates is related to β-cell mass dysfunction in type 2 diabetes mellitus (T2DM), and it may be involved in the development and progression of T2DM. In this study, we newly designed, synthesized, and evaluated a radiogallium-labeled pyridyl benzofuran derivative ([67/68Ga]GPBF) as an amylin imaging probe. Methods An in vitro competitive inhibition assay was performed to determine the binding affinity for amylin aggregates. An in vitro autoradiographic study was carried out using pancreatic sections from a T2DM patient. A biodistribution of [67Ga]GPBF in normal mice was evaluated. Finally, we carried out ex vivo autoradiography using mouse transplanted with amylin aggregates. Results GPBF exhibited binding affinity for amylin aggregates in vitro. In addition, [67Ga]GPBF clearly labeled islet amyloids in in vitro autoradiography of a T2DM pancreatic section. In a biodistribution study using normal mice, [67Ga]GPBF showed initial uptake into the pancreas, but non-specific accumulation in the liver, spleen, and pancreas was also observed. Furthermore, an ex vivo autoradiogram demonstrated that [67Ga]GPBF bound to amylin aggregates in the pancreas of the amylin aggregate-transplanted mice. Conclusions These results provide useful insights into the development of radiogallium labeled probes that target amylin aggregates in the T2DM pancreas.

The highly rigid and planar scaffolds with π-conjugated systems have been widely considered to be indispensable for β-amyloid (Aβ) binding ligands. In this study, a library of diphenoxy compounds with different types of more flexible linkers as Aβ ligands were synthesized and evaluated. Most of them displayed good affinity (Ki < 100 nM) for Aβ1-42 aggregates, and some ligands even showed values of Ki less than 10 nM. Structure-activity relationship analysis revealed that modification on the linkers or substituents tolerated great flexibility, which challenged the long-held belief that rigid and planar structures are exclusively favored for Aβ binding. Three ligands were labeled by iodine-125, and they exhibited good properties in vitro and in vivo, which further supported that this flexible scaffold was potential and promising for the development of Aβ imaging agents.

The deposition of islet amyloid is associated with β-cell mass dysfunction in type 2 diabetes mellitus (T2DM). Since the amylin aggregate is the main component of islet amyloid, in vivo imaging of amylin may be useful for diagnosis and elucidation of the pathogenic mechanism of T2DM. In the present study, we newly designed, synthesized, and evaluated two 18F labeled compounds ([18F]DANIR-F 2b and [18F]DANIR-F 2c) as positron emission tomography (PET) probes targeting amylin aggregates. In an in vitro binding study, DANIR-F 2b and DANIR-F 2c showed binding affinity for amylin aggregates (Ki = 160 and 29 nM, respectively). In addition, [18F]DANIR-F 2b and [18F]DANIR-F 2c clearly labeled islet amyloids in in vitro autoradiography of T2DM pancreatic sections. In the biodistribution study using normal mice, [18F]DANIR-F 2b and [18F]DANIR-F 2c displayed favorable pharamacokinetics in the pancreas and some organs located near the pancreas. Furthermore, in an ex vivo autoradiographic study, [18F]DANIR-F 2c also bound to amylin aggregates in the pancreas of the amylin transplanted mice. The results of this study suggest that [18F]DANIR-F 2c shows fundamental properties as a PET imaging probe targeting amylin aggregates in the T2DM pancreas.

Objectives To enable non-invasive real-time quantification of vasopressin 1A (V1A) receptors in peripheral organs, we sought to develop a suitable PET probe that would allow specific and selective V1A receptor imaging in vitro and in vivo. Methods We synthesized a high-affinity and -selectivity ligand, designated compound 17. The target structure was labeled with carbon-11 and tested for its utility as a V1A-targeted PET tracer by cell uptake studies, autoradiography, in vivo PET imaging and ex vivo biodistribution experiments. Results Compound 17 (PF-184563) and the respective precursor for radiolabeling were synthesized in an overall yield of 49% (over 7 steps) and 40% (over 8 steps), respectively. An inhibitory constant of 0.9 nM towards the V1A receptors was measured, while excellent selectivity over the related V1B, V2 and OT receptor (IC50 >10,000 nM) were obtained. Cell uptake studies revealed considerable V1A binding, which was significantly reduced in the presence of V1A antagonists. Conversely, there was no significant blockade in the presence of V1B and V2 antagonists. In vitro autoradiography and PET imaging studies in rodents indicated specific tracer binding mainly in the liver. Further, the pancreas, spleen and the heart exhibited specific binding of [11C]17 ([11C]PF-184563) by ex vivo biodistribution experiments. Conclusion We have developed the first V1A-targeted PET ligand that is suitable for subtype-selective receptor imaging in peripheral organs including the liver, heart, pancreas and spleen. Our findings suggest that [11C]PF-184563 can be a valuable tool to study the role of V1A receptors in liver diseases, as well as in cardiovascular pathologies.

Many diseases, including cancer, can lead to neuropathic pain (NP). NP is one of the accompanying symptoms of suffering in many conditions and the life quality of NP patient is seriously affected. Due to complex causes, the effects of clinical treatments have been very unsatisfactory. Many experts have found that neuron-microglia interaction plays an essential role in NP occurrence and development. Therefore, the activation of microglia, related inflammatory mediators and molecular and cellular signaling pathways have become the focus of NP research. With the help of modern functional imaging technology, advanced pre-and clinical studies have been carried out and NP interventions have been attempted by using the different pharmaceuticals and the extracted active components of various traditional herbal medicines. In this communication, we review the mechanism of microglia on NP formation and treatment and molecular imaging technology’s role in the clinical diagnosis and evaluation of NP therapies.

Background Alterations of dopamine D1 (D1R) and D2 receptor (D2R) are proposed in schizophrenia but brain neuroimaging and postmortem studies have shown controversial results in relation to D1R and D2R density. Besides, scarce information on the functionality of brain D1R and D2R is available. The present study characterized G-protein activation by D1R and D2R agonists in postmortem human brain. Furthermore, D2R functional status was compared between schizophrenia and control subjects. Methods G-protein receptor coupling was assessed in control caudate nucleus and frontal cortex by [35S]GTPγS-binding stimulation induced by increasing concentrations (10–10–10–3 M) of dopamine, and the selective dopaminergic agonists SKF38393 (D1R) and NPA (D2R). Concentration–response curves to NPA stimulation of [35S]GTPγS binding were analyzed in antipsychotic-free (n = 10) and antipsychotic-treated (n = 7) schizophrenia subjects and matched controls (n = 17). Results In caudate, [35S]GTPγS-binding responses to agonists were compatible with the existence of functional D2R. In contrast, stimulations in cortex showed responses that did not correspond to D1R or D2R. [35S]GTPγS-binding activation by NPA in caudate displayed biphasic curves with similar profile in schizophrenia (EC50H = 7.94 nM; EC50L = 7.08 μM) and control (EC50H = 7.24 nM; EC50L = 15.14 μM) subjects. The presence or absence of antipsychotic medication did not influence the pharmacological parameters. Conclusions Feasibility of functional evaluation of dopamine receptors in postmortem human brain by conventional [35S]GTPγS-binding assays appears to be restricted to signalling through inhibitory Gi/o proteins. These findings provide functional information about brain D2R status in subjects with schizophrenia and do not support the existence of D2R supersensitive in this mental disorder.

The sigma 1 receptor (S1R) is widely expressed in the CNS and is mainly located on the endoplasmic reticulum. The S1R is involved in the regulation of many neurotransmission systems and, indirectly, in neurodegenerative diseases. The S1R may therefore represent an interesting neuronal biomarker in neurodegenerative diseases such as Parkinson's (PD) or Alzheimer's diseases (AD). Here we present the characterisation of the S1R-specific 18F-labelled tracer 18F-IAM6067 in two animal models and in human brain tissue. Methods: Wistar rats were used for PET-CT imaging (60 min dynamic acquisition) and metabolite analysis (1, 2, 5, 10, 20, 60 min post-injection). To verify in vivo selectivity, haloperidol, BD1047 (S1R ligand), CM398 (S2R ligand) and SB206553 (5HT2B/C antagonist) were administrated for pre-saturation studies. Excitotoxic lesions induced by intra-striatal injection of AMPA were also imaged by 18F-IAM6067 PET-CT to test the sensitivity of the methods in a well-established model of neuronal loss. Tracer brain uptake was also verified by autoradiography in rats and in a mouse model of PD (intrastriatal 6-hydroxydopamine (6-OHDA) unilateral lesion). Finally, human cortical binding was investigated by autoradiography in three groups of subjects (control subjects with Braak ≤2, and AD patients, Braak >2 & ≤4 and Braak >4 stages). Results: We demonstrate that despite rapid peripheral metabolism of 18F-IAM6067, radiolabelled metabolites were hardly detected in brain samples. Brain uptake of 18F-IAM6067 showed differences in S1R anatomical distribution, namely from high to low uptake: pons-raphe, thalamus medio-dorsal, substantia nigra, hypothalamus, cerebellum, cortical areas and striatum. Pre-saturation studies showed 79-90% blockade of the binding in all areas of the brain indicated above except with the 5HT2B/C antagonist SB206553 and S2R ligand CM398 which induced no significant blockade, indicating good specificity of 18F-IAM6067 for S1Rs. No difference between ipsi- and contralateral sides of the brain in the mouse model of PD was detected. AMPA lesion induced a significant 69% decrease in 18F-IAM6067 uptake in the globus pallidus matching the neuronal loss as measured by NeuN, but only a trend to decrease (-16%) in the caudate putamen despite a significant 91% decrease in neuronal count. Moreover, no difference in the human cortical binding was shown between AD groups and controls. Conclusion: This work shows that 18F-IAM6067 is a specific and selective S1R radiotracer. The absence or small changes in S1R detected here in animal models and human tissue warrants further investigations and suggests that S1R might not be the anticipated ideal biomarker for neuronal loss in neurodegenerative diseases such as AD and PD.

Current synaptic vesicle 2A (SV2A) positron emission tomography (PET) imaging agents include the nanomolar affinity probes [11C]UCB-J and [18F]UCB-H derived from the anti-epileptic drug levitaracetam (Keppra®). An industry-utilized “de-risking” approach was used to carry out initial pharmacological characterization and to assess potential next-generation candidates amenable to F-18 radiolabeling for preliminary evaluation. Radioligand binding methods were employed in mammalian brain homogenates to determine the SV2A affinity (Kd) and maximal binding capacity (Bmax) of [3H]UCB-J. Novel leads were then screened to identify compounds minimally with comparable binding affinities with UCB-J in order to select a F-18-labeled candidate for subsequent in vivo assessment in rat. In parallel, mammalian brain tissue section autoradiography was performed to assess specific SV2A distribution. [3H]UCB-J bound with high affinity to a single population of sites in the rat brain (Kd = 2.6 ± 0.25 nM; Bmax = 810 ± 25 fmol/mg protein) and control human cortex (Kd = 2.9 ± 0.54 nM; Bmax = 10,000 ± 640 fmol/mg protein). Distribution of specific SV2A binding was shown to be homogeneous throughout the rodent brain and primarily in gray matter regions of rodent and human brain sections. Analog screening identified MNI-1038, MNI-1126/SDM-8, and SDM-2 as having comparable binding affinities with the currently available PET ligands. Subsequent [18F]MNI-1126/[18F]SDM-8 dynamic micro-PET imaging in rats revealed in vivo uptake and accumulation in the brain with favorable kinetics. Chase studies using 30 mg/kg levetiracetam confirmed that in vivo brain uptake of [18F]MNI-1126/[18F]SDM-8 was reversible. Taken together, these data suggest [18F]MNI-1126/[18F]SDM-8 (since renamed as [18F]SynVesT-1) characterized via an in vitro screening cascade provided a measurable in vivo SV2A specific signal in the rodent brain. This tracer as well as the close analog [18F]SDM-2 (since renamed as [18F]SynVesT-2) is currently undergoing further evaluation in preclinical and clinical studies.

Background Preclinical and some human data suggest allosteric modulation of the muscarinic M1 receptor (CHRM1) is a promising approach for the treatment of schizophrenia. However, it is suggested there is a subgroup of participants with schizophrenia who have profound loss of cortical CHRM1 (MRDS). This raises the possibility that some participants with schizophrenia may not respond optimally to CHRM1 allosteric modulation. Here we describe a novel methodology to measure positive allosteric modulation of CHRM1 in human CNS and the measurement of that response in the cortex, hippocampus, and striatum from participants with MRDS, non-MRDS and controls. Methods The cortex (Brodmann’s area 6), hippocampus, and striatum from 40 participants with schizophrenia (20 MRDS and 20 non-MRDS) and 20 controls were used to measure benzyl quinolone carboxylic acid-mediated shift in acetylcholine displacement of [3H]N-methylscopolamine using a novel in situ radioligand binding with autoradiography methodology. Results Compared with controls, participants with schizophrenia had lower levels of specific [3H]N-methylscopolamine binding in all CNS regions, whilst benzyl quinolone carboxylic acid-modulated binding was less in the striatum, Brodmann’s area 6, dentate gyrus, and subiculum. When divided by subgroup, only in MRDS was there lower specific [3H]N-methylscopolamine binding and less benzyl quinolone carboxylic acid-modulated binding in all cortical and subcortical regions studied. Conclusions In a subgroup of participants with schizophrenia, there is a widespread decreased responsiveness to a positive allosteric modulator at the CHRM1. This finding may have ramifications it positive allosteric modulators of the CHRM1 are used in clinical trials to treat schizophrenia as some participants may not have an optimal response.

Highly specific and sensitive biomarkers for pathologies related to dysfunctions in the basal ganglia circuit are of great value to assess therapeutic efficacy not only clinically to establish an early diagnosis, but also in terms of monitoring the efficacy of therapeutic interventions and decelerated neurodegeneration. The phosphodiesterase 10A (PDE10A) enzyme plays a central role in striatal signaling and is implicated in several neuropsychiatric disorders involving striatal pathology, such as Huntingtońs disease (HD) and schizophrenia. Inhibition of PDE10A activates the neurons in the striatum and consequently leads to alteration of behavioral aspects modulated by the striatal circuit. [18F]MNI-659, (2-(2-(3-(4-(2-[18F]fluoroethoxy)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione), is a newly developed PET radioligand that shows a high binding to PDE10A in the human brain in vivo. In the present study, we examined the in vitro binding of [18F]MNI-659 in human postmortem brain to gain a better understanding of the presence, density, disease-related alterations and therapy related to changes in PDE10A expression. The results show high specific binding of [18F]MNI-659 in the caudate nucleus, putamen and the hippocampal formation. Low specific [18F]MNI-659 binding was detected in nucleus accumbens in comparison to the caudate nucleus and putamen. In vitro binding studies with [18F]MNI-659 will facilitate in elucidating better understanding of the role of PDE10A activity in health and disease that may lead to new diagnostic opportunities in HD. This work was supported by the CHDI Foundation. The authors thank the members of the PET group at the Karolinska Institutet for excellent assistance; in particular Siv Eriksson for assistance with the autoradiography studies. We also thank Molecular Neuroimaging L.L.C. (MNI) for providing the [18F]MNI-659 precursor.

Therapies based on glucagon-like peptide-1 receptor (GLP-1R) agonism are highly effective in treating type-2 diabetes and obesity, but the localization of GLP-1 receptors mediating the antidiabetic and other possible actions of GLP-1 is still debated. The purpose with this study was to identify sites of GLP-1R mRNA and protein expression in the mouse gastrointestinal system by means of GLP-1R antibody immunohistochemistry, Glp-1r mRNA fluorescence in situ hybridization (FISH) and 125I-exendin (9-39) autoradiography. As expected GLP-1R staining was observed in almost all β cells in the pancreatic islets, but more rarely in α and δ cells. In the stomach, GLP-1R staining was found exclusively in the gastric corpus mucous neck cells, known to protect the stomach mucosa. The Brunner’s glands were strongly stained for GLP-1R, and pretreatment with GLP-1 agonist exendin-4 caused internalization of the receptor and mucin secretion, while pretreatment with PBS or antagonist exendin (9–39) did not. In the intestinal mucosa, GLP-1R staining was observed in intraepithelial lymphocytes, lamina propria lymphocytes, and enteroendocrine cells containing secretin, peptide YY, and somatostatin, but not cholecystokinin. GLP-1R staining was seen in nerve fibers within the CHAT- and NO-positive myenteric plexuses from the gastric corpus to the distal large intestine being strongest in the mid- and hindgut area. Finally, intraperitoneal administration of radiolabeled exendin (9–39) strongly labeled myenteric fibers. In conclusion, this study expands our knowledge of GLP-1 receptor localization and suggests that GLP-1 may serve an important role in modulating gastrointestinal health and mucosal protection.

Noradrenaline (NA) in the thalamus has important roles in physiological, pharmacological, and pathological neuromodulation. In this work, a complete characterization of NA axons and Alpha adrenoceptors distributions is provided. NA axons, revealed by immunohistochemistry against the synthesizing enzyme and the NA transporter, are present in all thalamic nuclei. The most densely innervated ones are the midline nuclei, intralaminar nuclei (paracentral and parafascicular), and the medial sector of the mediodorsal nucleus (MDm). The ventral motor nuclei and most somatosensory relay nuclei receive a moderate NA innervation. The pulvinar complex receives a heterogeneous innervation. The lateral geniculate nucleus (GL) has the lowest NA innervation. Alpha adrenoceptors were analyzed by in vitro quantitative autoradiography. Alpha-1 receptor densities are higher than Alpha-2 densities. Overall, axonal densities and Alpha adrenoceptor densities coincide; although some mismatches were identified. The nuclei with the highest Alpha-1 values are MDm, the parvocellular part of the ventral posterior medial nucleus, medial pulvinar, and midline nuclei. The nucleus with the lowest Alpha-1 receptor density is GL. Alpha-2 receptor densities are highest in the lateral dorsal, centromedian, medial and inferior pulvinar, and midline nuclei. These results suggest a role for NA in modulating thalamic involvement in consciousness, limbic, cognitive, and executive functions.

ObjectiveAlzheimer's disease (AD) is a neurodegenerative disease characterized by aggregation of Tau protein into paired helical filaments causing neurofibrillary tangles (NFT) in the brain. The aim of this study was to develop and evaluate the effectiveness of a novel radioiodinated tracer, 6-[125 I]iodo-3-(1H-pyrrolo[2,3-c]pyridine-1-yl)isoquinoline ([125 I]IPPI), for binding to Tau protein (Ki=0.75nM) in postmortem human brain (AD and cognitively normal (CN).MethodsRadiosynthesis of [125 I]IPPI was carried out by radioiododestannylation and purified chromatographically. Computational modeling studies of IPPI and MK-6240 binding on Tau fibril were evaluated. In vitro autoradiography studies were carried out with [3 H]PIB for Aβ plaques and [125 I]IPPI for Tau in AD and CN brains and evaluate drug effects.Results[125 I]IPPI was produced in >95% purity. Molecular modeling of IPPI revealed binding energies of IPPI (-7.8, -8.1, -8.2, -7.5 Kcal/mol) at the four sites were comparable to MK-6240 (-8.7, -8.5, -8.3, -7.5 Kcal/mol). Ratio of average grey matter (GM) [125 I]IPPI in AD versus CN was found to be 7.31 (p=.07) and AD GM/ white matter (WM) = 4.35 (p=.09). Ratio of average GM/WM [125 I]IPPI in CN was 1.21. Binding of [125 I]IPPI correlated with the presence of Tau, confirmed by anti-Tau Dako A0024. Specifically bound [125 I]IPPI to Tau in AD brains was displaced by MK-6240 and IPPI (>90%). Monoamine oxidase inhibitors (MAO) inhibitors deprenyl and clorgyline effected [125 I]IPPI binding at >1µM concentrations.Conclusion[125 I]IPPI exhibited high binding in human AD frontal cortex and anterior cingulate and is a suitable radioiodinated ligand for Tau imaging.

Significance Communication between cells in the brain relies on different types of transmitter receptors. Can we uncover organizational principles that harness the diversity of such signatures across the brain? We focus on the human cerebral cortex and demonstrate that the distribution of receptors forms a natural axis that stretches from association to sensory areas. Moreover, traversing this axis entails changes in the diversity, excitability, and mirrored density that reflect a basic division in receptor types, that is, ionotropic and metabotropic receptors. The unraveled principles offer explanatory depth for diverse phenomena and entail concrete, testable predictions., Transmitter receptors constitute a key component of the molecular machinery for intercellular communication in the brain. Recent efforts have mapped the density of diverse transmitter receptors across the human cerebral cortex with an unprecedented level of detail. Here, we distill these observations into key organizational principles. We demonstrate that receptor densities form a natural axis in the human cerebral cortex, reflecting decreases in differentiation at the level of laminar organization and a sensory-to-association axis at the functional level. Along this natural axis, key organizational principles are discerned: progressive molecular diversity (increase of the diversity of receptor density); excitation/inhibition (increase of the ratio of excitatory-to-inhibitory receptor density); and mirrored, orderly changes of the density of ionotropic and metabotropic receptors. The uncovered natural axis formed by the distribution of receptors aligns with the axis that is formed by other dimensions of cortical organization, such as the myelo- and cytoarchitectonic levels. Therefore, the uncovered natural axis constitutes a unifying organizational feature linking multiple dimensions of the cerebral cortex, thus bringing order to the heterogeneity of cortical organization.

Purpose The study demonstrates the use of Desorption Electrospray Ionization mass spectrometry imaging (DESI-MSI) for imaging of the PET tracer compound Cimbi-36 in brain tissue and compares imaging by DESI-MSI to imaging by autoradiography and PET. Procedures Rats were dosed intraperitoneally with 3 mg/kg of Cimbi-36 and euthanized at t = 5, 10, 15, 30, 60 and 120 min post-injection. The brains were removed, frozen and sectioned, and sagittal sections were imaged by DESI-MSI in positive ion mode. Additionally, brain sections from a non-dosed animal were incubated with C-14-labelled Cimbi-36 and imaged by autoradiography. Finally, PET images were acquired from an animal dosed with C-11-labelled Cimbi-36. Results DESI-MSI and autoradiography images of a sagittal brain sections showed similar distributions of Cimbi-36, with increased abundance in the frontal cortex and choroid plexus, regions which are high in 5-HT2A and 5-HT2C receptors. The PET image also showed increased abundance in cortex, but the spatial resolution was clearly inferior to DESI-MSI and autoradiography. The DESI-MSI results showed increased abundance of Cimbi-36 in brain tissue until 15 min, after which the abundance was declining. The PET-tracer was still clearly detectable at t = 120 min. Similar imaging of the kidneys showed the abundance of Cimbi-36 peaking at 30 min. Cimbi-36 was quantified in a t = 15 min brain section by quantitative DESI-MSI, resulting in tissue concentrations of 19.8 mu g/g in cortex, 15.4 mu g/g in cerebellum and 12.5 mu g/g in whole brain. Conclusions DESI imaging from an in vivo dosing experiment showed distribution of the PET tracer remarkably similar to what was obtained by autoradiography of an in vitro incubation experiment, indicating that the obtained results represent actual binding to certain receptors in the brain. DESI-MSI is suggested as a cost-effective screening tool, which does not rely on labelling of compounds.

Age‐related macular degeneration (AMD) is the leading cause of blindness in older people in the developed world while Stargardt's disease (SD) is a juvenile macular degeneration and an orphan disease. Both diseases are untreatable and are marked by accumulation of lipofuscin advancing to progressive deterioration of the retinal pigment epithelium (RPE) and retina and subsequent vision loss till blindness. We discovered that a small molecule belonging to the tetrahydropyridoether class of compounds, soraprazan renamed remofuscin, is able to remove existing lipofuscin from the RPE. This study investigated the drug penetration, distribution, and elimination into the eyes of a mouse model for increased lipofuscinogenesis, following a single intravitreal injection. We measured the time course of concentrations of remofuscin in different eye tissues using high‐performance liquid chromatography combined with mass spectroscopy (HPLC‐MS). We also visualized the penetration and distribution of 3H‐remofuscin in eye sections up to 20 weeks post‐injection using transmission electron microscopic (TEM) autoradiography. The distribution of silver grains revealed that remofuscin accumulated specifically in the RPE by binding to the RPE pigments (melanin, lipofuscin and melanolipofuscin) and that it was still detected after 20 weeks. Importantly, the melanosomes in choroidal melanocytes only rarely bind remofuscin emphasizing its potential to serve as an active ingredient in the RPE for the treatment of SD and dry AMD. In addition, our study highlights the importance of electron microscopic autoradiography as it is the only method able to show drug binding with a high intracellular resolution., TEM autoradiography shows: a depot effect of remofuscin that could be relevant for a local application of the drug. The RPE specificity of the drug. Only TEM autoradiography allows the investigation of drug binding with high intracellular resolution.

Significance Metal radiotracers exhibit beneficial properties that can improve in vivo positron emission tomography (PET) imaging over traditional radionuclides, such as longer half-lives, facile last-stage radiolabeling steps, and the potential for dual imaging-therapy applications. Among such radionuclides, 64 Cu exhibits a longer half-life (t 1/2 = 12.7 h) that allows for later PET imaging times and the ability to distribute 64 Cu imaging agents to facilities that do not have an on-site cyclotron. Herein, we report a 64 Cu PET imaging agent that shows appreciable in vivo brain uptake and exhibits high specific affinity for beta-amyloid aggregates, leading to the successful PET imaging of amyloid plaques in the brains of 5xFAD mice versus those of wild-type mice.

The aim of this report is to determine whether the times of neuron origin and neurogenetic gradients of PCs and Deep cerebellar nucli (DCN) glutamatergic neurons are different between mice and rats. Purkinje cells (PCs) were analyzed in each compartment of the cerebellar cortex (vermis, paravermis, medial, and lateral hemispheres), and deep glutamatergic neurons at the level of the medialis, interpositus, and lateralis nuclei. Tritiated thymidine ([3 H]TdR) autoradiography was applied on sections. The experimental rodents were the offspring of pregnant dams injected with [3 H]TdR on embryonic days (E) 11-12, E12-13, E13-14, E14-15, E15-16, and E16-17. Our results indicate that systematic differences exist in the pattern of neurogenesis and the spatial location of cerebellar PCs and deep glutamatergic neurons between mice and rats. In mice, PCs and deep glutamatergic neurons neurogenesis extend from E10 to E14, with a predominance of neurogenesis on E12 for PCs, and on E12, E11, and E10 for the medialis, interpositus, and lateralis neurons, respectively. When neurogenesis in rats was considered, the data reveal that PCs and deep glutamatergic neurons production extends from E12 to E16, with a peak of production on E14 for PCs, and on E14, E13, and E12 for the medialis, interpositus, and lateralis neurons, respectively. Current data also indicate that, both in mice and rats, both types of macroneurons are generated according to a lateral-to-medial gradient. Thus, the lateral hemisphere and the lateralis nucleus present more early-generated neurons than the vermis and the medialis nucleus, which in their turn have more late-produced neurons.

Albumin binding domain-Derived Affinity ProTeins (ADAPTs) are small (5 kDa) engineered scaffold proteins that are promising targeting agents for radionuclide-based imaging. A recent clinical study has demonstrated that radiolabeled ADAPTs can efficiently visualize human epidermal growth factor receptor 2 (HER2) expression in breast cancer using SPECT imaging. However, the use of ADAPTs directly labeled with radiometals for targeted radionuclide therapy is limited by their high reabsorption and prolonged retention of activity in kidneys. In this study, we investigated whether a co-injection of lysine or gelofusin, commonly used for reduction of renal uptake of radiolabeled peptides in clinics, would reduce the renal uptake of [99mTc]Tc(CO)3-ADAPT6 in NMRI mice. In order to better understand the mechanism behind the reabsorption of [99mTc]Tc(CO)3-ADAPT6, we included several compounds that act on various parts of the reabsorption system in kidneys. Administration of gelofusine, lysine, probenecid, furosemide, mannitol, or colchicine did not change the uptake of [99mTc]Tc(CO)3-ADAPT6 in kidneys. Sodium maleate reduced the uptake of [99mTc]Tc(CO)3-ADAPT6 to ca. 25% of the uptake in the control, a high dose of fructose (50 mmol/kg) reduced the uptake by ca. two-fold. However, a lower dose (20 mmol/kg) had no effect. These results indicate that common clinical strategies are not effective for reduction of kidney uptake of [99mTc]Tc(CO)3-ADAPT6 and that other strategies for reduction of activity uptake or retention in kidneys should be investigated for ADAPT6.

Molecular imaging agents targeting butyrylcholinesterase (BChE) have shown promise in other neurodegenerative disorders and may have utility in detecting changes to normal appearing white matter in multiple sclerosis (MS). BChE activity is present in white matter and localizes to activated microglia associated with MS lesions. The purpose of this study was to further characterize changes in the cholinergic system in MS pathology, and to explore the utility of BChE radioligands as potential diagnostic and treatment monitoring agents in MS. Cortical and white matter lesions were identified using myelin staining, and lesions were classified based on microglial activation patterns. Adjacent brain sections were used for cholinesterase histochemistry and in vitro autoradiography using phenyl 4-[123I]-iodophenylcarbamate (123I-PIP), a previously described small-molecule cholinesterase-binding radioligand. BChE activity is positively correlated with microglial activation in white matter MS lesions. There is no alteration in cholinesterase activity in cortical MS lesions. 123I-PIP autoradiography revealed uptake of radioactivity in normal white matter, absence of radioactivity within demyelinated MS lesions, and variable uptake of radioactivity in adjacent normal-appearing white matter. BChE imaging agents have the potential to detect MS lesions and subtle pathology in normal-appearing white matter in postmortem MS brain tissue. The possibility of BChE imaging agents serving to supplement current diagnostic and treatment monitoring strategies should be evaluated.

GluN2B is the most studied subunit of N-methyl-D-aspartate receptors (NMDARs) and implicated in the pathologies of various central nervous system disorders and neurodegenerative diseases. As pan NMDAR antagonists often produce debilitating side effects, new approaches in drug discovery have shifted to subtype-selective NMDAR modulators, especially GluN2B-selective antagonists. While positron emission tomography (PET) studies of GluN2B-selective NMDARs in the living brain would enable target engagement in drug development and improve our understanding in the NMDAR signaling pathways between normal and disease conditions, a suitable PET ligand is yet to be identified. Herein we developed an (18)F-labeled potent antagonist, 2-((1-(4-[(18)F]fluoro-3-methylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-methoxypyrimidine ([(18)F]13; also called [(18)F]N2B-0518) as a PET tracer for imaging the GluN2B subunit. The radiofluorination of [(18)F]13 was efficiently achieved by our spirocyclic iodonium ylide (SCIDY) method. In in vitro autoradiography studies, [(18)F]13 displayed highly region-specific binding in brain sections of rat and non-human primate, which was in accordance with the expression of GluN2B subunit. Ex vivo biodistribution in mice revealed that [(18)F]13 could penetrate the blood-brain barrier with moderate brain uptake (3.60% ID/g at 2 min) and rapid washout. Altogether, this work provides a GluN2B-selective PET tracer bearing new chemical scaffold and shows high specificity to GluN2B subunit in vitro, which may pave the way for the development of a new generation of GluN2B PET ligands.

To investigate the surface chemical changes in dynamic interactions of delmopinol on salivary films on oral mucosa in healthy participants after rinsing with an unbuffered water solution of delmopinol, and to examine the oral tissue disposition of delmopinol in rats after local administration.The contact angle technique was used to monitor the interaction of delmopinol with the salivary film coating the upper labial mucosa of 10 healthy participants through a 4 h period. The tissue disposition ofRinsing with delmopinol increased the polarity of the saliva coated mucosa during the time of observation. The binding of delmopinol was verified in the autoradiograms showing that radioactivity remained in the rat oral mucosa after 24 h. Delmopinol was however not irreversibly bound.The findings indicate that delmopinol interacts with the salivary film of the upper labial mucosa and affects its polarity. It appears that delmopinol assists in the maintenance of the hydrophilicity of the mucosal pellicle and thereby also reinforcing hydration of the mucosa. The rat autoradiograms, showed that radioactivity remains in the oral mucosa after 24 h, but diffuses through the mucosal membranes into the systemic circulation.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the human brain and plays a key role in several brain functions and neuropsychiatric disorders such as anxiety, epilepsy, and depression. The binding of benzodiazepines to the benzodiazepine receptor sites (BZR) located on GABAA receptors (GABAARs) potentiates the inhibitory effect of GABA leading to the anxiolytic, anticonvulsant and sedative effects used for treatment of those disorders. However, the function of GABAARs and the expression of BZR protein is determined by the GABAAR subunit stoichiometry (19 genes coding for individual subunits), and it remains to be established how the pentamer composition varies between brain regions and individuals.Here, we present a quantitative high-resolution in vivo atlas of the human brain BZRs, generated on the basis of [11C]flumazenil Positron Emission Tomography (PET) data. Next, based on autoradiography data, we transform the PET-generated atlas from binding values into BZR protein density. Finally, we examine the brain regional association with mRNA expression for the 19 subunits in the GABAAR, including an estimation of the minimally required expression of mRNA levels for each subunit to translate into BZR protein.This represents the first publicly available quantitative high-resolution in vivo atlas of the spatial distribution of BZR densities in the healthy human brain. The atlas provides a unique neuroscientific tool as well as novel insights into the association between mRNA expression for individual subunits in the GABAAR and the BZR density at each location in the brain.

Neuroinflammation is important in amyotrophic lateral sclerosis (ALS). The P2X7 receptor (P2X7R) is a promising target for neuroinflammation. The objective of this study was to compare 18F-DPA714, a second-generation translocator protein tracer, with 11C-JNJ717, a novel P2X7R tracer, in vitro and in vivo in ALS. Methods: For the in vitro portion of the study, autoradiography with 18F-DPA714 and 11C-JNJ717 was performed on human ALS brain sections in comparison to immunofluorescence with Iba1 and GFAP. For the in vivo portion, 3 male patients with early-stage ALS (59.3 ± 7.2 y old) and 6 healthy volunteers (48.2 ± 16.5 y old, 2 men and 4 women) underwent dynamic PET/MR scanning with 18F-DPA714 and 11C-JNJ717. Volume-of-distribution images were calculated using Logan plots and analyzed on a volume-of-interest basis. Results: Autoradiography showed no difference in 11C-JNJ717 binding but did show increased 18F-DPA714 binding in the motor cortex correlating with Iba1 expression (glial cells). Similar findings were observed in vivo, with a 13% increase in 18F-DPA714 binding in the motor cortex. Conclusion: In symptomatic ALS patients, 18F-DPA714 showed increased signal whereas 11C-JNJ717 was not elevated. ispartof: JOURNAL OF NUCLEAR MEDICINE vol:61 issue:4 pages:604-607 ispartof: location:United States status: published

Background Brain tumor vasculature can be significantly compromised and leakier than that of normal brain blood vessels. Little is known if there are vascular permeability alterations in the brain adjacent to tumor (BAT). Changes in BAT permeability may also lead to increased drug permeation in the BAT, which may exert toxicity on cells of the central nervous system. Herein, we studied permeation changes in BAT using quantitative fluorescent microscopy and autoradiography, while the effect of chemotherapy within the BAT region was determined by staining for activated astrocytes. Methods Human metastatic breast cancer cells (MDA-MB-231Br) were injected into left ventricle of female NuNu mice. Metastases were allowed to grow for 28 days, after which animals were injected fluorescent tracers Texas Red (625 Da) or Texas Red dextran (3 kDa) or a chemotherapeutic agent 14C-paclitaxel. The accumulation of tracers and 14C-paclitaxel in BAT were determined by using quantitative fluorescent microscopy and autoradiography respectively. The effect of chemotherapy in BAT was determined by staining for activated astrocytes. Results The mean permeability of texas Red (625 Da) within BAT region increased 1.0 to 2.5-fold when compared to normal brain, whereas, Texas Red dextran (3 kDa) demonstrated mean permeability increase ranging from 1.0 to 1.8-fold compared to normal brain. The Kin values in the BAT for both Texas Red (625 Da) and Texas Red dextran (3 kDa) were found to be 4.32 ± 0.2 × 105 mL/s/g and 1.6 ± 1.4 × 105 mL/s/g respectively and found to be significantly higher than the normal brain. We also found that there is significant increase in accumulation of 14C-Paclitaxel in BAT compared to the normal brain. We also observed animals treated with chemotherapy (paclitaxel (10 mg/kg), erubilin (1.5 mg/kg) and docetaxel (10 mg/kg)) showed activated astrocytes in BAT. Conclusions Our data showed increased permeation of fluorescent tracers and 14C-paclitaxel in the BAT. This increased permeation lead to elevated levels of activated astrocytes in BAT region in the animals treated with chemotherapy.

As antibiotic resistance rises, there is a need for strategies such as antibiotic adjuvants to conserve already-established antibiotics. A family of bacterial kinases known as the penicillin-binding-protein and serine/threonine kinase-associated (PASTA) kinases has attracted attention as targets for antibiotic adjuvants for β-lactams. Here, we report that the pyrazolopyridazine GW779439X sensitizes methicillin-resistant Staphylococcus aureus (MRSA) to various β-lactams through inhibition of the PASTA kinase Stk1. GW779439X potentiates β-lactam activity against multiple MRSA and MSSA isolates, including the sensitization of a ceftaroline-resistant isolate to ceftaroline. In silico modeling was used to guide the synthesis of GW779439X derivatives. The presence and orientation of GW779439X's methylpiperazine moiety was crucial for robust biochemical and microbiologic activity. Taken together, our data provide a proof of concept for developing the pyrazolopyridazines as selective Stk1 inhibitors which act across S. aureus isolates.

This study is a new analysis to obtain novel metabolic data on the functional connectivity of prefrontal-limbic regions in Pavlovian fear acquisition and extinction of tone-footshock conditioning. Mice were analyzed with the fluorodeoxyglucose (FDG) autoradiographic method to metabolically map regional brain activity. New FDG data were sampled from the nuclei of the habenula and other regions implicated in aversive conditioning, such as infralimbic cortex, amygdala and periaqueductal gray regions. The activity patterns among these regions were inter-correlated during acquisition, extinction or pseudorandom training to develop a functional connectivity model. Two subdivisions of the habenular complex showed increased activity after acquisition relative to extinction, with the pseudorandom group intermediate between the other two groups. Significant acquisition activation effects were also found in centromedial amygdala, dorsomedial and ventrolateral periaqueductal gray. FDG uptake increases during extinction were found only in dorsal and ventral infralimbic cortex. The overall pattern of activity correlations between these regions revealed extensive but differential functional connectivity during acquisition and extinction training, with less functional connectivity found after pseudorandom training. Interestingly, habenula nuclei showed a distinct pattern of inter-correlations with amygdala nuclei during extinction. The functional connectivity model revealed changing interactions among infralimbic cortex, amygdala, habenula and periaqueductal gray regions through the stages of Pavlovian fear acquisition and extinction. This study provided new data on the contributions of the habenula to fear conditioning, and revealed previously unreported infralimbic-amygdala-habenula-periaqueductal gray interactions implicated in acquisition and extinction of conditioned fear.

Introduction Receptor tyrosine kinase (RTK) platelet-derived growth factor receptor-alpha (PDGFRα) was recently identified as a molecular switch for dedifferentiation in thyroid cancer that predicts resistance to therapy as well as recurrence of disease in papillary thyroid cancer. Here we describe the radiolabeling and functional characterization of an imaging probe based on a PDGFRα-specific monoclonal antibody (mAb) for immuno-PET imaging of PDGFRα in papillary thyroid cancer. Methods Antibody D13C6 (Cell Signaling) was decorated with chelator NOTA using bioconjugation reaction with 2-(p-NCS-Bz)-NOTA. Radiolabeling was carried out using 40 μg of antibody-NOTA conjugate with 143–223 MBq of [64Cu]CuCl2 in 0.25 M NaOAc (pH 5.5) at 30 °C for 1 h. The reaction mixture was purified with size-exclusion chromatography (PD-10 column). PDGFRα and mock transfected B-CPAP thyroid cancer cells lines for validation of 64Cu-labeled immuno-conjugates were generated using LVX-Tet-On technology. PET imaging was performed in NSG mice bearing bilaterally-induced PDGFRα (+/−) B-CPAP tumors. Results Bioconjugation of NOTA chelator to monoclonal antibody D13C6 resulted in 2.8 ± 1.3 chelator molecules per antibody as determined by radiometric titration with 64Cu. [64Cu]Cu-NOTA-D13C6 was isolated in high radiochemical purity (>98%) and good radiochemical yields (19–61%). The specific activity was 0.9–5.1 MBq/μg. Cellular uptake studies revealed a specific radiotracer uptake in PDGFRα expressing cells compared to control cells. PET imaging resulted in SUVmean values of ~5.5 for PDGFRα (+) and ~2 for PDGFRα (−) tumors, after 48 h p.i.. After 1 h, radiotracer uptake was also observed in the bone marrow (SUVmean ~5) and spleen (SUVmean ~8.5). Conclusion Radiolabeled antibody [64Cu]Cu-NOTA-D13C6 represents a novel and promising radiotracer for immuno-PET imaging of PDGFRα in metastatic papillary thyroid cancer. Advances in knowledge and implications for patient care The presented work has the potential to allow physicians to identify papillary thyroid cancer patients at risk of metastases by using the novel immuno-PET imaging assay based on PDGFRα-targeting antibody [64Cu]Cu-NOTA-D13C6.

Cannabinoid administration modulates dopamine transmission via an indirect, multisynaptic mechanism that includes the activation of cannabinoid type-1 receptor (CB1R). The present study evaluated in rodents, the effects of acute and chronic (20 days) WIN55,212-2 administration, a non-selective CB1R agonist, on dopamine uptake and synthesis in the mesolimbic and nigrostriatal dopaminergic pathways and associate them to its effects on the endocannabinoid system. The effect of spontaneous withdrawal, after different abstinence periods (7 days, 20 days), was also assessed. Acute and chronic administration of WIN55,212-2 decreased dopamine transporter (DAT) binding and mRNA levels, as well as tyrosine hydroxylase (TH) mRNA expression in the substantia nigra (SN) and ventral tegmental area (VTA). In the striatum, chronic WIN55,212-2 administration led to decreased protein expression of DAT and TH, whereas no alterations were observed after acute administration, suggesting a diminished dopamine uptake and synthesis after chronic agonist treatment. Furthermore, after chronic agonist treatment, we observed reduced CB1R binding and mRNA levels in SN and striatum, providing evidence for a possible regulatory role of the endocannabinoid system on dopaminergic function. Seven days after WIN55,212-2 cessation, we observed a rebound increase in mRNA, binding and total protein levels of DAT and TH in VTA, SN and striatum proposing the existence of a biphasic expression pattern, which was also observed in CB1R binding levels. Within the 20-day period of abstinence, TH mRNA and protein levels and CB1R binding levels remain increased. The above results indicate that chronic CB1R agonist treatment induces long-lasting control of the mesostriatal dopaminergic activity.

Clinical and preclinical studies have shown dysfunctions in genetic expression and neurotransmission of γ-Aminobutyric acid (GABA), GABAA receptor subunits, and GABA-synthesizing enzymes GAD67 and GAD65 in schizophrenia. It is well documented that there is significant weight gain after chronic neuroleptic treatment in humans. While there are limited studies on the effects of diet on GABA signaling directly, a change in diet has been used clinically as an adjunct to treatment for schizophrenic relief. In this study, rats chronically consumed either a chow diet (CD) or a 60% high-fat diet (HFD) and drank from bottles that contained one of the following solutions: water, haloperidol (1.5 mg/kg), or olanzapine (10 mg/kg) for four weeks. Rats were then euthanized and their brains were processed for GABAA in-vitro receptor autoradiography using [3H] flunitrazepam. A chronic HFD treatment yielded significantly increased [3H] flunitrazepam binding in the rat cerebellum independent of neuroleptic treatment. The desynchronization between the prefrontal cortex and the cerebellum is associated with major cognitive and motor dysfunctions commonly found in schizophrenic symptomatology, such as slowed reaction time, motor dyscoordination, and prefrontal activations related to speech fluency and cognitive alertness. These data support the notion that there is a dietary effect on GABA signaling within the cerebellum, as well as the importance of considering nutritional intervention methods as an adjunct treatment for patients chronically treated with neuroleptics. Finally, we indicate that future studies involving the analysis of individual patient's genetic profiles will further assist towards a precision medicine approach to the treatment of schizophrenia.

A variety of physical fitness regimens have been shown to improve cognition, including executive function, yet our understanding of which parameters of motor training are important in optimizing outcomes remains limited. We used functional brain mapping to compare the ability of two motor challenges to acutely recruit the prefrontal-striatal circuit. The two motor tasks - walking in a complex running wheel with irregularly spaced rungs or walking in a running wheel with a smooth internal surface - differed only in the extent of skill required for their execution. Cerebral perfusion was mapped in rats by intravenous injection of [C]-iodoantipyrine during walking in either a motorized complex wheel or in a simple wheel. Regional cerebral blood flow (rCBF) was quantified by whole-brain autoradiography and analyzed in three-dimensional reconstructed brains by statistical parametric mapping and seed-based functional connectivity. Skilled or simple walking compared with rest, increased rCBF in regions of the motor circuit, somatosensory and visual cortex, as well as the hippocampus. Significantly greater rCBF increases were noted during skilled walking than for simple walking. Skilled walking, unlike simple walking or the resting condition, was associated with a significant positive functional connectivity in the prefrontal-striatal circuit (prelimbic cortex-dorsomedial striatum) and greater negative functional connectivity in the prefrontal-hippocampal circuit. Our findings suggest that the level of skill of a motor training task determines the extent of functional recruitment of the prefrontal-corticostriatal circuit, with implications for a new approach in neurorehabilitation that uses circuit-specific neuroplasticity to improve motor and cognitive functions.

GPR44 expression has recently been described as highly β-cell selective in the human pancreas and constitutes a tentative surrogate imaging biomarker in diabetes. A radiolabeled small-molecule GPR44 antagonist, [11C]AZ12204657, was evaluated for visualization of β-cells in pigs and nonhuman primates by positron emission tomography as well as in immunodeficient mice transplanted with human islets under the kidney capsule. In vitro autoradiography of human and animal pancreatic sections from subjects without and with diabetes, in combination with insulin staining, was performed to assess β-cell selectivity of the radiotracer. Proof of principle of in vivo targeting of human islets by [11C]AZ12204657 was shown in the immunodeficient mouse transplantation model. Furthermore, [11C]AZ12204657 bound by a GPR44-mediated mechanism in pancreatic sections from humans and pigs without diabetes, but not those with diabetes. In vivo [11C]AZ12204657 bound specifically to GPR44 in pancreas and spleen and could be competed away dose-dependently in nondiabetic pigs and nonhuman primates. [11C]AZ12204657 is a first-in-class surrogate imaging biomarker for pancreatic β-cells by targeting the protein GPR44.

Under certain conditions such as hypoxia-reoxygenation, the generation of reactive oxygen species (ROS) increases following hypoxia caused by a decreased oxygen supply. As another hypoxic condition, an excess neural activity status including epileptic seizure induces a decrease in tissue oxygen partial pressure (pO2) caused by enhanced oxygen utilization; however, whether ROS generation increases following the hypoxic status induced by transiently enhanced energy metabolism in brain tissue currently remains unknown. We herein investigated ROS-dependent chemiluminescence in cerebral cortex slices during the restoration of transiently enhanced energy metabolism induced by a high-potassium treatment with tissue pO2 changes and redox balance. ROS generation in the tissue was enhanced after high-potassium-induced hypoxia, but not by the reversed order of the treatment: control-potassium then high-potassium treatment, high-potassium treatment alone, and control-potassium treatment alone. The high-potassium treatment induced a transient decrease in tissue pO2 and a shift in the tissue redox balance towards reduction. The transient shift in the tissue redox balance towards reduction with enhanced metabolic activity and its recovery may correlate with ROS generation. This phenomenon may mimic ROS generation following the hypoxic status induced by transiently enhanced energy metabolism.

In the brain, the µ-opioid receptor (MOR) is involved in reward-seeking behaviors and plays a pivotal role in the mediation of opioid use disorders. Furthermore, reward-seeking behaviors and susceptibility to opioid addiction are particularly evident during the juvenile period, with a higher incidence of opioid use in males and higher sensitivity to opioids in females. Despite these age and sex differences in MOR-mediated behaviors, little is known regarding potential age and sex differences in the expression of MORs in the brain. Here, we used receptor autoradiography to compare MOR binding densities between juvenile and adult male and female rats. Age differences were found in MOR binding density in 12 out of 33 brain regions analyzed, with 11 regions showing higher MOR binding density in juveniles than in adults. These include the lateral septum, as well as sub-regions of the bed nucleus of the stria terminalis, hippocampus, and thalamus. Sex differences in MOR binding density were observed in only two brain regions, namely, the lateral septum (higher in males) and the posterior cortical nucleus of the amygdala (higher in females). Overall, these findings provide an important foundation for the generation of hypotheses regarding differential functional roles of MOR activation in juveniles versus adults. Specifically, we discuss the possibility that higher MOR binding densities in juveniles may allow for higher MOR activation, which could facilitate behaviors that are heightened during the juvenile period, such as reward and drug-seeking behaviors.

Purpose Mn2+ is used a contrast agent and marker for neuronal activity with magnetic resonance imaging (MRI) in rats and mice, but its accumulation is generally not assessed quantitatively. In this work, non-radioactive Mn and 52Mn are injected simultaneously in rats, and imaged with MRI, positron emission tomography (PET) and autoradiography (AR). Mn distributions are compared between modalities, to assess the potential and limitations on quantification of Mn with MRI, and to investigate the potential of multimodal measurement of Mn accumulation. Methods MRI (in vivo), PET (in vivo and post mortem), and AR (ex vivo) were acquired of rat brains, for which animals received simultaneous intraperitoneal (IP) or intracerebrovertricular (ICV) targeted injections containing the positron-emitting radionuclide 52Mn and additional non-radioactive MnCl2, which acts as an MRI contrast agent. Pre- and post-injection MR images were fit for the longitudinal relaxation rate (R1), coregistered, and subtracted to generate R1 difference maps, which are expected to be proportional to change in Mn concentration in tissue. AR and PET images were coregistered to smoothed R1 difference maps. Results Similar spatial distributions were seen across modalities, with Mn accumulation in the colliculus, near the injection site, and in the 4th ventricle. There was no 52Mn accumulation measurable with PET in the brain after IP injection. In areas of very highly localized and concentrated 52Mn accumulation in PET or AR, consistent increases of R1 were not seen with MRI. Scatter plots of corresponding voxel R1 difference and PET or AR signal intensity were generated and fit with least-squares linear models within anatomical regions. Linear correlations were observed, particularly in regions away from very highly localized and concentrated Mn accumulation at the injection site and the 4th ventricle. Accounting for radioactive decay of 52Mn, the MnCl2 longitudinal relaxivity was between 4.0 and 5.1 s−1/mM, which is within 22% of the in vitro relaxivity. Conclusions This proof-of-concept study demonstrates that MR has strong potential for quantitative assessment of Mn accumulation in the brain, although local discrepancies from linear correlation suggest limitations to this use of MR in areas of inflammation or very high concentrations of Mn. These discrepancies also suggest that a combination of modalities may have additional utility for discriminating between different pools of Mn accumulation in tissue. This article is protected by copyright. All rights reserved.

Minodronic acid is an aminobisphosphonate that is an antagonist of purinergic P2X2/3 receptors involved in pain. The aim of this study was to investigate the action and distribution of minodronic acid and the potential for P2X2/3 receptor antagonism based on the estimated concentration of minodronic acid. Microlocalization of radiolabeled minodronic acid was examined in the femur of neonatal rats. The bone-binding characteristics of minodronic acid and morphological changes in osteoclasts were analyzed in vitro. The minodronic acid concentration around bone resorption lacunae was predicted based on bone binding and the shape of lacunae. In microautoradiography, radioactive silver grains were abundant in bone-attached osteoclasts and were detected in calcified and ossification zones and in the cytoplasm of osteoclasts but not in the hypertrophic cartilage zone. In an osteoclast culture with 1 µM minodronic acid, 65% of minodronic acid was bound to bone, and C-terminal cross-linking telopeptide release was inhibited by 96%. Cultured osteoclasts without minodronic acid treatment formed ruffled borders and bone resorption lacunae and had rich cytoplasm, whereas those treated with 1 µM minodronic acid were not multinucleated, stained densely with toluidine blue, and were detached from the bone surface. In the 1 µM culture, the estimated minodronic acid concentration in resorption lacunae was 880 µM, which is higher than the IC50 for minodronic acid antagonism of P2X2/3 receptors. Thus, inhibition of P2X2/3 receptors around osteoclasts may contribute to the analgesic effect of minodronic acid.

Brain structure & function 225, 881-907 (2020). doi:10.1007/s00429-019-02022-4, Published by Springer, Berlin ; Heidelberg

We previously showed that 2 weeks of a severe food restricted (sFR) diet (40% of the caloric intake of the control (CT) diet) up‐regulated the circulating renin angiotensin (Ang) system (RAS) in female Fischer rats, most likely as a result of the fall in plasma volume. In this study, we investigated the role of the central RAS in the mean arterial pressure (MAP) and heart rate (HR) dysregulation associated with sFR. Although sFR reduced basal mean MAP and HR, the magnitude of the pressor response to intracerebroventricular (icv) microinjection of Ang‐[1‐8] was not affected; however, HR was 57 ± 13 bpm lower 26 min after Ang‐[1‐8] microinjection in the sFR rats and a similar response was observed after losartan was microinjected. The major catabolic pathway of Ang‐[1‐8] in the hypothalamus was via Ang‐[1‐7]; however, no differences were detected in the rate of Ang‐[1‐8] synthesis or degradation between CT and sFR animals. While sFR had no effect on the AT1R binding in the subfornical organ (SFO), the organum vasculosum laminae terminalis (OVLT) and median preoptic nucleus (MnPO) of the paraventricular anteroventral third ventricle, ligand binding increased 1.4‐fold in the paraventricular nucleus (PVN) of the hypothalamus. These findings suggest that sFR stimulates the central RAS by increasing AT1R expression in the PVN as a compensatory response to the reduction in basal MAP and HR. These findings have implications for people experiencing a period of sFR since an activated central RAS could increase their risk of disorders involving over activation of the RAS including renal and cardiovascular diseases., In this study, we investigated the role of the central RAS in the mean arterial pressure (MAP) and heart rate (HR) dysregulation associated with sFR. A major finding of this study was that central administration of the AT1R antagonist, losartan, reduced the blood pressure after 2 weeks on a sFR diet in Female rats and radioligand binding to the AT1R was selectively increased in the PVN of sFR rats.

International audience; Background: The 5-HT 6 receptor is one of the most recently identified serotonin receptors in the central nervous system. Because of its role in memory and cognitive process, this receptor might be implicated in Alzheimer's disease (AD) and associated disorders. Objective: The aim of this study was to investigate the binding of [ 18 F]2FNQ1P, a new specific radiotracer of 5-HT 6 receptors, and to quantify 5-HT 6 receptor density in caudate nucleus in a population of patients with different AD stages. Methods: Patients were classified according to the "ABC" NIA-AA classification. In vitro binding assays were performed in postmortem brain tissue from the healthy control (HC; n = 8) and severe AD ("High"; n = 8) groups. In vitro quantitative autoradiography was performed in human brain tissue (caudate nucleus) from patients with different stages of AD: HC (n = 15), "Low" (n = 18), "Int" (n = 20), and "High" (n = 15). Results: In vitro binding assays did not show significant differences for the K D and B max parameters between "High" and HC groups. In vitro quantitative autoradiography showed a significant difference between the "High" and HC groups (p = 0.0025). We also showed a progressive diminution in [ 18 F]2FNQ1P specific binding, which parallels 5-HT 6 receptors expression, according to increasing AD stage. Significant differences were observed between the HC group and all AD stages combined ("Low", "Intermediate", and "High") (p = 0.011). Conclusion: This study confirms the interest of investigating the role of 5-HT 6 receptors in AD and related disorders. [ 18 F]2FNQ1P demonstrated specific binding to 5-HT 6 receptors.

It is well settled that the amyloidogenic properties of the plasma protein transporter transthyretin (TTR) can be modulated by compounds that stabilize its native tetrameric conformation. TTR is also present in cerebrospinal fluid where it can bind to Aβ-peptides and prevent Aβ aggregation. We have previously shown that treatment of Alzheimer’s Disease (AD) model mice with iododiflunisal (IDIF), a TTR tetramer stabilizing compound, prevents AD pathologies. This evidence positioned IDIF as a new lead drug for AD. In dissecting the mechanism of action of IDIF, we disclose here different labeling strategies for the preparation of 131I-labeled IDIF and 131I- and 124I-labeled TTR, which have been further used for the preparation of IDIF-TTR complexes labeled either on the compound or the protein. The biodistribution of all labeled species after intravenous administration has been investigated in mice using ex vivo and in vivo techniques. Our results confirm the capacity of TTR to cross the blood brain barrier (BBB) and suggest that the formation of TTR-IDIF complexes enhances BBB permeability of both IDIF and TTR. The increased TTR and IDIF brain concentrations may result in higher Aβ-peptide sequestration capacity with the subsequent inhibition of AD symptoms as we have previously observed in mice. © 2019, The Author(s)., The work was supported by a grant from the Fundació Marató de TV3 (Neurodegenerative Diseases Call, Project Reference 20140330-31-32-33-34, http://www.ccma.cat/tv3/marato/en/ projectes-financats/2013/212/). The group at CIC biomaGUNE also acknowledges MINECO (Spain) for funding through Grant CTQ2017-87637-R. I. Cardoso worked under the Investigator FCT Program which is financed by national funds through the Foundation for Science and Technology (FCT, Portugal) and co-financed by the European Social Fund (ESF) through the Human Potential Operational Programme (HPOP), type 4.2 - Promotion of Scientific Employment.

Background Glutamate is an excitatory neurotransmitter binding to 3 classes of receptors, including the N-methyl, D-aspartate (NMDA) receptor. NMDA receptor binding is lower in major depression disorder and suicide. NMDA receptor blocking with ketamine can have antidepressant and anti-suicide effects. Early-life adversity (ELA) may cause glutamate-mediated excitotoxicity and is more common with major depression disorder and in suicide decedents. We sought to determine whether NMDA-receptor binding is altered with suicide and ELA. Methods A total 52 postmortem cases were organized as 13 quadruplets of suicide and non-suicide decedents matched for age, sex, and postmortem interval, with or without reported ELA (≤16 years). Tissue blocks containing dorsal prefrontal (BA8), dorsolateral prefrontal (BA9), or anterior cingulate (BA24) cortex were collected at autopsy. Psychiatrically healthy controls and suicide decedents underwent psychological autopsy to determine psychiatric diagnoses and details of childhood adversity. NMDA receptor binding was determined by quantitative autoradiography of [3H]MK-801 binding (displaced by unlabeled MK-801) in 20-µm-thick sections. Results [3H]MK-801 binding was not associated with suicide in BA8, BA9, or BA24. However, [3H]MK-801 binding with ELA was less in BA8, BA9, and BA24 independent of suicide (P Conclusions Less NMDA receptor binding with ELA is consistent with the hypothesis that stress can cause excitotoxicity via excessive glutamate, causing either NMDA receptor downregulation or less receptor binding due to neuron loss consequent to the excitotoxicity.

Quality control of tissues and organs for transplant is important to confirm their safety and effectiveness for regenerative medicine. However, quality evaluation is only carried out using a limited range of inspection criteria, because many of the available evaluation tests are invasive. In order to explore the potential of 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG)-bioradiography as a non-invasive test for estimation of the safety, soundness, and effectiveness of tissues for transplantation, [18F]FDG uptake and cell viability or metabolism were investigated using a reconstructed human epidermal model (RHEM). We developed an imaging system, and suitable bioradiographic image acquisition conditions and its effectiveness were investigated. [18F]FDG uptake increased in agreement with DNA content as a marker of cell numbers and for histological assessment during cell proliferation and keratinization. [18F]FDG uptake was significantly decreased in good agreement with the viability of tissues used with various hazardous chemical treatments. [18F]FDG uptake by the tissues was decreased by hypothermia treatment and increased by hypoxia treatment while maintaining cell viability in the tissue. Therefore, [18F]FDG-bioradiography can be useful to estimate cell viability or metabolism in this RHEM. This method might be utilized as a non-invasive test for quality evaluation of tissues for transplantation.

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Studies of brain functional activation during spatial navigation using electrophysiology and immediate-early gene responses have typically targeted a limited number of brain regions. Our study provides the first whole brain analysis of cerebral activation during retrieval of spatial memory in the freely-moving rat. Rats (LEARNERS) were trained in the Barnes maze, an allocentric spatial navigation task, while CONTROLS received passive exposure. After 19 days, functional brain mapping was performed during recall by bolus intravenous injection of [(14)C]-iodoantipyrine using a novel subcutaneous minipump triggered by remote activation. Regional cerebral blood flow (rCBF)-related tissue radioactivity was analyzed by statistical parametric mapping from autoradiographic images of the three-dimensionally reconstructed brains. Functional connectivity was examined between regions of the spatial navigation circuit through interregional correlation analysis. Significant rCBF increases were noted in LEARNERS compared to CONTROLS broadly across the spatial navigation circuit, including the hippocampus (anterior dorsal CA1, posterior ventral CA1–3), subiculum, thalamus, striatum, medial septum, cerebral cortex, with decreases noted in the mammillary nucleus, amygdala and insula. LEARNERS showed a significantly greater positive correlation of rCBF of the ventral hippocampus with retrosplenial, lateral orbital, parietal and primary visual cortex, and a significantly more negative correlation with the mammillary nucleus, amygdala, posterior entorhinal cortex, and anterior thalamic nucleus. The complex sensory component of the spatial navigation task was underscored by broad activation across visual, somatosensory, olfactory, auditory and vestibular circuits which was enhanced in LEARNERS. Brain mapping facilitated by an implantable minipump represents a powerful tool for evaluation of mammalian behaviors dependent on locomotion.

Excessive alcohol consumption is associated with neuroinflammation, which likely contributes to alcohol-related pathology. However, positron emission tomography (PET) studies using radioligands for the 18-kDa translocator protein (TSPO), which is considered a biomarker of neuroinflammation, reported decreased binding in alcohol use disorder (AUD) participants compared to controls. In contrast, autoradiographic findings in alcohol exposed rats reported increases in TSPO radioligand binding. To assess if these discrepancies reflected differences between in vitro and in vivo methodologies, we compared in vitro autoradiography (using [3 H]PBR28 and [3 H]PK11195) with in vivo PET (using [11 C]PBR28) in male, Wistar rats exposed to chronic alcohol-vapor (dependent n = 10) and in rats exposed to air-vapor (nondependent n = 10). PET scans were obtained with [11 C]PBR28, after which rats were euthanized and the brains were harvested for autoradiography with [3 H]PBR28 and [3 H]PK11195 (n = 7 dependent and n = 7 nondependent), and binding quantified in hippocampus, thalamus, and parietal cortex. Autoradiography revealed significantly higher binding in alcohol-dependent rats for both radioligands in thalamus and hippocampus (trend level for [3 H]PBR28) compared to nondependent rats, and these group differences were stronger for [3 H]PK11195 than [3 H]PBR28. In contrast, PET measures obtained in the same rats showed no group difference in [11 C]PBR28 binding. Our in vitro data are consistent with neuroinflammation associated with chronic alcohol exposure. Failure to observe similar increases in [11 C]PBR28 binding in vivo suggests the possibility that a mechanism mediated by chronic alcohol exposure interferes with [11 C]PBR28 binding to TSPO in vivo. These data question the sensitivity of PBR28 PET as a methodology to assess neuroinflammation in AUD.

The oxytocin receptor (OXTR) has been observed in the periphery of neonatal C57BL/6J mice (Mus musculus), including facial regions and the anogenital area. In those studies, ligand specificity was confirmed with a congenital OXTR knockout mouse as well as competitive binding techniques. The aim of this study was to determine if OXTR is present in the same peripheral sites in the neonatal prairie vole (Microtus ochrogaster) for cross-species comparisons. Receptor autoradiography was performed on 20 μm sagittal sections of whole postnatal day 0 (P0) male and female prairie voles using the 125iodinated-ornithine vasotocin ([125I]-OVTA) radioligand. A competition binding assay was used to assess the selectivity of [125I]-OVTA for peripheral OXTR. Radioactive ligand (0.05 nM [125I]-OVTA) was competed against concentrations of 0 and 1000 nM excess unlabeled oxytocin (OXT). Previously identified regions of significant OXTR ligand binding in the mouse were analyzed for comparison: rostral and lateral periodontium, olfactory epithelium, ciliary bodies of the eye, whisker pads, adrenal gland, and anogenital area. We also evaluated the liver and scapular brown adipose tissue, which displayed strong but non-specific signal on film in mice. While there were some areas that showed conserved OXTR ligand binding in the prairie vole (e.g., ciliary body of the eye and the anogenital area), areas showing OXTR ligand binding in the neonatal prairie vole were not identical to OXTR ligand binding in the periphery of the C57BL/6J neonatal mouse. Further, some of the regions measured in the prairie vole suggest sex differences in OXTR ligand binding. Collectively, as is well-established in the central nervous system, these data indicate that patterns of OXTR ligand binding in the infant periphery are species-specific.

International audience; [F-18]-MK-6240, a novel tau positron emission tomography (PET) tracer recently discovered for the in vivo detection of neurofibrillary tangles, has the potential to improve diagnostic accuracy in the detection of Alzheimer disease. We have examined regional and substrate-specific binding patterns as well as possible off-target binding of this tracer on human brain tissue to advance towards its validation. We applied [F-18]-MK-6240 phosphor screen and high resolution autoradiography to postmortem samples from patients with a definite pathological diagnosis of Alzheimer disease, frontotemporal lobar degeneration-tau (Pick’s disease, progressive supranuclear palsy and corticobasal degeneration), chronic traumatic encephalopathy, frontotemporal lobar degeneration-Tar DNA-binding protein 43 (TDP-43), dementia with Lewy bodies, cerebral amyloid angiopathy and elderly controls free of pathologic changes of neurodegenerative disease. We also directly compared the binding properties of [F-18]-MK-6240 and [F-18]-AV-1451 in human tissue, and examined potential nonspecific binding of both tau tracers to monoamine oxidases (MAO) by using autoradiography in the presence of selective monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) inhibitors. Our data indicate that MK-6240 strongly binds to neurofibrillary tangles in Alzheimer disease but does not seem to bind to a significant extent to tau aggregates in non-Alzheimer tauopathies, suggesting that it may have a limited utility for the in vivo detection of these pathologies. There is no evidence of binding to lesions containing β-amyloid, α-synuclein or TDP-43. In addition, we identified MK-6240 strong off-target binding to neuromelanin and melanin-containing cells, and some weaker binding to areas of hemorrhage. These binding patterns are nearly identical to those previously reported by our group and others for [F-18]-AV-1451. Of note, [F-18]-MK-6240 and [F-18]-AV-1451 autoradiographic binding signals were only weakly displaced by competing concentrations of selective MAO-B inhibitor deprenyl but not by MAO-A inhibitor clorgyline, suggesting that MAO enzymes do not appear to be a significant binding target of any of these two tracers. Together these novel findings provide relevant insights for the correct interpretation of in vivo [F-18]-MK-6240 PET imaging.

ObjectivePrenatal exposure to valproic acid (VPA) enhances the risk for later development of autism spectrum disorders (ASD). An altered gamma-aminobutyric acid (GABA) system may be a key factor in ASD. Here we investigated possible changes in the GABA system in rats exposed to a low dose of prenatal VPA.MethodWe performed autoradiography with [3H]muscimol, (a GABAA receptor agonist), and [11C]Ro15-4513 (a partial agonist of the GABAA α1+5 receptor subtypes), in brain sections containing amygdala, thalamus and hippocampus of rats treated prenatally with 20 mg/kg VPA or saline from the 12th day of gestation.ResultPrenatal VPA significantly increased [11C]Ro15-4513 binding in the left amygdala compared with controls (p3H]muscimol binding.ConclusionWe observed an asymmetric increase in GABAA receptor binding. Disturbances in the GABAA receptor system have also been detected in human autism with [11C]Ro15-4513.