The affinity of antipsychotic drugs to dopamine and serotonin 5-HT2 receptors determines their effects on prefrontal-striatal functional connectivity.

One of the major challenges of cross-species translation in psychiatry is the identification of quantifiable brain phenotypes linked to drug efficacy and/or side effects. A measure that has received increasing interest is the effect of antipsychotic drugs on resting-state functional connectivity (FC) in magnetic resonance imaging. However, quantitative comparisons of antipsychotic drug-induced alterations of FC patterns are missing. Consideration of receptor binding affinities provides a means for the effects of antipsychotic drugs on extended brain networks to be related directly to their molecular mechanism of action. Therefore, we examined the relationship between the affinities of three second-generation antipsychotics (amisulpride, risperidone and olanzapine) to dopamine and serotonin receptors and FC patterns related to the prefrontal cortex (PFC) and striatum in Sprague-Dawley rats. FC of the relevant regions was quantified by correlation coefficients and local network properties. Each drug group (32 animals per group) was subdivided into three dose groups and a vehicle control group. A linear relationship was discovered for the mid-dose of antipsychotic compounds, with stronger affinity to serotonin 5-HT2A, 5-HT2C and 5-HT1A receptors and decreased affinity to D3 receptors associated with increased prefrontal-striatal FC (p = 0.0004, r²â€¯= 0.46; p = 0.004, r²â€¯= 0.33; p = 0.002, r²â€¯= 0.37; p = 0.02, r²â€¯= 0.22, respectively). Interestingly, no correlation was observed for the low and high dose groups, and for D2 receptors. Our results indicate that drug-induced FC patterns may be linked to antipsychotic mechanism of action on the molecular level and suggest the technique's value for drug development, especially if our results are extended to a larger number of antipsychotics.

Effects of aripiprazole/OPC-14597 on motor activity, pharmacological models of psychosis, and brain activity in rats.

Aripiprazole (OPC-14597) is an antipsychotic with a unique pharmacology as a dopamine D2 receptor partial agonist, which has been demonstrated to reduce symptoms of schizophrenia. To further profile this compound in preclinical models, we examined aripiprazole-induced activity changes as measured by pharmacological magnetic resonance imaging (MRI) and characterized the drug in several rodent models of motor behaviors and of psychosis. Continuous arterial spin labeling MRI measuring blood perfusion (as an indirect measure of activity) reveals that aripiprazole dose-dependently decreased brain activity in the entorhinal piriform cortex, perirhinal cortex, nucleus accumbens shell, and basolateral amygdala. While no deficits were observed in the rotarod test for motor coordination in the simpler (8 RPM) version, in the more challenging condition (16 RPM) doses of 10 and 30mg/kg i.p. produced deficits. Catalepsy was seen only at the highest dose tested (30mg/kg i.p.) and only at the 3 and 6h time points, not at the 1h time point. In pharmacological models of psychosis, 1-30mg/kg aripiprazole i.p. effectively reduced locomotor activity induced by dopamine agonists (amphetamine and apomorphine), NMDA antagonists (MK-801 and phencyclidine (PCP)), and a serotonin agonist (2,5-dimethoxy-4-iodoamphetamine (DOI)). However, aripiprazole reversed prepulse inhibition deficits induced by amphetamine, but not by any of the other agents tested. Aripiprazole alters brain activity in regions relevant to schizophrenia, and furthermore, has a pharmacological profile that differs for the two psychosis models tested and does not match the typical or atypical psychotics. Thus, D2 partial agonists may constitute a new group of antipsychotics.

Natural abundance 13C-NMR spectroscopy for the quantitative determination of fecal fat.

OBJECTIVE: To evaluate 13C-NMR spectroscopy as a method for fat quantitation in human feces without time consuming or unpleasant preparation steps. DESIGN AND METHODS: Stool samples of seven healthy subjects were collected for 18 days before and during oral intake of the inhibitor of gastrointestinal lipases Orlistat. Fecal lipid content was determined first using 13C-NMR, then by conventional gravimetry after homogenization and Bligh & Dyer lipid extraction. RESULTS: The correlation between gravimetry and 13C-NMR was excellent (R2 = 0.91). In repeated measurements, the mean percentage error was 2.8%. On average, 13C-NMR yielded 1.27 g less fat than gravimetry. Orlistat efficacy for fat excretion assessed by 13C-NMR and by gravimetry was 34.3% and 33.9%, respectively. CONCLUSIONS: With a total measurement time of three minutes, 13C-NMR spectroscopy of unprocessed whole stool provides an accurate alternative to gravimetry for assessing total fecal fat excretion. 13C-NMR is superior with regard to practicability and speed.

Simultaneous in vivo monitoring of hepatic glucose and glucose-6-phosphate by (13)C-NMR spectroscopy.

Hepatic glucose-6-phosphate (G6P) was monitored non-invasively in rat liver by in vivo (13)C NMR spectroscopy after infusion of [1-(13)C] glucose. The phosphorylation of glucose to G6P yields small but characteristic displacements for all of its (13)C-NMR resonances relative to those of glucose. It is demonstrated that in vivo (13)C-NMR spectroscopy at 7 Tesla provides the spectral sensitivity and resolution to detect hepatic G6P present at sub-millimolar concentration as partially resolved low-field shoulders of the glucose C1 resonances at 96.86 ppm (C1beta) and 93. 02 ppm (C1alpha). Upon (13)C-labeling, the intracellular conversion of [1-(13)C] glucose to [1-(13)C] G6P could be monitored, which allowed the hepatic glucose-G6P substrate cycle to be assessed in situ. The close correlation found for the (13)C labeling patterns of glucose and G6P supports the concept of an active substrate cycle whose rate exceeds that of net hepatic glucose metabolism. High-resolution (13)C-NMR spectroscopy and biochemical analyses of tissue biopsies collected at the end of the experiments confirmed qualitatively the findings obtained in vivo.

19F-MRI of perfluorononane as a novel contrast modality for gastrointestinal imaging.

19F-Magnetic resonance imaging in conjunction with perfluorononane provides a new modality for gastrointestinal (GI) imaging as is demonstrated here with an animal model. Perfluorononane was found to be an ideal oral contrast agent since it is biologically inert, immiscible with water, and since it has a low viscosity and surface tension. Furthermore, its high fluorine content, together with the high sensitivity of 19F-MRI, allowed highly selective MR images of the GI tract of mice to be acquired. Due to the lack of 19F background signals, the contrast of the GI tract was only limited by the signal-to-noise ratio of the 19F-MR images. 19F-RARE images of 1-mm slices with an in-plane resolution of 0.23 x 0.23 mm2 were obtained from the GI tract after oral perfluorononane administration. The passage of perfluorononane through the entire GI tract was monitored by repetitive MR measurements with a maximal time resolution of 38 s. The three-dimensional surfaces of the GI tract were reconstructed and superimposed on corresponding 1H-MR images, which provided complementary anatomical information.

Ascorbic acid, a vitamin, is observed by in vivo 13C nuclear magnetic resonance spectroscopy of rat liver.

The first in vivo detection of a vitamin with nuclear magnetic resonance (NMR) is reported for mammalian liver. Vitamin C, also known as ascorbic acid, was monitored noninvasively in rat liver by "whole body" 13C NMR spectroscopy at high field after infusion of [1,2-13C2]glucose into anesthetized rats. Generally, the carbon resonances of ascorbic acid overlap with those of other highly abundant cellular metabolites, thus precluding their observation in situ. This problem was resolved by taking advantage of the 13C-13C spin couplings introduced by the two covalently bound 13C nuclei in [1,2-13C2]glucose. During glucose metabolism, [5,6-13C2]ascorbic acid was synthesized, which also exhibited characteristic 13C homonuclear spin couplings. This feature enabled the spectral discrimination of ascorbic acid from overlapping singlet resonances of other metabolites. Quantitative analysis of the spin-coupling patterns provided an estimate of the turnover rate of hepatic ascorbic acid in vivo (1.9 +/- 0.4 nmol.min-1.g-1) and a novel approach toward a better understanding of optimal ascorbic acid requirements in humans. The results obtained in vivo were confirmed with high-resolution proton and 13C NMR spectroscopy of liver extracts.

Proton magnetic resonance and human thyroid neoplasia III. Ex vivo chemical-shift microimaging.

Magnetic-resonance chemical-shift microimaging, with a spatial resolution of 40 x 40 microns, is a modality which can detect alterations to cellular chemistry and hence markers of pathological processes in human tissue ex vivo. This technique was used as a chemical microscope to assess follicular thyroid neoplasms, lesions which are unsatisfactorily investigated using standard histopathological techniques or water-based magnetic-resonance imaging. The chemical-shift images at the methyl frequency (0.9 ppm) identify chemical heterogeneity in follicular tumors which are histologically homogeneous. The observed changes to cellular chemistry, detectable in foci of approximately 100 cells or less, support the existence of a preinvasive state hitherto unidentified by current pathological techniques.

Adult-onset hypothyroidism and the cerebral metabolism of (1,2-13C2) acetate as detected by 13C nuclear magnetic resonance.

The effects of adult-onset hypothyroidism on the metabolic compartmentation of the cerebral tricarboxylic acid cycle and the gamma-aminobutyric acid (GABA) shunt have been investigated by 13C nuclear magnetic resonance spectroscopy. Rats thyroidectomized as adults and age-matched controls were infused in the right jugular vein with unlabeled or (1,2-13C2) acetate solutions for 60 min. At the end of the infusion, the brains were frozen in situ and perchloric acid extracts were prepared and analyzed by 13C nuclear magnetic resonance and reverse-phase HPLC. Thyroidectomized animals showed a decrease in the incorporation of 13C from (1,2-13C2) acetate in cerebral metabolites and an increase in the concentrations of unlabeled glutamate and GABA. Computer-assisted interpretation of the 13C multiplets observed for the carbons of glutamate, glutamine, and GABA indicated that adult-onset hypothyroidism produced 1) a decrease in the contribution of infused (1,2-13C2) acetate to the glial tricarboxylic acid cycle; 2) an increase in the contribution of unlabeled acetyl-CoA to the neuronal tricarboxylic acid cycle; and 3) impairments in the exchange of glutamate, glutamine, and GABA between the neuronal and glial compartments. Despite the fact that the adult brain has often been considered metabolically unresponsive to thyroid hormone status, present results show metabolic alterations in the neuronal and glial compartments that are reversible with substitution therapy.

Proton magnetic resonance and human cervical neoplasia. II. Ex vivo chemical-shift microimaging.

Proton chemical-shift imaging at 8.5 T has been used to detect malignant foci in small (6 mm3) biopsies from the human uterine cervix. Images based on the lipid resonances of frankly malignant cells discriminate between tumor tissue and host stroma and distinguish invasive from preinvasive cervical cancer (n = 7). With this method, foci of malignant cells were revealed in 500 micron slices with an in-plane resolution of 40 by 160 microns. The MR intensity maps reflected the local distribution of malignant cells as assessed by histopathology. The lower signal-to-noise ratio inherent for these non-water-based images was improved by applying postacquisitional matched Gaussian window functions, thus effecting a substantial increase in contrast with minimal loss in spatial resolution.

Cerebral metabolism of [1,2-13C2]glucose and [U-13C4]3-hydroxybutyrate in rat brain as detected by 13C NMR spectroscopy.

The metabolism of [1,2-13C2]glucose and [U-13C4]3-hydroxybutyrate was studied in rat brain with in vivo and in vitro 13C NMR spectroscopy, taking advantage, in particular, of homonuclear 13C-13C spin coupling patterns. After infusion of [1,2-13C2]glucose or [U-13C4]3-hydroxybutyrate into rats, the uptake of the substrates in brain and their metabolism to [1-13C]bicarbonate could be detected with in vivo 13C NMR spectroscopy. At the end of the infusion experiment, methanol/HCl/HClO4 extracts of the brain tissue were further analysed by high resolution 13C NMR spectroscopy. The 13C spin coupling patterns revealed entirely different isotopomer distributions for the closely related cerebral metabolites glutamate, glutamine and 4-aminobutyric acid. A quantitative analysis of the 13C spectra demonstrated (i) the existence of two kinetically distinct pools of glutamate, (ii) a pronounced CO2 fixation via pyruvate carboxylase in the glial cells accounting for as much as 38% of the oxaloacetate synthesis in the glial tricarboxylic acid cycle, (iii) a cerebral pyruvate recycling system contributing maximally 17% of the pyruvate metabolism through the pyruvate dehydrogenase in neurons, and (iv) a predominant production of 4-aminobutyric acid from glutamate synthesized in the neurons. In addition, the labelling pattern of N-acetyl aspartate upon infusion of labelled glucose or 3-hydroxybutyrate provided insight into the synthesis of this compound in mammalian brain. While the acetyl moiety originates from the metabolic equivalent of the C-1-C-2 part of cerebral glutamate, the aspartyl moiety is not in direct contact with the intermediates of glycolysis or of the tricarboxylic acid cycles.

Glycogen metabolism as detected by in vivo and in vitro 13C-NMR spectroscopy using [1,2-13C2]glucose as substrate.

The metabolism of glucose to glycogen in the liver of fasted and well-fed rats was investigated with 13C nuclear magnetic resonance spectroscopy using [1,2-(13)C2]glucose as the main substrate. The unique spectroscopic feature of this molecule is the 13C-13C homonuclear coupling leading to characteristic doublets for the C-1 and C-2 resonances of glucose and its breakdown products as long as the two 13C nuclei remain bonded together. The doublet resonances of [1,2-(13)C2]glucose thus provide an ideal marker to follow the fate of this exogenous substrate through the metabolic pathways. [1,2-(13)C2]Glucose was injected intraperitoneally into anesthetized rats and the in vivo 13C-NMR measurements of the intact animals revealed the transformation of the injected glucose into liver glycogen. Glycogen was extracted from the liver and high resolution 13C-NMR spectra were obtained before and after hydrolysis of glycogen. Intact [1,2-13C2]glucose molecules give rise to doublet resonances, natural abundance [13C]glucose molecules produce singlet resonances. From an analysis of the doublet-to-singlet intensities the following conclusions were derived. (i) In fasted rats virtually 100% of the glycosyl units in glycogen were 13C-NMR visible. In contrast, the 13C-NMR visibility of glycogen decreased to 30-40% in well-fed rats. (ii) In fed rats a minimum of 67 +/- 7% of the exogenous [1,2-(13)C2]glucose was incorporated into the liver glycogen via the direct pathway. No contribution of the indirect pathway could be detected. (iii) In fasted rats externally supplied glucose appeared to be consumed in different metabolic processes and less [1,2-(13)C2]glucose was found to be incorporated into glycogen (13 +/- 1%). However, the observation of [5,6-(13)C2]glucose in liver glycogen provided evidence for the operation of the so-called indirect pathway of glycogen synthesis. The activity of the indirect pathway was at least 9% but not more than 30% of the direct pathway. (vi) The pentose phosphate pathway was of little significance for glucose but became detectable upon injection of [1-(13)C]ribose.

Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR.

The metabolism of [1,2-13C2]acetate in rat brain was studied by in vivo and in vitro 13C NMR spectroscopy, in particular by taking advantage of the homonuclear 13C-13C spin coupling patterns. Well nourished rats were infused with [1,2-13C2]acetate or [1-13C]acetate in the jugular vein, and the in situ kinetics of 13C labeling during the infusion period was followed by 13C NMR techniques. The in vivo 13C NMR spectra showed signals from (i) the C-1 carbon of [1,2-13C2] acetate or [1-13C]acetate, (ii) 13CO3H-, and (iii) the natural abundance 13C carbons of sufficiently mobile fatty acids. Methanol/HCl/perchloric acid extracts of the brains were prepared and were further analyzed by high resolution 13C NMR. The homonuclear 13C-13C spin coupling patterns after infusion of [1,2-13C2]acetate showed very different isotopomer populations in glutamate, glutamine, and gamma-aminobutyric acid. Analyzing the relative proportions of these isotopomers revealed (i) two different glutamate compartments in the rat brain characterized by the presence and absence, respectively, of glutamine synthase activity, (ii) two different tricarboxylic acid cycles, one preferentially metabolizing [(1,2-13C2]acetate, the other mainly using unlabeled acetyl-coenzyme A, (iii) a hitherto unknown cerebral pyruvate recycling system associated with the tricarboxylic acid cycle, metabolizing primarily unlabeled acetyl-coenzyme A, and (iv) a predominant production of gamma-aminobutyric acid in the glutamate compartment lacking glutamine synthase.

Multilabeled 13C substrates as probes in in vivo 13C and 1H NMR spectroscopy.

The potential use of 13C multilabeled substrates has been studied in biological applications using in vivo and in vitro proton and 13C NMR spectroscopy. In 13C NMR spectroscopy, multilabeled compounds allow the simultaneous observation of several nuclei or increase distinctly the signal to noise ratio due to a higher degree of enrichment. Contiguous labeling of substrates leads to homonuclear 13C-13C spin couplings and provides a simple means to distinguish between endogenous stores of metabolites and metabolites derived from added substrates.

Monoclonal antibody-coated magnetite particles as contrast agents in magnetic resonance imaging of tumors.

A highly specific and powerful magnetic resonance imaging contrast agent has been prepared by coating magnetite (Fe3O4) particles with monoclonal antibodies directed against a tumor antigen. The preparation maintains both the immunoreactivity of the monoclonal antibody and the full relaxing capability of the magnetite particle. MRI image contrast by spin-echo methods can be easily induced in a concentration range of 1-10 nM of the antibody-coated magnetite particles.

In situ metabolism of 1,omega medium chain dicarboxylic acids in the liver of intact rats as detected by 13C and 1H NMR.

The hepatic metabolism of 1,omega-dodecanedioic acid, a physiologically relevant representative of the medium-chain dicarboxylic acid family, has been studied by a combination of in vivo and in vitro 13C and 1H NMR spectroscopic techniques. Rats in different nutritional or hormonal situations were infused with [1,12-13C2]- or [1,2,11,12-13C4]dodecanedioic acid, and the kinetics of 13C label appearance as well as the final relative concentrations of metabolic products were measured noninvasively in the liver of the intact rat by 13C NMR spectroscopy. Perchloric acid and chloroform/methanol extracts of liver biopsies obtained at the end of the infusion period were further analyzed by high resolution 13C NMR and one-dimensional and two-dimensional COSY and J-resolved 1H NMR. [1-13C]- and [1,2-13C2]adipic acids were the main end products of the in vivo metabolism of [1,12-13C2]- or [1,2,11,12-13C4]dodecanedioic acids, respectively, indicating that the beta-oxidation pathway of medium-chain dicarboxylic acids proceeds in situ monodirectionally. [1-13C]Adipic acid, the main product of peroxisomal beta-oxidation, could also be detected in situ. This finding, together with the in vivo and in vitro absence of signals characteristic of intramitochondrial oxidation of [1-13C]acetyl-coenzyme A, provide a strong evidence supporting a predominant contribution of the peroxisomal beta-oxidation system to the overall oxidation of these compounds in vivo. Homonuclear two-dimensional COSY 1H NMR spectra of acid extracts from rat liver provided a convenient method of analyzing the metabolic repercussions of dicarboxylic acid accumulation, revealing a decrease in the hepatic concentration of beta-hydroxybutyrate and an accumulation of adipic acid and the amino acid L-lysine.