M.T. Giralt

Profiling and Imaging of Lipids on Brain and Liver Tissue by Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry Using 2-Mercaptobenzothiazole as a Matrix

2-Mercaptobenzothiazole (MBT) is employed for the first time as a matrix for the analysis of lipids from tissue extracts using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We demonstrate that the performance of MBT is superior to that of the matrixes commonly employed for lipids, due to its low vapor pressure, its low acidity, and the formation of small crystals, although because of the strong background at low m/z, it precludes detection of species below approximately 500 Da. This inconvenience can be partly overcome with the formation of Cs adducts. Using a polymer-based dual calibration, a mass accuracy of approximately 10 ppm in lipid extracts and of approximately 80 ppm in tissues is achieved. We present spectra from liver and brain lipid extracts where a large amount of lipid species is identified, in both positive and negative ion modes, with high reproducibility. In addition, the above-mentioned special properties of MBT allow its employment for imaging mass spectrometry. In the present work, images of brain and liver tissues showing different lipid species are presented, demonstrating the advantages of the employment of MBT.

Matrix-assisted laser desorption ionization imaging mass spectrometry in lipidomics

The relevant structural, energetics, and regulatory roles of lipids are universally acknowledged. However, the high variability of lipid species and the large differences in concentrations make unraveling the role played by the different species in metabolism a titanic task. A recently developed technique, known as imaging mass spectrometry, may shed some light on the field, as it enables precise information to be obtained on the location of lipids in tissues. A review of the state of the art of the technique is presented in this manuscript, including detailed analysis of sample-preparation steps, data handling, and the identification of the species mapped so far.

Occlusal Disharmonies Modulate Central Catecholaminergic Activity in the Rat

Occlusal disharmonies have classically been thought to be involved in the etiopathogenesis of bruxism, as have, more recently, alterations in central neurotransmission, particularly dopaminergic neurotransmission. However, the connection between these two factors has still not been established. In this study, we assessed the effects of diverse occlusal disharmonies, maintained for either 1 day or 14 days, on neurochemical indices of dopaminergic and noradrenergic activity in the striatum, frontal cortex, and hypothalamus of the rat. The in vivo activity of tyrosine hydroxylase, determined as the accumulation of 3,4-dihydroxyphenylalanine (DOPA), 30 min after the administration of 3-hydroxybenzylhydrazine, a DOPA decarboxylase inhibitor, and dopamine and noradrenaline contents were quantified by high-performance liquid chromatography with electrochemical detection. The wearing of an acrylic cap on both lower incisors for 1 day induced a significant increase in DOPA accumulation in the regions analyzed, with parallel increases in dopamine levels in the hypothalamus and dopamine and noradrenaline in the frontal cortex. After the cap was maintained for 14 days, DOPA accumulation tended to return to control values, except in the left striatum, thereby causing an imbalance between hemispheres. In contrast, 1 or 14 days after the lower left and the upper right incisors were cut, less pronounced changes in catecholaminergic neurotransmission were found in the brain areas studied. Moreover, the cutting of one lower incisor did not modify either DOPA accumulation or dopamine and noradrenaline contents in the striatum or hypothalamus. These results provide experimental evidence of a modulation of central catecholaminergic neurotransmission by occlusal disharmonies, being dependent on the nature of the incisal alteration and on the time during which it was maintained.

Effects of Dopaminergic Drugs, Occlusal Disharmonies, and Chronic Stress on Non-functional Masticatory Activity in the Rat, Assessed by Incisal Attrition

Observational methods and the recording of nonspecific jaw movements or masticatory muscle activity have been used to evaluate oral parafunctional movements in animal models of bruxism. In this study, we have used a new approach in which the non-functional masticatory activity in the rat was assessed by the measurement of incisal attrition, with the aim of investigating the role of diverse factors involved in the etiology of bruxism. We quantified the attrition rate weekly by making superficial notches in the lower incisors and measuring the distances to the incisor edges. Repeated stimulation of the dopaminergic system with apomorphine led to an enhancement of the non-functional masticatory activity (p < 0.0001). The severity of the apomorphine-induced oral behavior was positively correlated (rs = 0.69, p < 0.01) with an increase in the incisal attrition rate (20.9%, p < 0.0001). Apomorphine-induced non-functional masticatory activity was strongly enhanced by the placement of an acrylic cap on both lower incisors (306%, p < 0.0001), but not by the cutting of a lower incisor. Repeated cocaine administration also increased the attrition rate (22.5%, p < 0.0001). However, neither chronic blockade of dopaminergic receptors with haloperidol, nor its withdrawal, modified attrition. In addition, since emotional disturbances are considered to be causal factors of bruxism, we tested whether experimental stress might accelerate tooth wear. Exposure to two different chronic stress regimes did not induce significant changes in incisal attrition. Moreover, exposure to chronic stress after the withdrawal of chronic haloperidol treatment did not alter attrition either. These results partially support the role of the central dopaminergic system in bruxism and suggest that stress, in general, may not be a relevant factor in tooth wear.

Imaging mass spectrometry (IMS) of cortical lipids from preclinical to severe stages of Alzheimer's disease

Alzheimers disease (AD) is a progressive neurodegenerative disease affecting millions of patients worldwide. Previous studies have demonstrated alterations in the lipid composition of lipid extracts from plasma and brain samples of AD patients. However, there is no consensus regarding the qualitative and quantitative changes of lipids in brains from AD patients. In addition, the recent developments in imaging mass spectrometry methods are leading to a new stage in the in situ analysis of lipid species in brain tissue slices from human postmortem samples. The present study uses the matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS), permitting the direct anatomical analysis of lipids in postmortem brain sections from AD patients, which are compared with the intensity of the lipid signal in samples from matched subjects with no neurological diseases. The frontal cortex samples from AD patients were classified in three groups based on Braaks histochemical criteria, ranging from non-cognitively impaired patients to those severely affected. The main results indicate a depletion of different sulfatide lipid species from the earliest stages of the disease in both white and gray matter areas of the frontal cortex. Therefore, the decrease in sulfatides in cortical areas could be considered as a marker of the disease, but may also indicate neurochemical modifications related to the pathogenesis of the disease. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Anatomical location of LPA1 activation and LPA phospholipid precursors in rodent and human brain

Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein‐coupled receptors: LPA1–LPA6. LPA evokes several responses in the CNS, including cortical development and folding, growth of the axonal cone and its retraction process. Those cell processes involve survival, migration, adhesion proliferation, differentiation, and myelination. The anatomical localization of LPA1 is incompletely understood, particularly with regard to LPA binding. Therefore, we have used functional [35S]GTPγS autoradiography to verify the anatomical distribution of LPA1 binding sites in adult rodent and human brain. The greatest activity was observed in myelinated areas of the white matter such as corpus callosum, internal capsule and cerebellum. MaLPA1‐null mice (a variant of LPA1‐null) lack [35S]GTPγS basal binding in white matter areas, where the LPA1 receptor is expressed at high levels, suggesting a relevant role of the activity of this receptor in the most myelinated brain areas. In addition, phospholipid precursors of LPA were localized by MALDI‐IMS in both rodent and human brain slices identifying numerous species of phosphatides and phosphatidylcholines. Both phosphatides and phosphatidylcholines species represent potential LPA precursors. The anatomical distribution of these precursors in rodent and human brain may indicate a metabolic relationship between LPA and LPA1 receptors. Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein‐coupled receptors (GPCR), LPA1 to LPA6. LPA evokes several responses in the central nervous system (CNS), including cortical development and folding, growth of the axonal cone and its retraction process. We used functional [35S]GTPγS autoradiography to verify the anatomical distribution of LPA1‐binding sites in adult rodent and human brain. The distribution of LPA1 receptors in rat, mouse and human brains show the highest activity in white matter myelinated areas. The basal and LPA‐evoked activities are abolished in MaLPA1‐null mice. The phospholipid precursors of LPA are localized by MALDI‐IMS. The anatomical distribution of LPA precursors in rodent and human brain suggests a relationship with functional LPA1 receptors.

Galanin activated Gi/o-proteins in human and rat central nervous systems.

The neuropeptide galanin (GAL) is involved in the control of hormone secretion, nociception, feeding behavior, attention, learning and memory. The anatomical localization of galanin receptors in the brain has been described using autoradiography and immunohistochemistry, but both techniques are limited by the availability of specific radioligands or antibodies. Functional autoradiography provides an alternative method by combining anatomical resolution and information of the activity mediated by G-protein coupled receptors. The present study analyzes the functional GAL receptors coupled to Gi/o-proteins in human and rat brain nuclei using [(35)S]GTPγS autoradiography. The results show the anatomical distribution of Gi/o-proteins activated by GAL receptors that trigger intracellular signaling mechanisms. The activity mediated by GAL receptors in human and rat brain showed a good correlation of the net stimulation in areas such as spinal cord, periaqueductal gray, putamen, CA3 layers of hippocampus, substantia nigra and diverse thalamic nuclei. The functional GAL receptors coupled to Gi/o-proteins showed a similar pattern for both species in most of the areas analyzed, but some discrete nuclei showed differences in the activity mediated by GAL, such as the ventroposteromedial thalamic nucleus, or areas that regulate learning and memory processes in the hippocampus. Taken into consideration the present results, the rat could be used as an experimental model for the study of the physiological role of GAL-mediated neurotransmission and the modulation of GAL receptors activity in the human CNS.

Increase in cortical endocannabinoid signaling in a rat model of basal forebrain cholinergic dysfunction

The basal forebrain cholinergic pathways progressively degenerate during the progression of Alzheimers disease, leading to an irreversible impairment of memory and thinking skills. The stereotaxic lesion with 192IgG-saporin in the rat brain has been used to eliminate basal forebrain cholinergic neurons and is aimed at emulating the cognitive damage described in this disease in order to explore its effects on behavior and on neurotransmission. Learning and memory processes that are controlled by cholinergic neurotransmission are also modulated by the endocannabinoid (eCB) system. The objective of the present study is to evaluate the eCB signaling in relation to the memory impairment induced in adult rats following a specific cholinergic lesion of the basal forebrain. Therefore, CB1 receptor-mediated signaling was analyzed using receptor and functional autoradiography, and cellular distribution by immunofluorescence. The passive avoidance test and histochemical data revealed a relationship between impaired behavioral responses and a loss of approximately 75% of cholinergic neurons in the nucleus basalis magnocellularis (NBM), accompanied by cortical cholinergic denervation. The decrease in CB1 receptor density observed in the hippocampus, together with hyperactivity of eCB signaling in the NBM and cortex, suggest an interaction between the eCB and cholinergic systems. Moreover, following basal forebrain cholinergic denervation, the presynaptic GABAergic immunoreactivity was reduced in cortical areas. In conclusion, CB1 receptors present in presynaptic GABAergic terminals in the hippocampus are down regulated, but not those in cortical glutamatergic synapses.

Descifrando la anatomía molecular de un tejido mediante análisis lipidómico por espectrometría de masas

Resumen La lipidomica esta emergiendo como una ciencia imprescindible para la comprension del funcionamiento de celulas y tejidos, dado que complementa el conocimiento obtenido mediante la genomica y la proteomica. La caracterizacion de los lipidos que componen una muestra biologica es una tarea compleja, debido a su enorme variedad y variabilidad, y por tanto necesita del empleo de tecnicas de alto rendimiento para su consecucion. La espectrometria de masas esta demostrando ser la tecnica ideal para llevar a cabo dicha tarea, especialmente desde el desarrollo de la imagen por espectrometria de masas. En el presente trabajo se presentan los resultados del empleo de una nueva matriz, 2-mercaptobenzotiazol, en el analisis lipidomico de extractos de higado quiescente y regenerante de roedor mediante espectrometria de masas MALDI-TOF. La buena reproducibilidad entre individuos, permite la observacion de diferencias en las especies lipidicas mayoritarias del higado normal y el afectado de esteatosis. Se muestran ademas imagenes de la disposicion anatomica de ciertas moleculas en el cerebro de rata, obtenidas representando la distribucion de las concentraciones de diversas especies de lipidos a lo largo de los cortes de tejido. Por ultimo, se discute la aplicacion de estas tecnicas a la investigacion experimental y clinica.

G protein coupling of galanin, muscarinic and cannabinoid receptors in areas related to the control of memory and cognitive functions of Alzheimer patients

was to determine the effects of beta amyloid 1-42 treatment on VDR expression in primary cortical neuron cultures. Methods: Cerebral cortex dissected from brains of Sprague Dawley rat embryos on the embryonic day 16 and cultured. The groups including 48 hours of 6 uM beta amyloid 1-42 treated group and control groups, were established. mRNA isolation and cDNA synthesis performed. The levels of VDR expressions were determined by RTqPCR. Localization of VDR was identified by immunofluorescent immunocytochemistry. Results: Expression of VDR in beta amyloid 1-42 treated group was found decreased when compared with control group. The immunolocalization of VDR was observed in both nucleus and cytoplasm of the primary cultured neurons of cerebral cortex. Conclusions: Relative attenuation in the levels of VDR expression by beta amyloid treatment in primary cortical neurons, might indicate potential role of beta amyloid to prevent probable neuroprotective effects of vitamin D in brain.