Distribution of lipids in human brain - Springer Link

Anal Bioanal Chem (2011) 401:89–101 DOI 10.1007/s00216-011-4882-x

ORIGINAL PAPER

Distribution of lipids in human brain Antonio Veloso & Roberto Fernández & Egoitz Astigarraga & Gabriel Barreda-Gómez & Iván Manuel & M. Teresa Giralt & Isidro Ferrer & Begoña Ochoa & Rafael Rodríguez-Puertas & José A. Fernández

Received: 30 December 2010 / Revised: 25 February 2011 / Accepted: 7 March 2011 / Published online: 26 March 2011 # Springer-Verlag 2011

Abstract The enormous abundance of lipid molecules in the central nervous system (CNS) suggests that their role is not limited to be structural and energetic components of cells. Over the last decades, some lipids in the CNS have been identified as intracellular signalers, while others are known to act as neuromodulators of neurotransmission through binding to specific receptors. Neurotransmitters of lipidic nature, currently known as neurolipids, are synthesized during the metabolism of phospholipid precursors present in cell membranes. Therefore, the anatomical identification of each of the different lipid species in human CNS by imaging mass spectrometry (IMS), in association

Published in the special issue MALDI Imaging with Guest Editor Olivier Laprévote. A. Veloso : R. Fernández : E. Astigarraga : J. A. Fernández (*) Department of Chemical Physics, Faculty of Science and Technology, University of the Basque Country, Barrio Sarriena s/n, 48940 Leioa, Spain e-mail: [email protected] G. Barreda-Gómez : I. Manuel : M. T. Giralt : R. Rodríguez-Puertas Department of Pharmacology, University of the Basque Country, Barrio Sarriena s/n, 48940 Leioa, Spain B. Ochoa Department of Physiology; Faculty of Medicine and Dentistry, University of the Basque Country, B Sarriena s/n, 48940 Leioa, Spain I. Ferrer Institute of Neuropathology, IDIBELL, University Hospital Bellvitge, Hospitalet de Llobregat, CIBERNED 08907 Barcelona, Spain

with other biochemical techniques with spatial resolution, can increase our knowledge on the precise metabolic routes that synthesize these neurolipids and their localization. The present study shows the lipid distribution obtained by MALDI-TOF IMS in human frontal cortex, hippocampus, and striatal area, together with functional autoradiography of cannabinoid and LPA receptors. The combined application of these methods to postmortem human brain samples may be envisioned as critical to further understand neurological diseases, in general, and particularly, the neurodegeneration that accompanies Alzheimer’s disease. Keywords Cannabinoid . Lysophosphatidic acid . MALDI-TOF . Mercaptobenzothiazole . Lipidomics . Imaging mass spectrometry Abbreviations 2-AG 2-Arachidonylglycerol AEA Anandamide CB Cannabinoids CNS Central nervous system eCB Endocannabinoids GPCR G-protein coupled receptor IMS Imaging mass spectrometry LPA Lysophosphatidic acid MALDI Matrix-assisted laser desorption/ionization MBT 2-Mercaptobenzothiazole MS Mass spectrometry PC Phosphatidylcholines PC(O) Plasmalogen PCA Principal component analysis PE Phosphatidylethanolamines PG Phosphatidylglycerols PI Phosphatidylinositols PLSA Probabilistic latent semantic analysis

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PS PUFA SF SM TOF

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Phosphatidylserines Polyunsaturated fatty acids Sulfatides Sphingomyelin Time of flight

Introduction Lipids represent 50–60% of the constituents of cell membranes, with the glycerophospholipid class being the most abundant. In some tissues and organs, such as the brain, they account for 20–25% of the dry weight and an enormous variety of molecular species exists [1]. The different glycerophospholipid species, together with cholesterol and sphingolipids, are the major components of cell membranes and determine the structure and fluidity of somatodendritic and axonal membranes of neurons, and also of those of the glia and organelles. Brain lipids are also involved in important metabolic pathways, usually related to the energetic homeostasis of cells. Whereas the role of brain lipids in intracellular signaling processes has been known for decades (e.g., phosphoinositides), their implication in neurotransmission is acquiring relevance due to their participation in cell proliferation, growth, and neuroprotection [2]. Some lipid molecules mediate their action by binding to specific receptors [3], e.g., cannabinoid receptors (CB), receptors for lysophosphatidic acid (LPA) or sphingomyelin (SM), and some orphan G-protein coupled receptors (GPCR) such as GPR55 [4]. Therefore, this type of lipids can be named “neurolipids”, in a similar way to the term “neuropeptides”, constituting a new class of neurotransmitters in the nervous system. The biosynthesis of neurolipids seems to be both ondemand, instead of the classical vesicular storage and release of monoaminergic neurotransmitters [5], and closely linked to the metabolism of membrane phospholipids, which is controlled by a myriad of specific enzymes, mainly phospholipases [6]. Thus, the specialization of the multiple types of phospholipases is acquiring a new significance. In this context, the diversity of brain lipid species is a further example of the central nervous system (CNS) specialization and complexity as a consequence of neurochemical evolution. Mammalian brains, especially the human brain, are at the top of this biochemical sophistication. The areas, nucleus, and even neurons of the CNS are highly specialized in controlling physiological processes which are reflected in specific neurotransmitter pathways, receptors and proteins present in each of the anatomically differentiated areas, and that are useful for their characterization. There are multiple examples, such as the dopaminergic cells in substantia nigra and the cholinergic pathways in

basal forebrain, whose impairment has frequently been associated with neurological diseases: Parkinson’s and Alzheimer’s, respectively. The anatomical specialization has been recognized and accepted for proteins and for the multiple neurotransmitters of different biochemical composition. The distribution pattern of each of the different lipid species in the brain may indicate not only a different cellular composition (neurons, glia, oligodendrocytes, nervous tracts, vascular pericytes, etc.), but also the specialization of these cells in discrete areas and nucleus in the brain. For example, it has been demonstrated [7] that some fatty acids are more abundant in the human CNS, like 16:0, 18:0, 18:1, 18:2, 20:4, 22:5 and 22:6. Despite the fact that some combinations are found more frequently, it is by no means obvious why so many species are necessary in the CNS. Most of the analytical techniques employed to determine lipid composition rely on the efficient extraction of lipids prior to their analysis by HPLC and/or mass spectrometry (MS), both of which provide precise data on the abundance of the different classes and species of lipids and acyl chains. However, the localization of such species is lost during the process of homogenization and purification. Recently, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has allowed scanning tissue slices to determine the distribution of peptides and metabolites [8]. This technique consists of obtaining slices of ∼10–20 μm of a tissue, covering them with a suitable MALDI matrix and scanning them in a MALDI-time of flight (TOF) mass spectrometer, thereby obtaining spectra in the nodes of a predefined mesh, reaching a spatial resolution of microns [9]. Each image represents separately the integral of a single mass channel against the coordinates of the mesh. So far, this technique has been applied to the study of the distribution of peptides [10], lipids [11], and administered drugs [12] in different tissues. In the present study, we use MALDI-IMS to determine and compare the distribution of lipids in three different areas of human brain: frontal cortex, hippocampus, and striatal area. Each sample is scanned using positive and negative detection, and the distribution of more than forty different lipid species is shown. The statistical analysis of the obtained spectra demonstrates that the most relevant differences in lipid composition are obtained when white and gray matters are compared. The cells and fibers that constitute both types of brain matter are very different. Among such differences, it is remarkable that the previously reported observation on the preferential distribution of phosphatidylcholine (PC) 32:0 in gray matter and PC 36:1 in white matter observed in rat brain by MALDI-IMS [13], is also found in human brain. Other cerebral substructures also present differences in lipid composition that could reflect their physiological specialization.

Distribution of lipids in human brain

Experimental methods Postmortem human brain samples The human postmortem samples used in the present study were obtained from the Brain Bank of the Institute of Neuropathology (University Hospital Bellvitge, Barcelona). Human brains were dissected at autopsy after prior informed consent and with the institutional approval of the Ethics Committees of the University Hospital Bellvitge and the University of the Basque Country (CEISH/28/ 2010). The samples were obtained from patients who died suddenly after infarct of the myocardium and who had shown no evidence of neurological or metabolic disease. The neuropathological study disclosed no abnormalities in the brain. Following autopsy, the brain samples were immediately frozen at −80 °C and stored. MALDI-TOF IMS The frozen tissue was brought to −25 °C, and 20 μm thick slices were obtained in a cryostat (Microm, HM550) and mounted on conducting slides (Bruker Daltonics). The slides containing the tissue sections were kept at −25 °C until they were introduced into a MALDI metal holder. A saturated solution of 2-mercaptobenzothiazole (MBT; Sigma-Aldrich, St. Louis, MO) in methanol was employed as matrix solution as described previously [14, 15]. A uniform coat of matrix was applied to each tissue section using a sprayer (DESAGA, model SG1B), loaded with an MBT/methanol saturated solution. Spectra were acquired in positive linear and reflectron modes using a Bruker Reflex IV TOF mass spectrometer (Bremen, Germany), equipped with a N2 laser (337 nm, 9 Hz, 100 μJ max. energy). Typical settings were: laser energy at 45–60%, repulsion plate 20.0 KV, extraction plate 16.35 KV, lens voltage 9.9 KV, reflectron field 23.0 KV, detector gain x5 and extraction pulse delay 200 ns. Thirty shots on each single location were accumulated to construct each individual spectrum, with a total acquisition time of 2–9 h, depending on the size of the scanned area defined in the tissue slice. The spatial resolution of the mass images shown in the present study varies from 100 to 500 μm. Acquired spectra were aligned maximizing correlation with the overall averaged spectrum, normalized by using the total ion current and exported to the computer program Histomass™ in a compatible format using scripts written in Mathematica™ 6.0. Data analysis and visualization was also carried out with Histomass™ (NorayBioinformatics). The average spectrum of each IMS experiment was recalibrated by comparison with the spectrum obtained from the corresponding extract, in which the peaks positions were determined with ∼20 ppm accuracy (1σ) using polyethylene glycol as external calibrant [15].

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Spectra were acquired in a 500–2,000 Da mass range. Previous tests for the detection of cholesterol demonstrated that no peak attributable to cholesterol or cholesterol–H2O can be found using our present combination of mass spectrometer and MBT. Thus, matrix deflection was set to 500 Da to avoid detector saturation. A number of peaks with reasonable s/n ratio was detected in the 1,400– 1,900 Da range, probably partly due to cardiolipins. However, owing to the lack of a previous report on cardiolipin identification in positive mode, we have omitted such results from this work. The data from the IMS experiments were analyzed using a modified version of the probabilistic latent semantic analysis (PLSA) algorithm [15–17], programmed using Mathematica 6.0 and with a common principal component analysis (PCA) algorithm present in Matlab 2010b. This kind of data analysis allows us to automatically group the spectra obtained from the tissue according to their similarity, and to obtain the average spectra of each of the regions of interest. Usually, both algorithms easily distinguish gray and white matter in brain tissue sections. Peak assignment The human brain is quite complex and contains a large number of lipids that share similar masses. Without using MS/MS it is not possible to differentiate among chemical variants of lipids with identical numbers of acyclic carbons and double bonds; that is, with identical masses. Therefore, the identity of the acyl chains and the position of their double bonds could not be specified in this study. The experimental values of the peaks in the mass spectra were compared with databases such as Lipid MAPS (http://www. lipidmaps.org/) and Madison Metabolomics Consortium (http://mmcd.nmrfam.wisc.edu/). Fifty parts per million was chosen as the tolerance window in order not to miss any likely candidate, as we use only the m/z value for the assignment. Only the assignments in accordance with those reported in previous studies are accepted and presented. [35S]GTPγS autoradiographic binding assay The brain tissue samples used for the autoradiography experiments were also kept frozen at −80 °C until they were processed in a Microm cryostat to obtain 20 μm sections that were mounted onto gelatin-coated slides and stored at −25 °C until used. Whenever possible, slices which were consecutive to those of the IMS experiments were used. Tissue sections were air-dried for 15 min prior to the autoradiographic assay, then immersed in a Tris–HCl buffer 50 mM (pH 7.4) with 100 mM NaCl, 3 mM MgCl2 and 0.2 mM EGTA, and subsequently preincubated in the same Tris–Cl buffer 50 mM, but supplemented with 2 mM GDP

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and 1 mM DL-dithiothreitol. Finally, the sections were incubated for 2 h at 30 °C in the same buffer containing 0.04 nM [35S]GTPγS (1250 Ci/mmol; Perkin Elmer, Boston, MA, USA). Agonist-stimulated binding was measured under the same conditions in the presence of the specific GPCR agonists: WIN 55212–2 (10−4 M) for CB receptors and LPA (10−5 M) for LPA receptors. Nonspecific binding was determined in presence of non-labeled GTPγS 10 mM. Sections were washed twice in Tris–Cl buffer 50 mM (pH 7.4), dipped in distilled water, and airdried at 4 °C. The sections were exposed for 48 h to Kodak Biomax MR films with previously calibrated 14C standards (Amersham). Once the films were developed, these were scanned in order to obtain digital autoradiograms showing images for the G-protein activation by CB and LPA receptors.

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mounted onto gelatin-coated slides and being hydrated after thawing. The hydration was performed by immersing the samples for 5 min in ethanol solutions diluted progressively in water (100%, 96%, 70% and 50%). Then, sections were submerged in thionine solution for 5 min. Later, tissues were washed with deionized water and dehydrated in ethanol solutions. Subsequently, the slides were covered with mounting media and coverslips for an adequate conservation at room temperature.

Results

A section consecutive to those used for IMS was counterstained with thionine (Nissl procedure), and a human brain atlas was used for the identification of the different areas analyzed by MS. Briefly, the process involved tissues being

The human frontal cortex sections used in the present study (Fig. 1) show two anatomically differentiated regions by Nissl staining, corresponding to a cortical circumvolution where the internal white matter (light blue in the staining slice in Fig. 1) is distinguished from the surrounding gray matter (dark blue) that forms the cerebral envelopment. Accordingly, in a consecutive section, the statistical analysis of the IMS data also identifies these regions automatically. As shown in Fig. 1, the lipid composition of gray and white matter is sufficiently different to be

Fig. 1 Stained section of human brain cortex (top-left corner) following the Nissl procedure, followed by a representation of the components obtained by PCA: gray matter in blue, white matter in red and the most internal layers of the cortex and the transition zone between both types of matter in white. The rainbow color-coded images (0–100 color-bar indicates the relative intensity) correspond to the distribution of some representative lipids in the tissue, each image constructed from a specific m/z value in daltons. The proposed

assignments are shown in Table 1. The experiment was carried out using MBT as the matrix, as described in the methods section and positive reflectron detection. The resolution measured as the distance between consecutive spectra, was 200 μm. The last two images are autoradiograms showing in gray scale the relative abundance of Gproteins activated by the specific agonist of cannabinoid receptors, WIN 55,212-2 and LPA receptors. The bar in the stained section corresponds to 5 mm

Thionine staining (Nissl procedure)


statistically significant. At the resolution employed to create the density maps (200 μm), the transition area between gray and white matter is also differentiated (white in the PCA image of Fig. 1). The images constructed integrating the m/z channels corresponding to the lipid species identified in positive mode (Table 1) are also shown in Fig. 1. A first view indicates that some lipids exhibit a uniform distribution across the tissue, while others are predominantly located in a certain type of matter. For example, it is well known [18, 19] that PC 32:0 and PC 38:6 are predominantly found in gray matter, while PC 36:1 is usually concentrated in white substance. Accordingly, the images constructed by integrating Table 1 Lipid species identified in reflectron positive mode m/z

Assignment [Ref]

706.5 718.6 725.5 732.6 734.6 758.6 760.6 768.6

PC 14:0/16:0+H+ [29] PE 16:0/18:1v+H+ [29] SM 34:1+Na+ [37], [38], [39], [40] PC 16:0/16:1+H+ [29], [41] PC 16:0/16:0+H+ [29], [39], [41], [19], [42], [31], [43] PC 16:0/18:2+H+ [29], [37] PC 16:0/18:1+H+ [37], [39], [41], [19], [42], [31], [43] PC(O) 34:1+Na+ [32] PE 38:4+H+ [29], [39], [31] SM 36:1+K+ [19], [42], [43]

769.6

813.7 815.5 826.6 828.6 830.6

PE 18:0/20:3+H+ [29] PC 16:0/16:1+K+, [43] PC 16:1/18:2+Na+ [37] PC 36:5+H+ [29] PC 16:0/18:0+Na+ [41], [32] PC 36:3+H+ [29], [41] PC 36:2+H+ [29], [41],[31] PC 18:0/18:0+H+ [29] PE 18:0/22:6+H+ [29], [31] PE 18:0/22:4+H+ [29], [19] PC 36:4+Na+ [37], [41], [43] PC 38:6+H+ [29], [37], [41], [31], [32], [43] PC 36:3+Na+ [41] SM 42:2+H+ [39], [40], [42] PG 36:1+K+ [39] PC 18:0/18:1+K+ [41], [19], [42], [43] PC 38:6+Na+ [41] PC 38:5+Na+ [41]

832.6 838.6 854.6 856.6

PC PC PC PC

770.6 780.6 784.6 786.6 790.6 792.6 796.6 804.6 806.6

38:4+Na+ [41], [32], [43] 18:0/22:4+H+ [29] 40:7+Na+ [41] 18:0/22:6+Na+ [41]

Assignment done by comparison of our assignment done following the procedure described in “Experimental methods” section with those in the references

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the mass-channels 734.6 (PC 32:0+H+), and 806.6 (PC 38:6+H+) respectively, show higher lipid density in gray matter; conversely, the image constructed by integrating m/z =826.6 (PC 36:1+K+) shows a higher intensity in white substance. It is also known that PC 34:1 gives the strongest intensity (indicative of its high abundance in brain tissue), and is evenly distributed in both types of matter; as it is also shown in the present work in the image constructed by integrating the m/z=760.6 (PC 34:1+H+). It is also worth noticing that some lipids predominantly found in white matter, such as m/z=718.6 (phosphatidylethanolamine (PE) 34:1+H+) are not homogeneously distributed along this matter, but they are found mainly in the central part, where the axon bundles are more concentrated, and therefore such lipid may be related to the myelin concentration. We have also tested if adducts of a given lipid species yield the same distribution map, as a further confirmation of the validity of the assignment. For example, the m/z=828.6, formerly assigned as PC 38:6+Na+ shows a preferential distribution in white matter, in contrast to what has previously been reported and what is observed for the proton adduct. Such an observation makes us to question such an assignment. Another likely candidate for this m/z channel is PC 36:0+K+ (m/z=828.6), which is preferentially located in white matter, as the distribution map of its proton adduct, m/z=790.6, shows . Nevertheless, it is necessary to record the MS/MS spectrum in order to establish the final assignment. The last two images in Fig. 1 are autoradiograms showing, in gray, the relative abundance of Gproteins activated by the specific agonist of cannabinoid receptors, WIN 55,212-2, and of LPA receptors. Human hippocampus exhibits a more complex anatomy than frontal cortex, as seen in the thionine-stained images in Fig. 2. The upper part includes the dentate gyrus, and shows several thin layers of white and gray matter. The base of the lateral ventricle is situated in the upper part of the dentate gyrus, where the cerebrospinal fluid circulates inside the brain. In the tissue used in the present study, the choroid plexus is located inside the lateral ventricle. This is a highly vascularized tissue, formed by ependymal cells whose structure is very different from the neuronal or glial cells. Two consecutive sections of human hippocampus were scanned. First, a scan at 500 μm resolution was recorded, to show the general lipid distribution over the hippocampus. Then, an area of 15×20 mm2 of the same section was scanned at 200 μm resolution to obtain a more detailed view of the lipid distribution. A third scan of a more restricted area of 8×18 mm2 covering the whole dentate gyrus was recorded at 100 μm resolution to obtain a more detailed lipid distribution. As the total number of spectra necessary to cover the area at such a resolution is 14,400, which exceeds the limit of

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Fig. 2

Stained section of human hippocampus (top-left corner). PCA representation of the experiment recorded at 500 μm resolution of an area of 30×20 mm2 of the section (2400 spectra, second image from the left). PCA representation of the experiment recorded at 200 μm resolution of an area of 15×20 mm2 of the same section (7200 spectra, third image from the left) and distribution of some representative lipids in the tissue with the corresponding m/z values in Da. The proposed assignments are shown in Table 1. An area of the same section centered in the dentate gyrus was scanned in two consecutive experiments (8×10 and 8×8 mm2 area), at 100 μm resolution, resulting in 8000 and 6400 spectra, respectively (left and right half and pasted together). The PCA analysis of the area and images built for each of the same selected lipids are also shown. The experiments were carried out using MBT as the matrix and reflectron positive detection. The autoradiograms (in this case from a different hippocampus sample) show in gray scale zone the relative abundance in the dentate gyrus of G-proteins activated by the specific agonist of cannabinoid receptors, and of LPA receptors. Bar 5 mm. CP choroid plexus; DG dentate gyrus; WM white matter; S subiculum

10,000 spectra of our mass spectrometer, two consecutive scans were needed to cover the entire region. The images were built selecting the same mass-channels as in Fig. 1. As it happens in the cortex, some lipids, such as PC 32:0 and

Fig. 3 Thionine-stained section of human striatum (top-left corner), corresponding mainly to the putamen nucleus (Pu) and the internal capsule white matter (ic). PCA representation of the components obtained by PLSA, showing the gray matter areas (caudate) in blue and the white matter (ic) in red. The rainbow color-coded distribution of some representative lipids in the tissue is shown, with the corresponding m/z values in Da. The proposed assignments are summarized in Table 1. The experiment was carried out using MBT

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PC 30:0, are present at a higher abundance in gray matter, while others such as phosphatidylglycerol (PG) 36:1, PC 36:0, PE 34:1, PE 40:4 and SM 42:2 are more abundant in white matter. The distribution of G-proteins activated by the specific agonist of cannabinoid receptors, WIN 55,212-2, and by LPA receptors is shown in the autoradiograms, where a higher density of CB receptors (presumably the CB1 subtype) in gray matter areas of the dentate gyrus zone is appreciated, specifically in the granular layer and also in the pyramidal layers of the subiculum, CA1 and CA3 hippocampal areas. The CB1-mediated activity is very scarce in white matter areas. Curiously, the anatomical distribution of LPA receptor-mediated G-protein activity in the hippocampus is similar to that observed for the CB1-mediated activity (Fig. 2, bottom). On the contrary, at the cerebral cortex and the striatal area, LPA receptor activity was recorded mainly in white matter-rich areas that are nervous tracts mainly composed of myelinated axons, e.g., the internal capsule. The distribution in the human striatum of some selected lipids is shown in Fig. 3, together with a consecutive

as matrix and reflectron positive detection. Dimensions of the scanned area are 17×15 mm2 and the distance between consecutive spectra was 200 μm, resulting in 6,375 spectra. The two autoradiograms (bottom-right corner) show in gray scale the relative abundance of Gproteins activated by the specific agonist of cannabinoid receptors, WIN 55,212-2, mainly located in the putamen and by LPA receptors in the white matter areas. Bar5 mm

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stained section for comparison, and with the autoradiograms showing the localization of the G-proteins activated by CB and LPA receptors. Two main areas can be distinguished in the striatum, which is mainly composed by white and gray matter, and that are clearly identified by the PCA image in red and blue, respectively. Accordingly, the lipid distribution in such areas matches that observed in cortex and in hippocampus (Figs. 1 and 2), demonstrating that the general lipid composition of white and gray matter is preserved all over the brain. In addition, the intensity of some lipid species show variations inside the putamen nucleus, as is the case of the m/z 734.6 (PC 32:0+H+) and the 760.6 (PC 34:1+H+) that could be related to the fiber tracts that cross this striatal area or even other substructures such as the striosomes. Two sections corresponding to the hippocampus and to the striatal area were also scanned with the MALDI-TOF configuration for negative ions detection. The images built with selected lipids are shown in Figs. 4 and 5. The statistical analysis gives a higher contrast between gray and white matter when the experiment is carried out with negative detection, probably because the strongest features in the negative spectra correspond to lipids that are almost exclusively located in white matter such as sulfatide (SF) 24:0 and SF 24:1. Figure 6 shows the average spectra in positive mode, corresponding to each of the three different brain areas that were analyzed: frontal cortex, hippocampus, and striatum. The difference in the PC 34:1 intensity between the three samples is very likely due to detector saturation. Apart from this, the overall aspect of all three average spectra is quite

similar, and the differences in the ratio of white/gray matter probably account for most of the variations in the signal intensity detected for each of the lipid species among the three different brain areas. The highest intensities were measured for PC 34:1 (m/z 760.6) that is evenly distributed in gray and white matter. Actually, the PC 34:1 signal is so strong in some spectra that it causes detector saturation. Although it is difficult to compare the different areas analyzed, the intensity of PC 32:0+H+ in all three tissues would indicate the relative abundance of gray/white matter in each brain area. PC 32:0 is considerably more abundant in gray than in white matter, and almost absent from choroid plexus cells (Fig. 7). The density maps presented in Figs. 1, 2, and 3 point to a relationship between PC 32:0 abundance and the presence of gray matter cells: neurons and their dendrites, and certain types of glial cells, mainly astrocytes. However, PC 36:1 shows the opposite anatomical distribution in hippocampus: its highest intensity is in white matter and its lowest in gray and in the choroid plexus. The cells of white matter are mainly composed of oligodendrocytes that form the myelinated axons, which are grouped in bundles. Therefore, the PC 36:1 must be an important lipid for the physiology of these myelin sheaths. Detection in negative mode (average spectra not presented) shows marked differences in the distribution of the detected lipid species. For example, all the detected SF species are clearly distributed in the white matter areas (Figs. 4 and 5), and therefore they must be associated to the kind of cells that constitute those tissues.

Fig. 4 Thionine-stained section of human striatum (top-left corner) showing the putamen nucleus (Pu), mainly composed by gray matter and the internal capsule (ic) of white matter. Representation of the components obtained by PCA (blue–red image), and distribution of 15 representative lipid species corresponding to different m/z values in

Da, obtained in reflectron negative mode. Proposed assignments are shown in Table 2. The experiment was carried out using MBT as matrix, and reflectron negative mode. Scanned area dimensions are 17×15 mm2 and the distance between consecutive spectra was 200 μm, resulting in 6,375 spectra. Bar 5 mm


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Fig. 5 Thionine-stained section of human striatum (top-left corner). The section was scanned at two different resolutions in reflectron negative mode: a 15×20 mm2 section was scanned at 200 μm (7500 spectra), and the PCA and the images (built integrating) constructed from 15 representative lipids are presented in the top three rows.

Another section of 8×15 mm2 was scanned at 150 mm2 (bottom three rows) corresponding to the dentate gyrus zone including the fimbria of the fornix region composed of white matter. The proposed assignments are shown in Table 2. The experiment was carried out using MBT as the matrix and reflectron negative detection. Bar 5 mm

Discussion

different detectability of lipids in complex samples has been published [21], which has demonstrated that the detection limit of, for example PE, is fifty times higher than for PC (1 ng compared to 0.020 ng) when DHB is used. Thus, in the present study, detection of PE species is possible thanks to, in part, the higher s/n ratio obtained, in comparison with DHB. Nevertheless, the number of detected species is greater when using both positive and negative detection, as can be seen from the data presented above. Another important issue is the radiation penetration into the sample. UV radiation penetrates less in the sample than other, longer wavelengths, such as IR, which also desorbs a larger amount of matter in each shot. Thus, if a representative spectrum of the sample is to be obtained, it is important to average a large enough number of shots in each point, Using MALDI-TOF IMS, we analyzed the anatomical distribution of 43 lipid species in three areas of the human brain: frontal cortex, hippocampus, and striatal area. In

It is worth mentioning that for several decades, the localization of lipids in the human brain has been based on techniques of lipid extraction after previous dissection of the target cerebral area, followed by lipid identification according to the mean molecular weight by using chromatographic separation methods [15]. The present results obtained by IMS avoid all the extraction/purification steps, which may result in modification of the original lipid composition and yield a more precise information on the anatomical localization of the lipids studied. However, a major drawback of IMS is that as there is no previous separation step, strong ion suppression may lead to a decrease in the total number of species detected. Indeed, PC species yield stronger signal intensities than other classes at equivalent concentration (e.g., PC(o), PE, or DAG), which hampers their detection [20]. Very recently, a study on the

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Fig. 6 Average spectra over the whole surface of the three tissue slices, recorded in positive mode. m/z channels assigned accordingly with Table 1 are labeled. Please note that the number of points in each spectrum has been divided by 4 in order to speed data handling

accordance with previous studies [15, 22], the main differences were observed between white matter and gray matter. To summarize the results, we will omit results from different adducts of the same species, unless they are pertinent for the discussion, as all the adducts should present the same distribution map. Frontal cortex O’Brien and Sampson [23], using chromatographic techniques, were pioneers in analyzing the lipid composition of human brain frontal lobe. They identified SM species in gray matter with a mass of m/z 802.1 Da and of 745.1 Da in white matter. In this work we show that some specific sphingolipids, such as SM 42:2+H+ (813.7), are located mainly in white matter. As for glycerophospholipids, a group of PC are clearly more abundant in white matter: PC(O) 34:1+Na+ (768.6), PC 36:2+H+ (786.6), 36:0+H+ (790.6), and also some PE species are preferentially located in white matter, e.g. PE 34:1+H+ (718.6), PE 40:6+H+ (792.6). As a matter of fact, all of the PE species we were able to identify are

more abundant in white matter. The SF species, which are exclusively detected in negative mode (Table 2), are also prominently distributed in white matter, as it could be expected from this class of myelin-specific sphingolipids. Nevertheless, SF could also be present in gray matter, as sulfatides are almost exclusively synthesized by oligodendrocytes in the CNS and are present predominantly in the myelin sheath surrounding axons and thus could also be present in gray matter, although in a lesser extent. On the other hand, as already mentioned, PC 30:0, PC 32:0 and PC 38:6 are the most abundant lipid species in the gray matter of the frontal cortex. Söderberg et al. [24] also reported differences in lipid distribution between white and gray matter. Additionally, they estimated that the ratio PE/PC was around 1.7 for the white matter and 0.8 for the gray matter. In the current work, although we have not used quantitative methodology, a similar trend was observed. Most of the glycerophospholipids are involved in cellular processes in the CNS, including regulation of cell growth, signal transduction, cell adhesion and neuronal plasticity. The


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Fig. 7 Average spectra over some areas of hippocampus, with the m/z channels labeled according to Table 1. The areas corresponding to the spectra are numbered in the tissue image. Note that dentate gyrus and gray matter are very similar in their lipid composition. Bar 5 mm. The number of points in each spectrum was divided by 4 to speed data handling

endocannabinoids (eCB) anandamide (AEA) and 2arachidonylglycerol (2-AG) that specifically activate CB receptors, as well as LPA that activate LPA receptors, are lipidic metabolites formed during the metabolism of certain glycerol-containing phospholipids through pathways that are not fully understood [25]. Consequently, eCB and LPA may be considered as signaling molecules that share the regulation of such metabolic processes involving glycerophospho-

lipids. It may be, thus, inferred that the distribution of CB receptors and LPA receptors and that of the glycerophospholipids, precursors of CB and LPA agonists might be similar. This anatomical information would further increase our knowledge about the location, biosynthesis, and catabolism of eCB and LPA. The current work shows that the highest density of active CB and LPA receptors is in the deepest layers of the frontal cortex, and that there is also a high density of

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A. Veloso et al.

Table 2 Lipid species identified in reflectron negative mode m/z

assignment

726.5 728.6 788.5 790.5 806.5 821.5 834.5 857.5

PE (O) 36:2-H− PE (O) 36:1-H− PS 36:1-H− PE 40:6-H− SF 18:0-H−/PS 38:6-H− PG 40:6-H− PS 40:6-H−/SF 20:0-H− PI 36:4-H−

862.6 878.6 885.6 888.6 890.6 904.6 906.6

SF 22:0-H− SF 22:0 (OH)-H− PI 38:4-H− SF 24:1-H− SF 24:0-H− SF 24:1 (OH)-H− SF 24:0 (OH)-H−

Assignment done by comparison with [19, 44]

LPA receptors in the adjacent white matter. So far, none of the lipids studied in this work present clearly a distribution similar to that of the eCB or LPA receptors. It well may be that what correlates with the receptor distribution is not a single lipid species but the distribution of specific fatty acids. Unfortunately, the lack of MS/MS capabilities of our spectrometer prevents us from carrying out such kind of studies. Hippocampus The analysis of the lipid composition in the hippocampus also allows us to distinguish between white and gray matter, which are distributed in thin layers depending on the localization of the neuronal somas and their fibers, with the main gray matter area being the layer of pyramidal cells distributed along the median part, going from the dentate gyrus to the subicular area. PC 32:0 and PC 30:0 seem to be specific of this layer of gray matter, whereas PE 40:4+H+ (796.6), SM 42:2+Na+, PC 36:0+H+ (790.6) and PG 36:1+K+ (815.5) are more abundant in white matter-rich areas. The present study analyzes in detail the distribution of dozens of lipid species present in the human hippocampus in both gray and white matter. Differences in the total amount of polyunsaturated fatty acids (PUFA) has previously been described between both types of tissues [26]: adrenic acid (22:4n−6) is reported to be ten times more abundant in white matter than in gray matter. On the contrary, gray matter is enriched in omega-3 PUFA, such as docosahexanoic acid (22:6n−3), between three and five times more than in white matter in the para-hippocampal cortex. Lipid species, especially phospholipids, could also be related to the presence in the hippocampal area of eCB and

their receptors, as it is shown in the present work when the localization of the G-proteins activated by a cannabinoid receptor agonist was analyzed. A high density of CB receptors has been reported in human hippocampus [27], which coincides with the distribution of the functional CB receptors presented in this work. The synthesis of eCB, that is accepted to be on-demand, could be anatomically coincident with the presence of some glycerophospholipids that could be eCB precursors. This could be the case of the PE 36:0+H+ (748.6), PE 40:4+H+ (796.6) in white matter and the PE 38:4+H+ (768.6) in gray matter. PE 36:4 and PE 38:4 have been described as possible precursors of AEA [28]. The lipid at m/z 768.8, which is present in both gray and white matter, has been assigned by some authors [29–31] to PE 38:4, while others [32] have assigned it as PC(O) 34:1+Na+. If the former assignment, which is backed up by more groups is correct, it might point to PE 38:4 as a possible precursor of AEA. Also the other eCB, 2-AG, is thought to be formed from the metabolism of arachidonic acid-containing phospholipid species [25]. Therefore, the most suitable candidates to be eCB precursors should be those located in gray matter, where the CB functional receptors are preferentially found. Further studies may indicate if the combination of the MALDI-TOF MS/MS-IMS with receptor autoradiography and/or other imaging methods can become a suitable way to elucidate metabolic routes. Finally, LPA activity in the hippocampus is not restricted to white matter areas and was quite similar to that of CB functional receptors. Striatum With regards to the caudate–putamen area, also known as the striatum, we have performed the study mainly in the putamen nucleus and the white matter area that is adjacent to this nucleus and separates it from the caudate, i.e., the internal capsule. PC 32:0+H+ (734.6), PC 34:1+H+ (760.6), SM 36:1+K+ (769.6), and PC 38:6+Na+ have proved to be characteristic of the putamen. Some of the most important neurotransmitters present in this area are acetylcholine and dopamine. There is also an elevated density of CB1 receptors, as previously reported [27, 33, 34]. The possible presence of choline- and arachidonic acid-containing precursor phospholipids in the striatum would also support the hypothesis of that there is an anatomical relationship between certain species of PL and some neurotransmitters and their receptors. The LPA activity recorded in the striatum was clearly distributed along the areas of white matter, in the internal capsule, and along the patches of fibers within the putamen nucleus. Although the exact LPA biosynthesis has still not been clarified, two different possibilities have been proposed, both of which involve the metabolism of phospholipids


(probably PC) and different subtypes of phospholipases (PLA1 and PLA2) [35]. The anatomical correlation of LPA activity and the location of specific phospholipases and certain lipid species, could shed some light on LPA production in the brain.

Conclusions IMS by MALDI-TOF allows the study of multiple lipid species in discrete areas of the human brain obtained from postmortem frozen samples at −80 °C. The method can be combined with other classical imaging techniques used in vitro to localize neurotransmitter receptors and their functionality, as is shown by [35S]GTPγS autoradiography. The optical resolution of both methods is quite similar for the moment. This combination is especially interesting for the elucidation of possible metabolic routes between phospholipid metabolism and neurotransmitter signaling that are of lipid nature, such as that of eCB, that mediate their action through GPCR, or LPA, SM or other fatty acid derivatives. Moreover, some N-acylamides have been classified as orphan lipids [36] and there is an increasing characterization of orphan GPCR as receptors for neurolipids. Therefore, the future development of IMS techniques is set to have a key role in the matching process between new isolated orphan lipids and GPCR for neurolipids. Acknowledgements This study was supported by the Spanish Ministry of Education and Science (SAF2007-60211), the Basque Government (IT-325-07, IT-336-10, IT-440-10, S-PE10UN50 and SAI07/46) and Carlos III Health Institute (FIS PI070628). A. V. is recipient of a UPV/EHU graduate fellowship.

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