B American Society for Mass Spectrometry, 2016 J. Am. Soc. Mass Spectrom. (2016) 27:1944Y1951DOI: 10.1007/s13361-016-1475-0

RESEARCH ARTICLE

Direct Visualization of Neurotransmitters in Rat Brain Slicesby Desorption Electrospray Ionization Mass SpectrometryImaging (DESI - MS)

Anna Maria A. P. Fernandes,1 Pedro H. Vendramini,1 Renan Galaverna,2

Nicolas V. Schwab,1 Luciane C. Alberici,3 Rodinei Augusti,4 Roger F. Castilho,5

Marcos N. Eberlin1

1Thomson Mass Spectrometry Laboratory, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil2Instituto de Química, UNICAMP, Campinas, SP, Brazil3Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, RibeirãoPreto, SP, Brazil4Departamento de Química, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil5Departamento de Patologia Clínica, Faculdade de Ciências Médicas, UNICAMP, Campinas, SP, Brazil

Abstract. Mass spectrometry imaging (MSI) of neurotransmitters has so far beenmainly performed by matrix-assisted laser desorption/ionization (MALDI) where de-rivatization reagents, deuterated matrix and/or high resolution, or tandem MS havebeen applied to circumvent problems with interfering ion peaks from matrix and fromisobaric species. We herein describe the application of desorption electrosprayionization mass spectrometry imaging (DESI)-MSI in rat brain coronal and sagittalslices for direct spatial monitoring of neurotransmitters and choline with no need ofderivatization reagents and/or deuterated materials. The amino acids γ-aminobutyric(GABA), glutamate, aspartate, serine, as well as acetylcholine, dopamine, andcholine were successfully imaged using a commercial DESI source coupled to a

hybrid quadrupole-Orbitrap mass spectrometer. The spatial distribution of the analyzed compounds in differentbrain regions was determined. We conclude that the ambient matrix-free DESI-MSI is suitable for neurotrans-mitter imaging and could be applied in studies that involve evaluation of imbalances in neurotransmitters levels.Keywords: Mass spectrometry imaging, Neurotransmitters, Desorption electrospray ionization, Rat brain

Received: 8 April 2016/Revised: 30 June 2016/Accepted: 31 July 2016/Published Online: 4 October 2016

Introduction

In the human body, arguably the brain is of most fundamentalimportance because it controls thinking, feelings, and mem-

ories, and also our major actions and reactions. The brain is alsoan incredibly complex organ integrating many parts. Althoughit comprises only ca. 2% of the body weight of an adult human,

it accounts for nearly 20% of the consumed energy [1]. Knowl-edge of brain operating systems is essential in order to seek acure for brain disorders such as drug addiction, depression, anda series of neurodegenerative diseases.

Neurotransmitters and neuromodulators play prominentroles in brain functioning: the former by promoting theintersynaptic signal transmission and the latter by modu-lating postsynaptic events. Of these two, neurotransmittersare the most common class of chemical messengers in thenervous system [2]. These relatively low molecular weightmolecules (<200 Da) are subdivided into two main groupsaccording to their chemical structure (i.e., biogenic aminesand amino acids). They have been studied in brain tissuemainly by indirect analyses [3], employing techniques thatare also time- and labor-consuming, and commonly withpoor specificity [4].

Electronic supplementary material The online version of this article (doi:10.1007/s13361-016-1475-0) contains supplementary material, which is availableto authorized users.

Correspondence to: Anna A. P. Fernandes;e-mail: annamabr@yahoo.com.br, Marcos N. Eberlin;e-mail: eberlin@iqm.unicamp.br

A major breakthrough in tissue analysis has been launchedby mass spectrometry imaging (MSI) [5, 6]. MSI combines thespeed, sensitivity, and selectivity of MS with spatial distribu-tion analysis at the molecular level to provide a new dimensionfor histology. MSI has enabled the 2D visualization of thearrangement of many types of biomolecules in different tissues[7]. Recently, MSI has also been applied to the detection andspatial localization of neurotransmitters [8, 9]. Matrix-assistedlaser desorption ionization (MALDI)-MSI, described initiallyby Caprioli and co-workers [10], has been the ionization tech-nique used in the majority of studies on this subject. Forexample, acetylcholine has been imaged by MALDI MS/MS-based MSI [11], MALDI-MSI using a deuterated matrix [12,13], and MALDI high-resolution and high-accuracy MS(HRMS) [14]. The biogenic amine dopamine [13, 15, 16] andthe amino acids γ-aminobutyric (GABA) [13, 15–19], glutamic[15, 16], and aspartic acids and serine [16] have also beenimaged by MSI using MALDI as the ionization technique inall but one of these studies [17].

MALDI operates under vacuum and requires a matrix as aproton donor that should be co-crystallized on the tissue sur-face. The quality of the crystals and the homogeneity of thematrix deposition are crucial for the image quality. Addition-ally, for analysis of low molecular weight compounds, such asneurotransmitters, the matrix represents a limiting factor be-cause it produces many other isobaric interferences [20].

As an alternative, desorption electrospray ionization (DESI)has offered an ambient desorption/ionization technique suitablefor MSI. As an ambient technique, DESI operates under atmo-spheric pressure [21]. DESI is also matrix-free and can utilizedifferent types of spray solvents to improve analyte selectivityand nondestructively analyze tissue [22]. The absence ofmatrixnot only simplifies the workflow but also enables the DESImethod to be more straightforward in neurotransmitter detec-tion because it is free from matrix interferences. Herein, wedemonstrate that DESI-MSI coupled to a high resolution andhigh accuracy mass spectrometer is able to provide properspatial distribution of GABA, glutamate, aspartate, serine, ace-tylcholine, dopamine and choline in rat brain slices. A prelim-inary report on this study has been presented [23].

ExperimentalMaterials and Methods

Unless otherwise stated, all chemicals and reagents were fromSigma-Aldrich, (Saint Louis, MO, USA) and used withoutfurther purification. Sodium pentobarbital was from CristáliaProdutos Químicos e Farmacêuticos Ltda (Campinas, SP,Brazil).

Mixture of Standards

Stock solutions of GABA (100 mM), choline chloride (120mM), acetylcholine acetate (90 mM), aspartic acid (20 mM),glutamic acid (14 mM), serine (100 mM), and dopamine

chloride (50 mM) were prepared using distilled and deionizedwater (dd water) and were kept at −20 °C until use. On the dayof the experiment, working solutions were made by diluting thestock solutions in dd water at a final concentration of 5 mM ofeach standard.

Animal Dissection

Male adult Wistar rats (300–400 g) were deeply anesthetizedwith sodium pentobarbital (75 mg/kg, i.p.) and were killed bydecapitation. All procedures were approved by the institutionalanimal care committee at UNICAMP (CEUA Protocol num-bers 2534–1 and 4123–1), and experiments were performed inaccordance with the guidelines for animal care. After decapi-tation, the brain was dissected within 1 min and was immedi-ately deeply frozen (for cryosectioning) in liquid nitrogen.Alternatively, the brain was kept in an ice bath and homoge-nized. The imaging experiments were conducted with brainsobtained from two different rats. One brain was used forsagittal sections and the other for coronal sections. The cerebraland cerebellar extracts were prepared from a third animal.

Tissue Section Preparation

The frozen brains were cut using a Leica CM 1900 cryostat-microtome (Leica Biosystems, Nussloch, Germany). Sagittaland coronal brain sections were cut at a thickness of 14 μm.Tissue sections were transferred by thaw mounting onto con-ventional microscope glass slides without any surface treat-ment and were stored at −80 °C. Sections were desiccated atroom temperature 15 min before use.

Brain Extracts

Cerebral and cerebellar homogenates were prepared accordingto the literature [24, 25] with modifications. After decapitation,the cerebrum (one hemisphere ~600 mg) and cerebellum (~400mg) were collected and placed in 2 and 1.5 mL, respectively, ofdd water. Tissues weremanually homogenized on ice in a 5mLGlass/PTFE Potter Elvehjem Tissue Grinder (Kimble Chase,Vineland, NJ, USA) until no chunks remained (20–30 strokesof the pestle) and were centrifuged (Smart R17; Hanil ScienceIndustrial Co. Ltd., (Gimpo, Gyeonggi Province, SouthKorea)) at 14,000 rpm and 10 °C for 10 min. Supernatantswere collected and stored at −20 °C until use.

Spot Analysis

Twomicroliters of cerebral or cerebellar homogenates or work-ing solution of standards were spotted in an Omni Slide(Prosolia Inc., Indianapolis, IN, USA) and analyzed underDESI conditions. Each homogenate or solution was analyzedin triplicate. Initially, spots were analyzed in the positive ionmode and were then subsequentely analyzed in the negative ionmode, which makes the images of the spots in the negative ionmode look splashed.

A. M. A. P. Fernandes et al.: Direct Imaging of Neurotransmitters by Matrix-Free DESI-MSI 1945

DESI-MS and MSI Analyses

The analyses were performed in a Q-Exactive (Thermo Scien-tific, Bremen, Germany) mass spectrometer with a resolutionof 140,000 at m/z 400 coupled with an Omni Spray Ion Source2-D (Prosolia Inc.) for data acquisition. Firefly v.1.3.0.0 dataconversion was used to generate the images, which were treat-ed in BioMAp3804. The step size was 0.15 μm, the flow ratewas 3.0 μL.min−1 and the surface scan rate was 600 μm.s−1.Analyses were performed in either the negative and or positiveion modes. The S-Lens RF level was set to 20 in order toincrease the transmission of low m/z ions, the capillary temper-ature was 280 °C and the spray voltage was 3.4 kV. The imageswere acquired with 75,000 resolution. The signals attributed tothe neurotransmitters were accurately assigned with errors lessthan 2 ppm (see Supporting Information for chemical structuresand details). MSI was performed with a spatial resolution [26]of 200 μm (See Supporting Information for details - Figure S1).

Synthesis of Acetylcholine

The acetylation of choline was performed according to theliterature [27] with slight modifications. Experimental synthe-sis and spectroscopic data (1H and 13C NMR) are available inthe Supporting Information.

Results and DiscussionNeurotransmitters in rat brain slices have been imaged byMSI since 2010 [17] exclusively using MALDI as theionization technique. One exception was the pioneeringwork on neurotransmitter imaging that used laser ablationelectrospray ionization MS to image GABA and choline[17]. In addition, DESI-MSI was applied to image nor-epinephrine and epinephrine in porcine and slices ofrabbit adrenal glands [28].

The relatively low concentration of neurotransmitters andthe presence of other easily ionizable and abundant biomole-cules such as lipids make the process of neurotransmitterimaging in brain slices similar to ‘looking for a needle in ahaystack’ [9, 29, 30]. Another challenge is the presence ofmany isobaric molecules from the tissues or from the matrix

in MALDI-MSI, which interfere with low-resolution MS anal-yses. In this scenario, DESI-MSI could contribute by specifi-cally circumventing the problem of isobaric low molecularweight interferences from the matrix.

We therefore first examined ion suppression under DESI-MSI conditions for neurotransmitter analysis. For that we usedan equimolar mixture of standards. Table 1 presents the ionsvia their m/z ratios observed in the spectra in Figure 1. Wefound the following preference for ionization in the positive ionmode: acetylcholine ofm/z 146.118 > choline ofm/z 104.107 >[dopamine + H]+ of m/z 154.086 > [GABA + H]+ of m/z104.071 (Figure 1a). Fortunately, acetylcholine and cholineare cationic species, making them readily ionized by DESI,resulting in the two more intense ion peaks. Dopamine is astrong base, is easily protonated, and produces a strong ionpeak. In contrast to the isolated molecule, which produces anintense DESI-MS ion peak, the peak intensity of protonatedGABA decreases to only 1% of the original value when presentin the equimolecular mixture tested (not shown). This decreaseindicates intense ion suppression [31, 32] suffered by GABAduring DESI-MSI analysis of neurotransmitters in tissues. Peakattribution for these neurotransmitters performed by DESI-MSoperating with a resolution of 140 K and a mass accuracy ofless than 2 ppm (Supplementary Table S1), in rat cerebral andcerebellar extracts. In the cerebellar extract, GABA, choline,and acetylcholine could be detected in the positive ion mode,with choline producing the most intense ion peak (Figure 1b).In the cerebral extract (Figure 1c), only acetylcholine andcholine were detected, with the strong prevalence of the ionpeak of choline over that of the acetylcholine neurotransmitter.The analysis of the mixture of standards in the negative ionmode revealed that serine suffers strong ion suppression fromboth the glutamic and aspartic acids (Figure 1d). The prefer-ence for DESI(−) ionization was: [aspartate – H]− of m/z132.029 > [glutamate – H]− of m/z 146.046 > [serine – H]− ofm/z 104.035. All of these amino acids could also be detected inthe cerebellar (Figure 1e) and cerebral (Figure 1f) extracts.

In addition to the use of HRMS Orbitrap for the unambig-uous DESI detection of these neurotransmitters in cerebellarand cerebral extracts, we performed simultaneous DESI-MSIof the mixture of the standards and the extracts. Figure 2 showsthe ion images generated in either the positive (Figure 2a–d) ornegative ion modes (Figure 2e–f). The concomitant detectionof all of these molecules in the mixtures of standards and in theextracts was observed. The direct comparison between thecerebellar (lines 2) and cerebral extracts (lines 3) shows thatacetylcholine (Figure 2c) seems to be more abundant in cere-bral extracts compared with cerebellar extracts. In contrast,glutamate (Figure 2g) is more abundant in cerebellar extracts(line 2). The abundance of choline is also notably larger in theextracts than in the mixture of the standards, which may occurbecause of better ionization of choline in the spots of theextracts due to low ionic suppression exerted by the otheranalytes, as by acetylcholine in particular. The increase in theconcentration of choline during the preparation of the extractsmay also exacerbate this effect [33].

Table 1. Mass‐To‐Charge Ratios (m/z) Observed in Figure 1

POSITIVE ION MODE[M + H]+ Observed m/zGABA 104.071Dopamine 154.086

[M]+ Observed m/zCholine 104.107Acetylcholine 146.118

NEGATIVE ION MODE[M – H]− Observed m/zGlutamate 146.046Aspartate 132.029Serine 104.035

1946 A. M. A. P. Fernandes et al.: Direct Imaging of Neurotransmitters by Matrix-Free DESI-MSI

The concern about misassignments of these low molecularweight neurotransmitter with a myriad of isobars (and isomers)in MSI has been previously raised [19], but the use of a HRMSOrbitrap analyzer and the concomitant image of the extractsand standards monitoring the samem/z value helps to minimizemisassignments. Indeed, Figure 2 confirms that neurotransmit-ters can be imaged by DESI-MSI with proper visualization oftheir spatial distributions.

An advantage of avoiding derivatization agents is the possibil-ity of imaging a broad range of molecules under the same condi-tions or in the same tissue slices while avoiding the risk of ionsuppression or interfering side reactions [34]. Figure 3a shows theimage of a sagittal rat brain slice generated from deprotonatedsulfatide 24:1 of m/z 888.600, which is found to be strictlyconnected to thewhite matter and brainstem [35]. Figure 3f showsthe image of a coronal rat brain slice generated from thedeprotonated dimer of docosahexaenoic acid (DHA) [36] of m/z654.567, which shows to be more abundant in the striatum. Theseimages show the versatility of DESI-MSI as an alternative for themore common Nissl stain protocol (Figure 3k and l) to definebrain structures. Figure 3b and g display images generated by theion of m/z 146.118 corresponding to the cationic acetylcholine inthe sagittal and coronal sections, respectively. These images show

greater relative abundance of this neurotransmitter in the striatum,thalamus, and midbrain. Despite the relatively high concentrationof cholinergic neurons in the brain, acetylcholine has beenmapped only indirectly via its receptors or by enzymes relatedto their synthesis or degradation [15]. MALDI-MS/MS imagingwas used to map acetylcholine in sagittal rat brain slices [11], andhighest concentrations were found in the hippocampus, thalamus,and striatum. A different spatial distribution of acetylcholine wasobtained by MALDI-MSI using D4-α-cyano-4-hydroxycinnamicacid (D4-CHCA) as the matrix [12], which indicated that acetyl-choline is most abundant in the cortex, corpus callosum, ventralhippocampal commissure, thalamus, and cerebellum. MALDI-HRMS for rat brain slices also show major localization of acetyl-choline in the cortex and brainstem [14], whereas high spatialresolution MALDI-MSI found acetylcholine mainly in the stria-tum, hippocampus, thalamus, pons, and other small structures.This MALDI-MSI distribution was corroborated by the intraper-itoneal administration of tacrine, a central cholinesterase inhibitorthat evoked a 7-fold increase in the concentration of acetylcholine-enriched regions [15]. There is, therefore, good agreement be-tween our DESI-MSI spatial distribution of acetylcholine andseveral previous MALDI-MSI data from sagittal rat brainsections.

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Figure 1. DESI(+)-MS of neurotransmitter standards and extracts. (a) DESI(+)-MS of an equimolar mixture (5 mM) of GABA (m/z104.071, [M + H]+), choline (m/z 104.107, [M]+), acetylcholine (m/z 146.118, [M]+), and dopamine (m/z 154.086, [M + H]+). (b) DESI(+)-MSof the cerebellar extract. (c)DESI(+)-MS of the cerebral extract. (d) DESI(−)-MSof an equimolarmixture (5mM) of serine (104.035,[M –H]−, aspartic acid (132.029, [M –H]−), and glutamic acid (146.046, [M –H]−). (e)DESI(−)-MS of the cerebellar extract. (f)DESI(−)-MS of the cerebral extract. Color bars were added to facilitate visualization of the ion peaks of interest

A. M. A. P. Fernandes et al.: Direct Imaging of Neurotransmitters by Matrix-Free DESI-MSI 1947

The DESI-MS coronal view at the striatum level (Figure 3g)shows the presence of the acetylcholine neurotransmitter in thisbrain region extending toward the olfactory tubercle. As far aswe could check, no profiles of acetylcholine in coronal sectionshave been reported. High spatial resolution MALDI-MSI im-ages of acetylcholine in a sagittal rat brain section have, how-ever, been reported, which corroborate our findings and is inaccordance with the extensive axonal arborization found in thecell bodies of the striatal cholinergic interneurons [15].

The images in Figure 3c and h are for the spatial distribution ofthe neurotransmitter GABA, detected as its protonated molecule[GABA + H]+ of m/z 104.071. The sagittal image of Figure 3creveals more pronounced abundances of GABA in the thalamus,midbrain, and basal forebrain. In accordance with what wasobserved in the sagittal section, this neurotransmitter is in thecoronal section at the striatum level found most notably in thecortex and basal forebrain (Figure 3h). GABA is an inhibitoryneurotransmitter that was the first to be imaged byMS [17]. Morerecently, the use of a derivatization strategy to obtain MALDIimproved images of GABA distribution that closely resemble theimages shown here has been reported [15, 16]. In these works,GABA MSI of sagittal rat brain sections revealed the prevalenceof this neurotransmitter in the hypothalamus, midbrain, and basalforebrain. GABA was also found in the medial septum/diagonalband region (MSDB) of the brain [15]. GABA predominates inthe hypothalamus and is also important in the midbrain and basalforebrain and its substructures [15, 37]. These data are in accor-dance with the DESI-MSI obtained herein.

Choline is not a neurotransmitter but is a precursor forthe synthesis of phosphatidylcholines (PC), which

comprises one-half of the total membrane lipid content.The increase in cell proliferation and cell membrane syn-thesis during tumorigenesis is known to affect the cholinemetabolism. Three-dimensional profiles of choline andcholine-containing compounds are commonly obtained byproton magnetic resonance spectroscopy (1H-MRS) andpositron emission tomography (PET) to follow tumor pro-gression [38]. Few studies have, however, described map-ping of choline in rat brains. The first image for cholinedistribution in a rat brain used HRMS to resolve theisobaric ions from protonated GABA and choline so as toproduce selective MSI but showed very diffuse imagesfrom a coronal rat brain section with a slight prevalenceof choline in the basal region [17]. Sagittal MSI of cholinewas further collected in two additional MSI studies [14, 39],but a discussion about the anatomical distribution of this me-tabolite in the brain sections was not provided. The MSI for thesagittal section of choline of m/z 104.107 in Figure 3d showsthat its concentration is greater in the cortex, striatum, thala-mus, midbrain, and cerebellum. In agreement with these find-ings, the coronal section in Figure 3i shows a high concentra-tion of choline in the cortex and striatum.

Dopamine was also imaged (Figure 3e and j). This neuro-transmitter was not observed in the sagittal rat brain sections(Figure 3e), but the coronal section showed dopamine mostlyconcentrated in the striatum, in agreement with previous re-ports (Figure 3j) [13, 15, 16, 40, 41].

Ionic suppression plays an important but not exclusive rolein the intensity of the images generated. Choline ions producethe most intense images in the sagittal sections compared with

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Figure 2. DESI-MSI of the spots of the standard solutions (first line in eachPanel), cerebellar extract (second line in each Panel), andcerebral extract (third line in each Panel). (a–d) DESI(+)-MSI for (a) GABA, (b) choline, (c) acetylcholine, and (d) dopamine. (e – g)DESI(−)-MSI for (e) serine, (f) aspartic acid, and (g) glutamic acid. Scale bar: 2 mm

1948 A. M. A. P. Fernandes et al.: Direct Imaging of Neurotransmitters by Matrix-Free DESI-MSI

the other images presented in Figure 3. In the mixture ofstandards, the cationic choline produces, however, an ion thatis approximately one-third as abundant as that from the cationicacetylcholine (Figure 1a). The intense image of choline (Fig-ure 3d) is related to the abundance of this cationic species in therat brain and also to post-mortem changes, including hydroly-ses of acetylcholine to produce choline, during the dissectionprocedure [11, 33].

The amino acidic neurotransmitters glutamate, aspartate,and serine were imaged via DESI in the negative ion mode(Figure 4). Again, the images of the ions of m/z 654.567 and

888.600 were used as references. For glutamic acid of m/z146.046, the sagittal view indicates the prevalence of thisneurotransmitter in the cortex, thalamus, and cerebellum, butit can also be observed in the striatum (Figure 4b). This imageis in good agreement with the coronal view (Figure 4f), whichpresents a greater concentration of this neurotransmitter in thecortex and in the striatum. Few MSI studies for rat brain slicesmonitoring this neurotransmitter have been reported. Using2,4-diphenyl-pyranylium tetrafluoroborate (DPP-TFB) as a de-rivatization reagent, MALDI-MSI showed a more prominentpresence of glutamate in the cortex and striatum in rat brain

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Figure 3. (+)-DESI-MSI of neurotransmitters andmetabolites in sagittal (b–e) and coronal (g–j) and (−)-DESI-MSI of lipids in sagittal(a) and coronal (f) rat brain sections. Relative abundance and spatial distribution of the ions ofm/z 888.600 and 654.567 (a) and (f),respectively; STR = striatum, HPF = hippocampal formation, TH = thalamus, HY = hypothalamus, MB = midbrain, CB = cerebellum,P = pons, MY = medulla, CC = corpus callosum, CTX = cortex; acetylcholine (b) and (g); GABA (c) and (h); choline (d) and (i), anddopamine (e) and (j). Sagittal (k) and coronal (l) adjacent rat brain slices stained using the Nissl protocol. Scale bar: 5 mm

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Figure 4. (−)-DESI-MSI of neurotransmitters and lipids in sagittal (a–d) and coronal (e–h) rat brain sections. Relative abundance andspatial distribution ofm/z 654.567 and 888.600 (a) and (e), respectively; STR = striatum, TH = thalamus, MB =midbrain, CC = corpuscallosum, PAL = pallidum; glutamic acid (b) and (f); aspartic acid (c) and (g); serine (d) and (h). Scale bar: 5 mm

A. M. A. P. Fernandes et al.: Direct Imaging of Neurotransmitters by Matrix-Free DESI-MSI 1949

coronal sections [15]. A similar profile was observed in sagittaland coronal rat brain sections using not only DPP-TFB but alsop-N,N,N-trimethylammonioanilyl N-hydroxysuccinimidyl car-bamate iodide (TAHS) and 4-hydroxy-3-methoxy-cinnamaldehyde (CA) as derivatization agents and MALDI-MSI [16]. The results from both of these MSI studies are ingood agreement with the images reported herein.

In the literature, MSI of aspartic acid and serine are alsoscarce. Figure 4c shows the sagittal view of aspartate via theion of m/z 132.029, which is more abundant in the thalamus,midbrain, and cerebellum. The coronal section in Figure 4gshows prevalence of aspartate in the region of the pallidumfrom the striatum. MALDI images of aspartic acid obtainedafter derivatization with TAHS and CA have been reported,and the results are in good agreement with our DESI-MSI data[16]. Serine (m/z 104.035) was found to be more abundant inthe cortex and corpus callosum of the sagittal sections (Fig-ure 4d), and this distribution matches what is observed in thecoronal section (Figure 4h). Serine derivatized with TAHS wasfound in the striatum in the coronal section and in the hypo-thalamus, thalamus, and cortex in the sagittal section [16].

ConclusionWe have shown that the more direct and simpler ambientDESI-MSI technique is also able to reveal the spatial distribu-tion of neurotransmitters in rat brain slices. Clear and well-resolved images were obtained, whereas the use of a high-resolution mass spectrometer was shown to be essential inorder to address isobars and to collect selective images.DESI-MSI can, therefore, be incorporated into neuroscienceinvestigations for the spatial screening of neurotransmitterssuch as, for instance, in cases in which the abundance and/ordistributions of these important biomolecules are expected tochange.

When the present study was in the final stage of preparation,Bergman et al. [42] reported the absolute quantitation of theneurotransmitters acetylcholine, GABA, and glutamate in ratbrain coronal slices by another ambient, matrix-free, anddesorption-based ionization technique named nano-DESI-MSI. Our study corroborates and extends these findings, de-scribing a detailed spatial distribution of the above-mentionedneurotransmitters as well as of aspartate, serine, and dopaminein coronal and sagittal rat brain slices. As we have anticipated[23], these results enlarge the application of atmospheric pres-sure ionization techniques to the field of neuroscience.

AcknowledgmentsResearch funding was provided by Fundação de Amparo àPesquisa do Estado de São Paulo (no. 11/50400-0 and no. 10/51677-2) and Conselho Nacional de Desenvolvimento Científ-ico e Tecnológico. A.M.A.P.F. was supported by a postdoctor-al CNPq fellowship (150781/2014-8). The authors thank thereviewers for valuable suggestions.

References1. Raichle, M.E., Gusnard, D.A.: Appraising the brain’s energy budget. Proc.

Natl. Acad. Sci. U. S. A. 99, 10237–10239 (2002)2. und Halbach, O.B., Dermietzel, R.: Neurotransmitters and

neuromodulators: Handbook of receptors and biological effects, 2nd edn,pp. 1–6. Wiley-VHC, Weinheim (2006)

3. Merighi, A., Carmignoto, G.: Cellular and molecular methods in neurosci-ence research, 1st edn. Springer-Verlag, New York (2002)

4. Manuel, I., Barreda-Gómez, G., González de San Román, E., Veloso, A.,Fernández, J.A., Giralt, M.T., Rodríguez-Puertas, R.: Neurotransmitterreceptor localization: from autoradiography to imaging mass spectrometry.ACS Chem. Neurosci. 6, 362–373 (2015)

5. Gessel, M.M., Norris, J.L., Caprioli, R.M.: MALDI imaging mass spec-trometry: spatial molecular analysis to enable a new age of discovery. J.Proteom. 107, 71–82 (2014)

6. Wu, C., Dill, A.L., Eberlin, L.S., Cooks, R.G., Ifa, D.R.: Mass spectrometryimaging under ambient conditions. Mass Spectrom. Rev. 32, 218–243(2013)

7. Tata, A., Fernandes, A.M., Santos, V.G., Alberici, R.M., Araldi, D., Parada,C.A., Braguini, W., Veronez, L., Silva Bisson, G., Reis, F.H., Alberici,L.C., Eberlin, M.N.: Nanoassisted laser desorption-ionization-MS imagingof tumors. Anal. Chem. 84, 6341–6345 (2012)

8. Gemperline, E., Chen, B., Li, L.: Challenges and recent advances in massspectrometric imaging of neurotransmitters. Bioanalysis 6, 525–540 (2014)

9. Shariatgorji, M., Svenningsson, P., Andrén, P.E.: Mass spectrometry im-aging, an emerging technology in neuropsychopharmacology.Neuropsychopharmacology 39, 34–49 (2014)

10. Caprioli, R.M., Farmer, T.B., Gile, J.: Molecular imaging of biologicalsamples: localization of peptides and proteins using MALDI-TOF MS.Anal. Chem. 69, 4751–4760 (1997)

11. Sugiura, Y., Zaima, N., Setou, M., Ito, S., Yao, I.: Visualization of acetyl-choline distribution in central nervous system tissue sections by tandemimaging mass spectrometry. Anal. Bioanal. Chem. 403, 1851–1861 (2012)

12. Shariatgorji,M., Nilsson, A., Goodwin, R.J., Svenningsson, P., Schintu, N.,Banka, Z., Kladni, L., Hasko, T., Szabo, A., Andren, P.E.: Deuteratedmatrix-assisted laser desorption ionization matrix uncovers masked massspectrometry imaging signals of small molecules. Anal. Chem. 84, 7152–7157 (2012)

13. Shariatgorji, M., Nilsson, A., Källback, P., Karlsson, O., Zhang, X.,Svenningsson, P., Andren, P.E.: Pyrylium salts as reactive matrices forMALDI-MS imaging of biologically active primary amines. J. Am. Soc.Mass Spectrom. 26, 934–939 (2015)

14. Ye, H., Wang, J., Greer, T., Strupat, K., Li, L.: Visualizing neurotransmit-ters and metabolites in the central nervous system by high resolution andhigh accuracy mass spectrometric imaging. ACSChem. Neurosci. 4, 1049–1056 (2013)

15. Shariatgorji, M., Nilsson, A., Goodwin, R.J., Källback, P., Schintu, N.,Zhang, X., Crossman, A.R., Bezard, E., Svenningsson, P., Andren, P.E.:Direct targeted quantitative molecular imaging of neurotransmitters in braintissue sections. Neuron 84, 697–707 (2014)

16. Esteve, C., Tolner, E.A., Shyti, R., Van den Maagdenberg, A.M.J.M.,McDonnell, L.A.: Mass spectrometry imaging of amino neurotransmitters:a comparison of derivatization methods and application in mouse braintissue. Metabolomics 12, 30 (2016)

17. Nemes, P., Woods, A.S., Vertes, A.: Simultaneous imaging of smallmetabolites and lipids in rat brain tissues at atmospheric pressure by laserablation electrospray ionization mass spectrometry. Anal. Chem. 82, 982–988 (2010)

18. Shrivas, K., Hayasaka, T., Sugiura, Y., Setou, M.:Method for simultaneousimaging of endogenous low molecular weight metabolites in mouse brainusing TiO2 nanoparticles in nanoparticle-assisted laser desorption/ionization-imaging mass spectrometry. Anal. Chem. 83, 7283–7289 (2011)

19. Manier, M.L., Spraggins, J.M., Reyzer, M.L., Norris, J.L., Caprioli, R.M.:A derivatization and validation strategy for determining the spatial locali-zation of endogenous amine metabolites in tissues using MALDI imagingmass spectrometry. J. Mass Spectrom. 49, 665–673 (2014)

20. Yalcin, E.B., de laMonte, S.M.: Review of matrix-assisted laser desorptionionization-imaging mass spectrometry for lipid biochemical histopatholo-gy. J. Histochem. Cytochem. 63, 762–771 (2015)

21. Takáts, Z., Wiseman, J.M., Gologan, B., Cooks, R.G.: Mass spectrometrysampling under ambient conditions with desorption electrospray ionization.Science 306, 471–473 (2004)

1950 A. M. A. P. Fernandes et al.: Direct Imaging of Neurotransmitters by Matrix-Free DESI-MSI

22. Eberlin, L.S., Ferreira, C.R., Dill, A.L., Ifa, D.R., Cheng, L., Cooks, R.G.:Nondestructive, histologically compatible tissue imaging by desorptionelectrospray ionization mass spectrometry. Chem. Biochem. 12, 2129–2132 (2011)

23. Fernandes, A.M.A.P., Schwab, N.V., Alberici, L.C., Eberlin, M.N.: Visu-alization of neurotransmitters in rat brain by desorption electrospray ioni-zation mass spectrometry imaging (DESI-MSI). Poster number ThP 672.Proceedings of the 63rd American Society for Mass Spectrometry AnnualConference, St. Louis, MO, 31 May–4 June (2015)

24. Monge-Acuña, A.A., Fornaguera-Trías, J.: A high performance liquidchromatography method with electrochemical detection of gamma-aminobutyric acid, glutamate, and glutamine in rat brain homogenates. J.Neurosci. Methods 183, 176–181 (2009)

25. Sancheti, J.S., Shaikh, M.F., Khatwani, P.F., Kulkarni, S.R., Sathaye, S.:Development and validation of a HPTLCmethod for simultaneous estima-tion of L-glutamic acid and γ-aminobutyric acid in mice brain. Ind. J.Pharm. Sci. 75, 716–721 (2013)

26. Campbell, D.I., Ferreira, C.R., Eberlin, L.S., Cooks, R.G.: Improved spatialresolution in the imaging of biological tissue using desorption electrosprayionization. Anal. Bioanal. Chem. 404, 389–398 (2012)

27. Lugemwa, F., Shaikh, K., Hochstedt, E.: Facile and efficient acetylation ofprimary alcohols and phenols with acetic anhydride catalyzed by driedsodium bicarbonate. Catalysts 3, 954–965 (2013)

28. Wu, C., Ifa, D.R., Manicke, N.E., Cooks, R.G.: Molecular imaging ofadrenal gland by desorption electrospray ionization mass spectrometry.Analyst 135, 28–32 (2010)

29. Nemes, P., Vertes, A.: Ambientmass spectrometry for in vivo local analysisand in situ molecular tissue imaging. Trends Anal. Chem. 34, 22–34 (2012)

30. Hanrieder, J., Phan, N.T., Kurczy, M.E., Ewing, A.G.: Imaging massspectrometry in neuroscience. ACS Chem. Neurosci. 4, 666–679 (2013)

31. Annesley, T.M.: Ion suppression in mass spectrometry. Clin. Chem. 49,1041–1044 (2003)

32. Jackson, A.U., Talaty, N., Cooks, R.G., Van Berkel, G.J.: Salt tolerance ofdesorption electrospray ionization (DESI). J. Am. Soc. Mass Spectrom. 18,2218–2225 (2007)

33. Dross, K., Kewitz, H.: Concentrations and origin of choline in rat brain.Nannyn-Schmiedeberg’s Arch. Pharmacol. 274, 91–106 (1972)

34. Wu, C., Ifa, D.R., Manicke, N.E., Cooks, R.G.: Rapid, direct analysis ofcholesterol by charge labeling in reactive desorption electrospray. Anal.Chem. 81, 7618–7624 (2009)

35. Eberlin, L., Ifa, D., Wu, C., Cooks, R.: Three-dimensional vizualization ofmouse brain by lipid analysis using ambient ionization mass spectrometry.Angew. Chem. Int. Ed. 49, 873–876 (2010)

36. Dill, A.L., Eberlin, L.S., Costa, A.B., Zheng, C., Ifa, D.R., Cheng, L.,Masterson, T.A., Koch, M.O., Vitek, O., Cooks, R.G.: Multivariate statis-tical identification of human bladder carcinomas using ambient ionizationimaging mass spectrometry. Chemistry 17, 2897–2902 (2011)

37. Ang, S.T., Lee, A.T., Foo, F.C., Ng, L., Low, C.M., Khanna, S.:GABAergic neurons of the medial septum play a nodal role in facilitationof nociception-induced affect. Sci. Rep. 5, 15419 (2015)

38. Wehrl, H.F., Schwab, J., Hasenbach, K., Reischl, G., Tabatabai, G.,Quintanilla-Martinez, L., Jiru, F., Chughtai, K., Kiss, A., Cay, F., Bukala,D., Heeren, R.M., Pichler, B.J., Sauter, A.W.: Multimodal elucidation ofcholine metabolism in a murine glioma model using magnetic resonancespectroscopy and 11C-choline positron emission tomography. Cancer Res.73, 1470–1480 (2013)

39. Tang, H.W., Wong, M.Y., Lam, W., Cheng, Y.C., Che, C.M., Ng, K.M.:Molecular histology analysis by matrix-assisted laser desorption/ionizationimaging mass spectrometry using gold nanoparticles as matrix. RapidCommun. Mass Spectrom. 25, 3690–3696 (2011)

40. Castilho, R.F., Hansson, O., Brundin, P.: Improving the survival of graftedembryonic dopamine neurons in rodent models of Parkinson’s disease.Prog. Brain Res. 127, 203–231 (2000)

41. Björklund, A., Dunnett, S.B.: Dopamine neuron systems in the brain: anupdate. Trends Neurosci. 30, 194–202 (2007)

42. Bergman, H.M., Lundin, E., Andersson, M., Lanekoff, I.: Quantitativemass spectrometry imaging of small-molecule neurotransmitters in rat braintissue sections using nanospray desorption electrospray ionization. Analyst141, 3686–3695 (2016)

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