Application & Background

The complex chemical signaling that occurs in the brain is responsible for almost all normal physiologic function (1). This signaling is carried out primarily by neurotransmitters (NTs), which are substances that carry chemical information between neurons in the brain. Due to their integral role in so many biological processes, researchers are continually striving to better understand how NTs function in various diseased states (1). Given the high degree of correlation between regions of the brain and their function, one very useful analytical method to characterize NT distribution within brain tissue is matrix-assisted laser desorption/ionization- (MALDI-) mass spectrometry imaging (MSI) (2, 3). The advantages of MALDI-MSI over more traditional analytical techniques, such as immunohistochemistry, is that MALDI-MSI is label-free, highly specific, and almost infinitely channeled, allowing researchers to distinguish between parent compounds and their metabolites and to detect thousands of analytes from a single experiment (3). However, analysis of NTs by MALDI-MSI can be difficult due to their poor ionization efficiency and their relative low abundance in brain tissue (3, 4). Thus, researchers have developed in situ chemical derivatization methods for easier detection and identification of NTs via MALDI-MSI (3).

In this study, a 2-fluoro-1-methyl pyridinium (FMP) cation was used as part of the structure of a reactive matrix that selectively target phenolic and primary amine groups that are present in many NTs. This reaction significantly increases their ionization efficiency. Using this novel compound and an optimized spraying protocol developed using the HTX TM-Sprayer, the distributions of several NTs in both healthy and diseased state rodent brain tissues were characterized via MALDI-MSI.

Experimental

experimental design

All animal experiments were conducted in agreement with the European Communities Council Direction of November 24, 1986 (86/609/EEC) on the ethical use of animals and were also approved by local ethics committee at the Karolinska Institute (N350/08 and N105/16). Male Sprague-Dawley rats (n = 8) were kept in separate, air-conditioned cages, under a 12-hour light/dark cycle with food and water provided ad libitum. Animals were injected with 6-hydroxydopamine (6-OHDA) under anesthesia to induce a loss of striatal dopamine (DA) to make them models for Parkinson’s disease (PD), a neurodegenerative disease characterized by the loss of dopaminergic neurons. Two weeks after 6-OHDA administration, animals in the treatment group received intraperitoneal administration of 10 mg/kg L-DOPA and 7.5 mg/ kg benserazide once daily for 21 days. Animals were sacrificed by decapitation 30 minutes after the last intraperitoneal injec-tion treatment, which was benserazide (7.5 mg/kg) and deuterated L-DOPA-d3 (10 mg/kg). Animals in the control group were sacrificed 4 weeks after the original 6-OHDA treatment. Two animals were un-treated. The brains of the animals were removed after decapitation and flash frozen and stored at -80°C.

Sample Preparation

Rat brain tissues were sectioned at 12 μm, using a Leica CM1900 microtome and thaw-mounted onto indium-tin-oxide-coated (ITO) coated glass slides. Slides were stored at -80⁰C until they were needed. Prior to internal standard and reactive matrix deposition, slides were placed under vacuum for 15 minutes to dry. To evaluate the quantitative response of the protocol for selected NTs, 0.2 μl of 10, 2.5, 0.63, 0.16, 0.039, and 0 ng/μl of homovanillic acid (HVA)-d2 and 13C6-DA were applied to the cortex region of the control rat brain tissue section. Before FMP-10 derivatization, internal standard solutions (DA-d4 33 μg/mL in 50% methanol and HVA-d5 33 μg/ mL) were sprayed over the tissue sections with the parameters presented in Table 1. Then, the reactive matrix, FMP-10, available from Tag-ON AB (Uppsala, Sweden) was applied using the HTX TM-Sprayer with the parameters also displayed in Table 1.

data collection and analysis

MALDI-MSI experiments were performed using either a MALDI-time- of-flight/time-of-flight (TOF/TOF) (rapifleX, Bruker Daltonik GmbH) or a MALDI Fourier transform ion cyclotron resonance (FTICR) (SolariX, 7T 2ω, Bruker Daltonik GmbH) mass spectrometer equipped with a Smartbeam II 2kHz laser or a Smartbeam 3D 10kHz laser (rapifleX) and operated in positive ion mode. MS/MS analysis was performed on a MALDI FTICR instrument with a collision energy voltage of 30V in order to confirm the identification of analytes. Both instruments were externally calibrated using red phosphorous and FTICR-MS was also internally calibrated with the lock mass of the FMP-10 cluster ion (m/z 555.2231).

Analyses were conducted in a random order, and flexImaging (Bruker Daltonik GmbH, v.4.1) was used to visualize the data. TOF/TOF spectra were normalized to total ion count (TIC), and FTICR spectra were normalized to the root mean square (RMS) of all data points. DataAnalysis (Bruker Daltonik GmbH, v.4.2) was used to examine the data, and msIQuant (v.2.0.1.15) was used for relative and absolute quantitation.

results

Optimization of FMP-10 Derivatization

Derivatization of analytes prior to MALDI-MSI analysis aims to both increase the ionization/desorption yield of the compounds of interest and to reduce ionization bias by adding the same chemical group and permanent charge to each analyte. The goal of these experiments was to image precursor, signaling, and metabolized compounds of catecholaminergic and serotonergic NTs by targeting their phenolic hydroxyl, primary amine, and secondary amine chemical moieties (Figure 1).

The optimized deposition of the FMP-10 derivatization agent, using an automated sprayer (HTX TM-Sprayer) provided very homogenous crystallization with an average crystal size between 15-20 μm. The crystallize matrix layer was also found to be very reproducible between technical replicates. Using the optimized condition, FMP-10 derivatization enabled high sensitivity imaging of NTs e.g. DA, serotonin (5-HT), and gamma-aminobutyric acid (GABA) with no need for any further sample treatment such as heating or incubation (Figure 1).

High Lateral Resolution Imaging of Neurotransmitters

The ability to control the temperature of the nozzle of the TM-Sprayer allowed for some tissues to be sprayed with FMP-10 at 90⁰C for highresolution imaging at 10 μm. Unlike most of derivatization agents, FMP-10 does not need incubation and heating to complete the reaction. The latter advantage facilitates high lateral resolution imaging of tissue sections without delocalization of target compounds. All major NTs e.g. DA and GABA were detected despite the smaller laser spot size, and the analytes localized to brain substructures with no significant sign of delocalization (Figure 2).