Quantitative Molecular Imaging of Neurotransmitters Direct Targeted Quantitative Molecular Imaging of Neurotransmitters in Brain Tissue Sections
Mohammadreza Shariatgorji,1 Anna Nilsson,1 Richard J.A. Goodwin,1,2 Patrik Ka® back,1 Nicoletta Schintu,3
Xiaoqun Zhang,3 Alan R. Crossman,4 Erwan Bezard,5 Per Svenningsson,3 and Per E. Andren1,
* 1Biomolecular Imaging and Proteomics, National Center for Mass Spectrometry Imaging, Department of Pharmaceutical Biosciences,
Uppsala University, P.O. Box 591 BMC, 75124 Uppsala, Sweden
2AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire SK10 4TF, UK
3Center for Molecular Medicine, Department of Neurology and Clinical Neuroscience, Karolinska Institutet and Karolinska University Hospital, 17176 Stockholm, Sweden
4Faculty of Life Sciences, University of Manchester, Manchester M13 9PL, UK
5Universite¥ de Bordeaux, Institut des Maladies Neurode¥ ge¥ ne¥ratives, UMR 5293 Bordeaux, France
*Correspondence: per.andren@farmbio.uu.se
http://dx.doi.org/10.1016/j.neuron.2014.10.011
SUMMARY
Current neuroimaging techniques have very limited abilities to directly identify and quantify neurotransmitters from brain sections. We have developed a molecular-specific approach for the simultaneous imaging and quantitation of multiple neurotransmitters, precursors, and metabolites, such as tyrosine, tryptamine, tyramine, phenethylamine, dopamine, 3-methoxytyramine, serotonin, GABA, glutamate, acetylcholine, and L-alpha-glycerylphosphorylcholine, in histological tissue sections at high spatial resolutions. The method is employed to directly measure changes in the absolute and relative levels of neurotransmitters in specific brain structures in animal disease models and in response to drug treatments, demonstrating the power of mass spectrometry imaging in neuroscience.
Quantitative Molecular Imaging of Neurotransmitters Introduction
Small-molecule neurotransmitters such as the catecholamine dopamine (DA), the amino acids g-aminobutyric acid (GABA) and glutamate (Glu), and acetylcholine (ACh) are critical chemical messengers that transmit signals between neurons. Changes in their concentrations are associated with numerous normal neuronal processes, such as sleep and aging, but also several disease states, including Alzheimerís disease, Parkinsonís disease (PD), depression, drug addiction, and attention deficit hyperactivity disorder. A better understanding of their relative abundance and distribution would provide insights into these complex neurological processes and disorders. At present, researchers rely on indirect histochemical, immunohistochemical (IHC), and ligand-based assays to detect these small-molecule transmitter substances (de Jong et al., 2005; Falck et al., 1962;
Jones and Beaudet, 1987). The antibodies used in IHC often have major limitations, such as an inability to distinguish between different transmitters (Keenan and Koopowitz, 1981).
Nuclear medicine imaging is a widely used tool for indirect visualization of the distribution, abundance, and activity of neurotransmitters in the brain, relying on radiolabeled tracers that emit gamma rays, which are detected using positron emission tomography (PET) or single photon emission computed tomography scanners (Pimlott and Sutherland, 2011). Developing appropriate labeled compounds is often complicated, particularly for studying endogenous chemical messengers (Badgaiyan, 2011). There is often an inability to discriminate between the labeled parent compound and a metabolite retaining the label. Imaging techniques that can directly visualize the localization of neurotransmitters and simultaneously quantitate them without relying on a label would thus represent a scientific breakthrough. Another significant advance in the molecular analysis of neurological tissues would be the development of a multiplex method that enables the simultaneous detection of multiple endogenous compounds and pharmaceutical agents.
MALDI mass spectrometry (MS) imaging has been used for direct molecule-specific compound detection, distribution mapping, and identifying molecular species in tissue sections (Caprioli et al., 1997; Cornett et al., 2007). It can generate multiplex full mass spectrum data at near-cellular spatial resolution.
Crucially, it provides a powerful tool for in situ visualization of target compounds and for determining their abundance and spatial distribution without requiring a great deal of a priori information.
Moreover, the data gathered during such experiments can be used to simultaneously identify unknown endogenous molecular changes due to disease or degeneration. MALDI-MS can be used for the direct analysis of targets, ranging from small molecules to large proteins (Schwamborn and Caprioli, 2010).
However, despite some notable successes, it is relatively insensitive toward certain compound classes and sometimes suffers from signal interference caused by matrix clusters/fragments (Sugiura et al., 2012; Ye et al., 2013). To date, it has found only limited applications in the study of neurotransmitters due to their poor ionization efficiencies. As such, the development of a reagent that would enable selective neurotransmitter derivatization while also facilitating their detection by MS and acting as an efficient matrix to assist their laser-induced desorption and ionization would be a significant breakthrough. Neuron 84, 697ñ707, November 19, 2014 ™2014 Elsevier Inc. 697
Here we describe a unique approach for the direct and absolute quantitative imaging of classical and common neurotransmitters in histological brain tissue sections. We report an example of the simultaneous mapping of biogenic amines and amino acid chemical messengers following in situ derivatization in brain tissue sections. The method is employed to simultaneously measure changes in the levels of neurotransmitters, including tyrosine, tryptamine, tyramine, phenethylamine, DA, 3-methoxytyramine (3-MT), serotonin (5-HT), GABA, and Glu, in specific brain structures of primates and rodents with and without DA depletion and L-3,4-dihydroxyphenylalanine (L-DOPA) treatment. In addition, we have developed a method that can be used to image and quantitate ACh and an ACh precursor, L-alpha-glycerylphosphoryl-choline (alpha-GPC) in brain sections, as well as to study activity within cholinergic pathways by measuring changes in ACh concentrations, imaged at a 15 mm spatial resolution. Together, these experiments demonstrate a significant advancement in the molecular imaging arsenal available to neuroscientists.
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