One of the main challenges in analyzing chemical messengers in the brain is the optimization of tissue sampling and preparation protocols. Limiting postmortem time and terminating enzyme activity is critical to identify low-abundance neurotransmitters and neuropeptides. Here, we used a rapid and uniform conductive heat transfer stabilization method that was compared with a conventional fresh freezing protocol. Together with a selective chemical derivatization method and an optimized quantitation approach using deuterated internal standards, we spatially mapped neurotransmitters and their related metabolites by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in rat brain tissue sections. Although the heat stabilization did not show differences in the levels of dopamine, norepinephrine, and serotonin, their related metabolites 3,4-dihydroxyphenylacetaldehyde, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3-methoxy-4-hydroxyphenylacetaldehyde, dihydroxyphenylethyleneglycol, and 5-hydroxyindoleacetic acid were all significantly lower, indicating reduced neurotransmitter postmortem turnover ratios. Heat stabilization enabled detection of an increased number and higher levels of prodynorphin, proenkephalin, and tachykinin-derived bioactive neuropeptides. The low-abundant C-terminal flanking peptide, neuropeptide-γ, and nociceptin remained intact and were exclusively imaged in heat-stabilized brains. Without heat stabilization, degradation fragments of full-length peptides occurred in the fresh frozen tissues. The sample preparation protocols were furthermore tested on rat brains affected by acute anesthesia induced by isoflurane and medetomidine, showing comparable results to non-anesthetized animals on the neurotransmitters level without significant changes. Our data provide evidence for the potential use of heat stabilization prior to MALDI-MSI analyses to improve the examination of the in vivo state of neuronal chemical messengers in brain tissues not impacted by prior acute anesthesia.

Department of Animal Biosciences Division of Anatomy Physiology Immunology and Pathology Swedish University of Agricultural Sciences Uppsala Sweden

Department of Pharmaceutical Biosciences Neuropharmacology and Addiction Uppsala University Uppsala Sweden

Department of Pharmaceutical Biosciences Spatial Mass Spectrometry Science for Life Laboratory Uppsala University Uppsala Sweden

Department of Pharmacy University of Salerno Fisciano SA Italy

Institute of Microbiology of the Czech Academy of Sciences Prague Czech Republic

See more in PubMed

Adachi, Y. U. , Yamada S., Satomoto M., Higuchi H., Watanabe K., and Kazama T.. 2005. “Isoflurane Anesthesia Induces Biphasic Effect on Dopamine Release in the Rat Striatum.” Brain Research Bulletin 67, no. 3: 176–181. 10.1016/j.brainresbull.2005.06.020. PubMed DOI

Arts, J. W. , Oosterhuis N. R., Kramer K., and Ohl F.. 2014. “Effects of Transfer From Breeding to Research Facility on the Welfare of Rats.” Animals 4, no. 4: 712–728. 10.3390/ani4040712. PubMed DOI PMC

Baijnath, S. , Kaya I., Nilsson A., Shariatgorji R., and Andren P. E.. 2022. “Advances in Spatial Mass Spectrometry Enable In‐Depth Neuropharmacodynamics.” Trends in Pharmacological Sciences 43, no. 9: 740–753. 10.1016/j.tips.2022.06.005. PubMed DOI

Balcom, G. J. , Lenox R. H., and Meyerhoff J. L.. 1975. “Regional Gamma‐Aminobutyric Acid Levels in Rat Brain Determined After Microwave Fixation.” Journal of Neurochemistry 24, no. 4: 609–613. 10.1111/j.1471-4159.1975.tb03835.x. PubMed DOI

Barre, F. P. Y. , Claes B. S. R., Dewez F., et al. 2018. “Specific Lipid and Metabolic Profiles of R‐CHOP‐Resistant Diffuse Large B‐Cell Lymphoma Elucidated by Matrix‐Assisted Laser Desorption Ionization Mass Spectrometry Imaging and In Vivo Imaging.” Analytical Chemistry 90, no. 24: 14198–14206. 10.1021/acs.analchem.8b02910. PubMed DOI PMC

Barton, D. A. , Esler M. D., Dawood T., et al. 2008. “Elevated Brain Serotonin Turnover in Patients With Depression: Effect of Genotype and Therapy.” Archives of General Psychiatry 65, no. 1: 38–46. 10.1001/archgenpsychiatry.2007.11. PubMed DOI

Bartus, R. T. , Dean R. L. 3rd, Beer B., and Lippa A. S.. 1982. “The Cholinergic Hypothesis of Geriatric Memory Dysfunction.” Science 217, no. 4558: 408–414. 10.1126/science.7046051. PubMed DOI

Belforte, N. A. , Moreno M. C., de Zavalia N., et al. 2010. “Melatonin: A Novel Neuroprotectant for the Treatment of Glaucoma.” Journal of Pineal Research 48, no. 4: 353–364. 10.1111/j.1600-079X.2010.00762.x. PubMed DOI

Bertrand, N. , Beley P., and Beley A.. 1994. “Brain Fixation for Acetylcholine Measurements.” Journal of Neuroscience Methods 53, no. 1: 81–85. 10.1016/0165-0270(94)90147-3. PubMed DOI

Blank, C. L. , Sasa S., Isernhagen R., et al. 1979. “Levels of Norepinephrine and Dopamine in Mouse Brain Regions Following Microwave Inactivation—Rapid Post‐Mortem Degradation of Striatal Dopamine in Decapitated Animals.” Journal of Neurochemistry 33, no. 1: 213–219. 10.1111/j.1471-4159.1979.tb11723.x. PubMed DOI

Cai, R. , Kalappa B. I., Brozoski T. J., Ling L. L., and Caspary D. M.. 2014. “Is GABA Neurotransmission Enhanced in Auditory Thalamus Relative to Inferior Colliculus?” Journal of Neurophysiology 111, no. 2: 229–238. 10.1152/jn.00556.2013. PubMed DOI PMC

Caprioli, R. M. , Farmer T. B., and Gile J.. 1997. “Molecular Imaging of Biological Samples: Localization of Peptides and Proteins Using MALDI‐TOF MS.” Analytical Chemistry 69, no. 23: 4751–4760. 10.1021/ac970888i. PubMed DOI

Che, F. Y. , Lim J., Pan H., Biswas R., and Fricker L. D.. 2005. “Quantitative Neuropeptidomics of Microwave‐Irradiated Mouse Brain and Pituitary.” Molecular & Cellular Proteomics 4, no. 9: 1391–1405. 10.1074/mcp.T500010-MCP200. PubMed DOI

Dienel, G. A. 2020. “Metabolomic and Imaging Mass Spectrometric Assays of Labile Brain Metabolites: Critical Importance of Brain Harvest Procedures.” Neurochemical Research 45, no. 11: 2586–2606. 10.1007/s11064-020-03124-w. PubMed DOI

Dienel, G. A. 2021. “Stop the Rot. Enzyme Inactivation at Brain Harvest Prevents Artifacts: A Guide for Preservation of the In Vivo Concentrations of Brain Constituents.” Journal of Neurochemistry 158, no. 5: 1007–1031. 10.1111/jnc.15293. PubMed DOI

Fallon, J. H. , and Leslie F. M.. 1986. “Distribution of Dynorphin and Enkephalin Peptides in the Rat Brain.” Journal of Comparative Neurology 249, no. 3: 293–336. 10.1002/cne.902490302. PubMed DOI

Goodwin, R. J. , Lang A. M., Allingham H., Boren M., and Pitt A. R.. 2010. “Stopping the Clock on Proteomic Degradation by Heat Treatment at the Point of Tissue Excision.” Proteomics 10, no. 9: 1751–1761. 10.1002/pmic.200900641. PubMed DOI

Greengard, P. 2001. “The Neurobiology of Slow Synaptic Transmission.” Science 294, no. 5544: 1024–1030. 10.1126/science.294.5544.1024. PubMed DOI

Groppetti, A. , Algeri S., Cattabeni F., et al. 1977. “Changes in Specific Activity of Dopamine Metabolites as Evidence of a Multiple Compartmentation of Dopamine in Striatal Neurons.” Journal of Neurochemistry 28, no. 1: 193–197. 10.1111/j.1471-4159.1977.tb07726.x. PubMed DOI

Guenther, S. , Römpp A., Kummer W., and Spengler B.. 2011. “AP‐MALDI Imaging of Neuropeptides in Mouse Pituitary Gland With 5 μm Spatial Resolution and High Mass Accuracy.” International Journal of Mass Spectrometry 305, no. 2–3: 228–237. 10.1016/j.ijms.2010.11.011. DOI

Hanrieder, J. , Ljungdahl A., and Andersson M.. 2012. “MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease.” Journal of Visualized Experiments 60, no. 60: e3445. 10.3791/3445. PubMed DOI PMC

Hattori, K. , Kajimura M., Hishiki T., et al. 2010. “Paradoxical ATP Elevation in Ischemic Penumbra Revealed by Quantitative Imaging Mass Spectrometry.” Antioxidants & Redox Signaling 13, no. 8: 1157–1167. 10.1089/ars.2010.3290. PubMed DOI PMC

Hulme, H. , Fridjonsdottir E., Gunnarsdottir H., et al. 2020. “Simultaneous Mass Spectrometry Imaging of Multiple Neuropeptides in the Brain and Alterations Induced by Experimental Parkinsonism and L‐DOPA Therapy.” Neurobiology of Disease 137: 104738. 10.1016/j.nbd.2020.104738. PubMed DOI

Irifune, M. , Sato T., Nishikawa T., et al. 1997. “Hyperlocomotion During Recovery From Isoflurane Anesthesia Is Associated With Increased Dopamine Turnover in the Nucleus Accumbens and Striatum in Mice.” Anesthesiology 86, no. 2: 464–475. 10.1097/00000542-199702000-00022. PubMed DOI

Kallback, P. , Nilsson A., Shariatgorji M., and Andren P. E.. 2016. “msIQuant—Quantitation Software for Mass Spectrometry Imaging Enabling Fast Access, Visualization, and Analysis of Large Data Sets.” Analytical Chemistry 88, no. 8: 4346–4353. 10.1021/acs.analchem.5b04603. PubMed DOI

Kallback, P. , Shariatgorji M., Nilsson A., and Andren P. E.. 2012. “Novel Mass Spectrometry Imaging Software Assisting Labeled Normalization and Quantitation of Drugs and Neuropeptides Directly in Tissue Sections.” Journal of Proteomics 75, no. 16: 4941–4951. 10.1016/j.jprot.2012.07.034. PubMed DOI

Kallback, P. , Vallianatou T., Nilsson A., et al. 2020. “Cross‐Validated Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Quantitation Protocol for a Pharmaceutical Drug and Its Drug‐Target Effects in the Brain Using Time‐Of‐Flight and Fourier Transform Ion Cyclotron Resonance Analyzers.” Analytical Chemistry 92, no. 21: 14676–14684. 10.1021/acs.analchem.0c03203. PubMed DOI PMC

Kanazawa, I. , and Jessell T.. 1976. “Post Mortem Changes and Regional Distribution of Substance P in the Rat and Mouse Nervous System.” Brain Research 117, no. 2: 362–367. 10.1016/0006-8993(76)90748-4. PubMed DOI

Kandel, E. R. , Koester J., Mack S., and Siegelbaum S.. 2021. Principles of Neural Science. Sixth ed. McGraw Hill.

Lotharius, J. , and Brundin P.. 2002. “Pathogenesis of Parkinson's Disease: Dopamine, Vesicles and Alpha‐Synuclein.” Nature Reviews. Neuroscience 3, no. 12: 932–942. 10.1038/nrn983. PubMed DOI

Macha, H. , Luptakova D., Juranek I., Andren P. E., and Havlicek V.. 2024. “Hypoxic‐Ischemic Insult Alters Polyamine and Neurotransmitter Abundance in the Specific Neonatal Rat Brain Subregions.” ACS Chemical Neuroscience 15, no. 15: 2811–2821. 10.1021/acschemneuro.4c00190. PubMed DOI PMC

Marin, C. , Bonastre M., Mengod G., Cortes R., and Rodriguez‐Oroz M. C.. 2015. “From Unilateral to Bilateral Parkinsonism: Effects of Lateralization on Dyskinesias and Associated Molecular Mechanisms.” Neuropharmacology 97: 365–375. 10.1016/j.neuropharm.2015.06.004. PubMed DOI

Norris, J. L. , and Caprioli R. M.. 2013. “Analysis of Tissue Specimens by Matrix‐Assisted Laser Desorption/Ionization Imaging Mass Spectrometry in Biological and Clinical Research.” Chemical Reviews 113, no. 4: 2309–2342. 10.1021/cr3004295. PubMed DOI PMC

Nylander, I. , Stenfors C., Tan‐No K., Mathe A. A., and Terenius L.. 1997. “A Comparison Between Microwave Irradiation and Decapitation: Basal Levels of Dynorphin and Enkephalin and the Effect of Chronic Morphine Treatment on Dynorphin Peptides.” Neuropeptides 31, no. 4: 357–365. 10.1016/s0143-4179(97)90072-x. PubMed DOI

O'Callaghan, J. P. , and Sriram K.. 2004. “Focused Microwave Irradiation of the Brain Preserves In Vivo Protein Phosphorylation: Comparison With Other Methods of Sacrifice and Analysis of Multiple Phosphoproteins.” Journal of Neuroscience Methods 135, no. 1–2: 159–168. 10.1016/j.jneumeth.2003.12.006. PubMed DOI

Paxinos, G. , and Watson C.. 2014. Paxino's and Watson's the Rat Brain in Stereotaxic Coordinates. Seventh ed. Elsevier/AP, Academic Press is an imprint of Elsevier.

Politis, M. , and Niccolini F.. 2015. “Serotonin in Parkinson's Disease.” Behavioural Brain Research 277: 136–145. 10.1016/j.bbr.2014.07.037. PubMed DOI

Russo, A. F. 2017. “Overview of Neuropeptides: Awakening the Senses?” Headache 57, no. Suppl 2: 37–46. 10.1111/head.13084. PubMed DOI PMC

Schmidt, D. E. , Speth R. C., Welsch F., and Schmidt M. J.. 1972. “The Use of Microwave Radiation in the Determination of Acetylcholine in the Rat Brain.” Brain Research 38, no. 2: 377–389. 10.1016/0006-8993(72)90720-2. PubMed DOI

Scifo, E. , Calza G., Fuhrmann M., Soliymani R., Baumann M., and Lalowski M.. 2017. “Recent Advances in Applying Mass Spectrometry and Systems Biology to Determine Brain Dynamics.” Expert Review of Proteomics 14, no. 6: 545–559. 10.1080/14789450.2017.1335200. PubMed DOI

Segerstrom, L. , Gustavsson J., and Nylander I.. 2016. “Minimizing Postsampling Degradation of Peptides by a Thermal Benchtop Tissue Stabilization Method.” Biopreservation and Biobanking 14, no. 2: 172–179. 10.1089/bio.2015.0088. PubMed DOI PMC

Sgroi, S. , Capper‐Loup C., Paganetti P., and Kaelin‐Lang A.. 2016. “Enkephalin and Dynorphin Neuropeptides Are Differently Correlated With Locomotor Hypersensitivity and Levodopa‐Induced Dyskinesia in Parkinsonian Rats.” Experimental Neurology 280: 80–88. 10.1016/j.expneurol.2016.03.024. PubMed DOI

Shariatgorji, M. , Nilsson A., Fridjonsdottir E., et al. 2019. “Comprehensive Mapping of Neurotransmitter Networks by MALDI‐MS Imaging.” Nature Methods 16, no. 10: 1021–1028. 10.1038/s41592-019-0551-3. PubMed DOI

Shariatgorji, M. , Svenningsson P., and Andren P. E.. 2014. “Mass Spectrometry Imaging, an Emerging Technology in Neuropsychopharmacology.” Neuropsychopharmacology 39, no. 1: 34–49. 10.1038/npp.2013.215. PubMed DOI PMC

Shariatgorji, R. , Nilsson A., Fridjonsdottir E., et al. 2021. “Spatial Visualization of Comprehensive Brain Neurotransmitter Systems and Neuroactive Substances by Selective In Situ Chemical Derivatization Mass Spectrometry Imaging.” Nature Protocols 16, no. 7: 3298–3321. 10.1038/s41596-021-00538-w. PubMed DOI

Shiaratgorji, M. N. A. , Goodwin R. J. A., Källback P., et al. 2014. “Direct Target Quantitative Molecular Imaging of Neurotransmitters in Brain Tissues Sections.” Neuron 84: 697–707. PubMed

Shults, C. W. , Quirion R., Chronwall B., Chase T. N., and O'Donohue T. L.. 1984. “A Comparison of the Anatomical Distribution of Substance P and Substance P Receptors in the Rat Central Nervous System.” Peptides 5, no. 6: 1097–1128. 10.1016/0196-9781(84)90177-3. PubMed DOI

Skold, K. , Svensson M., Kaplan A., Bjorkesten L., Astrom J., and Andren P. E.. 2002. “A Neuroproteomic Approach to Targeting Neuropeptides in the Brain.” Proteomics 2, no. 4: 447–454. 10.1002/1615-9861(200204)2:4<447::AID-PROT447>3.0.CO;2-A. PubMed DOI

Skold, K. , Svensson M., Nilsson A., et al. 2006. “Decreased Striatal Levels of PEP‐19 Following MPTP Lesion in the Mouse.” Journal of Proteome Research 5, no. 2: 262–269. 10.1021/pr050281f. PubMed DOI

Skold, K. , Svensson M., Norrman M., Sjogren B., Svenningsson P., and Andren P. E.. 2007. “The Significance of Biochemical and Molecular Sample Integrity in Brain Proteomics and Peptidomics: Stathmin 2‐20 and Peptides as Sample Quality Indicators.” Proteomics 7, no. 24: 4445–4456. 10.1002/pmic.200700142. PubMed DOI

Stenfors, C. , Bjellerup P., Mathe A. A., and Theodorsson E.. 1995. “Concurrent Analysis of Neuropeptides and Biogenic Amines in Brain Tissue of Rats Treated With Electroconvulsive Stimuli.” Brain Research 698, no. 1–2: 39–45. 10.1016/0006-8993(95)00784-n. PubMed DOI

Sturm, R. M. , Greer T., Woodards N., Gemperline E., and Li L.. 2013. “Mass Spectrometric Evaluation of Neuropeptidomic Profiles Upon Heat Stabilization Treatment of Neuroendocrine Tissues in Crustaceans.” Journal of Proteome Research 12, no. 2: 743–752. 10.1021/pr300805f. PubMed DOI PMC

Sugiura, Y. , Honda K., Kajimura M., and Suematsu M.. 2014. “Visualization and Quantification of Cerebral Metabolic Fluxes of Glucose in Awake Mice.” Proteomics 14, no. 7–8: 829–838. 10.1002/pmic.201300047. PubMed DOI

Sugiura, Y. , Katsumata Y., Sano M., et al. 2016. “Visualization of In Vivo Metabolic Flows Reveals Accelerated Utilization of Glucose and Lactate in Penumbra of Ischemic Heart.” Scientific Reports 6: 32361. 10.1038/srep32361. PubMed DOI PMC

Sugiura, Y. , Zaima N., Setou M., Ito S., and Yao I.. 2012. “Visualization of Acetylcholine Distribution in Central Nervous System Tissue Sections by Tandem Imaging Mass Spectrometry.” Analytical and Bioanalytical Chemistry 403, no. 7: 1851–1861. 10.1007/s00216-012-5988-5. PubMed DOI PMC

Svensson, M. , Boren M., Skold K., et al. 2009. “Heat Stabilization of the Tissue Proteome: A New Technology for Improved Proteomics.” Journal of Proteome Research 8, no. 2: 974–981. 10.1021/pr8006446. PubMed DOI

Svensson, M. , Skold K., Nilsson A., Falth M., Svenningsson P., and Andren P. E.. 2007. “Neuropeptidomics: Expanding Proteomics Downwards.” Biochemical Society Transactions 35, no. Pt 3: 588–593. 10.1042/BST0350588. PubMed DOI

Svensson, M. , Skold K., Svenningsson P., and Andren P. E.. 2003. “Peptidomics‐Based Discovery of Novel Neuropeptides.” Journal of Proteome Research 2, no. 2: 213–219. 10.1021/pr020010u. PubMed DOI

Taban, I. M. , Altelaar A. F., van der Burgt Y. E., et al. 2007. “Imaging of Peptides in the Rat Brain Using MALDI‐FTICR Mass Spectrometry.” Journal of the American Society for Mass Spectrometry 18, no. 1: 145–151. 10.1016/j.jasms.2006.09.017. PubMed DOI

Tjernstrom, N. , Li T. Q., Holst S., and Roman E.. 2022. “Functional Connectivity in Reward‐Related Networks Is Associated With Individual Differences in Gambling Strategies in Male Lister Hooded Rats.” Addiction Biology 27, no. 2: e13131. 10.1111/adb.13131. PubMed DOI

Vallianatou, T. , Angerer T. B., Kaya I., et al. 2024. “Applying Spatial Metabolomics to Investigate Age‐ and Drug‐Induced Neurochemical Changes.” ACS Chemical Neuroscience 15, no. 15: 2822–2829. 10.1021/acschemneuro.4c00199. PubMed DOI PMC

Vallianatou, T. , Shariatgorji M., Nilsson A., et al. 2019. “Molecular Imaging Identifies Age‐Related Attenuation of Acetylcholine in Retrosplenial Cortex in Response to Acetylcholinesterase Inhibition.” Neuropsychopharmacology 44, no. 12: 2091–2098. 10.1038/s41386-019-0397-5. PubMed DOI PMC

Vallianatou, T. , Shariatgorji R., Nilsson A., et al. 2021. “Integration of Mass Spectrometry Imaging and Machine Learning Visualizes Region‐Specific Age‐Induced and Drug‐Target Metabolic Perturbations in the Brain.” ACS Chemical Neuroscience 12, no. 10: 1811–1823. 10.1021/acschemneuro.1c00103. PubMed DOI PMC

Vallianatou, T. , Strittmatter N., Nilsson A., et al. 2018. “A Mass Spectrometry Imaging Approach for Investigating How Drug‐Drug Interactions Influence Drug Blood‐Brain Barrier Permeability.” NeuroImage 172: 808–816. 10.1016/j.neuroimage.2018.01.013. PubMed DOI

van der Heyden, J. A. , and Korf J.. 1978. “Regional Levels of GABA in the Brain: Rapid Semiautomated Assay and Prevention of Postmortem Increase by 3‐Mercapto‐Propionic Acid.” Journal of Neurochemistry 31, no. 1: 197–203. 10.1111/j.1471-4159.1978.tb12448.x. PubMed DOI

Verhage, M. , Maia A. S., Plomp J. J., et al. 2000. “Synaptic Assembly of the Brain in the Absence of Neurotransmitter Secretion.” Science 287, no. 5454: 864–869. 10.1126/science.287.5454.864. PubMed DOI

Vu, N. Q. , DeLaney K., and Li L.. 2021. “Neuropeptidomics: Improvements in Mass Spectrometry Imaging Analysis and Recent Advancements.” Current Protein & Peptide Science 22, no. 2: 158–169. 10.2174/1389203721666201116115708. PubMed DOI PMC

Wasek, B. , Arning E., and Bottiglieri T.. 2018. “The Use of Microwave Irradiation for Quantitative Analysis of Neurotransmitters in the Mouse Brain.” Journal of Neuroscience Methods 307: 188–193. 10.1016/j.jneumeth.2018.05.016. PubMed DOI

Whittington, R. A. , and Virag L.. 2006. “Isoflurane Decreases Extracellular Serotonin in the Mouse Hippocampus.” Anesthesia and Analgesia 103, no. 1: 92–98. 10.1213/01.ane.0000221488.48352.61. PubMed DOI