Abstract
The majority of research regarding the expression of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the brain has been conducted using histochemistry to identify enzymatic activity in frozen fixed tissue. However, retrospective human neurochemistry studies are generally restricted to formalin-fixed, paraffin-embedded (FFPE) tissues that are not suitable for histochemical procedures. The availability of commercially available antibody formulations provides the means to study such tissues by immunohistochemistry (IHC). In this study, we optimised IHC conditions for evaluating the expression of AChE and BuChE in the brainstem, focusing on the dorsal motor nucleus of the vagus, in human and piglet FFPE tissues, using commercially available antibodies. Our results were compared to published reports of histochemically determined AChE and BuChE expression. We varied antibody concentrations and antigen retrieval methods, and evaluated different detection systems, with the overall aim to optimise immunohistochemical staining. The primary findings, consistent across both species, are: (1) AChE and BuChE expression dominated in the neuronal somata, specifically in the neuronal cytoplasm; and (2) no change in the protocol resulted in axonal/neuropil expression of AChE. These results indicate that IHC is a suitable tool to detect AChE and BuChE in FFPE tissue using commercial antibodies, albeit the staining patterns obtained differed from those using histochemistry in frozen tissue. The underlying cause(s) for these differences are discussed in detail and may be associated with the principal components of the staining method, the antibody protein target and/or limitations to the detection of epitopes by tissue fixation.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Andrä J, Lachmann I, Luppa H (1988) A comparison of the localization of acetylcholinesterase in the rat brain as demonstrated by enzyme histochemistry and immunohistochemistry. Histochemistry 88:595–601. https://doi.org/10.1007/BF00570330
Barth F, Ghandour SM (1983) Cellular localization of butyrylcholinesterase in adult rat cerebellum determined by immunofluorescence. Neurosci Lett 39:149–153. https://doi.org/10.1016/0304-3940(83)90068-X
Beane M, Marrocco RT (2004) Norepinephrine and acetylcholine mediation of the components of reflexive attention: implications for attention deficit disorders. Prog Neurobiol 74:167–181. https://doi.org/10.1016/J.PNEUROBIO.2004.09.001
Bejjani C, Machaalani R, Waters KA (2013) The dorsal motor nucleus of the vagus (DMNV) in sudden infant death syndrome (SIDS): Pathways leading to apoptosis. Respir Physiol Neurobiol 185:203–210. https://doi.org/10.1016/j.resp.2012.09.001
Bhatt DK, Tewari HB (1978) Histochemical mapping of acetylcholinesterase and butyrylcholinesterase in the medulla oblongata and pons of squirrel (Funambulus palamarum). J Neurosci Res 3(5-6):419–439
Braid LR, Wood CA, Ford BN (2019) Human umbilical cord perivascular cells: A novel source of the organophosphate antidote butyrylcholinesterase. Chem Biol Interact 305:66–78. https://doi.org/10.1016/j.cbi.2019.03.022
Budantsev AY, Kornilova O, Medvedev B (2007) Microphotometric dynamic analysis of the histochemical acetylcholinesterase reaction. Biotech Histochem 82:311–317. https://doi.org/10.1080/10520290701797208
Calka J, Zalecki M, Wasowicz K, Arciszewski MB, Lakomy M (2008) A comparison of the distribution and morphology of ChAT-, VAChT-immunoreactive and AChE-positive neurons in the thoracolumbar and sacral spinal cord of the pig. Vet Med 53:434–444. https://doi.org/10.17221/1925-VETMED
Colovic MB, Krstic DZ, Lazarevic-Pasti TD, Bondzic AM, Vasic VM (2013) Acetylcholinesterase inhibitors: pharmacology and toxicology. Curr Neuropharmacol 11:315. https://doi.org/10.2174/1570159X11311030006
Dafferner AJ, Schopfer LM, Xiao G, Cashman JR, Yerramalla U, Johnson RC et al (2017) Immunopurification of acetylcholinesterase from red blood cells for detection of nerve agent exposure. Chem Res Toxicol 30:1897–1910. https://doi.org/10.1021/acs.chemrestox.7b00209
Darvesh S, Hopkins DA (2003) Differential distribution of butyrylcholinesterase and acetylcholinesterase in the human thalamus. J Comp Neurol 463:25–43. https://doi.org/10.1002/cne.10751
Darvesh S, Grantham DL, Hopkins DA (1998) Distribution of butyrylcholinesterase in the human amygdala and hippocampal formation’. J Comp Neurol 393:374–390. https://doi.org/10.1002/(SICI)1096-9861(19980413)393:33.0.CO;2-Z
Darvesh S, Hopkins DA, Geula C (2003) Neurobiology of butyrylcholinesterase. Nat Rev Neurosci 4:131–138. https://doi.org/10.1038/nrn1035
Dennie D, Louboutin JP, Strayer DS (2016) Migrating of bone marrow progenitor cells in the adult brain of rats and rabbits. World J Stem Cells 4:136–157. https://doi.org/10.4252/wjsc.v8.i4.136
Dobbing J, Sands J (1979) Comparative aspects of the brain growth spurt. Early Human Dev 3:79–83. https://doi.org/10.1016/0378-3782(79)90022-7
Dong H, Xiang YY, Farchi N, Ju W, Wu Y, Chen L et al (2004) Excessive expression of acetylcholinesterase impairs glutamatergic synaptogenesis in hippocampal neurons. J Neurosci 24:8950–8960. https://doi.org/10.1523/JNEUROSCI.2106-04.2004
Du A, Xie J, Guo K, Yang L, Wan Y, Ou YQ et al (2015) A novel role for synaptic acetylcholinesterase as an apoptotic deoxyribonuclease. Cell Discovery 1:15002. https://doi.org/10.1038/celldisc.2015.2
Duraiyan J, Govindarajan R, Kaliyappan K, Palanisamy M (2012) Applications of immunohistochemistry. J Pharm Bioallied Sci 4:307. https://doi.org/10.4103/0975-7406.100281
Garcia-Ayllon MS, Riba-Llena I, Serra-Basante C, Alom J, Boopathy R, Sáez-Valero J (2010) Altered levels of acetylcholinesterase in Alzheimer plasma. PLoS ONE 5(1):e8701. https://doi.org/10.1371/journal.pone.0008701
Glover L, Marques CA, Suska O, Horn D (2019) Persistent DNA damage foci and DNA replication with a broken chromosome in the African Trypansome. J Mol Biol Physiol. 10(4):e01252–19. https://doi.org/10.1128/mBio.01252-19
Hahn T, Desoye G, Lang I, Skofitsch G (1993) Location and activities of acetylcholinesterase and butyrylcholinesterase in the rat and human placenta. Anat Embryol 188:435–440. https://doi.org/10.1007/BF00190137
Harrington, CT, Hafid, N, & Waters, KA (2022) Butyrylcholinesterase is a potential biomarker for Sudden Infant Death Syndrome. EBioMedicine,80. doi: https://doi.org/10.1016/J.EBIOM.2022.104041.
Hayashi M, Miyata R, Tanuma N (2011) Decrease in acetylcholinergic neurons in the pedunculopontine tegmental nucleus in a patient with Prader-Willi syndrome. Neuropathology 31:280–285. https://doi.org/10.1111/j.1440-1789.2010.01157.x
Hayashi M, Ohto T, Shioda K, Fukatsu R (2012) Lesions of cortical GABAergic interneurons and acetylcholine neurons in xeroderma pigmentosum group A. Brain Develop 34:287–292. https://doi.org/10.1016/j.braindev.2011.06.015
Holzgrabe U, Kapková P, Alptuzun V, Scheiber J, Kugelmann E (2007) Targeting acetylcholinesterase to treat neurodegeneration. Expert Opin Ther Targets 11:161–179. https://doi.org/10.1517/14728222.11.2.161
Horio T, Ozawa A, Kamiie J, Sakaue M (2020) Immunohistochemical analysis for acetylcholinesterase and choline acetyltransferase in mouse cerebral cortex after traumatic brain injury. J Vet Med Sci 82:827–835. https://doi.org/10.1292/jvms.19-0551
Huang XF, Tork I, Paxinos G (1993) Dorsal motor nucleus of the vagus nerve: A cyto- and chemoarchitectonic study in the human. Journal of Comparative Neurology 330:158–182. https://doi.org/10.1002/cne.903300203
Jamur MC, Oliver C (2010) Permeabilization of cell membranes. Methods Mol Biol 588:63–66. doi: https://doi.org/10.1007/978-1-59745-324-0_9
Karnovsky MJ, Roots L (1964) Direct-coloring” thiocholine method for cholinesterases. J Histochem Cytochem 12:219–221. https://doi.org/10.1177/12.3.219
Koelle GB (1954) The histochemical localization of cholinesterases in the central nervous system of the rat. J Comp Neurol 100:211–235. https://doi.org/10.1002/cne.901000108
Krenacs L, Krenacs T, Stelkovics E, Raffeld M (2010) Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections. Methods Mol Biol 588:103–119. https://doi.org/10.1007/978-1-59745-324-0_14
Lavenex P, Lavenex PM, Bennett JL, Amaral DG (2009) Postmortem changes in the neuroanatomical characteristics of the primate brain: Hippocampal formation. J Comp Neurol 512:27–51. https://doi.org/10.1002/cne.21906
Liu SY, Xiong H, Yang JQ, Yang SH, Li Y, Yang WC, Yang GF (2018) Discovery of butyrylcholinesterase-activated near-infrared fluorogenic probe for live-cell and in vivo imaging. ACS Sensors 3:2118–2128. https://doi.org/10.1021/acssensors.8b00697
Luijerink L, Waters KA, Machaalani R (2021) Immunostaining for NeuN does not show all mature and healthy neurons in the human and pig brain: focus on the hippocampus. Appl Immunohistochem Mol Morphol 6:46–56. https://doi.org/10.1097/PAI.0000000000000925
Machaalani R, Waters KA (2003) Increased neuronal cell death after intermittent hypercapnic hypoxia in the developing piglet brainstem. Brain Res 985:127–134. https://doi.org/10.1016/S0006-8993(03)03003-8
Machaalani R, Waters KA (2008) Neuronal cell death in the Sudden Infant Death Syndrome brainstem and associations with risk factors. Brain 131(1):218–228. https://doi.org/10.1016/J.PRRV.2014.09.008
Machaalani R, Waters KA, Tinworth KD (2005) Effects of postnatal nicotine exposure on apoptotic markers in the developing piglet brain. Neuroscience 132:325–333. https://doi.org/10.1016/j.neuroscience.2004.12.039
Mack A, Robitzki A (2000) The key role of butyrylcholinesterase during neurogenesis and neural disorders: An antisense-5’butyrylcholinesterase-DNA study. Prog Neurobiol 60:607–628. https://doi.org/10.1016/S0301-0082(99)00047-7
Mallard C, Tolcos M, Leditschke J, Campbell P, Rees S (1999) Reduction in choline acetyltransferase immunoreactivity but not muscarinic-M2 receptor immunoreactivity in the brainstem of SIDS infants. J Neuropathol Exp Neurol 58:255–264. https://doi.org/10.1097/00005072-199903000-00005
Manuel SL, Johnson BW, Frevert CW, Duncan FE (2018) Revisiting the scientific method to improve rigor and reproducibility of immunohistochemistry in reproductive science. Biol Reprod 99:673–677. https://doi.org/10.1093/biolre/ioy094
Mesulam MM, Geula C (1991) Acetylcholinesterase-rich neurons of the human cerebral cortex: Cytoarchitectonic and ontogenetic patterns of distribution. J Comp Neurol 306(2):193–220. https://doi.org/10.1002/cne.903060202
Mesulam MM, Geula C, Cosgrove R, Mash D, Brimijoin S (1991) Immunocytochemical demonstration of axonal and perikaryal acetylcholinesterase in human cerebral cortex. Brain Res 539:233–238. https://doi.org/10.1016/0006-8993(91)91626-C
Mesulam M, Guillozet A, Shaw P, Quinn B (2002a) Widely spread butyrylcholinesterase can hydrolyze acetylcholine in the normal and Alzheimer brain. Neurobiol Dis 9:88–93. https://doi.org/10.1006/nbdi.2001.0462
Mesulam M, Guillozet A, Shaw P, Levey A, Duysen EG, Lockridge O (2002b) Acetylcholinesterase knockouts establish central cholinergic pathways and can use butyrylcholinesterase to hydrolyze acetylcholine. Neuroscience 110:627–639. https://doi.org/10.1016/S0306-4522(01)00613-3
Mizukami K, Akatsu H, Abrahamson EE, Mi Z, Ikonomovic MD (2016) Immunohistochemical analysis of hippocampal butyrylcholinesterase: Implications for regional vulnerability in Alzheimer’s disease. Neuropathology 36:135–145. https://doi.org/10.1111/neup.12241
Morán MA, Gómez-Ramos P (1992) Cholinesterase histochemistry in the human brain: effect of various fixation and storage conditions. J Neurosci Methods 43:49–54. https://doi.org/10.1016/0165-0270(92)90066-M
Nordberg A, Ballard C, Bullock R, Darreh-Shori T, Somogyi M (2013) A review of butyrylcholinesterase as a therapeutic target in the treatment of Alzheimer’s disease. Prim Care Companion CNS Disord 15(2):6731. https://doi.org/10.4088/PCC.12R01412
Otali D, Stockard C, Oelschlager D, Wan W, Manne U, Watts S et al (2009) Combined effects of formalin fixation and tissue processing on immunorecognition’. Biotech Histochem 84:223–247. https://doi.org/10.3109/10520290903039094
Patocka J, Kuca K, Jun D (2004) Acetylcholinesterase and butyrylcholinesterase–important enzymes of human body. Acta Med 47:215–228. https://doi.org/10.14712/18059694.2018.95
Paxinos G, Huang XF (1995) Atlas of the human brainstem. Academic Press, San Diego, CA
Pohanka M (2011) Cholinesterases, a target of pharmacology and toxicology. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 155:219–230. https://doi.org/10.5507/BP.2011.036
Rakonczay Z, Brimijoin S (1988) Monoclonal antibodies to human brain acetylcholinesterase: properties and applications. Cell Mol Neurobiol 8:85–93. https://doi.org/10.1007/BF00712914
Ramos-Vara JA (2005) Technical aspects of immunohistochemistry. Vet Pathol 42:405–426. https://doi.org/10.1354/VP.42-4-405
Reid GA, Chilukuri N, Darvesh S (2013) Butyrylcholinesterase and the cholinergic system. Neuroscience 234:53–68. https://doi.org/10.1016/j.neuroscience.2012.12.054
Rossi S, Laurino L, Furlanetto A, Chinellato S, Orvieto E, Canal F, Facchetti F, Dei Tos AP (2005) Rabbit monoclonal antibodies: a comparative study between a novel category of immunoreagents and the corresponding mouse monoclonal antibodies. Am J Clin Pathol 124:295–302. https://doi.org/10.1309/NR8H-N08G-DPVE-MU08
Rozzini L, Costardi D, Chilovi BV, Franzoni S, Trabucchi M, Padovani A (2007) Efficacy of cognitive rehabilitation in patients with mild cognitive impairment treated with cholinesterase inhibitors. Int J Geriatr Psychiatry 22:356–360. https://doi.org/10.1002/gps.1681
Ruberg M, Villageois A, Bonnet AM, Pillon B, Rieger F, Agid Y (1987) Acetylcholinesterase and butyrylcholinesterase activity in the cerebrospinal fluid of patients with neurodegenerative diseases involving cholinergic systems. J Neurol Neurosurg Psychiatry 50:538–543. https://doi.org/10.1136/JNNP.50.5.538
Shi SR, Liu C, Pootrakul L, Tang L, Young A, Chen R, Cote RJ, Taylor CR (2008) Evaluation of the value of frozen tissue section used as “gold standard” for immunohistochemistry. Am J Clin Pathol 129:358–366. https://doi.org/10.1309/7CXUYXT23E5AL8KQ
Tang S, Machaalani R, Kashem MA, Matsumoto I, Waters KA (2009) Intermittent hypercapnic hypoxia induced protein changes in the piglet hippocampus identified by MALDI-TOF-MS. Neurochem Res 34:2215–2225. https://doi.org/10.1007/S11064-009-0021-X
Uhlen M, Fagerberg L, Hallstrom BM, Lindskog C, Oksvold P, Mardinoglu A, Sivertsson A, Kampf C et al (2015) Tissue-based map of the human proteome. Science 347:126049. https://doi.org/10.1126/science.1260419
Uhlen M, Bandrowski A, Carr S, Edwards A, Ellenberg J, Lundberg E, Rimm DL, Rodriguez H et al (2016) A proposal for validation of antibodies. Nat Methods 13:823–827. https://doi.org/10.1038/nmeth.3995
Walczak-Nowicka LJ, Herbet M (2021) Acetylcholinesterase inhibitors in the treatment of neurodegenerative diseases and the role of acetylcholinesterase in their pathogenesis. Int J Mol Sci 22:9290. https://doi.org/10.3390/IJMS22179290
Zimmermann M (2013) Neuronal AChE splice variants and their non-hydrolytic functions: redefining a target of AChE inhibitors. Br J Pharmacol 170:953. https://doi.org/10.1111/BPH.12359
Zitella LM, Xiaol Y, Teplitzky BA, Kastl JK, Duchin Y, Baker KB et al (2015) In vivo 7T MRI of the non-human primate brainstem. PLoS ONE 10(5):e0127049. https://doi.org/10.1371/journal.pone.0127049
Zou D, Zhou Y, Liu L, Dong F, Shu T, Zhou Y, Tsai LH, Mao Y (2016) Transient enhancement of proliferation of neural progenitors and impairment of their long-term survival in p25 transgenic mice. Oncotarget 26:39148–39161. https://doi.org/10.18632/oncotarget.9834
Acknowledgements
This research was funded by the Miranda Belshaw Foundation, Australia. We thank Dr Carmel Harrington for introducing BuChE to us as a marker to pursue and for valuable discussions.
Author information
Authors and Affiliations
Contributions
MA conducted the experimental work, undertook the analyses, created the figures, searched the literature to create the summary tables and wrote the main manuscript. BH conducted the IF staining and microscopic imaging resulting in Fig. 9, and contributed to technical discussions. KW provided intellectual input. RM contributed to study design, tissue collection, staining analyses, intellectual input and drafting of manuscript. All authors reviewed the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Al Deleemy, M., Huynh, B., Waters, K.A. et al. Immunohistochemistry for acetylcholinesterase and butyrylcholinesterase in the dorsal motor nucleus of the vagus (DMNV) of formalin-fixed, paraffin-embedded tissue: comparison with reported literature. Histochem Cell Biol 159, 247–262 (2023). https://doi.org/10.1007/s00418-022-02164-3
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-022-02164-3