Use of Immunofluorescent and Genetic Labeling Strategies to Identify Cells Expressing Phenylethanolamine N-Methyltransferase in Mouse Cerebellum
Main Article Content
Abstract
Phenylethanolamine N-methyltransferase (Pnmt) catalyzes the N-methylation of norepinephrine to produce epinephrine, a potent stress hormone and neurotransmitter. Most of our knowledge about Pnmt is derived from its role in systemic production of epinephrine from adrenal chromaffin cells, but it is also known to be expressed in the central nervous system, including brainstem, retina, hypothalamus, and cerebellum. Of these regions, the cerebellum has been the least well-characterized with respect to Pnmt expression. Given the importance of the cerebellum for motor control and coordination, we sought to investigate cellular Pnmt expression in the cerebellum using a genetic-marking strategy with a Pnmt-Cre-recombinase knock-in driver strain (Pnmt+/Cre) and a β-galactosidase (βGal) reporter strain (R26R+/βGal) in parallel with Pnmt-specific immunofluorescent histochemical staining to identify Pnmt+ cells in sections of adult mouse cerebellum. Our results show active Pnmt protein expression in Purkinje neuron soma throughout the cerebellum, as demonstrated by positive Pnmt immunofluorescence and βGal expression. In contrast, the granular layer (GL) and Deep Cerebellar Nuclei (DCN) showed apparent historical expression with strong βGal expression in the absence of positive Pnmt immunofluorescence. These results suggest Pnmt+ cells may contribute more substantially to the cerebellum than previously appreciated and provide anatomical “blueprints” for investigating the role of cerebellar Pnmt expression in health and disease.
Keywords:
Cerebellum, mouse, adrenergic, Purkinje, Pnmt
Article Details
How to Cite
MEHTA, Meeti et al.
Use of Immunofluorescent and Genetic Labeling Strategies to Identify Cells Expressing Phenylethanolamine N-Methyltransferase in Mouse Cerebellum.
Medical Research Archives, [S.l.], v. 12, n. 7, aug. 2024.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/5392>. Date accessed: 05 aug. 2024.
doi: https://doi.org/10.18103/mra.v12i7.5392.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
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3 Hokfelt, T., Fuxe, K., Goldstein, M. & Johansson, O. Evidence for adrenaline neurons in the rat brain. Acta Physiol Scand 89, 286-288, Doi:10.1111/j.1748-1716.1973.tb05522.x (1973).
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5 Mezey, E. Phenylethanolamine N-methyltransferase-containing neurons in the limbic system of the young rat. Proc Natl Acad Sci U S A 86, 347-351, Doi:10.1073/pnas.86.1.347 (1989).
6 Andreassi, J. L., 2nd, Eggleston, W. B., Fu, G. & Stewart, J. K. Phenylethanolamine N-methyltransferase mRNA in rat hypothalamus and cerebellum. Brain Res 779, 289-291, Doi:10.1016/s0006-8993(97)01170-0 (1998).
7 Fujii, T., Sakai, M. & Nagatsu, I. Immunohistochemical demonstration of expression of tyrosine hydroxylase in cerebellar Purkinje cells of the human and mouse. Neurosci Lett 165, 161-163, Doi:10.1016/0304-3940(94)90734-x (1994).
8 Lew, J. Y. et al. Localization and characterization of phenylethanolamine N-methyl transferase in the brain of various mammalian species. Brain Res 119, 199-210, Doi:10.1016/0006-8993(77)90100-7 (1977).
9 Sjostedt, E. et al. An atlas of the protein-coding genes in the human, pig, and mouse brain. Science 367, Doi:10.1126/science.aay5947 (2020).
10 Ebert, S. N. et al. Targeted insertion of the Cre-recombinase gene at the phenylethanolamine n-methyltransferase locus: a new model for studying the developmental distribution of adrenergic cells. Dev Dyn 231, 849-858, Doi:10.1002/dvdy.20188 (2004).
11 Pfeifer, K., Boe, S. P., Rong, Q. & Ebert, S. N. Generating mouse models for studying the function and fate of intrinsic cardiac adrenergic cells. Ann N Y Acad Sci 1018, 418-423, Doi:10.1196/annals.1296.051 (2004).
12 D'Angelo, E. Physiology of the cerebellum. Handb Clin Neurol 154, 85-108, Doi:10.1016/B978-0-444-63956-1.00006-0 (2018).
13 Osuala, K. et al. Distinctive left-sided distribution of adrenergic-derived cells in the adult mouse heart. PLoS One 6, e22811, Doi:10.1371/journal.pone.0022811 (2011).
14 Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21, 70-71, Doi:10.1038/5007 (1999).
15 Ebert, S. N. & Thompson, R. P. Embryonic epinephrine synthesis in the rat heart before innervation: association with pacemaking and conduction tissue development. Circ Res 88, 117-124, Doi:10.1161/01.res.88.1.117 (2001).
16 Trifonov, S., Yamashita, Y., Kase, M., Maruyama, M. & Sugimoto, T. Overview and assessment of the histochemical methods and reagents for the detection of beta-galactosidase activity in transgenic animals. Anat Sci Int 91, 56-67, Doi:10.1007/s12565-015-0300-3 (2016).
17 Merkwitz, C., Blaschuk, O., Schulz, A. & Ricken, A. M. Comments on Methods to Suppress Endogenous beta-Galactosidase Activity in Mouse Tissues Expressing the LacZ Reporter Gene. J Histochem Cytochem 64, 579-586, Doi:10.1369/0022155416665337 (2016).
18 Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3, 1101-1108, Doi:10.1038/nprot.2008.73 (2008).
19 Pnmt images in GENSAT database, http://www.gensat.org/imagenavigator.jsp?imageID=26246
20 Diaz Borges, J. M. & Chavez, M. Regional changes in adrenaline of rat brain during development. J Neurosci Res 5, 465-468, Doi:10.1002/jnr.490050511 (1980).
21 Diaz Borges, J. M., Rodriguez, L. & Urbina, M. Regional changes in phenylethanolamine-N-methyltransferase of rat brain during development. J Neurosci Res 5, 363-367, Doi:10.1002/jnr.490050412 (1980).
22 Unsworth, B. R., Hayman, G. T., Carroll, A. & Lelkes, P. I. Tissue-specific alternative mRNA splicing of phenylethanolamine N-methyltransferase (PNMT) during development by intron retention. Int J Dev Neurosci 17, 45-55, Doi:10.1016/s0736-5748(98)00058-6 (1999).
23 Asan, E. Comparative single and double immunolabelling with antisera against catecholamine biosynthetic enzymes: criteria for the identification of dopaminergic, noradrenergic and adrenergic structures in selected rat brain areas. Histochemistry 99, 427-442, Doi:10.1007/BF00274095 (1993).
24 Carlton, S. M., Honda, C. N., Denoroy, L. & Willis, W. D., Jr. Descending phenylethanolamine-N-methyltransferase projections to the monkey spinal cord: an immunohistochemical double labeling study. Neurosci Lett 76, 133-139, Doi:10.1016/0304-3940(87)90704-x (1987).
25 Chamba, G. & Renaud, B. Distribution of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase activities in coronal sections of the rat lower brainstem. Brain Res 259, 95-102, Doi:10.1016/0006-8993(83)91069-7 (1983).
26 Ciriello, J., Caverson, M. M. & Park, D. H. Immunohistochemical identification of noradrenaline- and adrenaline- synthesizing neurons in the cat ventrolateral medulla. J Comp Neurol 253, 216-230, Doi:10.1002/cne.902530208 (1986).
27 Denoroy, L. et al. Catecholamine synthesizing enzyme activity in brainstem areas from victims of sudden infant death syndrome. Neuropediatrics 18, 187-190, Doi:10.1055/s-2008-1052477 (1987).
28 Gai, W. P., Geffen, L. B., Denoroy, L. & Blessing, W. W. Loss of C1 and C3 epinephrine-synthesizing neurons in the medulla oblongata in Parkinson's disease. Ann Neurol 33, 357-367, Doi:10.1002/ana.410330405 (1993).
29 Kalia, M. et al. Evidence for the existence of putative dopamine-, adrenaline- and noradrenaline-containing vagal motor neurons in the brainstem of the rat. Neurosci Lett 50, 57-62, Doi:10.1016/0304-3940(84)90462-2 (1984).
30 Li, A., Emond, L. & Nattie, E. Brainstem catecholaminergic neurons modulate both respiratory and cardiovascular function. Adv Exp Med Biol 605, 371-376, Doi:10.1007/978-0-387-73693-8_65 (2008).
31 Palkovits, M., Mezey, E., Skirboll, L. R. & Hokfelt, T. Adrenergic projections from the lower brainstem to the hypothalamic paraventricular nucleus, the lateral hypothalamic area and the central nucleus of the amygdala in rats. J Chem Neuroanat 5, 407-415, Doi:10.1016/0891-0618(92)90057-w (1992).
32 Vincent, S. R. Distributions of tyrosine hydroxylase-, dopamine-beta-hydroxylase-, and phenylethanolamine-N-methyltransferase-immunoreactive neurons in the brain of the hamster (Mesocricetus auratus). J Comp Neurol 268, 584-599, Doi:10.1002/cne.902680408 (1988).
33 Burke, W. J. et al. Evidence for retrograde degeneration of epinephrine neurons in Alzheimer's disease. Ann Neurol 24, 532-536, Doi:10.1002/ana.410240409 (1988).
34 Trocewicz, J., Oka, K. & Nagatsu, T. Highly sensitive assay for phenylethanolamine N-methyltransferase activity in rat brain by high-performance liquid chromatography with electrochemical detection. J Chromatogr 227, 407-413, Doi:10.1016/s0378-4347(00)80393-x (1982).
35 Wenk, G. L. & Stemmer, K. L. Activity of the enzymes dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase in discrete brain regions of the copper-zinc deficient rat following aluminum ingestion. Neurotoxicology 3, 93-99 (1982).
36 Yu, P. H. Phenylethanolamine N-methyltransferase from the brain and adrenal medulla of the rat: a comparison of their properties. Neurochem Res 3, 755-762, Doi:10.1007/BF00965998 (1978).
37 Altman, J. Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. J Comp Neurol 145, 399-463, Doi:10.1002/cne.901450402 (1972).
38 Heintz, N. Gene expression nervous system atlas (GENSAT). Nat Neurosci 7, 483, Doi:10.1038/nn0504-483 (2004).
39 Schmidt, E. F., Kus, L., Gong, S. & Heintz, N. BAC transgenic mice and the GENSAT database of engineered mouse strains. Cold Spring Harb Protoc 2013, Doi:10.1101/pdb.top073692 (2013).
40 Sved, A. F. PNMT-containing catecholaminergic neurons are not necessarily adrenergic. Brain Res 481, 113-118, Doi:10.1016/0006-8993(89)90490-3 (1989).
41 van der Heijden, M. E. & Sillitoe, R. V. Interactions Between Purkinje Cells and Granule Cells Coordinate the Development of Functional Cerebellar Circuits. Neuroscience 462, 4-21, Doi:10.1016/j.neuroscience.2020.06.010 (2021).
2 Hadjiconstantinou, M., Cohen, J. & Neff, N. H. Epinephrine: a potential neurotransmitter in retina. J Neurochem 41, 1440-1444, Doi:10.1111/j.1471-4159.1983.tb00843.x (1983).
3 Hokfelt, T., Fuxe, K., Goldstein, M. & Johansson, O. Evidence for adrenaline neurons in the rat brain. Acta Physiol Scand 89, 286-288, Doi:10.1111/j.1748-1716.1973.tb05522.x (1973).
4 Masana, M. I. & Mefford, I. N. Evidence for the presence of PNMT-containing cell bodies in the hypothalamus. Brain Res Bull 23, 477-482, Doi:10.1016/0361-9230(89)90193-7 (1989).
5 Mezey, E. Phenylethanolamine N-methyltransferase-containing neurons in the limbic system of the young rat. Proc Natl Acad Sci U S A 86, 347-351, Doi:10.1073/pnas.86.1.347 (1989).
6 Andreassi, J. L., 2nd, Eggleston, W. B., Fu, G. & Stewart, J. K. Phenylethanolamine N-methyltransferase mRNA in rat hypothalamus and cerebellum. Brain Res 779, 289-291, Doi:10.1016/s0006-8993(97)01170-0 (1998).
7 Fujii, T., Sakai, M. & Nagatsu, I. Immunohistochemical demonstration of expression of tyrosine hydroxylase in cerebellar Purkinje cells of the human and mouse. Neurosci Lett 165, 161-163, Doi:10.1016/0304-3940(94)90734-x (1994).
8 Lew, J. Y. et al. Localization and characterization of phenylethanolamine N-methyl transferase in the brain of various mammalian species. Brain Res 119, 199-210, Doi:10.1016/0006-8993(77)90100-7 (1977).
9 Sjostedt, E. et al. An atlas of the protein-coding genes in the human, pig, and mouse brain. Science 367, Doi:10.1126/science.aay5947 (2020).
10 Ebert, S. N. et al. Targeted insertion of the Cre-recombinase gene at the phenylethanolamine n-methyltransferase locus: a new model for studying the developmental distribution of adrenergic cells. Dev Dyn 231, 849-858, Doi:10.1002/dvdy.20188 (2004).
11 Pfeifer, K., Boe, S. P., Rong, Q. & Ebert, S. N. Generating mouse models for studying the function and fate of intrinsic cardiac adrenergic cells. Ann N Y Acad Sci 1018, 418-423, Doi:10.1196/annals.1296.051 (2004).
12 D'Angelo, E. Physiology of the cerebellum. Handb Clin Neurol 154, 85-108, Doi:10.1016/B978-0-444-63956-1.00006-0 (2018).
13 Osuala, K. et al. Distinctive left-sided distribution of adrenergic-derived cells in the adult mouse heart. PLoS One 6, e22811, Doi:10.1371/journal.pone.0022811 (2011).
14 Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21, 70-71, Doi:10.1038/5007 (1999).
15 Ebert, S. N. & Thompson, R. P. Embryonic epinephrine synthesis in the rat heart before innervation: association with pacemaking and conduction tissue development. Circ Res 88, 117-124, Doi:10.1161/01.res.88.1.117 (2001).
16 Trifonov, S., Yamashita, Y., Kase, M., Maruyama, M. & Sugimoto, T. Overview and assessment of the histochemical methods and reagents for the detection of beta-galactosidase activity in transgenic animals. Anat Sci Int 91, 56-67, Doi:10.1007/s12565-015-0300-3 (2016).
17 Merkwitz, C., Blaschuk, O., Schulz, A. & Ricken, A. M. Comments on Methods to Suppress Endogenous beta-Galactosidase Activity in Mouse Tissues Expressing the LacZ Reporter Gene. J Histochem Cytochem 64, 579-586, Doi:10.1369/0022155416665337 (2016).
18 Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3, 1101-1108, Doi:10.1038/nprot.2008.73 (2008).
19 Pnmt images in GENSAT database, http://www.gensat.org/imagenavigator.jsp?imageID=26246
20 Diaz Borges, J. M. & Chavez, M. Regional changes in adrenaline of rat brain during development. J Neurosci Res 5, 465-468, Doi:10.1002/jnr.490050511 (1980).
21 Diaz Borges, J. M., Rodriguez, L. & Urbina, M. Regional changes in phenylethanolamine-N-methyltransferase of rat brain during development. J Neurosci Res 5, 363-367, Doi:10.1002/jnr.490050412 (1980).
22 Unsworth, B. R., Hayman, G. T., Carroll, A. & Lelkes, P. I. Tissue-specific alternative mRNA splicing of phenylethanolamine N-methyltransferase (PNMT) during development by intron retention. Int J Dev Neurosci 17, 45-55, Doi:10.1016/s0736-5748(98)00058-6 (1999).
23 Asan, E. Comparative single and double immunolabelling with antisera against catecholamine biosynthetic enzymes: criteria for the identification of dopaminergic, noradrenergic and adrenergic structures in selected rat brain areas. Histochemistry 99, 427-442, Doi:10.1007/BF00274095 (1993).
24 Carlton, S. M., Honda, C. N., Denoroy, L. & Willis, W. D., Jr. Descending phenylethanolamine-N-methyltransferase projections to the monkey spinal cord: an immunohistochemical double labeling study. Neurosci Lett 76, 133-139, Doi:10.1016/0304-3940(87)90704-x (1987).
25 Chamba, G. & Renaud, B. Distribution of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase activities in coronal sections of the rat lower brainstem. Brain Res 259, 95-102, Doi:10.1016/0006-8993(83)91069-7 (1983).
26 Ciriello, J., Caverson, M. M. & Park, D. H. Immunohistochemical identification of noradrenaline- and adrenaline- synthesizing neurons in the cat ventrolateral medulla. J Comp Neurol 253, 216-230, Doi:10.1002/cne.902530208 (1986).
27 Denoroy, L. et al. Catecholamine synthesizing enzyme activity in brainstem areas from victims of sudden infant death syndrome. Neuropediatrics 18, 187-190, Doi:10.1055/s-2008-1052477 (1987).
28 Gai, W. P., Geffen, L. B., Denoroy, L. & Blessing, W. W. Loss of C1 and C3 epinephrine-synthesizing neurons in the medulla oblongata in Parkinson's disease. Ann Neurol 33, 357-367, Doi:10.1002/ana.410330405 (1993).
29 Kalia, M. et al. Evidence for the existence of putative dopamine-, adrenaline- and noradrenaline-containing vagal motor neurons in the brainstem of the rat. Neurosci Lett 50, 57-62, Doi:10.1016/0304-3940(84)90462-2 (1984).
30 Li, A., Emond, L. & Nattie, E. Brainstem catecholaminergic neurons modulate both respiratory and cardiovascular function. Adv Exp Med Biol 605, 371-376, Doi:10.1007/978-0-387-73693-8_65 (2008).
31 Palkovits, M., Mezey, E., Skirboll, L. R. & Hokfelt, T. Adrenergic projections from the lower brainstem to the hypothalamic paraventricular nucleus, the lateral hypothalamic area and the central nucleus of the amygdala in rats. J Chem Neuroanat 5, 407-415, Doi:10.1016/0891-0618(92)90057-w (1992).
32 Vincent, S. R. Distributions of tyrosine hydroxylase-, dopamine-beta-hydroxylase-, and phenylethanolamine-N-methyltransferase-immunoreactive neurons in the brain of the hamster (Mesocricetus auratus). J Comp Neurol 268, 584-599, Doi:10.1002/cne.902680408 (1988).
33 Burke, W. J. et al. Evidence for retrograde degeneration of epinephrine neurons in Alzheimer's disease. Ann Neurol 24, 532-536, Doi:10.1002/ana.410240409 (1988).
34 Trocewicz, J., Oka, K. & Nagatsu, T. Highly sensitive assay for phenylethanolamine N-methyltransferase activity in rat brain by high-performance liquid chromatography with electrochemical detection. J Chromatogr 227, 407-413, Doi:10.1016/s0378-4347(00)80393-x (1982).
35 Wenk, G. L. & Stemmer, K. L. Activity of the enzymes dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase in discrete brain regions of the copper-zinc deficient rat following aluminum ingestion. Neurotoxicology 3, 93-99 (1982).
36 Yu, P. H. Phenylethanolamine N-methyltransferase from the brain and adrenal medulla of the rat: a comparison of their properties. Neurochem Res 3, 755-762, Doi:10.1007/BF00965998 (1978).
37 Altman, J. Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. J Comp Neurol 145, 399-463, Doi:10.1002/cne.901450402 (1972).
38 Heintz, N. Gene expression nervous system atlas (GENSAT). Nat Neurosci 7, 483, Doi:10.1038/nn0504-483 (2004).
39 Schmidt, E. F., Kus, L., Gong, S. & Heintz, N. BAC transgenic mice and the GENSAT database of engineered mouse strains. Cold Spring Harb Protoc 2013, Doi:10.1101/pdb.top073692 (2013).
40 Sved, A. F. PNMT-containing catecholaminergic neurons are not necessarily adrenergic. Brain Res 481, 113-118, Doi:10.1016/0006-8993(89)90490-3 (1989).
41 van der Heijden, M. E. & Sillitoe, R. V. Interactions Between Purkinje Cells and Granule Cells Coordinate the Development of Functional Cerebellar Circuits. Neuroscience 462, 4-21, Doi:10.1016/j.neuroscience.2020.06.010 (2021).