Abstract
In the present work we characterized both the presynaptic and postsynaptic components of cholinergic transmission in a primary culture of corticostriatal neurons prepared from newborn rat brain. This culture preparation contains a small population of choline acetyltransferase (ChAT) immunoreactive neurons, corresponding to approximately 3% of the total cell number, and synthesizes increasing amounts of acetylcholine (ACh) from the third day in vitro (DIV), which reaches a plateau around the 10 day of culture. Muscarinic cholinergic receptors (mAChR), measured by the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB), are detectable from the fifth DIV and increase linearly during the time of culture. At the twelfth DIV, the density of mAChRs (approximately 600 fmol/mg protein) is comparable to the density of mAChR in adult rat cortex. These receptors are coupled to second messenger systems, since muscarinic agonists inhibit adenylate cyclase activity and stimulate phosphoinositide breakdown with efficacies and potencies similar to those found in adult rat cortex. Moreover, by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, we were able to demonstrate the presence of the m1, m3, and m4 mAChR subtype mRNAs in this neuronal culture at 12 DIV. Our data suggest that corticostriatal neuronal cultures develop in vitro ACh-synthesizing neurons and functionally active cholinergic receptors. This therefore makes them ideally suited to study the development and properties of brain mAChR subtypes.
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Alho, H., Ferrarese, C., Vicini, S., Vaccarino, F.M. (1988). Subsets of GABAergic neurons in dissociated cell cultures of neonatal rat cerebral cortex show colocalization with specific modulatory peptides. Dev. Brain Res. 39:193–204
Berridge, M.J., Downes, C.P., Hanley, M.R. (1982). Lithium amplifies agonist-dependent phosphatidil inositol responses in brain and salivary glands. Biochem. J. 206:587–595
Bertolino, M., Vicini, S., Mazzetta, J., Costa, E. (1988). Phencyclidine and glycine modulate NMDA-activated high conductance cationic channels by acting at different sites. Neurosci. Lett. 84:351–355
Birdsall, N.J.M., Burgen, A.S.V., Hulme, E.C. (1978). The binding of agonists to brain muscarinic receptors. Mol. Pharmacol. 14:723–736
Bonner, T.I. (1989). The molecuar basis of muscarinic receptors diversity. Trends Neurosci 12(4):148–151
Bonner, T.J., Buckley, N.J., Young, A.C., Brann, M.R. (1987). Identification of a family of muscarinic acetylcholine receptor genes. Science 237:527–532
Bonner, T.I., Young, A.C., Brann, M.R., Buckley, N.J. (1988). Cloning and expression of the human and rat m5 muscarinic receptor genes. Neuron 1:403–410
Buckley, N.J., Bonner, T.I., Brann, M.R. (1988). Localization of a family of muscarinic receptor mRNAs in rat brain. J. Neurosci 8(12):4646–4652
Candy, J.M., Perry, E.K., Perry, R.H., Bloxham, C.A., Thompson, J., Oakley, A.E., Edwardson, J.A. (1985). Evidence for the earlier prenatal development of cortical cholinergic afferents from the nucleus of Meynert in the human foetus. Neurosci. Lett. 61:91–95
Chirgwin, J.M., Przybyla, R.J., Macdonald, R.J., Rutter, W.J. (1979). Isolation of biologically active ribonucleic acid from soures enriched in ribonuclease. Biochemistry 18:5294–5299
Coyle, J.T., Yamamura, H.I. (1976). Neurochemical aspect of the ontogenesis of cholinergic neurons in the rat brain. Brain Res. 118:429–440
Eva, C., Hadjiconstantinou, M., Neff, N.H., Meek, J.L. (1984). Acetylcholine measurement by high-performance liquid chromatography using an enzyme-loaded postcolumn reactor. Anal. Biochem. 143:390–394
Fukuda, K., Higashida, H., Kubo, T., Maeda, A., Akida, I., Bujo, H., Mishina, M., Numa, S. (1988). Selective coupling with K+ currents of muscarinic acetylcholine receptor subtypes in NG108-15 cells. Nature 335:355–357
Ganong, W.F. (1975). The role of catecholamines and acetylcholine in the regulation of endocrine function. Life Sci 15:1401–1414
Goyal, R.K., Rattan, S. (1978). Neurohumoral, hormonal, and drug receptors for the lower esophageal sphincter. Gastroenterology 74:598–619
Grawford, G.H., Correa, L., Salvaterra, P.M. (1982). Interaction of monoclonal antibodies with mammalian choline acetyltransferase. Proc. Natl. Acad. Sci. U.S.A. 79:7031–7035
Hammer, R., Berrie, C.P., Birdsall, N.J.M., Burgen, A.S.V., Hulme, E.C. (1980). Pirenzepine distinguishes between different subclasses of muscarinic receptor. Nature 283:90–92
Harden, T.K., Tanner, L.I., Martin, M.W., Nakahata, N., Hughes, A.R., Hepler, J.R., Evans, T., Masters, S.B., Brown, J.H. (1986). Characteristics of two biochemical responses to stimulation of muscarinic cholinergic receptors. Trends Pharmacol. Sci., Suppl. 7:14–18
Itil, T., Fink, M. (1968). EEG and behavioral aspects of the interaction of anticholinergic hallucinogens and centrally active compound. Prog. Brain Res. 28:149–168
Jones, P.S.V., Barker, J.L., Buckley, N.J., Bonner, T.I., Collins, R.M., Brann, M.R. (1988). Cloned muscarinic receptor subtypes expressed in A9 L cells differ in their coupling to electrical responses. Mol. Pharmacol. 34:421–426
Jones, P.S.V., Murphy, T.J., Brann, M.R. (1989). Physiological comparison of cloned muscarinic receptors subtypes expressed in CHO cells. Trends Pharmacol. Sci., December Suppl., 72
Klawans, H.L. (1970). A pharmacologic analysis of Huntington’s chorea. Eur. Neurol. 4:148–163
Kubo, T., Maeda, A., Sugimoto, K., Akiba, I., Mikami, A., Takahashi, H., Haga, T., Haga, K., Ichiyama, A., Kangawa, K., Matsuo, H., Mishina, M., Hirose, T., Numa, S. (1986). Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the cDNA sequence. FEBS Lett. 209:367–372
Kuhar, M.J., Birdsall, N.J.M., Burgen, A.S.V., Hulme, E.C. (1980). Ontogeny of muscarinic receptor in rat brain. Brain Res. 184:375–383
Lai, J., Mei, L., Roeske, W.R., Chung, F.-Z., Yamamura, H.I., Venter, C.J. (1988). The cloned murine M1 muscarinic receptor is associated with the hydrolysis of phosphatidylinositols in transfected murine B82 cells. Life Sci. 422:2489–2502
Levi, G., Aloisi, F., Ciolti, M.T., and Gallo, V. (1984). Autoradiographic localization and depolarization-induced release of acidic aminoacids in differentiating cerebellar granule cell cultures. Brain Res. 290:77–86
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem 193:265–275
Maniatis, T., Fritsch, E.F., Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 211–212
Meek, J.L., Eva, C. (1984). Enzye adsorbed on an ion exchanger as a post-column reactor: application to acetylcholine measurement. J. Chromatog. 317:343–347
Nadler, J.V., Mathews, D.A., Cotman, C.W., Lynch, G.S. (1974). Development of cholinergic innervation in the hippocampal formation of the rat. Dev. Biol. 36:142–154
Nathanson, N.M. (1987). Molecular propertes of the muscarinic acetylcholine receptor. Annu. Rev. Neurosci 10:195–236
Olianas, M.C., Onali, P., Neff, N.H., Costa, E. (1983a). Adenylate cyclase activity of synaptic membranes from rat striatum. Inhibition by muscarinic receptor agonists. Mol. Pharmacol. 23:393–398
Olianas, M.C., Onali, P., Neff, N.H., Costa, E. (1983b). Muscarinic receptors modulate dopamine-activated adenylate cyclase from rat striatum. J. Neurochem. 411:364–369
Peralta, E.G., Ashkenazi, A., Winslow, J.W., Smith, D.H., Ramachandran, J., Capon, D.J. (1987). Distinct primary structures, ligand binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors. EMBO J. 6(13):3923–3929
Peralta, E.G., Ashkenazi, A., Winslow, J.W., Ramachandran, J., Capon, D.J. (1988). Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes. Nature 334:434–437
Rappolee, D.A., Brenner, C.A., Shultz, R., Mark, D., Werb, Z. (1988). Developmental expression of PDGF, TGF-α, and TGF-β genes preimplantation mouse embryos. Science 241:1823–1825
Roth, M.E., Lacy, M.J., McNeil, L.K., Kranz, D.M. (1988). Selection of variable-joining region combinations in the α chain of the T cell receptor. Science 241:1354–1358
Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Erlich, H.A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 230:487–491
Salomon, Y., Londos, D., Rodbell, M. (1974). A highly sensitive adenylate cyclase assay. Anal. Biochem. 58:541–548
Sternberg, L.A. (1979). Immunohistochemistry, 2nd ed., Wiley, New York, pp 104–169
Vaca, K. (1988). The development of cholinergic neurons. Brain Res. Rev. 13:261–286
Vicini, S., Alho, H., Costa, E., Mienville, J.M., Santi, M.R., Vaccarino, F.M. (1987). Modulation of aminobutyric acid (GABA) mediated inhibitory synaptic currents in dissociated cortical cell cultures. Proc. Natl. Acad. Sci. U.S.A.
Wang, A.M., Doyle, M.V., Mark, D.F. (1989). Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. U.S.A. 86:9717–9721
Weiner, D.M., Brann, M.R. (1989). Distribution of m1–m5 muscarinic receptor mRNAs in rat brain. Trends Pharmacol. Sci., December Suppl. 67
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Eva, C., Bovolin, P., Balzac, F. et al. Primary cultures of corticostriatal cells from newborn rats: A model to study muscarinic receptor subtypes regulation and function. J Mol Neurosci 2, 143 (1990). https://doi.org/10.1007/BF02896839
DOI: https://doi.org/10.1007/BF02896839