Abstract
Muscarinic receptors have been proposed to play an important role during brain development by regulating cell survival, proliferation, and differentiation. This study investigated the effect of muscarinic receptor activation on prenatal rat hippocampal pyramidal neuron differentiation and the signal transduction pathways involved in this effect. The cholinergic agonist carbachol, after 24 h in vitro, increased the length of the axon, without affecting the length of minor neurites. Carbachol-induced axonal growth was also observed in pyramidal neurons from the neocortex but not in granule neurons from the cerebellum. The effect of carbachol was mediated by the M1 subtype of muscarinic receptors. The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester, the two protein kinase C (PKC) inhibitors 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X) and 2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyridol[1,2-a]indol-3-yl}-3-(1-methylindol-3-yl)maleimide (Ro-32-0432), and the extracellular signal-regulated kinase (ERK)1/2 inhibitors 2′-amino-3′-methoxyflavone (PD98059) and 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) all blocked carbachol-induced axonal outgrowth. In addition, down-regulation of ERK1/2 with small interfering RNA abolished the neuritogenic effect of carbachol. These data suggest an involvement of Ca2+, PKC, and ERK1/2 in carbachol-induced axonal growth. Carbachol indeed increased the release of Ca2+ from intracellular stores and induced PKC and ERK1/2 activation. Additional experiments showed that PKC, but not Ca2+, is involved in carbachol-induced ERK1/2 activation. Together, these results show that cholinergic stimulation of prenatal hippocampal pyramidal neurons accelerates axonal growth through the induction of Ca2+ mobilization and the activation of PKC and especially of ERK1/2.
Footnotes
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This work was supported in part by the National Institutes of Health [Grants AA08154, ES07033, ES07032].
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K.L.V. and M.G. contributed equally to this work.
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The data presented here have in part appeared in abstract form as follows: VanDeMark K, Guizzetti M, Giordano G, and Costa LG (2006) Effect of ethanol on carbachol-induced neurite outgrowth in prenatal hippocampal neurons, in Proceedings of the 45th Meeting of the Society of Toxicology; 2006 March 5-9; San Diego, CA. Society of Toxicology, Reston, VA; VanDeMark KL, Guizzetti M, Giordano G, and Costa LG (2008) Ethanol inhibits carbachol-induced axon outgrowth in hippocampal neurons by inhibiting ERK1/2, in Proceedings of the 47th Annual Meeting of the Society of Toxicology; 2008 March 16-20; Seattle, WA, Society of Toxicology, Reston, VA; and VanDeMark KL, Guizzetti M, Giordano G, and Costa LG (2008) Ethanol inhibits hippocampal neuron differentiation induced by carbachol, in 2008 Joint ISBRA/RSA Scientific Conference; 2008 28 June-2 July; Washington, DC, International Society for Biomedical Research on Alcoholism, Denver, CO, and Research Society on Alcoholism, Austin, TX.
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doi:10.1124/jpet.108.150128.
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ABBREVIATIONS: PKC, protein kinase C; ERK, extracellular signal-regulated kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MAP, microtubule-associated protein; Fluo-3/AM, 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo-3H-xanthen-9-yl)]-2-(2′-amino-5′methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid pentaacetoxymethyl ester; BAY-11, 3-[(4-methylphenyl)sulfonyl]-2-propenenitrile; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; PD98059, 2′-amino-3′methoxyflavone; LY 294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; Gö 6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; siRNA, small interfering RNA; HBSS, Hanks' balanced salt solution; ACM, astrocyte-conditioned medium; CGC, cerebellar granule cell; MTT, thiazolyl blue tetrazolium bromide; CNS, central nervous system; IP3, inositol trisphosphate; DAG, diacylglycerol; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; Ro-32-0432, 2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyridol[1,2-a]indol-3-yl}-3-(1-methylindol-3-yl)maleimide; PI3K, phosphatidylinositol 3-kinase; PLD, phospholipase D; p70S6kinase, 70-kDa ribosomal S6 kinase; NF-κB, nuclear factor-κB.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received December 19, 2008.
- Accepted February 2, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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