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Molecular and Cellular Biology, October 2009, p. 5238-5250, Vol. 29, No. 19
0270-7306/09/$08.00+0     doi:10.1128/MCB.00245-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

Braulio M. de Castro,^1, Xavier De Jaeger,^1,2,3, Cristina Martins-Silva,^2,3, Ricardo D. F. Lima,⁴ Ernani Amaral,⁵ Cristiane Menezes,^2,3 Patricia Lima,⁵ Cintia M. L. Neves,⁶ Rita G. Pires,² Thomas W. Gould,^8, Ian Welch,⁹ Christopher Kushmerick,⁴ Cristina Guatimosim,⁵ Ivan Izquierdo,¹⁰ Martin Cammarota,¹⁰ R. Jane Rylett,^2,3 Marcus V. Gomez,^1,11 Marc G. Caron,¹² Ronald W. Oppenheim,⁸ Marco A. M. Prado,^1,2,3,7* and Vania F. Prado^1,2,3,6,7*

Programa de Farmacologia Molecular, UFMG, Belo Horizonte, Minas Gerais, Brazil,¹ Molecular Brain Research Group, Robarts Research Institute,² Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada,³ Departamento de Fisiologia e Biofísica,⁴ Departamento de Morfologia,⁵ Departamento de Bioquímica-Imunologia, UFMG, Belo Horizonte, Minas Gerais, Brazil,⁶ Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario, Canada,⁷ Department of Neurobiology and Anatomy, Wake Forest University, Winston-Salem, North Carolina,⁸ Animal Care and Veterinary Services, University of Western Ontario, London, Ontario, Canada,⁹ Centro de Memoria, Instituto de Pesquisas Biomedicas, PUC-RS, Porto Alegre, Brazil,¹⁰ Nucleo de Neurociencias, Faculdade de Medicina, UFMG, Belo Horizonte, Minas Gerais, Brazil,¹¹ Department of Cell Biology, Duke University, Durham, North Carolina,¹²

Received 24 February 2009/ Returned for modification 19 March 2009/ Accepted 8 July 2009

The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.

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* Corresponding author. Mailing address: Molecular Brain Research Group, Robarts Research Institute, University of Western Ontario, P.O. Box 5015, 100 Perth Drive, London, Ontario N6A 5K8, Canada. Phone: (519) 663-5777, ext. 24888. Fax: (519) 663-3789. E-mail for Marco A. M. Prado: mprado{at}robarts.ca. E-mail for Vania F. Prado: vprado{at}robarts.ca

Published ahead of print on 27 July 2009.

B.M.D.C., X.D.G., and C.M.-S. contributed equally to this work.

Present address: Department of Basic Medical Sciences, Oceania University of Medicine, P.O. Box 232, Apia, Samoa.

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Molecular and Cellular Biology, October 2009, p. 5238-5250, Vol. 29, No. 19
0270-7306/09/$08.00+0     doi:10.1128/MCB.00245-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.