The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

Abstract

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.

ACKNOWLEDGMENTS

We are indebted to Scott Zeitlin (Department of Neuroscience, University of Virginia School of Medicine) for the gift of Cre mice, Stanley Parsons (UCSB) for sharing unpublished data and for excellent suggestions for editing the manuscript, and Brian Collier (McGill University) for comments on the manuscript.

Research in our laboratory is supported by NIH-Fogarty grant R21 TW007800-02 (M.A.M.P., I.I., V.F.P., and M.G.C.), NIH grant RO1 NS53527 (R.W.O.), CIHR (MOP-89919 [V.F.P., R.J.R., and M.A.M.P.]), PRONEX-Fapemig (M.A.M.P. and V.F.P.), FINEP (M.A.M.P.), CNPq (Aging and Mental Health Programs [M.A.M.P., V.F.P., I.I., and M.C.]), FAPEMIG (V.F.P.), and Instituto do Milenio Toxins/MCT (M.V.G., M.A.M.P., and V.F.P.). C.M.-S. and C.M. received postdoctoral fellowships from DFAIT-Canada (Department of Foreign Affairs and International Trade Postdoctoral Fellowship Program). B.M.D.C., X.D.J., and R.L.F. received predoctoral student fellowships from CAPES and CNPq (Brazil).

M.G.C., M.A.M.P., V.F.P., I.I., C.M.-S., and B.M.D.C. have deposited a patent to cover the use of VAChT mutant mice in drug discovery.