SYNAPTIC EXOCYTOSIS AND NERVOUS SYSTEM DEVELOPMENT IMPAIRED IN CAENORHABDITIS ELEGANS UNC-13 MUTANTS H.MARUYAMA,T.L.RAKOW²andI.N.MARUYAMA* DepartmentofCellBiology,TheScrippsResearchInstitute,10550NorthTorreyPinesRoad,LaJolla,CA92037,USA Abstract ÐC. elegans mutants defective in unc-13 exhibited severe behavioral abnormalities including paralyzed loco- motion and slow pharyngeal pumping and irregular defecation cycle. Consistent with the phenotypes, the mutants accu- mulated abnormally high levels of the neurotransmitter acetylcholine and were resistant to acetylcholinesterase inhibitors. The unc-13 gene was expressed in most, if not all, neurons when analyzed by using chimeric constructs consisting of the unc-13 promoter and green ¯uorescence protein or b-galactosidase reporter gene. While Ca 21 -regulated acetylcholine releaseislacking,themutantswerestillabletoreleaseacetylcholine in vivo and in vitro atsimilarlevelstothatmediated by the regulated mechanism. Double mutants defective in both unc-13 and other genes involved in synaptic transmission showed the Unc-13 phenotype, rather than other mutant phenotypes, in terms of locomotion as well as of acetylcholine accumulation.Furthermore,electronmicroscopicreconstructionofthemutantnervoussystemuncoveredthatamajorityof neuronsdevelopedandconnectedasthoseinthewildtypeexceptforsubtleabnormalitiesincludinginappropriateconnec- tions through gap junctions and morphological alterations of neurons. Theseresultsdemonstratethatthe unc-13 geneproductplaysanessentialroleatalatestageinCa 21 -regulated synaptic exocytosis.NeurotransmittersreleasedthroughtheCa 21 -regulatedmechanismarerequiredfor,butdonotplaymajorroles inthenervoussystemdevelopment.ThelargeamountofCa 21 -independentneurotransmitterreleaseobservedinthe unc-13 mutantssuggeststhattheremaybeadistinctmechanismfromevokedorspontaneousreleaseinneurotransmission. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: acetylcholine, nematode, neuronal differentiation, neurotransmitter release. Analysisof C. elegans mutantswithabnormalbehavior, particularlyUncuncoordinated)phenotypes,hasproven to be useful towards understanding how the nervous system develops and functions. Many unc genes have been elucidated to be involved in nervous system development and function including neurotransmis- sion, 63 axonal guidance 4 and synaptic speci®city. 54,77 Mutants defective in unc-13 have also misplaced and misguided sensory and motor neurons. 68,69 Despite normal levels of acetylcholinesterase AChE), unc-13 mutants have long been known to be resistant to AChE inhibitors such as aldicarb and 2,2,2-trichloro-1-hydroxy- ethyl)-phosphonic acid dimethyl ester trichlorfon). 10,64 These mutants also accumulate abnormally high levels of acetylcholine ACh) without altered levels of choline or choline acetyltransferase activity. 33,35,55,58 The unc-13 gene encodes at least three distinct, one majorandtwominor,mRNAsproducedfromtwodiffer- ent promoters and by alternative splicing. 41,47 The func- tionofthetwominorproductsislessclearthanthemajor product, since deletion of an exon encoded only by the minormRNAsdoesnotcauseanobviousbehaviordefect in C. elegans.ThemajorproductUNC-13)hasaunique structure containing a C1 domain and three repeats of a C2 domain. The C1 domain is shared by other proteins such as protein kinase C 59 and n-chimaerin, 30 and is proposed to be responsible for membrane insertion of the domain after binding to diacylglycerol or tumor- promoting phorbol esters. 82 Indeed, UNC-13 is a high- af®nity receptor for diacylglycerol and phorbol esters in thepresenceofCa 21 in vitro, 1,47 andtheaf®nityisindis- tinguishable from that of protein kinase C enzymes. 40 The C2 domain is also shared by many proteins, and is responsible for its binding to phospholipids 23 and other proteins 83 in the case of synaptotagmin. UNC-13 physi- cally interacts with a complex between UNC-64 C. elegans syntaxin) and UNC-18 also known as Munc18, nSec1 or rbSec1). 67 The structure of UNC-13 is conserved in Drosophila 5 and vertebrates, 11 and a mammalian homolog of UNC-13, Munc13-1, also inter- acts with the synaptic vesicle-associated protein Doc2, 24,56 brain-speci®c b spectrin, 66 calmodulin 80 and the guanine nucleotide exchange factor msec7-1. 57 A human homolog of UNC-13, hmunc13, is also found in Neurotransmission in C. elegans unc-13 mutants 287 287 Neuroscience Vol.104,No.2,pp.287±297,2001 q 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/01 $20.00+0.00 PII: S0306-452201)00097-5 Pergamon www.elsevier.com/locate/neuroscience *Corresponding author. Tel.: 11-858-784-2012; fax: 11-858-784- 9740. E-mail address: ichi@scripps.edu I. N. Maruyama). ²Present address: Of®ce of Technology Administration, Baylor CollegeofMedicine,OneBaylorPlaza,Houston,TX77030,USA. Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; GFP, green ¯uorescence protein; HEPES, N-2-hydroxyethyl)pi- perazine-N 0 -2-ethanesulphonic acid); L, larval stage; levamisole, L[-]-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b]thiazole hydro- chloride; LG, linkage group; trichlorfon, 2,2,2-trichloro-1-hydro- xyethyl)-phosphonic acid dimethyl ester; Unc, uncoordinated; X- gal, 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside.