Reversible suppression of glutamatergic neurotransmission of cerebellar granule cells in vivo by genetically manipulated expression of tetanus neurotoxin light chain.
We developed a novel technique that allowed reversible suppression of glutamatergic neurotransmission in the cerebellar network. We generated two lines of transgenic mice termed Tet and TeNT mice and crossed the two transgenic lines to produce the Tet/TeNT double transgenic mice. In the Tet mice, the tetracycline-controlled reverse activator (rtTA) was expressed selectively in cerebellar granule cells by the promoter function of the GABA(A) receptor alpha6 subunit gene. In the TeNT mice, the fusion gene of tetanus neurotoxin light chain (TeNT) and enhanced green fluorescent protein (EGFP) was designed to be induced by the interaction of doxycycline (DOX)-activated rtTA with the tetracycline-responsive promoter. The Tet/TeNT mice grew normally even after DOX treatment and exhibited a restricted DOX-dependent expression of TeNT in cerebellar granule cells. Along with this expression, TeNT proteolytically cleaved the synaptic vesicle protein VAMP2 (also termed synaptobrevin2) and reduced glutamate release from granule cells. Both cleavage of VAMP2/synaptobrevin2 and reduction of glutamate release were reversed by removal of DOX. Among the four genotypes generated by heterozygous crossing of Tet and TeNT mice, only Tet/TeNT mice showed DOX-dependent reversible motor impairments as analyzed with fixed bar and rota-rod tests. Reversible suppression of glutamatergic neurotransmission thus can be manipulated with spatiotemporal accuracy by DOX treatment and removal. These transgenic mice will serve as an animal model to study the cerebellar function in motor coordination and learning.
Yamamoto
Mutsuya
M
Mitsubishi Pharma Corporation, Discovery Technology Laboratory, Yokohama, 227-0033, Japan. Yamamoto.Mutsuya@mf.m-pharma.co.jp
Wada
Norio
N
Kitabatake
Yasuji
Y
Watanabe
Dai
D
Anzai
Masayuki
M
Yokoyama
Minesuke
M
Teranishi
Yutaka
Y
Nakanishi
Shigetada
S
D000818:Animals; D001259:Ataxia / Q000139:chemically induced / Q000235:genetics; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003017:Clostridium tetani / Q000737:chemistry; D003433:Crosses, Genetic; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D018698:Glutamic Acid / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics* / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D023421:Models, Animal; D009043:Motor Activity / Q000187:drug effects / Q000235:genetics; D009928:Organ Specificity / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects; D021122:Protein Subunits / Q000235:genetics; D050683:R-SNARE Proteins; D012333:RNA, Messenger / Q000096:biosynthesis; D011963:Receptors, GABA-A / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology*; D013744:Tetanus Toxin / Q000235:genetics* / Q000494:pharmacology
C546172:Gabra6 protein, mouse; D008164:Luminescent Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; D050683:R-SNARE Proteins; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D008666:Metalloendopeptidases; C093556:zinc-endopeptidase, tetanus neurotoxin; D004318:Doxycycline