Vesicular glutamate transporter 3 is required for synaptic transmission in zebrafish hair cells.
Hair cells detect sound and movement and transmit this information via specialized ribbon synapses. Here we report that asteroid, a gene identified in an ethylnitrosourea mutagenesis screen of zebrafish larvae for auditory/vestibular mutants, encodes vesicular glutamate transporter 3 (Vglut3). A splice site mutation in exon 2 of vglut3 results in a severe truncation of the predicted protein product and morpholinos directed against the vglut3 ATG start site or the affected splice junction replicate the asteroid phenotype. In situ hybridization shows that vglut3 is exclusively expressed in hair cells of the ear and lateral line organ. A second transporter gene, vglut1, is also expressed in zebrafish hair cells, but the level of vglut1 mRNA is not increased in the absence of Vglut3. Antibodies against Vglut3 label the basal end of hair cells and labeling is not present in asteroid/vglut3 mutants. Based on the localization of Vglut3 in hair cells, we suspected that the lack of vestibulo-ocular and acoustic startle reflexes in asteroid/vglut3 mutants was attributable to a defect in synaptic transmission in hair cells. In support of this notion, action currents in postsynaptic acousticolateralis neurons are absent in asteroid/vglut3 mutants. At the ultrastructural level, mutant asteroid/vglut3 hair cells show a decrease in the number of ribbon-associated synaptic vesicles, indicating a role for Vglut3 in synaptic vesicle biogenesis and/or tethering to the ribbon body. Lack of postsynaptic action currents in the mutants suggests that the remaining hair-cell synaptic vesicles contain insufficient levels of glutamate for generation of action potentials in first-order neurons.
Obholzer
Nikolaus
N
Howard Hughes Medical Institute, Oregon Hearing Research Center and Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239, USA.
Wolfson
Sean
S
Trapani
Josef G
JG
Mo
Weike
W
Nechiporuk
Alex
A
Busch-Nentwich
Elisabeth
E
Seiler
Christoph
C
Sidi
Samuel
S
Söllner
Christian
C
Duncan
Robert N
RN
Boehland
Andrea
A
Nicolson
Teresa
T
D000161:Acoustic Stimulation / Q000379:methods; D000818:Animals; D030801:Animals, Genetically Modified; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006198:Hair Cells, Auditory / Q000502:physiology*; D007814:Larva; D046529:Microscopy, Electron, Transmission / Q000379:methods; D009154:Mutation / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics; D010812:Physical Stimulation / Q000379:methods; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012027:Reflex, Vestibulo-Ocular / Q000502:physiology; D013569:Synapses / Q000378:metabolism / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000502:physiology*; D050496:Vesicular Glutamate Transport Proteins / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish
D051792:Basic Helix-Loop-Helix Proteins; D049452:Green Fluorescent Proteins; D009419:Nerve Tissue Proteins; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D050496:Vesicular Glutamate Transport Proteins; C073804:FM1 43; C093564:Neurogenic differentiation factor 1