title: Identification of initially appearing glycine-immunoreactive neurons in the embryonic zebrafish brain.
abstract: Glycine is a major inhibitory neurotransmitter in the central nervous system of vertebrates. Here, we report the initial development of glycine-immunoreactive (Gly-ir) neurons and fibers in zebrafish. The earliest Gly-ir cells were found in the hindbrain and rostral spinal cord by 20 h post-fertilization (hpf). Gly-ir cells in rhombomeres 5 and 6 that also expressed glycine transporter 2 (glyt2) mRNA were highly stereotyped; they were bilaterally located and their axons ran across the midline and gradually turned caudally, joining the medial longitudinal fascicles in the spinal cord by 24 hpf. Gly-ir neurons in rhombomere 5 were uniquely identified, since there was one per hemisegment, whereas the number of Gly-ir neurons in rhombomere 6 were variable from one to three per hemisegment. Labeling of these neurons by single-cell electroporation and tracing them until the larval stage revealed that they became MiD2cm and MiD3cm, respectively. The retrograde labeling of reticulo-spinal neurons in Tg(glyt2:gfp) larva, which express GFP in Gly-ir cells, and a genetic mosaic analysis with glyt2:gfp DNA construct also supported this notion. Gly-ir cells were also distributed widely in the anterior brain by 27 hpf, whereas glyt2 was hardly expressed. Double staining with anti-glycine and anti-GABA antibodies demonstrated distinct distributions of Gly-ir and GABA-ir cells, as well as the presence of doubly immunoreactive cells in the brain and placodes. These results provide evidence of identifiable glycinergic (Gly-ir/glyt2-positive) neurons in vertebrate embryos, and they can be used in further studies of the neurons' development and function at the single-cell level.
authors: Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako-gun, Hyogo, 678-1297, Japan.
authors: Pricila Khan
authors: PK
authors: Moly
authors: Takanori
authors: T
authors: Ikenaga
authors: Chihiro
authors: C
authors: Kamihagi
authors: A F M Tariqul
authors: AF
authors: Islam
authors: Kohei
authors: K
authors: Hatta
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001369:Axons / Q000502:physiology; D001921:Brain / Q000166:cytology* / Q000196:embryology*; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D003911:Dextrans / Q000378:metabolism; D018274:Electroporation; D004625:Embryo, Nonmammalian; D005998:Glycine / Q000378:metabolism*; D050487:Glycine Plasma Membrane Transport Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009434:Neural Pathways / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D003911:Dextrans; C067661:Fluoro-Ruby; D050487:Glycine Plasma Membrane Transport Proteins; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D012235:Rhodamines; C507810:SLC6A5 protein, zebrafish; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins; D005680:gamma-Aminobutyric Acid; D002795:Choline O-Acetyltransferase; D005998:Glycine
keywords: GABA; GlyT2; MiD2cm; MiD3cm; electroporation; reticulospinal neuron