Identification of initially appearing glycine-immunoreactive neurons in the embryonic zebrafish brain
- Authors
- Moly, P.K., Ikenaga, T., Kamihagi, C., Tariqul Islam, A.F., and Hatta, K.
- ID
- ZDB-PUB-140203-3
- Date
- 2014
- Source
- Developmental Neurobiology 74(6): 616-32 (Journal)
- Registered Authors
- Hatta, Kohei
- Keywords
- GABA, GlyT2, MiD2cm, MiD3cm, electroporation, reticulospinal neuron
- MeSH Terms
-
- Animals
- Animals, Genetically Modified
- Axons/physiology
- Brain/cytology*
- Brain/embryology*
- Choline O-Acetyltransferase/metabolism
- Dextrans/metabolism
- Electroporation
- Embryo, Nonmammalian
- Glycine/metabolism*
- Glycine Plasma Membrane Transport Proteins/metabolism
- Green Fluorescent Proteins/genetics
- Green Fluorescent Proteins/metabolism
- Microtubule-Associated Proteins/metabolism
- Neural Pathways/physiology
- Neurons/cytology
- Neurons/metabolism*
- RNA, Messenger/metabolism
- Rhodamines/metabolism
- Zebrafish
- Zebrafish Proteins/metabolism
- gamma-Aminobutyric Acid/metabolism
- PubMed
- 24318965 Full text @ Dev. Neurobiol.
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.