Adult-type glycine receptors form clusters on embryonic rat spinal cord neurons developing in vitro

The time course of development and the cellular and subcellular distributions of adult-type, strychnine-binding glycine receptors (GlyRs) were examined on rat spinal cord neurons in vitro using both the GlyR antagonist strychnine and a specific antibody against the receptor. Spinal cord neurons in vivo had no detectable adult-type GlyRs at embryonic day 14, the age used to establish cell cultures. After being placed in culture, neurons continued for several days to show no expression of adult-type GlyRs. This contrasted with the observation that these neurons began to express receptors for substance P within 24 hr in culture. Beginning after 5-7 d in culture, however, neurons began to express adult-type GlyRs on their surfaces, and the number of such receptors increased abruptly thereafter. Ultimately, about 80% of the neurons in heterogeneous spinal cord cultures came to express adult-type GlyRs, with all of them beginning to express the GlyRs at approximately the same time. The subcellular distribution of adult- type GlyRs on neurons was observed by fluorescence microscopy using both anti-GlyR antibody and a fluorescent derivative of strychnine. On neurons that had been in culture for more than 10 d, GlyRs were localized in focal clusters, or aggregates, that were distributed over both cell bodies and neurites. The apparent location of these clusters on the cell surface was confirmed by the observation that GlyRs could be labeled with the antibody on living neurons. The nonuniform distribution of GlyRs was observed using the monovalent probe fluorescent strychnine on living neurons and using the antibody on fixed neurons and on living neurons at 4°C, thus showing that aggregation of GlyRs was not induced by the labeling procedures. Focal clusters of GlyRs were also seen on younger neurons that were in the early stages of expression of adult-type GlyRs. In addition to the clusters, however, such neurons showed regions where the GlyRs were more diffuse. These results show that adult-type GlyRs are expressed on developing spinal cord neurons only after several days in culture, and that such GlyRs typically form focal clusters on the surfaces of spinal cord neurons, even at very early stages of their expression. The results also suggest, however, that at the earliest stage of expression, at least some of the adult-type GlyRs on these neurons may have a different, more diffuse distribution.