Ventrally located commissural neurons express the GABAergic phenotype in developing rat spinal cord

Early-forming commissural neurons are studied intensively as a model of axo nal outgrowth and pathfinding, yet the neurotransmitter phenotype of the ma jority of these neurons is not known. The present study has determined that a substantial number of commissural neurons express the 65-kDa isoform of glutamic acid decarboxylase (GAD65) as early as embryonic day 12 (E12). Pat terns of GAD65 localization were compared with those of TAG-1, the Transien tly expressed Axonal Glycoprotein that is the best known marker of commissu ral axons. On E13, both GAD65- and TAG-1-labeled commissural axons emanate from similar lateral and ventromedial regions. However, dorsally located TA G-1-positive commissural axons were GAD65-negative. These results suggest t hat commissural neurons have both gamma-aminobutyric acid (GABA)ergic and n on-GABAergic phenotypes. The intensity of GAD65 staining within commissural somata and axons decreased between E14-15 and continued to decline during embryonic development, whereas terminal-like structures in surrounding neur opil increased dramatically. This sudden loss of somatic and axonal GAD65 s taining was unexpected and could be interpreted as commissural neurons only transiently expressing the GABAergic phenotype. Further experiments were u ndertaken to identify commissural neurons with other established GABAergic markers, GAD67 and GABA. When antibody labeling of the two GAD isoforms was compared, GAD67 was detected 1 day later than GAD65, and in a different su bcellular distribution. In contrast to GAD65, GAD67 intensely stained somat a but labeled few commissural axons. GABA immunoreactivity also was detecte d in commissural axons 1 day after GAD65, and the labeling pattern between E13 and E16 resembled that of GAD67 rather than GAD65. When GAD and GABA re sults were compared, it was clear that a number of ventrally located commis sural neurons expressed and maintained the GABAergic phenotype during embry onic development. However, the early expression and subcellular redistribut ion of GAD65 suggests that the GAD isoforms are differentially regulated. T he function of the transient GAD65 expression in commissural somata and axo ns is unknown, but its temporal expression pattern parallels the transient expression of TAG-1, as both are expressed during the early stages of commi ssural axon outgrowth and pathfinding. (C) 1999 Wiley-Liss, Inc.