Spinal commissural neurons serve as a model system for studying mechanisms that underlie axonal pathfinding during development, however little is known about their neurotransmitter phenotype or synaptic targets. Using antibodies specific for both forms of glutamic acid decarboxylase (GAD65 and GAD67) and y-aminobutyric acid (GABA), I determined that a substantial number of ventrally located commissural neurons in the developing rat spinal cord is GABAergic. During early embryonic ages (E12–17), both GAD65 and GABA were found in commissural neuron cell bodies, axons and their growth cones, whereas at older ages (E18–P28), they were localized primarily in terminal-like structures. To prove that these developmental changes in the localization of GAD65 and GABA were due to an intracellular translocation from cell bodies to axon terminals, axonal transport was blocked in organotypic slices. These perturbation experiments demonstrated that numerous GAD65- and GAD67-positive cell bodies were present and maintained throughout the developing spinal cord.; To show that the GABAergic commissural neurons expressed similar guidance molecules to previously described dorsal commissural neurons, I examined the expression of the L1 cell adhesion molecule on ventral commissural axons. L1 was detected on both the ipsi- and contralateral portions of the ventrally located commissural axons, including many GABAergic commissural axons. Numerous GAD65- and L1-positive commissural axons were identified coursing within the ventral marginal zone and ascended into the brainstem. To determine if these commissural neurons projected to targets outside of the spinal cord, embryos were injected with DiI in the unilateral ventral spinal cord. DiI-labeled axons in the contralateral ventral marginal zone projected rostrally into the brainstem at three consecutive ages and finally appeared to branch and terminate into small punctate structures. When DiI was injected into the presumptive lateral midbrain target, labeled axons traveled through the brainstem and into the spinal cord along a pathway similar to that seen with DiI injections in the ventral spinal cord and GAD65/L1 immunochemical labeled embryos. In addition, DiI injections in the lateral midbrain identified labeled cell bodies in the contralateral ventromedial spinal cord. Taken together, these findings suggest that GABAergic commissural neurons are present and maintained throughout development and are spinomesencephalic projection neurons that may terminate in the mesencephalic reticular formation.
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