Developmental Formation of the GABAergic and Glycinergic Networks in the Mouse Spinal Cord.

Gamma-aminobutyric acid (GABA) and glycine act as inhibitory neurotransmitters. Three types of inhibitory neurons and terminals, GABAergic, GABA/glycine coreleasing, and glycinergic, are orchestrated in the spinal cord neural circuits and play critical roles in regulating pain, locomotive movement, and respiratory rhythms. In this study, we first describe GABAergic and glycinergic transmission and inhibitory networks, consisting of three types of terminals in the mature mouse spinal cord. Second, we describe the developmental formation of GABAergic and glycinergic networks, with a specific focus on the differentiation of neurons, formation of synapses, maturation of removal systems, and changes in their action. GABAergic and glycinergic neurons are derived from the same domains of the ventricular zone. Initially, GABAergic neurons are differentiated, and their axons form synapses. Some of these neurons remain GABAergic in lamina I and II. Many GABAergic neurons convert to a coreleasing state. The coreleasing neurons and terminals remain in the dorsal horn, whereas many ultimately become glycinergic in the ventral horn. During the development of terminals and the transformation from radial glia to astrocytes, GABA and glycine receptor subunit compositions markedly change, removal systems mature, and GABAergic and glycinergic action shifts from excitatory to inhibitory.

Distinct development of the glycinergic terminals in the ventral and dorsal horns of the mouse cervical spinal cord.

In the spinal cord, glycine and γ-amino butyric acid (GABA) are inhibitory neurotransmitters. However, the ontogeny of the glycinergic network remains unclear. To address this point, we examined the developmental formation of glycinergic terminals by immunohistochemistry for glycine transporter 2 (GlyT2), a marker of glycinergic terminals, in developing mouse cervical spinal cord. Furthermore, the developmental localization of GlyT2 was compared with that of glutamic acid decarboxylase (GAD), a marker of GABAergic terminals, and vesicular GABA transporter (VGAT), a marker of inhibitory terminals, by single and double immunolabeling. GlyT2-positive dots (glycinergic terminals) were first detected in the marginal zone on embryonic day 14 (E14). In the ventral horn, they were detected at E16 and increased in observed density during postnatal development. Until postnatal day 7 (P7), GAD-positive dots (GABAergic terminals) were dominant and GlyT2 immunolabeling was localized at GAD-positive dots. During the second postnatal week, GABAergic terminals markedly decreased and glycinergic terminals became dominant. In the dorsal horn, glycinergic terminals were detected at P0 in lamina IV and P7 in lamina III and developmentally increased. GlyT2 was also localized at GAD-positive dots, and colocalizing dots were dominant at P21. VGAT-positive dots (inhibitory terminals) continued to increase until P21. These results suggest that GABAergic terminals first appear during embryonic development and may often change to colocalizing terminals throughout the gray matter during development. The colocalizing terminals may remain in the dorsal horn, whereas in the ventral horn, colocalizing terminals may give rise to glycinergic terminals.

Developmental localization of calcitonin gene-related peptide in dorsal sensory axons and ventral motor neurons of mouse cervical spinal cord.

Calcitonin gene-related peptide (CGRP) is a 37-amino-acid neuropeptide, synthesized by alternative splicing of calcitonin gene mRNA. CGRP is characteristically distributed in the nervous system, and its function varies depending on where it is expressed. To reveal developmental formation of the CGRP network and its function in neuronal maturation, we examined the immunohistochemical localization of CGRP in the developing mouse cervical spinal cord and dorsal root ganglion. CGRP immunolabeling (IL) was first detected in motor neurons on E13, and in ascending axons of the posterior funiculus and DRG neurons on E14. CGRP-positive sensory axon fibers entered Laminae I and II on E16, and Laminae I through IV on E18. The intensity of the CGRP-IL gradually increased in both ventral and dorsal horns during embryonic development, but markedly decreased in the ventral horn after birth. These results suggest that CGRP is expressed several days after neuronal settling and entry of sensory fibers, and that the CGRP network is formed in chronological and sequential order. Furthermore, because CGRP is markedly expressed in motor neurons when axons are vastly extending and innervating targets, CGRP may also be involved in axonal elongation and synapse formation during normal development.