Early in development, GABA and glycine exert excitatory action that turns to inhibition due to modification of the chloride equilibrium potential (E Cl ) controlled by the KCC2 and NKCC1 transporters. This switch is thought to be due to a late expression of KCC2 associated with a NKCC1 down-regulation. Here, we show in mouse embryonic spinal cord that both KCC2 and NKCC1 are expressed and functional early in development (E11.5-E13.5) when GABA A receptor activation induces strong excitatory action. After E15.5, a switch occurs rendering GABA unable to provide excitation. At these subsequent stages, NKCC1 becomes both inactive and less abundant in motoneurons while KCC2 remains functional and hyperpolarizes E Cl . In conclusion, in contrast to other systems, the cotransporters are concomitantly expressed early in the development of the mouse spinal cord. Moreover, whereas NKCC1 follows a classical functional extinction, KCC2 is highly expressed throughout both early and late embryonic life.

Rhythmic electrical activity is a hallmark of the developing embryonic CNS and is required for proper development in addition to genetic programs. Neurotransmitter release contributes to the genesis of this activity. In the mouse spinal cord, this rhythmic activity occurs after embryonic day 11.5 (E11.5) as waves spreading along the entire cord. At E12.5, blocking glycine receptors alters the propagation of the rhythmic activity, but the cellular source of the glycine receptor agonist, the release mechanisms, and its function remain obscure. At this early stage, the presence of synaptic activity even remains unexplored. Using isolated embryonic spinal cord preparations and whole-cell patch-clamp recordings of identified motoneurons, we find that the first synaptic activity develops at E12.5 and is mainly GABAergic. Using a multiple approach including direct measurement of neurotransmitter release (i.e., outside-out sniffer technique), we also show that, between E12.5 and E14.5, the main source of glycine in the embryonic spinal cord is radial cell progenitors, also known to be involved in neuronal migration. We then demonstrate that radial cells can release glycine during synaptogenesis. This spontaneous non-neuronal glycine release can also be evoked by mechanical stimuli and occurs through volume-sensitive chloride channels. Finally, we find that basal glycine release upregulates the propagating spontaneous rhythmic activity by depolarizing immature neurons and by increasing membrane potential fluctuations. Our data raise the question of a new role of radial cells as secretory cells involved in the modulation of the spontaneous electrical activity of embryonic neuronal networks.

A remarkable feature of early neuronal networks is their endogenous ability to generate spontaneous rhythmic electrical activity independently of any external stimuli. In the mouse embryonic SC, this activity starts at an embryonic age of ϳ12 d and is characterized by bursts of action potentials recurring every 2-3 min. Although these bursts have been extensively studied using extracellular recordings and are known to play an important role in motoneuron (MN) maturation, the mechanisms driving MN activity at the onset of synaptogenesis are still poorly understood. Because only cholinergic antagonists are known to abolish early spontaneous activity, it has long been assumed that spinal cord (SC) activity relies on a core network of MNs synchronized via direct cholinergic collaterals. Using a combination of whole-cell patch-clamp recordings and extracellular recordings in E12.5 isolated mouse SC preparations, we found that spontaneous MN activity is driven by recurrent giant depolarizing potentials. Our analysis reveals that these giant depolarizing potentials are mediated by the activation of GABA, glutamate, and glycine receptors. We did not detect direct nAChR activation evoked by ACh application on MNs, indicating that cholinergic inputs between MNs are not functional at this age. However, we obtained evidence that the cholinergic dependency of early SC activity reflects a presynaptic facilitation of GABA and glutamate synaptic release via nicotinic AChRs. Our study demonstrates that, even in its earliest form, the activity of spinal MNs relies on a refined poly-synaptic network and involves a tight presynaptic cholinergic regulation of both GABAergic and glutamatergic inputs.

To understand better the role of glycine and gamma-aminobutyric acid (GABA) in the mouse spinal cord during development, we previously described the ontogeny of GABA. Now, we present the ontogeny of glycine-immunoreactive (Gly-ir) somata and fibers, at brachial and lumbar levels, from embryonic day 11.5 (E11.5) to postnatal day 0 (P0). Spinal Gly-ir somata appeared at E12.5 in the ventral horn, with a higher density at the brachial level. They were intermingled with numerous Gly-ir fibers reaching the border of the marginal zone. By E13.5, at the brachial level, the number of Gly-ir perikarya sharply increased throughout the whole ventral horn, whereas the density of fibers declined in the marginal zone. In the dorsal horn, the first Gly-ir somata were then detected. From E13.5 to E16.5, at the brachial level, the density of Gly-ir cells remained stable in the ventral horn, and after E16.5 it decreased to reach a plateau. In the dorsal horn, the density of Gly-ir cells increased, and after E16.5 it remained stable. At the lumbar level, maximum expression was reached at E16.5 in both the ventral and dorsal horn. Finally, the co-localization of glycine and GABA was analyzed, in the ventral motor area, at E13.5, E15.5, and E17.5. The results showed that, regardless of developmental stage studied, one-third of the stained somata co-expressed GABA and glycine. Our data show that the glycinergic system matures 1 day later than the GABAergic system and follows a parallel spatiotemporal evolution, leading to a larger population of glycine cells in the ventral horn.