Anne-Emilie Allain

NKCC1 cotransporter inactivation underlies embryonic development of chloride-mediated inhibition in mouse spinal motoneuron

Early in development, GABA and glycine exert excitatory action that turns to inhibition due to modification of the chloride equilibrium potential (ECl) 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 GABAA 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 ECl. 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.

Glycine Release from Radial Cells Modulates the Spontaneous Activity and Its Propagation during Early Spinal Cord Development

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.

Ontogenic changes of the GABAergic system in the embryonic mouse spinal cord

Numerous studies have demonstrated an excitatory action of GABA early in development, which is likely to play a neurotrophic role. In order to better understand the role of GABA in the mouse spinal cord, we followed the evolution of GABAergic neurons over the course of development. We investigated, in the present study, the ontogeny of GABA immunoreactive (GABA-ir) cell bodies and fibers in the embryonic mouse spinal cord at brachial and lumbar levels. GABA-ir somata were first detected at embryonic day 11.5 (E11.5) exclusively at brachial level in the marginal zone. By E13.5, the number of GABAergic neurons sharply increased throughout the extent of the ventral horn both at brachial and lumbar level. Stained perikarya first appeared in the future dorsal horn at E15.5 and progressively invaded this area while they decreased in number in the presumed ventral gray matter. At E12.5, E13.5 and E15.5, we checked the possibility that ventral GABA-ir cells could belong to the motoneuronal population. Using a GABA/Islet-1/2 double labeling, we did not detect any double-stained neurons indicating that spinal motoneurons do not synthesize GABA during the course of development. GABA-ir fibers also appeared at the E11.5 stage in the presumptive lateral white matter at brachial level. At E12.5 and E13.5, GABA-ir fibers progressively invaded the ventral marginal zone and by E15.5 reached the dorsal marginal zone. At E17.5 and postnatal day 0 (P0), the number of GABA-ir fibers declined in the white matter. Finally, by P0, GABA immunoreactivity that delineated somata was mainly restricted to the dorsal gray matter and declined in intensity and extent. The ventral gray matter exhibited very few GABA-ir cell bodies at this neonatal stage of development. The significance of the migration of somatic GABA immunoreactivity from ventral to the dorsal gray matter is discussed.

Ontogenic Changes of the Spinal GABAergic Cell Population Are Controlled by the Serotonin (5-HT) System: Implication of 5-HT1 Receptor Family

During the development of the nervous system, the acquisition of the GABA neurotransmitter phenotype is crucial for neural networks operation. Although both intrinsic and extrinsic signals such as transcription factors and growth factors have been demonstrated to govern the acquisition of GABA, few data are available concerning the effects of modulatory transmitters expressed by axons that progressively invade emerging neuronal networks. Among such transmitters, serotonin (5-HT) is a good candidate because serotonergic axons innervate the entire CNS at very early stages of development. We have shown previously that descending 5-HT slows the maturation of inhibitory synaptic transmission in the embryonic mouse spinal cord. We now report that 5-HT also regulates the spatiotemporal changes of the GABAergic neuronal population in the mouse spinal cord. Using a quantitative confocal study performed on acute and cultured spinal cords, we find that the GABAergic population matures according to a similar rostrocaudal temporal gradient both in utero and in organotypic culture. Moreover, we show that 5-HT delays the appearance of the spinal GABAergic system. Indeed, in the absence of 5-HT descending inputs or exogenous 5-HT, the GABAergic population matures earlier. In the presence of exogenous 5-HT, the GABA population matures later. Finally, using a pharmacological approach, we show that 5-HT exerts its action via the 5-HT1 receptor family. Together, our data suggest that, during the course of the embryonic development, 5-HT descending inputs delay the maturation of lumbar spinal motor networks relative to brachial networks.

Acetylcholine Controls GABA-, Glutamate-, and Glycine-Dependent Giant Depolarizing Potentials that Govern Spontaneous Motoneuron Activity at the Onset of Synaptogenesis in the Mouse Embryonic Spinal Cord

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 ∼12 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.

Maturation of the GABAergic Transmission in Normal and Pathologic Motoneurons

γ-aminobutyric acid (GABA) acting on Cl−-permeable ionotropic type A (GABAA) receptors (GABAAR) is the major inhibitory neurotransmitter in the adult central nervous system of vertebrates. In immature brain structures, GABA exerts depolarizing effects mostly contributing to the expression of spontaneous activities that are instructive for the construction of neural networks but GABA also acts as a potent trophic factor. In the present paper, we concentrate on brainstem and spinal motoneurons that are largely targeted by GABAergic interneurons, and we bring together data on the switch from excitatory to inhibitory effects of GABA, on the maturation of the GABAergic system and GABAAR subunits. We finally discuss the role of GABA and its GABAAR in immature hypoglossal motoneurons of the spastic (SPA) mouse, a model of human hyperekplexic syndrome.

Serotonin controls the maturation of the GABA phenotype in the ventral spinal cord via 5‐HT1B receptors

Serotonin (5‐hydroxytryptamine or 5‐HT) is a pleiotropic neurotransmitter known to play a crucial modulating role during the construction of brain circuits. Descending bulbo‐spinal 5‐HT fibers, coming from the caudal medullary cell groups of the raphe nuclei, progressively invade the mouse spinal cord and arrive at lumbar segments at E15.5 when the number of ventral GABA immunoreactive (GABA‐ir) interneurons reaches its maximum. We thus raised the question of a possible interaction between these two neurotransmitter systems and investigated the effect of 5‐HT descending inputs on the maturation of the GABA phenotype in ventral spinal interneurons. Using a quantitative anatomical study performed on acute and cultured embryonic mouse spinal cord, we found that the GABAergic neuronal population matured according to a similar rostro‐caudal gradient both in utero and in organotypic culture. We showed that 5‐HT delayed the maturation of the GABA phenotype in lumbar but not brachial interneurons. Using pharmacological treatments and mice lacking 5‐HT1B or 5‐HT1A, we demonstrated that the 5‐HT repressing effect on the GABAergic phenotype was specifically attributed to 5‐HT1B receptors.

Nonsynaptic glycine release is involved in the early KCC2 expression.

The cation‐chloride co‐transporters are important regulators of the cellular Cl‐ homeostasis. Among them the Na+‐K+−2Cl− co‐transporter (NKCC1) is responsible for intracellular chloride accumulation in most immature brain structures, whereas the K+‐Cl− co‐transporter (KCC2) extrudes chloride from mature neurons, ensuring chloride‐mediated inhibitory effects of GABA/glycine. We have shown that both KCC2 and NKCC1 are expressed at early embryonic stages (E11.5) in the ventral spinal cord (SC). The mechanisms by which KCC2 is prematurely expressed are unknown. In this study, we found that chronically blocking glycine receptors (GlyR) by strychnine led to a loss of KCC2 expression, without affecting NKCC1 level. This effect was not dependent on the firing of Na+ action potentials but was mimicked by a Ca2+‐dependent PKC blocker. Blocking the vesicular release of neurotransmitters did not impinge on strychnine effect whereas blocking volume‐sensitive outwardly rectifying (VSOR) chloride channels reproduced the GlyR blockade, suggesting that KCC2 is controlled by a glycine release from progenitor radial cells in immature ventral spinal networks. Finally, we showed that the strychnine treatment prevented the maturation of rhythmic spontaneous activity. Thereby, the GlyR‐activation is a necessary developmental process for the expression of functional spinal motor networks.

Depolarizing GABA/glycine synaptic events switch from excitation to inhibition during frequency increases.

By acting on their ionotropic chloride channel receptors, GABA and glycine represent the major inhibitory transmitters of the central nervous system. Nevertheless, in various brain structures, depolarizing GABAergic/glycinergic postsynaptic potentials (dGPSPs) lead to dual inhibitory (shunting) and excitatory components, the functional consequences of which remain poorly acknowledged. Indeed, the extent to which each component prevails during dGPSP is unclear. Understanding the mechanisms predicting the dGPSP outcome on neural network activity is therefore a major issue in neurobiology. By combining electrophysiological recordings of spinal embryonic mouse motoneurons and modelling study, we demonstrate that increasing the chloride conductance (gCl) favors inhibition either during a single dGPSP or during trains in which gCl summates. Finally, based on this summation mechanism, the excitatory effect of EPSPs is overcome by dGPSPs in a frequency-dependent manner. These results reveal an important mechanism by which dGPSPs protect against the overexcitation of neural excitatory circuits.

VEGF modulates synaptic activity in the developing spinal cord

Although it has been documented that the nervous and the vascular systems share numerous analogies and are closely intermingled during development and pathological processes, interactions between the two systems are still poorly described. In this study, we investigated whether vascular endothelial growth factor (VEGF), which is a key regulator of vascular development, also modulates neuronal developmental processes. We report that VEGF enhances the gamma‐aminobutyric acid (GABA)/glycinergic but not glutamatergic synaptic activity in embryonic spinal motoneurons (MNs), without affecting MNs excitability. In response to VEGF, the frequency of these synaptic events but not their amplitude was increased. Blocking endogenous VEGF led to an opposite effect by decreasing frequency of synaptic events. We found that this effect occurred specifically at early developmental stages (E13.5 and E15.5) and vanished at the prenatal stage E17.5. Furthermore, VEGF was able to increase vesicular inhibitory amino acid transporter density at the MN membrane. Inhibition of single VEGF receptors did not modify electrophysiological parameters indicating receptor combinations or an alternative pathway. Altogether, our findings identify VEGF as a modulator of the neuronal activity during synapse formation and highlight a new ontogenic role for this angiogenic factor in the nervous system.