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, 38 (35), 7667-7682

Persistent Sodium Current Drives Excitability of Immature Renshaw Cells in Early Embryonic Spinal Networks

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Persistent Sodium Current Drives Excitability of Immature Renshaw Cells in Early Embryonic Spinal Networks

Juliette Boeri et al. J Neurosci.

Abstract

Spontaneous network activity (SNA) emerges in the spinal cord (SC) before the formation of peripheral sensory inputs and central descending inputs. SNA is characterized by recurrent giant depolarizing potentials (GDPs). Because GDPs in motoneurons (MNs) are mainly evoked by prolonged release of GABA, they likely necessitate sustained firing of interneurons. To address this issue we analyzed, as a model, embryonic Renshaw cell (V1R) activity at the onset of SNA (E12.5) in the embryonic mouse SC (both sexes). V1R are one of the interneurons known to contact MNs, which are generated early in the embryonic SC. Here, we show that V1R already produce GABA in E12.5 embryo, and that V1R make synaptic-like contacts with MNs and have putative extrasynaptic release sites, while paracrine release of GABA occurs at this developmental stage. In addition, we discovered that V1R are spontaneously active during SNA and can already generate several intrinsic activity patterns including repetitive-spiking and sodium-dependent plateau potential that rely on the presence of persistent sodium currents (INap). This is the first demonstration that INap is present in the embryonic SC and that this current can control intrinsic activation properties of newborn interneurons in the SC of mammalian embryos. Finally, we found that 5 μm riluzole, which is known to block INaP, altered SNA by reducing episode duration and increasing inter-episode interval. Because SNA is essential for neuronal maturation, axon pathfinding, and synaptogenesis, the presence of INaP in embryonic SC neurons may play a role in the early development of mammalian locomotor networks.SIGNIFICANCE STATEMENT The developing spinal cord (SC) exhibits spontaneous network activity (SNA) involved in the building of nascent locomotor circuits in the embryo. Many studies suggest that SNA depends on the rhythmic release of GABA, yet intracellular recordings of GABAergic neurons have never been performed at the onset of SNA in the SC. We first discovered that embryonic Renshaw cells (V1R) are GABAergic at E12.5 and spontaneously active during SNA. We uncover a new role for persistent sodium currents (INaP) in driving plateau potential in V1R and in SNA patterning in the embryonic SC. Our study thus sheds light on a role for INaP in the excitability of V1R and the developing SC.

Keywords: Renshaw cell; development; excitability; mouse embryo; persistent sodium current; spontaneous network activity.

Figures

Figure 1.
Figure 1.
V1R identification in the lumbar spinal cord of E12.5 embryos. A, Coronal slice of the lumbar spinal cord of E12.5 GA67-eGFP mouse embryo showing the distribution of eGFP neurons (A1), FoxD3 (A2), and calbindin (A3) -immunoreactive neurons. A4, Superimposed images showing the colocalization of eGFP, FoxD3 immunostaining, and calbindin immunostaining. B, Enlarged images from A showing that eGFP neurons localized in the ventrolateral area of the spinal cord (B1) are Foxd3-immunoreactive (B2), most of them being calbindin-immunoreactive (B3B4). C, Coronal slice of the ventrolateral part of the lumbar spinal cord of E12.5 GA67-eGFP mouse embryo showing the distribution of eGFP neurons (C1), FoxD3 (C2), and MafB (C3)-immunoreactive neurons. C4, Superimposed images showing the colocalization of eGFP FoxD3 immunostaining and MafB immunostaining. Note that all FoxD3-immunoreactive neurons localized in the marginal zone of the ventrolateral area are also MafB-positive, indicating that they are V1R neurons. D1, Example of a neuron filled with neurobiotin during the recording at the lumbar level of an embryonic spinal cord open book preparation of GAD67-GFP mice at E12.5. This eGFP neuron (D2) was immunoreactive to Foxd3 antibody (D3) as shown in the merged images (D4). Each image corresponds to a single confocal section.
Figure 2.
Figure 2.
V1R already produce GABA in the lumbar spinal cord of E12.5 embryos. A, Single confocal sections of coronal slice of the lumbar spinal cord of E12.5 GA67-eGFP mouse embryo showing the distribution of eGFP neurons (A1), calbindin (A2), and GABA (A3)-immunoreactive neurons. A4, Superimposed images showing the colocalization of eGFP, calbindin immunostaining, and GABA immunostaining. B1B3, Enlarged images from A1, A2, and A3 showing eGFP neurons (B1), calbindin-immunoreactive neurons (B2), and GABA-immunoreactive neurons (B3) in the ventrolateral area of the spinal cord. B4, Superimposed images showing the colocalization of calbindin immunostaining and GABA immunostaining with z projections within a stack. Note the colocalization of calbindin immunostaining and GABA immunostaining in the three axes, indicating that V1R already produce GABA at E12.5.
Figure 3.
Figure 3.
V1R make synaptic-like contacts with motoneurons (HB9-eGFP) at E12.5. A, Coronal slice of the lumbar spinal cord of E12.5 HB9-eGFP mouse embryo with cell nucleus staining (HOESCH) and synaptophysin immunostaining (A1). A1, Note that synaptophysin immunostaining is mainly restricted in the ventral funiculus. A2, Calbindin staining showing the distribution of V1R neurite extensions and synaptophysin immunostaining. A3, eGFP immunostaining showing the distribution of MN neurite extensions and synaptophysin immunostaining. Antibody against GFP was used to visualize MN morphology better (Czarnecki et al., 2014). A4, Superimposition of eGFP fluorescence, calbindin immunostaining and synaptophysin immunostaining (A1A4 are confocal stacks). B, Single confocal sections with z projections of enlarged images from A2 to A4 showing the colocalization of synaptophysin punctates with calbindin immunostaining opposed to eGFP immunostaining (B1B3; enlarged images in boxes). B2, Note that a synaptophysin punctate colocalized with calbindin immunostaining (B1, arrow) did not colocalize with eGFP immunostaining (B2, arrow). Note that synaptophysin punctate colocalized with eGFP immunostaining (B1, arrowheads) did not colocalize with calbindin immunostaining (B2, arrowhead). Barred arrow (B1) shows a colocalization of calbindin and synaptophysin immunostaining not opposed to eGFP immunostaining. B4, superimposed images (B1B3) with z projections showing calbindin immunostaining and eGFP appositions. C1, Confocal stacks showing neurobiotin-injected Foxd3-immunoreactive V1R, HB9-eGFP immunostaining and synaptophysin immunostaining in an SC open book preparation. C2C4, Single confocal sections with z projections of enlarged images from C1 (white box) showing the colocalization of synaptophysin punctates with neurobiotin staining (C2), the apposition of the same synaptophysin punctates to eGFP (C3) and the apposition of neurobiotin staining containing synaptophysin punctates to eGFP (C4; enlarged image in boxes in C2C4), indicating the presence of a V1R synaptic-like contact on an MN neurite.
Figure 4.
Figure 4.
Paracrine release of GABA detected by a sniffer outside-out patch. A1, Top drawing shows the location of the outside-out sniffer to detect paracrine release of GABA (left) and to obtain outside-out currents in response to GABA application (left). Bottom traces (purple): example of outside-out sniffer current evoked by the application of 30 mm KCl when the sniffer electrode was positioned in the dorsal area of the SC close to motor columns. Enlarged trace shows single-channel currents at the onset of the sniffer current. Right traces (green) show outside-out current evoked by the application of 3 and 10 μm GABA to a sniffer patch positioned outside the spinal cord. Purple and green traces are from the same outside-out patch. A2, Box plots of normalized maximum outside-out current evoked by KCl application (purple left) and by the application of 3 or 10 μm GABA (green right) on the same outside-out sniffer patch (n = 9). Amplitudes of the outside-out currents evoked by the application of 30 mm KCl, 3 or 10 μm GABA, were normalized to the amplitude of the outside-out currents evoked by the application of 30 μm GABA (data not shown). Note that the normalized amplitude of the outside-out current evoked by 30 mm KCl application (0.118 ± 0.097) was not significantly different (p > 0.9) from the normalized amplitude of the outside-out current evoked by the application of 3 μm GABA (0.152 ± 0.062). Normalized amplitudes of the outside-out currents evoked by the application of 30 mm KCl or of 3 μm GABA were significantly different (KCl: p = 0.0029; 3 μm GABA: p = 0.00665) from the normalized amplitude of the outside-out currents evoked by the application of 10 μm GABA (0.697 ± 0.073). **p < 0.01. B1, Example of motoneuron membrane potential depolarization evoked by the application of 3 μm GABA in the presence of 1 μm TTX (current-clamp recording: Vh = −60 mV; ECl = −30 mV). B2, The application of 3 μm GABA evoked a depolarizing response of 20.6 ± 6.1 mV (n = 7).
Figure 5.
Figure 5.
V1R display plateau potential, repetitive firing or generate a single AP during episodes of SNA in E12.5 spinal cord. Examples of spontaneous activities recorded in V1R being characterized by GDPs displaying plateau potential (A), repetitive firing (B), or a single AP (C) activity (Vh = −60 mV). Recordings shown in AC are from different cells.
Figure 6.
Figure 6.
V1R display different excitability patterns in E12.5 embryonic spinal cords. Excitability patterns were analyzed using depolarizing current step (2 s) and depolarizing current ramp (20 s). AD, Representative traces of voltage responses showing single-spiking activity (A), plateau potential activity (B), repetitive AP firing (C), and mixed repetitive-spiking/plateau potential activity (D). E, Proportions of V1R subtypes according to the observed discharge patterns. 28.6% of V1R could not sustain repetitive spiking, 41.7% were repetitive-spiking V1R, 8.5% were mixed V1R, 21.2% were plateau potential V1R (n = 164). F, Plateau potentials are blocked by TTX (1 μm) application (n = 5/5; F1, F2). F2, Plateau potentials are evoked by short (100 ms) pulses of depolarizing current.
Figure 7.
Figure 7.
INap is already expressed in V1R. A1, Representative trace of INap evoked by a slow depolarizing voltage ramp in a V1R (CsCl intracellular solution). INap (black trace) was isolated by subtracting the current elicited by a voltage ramp (70 mV/s) in the presence of TTX (inset, green trace) from the control current (inset, black trace). TTX-sensitive current was blocked by 5 μm riluzole (red trace). Left, Inset shows the protocol to generate voltage-dependent slow inward currents in control conditions (black), after 5 μm riluzole application (red) or 1 μm TTX application (green). Right, Inset shows the current evoked by a depolarizing voltage step from −100 to 20 mV in the absence and in the presence of 5 μm riluzole. A2, Voltage dependence of INap conductance calculated from the trace shown in A1. The activation curve was obtained by transforming the current evoked by a depolarizing voltage ramp from −100 to 20 mV (70 mV/s) using the following equation: GNaP = − INap/[(−Vh)+ENa+] where Vh is the holding potential at time t during a depolarizing voltage ramp and ENa+ is the equilibrium potential for sodium (ENa+ = 60 mV). The GNaP/Vh curve was fitted with a Boltzmann function (see Materials and Methods), where Vhalf is the Vh value for INap half activation, k the slope factor of the curve and Gmax the maximum conductance. B, Box plot showing Gmax density in V1R (n = 12). C, Box plots showing the variation of the percentage INap block by 5 μm riluzole in V1R.
Figure 8.
Figure 8.
Effect of 5 and 10 μm riluzole on APs. A, Effect of 5 μm (red trace) and 10 μm (blue trace) riluzole on the AP evoked by a depolarizing current step in an MN. B1, Box plot showing the percentage changes in AP amplitude (% control) in the presence of 5 μm riluzole and 10 μm riluzole. B2, Box plot showing the percentage changes in AP threshold (% control) in the presence of 5 and 10 μm riluzole. B3, Box plot showing the percentage changes in AP half-width (% control) in the presence of 5 and 10 μm riluzole. *p < 0.05, **p < 0.01.
Figure 9.
Figure 9.
Sustained discharge in embryonic V1R depends on INap. A, Representative traces of INap recorded in V1R that cannot sustain repetitive spiking (SS-V1R; A1), in a repetitive-spiking V1R (RS-V1R; A2) and in a plateau potential V1R (PP-V1R; A3). INap was isolated by subtracting the current elicited by a slow voltage ramp (−100 to + 20 mV; 70 mV/s) in the presence of 1 μm TTX from the current evoked in the absence of TTX. A4, Box plots showing Gmax density in SS-V1R (n = 13), RS-V1R (n = 8) and PP-V1R (n = 11). Note that Gmax density is significantly lower in SS-V1R (p < 0.01). B, Representative traces showing the effect of riluzole application (5 μm) on the intrinsic activity pattern evoked by suprathreshold current steps (left traces) or a suprathreshold current ramp (right traces) in an RS-V1R. Note that riluzole blocks repetitive spiking (n = 10/10). C, Representative traces showing the effect of riluzole application on plateau potential evoked by suprathreshold current steps (left traces) or by a suprathreshold current ramp (right traces) in a V1R. Note that riluzole blocks plateau potential activity (n = 7/7). ***p < 0.001.
Figure 10.
Figure 10.
Riluzole dramatically decreases the frequency and duration of episodes of SNA in E12.5 embryonic spinal cord. A1, Application of 5 μm riluzole inhibits spontaneous GIC activity in MNs (voltage-clamp recordings; Vh = −60 mV; ECl = −30 mV). A2, Enlarged trace from (A1) showing GIC before (1, black) and at the onset of riluzole application (2, red). Note that the amplitude and the duration of GICs were decreased on riluzole application. B1, Box plots showing the amplitude of the GIC in a control and on riluzole application (n = 8). B2, Box plots showing half amplitude durations of the GIC in control and on riluzole application (n = 8). Note that the amplitude (p < 0.05) and the duration (p < 0.01) of GICs were significantly reduced in the presence of riluzole. C, SNA recorded at the cervical (C) and lumbar (L) levels (see schematic drawing on the left) in extracellular configuration before (C1) and after 5 μm riluzole (C2). Note that one episode still occurred 35 min after riluzole application. C3C6, Box plots illustrating interburst interval, burst duration, intraburst spike frequency, and cervical-lumbar delay of episode propagation. *p < 0.05, **p < 0.01, ***p < 0.001.

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