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

Abstract

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.

Keywords: embryo; giant depolarizing potential; motoneuron; mouse; spinal cord; synaptogenesis.

Figure 1.

Spontaneous electrical activity recorded from MNs in the embryonic SC at E12.5. A, Simultaneous whole-cell current-clamp recording (top) and extracellular recording (bottom). The spontaneous activity recorded in MNs is characterized by GDPs occurring at low frequency (V_m = −60 mV). B, In the voltage-clamp mode (V_h = −60 mV), the spontaneous activity is characterized by slow GICs.

Figure 2.

Simultaneous activation of GABA_A, glutamate, and glycine receptors is involved in the generation of spontaneous GICs. A, The application of the GABA_A receptor antagonist gabazine (3 μm), the NMDA receptor antagonist dl-APV (200 μm), plus the AMPA/kainate receptor antagonist CNQX (20 μm) and the glycine receptor antagonist strychnine (3 μm) fully blocks sGICs. There is a presence of fast synaptic currents in the presence of gabazine in A2. V_h = −60 mV. B, Application of the glutamate receptor antagonists dl-APV (200 μm) and CNQX (20 μm) evoked a statistically significant reduction (p < 0.01) of sGIC amplitude as shown in C. C, Relative amplitude of sGICs (% of control) in different pharmacological conditions (test vs control). Error bars indicate SEM. *p < 0.05. **p < 0.01. n.s., Not significant. D, Recording of sGICs at different holding potentials with Echloride = −60 mV and Ecations = 0 mV. Setting V_h = 0 mV reveals the chloride conductance component of sGICs. Setting V_h = −60 mV reveals the cationic conductance component of sGICs. At V_h = 0 mV, sGICs were totally outward (top), whereas at V_h = −60 mV, sGICs were totally inward (bottom). At V_h = −40 mV, sGICs became biphasic and were composed of an early inward current followed by an outward current (middle).

Figure 3.

GICs evoked by neuronal network depolarization are dependent on vesicular release of neurotransmitters. A1, B2, Examples of inward currents evoked by the application of high K⁺ (30 mm). V_h = −60 mV. A1, eGICs are strongly inhibited in the presence of tetrodotoxin (0.3 μm TTX) or in the absence of external calcium. Decreasing external calcium concentration (0 mm [CaCl₂]_o + 4 mm [MgCl₂]_o) is more efficient than TTX in affecting the amplitude of the evoked current. Adding TTX in the absence of calcium had no additional effect. A2, Mean values of the amplitude of eGICs. B1, eGICs in control conditions and after preincubation of SCs with 4 μm bafilomycin A1. Preincubation with bafilomycin A1 resulted in the complete disappearance of eGICs. B2, Current density (pA/pF) of eGICs in control and after 1 h of preincubation with bafilomycin A1. *p < 0.5; **p < 0.01. n.s., Not significant.

Figure 4.

Evoked GICs reflect simultaneous release of GABA, glutamate, and glycine. A1, Example of inward currents evoked by the application of high K⁺ solutions (30 mm) in control conditions (black), in the presence of the GABA_AR antagonist gabazine (3 μm) (green) and in the presence of gabazine + glutamate receptor antagonists dl-APV (200 μm) and CNQX (20 μm) (light blue). V_h = −60 mV. A2, Example of inward currents evoked by the application of high K⁺ solutions (30 mm) in control conditions (black), in the presence of gabazine (3 μm) + glutamate receptor antagonists (200 μm APV and 20 μm CNQX) (light blue) and in the presence of gabazine, APV + CNQX, and the glycine receptor antagonist strychnine (3 μm) (dark blue). V_h = −60 mV. B, eGICs are almost abolished by the addition of strychnine to the solution containing both gabazine and the glutamate receptor antagonists dl-APV and CNQX. **p < 0.01.

Figure 5.

Expression of the vesicular transporters VIAAT for GABA and glycine and VGluT2 and VGluT1 for glutamate in the ventral horn of E12.5 HB9eGFP SCs. A1, B1, C2, MNs were visualized using GFP staining (white) and cell nuclei by DAPI staining (blue). A1, Vesicular transporter for GABA and glycine (VIAAT) immunostaining (red) in the SC of E12.5 HB9eGFP mouse embryos. A2, VIAAT and GFP coimmunostaining in the area of the median motor column. A2, An enlargement of the boxed area shown in A1. A3, Single confocal section showing VIAAT immunostaining (red) within the MN pools (box A3 in A2). VIAAT staining is punctiform and surrounds MN cell bodies (arrows). A4, Single confocal section showing VIAAT immunostaining (red) within the ventral funiculus (box A4 in A2). There is a presence of MN dendritic-like profiles (eGFP staining, white) within the ventral funiculus. VIAAT immunostaining (red) is also punctiform in this area. VIAAT-immunoreactive aggregates can be apposed to MN dendritic-like extensions (arrows). B1, Immunostaining of the glutamate vesicular transporter VGluT2 (red) in the ventral SC area of E12.5 HB9eGFP mouse embryos. B2, Confocal image showing VGluT2 immunostaining in the area of the median motor column. B2, An enlargement of the boxed area shown in B1. B3, Single confocal section showing VGluT2 immunostaining (red) within the MN pools (box B3 in B2). As observed for VIAAT immunostaining, VGluT2 staining is punctiform. There is a presence of VGluT2-immunoreactive aggregates within the MN area (arrows). B4, Single confocal section showing VGluT2 immunostaining (red) within the ventral funiculus (box B4 in B2). The VGluT2-immunoreactive aggregates are equally apposed to MN GFP⁺ extensions (arrows). C1, Immunostaining of the glutamate vesicular transporter VGluT1 (red) in the ventral SC area of E12.5 HB9eGFP mouse embryos. C2, Confocal image showing VGluT1 immunostaining in the area of the median motor column. An enlargement of the boxed area shown in C1. C3, Single confocal section showing VGluT1 immunostaining (red) within the MN pools (box C3 in C2). As observed for VGluT2 and VIAAT immunostaining, VGluT1 staining is punctiform. There is a presence of VGluT1-immunoreactive aggregates within the MN area (arrows). C4, Single confocal section showing VGluT1 immunostaining (red) within the ventral funiculus (box C4 in C2). The VGluT1-immunoreactive aggregates may be apposed to MN GFP⁺ extensions (arrows). A3, A4, B3, B4, C3, C4, Single confocal slices.

Figure 6.

Double immunostaining using VIAAT, VGluT2, VGluT1, and synaptophysin in the ventral horn of E12.5 SCs. A, VIAAT (red) and synaptophysin (green) immunostaining. B, VGluT2 (red) and synaptophysin (green) immunostaining. C, VGluT1 (red) and synaptophysin (green) immunostaining. Scale bar, 10 μm. A2, An enlargement of the boxed area shown in A1. B2, An enlargement of the boxed area shown in B1. C2, An enlargement of the boxed area shown in C1 (colocalized stainings: arrows). A–C, Single confocal slices.

Figure 7.

A simultaneous block of GABA_A, glutamate, and glycine receptors is required to fully inhibit both the SC local network activity and GDPs on MNs. A1, A2, Simultaneous extracellular recording of the SC spontaneous activity from lumbar motor networks and patch-clamp current-clamp recording of sGDPs on lumbar MNs (V_m = −60 mV). SC spontaneous activity and the occurrence of sGDPs are mildly affected in the presence of gabazine (3 μm). Adding the glutamate receptor antagonists dl-APV (200 μm) and CNQX (20 μm) evokes a decrease of sGDP amplitude. Adding APV (200 μm) and CNQX (20 μm) alone slightly reduces MN excitability. Right traces, Recovery of the SC and MN activities after antagonist washout. A3, Quantitative analysis of changes in membrane potential at the peak of sGDPs in the absence or presence of the antagonists (V_m = −60 mV) (test vs control). A4, Quantitative analysis of MN firing propensity, expressed as a percentage of spike occurrence on sGDPs (test vs control). A5, Depolarizing area of GDPs in the absence or presence of the antagonists (test vs control). B, Simultaneous extracellular recording of the SC spontaneous activity from lumbar motor networks and patch-clamp current-clamp recording of sGDPs on lumbar MNs (V_m = −60 mV) in the absence or presence of gabazine + dl-APV + CNQX + strychnine. Adding strychnine to the gabazine-APV-CNQX mixture completely but reversibly blocks the wave of propagating activity as well as the GDPs. **p < 0.01. ***p < 0.001.

Figure 8.

GAP junctions participate in the generation of sGICs and sGDPs. Application of the GAP junction blockers MFA (50 μm) (A) and 18β-GA (50 μm) (B) fully blocks sGICs. A1, B1, Continuous recording of MN activity before and during MFA and 18β-GA application, respectively (V_h = −60 mV). A2, B2, Examples of sGICs recorded in the presence or absence of MFA and 18β-GA, respectively (V_h = −60 mV). C, D, Simultaneous extracellular recording of the SC spontaneous activity and patch-clamp current-clamp recording of sGDPs (V_m = −60 mV). Application of the GAP junction blockers MFA (50 μm) (C) and 18β-GA (D) fully blocks sGDPs. C1, D1, Continuous recording of MN activity before and during MFA and 18β-GA application, respectively. C2, D2, Examples of sGDPs and extracellular recordings recorded in the presence or absence of MFA and 18β-GA, respectively.

Figure 9.

Spontaneous nAChR activation controls the spontaneous occurrence of GICs. A, The application of the general nAChR antagonists mecamylamine (Meca; 50 μm) + d-turbocurarine [dTC]; 5 μm) reversibly abolishes the spontaneous GIC activity. A1, Continuous recording of MN activity before, during, and after mecamylamine + d-turbocurarine application (V_h = −60 mV). A2, Example of sGICs recorded before and after the washout of mecamylamine + d-turbocurarine. B, As observed during mecamylamine + d-turbocurarine applications, 5 μm DHβE reversibly abolishes spontaneous GIC activity. B1, Continuous recording of MN activity before, during, and after DHβE application (V_h = −60 mV). B2, Example of sGICs recorded before and after the washout of DHβE.

Figure 10.

nAChR activation evoked GABA, glutamate, and glycine release. A1, Application of 100 μm ACh in control conditions (black), in the presence of 5 μm DHβE (red) or in the presence of 50 μm mecamylamine plus 10 μm dTC (orange). A2, Application of 100 μm ACh in control conditions (black), in the presence of 3 μm of gabazine (green). A3, Application of 100 μm ACh in control conditions (black), in the presence of gabazine + 200 μm dl-APV and 20 μm CNQX (light blue) and in the presence of 3 μm of gabazine + dl-APV, CNQX, and strychnine (3 μm) (dark blue). A4, Mean values of ACh amplitude evoke inward current in the presence of the different antagonists. V_h = −60 mV. B1, Example of inward currents evoked by the application of high K⁺ solutions (30 mm) in control conditions and in the presence of nAChR antagonists mecamylamine (50 μm) + d-turbocurarine (10 μm) (red) or DHβE (5 μm) (blue). B2, eGICs are insensitive to nAChR antagonists. V_h = −60 mV. *p < 0.05; **p < 0.01. n.s., Not significant.

Figure 11.

nAChR activation is not required for sGIC activity pattern but controls GABA and glutamate release via a presynaptic mechanism. A, Effect of an external K⁺ increase (up to 7 mm) in the presence of mecamylamine (50 μm) + dTC (10 μm) on spontaneous GIC activity (V_h = −60 mV). Increasing the external K⁺ concentration to 7 mm restores sGIC activity (A1, A2). The latter is then completely inhibited by the application of gabazine + dl-APV + CNQX. B1, Application of 100 μm ACh in control conditions (black) or in the presence of 1 μm TTX (red) or 5 μm DHβE (dark blue). V_h = − 60 mV. There is presence of synaptic currents in the presence of TTX. B2, Application of 100 μm ACh in the presence of 1 μm TTX (red), or in the presence of TTX and 3 μm gabazine (green). V_h = −60 mV. There is occurrence of fast synaptic currents in the presence of TTX plus gabazine. B3, Application of 100 μm ACh in the presence of 1 μm TTX plus 3 μm gabazine (green), or in the presence of TTX, gabazine, and 200 μm dl-APV plus 20 μm CNQX (orange). V_h = −60 mV. There is inhibition of fast synaptic currents after the application of APV + CNQX.