Jean-François Perrier

Subcellular distribution of L-type Ca2+ channels responsible for plateau potentials in motoneurons from the lumbar spinal cord of the turtle

L‐type calcium channels mediate the persistent inward current underlying plateau potentials in spinal motoneurons. Electrophysiological analysis shows that plateau potentials are generated by a persistent inward current mediated by low threshold L‐type calcium channels located in the dendrites. As motoneurons express L‐type calcium channels of the CaV1.2 and CaV1.3 subtypes, we have investigated the subcellular distribution of these channels using antibody labelling. The plateau generating a persistent inward current is modulated by the activation of metabotropic receptors. For this reason, we also examined the relationship between CaV1.2 and CaV1.3 subunits in motoneurons and presynaptic terminals labelled with antibodies against synapsin 1a. Motoneurons in the spinal cord of the adult turtle were identified as large neurons, immunopositive for choline acetyltransferase, located in the ventral horn. In these neurons, CaV1.2 subunits were present in the cell bodies and axons. Patches of CaV1.3 subunits were seen in association with the cell membrane of the somata and both the proximal and distal dendrites. Double labelling with an antibody against synapsin 1a showed that CaV1.3 subunits, but not CaV1.2 subunits, were always located at synaptic sites. The distribution of CaV1.2 and CaV1.3 strongly suggests that the persistent inward current underlying plateau potentials in spinal motoneurons is mediated by CaV1.3 and not by CaV1.2. Our findings also show that CaV1.3 may be located in the somatic and dendritic membrane adjacent to particular presynaptic terminals.

Spinal plasticity mediated by postsynaptic L-type Ca2+ channels

In the spinal cord, motoneurons and specific subgroups of interneurons express L-type Ca(2+) channels. As elsewhere, these dihydropyridine-sensitive channels mediate a slowly activating inward current in response to depolarisation and show little or no inactivation. The slow kinetics for activation and deactivation provide voltage-sensitive properties in a time range from hundreds of milliseconds to tens of seconds and lead to plateau potentials, bistability and wind-up in neurons in both sensory and motor networks. This slow dynamics is in part due to facilitation of L-type Ca(2+) channels by depolarisation. The voltage sensitivity of L-type Ca(2+) channels is also regulated by a range of metabotropic transmitter receptors. Up-regulation is mediated by receptors for glutamate, acetylcholine, noradrenaline and serotonin in motoneurons and by receptors for glutamate and substance P in plateau-generating dorsal horn interneurons. In both cell types, L-type Ca(2+) channels are down-regulated by activation of GABA(B) receptors. In this way, metabotropic regulation in cells expressing L-type Ca(2+) channels provides mechanisms for flexible adjustment of excitability and of the contribution of plateau currents to the intrinsic properties. This type of regulation also steers the magnitude and compartmental distribution of Ca(2+) influx during depolarisation, thus providing a signal for local synaptic plasticity.

Metabotropic synaptic regulation of intrinsic response properties of turtle spinal motoneurones

1 The effect of a brief train of electric stimuli in the dorsolateral funiculus on the intrinsic response properties of turtle motoneurones was investigated in transverse sections of the spinal cord in vitro. 2 Even when glutamatergic, GABAergic and glycinergic ionotropic synaptic transmission was blocked by antagonists of AMPA, NMDA, glycine and GABA receptors, dorsolateral funiculus (DLF) stimulation induced a facilitation of plateau potentials during current clamp and the underlying inward current in voltage clamp. This facilitation lasted more than 10 s. 3 The plateau potential and the facilitation by DLF stimulation was absent in the presence of 10 μm nifedipine. The DLF‐induced facilitation was reduced by antagonists of 5‐HT1a, group 1 metabotropic glutamate receptors and muscarine receptors. 4 These findings suggest that the intrinsic properties of spinal motoneurones are dynamically regulated by afferent synaptic activity. These afferents can be of spinal and extraspinal origin. Continuous regulation of intrinsic response properties could be a mechanism for motor flexibility.

Development and regulation of response properties in spinal cord motoneurons

The intrinsic response properties of spinal motoneurons determine how converging premotor neuronal input is translated into the final motor command transmitted to muscles. From the patchy data available it seems that these properties and their underlying currents are highly conserved in terrestrial vertebrates in terms of both phylogeny and ontogeny. Spinal motoneurons in adults are remarkably similar in many respects ranging from the resting membrane potential to pacemaker properties. Apart from the axolotls, spinal motoneurons from all species investigated have latent intrinsic response properties mediated by L-type Ca2+ channels. This mature phenotype is reached gradually during development through phases in which A-type potassium channels and T-type calcium channels are transiently expressed. The intrinsic response properties of mature spinal motoneurons are subject to short-term adjustments via metabotropic synaptic regulation of the properties of voltage-sensitive ion channels. Recent findings also suggest that regulation of channel expression may contribute to long-term changes in intrinsic response properties of motoneurons.

Mechanisms Causing Plateau Potentials in Spinal Motoneurones

Plateau potentials are generated by a voltage sensitive persistent inward current. In spinal motoneurones this current is predominantly mediated by influx of Ca2+ through L-type Ca2+ channels of the Ca(v)1.3 subtype. Depolarisation-induced facilitation of L-type Ca2+ channels is thought to be the mechanism for delayed activation (wind-up and warm-up) of the plateau potential and for the hysteresis in firing frequency and I-V relation during triangular depolarisation. L-type Ca2+ channels and plateau potentials in spinal motoneurones are facilitated by activation of metabotropic receptors for glutamate, acetylcholine, noradrenaline and serotonin and down regulated by activation of GABA(B) receptors. The facilitation has been shown to depend on activated calmodulin.

Synaptic Release of Serotonin Induced by Stimulation of the Raphe Nucleus Promotes Plateau Potentials in Spinal Motoneurons of the Adult Turtle

Serotonin (5-HT) is a major modulator of the CNS. In motoneurons recorded in slices of the spinal cord, 5-HT promotes plateau potentials mediated by the activity of low-threshold L-type calcium channels (CaV1.3). However, no direct evidence has shown that 5-HT actually promotes plateau potentials under physiological conditions. Here, we investigate how release of 5-HT induced by activation of the raphe nucleus modulates intrinsic properties of spinal motoneurons. We developed an integrated preparation of the brainstem left in continuity with the cervical segments of the spinal cord from adult turtles. Electrical stimulation of the raphe nucleus increased the excitability of motoneurons by decreasing the amplitude of the afterhyperpolarization following action potentials and by promoting plateau potentials. Antagonists of 5-HT2 receptors applied in the vicinity of motoneurons inhibited the facilitation of plateaus. In a slice preparation in which glutamatergic, GABAergic, and glycinergic ionotropic synaptic transmission was blocked, stimulation of the dorsolateral funiculus facilitated a plateau potential by promoting a voltage-sensitive persistent inward current. This effect was inhibited by the addition of antagonists for 5-HT2 receptors. Our study suggests that CaV1.3 channels are regulated by 5-HT released from raphe spinal synaptic terminals via 5-HT2 receptors.

Facilitation of plateau potentials in turtle motoneurones by a pathway dependent on calcium and calmodulin

1 The involvement of intracellular calcium and calmodulin in the modulation of plateau potentials in motoneurones was investigated using intracellular recordings from a spinal cord slice preparation. 2 Chelation of intracellular calcium with BAPTA‐AM or inactivation of calmodulin with W‐7 or trifluoperazine reduced the amplitude of depolarization‐induced plateau potentials. Inactivation of calmodulin also inhibited facilitation of plateau potentials by activation of group I metabotropic glutamate receptors or muscarinic receptors. 3 In low‐sodium medium and in the presence of tetraethylammonium and tetrodotoxin, calcium action potentials evoked by depolarization were followed by a short hyperpolarization ascribed to the calcium‐activated non‐selective cationic current (ICAN) and by a dihydropyridine‐sensitive afterdepolarization. The amplitude of the afterdepolarization depended on the number of calcium spikes and was mediated by L‐type calcium channels. 4 The dihydropyridine‐sensitive afterdepolarization induced by calcium spikes was reduced by blockade of calmodulin. 5 It is proposed that plateau potentials in spinal motoneurones are facilitated by activation of a calcium‐calmodulin‐dependent pathway.

Serotonin spillover onto the axon initial segment of motoneurons induces central fatigue by inhibiting action potential initiation

Motor fatigue induced by physical activity is an everyday experience characterized by a decreased capacity to generate motor force. Factors in both muscles and the central nervous system are involved. The central component of fatigue modulates the ability of motoneurons to activate muscle adequately independently of the muscle physiology. Indirect evidence indicates that central fatigue is caused by serotonin (5-HT), but the cellular mechanisms are unknown. In a slice preparation from the spinal cord of the adult turtle, we found that prolonged stimulation of the raphe-spinal pathway—as during motor exercise—activated 5-HT1A receptors that decreased motoneuronal excitability. Electrophysiological tests combined with pharmacology showed that focal activation of 5-HT1A receptors at the axon initial segment (AIS), but not on other motoneuronal compartments, inhibited the action potential initiation by modulating a Na+ current. Immunohistochemical staining against 5-HT revealed a high-density innervation of 5-HT terminals on the somatodendritic membrane and a complete absence on the AIS. This observation raised the hypothesis that a 5-HT spillover activates receptors at this latter compartment. We tested it by measuring the level of extracellular 5-HT with cyclic voltammetry and found that prolonged stimulations of the raphe-spinal pathway increased the level of 5-HT to a concentration sufficient to activate 5-HT1A receptors. Together our results demonstrate that prolonged release of 5-HT during motor activity spills over from its release sites to the AIS of motoneurons. Here, activated 5-HT1A receptors inhibit firing and, thereby, muscle contraction. Hence, this is a cellular mechanism for central fatigue.

Local facilitation of plateau potentials in dendrites of turtle motoneurones by synaptic activation of metabotropic receptors

1 The spatial distribution of synaptic facilitation of plateau potentials in dendrites of motoneurones was investigated in transverse sections of the spinal cord of the turtle using differential polarization by applied electric fields. 2 The excitability of motoneurones in response to depolarizing current pulses was increased following brief activation of either the dorsolateral funiculus (DLF) or the medial funiculus (MF) even when synaptic potentials were eliminated by antagonists of ionotropic receptors. 3 The medial and lateral compartments of motoneurones were differentially polarized by the electric field generated by passing current between two electrodes on either side of the preparation. In one direction of the field lateral dendrites were depolarized while the cell body and medial dendrites were hyperpolarized (S‐ configuration). With current in the opposite direction the cell body and medial dendrites were depolarized while lateral dendrites were hyperpolarized (S+ configuration). 4 Following brief activation of the DLF the excitability and the generation of plateau potentials were facilitated during differential depolarization of the lateral dendrites but not during differential depolarization of the cell body and medial dendrites. Following brief activation of the MF the excitability and generation of plateau potentials were facilitated during differential depolarization of the cell body and medial dendrites but not during differential depolarization of the lateral dendrites. 5 It is concluded that the synaptic facilitation of the dihydropyridine‐sensitive response to depolarization is compartmentalized in turtle motoneurones.

5‐HT1A receptors increase excitability of spinal motoneurons by inhibiting a TASK‐1‐like K+ current in the adult turtle

The modulatory effects of serotonin mediated by 5‐HT1A receptors in adult spinal motoneurons were investigated by intracellular recordings in a slice preparation from the turtle. In current‐clamp mode, activation of 5‐HT1A receptors by 8‐OH‐DPAT led to depolarization and an increase in input resistance in most motoneurons but caused hyperpolarization and a decrease in input resistance in the remaining smaller fraction of cells. When slices were preincubated in medium containing the 5‐HT1A receptor antagonist WAY‐100635, 8‐OH‐DPAT had no effect. In voltage‐clamp mode, with 1 mm CsCl in the bathing medium, 8‐OH‐DPAT consistently inhibited a leak current that was sensitive to extracellular acidification and anandamide, a TASK‐1 channel blocker. In medium with a low pH, as in the presence of anandamide, 8‐OH‐DPAT had no effect. Our results show that activation of 5‐HT1A receptors contributes to the excitatory effect of serotonin on spinal motoneurons by inhibition of a TASK‐1 potassium channel leading to depolarization and increased input resistance.