The development and the ionic nature of bistable behavior in lumbar motoneurons were investigated in rats. One week after birth, almost all (∼80%) ankle extensor motoneurons recorded in whole-cell configuration displayed self-sustained spiking in response to a brief depolarization that emerged when the temperature was raised >30°C. The effect of L-type Ca 2+ channel blockers on self-sustained spiking was variable, whereas blockade of the persistent sodium current (I NaP ) abolished them. When hyperpolarized, bistable motoneurons displayed a characteristic slow afterdepolarization (sADP). The sADPs generated by repeated depolarizing pulses summed to promote a plateau potential. The sADP was tightly associated with the emergence of Ca 2+ spikes. Substitution of extracellular Na + or chelation of intracellular Ca 2+ abolished both sADP and the plateau potential without affecting Ca 2+ spikes. These data suggest a key role of a Ca 2+ -activated nonselective cation conductance (I CaN ) in generating the plateau potential. In line with this, the blockade of I CaN by flufenamate abolished both sADP and plateau potentials. Furthermore, 2-aminoethoxydiphenyl borate (2-APB), a common activator of thermo-sensitive vanilloid transient receptor potential (TRPV) cation channels, promoted the sADP. Among TRPV channels, only the selective activation of TRPV2 channels by probenecid promoted the sADP to generate a plateau potential. To conclude, bistable behaviors are, to a large extent, determined by the interplay between three currents: L-type I Ca , I NaP , and a Na + -mediated I CaN flowing through putative TRPV2 channels.
Serotonin (5-HT) plays a critical role in locomotor pattern generation by modulating the rhythm and the coordinations. Pet-1, a transcription factor selectively expressed in the raphe nuclei, controls the differentiation of 5-HT neurons. Surprisingly, inactivation of Pet-1 (Pet-1−/− mice) that causes a 70% reduction in the number of 5-HT-positive neurons in the raphe does not impair locomotion in adult mice. The goal of the present study was to investigate the operation of the locomotor central pattern generator (CPG) in neonatal Pet-1−/− mice. We first confirmed, by means of immunohistochemistry, that there is a marked reduction of 5-HT innervation in the lumbar spinal cord of Pet-1−/− mice. Fictive locomotion was induced in the in vitro neonatal mouse spinal cord preparation by bath application of N-methyl-d,l-Aspartate (NMA) alone or together with dopamine and 5-HT. A locomotor pattern characterized by left–right and flexor–extensor alternations was observed in both conditions. Increasing the concentration of 5-HT from 0.5 to 5 μm impaired the pattern in Pet-1−/− mice. We tested the role of endogenous 5-HT in the NMA-induced fictive locomotion. Application of 5-HT2 or 5-HT7 receptor antagonists affected the NMA-induced fictive locomotion in both heterozygous and homozygous mice although the effects were weaker in the latter strain. This may be, at least partly, explained by the reduced expression of 5-HT2AR as observed by means of immunohistochemistry. These results suggest that compensatory mechanisms take place in Pet-1−/− mice that make locomotion less dependent upon 5-HT.
Spasticity is a disabling motor disorder affecting 70% of people with brain and spinal cord injury. The rate-dependent depression (RDD) of the H reflex is the only electrophysiological measurement correlated with the degree of spasticity assessed clinically in spastic patients. Several lines of evidence suggest that the mechanism underlying the H reflex RDD depends on the strength of synaptic inhibition through GABAA (GABAAR) and glycine receptors (GlyR). In adult rats with spinal cord transection (SCT), we studied the time course of the expression of GABAAR and GlyR at the membrane of retrogradely identified Gastrocnemius and Tibialis anterior motoneurons (MNs) 3, 8 and 16 weeks after injury, and measured the RDD of the H reflex at similar post lesion times. Three weeks after SCT, a significant decrease in the expression of GABAA and GlyR was observed compared to intact rats, and the H-reflex RDD was much less pronounced than in controls. Eight weeks after SCT, GlyR values returned to normal. Simultaneously, we observed a tendency to recover normal RDD of the H reflex at higher frequencies. We tested whether an anti-inflammatory treatment using methylprednisolone performed immediately after SCT could prevent alterations in GABAA/glycine receptors and/or the development of spasticity observed 3 weeks after injury. This treatment restored control levels of GlyR but not the expression of GABAAR, and it completely prevented the attenuation of RDD. These data strongly suggest that alteration of glycinergic inhibition of lumbar MNs is involved in the mechanisms underlying spasticity after SCI.
Maturation of inhibitory postsynaptic transmission onto motoneurons in the rat occurs during the perinatal period, a time window during which pathways arising from the brainstem reach the lumbar enlargement of the spinal cord. There is a developmental switch in miniature IPSCs (mIPSCs) from predominantly long-duration GABAergic to short-duration glycinergic events. We investigated the effects of a complete neonatal [postnatal day 0 (P0)] spinal cord transection (SCT) on the expression of Glycine and GABA A receptor subunits (GlyR and GABA A R subunits) in lumbar motoneurons. In control rats, the density of GlyR increased from P1 to P7 to reach a plateau, whereas that of GABA A R subunits dropped during the same period. In P7 animals with neonatal SCT (SCT-P7), the GlyR densities were unchanged compared with controls of the same age, while the developmental downregulation of GABA A R was prevented. Whole-cell patch-clamp recordings of mIPSCs performed in lumbar motoneurons at P7 revealed that the decay time constant of miniature IPSCs and the proportion of GABAergic events significantly increased after SCT. After daily injections of the 5-HT 2 R agonist DOI, GABA A R immunolabeling on SCT-P7 motoneurons dropped down to values reported in control-P7, while GlyR labeling remained stable. A SCT made at P5 significantly upregulated the expression of GABA A R 1 week later with little, if any, influence on GlyR. We conclude that the plasticity of GlyR is independent of supraspinal influences whereas that of GABA A R is markedly influenced by descending pathways, in particular serotoninergic projections.
Spontaneous activity is observed in most developing neuronal circuits, such as the retina, hippocampus, brainstem and spinal cord. In the spinal cord, spontaneous activity is important for generating embryonic movements critical for the proper development of motor axons, muscles and synaptic connections. A spontaneous bursting activity can be recorded in vitro from ventral roots during perinatal development. The depolarizing action of the inhibitory amino acids γ-aminobutyric acid and glycine is widely proposed to contribute to spontaneous activity in several immature systems. During development, the intracellular chloride concentration decreases, leading to a shift of equilibrium potential for Cl(-) ions towards more negative values, and thereby to a change in glycine- and γ-aminobutyric acid-evoked potentials from depolarization/excitation to hyperpolarization/inhibition. The up-regulation of the outward-directed Cl(-) pump, the neuron-specific potassium-chloride co-transporter type 2 KCC2, has been shown to underlie this shift. Here, we investigated whether spontaneous and locomotor-like activities are altered in genetically modified mice that express only 8-20% of KCC2, compared with wild-type animals. We show that a reduced amount of KCC2 leads to a depolarized equilibrium potential for Cl(-) ions in lumbar motoneurons, an increased spontaneous activity and a faster locomotor-like activity. However, the left-right and flexor-extensor alternating pattern observed during fictive locomotion was not affected. We conclude that neuronal networks within the spinal cord are more excitable in KCC2 mutant mice, which suggests that KCC2 strongly modulates the excitability of spinal cord networks.
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