The pre-Bötzinger complex (preBötC) in the ventrolateral medulla contains interneurons important for respiratory rhythm generation. Voltage-dependent sodium channels mediate transient current (I(NaT)), underlying action potentials, and persistent current (I(NaP)), contributing to repetitive firing, pacemaker properties, and the amplification of synaptic inputs. Voltage-clamp studies of the biophysical properties of these sodium currents were conducted on acutely dissociated preBötC region neurons. Reverse transcription-PCR demonstrated the presence of mRNA for Nav1.1, Nav1.2, and Nav1.6 alpha-subunits in individual neurons. A TTX-sensitive I(NaP) was evoked in all tested neurons by ramp depolarization from -80 to 0 mV. Including a constant in the Boltzmann equation for inactivation by estimating the steady-state fraction of Na+ channels available for inactivation allowed prediction of a window current that did not decay to 0 at voltages positive to -20 mV and closely matched the measured I(NaP). Riluzole (3 microM), a putative I(NaP) antagonist, reduced both I(NaP) and I(NaT) and produced a hyperpolarizing shift in the voltage dependence of steady-state inactivation. The latter decreased the predicted window current by an amount equivalent to the decrease in I(NaP). Riluzole also decreased the inactivation time constant at potentials in which the peak window/persistent currents are generated. Together, these findings imply that I(NaP) and I(NaT) arise from the same channels and that a simple modification of the Hodgkin-Huxley model can satisfactorily account for both currents. In the rostral ventral respiratory group (immediately caudal to preBötC), I(NaP) was also detected, but peak conductance, current density, and input resistance were smaller than in preBötC region cells.
[Show abstract][Hide abstract] ABSTRACT: Riluzole is the sole treatment for amyotrophic lateral sclerosis (ALS), but its therapeutically relevant actions on motor neurons are not well defined. Whole cell patch clamp recordings from hypoglossal motor neurons (HMs, n=25) in brainstem slices from 10-23 day old rats anesthetised with sodium pentobarbitone, to investigate the hypothesis that riluzole inhibited HMs by multiple mechanisms. Riluzole (20 μM) hyperpolarized HMs by decreasing an inward current, inhibited voltage-gated persistent Na(+) and Ca(2+) currents activated by slow voltage ramps, and negatively shifted activation of IH. Repetitive firing of HMs was strongly inhibited by riluzole, which also increased action potential threshold voltage and rheobase and decreased amplitude and maximum rise slope, but did not alter the maximal afterhyperpolarization amplitude or decay time constant. HM rheobase was inversely correlated with persistent Na(+) current density. Glutamatergic synaptic transmission was inhibited by riluzole by both pre- and postsynaptic effects. Riluzole decreased activity-dependent glutamate release, as shown by decreased amplitude of evoked and spontaneous EPSCs, decreased paired pulse ratio and decreased spontaneous, but not miniature, EPSC frequency. However, riluzole also decreased miniature EPSC amplitude and the inward current evoked by local application of glutamate onto HMs, suggesting a reduction of postsynaptic glutamate receptor sensitivity. Riluzole thus has a marked inhibitory effect on HM activity by membrane hyperpolarization, decreasing firing and inhibiting glutamatergic excitation by both pre- and postsynaptic mechanisms. These results broaden the range of mechanisms controlling motor neuron inhibition by riluzole, and are relevant to researchers and clinicians interested in understanding ALS pathogenesis and treatment.
Journal of Neurophysiology 06/2013; 110(5). DOI:10.1152/jn.00587.2012 · 2.89 Impact Factor
"Albeit through different mechanisms, both markers produce consistent and widespread labeling of somatic MNs allowing for a direct comparison with the viral labeling pattern. Thus, in some experiments animals were injected with FG 24–48 h before the viral injection to produce global labeling of somatic motoneurons , . The marker was first diluted in 0.9% physiological saline and administered intraperitoneally (IP, 50 µg/g). "
[Show abstract][Hide abstract] ABSTRACT: The neonatal mouse has become a model system for studying the locomotor function of the lumbar spinal cord. However, information about the synaptic connectivity within the governing neural network remains scarce. A neurotropic pseudorabies virus (PRV) Bartha has been used to map neuronal connectivity in other parts of the nervous system, due to its ability to travel trans-neuronally. Its use in spinal circuits regulating locomotion has been limited and no study has defined the time course of labelling for neurons known to project monosynaptically to motoneurons.
Here we investigated the ability of PRV Bartha, expressing green and/or red fluorescence, to label spinal neurons projecting monosynaptically to motoneurons of two principal hindlimb muscles, the tibialis anterior (TA) and gastrocnemius (GC). As revealed by combined immunocytochemistry and confocal microscopy, 24-32 h after the viral muscle injection the label was restricted to the motoneuron pool while at 32-40 h the fluorescence was seen in interneurons throughout the medial and lateral ventral grey matter. Two classes of ipsilateral interneurons known to project monosynaptically to motoneurons (Renshaw cells and cells of origin of C-terminals) were consistently labeled at 40 h post-injection but also a group in the ventral grey matter contralaterally. Our results suggest that the labeling of last order interneurons occurred 8-12 h after motoneuron labeling and we presume this is the time taken by the virus to cross one synapse, to travel retrogradely and to replicate in the labeled cells.
The study establishes the time window for virally-labelling monosynaptic projections to lumbar motoneurons following viral injection into hindlimb muscles. Moreover, it provides a good foundation for intracellular targeting of the labeled neurons in future physiological studies and better understanding the functional organization of the lumbar neural networks.
PLoS ONE 07/2010; 5(7):e11743. DOI:10.1371/journal.pone.0011743 · 3.23 Impact Factor
"The kinetics of this transient Na+ current were significantly slower than those found in neurons (Fig. 1 B). We found that, when a recording was made at membrane potentials more positive than −40 mV, bath-application of phenytoin or riluzole, antagonists of persistent component of Na+ channels (Kononenko et al., 2004; Ptak et al., 2005; Zeng et al., 2005), induced a sustained outward current in the NG2 cell (Fig. 1, C–F), which is consistent with the removal of a noninactivating Na+ conductance (Stys et al., 1993; Kononenko et al., 2004; Ptak et al., 2005). Furthermore, when the recording pipette contained N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX314), an intracellular sodium channel blocker, phenytoin-induced outward currents were absent (Fig. 1, C and D), which confirms the notion that phenytoin-induced outward currents were caused by the blockade of persistent Na+ currents. "
[Show abstract][Hide abstract] ABSTRACT: NG2 cells originate from various brain regions and migrate to their destinations during early development. These cells express voltage-gated Na(+) channels but fail to produce typical action potentials. The physiological role of Na(+) channels in these cells is unclear. We found that GABA induces membrane depolarization and Ca(2+) elevation in NG2 cells, a process requiring activation of GABA(A) receptors, Na(+) channels, and Na(+)/Ca(2+) exchangers (NCXs), but not Ca(2+) channels. We have identified a persistent Na(+) current in these cells that may underlie the GABA-induced pathway of prolonged Na(+) elevation, which in turn triggers Ca(2+) influx via NCXs. This unique Ca(2+) signaling pathway is further shown to be involved in the migration of NG2 cells. Thus, GABAergic signaling mediated by sequential activation of GABA(A) receptors, noninactivating Na(+) channels, and NCXs may play an important role in the development and function of NG2 glial cells in the brain.
The Journal of Cell Biology 08/2009; 186(1):113-28. DOI:10.1083/jcb.200811071 · 9.83 Impact Factor
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