NMDA receptors in preBötzinger complex neurons can drive respiratory rhythm independent of AMPA receptors

The role of AMPA receptors (AMPARs) in generation and propagation of respiratory rhythm is well documented both in vivo and in vitro , whereas the functional significance of NMDA receptors (NMDARs) in preBötzinger complex (preBötC) neurons has not been explored. Here we examined the interactions between AMPARs and NMDARs during spontaneous respiratory rhythm generation in slices from neonatal rats in vitro . We tested the hypothesis that activation of NMDARs can drive respiratory rhythm in the absence of other excitatory drives. Blockade of NMDARs with dizocilpine hydrogen maleate (MK-801, 20 μ m ) had a negligible effect on respiratory rhythm and pattern under standard conditions in vitro , whereas blockade of AMPARs with NBQX (0.5 μ m ) completely abolished respiratory activity. Removal of extracellular Mg²⁺ to relieve the voltage-dependent block of NMDARs maintained respiratory rhythm without a significant effect on period, even in the presence of high NBQX concentrations (≤ 100 μ m ). Removal of Mg²⁺ increased inspiratory-modulated inward current peak ( I _I) and charge ( Q _I) in preBötC neurons voltage-clamped at −60 mV by 245% and 309%, respectively, with respect to basal values. We conclude that the normal AMPAR-mediated postsynaptic current underlying respiratory drive can be replaced by NMDAR-mediated postsynaptic current when the voltage-dependent Mg²⁺ block is removed. Under this condition, respiratory-related frequency is unaffected by changes in I _I, suggesting that the two can be independently regulated.     

Respiratory rhythm generation during gasping depends on persistent sodium current

In severe hypoxia, homeostatic mechanisms maintain function of the brainstem respiratory network. We hypothesized that hypoxia involves a transition from neuronal mechanisms of normal breathing (eupnea) to a rudimentary pattern of inspiratory movements (gasping). We provide evidence for hypoxia-driven transformation within the central respiratory oscillator, in which gasping relies on persistent sodium current, whereas eupnea does not depend on this cellular mechanism.     

Role of persistent sodium current in bursting activity of mouse neocortical networks in vitro

Most types of electrographic epileptiform activity can be characterized by isolated or repetitive bursts in brain electrical activity. This observation is our motivation to determine mechanisms that underlie bursting behavior of neuronal networks. Here we show that the persistent sodium (Na(P)) current in mouse neocortical slices is associated with cellular bursting and our data suggest that these cells are capable of driving networks into a bursting state. This conclusion is supported by the following observations. 1) Both low concentrations of tetrodotoxin (TTX) and riluzole reduce and eventually stop network bursting while they simultaneously abolish intrinsic bursting properties and sensitivity levels to electrical stimulation in individual intrinsically bursting cells. 2) The sensitivity levels of regular spiking neurons are not significantly affected by riluzole or TTX at the termination of network bursting. 3) Propagation of cellular bursting in a neuronal network depended on excitatory connectivity and disappeared on bath application of CNQX (20 microM) + CPP (10 microM). 4) Voltage-clamp measurements show that riluzole (20 microM) and very low concentrations of TTX (50 nM) attenuate Na(P) currents in the neural membrane within a 1-min interval after bath application of the drug. 5) Recordings of synaptic activity demonstrate that riluzole at this concentration does not affect synaptic properties. 6) Simulations with a neocortical network model including different types of pyramidal cells, inhibitory interneurons, neurons with and without Na(P) currents, and recurrent excitation confirm the essence of our experimental observations that Na(P) conductance can be a critical factor sustaining slow population bursting.     

Role of synaptic inhibition in turtle respiratory rhythm generation

In vitro brainstem and brainstem-spinal cord preparations were used to determine the role of synaptic inhibition in respiratory rhythm generation in adult turtles. Bath application of bicuculline (a GABA_A receptor antagonist) to brainstems increased hypoglossal burst frequency and amplitude, with peak discharge shifted towards the burst onset. Strychnine (a glycine receptor antagonist) increased amplitude and frequency, and decreased burst duration, but only at relatively high concentrations (10-100 μM). Rhythmic activity persisted during combined bicuculline and strychnine application (50 μM each) with increased amplitude and frequency, decreased burst duration, and a rapid onset-decrementing burst pattern. The bicuculline-strychnine rhythm frequency decreased during μ-opioid receptor activation or decreased bath P _CO2. Synaptic inhibition blockade in the brainstem of brainstem-spinal cord preparations increased burst amplitude in spinal expiratory (pectoralis) nerves and nearly abolished spinal inspiratory activity (serratus nerves), suggesting that medullary expiratory motoneurons were mainly active. Under conditions of synaptic inhibition blockade in vitro , the turtle respiratory network was able to produce a rhythm that was sensitive to characteristic respiratory stimuli, perhaps via an expiratory (rather than inspiratory) pacemaker-driven mechanism. Thus, these data indicate that the adult turtle respiratory rhythm generator has the potential to operate in a pacemaker-driven manner.     

Inspiratory bursts in the preBötzinger complex depend on a calcium-activated non-specific cation current linked to glutamate receptors in neonatal mice

Inspiratory neurons of the preBötzinger complex (preBötC) form local excitatory networks and display 10–30 mV transient depolarizations, dubbed inspiratory drive potentials , with superimposed spiking. AMPA receptors are critical for rhythmogenesis under normal conditions in vitro but whether other postsynaptic mechanisms contribute to drive potential generation remains unknown. We examined synaptic and intrinsic membrane properties that generate inspiratory drive potentials in preBötC neurons using neonatal mouse medullary slice preparations that generate respiratory rhythm. We found that NMDA receptors, group I metabotropic glutamate receptors (mGluRs), but not group II mGluRs, contributed to inspiratory drive potentials. Subtype 1 of the group I mGluR family (mGluR1) probably regulates a K⁺ channel, whereas mGluR5 operates via an inositol 1,4,5-trisphosphate (IP₃) receptor-dependent mechanism to augment drive potential generation. We tested for and verified the presence of a Ca²⁺-activated non-specific cation current ( I _CAN) in preBötC neurons. We also found that high concentrations of intracellular BAPTA, a high-affinity Ca²⁺ chelator, and the I _CAN antagonist flufenamic acid (FFA) decreased the magnitude of drive potentials. We conclude that I _CAN underlies robust inspiratory drive potentials in preBötC neurons, and is only fully evoked by ionotropic and metabotropic glutamatergic synaptic inputs, i.e. by network activity.     

Upregulation of persistent and ramp sodium current in dorsal horn neurons after spinal cord injury

Traumatic spinal cord injury (SCI) results not only in motor impairment, but also in chronic central neuropathic pain, which often is refractory to conventional treatment approaches. Upregulated expression of sodium channel Nav1.3 has been observed within the spinal dorsal horn neurons after SCI, and appears to contribute to neuronal hyperresponsiveness and pain-related behaviors. In this study we characterized the changes in sodium current properties within dorsal horn neurons after contusive SCI. Four weeks after adult male Sprague-Dawley rats underwent T9 spinal cord contusion injury, when behavioral nociceptive thresholds were decreased to both mechanical and thermal stimuli, whole-cell patch-clamp recordings were performed on acutely dissociated lumbar dorsal horn neurons. The cells demonstrated characteristic fast-activating and fast-inactivating sodium currents. SCI led to a shift of the steady-state activation and inactivation of the sodium current towards more depolarized potentials. The shifted steady-state inactivation shows similarities to that obtained from axotomized dorsal root ganglions, which were shown to upregulate Nav1.3. Small slow depolarizations below action potential threshold produced ramp currents, which were markedly enhanced by SCI (from 182 ± 41 to 338 ± 55 pA). The density of the noninactivating persistent sodium current was also significantly enhanced in neurons from SCI animals (from 17.4 ± 3.2 to 27.7 ± 4.4 pA/pF at 50–70 ms of depolarization). The increased persistent sodium current and ramp current, which are consistent with upregulation of Nav1.3 within dorsal horn neurons, suggest a basis for the hyperresponsiveness of these neurons following SCI.     

Development of respiratory rhythm generation in ectothermic vertebrates

Compared with birds and mammals, very little is known about the development and regulation of respiratory rhythm generation in ectothermic vertebrates. The development and regulation of respiratory rhythm generation in ectothermic vertebrates (fish, amphibians and reptiles) should provide insight into the evolution of these mechanisms. One useful model for examining the development of respiratory rhythm generation in ectothermic vertebrates has emerged from studies with the North American bullfrog (Rana catesbeiana). A major advantage of bullfrogs as a comparative model for respiratory rhythm generation is that respiratory output may be measured at all stages of development, both in vivo and in vitro. An emerging view of recent studies in developing bullfrogs is that many of the mechanisms of respiratory rhythm generation are very similar to those seen in birds and mammals. The overall conclusion from these studies is that respiratory rhythm generation during development may be highly conserved during evolution. The development of respiratory rhythm generation in mammals may, therefore, reflect the antecedent mechanisms seen in ectothermic vertebrates. The main focus of this brief review is to discuss recent data on the development of respiratory rhythm generation in ectothermic vertebrates, with particular emphasis on the North American bullfrog (R. catesbeiana) as a model.     

Tetrodotoxin-, dihydropyridine-, and riluzole-resistant persistent inward current: novel sodium channels in rodent spinal neurons

Recently, we reported the tetrodotoxin (TTX)- and dihydropyridine (DHP)-resistant (TDR) inward currents in neonatal mouse spinal neurons. In this study, we further characterized these currents in the presence of 1-5 μM TTX and 20-30 μM DHP (nifedipine, nimodipine, or isradipine). TDR inward currents were recorded by voltage ramp (persistent inward current, TDR-PIC) and step (TDR-I(p)) protocols. TDR-PIC and TDR-I(p) were found in 80.2% of recorded neurons (101/126) crossing laminae I to X from T12 to L6. TDR-PIC activated at -28.6 ± 13 mV with an amplitude of 80.6 ± 75 pA and time constant of 470.6 ± 240 ms (n = 75). TDR-I(p) had an amplitude of 151.2 ± 151 pA and a voltage threshold of -17.0 ± 9 mV (n = 54) with a wide range of kinetics parameters. The half-maximal activation was -21.5 ± 8 mV (-37 to -12 mV, n = 29) with a time constant of 5.2 ± 2 ms (1.2-11.2 ms, n = 19), whereas the half-maximal inactivation was -26.9 ± 9 mV (-39 to -18 mV, n = 14) with a time constant of 1.4 ± 0.4 s (0.5-2.2 s, n = 19). TDR-PIC and TDR-I(p) could be reduced by 60% in zero calcium and completely removed in zero sodium solutions, suggesting that they were mediated by sodium ions. Furthermore, the reversal potential of TDR-I(p) was estimated as 56.6 ± 3 mV (n = 10). TDR-PIC and TDR-I(p) persisted in 1-205 μM TTX, 20-100 μM DHP, 3-30 μM riluzole, 50-300 μM flufenamic acid, and 2-30 mM intracellular BAPTA. They also persisted with T-, N-, P/Q-, and R-type calcium channel blockers. In conclusion, we demonstrated novel TTX-, DHP-, and riluzole-resistant sodium channels in neonatal rodent spinal neurons. The unique pharmacological and electrophysiological properties would allow these channels to play a functional role in spinal motor system.