Synaptically activated burst-generating conductances may underlie a group-pacemaker mechanism for respiratory rhythm generation in mammals.

Breathing, chewing, and walking are critical life-sustaining behaviors in mammals that consist essentially of simple rhythmic movements. Breathing movements in particular involve the diaphragm, thorax, and airways but emanate from a network in the lower brain stem. This network can be studied in reduced preparations in vitro and using simplified mathematical models that make testable predictions. An iterative approach that employs both in vitro and in silico models argues against canonical mechanisms for respiratory rhythm in neonatal rodents that involve reciprocal inhibition and pacemaker properties. We present an alternative model in which emergent network properties play a rhythmogenic role. Specifically, we show evidence that synaptically activated burst-generating conductances-which are only available in the context of network activity-engender robust periodic bursts in respiratory neurons. Because the cellular burst-generating mechanism is linked to network synaptic drive we dub this type of system a group pacemaker.

AMPA and metabotropic glutamate receptors cooperatively generate inspiratory-like depolarization in mouse respiratory neurons in vitro.

Excitatory transmission mediated by AMPA receptors is critical for respiratory rhythm generation. However, the role of AMPA receptors has not been fully explored. Here we tested the functional role of AMPA receptors in inspiratory neurons of the neonatal mouse preBötzinger complex (preBötC) using an in vitro slice model that retains active respiratory function. Immediately before and during inspiration, preBötC neurons displayed envelopes of depolarization, dubbed inspiratory drive potentials, that required AMPA receptors but largely depended on the Ca(2+)-activated non-specific cation current (I(CAN)). We showed that AMPA receptor-mediated depolarization opened voltage-gated Ca(2+) channels to directly evoke I(CAN). Inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca(2+) release also evoked I(CAN). Inositol 1,4,5-trisphosphate receptors acted downstream of group I metabotropic glutamate receptor activity but, here too, AMPA receptor-mediated Ca(2+) influx was essential to trigger the metabotropic glutamate receptor contribution to inspiratory drive potential generation. This study helps to elucidate the role of excitatory transmission in respiratory rhythm generation in vitro. AMPA receptors in preBötC neurons initiate convergent signaling pathways that evoke post-synaptic I(CAN), which underlies inspiratory drive potentials. The coupling of AMPA receptors with I(CAN) suggests that latent burst-generating intrinsic conductances are recruited by excitatory synaptic interactions among preBötC neurons in the context of respiratory network activity in vitro, exemplifying a rhythmogenic mechanism based on emergent properties of the network.

What role do pacemakers play in the generation of respiratory rhythm?

The pacemaker hypothesis that specialized neurons with conditional oscillatory- bursting properties are obligatory for respiratory rhythm generation in vitro has gained widespread acceptance, despite lack of direct proof. Here we critique the pacemaker hypothesis and provide an alternative explanation for rhythmogenesis based on emergent network properties. Pacemaker neurons in the preBötC depend on either persistent Na+ current I(NaP) or Ca(2+)-activated nonspecific cationic current (I(CAN)). Activity in slice preparations and synaptically- isolated pacemaker neurons undergo similar frequency modulation by perturbations including hypoxia and changes in external K+. These data have been used to argue that pacemaker cells must be rhythmogenic, but may simply reflect the action of these perturbations on intrinsic membrane properties throughout the preBötC and does not constitute proof that pacemakers necessarily drive the rhythm with synaptic coupling in place. Likewise, bath-applied drugs, such as riluzole (RIL) and flufenamic acid (FFA), attenuate I(NaP) and I(CAN), respectively, throughout the slice. Thus, when these drugs stop the rhythm, a widespread depression of excitability is likely the underlying cause, not selective blockade of bursting-pacemaker activity. We propose that rhythmogenesis is an emergent network property, wherein recurrent synaptic excitation initiates a positive feedback cycle among interneurons and that intrinsic currents like I(CAN) and I(NaP) promote inspiratory burst generation by augmenting synaptic excitation in the context of network activity. In this group-pacemaker framework, individual pacemaker neurons can be embedded but play the same role as every other network constituent.

Phosphatidylinositol 4,5-bisphosphate regulates inspiratory burst activity in the neonatal mouse preBötzinger complex.

Neurons of the preBötzinger complex (preBötC) form local excitatory networks and synchronously discharge bursts of action potentials during the inspiratory phase of respiratory network activity. Synaptic input periodically evokes a Ca(2+)-activated non-specific cation current (I(CAN)) postsynaptically to generate 10-30 mV transient depolarizations, dubbed inspiratory drive potentials, which underlie inspiratory bursts. The molecular identity of I(CAN) and its regulation by intracellular signalling mechanisms during inspiratory drive potential generation remains unknown. Here we show that mRNAs coding for two members of the transient receptor potential (TRP) family of ion channels, namely TRPM4 and TRPM5, are expressed within the preBötC region of neonatal mice. Hypothesizing that the phosphoinositides maintaining TRPM4 and TRPM5 channel sensitivity to Ca(2+) may similarly influence I(CAN) and thus regulate inspiratory drive potentials, we manipulated intracellular phosphatidylinositol 4,5-bisphosphate (PIP(2)) and measured its effect on preBötC neurons in the context of ongoing respiratory-related rhythms in slice preparations. Consistent with the involvement of TRPM4 and TRPM5, excess PIP(2) augmented the inspiratory drive potential and diminution of PIP(2) reduced it; sensitivity to flufenamic acid (FFA) suggested that these effects of PIP(2) were I(CAN) mediated. Inositol 1,4,5-trisphosphate (IP(3)), the product of PIP(2) hydrolysis, ordinarily causes IP(3) receptor-mediated I(CAN) activation. Simultaneously increasing PIP(2) while blocking IP(3) receptors intracellularly counteracted the reduction in the inspiratory drive potential that normally resulted from IP(3) receptor blockade. We propose that PIP(2) protects I(CAN) from rundown by interacting directly with underlying ion channels and preventing desensitization, which may enhance the robustness of respiratory rhythm.

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.

Role of persistent sodium current in mouse preBötzinger Complex neurons and respiratory rhythm generation.

Breathing movements in mammals depend on respiratory neurons in the preBötzinger Complex (preBötC), which comprise a rhythmic network and generate robust bursts that form the basis for inspiration. Persistent Na(+) current (I(NaP)) is widespread in the preBötC and is hypothesized to play a critical role in rhythm generation because of its subthreshold activation and slow inactivation properties that putatively promote long-lasting burst depolarizations. In neonatal mouse slice preparations that retain the preBötC and generate a respiratory-related rhythm, we tested the role of I(NaP) with multiple Na(+) channel antagonists: tetrodotoxin (TTX; 20 nM), riluzole (RIL; 10 microM), and the intracellular Na(+) channel antagonist QX-314 (2 mM). Here we show that I(NaP) promotes intraburst spiking in preBötC neurons but surprisingly does not contribute to the depolarization that underlies inspiratory bursts, i.e. the inspiratory drive potential. Local microinjection in the preBötC of 10 microM RIL or 20 nM TTX does not perturb respiratory frequency, even in the presence of bath-applied 100 microM flufenamic acid (FFA), which attenuates a Ca(2+)-activated non-specific cation current (I(CAN)) that may also have burst-generating functionality. These data contradict the hypothesis that I(NaP) in preBötC neurons is obligatory for rhythmogenesis. However, in the presence of FFA, local microinjection of 10 microM RIL in the raphe obscurus causes rhythm cessation, which suggests that I(NaP) regulates the excitability of neurons outside the preBötC, including serotonergic raphe neurons that project to, and help maintain, rhythmic preBötC function.

Sodium and calcium current-mediated pacemaker neurons and respiratory rhythm generation.

The breathing motor pattern in mammals originates in brainstem networks. Whether pacemaker neurons play an obligatory role remains a key unanswered question. We performed whole-cell recordings in the preBotzinger Complex in slice preparations from neonatal rodents and tested for pacemaker activity. We observed persistent Na+ current (I(NaP))-mediated bursting in approximately 5% of inspiratory neurons in postnatal day 0 (P0)-P5 and in P8-P10 slices. I(NaP)-mediated bursting was voltage dependent and blocked by 20 mum riluzole (RIL). We found Ca2+ current (I(Ca))-dependent bursting in 7.5% of inspiratory neurons in P8-P10 slices, but in P0-P5 slices these cells were exceedingly rare (0.6%). This bursting was voltage independent and blocked by 100 microm Cd2+ or flufenamic acid (FFA) (10-200 microm), which suggests that a Ca2+-activated inward cationic current (I(CAN)) underlies burst generation. These data substantiate our observation that P0-P5 slices exposed to RIL contain few (if any) pacemaker neurons, yet maintain respiratory rhythm. We also show that 20 nm TTX or coapplication of 20 microm RIL + FFA (100-200 microm) stops the respiratory rhythm, but that adding 2 mum substance P restarts it. We conclude that I(NaP) and I(CAN) enhance neuronal excitability and promote rhythmogenesis, even if their magnitude is insufficient to support bursting-pacemaker activity in individual neurons. When I(NaP) and I(CAN) are removed pharmacologically, the rhythm can be maintained by boosting neural excitability, which is inconsistent with a pacemaker-essential mechanism of respiratory rhythmogenesis by the preBotzinger complex.