Christopher A. Del Negro

Looking for inspiration: new perspectives on respiratory rhythm.

Recent experiments in vivo and in vitro have advanced our understanding of the sites and mechanisms involved in mammalian respiratory rhythm generation. Here we evaluate and interpret the new evidence for two separate brainstem respiratory oscillators and for the essential role of emergent network properties in rhythm generation. Lesion studies suggest that respiratory cell death might explain morbidity and mortality associated with neurodegenerative disorders and ageing.

Respiratory rhythm generation in neonatal and adult mammals: the hybrid pacemaker–network model

We review a new unified model of respiratory rhythm generation - the hybrid pacemaker-network model. This model represents a comprehensive synthesis of cellular and network mechanisms that can theoretically account for rhythm generation in different functional states, from the most reduced states in the neonatal nervous system in vitro to the intact adult system in vivo. The model incorporates a critical neuronal kernel consisting of a network of excitatory neurons with state-dependent, oscillatory bursting or pacemaker properties. This kernel, located in the pre-Bötzinger complex of the ventrolateral medulla, provides a rudimentary pacemaker network mechanism for generating an inspiratory rhythm, revealed predominately in functionally reduced states in vitro. In vivo the kernel is embedded in a larger network that interacts with the kernel via inhibitory synaptic connections that provide the dynamic control required for the evolution of the complete pattern of inspiratory and expiratory network activity. The resulting hybrid of cellular pacemaker and network properties functionally endows the system with multiple mechanisms of rhythm generation. New biophysically realistic mathematical models of the hybrid pacemaker-network have been developed that illustrate these concepts and provide a computational framework for investigating interactions of cellular and network processes that must be analyzed to understand rhythm generation.

Understanding the Rhythm of Breathing: So Near, Yet So Far

Breathing is an essential behavior that presents a unique opportunity to understand how the nervous system functions normally, how it balances inherent robustness with a highly regulated lability, how it adapts to both rapidly and slowly changing conditions, and how particular dysfunctions result in disease. We focus on recent advancements related to two essential sites for respiratory rhythmogenesis: (a) the preBötzinger Complex (preBötC) as the site for the generation of inspiratory rhythm and (b) the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) as the site for the generation of active expiration.

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 preBötzinger Complex in slice preparations from neonatal rodents and tested for pacemaker activity. We observed persistent Na+ current (INaP)-mediated bursting in ∼5% of inspiratory neurons in postnatal day 0 (P0)-P5 and in P8-P10 slices. INaP-mediated bursting was voltage dependent and blocked by 20 μm riluzole (RIL). We found Ca2+ current (ICa)-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 μm Cd2+ or flufenamic acid (FFA) (10-200 μm), which suggests that a Ca2+-activated inward cationic current (ICAN) 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 μm RIL + FFA (100-200 μm) stops the respiratory rhythm, but that adding 2 μm substance P restarts it. We conclude that INaP and ICAN enhance neuronal excitability and promote rhythmogenesis, even if their magnitude is insufficient to support bursting-pacemaker activity in individual neurons. When INaP and ICAN 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 preBötzinger complex.

Respiratory rhythm: an emergent network property?

We tested the hypothesis that pacemaker neurons generate breathing rhythm in mammals. We monitored respiratory-related motor nerve rhythm in neonatal rodent slice preparations. Blockade of the persistent sodium current (I(NaP)), which was postulated to underlie voltage-dependent bursting in respiratory pacemaker neurons, with riluzole (< or =200 microM) did not alter the frequency of respiratory-related motor output. Yet, in every pacemaker neuron recorded (50/50), bursting was abolished at much lower concentrations of riluzole (< or =20 microM). Thus, eliminating the pacemaker population (our statistics confirm that this population is reduced at least 94%, p < 0.05) does not affect respiratory rhythm. These results suggest that voltage-dependent bursting in pacemaker neurons is not essential for respiratory rhythmogenesis, which may instead be an emergent network property.

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 (ICAN) in preBötC neurons. We also found that high concentrations of intracellular BAPTA, a high‐affinity Ca2+ chelator, and the ICAN antagonist flufenamic acid (FFA) decreased the magnitude of drive potentials. We conclude that ICAN 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.

Calcium-activated nonspecific cation current and synaptic depression promote network-dependent burst oscillations

Central pattern generators (CPGs) produce neural-motor rhythms that often depend on specialized cellular or synaptic properties such as pacemaker neurons or alternating phases of synaptic inhibition. Motivated by experimental evidence suggesting that activity in the mammalian respiratory CPG, the preBötzinger complex, does not require either of these components, we present and analyze a mathematical model demonstrating an unconventional mechanism of rhythm generation in which glutamatergic synapses and the short-term depression of excitatory transmission play key rhythmogenic roles. Recurrent synaptic excitation triggers postsynaptic Ca2+-activated nonspecific cation current (ICAN) to initiate a network-wide burst. Robust depolarization due to ICAN also causes voltage-dependent spike inactivation, which diminishes recurrent excitation and thus attenuates postsynaptic Ca2+ accumulation. Consequently, activity-dependent outward currents—produced by Na/K ATPase pumps or other ionic mechanisms—can terminate the burst and cause a transient quiescent state in the network. The recovery of sporadic spiking activity rekindles excitatory interactions and initiates a new cycle. Because synaptic inputs gate postsynaptic burst-generating conductances, this rhythm-generating mechanism represents a new paradigm that can be dubbed a ‘group pacemaker’ in which the basic rhythmogenic unit encompasses a fully interdependent ensemble of synaptic and intrinsic components. This conceptual framework should be considered as an alternative to traditional models when analyzing CPGs for which mechanistic details have not yet been elucidated.

Developmental Origin of PreBötzinger Complex Respiratory Neurons

A subset of preBötzinger Complex (preBötC) neurokinin 1 receptor (NK1R) and somatostatin peptide (SST)-expressing neurons are necessary for breathing in adult rats, in vivo. Their developmental origins and relationship to other preBötC glutamatergic neurons are unknown. Here we show, in mice, that the “core” of preBötC SST+/NK1R+/SST 2a receptor+ (SST2aR) neurons, are derived from Dbx1-expressing progenitors. We also show that Dbx1-derived neurons heterogeneously coexpress NK1R and SST2aR within and beyond the borders of preBötC. More striking, we find that nearly all non-catecholaminergic glutamatergic neurons of the ventrolateral medulla (VLM) are also Dbx1 derived. PreBötC SST+ neurons are born between E9.5 and E11.5 in the same proportion as non-SST-expressing neurons. Additionally, preBötC Dbx1 neurons are respiratory modulated and show an early inspiratory phase of firing in rhythmically active slice preparations. Loss of Dbx1 eliminates all glutamatergic neurons from the respiratory VLM including preBötC NK1R+/SST+ neurons. Dbx1 mutant mice do not express any spontaneous respiratory behaviors in vivo. Moreover, they do not generate rhythmic inspiratory activity in isolated en bloc preparations even after acidic or serotonergic stimulation. These data indicate that preBötC core neurons represent a subset of a larger, more heterogeneous population of VLM Dbx1-derived neurons. These data indicate that Dbx1-derived neurons are essential for the expression and, we hypothesize, are responsible for the generation of respiratory behavior both in vitro and in vivo.

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 (INaP) 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 INaP with multiple Na+ channel antagonists: tetrodotoxin (TTX; 20 nm), riluzole (RIL; 10 μm), and the intracellular Na+ channel antagonist QX‐314 (2 mm). Here we show that INaP 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 μm RIL or 20 nm TTX does not perturb respiratory frequency, even in the presence of bath‐applied 100 μm flufenamic acid (FFA), which attenuates a Ca2+‐activated non‐specific cation current (ICAN) that may also have burst‐generating functionality. These data contradict the hypothesis that INaP in preBötC neurons is obligatory for rhythmogenesis. However, in the presence of FFA, local microinjection of 10 μm RIL in the raphe obscurus causes rhythm cessation, which suggests that INaP regulates the excitability of neurons outside the preBötC, including serotonergic raphe neurons that project to, and help maintain, rhythmic preBötC function.

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 Ca2+‐activated non‐specific cation current (ICAN) postsynaptically to generate 10–30 mV transient depolarizations, dubbed inspiratory drive potentials, which underlie inspiratory bursts. The molecular identity of ICAN 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 Ca2+ may similarly influence ICAN and thus regulate inspiratory drive potentials, we manipulated intracellular phosphatidylinositol 4,5‐bisphosphate (PIP2) 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 PIP2 augmented the inspiratory drive potential and diminution of PIP2 reduced it; sensitivity to flufenamic acid (FFA) suggested that these effects of PIP2 were ICAN mediated. Inositol 1,4,5‐trisphosphate (IP3), the product of PIP2 hydrolysis, ordinarily causes IP3 receptor‐mediated ICAN activation. Simultaneously increasing PIP2 while blocking IP3 receptors intracellularly counteracted the reduction in the inspiratory drive potential that normally resulted from IP3 receptor blockade. We propose that PIP2 protects ICAN from rundown by interacting directly with underlying ion channels and preventing desensitization, which may enhance the robustness of respiratory rhythm.