John A. Hayes

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.

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.

Cumulative lesioning of respiratory interneurons disrupts and precludes motor rhythms in vitro

How brain functions degenerate in the face of progressive cell loss is an important issue that pertains to neurodegenerative diseases and basic properties of neural networks. We developed an automated system that uses two-photon microscopy to detect rhythmic neurons from calcium activity, and then individually laser ablates the targets while monitoring network function in real time. We applied this system to the mammalian respiratory oscillator located in the pre-Bötzinger Complex (preBötC) of the ventral medulla, which spontaneously generates breathing-related motor activity in vitro. Here, we show that cumulatively deleting preBötC neurons progressively decreases respiratory frequency and the amplitude of motor output. On average, the deletion of 120 ± 45 neurons stopped spontaneous respiratory rhythm, and our data suggest ≈82% of the rhythm-generating neurons remain unlesioned. Cumulative ablations in other medullary respiratory regions did not affect frequency but diminished the amplitude of motor output to a lesser degree. These results suggest that the preBötC can sustain insults that destroy no more than ≈18% of its constituent interneurons, which may have implications for the onset of respiratory pathologies in disease states.

Asymmetric control of inspiratory and expiratory phases by excitability in the respiratory network of neonatal mice in vitro

Rhythmic motor behaviours consist of alternating movements, e.g. swing‐stance in stepping, jaw opening and closing during chewing, and inspiration–expiration in breathing, which must be labile in frequency, and in some cases, in the duration of individual phases, to adjust to physiological demands. These movements are the expression of underlying neural circuits whose organization governs the properties of the motor behaviour. To determine if the ability to operate over a broad range of frequencies in respiration is expressed in the rhythm generator, we isolated the kernel of essential respiratory circuits using rhythmically active in vitro slices from neonatal mice. We show respiratory motor output in these slices at very low frequencies (0.008 Hz), well below the typical frequency in vitro (∼0.2 Hz) and in most intact normothermic mammals. Across this broad range of frequencies, inspiratory motor output bursts remained remarkably constant in pattern, i.e. duration, peak amplitude and area. The change in frequency was instead attributable to increased interburst interval, and was largely unaffected by removal of fast inhibitory transmission. Modulation of the frequency was primarily achieved by manipulating extracellular potassium, which significantly affects neuronal excitability. When excitability was lowered to slow down, or in some cases stop, spontaneous rhythm, brief stimulation of the respiratory network with a glutamatergic agonist could evoke (rhythmic) motor output. In slices with slow (<0.02 Hz) spontaneous rhythms, evoked motor output could follow a spontaneous burst at short (≤1 s) or long (∼60 s) intervals. The intensity or timing of stimulation determined the latency to the first evoked burst, with no evidence for a refractory period greater than ∼1 s, even with interburst intervals >60 s. We observed during inspiration a large magnitude (∼0.6 nA) outward current generated by Na+/K+ ATPase that deactivated in 25–100 ms and thus could contribute to burst termination and the latency of evoked bursts but is unlikely to control the interburst interval. We propose that the respiratory network functions over a broad range of frequencies by engaging distinct mechanisms from those controlling inspiratory duration and pattern that specifically govern the interburst interval.

Laser ablation of Dbx1 neurons in the pre-Bötzinger complex stops inspiratory rhythm and impairs output in neonatal mice

To understand the neural origins of rhythmic behavior one must characterize the central pattern generator circuit and quantify the population size needed to sustain functionality. Breathing-related interneurons of the brainstem pre-Bötzinger complex (preBötC) that putatively comprise the core respiratory rhythm generator in mammals are derived from Dbx1-expressing precursors. Here, we show that selective photonic destruction of Dbx1 preBötC neurons in neonatal mouse slices impairs respiratory rhythm but surprisingly also the magnitude of motor output; respiratory hypoglossal nerve discharge decreased and its frequency steadily diminished until rhythm stopped irreversibly after 85±20 (mean ± SEM) cellular ablations, which corresponds to ∼15% of the estimated population. These results demonstrate that a single canonical interneuron class generates respiratory rhythm and contributes in a premotor capacity, whereas these functions are normally attributed to discrete populations. We also establish quantitative cellular parameters that govern network viability, which may have ramifications for respiratory pathology in disease states. DOI: http://dx.doi.org/10.7554/eLife.03427.001

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.

Dendritic Calcium Activity Precedes Inspiratory Bursts in preBötzinger Complex Neurons

Medullary interneurons of the preBötzinger complex assemble excitatory networks that produce inspiratory-related neural rhythms, but the importance of somatodendritic conductances in rhythm generation is still incompletely understood. Synaptic input may cause Ca2+ accumulation postsynaptically to evoke a Ca2+-activated inward current that contributes to inspiratory burst generation. We measured Ca2+ transients by two-photon imaging dendrites while recording neuronal somata electrophysiologically. Dendritic Ca2+ accumulation frequently precedes inspiratory bursts, particularly at recording sites 50–300 μm distal from the soma. Preinspiratory Ca2+ transients occur in hotspots, not ubiquitously, in dendrites. Ca2+ activity propagates orthodromically toward the soma (and antidromically to more distal regions of the dendrite) at rapid rates (300–700 μm/s). These high propagation rates suggest that dendritic Ca2+ activates an inward current to electrotonically depolarize the soma, rather than propagate as a regenerative Ca2+ wave. These data provide new evidence that respiratory rhythmogenesis may depend on dendritic burst-generating conductances activated in the context of network activity.

A 'group pacemaker' mechanism for respiratory rhythm generation.

Central pattern generator (CPG) networks emit neural rhythms that drive behaviours such as breathing, locomotion and mastication. Understanding their mechanisms of rhythmogenesis is a longstanding problem. One contemporary debate focuses on whether pacemaker neurons that encapsulate network activity in their intrinsic autorhythmicity or emergent network properties are the fundamental basis for CPG function. Pacemakerdriven mechanisms are intuitive and straightforward – examples in the CNS of many animals from invertebrates to mammals abound – but emergent properties can be abstruse and ill-defined. In this issue of The Journal of Physiology, Mironov (2008) presents a breakthrough analysis of emergent network properties – and emphasizes their importance – using the respiratory oscillator in the pre-Botzinger complex (preBotC) of the ventral medulla in mammals as a model system (Smith et al. 1991; Feldman & Del Negro, 2006). The major proposal for emergent network properties in the preBotC – dubbed the group-pacemaker hypothesis – posited that periodic inspiratory bursts originated due to intrinsic currents, which are ordinarily latent and unavailable, except when evoked synaptically in the context of network function (Rekling & Feldman, 1998). Ca2+-activated non-specific cation current (ICAN) has been recognized as a predominant inspiratory burst-generating current coupled to metabotropic glutamate receptors (mGluRs) (Pace et al. 2007) that could satisfy the group-pacemaker mechanism. Mironov now validates the group-pacemaker hypothesis; he shows the subcellular machinery for synaptically evoking ICAN, and then manipulates this machinery to perturb and/or abolish respiratory rhythms in vitro. Performing on-cell recordings in the preBotC while monitoring inspiratory motor output from the hypoglossal (XII) nerve, Mironov recorded ion channel activity prior to – and during – the inspiratory burst. TRPM4, and its close relative TRPM5 (subtypes of the transient receptor potential, i.e. TRP, family) are monovalent cation channels activated by intracellular Ca2+. Both subtypes were previously suggested to constitute ICAN (Crowder et al. 2007). Mironov establishes that the underlying channels in the soma are TRPM4 by testing their sensitivity to Ca2+ and phosphatidylinositol 4,5-bisphosphate (PIP2) as well as blockade by flufenamate (FFA) and Gd3+. At the systems level, bath-applied Gd3+ stopped the respiratory rhythm within several minutes, which suggests that TRPM4-mediated ICAN is essential for rhythmogenesis, while avoiding the pharmacological caveats associated with FFA. To examine the role of ICAN in networks, Mironov performs two-photon imaging combined with on-cell patch recordings in pairs of connected preBotC neurons. Synaptic excitation gives rise to propagating Ca2+ waves in dendrites, which then evoke TRPM4 in the soma (Fig. 1A). During endogenous network activity these same propagating Ca2+ waves cause vigorous TRPM4 activity in the prelude to, and during, the inspiratory burst. Additionally, the mGluR agonist DHPG, which modulates respiratory frequency and augments TRPM4 activity, here is shown to accelerate the propagation speed of the Ca2+ wave. Interestingly, local application of thapsigargin to deplete the Ca2+ stores halts the wave in mid-dendrite, and occludes the inspiratory burst altogether. These observations suggest that group 1 mGluRs contribute to inspiratory bursts via inositol 1,4,5-trisphosphate (IP3) and Ca2+-induced Ca2+ release, and may influence respiratory frequency by regulating the speed of Ca2+ wave propagation. Figure 1 The cellular mechanisms underlying a ‘group pacemaker’ in the respiratory CPG contained in the preBotC To test this novel idea regarding frequency modulation Mironov turns to simulations. He creates Ca2+ release compartments coupled to soma-like compartments with ICAN, which he couples in a ring of alternating compartment types (Fig. 1B). This ring system generates coherent rhythmicity only when propagation speed is held near ∼70 μm s−1, matching the measured value. The range of propagation speeds that support coherent rhythmicity can be extended when ring oscillators are sparsely coupled to one another, roughly representing distinct clusters of rhythmic neurons observed in the preBotC (Hartelt et al. 2008). This arrangement may be advantageous to synchronize preBotC neurons, which may be sparsely connected (Rekling et al. 2000), and by ‘clamping’ the frequency of the network rhythm on the basis of wave propagation speed. This innovative new idea suggests that frequency modulation may not necessarily depend on regulating somatic ion channels to hyper- or depolarize baseline membrane potential, but could be tuned by metabotropic receptors that modify dendritic cable properties or Ca2+ release mechanisms and influence the speed of Ca2+ waves. Mironovs analysis establishes a viable model for emergent network properties in a CPG. Post-synaptic ion channels become active in the context of network function; bursts of channel activity depend on metabotropic receptors and intracellular signalling. While some uncertainties remain, i.e. regarding the specific role of ionotropic AMPA receptors (AMPARs) and whether TRPM4 is expressed in dendrites, this demonstration of a group pacemaker in the preBotC should cause us to re-examine the underlying mechanism in other important CPGs (e.g. locomotion, swimming and oral-motor activity) as well as rhythmic brain networks in general.

4-Aminopyridine-sensitive outward currents in preBötzinger complex neurons influence respiratory rhythm generation in neonatal mice

We measured a low‐threshold, inactivating K+ current, i.e. A‐current (IA), in respiratory neurons of the preBötzinger complex (preBötC) in rhythmically active slice preparations from neonatal C57BL/6 mice. The majority of inspiratory neurons (21/34 = 61.8%), but not expiratory neurons (1/8 = 12.5%), expressed IA. In whole‐cell and somatic outside‐out patches IA activated at −60 mV (half‐activation voltage measured −16.3 mV) and only fully inactivated above −40 mV (half‐inactivation voltage measured −85.6 mV), indicating that IA can influence membrane trajectory at baseline voltages during respiratory rhythm generation in vitro. 4‐Aminopyridine (4‐AP, 2 mm) attenuated IA in both whole‐cell and somatic outside‐out patches. In the context of rhythmic network activity, 4‐AP caused irregular respiratory‐related motor output on XII nerves and disrupted rhythmogenesis as detected with whole‐cell and field recordings in the preBötC. Whole‐cell current‐clamp recordings showed that 4‐AP changed the envelope of depolarization underlying inspiratory bursts (i.e. inspiratory drive potentials) from an incrementing pattern to a decrementing pattern during rhythm generation and abolished current pulse‐induced delayed excitation. These data suggest that IA opposes excitatory synaptic depolarizations at baseline voltages of approximately −60 mV and influences the inspiratory burst pattern. We propose that IA promotes orderly recruitment of constituent rhythmogenic neurons by minimizing the activity of these neurons until they receive massive coincident synaptic input, which reduces the periodic fluctuations of inspiratory activity.