A ‘group pacemaker’ mechanism for respiratory rhythm generation

Abstract

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. Mironov's 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.