Neuronal pacemaker for breathing visualized in vitro

Abstract

Breathing movements in mammals arise from a rhythmic pattern of neural activity, thought to originate in the pre-Bötzinger complex in the lower brainstem. The mechanisms generating the neural rhythm in this region are unknown. The central question is whether the rhythm is generated by a network of bursting pacemaker neurons coupled by excitatory synapses that synchronize pacemaker activity. Here we visualized the activity of inspiratory pacemaker neurons at single-cell and population levels with calcium-sensitive dye. We developed methods to label these neurons retrogradely with the dye in neonatal rodent brainstem slices that retain the rhythmically active respiratory network. We simultaneously used infrared structural imaging to allow patch-clamp recording from the identified neurons. After we pharmacologically blocked glutamatergic synaptic transmission, a subpopulation of inspiratory neurons continued to burst rhythmically but asynchronously. The intrinsic bursting frequency of these pacemaker neurons depended on the baseline membrane potential, providing a cellular mechanism for respiratory frequency control. These results provide evidence that the neuronal kernel for rhythm generation consists of a network of synaptically-coupled pacemaker neurons.

Emergence of the Pre-Bötzinger Respiratory Rhythm Generator in the Mouse Embryo

“…This group is readily identifiable and amenable to analysis in in vitro preparations. The respiratory neural network in the PBC region contains rhythmic neurons connected by glutamatergic synapses, some of them exhibiting pacemaker properties (Johnson et al, 1994;Koshiya and Smith, 1999;Thoby-Brisson et al, 2000;Thoby-Brisson and Ramirez, 2001;Pena et al, 2004). The destruction of neurons expressing the NK1 receptor (NK1R) leading to an ataxic respiration (Gray et al, 2001) supports the view that the PBC plays a primary role in respiratory rhythmogenesis.…”

“…Second, rhythm generation is concomitant to establishment of NK1R immunoreactivity in this region (Gray et al, 1999). Third, glutamatergic transmission through AMPA/kainate receptors conditions rhythm generation (Smith et al, 1991;Koshiya and Smith, 1999). Fourth, SP and DAMGO exert respective excitatory and depressing neuromodulatory influences on the rhythm generated (Gray et al, 1999;Janczewski et al, 2002;Manzke et al, 2003;Mellen et al, 2003), although the level of -opioids and NK1 receptors expression might not be fully developed at this stage (Pagliardini et al, 2003;Kivell et al, 2004).…”

Postnatal expression of neurotransmitters, receptors, and cytochrome oxidase in the rat pre-Bötzinger complex

“…Removal of only the PBC in the brain stem eliminated respiratory rhythm generation in neonatal rats (42). All six basic types of respiratory neurons were identified in the PBC (9, 42), and neurons with voltagedependent pacemaker-like properties were also found there (6,7,17,22,47). It has been implied that the PBC functions as a central hypoxia chemosensor for respiration (44,45).…”

Robust network oscillations during mammalian respiratory rhythm generation driven by synaptic dynamics

“…One such mammalian CPG, the preBötzinger complex (preBötC), gives rise to the eupneic respiratory rhythm (2,3). Located in the medulla, the preBötC preserves a spontaneous respiratory-like rhythm when isolated in transverse slices, but the precise nature of the cellular and synaptic mechanisms underlying rhythmogenesis remains elusive (3)(4)(5)(6)(7). An early hypothesis was that the neuronal activity is driven by intrinsically bursting pacemaker neurons synchronized via excitatory synaptic connections (2,6,8,9).…”

“…Located in the medulla, the preBötC preserves a spontaneous respiratory-like rhythm when isolated in transverse slices, but the precise nature of the cellular and synaptic mechanisms underlying rhythmogenesis remains elusive (3)(4)(5)(6)(7). An early hypothesis was that the neuronal activity is driven by intrinsically bursting pacemaker neurons synchronized via excitatory synaptic connections (2,6,8,9). However, electrophysiological and modeling studies (7,(10)(11)(12) now suggest the rhythm emerges through stochastic activation of intrinsic currents conveyed by recurrent synaptic connections, without the need for pacemaker neurons (3,4,11,13,14).…”