Synaptic Depression Influences Inspiratory-Expiratory Phase Transition in Dbx1 Interneurons of the preBotzinger Complex in Neonatal Mice

The brainstem preBotzinger complex (preBotC) generates the rhythm underlying inspiratory breathing movements and its core interneurons are derived from Dbx1-expressing precursors. Recurrent synaptic excitation is required to initiate inspiratory bursts, but whether excitatory synaptic mechanisms also contribute to inspiratory-expiratory phase transition is unknown. Here, we examined the role of short-term synaptic depression using a rhythmically active neonatal mouse brainstem slice preparation. We show that afferent axonal projections to Dbx1 preBotC neurons undergo activity-dependent depression and we identify a refractory period (similar to 2 s) after endogenous inspiratory bursts that precludes light-evoked bursts in channelrhodopsin-expressing Dbx1 preBotC neurons. We demonstrate that the duration of the refractory period-but neither the cycle period nor the magnitude of endogenous inspiratory bursts-is sensitive to changes in extracellular Ca2+. Further, we show that postsynaptic factors are unlikely to explain the refractory period or its modulation by Ca2+. Our findings are consistent with the hypothesis that short-term synaptic depression in Dbx1 preBotC neurons influences the inspiratory-expiratory phase transition during respiratory rhythmogenesis

Fate mapping neurons and glia derived from Dbx1-expressing progenitors in mouse preBotzinger complex

The brainstem preBotzinger complex (preBotC) generates the inspiratory breathing rhythm, and its core rhythmogenic interneurons are derived from Dbx1-expressing progenitors. To study the neural bases of breathing, tamoxifen-inducible Cre-driver mice and Cre-dependent reporters are used to identify, record, and perturb Dbx1 preBotC neurons. However, the relationship between tamoxifen administration and reporter protein expression in preBotC neurons and glia has not been quantified. To address this problem, we crossed mice that express tamoxifen-inducible Cre recombinase under the control of the Dbx1 gene (Dbx1(CreERT2)) with Cre-dependent fluorescent reporter mice (Rosa26(tdTomato)), administered tamoxifen at different times during development, and analyzed tdTomato expression in the preBotC of their offspring. We also crossed Rosa26(tdTomato) reporters with mice that constitutively express Cre driven by Dbx1 (Dbx1(Cre)) and analyzed tdTomato expression in the preBotC of their offspring for comparison. We show that Dbx1-expressing progenitors give rise to preBotC neurons and glia. Peak neuronal tdTomato expression occurs when tamoxifen is administered at embryonic day 9.5 (E9.5), whereas tdTomato expression in glia shows no clear relationship with tamoxifen timing. These results can be used to bias reporter protein expression in neurons (or glia). Tamoxifen administration at E9.5 labels 91% of Dbx1-derived neurons in the preBotC, yet only 48% of Dbx1-derived glia. By fate mapping Dbx1-expressing progenitors, this study illustrates the developmental assemblage of Dbx1-derived cells in preBotC, which can be used to design intersectional Cre/lox experiments that interrogate its cellular composition, structure, and function

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

Dendritic Calcium Activity Precedes Inspiratory Bursts in preBotzinger Complex Neurons

Medullary interneurons of the preBotzinger 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 Ca(2+) accumulation postsynaptically to evoke a Ca(2+)-activated inward current that contributes to inspiratory burst generation. We measured Ca(2+) transients by two-photon imaging dendrites while recording neuronal somata electrophysiologically. Dendritic Ca(2+) accumulation frequently precedes inspiratory bursts, particularly at recording sites 50-300 mu m distal from the soma. Preinspiratory Ca(2+) transients occur in hotspots, not ubiquitously, in dendrites. Ca(2+) activity propagates orthodromically toward the soma (and antidromically to more distal regions of the dendrite) at rapid rates (300-700 mu m/s). These high propagation rates suggest that dendritic Ca(2+) activates an inward current to electrotonically depolarize the soma, rather than propagate as a regenerative Ca(2+) wave. These data provide new evidence that respiratory rhythmogenesis may depend on dendritic burst-generating conductances activated in the context of network activity

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 preBotzinger Complex (preBotC) 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

Oscillation Regularity in Noise-Driven Excitable Systems with Multi-Time-Scale Adaptation

We investigate oscillation regularity of a noise-driven system modeled with a slow after-hyperpolarizing adaptation current (AHP) composed of multiple-exponential relaxation time scales. Sufficiently separated slow and fast AHP time scales (biphasic decay) cause a peak in oscillation irregularity for intermediate input currents I, with relatively regular oscillations for small and large currents. An analytic formulation of the system as a stochastic escape problem establishes that the phenomena is distinct from standard forms of coherence resonance. Our results explain data on the oscillation regularity of the pre-Bötzinger complex, a neural oscillator responsible for inspiratory breathing rhythm generation in mammals

Oscillation Regularity in Noise-Driven Excitable Systems with Multi-Time-Scale Adaptation

We investigate oscillation regularity of a noise-driven system modeled with a slow after-hyperpolarizing adaptation current (AHP) composed of multiple-exponential relaxation time scales. Sufficiently separated slow and fast AHP time scales (biphasic decay) cause a peak in oscillation irregularity for intermediate input currents I, with relatively regular oscillations for small and large currents. An analytic formulation of the system as a stochastic escape problem establishes that the phenomena is distinct from standard forms of coherence resonance. Our results explain data on the oscillation regularity of the pre-Bötzinger complex, a neural oscillator responsible for inspiratory breathing rhythm generation in mammals

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 (similar to 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 (\u3c 0.02 Hz) spontaneous rhythms, evoked motor output could follow a spontaneous burst at short ( 60 s. We observed during inspiration a large magnitude (similar to 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

Organotypic slice cultures containing the preBotzinger complex generate respiratory-like rhythms

Study of acute brain stem slice preparations in vitro has advanced our understanding of the cellular and synaptic mechanisms of respiratory rhythm generation, but their inherent limitations preclude long-term manipulation and recording experiments. In the current study, we have developed an organotypic slice culture preparation containing the preBotzinger complex (preBotC), the core inspiratory rhythm generator of the ventrolateral brain stem. We measured bilateral synchronous network oscillations, using calcium-sensitive fluorescent dyes, in both ventrolateral (presumably the preBotC) and dorsomedial regions of slice cultures at 7-43 days in vitro. These calcium oscillations appear to be driven by periodic bursts of inspiratory neuronal activity, because whole cell recordings from ventrolateral neurons in culture revealed inspiratory-like drive potentials, and no oscillatory activity was detected from glial fibrillary associated protein-expressing astrocytes in cultures. Acute slices showed a burst frequency of 10.9 +/- 4.2 bursts/min, which was not different from that of brain stem slice cultures (13.7 +/- 10.6 bursts/min). However, slice cocultures that include two cerebellar explants placed along the dorsolateral border of the brainstem displayed up to 193% faster burst frequency (22.4 +/- 8.3 bursts/min) and higher signal amplitude (340%) compared with acute slices. We conclude that preBotC-containing slice cultures retain inspiratory-like rhythmic function and therefore may facilitate lines of experimentation that involve extended incubation (e.g., genetic transfection or chronic drug exposure) while simultaneously being amenable to imaging and electrophysiology at cellular, synaptic, and network levels

Mechanisms Leading to Rhythm Cessation in the Respiratory PreBotzinger Complex Due to Piecewise Cumulative Neuronal Deletions

The brainstem pre-Botzinger complex (preBotC) generates the rhythm underlying inspiratory breathing movements and its core interneurons are derived from Dbx-1 expressing precursors. Recurrent synaptic excitation is required to initiate inspiratory bursts, but whether excitatory synaptic mechanisms also contribute to inspiratory–expiratory phase transition is unknown. Here, we examined the role of short-term synaptic depression using a rhythmically active neonatal mouse brainstem slice preparation. We show that different axonal projections to Dbx-1 PreBotC neurons undergo activity-dependent depression and we identify a refractory period (approx. 2 s) after endogenous inspiratory bursts that precludes light-evoked bursts in channelrhodopsin-expressing Dbx1 Pre-BotC neurons. We demonstrate that the duration of the refractory period---but neither the cycle period nor the magnitude of endogenous inspiratory burst---is sensitive to changes in extracellular Ca^2+. Further, we show that postsynaptic factors are unlikely to explain the refractory period or its modulation by Ca^2+. Our findings are consistent with the hypothesis that short-term synaptic depression in Dbx-1 Pre-BotC neurons influences the inspiratory-expiratory phase transition during respiratory rhythmogenesis