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Title: Putting the theory into ‘burstlet theory’ with a biophysical model of burstlets and bursts in the respiratory preBötzinger complex
Inspiratory breathing rhythms arise from synchronized neuronal activity in a bilaterally distributed brainstem structure known as the preBötzinger complex (preBötC). In in vitro slice preparations containing the preBötC, extracellular potassium must be elevated above physiological levels (to 7–9 mM) to observe regular rhythmic respiratory motor output in the hypoglossal nerve to which the preBötC projects. Reexamination of how extracellular K + affects preBötC neuronal activity has revealed that low-amplitude oscillations persist at physiological levels. These oscillatory events are subthreshold from the standpoint of transmission to motor output and are dubbed burstlets. Burstlets arise from synchronized neural activity in a rhythmogenic neuronal subpopulation within the preBötC that in some instances may fail to recruit the larger network events, or bursts, required to generate motor output. The fraction of subthreshold preBötC oscillatory events (burstlet fraction) decreases sigmoidally with increasing extracellular potassium. These observations underlie the burstlet theory of respiratory rhythm generation. Experimental and computational studies have suggested that recruitment of the non-rhythmogenic component of the preBötC population requires intracellular Ca 2+ dynamics and activation of a calcium-activated nonselective cationic current. In this computational study, we show how intracellular calcium dynamics driven by synaptically triggered Ca 2+ influx as well as Ca 2+ release/uptake by the endoplasmic reticulum in conjunction with a calcium-activated nonselective cationic current can reproduce and offer an explanation for many of the key properties associated with the burstlet theory of respiratory rhythm generation. Altogether, our modeling work provides a mechanistic basis that can unify a wide range of experimental findings on rhythm generation and motor output recruitment in the preBötC.  more » « less
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National Science Foundation
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  1. Previously our computational modeling studies (Phillips et al., 2019) proposed that neuronal persistent sodium current (I NaP ) and calcium-activated non-selective cation current (I CAN ) are key biophysical factors that, respectively, generate inspiratory rhythm and burst pattern in the mammalian preBötzinger complex (preBötC) respiratory oscillator isolated in vitro. Here, we experimentally tested and confirmed three predictions of the model from new simulations concerning the roles of I NaP and I CAN : (1) I NaP and I CAN blockade have opposite effects on the relationship between network excitability and preBötC rhythmic activity; (2) I NaP is essential for preBötC rhythmogenesis; and (3) I CAN is essential for generating the amplitude of rhythmic output but not rhythm generation. These predictions were confirmed via optogenetic manipulations of preBötC network excitability during graded I NaP or I CAN blockade by pharmacological manipulations in slices in vitro containing the rhythmically active preBötC from the medulla oblongata of neonatal mice. Our results support and advance the hypothesis that I NaP and I CAN mechanistically underlie rhythm and inspiratory burst pattern generation, respectively, in the isolated preBötC. 
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  2. Abstract

    Breathing behaviour involves the generation of normal breaths (eupnoea) on a timescale of seconds and sigh breaths on the order of minutes. Both rhythms emerge in tandem from a single brainstem site, but whether and how a single cell population can generate two disparate rhythms remains unclear. We posit that recurrent synaptic excitation in concert with synaptic depression and cellular refractoriness gives rise to the eupnoea rhythm, whereas an intracellular calcium oscillation that is slower by orders of magnitude gives rise to the sigh rhythm. A mathematical model capturing these dynamics simultaneously generates eupnoea and sigh rhythms with disparate frequencies, which can be separately regulated by physiological parameters. We experimentally validated key model predictions regarding intracellular calcium signalling. All vertebrate brains feature a network oscillator that drives the breathing pump for regular respiration. However, in air‐breathing mammals with compliant lungs susceptible to collapse, the breathing rhythmogenic network may have refashioned ubiquitous intracellular signalling systems to produce a second slower rhythm (for sighs) that prevents atelectasis without impeding eupnoea.image

    Key points

    A simplified activity‐based model of the preBötC generates inspiratory and sigh rhythms from a single neuron population.

    Inspiration is attributable to a canonical excitatory network oscillator mechanism.

    Sigh emerges from intracellular calcium signalling.

    The model predicts that perturbations of calcium uptake and release across the endoplasmic reticulum counterintuitively accelerate and decelerate sigh rhythmicity, respectively, which was experimentally validated.

    Vertebrate evolution may have adapted existing intracellular signalling mechanisms to produce slow oscillations needed to optimize pulmonary function in mammals.

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  3. Abstract

    Breathing depends on interneurons in the preBötzinger complex (preBötC) derived fromDbx1-expressing precursors. Here we investigate whether rhythm- and pattern-generating functions reside in discrete classes of Dbx1 preBötC neurons. In a slice model of breathing with ~ 5 s cycle period, putatively rhythmogenic Type-1 Dbx1 preBötC neurons activate 100–300 ms prior to Type-2 neurons, putatively specialized for output pattern, and 300–500 ms prior to the inspiratory motor output. We sequenced Type-1 and Type-2 transcriptomes and identified differential expression of 123 genes including ionotropic receptors (Gria3, Gabra1) that may explain their preinspiratory activation profiles and Ca2+signaling (Cracr2a,Sgk1) involved in inspiratory and sigh bursts. Surprisingly, neuropeptide receptors that influence breathing (e.g., µ-opioid and bombesin-like peptide receptors) were only sparsely expressed, which suggests that cognate peptides and opioid drugs exert their profound effects on a small fraction of the preBötC core. These data in the public domain help explain the neural origins of breathing.

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  4. Key points

    The functional neuroanatomy of the mammalian respiratory network is far from being understood since experimental tools that measure neural activity across this brainstem‐wide circuit are lacking.

    Here, we use silicon multi‐electrode arrays to record respiratory local field potentials (rLFPs) from 196–364 electrode sites within 8–10 mm3of brainstem tissue in single arterially perfused brainstem preparations with respect to the ongoing respiratory motor pattern of inspiration (I), post‐inspiration (PI) and late‐expiration (E2).

    rLFPs peaked specifically at the three respiratory phase transitions, E2–I, I–PI and PI–E2.

    We show, for the first time, that only the I–PI transition engages a brainstem‐wide network, and that rLFPs during the PI–E2 transition identify a hitherto unknown role for the dorsal respiratory group.

    Volumetric mapping of pontomedullary rLFPs in single preparations could become a reliable tool for assessing the functional neuroanatomy of the respiratory network in health and disease.


    While it is widely accepted that inspiratory rhythm generation depends on the pre‐Bötzinger complex, the functional neuroanatomy of the neural circuits that generate expiration is debated. We hypothesized that the compartmental organization of the brainstem respiratory network is sufficient to generate macroscopic local field potentials (LFPs), and if so, respiratory (r) LFPs could be used to map the functional neuroanatomy of the respiratory network. We developed an approach using silicon multi‐electrode arrays to record spontaneous LFPs from hundreds of electrode sites in a volume of brainstem tissue while monitoring the respiratory motor pattern on phrenic and vagal nerves in the perfused brainstem preparation. Our results revealed the expression of rLFPs across the pontomedullary brainstem. rLFPs occurred specifically at the three transitions between respiratory phases: (1) from late expiration (E2) to inspiration (I), (2) from I to post‐inspiration (PI), and (3) from PI to E2. Thus, respiratory network activity was maximal at respiratory phase transitions. Spatially, the E2–I, and PI–E2 transitions were anatomically localized to the ventral and dorsal respiratory groups, respectively. In contrast, our data show, for the first time, that the generation of controlled expiration during the post‐inspiratory phase engages a distributed neuronal population within ventral, dorsal and pontine network compartments. A group‐wise independent component analysis demonstrated that all preparations exhibited rLFPs with a similar temporal structure and thus share a similar functional neuroanatomy. Thus, volumetric mapping of rLFPs could allow for the physiological assessment of global respiratory network organization in health and disease.

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  5. Key points

    Heart failure (HF), the leading cause of death in developed countries, occurs in the setting of reduced (HFrEF) or preserved (HFpEF) ejection fraction. Unlike HFrEF, there are no effective treatments for HFpEF, which accounts for ∼50% of heart failure.

    Abnormal intracellular calcium dynamics in cardiomyocytes have major implications for contractility and rhythm, but compared to HFrEF, very little is known about calcium cycling in HFpEF.

    We used rat models of HFpEF and HFrEF to reveal distinct differences in intracellular calcium regulation and excitation‐contraction (EC) coupling.

    While HFrEF is characterized by defective EC coupling at baseline, HFpEF exhibits enhanced coupling fidelity, further aggravated by a reduction in β‐adrenergic sensitivity.

    These differences in EC coupling and β‐adrenergic sensitivity may help explain why therapies that work in HFrEF are ineffective in HFpEF.


    Heart failure with reduced or preserved ejection fraction (respectively, HFrEF and HFpEF) is the leading cause of death in developed countries. Although numerous therapies improve outcomes in HFrEF, there are no effective treatments for HFpEF. We studied phenotypically verified rat models of HFrEF and HFpEF to compare excitation‐contraction (EC) coupling and protein expression in these two forms of heart failure. Dahl salt‐sensitive rats were fed a high‐salt diet (8% NaCl) from 7 weeks of age to induce HFpEF. Impaired diastolic relaxation and preserved ejection fraction were confirmed in each animal echocardiographically, and clinical signs of heart failure were documented. To generate HFrEF, Sprague‐Dawley (SD) rats underwent permanent left anterior descending coronary artery ligation which, 8–10 weeks later, led to systolic dysfunction (verified echocardiographically) and clinical signs of heart failure. Calcium (Ca2+) transients were measured in isolated cardiomyocytes under field stimulation or patch clamp. Ultra‐high‐speed laser scanning confocal imaging captured Ca2+sparks evoked by voltage steps. Western blotting and PCR were used to assay changes in EC coupling protein and RNA expression. Cardiomyocytes from rats with HFrEF exhibited impaired EC coupling, including decreased Ca2+transient (CaT) amplitude and defective couplon recruitment, associated with transverse (t)‐tubule disruption. In stark contrast, HFpEF cardiomyocytes showed saturated EC coupling (increasedICa, high probability of couplon recruitment with greater Ca2+release synchrony, increased CaT) and preserved t‐tubule integrity. β‐Adrenergic stimulation of HFpEF myocytes with isoprenaline (isoproterenol) failed to elicit robust increases inICaor CaT and relaxation kinetics. Fundamental differences in EC coupling distinguish HFrEF from HFpEF.

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