Abstract:Key points• The transcription factor Dbx1 gives rise to putatively respiratory rhythm-generating neurons in the pre-Bötzinger complex. Comparative analysis of Dbx1-derived (Dbx1 + ) and non-Dbx1-derived (Dbx1 − ) neurons can help elucidate the cellular bases of respiratory rhythm generation.
• In vitro, Dbx1+ neurons activate earlier in the respiratory cycle, discharge larger magnitude inspiratory bursts and exhibit a lower rheobase compared with Dbx1 − neurons.+ neurons tend to express the intrinsic currents … Show more
“…We observed three types of neuronal firing patterns: preinspiratory (pre-I), inspiratory (I), and postinspiratory (post-I) (Figure 2). In Dbx1-ChR2 mice (Figure 2, middle column), in response to bilateral preBötC short pulse photostimulation (SPP; 100–300 ms pulse; 473 nm) at any point in the respiratory cycle, we observed increased firing in all preinspiratory (n=12/12 units in 3 mice) and most inspiratory (n=4/7 units in 3 mice) preBötC neurons, with no responsive postinspiratory (n=0/9 units in 3 mice) preBötC neurons; these results are consistent with the firing patterns of identified preBötC Dbx1 + neurons in vitro (Picardo et al, 2013). In SST-ChR2 mice (Figure 2, right column), all recorded inspiratory (n=5/5 units in 2 mice) and all postinspiratory (n=5/5 units in 2 mice) preBötC neurons, but no preinspiratory (n=0/15 units in 2 mice) preBötC neurons were responsive to bilateral preBötC SPP at any point in the respiratory cycle.…”
Section: Resultssupporting
confidence: 85%
“…The homeobox gene Dbx1 controls the fate of glutamatergic interneurons required for preBötC development that likely include respiratory rhythmogenic neurons (Bouvier et al, 2010; Gray et al, 2010; Picardo et al, 2013). ~15% of preBötC Dbx1 + neurons are preBötC SST + neurons, necessary for normal breathing in anesthetized or awake adult rats (Gray et al, 2010; Tan et al, 2008).…”
Section: Discussionmentioning
confidence: 99%
“…preBötC Dbx1 + neurons are strong candidates as generators of inspiratory rhythm (Wang et al, 2014). In rhythmically active slice preparations from neonatal rodents, preBötC Dbx1 + neurons have a preinspiratory firing pattern, initiating spike activity before the onset of each inspiratory hypoglossal nerve burst, and continue to fire throughout inspiration (Picardo et al, 2013). In vitro , a subset of Dbx1 + neurons are premotoneurons, projecting directly to motoneurons, transmitting bursts of inspiratory activity that ultimately pattern inspiratory muscle contraction (Kam et al, 2013a; Revill et al, 2015; Wang et al, 2014).…”
Summary
Normal breathing in rodents requires activity of glutamatergic Dbx1-derived (Dbx1+) preBötzinger Complex (preBötC) neurons expressing somatostatin (SST). We combined in vivo optogenetic and pharmacological perturbations to elucidate the functional roles of these neurons in breathing. In transgenic adult mice expressing channelrhodopsin (ChR2) in Dbx1+ neurons, photoresponsive preBötC neurons had preinspiratory or inspiratory firing patterns associated with excitatory effects on burst timing and pattern. In transgenic adult mice expressing ChR2 in SST+ neurons, photoresponsive preBötC neurons had inspiratory or postinspiratory firing patterns associated with excitatory responses on pattern or inhibitory responses that were largely eliminated by blocking synaptic inhibition within preBötC or by local viral infection limiting ChR2 expression to preBötC SST+ neurons. We conclude that: i) preinspiratory preBötC Dbx1+ neurons are rhythmogenic; ii) inspiratory preBötC Dbx1+ and SST+ neurons primarily act to pattern respiratory motor output, and; iii) SST+ neuron-mediated pathways and postsynaptic inhibition within preBötC modulate breathing pattern.
“…We observed three types of neuronal firing patterns: preinspiratory (pre-I), inspiratory (I), and postinspiratory (post-I) (Figure 2). In Dbx1-ChR2 mice (Figure 2, middle column), in response to bilateral preBötC short pulse photostimulation (SPP; 100–300 ms pulse; 473 nm) at any point in the respiratory cycle, we observed increased firing in all preinspiratory (n=12/12 units in 3 mice) and most inspiratory (n=4/7 units in 3 mice) preBötC neurons, with no responsive postinspiratory (n=0/9 units in 3 mice) preBötC neurons; these results are consistent with the firing patterns of identified preBötC Dbx1 + neurons in vitro (Picardo et al, 2013). In SST-ChR2 mice (Figure 2, right column), all recorded inspiratory (n=5/5 units in 2 mice) and all postinspiratory (n=5/5 units in 2 mice) preBötC neurons, but no preinspiratory (n=0/15 units in 2 mice) preBötC neurons were responsive to bilateral preBötC SPP at any point in the respiratory cycle.…”
Section: Resultssupporting
confidence: 85%
“…The homeobox gene Dbx1 controls the fate of glutamatergic interneurons required for preBötC development that likely include respiratory rhythmogenic neurons (Bouvier et al, 2010; Gray et al, 2010; Picardo et al, 2013). ~15% of preBötC Dbx1 + neurons are preBötC SST + neurons, necessary for normal breathing in anesthetized or awake adult rats (Gray et al, 2010; Tan et al, 2008).…”
Section: Discussionmentioning
confidence: 99%
“…preBötC Dbx1 + neurons are strong candidates as generators of inspiratory rhythm (Wang et al, 2014). In rhythmically active slice preparations from neonatal rodents, preBötC Dbx1 + neurons have a preinspiratory firing pattern, initiating spike activity before the onset of each inspiratory hypoglossal nerve burst, and continue to fire throughout inspiration (Picardo et al, 2013). In vitro , a subset of Dbx1 + neurons are premotoneurons, projecting directly to motoneurons, transmitting bursts of inspiratory activity that ultimately pattern inspiratory muscle contraction (Kam et al, 2013a; Revill et al, 2015; Wang et al, 2014).…”
Summary
Normal breathing in rodents requires activity of glutamatergic Dbx1-derived (Dbx1+) preBötzinger Complex (preBötC) neurons expressing somatostatin (SST). We combined in vivo optogenetic and pharmacological perturbations to elucidate the functional roles of these neurons in breathing. In transgenic adult mice expressing channelrhodopsin (ChR2) in Dbx1+ neurons, photoresponsive preBötC neurons had preinspiratory or inspiratory firing patterns associated with excitatory effects on burst timing and pattern. In transgenic adult mice expressing ChR2 in SST+ neurons, photoresponsive preBötC neurons had inspiratory or postinspiratory firing patterns associated with excitatory responses on pattern or inhibitory responses that were largely eliminated by blocking synaptic inhibition within preBötC or by local viral infection limiting ChR2 expression to preBötC SST+ neurons. We conclude that: i) preinspiratory preBötC Dbx1+ neurons are rhythmogenic; ii) inspiratory preBötC Dbx1+ and SST+ neurons primarily act to pattern respiratory motor output, and; iii) SST+ neuron-mediated pathways and postsynaptic inhibition within preBötC modulate breathing pattern.
“…Figure 7 shows representative data from such a Dbx1 neuron whose XII-evoked antidromic spike was extinguished by collision with an orthodromic spike triggered by a somatic current pulse (Figure 7E). Most Dbx1 preBötC neurons are inspiratory and show commissural axons that cross the midline and innervate the contralateral preBötC (Figure 8A–C), as shown previously (Bouvier et al, 2010; Picardo et al, 2013). Here, we identify Dbx1 preBötC neurons that are also inspiratory modulated but send axons ipsilaterally toward the XII nucleus (Figure 8D–F and Figure 8—figure supplement 1), consistent with a role related to premotor transmission of inspiratory drive from preBötC to XII motoneurons.10.7554/eLife.03427.015Figure 7.Dbx1 preBötC neurons with premotor function.( A ) Fluorescence and ( B ) bright field images of a slice preparation.…”
Section: Resultssupporting
confidence: 84%
“…These spatial relationships visible in bright field or epifluorescence allow us to pinpoint the preBötC (Figure 1A). At the cellular level, identifying putative rhythmogenic neurons on the basis of fluorescent protein expression alone is acceptable because the overwhelming majority of Dbx1 preBötC neurons are inspiratory (e.g., Figure 1B) (Picardo et al, 2013). 10.7554/eLife.03427.003Figure 1.Dbx1 preBötC neurons.( A ) Bright field (left) and fluorescence (right) images of the right half of a preBötC-surface slice preparation.…”
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
Opioid overdose is the leading cause of drug overdose lethality, posing an urgent need for investigation. The key brain region for inspiratory rhythm regulation and opioid‐induced respiratory depression (OIRD) is the preBötzinger Complex (preBötC) and current knowledge has mainly been obtained from animal systems. This study aims to establish a protocol to generate human preBötC neurons from induced pluripotent cells (iPSCs) and develop an opioid overdose and recovery model utilizing these iPSC‐preBötC neurons. A de novo protocol to differentiate preBötC‐like neurons from human iPSCs is established. These neurons express essential preBötC markers analyzed by immunocytochemistry and demonstrate expected electrophysiological responses to preBötC modulators analyzed by patch clamp electrophysiology. The correlation of the specific biomarkers and function analysis strongly suggests a preBötC‐like phenotype. Moreover, the dose‐dependent inhibition of these neurons’ activity is demonstrated for four different opioids with identified IC50's comparable to the literature. Inhibition is rescued by naloxone in a concentration‐dependent manner. This iPSC‐preBötC mimic is crucial for investigating OIRD and combating the overdose crisis and a first step for the integration of a functional overdose model into microphysiological systems.
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