Abstract only
Breathing is composed of three phases: inspiration, postinspiration, and active expiration. Excitatory rhythm generating networks have been identified for the generation of inspiration, ...the preBötzinger complex (preBötC), and for active expiration, the lateral parafacial (pF
L
). By utilizing a combination of a new in vitro horizontal slice preparation, optogenetics, and in vivo techniques, we have recently identified and characterized a novel population of neurons that we show to be necessary and sufficient for the generation of postinspiration (Anderson et al. 2016). This network, located dorsomedial to the nucleus ambiguus and caudal to the facial nucleus, is referred to as the postinspiratory complex, or PiCo. PiCo can function as an independent oscillator dependent on excitatory, non‐NMDA mechanisms. Here we show that a subpopulation of PiCo neurons are endowed with I
CAN
and I
NaP
dependent pacemaker properties, suggesting that PiCo has similar rhythm generating properties to the preBötC. Additionally, we observed differential effects of inhibitory antagonists. When strychnine, a glycine receptor antagonist, was bath applied to the horizontal slice in vitro, the burst duration of fictive inspiratory preBötC bursts significantly decreased while PiCo burst duration was unchanged. Upon the additional application of gabazine, a GABAergic receptor antagonist, the two rhythms progressively synchronized. Furthermore, in the presence of gabazine, the average burst area and amplitude significantly increased for both preBötC and PiCo rhythms. Based on our discovery, we propose a triple‐oscillator hypothesis that states that the three phases of respiration are generated by three, anatomically distinct, excitatory, rhythmogenic and interactive networks: the preBötC, PiCo, and PF
L
. We further conclude that network inhibition serves to coordinate the timing, phasing and patterning of these rhythms.
Support or Funding Information
1F31NS087828‐01
RO1 HL107084‐01
The preBötzinger Complex (preBötC), a medullary network critical for breathing, relies on excitatory interneurons to generate the inspiratory rhythm. Yet, half of preBötC neurons are inhibitory, and ...the role of inhibition in rhythmogenesis remains controversial. Using optogenetics and electrophysiology in vitro and in vivo, we demonstrate that the intrinsic excitability of excitatory neurons is reduced following large depolarizing inspiratory bursts. This refractory period limits the preBötC to very slow breathing frequencies. Inhibition integrated within the network is required to prevent overexcitation of preBötC neurons, thereby regulating the refractory period and allowing rapid breathing. In vivo, sensory feedback inhibition also regulates the refractory period, and in slowly breathing mice with sensory feedback removed, activity of inhibitory, but not excitatory, neurons restores breathing to physiological frequencies. We conclude that excitation and inhibition are interdependent for the breathing rhythm, because inhibition permits physiological preBötC bursting by controlling refractory properties of excitatory neurons.
A prolonged reduction in central neural respiratory activity elicits a form of plasticity known as inactivity-induced phrenic motor facilitation (iPMF), a 'rebound' increase in phrenic burst ...amplitude apparent once respiratory neural activity is restored. iPMF requires atypical protein kinase C (aPKC) activity within spinal segments containing the phrenic motor nucleus to stabilize an early transient increase in phrenic burst amplitude and to form long-lasting iPMF following reduced respiratory neural activity. Upstream signal(s) leading to spinal aPKC activation are unknown. We tested the hypothesis that spinal tumour necrosis factor-α (TNFα) is necessary for iPMF via an aPKC-dependent mechanism. Anaesthetized, ventilated rats were exposed to a 30 min neural apnoea; upon resumption of respiratory neural activity, a prolonged increase in phrenic burst amplitude (42 ± 9% baseline; P < 0.05) was apparent, indicating long-lasting iPMF. Pretreatment with recombinant human soluble TNF receptor 1 (sTNFR1) in the intrathecal space at the level of the phrenic motor nucleus prior to neural apnoea blocked long-lasting iPMF (2 ± 8% baseline; P > 0.05). Intrathecal TNFα without neural apnoea was sufficient to elicit long-lasting phrenic motor facilitation (pMF; 62 ± 7% baseline; P < 0.05). Similar to iPMF, TNFα-induced pMF required spinal aPKC activity, as intrathecal delivery of a ζ-pseudosubstrate inhibitory peptide (PKCζ-PS) 35 min following intrathecal TNFα arrested TNFα-induced pMF (28 ± 8% baseline; P < 0.05). These data demonstrate that: (1) spinal TNFα is necessary for iPMF; and (2) spinal TNFα is sufficient to elicit pMF via a similar aPKC-dependent mechanism. These data are consistent with the hypothesis that reduced respiratory neural activity elicits iPMF via a TNFα-dependent increase in spinal aPKC activity.
Abstract only
Mammalian respiration consists of three phases: (1) inspiration, (2) an early phase of expiration, postinspiration, and (3) a late phase of expiration. Two excitatory oscillators have ...been proposed for the generation of inspiration and active expiration, a conditional expiration that occurs during the late expiratory phase. However, the cellular and circuit mechanisms underlying the generation of postinspiration are largely unknown. Here we describe a third, previously undiscovered excitatory rhythm generator, the Postinspiratory Complex, or PIC, located immediately caudal to the facial nucleus and just medial and dorsal to the rostral‐most portion of the nucleus ambiguus. In this study a variety of in vitro and in vivo approaches are employed to demonstrate that this rhythmogenic kernel generates activity in the postinspiratory phase. Immunohistochemical and optogenetic techniques are utilized to reveal that PIC neurons co‐express acetylcholine and glutamate. Differential responses to neuromodulators and a distinct anatomical location distinguish the PIC from other described respiratory oscillators. Inhibitory interactions between this rhythmogenic network and the preBötzinger Complex, the network responsible for generating inspiration, establish the relative timing, but are not responsible for the generation of the burst underlying inspiratory and postinspiratory activity. Light stimulation of cholinergic PIC neurons in Chat‐cre:Ai27 mice evokes postinspiratory vagal activity in vivo that can reset the inspiratory rhythm. We therefore propose that the three phases of mammalian breathing are generated by three excitatory rhythmogenic medullary networks that are coordinated by inhibitory mechanisms.
Support or Funding Information
NIH NS087828‐01
NIH HL107084‐01
Abstract only
An acute disruption in descending synaptic inputs to the phrenic motor nucleus, elicits a form of spinal plasticity called inactivity‐induced phrenic motor facilitation (iPMF). iPMF is ...manifested as a rebound increase in phrenic amplitude that is apparent when synaptic inputs are restored. Here we tested the hypothesis that chronic withdrawal of phrenic synaptic inputs also elicits plasticity. Rats received a unilateral injection of aCSF or botulinum toxin A (BoNT/A;10ng) into the C4 phrenic motor pool. Three days post‐unilateral BoNT/A, cleaved SNAP‐25 was localized to ipsilateral C4 spinal segments, suggesting impairment of synaptic inputs. In a separate group of rats, ventral spinal segments (C3‐C5) were harvested and membrane proteins were biotinylated to determine membrane expression of GluA1/A2 receptors. No change in GluA1 was detected in spinal segments ipsilateral or contralateral to BoNT/A (ipsi: 14±15; contra: 19±10% sham; p<0.05). By contrast, membrane GluA2 expression was significantly increased in ventral segments ipsilateral, but not contralateral to BoNT/A (ipsi: 70±24; contra: 36±26% sham; p<0.05). Collectively, our data suggest that chronic withdrawal of phrenic synaptic inputs increases ipsilateral membrane expression of GluA2‐containing AMPA receptors. We hypothesize that iPMF is a compensatory mechanism to maintain respiratory motor output throughout life. NIH‐HL105511.
Breathing's remarkable ability to adapt to changes in metabolic, environmental, and behavioral demands stems from a complex integration of its rhythm-generating network within the wider nervous ...system. Yet, this integration complicates identification of its specific rhythmogenic elements. Based on principles learned from smaller rhythmic networks of invertebrates, we define criteria that identify rhythmogenic elements of the mammalian breathing network and discuss how they interact to produce robust, dynamic breathing.
Abstract only
The analgesic utility of opiates is limited by the risk for adverse and life‐threatening side effects, including respiratory depression. Opioid‐induced respiratory depression (OIRD) is ...characterized by a pronounced decrease in the frequency and regularity of inspiratory efforts. The inspiratory rhythm originates from the preBotzinger Compex (preBötC), a region in the ventral medulla that contains neurons that express the μ‐opioid receptor (μOR) and is intrinsically sensitive to opioids, including the synthetic μOR agonist DAMGO. However, the cellular‐level mechanisms of opioid‐induced suppression of rhythmogenesis within the preBötC are controversial. Some evidence suggests opiates regulate synaptic transmission while other evidence suggests membrane potential (V
m
) is the primary effector. At the network level, the frequency and regularity of the inspiratory rhythm is primarily determined by the interburst interval IBI which is regulated, in part, by excitatory neurons with “pre‐inspiratory” (pre‐I) spiking activity, but the network‐level effects of opiates have not been well defined. To shed light on these cellular‐ and network‐level processes, we utilized Oprm1
Cre
mice to optogenetically manipulate μOR neurons
in vitro
and
in vivo
. In brainstem slice preparations from Oprm1
Arch
neonatal mice, unit recordings from pre‐I neurons revealed that 37% of these neurons express μOR. In the presence of increasing concentrations of DAMGO, pre‐I spiking was eliminated initially, followed by suppression of spiking activity during inspiratory bursts. Next, using intracellular recordings, laser power was tuned such that photoinhibition hyperpolarized μOR neurons by ~5–10mV, mimicking the effect of DAMGO on V
m
when bath applied at concentrations (100–300nm) that dramatically suppress inspiratory frequency (>90%). However, bilateral photoinhibition of μOR neurons reduced frequency by 36%, only partially mimicking the suppression of frequency by DAMGO. In slices from Oprm1
ChR2
mice, photostimuation of μOR neurons under control conditions increased frequency by 204%, indicating that these neurons are integrated within this rhythmogenic network. However, following suppression of the rhythm with DAMGO, ChR2‐mediated depolarization of μOR neurons could not rescue inspiratory frequency. These findings
in vitro
were largely corroborated by experiments in anesthetized mice
in vivo
, and suggest that hyperpolarization of μOR neurons at the cellular‐level only partially contributes to OIRD in the preBötC. Based on these initial results, we propose a model of preBötC OIRD in which the effects of opioids are twofold: 1) a modest hyperpolarization of μOR neurons at the cellular‐level reduces pre‐I spiking activity within the network and causes the duration between inspirations to become longer and more irregular, followed by 2) a crash in network‐driven rhythmogenesis, potentially through a μOR‐mediated reduction in the efficacy of excitatory synaptic transmission.
Support or Funding Information
K99 HL145004 (Baertsch)R01 HL144801 (Ramirez)
Subacute necrotizing encephalopathy, or Leigh syndrome (LS), is the most common pediatric presentation of genetic mitochondrial disease. LS is a multi‐system disorder with severe neurologic, ...metabolic, and musculoskeletal symptoms. The presence of progressive, symmetric, and necrotizing lesions in the brainstem are a defining feature of the disease, and the major cause of morbidity and mortality, but the mechanisms underlying their pathogenesis have been elusive. Recently, we demonstrated that high‐dose pexidartinib, a CSF1R inhibitor, prevents LS CNS lesions and systemic disease in the Ndufs4(−/−) mouse model of LS. While the dose–response in this study implicated peripheral immune cells, the immune populations involved have not yet been elucidated. Here, we used a targeted genetic tool, deletion of the colony‐stimulating Factor 1 receptor (CSF1R) macrophage super‐enhancer FIRE (Csf1rΔFIRE), to specifically deplete microglia and define the role of microglia in the pathogenesis of LS. Homozygosity for the Csf1rΔFIRE allele ablates microglia in both control and Ndufs4(−/−) animals, but onset of CNS lesions and sequalae in the Ndufs4(−/−), including mortality, are only marginally impacted by microglia depletion. The overall development of necrotizing CNS lesions is not altered, though microglia remain absent. Finally, histologic analysis of brainstem lesions provides direct evidence of a causal role for peripheral macrophages in the characteristic CNS lesions. These data demonstrate that peripheral macrophages play a key role in the pathogenesis of disease in the Ndufs4(−/−) model.
Microglia and peripheral macrophages contribute to CNS pathology in the Ndufs4(−/−) mouse model of Leigh syndrome, while peripheral macrophages alone are sufficient to drive disease in microglia−deficient Ndufs4(−/−) animals. (A−B) Cortex of Ndufs4(−/−)/Csf1r (wt/wt) (A) and Ndufs4(−/−)/Csf1r (fr/fr) (B) mice stained for the pan−macrophage marker IBA1 (red) and the microglia−specific marker P2YR12 (green); DNA is co−stained with DAPI (blue). Microglia are absent by both IBA1 and P2YR12 staining in the Ndufs4(−/−)/Csf1r (fr/fr) cortex. (C−D) Brainstem lesions from Ndufs4(−/−)/Csf1r (wt/wt) (C) and Ndufs4(−/−)/Csf1r (fr/fr) (D) mice stained for the pan−macrophage marker IBA1 and the microglia−specific marker P2YR12. Cells positive for IBA1, the pan−macrophage marker, are present in both genotypes, while P2YR12 staining is absent in cells in the brainstem lesions of Ndufs4(−/−)/Csf1r (fr/fr) animals. Note: the P2YR12 staining surrounding the lesion site does not appear to be cellular in origin, and is thought to reflect the presence of aggregated platelets, which are P2YR12 positive. (E−F) Cortex of Ndufs4(−/−)/Csf1r (wt/wt) (E) and Ndufs4(−/−)/Csf1r (fr/fr) (F) mice stained for the pan−macrophage marker IBA1 (red) and the peripheral leukocyte marker CD45 (green); DNA is co−stained with DAPI (blue). Microglia are absent in the Ndufs4(−/−)/Csf1r (fr/fr) cortex, and microglia (by IBA1 posivitivy and morphology) do not express CD45 (E). A few compact cells with CD45 positivity are present in both genotypes, presumed to be circulating leukocytes. (G−H) Brainstem lesions in Ndufs4(−/−)/Csf1r (wt/wt) (G) and Ndufs4(−/−)/Csf1r (fr/fr) (H) mice stained for the pan−macrophage marker IBA1 and the peripheral leukocyte marker CD45. CD45 positive cells are present in both in the Ndufs4(−/−)/Csf1r (fr/fr) and Ndufs4(−/−)/Csf1r (wt/wt) lesions, while most or all IBA1 positive cells in the Ndufs4(−/−)/Csf1r (fr/fr) lesion appear to be positive for the peripheral leukocyte marker CD45. Co−staining of CD45 and IBA1 is indicative of peripheral macrophages.
The triple oscillator hypothesis posits that breathing is generated by three rhythmogenic microcircuits contained within the ventrolateral medulla. The preBötzinger Complex (preBötC) generates the ...inexorable inspiratory phase of breathing, whereas the conditional postinspiratory and active expiratory phases of breathing are generated by the Postinspiratory Complex (PiCo) and lateral parafacial respiratory group (PFL), respectively. Neuromodulators including norepinephrine, opioids, and somatostatin are known to differentially modulate the activity of each rhythmic circuit, likely contributing to the independence of each breathing phase and the flexibility of the respiratory rhythm. However, whether the functional boundaries of each of these microcircuits are static or can change dynamically based on the neuromodulatory state of the network is unknown. The neuromodulator Substance P (SP) facilitates breathing frequency in vivo and also in medullary slices that isolate the preBötC in vitro. Using a neonatal mouse horizontal brainstem slice preparation that preserves the rostrocaudal extent of the ventral medulla, we tested the hypothesis that SP differentially modulates bursting activity in the preBötC and PiCo and explored how SP may reshape the distribution of inspiratory activity within the wider medullary network. Rhythmic bursting activity was simultaneously recorded from the preBötC and PiCo (n=5). As expected, recordings from the PiCo, located ~500μm rostral to the preBötC, revealed a slow postinspiratory rhythm with robust bursts occurring after only ~1/12 preBötC bursts under control conditions. Following introduction of 0.5–1.0μM SP into the bath superfusion solution, PiCo bursts decreased in amplitude (~40%) and accelerated such that they occurred 1:1 with preBötC bursts without changing the time interval between peak preBötC and PiCo activity. In some PiCo recordings, we noted a small amount of inspiratory activity synchronized with preBötC bursts that was eliminated in the presence of SP. To test whether SP alters the spatial distribution of inspiratory activity along the ventral medulla, we simultaneously recorded inspiratory population activity generated at the preBötC and 400–600μm rostral (n=8). Following application of 1μM SP frequency was increased (~90%) and inspiratory burst amplitude was reduced by ~20% at the preBötC, whereas burst amplitude decreased by ~50% in rostral recordings. Based on these preliminary experiments, we hypothesize that SP reshapes breathing activity generated within the medulla by facilitating postinspiratory activity and reducing the extent of inspiratory activity rostral of the preBötC to shrink the size of the active inspiratory network. Differential neuromodulatory properties and dynamic functional boundaries of the rhythmogenic circuits that generate breathing may contribute to the remarkable flexibility and robustness of this vital physiological behavior.
Support or Funding Information
NIH F32 HL134207
NIH R01 HL126523
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
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