Peritoneal sepsis caused by Escherichia coli triggers brainstem inflammation and alters the function of sympatho-respiratory control circuits.

BACKGROUND: Sepsis has a high mortality rate due to multiple organ failure. However, the influence of peripheral inflammation on brainstem autonomic and respiratory circuits in sepsis is poorly understood. Our working hypothesis is that peripheral inflammation affects central autonomic circuits and consequently contributes to multiorgan failure in sepsis. METHODS: In an Escherichia coli (E. coli)-fibrin clot model of peritonitis, we first recorded ventilatory patterns using plethysmography before and 24 h after fibrin clot implantation. To assess whether peritonitis was associated with brainstem neuro-inflammation, we measured cytokine and chemokine levels in Luminex assays. To determine the effect of E. coli peritonitis on brainstem function, we assessed sympatho-respiratory nerve activities at baseline and during brief (20 s) hypoxemic ischemia challenges using in situ-perfused brainstem preparations (PBPs) from sham or infected rats. PBPs lack peripheral organs and blood, but generate vascular tone and in vivo rhythmic activities in thoracic sympathetic (tSNA), phrenic and vagal nerves. RESULTS: Respiratory frequency was greater (p < 0.001) at 24 h post-infection with E. coli than in the sham control. However, breath-by-breath variability and total protein in the BALF did not differ. IL-1ß (p < 0.05), IL-6 (p < 0.05) and IL-17 (p < 0.04) concentrations were greater in the brainstem of infected rats. In the PBP, integrated tSNA (p < 0.05) and perfusion pressure were greater (p < 0.001), indicating a neural-mediated pathophysiological high sympathetic drive. Moreover, respiratory frequency was greater (p < 0.001) in PBPs from infected rats than from sham rats. Normalized phase durations of inspiration and expiration were greater (p < 0.009, p < 0.015, respectively), but the post-inspiratory phase (p < 0.007) and the breath-by-breath variability (p < 0.001) were less compared to sham PBPs. Hypoxemic ischemia triggered a biphasic response, respiratory augmentation followed by depression. PBPs from infected rats had weaker respiratory augmentation (p < 0.001) and depression (p < 0.001) than PBPs from sham rats. In contrast, tSNA in E. coli-treated PBPs was enhanced throughout the entire response to hypoxemic ischemia (p < 0.01), consistent with sympathetic hyperactivity. CONCLUSION: We show that peripheral sepsis caused brainstem inflammation and impaired sympatho-respiratory motor control in a single day after infection. We conclude that central sympathetic hyperactivity may impact vital organ systems in sepsis.

Noncovalent Peptide Stapling Using Alpha-Methyl-l-Phenylalanine for α-Helical Peptidomimetics.

Peptides and peptidomimetics are attractive drug candidates because of their high target specificity and low-toxicity profiles. Developing peptidomimetics using hydrocarbon (HC)-stapling or other stapling strategies has gained momentum because of their high stability and resistance to proteases; however, they have limitations. Here, we take advantage of the α-methyl group and an aromatic phenyl ring in a unique unnatural amino acid, α-methyl-l-phenylalanine (αF), and propose a novel, noncovalent stapling strategy to stabilize peptides. We utilized this strategy to create an α-helical B-chain mimetic of a complex insulin-like peptide, human relaxin-3 (H3 relaxin). Our comprehensive data set (in vitro, ex vivo, and in vivo) confirmed that the new high-yielding B-chain mimetic, H3B10-27(13/17αF), is remarkably stable in serum and fully mimics the biological function of H3 relaxin. H3B10-27(13/17αF) is an excellent scaffold for further development as a drug lead and an important tool to decipher the physiological functions of the neuropeptide G protein-coupled receptor, RXFP3.

Advancing respiratory-cardiovascular physiology with the working heart-brainstem preparation over 25 years.

Twenty-five years ago, a new physiological preparation called the working heart-brainstem preparation (WHBP) was introduced with the claim it would provide a new platform allowing studies not possible before in cardiovascular, neuroendocrine, autonomic and respiratory research. Herein, we review some of the progress made with the WHBP, some advantages and disadvantages along with potential future applications, and provide photographs and technical drawings of all the customised equipment used for the preparation. Using mice or rats, the WHBP is an in situ experimental model that is perfused via an extracorporeal circuit benefitting from unprecedented surgical access, mechanical stability of the brain for whole cell recording and an uncompromised use of pharmacological agents akin to in vitro approaches. The preparation has revealed novel mechanistic insights into, for example, the generation of distinct respiratory rhythms, the neurogenesis of sympathetic activity, coupling between respiration and the heart and circulation, hypothalamic and spinal control mechanisms, and peripheral and central chemoreceptor mechanisms. Insights have been gleaned into diseases such as hypertension, heart failure and sleep apnoea. Findings from the in situ preparation have been ratified in conscious in vivo animals and when tested have translated to humans. We conclude by discussing potential future applications of the WHBP including two-photon imaging of peripheral and central nervous systems and adoption of pharmacogenetic tools that will improve our understanding of physiological mechanisms and reveal novel mechanisms that may guide new treatment strategies for cardiorespiratory diseases.

Volumetric mapping of the functional neuroanatomy of the respiratory network in the perfused brainstem preparation of rats.

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 mm3 of 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. ABSTRACT: 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.

Effects of ion channel noise on neural circuits: an application to the respiratory pattern generator to investigate breathing variability.

Neural activity generally displays irregular firing patterns even in circuits with apparently regular outputs, such as motor pattern generators, in which the output frequency fluctuates randomly around a mean value. This "circuit noise" is inherited from the random firing of single neurons, which emerges from stochastic ion channel gating (channel noise), spontaneous neurotransmitter release, and its diffusion and binding to synaptic receptors. Here we demonstrate how to expand conductance-based network models that are originally deterministic to include realistic, physiological noise, focusing on stochastic ion channel gating. We illustrate this procedure with a well-established conductance-based model of the respiratory pattern generator, which allows us to investigate how channel noise affects neural dynamics at the circuit level and, in particular, to understand the relationship between the respiratory pattern and its breath-to-breath variability. We show that as the channel number increases, the duration of inspiration and expiration varies, and so does the coefficient of variation of the breath-to-breath interval, which attains a minimum when the mean duration of expiration slightly exceeds that of inspiration. For small channel numbers, the variability of the expiratory phase dominates over that of the inspiratory phase, and vice versa for large channel numbers. Among the four different cell types in the respiratory pattern generator, pacemaker cells exhibit the highest sensitivity to channel noise. The model shows that suppressing input from the pons leads to longer inspiratory phases, a reduction in breathing frequency, and larger breath-to-breath variability, whereas enhanced input from the raphe nucleus increases breathing frequency without changing its pattern. NEW & NOTEWORTHY: A major source of noise in neuronal circuits is the "flickering" of ion currents passing through the neurons' membranes (channel noise), which cannot be suppressed experimentally. Computational simulations are therefore the best way to investigate the effects of this physiological noise by manipulating its level at will. We investigate the role of noise in the respiratory pattern generator and show that endogenous, breath-to-breath variability is tightly linked to the respiratory pattern.

Kölliker-Fuse nuclei regulate respiratory rhythm variability via a gain-control mechanism.

Respiration varies from breath to breath. On the millisecond timescale of spiking, neuronal circuits exhibit variability due to the stochastic properties of ion channels and synapses. Does this fast, microscopic source of variability contribute to the slower, macroscopic variability of the respiratory period? To address this question, we modeled a stochastic oscillator with forcing; then, we tested its predictions experimentally for the respiratory rhythm generated by the in situ perfused preparation during vagal nerve stimulation (VNS). Our simulations identified a relationship among the gain of the input, entrainment strength, and rhythm variability. Specifically, at high gain, the periodic input entrained the oscillator and reduced variability, whereas at low gain, the noise interacted with the input, causing events known as "phase slips", which increased variability on a slow timescale. Experimentally, the in situ preparation behaved like the low-gain model: VNS entrained respiration but exhibited phase slips that increased rhythm variability. Next, we used bilateral muscimol microinjections in discrete respiratory compartments to identify areas involved in VNS gain control. Suppression of activity in the nucleus tractus solitarii occluded both entrainment and amplification of rhythm variability by VNS, confirming that these effects were due to the activation of the Hering-Breuer reflex. Suppressing activity of the Kölliker-Fuse nuclei (KFn) enhanced entrainment and reduced rhythm variability during VNS, consistent with the predictions of the high-gain model. Together, the model and experiments suggest that the KFn regulates respiratory rhythm variability via a gain control mechanism.

The pre-Bötzinger complex is necessary for the expression of inspiratory and post-inspiratory motor discharge of the vagus.

The mammalian three-phase respiratory motor pattern of inspiration, post-inspiration and expiration is expressed in spinal and cranial motor nerve discharge and is generated by a distributed ponto-medullary respiratory pattern generating network. Respiratory motor pattern generation depends on a rhythmogenic kernel located within the pre-Bötzinger complex (pre-BötC). In the present study, we tested the effect of unilateral and bilateral inactivation of the pre-BötC after local microinjection of the GABAA receptor agonist isoguvacine (10 mM, 50 nl) on phrenic (PNA), hypoglossal (HNA) and vagal nerve (VNA) respiratory motor activities in an in situ perfused brainstem preparation of rats. Bilateral inactivation of the pre-BötC triggered cessation of phrenic (PNA), hypoglossal (HNA) and vagal (VNA) nerve activities for 15-20 min. Ipsilateral isoguvacine injections into the pre-BötC triggered transient (6-8 min) cessation of inspiratory and post-inspiratory VNA (p < 0.001) and suppressed inspiratory HNA by - 70 ± 15% (p < 0.01), while inspiratory PNA burst frequency increased by 46 ± 30% (p < 0.01). Taken together, these observations confirm the role of the pre-BötC as the rhythmogenic kernel of the mammalian respiratory network in situ and highlight a significant role for the pre-BötC in the transmission of vagal inspiratory and post-inspiratory pre-motor drive to the nucleus ambiguus.

Neuroanatomical frameworks for volitional control of breathing and orofacial behaviors.

Breathing is the only vital function that can be volitionally controlled. However, a detailed understanding how volitional (cortical) motor commands can transform vital breathing activity into adaptive breathing patterns that accommodate orofacial behaviors such as swallowing, vocalization or sniffing remains to be developed. Recent neuroanatomical tract tracing studies have identified patterns and origins of descending forebrain projections that target brain nuclei involved in laryngeal adductor function which is critically involved in orofacial behavior. These nuclei include the midbrain periaqueductal gray and nuclei of the respiratory rhythm and pattern generating network in the brainstem, specifically including the pontine Kölliker-Fuse nucleus and the pre-Bötzinger complex in the medulla oblongata. This review discusses the functional implications of the forebrain-brainstem anatomical connectivity that could underlie the volitional control and coordination of orofacial behaviors with breathing.

Persistent glossopharyngeal nerve respiratory discharge patterns after ponto-medullary transection.

Shape and size of the nasopharyngeal airway is controlled by muscles innervated facial, glossopharyngeal, vagal, and hypoglossal cranial nerves. Contrary to brainstem networks that drive facial, vagal and hypoglossal nerve activities (FNA, VNA, HNA) the discharge patterns and origins of glossopharyngeal nerve activity (GPNA) remain poorly investigated. Here, an in situ perfused brainstem preparation (n=19) was used for recordings of GPNA in relation to phrenic (PNA), FNA, VNA and HNA. Brainstem transections were performed (n=10/19) to explore the role of pontomedullary synaptic interactions in generating GPNA. GPNA generally mirrors FNA and HNA discharge patterns and displays pre-inspiratory activity relative to the PNA, followed by robust inspiratory discharge in coincidence with PNA. Postinspiratory (early expiratory) discharge was, contrary to VNA, generally absent in FNA, GPNA or HNA. As described previously FNA and HNA discharge was virtually eliminated after pontomedullary transection while an apneustic inspiratory motor discharge was maintained in PNA, VNA and GPNA. After brainstem transection GPNA displayed an increased tonic activity starting during mid-expiration and thus developed prolonged pre-inspiratory activity compared to control. In conclusion respiratory GPNA reflects FNA and HNA which implies similar function in controlling upper airway patency during breathing. That GPNA preserved its pre-inspiratory/inspiratory discharge pattern in relation PNA after pontomedullary transection suggest that GPNA premotor circuits may have a different anatomical distribution compared HNA and FNA and thus may therefore hold a unique role in preserving airway patency.