The gut microbiome consists of a multi-kingdom microbial community. Whilst the role of bacteria as causal contributors governing host physiological development is well established, the role of fungi remains to be determined. Here, we use germ-free mice colonized with defined species of bacteria, fungi, or both to differentiate the causal role of fungi on microbiome assembly, immune development, susceptibility to colitis, and airway inflammation. Fungal colonization promotes major shifts in bacterial microbiome ecology, and has an independent effect on innate and adaptive immune development in young mice. While exclusive fungal colonization is insufficient to elicit overt dextran sulfate sodium-induced colitis, bacterial and fungal co-colonization increase colonic inflammation. Ovalbumin-induced airway inflammation reveals that bacterial, but not fungal colonization is necessary to decrease airway inflammation, yet fungi selectively promotes macrophage infiltration in the airway. Together, our findings demonstrate a causal role for fungi in microbial ecology and host immune functionality, and therefore prompt the inclusion of fungi in therapeutic approaches aimed at modulating early life microbiomes.

In Rana catesbeiana the upper airways are used for two distinct yet highly coordinated ventilatory behaviours: buccal ventilation and lung inflation cycles. How these behaviours are generated and coordinated is unknown. The purpose of this study was to identify putative rhythmogenic brainstem loci involved in these ventilatory behaviours. We surveyed the isolated postmetamorphic brainstem to determine sites where local depolarization, produced by microinjecting the non-NMDA glutamate receptor agonist, AMPA, augmented the ventilatory motor patterns. Two sites were identified: a caudal site, at the level of cranial nerve (CN) X, where AMPA injections caused increased buccal burst frequency but abolished lung bursts, and a rostral site, between the levels of CN VIII and IX, where injections increased the frequency of both types of ventilatory bursts. These two sites were further examined using GABA microinjections to locally inhibit cells. GABA injected into the caudal site suppressed the buccal rhythm but the lung rhythm continued, albeit at a different frequency. When GABA was injected into the rostral site the lung bursts were abolished but the buccal rhythm continued. When the two sites were physically separated by transection, both rostral and caudal brainstem sections were capable of rhythmogenesis. The results suggest the respiratory network within the amphibian brainstem is composed of at least two distinct but interacting oscillators, the buccal and lung oscillators. These putative oscillators may provide a promising experimental model for studying coupled oscillators in vertebrates. www.jphysiol.org unpublished observations). Finally, hypercapnic challenge in preparations from postmetamorphic animals causes lung burst frequency to increase but has no effect on buccal frequency (Torgerson et al. 1997b). Transection studies have so far been suggestive but ultimately inconclusive in determining whether lung and buccal rhythm generating circuits are spatially separated. In premetamorphic animals, transection studies indicate that the only region of the brainstem capable of rhythmogenesis resides caudal to CN IX (Gdovin et al. 1999;Torgerson et al. 2001b). In postmetamorphic animals, brainstem sections were capable of rhythmogenesis as long as they included the region between CN VII and IX, suggesting that this region alone was essential for rhythmogenesis (Torgerson et al. 2001b). Thus, transection studies to date have failed to demonstrate the presence of multiple rhythmogenic brainstem sites in the same animal. Here we report the results of a drug microinjection and transection study. Our rationale was to use drug microinjections to identify important sites for rhythmogenesis and then use transection to determine whether rhythmogenesis persisted after the sites were physically separated. Similar techniques have been used previously to identify brain regions important for respiration and other behaviours (e.g. Smith et al. 1991;Coles & Dick, 1996;Ramirez et al. 1998;Solomon et al. 1999;McCrimmon et al. 2000;...

Inputs from central (brainstem) and peripheral (carotid body) respiratory chemoreceptors are coordinated to protect blood gases against potentially deleterious fluctuations. However, the mathematics of the steady-state interaction between chemoreceptors has been difficult to ascertain. Further, how this interaction affects time-dependent phenomena (in which chemoresponses depend upon previous experience) is largely unknown. To determine how central P CO 2 modulates the response to peripheral chemostimulation in the rat, we utilized an in situ arterially perfused, vagotomized, decerebrate preparation, in which central and peripheral chemoreceptors were perfused separately (i.e. dual perfused preparation (DPP)). We carried out two sets of experiments: in Experiment 1, we alternated steady-state brainstem P CO 2 between 25 and 50 Torr in each preparation, and applied specific carotid body hypoxia (60 Torr P O 2 and 40 Torr P CO 2 ) under both conditions; in Experiment 2, we applied four 5 min bouts (separated by 5 min) of specific carotid body hypoxia (60 Torr P O 2 and 40 Torr P CO 2 ) while holding the brainstem at either 30 Torr or 50 Torr P CO 2 . We demonstrate that the level of brainstem P CO 2 modulates (a) the magnitude of the phrenic responses to a single step of specific carotid body hypoxia and (b) the magnitude of time-dependent phenomena. We report that the interaction between chemoreceptors is negative (i.e. hypo-additive), whereby a lower brainstem P CO 2 augments phrenic responses resulting from specific carotid body hypoxia. A negative interaction may underlie the pathophysiology of central sleep apnoea in populations that are chronically hypocapnic.

Though the mechanics of breathing differ fundamentally between amniotes and "lower" vertebrates, homologous rhythm generators may drive air breathing in all lunged vertebrates. In both frogs and rats, two coupled oscillators, one active during the inspiratory (I) phase and the other active during the preinspiratory (PreI) phase, have been hypothesized to generate the respiratory rhythm. We used opioids to uncouple these oscillators. In the intact rat, complete arrest of the external rhythm by opioid-induced suppression of the putative I oscillator, that is, pre-Bötz-inger complex (PBC) oscillator, did not arrest the putative PreI oscillator. In the unanesthetized frog, the comparable PreI oscillator, that is, the putative buccal/ gill oscillator, was refractory to opioids even though the comparable I oscillator, the putative lung oscillator, was arrested. Studies in en bloc brainstem preparations derived from both juvenile frogs and metamorphic tadpoles confirmed these results and suggested that opioids may play a role in the clustering of lung bursts into episodes. As the frog and rat respiratory circuitry produce functionally equivalent motor outputs during lung inflation, these data argue for a close homology between the frog and rat oscillators. We suggest that the respiratory rhythm of all lunged vertebrates is generated by paired coupled oscillators. These may have originated from the gill and lung oscillators of the earliest air breathers.

Asthma accounts for 380,000 deaths a year. Carotid body denervation has been shown to have a profound effect on airway hyper-responsiveness in animal models but a mechanistic explanation is lacking. Here we demonstrate, using a rat model of asthma (OVA-sensitized), that carotid body activation during airborne allergic provocation is caused by systemic release of lysophosphatidic acid (LPA). Carotid body activation by LPA involves TRPV1 and LPA-specific receptors, and induces parasympathetic (vagal) activity. We demonstrate that this activation is sufficient to cause acute bronchoconstriction. Moreover, we show that prophylactic administration of TRPV1 (AMG9810) and LPA (BrP-LPA) receptor antagonists prevents bradykinin-induced asthmatic bronchoconstriction and, if administered following allergen exposure, reduces the associated respiratory distress. Our discovery provides mechanistic insight into the critical roles of carotid body LPA receptors in allergen-induced respiratory distress and suggests alternate treatment options for asthma.

Interaction between central (brainstem) and peripheral (carotid body) respiratory chemosensitivity is vital to protect blood gases against potentially deleterious fluctuations, especially during sleep. Previously, using an in situ arterially perfused, vagotomized, decerebrate preparation in which brainstem and peripheral chemoreceptors are perfused separately (i.e. dual perfused preparation; DPP), we observed that the phrenic response to specific carotid body hypoxia was larger when the brainstem was held at 25 Torr P CO 2 compared to 50 Torr P CO 2 . This suggests a negative (i.e. hypo-additive) interaction between chemoreceptors. The current study was designed to (a) determine whether this observation could be generalized to all carotid body stimuli, and (b) exclude the possibility that the hypo-additive response was the simple consequence of ventilatory saturation at high brainstem P CO 2 . Specifically, we tested how steady-state brainstem P CO 2 modulates peripheral chemoreflex magnitude in response to carotid body P CO 2 and P O 2 perturbations, both above and below eupnoeic levels. We found that the peripheral chemoreflex was more responsive the lower the brainstem P CO 2 regardless of whether the peripheral chemoreceptors received stimuli which increased or decreased activation. These findings demonstrate a negative interaction between brainstem and peripheral chemosensitivity in the rat in the absence of ventilatory saturation. We suggest that a negative interaction in humans may contribute to increased controller gain associated with sleep-related breathing disorders and propose that the assumption of simple addition between chemoreceptor inputs used in current models of the respiratory control system be reconsidered.

Oxygen (O(2)) is one of the most important environmental factors that affects both physiological processes and development of aerobic animals, yet little is known about the neural mechanism of O(2) sensing and adaptive responses to low O(2) (hypoxia) during development. In the pond snail, Helisoma trivolvis, the first embryonic neurons (ENC1s) to develop are a pair of serotonergic sensory-motor cells that regulate a cilia-driven rotational behavior. Here, we report that the ENC1-ciliary cell circuit mediates an adaptive behavioral response to hypoxia. Exposure of egg masses to hypoxia elicited a dose-dependent and reversible acceleration of embryonic rotation that mixed capsular fluid, thereby facilitating O(2) diffusion to the embryo. The O(2) partial pressures (Po(2)) for threshold, half-maximal, and maximal rotational response were 60, 28, and 13 mm Hg, respectively. During hypoxia, embryos relocated to the periphery of the egg masses where higher Po(2) levels occurred. Furthermore, intermittent hypoxia treatments induced a sensitization of the rotational response. In isolated ciliary cells, ciliary beating was unaffected by hypoxia, suggesting that in the embryo, O(2) sensing occurs upstream of the motile cilia. The rotational response of embryos to hypoxia was attenuated by application of the serotonin receptor antagonist, mianserin, correlated to the development of ENC1-ciliary cell circuit, and abolished by laser-ablation of ENC1s. Together, these data suggest that ENC1s are unique oxygen sensors that may provide a good single cell model for the examination of mechanistic, developmental, and evolutionary aspects of O(2) sensing.

A debate has raged since the discovery of central and peripheral respiratory chemoreceptors as to whether the reflexes they mediate combine in an additive (i.e., no interaction), hypoadditive or hyperadditive manner. Here we critically review pertinent literature related to O2 and CO2 sensing from the perspective of system integration and summarize many of the studies on which these seemingly opposing views are based. Despite the intensity and quality of this debate, we have yet to reach consensus, either within or between species. In reviewing this literature, we are struck by the merits of the approaches and preparations that have been brought to bear on this question. This suggests that either the nature of combination is not important to system responses, contrary to what has long been supposed, or that the nature of the combination is more malleable than previously assumed, changing depending on physiological state and/or respiratory requirement.