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Spatially distinct, interacting oscillators in the bullfrog medulla generate and coordinated buccal and lung ventilatory rhythms, but how these rhythms are transmitted onto trigeminal and hypoglossal motor neurons is unknown. Using a vertically-mounted isolated brainstem preparation, the Sheep Dip, we identified the regions of the brainstem containing motor nuclei using a solution capable of blocking synaptic release and, following washout, locally exposed these regions to 5 μM NBQX and/or 50 μM AP5. Local application of NBQX significantly reduced the amplitude of buccal and lung bursts on the trigeminal nerve, and lung bursts on the hypoglossal nerve. Local AP5 caused a significant reduction in lung burst amplitude on both nerves, but for buccal bursts, hypoglossal amplitude increased and trigeminal amplitude was unchanged. Local co-application of NBQX and AP5 eliminated fictive respiratory motor output completely in both nerves. These results are consistent with mammalian data, suggesting a critical role for glutamate in transmission of respiratory activity from oscillators to motor neurons., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Pituitary adenylate cyclase-activating peptide (PACAP) is an important mediator of the stress response and is crucial in maintaining breathing in neonates. Here we investigate the role of exogenously applied PACAP in neonatal breathing using the neonatal rat in situ working heart-brainstem preparation. A 1-min bolus of 250 nM PACAP-38 caused an increased in respiratory frequency that was rapid and transient, but had no effect on neural tidal volume or neural minute ventilation. Denervation of the carotid body abolished this effect. PACAP had a persistent effect on breathing stability in both carotid body-intact and -denervated preparations, as shown by decreases in respiratory variability 5 min following application. These data suggest that PACAP released during stress acts via carotid body dependent and independent mechanisms to stimulate and stabilize breathing. These mechanisms may account for PACAP's critical role in defending neonatal breathing against environmental stress., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Abnormal respiratory chemosensitivity is implicated in recurrent apnea syndromes, with the peripheral chemoreceptors, the carotid bodies, playing a particularly important role. Previous work suggests that supraphysiological concentrations of the endocannabinoid endovanilloid and TASK channel blocker anandamide (ANA) excite carotid bodies, but the mechanism(s) and physiological significance are unknown. Given that carotid body output is temperature-sensitive, we hypothesized that ANA stimulates carotid body chemosensory afferents via temperature-sensitive vanilloid (TRPV1) receptors. To test this hypothesis, we used the dual-perfused in situ rat preparation to confirm that independent perfusion of carotid arteries with supraphysiological concentrations of ANA strongly excites carotid sinus nerve afferents and that this activity is sufficient to increase phrenic activity. Next, using ex vivo carotid body preparations, we demonstrate that these effects are mediated by TRPV1 receptors, not CB1 receptors or TASK channels: in CB1-null mouse preparations, ANA increased afferent activity across all levels of Po(2), whereas in TRPV1-null mouse preparations, the stimulatory effect of ANA was absent. In rat ex vivo preparations, ANA's stimulatory effects were mimicked by olvanil, a nonpungent TRPV1 agonist, and suppressed by the TRPV1 antagonist AMG-9810. The specific CB1 agonist oleamide had no effect. Physiological levels of ANA had no effect alone but increased sensitivity to mild hyperthermia. AMG-9810 blocked ANA's effect on the temperature response. Immunolabeling and RT-PCR demonstrated that TRPV1 receptors are not expressed in carotid body glomus cells but reside in petrosal sensory afferents. Together, these results suggest that ANA plays a physiological role in augmenting afferent responses to mild hyperthermia by activating TRPV1 receptors on petrosal afferents.

Pyrolysis mass spectrometry is a data rich analysis technique now becoming widely applied in microbiology. Data analysis is a key step in the exploitation of the technique and the application of neural network analysis to pyrolysis mass spectrometric data offers new opportunities for classification, identification and inter-strain comparison of microorganisms in biotechnology and clinical microbiology. The use of a supervised neural network for the identification of members of a streptomycete species-group is described.

Curie-point pyrolysis mass spectrometry was used to evaluate the taxonomic integrity of three subclusters, provisionally labelled S. albidoflavus, S. anulatus and S. halstedii, which formed a species-group in an extensive numerical phenetic survey of the genus Streptomyces. Excellent agreement was found between the results of the triplicate analyses of each strain while the duplicated strains clustered adjacent to one another. Sequential principal component-canonical variates analysis of the experimental data collected on the 32 representative organisms showed that the S. albidoflavus strains formed a distinct group. This result taken together with earlier chemical, molecular and numerical taxonomic data indicates that the S. albidoflavus subcluster corresponds to a distinct species. In contrast, the subclusters equated with S. anulatus and S. halstedii were found to be heterogeneous and hence in need of further study. However, it is evident from the present investigation that Curie-point pyrolysis mass spectrometry provides a rapid and reproducible way of evaluating the taxonomic integrity of Streptomyces species-groups.

Evidence is presented to show that numerical taxonomy is of proven value both for the circumscription and identification of Streptomyces species. In addition, 252 representatives of numerically defined species and species-groups of this taxon were examined for 273 unit characters and the resultant data analysed using conventional statistics. Clustering was only marginally affected by the proximity coefficients used or by test error, estimated at 3.37%. The numerical classification obtained confirmed and extended the results of previous taxometric surveys, notably by showing that the Streptomyces albidoflavus species-group encompassed taxospecies corresponding to S. albidoflavus, Streptomyces anulatus and Streptomyces halstedii. Rapid enzyme tests based upon the fluorophores, 7-amino-4-methylcoumarin and 4-methylumbelliferone, provide useful data for streptomycete systematics. It can be concluded that the genus Streptomyces is currently well circumscribed and underspeciated.

Pilot experiments were designed to determine the potential of Curie-point pyrolysis mass spectrometry (PyMS) in the classification, identification and typing of industrially significant actinomycetes, and for the detection of target and novel actinomycetes needed for pharmacological screening programmes. The results indicate that the method is of value for the separation of actinomycetes at and below the species level, in the detection and circumscription of novel actinomycetes, and for the detection of identical and duplicated strains. There is also evidence that the pyrolysis system will permit the identification of target actinomycetes directly from selective isolation plates. PyMS is one of the methods that should be used to generate polyphasic taxonomies of actinomycete genera.