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Ectoaminopeptidase activity (EAA), 14 C-glutamic acid assimilation (GA) and respiration (GR) rates were measured over spring and fall, whilst maintaining in situ hydrostatic pressure condi- tion through a 2000 m water column in the NW Mediterranean. Depth-integrated EAA was maximal in the deep-sea layer due to the lack of ready-to-use compounds. Conversely, depth-integrated fluxes for GA and GR in the bathypelagic zone, though far from negligible, were much lower than in the photic layer, the exception being in the 1000 to 2000 m layer during fall when respiration fluxes and particle fluxes were optimal. Metabolic rates obtained from samples recovered and incubated under in situ pressure conditions were 4.53 ± 4.45 (mean ± SD; n = 19) times higher than their decompressed counterparts, proving that deep-sea bacteria were adapted to high pressure. 14 C-glutamic acid uptake rates (GU = GA + GR) measured under in situ pressure conditions were used to calculate the bacterial 14 C-glutamic acid assimilation yield (GYG = GA/GU). In decompressed samples this yield was always lower by around 20% than in those measured under in situ pressure conditions. Because experiments currently used to estimate bacterial growth efficiency (BGE) are not carried out under in situ pressure conditions, we hypothesize that pressure affects BGE in the same way that it affects GYG. Thus, the stress caused by decompression induces an increase in energy cost, and so under- estimates BGE. Growth of deep-sea bacteria is highly dependent on the quantity and quality of sink- ing particles reaching 1000 m. During mesotrophic conditions (spring), high fluxes of relatively fresh particulate organic carbon generated relatively small depth-integrated EAA rates (20.6 mgC m -2 h -1 ) and high GYG (65%). Meanwhile, during oligotrophic conditions (fall), a minimal flux of old organic matter generated maximal depth-integrated EAA rates (62.2 mgC m -2 h -1 ) and lower GYG (12%). From these observations and from the literature, we explore the relationship between the nutritive sources available to deep-sea bacteria, their role in the mineralization of peptide compounds and consequently the implications for the global carbon cycle.

Microbiological and biogeochemical studies were carried out in sediments and near bottom waters in the upwelling area off the Malaga coast and in the Almeria-Oran frontal zone. In these nutrient-rich conditions, metabolic activity is potentially limited by oxygen availability through the sediment depth. In the surficial sediments of the frontal zone, however, oxygen penetrated to a depth of 4 cm, allowing oxic mineralisation processes to occur throughout the layer. In the surficial sediments of the upwelling area, oxygen penetration was limited to the top 2.5 cm, leading to anoxic conditions. Glutamate respiration and global oxygen uptake rates were clearly higher than in the frontal zone. In the superficial sediments of the frontal zone, bacteria were less abundant and showed the lowest potential rate for mineralisation processes, but the highest rate for proteolysis. This discrepancy is probably due to differences in the quality of organic inputs into the two areas, with labile organic compounds reaching the sea bottom in the shallower upwelling zone. Such inputs enhance the mineralisation of low molecular weight monomers, whilst inhibiting the polymer hydrolysis processes. Conversely, in the deeper frontal zone, labile monomers become exhausted, decreasing the mineralisation rates. Concomitantly, bacteria have to develop ectoenzymatic activities in order to extract their carbon and energy from the available semi-labile polymers. Consequently, the theoretical relationship between the mineralisation and hydrolysis processes are tightly coupled in the upwelling area, and uncoupled in the frontal zone., La distribution de facteurs biogéochimiques et de divers processus microbiens a été étudiée dans les eaux proches du fond et les sédiments superficiels dans la zone de l'upwelling de Malaga et au niveau du front Almeria-Oran. Dans ces conditions relativement eutrophes, les activités métaboliques sont limitées par la disponibilité de l'oxygène moléculaire dans l'épaisseur sédimentaire. Toutefois, dans les sédiments de la zone frontale l'oxygène pénètre jusqu'à 4 cm de profondeur, permettant le développement de processus oxiques à travers cette couche. Par contre, dans la zone d'upwelling, où la vitesse potentielle de respiration du glutamate et la consommation globale d'oxygène sont nettement supérieures à celles de la zone frontale, la pénétration de l'oxygène est limitée à 2,5 cm, conduisant à l'anoxie à partir de ce niveau. Dans les sédiments superficiels de la zone frontale, les bactéries sont moins abondantes, et moins actives dans le processus de respiration du glutamate, que leurs homologues de la zone d'upwelling. Par contre, leur potentiel d'hydrolyse des polypeptides est plus développé. Cette divergence est probablement due à la différence de qualité des apports en matières organiques dans les deux zones. Par rapport à la zone frontale, dans la zone d'upwelling moins profonde les apports au domaine benthique incluent une fraction plus labile. Ces apports en matériel labile dopent les processus de minéralisation des monomères, alors que par un effet de retro-inhibition ils ralentissent l'hydrolyse des polymères. À l'opposé, dans la zone frontale plus profonde, le matériel atteignant le domaine benthique a été épuisé de sa fraction labile, ce qui provoque un ralentissement des vitesses de minéralisation des monomères. Simultanément, les bactéries doivent développer des activités ectoenzymatiques pour pouvoir extraire des polymères semi-réfractaires disponibles le carbone nécessaire à leur métabolisme. En conséquence, les relations entre les processus d'hydrolyse et de minéralisation sont couplés dans la zone d'upwelling et découplés dans la zone frontale.

International audience; Kinetic parameters for aminopeptidase, phosphatase, and bacterial production rates were studied during spring and fall through a 2000 m water column in the NW Mediterranean. Bacterial production ranged from 60.4 ng at 30 m to 0.2 ng C l -1 h -1 at 2000 m. For both ectoenzymatic activities, the Km values ranged from 0.44 to 1.13 μM for aminopeptidase activity and from 0.05 to 1.23 μM for phosphatase activity. Depth profiles of the potential activity of aminopeptidase and phosphatase activity drastically decreased below depths of 100 m. At 1000 m, hydrolytic activities were one order of magnitude lower than the maximal rate measured in the surface layer. Despite this decrease, depth-integrated rates through the thickness of different water masses showed that the potential hydrolysis fluxes within the productive surface layer (10-200 m), through the twilight zone (200-1000 m depth), and through the deep water mass (1000-2000 m) were roughly the same order of magnitude. This study used the first assay for measuring ectoenzymatic activities of deep-sea microbial populations where pressure stresses were eliminated during sampling and incubation. The results showed that prokaryotic induced ectoenzymatic activities are affected by pressure conditions. Generally, aminopeptidase and phosphatase rates measured in samples maintained under in situ pressure conditions were 2.3 times higher than those measured in their decompressed counterparts.

Membrane potential changes and discharges from 28 laryngeal motoneurons were recorded intracellularly in the caudal nucleus ambiguus of decerebrate, paralyzed and ventilated cats. Electrical activities were recorded from 17 expiratory laryngeal motoneurons (ELMs) with maximal depolarizing membrane potential in early expiration, and from 11 inspiratory laryngeal motoneurons (ILMs) with maximal depolarizing membrane potential in inspiration. Activities during breathing were compared with those observed during fictive coughing and swallowing evoked by electrical stimulation of the superior laryngeal nerves. These non-respiratory behaviors were evidenced in paralyzed animals by characteristic discharge patterns of the phrenic, abdominal nerves and pharyngeal branch of the vagus nerve. We recorded the activity of 11 ELMs and 5 ILMs during coughing in which ELMs, but not ILMs, exhibited increased membrane depolarization and discharge frequencies. Membrane depolarization and discharge frequencies of all ELMs were also significantly increased during swallowing. In addition, membrane depolarization of most ELMs (15/17) was preceded by a short-lasting hyperpolarization due to chloride-dependent inhibitory mechanisms occurring at the onset of swallowing. Out of 10 ILMs tested during swallowing, 7 exhibited membrane depolarization, preceded in 5 cases by a short-lasting hyperpolarization. Discharge frequencies of ILMs were significantly reduced during swallowing. The same pattern of phasic activities of ILMs and ELMs was observed during coughing and breathing, suggesting the involvement of similar excitatory pathways in both behaviors. These results imply that the duration of activation and the discharge frequency of neurons of the central generator for breathing that drive laryngeal motoneurons are enhanced during coughing. During swallowing, in addition to central excitatory mechanisms, laryngeal motoneurons are subjected to an initial inhibition of unknown origin. This inhibition probably contributes to the temporal organization of the swallowing motor sequence.

Twenty-seven samples of undecompressed seawater were collected between 800 and 2000 m depth in the NW Mediterranean. Most of the samples (24) were collected at the same sampling station. Potential 14 C-glucose uptake rates and potential bacterial production rates were measured concomitantly under natural pressure conditions and under atmospheric pressure conditions. Compression of 5 surficial water samples suggested that metabolic activity of surface-borne bacteria would decrease when carried down by particle sedimentation or by a cascading seawater mass. Conversely, during the stratified water period, intermediate and deep-sea bacteria appeared adapted to the natural pressure conditions; for 90 % of the samples metabolic rates were higher in samples kept in their natural pressure conditions than in their decompressed counterparts. Even if variable with time and depth, a global estimation of this adaptation to the natural pressure conditions resulted in a 3.5-fold increase relative to the measures done on decompressed samples. The pressure effect appeared at 800 m depth, a relatively shallow depth comparative to the average depth of the global ocean. In deep waters, potential glucose uptake rate and potential bacterial production rate varied greatly as a function of time and depth, ranging from 2 to 80 pmol C metabolised l -1 h -1 , and from 4 to over 400 pmol bacterial C produced l -1 h -1 , respectively.

We studied the distribution of bacterial populations and their catabolic activities [I4C-glutamate respirat~on and Leu-MCA (L-leucine-7-amido-4-methyl-coumarin hydrochloride) hydrolysis] in a set of samples collected in the near bottom waters and in the superficial sediments (0 to 41 cm depth) originating from 9 cores collected between 585 and 2065 m depth in diverse NW Mediterranean continental margins. Bacterial densities and respiratlon rates in the sedlment generally exceeded counts and rates in the near bottom water by 4 orders of magnitude. The relative contributions of benthic and pelagic bacteria to the organic matter turnover in this oceanic area are discussed. Data showed that the highest bacterial densities, as well as the highest rates of glutamate mineralizat~on, were usually in the most superficial layers of sediment, whereas proteolysls rates were frequently maxlmal nearly 5 cm deeper. This pattern for proteolysis differs from the decreasing gradient usually described.

Seventy-four bacterial strains were freshly isolated from a mine gallery. Using these bacteria, we have investigated how a molecular identification based on the analysis of small subunit rDNA sequences would compare in terms of precision and reliability to a more classical comparison of phenotypical descriptions (100 morphological and physiological traits). Our data clearly showed that a phylogenetic analysis of small subunit rDNA sequences is more efficient than classical phenotypic methods for the identification of bacterial strains freshly isolated from a natural environment, because occurrences of misidentification are very much decreased by this method. The lack of rDNA sequences for many described species is probably the major cause of a few failures in molecular identification, as the completeness of the database of small subunit rDNA sequences holds much importance in the degree of uncertainty in such identifications.

The distribution of retrogradely doubly labeled brainstem neurons were analyzed in the cat after injection of two different fluorescent markers into the phrenic and abdominal motor nuclei. Diamidino Yellow (DY) was first injected either ipsilaterally or bilaterally into the ventral horn of lumbar spinal cord, and then Fast Blue (FB) into the right ventral horn of cervical spinal cord. Doubly labeled neurons were mainly found in the caudal ventrolateral medulla (retroambiguus region), in the dorsomedial and dorsolateral regions of the nucleus of the tractus solitarius (NTS) and in the raphe nuclei. In addition, doubly labeled neurons were found in the parabrachial and Kölliker-Fuse nuclei. Our results give anatomical evidence that pontine and medullary neurons are the source of a common pathway to both phrenic and abdominal motor nuclei. These neurons might be involved in strain efforts for expulsion such as vomiting or defecation.

In decerebrate, paralyzed, and ventilated cats, we recorded the activity of 100 spontaneously active phrenic motor axons during the increased phrenic discharges characteristic of fictive vomiting (FV) and coughing (FC). During control respiratory cycles, approximately one-half the neurons were recruited in the first decile of inspiration; recruitment continued throughout inspiration. During FV, the duration of phrenic discharge was halved; 20 of 26 motoneurons studied were recruited in the first decile of the burst. During FC, recruitment times did not change compared with control, although the duration of the phrenic burst doubled. Discharge frequencies increased and recruitment order of phrenic motoneurons was virtually unaffected during FC and FV. Limited recruitment of previously inactive neurons in the filaments from which we recorded was found during FV and FC. During FV, 1 previously inactive motoneuron was recruited in 16 filaments containing 25 spontaneously active motor axons. During FC, 3 new motoneurons were recruited in addition to the 64 already active in 35 filaments. Recruitment during FV and FC was absent even when recording from filaments known, on the basis of antidromic activation, to contain inactive motor axons. During FV, 10 of 26 motoneurons began their discharges with doublets (interspike interval < 10 ms); doublets occurred in only 4 of 67 motoneurons during FC. Already active phrenic motoneurons contributed to the intense phrenic activity associated with both respiratory (coughing) and nonrespiratory (vomiting) behavior by increases in discharge frequency, earlier recruitment, and doublets; the contribution of previously quiescent motoneurons remains uncertain.

Ammonium and nitrite oxidizing bacteria were enriched in separate cultures using a primary inoculum of water from an enclosed shrimp rearing facility. Each enrichment was continued by transfer to increasing volumes of medium up to 151. Seventy four strains of heterotrophic bacteria were isolated; seventeen from each primary inoculum, and twenty from each of the 151 enrichments at the time when all the ammonium or nitrite had been utilized. All the isolates, described by 101 characters, were subjected to numerical taxonomy. During the enrichment for ammonium oxidizers, a strong selective pressure led to the disappearance of some strains, mostly enterobacteria. Two taxonomic groups composed of pseudomonads and asporogenous Gram positive rods were associated with the ammonium oxidizers. The population was characterized by an inability to use amino acids as carbon and energy sources. In contrast, the strains associated with nitrite oxidizers were able to use all the carbon and energy sources tested. Many of these strains were Gram positive, asporogenous aerobic rods, others were Gram negative pseudomonads.

In marine sediments, where soluble gases diffuse only very slightly, many organisms struggle for molecular oxygen. Microaerophilic bacteria, able to grow at reduced pO2 between 0.2 and 12%, have an advantage. Distribution of aerobes, microaerophiles and anaerobes was compared with the oxygen gradient in seawater and sediment samples collected in a northern Mediterranean lagoon. In the near bottom seawater and in the 0–10 mm upperlayer of sediment, the microaerophilic counts were less than 1% of aerobe densities. In the 10–15 mm zone, these two groups were equivalent in density (1 × 105 cells ml−1). As expected, the microaerophiles took advantage of their low oxygen tension requirements in the subsurface sediments, between the well aerated zone (0–5 mm depth) and the low redox potential zone. Then, beyond a depth of 20 mm, the anaerobes prevailed in this sandy clay.

In decerebrate, paralyzed and ventilated cats, we monitored the intracellular responses of 30 inspiratory neurons of the dorsal respiratory group (DRG) to stimulation of vagal and expiratory muscle (internal intercostal and abdominal) afferents. We hypothesized that the inhibitory effects of stimulation of expiratory muscle afferents, previously reported, would block the excitatory responses of inspiratory neurons of the DRG to vagal stimulation. Although prolonged stimulus trains to expiratory muscle afferents caused respiratory phase-switching, single shocks or short trains elicited no responses in 17 bulbospinal neurons, excitatory responses in 6, and inhibitory responses in 2. Of the 4 propriobulbar neurons tested, 2 had inhibitory responses and 2 did not respond. In only 2 neurons, both bulbospinal, did conditioning stimuli to expiratory muscle afferents block or reduce the excitatory effects of vagal stimulation. These results suggest that interaction of vagal and expiratory muscle afferents, which might account for the absence of a change in inspiratory duration despite increased vagal afferent feedback at elevated end-expiratory lung volumes, does not occur within the DRG.

International audience; During the Aegean Sea component of the EU MTP-MATER project, benthic samples were acquired along a depth gradient from two continental margins in the Aegean Sea. Sampling was undertaken during spring and summer 1997 and the microbial metabolic activities measured (Vmax for aminopeptidase activity, 14C-glutamate respiration and assimilation) displayed seasonal variability even in deep-sea conditions. The metabolic rates encountered in the North Aegean (average depth 566±234 m), were approximately five-fold higher than in the deeper (1336±140 m) Southern part of the Aegean. The aminopeptidase rates, however, were the exception with higher values recorded in the more oligotrophic sediments of the Southern stations (1383±152 vs. 766±297 nmol MCA cm-2 h-1). A discrepancy in bacterial metabolism also appeared in the near bottom waters. In the Southern stations, 80% of the glutamate uptake was used for energy yielding processes and only 20% devoted to biomass production, while in the North Aegean, most of the used glutamate was incorporated into bacterial cells. During the early burial stages, bacterial mineralization rates estimated from 14C-glutamate respiration decreased drastically compared to the rates of biopolymer hydrolysis estimated by aminopeptidase assays. Thus, at the 2-cm depth layer, these rates were only 32 and up to 77% of the corresponding average values, respectively, in the superficial layer. Such a discrepancy between the evolution of these two metabolic activities is possibly due to the rapid removal of readily utilizable monomers in the surface deposits. The correlation between bacterial respiration and total organic carbon, or total organic nitrogen, is higher in the surficial sediment (0-2 and 2-4 cm) than in the underlying layer. Conversely, it is only at 4-cm depth layer that the hydrolysis rates appear correlated with organic carbon and nitrogen concentrations. This pattern confirms the drastic degradation of organic matter during the early burial stages.

In this study, we used the fact that bacteria are able to cleave a fluorogenic substrate analog ( l -leucine-7-amido-4-methylcoumarin) to determine the maximal ectoproteolytic activities ( V m ) and affinities ( K m ) of natural benthic microbial communities by the multiconcentration kinetic method. This investigation was performed during the winter and summer of 1997 with a set of 36 samples of near-bottom water and sediment collected from a coastal area and an offshore area in the western part of the Gulf of Lions. The existence of biphasic microbial ectoproteolysis was statistically confirmed for both the near-bottom water and the sediment, regardless of the spatial and seasonal conditions. Globally, 72.2% of the entire set of bacterial consortia collected at the water-sediment boundary layer showed biphasic microbial kinetics. A specific estimator of the biphasicity indicated that deep benthic bacterial consortia responded better with episodic nutrient supplies than shallower benthic bacterial consortia responded.

We used the expression of the immediate-early gene c-fos, a marker of neuronal activation, to localize brainstem neuronal populations functionally related to fictive cough (FC). In decerebrate, paralyzed, and ventilated cats, the level of Fos-like immunoreactivity (FLI) was examined in five groups of animals: (1) controls, sham-operated unstimulated animals; (2) coughing cats, including both animals in which FC was elicited by unilateral electrical stimulation of the superior laryngeal nerve (SLN) and (3) those in which FC was elicited by bilateral SLN stimulation; (4) stimulated–treated cats, in which bilateral SLN stimulation was applied after selective blockade of FC by codeine; and (5) codeine controls, sham-operated unstimulated cats subjected to administration of codeine. Fifteen brainstem structures were compared for numbers of labeled cells. Because codeine selectively blocks FC, brainstem nuclei activated specifically during FC were identified as regions showing increased FLI after FC and significant reductions in FLI after FC suppression by codeine in stimulated–treated cats. In coughing animals, we observed a selective immunoreactivity in the interstitial and ventrolateral subdivisions of the nucleus of the tractus solitarius, the medial part of the lateral tegmental field, the internal division of the lateral reticular nucleus, the nucleus retroambiguus, the para-ambigual region, the retrofacial nucleus, and the medial parabrachial nucleus. FLI in all these nuclei was significantly reduced in stimulated–treated cats. Our results are consistent with the involvement of neurons overlapping the main brainstem respiratory-related regions as well as the lateral tegmental field and the lateral reticular nucleus in the neural processing of laryngeal-induced FC.

Membrane potential changes and/or discharges from 36 inspiratory neurons were recorded intracellularly in the dorsal respiratory group (DRG; i.e., the ventrolateral subdivision of the nucleus tractus solitarii) in decerebrate, paralyzed, and ventilated cats. Electrical activities were recorded from both somata (n = 10) and axons (n = 26). Activities during quiet breathing were compared with those observed during fictive coughing and swallowing evoked by repetitive electrical stimulation of afferent fibers of the superior laryngeal nerve (SLN). These nonrespiratory behaviors were evident in paralyzed animals as characteristic discharge patterns of the phrenic, abdominal, and hypoglossal nerves. Twenty-six neurons exhibiting antidromic action potentials in response to electrical stimuli applied to the cervical (C3-5) spinal cord were classified as inspiratory bulbospinal neurons (IBSNs). These neurons were considered as premotoneurons. The remaining 10 inspiratory neurons (I-NAA) were not antidromically activated by electrical stimuli applied to either cervical spinal cord or ipsilateral cervical vagus. These neurons are thought to be propriobulbar neurons. We recorded the activity of 31 DRG inspiratory neurons (24 IBSNs and 7 I-NAA) during coughing. All but one (a late-recruited IBSN) discharged a burst of action potentials during the coughing-related phrenic nerve activity. Typically, ramp-like membrane depolarization trajectories and discharge frequencies during coughing were similar to those observed during inspiration. We recorded the activity of 33 DRG inspiratory neurons (23 IBSNs and 10 I-NAA) during swallowing. Most (28/33) neurons were briefly activated, i.e., discharged a burst of action potentials during swallowing, but peak discharge frequency decreased compared with that measured during inspiration. The membrane potentials of nine somata exhibited a brief bell-shaped depolarization during swallowing, the amplitude of which was similar to that observed during inspiration. These results suggest that some inspiratory premotoneurons and propriobulbar neurons of the DRG might be involved in nonrespiratory motor activities, even if clearly antagonistic to breathing (e.g., swallowing). We postulate the existence in the medulla oblongata of adult mammals of neurons exhibiting a "functional flexibility".

Intracellular recordings and labelings with horseradish peroxidase (HRP) of expiratory (E) neurons were performed in decerebrate, paralyzed, and ventilated rats. A total of 37 neurons were recorded, from which 4 cells and 1 axon were labeled. They were located in two regions of the ventrolateral medulla. One was in the rostral portion of the nucleus ambiguus just caudal to the facial nucleus, and the other in the nucleus retroambiguus at the level of the caudal medulla. These expiratory neurons had rhythmical changes in membrane potential similar to those reported in cat, i.e., a depolarization in the intervals between phrenic bursts which evolved in an augmenting (E-aug, n = 15), or bell-shaped or ‘plateau’ (E-all, n = 22) pattern until a rapid hperpolarization at the start of inspiration. Both types were hyperpolarized during inspiration by chloride-dependent, inhibitory postsynaptic potenials (IPSPs) which were demonstrated in 17 neurons (10 E-aug and 7 E-all) from which reversal was obtained. Such IPSPs also existed during post-inspiration (stage I of expiration) in 4 of the 10 augmenting E neurons. They were identified by antidromic stimulation or HRP labeling, or both, as bulbospinal neurons (n = 2), cranial motoneurons (n = 4), or not antidromically activated (NAA) neurons (n = 31). All the identified bulbospinal neurons and the motoneurons bulbospinal neuron with ipsilateral axon giving off intramedullary collaterals, and NAA neurons with rostral medullary projections or with axons crossing the midline. These results indicate that the distribution and the electrophysiological properties of the expiratory neurons in the rat medulla are similar to those described in the cat.

Intracellular recordings and labelings with horseradish peroxidase (HRP) of inspiratory neurons were performed in decerebrate, paralyzed and ventilated rats. A total of 58 neurons were located within the ventrolateral medulla. They were identified as bulbospinal neurons (n = 15), cranial motoneurons (n = 9) and not antidromically activated (NAA) neurons (n = 34) by antidromic stimulation or HRP labeling, or both. These inspiratory neurons had rhythmical changes in membrane potentials similar to those reported in cats, i.e. an abrupt depolarization at the onset of phrenic discharge followed by trajectories of depolarization which evolved into augmenting I, bell-shaped I or decrementing I patterns until a rapid repolarization at the start of expiration. All types were hyperpolarized during expiration by chloride-dependent inhibitory postsynaptic potentials (IPSPs) which were demonstrated in 13 neurons from which the reversal was obtained. Such IPSPs were apparent in two waves throughout expiration, an early one in post-inspiration (stage I of expiration) and a late one in late expiration (stage II of expiration). These properties are also similar to those of feline inspiratory medullary neurons. Four labeled bulbospinal neurons had axonal collaterals which were ipsi- and contralateral to the site of their somata. Two of 6 labeled NAA neurons exhibited profuse axonal arborizations within various medullary nuclei. No medullary axonal collateral was seen from 6 labeled motoneurons. These results indicate that even though in the rat a single concentration of inspiratory neurons within the ventrolateral medulla has been demonstrated, there is no fundamental difference in the organization of the inspiratory neuronal network compared to that of the cat.

The topographical neuronal distribution within the rat nucleus ambiguus has been studied with the simultaneous retrograde labeling technique by means of four different fluorochromes injected within the various muscles and/or nerves of the oro-pharyngeal region. This technique has permitted the identification of several types of neurons along the same coronal plane. Most were motoneurons innervating the various muscles of the upper airway, including pharyngeal constrictor, stylopharyngeal, intrinsic laryngeal and the upper portion of the esophagus. Some neurons may have been preganglionic parasympathetic neurons. No evidence of axonal branching of any of the labeled motoneurons or parasympathetic neurons was found.

The location of neurons within the ventral respiratory group (VRG) of rat was mapped following injections of 3 different fluorochrome tracers into different sites known to receive projections from VRG neurons. Injection sites included muscles innervated by the vagus (X) and glossopharyngeal (IX) nerves, and the sites of expiratory activity in the caudal medulla and of inspiratory activity in the spinal cord at the C4 level. Labeling of vagal motoneurons resulting from fluorochrome injections into muscles innervated by X and IX nerves was always ipsilateral to the site of injection. Both propriobulbar and bulbospinal neurons had primarily ipsilateral projections. No double-labeled cell bodies were observed. The cell bodies of the 3 types of neurons, propriobulbar, bulbospinal and vagal/glossopharyngeal, were unevenly distributed along the rostrocaudal axis of the VRG, suggesting a complex mosaic of neurons which regulate respiratory-related functions such as swallowing and vocalization.

We examined the membrane potentials of 63 respiratory neurons in the ventrolateral medulla of decerebrate rats, whose trajectories had the characteristics of the post-inspiratory neurons, i.e. exhibiting hyperpolarization during inspiration, rapid depolarization at end-inspiration and progressive repolarization with a decrementing pattern during the intervals between phrenic bursts. Synaptic responses of 6 post-inspiratory neurons which were tested by stimulation of cervical vagus or superior laryngeal nerves were excitatory. Eleven of these 63 post-inspiratory neurons were labeled by intracellular injection of horseradish peroxidase (HRP). Ten of these 11 labeled neurons were motoneurons since their axons exited the medulla after joining the roots of cranial nerves. However, only one of these motoneurons was antidromically activated by stimulation of the ipsilateral cervical vagus nerve. We assumed that most of the post-inspiratory medullary neurons of the present study were motoneurons, but not interneurons, although antidromic invasion was not possible after stimulation of the cervical vagus and superior laryngeal nerves. Two post-inspiratory neurons of this sample had bulbospinal axons, which were revealed by antidromical activation of spinal cord and HRP labeling, respectively. The axon of the labeled bulbospinal neuron had axonal collaterals which were distributed within the region of the nucleus ambiguus of the ipsilateral medulla. The functional significance of this type of post-inspiratory neuron is discussed.

We analyzed the membrane potential of 161 respiratory neurons in the medulla of decerebrate rats which were paralyzed and ventilated. Three types of inspiratory (I) neurons were observed: those displaying progressive depolarization in inspiration (augmenting I neurons), those which gradually repolarized after maximal depolarization at the onset of inspiration (decrementing I neurons) and those exhibiting a plateau or bell-shaped membrane potential trajectory throughout inspiration (I-all neurons). Three types of expiratory (E) neurons were also encountered: those in which the membrane potential progressively depolarized (augmenting E neurons), those in which the membrane potential repolarized during the interval between phrenic bursts (decrementing E or post-I neurons) and those exhibiting a plateau or bell-shaped membrane potential trajectory throughout expiration (E-all neurons). Axonal projections of these medullary neurons were identified in the cranial nerves (n = 34), or in the spinal cord (n = 19) as revealed by antidromic stimulation and/or by reconstruction following horseradish peroxidase (HRP) labeling. The other 108 neurons were not antidromically activated (NAA) by the stimulations tested, or had their axons terminating inside the medulla as revealed by HRP labeling. All these respiratory neurons, except for 3 which were hypoglossal motoneurons, had their somata within the ventrolateral medulla, in the region of the nucleus ambiguus, homologous to the ventral respiratory group (VRG) of the cat. No dorsal respiratory group (DRG) was detected within the medulla of the rats. Due to this absence of a DRG, it is concluded that the neural organization of respiratory centers is quite different in cats and rats.

Cancer therapy with cytotoxic drugs such as cisplatin or cyclophosphamide is usually associated with violent crisis of vomiting. Recently, it was shown that 5-HT 3 receptor antagonists block cisplatin-induced vomiting but the mechanisms and their sites of action remain unknown. We tested the hypothesis that these agents act on structures within the central nervous system by evaluating the effectiveness of vagal stimulation in eliciting fictive vomiting in decerebrate, paralyzed and ventilated cats before and after administration of such agents. Fictive vomiting was defined as a series of large bursts of synchronous activity in the phrenic and abdominal (expiratory) nerves (retching) followed by a burst in which the abdominal activity was prolonged (expulsion). The latency and number of these co-activations were measured before and after intravenous administration of three 5-HT 3 receptor antagonists (GR 38032F (Ondansetron), Zacopride, and BRL 43694A (Granisetron)). All compounds, administered at doses of 1 and 2 mg/kg failed to block vomiting behaviour in 100% and 68% of trials, respectively. Nor did their administration affect the latency and number of co-activations. We conclude that intravenous administration of 5-HT 3 receptor antagonists do not act centrally on either the brainstem neuronal network known as the “vomiting center” or related neuronal structures. Our results suggest that the anti-emetic effect of 5-HT 3 receptor antagonists in cisplatin-induced vomiting is mediated peripherally rather than centrally.

Activities of respiratory laryngeal and oropharyngeal respiratory nerves were studied during fictive vomiting elicited by supradiaphragmatic vagus nerve stimulation in the decerebrate cat. Inspiratory laryngeal nerves were strongly inhibited throughout the retching and expulsion phase. Glossopharyngeal, hypoglossal and expiratory laryngeal nerves were coactivated with the phrenic and abdominal nerve bursts. The pharyngeal branch of the vagus nerve discharged during the phrenic and abdominal inter-burst of the retching phase, and was silent during the abdominal expulsion. These activities permit speculation about the role of upper airway muscles during vomiting.

In decerebrate cats, stable intracellular recordings were made from 37 laryngeal motoneurones, the membrane potentials of which varied in relation to respiration. These motoneurones were identified as laryngeal since all were antidromically activated by stimulation of the recurrent laryngeal nerve, but in two, the antidromic activity could only be elicited by vagal stimulation (vagotomized cats). The cell bodies were all located within the nucleus ambiguus. Sixteen cells were depolarized during the phrenic burst and were classified as inspiratory laryngeal motoneurones (ILM). Thet repolarized at end-inspiration and received two successive waves of postsynaptic inhibition during expiration: an early, strong one and a late (end-expiratory), weaker one. The decay of the first wave was related to the duration of postinspiratory phrenic activity. Twenty-one cells depolarized abruptly in early expiration followed by a more-or-less gradual repolarization. They were classified as expiratory laryngeal motoneurones (ELM). All ELM were strongly inhibited during inspiration. Some of them received weak inhibition during end expiratory phase. The rapid and large depolarization observed during early expiration (and consequent maximal discharge frequency) can be explained by two summating mechanisms: a postinhibitory rebound resulting from the removal of inhibition during inspiration, and an excitatory phenomenon of unknown origin. The amplitude of this excitatory phenomenon was largest in cats with the most residual (early expiratory) phrenic activity. To explain the hyperpolarizations occurring in ELM during late expiratory and inspiratory phases and those occurring in ILM during early expiration, we hypothesize that reciprocal inhibition exists between networks controlling ILM and ELM activities or between these motoneurones themselves. The cause of the relationship between residual phrenic nerve activity and the events in early expiration in both ILM (hyperpolarization) and ELM (depolarization) remains to be elucidated. Our observations are consistent with the braking effect on expiratory flow in postinspiration caused by the activation of the adductor muscles driven by the expiratory laryngeal motoneurones, and the release of the abductor muscles driven by the inspiratory laryngeal motoneurones.

We have studied in cats the discharge pattern in response to lung inflation and deflation of 283 medullary respiratory neurones, 173 being inspiratory and 110 expiratory. The ventral respiratory nucleus, near the nucleus ambiguus, was particularly investigated. The neurones were classified into bulbo-spinal neurones, laryngeal motoneurones and propriobulbar neurones by antidromic invasion from the spinal cord or the vagus nerve (collision test). The bulbo-spinal neurones responded in the same direction as spinal motoneurones in the Hering-Breuer reflexes: depression of inspiratory neurones and facilitation of expiratory neurones by inflation of the lungs. All the expiratory laryngeal motoneurones and some inspiratory laryngeal motoneurons responded in the opposite direction to the Hering-Breuer reflexes: depression of expiratory motoneurones and facilitation of inspiratory motoneurones. The function of propriobular neurones could be inferred from their response to Hering-Breuer reflexes: one group of propriobulbar neurones responded in such a manner as to be linked to the activity of the bulbo-spinal neurones; the other group responded in accordance with the activity of the laryngeal motoneurones.

Agar-mixed cultures in semi-solid media provide a range of oxygen concentrations which fulfill microaerophilic bacterial requirements. Such conditions are more suitable than those provided by liquid media since, in broth cultures, the oxygen concentration must be determined at a level which might later show a lack of or excess of sensitivity to microaerophilic bacteria, thereby giving an erroneous count. A high content of organic substrates appears to be disadvantageous. It can prevent enumeration of microaerophilic growing rings or even induce false results. Minielectrodes permit the control of the oxygen concentration in each growing ring so as to confirm the efficiency of the culture method used for a selective count of microaerophilic bacteria. Semi-solid seawater culture medium supplemented with calcium succinate at 0.5 gram per litre appears to be an adequate culture medium for counting microaerophilic bacteria in marine sediments.

In anesthetized cats, electrical stimulation of the cerebral cortex and subcortical structures with short trains could induce onset or termination of activity of the phrenic nerve. Onset of phrenic discharge was obtained following stimulation of the primary sensory areas of the cerebral cortex, the periaqueductal gray, the ponto-mesencephalic reticular formation, the parabrachial nucleus, the vestibular nucleus, and the fastigial nucleus. When these structures were stimulated by repetitive trains at frequencies of 1–5 Hz, the respiratory rhythm was entrained to these same frequencies. The phrenic discharge during these repetitive stimulations exhibited a decrementing pattern similar to that during thermal polypnea. Termination of phrenic activity was obtained upon stimulation of the motor and visceral cortices, the internal capsule, the pyramidal tract, the red nucleus, the rubrospinal tract and the motor nucleus of the trigeminal nerve. Stimulation of these latter structures by repetitive trains resulted in a temporary or permanent arrest of phrenic discharge. These results are discussed in the context of the possible roles of medullary respiratory neurons in the onset or termination of inspiratory activity.

Intracellular recordings of the activities of 10 inspiratory bulbospinal neurones of the medulla were performed in decerebrate cats. Fictive vomiting was induced by repetitive stimulation of the supra-diaphragmatic vagus nerves and was defined by series of synchronous large bursts in both the phrenic (inspiratory) and abdominal (expiratory) nerves. During these synchronous bursts the inspiratory bulbospinal neurones of both the dorsal and ventral respiratory groups were strongly hyperpolarized by chloride-dependent inhibitory postsynaptic potentials (IPSPs). We concluded that during vomiting the central pattern generator is inhibited, and that another pattern generator drives the spinal respiratory motoneurones.

Intracellular recordings of the activities of 16 bulbar expiratory neurones of the rostral medulla were performed in decerebrate cats. Seven of these identified neurones were intracellularly injected with horseradish peroxidase for morphological examination. We observed 3 categories of expiratory neurones including two pharyngeal motoneurones of the retrofacial nucleus, one with an augmenting, the other with a decrementing discharge pattern. Augmenting patterns were also observed in neurones ventromedial to the retrofacial nucleus and in another located 320 μm from the ventral surface of medulla. Their possible functions are discussed in relation to their anatomical location and morphology.

The central distributions of efferent and afferent components of the pharyngeal branches of the vagus (PH-X) and glossopharyngeal (PH-IX) nerves in the cat were studied by soaking their central cut ends in a horseradish peroxidase (HRP) solution. HRP-labelled PH-X neurones were distributed ipsilaterally in the rostral part of the nucleus ambiguus (NA) and the retrofacial nucleus (RFN); HRP-labelled PH-IX neurones were found in the ipsilateral RFN and the bulbopontine lateral reticular formation (RF). Vagal pharyngeal neurones constituted a large population of brainstem motoneurones. The population of HRP-labelled glossopharyngeal neurones was divided into two components. Indeed, on the basis of their location and somal morphology, the most ventral cells were identified as cranial motoneurones and those scattered in the lateral RF as parasympathetic preganglionic neurones. Application of HRP to the PH-IX nerve resulted also in the labelling of fibres and terminals in the alaminar spinal trigeminal nucleus and the nucleus of the solitary tract (NTS). The afferent fibres entered the lateral medulla with the glossopharyngeal roots, ran dorsomedially, then turned caudally toward the NTS and the caudal part of the alaminar spinal trigeminal motor (V) nucleus. In the NTS, labelled fibres ran mainly along the solitary tract, projecting to terminals in the dorsal and dorsolateral nuclei of the NTS.

In anesthetized or decerebrated cats, extracellular activities of pairs of respiratory neurons located in the regions of the dorsal (DRN), ventral (VRN) and retrofacial (RFN) medullary respiratory nuclei were recorded using two separate microelectrodes. Neurons were classified as bulbospinal or laryngeal if stimulation of the spinal cord or vagus nerve elicited antidromic action potentials, or as propriobulbar if they were not antidromically activated. Of 163 pairs of single unit activities, either inspiratory (143 pairs) or expiratory (20 pairs), cross-correlation analyses indicated that 23% had short latency peaks, either broad (12%) or sharp (1%) in their cross-correlograms, 3% had short latency troughs and 74% had flat cross-correlograms. When the two neurons were located in the DRN (68 pairs) the probability of obtaining a positive cross-correlogram was high for inspiratory bulbospinal neurons, indicating shared inputs and excitatory relationships. When one neuron of the pair was located in the RFN and the other in either the DRN or VRN (95 pairs), cross-correlation analysis revealed shared inputs, excitatory and inhibitory relationships. Among expiratory neurons interactions were only inhibitory with a more frequent incidence (3/20) than between inspiratory neurons (2/143). Our results indicate that: short time scale synchrony due to shared inputs (broad peaks) are largely distributed in the respiratory neuronal network and operate over long distance (i.e. RFN, caudal medulla); excitatory coupling may exist between remote neurons but is more frequent between inspiratory bulbospinal neurons located in the DRN.

1. We recorded the membrane potentials of sixty-three respiratory neurones in the rostral, ventral medulla of decerebrate vagotomized cats. Stable recordings were obtained in thirty-eight expiratory and twenty-five inspiratory neurones. Axonal projections were identified by antidromic invasion after electrical stimulation of the region of the dorsal respiratory group (DRG), spinal cord, and the cervical vagus, superior laryngeal and pharyngeal nerves. 2. Two types of expiratory neurones were encountered: those in which the membrane potential progressively depolarized (augmenting neurons, n = 22) and those in which the membrane potential repolarized (decrementing or post-inspiratory neurones, n = 16) during the interval between phrenic bursts. Both types were hyperpolarized during inspiration by chloride-dependent, inhibitory postsynaptic potentials (IPSPs) which decreased membrane resistance. In augmenting neurones two waves of IPSPs appeared, one early and one late in inspiration. 3. Five out of seventeen augmenting expiratory neurones tested were antidromically activated by contralateral stimulation of the spinal cord (n = 3) or the DRG (n = 2). Spinal axons were not detected in any of the sixteen decrementing expiratory neurones tested. Of thirteen expiratory neurones tested with pharyngeal nerve stimulation, one (an augmenting type) was antidromically activated. Superior laryngeal or vagal axons could not be demonstrated for any expiratory neurones. 4. Two types of inspiratory neurones were also encountered: those displaying progressive depolarization throughout inspiration (n = 5) and those which gradually repolarized after maximal depolarization at the onset of inspiration (n = 10). None of the former had identifiable spinal or medullary axons, but superior laryngeal axons were demonstrated in three and pharyngeal axons were found in three. None of the latter was antidromically activated from any of the sites stimulated. 5. Stimulation of the superior laryngeal or pharyngeal nerves evoked excitatory postsynaptic potentials (EPSPs) in all neurones except in post-inspiratory neurones. In these, stimulation of the superior laryngeal or pharyngeal nerves evoked IPSPs in five of twelve neurones tested. 6. We conclude that a spectrum of respiratory neurones lie within or ventral to the retrofacial nucleus. These neurones may control upper-airway muscles or may play a role in chemoreception.

In decerebrate, paralyzed and artificially ventilated cats, we recorded the discharge of 64 motor axons supplying the pharyngeal muscles. Filaments containing motor axons, with discharges related to the respiratory cycle (phrenic nerve activity), were teased from the pharyngeal branches of the vagus and glossopharyngeal nerves. Most units (n = 41) fired only during expiration and exhibited a steady, a decreasing or a late augmenting discharge pattern. These units were found only in vagal filaments. Twenty three units discharged during inspiration and exhibited a steady, a late augmenting or a tonic discharge pattern. The inspiratory-related units were present in both the vagus (n = 13) and glossopharyngeal (n = 10) nerves. Nineteen of 20 pharyngeal inspiratory-related units tested were activated at short latency (range 3.4 to 8.0 ms) by stimulation of afferents in the superior laryngeal nerve (SLN). In 13 of these, such stimulation also suppressed their spontaneous activity, SLN stimulation elicited in all 17 pharyngeal expiratory-related units tested a short latency (range 0 to 8 ms) reduction of activity, followed in 7 units by an increase in activity. SLN stimulation occasionally evoked single or rhythmic multifibre bursts in the vagal pharyngeal filaments. These bursts, involving expiratory-related units, likely correspond to the buccopharyngeal stage of swallowing.

The effects of inhibitory (γ-aminobutyric acid (GABA) and glycine) and excitatory ( l -glutamate and dl -homocysteate, DLH) amino acids on the excitability of respiratory bulbospinal neurons were studied in decerebrate, paralyzed, bilaterally vagotomized, artificially ventilated cats. Unit activities were recorded extracellularly in the medulla in both the ventrolateral portion of the nucleus tractus solitarius and the para-ambigual region in the vicinity of the nucleus ambiguus (dorsal and ventral respiratory groups, respectively). All neurons were bulbospinal since they could be antidromically activated by electrical stimuli to the spinal cord. We used variations in antidromic latency (ADL) as a measure of changes in excitability of the soma. All neurons exhibited variations in ADL related to the respiratory cycle, being shortest (minimum ADL) during neural activity and longest (maximum ADL) in the silent period. Neurons whose discharge frequencies fell during application of putative inhibitory amino acids showed an increase of minimum ADL compared to control, indicating hyperpolarization. Minimum ADL, in some cells, became shorter during application of excitatory amino acids, indicating depolarization; in others, mechanisms secondary to increased neuronal firing likely obscured their effects. The transient maximum ADL usually present at the onset of the silent period was increased by excitatory amino acids and, in some units, was reduced or eliminated by inhibitory amino acids. These effects are discussed in terms of a modulation by synaptic inputs and neurotransmitters of the cumulative afterhyperpolarization which follows bursts of action potentials.

Activities of respiratory laryngeal motoneurones were recorded intracellularly in the nucleus ambiguus of the cat. Some of them were intracellularly injected with peroxidase for morphological reconstruction. Stimulation of the superior laryngeal nerve (SLN) evoked excitatory responses in both somata and axons of expiratory laryngeal motoneurones. In inspiratory laryngeal motoneurones, the responses induced by the SLN depended on the site of recording: inhibition and decrease of excitatory input in somata, excitation in axons. We conclude that excitatory synaptic effects of SLN stimulation acts mainly on, or close to, the initial segment of inspiratory motoneurones, while inhibition reaches the somato-dendritic region.

The influence of halothane anesthesia on respiratory neurons of the region of the retrofacial nucleus were studied in decerebrate, bivagotomized, paralyzed and artificially ventilated cats. Compared with the control situation without anesthesia, the infusion of halothane induced an important decline of spike activity of inspiratory neurons, at times abolishing their activity, but had no significant effect on the expiratory neurons. The functional significance of these findings is briefly discussed.

The presence of bacteria in a deep clay sediment was analyzed in a 20-m-long core horizontally drilled from a mine gallery at a depth of 224 m in the Boom clay formation (Mol, Belgium). This clay deposit is the result of a marine sedimentary process that occurred 35 million years ago. Bacterial activities were estimated by measuring respiration on [14C]glucose. Using the same samples, universal primers for the genes coding for eubacterial 16S rRNA were used to amplify extracted DNA. PCR products were then cloned, sequenced, and analyzed by molecular phylogeny. Our data showed a decrease in bacterial densities as a function of distance from the gallery, with few bacteria detectable by culture at more than 80 cm from the gallery wall. PCR experiments showed the presence of bacteria in all samples, and phylogenetic analyses were then used to tentatively identify these organisms. Because of low bacterial densities in deep clay samples, direct counts and enumeration of viable bacteria on diverse culture media remained negative. All experiments, both cultures and PCR, demonstrated the difficulty of analyzing samples that contain only a few poorly active bacteria as it is difficult to avoid a small contamination by active bacteria during sampling. Since the porosity of the Boom clay formation is less than the expected size of bacteria, it is possible that some of the bacteria present in this 35-million-year-old deep clay deposit derive from cells initially trapped during the sedimentation process.