Determinants of functional coupling between astrocytes and respiratory neurons in the pre-Bötzinger complex

Respiratory neuronal network activity is thought to require efficient functioning of astrocytes. Here, we analyzed neuron-astrocyte communication in the pre-Bötzinger Complex (preBötC) of rhythmic slice preparations from neonatal mice. In astrocytes that exhibited rhythmic potassium fluxes and glutamate transporter currents, we did not find a translation of respiratory neuronal activity into phase-locked astroglial calcium signals. In up to 20% of astrocytes, 2-photon calcium imaging revealed spontaneous calcium fluctuations, although with no correlation to neuronal activity. Calcium signals could be elicited in preBötC astrocytes by metabotropic glutamate receptor activation or after inhibition of glial glutamate uptake. In the latter case, astrocyte calcium elevation preceded a surge of respiratory neuron discharge activity followed by network failure. We conclude that astrocytes do not exhibit respiratory-rhythmic calcium fluctuations when they are able to prevent synaptic glutamate accumulation. Calcium signaling is, however, observed when glutamate transport processes in astrocytes are suppressed or neuronal discharge activity is excessive.     

Raphe modulation of the pre-Bötzinger complex respiratory bursts in in vitro medullary half-slice preparations of neonatal mice

Spontaneous respiratory bursts which begin in the pre-Bötzinger complex were recorded from the hypoglossal (XIIth) nerve rootlets of in vitro slices prepared from newborn mice. First, we examined the respiratory bursts before and after a midline or para-midline transection which spared the caudal raphe nuclei: the raphe obscurus and raphe pallidus. After a midline transection, the respiratory bursts in both half-slices were desynchronized and had slightly decreased amplitudes and frequencies. After a para-midline transection, the bursts continued with similar frequencies in the half slice containing the raphe obscurus and raphe pallidus. Second, to analyze the effects of modulation by the raphe obscurus and raphe pallidus, a dorsal or ventral midline lesion was used to damage either the raphe obscurus or raphe pallidus. After a dorsal lesion, the synchronized respiratory bursts persisted with slightly decreased frequencies. In contrast, after a ventral lesion, the bursts were almost completely abolished, but recovered significantly after the addition of 5-HT. The present results demonstrated that the pre-Bötzinger complex on each side of the medulla can independently generate rhythmic respiratory activity. It is suggested that the 5-HT released from the ventral part of the raphe nuclei (predominantly the raphe pallidus) plays a critical role in sustaining rhythmic respiratory bursts.     

Neuroanatomical and functional organization of the pre-Bötzinger complex in the cat

The technique of horseradish peroxidase retrograde axonal transport and local electrical stimulation of the pre-Botzinger complex was used to study the connections between neurones of the bulbar respiratory nucleus and descending pathways from bulbar nuclei in the cat spinal cord. A possible role of the nuclei under study for rhythmogenesis of breathing and respiratory control, is discussed.     

Respiratory reactions on microinjections of GABA and baclofen into the Bötzinger complex and the pre-Bötzinger complex in rats

In adult anaesthetized rats the respiratory reactions to microinjections of GABA (10(-5) M) and baclofen (10(-6) M) into Botzinger complex (BC) and pre-Botzinger complex (PBC) were investigated. It was shown, that GABA microinjections into BC shortened inspiratory time and extended expiratory time while respiratory rate was not changed essentially, under this conditions the tidal volume and ventilation were increased. GABA microinjections into PBC significantly inhibited respiratory rhythm due to inspiratory and expiratory time prolongations and reduced tidal volume. The microinjections of baclofen into BC reduced expiration time and ventilation, and increased respiratory frequency whereas microinjections into PBC increased tidal volume without respiratory rate and expiratory time changes. It is suggested that the reactions observed demonstrate the various contribution of GABAergic mechanisms, including GABA(B)-receptors within BC and PBC, in control of respiratory pattern parameters.     

Dynamics of in-phase and anti-phase bursting in the coupled pre-Bötzinger complex cells

Activity of neurons in the pre-Bötzinger complex within the mammalian brain stem has an important role in the generation of respiratory rhythms. Previous experimental results have shown that the dynamics of sodium and calcium within each cell may be responsible for various bursting mechanisms. In this paper, we study the bursting dynamics of the two-coupled pre-Bötzinger complex neurons. Using a combination of fast-slow decomposition and two-parameter bifurcation analysis, we explore the possible forms of dynamics that the model network can produce as well the transitions of in-phase and anti-phase bursting respectively.     

Computational study on neuronal activities arising in the pre-Bötzinger complex

Experimental investigations have shown that the pre-Bötzinger complex (pre-BötC) within the mammalian brainstem generates the inspiratory phase of respiratory rhythm. Based on a single-compartment model of a pre-BötC inspiratory neuron, we, in this paper, use semi-analytical, numerical as well as fast-slow dynamical methods to investigate the effects of sodium conductance (\(g_{\text{Na}}\)) and potassium conductance (\(g_{{\text{K}}}\)) on the firing activities of pre-BötC and try to reveal the dynamical mechanisms behind them. We show how \(g_{{\text{Na}}}\) and \(g_{\text{K}}\) affect the bifurcations of the fast-subsystem and how the the firing patterns of pre-BötC transit according to the bifurcations.     

Modeling the effects of extracellular potassium on bursting properties in pre-Bötzinger complex neurons

There are many types of neurons that intrinsically generate rhythmic bursting activity, even when isolated, and these neurons underlie several specific motor behaviors. Rhythmic neurons that drive the inspiratory phase of respiration are located in the medullary pre-Bötzinger Complex (pre-BötC). However, it is not known if their rhythmic bursting is the result of intrinsic mechanisms or synaptic interactions. In many cases, for bursting to occur, the excitability of these neurons needs to be elevated. This excitation is provided in vitro (e.g. in slices), by increasing extracellular potassium concentration (K _out ) well beyond physiologic levels. Elevated K _out shifts the reversal potentials for all potassium currents including the potassium component of leakage to higher values. However, how an increase in K _out , and the resultant changes in potassium currents, induce bursting activity, have yet to be established. Moreover, it is not known if the endogenous bursting induced in vitro is representative of neural behavior in vivo. Our modeling study examines the interplay between K _out , excitability, and selected currents, as they relate to endogenous rhythmic bursting. Starting with a Hodgkin-Huxley formalization of a pre-BötC neuron, a potassium ion component was incorporated into the leakage current, and model behaviors were investigated at varying concentrations of K _out . Our simulations show that endogenous bursting activity, evoked in vitro by elevation of K _out , is the result of a specific relationship between the leakage and voltage-dependent, delayed rectifier potassium currents, which may not be observed at physiological levels of extracellular potassium.     

CO(2)/H(+) chemoreception in the cat pre-B?tzinger complex in vivo.

We examined the effects of focal tissue acidosis in the pre-B?tzinger complex (pre-B?tC; the proposed locus of respiratory rhythm generation) on phrenic nerve discharge in chloralose-anesthetized, vagotomized, paralyzed, mechanically ventilated cats. Focal tissue acidosis was produced by unilateral microinjection of 10-20 nl of the carbonic anhydrase inhibitors acetazolamide (AZ; 50 microM) or methazolamide (MZ; 50 microM). Microinjection of AZ and MZ into 14 sites in the pre-B?tC reversibly increased the peak amplitude of integrated phrenic nerve discharge and, in some sites, produced augmented bursts (i.e., eupneic breath ending with a high-amplitude, short-duration burst). Microinjection of AZ and MZ into this region also reversibly increased the frequency of eupneic phrenic bursts in seven sites and produced premature bursts (i.e., doublets) in five sites. Phrenic nerve discharge increased within 5-15 min of microinjection of either agent; however, the time to the peak increase and the time to recovery were less with AZ than with MZ, consistent with the different pharmacological properties of AZ and MZ. In contrast to other CO(2)/H(+) brain stem respiratory chemosensitive sites demonstrated in vivo, which have only shown increases in amplitude of integrated phrenic nerve activity, focal tissue acidosis in the pre-B?tC increases frequency of phrenic bursts and produces premature (i.e., doublet) bursts. These data indicate that the pre-B?tC has the potential to play a role in the modulation of respiratory rhythm and pattern elicited by increased CO(2)/H(+) and lend additional support to the concept that the proposed locus for respiratory rhythm generation has intrinsic chemosensitivity.     

Synaptic relationship between somatostatin- and neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats

J. Neurochem. (2012) 122, 923-933. ABSTRACT: The pre-B?tzinger complex (pre-B?tC) in the ventrolateral medulla oblongata is critical for the generation of respiratory rhythm in mammals. Somatostatin (SST) and neurokinin 1 receptor (NK1R) immunoreactivity have been used as markers of the pre-B?tC. SST immunoreactivity almost completely overlaps with small fusiform NK1R-immunoreactive (ir) neurons, the presumed rhythmogenic neurons, but not with large multipolar NK1R-ir neurons. Understanding the neurochemical characteristics, especially the synaptic relationship of SST/NK1R-ir neurons within the pre-B?tC network is essential in providing cellular and structural bases for understanding their physiological significance. This work has not been documented so far. We found that SST immunoreactivity was highly expressed in terminals, somas, and primary dendrites in the pre-B?tC. Besides the small fusiform neurons, a small population of medium-sized NK1R-ir neurons also colocalized with SST. Large NK1R-ir neurons were not SST-ir, but received somatostatinergic inputs. SST-ir terminals were glutamatergic or GABAergic, and synapsed with NK1R-ir neurons. Most of synapses between them were of the symmetric type, indicating their inhibitory nature. Asymmetric synapses were evident between SST-ir terminals and NK1R-ir dendrites, strongly suggesting an excitatory innervation from the presumed rhythmogenic neurons as these neurons are glutamatergic. We speculate that SST-mediated excitatory and inhibitory synaptic transmission onto NK1R-ir rhythmogenic and follower neurons synchronizes their activity to contribute to respiratory rhythmogenesis and control.     

Anatomical and functional development of the pre-B?tzinger complex in prenatal rodents.

Developmental anomalies of central respiratory neural control contribute to newborn mortality and morbidity. Elucidation of the cellular, molecular, trophic, and genetic mechanisms involved in the formation and function of respiratory nuclei during prenatal development will provide a foundation for understanding pathologies. The pre-B?tzinger Complex (pre-B?tC) is a specific group of neurons located in the ventrolateral medulla that is critical for respiratory rhythmogenesis. Thus it has become a major focus of research. Here, we provide an overview of current knowledge regarding the anatomical and functional emergence of the rodent pre-B?tC during the prenatal period.     

Cooperation of intrinsic bursting and calcium oscillations underlying activity patterns of model pre-Bötzinger complex neurons

Activity of neurons in the pre-Bötzinger complex (pre-BötC) within the mammalian brainstem drives the inspiratory phase of the respiratory rhythm. Experimental results have suggested that multiple bursting mechanisms based on a calcium-activated nonspecific cationic (CAN) current, a persistent sodium (NaP) current, and calcium dynamics may be incorporated within the pre-BötC. Previous modeling works have incorporated representations of some or all of these mechanisms. In this study, we consider a single-compartment model of a pre-BötC inspiratory neuron that encompasses particular aspects of all of these features. We present a novel mathematical analysis of the interaction of the corresponding rhythmic mechanisms arising in the model, including square-wave bursting and autonomous calcium oscillations, which requires treatment of a system of differential equations incorporating three slow variables.     

Postnatal expression of neurotransmitters, receptors, and cytochrome oxidase in the rat pre-B?tzinger complex.

The pre-B?tzinger complex (PBC) is postulated as the center of respiratory rhythmogenesis. Previously, we found a reduction or plateau of cytochrome oxidase (CO) activity in the PBC and other respiratory nuclei at postnatal days 3-4, despite a general increase of CO with age, suggesting a period of synaptic readjustment. The present study examined the expression of CO and a number of neurochemicals in the PBC at closer time intervals. At postnatal days 3-4 and, more prominently, at postnatal day 12, expression of CO, glutamate, and N-methyl-D-aspartate receptor subunit 1 was reduced, whereas expression of GABA, GABA(B) receptor, glycine receptor, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit 2 was increased. These findings are consistent with our hypothesis that decreased CO activity is associated with an increase in inhibitory drive (mediated by GABA and glycine, their receptors, and possibly blockage of Ca(2+) entry by glutamate receptor subunit 2) and a decrease in excitatory drive (mediated by glutamate and its receptors). Our findings point to two critical periods during postnatal development of the rat when their respiratory system may be more vulnerable to respiratory insults.     

Intrinsic bursting activity in the pre-Bötzinger Complex: Role of persistent sodium and potassium currents

Computational models of single pacemaker neuron and neural population in the pre-Bötzinger Complex (pBC) were developed based on the previous models by Butera et al. (1999a,b). Our modeling study focused on the conditions that could define endogenous bursting vs. tonic activity in single pacemaker neurons and population bursting vs. asynchronous firing in populations of pacemaker neurons. We show that both bursting activity in single pacemaker neurons and population bursting activity may be released or suppressed depending on the expression of persistent sodium (I_NaP) and delayed-rectifier potassium (I_K) currents. Specifically, a transition from asynchronous firing to population bursting could be induced by a reduction of I_K via a direct suppression of the potassium conductance or through an elevation of extracellular potassium concentration. Similar population bursting activity could be triggered by an augmentation of I_NaP. These findings are discussed in the context of the possible role of population bursting activity in the pBC in the respiratory rhythm generation in vivo vs. in vitro and during normal breathing in vivo vs. gasping.     

Small reduction of neurokinin-1 receptor-expressing neurons in the pre-B?tzinger complex area induces abnormal breathing periods in awake goats.

In awake rats, >80% bilateral reduction of neurokinin-1 receptor (NK1R)-expressing neurons in the pre-B?tzinger complex (pre-B?tzC) resulted in hypoventilation and an "ataxic" breathing pattern (Gray PA, Rekling JC, Bocchiaro CM, Feldman JL, Science 286: 1566-1568, 1999). Accordingly, the present study was designed to gain further insight into the role of the pre-B?tzC area NK1R-expressing neurons in the control of breathing during physiological conditions. Microtubules were chronically implanted bilaterally into the medulla of adult goats. After recovery from surgery, the neurotoxin saporin conjugated to substance P, specific for NK1R-expressing neurons, was bilaterally injected (50 pM in 10 microl) into the pre-B?tzC area during the awake state (n = 8). In unoperated goats, 34 +/- 0.01% of the pre-B?tzC area neurons are immunoreactive for the NK1R, but, in goats after bilateral injection of SP-SAP into the pre-B?tzC area, NK1R immunoreactivity was reduced to 22.5 +/- 2.5% (29% decrease, P < 0.01). Ten to fourteen days after the injection, the frequency of abnormal breathing periods was sixfold greater than before injection (107.8 +/- 21.8/h, P < 0.001). Fifty-six percent of these periods were breaths of varying duration and volume with an altered respiratory muscle activation pattern, whereas the remaining were rapid, complete breaths with coordinated inspiratory-expiratory cycles. The rate of occurrence and characteristics of abnormal breathing periods were not altered during a CO2 inhalation-induced hyperpnea. Pathological breathing patterns were eliminated during non-rapid eye movement sleep in seven of eight goats, but they frequently occurred on arousal from non-rapid eye movement sleep. We conclude that a moderate reduction in pre-B?tzC NK1R-expressing neurons results in state-dependent transient changes in respiratory rhythm and/or eupneic respiratory muscle activation patterns.     

Stimulation of bursting in pre-Bötzinger neurons by Epac through calcium release and modulation of TRPM4 and K-ATP channels

The exchange factor directly activated by cAMP (Epac) can couple cAMP production to the activation of particular membrane and cytoplasmic targets. Using patch-clamp recordings and calcium imaging in organotypic brainstem slices, we examined the role of Epac in pre-B?tzinger complex, an essential part of the respiratory network. The selective agonist 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT) sensitized calcium mobilisation from inositol-1,4,5-trisphosphate-sensitive internal stores that stimulated TRPM4 (transient receptor potential cation channel, subfamily M, Melastatin) channels and potentiated the bursts of action potentials. 8-pCPT actions were abolished after inhibition of phospholipase C with U73122 and depletion of calcium stores with thapsigargin. Caffeine-sensitive release channels were not modulated by 8-pCPT. Epac inhibited ATP-sensitive K(+) channels that also led to the enhancement of bursting by 8-pCPT. Bursting activity, spontaneous calcium transients and activity of TRPM4 and ATP-sensitive K(+) channels were potentiated after brief exposures to bradykinin and incubation with wortmannin produced opposite effects that can be explained by changes in phosphatidylinositol 4,5-bisphosphate levels. 8-pCPT stimulated the respiratory motor output in functionally intact preparations and the effects of bradykinin and wortmannin were identical to those observed in organotypic slices. The data thus indicate a novel pathway of controlling bursting activity in pre-B?tzinger complex neurons through Epac that can involved in reinforcement of the respiratory activity by cAMP.     

Function of the IsiA pigment–protein complex in vivo

Photosynthesis Research - The acclimation of cyanobacterial photosynthetic apparatus to iron deficiency is crucial for their performance under limiting conditions. In many cyanobacterial species,...     

Are network motifs the spandrels of cellular complexity?

Cellular networks display modular organization at different levels, from small sets of genes exchanging signals in morphogenesis to large groups of proteins involved in cell death. At the smallest scale, minute groups of interacting proteins or genes, so-called 'network motifs', have been suggested to be the functional building blocks of network biology. In this context, the relative abundance of a network motif would reflect its adaptive value. However, although the overabundance of motifs is non-random, recent studies by Mazurie et al. and by Kuo et al. show that motif abundance does not reflect their true adaptive value. Just as some architectural components emerge as a byproduct of a prior decision, common motifs might be a side effect of inevitable rules of genome growth and change.     

Involvement of Erks activation in cadmium‐induced AP‐1 transactivation in vitro and in vivo

Cadmium is a potent and effective carcinogen in rodents and has recently been accepted by IARC (International Agency for Research on Cancer) as a category 1 carcinogen. Cadmium-induced upregulation of intracellular signaling pathways leading to increased mitogenesis is thought to be a major mechanism for the carcinogenic activity following chronic cadmium exposure. In the present study, we found that exposure of cells to cadmium induced significant activation of AP1 and all three members of the MAP kinase family in mouse epidermal JB6 cells. The induction of AP1 activity by cadmium appears to involve activation of Erks, since the induction of AP1 activity by cadmium was blocked by pretreatment of cells with PD98058. Interestingly, the induction of AP1 by cadmium was greatly enhanced by the chemical tumor promoter, TPA and the growth factor EGF, but not by ultraviolet C radiation. In vivo studies demonstrated that cadmium could also induce transactivation of AP1 in AP1luciferase report transgenic mice. Considering the role of AP1 activation in tumor promotion, the results presented in this study provide a possible molecular mechanism for cadmiuminduced carcinogenesis.