Calcium imaging of neuronal activity in the most rostral parafacial respiratory group of the newborn rat

Onimaru, Hiroshi; Dutschmann, Mathias

doi:10.1007/s12576-011-0179-2

Cited by 20 publications

(14 citation statements)

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“…Several fluorescent Ca ++ sensitive dyes were surveyed before selecting Fluo-8, a bright dye that overcame quenching by haemoglobin and yielded suitable detection in erythrocytes [30,31]. The Ca ++ affinity of Fluo-8, reported at 389 nM, was also attractive because it should produce a low signal from uninfected erythrocytes, which maintain low intracellular free Ca ++ concentrations through the action of a Ca ++ ATPase pump [32].…”

Section: Resultsmentioning

confidence: 99%

A kinetic fluorescence assay reveals unusual features of Ca++ uptake in Plasmodium falciparum-infected erythrocytes

Zipprer

Neggers

Kushwaha

et al. 2014

Malar J

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BackgroundTo facilitate development within erythrocytes, malaria parasites increase their host cell uptake of diverse solutes including Ca++. The mechanism and molecular basis of increased Ca++ permeability remains less well studied than that of other solutes.MethodsBased on an appropriate Ca++ affinity and its greater brightness than related fluorophores, Fluo-8 was selected and used to develop a robust fluorescence-based assay for Ca++ uptake by human erythrocytes infected with Plasmodium falciparum.ResultsBoth uninfected and infected cells exhibited a large Ca++-dependent fluorescence signal after loading with the Fluo-8 dye. Probenecid, an inhibitor of erythrocyte organic anion transporters, abolished the fluorescence signal in uninfected cells; in infected cells, this agent increased fluorescence via mechanisms that depend on parasite genotype. Kinetic fluorescence measurements in 384-well microplates revealed that the infected cell Ca++ uptake is not mediated by the plasmodial surface anion channel (PSAC), a parasite nutrient channel at the host membrane; it also appears to be distinct from mammalian Ca++ channels. Imaging studies confirmed a low intracellular Ca++ in uninfected cells and higher levels in both the host and parasite compartments of infected cells. Parasite growth inhibition studies revealed a conserved requirement for extracellular Ca++.ConclusionsNondestructive loading of Fluo-8 into human erythrocytes permits measurement of Ca++ uptake kinetics. The greater Ca++ permeability of cells infected with malaria parasites is apparent when probenecid is used to inhibit Fluo-8 efflux at the host membrane. This permeability is mediated by a distinct pathway and may be essential for intracellular parasite development. The miniaturized assay presented here should help clarify the precise transport mechanism and may identify inhibitors suitable for antimalarial drug development.

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Section: Resultsmentioning

confidence: 99%

A kinetic fluorescence assay reveals unusual features of Ca++ uptake in Plasmodium falciparum-infected erythrocytes

Zipprer

Neggers

Kushwaha

et al. 2014

Malar J

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“…Onimaru & Homma, 1992). They received heterogeneous synaptic inputs during the inspiratory phase and more than 70% of them in this region showed only weak inspiratory inhibition (Onimaru et al 2008; Onimaru & Dutschmann, 2012). The electrodes (inner tip diameter, 1.2–2.0 μm; resistance, 4–8 MΩ) were filled with the following pipette solution (in m m ): 130 potassium gluconate, 10 EGTA, 10 Hepes, 2 Na 2 ‐ATP, 1 CaCl 2 and 1 MgCl 2 , with pH 7.2–7.3 adjusted with KOH.…”

Section: Methodsmentioning

confidence: 99%

Postsynaptic mechanisms of CO₂ responses in parafacial respiratory neurons of newborn rats

Onimaru

Ikeda

Kawakami

2012

The Journal of Physiology

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Key points• The central chemoreceptors for respiratory control in the medulla sense changes in CO 2 concentration and regulate respiratory activity.• Neurons that express a transcription factor, Phox2b, in the parafacial region of the rostral and ventrolateral medulla are excited by hypercapnic stimulation and are proposed to play an important role in central chemoreception.• In this study, we show evidence that Phox2b-expressing parafacial neurons in neonatal rats were sensitive to hypercapnia via direct action on the postsynaptic membrane without contribution of putative presynaptic or other calcium-dependent components.• Since these parafacial neurons are also a part of the respiratory rhythm generator in neonates, they are essential for postnatal survival, which is probably due to their contribution to central chemoreception as well as respiratory rhythm generation. AbstractThe parafacial respiratory group (pFRG) in the rostral ventrolateral medulla of the newborn rat is predominantly composed of pre-inspiratory (Pre-I) neurons and is involved in respiratory rhythm generation. The subgroup located close to the ventral surface (at least partially overlapping the retrotrapezoid nucleus, RTN) expresses the Phox2b transcription factor and responds to hypercapnic stimulation with strong depolarization, which suggests it has a role in central chemoreception. Although a CO 2 response of pFRG/RTN neurons has been confirmed in the presence of tetrodotoxin (TTX), it is unknown whether the depolarization involved in this response is induced by a direct postsynaptic response of pFRG/RTN neurons or by any presynaptic components mediated by Ca 2+ -dependent mechanisms. In this study, we examined the effects of ATP or substance P receptor antagonists on hypercapnic responses of rostral pFRG/RTN neurons. We tested effects of Cd 2+ and low Ca 2+ -high Mg 2+ in the presence of TTX. The experiments were performed in in vitro brainstem-spinal cord preparations from newborn rats in which Pre-I neurons reflect the discharge pattern of the pFRG. We found that ATP receptor and substance P receptor antagonists do not block membrane potential responses to hypercapnic stimulation (2% → 8%) of pFRG/RTN neurons in the rostral parafacial region. Moreover, rostral pFRG/RTN neurons were depolarized by hypercapnia under conditions where the contribution of presynaptic components was inhibited in the presence of TTX and Cd 2+ or in a low Ca 2+ -high Mg 2+ solution containing TTX and Cd 2+ . All cases (except some cases in a low Ca 2+ -high Mg 2+ solution) of membrane depolarization by hypercapnic stimulation were accompanied with an increase in input resistance. These neurons were predominantly Phox2b immunoreactive. suggest that the response of pFRG/RTN neurons to hypercapnia is induced by direct action on the postsynaptic membrane via closing of K + channels.

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“…The distribution of pFRG-Pre-I neurons overlaps with that of Phox2b-expressing cells (Figs. 1, 2) [10–12]. It is of note that pFRG-Pre-I neurons in the deeper ventral medulla at the caudal area are Phox2b-negative [10].…”

Section: Section 1: Identification Of the Pfrg In The Respiratory Rhymentioning

confidence: 99%

The respiratory control mechanisms in the brainstem and spinal cord: integrative views of the neuroanatomy and neurophysiology

et al. 2016

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Respiratory activities are produced by medullary respiratory rhythm generators and are modulated from various sites in the lower brainstem, and which are then output as motor activities through premotor efferent networks in the brainstem and spinal cord. Over the past few decades, new knowledge has been accumulated on the anatomical and physiological mechanisms underlying the generation and regulation of respiratory rhythm. In this review, we focus on the recent findings and attempt to elucidate the anatomical and functional mechanisms underlying respiratory control in the lower brainstem and spinal cord.Electronic supplementary materialThe online version of this article (doi:10.1007/s12576-016-0475-y) contains supplementary material, which is available to authorized users.

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Calcium imaging of neuronal activity in the most rostral parafacial respiratory group of the newborn rat

Cited by 20 publications

References 23 publications

A kinetic fluorescence assay reveals unusual features of Ca++ uptake in Plasmodium falciparum-infected erythrocytes

A kinetic fluorescence assay reveals unusual features of Ca++ uptake in Plasmodium falciparum-infected erythrocytes

Postsynaptic mechanisms of CO₂ responses in parafacial respiratory neurons of newborn rats

The respiratory control mechanisms in the brainstem and spinal cord: integrative views of the neuroanatomy and neurophysiology

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Calcium imaging of neuronal activity in the most rostral parafacial respiratory group of the newborn rat

Cited by 20 publications

References 23 publications

A kinetic fluorescence assay reveals unusual features of Ca++ uptake in Plasmodium falciparum-infected erythrocytes

A kinetic fluorescence assay reveals unusual features of Ca++ uptake in Plasmodium falciparum-infected erythrocytes

Postsynaptic mechanisms of CO2 responses in parafacial respiratory neurons of newborn rats

The respiratory control mechanisms in the brainstem and spinal cord: integrative views of the neuroanatomy and neurophysiology

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Postsynaptic mechanisms of CO₂ responses in parafacial respiratory neurons of newborn rats