Michael J. Devinney

Hypoxia‐induced phrenic long‐term facilitation: emergent properties

As in other neural systems, plasticity is a hallmark of the neural system controlling breathing. One spinal mechanism of respiratory plasticity is phrenic long‐term facilitation (pLTF) following acute intermittent hypoxia. Although cellular mechanisms giving rise to pLTF occur within the phrenic motor nucleus, different signaling cascades elicit pLTF under different conditions. These cascades, referred to as Q and S pathways to phrenic motor facilitation (pMF), interact via cross‐talk inhibition. Whereas the Q pathway dominates pLTF after mild to moderate hypoxic episodes, the S pathway dominates after severe hypoxic episodes. The biological significance of multiple pathways to pMF is unknown. This review will discuss the possibility that interactions between pathways confer emergent properties to pLTF, including pattern sensitivity and metaplasticity. Understanding these mechanisms and their interactions may enable us to optimize intermittent hypoxia‐induced plasticity as a treatment for patients that suffer from ventilatory impairment or other motor deficits.

The interaction of biological and noxious transition metals with the zinc probes FluoZin-3 and Newport Green.

Zinc-sensitive fluorescent probes have become increasingly important in the investigation of the cellular roles of zinc. There is, however, little information on how the other transition metals in cells may influence the measurement of zinc. We have characterized in vitro the interaction of the nominal zinc indicators FluoZin-3 and Newport Green with all the cationic transition metals found within cells, Cr, Mn, Fe, Co, and Cu, as well as Ni and Cd, by measuring their dissociation constants. In addition, we have shown how FluoZin-3 can be used to quantify the concentration of copper in a cell-free assay and report that the fluorescence of Newport Green is boosted by both Cu(I) and Fe(II). Furthermore, we have introduced diagnostics for detecting the interference of metals other than zinc with its measurement within cells.

Glutamate mobilizes [Zn2+]i through Ca2+-dependent reactive oxygen species accumulation

Liberation of zinc from intracellular stores contributes to oxidant‐induced neuronal injury. However, little is known regarding how endogenous oxidant systems regulate intracellular free zinc ([Zn2+]i). Here we simultaneously imaged [Ca2+]i and [Zn2+]i to study acute [Zn2+]i changes in cultured rat forebrain neurons after glutamate receptor activation. Neurons were loaded with fura‐2FF and FluoZin‐3 to follow [Ca2+]i and [Zn2+]i, respectively. Neurons treated with glutamate (100 μM) for 10 min gave large Ca2+ responses that did not recover after termination of the glutamate stimulus. Glutamate also increased [Zn2+]i, however glutamate‐induced [Zn2+]i changes were completely dependent on Ca2+ entry, appeared to arise entirely from internal stores, and were substantially reduced by co‐application of the membrane‐permeant chelator TPEN during the glutamate treatment. Pharmacological maneuvers revealed that a number of endogenous oxidant producing systems, including nitric oxide synthase, phospholipase A2, and mitochondria all contributed to glutamate‐induced [Zn2+]i changes. We found no evidence that mitochondria buffered [Zn2+]i during acute glutamate receptor activation. We conclude that glutamate‐induced [Zn2+]i transients are caused in part by [Ca2+]i‐induced reactive oxygen species that arises from both cytosolic and mitochondrial sources.

Phrenic Long-Term Facilitation Requires PKCθ Activity within Phrenic Motor Neurons

Acute intermittent hypoxia (AIH) induces a form of spinal motor plasticity known as phrenic long-term facilitation (pLTF); pLTF is a prolonged increase in phrenic motor output after AIH has ended. In anesthetized rats, we demonstrate that pLTF requires activity of the novel PKC isoform, PKCθ, and that the relevant PKCθ is within phrenic motor neurons. Whereas spinal PKCθ inhibitors block pLTF, inhibitors targeting other PKC isoforms do not. PKCθ is highly expressed in phrenic motor neurons, and PKCθ knockdown with intrapleural siRNAs abolishes pLTF. Intrapleural siRNAs targeting PKCζ, an atypical PKC isoform expressed in phrenic motor neurons that underlies a distinct form of phrenic motor plasticity, does not affect pLTF. Thus, PKCθ plays a critical role in spinal AIH-induced respiratory motor plasticity, and the relevant PKCθ is localized within phrenic motor neurons. Intrapleural siRNA delivery has considerable potential as a therapeutic tool to selectively manipulate plasticity in vital respiratory motor neurons.

A comparison of Zn2+- and Ca2+-triggered depolarization of liver mitochondria reveals no evidence of Zn2+-induced permeability transition

Intracellular Zn(2+) toxicity is associated with mitochondrial dysfunction. Zn(2+) depolarizes mitochondria in assays using isolated organelles as well as cultured cells. Some reports suggest that Zn(2+)-induced depolarization results from the opening of the mitochondrial permeability transition pore (mPTP). For a more detailed analysis of this relationship, we compared Zn(2+)-induced depolarization with the effects of Ca(2+) in single isolated rat liver mitochondria monitored with the potentiometric probe rhodamine 123. Consistent with previous work, we found that relatively low levels of Ca(2+) caused rapid, complete and irreversible loss of mitochondrial membrane potential, an effect that was diminished by classic inhibitors of mPT, including high Mg(2+), ADP and cyclosporine A. Zn(2+) also depolarized mitochondria, but only at relatively high concentrations. Furthermore Zn(2+)-induced depolarization was slower, partial and sometimes reversible, and was not affected by inhibitors of mPT. We also compared the effects of Ca(2+) and Zn(2+) in a calcein-retention assay. Consistent with the well-documented ability of Ca(2+) to induce mPT, we found that it caused rapid and substantial loss of matrix calcein. In contrast, calcein remained in Zn(2+)-treated mitochondria. Considered together, our results suggest that Ca(2+) and Zn(2+) depolarize mitochondria by considerably different mechanisms, that opening of the mPTP is not a direct consequence of Zn(2+)-induced depolarization, and that Zn(2+) is not a particularly potent mitochondrial inhibitor.

Phrenic motor neuron TrkB expression is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation

ABSTRACT Phrenic long‐term facilitation (pLTF) is a form of hypoxia‐induced spinal respiratory motor plasticity that requires new synthesis of brain derived neurotrophic factor (BDNF) and activation of its high‐affinity receptor, tropomyosin receptor kinase B (TrkB). Since the cellular location of relevant TrkB receptors is not known, we utilized intrapleural siRNA injections to selectively knock down TrkB receptor protein within phrenic motor neurons. TrkB receptors within phrenic motor neurons are necessary for BDNF‐dependent acute intermittent hypoxia‐induced pLTF, demonstrating that phrenic motor neurons are a critical site of respiratory motor plasticity. HighlightsAcute intermittent hypoxia‐induced respiratory motor plasticity requires BDNF signaling.Phrenic motor neurons express the high affinity BDNF receptor TrkB.Intrapleural siRNA targeting TrkB receptors decreased TrkB receptor expression selectively on phrenic motor neurons.Intrapleural siRNA targeting TrkB receptors abolished BDNF dependent, hypoxia‐induced respiratory motor plasticity.Phrenic motor neurons are a critical site for spinal respiratory motor plasticity.

Sustained Hypoxia Elicits Competing Spinal Mechanisms of Phrenic Motor Facilitation.

Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF), a form of spinal motor plasticity. Competing mechanisms give rise to phrenic motor facilitation (pMF; a general term including pLTF) depending on the severity of hypoxia within episodes. In contrast, moderate acute sustained hypoxia (mASH) does not elicit pMF. By varying the severity of ASH and targeting competing mechanisms of pMF, we sought to illustrate why moderate AIH (mAIH) elicits pMF but mASH does not. Although mAIH elicits serotonin-dependent pLTF, mASH does not; thus, mAIH-induced pLTF is pattern sensitive. In contrast, severe AIH (sAIH) elicits pLTF through adenosine-dependent mechanisms, likely from greater extracellular adenosine accumulation. Because serotonin- and adenosine-dependent pMF interact via cross talk inhibition, we hypothesized that pMF is obscured because the competing mechanisms of pMF are balanced and offsetting during mASH. Here, we demonstrate the following: (1) blocking spinal A2A receptors with MSX-3 reveals mASH-induced pMF; and (2) sASH elicits A2A-dependent pMF. In anesthetized rats pretreated with intrathecal A2A receptor antagonist injections before mASH (PaO2 = 40–54 mmHg) or sASH (PaO2 = 25–36 mmHg), (1) mASH induced a serotonin-dependent pMF and (2) sASH induced an adenosine-dependent pMF, which was enhanced by spinal serotonin receptor inhibition. Thus, competing adenosine- and serotonin-dependent mechanisms contribute differentially to pMF depending on the pattern/severity of hypoxia. Understanding interactions between these mechanisms has clinical relevance as we develop therapies to treat severe neuromuscular disorders that compromise somatic motor behaviors, including breathing. Moreover, these results demonstrate how competing mechanisms of plasticity can give rise to pattern sensitivity in pLTF. SIGNIFICANCE STATEMENT Intermittent hypoxia elicits pattern-sensitive spinal plasticity and improves motor function after spinal injury or during neuromuscular disease. Specific mechanisms of pattern sensitivity in this form of plasticity are unknown. We provide evidence that competing mechanisms of phrenic motor facilitation mediated by adenosine 2A and serotonin 2 receptors are differentially expressed, depending on the pattern/severity of hypoxia. Understanding how these distinct mechanisms interact during hypoxic exposures differing in severity and duration will help explain interesting properties of plasticity, such as pattern sensitivity, and may help optimize therapies to restore motor function in patients with neuromuscular disorders that compromise movement.

Climbing the delirium mountain: is alpine anaesthesia the perioperative cause?

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Spinal activation of protein kinase C elicits phrenic motor facilitation

The protein kinase C family regulates many cellular functions, including multiple forms of neuroplasticity. The novel PKCθ and atypical PKCζ isoforms have been implicated in distinct forms of spinal, respiratory motor plasticity, including phrenic motor facilitation (pMF) following acute intermittent hypoxia or inactivity, respectively. Although these PKC isoforms are critical in regulating spinal motor plasticity, other isoforms may be important for phrenic motor plasticity. We tested the impact of conventional/novel PKC activator, phorbol 12-myristate 13-acetate (PMA) on pMF. Rats given cervical intrathecal injections of PMA exhibited pMF, which was abolished by pretreatment of broad-spectrum PKC inhibitors bisindolymalemide 1 (BIS) or NPC-15437 (NPC). Because PMA fails to activate atypical PKC isoforms, and NPC does not block PKCθ, this finding demonstrates that classical/novel PKC isoforms besides PKCθ are sufficient to elicit pMF. These results advance our understanding of mechanisms producing respiratory motor plasticity, and may inspire new treatments for disorders that compromise breathing, such as ALS, spinal injury and obstructive sleep apnea.

Plasma self-bias and ion acceleration in the madhex helicon source

Summary form only given. Helicon and magnetized radio-frequency (RF) plasmas have been studied for some time. It is often claimed that the ion acceleration observed in these sources is due to double layers. Recently strong (160 V) time averaged self-bias has been observed in MadHeX1, resulting in a large potential difference between the plasma source and expansion regions2, has piqued our interest to further examine and fully realize the ion acceleration process. The modified MadHeX experimental facility consists of a 120 cm long, 10 cm inner diameter Pyrex tube attached to a stainless steel expansion chamber, which is 60 cm long and 45 cm in diameter (expansion ratio RE = 4.5) with an axial magnetic field, variable up to 1.2 kG at the source region that can be operated in flat or nozzle field configurations. An 18 cm long, 12 cm diameter half-turn double-helix antenna is used to excite helicon waves in the source. A new double magnetic mirror and an additional magnetic coil placed between the transition region between plasma source and expansion chamber are used to increase plasma ionization rate and reduce ion-electron recombination and neutral reflux in the expansion region. The effect of RF power, magnetic field strength and gas flow rate on the plasma parameters including electron temperature, density, plasma potential and ion beam acceleration are explored by probe diagnostics (Langmuir probe, emissive probe and retarding potential analyzer) and non-invasive optical techniques (laser induced fluorescence and optical emission spectroscopy). The role of substantial RF fluctuations in the plasma potential and the upstream end-plate boundary condition are addressed. The effect of the electron energy distribution that may include substantial tails on plasma self-bias and the ion beam formation and acceleration is examined by optical emission spectroscopy and cross-checked with the results via using a retarding potential analyzer. Also, its effect on the ion energy distribution is verified by using argon 668 nm laser induced fluorescence.